Chaoyong Yang

Using cell-based aptamer selection, we have developed a strategy to use the differences at the molecular level between any two types of cells for the identification of molecular signatures on the surface of targeted cells. A group of aptamers have been generated for the specific recognition of leukemia cells. The selected aptamers can bind to target cells with an equilibrium dissociation constant (K(d)) in the nanomolar-to-picomolar range. The cell-based selection process is simple, fast, straightforward, and reproducible, and, most importantly, can be done without prior knowledge of target molecules. The selected aptamers can specifically recognize target leukemia cells mixed with normal human bone marrow aspirates and can also identify cancer cells closely related to the target cell line in real clinical specimens. The cell-based aptamer selection holds a great promise in developing specific molecular probes for cancer diagnosis and cancer biomarker discovery.

Abstract Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single‐nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal‐transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem–loop structure holds the fluorescence‐donor and fluorescence‐acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein–DNA interaction studies, and protein recognition.

Make it a double: Complementary probes with two pyrene labels were engineered for the amplification of a target DNA sequence. In the stem-closed conformation of the probes in the absence of the target, the two pyrene moieties were separated. The target propagated hybridization chain reactions to bring pyrene moieties on neighboring probes close together to form fluorescent pyrene excimers (see picture).

Fluorescent labeling based on silica nanoparticles facilitates unique applications in bioanalysis and bioseparation. Dye-doped silica nanoparticles have significant advantages over single-dye labeling in signal amplification, photostability and surface modification for various biological applications. We have studied the formation of tris(2,2‘-bipyridyl)dichlororuthenium(II) (Ru(bpy)) dye-doped silica nanoparticles by ammonia-catalyzed hydrolysis of tetraethyl orthosilicate (TEOS) in water-in-oil microemulsion. The fluorescence spectra, particle size, and size distribution of Ru(bpy) dye-doped silica nanoparticles were examined as a function of reactant concentrations (TEOS and ammonium hydroxide), nature of surfactant molecules, and molar ratios of water to surfactant (R) and cosurfactant to surfactant (p). The particle size and fluorescence spectra were dependent upon the type of microemulsion system chosen. The particle size was found to decrease with an increase in concentration of ammonium hydroxide and increase in water to surfactant molar ratio (R) and cosurfactant to surfactant molar ratio (p). This optimization study of the preparation of dye-doped silica nanoparticles provides a fundamental knowledge of the synthesis and optical properties of Ru(bpy) dye-doped silica nanoparticles. With this information, these nanoparticles can be easily manipulated, with regard to particle size and size distribution, and bioconjugated as needed for bioanalysis and bioseparation applications.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38 ± 9 and 67 ± 8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

Direct, label-free detection of unmodified DNA is a great challenge for DNA analyses. Surface-enhanced Raman spectroscopy (SERS) is a promising tool for DNA analyses by providing intrinsic chemical information with a high sensitivity. To address the irreproducibility in SERS analysis that hampers reliable DNA detection, we used iodide-modified Ag nanoparticles to obtain highly reproducible SERS signals of single- and double-strand DNA in aqueous solutions close to physiological conditions. The phosphate backbone signal was used as an internal standard to calibrate the absolute signal of each base for a more reliable determination of the DNA structure, which has not been achieved before. Clear identification of DNA with single-base sensitivity and the observation of a hybridization event have been demonstrated.

Cocaine cracked: The gel–sol transition of an enzyme-caged hydrogel has been efficiently controlled by target binding events, which trigger release of the enzyme to take part in its catalytic role for signal amplification (see picture). As low as 20 ng of cocaine can be visually detected within 10 min without any aid of sophisticated instrumentation. Visual detection is an increasingly attractive method in many fields because both qualitative and semiquantitative assessment can be performed in real time without any advanced or complicated instrumentation. It is especially useful for rapid diagnostics in disaster situations, home healthcare settings, and in poorly equipped rural areas, where low cost, rapidity, and simplicity are essential. A variety of colorimetric reagents, such as visible dyes,1 polymers,2 enzymes,3 and gold nanoparticles (AuNPs),4–9 can be used for visual detection of specific targets. The color change of these reagents is based on diverse, yet selective, molecular interactions. Examples include stimuli-induced release or absorbance of dye molecules, polymers whose color changes are initiated by target binding, or enzymatic reactions triggered by molecular recognition. Meanwhile, “stimuli-responsive” or “smart” hydrogels have attracted particular attention in the development of biosensor devices that utilize a broad spectrum of triggers, including temperature, pH, ionic strength, and electric field. However, most biosensing devices operate on the basis of mechanical work performed by gel swelling and shrinking, or property changes of free-swelling gels, such as changes in optical transmission,10 refractive index,11 or resonance frequency,12 most of which must rely on time-consuming manipulation and sophisticated instruments. Herein, we propose a colorimetric agent-caging hydrogel as a novel visual detection platform that relies on DNA base-pair recognition and aptamer–target interactions for simple and rapid target detection with the naked eye. Figure 1 illustrates the working principle of our visual detection method. Two pieces of DNA, strand A and strand B, are grafted onto linear polyacrylamide polymers to form polymer strands A and B (PS-A and PS-B), respectively. The sequences of DNA strands A and B are complementary to an adjacent area of a DNA aptamer sequence. When mixed in equal amounts, the polymers grafted with strand A and strand B are in transparent liquid form. The addition of aptamer linker-Apt initiates hybridization of strand A and strand B with the aptamer sequence, thus cross-linking the linear polyacrylamide polymers. As the hybridization proceeds, the cross-linking ratio of polyacrylamide increases, which results in the increase of viscosity of the polymer solution. The polymer will finally transform into a gel.13 Upon introduction of a target, the aptamer will bind with it, and the gel will be dissolved as a result of reducing the cross-linking density by competitive target–aptamer binding.14 If an enzyme is added prior to the addition of the aptamer, the enzyme will be trapped inside the 3D network of the hydrogel (represented as pink symbols in Figure 1). When target molecules are introduced to dissolve the gel, the enzyme is released and can take part in its catalytic role for signal amplification. A cascade of events is thus set in motion, whereby target binding triggers an enzymatic reaction, which, in turn, changes the substrate color, thus allowing visual detection. Because our aptamer cross-linked hydrogel colorimetric platform can be targeted to any ligand for which there is a corresponding aptamer,15 we anticipate that it will find many visual detection applications in a wide variety of fields. Working principle of DNA cross-linked hydrogel for signal amplification and visual detection. There can be no argument against the statement that drug misuse is a major challenge confronting public health and law enforcement. In this work, cocaine was used as the model target to test our new visual sensing method. A cocaine aptamer has previously been obtained by Landry’s research group through an in vitro selection process,17 and has been already used for the design of several aptasensors.18 Our design of cocaine strands A and B and linker-Apt have been adopted from the recent report of the Lu group using gold nanoparticles and an aptamer for colorimetric cocaine sensors.18b, 18c To systematically study the principles of the hydrogel platform and to optimize the system, we trapped gold nanoparticles (AuNPs) inside the hydrogel. AuNPs were adopted as indicating reagents or signal-amplifying agents based on their unique optical properties and chemical stability. Firstly, gold nanoparticles with diameters of only a few nanometers can be easily obtained. Such a diameter range is equivalent to that of most enzymes (3–15 nm); therefore, the behavior of hydrogel-trapped enzymes can be extrapolated by studying that of gold nanoparticles. Secondly, and more importantly, the remarkably large extinction coefficient of AuNPs at the visible wavelength (around 520 nm) makes them a sensitive indicating reagent for visual detection. Thus, either trapping or release of AuNPs by the aptamer cross-linked hydrogel through molecular recognition can be directly visualized by their characteristic red color. In our experiment, 13 nm water-soluble AuNPs were prepared by following an established protocol,19 and modified with bovine serum albumin (BSA) to avoid aggregation caused by the high salt concentration. Before addition of linker-Apt, the modified AuNPs were added into the sol system, and were mixed thoroughly with PS-A and PS-B. After introduction of linker-Apt, a homogeneous red-colored hydrogel formed with evenly dispersed AuNPs trapped inside. After washing three times with buffer solution to remove surface-bound AuNPs, the gel was placed in a buffer solution and was found to remain in gel form. In buffer solution, the gel appeared red, while the upper buffer solution layer remained colorless (Figure 2 a). Upon addition of the target, the gel dissolved and released AuNPs to the upper layer of the buffer solution. As a result, the buffer solution turned from colorless to intense red, a change that can be easily seen with the naked eye. Release of AuNPs from the hydrogel upon introduction of cocaine. a) Photograph of the hydrogel before (left) and 30 min after (right) addition of cocaine. Four hydrogels with different DNA cross-linking densities (0.1–0.7 mM) were prepared to study cargo release kinetics. AuNPs were trapped in the DNA hydrogel with a cover layer of 10 mM tris(hydroxymethyl)aminomethane (Tris-HCl) buffer (pH 8.0, 200 mM NaCl). Upon introduction of 1 mM cocaine, AuNPs were released from the DNA hydrogel to form a uniform red solution. b) Release kinetics of AuNPs from four types of hydrogels upon introduction of cocaine. The encapsulating stability of each hydrogel was examined for 30 min before addition of 1 mM cocaine. The greatest response sensitivity in such a sensing scheme relies on optimizing the hydrogel pore size to maximize the diffusion rate of target molecules into the gel for target recognition and rapid detection, while minimizing the nonspecific leaking of cargoes to avoid false positive results. The pore size of the gel is determined by the cross-linking ratio of DNA. Accordingly, four hydrogels with different DNA cross-linking densities (0.1, 0.3, 0.5, 0.7 mM) were prepared, and the kinetics of target-triggered release of AuNPs from hydrogels was investigated by both the naked eye and UV/Vis spectrometry (Figure 2). The gel was prepared with AuNPs that were encapsulated and placed at the bottom of a quartz microcell with a buffer solution on top. The release of AuNPs to the buffer solution over time could be quantitatively monitored through the strong AuNP absorption at 520 nm. The absorption curves on the buffer solution from the four types of hydrogels during the release of AuNPs are shown in Figure 2 b. The gels were monitored for 30 min before the introduction of 1 mM cocaine in order to check the encapsulating stability of the hydrogel. The 0.1 mM hydrogel showed the fastest response, but the lowest encapsulating stability. The 0.3 mM and 0.5 mM hydrogels gave a similar response; the 0.5 mM hydrogel had a lower background, as well as somewhat slower kinetics. As for the 0.7 mM hydrogel, the response was much slower and did not reach equilibrium during the monitoring period. The quantitative results indicate a 3.7 times signal-to-background difference for the 0.1 mM hydrogel, 8.1 times for the 0.3 mM hydrogel, 11 times for the 0.5 mM hydrogel, and 7.7 times for the 0.7 mM hydrogel. In particular, if the readable signal was set to be three times higher than the background signal, it took less than 10 min for all these four types of gel to reach their three-times signal-to-background difference, which indicated fast detection. Figure 2 a shows photographs taken 30 min after introducing 1 mM cocaine, when the reactions were almost completed. The tubes on the left are the control experiments under the same working conditions without cocaine. By correlating with the spectrometric data, leaking is a problem for the 0.1 mM hydrogel, and the 0.7 mM hydrogel has a slower reaction rate. In contrast, the 0.3 mM and 0.5 mM hydrogels gave the best results. This difference among four hydrogels clearly demonstrated the concentration-dependent encapsulation and release capability upon target binding. That is, low-concentration cross-linking hydrogels tend to dissolve much faster and easier than high-concentration cross-linking hydrogels, but have a stability problem. On the other hand, high-concentration cross-linking hydrogels might have slower kinetics for the gel–sol transition, and thus prolong the detection time. As a consequence, the optimal condition was determined empirically to be the 0.5 mM DNA cross-linker concentration, which was used in the next step. The AuNP model also suggested that nanoparticles or molecules with dimensions of approximately 10 nm can be doped inside the hydrogel and then released. As a further step, we attempted to introduce an enzyme into the gel system. A common test for amylose is to mix it with a small amount of iodine solution. The amylose induces a color change from yellow to dark blue. On the other hand, amylase can break amylose down into sugar, which is colorless in the presence of iodine. Even though these two phenomena are well known, they have not, to the best of our knowledge, been combined into a colorimetric sensing platform. Therefore, we chose the amylose–I2–amylase system because of the specificity of its color change, the fact that no toxic reagents are involved, and the simplicity and cost-effectiveness of its operation. More importantly, both amlyose and amylase are large polymers with high molecular weight. As a result, they can be separated physically by the hydrogel, with amylase trapped inside the gel and amylose outside the gel. Therefore, no amylose is digested by amylase unless the enzyme is released as a result of gel dissolution upon target recognition. However, once the target dissolves a certain area of the hydrogel and releases enough amylase, the color change would be sufficiently distinguishable to draw a clinically sound conclusion, even though the whole gel is not completely dissolved. Hence, the use of enzymes for signal amplification and colorimetric reaction delivers a method for visual detection with high sensitivity. Because the complex formed between amylose and I2 might affect the enzyme function, I2 solution was introduced 10 min later as the last step in order to evaluate the results of the reaction. Similar to the trapping procedure for AuNPs, an amylase-caged hydrogel was prepared by adding linker-Apt into a well-mixed solution containing PS-A, PS-B, and amylase. The loading capacity of amylase for hydrogels was found to be as high as 2 μg per 10 μL gel. After introduction of linker-Apt, a homogeneous colorless hydrogel formed with evenly dispersed enzyme trapped inside. No change of catalytic activity of the enzyme was observed after trapping, thus suggesting that the trapping process is very mild. The enzyme–hydrogel response to cocaine was investigated by visually observing the reaction in an Eppendorf tube (Figure 3 a). Several tubes were prepared: In tubes 1 and 2, no amylase was trapped in the gel. Tube 1 had gel on the bottom and a blue solution of the amylose–I2 complex on top. No color change or gel dissolution was observed. 1 mM cocaine was introduced into tube 2, and the gel was totally dissolved. Since no enzyme was trapped, only a homologous blue solution was obtained. The gel in tube 3 was preloaded with amylase. However, without the target, tube 3 behaved in a manner similar to tube 1, where amylase and amylose blue solutions were well separated by the gel. Then, different amounts of cocaine were introduced into the upper solution of tubes 4–7. In tube 4 with cocaine, the gel dissolved and the solution was colorless. In tubes 5 and 6, a much smaller amount of cocaine was added, which was not enough to completely dissolve the gel, and the solution was colorless after introduction of I2. This result occurred because the gel partially dissolved and released enough enzyme to hydrolyze the amylose. In this regard, even 10 μM cocaine, which was only 100 ng in our experimental conditions, could be detected directly with the naked eye. We also tried to lower the cocaine concentration to 2 μM in tube 7. Although the blue color did not fade completely, it could still be distinguished from tube 3. From the comparison of tubes 1–7, we demonstrated how the introduction of an enzyme reaction into this system amplifies the signal and enables the direct detection of lower amounts of target with the naked eye, thus improving the overall sensitivity of this visual detection method. Photograph of gels with enzymatic reaction for visual detection of cocaine, I2 solution was always introduced 10 min later as the last step to evaluate the results of the reaction. a) Gel response to different amounts of cocaine. b) Control tests for two cocaine analogues, benzoylecgonine (BE) and ecgonine methyl ester (EME). It has been reported that two cocaine metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME) have no affinity for the cocaine aptamer,17 and should therefore not cause hydrogel dissolution. We then used these two metabolites as negative controls. Our results indicated that even at a concentration of 1 mM, neither benzoylecgonine (BE) nor ecgonine methyl ester (EME) caused gel dissolution or color fading (Figure 3 b), thus confirming that the gel–sol transition and enzymatic reaction were indeed triggered by cocaine–aptamer recognition. It should be noted that this aptamer sequence has been found to bind with steroids20 and quinine.21 To use the sensor developed for cocaine detection, one should consider the potential false positive signal caused by these interferences. An aptamer with better selectivity is thus much desirable. In conclusion, we have demonstrated the general design for a colorimetric visual detection platform based on an aptamer cross-linked hydrogel. Competitive binding of the target to the aptamer causes the reduction of cross-linking density and therefore induces gel dissolution. We were able to use this simple system to detect less than 20 ng of cocaine with the naked eye within 10 min. This result is comparable to the most sensitive methods18 reported to date, but can be achieved without the aid of sophisticated instrumentation. As no special features on the aptamers are required, our technique might be a generic approach that can be applied with different aptamer sequences for the detection of other molecules. Since the hydrogel is convenient for either micro- or nanopatterning, this colorimetric visual detection platform can be further developed into lab-on-a-chip devices for diversified applications, such as forensic analysis, medical diagnostics, and environmental monitoring. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from approximately 400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (approximately 40 ns) than that of the background (approximately 5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.

