Fluorescence-based array biosensors for detectionof biohazards

K.E. Sapsford1, Y.S. Shubin2, J.B. Delehanty3, J.P. Golden3, C.R. Taitt3,L.C. Shriver-Lake3 and F.S. Ligler3

1Center 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. SUMMARY

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 wave-

guides 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.

2. INTRODUCTION

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 pretreat-

ment.

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 tech-

niques (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),

1. Summary, 47

2. Introduction, 47

3. Total internal reflection fluorescence transduction, 48

4. The molecular recognition element, 49

5. Immobilization of the biomolecule onto the wave-

guide, 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 biosen-

sors, 52

9. The future, 54

10. Acknowledgements, 55

11. References, 55

Correspondence to: F.S. Ligler, Center for Bio/Molecular Science and Engineering,

Naval Research Laboratory, Washington DC 20375-5348, USA

(e-mail: fligler@cbmse.nrl.navy.mil).

ª 2004 The Society for Applied Microbiology

Journal of Applied Microbiology 2004, 96, 47–58 doi:10.1046/j.1365-2672.2003.02115.x

although the field of electrochemical micro-array transduc-

tion 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 intro-

duction 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.

3. TIRF transduction

Fluorescence, absorbance, bioluminescence, chemilumines-

cence 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. Tech-

niques that can be grouped under the principle of reflectance

include: attenuated total reflectance which monitors altera-

tions 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 instrumenta-

tion. 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 require-

ments of the optical components which could reduce the

robustness of a device should it be intended for field

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)

Bacteria Francisella tularensis, Brucella abortus,

Bacillus anthracis (anthrax), Listeria monocytogenes,

Campylobacter jejuni, Yersinia pestis (F1 is an antigen),

Escherichia coli, Salmonella, Shigella spp.

Virus Hepatitis, rotavirus, Norwalk virus group

Detector 2Light source

n1

Filter

r

Detector

n1θ

n2

Detector 1

Filter

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)

48 K.E. SAPSFORD ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

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), photodi-

odes (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,

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 devel-

opment, 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 biomoleculeonto the waveguide

One important prerequisite for all immobilization tech-

niques 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

BIOSENSORS FOR DETECTION OF BIOHAZARDS 49

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

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 develop-

ing 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 wave-

guide 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 lm 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 lm 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 biomol-

ecules onto a Ta2O5 waveguide using (3-glycidoxypropyl)

trimethoxysilane. Noncontact printing has also been suc-

cessfully 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.

7. TIRF array biosensors: state of the art

Much of the initial investigation into the use of planar

waveguides in TIRF biosensors has centered on both

instrumentation development and reproducible immobiliza-

tion of the capture biomolecules. However, once the

hardware and biochemistry are optimized, the question of

application becomes the driving force behind further

development.

50 K.E. SAPSFORD ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

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 sequen-

tially and the slide imaged. Later the use of a PDMS flow

cell for the patterning of capture biomolecules was devel-

oped 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 detec-

tion 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

DD-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

Flow cells

(a) (b)

Planar waveguidesImmobilized capture

biomolecules

Fig. 2 The patterning of capture biomole-

cules 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

Table 2 Summary of research groups currently involved in multi-capture biomolecule patterning studied using TIRF

Research group Analytes measured

Single- or

multi-analyte detection References

Ligler et al. Ricin, Yersinia pestis F1, staphylococcal enterotoxin

B, ovalbumin, mouse and human IgG, DD-dimer,

Bacillus globigii (anthrax simulant), MS2 bacteriophage

(virus simulant), cholera toxin, botulinum toxoids

A and B, B. anthracis, F. tularensis, B. abortus

Single and multi Wadkins et al. 1997, 1998; Ligler et al.

1998; Feldstein et al. 1999;

Golden et al. 1999; Rowe

et al. 1999a,b; Rowe-Taitt et al.

2000a,–c; Shriver-Lake et al. 2003;

Taitt et al. 2002

Reichert et al. Four different human IgG subclasses Multi Silzel et al. 1998

Plowman et al. Various IgGs, creatine kinase MB, cardiac troponin

I, myoglobin

Single and multi Plowman et al. 1999

Abel et al. 16-Mer and 22-mer oligonucleotides Single Budach et al. 1999

Zeller et al. Mouse and rabbit IgG Single Zeller et al. 2000

BIOSENSORS FOR DETECTION OF BIOHAZARDS 51

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

into commercial applications. The impact of potential

environmental interferents has also been addressed by

Rowe-Taitt et al. (2000c). Analyte samples of Bacillusanthracis, 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

0Æ5-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 pretreat-

ment 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 mMM sodium phosphate/1Æ5 MM NaCl/0Æ5% 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 DD-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 0Æ5 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 anti-

bodies 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.

