Lisa C. Shriver‐Lake

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional crosslinkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2–6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immonilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.

Covalent attachment of functional proteins to a solid support is important for biosensors. One method employs thiol-terminal silanes and heterobifunctional crosslinkers such as N-succinimidyl 4-maleimidobutyrate (GMBS) to immobilize proteins through amino groups onto glass, silica, silicon or platinum surfaces. In this report, several heterobifunctional crosslinkers are compared to GMBS for their ability to immobilize active antibodies onto glass cover slips at a high density. Antibodies were immobilized at densities of 74–220 ng/cm2 with high levels of specific antigen binding. Carbohydrate-reactive crosslinkers were also compared to GMBS using a fiber optic biosensor to detect fluorescently-labeled antigen. At the concentrations tested, the antibodies immobilized with carbohydrate-reactive crosslinkers bound more antigen than GMBS immobilized antibodies as indicated by the fluorescence signal.

With recent advances in surface chemistry, microfluidics, and data analysis, there are ever increasing reports of array-based methods for detecting and quantifying multiple targets. However, only a few systems have been described that require minimal preparation of complex samples and possess a means of quantitatively assessing matrix effects. The NRL Array Biosensor has been developed with the goal of rapid and sensitive detection of multiple targets from multiple samples analyzed simultaneously. A key characteristic of this system is its two-dimensional configuration, which allows controls and standards to be analyzed in parallel with unknowns. Although the majority of our work has focused on instrument automation and immunoassay development, we have recently initiated efforts to utilize alternative recognition molecules, such as peptides and sugars, for detection of a wider variety of targets. The array biosensor has demonstrated utility for a variety of applications, including food safety, disease diagnosis, monitoring immune response, and homeland security, and is presently being transitioned to the commercial sector for manufacturing.

Contamination of food by mycotoxins occurs in minute quantities, and therefore, there is a need for a highly sensitive and selective device that can detect and quantify these organic toxins. We report the development of a rapid and highly sensitive array biosensor for the detection and quantitation of ochratoxin A (OTA). The array biosensor utilizes a competitive immunoassay format. Immobilized OTA derivatives compete with toxin in solution for binding to fluorescent anti-OTA antibody spiked into the sample. This competition is quantified by measuring the formation of the fluorescent immunocomplex on the waveguide surface. The fluorescent signal is inversely proportional to the concentration of OTA in the sample. Analyses for OTA in buffer and a variety of food and beverage samples were performed. Samples were extracted with methanol, without any sample cleanup or preconcentration step prior to analysis. The limit of detection for OTA in several cereals ranged from 3.8 to 100 ng/g, while in coffee and wine, detection limits were 7 and 38 ng/g, respectively.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetection of TNT in Water Using an Evanescent Wave Fiber-Optic BiosensorLisa C. Shriver-Lake, Kristen A. Breslin, Paul T. Charles, David W. Conrad, Joel P. Golden, and Frances S. LiglerCite this: Anal. Chem. 1995, 67, 14, 2431–2435Publication Date (Print):July 15, 1995Publication History Published online1 May 2002Published inissue 15 July 1995https://doi.org/10.1021/ac00110a018RIGHTS & PERMISSIONSArticle Views725Altmetric-Citations99LEARN 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 PDF (1 MB) Get e-Alertsclose Get e-Alerts

Because of the potential health risks of aflatoxin B1 (AFB1), it is essential to monitor the level of this mycotoxin in a variety of foods. An indirect competitive immunoassay has been developed using the NRL array biosensor, offering rapid, sensitive detection and quantification of AFB1 in buffer, corn and nut products. AFB1-spiked foods were extracted with methanol and Cy5-anti-AFB1 added to the resulting sample. The extracted sample/antibody mix was passed over a waveguide surface patterned with immobilized AFB1. The resulting fluorescence signal decreased as the concentration of AFB1 in the sample increased. The limit of detection for AFB1 in buffer, 0.3 ng/ml, was found to increase to between 1.5 and 5.1 ng/g and 0.6 and 1.4 ng/g when measured in various corn and nut products, respectively.

Proteins were attached in defined geometric patterns on a surface. A prerequisite to making a pattern of proteins is generation of surfaces resistant to nonspecific protein adsorption. This was accomplished via oxidation of the thiol terminus of an organosilane self-assembled monolayer film by deep ultraviolet (DUV) irradiation. The resultant surface exhibited marked resistance to protein adsorption. Using a mask to protect regions of the silanized surface from irradiation, proteins were selectively adsorbed or attached via covalent linkage at locations protected from the DUV light. Antibodies immobilized in patterns using this procedure retained their antigen-binding capability. Thus chemistry and DUV lithography were combined to create patterns of active biomolecules which could be used in the microfabrication of electronic devices and biosensors.

Foodborne contaminants come in a variety of sizes ranging from simple chemical compounds to entire bacterial cells. Due to public health concerns, there is a current need in the food industry for a sensitive, specific and rapid method to monitor for the presence of these toxic species, either as a result of natural or deliberate contamination. The Array Biosensor developed at the NRL encompasses these qualities, including the ability to measure multiple analytes simultaneously on a single substrate. In this study, we demonstrate the Array Biosensor's ability to measure both large pathogens, such as the bacteria Campylobacter jejuni (C. jejuni), and small toxins, including the mycotoxins ochratoxin A, fumonisin B, aflatoxin B1 and deoxynivalenol. Sandwich immunoassays were used to measure C. jejuni in buffer and a number of food matrices, while competitive immunoassays, taking only 15 min, were developed for the simultaneous detection of multiple mycotoxins. The combination of sandwich and competitive immunoassay formats on a single substrate was demonstrated, allowing the simultaneous detection of both large (C. jejuni) and small (aflatoxin B1) food contaminants.

Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.

Biosensors have successfully demonstrated the capability to detect multiple pathogens simultaneously at very low levels. Miniaturization of biosensors is essential for use in the field or at the point of care. While microfluidic systems reduce the footprint for biochemical processing devices and electronic components are continually becoming smaller, optical components suitable for integration--such as LEDs and CMOS chips--are generally still too expensive for disposable components. This paper describes the integration of polymer diodes onto a biosensor chip to create a disposable device that includes both the detector and the sensing surface coated with immobilized capture antibody. We performed a chemiluminescence immunoassay on the OPD substrate and measured the results using a hand-held reader attached to a laptop computer. The miniaturized biosensor with the disposable slide including the organic photodiode detected Staphylococcal enterotoxin B at concentrations as low as 0.5 ng/mL.

A portable fiber optic biosensor was employed for on-site analysis of the explosive trinitrotoluene (TNT) in groundwater. Two military bases, Umatilla Army Depot and Naval Submarine Base Bangor, contain sites where the groundwater has been contaminated with TNT. Samples from monitoring wells at these sites were split and analyzed on-site using the fiber optic biosensor and in a laboratory by U.S. EPA SW-846 Method 8330 (reverse-phase high-performance liquid chromatography). The fiber optic biosensor is small (6.5 in. × 4.5 in. × 3.5 in., 2.5 lb), portable, and able to monitor four optical probes simultaneously. A competitive, fluorescent immunoassay was performed on the surface of the optical fiber probe. The fiber optic biosensor was able to detect 20 μg/L TNT in less than 16 min simultaneously on four probes.

A fiber optic immunosensor has been developed for simultaneous detection of the most common explosives, 2,4,6,-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It employs a competitive immunoassay on the antibody-coated fiber optic probes, in which a fluorescent antigen competes with free antigen of unknown concentration for binding sites on the fiber surface. To achieve dual explosive detection, two α-TNT fiber probes and two α-RDX fiber probes are connected in series. The sample is mixed with fluorescent analogs, Cy5-ethylenediamine-trinitrobenzene (Cy5-EDA-TNB) and Cy5-ethylenediamine-RDX hapten (Cy5-EDA-RDH). Inhibition of the maximum signal in the presence of the sample is proportional to the concentration of the explosive. The detection limits for the multi-analyte assays are equivalent (6 ng/ml for both TNT and RDX) to those of the individual assays (5 ng/ml for both TNT and RDX). The standard curves for TNT and RDX represent a linear relationship between percent signal inhibition and the natural logarithm of analyte concentration in the multi-analyte format, as well as in single analyte assays, thus allowing a simple and precise method of quantification. There is minimal cross-reactivity for the two antigens in the multi-analyte immunosensor, so it is also an effective means in analyzing samples containing mixtures of RDX and TNT.

Deoxynivalenol (DON), a mycotoxin produced by several Fusaruim species, is a worldwide contaminant of foods and feeds. Because of the potential dangers due to accidental or intentional contamination of foods with DON, there is a need to develop a rapid and highly sensitive method for easy identification and quantification of DON. In this study, we have developed and utilized a competitive immunoassay technique to detect DON in various food matrixes and indoor air samples using an array biosensor. A DON-biotin conjugate, immobilized on a NeutrAvidin-coated optical waveguide, competed with the DON in the sample for binding to fluorescently labeled DON monoclonal antibodies. To demonstrate a simple procedure amenable for on-site use, DON-spiked cornmeal, cornflakes, wheat, barley, and oats were extracted with methanol-water (3:1) and assayed without cleanup or preconcentration. The limits of detection ranged from 0.2 ng/mL in buffer to 50 ng/g in oats. The detection limit of DON spiked into an aqueous effluent from an air sampler was 4 ng/mL.