The past decade has witnessed ongoing progress in precision medicine to improve human health. As an emerging diagnostic technique, liquid biopsy can provide real-time, comprehensive, dynamic physiological and pathological information in a noninvasive manner, opening a new window for precision medicine. Liquid biopsy depends on the sensitive and reliable detection of circulating targets (e.g., cells, extracellular vesicles, proteins, microRNAs) from body fluids, the performance of which is largely governed by recognition ligands. Aptamers are single-stranded functional oligonucleotides, capable of folding into unique tertiary structures to bind to their targets with superior specificity and affinity. Their mature evolution procedure, facile modification, and affinity regulation, as well as versatile structural design and engineering, make aptamers ideal recognition ligands for liquid biopsy. In this review, we present a broad overview of aptamer-based liquid biopsy techniques for precision medicine. We begin with recent advances in aptamer selection, followed by a summary of state-of-the-art strategies for multivalent aptamer assembly and aptamer interface modification. We will further describe aptamer-based micro-/nanoisolation platforms, aptamer-enabled release methods, and aptamer-assisted signal amplification and detection strategies. Finally, we present our perspectives regarding the opportunities and challenges of aptamer-based liquid biopsy for precision medicine.

Portable devices with the advantages of rapid, on-site, user-friendly, and cost-effective assessment are widely applied in daily life. However, only a limited number of quantitative portable devices are commercially available, among which the personal glucose meter (PGM) is the most successful example and has been the most widely used. However, PGMs can detect only blood glucose as the unique target. Here we describe a novel design that combines a glucoamylase-trapped aptamer-cross-linked hydrogel with a PGM for portable and quantitative detection of non-glucose targets. Upon target introduction, the hydrogel collapses to release glucoamylase, which catalyzes the hydrolysis of amylose to produce a large amount of glucose for quantitative readout by the PGM. With the advantages of low cost, rapidity, portability, and ease of use, the method reported here has the potential to be used by the public for portable and quantitative detection of a wide range of non-glucose targets.

The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The Kd values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, respectively. Simulated interaction modeling, along with competitive experiments, suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2 and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.

Molecular beacon DNA probes, containing 1-4 pyrene monomers on the 5' end and the quencher DABCYL on the 3' end, were engineered and employed for real-time probing of DNA sequences. In the absence of a target sequence, the multiple-pyrene labeled molecular beacons (MBs) assumed a stem-closed conformation resulting in quenching of the pyrene excimer fluorescence. In the presence of target, the beacons switched to a stem-open conformation, which separated the pyrene label from the quencher molecule and generated an excimer emission signal proportional to the target concentration. Steady-state fluorescence assays resulted in a subnanomolar limit of detection in buffer, whereas time-resolved signaling enabled low-nanomolar target detection in cell-growth media. It was found that the excimer emission intensity could be scaled by increasing the number of pyrene monomers conjugated to the 5' terminal. Each additional pyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields, and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime ( approximately 40 ns), large Stokes shift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternative to traditional fluorophore labeling in nucleic acid probes.

Abstract Point‐of‐care testing (POCT) with the advantages of speed, simplicity, portability, and low cost is critical for the measurement of analytes in a variety of environments where access to laboratory infrastructure is lacking. While qualitative POCTs are widely available, quantitative POCTs present significant challenges. Here we describe a novel method that integrates an Au core/Pt shell nanoparticle (Au@PtNP) encapsulated target‐responsive hydrogel with a volumetric bar‐chart chip (V‐Chip) for quantitative POCT. Upon target introduction, the hydrogel immediately dissolves and releases Au@PtNPs, which can efficiently catalyze the decomposition of H 2 O 2 to generate a large volume of O 2 to move of an ink bar in the V‐Chip. The concentration of the target introduced can be visually quantified by reading the traveling distance of the ink bar. This method has the potential to be used for portable and quantitative detection of a wide range of targets without any external instrument.

Abstract Circulating tumor cell (CTC)‐enrichment by using aptamers has a number of advantages, but the issue of compromised binding affinities and stabilities in real samples hinders its wide applications. Inspired by the high efficiency of the prey mechanism of the octopus, we engineered a deterministic lateral displacement (DLD)‐patterned microfluidic chip modified with multivalent aptamer‐functionalized nanospheres (AP‐Octopus‐Chip) to enhance capture efficiency. The multivalent aptamer–antigen binding efficiency improves 100‐fold and the capture efficiency is enhanced more than 300 % compared with a monovalent aptamer‐modified chip. Moreover, the captured cancer cells can be released through a thiol exchange reaction with up to 80 % efficiency and 96 % viability, which is fully compatible with downstream mutation detection and CTC culture. Using the chip, we were able to find CTCs in all cancer samples analyzed.

Based on graphene oxide-protected DNA probes, we have developed a cyclic enzymatic amplification method for sensitive miRNA detection in complex biological samples. By using the quenching nature of graphene oxide for multiple fluorophores, this method can distinguish highly similar miRNA sequences and detect them simultaneously.

A disposable, equipment-free, versatile point-of-care testing platform, microfluidic distance readout sweet hydrogel integrated paper-based analytical device (μDiSH-PAD), was developed for portable quantitative detection of different types of targets. The platform relies on a target-responsive aptamer cross-linked hydrogel for target recognition, cascade enzymatic reactions for signal amplification, and microfluidic paper-based analytic devices (μPADs) for visual distance-based quantitative readout. A "sweet" hydrogel with trapped glucoamylase (GA) was synthesized using an aptamer as a cross-linker. When target is present in the sample, the "sweet" hydrogel collapses and releases enzyme GA into the sample, generating glucose by amylolysis. A hydrophilic channel on the μPADs is modified with glucose oxidase (GOx) and colorless 3,3'-diaminobenzidine (DAB) as the substrate. When glucose travels along the channel by capillary action, it is converted to H2O2 by GOx. In addition, DAB is converted into brown insoluble poly-3,3'-diaminobenzidine [poly(DAB)] by horseradish peroxidase, producing a visible brown bar, whose length is positively correlated to the concentration of targets. The distance-based visual quantitative platform can detect cocaine in urine with high selectivity, sensitivity, and accuracy. Because the target-induced cascade reaction is triggered by aptamer/target recognition, this method is widely suitable for different kinds of targets. With the advantages of low cost, ease of operation, general applicability, and disposability with quantitative readout, the μDiSH-PAD holds great potential for portable detection of trace targets in environmental monitoring, security inspection, personalized healthcare, and clinical diagnostics.

Simultaneous targeted cancer imaging, therapy and real-time therapeutic monitoring can prevent over- or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near-infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH-activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer-specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real-time imaging. Upon NIR irradiation, R16FP-mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.

Abstract Immunotherapy has revolutionized cancer treatment, but its efficacy is severely hindered by the lack of effective predictors. Herein, we developed a homogeneous, low‐volume, efficient, and sensitive exosomal programmed death‐ligand 1 (PD‐L1, a type of transmembrane protein) quantitation method for cancer diagnosis and immunotherapy response prediction (HOLMES‐Exo PD‐L1 ). The method combines a newly evolved aptamer that efficiently binds to PD‐L1 with less hindrance by antigen glycosylation than antibody, and homogeneous thermophoresis with a rapid binding kinetic. As a result, HOLMES‐Exo PD‐L1 is higher in sensitivity, more rapid in reaction time, and easier to operate than existing enzyme‐linked immunosorbent assay (ELISA)‐based methods. As a consequence of an outstanding improvement of sensitivity, the level of circulating exosomal PD‐L1 detected by HOLMES‐Exo PD‐L1 can effectively distinguish cancer patients from healthy volunteers, and for the first time was found to correlate positively with the metastasis of adenocarcinoma. Overall, HOLMES‐Exo PD‐L1 brings a fresh approach to exosomal PD‐L1 quantitation, offering unprecedented potential for early cancer diagnosis and immunotherapy response prediction.

Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single molecule/cell analysis. The hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment for long-term single cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol-gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol-gel regulation provides a new way to maintain phenotype-genotype linkages in the hydrogel matrix for high throughput molecular evolution. In this Account, we will review the hydrogel droplet generation on microfluidics, single molecule/cell encapsulation in hydrogel droplets, as well as the progress made by our group and others in the application of hydrogel droplet microfluidics for single molecule/cell analysis, including single cell culture, single molecule/cell detection, single cell sequencing, and molecular evolution.

The COVID-19 pandemic caused by SARS-CoV-2 is threating global health. Inhibiting interaction of the receptor-binding domain of SARS-CoV-2 S protein (SRBD ) and human ACE2 receptor is a promising treatment strategy. However, SARS-CoV-2 neutralizing antibodies are compromised by their risk of antibody-dependent enhancement (ADE) and unfavorably large size for intranasal delivery. To avoid these limitations, we demonstrated an aptamer blocking strategy by engineering aptamers' binding to the region on SRBD that directly mediates ACE2 receptor engagement, leading to block SARS-CoV-2 infection. With aptamer selection against SRBD and molecular docking, aptamer CoV2-6 was identified and applied to prevent, compete with, and substitute ACE2 from binding to SRBD . CoV2-6 was further shortened and engineered as a circular bivalent aptamer CoV2-6C3 (cb-CoV2-6C3) to improve the stability, affinity, and inhibition efficacy. cb-CoV2-6C3 is stable in serum for more than 12 h and can be stored at room temperature for more than 14 days. Furthermore, cb-CoV2-6C3 binds to SRBD with high affinity (Kd =0.13 nM) and blocks authentic SARS-CoV-2 virus with an IC50 of 0.42 nM.

Up to 90% of cancer-related deaths are caused by metastatic cancer. Circulating tumor cells (CTCs), a type of cancer cell that spreads through the blood after detaching from a solid tumor, are essential for the establishment of distant metastasis for a given cancer. As a new type of liquid biopsy, analysis of CTCs offers the possibility to avoid invasive tissue biopsy procedures with practical implications for diagnostics. The fundamental challenges of analyzing and profiling CTCs are the extremely low abundances of CTCs in the blood and the intrinsic heterogeneity of CTCs. Various technologies have been proposed for the enrichment and single-cell analysis of CTCs. This review aims to provide in-depth insights into CTC analysis, including various techniques for isolation of CTCs with capture methods based on physical and biochemical principles, and single-cell analysis of CTCs at the genomic, proteomic and phenotypic level, as well as current developmental trends and promising research directions.

Equipment-free devices with quantitative readout are of great significance to point-of-care testing (POCT), which provides real-time readout to users and is especially important in low-resource settings.

The popularization of point-of-care (POC) testing is of great importance in healthcare diagnostics and other fields in the developing world, especially in resource-limited settings. To date, there are still great challenges in the development of simple, quick, affordable, yet highly effective and selective biosensors for POC testing. To meet the increasing need for POC testing, researchers need to consider biosensor miniaturization. In this review, we focus on recent advances in miniaturized biosensors for POC testing. We first review the miniaturization strategies, including handheld instruments and microfluidics-assisted miniaturized biosensing systems. Recent progress in recognition biosensing interactions for miniaturized biosensing systems is then summarized. We further describe how POC testing applications can be realized based on developments in integration, automation and multiplexing. Finally, the future prospects and remaining challenges of miniaturized biosensors for POC testing are discussed.