8. Miniaturization and automation of TIRF arraybiosensors

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 wave-

guide 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-

52 K.E. SAPSFORD ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

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.

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.

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 possi-

bility 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 collec-

tion capability of uncooled low light cameras was sufficient,

the backgrounds were too variable for high sensitivity

measurements. Recent advances in CCD technology made it

Flow cell channels

Patterned

Antibodies

ReflectivecladdingWaveguide

(a) Topview (b) Side view

Capture

Fig. 3 (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)

BIOSENSORS FOR DETECTION OF BIOHAZARDS 53

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

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, simpli-

fying 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 5Æ5 kg.

9. The future

This review has discussed the use of optical array biosen-

sors, 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.

(a) (b)

Fig. 4 (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)

Fig. 5 The latest version of the portable array biosensor

54 K.E. SAPSFORD ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

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 attach-

ment 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; Deleh-

anty 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 concentra-

tions 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 monit-

oring. The speed of signal transduction and relative

resistance to matrix effects and other interfering influences

are two key advantages of planar waveguide TIRF biosen-

sors. In conclusion, the future looks bright for biohazard

monitoring using planar waveguide TIRF.

10. ACKNOWLEDGEMENTS

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.

11. REFERENCES

Altin, J.G., White, F.A.J. and Easton, C.J. (2001) Synthesis of the

chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA)

and its use with the IAsys biosensor to study receptor-ligand

interactions on model membranes. Biochimica et Biophysica Acta

1513, 131–148.

Arenkov, P., Kukhtin, A., Gemmell, A., Voloshchuk, S., Chupeeva, V.

and Mirzabekov, A. (2000) Protein microchips: use for immuno-

assay and enzymatic reactions. Analytical Biochemistry 278, 123–

131.

Asanov, A.N., Wilson, W.W. and Oldham, P.B. (1998) Regenerable

biosensor platform: a total internal reflection fluorescence cell with

electrochemical control. Analytical Chemistry 70, 1156–1563.

Askari, M., Alarie, J.P., Moreno-Bondi, M. and Vo-Dinh, T. (2001)

Application of an antibody biochip for p53 detection and cancer

diagnosis. Biotechnology Progress 17, 543–552.

Bernard, A., Michel, B. and Delamarche, E. (2001) Micromosaic

immunoassays. Analytical Chemistry 73, 8–12.

Bhatia, S.K., Hickman, J.J. and Ligler, F.S. (1992) New approach to

producing patterned biomolecular assemblies. Journal of the Ameri-

can Chemical Society 114, 4432–4433.

Bhatia, S.K., Teixeira, J.L., Anderson, M., Shriver-Lake, L.C.,

Calvert, J.M., Georger, J.H., Hickman, J.J., Dulcey, C.S. et al.

(1993) Fabrication of surfaces resistant to protein adsorption and

application to two-dimensional protein patterning. Analytical Bio-

chemistry 208, 197–205.

Birkert, O., Haake, H.-M., Schutz, A., Mack, J., Brecht, A., Jung, G.

and Gauglitz, G. (2000) A streptavidin surface on planar glass

substrates for the detection of biomolecular interaction. Analytical

Biochemistry 282, 200–208.

Blawas, A.S. and Reichert, W.M. (1998) Protein patterning. Bioma-

terials 19, 595–609.

Braguglia, C.M. (1998) Biosensors: an outline of general principles and

application. Chemical and Biochemical Engineering Quarterly 12, 183–

190.

Brecht, A., Klotz, A., Barzen, C., Gauglitz, G., Harris, R.D., Quigley,

G.R., Wilkinson, J.S., Sztajnbok, P. et al. (1998) Optical immuno-

probe development for multiresidue monitoring in water. Analytica

Chimica Acta 362, 69–79.

Brown, P.O. and Botstein, D. (1999) Exploring the new world of the

genome with DNA microarrays. Nature Genetics Supplement 21,

33–37.

