multiplexed optical pathogen detection with lab-on-a-chip devices

REVIEW ARTICLE

Multiplexed optical pathogen detectionwith lab-on-a-chip devices

Holger Schulze1, Gerard Giraud2, Jason Crain2; 3, and Till T. Bachmann*; 1

1 Division of Pathway Medicine, Medical School, The University of Edinburgh, Chancellor’s Building, Little France Crescent,Edinburgh EH16 4SB, Scotland (UK)

2 School of Physics and Astronomy, The University of Edinburgh, The King’s Buildings, Edinburgh EH9 3JZ, Scotland (UK)3 National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, UK

Received 20 February 2009, accepted 27 February 2009Published online 15 April 2009

Key words: infectious diseases, microTAS, in vitro diagnostics, point of care testing

# 2009 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1. Introduction

Since early pioneers such as Antonie van Leuwen-hoek, Louis Pasteur, and Robert Koch laid theground in infectious disease diagnostics, significantimprovements have been made regarding isolationof causative pathogens, their amplification and visua-lisation. Today, the majority of detection technolo-

gies are still relying on these basic steps of isolation,cultivation and optical detection. This is especiallytrue for bacterial and fungal infections as cultivationprocedures are comparably simple, hence lengthy.Great improvements in terms of assay speed andsensitivity were made by the introduction of immu-nological assays such as ELISA, monoclonal anti-bodies and lateral flow immunoassays. Then, mole-

# 2009 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Infectious diseases are still a main cause of human mor-bidity and mortality. Advanced diagnostics is consideredto be a key driver to improve the respective therapeuticoutcome. The main factors influencing the impact of di-agnostics include: assay speed, availability, informationcontent, in-vitro diagnostics and cost, for which molecu-lar assays are providing the most promising opportu-nities. Miniaturisation and integration of assay steps intolab-on-a-chip devices has been described as an appropri-ate way to speed up assay time and make assays avail-able onsite at competitive costs. As meaningful assaysfor infectious diseases need to include a whole range ofclinical relevant information about the pathogen, multi-plexed functionality is often required for which opticaltransduction is particularly well suited. The aim of thisreview is to assess existing developments in this fieldand to give an outlook on future requirements and solu-tions.

Fully integrated microsystem for influenza virus detectionincluding sample preparation, RT-PCR and fluorescencedetection developed by Juergen Pipper and colleagues[64] (Image was kindly provided by Juergen Pipper).

* Corresponding author: e-mail: [email protected], Phone: +44 (0)131 242 9438, Fax: +44 (0)131 242 6244

J. Biophoton. 2, No. 4, 199–211 (2009) / DOI 10.1002/jbio.200910009

cular detection was introduced mainly for the diag-nostics of viral diseases such as HIV and Hepatitis ashere very demanding assay sensitivities of a few par-ticles per sample are required and suitable targetamplification procedures like the polymerase chainreaction (PCR) have been developed. Today, quanti-tative PCR which is preceded by reverse transcrip-tion in case of RNA viruses is the standard detectionmethod in virology and is finding increasing applica-tion in other disease areas.

While initial approaches to identify the etiologi-cal agent of an infectious disease were primarilylimited to single targets, there is an increasing de-mand for multiplexed detection. This is understand-able as a plethora of microorganism species cancause pathogenic events in humans and an increas-ing knowledge about their genetic armour is gener-ated. As an example, E. coli is a commonly knownorganism which readily acquires genetic informationsuch as virulence factors and antibiotic resistancemarkers [1]. Future diagnostics will have to adaptto this situation. Highly multiplex molecular detec-tion technologies were developed to sense and clas-sify pathogenic microorganisms. Because of its par-ticular suitability for multiplexed detection, themajority of these methods use optical methodsamong which fluorescence intensity is the dominanttechnology. Systems to quantify pathogens usingreal time PCR were described repeatedly [2] andcommercial systems are offered by a large numberof vendors. Some of them developed systems with atremendous multiplexing capability (Applied Bio-systems (ABI PRISM 7900HT, up to 384plex),Roche (LightCycler 480 Real-Time PCR System, upto 384plex), Fluidigm (BioMark, up to 9,216plex))and their suitability for pathogen typing was de-monstrated by various academic groups. The tech-nology which provided the key step change in en-abling highly multiplexed molecular analysis isDNA microarrays. It has been used for bacterialand viral typing since their early days and systemsfor microbial species typing [3, 4], antibiotic resis-tance detection [5–8] and virulence assessment[9, 10] have been described. For a review closest tothe field see [11] and [12]. DNA microarray typingtechniques nearly entirely rely on a target amplifi-cation step to enhance the system’s sensitivity. Thisstep is predominantly achieved by PCR. As PCRcan be carried out in a consensus format or usingindividual target specific primers, this technique canalso be used as an end point method in a multi-plexed fashion whereby the amplification productsare analysed by standard methods such as slab gelanalysis, capillary electrophoresis, lab-on-a-chip(Agilent, Bioanalyser 2100) or lab-on-a-tape(Seegene/Lab901, ScreenTape) analysis. Eppendorfrecently announced a new method, termed rapID,whereby real-time PCR and low density DNA mi-