Summary, 47 Introduction, 47 Total internal reflection fluorescence transduction, 48 The molecular recognition element, 49 Immobilization of the biomolecule onto the waveguide, 49 Creation of low-density biomolecular arrays, 50 TIRF array biosensors: state of the art, 50 Miniaturization and automation of TIRF array biosensors, 52 The future, 54 Acknowledgements, 55 References, 55 Total internal reflection fluorescence (TIRF) is a process whereby fluorophores that are either attached to or are in close proximity with the surface of a waveguide are selectively excited via an evanescent wave. Planar waveguides provide the possibility of immobilizing multiple capture biomolecules onto a single surface and therefore, offer the exciting prospect of multi-analyte detection. The production of arrays and the results of various groups which use TIRF to interrogate such surfaces is reviewed, along with a look at how far the field has advanced toward the production of an automated, portable, multi-analyte array biosensor for real-time biohazard detection. In particular, a miniaturized, fully automated, stand-alone array biosensor developed at the Naval Research Laboratory is reported that monitors interactions between binding partners either as the final image or in real-time. A variety of analytes including toxins, bacteria and viruses have been detected both in buffer and complex matrices, such as blood and soil suspensions, with comparable detection limits. A number of developments have led to a TIRF array biosensor weighing only 5·5 kg which is automated for environmental, clinical and food monitoring or for detection of bioterrorist agents. Potential biohazards, that present a threat to human health, are numerous and encompass protein, bacterial and viral analytes, some examples of which are given in Table 1. Whether the analytes are food-, water- or air-borne, there is a current need for rapid detection and agent identification. A reliable sensor would have applications in areas such as environmental monitoring of pollutants, emergency room medical diagnostics and health care, process monitoring in the chemical, food and beverage industries, and early warning biological warfare (BW) agent detection for military and homeland defense. Clearly, such a device should be small, lightweight and portable, highly sensitive, capable of multi-analyte discrimination, and able to measure analytes in complex sample matrices with little or no sample pretreatment. Due to the sensitivity and specificity of biological molecules, biosensors are ideal candidates for such a system, offering the possibility of rapid, continuous field monitoring not currently provided by established measurement techniques (Hall 1990; Braguglia 1998; Eggins 1998; Pearson et al. 2000). The development of array biosensors, which provide multi-analyte detection capability, is a relatively new field for both optical and electrochemical transduction. The technology owes much to advances in microfabrication and the human genome project, which has led to the ability to immobilize arrays of biomolecules in discrete regions on a transducer surface. This review will deal with optical transduction, in particular total internal reflection fluorescence (TIRF), although the field of electrochemical micro-array transduction is also generating much interest and research (Wang et al. 1997; Kukla et al. 1999; Wu 1999; Zhang et al. 2000; Gray et al. 2001; Krantz-Rulcker et al. 2001; Pancrazio 2001; Young et al. 2001). A brief introduction into the principle and typical instrumentation used in the TIRF transduction mechanism will be given. The choice of biomolecule and methods by which it is immobilized onto a planar waveguide will be discussed followed by an introduction to the various techniques used to create low-density arrays. The production of arrays and the results of various groups which use TIRF to interrogate such surfaces is reviewed, along with a look at how far the field has advanced toward the production of an automated, portable, multi-analyte array biosensor for real-time biohazard detection. Fluorescence, absorbance, bioluminescence, chemiluminescence and refractive index changes can all be exploited for the development of optical biosensors. However, in terms of array biosensors, fluorescence and refractive index change using reflectance transduction are the most popular. Techniques that can be grouped under the principle of reflectance include: attenuated total reflectance which monitors alterations in the infrared, visible and u.v. regions; surface plasmon resonance (SPR) (Homola et al. 2002) and interferometric techniques (Campbell and McCloskey 2002), which measure variations in refractive index; and TIRF (Sapsford et al. 2002a), which monitors generation of a fluorescence signal. SPR imaging (Homola et al. 2001a,b; Lee et al. 2001; Lu et al. 2001; Nelson et al. 2001; O'Brien et al. 2001; Wegner et al. 2002), interferometry (Schipper et al. 1997, 1998; Schneider et al. 1997, 2000; Campbell et al. 1998, 1999; Plowman et al. 1998; Edwards et al. 1999) and TIRF have all been developed as transduction methods to investigate the interactions of arrays of biomolecules immobilized onto a sensing surface. In TIRF measurements, the evanescent wave interacts with and excites the fluorophore near the surface of the waveguide, and the resulting fluorescence is measured by the detector (Lu et al. 1992; Chittur 1998; Plowman et al. 1998; Wadkins et al. 1998). There has been extensive research into improving the optics and sensitivity of TIRF instrumentation. Most of the final systems described consist of a number of similar components (see Fig. 1), such as the light source and detector and a variety of focusing lenses to improve detector response (Duveneck et al. 1995; Herron et al. 1996, 1997; Golden 1998; Feldstein et al. 1999). The basic experimental arrangement of a system based on the principle of total internal reflection fluorescence (TIRF) (adapted from Sapsford et al. 2002a) Coherent light in the form of lasers is typically used as the excitation source in TIRF studies. The exact choice of the laser is dependent upon the fluorescent label used. The most commonly used lasers include the argon-ion (488 nm) laser for the fluorescein label and a helium–neon (633 nm) or diode laser (635 nm) for the cyanine dye (Cy5) and Alexafluor labels. The laser light is typically coupled into the waveguide using either lens or grating techniques. One effect of using bulk internal reflection element (IRE) waveguides and collimated light is the production of sensing ‘hot spots’ along the planar surface. These occur where the light beam is reflected, illuminating only discrete regions of the waveguide sensing surface. These hot spots have been successfully utilized as sensing regions by Brecht et al. (1998) and Klotz et al. (1998) in the development of an immunofluorescence sensor for water analysis. In contrast, there are a number of methods for achieving uniform longitudinal excitation of the sensing region. A popular technique involves the use of integrated optical waveguides (IOWs) which consist of thin films of inorganic metal oxide compounds such as tin oxide (Duveneck et al. 1995), indium tin oxide (Asanov et al. 1998), silicon oxynitride (Plowman et al. 1999; Hofmann et al. 2002) and tantalium pentoxide (Duveneck et al. 1997; Pawlak et al. 1998). The light is then coupled into these IOWs via a prism or grating arrangement; however, this results in increased constraints and requirements of the optical components which could reduce the robustness of a device should it be intended for field applications. Feldstein et al. (1999, 2000) used an alternate approach by incorporating a line generator and a cylindrical lens to focus the beam into the multi-mode bulk waveguide that included a propagation and distribution region prior to the sensing surface, thereby producing both uniform lateral and longitudinal excitation of the microscope slide. Herron et al. (1996, 1997) also utilized a cylindrical lens to focus the laser beam; however, in their system the lens was molded as part of the planar waveguide holder. Golden (1998) used a two-dimensional graded index lens to focus the fluorescence from the planar waveguide onto a charge coupled device (CCD), providing a shorter working distance than a standard lens with a concomitant decrease in overall instrument size. The introduction of bandpass and longpass filters was found to improve the rejection of scattered laser light and hence reduce the background of the system (Feldstein et al. 1999). A number of devices have been used for detection of the resulting fluorescence emission, in particular CCD cameras (Silzel et al. 1998; Feldstein et al. 1999; Plowman et al. 1999), multiple photomultiplier tubes (PMT) (Schult et al. 1999), photodiodes (Brecht et al. 1998; Golden and Ligler 2002), a single PMT (Lundgren et al. 2000; Schuderer et al. 2000) and more recently a complementary metaloxide-semiconductor (CMOS) camera (Vo-Dinh et al. 1999; Golden and Ligler 2002). The choice of biomolecule used in the development of an array biosensor is largely dependent on the availability of the bio-recognition molecule for the analyte of interest and the application required (Iqbal et al. 2000). To date, antibody–antigen interactions, nucleic acid hybridization (DNA/RNA), and to a lesser extent, receptor–ligand binding have been monitored via TIRF. Although these biomolecules typically contain intrinsic fluorescence, in the form of amino acid residues or cofactors, extrinsic fluorescence labels which preferably excite at a different wavelength are normally introduced to one of the binding partners. These extrinsic fluorescence labels take the form of dyes, such as rhodamine, coumarin, cyanine, or fluorescein, and allow the use, through spectral selection, of visible wavelength excitation sources, such as laser diodes. Antibody–antigen binding interactions are the most well characterized systems employed in TIRF-based sensors. The assays are carried out using antibody–antigen systems and can be divided into four main categories: direct, competitive, displacement and sandwich immunoassays (Rabbany et al. 1994; Sapsford et al. 2002b). The direct assay is the simplest method to perform; however, it requires that the antigen contain some form of intrinsic fluorescence that can be detected. In the absence of a fluorescent antigen, the preferred formats are competitive and sandwich assays. Competitive formats are especially useful in the detection of molecules, such as 2,4,6-trinitriotoluene (TNT; MW 213 Da), not large enough to possess two distinct epitopes (e.g. haptens) as required for the sandwich assays (Silzel et al. 1998; Plowman et al. 1999; Rowe et al. 1999a,b; Schult et al. 1999; Sapsford et al. 2002b). The displacement assay format has only recently been demonstrated in planar waveguide TIRF for measurement of the explosive TNT (Sapsford et al. 2002b). To date only electrochemical transduction mechanisms have been extensively used for DNA biosensors in both environmental monitoring and BW agent detection, as reviewed in the literature (Wang et al. 1997; Iqbal et al. 2000). Currently, high-density DNA/RNA microarray biosensors based on optical transduction, such as confocal microscopy and TIRF (Duveneck et al. 1997; Budach et al. 1999; Schuderer et al. 2000) have simply monitored DNA hybridization between an immobilized strand and its fluorescent-labelled complement. Clearly, more has to be performed towards the development of portable, biohazard, optical-based sensing systems using DNA arrays. Currently, only a limited number of studies describing receptor–ligand binding using planar waveguide TIRF have been reported (Schmid et al. 1997, 1998; Pawlak et al. 1998; Rowe-Taitt et al. 2000a). One major problem with studying receptor–ligand binding has been the immobilization of the receptor protein such that it remains active on the surface. When successful, receptor–ligand binding studies offer applications in the pharmaceutical industry for drug development, for investigating membrane processes and also in biohazard monitoring applications, as demonstrated by Rowe-Taitt et al. (2000a) for toxin binding to ganglioside. One important prerequisite for all immobilization techniques is that the integrity of the biomolecule be preserved and that the active site remain accessible to the binding partner. There are various methods in which the biological component of a biosensing system can be immobilized onto the surface of the transducer, including physical adsorption, covalent immobilization, and entrapment in polymer matrices (Hall 1990). Physical adsorption and covalent binding to functionalized surfaces are the most commonly used in TIRF measurements. There are a number of different planar surfaces used in the immobilization of biomolecules for study with TIRF. These include simple bulk waveguides such as glass, silica and polystyrene, and the slightly more complicated IOW waveguides such as tantalium pentoxide (Ta2O5). There are, likewise, a variety of different surface chemistries used to modify these waveguides in order to facilitate biomolecule immobilization. Hofmann et al. (2002), for example, used a dextran-based photo-immobilization procedure to produce a network-like multilayer structure of immobilized rabbit IgG capture antibodies. Silanization of the waveguide, whether it be the bulk glass or an IOW, is a popular method of functionalizing the surface for further chemistry, whether physical (Plowman et al. 1999) or covalent (Asanov et al. 1998). The avidin-biotin interaction is also extensively used in the immobilization of biotinylated molecular recognition elements. This noncovalent protein–ligand interaction is commonly used either via the physical adsorption of avidin onto the surface (Herron et al. 1993, 1996; Silzel et al. 1998; Schult et al. 1999; Schuderer et al. 2000) or in the production of multi-layers; often involving the use of both covalent and noncovalent interactions (Rowe et al. 1999a; Birkert et al. 2000). A number of the researchers currently involved in developing planar waveguide TIRF focused much of their initial research in the field of fiber optics. Planar waveguides offer a number of advantages compared with fiber optic technology, including the relative ease of preparation and integration into fluidic systems. As a precedent to patterning arrays, researchers immobilized capture biomolecules uniformly over the planar surface and monitored the fluorescent signal intensity either as a function of time or the concentration of the labelled binding partner (Herron et al. 1993; Duveneck et al. 1997; Brecht et al. 1998; Pawlak et al. 1998; Schult et al. 1999; Hofmann et al. 2002). The most important advantage of using a planar waveguide is the possibility of creating patterns of immobilized biomolecules leading to multiple, parallel assays on a single waveguide. A number of techniques have been used in the creation of patterned biomolecular assemblies on planar surfaces, as reviewed by Blawas and Reichert (1998). In terms of fluorescence studies, the production of these patterned surfaces has been investigated using either the fluorescence microscope or TIRF instrumentation. The patterns are typically created using either photolithography or by depositing the recognition molecules in physically separate locations on the waveguide. Photolithographic patterning of proteins on surfaces has been utilized by a number of researchers (Conrad et al. 1997, 1998; Guschin et al. 1997; Wadkins et al. 1997; Schwarz et al. 1998; Arenkov et al. 2000; Liu et al. 2000) and typically involves conversion of a surface species in order to create patterns, which can be used to immobilize the capture biomolecule in specific regions. For example, Bhatia et al. (1992, 1993) used ultraviolet light to pattern (3-mercaptopropyl) trimethoxysilane on a glass surface. Exposed regions of the surface became protein resistant through the conversion of the thiol group to a sulphonate species, while the masked areas were subsequently used to bind the biomolecule. This proved to be a convenient method of creating high resolution patterns (<10 μm in width) of immobilized capture biomolecules. Unfortunately, this method had the disadvantage that only a single biomolecule could be immobilized in a pattern. The use of ink jet printing is another popular choice for the production of patterned biomolecular surfaces. Silzel et al. (1998) ink jet printed either the capture antibodies or avidin in 200 μm diameter zones on the surface of polystyrene waveguides. Biotinylated antibodies were later immobilized on the avidin spots. A checkerboard pattern of two different oligonucleotides was produced by Budach et al. (1999) using the ink jet printing of capture biomolecules onto a Ta2O5 waveguide using (3-glycidoxypropyl) trimethoxysilane. Noncontact printing has also been successfully used by Delehanty and Ligler (2002, 2003) in the production of arrays of capture antibodies and has the capability for patterning different capture biomolecules onto a single waveguide surface. Physically isolated patterning has been accomplished using flow cells constructed from a variety of materials, including polydimethylsiloxane (PDMS) (Feldstein et al. 1999; Bernard et al. 2001), a rubber gasket (Plowman et al. 1999), a Teflon block fitted with O-rings (Schuderer et al. 2000) and a microfluidics network made of silicon (Bernard et al. 2001). Typically the flow cell, containing a number of channels, is temporarily attached to the surface of the planar waveguide and each channel filled with a solution of the capture biomolecule, as shown in Fig. 2a. In this example (Feldstein et al. 1999; Bernard et al. 2001), the resulting waveguide was patterned with stripes of immobilized biomolecules (Fig. 2b). The sample and fluorescent-labelled antibody were then passed over the surface using a second flow cell orientated perpendicular to the immobilized capture biomolecule columns, as shown in Fig. 2b. Physically isolated patterning allows the immobilization of a number of different capture biomolecules, i.e. one type in each flow cell channel, onto a single surface, creating an array of recognition sites with no cross contamination. The patterning of capture biomolecules using flow cells (adapted from Feldstein et al. 1999). (a) A multichannel flow cell is pressed onto the planar waveguide and each channel filled with a solution of the capture biomolecule. (b) Sample and fluorescent tracer antibodies are passed over the waveguide surface perpendicular to the immobilized capture biomolecule channels using a second flow cell Much of the initial investigation into the use of planar waveguides in TIRF biosensors has centered on both instrumentation development and reproducible immobilization of the capture biomolecules. However, once the hardware and biochemistry are optimized, the question of application becomes the driving force behind further development. A number of studies have investigated the use of a single capture biomolecule-analyte assay (Brecht et al. 1998; Klotz et al. 1998). However, as previously stated, one of the major advantages of using a planar substrate is the ability to create arrays of different capture biomolecules for multi-analyte sensing. To date, there are at least five research groups involved in the immobilization of patterns of multiple capture biomolecules onto planar waveguides (Table 2), although only three of these groups have demonstrated multi-analyte measurements. Zeller et al. (2000) have developed a unique TIRF system in which the planar waveguide consists of multiple, single pad, sensing units. Each of these single pads has its own laser light input, background suppression, and coupling of the fluorescence emission to the detector. The authors demonstrated a two-pad sensing device in which one pad was modified with mouse immunoglobulin (IgG) and the other with rabbit IgG. However, only one Cy5-labelled antibody directed against each immobilized antigen was assayed at a time. It was suggested that the laser light could be split into spatially different parts in multi-analyte measurements, using multiple single sensing pads. Such a process would probably involve a number of optical components, and therefore the robustness of the system for use outside the laboratory is still in question. The long-term aim of most biosensor research is the development of a fully-automated instrument geared towards portability and low cost, essential considerations for biohazard monitoring applications. Ligler's group at the Naval Research Laboratory (NRL) group first published papers on TIRF studies in 1997 measuring various different IgG species; this work was extended in 1998 for the detection of three analytes ricin, Yersinia pestis F1 and staphylococcal enterotoxin B (SEB) (Wadkins et al. 1997, 1998). In the latter study (Wadkins et al. 1998), the antigens and the Cy5-labelled tracer antibodies were added sequentially and the slide imaged. Later the use of a PDMS flow cell for the patterning of capture biomolecules was developed and the immunoassay, which was run prior to imaging, was carried out using a fixed polymethylmethacrylate (PMMA) flow cell aligned perpendicular to the patterned antibody channels (Ligler et al. 1998). Simultaneous detection of analytes was demonstrated. Yersinia pestis F1 was detected and measured in clinical fluids such as whole blood, plasma, urine, saliva and nasal secretions, as well as SEB and d-dimer. All had detection limits suitable for clinical analysis requirements (Rowe et al. 1999a). Studies that evaluate potential matrix interferents are essential not only for system development but are also a requirement if the instrumentation is to make the transition into commercial applications. The impact of potential environmental interferents has also been addressed by Rowe-Taitt et al. (2000c). Analyte samples of Bacillus anthracis, Francisella tularensis LVS, Brucella abortus, SEB, cholera toxin and ricin were assayed in the presence of interferents such as sand, clay, pollen and smoke extracts, and results were compared with buffer controls. No false-positive or false-negative responses were caused by the potential interferents; however, in some cases the signal amplitude was affected. More recently the detection of SEB spiked into food samples, such as ham extract, ground beef, milk, egg and cantaloupe, was demonstrated, all with a 0·5-ng ml−1 limit of detection (Shriver-Lake et al. 2003). Most methods for the detection and identification of biohazards in real-world samples require extensive pretreatment or concentration prior to analysis. Samples measured using the NRL array biosensor were prepared with the minimum of manipulations. Liquid samples for example, such as milk, were typically buffered with 10x PBSTB (100 mm sodium phosphate/1·5 m NaCl/0·5% Tween/10 mg ml−1 BSA) prior to spiking, incubation and analysis. Solid samples, such as beef and cantaloupe, where mixed with a 1 : 1 (w/v) ratio of food to buffer (PBSTB). This was then homogenized in a Waring blender, the homogenate spiked, and the sample incubated at room temperature for 2 h. The sample was then centrifuged at 1000 g for 5 min and the resulting liquid collected for analysis on the NRL array biosensor. Assays for Salmonella typhimurium have also been developed for analysis of foodstuffs (whole egg, sausage, chicken washings, alfalfa sprouts and cantaloupe). In contrast to results obtained with SEB, some sensitivity was lost when S. typhimurium was spiked into these foods. However, little effect was observed by large excesses of other bacteria often found in the same foodstuffs (Campylobacter, Escherichia coli); the lack of false-positives in the presence of E. coli is especially surprising, given the close relationship between E. coli and Salmonella. During the development of multi-analyte assays for SEB, Y. pestis F1 and d-dimer, the room temperature CCD camera was replaced with a thermoelectrically cooled version, which resulted in a reduction of the background because of electronic noise fluctuations (Golden et al. 1999) and an observed decrease in the limit of detection from 5 ng ml−1 (Wadkins et al. 1998) to 1 ng ml−1 for SEB (Rowe et al. 1999a). This limit of detection was further reduced to 0·5 ng ml−1 by switching from a polyclonal to a monoclonal capture SEB antibody and a Cy5 to Alexafluor 647 labelled tracer antibody (Shriver-Lake et al. 2003). Another variation on previous experiments was the use of a temporary, removable PDMS flow cell in the immunoassays as compared with the permanently mounted PMMA flow cell. In order to further develop the array technology, an automated image analysis program was developed and some of the optical and fluidic components were miniaturized (Feldstein et al. 1999). To test the ability of the array biosensor to measure three diverse classes of analytes, assays were carried out using bacterial, viral and protein analytes (Rowe et al. 1999b). Single- or multi-analyte samples were run through the assay channels followed by either individual Cy5-labelled tracer antibodies or a mixture of tracer antibodies. The array biosensor was used in the study of 126 blind samples and the automated image analysis proved reliable in the discrimination of fluorescent signals, with detection limits in the mid ng/ml range, equivalent to ELISA results using the same antibodies. Assays, which used mixtures of fluorescent antibodies, gave the same results as those in which the fluorescent antibodies were run individually. The approach using mixtures of tracer antibodies was later extended to monitor the six biohazardous analytes B. anthracis, F. tularensis, B. abortus, botulinum toxoids, cholera toxin and ricin, demonstrating simultaneous analysis of six samples for six analytes in 12 min (Rowe-Taitt et al. 2000b,c). All the above immunoassays were carried out using a standard 6 × 6 array format; however, Taitt et al. (2002) has demonstrated that with the use of complementary mixtures of capture and tracer antibodies, up to nine analytes can be detected using a single 3 × 3 array format. For many applications it is important that the biosensor be fully automated, portable and stand alone; therefore recent studies by the NRL group have concentrated on realizing this goal. This has involved miniaturization of the system and the combination of an automated fluidics system with an automated image analysis program (Feldstein et al. 1999, 2000; Rowe-Taitt et al. 2000b). The sensing component, in the form of a microscope slide, does not limit the minimum size and weight of the biosensor. Typically the limiting factors are the associated optics, electronics and fluidics. Two inventions made it possible to automate and miniaturize the fluidic system for the NRL array biosensor, leading to a small, fully automated prototype array biosensor, which fitted into a tackle box and weighed 16 kg (Ligler et al. 2001). The first was a method for attaching flow channels to the waveguide without stripping evanescent light from the surface (Feldstein et al. 1999, 2000). This was achieved using a unique patterned reflective cladding (see Fig. 3), which insulated the waveguide optically from the flow cell, yet allowed the evanescent excitation of fluorophores within the channels of the flow cell (Feldstein et al. 1999). The signal loss because of flow cell attachment onto unclad planar waveguides was 90% of the original signal. The silver-clad waveguides, by comparison, maintained 85–90% of their signal for the same assays after attachment (Feldstein et al. 1999). The silver cladding allowed for the possibility of studying binding kinetics in real-time. Such kinetics measurements have been demonstrated by Sapsford et al. (2001) and K.E. Sapsford and F.S. Ligler (unpublished data) for both specific and nonspecific binding. (a) The pattern of the reflective cladding covers the area where the six-channel flow cell makes contact with the waveguide (adapted from Feldstein et al. 1999). The rest of the waveguide surface is left unclad, and the array of biomolecules is subsequently immobilized onto the exposed glass. The cladding is deposited using vacuum deposition through a metal mask, which acts like a stencil and allows only selected areas to be coated. (b) The silver-based cladding consists of three layers: a thin, transparent dielectric material to promote adhesion, a silver film for reflectance, and a thin chromium film to protect the silver from dissolution in the saline buffers required for bioassays (Feldstein et al. 2000) The second invention was an integrated fluidics unit whereby fluid flow was controlled using a small air relief valve (Dodson et al. 2001; Feldstein 2002). This unit consists of a plastic cube containing two sets of six reservoirs: one set for samples and one for fluorescent tracer reagent. Each set of reservoirs is connected at the top by a conduit leading to a single external air relief valve. At the bottom of each reservoir is an outlet that leads, via a J-tube, to one of the channels on the flow cell; each channel is connected to a single sample reservoir and a single tracer reservoir (Fig. 4a). A small peristaltic pump is connected to the opposite end of each flow channel and suction is applied. By switching the air relief valve on the fluidics cube, one can selectively flow solutions from either the sample or tracer reservoirs over the patterned substrate. This system has reduced the size, weight and power requirements of the fluidic components. Several prototypes of this system have been constructed by fabricating a layered fluidic system in plastic (Dodson et al. 2001). Initial fluidic components were designed, milled in polycarbonate sheets, assembled and tested. Once a design has been fully optimized, the entire fluidics cube can be produced inexpensively and in large quantities by injection molding. (a) Schematic of a portion of the fluidics cube. Groups of reservoirs (two reservoirs shown here out of a group of six actually fabricated) are connected at the top by conduit leading to a single air vent. At the bottom, each reservoir is connected to a J-tube which prevents release during the filling process. The J-tube empties into a conduit leading to a flow channel attached to the planar waveguide. The exit from the reservoirs empties through the flow channels across the waveguide as follows; a negative pressure is exerted on the reservoirs using a finger-sized peristaltic pump placed after the waveguide. When one air valve is opened, the pump pulls the fluid from all reservoirs attached to that valve (e.g. all sample reservoirs) through the flow channels and out to waste. When the sample valve is closed and the tracer valve opened, all the reservoirs containing fluorescent reagents empty their contents into the flow channels. (b) The conventional automated fluidics system (left) with a four-way valve, six-channel pump and six sample reservoirs compared with the fluidics cube attached to the flow channel and waveguide, and small valves and pumps (right) The reduction in size resulting from incorporation of the fluidics cube is shown in Fig. 4b. The system on the left uses a large multi-head peristaltic pump, large switching valves, and reservoirs; not shown are the containers required for buffer and tracer reagent. The system on the right uses the fluidics cube, attached to the flow channels and waveguide, and three small pumps and several Lee valves. The fluidics cube attaches directly to the flow channels and waveguide with no intervening tubing. Connections to the plastic block are limited to an air line, and two air relief vents, all of which are inserted simultaneously through a gasket at the top of the cube. The combination of these inventions allowed the miniaturization and automation of the fluidics system in the NRL array biosensor; however, the electronics and optics of the original system were still a bit cumbersome for field use. In order to miniaturize the array biosensor further, Golden and Ligler (2002) considered the possibility of replacing the CCD with the less expensive alternatives for image capture, a CMOS camera or a photodiode array. Both would provide smaller systems more amenable for portable sensors. The CMOS cameras, in particular, would simplify data acquisition. However, careful comparison revealed that the Peltier-cooled CCDs still have an order of magnitude better signal-to-noise ratio than either of the other two imaging systems (Golden and Ligler 2002). The original tackle-box system included a small Pentium computer (Ligler et al. 2001). In the next version, the Pentium was removed and the data recorded using a laptop computer. In addition to the Pentium with its keyboard and screen, the breadboard included a rather large and expensive (ca$24 000) Peltier-cooled CCD. While the photon collection capability of uncooled low light cameras was sufficient, the backgrounds were too variable for high sensitivity measurements. Recent advances in CCD technology made it possible to replace the original CCD with a smaller, lighter, and less expensive (ca$7000) CCD; for applications requiring less sensitivity, even smaller, cheaper, uncooled CCDs may now be adequate. Furthermore, the ‘fire wire’ technology used in the new CCD cameras eliminates the requirement for a specialized data interface board, simplifying the electronics. Currently, the NRL group has assembled a smaller array biosensor for sample monitoring and is in the process of optimizing its operation. It uses a relatively small, inexpensive CCD, the fluidics cube, and the small pumps and valves (Fig. 5). Image display and analysis are performed using a laptop computer (not shown). The device fits into a tackle box which is about one-third the size of the previous version and weighs only 5·5 kg. The latest version of the portable array biosensor This review has discussed the use of optical array biosensors, based on TIRF, for the detection of biohazards and BW agents, including background, history and current developments in the TIRF field. Prototypes exist for a device that is fully automated and portable, characteristics essential for continuous monitoring applications (Ligler et al. 2001). However, with the development of smaller electronics and components, such as the CMOS camera currently used in the confocal microscopy studies of microarrays (Vo-Dinh et al. 1999; Askari et al. 2001), the potential for even smaller, handheld planar waveguide TIRF devices could become reality. Although the majority of studies have centered on antibody–antigen type systems, expansion of the work concerned with DNA/mRNA and receptor–ligand binding interactions (Rogers et al. 1989; Fisher and Tjarnhage 2000; Lang et al. 2000; Altin et al. 2001; Puu 2001), as well as expansion into the use of enzymes (Wilson and Hu 2000), DNA aptamers (Potyrailo et al. 1998; Li and Li 2000; Wang et al. 2001; Liss et al. 2002) and other types of recognition biomolecules (Mauro et al. 2000) could lead to a much wider field of applications. Advances in biomolecule immobilization technology, such as site directed mutagenesis, which allows for unique attachment sites to be generated on the protein surface and hence orientational control upon immobilization (McLean et al. 1993; Vigmond et al. 1994; Lu et al. 1995; Huang et al. 1996), and the prevention of nonspecific interactions could improve the sensitivity of TIRF measurements (Conrad et al. 1997). Regeneration of the surface would be useful for commercial applications as an alternative to disposable substrates (Duveneck et al. 1997; Asanov et al. 1998; Budach et al. 1999). In addition, the power of the larger scale arrays has been demonstrated using both DNA chips (Brown and Botstein 1999; Lockhart and Winzeler 2000) and antibody chips (MacBeath and Schreiber 2000; Delehanty and Ligler 2002, 2003; Yang et al. 2002). The methods used to deposit the spots in high-density arrays are currently being improved to attain reproducible surface concentrations of the capture biomolecules (Delehanty and Ligler 2002, 2003) for future use in TIRF. The use of planar waveguide TIRF for the detection of multiple analytes has been demonstrated as well as the ability to miniaturize the instrumentation making it a promising device for real-time field monitoring. The benefit of spatially distinct sensing regions is enabling these systems to gain advantage over single-analyte sensing systems, particularly in terms of environmental and clinical monitoring. The speed of signal transduction and relative resistance to matrix effects and other interfering influences are two key advantages of planar waveguide TIRF biosensors. In conclusion, the future looks bright for biohazard monitoring using planar waveguide TIRF. The authors would like to thank Dr Caroline Schauer for her useful discussions regarding manuscript preparation. This work was supported by funding from N.A.S.A. and the Office of Naval Research. The views expressed here are those of the authors and do not represent those of the US Navy, the US Department of Defense, or the US Government.

