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Analytica Chimica Acta 559 (2006) 137–151 Review Biosensor technology for detecting biological warfare agents: Recent
Analytica Chimica Acta 559 (2006) 137–151 Review Biosensor technology for detecting biological warfare agents: Recent

Analytica Chimica Acta 559 (2006) 137–151

Analytica Chimica Acta 559 (2006) 137–151 Review Biosensor technology for detecting biological warfare agents: Recent

Review

Biosensor technology for detecting biological warfare agents:

Recent progress and future trends

J. Justin Gooding

School of Chemistry, The University of New South Wales, Sydney 2052, Australia

Received 2 August 2005; received in revised form 30 November 2005; accepted 5 December 2005 Available online 24 January 2006

Abstract

This review paper outlines the important issues with regards to the development of biosensors for the monitoring of biological warfare agents (BWAs) starting with the basic components of biosensors and the features of BWAs, which are compatible with detection using biological recognition molecules. The advantages and limitations of biosensors are discussed followed by the current state of the art in biosensors for detecting BWAs. Finally the developments required to enable biosensors to become more effective at providing early warning of possible biological attack and advances towards these developments are covered. © 2005 Elsevier B.V. All rights reserved.

Keywords: Biosensors; Biological agents; Affinity sensors; Bioweapon detection; Detect-to-protect

Contents

1. Introduction

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137

2. Biosensors for

detecting biological agents

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139

2.1. Nucleic

acid-based biosensing technologies—detect-to-treat biosensors

 

139

2.2. Detection using surface features—detect-to-protect biosensors

 

141

3. Cutting edge of biosensor technology which may be applicable

to

bioweapons

detection .

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146

Air sampling

3.1. .

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3.2. Reducing the

detection limit and providing a

more rapid

response .

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3.3. Decreasing the response

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Acknowledgements

References

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4. Conclusions

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time—the needle in . . .

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148

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1. Introduction

With the increased threat of terrorist attacks, an attack using a BWA is a conceivable event. We have already seen evidence of the destructiveness of such attacks, and the associated fear they create, through episodes such as the Sarin gas and anthrax attacks by the Aum Shinrikyo cult in Tokyo in 1995, the use of Salmonella in Oregon restaurants in 1984 by the Rajneeshee cult and the anthrax letter attacks soon after September 11th.

Tel.: +61 2 9385 5384; fax: +61 2 9385 6141. E-mail address: Justin.gooding@unsw.edu.au.

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.aca.2005.12.020

One of the problems with biological attacks is actually deter- mining whether an attack has occurred. The difficulty arises because often the initial symptoms after infection from BWAs are difficult to distinguish from symptoms from infections from more benign biological agents. The solution to this detection problem is to employ molecular techniques, which can identify chemical markers from known biological agents. Two classes of such devices can be envisaged. These classes of devices can be referred to as “detect-to-treat”, thus allowing early identifica- tion of an infection, and “detect-to-protect” to provide a warning that a site is contaminated by BWAs to prevent infection [1]. The criteria for such analytical methods are quite different. A detect-

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J.J. Gooding / Analytica Chimica Acta 559 (2006) 137–151

to-treat system must be able to identify a BWA, from a biological sample, within a few hours of infection. Hence detect-to-treat biosensors are compatible with an analytical laboratory using personnel trained to perform the analysis. A detect-to-protect system must be able to provide a warning within a couple of minutes from, most frequently, an airborne sample without user intervention. Analytical devices, which could satisfy both sets of criteria, and are possibly the only answer to the detect-to-protect criteria, are biosensors. A biosensor comprises a biorecognition molecule immo- bilised over a signal transducer to give a reagentless analyti- cal device. The biorecognition molecule, such as an enzyme, antibody, sequence of DNA, peptide or even a microorganism, provides the biosensor with its selectivity for the target analyte so that the molecule of interest can be picked out by the biosensor from a matrix of many other molecules. The signal transducer determines the extent of the biorecognition event and converts it into an electronic signal, which can be outputted to the end user. Common transducers include amperometric electrodes, optical waveguides or mass sensitive piezoelectric crystals. Biosensors can be subdivided into two classes based on the type of biorecognition molecule. Catalytic biosensors employ enzymes and microorganisms as the biorecognition molecule which catalyses a reaction involving the analyte to give a product. Common analytes for catalytic biosensors are small organic molecules like glucose. Although some BWAs are small molecules, such as aflatoxin, most frequently they are large macromolecules and microorganisms, which cannot usu- ally be detected using catalytic biorecognition molecules. The other category of biosensors is affinity biosensors. Biorecog- nition molecules commonly used in affinity biosensors include antibodies, DNA, peptides and lectins. Affinity biosensors are characterised by a binding event between the biorecognition molecule and the analyte (the affinity reaction) often with no further reaction occurring. Hence the challenge then becomes transducing the biorecognition event. As this class of biosen- sor is compatible with the detection of virtually all biological agents it is this challenge that faces researchers attempting to develop portable devices for detecting toxins, microbes and viruses. Transduction of affinity biosensors has been achieved using labelled species and using label free approaches. If transduction is achieved using labelled species the principles are very simi- lar to immunoassay with the amount of analyte detected being inferred from the amount of label which binds to the interface. The most common transducers for detecting labelled species are optical, where an optically active label is detected [2–4], or elec- trochemical, where the label is electroactive [3,5]. Label-free methods most frequently involve evanescent wave based optical methods [6–8] or using mass sensitive acoustic wave devices [9] which monitor molecules binding to, or desorbing from, a trans- ducer surface. Any change in species adsorbed to this surface will give a response. In the case of both label and label free meth- ods one of the key factors, which limits biosensor performance is non-specific binding. The problem of non-specific binding highlights the importance of interfacial design in a biosensor

[10,11].