croarray technology was merged for pathogen de-tection [13, 14].

The ultimate goal would be to acquire the com-plete DNA sequence information from a sample toarrive at an unbiased diagnostic platform. However,mainly cost and assay time barriers make this a goalunlikely to be met in the near future. Advances in no-vel sequencing technologies, e.g. from establishedplayers like 454/Roche (Genome Sequencer FLXSystem) or earlier stage (Pacific Bioscience (SingleMolecule Real Time (SMRT) DNA sequencing tech-nology) and young companies (DNAe (Genalysis)are promising and combinations with establishedtechnologies such as DNA microarray technologycould lead to more cost effective approaches (e.g.NimbleGen (Sequence Capture), Febit (Hybselse-lect), Agilent (SureSelect)). Several projects are un-derway to use rapid sequencing for pathogen detec-tion. Similarly, US Genomics was awarded $8.6 and$9.1m by the US Department of Homeland Securityfor developing their DLA technology with opticalreadout for airborne pathogen detection. This com-pany is also teaming up with Becton Dickinson to-wards clinical pathogen diagnostics. Multiplexed beadbased molecular assays are offered commerciallybased on the Luminex xMAP technology (e.g. for Sal-monella). However, these systems require large scaleinstrumentation and are not immediately suitable forapplications targeted by integrated miniaturised sys-tems. Further promising technologies employ proteinbinders, peptides, aptamers, etc. as recognition ele-ments often in an arrayed format to increase the mul-tiplexing capability [10, 15, 16]. Diagnostics of infec-tious diseases can be also approached from the hostresponse to infection by measurement of nucleic acid[17, 18] and metabolomic biomarkers [19].

Pathogen detection technologies are a field whichgained remarkable attention in recent years due tothe rise of antimicrobial resistance (e.g. MRSA) andhospital acquired infections (HAI) in industrial coun-tries and the need for advanced diagnostics in devel-oping countries. Here, the impact is the greatest inenvironments with minimum resources [20, 21]. Totackle those applications, the technologies and ap-proaches described above require significant simplifi-cation and integration. Microfluidic systems such asmTAS, lab-on-a-chip, or BioMEMS offer the great ad-vantage to integrate a large number of assay handlingsteps into a single device which makes the system in-dependent from user intervention and, thereby, lessprone to contamination. The main steps which can beidentified for these systems are sampling, sample pre-paration [22], isolation of compound of interest, tar-get amplification, detection/binding of analyte, andreadout. Concepts for these devices have been pub-lished repeatedly [23, 24] and surrounding technologydevelopment was reviewed elsewhere [25, 26]. Tech-nologies to identify pathogens and infectious events

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in integrated devices cross benefit from other bioana-lytical fields such as integrated biomarker detection[27] and nanodiagnostics [28, 29]. In the followingparagraphs we review recent developments specifi-cally towards integrated systems for multiplexedoptical bacterial and viral pathogen detection andconclude with a specific discussion on innovativefluorescent transduction techniques.