Abstract Tumor‐derived exosomal proteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is hindered by large numbers of normal cell‐derived exosomes. Herein, we developed a dual‐ t arget‐specific aptamer r ecognition a ctivated in situ c onnection system on e xosome membrane combined with droplet digital PC R (ddPCR) (TRACER) for quantitation of tumor‐derived exosomal PD‐L1 (Exo‐ PD‐L1 ). Leveraging the high binding affinity of aptamers, excellent selectivity of dual‐aptamer recognition, and the high sensitivity of ddPCR, this method exhibits significant sensitivity and selectivity for tracing tumor‐derived Exo‐ PD‐L1 in a wash‐free manner. Due to the excellent sensitivity, the level of tumor‐derived Exo‐ PD‐L1 detected by TRACER can distinguish cancer patients from healthy donors, and for the first time was identified as a more reliable tumor diagnostic marker than total Exo‐ PD‐L1 . The TRACER strategy holds great potential for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes.

The ubiquitous biomembrane interface, with its dynamic lateral fluidity, allows membrane-bound components to rearrange and localize for high-affinity multivalent ligand–receptor interactions in diverse life activities. Inspired by this, we herein engineered a fluidic multivalent nanointerface by decorating a microfluidic chip with aptamer-functionalized leukocyte membrane nanovesicles for high-performance isolation of circulating tumor cells (CTCs). This fluidic biomimetic nanointerface with active recruitment-binding afforded significant affinity enhancement by 4 orders of magnitude, exhibiting 7-fold higher capture efficiency compared to a monovalent aptamer functionalized-chip in blood. Meanwhile, this soft nanointerface inherited the biological benefits of a natural biomembrane, minimizing background blood cell adsorption and maintaining excellent CTC viability (97.6%). Using the chip, CTCs were successfully detected in all cancer patient samples tested (17/17), suggesting the high potential of this fluidity-enhanced multivalent binding strategy in clinical applications. We expect this bioengineered interface strategy will lead to the design of innovative biomimetic platforms in the biomedical field by leveraging natural cell–cell interaction with a natural biomaterial.

A simple microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay was developed for rapid COVID-19 detection.

Liquid biopsy offers non-invasive and real-time molecular profiling of individual patients, and is thus considered a revolutionary technology in precision medicine. Exosomes have been acknowledged as significant biomarkers in liquid biopsy, as they play a central role in cell-cell communication and are closely related to the pathogenesis of most human malignancies. Nevertheless, in biofluids exosomes always co-exist with other particles, and the cargo components of exosomes are highly heterogeneous. Thus, the isolation and molecular characterization of exosomes are still technically challenging. Microfluidics technology effectively addresses this challenge by virtue of its inherent advantages, such as precise manipulation of fluids, low consumption of samples and reagents, and a high level of integration. Recent advances in microfluidics allow in situ exosome capture and molecular detection with unprecedented selectivity and sensitivity. In this review, the state-of-the-art developments in microfluidics-based exosome research, including exosome isolation approaches and molecular detection strategies, with highlights of the characterization of exosomal biomarkers in cancer liquid biopsy is summarized. The major challenges are also discussed and some perspectives for the future directions of exosome-based liquid biopsy in microfluidic systems are presented.

Recently, lysosome targeting chimeras (LYTACs) have emerged as a promising technology that expands the scope of targeted protein degradation to extracellular targets. However, the preparation of chimeras by conjugation of the antibody and trivalent N-acetylgalactosamine (tri-GalNAc) is a complex and time-consuming process. The large uncertainty in number and position and the large molecular weights of the chimeras result in low internalization efficiency. To circumvent these problems, we developed the first aptamer-based LYTAC (Apt-LYTAC) to realize liver-cell-specific degradation of extracellular and membrane proteins by conjugating aptamers to tri-GalNAc. Taking advantage of the facile synthesis and low molecular weight of the aptamer, the Apt-LYTACs can efficiently and quickly degrade the extracellular protein PDGF and the membrane protein PTK7 through a lysosomal degradation pathway. We anticipate that the novel Apt-LYTACs will expand the usage of aptamers and provide a new dimension for targeted protein degradation.

We have synthesized dual-luminophore-doped silica nanoparticles for multiplexed signaling in bioanalysis. Two luminophores, Tris(2,2‘-bipyridyl)osmium(II)bis(hexafluorophosphate) (OsBpy) and Tris(2,2‘-bipyridyl)dichlororuthenium(II)hexahydrate (RuBpy), were simultaneously entrapped inside silica nanoparticles at precisely controlled ratios, with desirable sizes and required surface functionality. Single-wavelength excitation with dual emission endows the nanoparticles with optical encoding capability for rapid and high-throughput multiplexed detection. The nanoparticles can be prepared with sizes ranging from a few nanometers to a few hundred nanometers, with specific ratios of luminescence intensities at two well-resolved wavelengths and with excellent reproducibility. These nanoparticles also possess unique properties of high signal amplification, excellent photostability, and easy surface bioconjugation for highly sensitive measurements when used as signaling markers. A simplified ligand binding system using avidin−biotin and an application extension to immunoassays have been explored, demonstrating the potential use of these easily obtainable bioconjugated nanoparticles for multiplexed signaling and bioassays.

A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (microDG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the microDG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation. After bulk PCR amplification, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry. DNA targets ranging in size from 380 to 1139 bp at single molecule concentrations are quantitatively amplified using SCGA. Long-range sequencing results from beads each carrying approximately 100 amol of a 624 bp product demonstrate that these amplicons are competent for achieving attomole-scale Sanger sequencing from a single bead and for advancing pyrosequencing read-lengths. Successful single cell analysis of the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene in human lymphocyte cells and of the gyr B gene in bacterial Escherichia coli K12 cells establishes that SCGA will also be valuable for performing high-throughput genetic analysis on single cells.

A novel LNA-MB (molecular beacon based on locked nucleic acid bases) has been designed and investigated. It exhibits very high melting temperature and is thermally stable, shows superior single base mismatch discrimination capability, and is stable against digestion by nuclease and has no binding with single-stranded DNA binding proteins. The LNA-MB will be widely useful in a variety of areas, especially for in vivo hybridization studies.

The active targeting of drugs in a cell-, tissue- or disease-specific manner represents a potentially powerful technology with widespread applications in medicine, including the treatment of cancers. Aptamers have properties such as high affinity and specificity for targets, easy chemical synthesis and modification, and rapid tissue penetration. They have become attractive molecules in diagnostics and therapeutics rivaling and, in some cases, surpassing other molecular probes, such as antibodies. In this review, we highlight the recent progress in aptamer-mediated delivery for therapeutics and disease-targeting based on aptamer integration with a variety of nanomaterials, such as gold nanorods, DNA micelles, DNA hydrogels and carbon nanotubes.

Noninvasive and accurate measurement of intracellular temperature is of great significance in biology and medicine. This paper describes a safe, stable, and accurate intracellular nano-thermometer based on an l-DNA molecular beacon (l-MB), a dual-labeled hairpin oligonucleotide built from the optical isomer of naturally occurring d-DNA. Relying on the temperature-responsive hairpin structure and the FRET signaling mechanism of MBs, the fluorescence of l-MBs is quenched below the melting temperature and enhanced with increasing temperature. Because of the excellent reversibility and tunable response range, l-MBs are very suitable for temperature sensing. More importantly, the non-natural l-DNA backbone prevents the l-MBs from binding to cellular nucleic acids and proteins as well as from being digested by nucleases inside the cells, thus ensuring excellent stability and accuracy of the nano-thermometer in a complex cellular environment. The l-MB nano-thermometer was used for the photothermal study of Pd nanosheets in living cells, establishing the nano-thermometer as a useful tool for intracellular temperature measurement.

Because of the severe health risks associated with lead pollution, rapid, sensitive, and portable detection of low levels of Pb2+ in biological and environmental samples is of great importance. In this work, a Pb2+-responsive hydrogel was prepared using a DNAzyme and its substrate as cross-linker for rapid, sensitive, portable, and quantitative detection of Pb2+. Gold nanoparticles (AuNPs) were first encapsulated in the hydrogel as an indicator for colorimetric analysis. In the absence of lead, the DNAzyme is inactive, and the substrate cross-linker maintains the hydrogel in the gel form. In contrast, the presence of lead activates the DNAzyme to cleave the substrate, decreasing the cross-linking density of the hydrogel and resulting in dissolution of the hydrogel and release of AuNPs for visual detection. As low as 10 nM Pb2+ can be detected by the naked eye. Furthermore, to realize quantitative visual detection, a volumetric bar-chart chip (V-chip) was used for quantitative readout of the hydrogel system by replacing AuNPs with gold–platinum core–shell nanoparticles (Au@PtNPs). The Au@PtNPs released from the hydrogel upon target activation can efficiently catalyze the decomposition of H2O2 to generate a large volume of O2. The gas pressure moves an ink bar in the V-chip for portable visual quantitative detection of lead with a detection limit less than 5 nM. The device was able to detect lead in digested blood with excellent accuracy. The method developed can be used for portable lead quantitation in many applications. Furthermore, the method can be further extended to portable visual quantitative detection of a variety of targets by replacing the lead-responsive DNAzyme with other DNAzymes.

Poly(methyl methacrylate) (PMMA) is gaining in popularity in microfluidic devices because of its low cost, excellent optical transparency, attractive mechanical/chemical properties, and simple fabrication procedures. It has been used to fabricate micromixers, PCR reactors, CE and many other microdevices. Here we present the design, fabrication, characterization and application of pneumatic microvalves and micropumps based on PMMA. Valves and pumps are fabricated by sandwiching a PDMS membrane between PMMA fluidic channel and manifold wafers. Valve closing or opening can be controlled by adjusting the pressure in a displacement chamber on the pneumatic layer via a computer regulated solenoid. The valve provides up to 15.4 microL s(-1) at 60 kPa fluid pressure and seals reliably against forward fluid pressure as high as 60 kPa. A PMMA diaphragm pump can be assembled by simply connecting three valves in series. By varying valve volume or opening time, pumping rates ranging from nL to microL per second can be accurately achieved. The PMMA based valves and pumps were further tested in a disposable automatic nucleic acid extraction microchip to extract DNA from human whole blood. The DNA extraction efficiency was about 25% and the 260 nm/280 nm UV absorption ratio for extracted DNA was 1.72. Because of its advantages of inexpensive, facile fabrication, robust and easy integration, the PMMA valve and pump will find their wide application for fluidic manipulation in portable and disposable microfluidic devices.

We have designed a novel molecular assembly of quencher molecules to form superquenchers with excellent quenching efficiency. The superquencher can be engineered as desired by assembling different types and different numbers of quencher molecules. By labeling a superquencher to a molecular beacon, a 320-fold enhancement of fluorescent signal was achieved, compared to about 14-fold from a molecular beacon prepared with the same monomer quencher. Our molecular assembly approach can effectively improve the sensitivity of a variety of fluorescent assays and can be widely useful for molecular interaction studies.

Abstract Herein, we demonstrate that a very familiar, yet underutilized, physical parameter—gas pressure—can serve as signal readout for highly sensitive bioanalysis. Integration of a catalyzed gas‐generation reaction with a molecular recognition component leads to significant pressure changes, which can be measured with high sensitivity using a low‐cost and portable pressure meter. This new signaling strategy opens up a new way for simple, portable, yet highly sensitive biomedical analysis in a variety of settings.

Abstract Targeted drug delivery is important in cancer therapy to decrease the systemic toxicity resulting from nonspecific drug distribution and to enhance drug delivery efficiency. We have developed an aptamer-based DNA dendritic nanostructure as a multifunctional vehicle for targeted cancer cell imaging and drug delivery. The multifunctional DNA dendrimer is constructed from functional Y-shaped building blocks with predesigned base-pairing hybridization including fluorophores, targeting DNA aptamers and intercalated anticancer drugs. With controllable step-by-step self-assembly, the programmable DNA dendrimer has several appealing features, including facile modular design, excellent biostability and biocompatibility, high selectivity, strong binding affinity, good cell internalization efficiency and high drug loading capacity. Due to the unique structural features of DNA dendrimers, multiple copies of aptamers can be incorporated into each dendrimer, generating a multivalent aptamer-tethered nanostructure with enhanced binding affinity. A model chemotherapeutic anticancer drug, doxorubicin, was delivered via these aptamer-based DNA dendrimers and exerted a potent toxicity for target cancer cells (human T cell acute lymphoblastic leukemia cell line) with low side effects for the non-target cells (human Burkitt’s lymphoma cell line). This controllable aptamer-based DNA dendrimer is a promising candidate for biomedical applications.