This review will outline the developments in biosensors for detecting biological warfare agents before discussing what prob- lems require solutions for biosensors for detecting BWAs to become common. The emphasis will be on the development towards handheld analytical devices, which provide warning of BWA attacks rather than laboratory instruments where impres- sive advances have been made with automated PCR and mass spectrometry instruments [12]. The analytes, anthrax, tularemia, botulinum toxin, plague (aerosol version only), smallpox and hemorrhagic fever will be treated as indicative analytes of a wider class of potential bioterrorist agents. They are all cat- egory A agents according to the Center for Disease Control and Prevention (CDC) in the United States [13]. They cover the main types of biological agents from Gram-positive bacte- ria, which form spores (Bacillus anthracis, Anthrax; Yersinia pestis, Pneumonic plague), gram-negative (Francisella tularen- sis, tularemia), toxins derived from bacterial species (botulinum toxin from Clostridium botulinum) and viruses (Smallpox). For most of these analytes the current preferred methods of detection are classical taxonomic tests. In the case of the microorganism derived biological agents, microbiological tests where different growth media, different shapes and responsiveness to different stains are used to identify the species [14]. Such tests typically take about 48 h and therefore are not applicable to detect-to- protect technologies and may take too long for some detect-to- treat applications. For the viral species typically scrapings from lesions are examined using microscopic techniques for any tell tale taxonomy before samples are analysed by nucleic acid tech- niques [13]. To develop biosensors for these analytes the important con- siderations are the matrix in which the analyte will be found (for example, air, water, food, person), the form the analyte will be in (spore, bacteria, virus, toxin) and the possible features of the analyte, which could be recognised with an appropriate biorecognition molecule. Identification of such features requires some knowledge of the biological species and the forms in which they are found. In the case of the bacterial species and viruses, nucleic acid techniques can be used to identify the organisms or affinity molecule can be used to detect surface sites on these organisms. An alternative for bacterial species is to monitor the toxins they release. Looking at the analytes gram-positive bacteria, gram-negative bacteria, toxins and viruses in turn will provide some insight into the sorts of recognition molecules that can be used as the basis of a biosensor.

(1) Gram-positive bacteria: The organism causing anthrax is an example of such bacteria and is one of the very few pathogenic organisms, which is both aerobic and spore forming [15]. The bioterrorist threat from anthrax is most frequently as a spore, which are very resistant to degradation [13]. Upon inhalation of spores, in 75% of cases infection is lethal with the number of spores required being between about 10000 and 1000. The opportunities exist to detect either the spores or the toxins (endema toxin and lethal toxin [13]) after infection. Detecting the spores would be required if a biosensor was to be a detect-to-protect system for the presence of anthrax spores in the atmosphere whilst

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139

the presence of the toxins would be used for a detect-to-treat device. The problem with detecting the toxins after release is that approaches such as immunoassays are too insensi- tive to detect toxins in infected patients unless the levels are so high that death will be the result [16]. To detect anthrax spores there are essentially three options. Firstly to detect dipicolinic acid (DPA) which is a major constituent of all spores [17,18]. The drawback of detecting DPA is it is non- specific, it only informs that bacterial spores are present [17]. The second option is to detect the DNA from a spore. Using polymerase chain reaction to amplify the DNA as lit- tle as one spore has been detected [16]. The third option is to detect surface features (epitopes) on the spores, which are specific to anthrax spores using affinity molecules such as antibodies. For example, the surface of spores have gly- coproteins present, which differ between species and hence can be used as the epitopes to which antibodies are raised

[15].

(2) Gram-negative bacteria: Francisella tularensis is an exam- ple of such bacteria which are non-spore forming. Tularemia can cause infection by inhalation of contaminated dust, con- tact with the mucous membrane or from insect bites [14]. As little as 25 organisms can cause infection [19]. Immuno- and nucleic acid markers are the most likely recognition molecules. (3) Toxins: The Botulinum neurotoxins (BoNT) and cholera toxin (CT) are perhaps the two most important analytes of this type of biological agent. With botulism the infection can come from the bacteria Clostridium botulinum. Both the spores or the toxin can be aerosolised and aerosolised is the most likely weaponised form [20]. Similar symptoms are observed if BoNT is ingested or inhaled. The toxins are zinc endopeptidases of 150,000 Da, which disrupt ner- vous function and are the most toxic compounds known [13]. Being proteins antibodies can be raised to them as the basis of an immunosensor [20] or nucleic acid techniques could be used to identify the microorganism. An alternative biorecognition molecule, which has found use for both these toxins are lipids with carbohydrate heads known as ganglio- sides. Both CT and BoNT penetrate cell walls by binding at gangliosides on the outer cell membrane. (4) Viruses: Smallpox is a brick shaped DNA virus (variola major) 200 nm in diameter, which is aerosol infective and has approximately 30% mortality rate whilst hemorrhagic fever is an RNA virus. Most viruses can simplistically be thought of as a bundle of nucleic acids in a protein or gly- coprotein bag. The detection options are to identify nucleic acid markers or to use affinity ligands raised to the protein coat of the virus.

other types of recognition molecules such as peptides, glycol- ipids and aptamers have also been used [2,13,15,21] with similar transduction schemes. The most commonly used biosensing methodologies for detecting BWAs will be described in detail.

2.1. Nucleic acid-based biosensing

technologies—detect-to-treat biosensors

For the detection and identification of viruses and bacteria, but not toxins, nucleic acid techniques are exceedingly attrac- tive because they can provide unambiguous identification a characteristic sequence, are highly specific and because using polymerase chain reaction (PCR) amplification techniques the detection of femto or even attograms of nucleic acids is not uncommon [22]. The exquisite sensitivity of advanced nucleic acid techniques are demonstrated by the work of Hartley and Baeumner [16] who have demonstrated the detection of DNA from a single anthrax spore. Similarly, Versage et al. [23] have detected a single Francisella tularensis organism using real time PCR. Analyses can be performed relatively quickly when com- pared with microbiological techniques, but typically still take several hours. The speed of the assays have been reduced to an hour using real time PCR where the amplification reaction is fol- lowed as it progresses [24]. Furthermore, with the use of multiple target sequences exquisite selectivity as well as sensitivity can be achieved. For example, a single Francisella tularensis bac- teria was differentiated from not only non-Francisella bacteria but from Francisella philomiragia [23]. Nucleic acid detection still suffers from a number of draw- backs. Firstly, the ability to amplify minute quantities of DNA means that contamination can be a big problem with the contam- inant also being amplified. As a consequence, nucleic acid tech- niques require pure un-degraded nucleic acids, which implies significant sample preparation and hence is contrary to the entire concept of a biosensor being a reagentless system. The sample preparation steps required to perform a nucleic acid analysis in the simplest case require:

(1) The release of the DNA from the target organism in suffi- cient quantity to allow amplification. In essence this means the removal of the DNA from the nucleoid, which means lysing the microorganism. (2) The removal of PCR inhibitory contaminants such as RNases or DNase enzymes and other proteins. This step may be influenced by many factors including the cell type, the stage of the cell cycle whether it is a cell or spore and the sample matrix. (3) The type of nucleic acid being amplified as RNA is far less stable than DNA.