The following review focuses on progress made in the last five years with the NRL Array Biosensor, a portable instrument for rapid and simultaneous detection of multiple targets. Since 2003, the Array Biosensor has been automated and miniaturized for operation at the point-of-use. The Array Biosensor has also been used to demonstrate (1) quantitative immunoassays against an expanded number of toxins and toxin indicators in food and clinical fluids, and (2) the efficacy of semi-selective molecules as alternative recognition moieties. Blind trials, with unknown samples in a variety of matrices, have demonstrated the versatility, sensitivity, and reliability of the automated system.

Food poisoning causes untold discomfort to many people each year. One of the primary culprits in food poisoning is Escherichia coli O157:H7. While most cases cause intestinal discomfort, up to 7% of the incidences lead to a severe complication called hemolytic uremic syndrome which may be fatal. The traditional method for detection of E. coli O157:H7 in cases of food poisoning is to culture the food matrices and/or human stool. Additional performance-based antibody methods are also being used. The NRL array biosensor was developed to detect multiple antigens in multiple samples with little sample pretreatment in under 30 min. An assay for the specific detection of E. coli O157:H7 was developed, optimized and tested with a variety of spiked food matrices in this study. With no sample pre-enrichment, 5 × 103 cells mL−1 were detected in buffer in less than 30 min. Slight losses of sensitivity (1–5 × 10−4 cell mL−1) but not specificity occur in the presence of high levels of extraneous bacteria and in various food matrices (ground beef, turkey sausage, carcass wash, and apple juice). No significant difference was observed in the detection of E. coli O157:H7 in typical culture media (Luria Broth and Tryptic Soy Broth).

Single domain antibodies derived from the variable region of the unique heavy chain antibodies found in camelids yield high affinity and regenerable recognition elements. Adding an additional disulfide bond that bridges framework regions is a proven method to increase their melting temperature, however often at the expense of protein production. To fulfill their full potential it is essential to achieve robust protein production of these stable binding elements. In this work, we tested the hypothesis that decreasing the isoelectric point of single domain antibody extra disulfide bond mutants whose production fell due to the incorporation of the extra disulfide bond would lead to recovery of the protein yield, while maintaining the favorable melting temperature and affinity.Introduction of negative charges into a disulfide bond mutant of a single domain antibody specific for the L1 antigen of the vaccinia virus led to approximately 3.5-fold increase of protein production to 14 mg/L, while affinity and melting temperature was maintained. In addition, refolding following heat denaturation improved from 15 to 70 %. It also maintained nearly 100 % of its binding function after heating to 85 °C for an hour at 1 mg/mL. Disappointingly, the replacement of neutral or positively charged amino acids with negatively charged ones to lower the isoelectric point of two anti-toxin single domain antibodies stabilized with a second disulfide bond yielded only slight increases in protein production. Nonetheless, for one of these binders the charge change itself stabilized the structure equivalent to disulfide bond addition, thus providing an alternative route to stabilization which is not accompanied by loss in production.The ability to produce high affinity, stable single domain antibodies is critical for their utility. While the addition of a second disulfide bond is a proven method for enhancing stability of single domain antibodies, it frequently comes at the cost of reduced yields. While decreasing the isoelectric point of double disulfide mutants of single domain antibodies may improve protein production, charge addition appears to consistently improve refolding and some charge changes can also improve thermal stability, thus providing a number of benefits making the examination of such mutations worth consideration.

Electroanalytical techniques are useful for detection and identification because the instrumentation is simple and can support a wide variety of assays. One example is cyclic square wave voltammetry (CSWV), a practical detection technique for different classes of compounds including explosives, herbicides/pesticides, industrial compounds, and heavy metals. A key barrier to the widespread application of CSWV for chemical identification is the necessity of a high performance, generalizable classification algorithm. Here, machine and deep learning models were developed for classifying samples based on voltammograms alone. The highest performing models were Long Short-Term Memory (LSTM) and Fully Convolutional Networks (FCNs), depending on the dataset against which performance was assessed. When compared to other algorithms, previously used for classification of CSWV and other similar data, our LSTM and FCN-based neural networks achieve higher sensitivity and specificity with the area under the curve values from receiver operating characteristic (ROC) analyses greater than 0.99 for several datasets. Class activation maps were paired with CSWV scans to assist in understanding the decision-making process of the networks, and their ability to utilize this information was examined. The best-performing models were then successfully applied to new or holdout experimental data. An automated method for processing CSWV data, training machine learning models, and evaluating their prediction performance is described, and the tools generated provide support for the identification of compounds using CSWV from samples in the field.