Angewandte Chemie International EditionVolume 52, Issue 5 p. 1472-1476 Communication DNA Aptamer-Mediated Cell Targeting† Xiangling Xiong, Xiangling Xiong Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorDr. Haipeng Liu, Dr. Haipeng Liu Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorDr. Zilong Zhao, Dr. Zilong Zhao Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology and College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (P.R. China)Search for more papers by this authorDr. Meghan B. Altman, Dr. Meghan B. Altman Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorDr. Dalia Lopez-Colon, Dr. Dalia Lopez-Colon Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorProf. Chaoyong James Yang, Corresponding Author Prof. Chaoyong James Yang [email protected] State Key Laboratory for Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China) Chaoyong James Yang, State Key Laboratory for Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China) Weihong Tan, Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorProf. Lung-Ji Chang, Prof. Lung-Ji Chang Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorProf. Chen Liu, Prof. Chen Liu Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorProf. Weihong Tan, Corresponding Author Prof. Weihong Tan [email protected] Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA) Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology and College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (P.R. China) Chaoyong James Yang, State Key Laboratory for Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China) Weihong Tan, Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this author Xiangling Xiong, Xiangling Xiong Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorDr. Haipeng Liu, Dr. Haipeng Liu Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorDr. Zilong Zhao, Dr. Zilong Zhao Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology and College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (P.R. China)Search for more papers by this authorDr. Meghan B. Altman, Dr. Meghan B. Altman Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorDr. Dalia Lopez-Colon, Dr. Dalia Lopez-Colon Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorProf. Chaoyong James Yang, Corresponding Author Prof. Chaoyong James Yang [email protected] State Key Laboratory for Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China) Chaoyong James Yang, State Key Laboratory for Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China) Weihong Tan, Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorProf. Lung-Ji Chang, Prof. Lung-Ji Chang Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorProf. Chen Liu, Prof. Chen Liu Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this authorProf. Weihong Tan, Corresponding Author Prof. Weihong Tan [email protected] Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA) Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology and College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (P.R. China) Chaoyong James Yang, State Key Laboratory for Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China) Weihong Tan, Departments of Chemistry, of Physiology and Functional Genomics, of Molecular Genetics and Microbiology, and of Pathology and Laboratory Medicine, Shands Cancer Center, Center for Research at the Bio/nano Interface, University of Florida, Gainesville, FL 32611-7200 (USA)Search for more papers by this author First published: 11 December 2012 https://doi.org/10.1002/anie.201207063Citations: 122 † This work is supported by grants awarded by the National Institutes of Health (GM066137, GM079359 and CA133086), by the National Key Scientific Program of China (2011CB911000), the China National Instrumentation Program (2011YQ03012412), and the Foundation for Innovative Research Groups of NSFC (21221003). Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract An apt modification: A simple and effective way to modify the cell surface with target-specific ligands, such as DNA aptamers, while minimizing the effects on the modified cells has been developed. After incubating with lipo–aptamer probes, immune cells (red, see scheme) recognize and kill cancer cells (blue) in the cell mixture. Citing Literature Supporting Information As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Filename Description anie_201207063_sm_miscellaneous_information.pdf1.8 MB miscellaneous_information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume52, Issue5January 28, 2013Pages 1472-1476 RelatedInformation

A target-responsive aptamer-cross-linked hydrogel was designed and synthesized for portable and visual quantitative detection of the toxin Ochratoxin A (OTA), which occurs in food and beverages. The hydrogel network forms by hybridization between one designed DNA strand containing the OTA aptamer and two complementary DNA strands grafting on linear polyacrylamide chains. Upon the introduction of OTA, the aptamer binds with OTA, leading to the dissociation of the hydrogel, followed by release of the preloaded gold nanoparticles (AuNPs), which can be observed by the naked eye. To enable sensitive visual and quantitative detection, we encapsulated Au@Pt core-shell nanoparticles (Au@PtNPs) in the hydrogel to generate quantitative readout in a volumetric bar-chart chip (V-Chip). In the V-Chip, Au@PtNPs catalyzes the oxidation of H2O2 to generate O2, which induces movement of an ink bar to a concentration-dependent distance for visual quantitative readout. Furthermore, to improve the detection limit in complex real samples, we introduced an immunoaffinity column (IAC) of OTA to enrich OTA from beer. After the enrichment, as low as 1.27 nM (0.51 ppb) OTA can be detected by the V-Chip, which satisfies the test requirement (2.0 ppb) by the European Commission. The integration of a target-responsive hydrogel with portable enrichment by IAC, as well as signal amplification and quantitative readout by a simple microfluidic device, offers a new method for portable detection of food safety hazard toxin OTA.

Abstract Even though the diagnostic and prognostic value of circulating tumor cells (CTCs) has been demonstrated, their clinical utility and widespread adoption have been limited. Herein, we describe a new device, size‐dictated immunocapture chip (SDI‐Chip), for efficient, sensitive, and spatially resolved capture and detection of CTCs. SDI‐Chip enables selective, frequent, and extended interaction of CTCs with hydrodynamically optimized immunocoated micropillar surfaces. CTCs with different antigen expression levels can be efficiently captured and spatially resolved around the micropillars. Capture efficiency greater than 92 % with a purity of 82 % was achieved with blood samples. CTCs were detected in non‐metastasis colorectal (CRC) patients, while none was detected from healthy volunteers. We believe that SDI‐Chip will facilitate the transition of tumor diagnosis from anatomical pathology to molecular pathology in localized CRC patients.

A versatile point-of-care assay platform was developed for simultaneous detection of multiple targets based on a microfluidic paper-based analytic device (μPAD) using a target-responsive hydrogel to mediate fluidic flow and signal readout. An aptamer-cross-linked hydrogel was used as a target-responsive flow regulator in the μPAD. In the absence of a target, the hydrogel is formed in the flow channel, stopping the flow in the μPAD and preventing the colored indicator from traveling to the final observation spot, thus yielding a "signal off" readout. In contrast, in the presence of a target, no hydrogel is formed because of the preferential interaction of target and aptamer. This allows free fluidic flow in the μPAD, carrying the indicator to the observation spot and producing a "signal on" readout. The device is inexpensive to fabricate, easy to use, and disposable after detection. Testing results can be obtained within 6 min by the naked eye via a simple loading operation without the need for any auxiliary equipment. Multiple targets, including cocaine, adenosine, and Pb(2+), can be detected simultaneously, even in complex biological matrices such as urine. The reported method offers simple, low cost, rapid, user-friendly, point-of-care testing, which will be useful in many applications.

A facile one-pot sonochemical approach is presented to prepare highly blue-emitting Ag nanoclusters (AgNCs) using glutathione as a stabilizing agent in aqueous solution. The as-prepared AgNCs can be applied in the selective detection of S2− with a limit of detection of 2 nM based on fluorescence quenching.

MicroRNAs are a class of small noncoding RNAs that regulate gene expression post-transcriptionally either by inhibiting protein translation or by causing the degradation of target mRNAs. Current evidence indicates that miR-33b is involved in the regulation of lipid metabolism, cholesterol homeostasis, glucose metabolism and several human diseases; however, whether miR-33b contributes to the pathogenesis of human cancers and participates in the regulation of self-renewal of human cancer stem cells remains unknown. Here, we report the identification of miR-33b as a negative regulator of cell stemness and metastasis in breast cancer. Compared with paired normal breast tissues, miR-33b expression is downregulated in breast tumor samples and is inversely correlated with lymph node metastatic status. Ectopic overexpression of miR-33b in highly metastatic breast cancer cells suppresses cell self-renewal, migration and invasion in vitro and inhibits lung metastasis in vivo. Conversely, miR-33b knockdown promotes the self-renewal, migration and invasion capabilities of noncancerous mammary epithelial cells. The mechanism through which miR-33b inhibits the stemness, migration and invasion of breast cancer cells is by targeting HMGA2, SALL4 and Twist1. These data indicate that miR-33b acts as an onco-suppressive microRNA in breast cancer progression by inhibiting the stemness and metastasis of breast cancer cells.

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5′-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.

A facile approach was developed to prepare positively charged and red-emitting lysozyme-stabilized Ag nanoclusters (Lys-AgNCs) using NaBH4 as a reducing agent at room temperature. The Lys-AgNCs can be applied in the highly selective detection of Hg2+.

Abstract Artificial multi‐enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH–MDH and G6pDH–LDH) through the control of NAD + substrate channeling by specifically shifting NAD + between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine.

We propose the use of DNAzyme as a crosslinker of hydrogel to develop a catalytic platform for the sensing of metal ions. The DNAzyme crosslinked hydrogel can undergo gel–sol transition in response to Cu2+ ions, which enables sensitive visual detection of Cu2+ by observing the release of pre-trapped AuNPs.

Measuring distances at molecular length scales in living systems is a significant challenge. Methods like Förster resonance energy transfer (FRET) have limitations due to short detection distances and strict orientations. Recently, surface energy transfer (SET) has been used in bulk solutions; however, it cannot be applied to living systems. Here, we have developed an SET nanoruler, using aptamer−gold nanoparticle conjugates with different diameters, to monitor the distance between binding sites of a receptor on living cells. The nanoruler can measure separation distances well beyond the detection limit of FRET. Thus, for the first time, we have developed an effective SET nanoruler for live cells with long distance, easy construction, fast detection, and low background. This is also the first time that the distance between the aptamer and antibody binding sites in the membrane protein PTK7 was measured accurately. The SET nanoruler represents the next leap forward to monitor structural components within living cell membranes.

Simultaneous monitoring of the expression, distribution, and dynamics of biological molecules in living cells is one of the most challenging tasks in the analytical sciences. The key to effective and successful intracellular imaging is the development of delivery platforms with high efficiency and ultrasensitive molecular probes for specific targets of interest. To achieve these goals, many nanomaterials are widely used as carriers to introduce nucleic acid probes into living cells for real-time imaging of biomolecules. However, limitations on their use include issues of cytotoxicity and delivery efficiency. Herein, we propose a switchable aptamer micelle flare (SAMF), formed by self-assembly of an aptamer switch probe-diacyllipid chimera, to monitor ATP molecules inside living cells. Similarity of hydrophobic composition between diacyllipids in the micelle flares and phospholipid bilayers in the dynamic membranes of living cells allows SAMFs to be uptaken by living cells more efficiently than aptamer switch probes without external auxiliary. Switchable aptamers were found to bind target ATP molecules with high selectivity and specificity, resulting in restoration of the fluorescence signal from "OFF" to "ON" state, thus indicating the presence of the analyte. These switchable aptamer micelle flares, which exhibit cell permeability and nanoscale controllability, show exceptional promise for molecular imaging in bioanalysis, disease diagnosis, and drug delivery.

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads–antibody–antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (<1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases.

Transport of PEGylated silica nanoparticles (PSiNPs) with diameters of 100, 50, and 25 nm across the blood–brain barrier (BBB) was evaluated using an in vitro BBB model based on mouse cerebral endothelial cells (bEnd.3) cultured on transwell inserts within a chamber. In vivo animal experiments were further performed by noninvasive in vivo imaging and ex vivo optical imaging after injection via carotid artery. Confocal fluorescence studies were carried out to evaluate the uptake of PSiNPs by brain endothelial cells. The results showed that PSiNPs can traverse the BBB in vitro and in vivo. The transport efficiency of PSiNPs across BBB was found to be size-dependent, with increased particle size resulting in decreased efficiency. This work points to the potential application of small sized silica nanoparticles in brain imaging or drug delivery.

A distance-readout microfluidic chip was combined with an AFB₁-responsive hydrogel for rapid, portable, selective, and quantitative detection of AFB₁ in real samples.

Substrate channeling, in which a metabolic intermediate is directly passed from one enzyme to the next enzyme in an enzyme cascade, accelerates the processing of metabolites and improves substrate selectivity. Synthetic design and precise control of channeling outside the cellular environment are of significance in areas such as synthetic biology, synthetic chemistry, and biomedicine. In particular, the precise control of synthetic substrate channeling in response to light is highly important, but remains a major challenge. Herein, we develop a photoresponsive molecule-based synthetic substrate channeling system on DNA origami to regulate enzyme cascade activity. The photoresponsive azobenzene molecules introduced into DNA strands enable reversible switching of the position of substrate channeling to selectively activate or inhibit the enzyme cascade activity. Moreover, DNA origami allows precise control of interenzyme distance and swinging range of the swing arm to optimize the regulation efficiency. By combining the accurate and addressable assembly ability of DNA origami and the clean, rapid, and reversible regulation of photoresponsive molecules, this light-driven substrate channeling system is expected to find important applications in synthetic biology and biomedicine.

Based on backbone-modified molecular beacons and duplex-specific nuclease, we have developed a target recycling amplification method for highly sensitive and selective miRNA detection. The combination of a low fluorescence background of 2-OMe-RNA modified MB and nuclease-assisted signal amplification leads to ultrahigh assay sensitivity, and the powerful discriminating ability of MB enables the differentiation of highly similar miRNAs with one-base difference, both of which are of great significance to miRNA detection.

Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective detection of small molecules by means of FA in complex biological samples.

Cancer is a major public health issue, with metastatic cancer accounting for the overwhelming majority of cancer deaths. Early diagnosis and appropriate treatment of metastatic cancer may largely prolong the survival rate and improve the quality of life for patients. In this study, we have identified a panel of DNA aptamers specifically binding to MDA-MB-231 cells derived from metastatic site-pleural effusion, with high affinity after 15 rounds of selections using the cell-based systematic evolution of ligands by exponential enrichment (SELEX) method. The selected aptamers were subjected to flow cytometry and laser confocal fluorescence microscopy to evaluate their binding affinity and selectivity. The aptamer LXL-1 with the highest abundance in the enriched library demonstrated a low Kd value and excellent selectivity for the recognition of the metastatic breast cancer cells. Tissue imaging results showed that truncated aptamer sequence LXL-1-A was highly specific to the corresponding tumor tissue and displayed 76% detection rate against breast cancer tissue with metastasis in regional lymph nodes. Therefore, on the basis of its excellent targeting properties and functional versatility, LXL-1-A holds great potential to be used as a molecular imaging probe for the detection of breast cancer metastasis. Our result clearly demonstrates that metastatic-cell-based SELEX can be used to generate DNA ligands specifically recognizing metastatic cancer cells, which is of great significance for metastatic cancer diagnosis and treatment.

Accurate sensing of the extracellular pH is a very important yet challenging task in biological and clinical applications. This paper describes the development of an amphiphilic lipid–DNA molecule as a simple yet useful cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing. The lipid–DNA probe, which consists of a hydrophobic diacyllipid tail and a hydrophilic DNA strand, is modified with two fluorescent dyes; one is pH-sensitive as pH indicator and the other is pH-insensitive as an internal reference. The lipid–DNA probe showed sensitive and reversible response to pH change in the range of 6.0–8.0, which is suitable for most extracellular studies. In addition, based on simple hydrophobic interactions with the cell membrane, the lipid–DNA probe can be easily anchored on the cell surface with negligible cytotoxicity, excellent stability, and unique ratiometric readout, thus ensuring its accurate sensing of extracellular pH. Finally, this lipid–DNA-based ratiometric pH indicator was successfully used for extracellular pH sensing of cells in 3D culture environment, demonstrating the potential applications of the sensor in biological and medical studies.