2. Biosensors for detecting biological agents

The majority of existing technologies for detecting BWAs such as those listed above have relied on either antibodies as the recognition molecule, which bind with a surface feature of the BWA, or have used nucleic acids and determined a nucleic acid sequence known to be found in the BWA being tested. A few

Despite these potential problems nucleic acid techniques, which employ PCR have been used for the detection of anthrax spores [16,25], Francisellas tularensis [19,23] and orthopox viruses [26,27]. Furthermore, nucleic acid detection methods employing PCR for the identification of disease agents have been shown to be reliably performed under field conditions by enlisted personnel [22]. Recent developments in miniaturisation of PCR

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J.J. Gooding / Analytica Chimica Acta 559 (2006) 137–151 Fig. 1. The principle of the biosensor

Fig. 1. The principle of the biosensor described by Baeumner et al. [30]. The biosensor consists of sulforhodamine B dye-entrapped liposomes, which contain covalently attached probe DNA on their surface and capture probes immobilised onto the surface of polyethersulfone membranes. To perform the assay the lipo- somes are mixed with the sample containing the potential target DNA or RNA sequence. Placing the membrane into the sample results in mixture migrating up the membrane. Capture of the target species by hybridisation, results in the liposomes also being captured and the capture zone will fluoresce. Reproduced with permission from [30], Copyright Springer (2004).

thermocyclers has seen hand held PCR thermocyclers close to commercialisation [19,28], which perform well in comparison to established laboratory based technology when analysing for Francisella tularensis [19]. The influence of toxins on gene expression monitored using a gene chip has also been proposed as an approach to detecting the presence of cholera toxin [29]. This paradigm shift in the use of DNA microarrays for detecting biological agents relies on the knowledge of gene markers, which respond when a cell is exposed to a toxin. In the case of cholera toxin cell lines, which contained ganglioside GM1, the glycolipid responsible for transfer of the cholera toxin A subunit across the cell mem- brane, were used. The power of the approach is that if a variety of genetic markers are known then identification of a previously unknown toxin is possible. Nucleic acid techniques can also detect artificially created pathogens or unknown pathogens by detecting DNA sequences unchanged from the original organ- ism [22]. Affinity molecules, which bind to surface sites on the known species will not possess this ability if the surface epitope used for recognition is changed by the genetic modification. The strengths and drawbacks of nucleic acid based biosen- sors for the detection of BWAs is exemplified by a recent paper by Bauemner et al. [30] for the detection of mRNA from ger- minated spores. The detection is achieved using a sandwich assay where oligonucleotides specific for a sequence indicative of anthrax are immobilised onto a polyethersulfone membrane (the capture probe), see Fig. 1. A reporter probe is also synthe- sized which is complementary to a different part of the target sequence. The reporter probes are conjugated with a liposome containing the fluorescent dye sulforhodamine B. Placing the membrane containing the capture probe into a solution of the amplified target and the reporter probes results in a sandwich

being formed and that part of the membrane emitting light.

The advantage of the biosensor is its capability of detecting as little as a femtomole of target in 4 h with no cross-reactivity with 11 other similar microorganisms tested. Furthermore, the detection of mRNA, being the nucleic acid, which is the active component of genes, indicates that the spores are viable and no cross-reactivity is observed from nonviable spores. Greater sen- sitivity could be achieved with longer sample preparation time down to the level of detecting mRNA from a single spore. These clear advantages are counterbalanced by the 4 h to perform the entire assay, although the actual biosensing part of the assay takes only 15 min. The rest of the assay time is involved in gem- inating the spores, extracting the mRNA and finally amplifying the mRNA. These sample preparation steps require considerable skill and care to perform, especially when handling RNA, which

is about 100,000 times less stable than DNA.

Integration of automated sample preparation with DNA amplification and detection has been achieved to produce bench- top size instruments [12] such as the Biohazard Detection System used by the US Postal service and developed in con- junction with Northrop Grumman and the US Army Soldier and Biological Chemical Command. For truly portable hand held devices, which require the user to simply contact the sam- ple with the biosensor the entire detection procedure must be automated (handheld thermocyclers still require the preparation

of the DNA sample). There are essentially three steps to this process. Firstly, the preparation of the nucleic acids for ampli- fication, secondly the amplification and labelling of the nucleic acids if required and finally the detection, typically using sur- face immobilised probe sequences. The last step of this process has been the subject of considerable research effort with some impressive technologies being developed using both electro- chemical [31,32] and optical transduction [33,34]. Researchers have focussed on better surface chemistry for DNA immobili- sation [35–38], new types of labels [39–41], novel transduction methods [42–49] and miniaturisation [50]. These studies have made significant advances with regards to some known stum- bling blocks to the widespread use of DNA biosensors such as sensitivity [43,51,52] and the ability to differentiate between a perfectly complementary sequence and a sequence containing

a single base-pair mismatch [53,54]. Advances in performing

PCR on a chip have been briefly mentioned above with regards to handheld PCR thermocyclers [55–57]. There has been con- siderably less research on sample pre-treatment, which proves to be the stumbling block for the in-field use DNA biosen- sors, although some impressive advances have been made on microfluidic platforms [58]. For example, Nanogen has shown the application of electric fields for lysing cells and manipu- lating DNA [59–61]. Similarly, Taylor et al. [62] have shown sonication can be used to lyse anthrax spores. Without having the sample preparation steps automated, and more importantly, without being able to reduce the entire assay time to a few minutes, DNA based biosensors will never fulfil the criteria of detect-to-protect devices. They will, however, prove highly use- ful in laboratory based detect-to-treat testing of infection and identification of suspect organisms. This view is supported in a recent article in the journal Science [1] where the head of the

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US chemical and biological national security at Lawrence Liv- ermore National Laboratory is quoted as saying “if I go from wanting an answer in hours to wanting one in 2 min, I have eliminated all kinds of technologies, like PCR”.