We demonstrate the viability of using ultra-thin sheets of microbially grown nanocellulose to build functional medical sensors. Microbially grown nanocellulose is an interesting alternative to plastics, as it is hydrophilic, biocompatible, porous, and hydrogen bonding, thereby allowing the potential development of new application routes. Exploiting the distinguishing properties of this material enables us to develop solution-based processes to create nanocellulose printed circuit boards, allowing a variety of electronics to be mounted onto our nanocellulose. As proofs of concept, we have demonstrated applications in medical sensing such as heart rate monitoring and temperature sensing—potential applications fitting the wide-ranging paradigm of a future where the Internet of Things is dominant.

Contamination of food with infectious agents, intentional or not, is a global concern with far-reaching economic and social impact. Staphylococcal enterotoxins are a major cause of food poisoning, but most methods for the identification of these agents in food require extensive pretreatment or concentration of the sample prior to analysis. The array biosensor was developed as a portable device for the simultaneous analysis of multiple complex samples for multiple targets with minimal sample preparation. In this study, we use an array biosensor to expand and improve on a staphylococcal enterotoxin B (SEB) assay with the ultimate intent of incorporating testing for SEB into a battery of sensitive and convenient assays for food safety validation. In addition to buffer studies, six different types of food samples, including beverages, homogenates of fruit and meat, and carcass washings, were spiked with SEB, incubated for at least 2 h to permit antigen sequestration, and assayed. For all samples, there were differences in fluorescence intensity, but 0.5 ng of SEB per ml could be detected in <20 min with little if any pretreatment and no sample preconcentration.

The array biosensor employs an array of capture molecules on a planar optical waveguide to interrogate multiple samples simultaneously for multiple targets. In assay development and demonstration studies published previously, we have quantified this biosensor's capability for rapid identification of a wide variety of targets in complex sample media. This paper describes the miniaturization and automation of the array biosensor for portability and on-site use. The fluidics have been redesigned and constructed with reliability and commercial production of disposable components in mind. To demonstrate the automated operation, simultaneous assays were automatically conducted on samples containing both ovalbumin and staphylococcal enterotoxin B. Results demonstrate the capability of the biosensor for detection and quantification.

The array biosensor provides the capability for simultaneously measuring titers of antibody against multiple antigens. Human antibodies against four different targets, tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B (SEB) and hepatitis B, were measured simultaneously in sera from eight different donors in a single assay and titers were determined. The assays could measure amounts of bound antibody as low as approximately 100 fg. Each individual serum exhibited a different pattern of reactivity against the four target antigens. Applications of this biosensor capability include monitoring for exposure to pathogens and for efficacy of vaccination.

An integrated system with automated immunomagnetic separation and processing of fluidic samples was demonstrated for multiplexed optical detection of bacterial targets. Mixtures of target-specific magnetic bead sets were processed in the NRL MagTrap with the aid of rotating magnet arrays that entrapped and moved the beads within the channel during reagent processing. Processing was performed in buffer and human serum matrixes with 10-fold dilutions in the range of 102–106 cells/mL of target bacteria. Reversal of magnets’ rotation post-processing released the beads back into the flow and moved them into the microflow cytometer for optical interrogation. Identification of the beads and the detection of PE fluorescence were performed simultaneously for multiplexed detection. Multiplexing was performed with specifically targeted bead sets to detect E. coli 0157.H7, Salmonella Common Structural Antigen, Listeria sp., and Shigella sp., dose–response curves were obtained, and limits of detection were calculated for each target in the buffer and clinical matrix. Additional tests demonstrated the potential for using the MagTrap to concentrate target from larger volumes of sample prior to the addition of assay reagents.

Traumatic brain injury (TBI) results from an event that causes rapid acceleration and deceleration of the brain or penetration of the skull with an object. Responses to stimuli and questions, loss of consciousness, and altered behavior are symptoms currently used to justify brain imaging for diagnosis and therapeutic guidance. Tests based on such symptoms are susceptible to false-positive and false-negative results due to stress, fatigue, and medications. Biochemical markers of neuronal damage and the physiological response to that damage are being identified. Biosensors capable of rapid measurement of such markers in the circulation offer a solution for on-site triage, as long as three criteria are met: (a) Recognition reagents can be identified that are sufficiently sensitive and specific, (b) the biosensor can provide quantitative assessment of multiple markers rapidly and simultaneously, and (c) both the sensor and reagents are designed for use outside the laboratory.

Biosensors are uniquely qualified to meet the need for rapid, inexpensive analytical procedures. The authors' intent was to develop a simple, real-time immunoassay that could process multiple samples in a semi-automated manner, while maintaining maximum versatility to permit its application under various conditions. To achieve this goal, the authors have developed a biosensor which detects antibody-antigen binding within the evanescent wave of an optical biosensor.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

The fiber-optic biosensor combines evanescent wave sensing, fiber-optic technology, and fluorescence immunoassays to achieve a rapid, sensitive, and simple detection system. The response time varies from 2 to 15 min, depending on the type of assay performed. Sensitivity in the 1-10 ng/ml or 1000-cell range has been achieved. Fiber-optic biosensors can be adapted to detect the presence of toxic agents in a wide variety of samples.

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity. An immune phage display library containing more than 107 unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15–17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies. SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.

Botulinum neurotoxin (BoNT) is a highly potent and lethal toxin, which even in minute quantities can lead to death. BoNT occurs in seven well described serotypes, A-G, and it is critical to not only detect the presence of BoNT, but also to determine the serotype to which a person has been exposed, as the degree of toxicity and persistence of symptoms varies greatly between the various types. Recently, Conway et al. 2010 developed single domain antibodies (sdAb), the recombinant variable domains of heavy-chain-only antibodies derived from camelids, for the detection of all seven serotypes of BoNT; identifying pairs of sdAb for each serotype they demonstrated the sensitive detection of each toxin. Using the sequence information provided in that work, fourteen of their sdAb were recreated with one goal being confirmation of their binding ability and specificity for the seven serotypes of BoNT. This was accomplished using a direct binding assay with the toxins immobilized on microtiter plates. In addition, the melting temperatures and production yields from E. coli shake flask fermentation were determined for each of the sdAb produced. In several instances, alternatives or variants of the previously described sdAb were prepared, either to improve the stability or production yields of the anti-BoNT sdAb. Insertion of four framework 1 point mutations (1E or D, 3Q, 5V, and 6E) gave repeated improvement in thermal stability by 5–9 °C, offering a method for increasing sdAb melting temperatures. This work provides for the independent verification of the ability of these sdAb to recognize all seven serotypes of BoNT, furnishing melting temperature, relative affinity, and production yield information that will allow for their future utilization with increased confidence.

We describe the use of a paper-based probe impregnated with a vanadium-containing polyoxometalate anion, [PMo11VO40]5-, on screen-printed carbon electrodes for the electrochemical determination of chlorate. Cyclic voltammetry (CV) and chronocoulometry were used to characterize the ClO₃- response in a pH = 2.5 solution of 100 mM sodium acetate. A linear CV current response was observed between 0.156 and 1.25 mg/mL with a detection limit of 0.083 mg/mL (S/N > 3). This performance was reproducible using [PMo11VO40]5--impregnated filter paper stored under ambient conditions for as long as 8 months prior to use. At high concentration of chlorate, an additional catalytic cathodic peak was seen in the reverse scan of the CVs, which was digitally simulated using a simple model. For chronocoulometry, the charge measured after 5 min gave a linear response from 0.625 to 2.5 mg/mL with a detection limit of 0.31 mg/mL (S/N > 3). In addition, the slope of charge vs. time also gave a linear response. In this case the linear range was from 0.312 to 2.5 mg/mL with a detection limit of 0.15 mg/mL (S/N > 3). Simple assays were conducted using three types of soil, and recovery measurements reported.

We report a simple and inexpensive electrochemical assay using a custom built hand-held potentiostat for the identification of explosives. The assay is based on a wipe test and is specifically designed for use in the field. The prototype instrument designed to run the assay is capable of performing time-resolved electrochemical measurements including cyclic square wave voltammetry using an embedded microcontroller with parts costing roughly $250 USD. We generated an example library of cyclic square wave voltammograms of 12 compounds including 10 nitroaromatics, a nitramine (RDX), and a nitrate ester (nitroglycine), and designed a simple discrimination algorithm based on this library data for identification.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes an arthralgia febrile illness that has affected millions of people on three continents. Previously, neutralizing monoclonal antibodies that have prophylactic and therapeutic activity were found to remove virus in joint tissues, thereby reducing the severity of symptoms in mice and non-human primates. In this study, we sought to develop thermostable small recombinant antibodies against CHIKV for future diagnostic, prophylactic and therapeutic applications. To develop these single domain antibodies (sdAb) a CHIKV immune library was constructed by displaying the consortium of variable heavy domains (VHH) amplified from peripheral white blood cells isolated from llamas immunized with CHIKV virus-like particles (VLPs). Five anti-CHIKV sdAb isolated using bio-panning were evaluated for their affinity and thermal stability. Their ability to detect CHIKV VLPs was demonstrated in both MagPlex- and ELISA- based assays. Finally, the ability of two sdAb, CC3 and CA6, to inhibit CHIKV infection were tested using a plaque reduction and neutralization test (PRNT), yielding PRNT50 values of 0.6 and 45.6 nM, respectively.

Reliable detection and diagnosis of dengue virus (DENV) is important for both patient care and epidemiological control. Starting with a llama immunized with a mixture of recombinant nonstructural protein 1 (NS1) antigen from the four DENV serotypes, a phage display immune library of single domain antibodies was constructed and binders selected which exhibited specificity and affinity for DENV NS1. Each of these single domain antibodies was evaluated for its binding affinity to NS1 from the four serotypes, and incorporated into a sandwich format for NS1 detection. An optimal pair was chosen that provided the best combination of sensitivity for all four DENV NS1 antigens spiked into 50% human serum while showing no cross reactivity to NS1 from Zika virus, yellow fever virus, tick-borne encephalitis virus, and minimal binding to NS1 from Japanese encephalitis virus and West Nile virus. These rugged and robust recombinant binding molecules offer attractive alternatives to conventional antibodies for implementation into immunoassays destined for resource limited locals.

Abstract We present the first demonstration of a fully-flexible, self-powered glucose indicator system that synergizes two flexible electronic technologies: a flexible self-powering unit in the form of a biofuel cell, with a flexible electronic device - a circuit-board decal fabricated with biocompatible microbial nanocellulose. Our proof-of-concept device, comprising an enzymatic glucose fuel cell, glucose sensor and a LED indicator, does not require additional electronic equipment for detection or verification; and the entire structure collapses into a microns-thin, self-adhering, single-centimeter-square decal, weighing less than 40 mg. The flexible glucose indicator system continuously operates a light emitting diode (LED) through a capacitive charge/discharge cycle, which is directly correlated to the glucose concentration. Our indicator was shown to operate at high sensitivity within a linear glucose concentration range of 1 mM–45 mM glucose continuously, achieving a 1.8 VDC output from a flexible indicator system that deliver sufficient power to drive an LED circuit. Importantly, the results presented provide a basis upon which further development of indicator systems with biocompatible diffusing polymers to act as buffering diffusion barriers, thereby allowing them to be potentially useful for low-cost, direct-line-of-sight applications in medicine, husbandry, agriculture, and the food and beverage industries.

Although food is a necessity, compounds within food products can be dangerous and life-threatening for people with food allergies. These allergy-causing compounds, such as proteins from eggs and milk, must be identified on the labels of commercial products. Unintentional contamination of food with these compounds occurs as a result of storage, manufacturing procedures, or cleaning procedures. A sensitive, specific, and rapid method to identify foods containing allergens is required by the food industry. The array biosensor, a rapid detection system, may provide a solution to this need. The array biosensor performs fluorescent immunoassays on the surface of a planar waveguide by first running samples, then fluorescently labeled antibodies, over a surface patterned with capture antibodies. An optical image is captured by a charged-coupled device camera and converted into fluorescence values. Signal intensity and spot location provide information on the compound and its concentration. The array biosensor has been successfully demonstrated for toxin, bacteria, and virus detection at low levels in under 20 min in food, clinical samples, and environmental matrixes. An assay for detection of ovalbumin as an indicator of egg contamination has been developed with limits of detection of 25 pg/mL in buffer and 1.3 ng/mL (13 ng/g) in non-egg pasta extract (buffer:pasta 10:1, v/w).

A spinning magnetic trap (MagTrap) for automated sample processing was integrated with a microflow cytometer capable of simultaneously detecting multiple targets to provide an automated sample-to-answer diagnosis in 40 min. After target capture on fluorescently coded magnetic microspheres, the magnetic trap automatically concentrated the fluorescently coded microspheres, separated the captured target from the sample matrix, and exposed the bound target sequentially to biotinylated tracer molecules and streptavidin-labeled phycoerythrin. The concentrated microspheres were then hydrodynamically focused in a microflow cytometer capable of 4-color analysis (two wavelengths for microsphere identification, one for light scatter to discriminate single microspheres and one for phycoerythrin bound to the target). A three-fold decrease in sample preparation time and an improved detection limit, independent of target preconcentration, was demonstrated for detection of Escherichia coli 0157:H7 using the MagTrap as compared to manual processing. Simultaneous analysis of positive and negative controls, along with the assay reagents specific for the target, was used to obtain dose-response curves, demonstrating the potential for quantification of pathogen load in buffer and serum.

Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together β-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process. Here, we demonstrate that inclusion of the helper plasmid pTUM4, which results in the expression of four chaperones, DsbA, DsbC, FkpA, and SurA, increased yield on average 3.5-fold for the nine multi-disulfide bond single domain antibodies evaluated. No increase in production was observed for single domain antibodies containing only the canonical disulfide bond.

Two primary explosives involved in groundwater contamination, 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), were detected on-site at low ppb levels with a semiautomated fiber optic biosensor. Validation of the Analyte 2000 (manufactured by Research International Inc., Woodinville, WA) for TNT and RDX detection was performed at two Superfund sites, Umatilla Army Depot and Naval Surface Weapons Center Crane. Samples from monitoring wells were split for analysis using the fiber optic biosensor on-site and using U.S. EPA SW-846 Method 8330 (reverse-phase high performance liquid chromatography) in an offsite laboratory. The Analyte 2000, a multifiber probe fluorimeter, was coupled to a fluidics unit for semiautomated operation. The fiber optic biosensor assay is based on a competitive fluorescent immunoassay performed on the silica core of a fiber probe. From these studies, the limit of detection was determined to be 5 μg/L for both TNT and RDX. In addition to the field samples, extensive laboratory analyses were performed to determine cross-reactivity, matrix effects, and false positive/negative rates.

