Journal of Applied Microbiology 2004, 96, 47–58
doi:10.1046/j.1365-2672.2003.02115.x
Fluorescence-based array biosensors for detection of biohazards K.E. Sapsford1, Y.S. Shubin2, J.B. Delehanty3, J.P. Golden3, C.R. Taitt3, L.C. Shriver-Lake3 and F.S. Ligler3 1
Center for Bioresource Development, George Mason University, Fairfax, VA , USA, 2Geo-Centers, Inc., Lanham, MD, USA, and 3Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USA Presented at the Lab on a Chip Conference 8–9 January 2003
1. 2. 3. 4. 5.
Summary, 47 Introduction, 47 Total internal reflection fluorescence transduction, 48 The molecular recognition element, 49 Immobilization of the biomolecule onto the waveguide, 49 6. Creation of low-density biomolecular arrays, 50
7. TIRF array biosensors: state of the art, 50 8. Miniaturization and automation of TIRF array biosensors, 52 9. The future, 54 10. Acknowledgements, 55 11. References, 55
1. SUMMARY
2. INTRODUCTION
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 5Æ5 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),
Correspondence to: F.S. Ligler, Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington DC 20375-5348, USA (e-mail:
[email protected]).
ª 2004 The Society for Applied Microbiology
48 K . E . S A P S F O R D ET AL.
Table 1 Potential biohazards of interest. Examples were taken from the US Food and Drug Administration (FDA) website at http:// www.cfsan.fda.gov/mow/intro.html Type
Species
Protein
Cholera toxin, Staphylococcus enterotoxin (SEB), botulinum toxin, Ricinus communis agglutinin II (Ricin) Francisella tularensis, Brucella abortus, Bacillus anthracis (anthrax), Listeria monocytogenes, Campylobacter jejuni, Yersinia pestis (F1 is an antigen), Escherichia coli, Salmonella, Shigella spp. Hepatitis, rotavirus, Norwalk virus group
Bacteria
Virus
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, multianalyte array biosensor for real-time biohazard detection. 3. TIRF transduction 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). 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
n2
θ
n11 Filter Filter r
Detector 1 Light source
Detector 2
Fig. 1 The basic experimental arrangement of a system based on the principle of total internal reflection fluorescence (TIRF) (adapted from Sapsford et al. 2002a)
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x
BIOSENSORS FOR DETECTION OF BIOHAZARDS
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). 4. The molecular recognition element 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,
49
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. 5. Immobilization of the biomolecule onto the waveguide 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
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x
50 K . E . S A P S F O R D ET AL.
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). 6. Creation of low-density biomolecular arrays 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 (