Biofabricated semiconductor arrays exhibit smaller channel pitches than those created using existing lithographic methods. However, the metal ions within biolattices and the submicrometer dimensions of typical biotemplates result in both poor transport performance and a lack of large-area array uniformity. Using DNA-templated parallel carbon nanotube (CNT) arrays as model systems, we developed a rinsing-after-fixing approach to improve the key transport performance metrics by more than a factor of 10 compared with those of previous biotemplated field-effect transistors. We also used spatially confined placement of assembled CNT arrays within polymethyl methacrylate cavities to demonstrate centimeter-scale alignment. At the interface of high-performance electronics and biomolecular self-assembly, such approaches may enable the production of scalable biotemplated electronics that are sensitive to local biological environments.

Paper based microfluidics (µPADs) with advantages of portability, low cost, and ease of use have attracted extensive attention. Here we describe a novel method that integrates glucoamylase-trapped aptamer-crosslinked hydrogel for molecular recognition with cascaded enzymatic reactions for signal amplification and a µPAD for portable readout. Upon target introduction, the hydrogel decomposes to release glucoamylase, which catalyzes the hydrolysis of amylose to produce a large amount of glucose. With a simple folding of the µPAD, the sample solution containing glucose product wicks and diffuses in parallel to each test-zone to carry out homogeneous assays, where glucose is used to produce I2 for brown color visualization through multiple enzymatic and chemical cascade reactions. Through color gradient changes based on different concentrations of the target, a semiquantitative assay is achieved by the naked eye, and quantitation can be obtained by handheld devices. Detection of cocaine in buffer and urine was performed to demonstrate the utility of the hydrogel-µPAD system. More importantly, the hydrogel-µPAD system can be extended to the detection of various targets by incorporating the corresponding aptamer into the hydrogel. The hydrogel-µPAD system reported here provides a new platform for portable, disposable and visual detection of a wide range of targets.

Nucleic acids are natural biopolymers of nucleotides that store, encode, transmit and express genetic information, which play central roles in diverse cellular events and diseases in living things. The analysis of nucleic acids and nucleic acids-based analysis have been widely applied in biological studies, clinical diagnosis, environmental analysis, food safety and forensic analysis. During the past decades, the field of nucleic acids analysis has been rapidly advancing with many technological breakthroughs. In this review, we focus on the methods developed for analyzing nucleic acids, nucleic acids-based analysis, device for nucleic acids analysis, and applications of nucleic acids analysis. The representative strategies for the development of new nucleic acids analysis in this field are summarized, and key advantages and possible limitations are discussed. Finally, a brief perspective on existing challenges and further research development is provided.

Point-of-care testing (POCT) with the advantages of speed, simplicity, and low cost, as well as no need for instrumentation, is critical for the measurement of analytes in a variety of environments lacking access to laboratory infrastructure. In the present study, a hydrogel pressure-based assay for quantitative POCT was developed by integrating a target-responsive hydrogel with pressuremeter readout. The target-responsive hydrogels were constructed with DNA grafted linear polyacrylamide and the cross-linking DNA for selective target recognition. The hydrogel response to the target substance allows release of the preloaded Pt nanoparticles, which have good stability and excellent catalytic ability for decomposing H2O2 to O2. Then, the generated O2 in a sealed environment leads to significant pressure increase, which can be easily read out by a handheld pressuremeter. Using this target-responsive hydrogel pressure-based assay, portable and highly sensitive detection of cocaine, ochratoxin A, and lead ion were achieved with excellent accuracy and selectivity. With the advantages of portability, high sensitivity, and simple sample processing, the hydrogel pressure-based assay shows great potential for quantitative POCT of a broad range of targets in resource-limited settings.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTRecent Progress in Microfluidics-Based BiosensingYanling SongYanling SongInstitute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, ChinaMore by Yanling SongView Biography, Bingqian LinBingqian LinMOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, Department of Chemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, ChinaMore by Bingqian LinView Biography, Tian TianTian TianMOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, Department of Chemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, ChinaMore by Tian TianView Biography, Xing XuXing XuMOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, Department of Chemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, ChinaMore by Xing XuView Biography, Wei WangWei WangInstitute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, ChinaMore by Wei WangView Biography, Qingyu RuanQingyu RuanMOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, Department of Chemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, ChinaMore by Qingyu RuanView Biography, Jingjing GuoJingjing GuoMOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, Department of Chemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, ChinaMore by Jingjing GuoView Biography, Zhi ZhuZhi ZhuMOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, Department of Chemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, ChinaMore by Zhi ZhuView Biographyhttp://orcid.org/0000-0002-3287-4920, and Chaoyong Yang*Chaoyong YangInstitute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, ChinaMOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, Department of Chemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China*Phone: (+86) 21-683-83993. E-mail: [email protected]More by Chaoyong YangView Biographyhttp://orcid.org/0000-0002-2374-5342Cite this: Anal. Chem. 2019, 91, 1, 388–404Publication Date (Web):November 9, 2018Publication History Published online9 November 2018Published inissue 2 January 2019https://doi.org/10.1021/acs.analchem.8b05007Copyright © 2018 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views5176Altmetric-Citations69LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (3 MB) Get e-AlertsSUBJECTS:Biosensing,Biotechnology,Fluid dynamics,Liquids,Sensors Get e-Alerts

Enzyme-linked immunosorbent assay (ELISA) is a popular laboratory technique for detection of disease-specific protein biomarkers with high specificity and sensitivity. However, ELISA requires labor-intensive and time-consuming procedures with skilled operators and spectroscopic instrumentation. Simplification of the procedures and miniaturization of the devices are crucial for ELISA-based point-of-care (POC) testing in resource-limited settings. Here, we present a fully integrated, instrument-free, low-cost and portable POC platform which integrates the process of ELISA and the distance readout into a single microfluidic chip. Based on manipulation using a permanent magnet, the process is initiated by moving magnetic beads with capture antibody through different aqueous phases containing ELISA reagents to form bead/antibody/antigen/antibody sandwich structure, and finally converts the molecular recognition signal into a highly sensitive distance readout for visual quantitative bioanalysis. Without additional equipment and complicated operations, our integrated ELISA-Chip with distance readout allows ultrasensitive quantitation of disease biomarkers within 2 h. The ELISA-Chip method also showed high specificity, good precision and great accuracy. Furthermore, the ELISA-Chip system is highly applicable as a sandwich-based platform for the detection of a variety of protein biomarkers. With the advantages of visual analysis, easy operation, high sensitivity, and low cost, the integrated sample-in-answer-out ELISA-Chip with distance readout shows great potential for quantitative POCT in resource-limited settings.

Abstract The commonly existing cellular heterogeneity plays a critical role in biological processes such as embryonic development, cell differentiation, and disease progress. Single‐cell omics‐based heterogeneous studies have great significance for identifying different cell populations, discovering new cell types, revealing informative cell features, and uncovering significant interrelationships between cells. Recently, microfluidics has evolved to be a powerful technology for single‐cell omics analysis due to its merits of throughput, sensitivity, and accuracy. Herein, the recent advances of microfluidic single‐cell omics analysis, including different microfluidic platform designs, lysis strategies, and omics analysis techniques, are reviewed. Representative applications of microfluidic single‐cell omics analysis in complex biological studies are then summarized. Finally, a few perspectives on the future challenges and development trends of microfluidic‐assisted single‐cell omics analysis are discussed.

Due to its large enhancement effect, nanostructure-based surface-enhanced Raman scattering (SERS) technology had been widely applied for bioanalysis and cell imaging. However, most SERS nanostructures suffer from poor signal reproducibility, which hinders the application of SERS nanostructures in quantitative detection. We report an etching-assisted approach to synthesize SERS-active plasmonic nanoparticles with 1 nm interior nanogap for multiplex quantitative detection and cancer cell imaging. Raman dyes and methoxy poly(ethylene glycol) thiol (mPEG-SH) were attached to gold nanoparticles (AuNPs) to prepare gold cores. Next, Ag atoms were deposited on gold cores in the presence of Pluronic F127 to form a Ag shell. HAuCl4 was used to etch the Ag shell and form an interior nanogap in Au@AgAuNPs, leading to increased Raman intensity of dyes. SERS intensity distribution of Au@AgAuNPs was found to be more uniform than that of aggregated AuNPs. Finally, Au@AgAuNPs were used for multiplex quantitative detection and cancer cell imaging. With the advantages of simple and rapid preparation of Au@AgAuNPs with highly uniform, stable, and reproducible Raman intensity, the method reported here will widen the applications of SERS-active nanoparticles in diagnostics and imaging.

Due to uranium's increasing exploitation in nuclear energy and its toxicity to human health, it is of great significance to detect uranium contamination. In particular, development of a rapid, sensitive and portable method is important for personal health care for those who frequently come into contact with uranium ore mining or who investigate leaks at nuclear power plants. The most stable form of uranium in water is uranyl ion (UO2(2+)). In this work, a UO2(2+) responsive smart hydrogel was designed and synthesized for rapid, portable, sensitive detection of UO2(2+). A UO2(2+) dependent DNAzyme complex composed of substrate strand and enzyme strand was utilized to crosslink DNA-grafted polyacrylamide chains to form a DNA hydrogel. Colorimetric analysis was achieved by encapsulating gold nanoparticles (AuNPs) in the DNAzyme-crosslinked hydrogel to indicate the concentration of UO2(2+). Without UO2(2+), the enzyme strand is not active. The presence of UO2(2+) in the sample activates the enzyme strand and triggers the cleavage of the substrate strand from the enzyme strand, thereby decreasing the density of crosslinkers and destabilizing the hydrogel, which then releases the encapsulated AuNPs. As low as 100nM UO2(2+) was visually detected by the naked eye. The target-responsive hydrogel was also demonstrated to be applicable in natural water spiked with UO2(2+). Furthermore, to avoid the visual errors caused by naked eye observation, a previously developed volumetric bar-chart chip (V-Chip) was used to quantitatively detect UO2(2+) concentrations in water by encapsulating Au-Pt nanoparticles in the hydrogel. The UO2(2+) concentrations were visually quantified from the travelling distance of ink-bar on the V-Chip. The method can be used for portable and quantitative detection of uranium in field applications without skilled operators and sophisticated instruments.

An integrated distance-based origami paper analytical device (ID-oPAD) is developed for simple, user friendly and visual detection of targets of interest. The platform enables complete integration of target recognition, signal amplification, and visual signal output based on aptamer/invertase-functionalized sepharose beads, cascaded enzymatic reactions, and a 3D microfluidic paper-based analytical device with distance-based readout, respectively. The invertase–DNA conjugate is released upon target addition, after which it permeates through the cellulose and flows down into the bottom detection zone, whereas sepharose beads with larger size are excluded and stay in the upper zone. Finally, the released conjugate initiates cascaded enzymatic reactions and translates the target signal into a brown bar chart reading. By simply closing the device, the ID-oPAD enables a sample-in-answer-out assay within 30 min with visual and quantitative readout. Importantly, bound/free probe separation is achieved by taking advantage of the size difference between sepharose beads and cellulose pores, and the downstream enzymatic amplification is realized based on the compatibility of multiple enzymes with corresponding substrates. Overall, with the advantages of low-cost, disposability, simple operation, and visual quantitative readout, the ID-oPAD offers an ideal platform for point-of-care testing, especially in resource-limited areas.

Abstract Microfluidic chips with nano‐scale structures have shown great potential, but the fabrication and cost issues restrict their application. Herein, we propose a conceptually new “DNA nanolithography in a microfluidic chip” by using sub‐10 nm three‐dimensional DNA structures (TDNs) as frameworks with a pendant aptamer at the top vertex (ApTDN‐Chip). The nano‐scale framework ensures that the aptamer is in a highly ordered upright orientation, avoiding the undesired orientation or crowding effects caused by conventional microfluidic interface fabrication processes. Compared with a monovalent aptamer modified chip, the capture efficiency of ApTDN‐Chip was enhanced nearly 60 % due to the highly precise dimension and rigid framework of TDNs. In addition, the scaffolds make DNase I more accessible to the aptamer with up to 83 % release efficiency and 91 % cell viability, which is fully compatible with downstream molecular analysis. Overall, this strategy provides a novel perspective on engineering nano‐scaffolds to achieve a more ordered nano‐topography of microfluidic chips.

Abstract Deepening our understanding of mammalian gut microbiota has been greatly hampered by the lack of a facile, real‐time, and in vivo bacterial imaging method. To address this unmet need in microbial visualization, we herein report the development of a second near‐infrared (NIR‐II)‐based method for in vivo imaging of gut bacteria. Using d ‐propargylglycine in gavage and then click reaction with an azide‐containing NIR‐II dye, gut microbiota of a donor mouse was strongly labeled with NIR‐II fluorescence on their peptidoglycan. The bacteria could be readily visualized in recipient mouse gut with high spatial resolution and deep tissue penetration under NIR irradiation. The NIR‐II‐based metabolic labeling strategy reported herein, provides, to the best of our knowledge, the first protocol for facile in vivo visualization of gut microbiota within deep tissues, and offers an instrumental tool for deciphering the complex biology of these gut “dark matters”.

Abstract Currently, there are more than 200 fecal microbiota transplantation (FMT) clinical trials worldwide. However, our knowledge of this microbial therapy is still limited. Here we develop a strategy using sequential tagging with D-amino acid-based metabolic probes (STAMP) for assessing the viabilities of transplanted microbiotas. A fluorescent D-amino acid (FDAA) is first administered to donor mice to metabolically label the gut microbiotas in vivo. The labeled microbiotas are transplanted to recipient mice, which receive a second FDAA with a different color. The surviving transplants should incorporate both FDAAs and can be readily distinguished by presenting two colors simultaneously. Isolation of surviving bacteria and 16S rDNA sequencing identify several enriched genera, suggesting the importance of specific bacteria in FMT. In addition, using STAMP, we evaluate the effects on transplant survival of pre-treating recipients using different antibiotics. We propose STAMP as a versatile tool for deciphering the complex biology of FMT, and potentially improving its treatment efficacy.