Several covalent immobilization methods, which have been routinely used with proteins and antibodies, were studied for their ability to immobilize genetically engineered Escherichia coli cells to glass beads. The cells used in this study expressed a metal binding peptide that binds cadmium (Cd) and mercury (Hg). The initial work focused on a method employing 2.5% aminopropyltrimethoxy silane and 2.5% glutaraldehyde for covalent immobilization of cells onto porous glass beads. Scanning electron microscopy (SEM) demonstrated cell attachment (average of 3.0×108 cells per bead) to the irregular surface. Columns containing cells immobilized with the 2.5% aminosilane and 2.5% glutaraldehyde removed more than 90% of the Cd from solutions with 50 ppb and 1 ppm levels. Following removal of the bound Cd with HCl elution and regeneration to pH 6.0, the columns were shown to effectively bind additional cadmium. Various concentrations of aminosilane and glutaraldehyde were tested for improved cell density. Glutaraldehyde is a universal and convenient cross-linker, but there are some concerns with its effects on the cells and proteins, therefore, two additional covalent techniques were examined. One method employed the aminopropyltrimethoxy silane and carbodiimide, and the other used mercaptopropyltrimethoxy silane and the heterobifunctional cross-linker GMBS. Some comparisons of these two immobilization methods to the method employing glutaraldehyde are described.

Abstract Optical biosensors are analytical tools employed in environmental, biotechnology and clinical fields. For remote evanescent wave detection with optical fibers, the majority of the fiber remains cladded while only a small portion of the core is exposed to perform the analysis. To improve the sensitivity of this type of fiber optic biosensor, tapering the sensing region of the fiber was investigated. Silica fibers were tapered into two geometries: step and continuous tapers. To determine the effect of tapering on detection levels, a fluoroimmunoassay was performed on fibers with nontapered, step-tapered and continuously tapered geometries. Solutions containing a fluorescent analyte were circulated over an antibody-coated fiber and the fluoresence signal measured The minimum detection limit for fibers with 10 cm sensing region was 1.56 nM, 0.31 nM and 0.16 nM for nontapered, step-tapered and continuously tapered fibers, respectively. Continuous tapering of the sensing region of an optical fiber offered a 10-fold increase in the sensitivity of the optical biosensor.

The electrochemical detection of heavy metal ions is reported using an inexpensive portable in-house built potentiostat and epitaxial graphene. Monolayer, hydrogen-intercalated quasi-freestanding bilayer, and multilayer epitaxial graphene were each tested as working electrodes before and after modification with an oxygen plasma etch to introduce oxygen chemical groups to the surface. The graphene samples were characterized using X-ray photoelectron spectroscopy, atomic force microscopy, Raman spectroscopy, and van der Pauw Hall measurements. Dose-response curves in seawater were evaluated with added trace levels of four heavy metal salts (CdCl2, CuSO4, HgCl2, and PbCl2), along with detection algorithms based on machine learning and library development for each form of graphene and its oxygen plasma modification. Oxygen plasma-modified, hydrogen-intercalated quasi-freestanding bilayer epitaxial graphene was found to perform best for correctly identifying heavy metals in seawater.

The regeneration of antibodies covalently immobilized to an optical fibre surface was investigated by dissociation of the antibody-antigen complex with three different solvents: (a) an acidic solution (0·1 M glycine hydrochloride in 50% (v/v) ethylene glycol, pH 1·75), (b) a basic solution (0·05 M tetraethylamine in 50% (v/v) ethylene glycol, pH 11·0) and (c) 50% (v/v) ethanol in PBS. The fibres coated with polyclonal rabbit anti-goat antibody against a large protein retained 70% and 65% of the original signal after five consecutive regenerations with acidic and basic solvent systems, respectively. The fibres coated with monoclonal mouse anti-trinitrobenzene antibody specific for a small organic molecule, retained over 90% of the original signal when regenerated with basic and ethanol solutions. This study evaluated regeneration and reuse of antibody-coated fibre optic biosensors as a means of reducing routine laboratory analysis costs and time.

A field method for quantitative analysis of explosives in contaminated soil samples is described. The method is based on a displacement immunoassay performed in a commercial instrument, the FAST 2000, engineered by Research International Inc. The method can be used on-site to measure 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) within 5min. For this study, replicate analyses were performed on soil extracts prepared from each field sample as well as appropriate controls, blanks, and laboratory standards. Statistical analyses were done to assess accuracy, bias, and predictability of the method. The results demonstrated that the immunosensor could be used effectively to screen environmental samples for the presence or absence of explosives. In most samples, the method also provided quantitative values that were in good agreement with standard laboratory analyses using HPLC. A limited number of sample matrices interfered with the immunoassay and produced results that varied significantly from the laboratory data. In each case, the compounds causing the problem have been identified and efforts are being made to minimize these matrix interferences in future field evaluations.

We have developed an array biosensor for the simultaneous detection of multiple targets in multiple samples within 15–30 min. The biosensor is based on a planar waveguide, a modified microscope slide, with a pattern of small (mm2) sensing regions. The waveguide is illuminated by launching the emission of a 635 nm diode laser into the proximal end of the slide via a line generator. The evanescent field excites fluorophores bound in the sensing region and the emitted fluorescence is measured using a Peltier-cooled CCD camera. Assays can be performed on the waveguide in multichannel flow chambers and then interrogated using the detection system described here. This biosensor can detect many different targets, including proteins, toxins, cells, virus, and explosives with detection limits rivaling those of the ELISA detection system.

Detection of toxins and other large molecules is important for both clinical and environmental analyses. A significant factor in analyses of these compounds is the safety of the operator. The NRL fiber-optic biosensors has been created to utilize long optical fibers to prevent the contamination of the operator and the optical components from hazrdous materials. The optical fiber is clad for the majority of its length, with only a small portion of the core exposed. Capture antibodies are immobilized on the exposed core, which has been tapered for improved sensitivity. The toxins from Clostridium botulinum and pseudexin have been detected in the evanescent wave in concentrations as low as 30 pM in under 1 min.

Egg is one of the 5 major allergenic foods that are responsible for more than 3/4 of food allergies in children. Food-allergic responses can be controlled by avoidance of the offending foods. The applicability of a commercial enzyme-linked immunosorbent assay (ELISA) kit for the detection of egg in food products such as cookies, crackers, pretzels, salad dressings, and raw and cooked noodles was evaluated. A preliminary evaluation of an antibody-based biosensor was also performed. A National Institute of Standards and Technology (NIST) whole dried egg powder reference material, SRM 8415, was used as a standard. A homogeneous and stable aqueous egg suspension was prepared for the evaluation of the performance of the Veratox for Egg Allergen Test (Neogen Corp., Lansing, MI). This test does not detect egg yolk proteins. Each gram of the aqueous dried egg suspension contained 643 microg whole dried egg, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. When cookies, crackers, salad dressings, noodles, and ice cream were spiked at a level of 24 mg/kg SRM 8415, recoveries for whole egg averaged about 28%. All foods containing egg as indicated on the ingredient label were found positive by the Veratox test. No false positives occurred in samples that did not contain eggs. Similar results were obtained using the Naval Research Laboratory (NRL) array biosensor, an evanescent wave fluoroimmunosensor. Results for cooked noodles showed that they contained <1% of the egg found in uncooked noodles. A comparison of extracts from cooked and uncooked noodles by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in protein profiles. The boiling of the noodles could have reduced the immunoreactivity of the egg proteins to the antibodies used in the kit or rendered the egg proteins nonextractable.

In this paper are the experimental results used to characterize four distinct monoclonal anti-TNT antibodies (in vivo and in vitro cloned) for potential use in a field-portable immunosensor. Direct and competitive enzyme-linked immunosorbent assays (ELISA) were performed to determine their affinity for TNT and a fluorescently labeled analog of TNT for use in an immunosensor. Effective concentrations (EC(50)), inhibition concentration (IC(50)) and cross-reactivity measurements to related nitroaromatics (e.g., 2,4,6-trinitrobenzene [TNB], methyl-2,4,6-trinitrophenyl nitramine [tetryl], 2-amino-4,6-dinitrotoluene [2A-4,6-DNT], 2,4-dinitrotoluene [2,4-DNT] and 1,3-dinitrotoluene [1,3-DNT]) were measured. Final characterization of the monoclonal antibodies was based on performance (measured by fluorescence dose response) using a fluorescence-based microcapillary displacement assay. Analytical techniques showed a high degree of affinity for TNT and varying degrees of cross-reactivity with each respective monoclonal antibody. Microcapillary displacement immunoassays with each of the antibodies resulted in detection capabilities at the lowest applied TNT concentration (10 ng/ml).

Timely identification of biothreat organisms from large numbers of clinical or environmental samples in potential outbreak or attack scenario is critical for effective diagnosis and treatment. This study aims to evaluate the potential of resequencing arrays for this purpose. Albeit suboptimal, this report demonstrated that respiratory pathogen microarray version 1 can identify Bacillus anthracis, Francisella tularensis, Yersinia pestis and distinguish them from benign 'near neighbor' species in a single assay. Additionally, the sequence information can discriminate strains and possibly the sources of the strains. With further development, it is possible to use resequencing microarrays for biothreat surveillance.

Microspheres and nanospheres are being used in many of today’s biosensing applications for automated sample processing, flow cytometry, signal amplification in microarrays, and labeling in multiplexed analyses. The surfaces of the spheres/particles need to be modified with proteins and other biomolecules to be used in these sensing applications. This chapter contains protocols to modify carboxyl- and amine-coated polymer spheres with proteins and peptides.

Development of antimicrobial peptide (AMP)-functionalized materials has renewed interest in using poly(ethylene glycol) (PEG)-mediated linking to minimize unwanted interactions while engendering the peptides with sufficient flexibility and freedom of movement to interact with the targeted cell types. While PEG-based linkers have been used in many AMP-based materials, the role of the tether length has been minimally explored. Here, we assess the impact of varying the length of PEG-based linkers on the binding of bacterial cells by surface-immobilized AMPs. While higher surface densities of immobilized AMPs were observed using shorter PEG linkers, the increased density was insufficient to fully account for the increased binding activity of peptides. Furthermore, effects were specific to both the peptide and cell type tested. These results suggest that simple alterations in linking strategies-such as changing tether length-may result in large differences in the surface properties of the immobilized AMPs that are not easily predictable.

Protein scaffolds have proven useful for co-localization of enzymes, providing control over stoichiometry and leading to higher local enzyme concentrations, which have led to improved product formation. To broaden their usefulness, it is necessary to have a wide choice of building blocks to mix and match for scaffold generation. Ideally, the scaffold building blocks should function at any location within the scaffold and have high affinity interactions with their binding partners. We examined the utility of orthogonal synthetic coiled coils (zippers) as scaffold components. The orthogonal zippers are coiled coil domains that form heterodimers only with their specific partner and not with other zipper domains. Focusing on two orthogonal zipper pairs, we demonstrated that they are able to function on either end or in the middle of a multiblock assembly. Surface plasmon resonance was employed to assess the binding kinetics of zipper pairs placed at the start, middle, or end of a construct. Size-exclusion chromatography was used to demonstrate the ability of a scaffold with two zipper domains to bind their partners simultaneously. We then expanded the study to examine the binding kinetics and cross-reactivities of three additional zipper pairs. By validating the affinities and specificities of synthetic zipper pairs, we demonstrated the potential for zipper domains to provide an expanded library of scaffolding parts for tethering enzymes in complex pathways for synthetic biology applications.

Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.

This paper investigated the effects of diffusion between non-conductive sheath and conductive sample fluids in an impedance-based biosensor. Impedance measurements were made with 2- and 4-electrode configurations. The 4-electrode design offers the advantage of impedance measurements at low frequencies (<1 kHz) without the deleterious effects of double layer impedance which are present in the 2-electrode design. Hydrodynamic flow focusing was achieved with a modified T-junction design with a smaller cross-section for the sample channel than for the focusing channel, which resulted in 2D focusing of the sample stream with just one sheath stream. By choosing a non-conductive sheath fluid and a conductive sample fluid, the electric field was confined to the focused stream. In order to utilize this system for biosensing applications, we characterized it for electrical and flow parameters. In particular, we investigated the effects of varying flow velocities and flow-rate ratios on the focused stream. Increasing flow-rate ratios reduced the cross-sectional area of the focused streams as was verified by finite element modeling and confocal microscopy. Antibody mediated binding of Escherichia coli to the electrode surface caused an increase in solution resistance at low frequencies. The results also showed that the diffusion mass transport at the interface of the two streams limited the benefits of increased flow focusing. Increasing flow velocities could be used to offset the diffusion effect. To optimize detection sensitivity, flow parameters and mass transport must be considered in conjunction, with the goal of reducing diffusion of conducting species out of the focused stream while simultaneously minimizing its cross-sectional area.

A 4-electrode impedance-based microfluidic sensor was designed to achieve tunable sensitivity, while simultaneously decreasing the channel clogging and nonspecific binding that often adversely impact device function. Hydrodynamic focusing – a characteristic of laminar flow at low Reynold's number – provides highly controllable sensitivity in impedance-based microfluidic biosensors, by creating a “virtual” microchannel with soft walls and adjustable dimensions. Enhanced sensitivity, limited nonspecific binding, and a reduction in microchannel clogging have been achieved using hydrodynamic focusing within a 250 μm deep by 1 mm wide microchannel. The microfluidic sensor was able to detect microparticles as small as 5 μm in diameter specifically bound between co-planar platinum micro-electrodes. However, detection of submicron particles and E. coli cells in similar fashion proved to be challenging. A theoretical analysis of forces exerted by the fluids within the microchannel allowed specification of flow rates amenable to decreased nonspecific binding of interfering cells or microparticles, while ensuring minimal disruption to specifically bound targets. Simulations revealed that complex flow behavior of the hydrodynamically focused streams around specifically bound micron-sized particles reduces sensitivity. An improved understanding is thus presented of the parameters that impact the sensitivity of impedance-based biosensors implementing hydrodynamic focusing and specific target binding.

We describe the use and characterization of a multilayer film, prepared via layer-by-layer deposition of cationic para-rosaniline acetate dye (i.e., PR) and the vanadium-containing Keggin-type polyoxometalate anion, [PMo11VO40]5− (i.e., PVMo11), on indium tin oxide (ITO) as an electrode for determination of chlorate. A linear cyclic voltammetry (CV) current response was observed for the 0 μM ≤ [ClO3−] ≤ 1000 μM range with a detection limit of ∼220 μM ClO3− (S/N > 3) in a pH = 2.5 solution of 100 mM sodium acetate. Electrode response was insensitive to interference by oxygen and nitrogen-based explosives like TNT, a prerequisite for use in the field. A Taguchi L16 array was used to investigate the performance of the electrode as functions of number of PVMo11/PR bilayers (L; 3–6 bilayers), solution pH (H; pH ∼1.32–2.85), solution [ClO3−] (C; 250–1000 μM), CV scan rate (S; 50–200 mV s−1), and film age (A; 1–8 weeks). Maximum current response was obtained for a 1 week old 5 bilayer film immersed in a pH 2.85 0.10 M NaCl (aq) solution containing 1000 μM ClO3−. However, current response fell as films aged, requiring aging of films for approximately 8 weeks prior to use to obtain reproducible analyses. A two-level full factorial design using films aged 8 weeks identified the variables S, L, H, and C and variable interactions LS and HS as statistically significant contributors to the film current response and provided a model describing film performance.