Budach, W., Abel, A.P., Bruno, A.P. and Neuschafer, D. (1999) Planar

waveguides as high performance sensing platforms for fluorescence-

based multiplexed oligonucleotide hybridization assays. Analytical

Chemistry 71, 3347–3355.

Campbell, D.P. and McCloskey, C.J. (2002) Interferometric biosen-

sors. In Optical Biosensors: Present and Future ed. Ligler, F.S. and

Rowe Taitt, C.A. pp. 277–304. Amsterdam, The Netherlands:

Elsevier Science.

Campbell, D.P., Hartman, N.F., Moore, J.L., Suggs, J.V. and Cobb,

J.M. (1998) Reversible integrated optic evanescent field biosensor

using chemical amplification for added sensitivity. Proceedings of

SPIE 3253, 20–26.

Campbell, D.P., Moore, J.L., Cobb, J.M., Hartman, N.F., Schneider,

B.H. and Venugopal, M.G. (1999) Optical system-on-a-chip for

chemical and biochemical sensing: the chemistry. Proceedings of

SPIE 3540, 153–163.

Chittur, K.K. (1998) FTIR/ATR for protein adsorption to biomate-

rials surfaces. Biomaterials 19, 357–369.

Conrad, D.W., Davis, A.V., Golightley, S.K., Bart, J.C. and Ligler,

F.S. (1997) Photoactivatable silanes for the site specific immobiliza-

tion of antibodies. Proceedings of SPIE 2978, 12–21.

BIOSENSORS FOR DETECTION OF BIOHAZARDS 55

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

Conrad, D.W., Golightley, S.K. and Bart, J.C. (1998) Photoactivatable

o-nitrobenzyl polyethylene glycol – silane for the production of

patterned biomolecular arrays. US Patent 5,773,308.

Delehanty, J.B. and Ligler, F.S. (2002) A microarray immunoassay for

simultaneous detection of proteins and bacteria. Analytical Chemistry

74, 5681–5687.

Delehanty, J.B. and Ligler, F.S. (2003) A method for printing

functional protein microarrays. BioTechniques 34, 380.

Dodson, J.M., Feldstein, M.J., Leatzow, D.M., Flack, L.K., Golden,

J.P. and Ligler, F.S. (2001) Fluidics cube for biosensor miniatur-

ization. Analytical Chemistry 73, 3776–3780.

Duveneck, G.L., Neuschafer, D. and Ehrat, M. (1995) Process for

detecting evanescently excited luminescence. International Patent

Go1N 21/77, 21/64.

Duveneck, G.L., Pawlak, M., Neuschafer, D., Bar, E., Budach, W.,

Pieles U. and Ehrat, M. (1997) Novel bioaffinity sensors for trace

analysis based on luminescence excitation by planar waveguides.

Sensors and Actuators B 38–39, 88–95.

Edwards, J.G., Campbell, D.P. and Moore, J.L. (1999) Integrated optic

chemical sensor for environmental monitoring. Proceedings of SPIE

3534, 614–619.

Eggins, B.R. (1998) Biosensors: an Introduction. New York: John Wiley

& Sons.

Feldstein, M.J. (2002) US Patent Application No. 09/917,649, filed

08/07/01.

Feldstein, M.J., Golden, J.P., Rowe, C.A., MacCraith, B.D. and

Ligler, F.S. (1999) Array biosensor: optical and fluidics systems.

Journal of Biomedical Microdevices 1, 139–153.

Feldstein, M.J., Golden, J.P., Ligler, F.S. and Rowe, C.A. (2000)

Reflectivity coated optical waveguide and fluidics integration. US

Patent 6, 192, 168.

Fisher, M.I. and Tjarnhage, T. (2000) Structure and activity of lipid

membrane biosensor surfaces studied with atomic force microscopy

and resonant mirror. Biosensors & Bioelectronics 15, 463–471.

Golden, J.P. (1998) Chemical sensor using two-dimensional lens array.

US Patent 5,827,748.

Golden, J.P. and Ligler, F.S. (2002) A comparison of imaging methods

for use in an array biosensor. Biosensors & Bioelectronics 17, 719–725.

Golden, J.P., Rowe, C.A., Feldstein, M.J. and Ligler, F.S. (1999)

Array biosensor-recent developments. Proceedings of SPIE 3602,

132–139.

Gray, S.A., Kusel, J.K., Shaffer, K.M., Shubin, Y.S., Stenger, D.A.

and Pancrazio, J.J. (2001) Design and demonstration of an automated

cell-based biosensor. Biosensors & Bioelectroics 16, 535–542.