2.2. Detection using surface features—detect-to-protect

biosensors

Biosensors, which use surface features of the BWA, rather than requiring extraction of a molecular marker indicative of the agent, are compatible with the development of detect-to- protect devices. To satisfy the ideal criteria for such devices, these biosensors must give a rapid response, must require no user intervention, must not give false positives, must have the sensitivity to detect just a few of the biological agents and must be able to sample in air despite biological molecules having evolved to operate in an aqueous environment. A representative list of affinity biosensing concepts that are motivated towards producing detect-to-protect devices are listed in Table 1 with some of their attributes. Some of the important developments are discussed in terms below. The first commercial devices for detecting biological warfare agents such as those by BioVeris [63], Response Biomedical Corporation [64] and QTL [65] are an attempt to automate a sandwich immunoassay by immobilising capture antibodies on beads. The main differences between these three companies are in assay formats and the types of labels used. The QTL system exemplifies the approach. The user has an instrument smaller than a laptop computer weighing three pounds, which is sold with self-sealing cartridges. The cartridges are swabbed onto the area of interest, placed in the meter, which gives an answer within 10 min. The main direction in the development of automated immunoassays is for more sensitive labels to reduce detection limit. BioVeris employs electrochemiluminescent labels where once a sandwich of the capture antibody, the analyte and the reporter antibody is formed on magnetic beads, the beads are trapped over an electrode and a potential applied to the electrode to stimulate emission of light from an electrochemiluminescent label. The ability to repeatedly stimulate the electrochemilu- minescent label provides the potential for enhanced sensitivity with this transduction principle. QTL use a newly discovered phenomenon of fluorescence superquenching. In fluorescence superquenching, a fluorescent polymer is employed. Associa- tion of this polymer with an energy or electron transfer quencher will cause the fluorescence of the whole polymer strand to be quenched in an analogous manner to the breaking of one Christ- mas light bulb turning off the entire circuit of Christmas lights. The way QTL configure their immunoassay the fluorescent poly- mers are coated around magnetic beads. The presence of the BWA typically causes the quencher to dissociate from the poly- mer with a concomitant increase in fluorescence. The potential sensitivity of fluorescence superquenching over using conven- tional fluorophores is apparent but it is possible to envisage other energy transfer species in a biological sample, which could interfere. Despite the sensitivity of the label over conventional fluorescence Table 1 shows the Response Biomedical Corpo- ration device, which employs a conventional fluorophore has a

lower detection limit than QTL for anthrax spores. The higher detection limit of the QTL system may reflect interferences or the surface immobilisation chemistry employed as the surface chemistry is vital in enabling thermodynamically favourable sur- face binding and to limit non-specific binding. In the screening of a sample for several potential bioagents using immunoassay approaches either multiple labels could be used, say with different coloured quantum dots [66] or alter- natively, protein chips with biorecognition molecules for cer- tain bioagents are deposited at defined locations. Ligler and co-workers from the Naval Research Laboratory have worked extensively on the latter concept [67–77] employing a pragmatic approach of only using reagents, which are commercially avail- able. They have developed two different approaches. The first [67–71] has been commercialised by Research International, Monroe WA, USA, as the Analyte 2000 and then as a portable, automated version as the RAPTOR [78]. A monolayer of capture antibodies are immobilised on optical fibres with the RAPTOR instrument having four channels, thus allowing four analytes to be detected. Binding of the antigen is then transduced by fluorescently labelled reporter antibodies in a sandwich assay format. An important innovation in this work is the use of a specially tapered optical fibres which allow the excitation of the fluorophore label on the reporter antibody, via the evanescent wave generated by light propagating down the fibre, as well as the collection of the fluorescent light emitted by the label. The advantage of using evanescent electromagnetic radiation is that analytes can be detected in real time as only fluorophores near the surface of the optical fibre will be excited, thus relax- ing the requirements for separation of bound from free reporter antibodies. The more recent technology of the Naval Research Laboratory uses a planar waveguide to allow arrays to be devel- oped. The biosensor interface is prepared on glass substrates, which are modified with a silane based self-assembled mono- layer to which antibodies and gangliosides for the target analytes are immobilised. Six stripes containing six different biorecog- nition molecules (Fig. 2) are immobilised using a microfluidic block containing six channels. In the latest publication, [77] on working towards a desktop type instrument, the sample is then delivered using a similar microfluidic block to deliver sam- ples at right angles to the stripes containing the biorecognition molecules (Fig. 2B). Exposure to reporter antibodies with flu- orescent labels allows the presence of BWAs to be detected using evanescent wave excitation monitored with a CCD camera (Fig. 2D). The higher detection limits relative to the BioVeris immunoassay are most likely a consequence of the antibodies being immobilised on a flat surface but the clear advantage is up to nine analytes have been measured at the same time [75]. Portable biosensors where the automation of sandwich immunoassays has been achieved without requiring batteries are currently on the market using the same principle as the pregnancy test kits. A typical pregnancy biosensor works by detecting the hormone Human Chronionic Gonadotrophin (hCG) in urine [79]. The biosensor has an absorbent swab to which the sample is applied and a plastic holder with two windows; the measurement window reports the results of the test and the validation window reveals whether the actual device is operating normally. Wetting

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Table 1 Representative detect-to-protect biosensors for different BWAs or BWA type analytes

Analytes

Detection limit

Assay time

Reagent free

Real samples

Comment

Refs.

1 Anthrax spores

200 spores/mL

5 min

Yes

No

Love wave acoustic sensor. Non-specific binding can be a problem.

[84]

Anthrax

2 spores

4000 spores

15 min

No

Surfaces

Commercialised by Response Biomedical, a fluorescent bead immunoassay commercialised as a benchtop instrument

[64]

BoNT

5 ng

Ricin

10 ng

Smallpox

100000 pfu

3 BoNT B

5 pg/mL (8 nM)

15 min

Yes

No

Bead linked to the end of a cantilever using a linkage which is attacked by the BoNT B which is a zinc endopeptidase

[20]

4 BoNT

15 pg/mL

20 s

Yes

No

Fluorescent dyes laden liposomes containing ganglioside GT 1b as reporters. Similar to pregnancy tests

[81]

5 CT

1 fg/mL

20 s

No

No

Liposomes with ganglioside GM1 act as reporters in a sandwich assay where formation of the sandwich was monitored using impedance

[85]

6 CT

0.1 pg/mL

1 min

Yes

No

ISFET where CT binds directly to surface immobilised anti-CT antibody. Problems are reproducibility and non-specific binding

[86]

7 CT

12 pg/mL

Few minutes

Yes

No

Bilayer with donor labelled GM1 and acceptor labelled GM1. Binding of CT brings the donor and acceptor labelled GM1s closer together and increased

[127,128]

 

Forster¨

energy transfer

8 CT

120 pg/mL

5 min

Yes

No

Uses redox active lipid microstructures containing ganglioside GM1 on an electrode surface. Binding of CT to GM1 reduces current

[91–93]

9 CT

90 g/mL

NA

Yes

No

Use liposomes containing polydiacetylene and ganglioside GM1. Binding of the CT to the GM1 results in a change in liposome colour

[90]

10 CT

160 g/mL

90 min

Yes

No

Ion selective field effect transistor (ISFET) which detects enzymatically the hydrolysis of NAD + by cholera toxin

[129]

11 Anti-CT antibody

200 ng/mL

70 min

Yes

No

Chemiluminescent immunometric assay to detect labelled antibodies

[130,131]

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Table 1 (Continued )

143

Analytes

Detection limit

Assay time

Reagent free

Real samples

Comment

Refs.