A method for covalent attachment of the enzyme acetylcholinesterase (E.C. 3.1.1.7, Electric Eel) on two different solid surfaces (glass and platinum) is described. The chemistry includes covalent bond formation between a hydroxyl group on the surface, a thiol-terminal silane, a heterobifunctional crosslinker, and the enzyme. The activity of the immobilized enzyme has been determined spectrophotometrically using two different substrates, acetylcholine and butyrylthiocholine. It is determined that after immobilization, acetylcholinesterase retains at least 50% of its activity on glass and 15% of its activity on platinum surfaces when assayed using butyrylthiocholine as a substrate. The general procedure described here for the immobilization of enzymes should find broad use in the fabrication of enzyme electrodes and in biosensor technology.

The morphology and composition of tissue located within parietal shell canals of the barnacle Amphibalanus amphitrite are described. Longitudinal canal tissue nearly spans the length of side shell plates, terminating near the leading edge of the specimen basis in proximity to female reproductive tissue located throughout the peripheral sub-mantle region, i.e. mantle parenchyma. Microscopic examination of stained longitudinal canal sections reveal the presence of cell nuclei as well as an abundance of micron-sized spheroids staining positive for basic residues and lipids. Spheroids with the same staining profile are present extensively in ovarioles, particularly within oocytes which are readily identifiable at various developmental stages. Mass spectrometry analysis of longitudinal canal tissue compared to tissue collected from the mantle parenchyma reveals a nearly 50% overlap of the protein profile with the greatest number of sequence matches to vitellogenin, a glycolipoprotein playing a key role in vitellogenesis-yolk formation in developing oocytes. The morphological similarity and proximity to female reproductive tissue, combined with mass spectrometry of the two tissues, provides compelling evidence that one of several possible functions of longitudinal canal tissue is supporting the female reproductive system of A. amphitrite, thus expanding the understanding of the growth and development of this sessile marine organism.

Abstract Recently Bekker et al. [Bekker G‐J et al. Protein Sci. 2019;28:429–438.] described a computational strategy of applying molecular‐dynamics simulations to estimate the relative stabilities of single‐domain antibodies, and utilized their method to design changes with the aim of increasing the stability of a single‐domain antibody with a known crystal structure. The structure from which they generated potentially stabilizing mutations is an anti‐cholera toxin single domain antibody selected from a naïve library which has relatively low thermal stability, reflected by a melting point of 48°C. Their work was purely theoretical, so to examine their predictions, we prepared the parental and predicted stabilizing mutant single domain antibodies and examined their thermal stability, ability to refold and affinity. We found that the mutation that improved stability the most (~7°C) was one which changed an amino acid in CDR1 from an asparagine to an aspartic acid. This change unfortunately was also accompanied by a reduction in affinity. Thus, while their modeling did appear to successfully predict stabilizing mutations, introducing mutations in the binding regions is problematic. Of further interest, the mutations selected via their high temperature simulations, did improve refolding, suggesting that they were successful in stabilizing the structure at high temperatures and thereby decrease aggregation. Our result should permit them to reassess and refine their model and may one day lead to a useful in silico approach to protein stabilization.

Field Analytical Chemistry & TechnologyVolume 4, Issue 5 p. 239-245 Original Research Articles New horizons: Explosive detection in soil extracts with a fiber-optic biosensor† Lisa C. Shriver-Lake, Corresponding Author Lisa C. Shriver-Lake Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375-55348Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375-55348Search for more papers by this authorCharles H. Patterson, Charles H. Patterson George Mason University, 4400 University Drive, Fairfax, Virginia 22030-4444Search for more papers by this authorSaskia K. van Bergen, Saskia K. van Bergen George Mason University, 4400 University Drive, Fairfax, Virginia 22030-4444Search for more papers by this author Lisa C. Shriver-Lake, Corresponding Author Lisa C. Shriver-Lake Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375-55348Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375-55348Search for more papers by this authorCharles H. Patterson, Charles H. Patterson George Mason University, 4400 University Drive, Fairfax, Virginia 22030-4444Search for more papers by this authorSaskia K. van Bergen, Saskia K. van Bergen George Mason University, 4400 University Drive, Fairfax, Virginia 22030-4444Search for more papers by this author First published: 25 October 2000 https://doi.org/10.1002/1520-6521(2000)4:5<239::AID-FACT3>3.0.CO;2-ZCitations: 14 † This article is a U.S. Government work, and, as such, is in the public domain in the United States of America. AboutPDF 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 Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Contamination of soils with the explosives TNT and RDX is a worldwide problem that has resulted from inadequate disposal methods. Many of these contamination sites are currently being characterized or are undergoing remediation. The ability to obtain real-time, on-site results would save remediation time, reduce cost, and provide for efficient use of labor during cleanup. The NRL fiber-optic biosensor, which has been demonstrated for the on-site detection of explosives in ground water, has expanded its horizons to include detection in soil extracts. Soil samples from several sites in the United States were analyzed for TNT and RDX. The explosives were removed from the soil with a 3-min acetone extraction. The extract was mixed with buffer containing a fluorescent explosive analog and exposed to the antibody-coated optical probes. In the presence of either TNT or RDX, a decrease in the fluorescence signal, proportional to the explosive concentration, was observed. In less than 20 min, analysis on four optical probes was completed. Extract results from the fiber-optic biosensor were compared to those from U.S. EPA SW 846 Method 8330 (reverse-phase high-performance chromatography). Detection limits of 0.5 mg/kg (0.1 mg/l) of TNT and RDX in soil acetone extracts were obtained. © 2000 John Wiley & Sons, Inc.* Field Analyt Chem Technol 4: 239–245, 2000 Citing Literature Volume4, Issue52000Pages 239-245 RelatedInformation

Abstract Previously developed assays for Salmonella typhimurium and staphylococcal enterotoxin B (SEB) were combined into a single multiplexed test and integrated into a fully automated prototype of the NRL Array Biosensor. Tests were performed on 216 blind samples of water, apple juice, and milk spiked with SEB (1–10,000 pg/ml) and S. typhimurium (5×103−5×107 colony‐forming units/ml). SEB and S. typhimurium were routinely detected in both water and apple juice at 100 pg/ml and 5×105 colony‐forming units/ml respectively. Inclusion of milk as a sample matrix decreased the sensitivity of the assays by an order of magnitude. Keywords: ImmunosensorbiosensorsalmonellaSEBpathogen detection The authors thank Dr. John Callahan (FDA‐CFSAN) for preparing the spiked samples. This work was funded by the Food and Drug Administration (grant # FDU‐002245‐01). The views expressed here are those of the authors and do not represent those of the U.S. Navy, the U.S. Department of Defense or the U.S. Government.

In this work, we describe the selection and characterization of single-domain antibodies (sdAb) towards the E2/E3E2 envelope protein of the Western equine encephalitis virus (WEEV). Our purpose was to identify novel recognition elements which could be used for the detection, diagnosis, and perhaps treatment of western equine encephalitis (WEE). To achieve this goal, we prepared an immune phage display library derived from the peripheral blood lymphocytes of a llama that had been immunized with an equine vaccine that includes killed WEEV (West Nile Innovator + VEWT). This library was panned against recombinant envelope (E2/E3E2) protein from WEEV, and seven representative sdAb from the five identified sequence families were characterized. The specificity, affinity, and melting point of each sdAb was determined, and their ability to detect the recombinant protein in a MagPlex sandwich immunoassay was confirmed. Thus, these new binders represent novel recognition elements for the E2/E3E2 proteins of WEEV that are available to the research community for further investigation into their applicability for use in the diagnosis or treatment of WEE.

Marine toxins, such as saxitoxin and domoic acid are associated with algae blooms and can bioaccumulate in shell fish which present both health and economic concerns. The ability to detect the presence of toxin is paramount for the administration of the correct supportive care in case of intoxication; environmental monitoring to detect the presence of toxin is also important for prevention of intoxication. Immunoassays are one tool that has successfully been applied to the detection of marine toxins. Herein, we had the variable regions of two saxitoxin binding monoclonal antibodies sequenced and used the information to produce recombinant constructs that consist of linked heavy and light variable domains that make up the binding domains of the antibodies (scFv). Recombinantly produced binding elements such as scFv provide an alternative to traditional antibodies and serve to “preserve” monoclonal antibodies as they can be easily recreated from their sequence data. In this paper, we combined the anti-saxitoxin scFv developed here with a previously developed anti-domoic acid scFv and demonstrated their utility in a microsphere-based competitive immunoassay format. In addition to detection in buffer, we demonstrated equivalent sensitivity in oyster and scallop matrices. The potential for multiplexed detection using scFvs in this immunoassay format is demonstrated.

With the availability of fluorescent dyes that are excited above 600 nm and couple easily to proteins, portable biosensors are becoming a reality. The use of diode lasers to excite these days permits systems to be made small and lightweight. An added benefit to switching to the near-infrared (NIR) regime is the reduction in background fluorescence from environmental and clinical samples. Previously, an evanescent wave biosensor was developed at the Naval Research Laboratory (NRL) using tetramethylrhodamine isothiocyanate as the fluorescent tag. Using this biosensor, toxins and other proteins have been detected down to 1 ng ml−1 and bacteria to 3000 cells ml−1. In this study, two new biosensors have been constructed using the same design with only selected components changed for detection of other fluorophores. An antigen (goat IgG) is labelled with one of three fluorophores: tetramethylrhodamine isothiocyanate, Cy5 and a near-infrared dye. The direct binding of each labelled antigen to an antibody-coated fiber-optic probe is measured. Comparisons of signal magnitudes, level of detection achieved, and photo-bleaching have been performed.

The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (β-gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with β-gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores.

Individual assays for the detection of the explosives hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6,-trinitrotoluene (TNT) with a fiber optic biosensor have been developed in our laboratory. The next step is to develop an assay for simultaneous detection of RDX and TNT. To obtain this goal, a mixture of the fluorescently-labeled analogs Cy5-EDA-RDH and Cy5-EDA-TNB used in the individual assays is run over anti-RDX and anti-TNT fiber probes connected in series. There is an increased total fluorophore concentration in the mixture which results in a significant increase in the normalized signal compared to that produced by a single fluorophore. Aggregation of the amphiphilic fluorophores in the mixture on the surface of the fiber optic probe is a possible reason for the observed effect. We explored the use of nonionic (Tween 20) and ionic detergents (deoxycholic acid) to block aggregate formation and allow analytical determination of RDX and TNT. Conditions are reported under which this is possible.

The war against terrorism requires numerous sensor technologies for homeland security. For the detection of bioterrorist agents, 1) generic detectors that indicate something amiss, 2) biosensors for rapid screening and presumptive identification, and 3) identification systems for definitive confirmation are all required. The Array Biosensor addresses the second need. This biosensor can detect multiple agents simultaneously in less than 15 min, is automated, and can be used on site. This paper reports on the current version of the automated system and reviews data obtained in assays for bioterrorist agents.

Several methods are described in which a biological recognition molecule--a critical element in any biosensor--is immobilized onto a silica or silica-based sensing substrate. Although several variations are described, the methods for covalent immobilization share a common theme and are generally composed of three steps: modification of the surface to add specific functional groups (using appropriate silanes or an amine or carboxyl-containing hydrogel), covalent attachment of a crosslinker through one of its reactive moieties, and finally, covalent linking of the biomolecule (recognition element) to the remaining reactive moiety of the crosslinker. One final method is presented in which the surface is modified with a highly hydrophobic silane and a glycolipid recognition element immobilized, essentially irreversibly, by hydrophobic interactions. All of the methods described have been successfully used to immobilize biological recognition molecules onto sensing surfaces, with full functionality in biosensor-binding assays.

The electrochemical response of multilayer epitaxial graphene electrodes on silicon carbide substrates was studied for use as an electrochemical sensor for seawater samples spiked with environmental contaminants using cyclic square wave voltammetry. Results indicate that these graphene working electrodes are more robust and have lower background current than either screen-printed carbon or edge-plane graphite in seawater. Identification algorithms developed using machine learning techniques are described for several heavy metals, herbicides, pesticides, and industrial compounds. Dose-response curves provide a basis for quantitative analysis.

Using an evanescent wave fiber optic-based biosensor developed at Naval Research Laboratory, ricin toxin can be detected in the low ng/ml range. Sensitivity was established at 1 - 5 ng/ml using a two-step assay. The two-step assay showed enhanced signal levels in comparison to a one-step assay. A two-step assay utilizes a 10 minute incubation of an immobilized affinity purified anti-ricin antibody fiber optic probe in the ricin sample before placement in a solution of fluorophore-labeled goat anti-ricin antibodies. The specific fluorescent signal is obtained by the binding of the fluorophore-labeled antibodies to ricin which is bound by the immobilized antibodies on the fiber optic probe. The toxin can be detected directly from urine and river water using this fiber optic assay.

Explosives are one of many hazardous waste problems of concern to the Department of Defense. Defective storage facilities or byproducts of weapons manufacture have led to contamination of soil and water with explosives. Most explosives are toxic, thus posing an ecological and human health hazard. The ability to do on-site or down-stream detection of explosives will be invaluable for site characterization and remediation by saving both time and money. The evanescent wave fiber optic biosensor that was developed at NRL has been modified for the detection of trinitrotoluene (TNT), by developing a competitive immunoassay on the surface of an optical probe. A fluorescently labelled analog of TNT, trinitrobenzenesulfonic acid (TNB), was used as the competitor. Enzyme-linked immunosorbent assays were performed to determine the best fluorescently labeled competitor available to be able to achieve high sensitivity in the fiber optic assay. For the competition assay, 7.5 ng/ml Cyanine 5-ethylenediamine-labelled TNB (Cy5-EDA-TNB) was exposed to an antibody-coated optical fiber generating specific signal above background that corresponds to the 100% or reference signal. Inhibition of this signal was observed in the presence of TNT with the percent inhibition proportional to the TNT concentration in the sample. Detection sensitivities in aqueous solutions containing 10 ng/ml TNT (8 ppb) have been achieved using this system.

An array biosensor developed for performing simultaneous analysis of multiple samples for multiple analytes has been miniaturized and fully automated. The biochemical component of the multi-analyte biosensor consists of a patterned array of biological recognition elements ("capture" antibodies) immobilized on the surface of a planar waveguide. A fluorescence assay is performed on the patterned surface, yielding an array of fluorescent spots, the locations of which are used to identify what analyte is present. Signal transduction is accomplished by means of a diode laser for fluorescence excitation, optical filters and a CCD camera for image capture. A laptop computer controls the miniaturized fluidics system and image capture. Data analysis software has been developed to locate each spot and quantify the fluorescent signal in each spot. The array biosensor is capable of detecting a variety of analytes including toxins, bacteria and viruses and shows minimal interference from complex physiological sample matrices such whole blood and blood components, fecal matter, saliva, nasal secretions, and urine. Some results from recent field trials are presented.