2. mTAS for bacterial detection

2.1 Integrated preanalytics

The realisation of a totally integrated pathogen assayis dependent on successful implementation of all cri-tical process steps. Still, sample preparation is thekey bottleneck. An early example using soft litho-graphy and PDMS in a technology developed in thegroup of Stephen Quake at Caltech was describedby Hong [30]. Here, they described a genomic DNAchip which enabled them to extract DNA from 28E. coli cells which were added to the system sus-pended in diluted culture media. The extractedDNA was detected subsequently by off chip PCRand slab gel analysis. The system contained a ringmixer and structure in which cell lysis and subse-quent binding and elution of the genomic DNA waselegantly accomplished. From a larger programme atthe Bio Lab, Samsung Advanced Institute of Tech-nology several studies on integration of prenalyticswere published. Lee et al. [31] described a portablesample preparation device which used Laser Irra-diated Magnetic Bead System LIMBS for cell lysisand DNA isolation using a 40 s laser pulse. Theauthors used E. coli cells to demonstrate the feasibil-

ity of the system and achieved lower limits of detec-tion of 10 E. coli cells/mL starting from a 9 mL of cellsuspension (105 cells/mL) mixed with 1 mL of mag-netic beads. The extracted DNA was confirmed bysubsequent PCR/qPCR amplification and analysison an Agilent Bioanalyser 2100. Further targetpathogens were Streptococcus mutans (tooth decay),Staphylococcus epidermidis (opportunistic pathogen,mainly from skin) and Hepatitis B virus. In prelimin-ary experiments the authors demonstrated the possi-bility to perform real time PCR directly in the cham-ber after LIMBS in 32 minutes. The principle wasfurther developed and described by Cheoung et al.[32]. Here, the authors used Au nanorods to employan optothermal effect after 30 s laser irradiation forlysis of E. coli cells (10,000/mL) followed by real timePCR detection without removing the nanoparticles.The papers describe very useful and interesting stepsto accomplish rapid DNA extraction from bacterialcells followed by optical detection using an off chipreader. The system’s performance with clinical sam-ples, however, was not reported. A key developmenttowards this was reported from the same group [33].They transferred the LIMBS principle onto a poly-mer based CD (with preloaded reagents) employingcentrifugal microfluidics. The system was tested withE. coli and HBV spiked in whole blood. The processwas demonstrated with 103–105 cell/mL and could becompleted in 12 min.

2.2 Integrated PCR-CE systems

The integration of PCR in microfluidic chip basedsystems is a wide field of activities [25]. As an exam-ple, Liao et al. described a PCR chip with subse-quent standard slab gel detection which could be

Figure 1 (online colour at: www.biophotonics-journal.org) Concepts for optical mTAS devices for pathogen detection from2004 [24] and 2009 [23]. Reprinted from Ref. [23] with permission from the American Chemical Society (ACS) (Copyright2009) and from Ref. [24] with permission from Elsevier (Copyright 2005).

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used to amplify several genes to identify S. pneumo-niae (lytA, pbp2b), Haemophilus influenzae (HiGI1),Staphylococcus aureus (coag), Streptococcus pyo-genes (speB), and Neisseria meningitides (16SrDNA) [34]. In addition the authors showed amplifi-cation of antibiotic resistance genes (F2-R1 (HF),mecA (SA), ermB (SP), blaTEM (NM)). Prakash andcolleagues [35] described an example of a combinedPCR-CE system on chip. It was designed to detectBordetella pertussis the causative agent of whoopingcough by PCR amplification of a 167 bp fragment ofthe Is481 region. B. pertussis carries 50 copies of thisregion which makes it a useful target. The systemcontained 9 PCR chambers which could be used toamplify DNA from different samples and enable the-oretically a nineplexed detection. The ampliconswere labelled during PCR and subsequently ana-lysed by CE. The authors loaded the system with3 mL PCR mastermix per chamber containing the ex-tracted B. pertussis DNA. The authors claim a detec-tion limit of 2 cfu which they archived by extractingDNA from pure cultures which were suspended insterile PCR grade water. An example of an inte-grated PCR-CE device was described by Lagally[36] and [37–41]. Here, the authors performed a spe-cies specific detection of E. coli and S. aureus by am-plification of 16SrRNA and femA targets, respec-tively. In addition antibiotic resistance and virulencemarkers (fliC, sltI, mecA) were detected in a multi-plexed fashion in 30 min. The paper also gives inter-esting data on the detection of virulence markers ofE.coli O157 : H7 in front of a serial dilution of E. coliK12 cells. The device was able to detect both mar-kers until a ratio of 0.25% (25 E. coli O157 : H7 cellsin a background of 99,975 E. coli K12 cells).