12 Anthrax spores

<10000 spores

10 min

Yes

Surfaces liquids

Fluorescence polymer superquenching where magnetic beads are coated with fluorescent polymers. Binding of analyte removed quencher. All reagents included in one use cartridge. This is the technology of QTL

[65,132]

BoNT

NA

Ricin

NA

SEB

NA

13 Anthrax spores

1 g/mL 400 ng/mL 400 ng/mL

15–25 min

Yes

Surfaces, liquids

Pregnancy type test using flow immunosensor systems in a single cartridge. Commercialised by Biowarfare Agent Detection Devices (BADD)

[80]

BoNT

 

Ricin

14 BoNT

0.05 ng/mL 1 ng/mL 10 pg/mL 10 5 pfu/mL

30 min

No

Surfaces, milk, food

BioVeris has commercialised an electrochemiluminescence coupled with magnetic beads, essentially a sandwich immunoassay for bioweapons agents

[22,63]

Ricin

 

SEB

Smallpox

15 Anthrax

30 cfu/mL 1–10 ng/mL 0.1–1.0 ng/mL <0.5 ng/mL

15 min

Yes

Liquids, blood

Optical fibre based fluorescent inmmunoassay. Field instruments called The Analyte 2000 and the Raptor have been developed from this technology by Research International

[67–71]

BoNT

 

CT

Ricin

F.

tularensis

5 × 10 4 cfu/mL

16 Anthrax

10 4 –10 5 cfu/mL 40 ng/mL 200 ng/mL 1.6 ng/mL (antibody) 40 ng/mL (ganglioside) 8 ng/mL 3 ng/mL

12

min

No

Nasal swabs, plasma, serum, airborne

Multianalyte arrays using sandwich assays with fluorescent labels where the capture antibodies are immobilised on a wave-guide. Excitation is by evanescent wave and microfluidics are used to deliver samples

[72–77]

BoNT

A

 

BoNT

B

 

CT

Ricin

SEB

Bacillus globigii

F.

tularensis

2.5 × 10 4 cfu/mL

17 BoNT

100 ng/mL <1 ng/mL 5 ng/mL

15 min

No

No

Uses an evanescent wave device to detect binding label free. Non-specific binding will be a problem

[82]

SEB

Yes

Ricin

Yes

F.

tularensis

1 × 10 4.5 cfu/mL

No

18 SEB

2 ng/mL

15 min

No

Urine

A portable 2 channel SPR device, which has been fitted to aircraft for airborne monitoring. Detection limts are decreased with a one-step and two-step amplification

[87–89]

 

60 min

Seawater

 

0.56 ng/mL, one step 2.8 pg/mL, two steps

60 min

 

19 CT

10 ng/mL

2 h

No

No

Simple immunoassay using quantum dots as the label. Using different size dots can do multiplexing in the same sample

[66]

Ricin

30 ng/mL

SEB

3 ng/mL

20 Bacteriophages

 

8 min

No

No

Sandwich assay which employed interdigitated electrodes and redox cycling. Non-specific binding becomes the limiting factor

[133]

Viruses

90 ng/mL 1.5 × 10 10 viruses/mL

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Table 1 (Continued )

 

Analytes

Detection limit

Assay time

Reagent free

Real samples

Comment

Refs.

21

Anthrax

1000 cfu

3 min

Yes

Nasal swabs

Genetically modified B lymphocytes engineered with luminescent proteins and membrane bound recognition antibodies

[94]

Y.

pestis

50 cfu

22

Anthrax

1–100 ng/mL

2 h

No

Airborne

Benchtop instrument combining immunoassay and PCR based DNA detection to verify immunoassay results. Can monitor air continuously for more than 1 week. Referred to as Automatic Pathogen Detection System (APDS)

[96–100]

BoNT

 

Y.

pestis

All entries consider the biosensor alone rather than the entire sample collection and preparation system to allow comparison between the actual sensing components of each device. All detection limits refer to measurements in buffer to allow comparisons between systems. With regards to using a biosensor, reagent free refers when no reagents need to be added by the user to operate the biosensor but does not consider any sample preparation. Real sample refers to where the analysis of natural samples with the biosensor was reported with the types of samples analysed as listed. BoNT refers to Botulinum toxin, CT is Cholera toxin, SEB is Staphylococcal enterotoxin B, NA refers to the information not being available, and the shaded rows indicate technologies which are or will soon be commercially available.

a swab with urine results in it wicking up the swab towards the capture antibodies. The urine also solubilizes reporter antibod- ies, which are labelled with a coloured bead. As the hormone is smaller than the reporter antibody it wicks up the swab quicker and reaches the capture antibody first. If the hormone is present, and is captured the reporter antibody with its coloured bead will also bind to the antibody and a coloured band will appear in the test window. The reporter antibody also flows past the upstream validation window where a coloured line reveals a negative result is not a consequence of the biosensor not work- ing. This biosensor format has been applied to other analytes such as cryptosporidium in water and is the basis of the Biowar- fare Agent Detection Devices (BADD) technology [80] for the detection of anthrax, botulinum toxin (BoNT) and ricin. In the case of anthrax the biosensor can detect as low as 1 g/mL in solution or 0.25 g off a surface in 15–25 min. Slightly bet- ter performances are obtained with botulinum toxin and ricin biosensors (detection limits of 400 ng/mL or 50 ng from a surface in 5–10 min). The advantage of these biosensors is simplicity.

