Fluorescence-based array biosensors for detection of biohazards

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 This to be a method of creating patterns in of immobilized capture this method the that only a single biomolecule could be immobilized in a The use of is popular choice for the production of patterned biomolecular Silzel et al. (1998) either the capture or avidin in on the surface of were immobilized on the avidin A pattern of two different was by Budach et al. using the of capture biomolecules onto a waveguide using has also been successfully used by and Ligler in the production of arrays of capture and has the for patterning different capture biomolecules onto a single waveguide surface. patterning has been using from a variety of including (Feldstein et al. 1999; et al. a (Plowman et al. 1999), a with et al. 2000) and a of silicon et al. 2001). the a number of is attached to the surface of the planar waveguide and with a of the capture as in Fig. In this (Feldstein et al. 1999; et al. the resulting waveguide was patterned with of immobilized biomolecules The sample and fluorescent-labelled were then over the surface using a to the immobilized capture biomolecule as in Fig. patterning the immobilization of a number of different capture one in onto a single surface, creating an array of recognition with no The patterning of capture biomolecules using (adapted from Feldstein et al. 1999). A is onto the planar waveguide and with a of the capture and fluorescent are over the waveguide surface to the immobilized capture biomolecule using a of the initial into the use of planar waveguides in TIRF biosensors has on both instrumentation development and immobilization of the capture However, the and are the of application the further A number of studies have investigated the use of a single capture assay (Brecht et al. 1998; Klotz et al. 1998). However, as one of the major advantages of using a planar is the ability to create arrays of different capture biomolecules for multi-analyte To date, there are at research groups involved in the immobilization of patterns of multiple capture biomolecules onto planar waveguides although only of these groups have demonstrated multi-analyte measurements. et al. have developed a TIRF system in which the planar waveguide of multiple, single sensing of these single has its laser light background and of the fluorescence to the The demonstrated a sensing device in which one was with and the with rabbit However, only one immobilized antigen was at a was that the laser light could be into different in multi-analyte measurements, using multiple single sensing a process would a number of optical and the robustness of the system for use the is in The of most biosensor research is the development of a instrument towards and for biohazard monitoring applications. group at the Naval Research Laboratory group on TIRF studies in various different IgG this was in for the detection of analytes and et al. 1997, 1998). In the study et al. 1998), the and the were and the the use of a for the patterning of capture biomolecules was developed and the which was prior to was carried out using a to the patterned et al. 1998). detection of analytes was was detected and measured in clinical such as and as well as and detection for clinical requirements (Rowe et al. that are not only for system development are also a the instrumentation is to the into applications. The of environmental has also been by Rowe-Taitt et al. of toxin and were in the of such as and and results were compared with buffer or were by the however, in some the signal was recently the detection of into food such as and was all with a of detection et al. Most methods for the detection and of in extensive or concentration prior to analysis. measured using the array biosensor were with the of for example, such as were typically with prior to and analysis. such as and where with a of food to buffer This was then in a the and the sample at room for The sample was then at for and the resulting for on the array for have also been developed for of and In to results with some sensitivity was was into these However, little effect was by large of bacteria often found in the the of in the of is especially given the close between and the development of multi-analyte assays for and the room CCD camera was with a which in a of the background of et al. 1999) and an decrease in the of detection from et al. to for (Rowe et al. This of detection was further to by from a to a capture and a to Alexafluor labelled et al. on was the use of a in the immunoassays as compared with the In order to further the array technology, an automated image was developed and some of the optical and fluidic components were (Feldstein et al. 1999). To the ability of the array biosensor to measure of analytes, assays were carried out using viral and protein analytes (Rowe et al. or multi-analyte were through the assay followed by either or a of antibodies. The array biosensor was used in the study of and the automated image reliable in the of fluorescent with detection in the to