Exosomal glycoproteins play important roles in many physiological and pathological functions. Herein, we developed a dual labeling strategy based on a protein-specific aptamer tagging and metabolic glycan labeling for visualizing glycosylation of specific proteins on exosomes. The glycosylation of exosomal PD-L1 (exoPD-L1) was imaged in situ using intramolecular fluorescence resonance energy transfer (FRET) between fluorescent PD-L1 aptamers bound on exoPD-L1 and fluorescent tags on glycans introduced via metabolic glycan labeling. This method enables in situ visualization and biological function study of exosomal protein glycosylation. Exosomal PD-L1 glycosylation was confirmed to be required in interaction with PD-1 and participated in inhibiting of CD8+ T cell proliferation. This is an efficient and non-destructive method to study the presence and function of exosomal protein-specific glycosylation in situ, which provides a powerful tool for exosomal glycoproteomics research.

This review highlights the developments, accomplishments and challenges of integrated μPADs, including sample pretreatment, signal transduction/amplification and results output.

DNA hydrogels have received considerable attention in analytical science, however, some limitations still exist in the applications of intelligent hydrogels. In this paper, we describe a way to prepare gel film in a capillary tube based on the thermal reversible principle of DNA hydrogel and the principle of capillary action. Because of the slight change in the internal structure of gel, its permeability can be increased by the addition of some specific targets. The capillary behavior is thus changed due to the different permeability of the hydrogel film. The duration time of the target solution flowing through the capillary tube with a specified length is used to quantify this change. With this proposed method, ultra-trace DNA hydrogel (0.01 μL) is sufficient to realize the sensitive detection of cocaine without the aid of other instruments, which has a low detection limit (1.17 nM) and good selectivity.

The analysis of circulating tumor cells (CTCs) holds great significance for cancer diagnosis, prognosis, and personalized therapy. However, the rarity, vulnerability and heterogeneity of CTCs bring daunting technical challenges to their isolation, release and analysis. Recent exciting advances in microfluidics have greatly promoted CTC isolation and analysis due to its merits of precise control of fluid behavior, integration, and automation. Especially, aptamer-based microfluidic chip is considered as promising platform, because aptamers as recognition ligands have inherent superiority in CTC isolation and release for their convenient modification and controllable recognition ability. This review focuses on recent progresses in aptamer-based microfluidics for isolation, release and analysis of CTCs. First, existing CTC-related aptamers and their selection methods are briefly introduced. Strategies for conjugating aptamers onto microfluidic chips are also reviewed. Then, aptamer-based microfluidic chips for CTC isolation, release and analysis are summarized. Finally, future research directions and challenges in this field are discussed.

Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.

New neutralizing agents against SARS-CoV-2 and associated mutant strains are urgently needed for the treatment and prophylaxis of COVID-19. Herein, we develop a spherical cocktail neutralizing aptamer-gold nanoparticle (SNAP) to block the interaction between the receptor-binding domain (RBD) of SARS-CoV-2 and host ACE2. With the multivalent aptamer assembly as well as the steric hindrance effect of the gold scaffold, SNAP exhibits exceptional binding affinity against the RBD with a dissociation constant of 3.90 pM and potent neutralization against authentic SARS-CoV-2 with a half-maximal inhibitory concentration of 142.80 fM, about 2 or 3 orders of magnitude lower than that of the reported neutralizing aptamers and antibodies. More importantly, the synergetic blocking strategy of multivalent multisite binding and steric hindrance ensures broad neutralizing activity of SNAP, almost completely blocking the infection of three mutant pseudoviruses. Overall, the SNAP strategy provides a new direction for the development of antivirus agents against SARS-CoV-2 and other emerging coronaviruses.

Broad-spectrum anti-SARS-CoV-2 strategies that can inhibit the infection of wild-type and mutant strains would alleviate their threats to global public health. Here, we propose an icosahedral DNA framework for the assembly of up to 30 spatially arranged neutralizing aptamers (IDNA-30) to inhibit viral infection. Each triangular plane of IDNA-30 is composed of three precisely positioned aptamers topologically matching the SARS-CoV-2 spike trimer, thus forming a multivalent spatially patterned binding. Due to its multiple binding sites and moderate size, multifaced IDNA-30 induces aggregation of viruses. The rigid icosahedron framework afforded by four helixes not only forms a steric barrier to prevent the virus from binding to the host but also limits the conformational transformation of the SARS-CoV-2 spike trimer. Combining multivalent topologically patterned aptamers with structurally well-defined nanoformulations, IDNA-30 exhibits excellent broad-spectrum neutralization against SARS-CoV-2, including almost completely blocking the infection of Omicron pseudovirus. Overall, this multidimensional neutralizing strategy provides a new direction for the assembly of neutralizing reagents to enhance their inhibitory effect against SARS-CoV-2 infection and combat other disease-causing viruses.

Single-cell genome and transcriptome sequencing investigates how genotype influences the phenotype of single cells, so as to comprehensively interpret biological inheritance and explain functional heterogeneity at the single-cell level. Current sample preparation technologies for simultaneous DNA and RNA sequencing of the same cell are cumbersome, expensive, and suffer from cross-contamination and limited sensitivity. Herein we describe DMF-DR-seq, a nanoliter-scale single-cell multi-omics sample preparation platform based on digital microfluidics. DMF-DR-seq integrates the major steps of single-cell isolation, DNA/RNA separation, and nucleic acid amplification in situ. The results confirm the enhanced ability of DMF-DR-seq relative to current state-of-the-art technology, with lower amplification bias, higher genome-wide coverage in DNA sequencing and better gene detection ability in RNA sequencing results. By using DMF-DR-seq, we identified the genome variation-induced abnormal transcriptome expression of single circulating tumor cells (CTCs) and cancer cells from multiple myeloma patients. The results identified potentially essential genes, known as transporters associated with antigen presentation (TAP1 and TAP2), that participate in the pathologic progress. The unique flexibility, sensitivity, and accuracy of DMF-DR-seq suggest its potential utility in deeper multi-omics analysis for inheritance mechanism study in single-cell biology.

Outbreaks of both influenza virus and the novel coronavirus SARS-CoV-2 are serious threats to human health and life. It is very important to establish a rapid, accurate test with large-scale detection potential to prevent the further spread of the epidemic. An optimized RPA-Cas12a-based platform combined with digital microfluidics (DMF), the RCD platform, was established to achieve the automated, rapid detection of influenza viruses and SARS-CoV-2. The probe in the RPA-Cas12a system was optimized to produce maximal fluorescence to increase the amplification signal. The reaction droplets in the platform were all at the microliter level and the detection could be accomplished within 30 min due to the effective mixing of droplets by digital microfluidic technology. The whole process from amplification to recognition is completed in the chip, which reduces the risk of aerosol contamination. One chip can contain multiple detection reaction areas, offering the potential for customized detection. The RCD platform demonstrated a high level of sensitivity, specificity (no false positives or negatives), speed (≤30 min), automation and multiplexing. We also used the RCD platform to detect nucleic acids from influenza patients and COVID-19 patients. The results were consistent with the findings of qPCR. The RCD platform is a one-step, rapid, highly sensitive and specific method with the advantages of digital microfluidic technology, which circumvents the shortcomings of manual operation. The development of the RCD platform provides potential for the isothermal automatic detection of nucleic acids during epidemics.Supplementary material is available in the online version of this article at 10.1007/s11426-021-1169-1.

Recently, the SARS-CoV-2 Omicron has spread very quickly worldwide. Several studies have indicated that the Omicron variant causes a substantial evasion of the humoral immune response and the majority of existing SARS-CoV-2 neutralizing antibodies. Here we address this challenge by applying a spherical cocktail neutralizing aptamer-gold nanoparticle (SNAP) to block the interaction of Omicron receptor binding domain (RBD) and host Angiotensin-Converting Enzyme 2 (ACE2). With the synergetic blocking strategy based on multivalent multisite aptamer binding and steric hindrance by the size-matched gold scaffold, the SNAP conjugate tightly binds to Omicron RBD with a dissociation constant of 13.6 pM, almost completely blocking the infection of Omicron pseudovirus with a half-maximal inhibitory concentration of 35.9 pM. Overall, the SNAP strategy not only fills the gap of the humoral immune evasion caused by clustered mutations on Omicron, but also provides a clue for the development of new broad neutralizing reagents against future variants.

Digital microfluidic (DMF) bioassays with the benefits of automation, addressability, integration and dynamic configuration ability for nucleic acids, proteins, immunoreaction and cell analysis are presented in this review.

Abstract Extracellular vesicles (EVs) play an important role in many physiological processes. Thus, EV analysis has a great value for the understanding of mechanisms underlying disease progress or diagnosis, prognosis and therapy. The overlapped physical and immune properties between EVs and events in body fluids, as well as the phenotypic heterogeneity of EVs, require efficient isolation and analysis methods. The unique properties of aptamers, such as facile modification and programmability, make them easily assembled as powerful platforms for EV isolation and analysis. EVs can also be used as vehicles for drug delivery, benefiting from the properties of homing ability, hypo‐immunogenicity, and strong tolerance. The affinity recognition ability to targets and the feature of single stranded DNA of aptamers make them useful in promoting the targetability of EVs and delivery of nucleic acid drugs. This review summarizes recent progress in aptamer‐based EV isolation, analysis, and aptamer‐functionalized EVs for therapeutics.

Microfluidic biosensors integrating fluid control, target recognition, as well as signal transduction and output, have been widely used in the field of disease diagnosis, drug screening, food safety and environmental monitoring in the past two decades. As the central part and technical characteristics of microfluidic biosensors, the fluid control is not only associated with accuracy and convenience of the sensors, but also affects the material selection and working mode of the sensors. This review summarizes the fluid driving forces for microfluidic biosensors, including gravity, capillary force, centrifugal force, pressure, light, sound, electrical, and magnetic forces. Then, the recent advances in microfluidic biosensors for the detection of viruses, cells, nucleic acids, proteins and small molecules are discussed. Finally, we propose the current challenges and future perspectives of microfluidic biosensors. We hope this review can provide readers with a new perspective to understand the technical characteristics and application potential of microfluidic biosensors.

Isolation and analysis of tumor-derived extracellular vesicles (T-EVs) are important for clinical cancer management. Here, we develop a fluid multivalent magnetic interface (FluidmagFace) in a microfluidic chip for high-performance isolation, release, and protein profiling of T-EVs. The FluidmagFace increases affinity by 105-fold with fluidity-enhanced multivalent binding to improve isolation efficiency by 13.9 % compared with a non-fluid interface. Its anti-adsorption property and microfluidic hydrodynamic shear minimize contamination, increasing detection sensitivity by two orders of magnitude. Moreover, its reversibility and expandability allow high-throughput recovery of T-EVs for mass spectrometric protein analysis. With the chip, T-EVs were detected in all tested cancer samples with identification of differentially expressed proteins compared with healthy controls. The FluidmagFace opens a new avenue to isolation and release of targets for cancer diagnosis and biomarker discovery.

Unlike plant and microbial cells having cell walls, the outermost layer of mammalian cell is a delicate, two-layered structure of phospholipids with proteins embedded, which is susceptible to environmental changes. It is necessary to create an "armor" on cell surface to protect cell integrity. Here, we propose an Auto-assembled Resilient bioMimetic calcified ORnaments (ARMOR) strategy driven by dual-aptamer-based hybridization chain reaction (HCR) and Ca2+ assisted calcification for selective cell protection. This co-recognition design enhances the selectivity and leverages robust in situ signal amplification by HCR to improve the sensitivity. The calcified shell is cogenerated by crosslinking the alginate-HCR product with Ca2+ ion. ARMOR has high efficiency for shielding cells from environmental assaults, which can be applied to circulating tumor cell (CTC) protection, isolation, and identification, maintaining the native state and intact genetic information for downstream analysis.

Single-cell RNA sequencing (scRNA-seq) reveals the transcriptional heterogeneity of cells, but the static snapshots fail to reveal the time-resolved dynamics of transcription. Herein, we develop Well-TEMP-seq, a high-throughput, cost-effective, accurate, and efficient method for massively parallel profiling the temporal dynamics of single-cell gene expression. Well-TEMP-seq combines metabolic RNA labeling with scRNA-seq method Well-paired-seq to distinguish newly transcribed RNAs marked by T-to-C substitutions from pre-existing RNAs in each of thousands of single cells. The Well-paired-seq chip ensures a high single cell/barcoded bead pairing rate (~80%) and the improved alkylation chemistry on beads greatly alleviates chemical conversion-induced cell loss (~67.5% recovery). We further apply Well-TEMP-seq to profile the transcriptional dynamics of colorectal cancer cells exposed to 5-AZA-CdR, a DNA-demethylating drug. Well-TEMP-seq unbiasedly captures the RNA dynamics and outperforms the splicing-based RNA velocity method. We anticipate that Well-TEMP-seq will be broadly applicable to unveil the dynamics of single-cell gene expression in diverse biological processes.

Protein bioassay is a critical tool for the screening and detection of protein biomarkers in disease diagnostics and biological applications. However, the detection sensitivity and system automation of current immunoassays do not meet the emerging demands of clinical applications. Here, we developed a dissolution-enhanced luminescence-enhanced digital microfluidics immunoassay (DEL-DMF), which significantly improves the sensitivity and automation of the protein bioassay. In DEL-DMF, the sample and reagent droplets are controlled to complete the processes of sample transport, immunoreaction, and buffer washing, which not only minimizes sample consumption to 2 μL and enhances the binding efficiency of immunoreaction but also streamlines all the procedures and simplifies the process of immunoassay. Moreover, dissolution-enhanced luminescence using NaEuF4 NPs as nanoprobes boosts the fluorescence and increases the sensitivity of the bioassay. We demonstrate the enhanced analytical performance of our DEL-DMF immunoassay to detect H5N1 hemagglutinin in human serum and saliva. A limit of detection of 1.16 pM was achieved in less than 0.5 h with only 2 μL sample consumption. Overall, our DEL-DMF immunoassay combines the merits of the microfluidics platform and dissolution-enhanced luminescence, thus affording superior detection sensitivity and system automation for protein biomarkers. This novel immunoassay microsystem holds great potential in clinical and biological applications.