Guschin, D., Yershov, G., Zaslavsky, A., Gemmell, A., Shick, V.,

Proudnikov, D., Arenkov, P. and Mirzabekov, A. (1997) Manual

manufacturing of oligonucleotide, DNA and protein microchips.

Analytical Biochemistry 250, 203–211.

Hall, E.A.H. (1990) Biosensors. Milton Keynes, UK: Open University

Press.

Herron, J.N., Caldwell, K.D., Christensen, D.A., Dyer, S., Hlady, V.,

Huang, P., Janatova, V., Wang, H.-K. et al. (1993) Fluorescent

immunosensors using planar waveguides. Proceedings of SPIE 1885,

28–39.

Herron, J.N., Christensen, D.A., Caldwell, K.D., Janatova, V., Huang,

S.-C. and Wang, H.-K. (1996) Waveguide immunosensor with coating

chemistry providing enhanced sensitivity. US Patent 5,512,492.

Herron, J.N., Christensen, D.A., Wang, H.-K. and Caldwell,

K.D.(1997) Apparatus and methods for multi-analyte homogeneous

fluoro-immunoassays. US Patent 5,677,196.

Hofmann, O., Voirin, G., Niedermann, P. and Manz, A. (2002) Three-

dimensional microfluidic confinement for efficient sample delivery to

biosensor surfaces. Application to immunoassays on planar optical

waveguides. Analytical Chemistry 74, 5243–5250.

Homola, J., Dostalek, J. and Ctyroky, J. (2001a) Novel approach to

surface plasmon resonance multichannel sensing. Proceedings of

SPIE 4416, 86–89.

Homola, J., Lu, H.B., Nenninger, G.G., Dostalek, J. and Yee, S.S.

(2001b) A novel multichannel surface plasmon resonance biosensor.

Sensors and Actuators B 76, 403–410.

Homola, J., Yee, S.S. and Myszka, D. (2002) Surface plasmon

resonance biosensors. In Optical Biosensors: Present and Future ed.

Ligler, F.S. and Rowe Taitt, C.A. pp. 207–252. Amsterdam, The

Netherlands: Elsevier Science.

Huang, S.-C., Caldwell, K.D., Lin, J.-N., Wang, H.-K. and Herron,

J.N. (1996) Site-specific immobilization of monoclonal antibodies

using spacer-mediated antibody attachment. Langmuir 12, 4292–

4298.

Iqbal, S.S., Mayo, M.W., Bruno, J.G., Bronk, B.V., Batt, C.A. and

Chambers, J.P. (2000) A review of molecular recognition technol-

ogies for detection of biological threat agents. Biosensors &

Bioelectronics 15, 549–578.

Klotz, A., Brecht, A., Barzen, C., Gauglitz, G., Harris, R.D., Quigley,

G.R., Wilkinson, J.S. and Abuknesha, R.A. (1998) Immunofluores-

cence sensor for water analysis. Sensors and Actuators B 51, 181–187.

Krantz-Rulcker, C., Stenberg, M., Winquist, F. and Lundstrom, I.

(2001) Electronic tongues for environmental monitoring based on

sensor arrays and pattern recognition: a review. Analytica Chimica

Acta 426, 217–226.

Kukla, A.L., Kanjuk, N.I., Starodub, N.F. and Shirshov, Y.M. (1999)

Multienzyme electrochemical sensor array for determination of

heavy metal ions. Sensors and Actuators B 57, 213–218.

Lang, S., Xu, J., Stuart, F., Thomas, R.M., Vrijbloed, J.W. and

Robinson, J.A. (2000) Analysis of antibody A6 binding to the

extracellular interferon gamma receptor alpha-chain by alanine-

scanning mutagenesis and random mutagenesis with phage display.

Biochemistry 39, 15674–15685.

Lee, H.J., Goodrich, T.T. and Corn, R.M. (2001) Surface plasmon

resonance imaging measurements of 1-D and 2-D DNA microarrays

created from microfluidics channels on gold thin films. Analytical

Chemistry 73, 5525–5531.

Li, J. and Li, Y. (2000) A highly sensitive and selective catalytic DNA

biosensor for lead ions. Journal of the American Chemical Society 122,

10466–10467.