Abstract Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to refold following denaturation, and inexpensive production in Escherichia coli or yeast. However, their small size does have drawbacks, one being that they can lose activity upon attachment or adsorption to surfaces, or may fail to adsorb efficiently, as they are highly soluble. This can make the transition from using conventional antibodies to sdAb nontrivial for assay development. Specifically, it is often necessary to re-optimize the protocols and tailor the recombinant sdAb through protein engineering to function efficiently in handheld assays, which currently are utilized for point of care testing and field applications. This work focuses on optimizing the integration of sdAb into rapid vertical flow assays. To achieve this goal, we engineered sdAb-based constructs and developed general protocols for the attachment of the sdAb to both gold nanoparticles and a support membrane. We achieved a limit of detection of 0.11 µg/mL for toxins staphylococcal enterotoxin B and ricin, both potential biothreat agents. Additionally, we demonstrated the ability to detect the nucleocapsid protein of SARS-CoV-2, a common target of antigen tests for COVID-19.

A fiber optic-based biosensor which integrates a novel array of optical and electrical components, together with long fused silica fibers and proteins for detection of analyte in solution has been put into limited production. The optical fiber core near the distal end is tapered and coated with either antibodies or DNA binding proteins. Assays are performed by flowing a solution containing the fluorescently tagged ligand molecules over the coated fiber. Within seconds, analyte recognition occurs and a fluorescence signal is transmitted back up the fiber. Applications for the biosensor include clinical diagnostics, pollution control, and environmental monitoring. Ten of the laboratory devices described in this article have been constructed and are being used for assay development. In addition, the prototype of a truly portable device has recently been built and is now being tested.

Anti-Staphylococcal Enterotoxin B single domain antibodies were engineered to include the N-terminal peptide sequence of the major outer membrane lipoprotein from Escherichia coli, which directs the N-terminal addition of lipid to the single domain antibody. We produced and purified two different single domain antibodies as well as a variant and dimer construct of one of the two, all with and without the added lipid. Their ability to function as the capture antibody in standard enzyme-linked immunosorbent assays were evaluated, finding that coating polystyrene microtiter plates with the lipid-tagged single domain antibodies gave a 3-fold improvement in the observed limit of detection. This increase was likely due to an increased amount of single domain antibody adsorbed to the microtiter plate, which translated to improved limits of detection of Staphylococcal Enterotoxin B over using the same single domain antibody sans lipid-tag. However, improved orientation may also play a role. Regardless of the mechanism, the biosynthetic lipid-tagging of single domain antibodies represent a facile modality that can enhance their ability to be utilized as immunoassay capture reagent as well as facilitate their incorporation into liposome targeting applications in the future.

Lassa virus is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. It is important to develop new reagents applicable either for the specific diagnosis or as improved therapeutics for the treatment of Lassa fever. Here, we describe the development and initial testing of llama-derived single-domain antibodies that are specific for the Lassa virus nucleoprotein. Four sequence families based on complementarity-determining region (CDR) homology were identified by phage-based enzyme-linked immunosorbent assays, however, the highest affinity clones all belonged to the same sequence family which possess a second disulfide bond between Framework 2 and CDR3. The affinity and thermal stability were evaluated for each clone. A MagPlex-based homogeneous sandwich immunoassay for Lassa virus-like particles was also demonstrated to show their potential for further development as diagnostic reagents.

The thermal stabilization of enzymes is a critical factor in the development and reliability of enzyme-based processes and functional materials. Using a simple amine coupling approach for enzyme immobilization onto magnetic microbeads, followed by encasement of the beads in a hydrogel, we demonstrate that the thermal stability of the enzyme acetylcholinesterase can be increased dramatically. For example, when free and microbead-immobilized enzyme (“EM Conjugate”) are incubated overnight in a dry state at 63 °C (140 °F), the catalytic efficiency (kcat/Km) of the latter is higher than the former by six orders of magnitude (a factor of 2.16 × 106). This effect arises mostly through a ∼29,700-fold decrease in Km experienced by the EM Conjugate, relative to that of the free enzyme. Encapsulation of the EM Conjugate in a hydrogel based on poly(N-(3-aminopropyl methacrylamide)), which contains a primary amine, affords the enzyme additional stability when incubated overnight at 63 °C in an aqueous state. For example, its catalytic efficiency is four orders of magnitude higher than that of both the free enzyme (a factor of 4.34 × 104) and that of the EM Conjugate alone (a factor of 1.78 × 104) after all are incubated overnight at 63 °C. The presence of the hydrogel also caused the Michaelis constant to decrease by 1.38 × 104 relative to that of the EM Conjugate, reaching a value of 2.18 × 10−3 M. Thus the hydrogel enables the AChE substrate binding site to retain a significant amount of its natural affinity for the substrate, after heating. This effect may occur via ion-pairing by the primary amines in the hydrogel polymer repeat unit, which are protonated and positively-charged at the assay pH. To the best of our knowledge, this simple method for enzyme thermal stabilization is novel and has not yet been investigated.

This chapter describes an evanescent wave fiber optic biosensor and its application to immunoassays for rapid detection of bacterial cells and pollutants. Whole cells of Burkholderia cepacia G4 5223-PR1 (G4) are of interest for their ability to degrade trichloroethylene (TCE) which is one of the most prevalent contaminants of ground water in the United States. The lower limit of detection of the G4 with this system is 104 -105 cells/ml. In addition to TCE, the explosive trinitrotoluene (TNT) is a known contaminant of ground water. Limits of detection of TNT with this system is 10 ng/ml.

Abstract Immunoassays are widely used in various fields, including biomedical research, clinical diagnostics, and environmental monitoring. Single domain antibodies (sdAbs) provide small, tailorable, recognition elements that have been integrated into immunoassays for detecting a myriad of targets. Deoxyribonucleic acid (DNA) circuits are synthetic molecular devices composed of DNA strands that can perform logical or computational operations. They have a range of applications, including biosensing, diagnostics, and drug delivery. Recently, an immunoassay method was reported that used catalytic hairpin assemblies (CHA) with antibodies for homogeneous protein detection. The CHA process uses DNA hairpins that interact in the presence of a catalytic DNA sequence. This paper presents a new strategy to couple the recognition of sdAbs with CHA circuits using genetic fusions of sdAbs with rhizavidin (rz), a dimeric biotin binding protein. A pair of sdAb‐rz constructs is each functionalized with a biotinylated DNA sequence that represents half of the catalyst. When both sdAbs bind to the target protein, a signal is generated through the CHA circuit. The split catalyst approach amplifies signals through a DNA circuit without washing steps. Furthermore, this method distinctively utilizes programmable DNA circuits, which are highly modular and can accommodate new targets without disrupting the assay.

The detection of the explosive TNT using a fiber optic biosensor has successfully undergone the transition from laboratory analysis to on-site, real time sample monitoring due to advances in miniaturization and portability. A larger "breadboard" system capable of monitoring a single probe was replaced by a lightweight, portable sensor that can monitor 4 optical probes simultaneously. This new fiber optic biosensor was taken to two contaminated military bases for on-site testing for TNT in groundwater. Prior to on-site testing, assay variables, including buffers and fluorescence analog concentration, were optimized to permit TNT detection in the 5-20 μg/L range. In addition, several cross reactivity studies were performed demonstrating interference from TNB, a degradation product of TNT but not the explosive RDX or its degradation product, HMX.

Reproducible immobilization of peptides and proteins on microsphere surfaces is a critical factor for optimal sensitivity and selectivity in bead-based assays. However, peptides with unusually large numbers of lysine residues—whose amines are targeted in the most common microsphere immobilization chemistries—may be particularly challenging to use in bead-based arrays, as they may lose activity through multipoint attachments and incorrect presentation. For this reason, it is imperative to achieve site-directed attachment chemistry, such that a single site of attachment provides reproducibly oriented peptides on the microsphere surface. This can be achieved by inserting a unique targetable residue, such as a cysteine. Here, we present methods for attaching cysteine-containing peptides to standard carboxy-functionalized microsphere surfaces using thiol- rather than amine-directed chemistries. We show that the presence of a cationic detergent (CTAB) and a “passivating” agent such as β-mercaptoethanol facilitates improved bead recovery after peptide immobilization and may enhance functionality of the attached peptides.

BioTechniquesVol. 55, No. 6 BioFeedback: [Letter to the editor]Open AccessLoss of cationic peptides with agarose gel-immobilized tris[2- carboxyethyl]phosphine (TCEP)Lisa C. Shriver-Lake, Stella H. North & Chris Rowe TaittLisa C. Shriver-LakeCenter for Bio/Molecular Science & Engineering, US Naval Research Laboratory, Washington, DCSearch for more papers by this author, Stella H. NorthCenter for Bio/Molecular Science & Engineering, US Naval Research Laboratory, Washington, DCSearch for more papers by this author & Chris Rowe Taitt*Address correspondence to Chris Rowe Taitt, Center for Bio/Molecular Science & Engineering, US Naval Research Laboratory, Washington, DC. E-mail: E-mail Address: chris.taitt@nrl.navy.milCenter for Bio/Molecular Science & Engineering, US Naval Research Laboratory, Washington, DCSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114112AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: Tris[2-carboxyethyl]phosphineTCEPpeptide/protein reductionimmobilized TCEPantimicrobial peptideTrivalent phosphines have been used for disulfide reduction for many years (1, 2). Tris[2-carboxyethyl]phosphine (TCEP) has the added advantages of water solubility, broad pH stability, and minimal reactivity toward other functional groups found in proteins. In contrast to reducing agents such as dithiothreitol, TCEP does not contain thiols, and thus its removal is not required before subsequent modification of reduced proteins and peptides. However, TCEP can react with maleimidyl, iodoacetic acid and iodoacetamide compounds commonly used for protein and peptide modification (3, 4). For this reason, agarose gel-immobilized TCEP has been commercially developed (e.g., Pierce, G-Biosciences) to allow immediate use of TCEP-reduced proteins and peptides without requiring gel filtration or dialysis. As gel-immobilized TCEP has been used to minimize TCEP interference, we have applied it to reduction of various proteins with great success. However, when we used this gel to reduce a number of cysteine-containing cationic peptides, we recently observed a potential side effect of this matrix—namely significant sample loss.As part of our current research using antimicrobial peptides (AMPs) to capture, detect, and classify various microbial targets (5–10), we have used a number of AMPs with engineered cysteine residues for accurate quantification or to allow oriented immobilization onto surfaces (9, 10). For the present study, a series of eight custom peptides based on the cationic AMPs cecropin A (KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK), cecropin B (KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL), melittin (GIGAVLKVLTTGLPALISWIKRKRQQ), andcecropin A (1–8)-melittin (1–18) hybrid peptide (KWKLFKKIGIGAVLKVLTTGLPALIS) were synthesized to possess a unique cysteine at various loci without changing the peptides' net charge (90% purity, Biosynthesis, Inc. Lewisville, TX)(Figure 1). Each peptide was diluted to 0.7–1.0 µg/mL in phosphate-buffered saline (PBS), pH 7.4 and then treated with 5 mM neutralized TCEP or 100 µL immobilized-TCEP (catalog # 20490 and 77712, respectively; Pierce, Rockford, IL) for 20 min to ensure complete reduction of any dithiols. Given TCEP's instability in phosphate buffers (11), TCEP gel and TCEP stock solutions were made up immediately prior to use. After treatment, the agarose gel was removed from peptides by centrifugation and/or filtration. Peptide concentrations of TCEP- and TCEP-agarose-treated solutions were determined by absorbance at 280 nm (Thermo Scientific NanoDrop 2000, Thermo Scientific, Waltham, MA USA, cat no. ND-2000). Each sample was prepared in triplicate. Sepharose 4B (agarose gel, catalog # 17-0120-01; GE Healthcare, Piscataway, NJ) was used as a control to determine the extent of loss (if any) attributable to the agarose matrix itself.Figure 1. Recovery of cysteine-containing peptides after treatment with immobilized TCEP gel.Construct sequences are shown above the graph with sequences of the base peptides shown in gray: cecropin A (constructs A-C, striped bars), cecropin B (construct D, stippled bar), melittin (constructs E-H, gray bars), and cecropin A (1–8)-melittin (1–18) hybrid peptide (construct I, black bar). The position of the unique cysteine in each construct is indicated as the underlined residue.As expected, treatment of peptides with 5 mM TCEP solution or buffer (no TCEP) resulted in no significant peptide loss or precipitation when compared with untreated controls (P > 0.15). Small but statistically significant losses (3%–15%) were incurred when treating peptides with Sepharose (P < 0.005); similar losses have been observed with protein controls (data not shown). However, significantly greater losses were observed when peptides were treated with agarose gel functionalized with TCEP (P < 0.00001; Figure 1).The degree of loss on the gel-immobilized TCEP appeared to be peptide-specific for the four base peptides tested. The highest losses (up to 85%) were observed with the 4 different melittin constructs, independent of the locations of the cysteinyl residues, whereas 35%–40% losses were observed with analogous cecropin A constructs. Losses intermediate between those for these two peptides were observed with a hybrid peptide comprising domains from melittin and cecropin. Interestingly, the degree of loss could not be directly related to the net charge at neutral pH, mean hydrophobicity, mean or relative hydrophobic moments, or position of the unique cysteinyl residues.In spite of the lack of direct relationship between losses and net peptide charge, we anticipated that there might be an interaction between the cationic peptides and immobilized TCEP, which displays one or more carboxyl moieties. We therefore incubated the series of cysteinyl peptides, demonstrating the highest losses with immobilized TCEP gel under conditions of varying ionic strengths (10 mM phosphate buffer with 0.15 M NaCl [PBS] to 1 M NaCl, final pH = 7.4) (Figure 2). While recoveries of 2 constructs were improved by increasing the ionic strength to > 0.3 M, inclusion of 1 M NaCl significantly improved peptide recovery for all 4 peptides tested (P < 0.05). Thus, peptide losses due to electrostatic interactions could be partially (constructs E and H) or fully (constructs F and G) mitigated by use of higher ionic strength buffers.Figure 2. Recovery of cysteine-containing melittin constructs.Constructs were incubated with immobilized TCEP gel in phosphate buffer containing 0.15 M NaCl (PBS, white), 0.3 M NaCl (light gray), 0.5 M NaCl (medium gray), or 1 M NaCl (dark gray). Construct sequences are shown above the graph with sequences of the base peptide (melittin) shown in gray. The positions of the unique cysteines are indicated as the underlined residue. Asterisks above the bars indicate a significant (P < 0.05) improvement over recoveries obtained with PBS.The utility of a one-pot reaction for both reduction and labeling of protein and peptide thiols—with minimal manipulation required to remove TCEP—is appealing, and high-efficiency labeling of proteins following treatment with immobilized TCEP can be accomplished (12). In our hands, however, significantly greater losses of cationic peptides were observed after treatment with agarose-immobilized TCEP than with naked agarose. While this loss could be mitigated with higher ionic strength buffers, the full extent of recovery still depended on the peptide used; even in the presence of 1 M NaCl,some peptides were still not recovered at levels approaching 100%.Our results emphasize a potential pitfall associated with use of immobilized TCEP-reagents, namely sample loss during reduction. Our observations additionally highlight the need to quantify peptides after treatment with these matrices. While a 37%–56% decrease in peptide immobilization on maleimide-activated surfaces was observed if soluble TCEP was not removed (determined by far-UV circular dichroism spectroscopy; data not shown) (13), losses of similar magnitude were observed with samples that were reduced with immobilized TCEP. In some cases, these losses could be only partially recovered by high salt concentrations. This trade-off should be carefully considered when samples are precious, low ionic strength is needed, or purification of reduced species after TCEP treatment is not feasible.Author contributionsL.C.S.-L. performed the reductions, quantified the peptides, analyzed the data, and edited the manuscript. S.H.N. contributed to conception, design of the experiments, analyzed the data, performed immobilization experiments (efficiency of covalent modification after treatment), and edited the manuscript. C.R.T conceived and designed the experiments, performed controls, analyzed the data (statistics), and prepared the manuscriptAcknowledgmentsThis work was supported by the Office of Naval Research and the Naval Research Laboratory. The opinions expressed herein are those of the authors and do not reflect those of Office of Naval Research, the Naval Research Laboratory, the US Navy or Department of Defense, or the US Government.Competing interestsThe authors declare no competing interests.References1. Burns, J., J. Butler, J. Moran, and G. Whitesides. 1991. Selective reduction of disulfides by tris(2-carboxyethyl)phosphine. J. Org. Chem. 56:2648–2650.Crossref, CAS, Google Scholar2. Levison, M.E., A.S. Josephson, and D.M. Kirschenbaum. 1969. Reduction of biological substances by water-soluble phosphines: Gamma-globulin (IgG). Experientia 25:126–127.Crossref, Medline, CAS, Google Scholar3. Shafer, D.E., J.K. Inman, and A. Lees. 2000. Reaction of tris(2-carboxyethyl)phosphine (TCEP) with maleimide and α-haloacyl groups: Anomalous elution of TCEP by gel filtration. Anal. Biochem. 282:161–164.Crossref, Medline, CAS, Google Scholar4. Schumacher, F.F., M. Nobles, C. Ryan, M. Smith, A. Tinker, S. Caddick, and J. Baker. 2011. In situ maleimide bridging of disulfides and a new approach to protein PEGylation. Bioconjug. Chem. 22:132–136.Crossref, Medline, CAS, Google Scholar5. Kulagina, N., C. Taitt, G.P. Anderson, and F.S. Ligler. Affinity-based detection of biological targets. Allowed US Patent Appl Pub. No. 2006/0281074.Google Scholar6. Kulagina, N.V., M.E. Lassman, F.S. Ligler, and C.R. Taitt. 2005. Antimicrobial peptides for detection of bacteria in biosensor assays. Anal. Chem. 77:6504–6508.Crossref, Medline, CAS, Google Scholar7. Kulagina, N.V., K.M. Shaffer, G.P. Anderson, F.S. Ligler, and C.R. Taitt. 2006. Antimicrobial peptide-based array for Escherichia coli and Salmonella screening. Anal. Chim. Acta 575:9–15.Crossref, Medline, CAS, Google Scholar8. Kulagina, N.V., K.M. Shaffer, F.S. Ligler, and C.R. Taitt. 2007. Antimicrobial peptides as new recognition molecules for screening challenging species. Sens. Actuators B Chem. 121:150–157.Crossref, Medline, CAS, Google Scholar9. North, S.H., J. Wojciechowski, V.M. Chu, and C.R. Taitt. 2012. Surface immobilization chemistry influences peptide-based detection of lipopolysaccharide and lipoteichoic acid. J. Pept. Sci. 18:366–372.Crossref, Medline, CAS, Google Scholar10. Shriver-Lake, L.C., S.H. North, S.N. Dean, and C.R. Taitt. 2013. Antimicrobial peptides for detection and diagnostic assays, p. 85–104. In S.A. Piletsky and M.J. Whitcomb (Eds.), Designing receptors for the next generation of biosensors. Springer, Heidelberg.Google Scholar11. Han, J.C. and G. Han. 1994. A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Anal. Biochem. 220:5–10.Crossref, Medline, CAS, Google Scholar12. Tzanavaras, P.D., C. Mitani, A. Anthemidis, and D.G. Themelis. 2012. On-line cleavage of disulfide bonds by soluble and immobilized tris- (2-carboxyethyl)phosphine using sequential injection analysis. Talanta 96:21–25.Crossref, Medline, CAS, Google Scholar13. Fears, K.P. and R. Latour. 2009. Assessing the influence of adsorbed-state conformation on the bioactivity of adsorbed enzyme layers. Langmuir 25:13926–13933.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByProtein Reduction and Dialysis-Free Work-Up through Phosphines Immobilized on a Magnetic Support: TCEP-Functionalized Carbon-Coated Cobalt Nanoparticles5 June 2017 | Chemistry - A European Journal, Vol. 23, No. 36 Vol. 55, No. 6 Follow us on social media for the latest updates Metrics History Received 4 September 2013 Accepted 1 November 2013 Published online 3 April 2018 Published in print December 2013 Information© 2013 Author(s)KeywordsTris[2-carboxyethyl]phosphineTCEPpeptide/protein reductionimmobilized TCEPantimicrobial peptideAuthor contributionsL.C.S.-L. performed the reductions, quantified the peptides, analyzed the data, and edited the manuscript. S.H.N. contributed to conception, design of the experiments, analyzed the data, performed immobilization experiments (efficiency of covalent modification after treatment), and edited the manuscript. C.R.T conceived and designed the experiments, performed controls, analyzed the data (statistics), and prepared the manuscriptAcknowledgmentsThis work was supported by the Office of Naval Research and the Naval Research Laboratory. The opinions expressed herein are those of the authors and do not reflect those of Office of Naval Research, the Naval Research Laboratory, the US Navy or Department of Defense, or the US Government.Competing interestsThe authors declare no competing interests.PDF download