Abstract Recently, lysosome targeting chimeras (LYTACs) have emerged as a promising technology that expands the scope of targeted protein degradation to extracellular targets. However, the preparation of chimeras by conjugation of the antibody and trivalent N‐acetylgalactosamine (tri‐GalNAc) is a complex and time‐consuming process. The large uncertainty in number and position and the large molecular weights of the chimeras result in low internalization efficiency. To circumvent these problems, we developed the first aptamer‐based LYTAC (Apt‐LYTAC) to realize liver‐cell‐specific degradation of extracellular and membrane proteins by conjugating aptamers to tri‐GalNAc. Taking advantage of the facile synthesis and low molecular weight of the aptamer, the Apt‐LYTACs can efficiently and quickly degrade the extracellular protein PDGF and the membrane protein PTK7 through a lysosomal degradation pathway. We anticipate that the novel Apt‐LYTACs will expand the usage of aptamers and provide a new dimension for targeted protein degradation.

Periostin, a multifunctional extracellular protein, plays an important role in inflammatory disorders and tumorigenesis. Our previous work has demonstrated that periostin deficiency inhibits colorectal cancer (CRC) progression. Here, we aim to clarify the role of periostin in the immune microenvironment of CRC. We find that periostin deficiency significantly decreases the infiltration of programmed death receptor 1 (PD-1)+ tumor-associated macrophages (TAMs) in CRC tissues. Periostin promotes the expression of PD-1 on TAMs by integrin-ILK-nuclear factor κB (NF-κB) signaling, and PD-1+ TAMs produce interleukin-6 (IL-6) and interferon γ (IFN-γ) to induce the expression of PD-L1 on colorectal tumor cells. Moreover, combined inhibition of periostin and PD-1 significantly suppresses CRC progression compared with the inhibition of periostin or PD-1 alone. In summary, our results suggest that periostin deficiency reduces the infiltration of PD-1+ TAMs and enhances the efficacy of anti-PD-1 treatment in CRC.

Open AccessCCS ChemistryRESEARCH ARTICLES3 Feb 2024Green Synthesized Liquid-like Dynamic Polymer Chains with Decreased Nonspecific Adhesivity for High-Purity Capture of Circulating Tumor Cells Feng Wu†, Xiaofeng Chen†, Shuli Wang, Ruimin Zhou, Chunyan Wang, Lejian Yu, Jing Zheng, Chaoyong Yang and Xu Hou Feng Wu† State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 College of Physical Science and Technology, Xiamen University, Xiamen, Fujian 361005 College of Physics and New Energy, Xuzhou University of Technology, Xuzhou, Jiangsu 221018 , Xiaofeng Chen† State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 , Shuli Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 Fujian Engineering Research Center for Solid-State Lighting, Department of Electronic Science, School of Electronic Science and Engineering, Xiamen University, Xiamen, Fujian 361005 Institute of Artificial Intelligence, Xiamen University, Xiamen, Fujian 361005 , Ruimin Zhou College of Physical Science and Technology, Xiamen University, Xiamen, Fujian 361005 , Chunyan Wang State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 , Lejian Yu State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 , Jing Zheng State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 , Chaoyong Yang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 Innovation Laboratory for Sciences and Technologies of Energy Materials of Fujian Province (IKKEM), Xiamen, Fujian 361102 and Xu Hou *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005 College of Physical Science and Technology, Xiamen University, Xiamen, Fujian 361005 Institute of Artificial Intelligence, Xiamen University, Xiamen, Fujian 361005 Innovation Laboratory for Sciences and Technologies of Energy Materials of Fujian Province (IKKEM), Xiamen, Fujian 361102 State Key Laboratory of Bio-Fibers and Eco-Textiles, Qingdao University, Qingdao, Shandong 266071 https://doi.org/10.31635/ccschem.023.202302731 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The capture of circulating tumor cells (CTCs) is of great significance in reducing cancer mortality and complications. However, the nonspecific binding of proteins and white blood cells (WBCs) weakens the targeting capabilities of the capture surfaces, which critically hampers the efficiency and purity of the captured CTCs. Herein, we propose a liquid-like interface design strategy that consists of liquid-like polymer chains and anti-EpCAM modification processes for high-purity and high-efficiency capture of CTCs. The dynamic flexible feature of the liquid-like chains endows the modified surfaces with excellent antiadhesion property for proteins and blood cells. The liquid-like surfaces can capture the target CTCs and show high cell viability due to the environment-friendly surface modification processes. When liquid-like surface designs were introduced in the deterministic lateral displacement (DLD)-patterned microfluidic chip, the nonspecific adhesion rate of WBCs was reduced by more than fivefold compared to that in the DLD chip without liquid-like interface design, while maintaining comparable capture efficiency. Overall, this strategy provides a novel perspective on surface design for achieving high purity and efficient capture of CTCs. Download figure Download PowerPoint Introduction Metastasis is the primary factor in fatalities from cancer. The majority of cancer patients receive a cancer metastasis diagnosis and get a dismal prognosis. Circulating tumor cells (CTCs), shed from solid tumors into the vasculature, have been studied as a key class of cancer biomarkers for a deeper understanding of cancer metastasis. Therefore, the capture of CTCs from blood samples facilitates monitoring of cancer progression and treatment response and is of great significance in reducing cancer mortality and complications.1–4 The existing technologies for separating CTCs from the peripheral blood can be classified into two categories: (1) physical force-based separation methods5–9 that depend on the size, deformability, density, and inertial forces and (2) affinity-based separation methods10–15 that depend on the specific interaction between the biomarkers on the CTC membrane and the recognition ligands of aptamer/antibody/peptide on the capture interface. In recent years, affinity-based microfluidic technologies, which utilize the synergistic effect of both physical forces and affinity for sensitive and specific capture of CTCs, have aroused extensive research interest.16–20 For example, Nagrath et al. reported a microfluidic device with specific antibody-modified micropost arrays, where the micropost arrays increase the contact chance of CTCs with the target antibody through improving the mixing efficiency, and the antibody can specifically capture the target CTCs.18 However, the increased mixing efficiency also led to the increase of the frequency of nontargeted cells, such as white blood cells (WBCs), which nonspecifically adsorb on the microchannel surfaces and further decrease the purity of the captured CTCs and reduce the accuracy of molecular analysis. Therefore, it is extremely urgent to find new CTC capture technologies with high efficiency and purity. Aiming at the above-mentioned problem, a series of microfluidic-based CTC capture devices were developed by integrating solid nanointerface design strategies with deterministic lateral displacement (DLD) patterned structures.21,22 In DLD-patterned microfluidic devices, the large-sized CTCs show continuous and frequent collisions with immune-modified patterned structures, while small-sized blood cells migrate in the original flow direction, which minimizes their interaction with the patterned structures and further results in the decreased nonspecific WBC adsorption and high purity CTC capture. By modifying the structures with enhanced interface affinity nanointerface, the capture efficiency of the microfluidic devices was significantly improved compared to that without nanointerface designs.16 In addition, through high-affinity modifications, such as multivalent nanointerface designs, the capture efficiency of CTCs was further improved. However, some proteins such as fibrinogen (Fn) can adsorb on the solid surfaces of the microfluidic devices, which may promote eukaryotic cell adhesion and further reduce the purity of the captured CTCs.23 Therefore, it is urgently needed to develop novel approaches to further minimize the nonspecific interaction and maximize the specific binding. Inspired by the blood cell-resistant capability of the leukocyte membrane, leukocyte membrane-cloaked nanoparticles were reported to decrease their nonspecific adsorption to blood cells.24,25 Recently, we proposed a bioinspired fluidic multivalent nanointerface design strategy by modifying the DLD-patterned microfluidic chip with aptamer-functionalized leukocyte membrane nanovesicles by biotin-streptavidin interaction for high-performance isolation of CTCs with minimized background blood cell adsorption.17 Therefore, soft and flexible interfaces provide a novel design strategy for the high-efficient and purity capture of CTCs for further clinical application of cell-based liquid biopsy.26 Liquid-like surfaces are a kind of surfaces that are modified by flexible polymer brushes, typically polydimethylsiloxane (PDMS) or perfluoropolyether brushes/film, with an extremely low glass transition temperature, and their chemical bonds have a very low rotational conformation transition energy barrier, generally comparable to the thermal motion energy.27–31 Therefore, the surface molecules are highly mobile at room temperature, and the molecular chains have a high dynamic characteristic similar to that of the fluids.32,33 In recent years, liquid-like surfaces have shown great potential applications in areas such as hydro/oleophobic,27 lossless/directional liquid transport,34 antifouling,35 antiicing,36 and condensation heat transfer37 due to their unique interfacial physicochemical properties. However, the applications of liquid-like surfaces for CTC isolation and detection were rarely investigated.38–40 The authors assumed that this resulted from the currently reported preparation processes for liquid-like surfaces that usually use cytotoxic organic solvents, such as toluene, acetone, tetrahydrofuran (THF), etc. When liquid-like molecules are modified on the surfaces of polymers such as PDMS, the residual organic solvents in the networks of polymers lead to cytotoxicity, which results in the death of the target cells and influences the downstream biomedical analysis. Thus, the development of a green and biocompatible preparation method that utilizes nontoxic solvents for liquid-like surfaces seems to be urgently required for the exploration of liquid-like surfaces in the application of the isolation CTCs. The highly flexible liquid-like molecule chains hold the potential of the reduction of the nonspecific adsorption of proteins through the weak interactions between the surface and the proteins (such as hydrogen bond, ionic bond, and ligand/receptor interactions) and the nonspecific adhesion of WBCs, which is important for the CTC isolation with high purity. Herein, we propose a green liquid-like interface design strategy for antibiofouling and high-purity capture of CTCs in affinity-modified DLD-patterned microfluidic devices. Flexible PDMS brushes are modified on the selected substrate through an ethanol solution-based preparation process, and further functionalized by biotinylated anti-EpCAM for specific capture of CTCs. In addition, due to the dynamic flexible feature of the liquid-like chains, the nonspecific adsorption of proteins and WBCs is significantly decreased on the liquid-like surfaces compared to that on solid surfaces. Moreover, by integrating the green liquid-like surface designs with the DLD patterns, high-efficiency and high-purity capture of CTC was achieved, with nonspecific WBC adsorption decreased more than fivefold compared to traditional solid surface designs. Hence, our designed liquid-like interface is an attractive candidate for highly efficient and specific capture of CTCs in clinical settings. Experimental Methods Materials The quartz crystals were obtained from Jiaxing Crystal Electronics Co. (China). Bis(3-aminopropyl) terminated PDMS (μPDMS) with a molecular weight of 3000, N,N'-disuccinimidyl carbonate (DSC), and 3-aminopropyl triethoxysilane (APTES) were purchased from Aladdin (China). Streptavidin (SA), and acid orange was purchased from Sigma-Aldrich (United States). Biotinylated anti-EpCAM and 6-diamidino-2-phenylindole (DAPI) were purchased from Thermo Fisher Scientific Inc. (United States). Fluorescein isothiocyanate isomer (FITC)-labeled antihuman fibrinogen was purchased from Bioss (China). Ethanol, toluene, n-hexane, N,N-dimethylformamide (DMF), THF, and dimethyl sulfoxide (DMSO) were used as received. Deionized water with a resistivity of 18.2 MΩ cm was obtained from Milli-Q system. Preparation of liquid-like polymer chains on the substrates Ethanol solution of DSC (1 μg mL−1) and ethanol solution of μPDMS (1 mg mL−1) were prepared for the grafting of liquid-like polymer chains. The amino-functionalized substrates were then dipped in ethanol solution of DSC for 60 min at room temperature, and rinsed in ethanol and washed rapidly. The sample was then dipped in the ethanol solution of μPDMS for another 60 min at room temperature, rinsed in ethanol, and then dried by compressed air. The above two processes were repeated for several cycles to obtain longer μPDMS polymer brushes. Next, the samples were immersed in the phosphate-buffered saline (PBS) solution of SA (10 μg mL−1) at room temperature for 1 h. After the samples were ultrasonically washed with PBS and dried by nitrogen gas, the samples were immersed in PBS solution of anti-EpCAM (20 μg mL−1) at 4 °C overnight. The preparation of liquid-like interface modified microfluidic inner surface was similar to that described above. CTC viability SW480 colorectal tumor cells were employed as representative CTCs for the cell counting kit-8 (CCK-8) assay to confirm whether liquid-like interface was toxic to CTCs. SW480 cells were cultured on the pure PDMS and liquid-like interface for 24 h, respectively. The medium was removed, and the percentage of the survival of CTCs was quantified by CCK-8 assay. To investigate the apoptosis of CTCs on each surface, a cell permeable acridine orange (AO) in combination with a plasma membrane-impermeable DNA-binding dye propidium iodide (PI) was applied. AO and PI excite green and red fluorescence respectively when they are intercalated into DNA. Only AO but not PI can cross the plasma membrane of a normal cell. In brief, CTCs were cultured onto the samples for 1 h. Subsequently, the cells were stained with a 1:1 mixture of AO (100 mg mL−1) and PI (100 mg mL−1) at 37 °C for 5 min, and then inspected in a fluorescence microscope immediately. Characterization of the liquid-like interface The self-assembly process of the liquid-like polymer was monitored using quartz crystal microbalance with dissipation (QCM-D) (Q-Sense AB, Sweden). The QCM-D has the ability of simultaneously measuring the normalized resonant frequency and energy dissipation shifts. First, the gold-coated crystals were modified with amine groups. Second, the ethanol solution of DSC was injected into the measurement cell at a proper flow rate using a peristaltic pump. Then, the ethanol solution of μPDMS was injected into the measurement cell. All the experiments were conducted at room temperature. The acid orange II (AO II) colorimetric method was used to determine the density of amine groups. In detail, the samples were immersed in an aqueous solution of AO II (pH 4.0) for 4 h. Then the samples were washed with aqueous solution of HCl (pH 4.0) to remove unreacted AO II. Afterward, the AO II on the sample was eluted with 200 μL of aqueous solution of NaOH (pH 11.0), and the amount of AO II was measured by the fluorescence signal with excitation at 485 nm and emission at 520 nm using a Molecular Devices SpectraMax ID5. The density of amine groups was calculated based on a standard curve. The chemical compositions of the liquid-like polymer layer were analyzed by X-ray photoelectron spectroscopy (XPS). The instrument (PHI Quantum 2000 Scanning ESCA Microprobe, Physical Electronics, Eden Prairie, Minnesota, United States) was equipped with a monochromatic Al Kα (1486.6 eV) X-ray source operated at 15 kV and 35 W at a pressure of 5 × 10−7 Pa. The carbon peak (284.4 eV) was designated as the reference for charge calibration. The thickness of the grafted liquid-like polymer layer was measured by a spectroscopic ellipsometer M-2000U. The surface topologies of the grafted liquid-like polymer layer were analyzed by an atomic force microscope (Cypher S, Asylum Research-Oxford Instruments, Santa Barbara, California, United States) using the tapping mode in air. Sliding angle measurements were carried out using OCA20 equipment (Data Physics, Filderstadt, Germany) under ambient conditions. The surface was tilted with respect to the horizontal plane until the liquid droplet started to slide along the surface. Adsorption of protein on the surfaces The samples were covered with 50 μL of fresh human platelet poor plasma extracted from fresh venous blood obtained from a healthy adult volunteer for 1 h and then rinsed three times with PBS to remove the nonadhered Fn. Subsequently, the samples were blocked with 1 wt % bovine serum albumin (BSA) in PBS at 37 °C for 30 min and then rinsed with PBS. Finally, FITC-labeled antihuman fibrinogen was added and incubated at 37 °C for 1 h. After rinsing with PBS again, the stained samples were observed under an inverted fluorescence microscope (TH4-200, Olympus, Tokyo, Japan). Quantitation of the protein adsorption was measured using a QCM-D. After construction the liquid-like interface on sensors, a PBS solution (pH 7.4) of BSA (50 μg mL−1) and Fn (1 μg mL−1) was introduced to the axial flow sample chamber and kept at a constant flow rate of 1 mL h−1 for 60 min in sequence. Adhesion of WBCs on the surfaces Fresh whole blood of a volunteer was added to the sample surfaces for 1 h. Then, the adherent blood cells were fixed with 2.5% glutaraldehyde solution at 4 °C for 2 h. Next, the morphology of the adhered blood cells was examined using by a scanning electron microscope (SEM, 2400 s, Hitachi, Tokyo, Japan). Adhesion of CTCs on the surfaces SW480 colorectal tumor cells were cultured on the samples for 1 h. After washing, SW480 cells were fixed by 4% polyformaldehyde, and then blocked by goat serum, followed by immunofluorescence staining with anti-panCK. The immunofluorescence results were scanned by a fluorescence microscope (Nikon, Tokyo, Japan). Capture of CTCs in the DLD patterned microfluidic chips 100 LNCap prostate and SW480 colorectal cells were suspended in 1 mL of whole blood obtained from healthy donors, respectively. All the blood samples were injected into the chip at a flow rate range from 0.5 to 3 mL h−1. After capture and washing, cells on the chip were fixed by 4% polyformaldehyde and then blocked by goat serum, followed by immunofluorescence staining with DAPI and anti-panCK. The immunofluorescence results were scanned by a fluorescence microscope (Nikon, Japan). Results and Discussion Design and working principle of liquid-like interface The schematic illustration of the working mechanism of liquid-like surface design for reduced nonspecific adhesion of proteins and WBCs and specific capture of CTCs is shown in Figure 1a. The liquid-like surfaces are composed of PDMS polymer brushes and terminal-modified biotinylated anti-EpCAM. The PDMS polymer brushes grafted on the surfaces are highly mobile at room temperature because of their highly melted state resulting from their extremely low glass transition temperature, and they act as a liquid-like slippery layer.37 When the proteins contact the liquid-like surfaces, it is difficult for them to nonspecifically adhere on the surfaces due to the highly mobile polymer chains, which further reduce the adhesion of WBCs. Though at a highly mobile state, the terminal biotinylated anti-EpCAM on the liquid-like molecule chains offer the affinity sites for the target CTCs and can specifically capture them from the biological fluids. Figure 1 | Design and working principle of the liquid-like interfaces. (a) Schematic diagram showing the "liquid-like" interface for decreased nonspecific adsorption of proteins and WBCs and specific capture of CTCs. (b) Schematic of the green and environmentally friendly design processes of liquid-like interface. (c) Biocompatibility of the liquid-like interfaces. Using ethanol as the solvent for liquid-like interface design, the PDMS film does not swell and shows low cytotoxicity. Using toluene as the solvent, the PDMS swells and shows high cytotoxicity. AO in combination with a plasma membrane, impermeable DNA-binding dye PI was applied to detect the vitality of CTCs. Only AO but not PI can pass through the plasma membrane of normal CTCs. Normal CTCs emit green fluorescence (AO), and dead CTCs emit red fluorescence (PI). Download figure Download PowerPoint The green and noncytotoxic liquid-like interface modification process by a covalent layer-by-layer method is illustrated in Figure 1b. To fulfill the requirements of nontoxic organic solvents in the whole process, we use ethanol and deionized water as the solvent in the whole solution-based modification process, and chose the ethanol soluble monomers DSC and μPDMS to graft the polymer brushes. First, the substrates were grafted a monolayer of amino groups by oxygen plasma treatment and immersed in the ethanol solution of APTES. Second, the amino modified surface was immersed in the ethanol solution of DSC and μPDMS subsequently to obtain liquid-like polymer chains via the ring-opening reaction between succinimide groups and the amino groups.41 Third, the above process was repeated for several cycles to obtain longer liquid-like μPDMS polymer brushes. Fourth, the amino-group terminated μPDMS brushes were further modified by the SA and anti-EpCAM to endow the liquid-like surfaces with specific binding capability to CTCs. Compared to traditional cytotoxic organic solvent-based preparation processes, the liquid-like surfaces prepared through our green synthesis processes here are more suitable to modify a broad range of materials (such as glass, silicon, PDMS, etc.) and show significant high cell viability. Due to the ethanol solution-based modification processes, the PDMS (mostly used material in microfluidic devices) film was not swelled and maintained its flatness after the modification processes. In contrast, PDMS film was swelled and deformed when immersed in solvents such as toluene, acetone, THF, etc. (Figure 1c and Supporting Information Figure S1), which indicate that a significant amount of residual toxic solvent is left in the PDMS networks. The residual toxic solvent in the PDMS networks can be released onto the surface and into the cell culture medium, which brings about cytotoxicity and results in the ratio of vital CTCs as low as 3.5%. However, the ratio of vital CTCs on the liquid-like surfaces prepared through green synthesis processes reached up to 96.3% ( Supporting Information Figure S2a), demonstrating the high viability of the surfaces and the biocompatibility of our method, which is critical for the subsequent cell culture and genetic analysis. Supporting Information Figure S2b shows that the CTC viability of the liquid-like interface was better than the common PDMS (well-known as biocombatible polymer employed for many applications in the biomedical field), indicating that the liquid-like interface is favorable, biocompatible, and exhibits no obvious cytotoxicity. Characterization of controllable liquid-like surface To precisely control the reaction conditions in our green liquid-like interface design processes, the self-assembly processes of DSC and μPDMS polymer chains were characterized by quartz crystal microbalance with dissipation mode (QCM-D), which can detect mass changes at the surface in a sensitive and real-time manner. As shown in Figure 2a, the grafting of the DSC on the APTES-modified surface was completed in a short time, and the grafting density of DSC on the surface was increased to 15.38 ng cm−2, while the grafting density on the surface was gradually increased to 53.76 ng cm−2 after grafting of μPDMS chains on the DSC surface. The amino group density of the surfaces also provides an alternative characterization method for the modification process. The surface amino density gradually decreased from the 6.63 to minimal 0.56 nmol cm−2 after DSC modification with a reaction time of 80 min, and then recovered to a density approximating the original state (6.30 nmol cm−2) after μPDMS modification with a reaction time of 80 min (Figure 2b), indicating a relatively complete reaction. The surface chemical properties after each modification process were also characterized by XPS, and the fitting curve of peaks of C1s indicates the successful modification of DSC and μPDMS chains (Figure 2c). Figure 2 | Physicochemical characterizations of the liquid-like interface. (a) Monitoring the self-assembly process by measuring the grafting density of DSC and μPDMS using QCM-D. (b) The amount of amine groups was determined by the acid orange method. (c) The fitting curve of peaks of C1s of XPS measurement before and after modification of DSC and μPDMS. (d) Ellipsometry thicknesses of liquid-like polymer as a function of the number of modification cycles. (e) Sliding angles of a droplet of blood (10 μL) on the liquid-like interface with different numbers of modification cycles. (f) The surface roughness of the liquid-like interface as a function of the number of modification cycles. (g) Sliding angle of liquids with different surface tension on the liquid-like interface with four modification cycles. (h) Sliding of 20 μL of blood on the solid interface and liquid-like interface with a slanting angle of 10°. Download figure Download PowerPoint The thickness of the modified flexible polymer brushes is increased with the increment of the cycle number of the layer-by-layer modification process. As shown in Figure 2d, the thickness of the self-assembled layer increases from 3.45 to 17.6 nm when the modification cycle increases from 1 to 6. The thickness increase is beneficial for enhancing the flexibility of the liquid-like molecules. The liquid-like property of the surfaces is characterized by measuring the sliding angle of the droplets of blood, water, ethanol, and organic solvents such as n-hexane, toluene, DMF, and DMSO on the modified surfaces. As shown in Figure 2e, the sliding angle of the blood droplet (10 μL) decreases from 36.6° to 16.3° when the modification cycle increased to 4, indicating the enhancement of the flexibility of the liquid-like molecules. However, it was increased to a small extent when the modification cycle was further increased, mainly due to the increase in the roughness of the modified surface (Figure 2f and Supporting Information Figure S3). Therefore, four cycles of modification is proper for the liquid-like surface design. Figure 2g shows the sliding angle of the different droplets on the liquid-like surface with four modification cycle. Due to the high viscosity of blood, the sliding angle of blood is higher than water and organic solvents. Nevertheless, the blood droplet (20 μL), ethanol droplet, and oil droplet dropped onto the liquid-like surface slid off readily without leaving any traces on the liquid-like surface and pinned on the conventional solid surface at a same slant angle of 10° (Figure 2h and Supporting Information Figure S4). The above results indicate the flexibility of the polymer brush and liquid-like property of the modified surface. Reduced nonspecific adhesion of proteins and WBCs, and the specific adhesion of CTCs In addition, the high-flexibility of the polymer chains also endows the liquid-like surface with reduced nonspecific adhesion of protein and WBCs. The quantity of plasma protein adsorbed on the surface has a significant impact on the blood compatibility since protein adsorption is the first event that initiates the following bioresponses such as cell adhesion. Here, we choose blood BSA and Fn as the representative proteins to study the antiadhesion performance of the liquid-like interfaces. The BSA adsorption occurs immediately when the blood samples make contact with the capture surface. Then the surface-adsorbed BSA is substituted by Fn with higher surface affinity based on the Vroman effect.42 The Fn was labeled with FITC (red) to characterize their adhesion on the surfaces by the fluorescence microscope. As shown in Figure 3a, strong re