Ligler, F.S., Conrad, D.W., Golden, J.P., Feldstein, M.J., MacCraith,

B.D., Balderson, S.D., Czarnaski, J. and Rowe, C.A. (1998) Array

biosensor for multi-analyte sensing. Proceedings of SPIE 3258,

50–55.

Ligler, F.S., Golden, J.P., Rowe-Taitt, C.A. and Dodson, J.P. (2001)

Array biosensor for simultaneous detection of multiple analytes.

Proceedings of SPIE 4252, 32–36.

Liss, M., Peterson, B., Wolf, H. and Prohaska, E. (2002) An aptamer-

based quartz crystal protein biosensor. Analytical Chemistry 74,

4488–4495.

56 K.E. SAPSFORD ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

Liu, X.H., Wang, H.K., Herron, J.N. and Prestwich, G.D. (2000)

Photopatterning of antibodies on biosensors. Bioconjugate Chemistry

11, 755–761.

Lockhart, D.J. and Winzeler, E.A. (2000) Genomics, gene expression

and DNA arrays. Nature 405, 827–836.

Lu, B., Lu, C. and Wei, Y. (1992) A planar quartz waveguide

immunosensor based on the total internal reflection fluorescence

principle. Analytical Letters 25, 1–10.

Lu, E.J., Xie, J., Lu, C., Wu, C. and Wei, Y. (1995) Oriented

immobilization of Fab’ fragments on silica surfaces. Analytical

Chemistry 67, 83–87.

Lu, H.B., Campbell, C.T., Nenninger, G.G., Yee, S.S. and Ratner,

B.D. (2001) Protein contact printing for a surface plasmon resonance

biosensor with on-chip referencing. Sensors and Actuators B 74,

91–99.

Lundgren, J.S., Watkins, N., Racz, D. and Ligler, F.S. (2000) A liquid

crystal pixel array for signal discrimination in array biosensors.

Biosensors & Bioelectronics 15, 417–421.

MacBeath, G. and Schreiber, S.L. (2000) Printing proteins as

microarrays for high-throughput function determination. Science

289, 1760–1763.

McLean, M.A., Stayton, P.S. and Sligar, S.G. (1993) Engineering

protein orientation at surfaces to control macromolecular recognition

events. Analytical Chemistry 65, 2676–2678.

Mauro, J.M., Cao, L.K., Kondracki, L.M., Walz, S.E. and Campbell,

J.R. (2000) Fiber-optic fluorometric sensing of polymerase chain-

reaction amplified DNA using an immobilized DNA capture

protein. Analytical Biochemistry 235, 61–72.

Nelson, B.P., Grimsrud, T.E., Liles, M.R., Goodman, M. and Corn,

R.M. (2001) Surface plasmon resonance imaging measurements of

DNA and RNA hybridization adsorption onto DNA microarrays.

Analytical Chemistry 73, 1–7.

O’Brien, M.J., Perez-Luna, V.H., Brueck, S.R.J. and Lopez, G.P.

(2001) A surface plasmon resonance array biosensor based on

spectroscopic imaging. Biosensors & Bioelectronics 16, 97–108.

Pancrazio, J. (2001) Cell-based biosensors – Preface. Biosensors &

Bioelectronics, Special issue. 16, 427–428.

Pawlak, M., Grell, E., Schick, E., Anselmetti, D. and Ehrat, M.

(1998) Functional immobilization of biomembrane fragments on

planar waveguides for the investigation of site-directed ligand

binding by surface confined fluorescence. Faraday Discussions 111,

273–288.

Pearson, J.E., Gill, A. and Vadgama, P. (2000) Analytical aspects of

biosensors. Annals of Clinical Biochemistry 37, 119–145.

Plowman, T.E., Saavedra, S.S. and Reichert, W.H. (1998) Planar

integrated optical methods for examining thin-films and their surface

adlayers. Biomaterials 19, 341–355.

Plowman, T.E., Durstchi, J.D., Wang, H.K., Christensen, D.A.,

Herron, J.N. and Reichert, W.M. (1999) Multiple-analyte fluoro-

immunoassay using an intergrated optical waveguide sensor. Ana-

lytical Chemistry 71, 4344–4352.

Potyrailo, R.A., Conrad, R.C., Ellington, A.D. and Hieftje, G.M.

(1998) Adapting selected nucleic acid ligands (aptamers) to biosen-

sors. Analytical Chemistry 70, 3419–3425.