No power is required for the device as the transducer of the opti- cal signal is the users eye. The drawbacks are the need to wet the device and the poor detection limits. The high detection limit is a consequence of the low sensitivity of the human eye with a con- siderable number of reporter antibodies needing to be captured to generate a discernable colour change for our eyes to see. The problem of detection limit with these devices appears to have been solved for BoNT by Durst and co-workers [81] where the reporter is a liposome containing a fluorescent rhodamine dye. Incorporated into the lipid bilayer of the liposome is the gan- glioside GT 1b, which specifically binds to BoNT. The device has an incredibly low detection limit of 15 pg/mL. Coupled with the simplicity and power free needs of the device this is a very attractive technology. In common with the BADD technology, the requirement for wetting the sample for the detection scheme to work remains and is therefore incompatible with detecting airborne analytes. The biosensing concepts discussed above all use innova- tive approaches to performing a sandwich immunoassay, which

tive approaches to performing a sandwich immunoassay, which Fig. 2. Orthogonal patterning and assay method. Biotinylated

Fig. 2. Orthogonal patterning and assay method. Biotinylated capture antibodies are immobilized in stripes on a NeutrAvidin-coated slide/waveguide via avidin–biotin interactions (A). For analysis, samples are loaded onto the slide using a PDMS flow guide module with channels oriented orthogonal to the stripes of immobilized capture antibodies (B). After incubation with fluorescent sample (direct assay) or after interrogation of the array with fluorescent tracer antibody (sandwich assay), fluorescent complexes are formed in the appropriate loci (C). The pattern of fluorescent spots are imaged and digitised (D). Reproduced with permission from [77], Copyright Springer (2004).

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145

requires little or no user intervention to perform. The difficulty with making a sandwich immunosensor reagentless arises from the need for two recognition molecules, the capture species and

the reporter which must not only bind to different epitopes of the antigen but the two binding events must be separated temporally.

In recent times other biosensing concepts have been developed

which do not employ the sandwich assay concept, many of which are label free. Truly label free methods for detecting biological agents have been developed using optical based evanescent wave devices [82] such as surface plasmon resonance (SPR) and the reso- nant mirror, reflectometry [7,83], acoustic wave devices such as the Love wave acoustic sensors [84] and quartz crystal microbal- ances [85] and ion selective field effect transistors (ISFET) [86].

As can be seen from Table 1 incredibly low detection limits and rapid responses can be obtained with such devices. The key

drawback of all these devices is that they all detect a change at the transducer surface rather than use a signal which requires actual binding between the biorecognition molecule and the analyte. Any change at the interface will cause a similar response and therefore non-specific binding becomes a problem; a problem exacerbated by the high sensitivity of the devices. This problem highlights one of the advantages of the sandwich assay format

in using two different biorecognition molecules. Two biorecog-

nition molecules provide two layers of selectivity which helps reduce the problem of non-specific binding. The problem of non-specific binding with label free methods has however been diminished significantly by a two channel SPR device developed by Texas Instruments and using for the detec- tion of BWAs by Naimushin et al. [87–89]. One channel was modified with antibodies for Staphylococcus aureus enterotoxin

B (SEB) and the other channel is used as a reference channel.

The reference channel compensates for changes in refractive index due to non-specific binding, if any, and changes due to dif- ferent solutions. Without any amplification steps the biosensor can detect 70 pM (2 ng/mL) SEB in 15 min and can run continu- ously for hours. Amplification of the SPR signal using secondary antibodies which bind to the captured SEB and using a third anti- body to bind to the secondary antibody detection limits down to 100 fM (2.8 pg/mL) can be achieved in buffer [87]. These detec- tion limits are somewhat elevated when analysis is performed in urine but 50 pM SEB can still be detected in urine [87]. The SPR system is a portable SPR and the same workers have recently reported its incorporation within an aircraft for air monitoring

[89].

There are a number of new biosensing concepts which are effectively reagentless which are perhaps more robust then the completely label free methods discussed above. Lipid bilayers have been used for a number of reagentless biosensing concepts.

The attractiveness of lipid bilayers is that gangliosides can easily

be incorporated. Gangliosides are lipids with carbohydrate heads

found on cell surfaces. Gangliosides are typically the location on

the cell surface where toxins bind to gain entry into the cell [90]. Hence gangliosides have been shown to be selective for a variety

of toxins [72]. Pan and Charych [90] reported on polydiacetylene

liposomes containing ganglioside GM1, which is selective for cholera toxin (CT). Binding of the CT causes a conformational

toxin (CT). Binding of the CT causes a conformational Fig. 3. A schematic of the proposed

Fig. 3. A schematic of the proposed mechanism for redox microstructure sensor. (A) Without binding of analyte electrons can hop from one ferrocene to another whilst if cholera binds (B) the hopping pathway is blocked. Reproduced with permission of The Royal Society of Chemistry from [93], Copyright Royal Society of Chemistry (2004).

change to the polydiacetylene backbone in the liposome with a resultant colour change from purple-blue to orange. A similar idea has been converted to an electrochemical for- mat where lipid microstructures are adsorbed onto electrode surfaces [91–93]. Apart from the normal lipids and ganglio- side GM1 the lipid structures also contained ferrocene labelled lipids. Ferrocene molecules on the other side of the lipid struc- ture from the electrode would normally be regarded as too far from the electrode to be oxidised or reduced. However, all fer- rocene molecules can be interrogated electrochemically in this system due to electron hopping from ferrocene to ferrocene (Fig. 3). Binding of CT interrupted the electron-hopping path- way and a reduced current was observed. A reduced signal on binding of an analyte is usually disfavoured as false positives are more likely than with a transduction method where an enhanced signal arises from a binding event. Swanson and co-workers have used the multivalent character of CT to allow multiple ganglioside GM1 molecules to bind to the same toxin in a fluorescent biosensor. The gangliosides are labelled with either a donor or an acceptor. Binding of CT brings the donor and acceptor closer together, which increases the amount of Forster¨ Resonance Energy Transfer (FRET) and

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J.J. Gooding / Analytica Chimica Acta 559 (2006) 137–151 Fig. 4. A microcantilever based biosensor developed

Fig. 4. A microcantilever based biosensor developed by Parpura and co-workers [20] for the detection of botulinum toxin. (A) Top view of a cantilever with a bead functionalised tip. (B) Schematic of how the bead is attached to the tip and the resultant frequency response of the cantilever. Recombinant synaptobrevin 2 (red) is attached to bead surface where it binds to recombinant syntaxin 1a, which is attached to the cantilever tip. The synaptobrevin 2-syntaxin intermolecular interaction leads to the bead’s being tethered to the cantilever tip. The amplitude of oscillation of the tip with frequency is shown below the cartoon. BoNT-B in the presence of zinc ions (Zn 2+ ) cleaves synaptobrevin 2 in such manner that the short fragment of synaptobrevin 2 remaining on the bead has no ability to interact with syntaxin 1a on the cantilever tip, leading to the detachment of the bead from the tip (double-headed vertical arrow) and a more dramatic change in amplitude with frequency (panel below cartoon). Reproduced with permission from [20], Copyright National Academy of Sciences, USA (2003).