A field demonstration was conducted to assess the performance of eight commercially-available and emerging colorimetric, immunoassay, and biosensor on-site analytical methods for explosives 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in ground water and leachate at the Umatilla Army Depot Activity, Hermiston, Oregon and US Naval Submarine Base, Bangor, Washington, Superfund sites. Ground water samples were analyzed by each of the on-site methods and results compared to laboratory analysis using high performance liquid chromatography (HPLC) with EPA SW-846 Method 8330. The commercial methods evaluated include the EnSys, Inc., TNT and RDX colorimetric test kits (EPA SW-846 Methods 8515 and 8510) with a solid phase extraction (SPE) step, the DTECH/EM Science TNT and RDX immunoassay test kits (EPA SW-846 Methods 4050 and 4051), and the Ohmicron TNT immunoassay test kit. The emerging methods tested include the antibody-based Naval Research Laboratory (NRL) Continuous Flow Immunosensor (CFI) for TNT and RDX, and the Fiber Optic Biosensor (FOB) for TNT. Accuracy of the on-site methods were evaluated using linear regression analysis and relative percent difference (RPD) comparison criteria. Over the range of conditions tested, the colorimetric methods for TNT and RDX showed the highest accuracy of the emerging methods for TNT and RDX. The colorimetric methodmore » was selected for routine ground water monitoring at the Umatilla site, and further field testing on the NRL CFI and FOB biosensors will continue at both Superfund sites.« less

The NRL fiber-optic biosensor was developed with the specific goal of using long fibers (1-20 m) to facilitate the analysis of environmental and clinical samples for hazardous materials. The long fibers were deemed important for preventing contamination of the operator and the optical components by potentially hazardous materials. The use of the biosensor is described here in terms of the chemistry for protein immobilization, stability of antibody-coated fibers, and sensitivity for detection of protein toxins. Antibodies coated on the fiber are stable for over 1 year of storage prior to use. The fiber optic biosensor has been used to measure ng/ml (pM) concentration of toxins in under a minute.

An array biosensor developed for simultaneous analysis of multiple samples has been utilized to develop assays for toxins and pathogens in a variety of foods. The biochemical component of the multi-analyte biosensor consists of a patterned array of biological recognition elements immobilized on the surface of a planar waveguide. A fluorescence assay is performed on the patterned surface, yielding an array of fluorescent spots, the locations of which are used to identify what analyte is present. Signal transduction is accomplished by means of a diode laser for fluorescence excitation, optical filters and a CCD camera for image capture. A laptop computer controls the miniaturized fluidics system and image capture. Results for four mycotoxin competition assays in buffer and food samples are presented.

Array biosensors provide the capability of immobilizing multiple capture biomolecules onto a single surface and therefore offer the exciting prospect of multi-analyte detection. A miniaturized, fully automated, stand-alone biosensor is reported which can simultaneously test multiple samples for multiple analytes. This portable system (< 10 lbs) is particularly appropriate for on-site monitoring for food safety, infectious disease detection, and biological warfare defense. The surface-selective nature of this technology allows determination of binding constants and tracking of both specific and non-specific binding events as they occur. Thus, it provides an exciting new research tool for characterizing the interactions of biomolecules with surfaces or immobilized receptors in real time. This capability has important implications for development of new materials and sensors.

Immobilization of functional antibodies on optical fibers at high density and configuration of optical components so as to maximize the signal/noise (S/N) are key elements in the development of fiber optic-based immunosensors. Functional antibodies were immobilized on silica substrates using thiol-terminal silanes and heterobifunctional crosslinkers. Anti-IgG antibody was routinely immobilized on glass cover slips at 0.66 ng/mm2 and on silica fibers at 0.96 ng/mm2 using this procedure. The immobilized antibody bound 0.37-to-0.55 moles IgG antigen per mole antibody. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was only 2-6% of the total antigen binding. Initial assessments of antibody immobilization and function were performed using radiolabelled proteins. In order to convert the system to fluorescence, antigens were labelled with a variety of fluorophores and optical components were tested in a variety of configurations. Problems with background fluorescence were identified stemming from fluorescence of the glass substrates, stickiness of fluorescent proteins, and fluorescence from optical components. The latter problem was addressed by testing a variety of components and configurations in a sensor testbed to determine which provided the greatest sensitivity. Using this iterative approach, we were able to identify factors which degrade sensitivity and to detect 100 pM fluorophor in a distal cuvette configuration with S/N of 14.

In the Center for Bio/Molecular Science and Engineering at the Naval Research Laboratory (NRL), two different types of immunosensors are being developed for detection of environmental pollutants and monitoring of bacteria for bioremediation. Both biosensors are rapid, sensitive and easy to operate. The first sensor, the continuous flow immunosensor, is based on displacement of fluorescently-labeled antigen from antibodies immobilized on beads. Antigen is injected into a flow stream that passes over a 100 μL bed volume of antibody-coated beads saturated with fluorescently-labeled antigen. The displacement of the labeled antigen causes an increase in fluorescence, proportional to the antigen concentration, to be observed downstream. The other sensor, the fiber optic biosensor, utilizes long, partially clad optical fibers. Antibodies are immobilized onto the fiber core in the unclad region at the distal end of the fiber. Upon binding of antigen and a fluorescent molecule, a change in the fluorescence signal is observed. For small molecules, competitive immunoassays are performed in which a decrease in the fluorescence signal is observed which is proportional to the antigen concentration. For bacterial cells, sandwich or direct immunoassays are performed which generate an increase in the fluorescence signal proportional to the specific cell concentration.

The utility of pathogen and toxin detection systems depends not only upon sensitivity, specificity and capability for multiplexed recognition, but also on access at the point of need, ease of use, and response time. Combining microfluidics and optical biosensors facilitates miniaturization and automation, while careful design makes it possible to test variable quantities of complex matrices such as food (mL) and clinical samples (μL). We report on progress towards an optofluidic system that combines a rotating magnet trap for automated sample processing and a microflow cytometer capable of 4-color analysis to achieve multi-analyte diagnostics. Sample and antibody-coated magnetic beads are introduced into a moving magnetic field created by magnets rotating in the opposite direction of the flow. The color-coded magnetic beads with captured target remain in suspension while fluorescent tracer reagents are introduced to the flow, thus optimizing the binding kinetics and minimizing aggregation. Magnetic beads with bound target and tracer reagents are released for multiplexed analysis by reversing the direction of spinning magnets.

Based upon a biosensor design which utilizes standard fluorescent dyes (FITC, TRITC), a new device has been developed which incorporates a laser diode light source to excite novel near infrared (NIR) dyes. The purpose of switching to the NIR regime is to decrease the background fluorescence of biological samples and to decrease the size and power requirements of the biosensor. New dyes which fluoresce in the NIR have been conjugated to protein antigen and immunoassays performed. Assay results using excitation at 780 nm are shown.

Abstract : Fluorescence-based flow cytometry dates back to the 1960s. Essentially, cells or particles are aligned in a flow stream and optically interrogated. Size, density, and fluorescence at multiple wavelengths can be quantified. In many cases, tags, such as fluorescently labeled antibodies, are mixed with the samples prior to analysis so that specific targets or cell functions can be identified. Currently, large, complex, laboratory- based flow cytometers are required to perform medical diagnostics, such as white blood cell counts and immunoassays to detect infection, or for environmental monitoring applications, such as classification of marine algae. In the traditional design, the sample containing the particles or cells is pumped out of a small tube into a much larger, concentric pipe that is carrying filtered water. This hydrodynamic focusing puts all the particles into the center of the wider stream, which then is tapered to a smaller diameter. The particles are thus focused and pass single-file through the laser beams for analysis. Over the last decade, flow cytometers have become smaller in size and less expensive, but this sheath flow design is not amenable to miniaturization to the point that the systems are portable. NRL's Center for Bio/Molecular Science and Engineering has developed a microfluidic sheath flow system that is robust, simple to fabricate, and very compact. This sheath flow device forms the basis of a microflow cytometer that has demonstrated the capability for 4-color analysis that is competitive with the larger, commercial systems.

Abstract Biosensors, affinity chromatography, and many bio analytical methods require a high density of functional molecules, low non-specific protein adsorption, long-term stability, and durability (1). Proteins, including antibodies and enzymes, and cells have been immobilized onto solid supports for these and other applications over the last 30 years. In the biosensor arena, optical transduction methods are increasingly employed. With this increase, solid supports such as fused silica and quartz are appropriate substrates for immobilization. Non-specific protein adsorption to these substrates has been a major problem.

You have accessJournal of UrologyPodium, Monday, May 22, 2006, 3:30 - 5:30 pm1 Apr 2006982: A Multianalyte Biosensor for Bladder Cancer Diagnostics Marta Sanchez-Carbayo, Lisa C. Shriver-Lake, Carlos Cordon-Cardo, and Frances S. Ligler Marta Sanchez-CarbayoMarta Sanchez-Carbayo More articles by this author , Lisa C. Shriver-LakeLisa C. Shriver-Lake More articles by this author , Carlos Cordon-CardoCarlos Cordon-Cardo More articles by this author , and Frances S. LiglerFrances S. Ligler More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)33207-5AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "982: A Multianalyte Biosensor for Bladder Cancer Diagnostics." The Journal of Urology, 175(4S), pp. 316–317 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 175Issue 4SApril 2006Page: 316-317 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Marta Sanchez-Carbayo More articles by this author Lisa C. Shriver-Lake More articles by this author Carlos Cordon-Cardo More articles by this author Frances S. Ligler More articles by this author Expand All Advertisement PDF DownloadLoading ...