Puu, G. (2001) An approach for analysis of protein toxins based on

thin-films of lipid mixtures in an optical biosensor. Analytical

Chemistry 73, 72–79.

Rabbany, S.Y., Donner, B.L. and Ligler, F.S. (1994) Optical

immunosensors. Critical Reviews in Biomedical Engineering 22, 307–

346.

Rogers, K.R., Valdes, J.J. and Eldefrawi, M.E. (1989) Acetylcholine-

receptor fiber optic evanescent fluorosensor. Analytical Biochemistry

182, 353–359.

Rowe, C.A., Scruggs, S.B., Feldstein, M.J., Golden, J.P. and Ligler,

F.S. (1999a) An array immunosensor for simultaneous detection of

clinical analytes. Analytical Chemistry 71, 433–439.

Rowe, C.A., Tender, L.M., Feldstein, M.J., Golden, J.P., Scruggs,

S.B., MacCraith, B.D., Cras, J.J. and Ligler, F.S. (1999b) Array

biosensor for simultaneous identification of bacterial, viral and

protein analytes. Analytical Chemistry 71, 3846–3852.

Rowe-Taitt, C.A., Cras, J.J., Patterson, C.H., Golden, J.P. and Ligler,

F.S. (2000a) A ganglioside-based assay for cholera toxin using an

array biosensor. Analytical Biochemistry 281, 123–133.

Rowe-Taitt, C.A., Golden, J.P., Feldstein, M.J., Cras, J.J., Hoffman,

K.E. and Ligler, F.S. (2000b) Array biosensor for detection of

biohazards. Biosensors & Bioelectronics 14, 785–794.

Rowe-Taitt, C.A., Hazzard, J.W., Hoffman, K.E., Cras, J.J., Golden,

J.P. and Ligler, F.S. (2000c) Simultaneous detection of six

biohazardous agents using a planar waveguide array biosensor.

Biosensors & Bioelectronics 15, 579–595.

Sapsford, K.E., Liron, Z., Shubin, Y.S. and Ligler, F.S. (2001)

Kinetics of antigen binding to arrays of antibodies in different sized

spots. Analytical Chemistry 73, 5518–5524.

Sapsford, K.E., Rowe-Taitt, C.A. and Ligler, F.S.(2002a) Planar

waveguides for fluorescence biosensors. In Optical Biosensors: Present

and Future ed. Ligler, F.S. and Rowe-Taitt, C.A. pp. 95–122.

Amsterdam, The Netherlands: Elsevier Science.

Sapsford, K.E., Charles, P.T., Patterson, C.H. Jr and Ligler, F.S.

(2002b) Demonstration of four immunoassay formats using the array

biosensor. Analytical Chemistry 74, 1061–1068.

Schipper, E.F., Brugman, A.M., Dominguez, C., Lechuga, L.M.,

Kooyman, R.P.H. and Greve, J. (1997) The realization of an

integrated Mach-Zehnder waveguide immunosensor in silicon

technology. Sensors and Actuators B 40, 147–153.

Schipper, E.F., Rauchalles S.R., Kooyman, P.H., Hock, B. and Greve,

J. (1998) The waveguide Mach-Zehnder interferometer as atrazine

sensor. Analytical Chemistry 70, 1192–1197.

Schmid, E.L., Keller, T.A., Dienes, Z. and Vogel, H. (1997) Reversible

oriented surface immobilization of functional proteins on oxide

surfaces. Analytical Chemistry 69, 1979–1985.

Schmid, E.L., Tairi, A.-P., Hovius, R. and Vogel, H. (1998) Screening

ligands for membrane protein receptors by total internal reflection

fluorescence: the S-HT3 serotonin receptor. Analytical Chemistry 70,

1331–1338.

Schneider, B.H., Edwards, J.G. and Hartman, N.F. (1997) Hartman

interferometer: a versatile integrated optic sensor for label-free, real-

time quantification of nucleic acids, proteins, and pathogens. Clinical

Chemistry 43, 1757–1763.

Schneider, B.H., Dickinson, E.L., Vach, M.D., Hoijer, J.V. and

Howard, L.V. (2000) Optical chip immunoassay for hCG in human

whole blood. Biosensors & Bioelectronics 15, 597–604.

Schuderer, J., Akkoyun, A., Brandenburg, A., Bilitewski, U. and

Wagner, E. (2000) Development of a multichannel fluorescence

affinity sensor system. Analytical Chemistry 72, 3942–3948.