so increased acceptor fluorescence is observed upon binding of the toxin. Parpura and co-workers [20] from the University of Califor- nia at Riverside have recently published an idea for detecting botulinum toxin (BoNT), which is a complete departure from the use of affinity biosensors for detecting BWAs. The beauty of the idea is to exploit the biological action of the toxin to allow its detection. BoNT B is a zinc endopeptidase enzyme which disrupt the synaptic nerves by cause a cleavage in synap- tobrevin 2 if zinc is present. The biosensor monitors the change resonant oscillation microcantilever. A bead is attached to the cantilever using an affinity complex between synaptobrevin 2 attached to the bead and syntaxin 1A attached to the cantilever. BoNT causes a break in the affinity complex, thus releasing the bead and causing a change in the resonant oscillation (Fig. 4). The detection limit of the biosensor is 5 pg/mL if the assay is left for 15 min (Table 1). Unlike all the affinity biosensors discussed above where the binding reaches equilibrium this biosensor is a kinetic assay reliant on the biological activity of the toxin. Therefore, if the user was willing to wait longer then the detec- tion limits could be further reduced or alternatively if a faster response is required this is achievable but at the cost to the detec- tion limit. Another novel technology applicable to the detection of BWAs is CANARY (cellular analysis and notification of antigen

risks and yields). Developed by Rider et al. [94], CANARY uses

B lymphocyte cells as the detector, and as the reporter, of the

presence of pathogens with response times of a few seconds. The lymphocytes were genetically engineered to express both cytosolic aequorin, a calcium sensitive bioluminescent protein from the jellyfish Aequoria victoria, as well as membrane bound

antibodies specific for the pathogen of interest. Cross-linking

of the antibodies during recognition of the pathogen results in

elevated intracellular calcium with a concomitant emission of light. With regards to bacteria of interest in biological warfare, cells engineered for Yersinia pestis (plague) could detect as little

as 50 cfu in 3 min whilst suffering no interference from a large

excess of Francisella tularensis. Similarly, cells were also devel- oped to detect anthrax spores from seeded nasal swabs down to a level 1000 cfu.

3. Cutting edge of biosensor technology which may be

applicable to bioweapons detection

None of the existing biosensors discussed above for detect- ing biological warfare agents satisfy the most demanding of requirements, a detect-to-protect biosensor which could be worn by a soldier or civilian in an environment where the possibil- ity of a biological attack is prevalent. There are a number of impediments to the development of such a biosensor including

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147

(1) transferring an airborne sample to an aqueous environment where the biosensor can operate, (2) reducing the detection limit, and (3) obtaining more rapid responses. There are a number of advances with regards to both novel biosensing components and novel biosensors, which will enable these problems to be overcome.

3.1. Air sampling

The transferral of airborne samples to an aqueous envi- ronment is necessary because the function of biorecognition molecule is optimised for aqueous environments. There are very few options currently available for performing this task. The demands on any air sample technology are quite stringent. Extremely low levels of airborne BWAs can still have a severe impact and therefore it is desirable for the sampling technique to not only transfer biological material into the aqueous phase but also concentrate it. The average person breathes roughly 6 L/min. In this large volume sample there need be only a BWA or two to do harm [95] and therefore this must be concentrated down to a few millilitres at most. The concentration of the analyte must be achieved without harming the delicate biological sample and be rapid so the response time of the biosensor is not lim- ited by this step. There are currently two main technologies for achieving this at present. These are the wetted cyclone samplers and virtual impactors. At present neither of these types of sam- plers is compatible with a wearable device but are highly suitable for sampling in confined spaces. Therefore, if the biosensor is required to provide early warning of biological agents entering a building then these types of concentrators are ideal as virtually all the air entering a building can be sampled in a similar manner to the BioWatch filtering system used in some state buildings in the United States. A technology has been developed by the Lawrence Livermore National Laboratory in the United States, which employs both a virtual impactor and a cyclone sampler in series to concentrate aerosol for the multiplex detection of BWAs by immunoassay and PCR [96,97]. The benchtop device is called the autonomous pathogen detection system (APDS). The APDS is designed to sample air for BWAs over extended periods (at present a week) in domestic situations (such as airport terminals) where the public may be at risk. The virtual impactor collects particles of 1–10 m in size, which are concentrated in the cyclone sampler. Once an hour the aerosol collector dispenses liquid to an automated fluidics module, which performs a bead based immunoassay, which requires 2 h to complete [98]. The sandwich immunoassay employs a flow cytometer and antibody modified microspheres labelled with a bar code of fluorescent dyes [98–100]. The bar code allows up to 100 different analytes to be monitored with the APDS currently monitoring eight including anthrax, botulinum toxin and pneumonic plague [96,97]. Detection limits are typ- ically between 1 and 100 g/L depending on the analyte [12]. Part of each sample analysed by immunoassay is archived. If a positive signal is recorded, and the BWA contains DNA, part of the archived sample is retrieved for DNA purification and anal- ysis by a real-time flow-through PCR detection system based on an earlier technology developed by Belgrader et al. [25]. In

this way, nucleic acid confirmation of the immunoassay result is obtained. APDS instruments have been deployed in at strategic locations where they have reliably operated for weeks or months

[96].

At this point in time it is not evident if alternatives to the cyclone sampler and virtual impactor are being explored, with a view to personalised biosensors for sampling airborne pathogens carried by individuals. The low concentration of biological agents required to cause infection does create a problem as it means that a small biosensor placed in a single location could quite easily miss interacting with a BWA. An alternative to a sampler, which concentrates the air sample is to use a high surface area biosensor. The research interest in electronic tex- tiles and other smart fabrics, which could perform additional tasks such as energy conversion or sense environmental stim- uli is highly compatible with a high surface area biosensor [101–104]. Thus it is possible to envisage an electronic textile where biorecognition molecules are incorporated within the tex- tile and the electronic fibres act as the transducer. A high surface area biosensor however requires the biosensor to either work in the airborne environment or for the biorecognition molecules to be in a water-like environment.