results using the antibodies. which used of fluorescent the results as in which the fluorescent were The approach using of was to the analytes toxin and of for analytes in et al. the immunoassays were carried out using a standard array however, et al. has demonstrated that with the use of complementary of capture and to analytes can be detected using a single array For applications it is important that the biosensor be fully automated, and studies by the group have on this This has involved of the system and the of an automated system with an automated image (Feldstein et al. 2000; Rowe-Taitt et al. The sensing in the form of a microscope not the and of the the are the and it to and the fluidic system for the array leading to a small, fully automated array which into a and kg et al. 2001). The was a method for to the waveguide evanescent light from the surface (Feldstein et al. 2000). This was using a patterned (see Fig. which the waveguide from the the evanescent excitation of fluorophores the of the (Feldstein et al. 1999). The signal of onto planar waveguides was of the signal. The by of their signal for the assays (Feldstein et al. 1999). The for the possibility of studying binding in real-time. have been demonstrated by Sapsford et al. and Sapsford and Ligler for both specific and The pattern of the the where the with the waveguide (adapted from Feldstein et al. 1999). The of the waveguide surface is and the array of biomolecules is subsequently immobilized onto the The is using through a metal which a and only areas to be The of a to a for and a thin to the from in the required for (Feldstein et al. 2000) The was an integrated whereby was using a et al. 2001; Feldstein 2002). 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When one is the the from all attached to that (e.g. all sample through the and out to When the sample is and the all the fluorescent their into the The automated system with a and sample compared with the attached to the and waveguide, and and The in resulting from of the is in Fig. The system on the a large large and not are the required for buffer and The system on the the attached to the and waveguide, and and Lee The to the and waveguide with no to the are limited to an and two all of which are through a at the of the The of these the and automation of the system in the array however, the and optics of the system were a for field In order to the array biosensor Golden and Ligler the possibility of the CCD with the for image a camera or a would provide systems more for sensors. The in particular, would However, that the have an order of than either of the two imaging systems and Ligler 2002). The system included a et al. 2001). In the the was and the using a In to the with its and the included a large and the of light cameras was the were for sensitivity measurements. advances in CCD technology it to the CCD with a and for applications be the technology used in the new CCD cameras the for a the Currently, the group has a array biosensor for sample monitoring and is in the process of its a relatively small, the and the and and are performed using a The device into a which is the of the and only 5·5 The of the array biosensor This review has discussed the use of optical array biosensors, based on for the detection of and BW including and current developments in the TIRF for a device that is fully automated and portable, for continuous monitoring applications et al. 2001). However, with the development of and such as the camera currently used in the confocal microscopy studies of (Vo-Dinh et al. 1999; et al. the for planar waveguide TIRF devices could Although the of studies have on antibody–antigen of the with and receptor–ligand binding interactions et al. and 2000; et al. 2000; et al. 2001; as well as into the use of and DNA et al. 1998; and 2000; et al. 2001; et al. 2002) and of recognition biomolecules et al. 2000) could to a much field of applications. in biomolecule immobilization technology, such as site which for to be on the protein surface and hence upon immobilization et al. 1993; et al. 1994; Lu et al. 1995; et al. and the of interactions could improve the sensitivity of TIRF (Conrad et al. of the surface would be useful for applications as an to (Duveneck et al. 1997; et al. 1998; Budach et al. 1999). In the of the arrays has been demonstrated using both DNA and 1999; and 2000) and and 2000; and Ligler et al. 2002). The methods used to the spots in high-density arrays are currently to surface of the capture biomolecules and Ligler for use in TIRF. The use of planar waveguide TIRF for the detection of multiple analytes has been demonstrated as well as the ability to the instrumentation it a device for real-time field The of distinct sensing regions is these systems to advantage over sensing in terms of environmental and clinical The of signal transduction and relative to and are two advantages of planar waveguide TIRF biosensors. In the for biohazard monitoring using planar waveguide TIRF. The would to for useful This was by from and the of Naval The are of the and not of the the of or the