BIOSENSORS FOR DETECTION OF BIOHAZARDS 57

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x

Schult, K., Katerkamp, A., Trau, D., Grawe, F., Cammann, K. and

Meusel, M. (1999) Disposable optical sensor chip for medical

diagnostics: new ways in bioanalysis. Analytical Chemistry 71, 5430–

5435.

Schwarz, A., Rossier, J.S., Roulet, Mermod, E.N., Roberts, M.A. and

Girault, H.H. (1998) Micropatterning of biomolecules on polymer

substrates. Langmuir 14, 5526–5531.

Shriver-Lake, L.C., Shubin, Y.S. and Ligler, F.S. (2003) Detection of

staphylococcal enterotoxin B in spiked food samples. Journal of Food

Protection 66, 1851–1856.

Silzel, J.W., Cercek, B., Dodson, C., Tsay, T. and Obremski, R.J.

(1998) Mass-sensing, multianalyte microarray immunoassay with

image detection. Clinical Chemistry 44, 2036–2043.

Taitt, C.A., Anderson, G.P., Lingerfelt, B.M., Feldstein, M.J. and

Ligler, F.S. (2002) Nine analyte detection using an array-based

biosensor. Analytical Chemistry 74, 6114–6125.

Vigmond, S.J., Iwakura, M., Mizutani, F. and Katsura, T. (1994) Site-

specific immobilization of molecularly engineered dihydrofolate-

reductase to gold-surfaces. Langmuir 10, 2860–2862.

Vo-Dinh, T., Alarie, J.P., Isola, N., Landis, D., Wintenberg, A.L. and

Ericson, M.N. (1999) DNA biochip using a phototransistor

intergrated circuit. Analytical Chemistry 71, 358–363.

Wadkins, R.M., Golden, J.P. and Ligler, F.S. (1997) Patterned planar

array immunosensor for multianalyte detection. Journal of Biomedical

Optics 2, 74–79.

Wadkins, R.M., Golden, J.P., Pritsiolas, L.M. and Ligler, F.S. (1998)

Detection of multiple toxic agents using a planar array immunosen-

sor. Biosensors & Bioelectronics 13, 407–415.

Wang, J., Rivas, G., Cai, X., Palecek, E., Nielsen, P., Shiraishi, H.,

Dontha, N., Luo, D. et al. (1997) DNA electrochemical biosensors

for environmental monitoring: a review. Analytica Chimica Acta 347,

1–8.

Wang, L., Carrasco, C., Kumar, A., Stephens, C.E., Bailly, C., Boykin,

D.W. and Wilson, W.D. (2001) Evaluation of the influence of

compound structure on stacked-dimer formation in the DNA minor

groove. Biochemistry 40, 2511–2521.

Wegner, G.L., Lee, H.J. and Corn, R.M. (2002) Characterization and

optimization of peptide arrays for the study of epitope-antibody

interactions using surface plasmon resonance imaging. Analytical

Chemistry 74, 5161–5168.

Wilson, G.S. and Hu, Y. (2000) Enzyme based biosensors for in vivo

measurements. Chemical Reviews 100, 2693–2704.

Wu, T.-Z. (1999) A piezoelectric biosensor as an olfactory receptor

for odour detection: electronic nose. Biosensors & Bioelectronics 14,

9–18.

Yang, J.M., Bell, J., Huang, Y., Tirado, M., Thomas, D., Forster, A.

H., Haigis, R.W., Swanson, P.D. et al. (2002) An integrated, stacked

microlaboratory for biological agent detection with DNA and

immunoassays. Biosensors & Bioelectronics 17, 605–618.

Young, S.J., Hart, J.P., Dowman, A.A. and Cowell, D.C. (2001)

The non-specific inhibition of enzymes by environmental pollut-

ants: a study of a model system towards the development of

electrochemical biosensor arrays. Biosensors & Bioelectronics 16,

887–894.

Zeller, P.N., Voirin, G. and Kunz, R.E. (2000) Single-pad scheme for

integrated optical fluorescence sensing. Biosensors & Bioelectronics

15, 591–595.

Zhang, S., Zhao, H. and John, R. (2000) Development of a generic

microelectrode array biosensing system. Analytica Chimica Acta 421,

175–188.

58 K.E. SAPSFORD ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 47–58, doi:10.1046/j.1365-2672.2003.02115.x