3.2. Reducing the detection limit and providing a more rapid response

Any innovative biosensor, which can provide a warning of airborne analytes will be required to have very low detection limits. The detection limit of biosensors can be lowered in one of three ways. Firstly, to use biorecognition molecules or surface chemistry which have higher equilibrium constants of binding, secondly to limit non-specific binding or finally to use more sensitive transducers. Novel biorecognition molecules with high affinity for pro- teinaceous species include aptamers [105], peptides and antibod- ies developed from phage display libraries. Aptamers [106–108] are sequences of DNA, which bind to a large macromolecules in a similar manner to an antibody. New aptamers can be found by starting with libraries of random oligonucleotides, which are passed down an affinity column, which contains an analogue of a surface epitope of the target biological agent. Removal of the oligonucleotides, which do not bind followed by elution of the bound ones allows a refinement of the library. Cloning of the eluted oligonucleotides followed by repeated passages through the column allows further refinement until only the most strongly binding aptamers with the highest affinity for the analyte are left. The selected aptamer is then amplified and a new affinity molecule is identified. This procedure for produc- ing aptamers is called the SELEX method and can isolate new biorecognition molecules with affinities higher than natural anti- bodies. Similar principles have been used to identify peptide sequences for microorganisms [109,110] and new antibodies

[111].

Using biorecognition molecules with higher affinity has its limitations however. The strongest known of all bioaffinity reac- tions is avidin with biotin and it is hard to envisage a K a higher than 10 15 M 1 . Recently some exciting work where binding

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of aptamers to the proteins allows amplification of the bound aptamers only which has allowed detection limits significantly below the affinity constant to be achieved [112]. However, this currently requires user intervention through amplification of nucleic acids using PCR. To get lower detection limits still will then require, preventing any non-specific binding and using more sensitive transducers. Higher sensitivity transducers are probably the major areas of focus into biosensor research. For labelled biosensors, which employ the sandwich assay format the most common approach to increasing the sensitivity of transduction is to use brighter labels, that is label systems that require fewer labels to be present for the transducer to be able to detect them. Examples of brighter labels including fluorescence superquenching [46,47], quantum dots [39,66], electrochemiluminescent molecules [63] and dye labelled liposomes [81] were discussed above and show consid- erable promise. The alternative to brighter labels is to use more sensitive trans- ducers, which can detect just a few molecules that have bound without using labels. The microcantilever transducer used by Parpura and co-workers [20] is one example. Microcantilevers transducers either use the sensitivity of the characteristic fre- quency of resonance to changes in mass [113] or bending of the cantilever due to changes in surface stress [114,115] to moni- tor affinity reactions. Recently Gupta et al. [116] have used the resonance of a microcantilever to detect the binding of a single virus particle. It is proposed that the biosensing concept could be used to detect airborne viruses although the applicability of the biosensor has yet to be demonstrated for the detection of viruses in samples other than pure buffer solutions. The detection of a single virus has been demonstrated by Lieber and co-workers using a silicon nanowire field effect tran- sistor (FET), see Fig. 5. Nanowire or nanotube based FETs have been demonstrated by a number of workers [117] for the detec- tion of, protein binding [118,119], enzyme activity [120] and even for detecting small molecules such as oxygen [121]. All of these biosensors work on the same transduction principle of a change in resistance through the nanotube or nanowire as a consequence of the biorecognition event. The Lieber group’s recent contribution is the first example of this highly attractive transduction technology being employed for biological agents. Furthermore, the ability of the biosensor to detect viruses has been performed with both pure and unpurified virus solutions with similar results. Recent work but this group has also shown these nanowire based FETs can work in protein samples with no evidence of non-specific binding [122].

3.3. Decreasing the response time—the needle in a

haystack problem.

When the potential stumbling blocks with biosensors that detect just a few molecules are solved there is an additional challenge, which relates to detecting single particles or a few particles. How long must one wait for a response? Expressed in another way, is the absence of a positive signal evidence that there is no biological agent or is it a false negative due to the fact that the biological agent did not cross the nano-dimensional

that the biological agent did not cross the nano-dimensional Fig. 5. Schematic of the nano-wire based

Fig. 5. Schematic of the nano-wire based detection of a single virus showing the two nanowire devices in parallel (left) and the expected response (right). Only nanowire 2 is modified with antibodies, which are specific for the virus. Binding of the virus reduces the conductance. Reproduced with permission from [126], Copyright National Academy of Sciences, USA (2004).

space occupied by the biosensor? An array of the nanoscale biosensors could reduce this problem but still presents a related

problem, which can be referred to as ‘the needle in a haystack problem’. When a biosensor is in a defined location in space, on

a surface, even if the analyte is present it must still diffuse to the sensing surface for recognition to occur. Even in a small volume this could represent diffusing a long distance on the molecular scale. Hence the analyte may never be found by the biosensor in

a reasonable time in a similar manner to a needle in a haystack

never being found. One solution to the needle in the haystack problem is to use

a highly porous, high surface area biosensor where there is inti-

mate mixing of the sample for analysis with the biosensor. This idea is exploited by the company PamGene [123] for the detec- tion of DNA using fluorescently labelled reporter sequences. Although labels may complicate the assay the short diffusion path-lengths as a consequence of the intimate mixing of sam- ple and transducer allows the hybridisation of target DNA in real time. The attractiveness of highly porous transducers with- out using conventional labels is the subject of intense research activity via porous silicon as a transducer [7,8,83,124]. Porous silicon photonic crystals can be used as mirrors where bind- ing events cause changes in reflectivity. The high porosity can potentially provide rapid responses and the mirrors that can be fabricated can have reflectance peaks with subnanometer full

J.J. Gooding / Analytica Chimica Acta 559 (2006) 137–151

149

width half maximum [125]. Such sharp peaks mean that even a few molecules bound can cause a large shift.

4. Conclusions

Biosensors have incredible potential for achieving the very difficult task of producing detect-to-protect devices once a num- ber of criteria are met. These criteria include having sufficiently low detection limits to detect just a few biological warfare agents, being able to sample analytes from the air and from complex biological matrices, giving responses within a minute or two, giving very few false positives and being light, portable devices which require no power to operate. No single device will fulfil all these criteria in the short term and to do so will require a long term focussed scientific and financial effort if this exciting technology is to fulfil its promise. In the short term however devices, which meet some of these goals have been and will be developed.

Acknowledgements

I would like to thank the Alexander von Humboldt Founda- tion fellowship support during part of the time this review was written.

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