Polymer probes light up: Amplifying fluorescent polymers conjugated with biomolecules have excellent light-harvesting and superquenching properties (see scheme). A novel synthetic method is reported for the highly efficient, simple, fast, and readily controllable conjugation of a water-soluble poly(phenylene ethynylene) with an oligonucleotide.

Speedy delivery: A self-assembled bifunctional unit incorporates a DNA aptamer for target recognition (see picture, green helix) and a G-quadruplex for drug loading. The modified DNA selectively delivered a photosensitizer (red bars) to cancer cells; irradiation with visible light generated high toxicity. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Simple, fast and direct analysis or monitoring of significant molecules in complex biological samples is important for many biological study, clinical diagnosis and forensic investigations. Herein we highlight a general method to tailor aptamer sequence into functional subunits to design target-induced light-switching excimer sensors for rapid, sensitive and selective detection of important molecules in complex biological fluids. Our approach is to split one single strand aptamer into two pieces and each terminally labeled with a pyrene molecule while maintaining their binding affinity to target molecules. In the presence of target molecules, two aptamer fragments are induced to self-assemble to form aptamer-target complex and bring two pyrene molecules into a close proximity to form an excimer, resulting in fluorescent switching from approximately 400 nm to 485 nm. With an anti-cocaine sensor, as low as 1 microM of cocaine can be detected using steady-state fluorescence assays and more importantly low picomole level of target can be directly visualized with naked eyes. Because the excimer has a long fluorescence lifetime, time-resolved measurements were used to directly detect as low as 5 microM cocaine in urine samples quantitatively without any sample pretreatment.

This paper reports a nontoxic, rapid, one-pot and template-free synthesis of three-dimensional (3D) Pt nanoflowers (PtNFs) with high yield and good size monodispersity supported on graphene oxide (GO) nanosheets. The key synthesis strategy employed a low-cost, green solvent, ethanol as the reductant and an advanced, powerful 2D carbon material, GO nanosheets as the stabilizing material. The resulting PtNFs-GO nanosheets were characterized by transmission electron microscopy (TEM), high-resolution TEM, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and electrochemical techniques. It was found that the monodispersed, porous PtNFs supported on GO nanosheets were a uniform size of 30 nm and each was composed of numerous “clean” and small (4 nm) Pt nanoparticles, which revealed an unusually high activity for methanol oxidation reaction compared to commercial Pt black. Furthermore, based on a systematic study of the PtNFs growth conditions, a possible mechanism, and especially the importance of GO in the formation was proposed. Our study demonstrates that GO is a promising support material for developing next-generation advanced Pt based fuel cells and their relevant electrodes in the field of energy.

A T7 exonuclease-assisted cyclic enzymatic amplification method (CEAM) was combined with rolling circle amplification (RCA) to develop a RCA–CEAM dual amplification method for ultrasensitive detection of microRNA with excellent selectivity.