2.3 Sample-to-answer systems

Easley and colleagues described a true sample-to-an-swer mTAS (Figure 2) which could be used withwhole blood [42]. The device integrated on chipDNA purification by solid phase extraction (SPE),PCR amplification, and separation and detection bymicrochip electrophoresis. The authors demonstratedthe performance of their chip by detecting Bacillusanthracis from infected asymptomatic mice in lessthan 30 min (10 min extraction, 11 min amplifica-tion). In a second example, Bordetella pertussis wasdetected from nasal aspirate of a patient with sus-pected whooping cough. This study is a rare exampleof a lab-on-a-chip device for pathogen detectionwhich integrated all necessary steps for pathogen de-tection, was demonstrated with real samples, andcould be performed in reasonable time. In a differ-ent publication the same authors described a similarsystem with infrared-mediated DNA amplification

for the detection of Salmonella typhimurium [43].One of the highly cited landmark publications in thefield of integrated (electrochemical) pathogen detec-tion devices was made by Piotr Grodzinski’s groupat Motorola Labs [44]. They described a completesample-to-answer system capable of detecting E. colicells which were spiked into rabbit blood from which1ml was loaded in the biochip device. The systemused the Motorola eSensor (now Osmetech) forelectrochemical detection of hybridisation events. Asit contained 16 electrodes, multiplexing would bepossible if a suitable amplification process could beimplemented. The biochip device remarkably inte-grated all necessary functions for the sample-to-an-swer process including immunoseparation by mag-netic beads of E. coli cells, thermal lysis and PCRamplification followed by electrochemical detection.

Figure 2 (online colour at: www.biophotonics-journal.org)Example of a fully integrated PCR-CE microfluidic chipfor bacterial detection [42]. Reprinted from Ref. [42].Copyright (2006) to this figure retained by the NationalAcademy of Sciences, USA.


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This was accomplished by an arrangement of onchipthermopneumatic and electrochemical pumps andparaffin valves. This study is one of the few exam-ples of totally integrated systems, however, more re-cent directly related publications or follow-up activ-ities, were not reported but key staff moved on toCombimatrix [45–47]. Similarly, researchers at HongKong University of Science and Technology have de-veloped an integrated glass/silicon DNA biochip formultiplexed electrochemical pathogen detection [48].With a view on Global Health Diagnostics the Pro-gram for Appropriate Technology in Health (PATH)in Seattle made significant contributions especiallyfor non instrumental diagnostics. As an exampletheir disposable Enterics Card contains a fully inte-grated multiplexed lab-on-a-card assay for entericpathogens to diagnose and guide therapy forpatients with acute diarrhoea (Figure 3) [49, 50]. Thesystem, termed lab-on-a-card, remarkably includesall necessary reagents and the samples can bedirectly loaded. Pathogens are immunocaptured, se-

parated and subjected to a multiplexed nucleic acidamplification and visual on-chip detection by thenaked eye. The target species are Shigella dysenter-iae serotype 1 (ipaH, malB, stx1), Shigella toxin-pro-ducing E. coli/E. coli 0157 : H7 (stx2, stx1, rfb, malB),Campylobacter jejuni (gyrA), Salmonella (invA), andShigella species. The detection of the labelled PCRamplicons is conducted in a Micronics MagnaFlowdevice which contains a visual detection method fornucleic acids comprising a fluid flow over capturezones resulting in visible lines for each pathogenvery similar to a lateral flow immunoassay. The sys-tem is a good example of consequent integration ofall necessary steps and the choice of the right targetsto give meaningful information to guide medical in-terventions. The system is currently tested in a clini-cal study in Brazil involving over 1,100 patients (ex-pected finalisation in February 2009).

2.4 Other Systems

Several reports were published where groups used offchip assays followed by microfluidic analysis systemssuch as the Bioanalyser 2100 from Agilent. Ikeda [51]demonstrated a study where they immobilised 5 differ-ent capture oligonucleotides to fluorescently labelledbeads which were used to bind to ribosomal RNA iso-lated from pure cultures of food poisoning bacteria.The rRNA itself was labelled using a commercial kit(Oligreen) and hybridisation products were analysedusing a commercial chip (Cell Fluorescence LabChip).Using this assay, the authors described lower detectionlimits of 2 mg rRNA which would require significanttarget amplification e.g. by precultivation to meet de-sired sensitivities of pathogen detection.

In a fundamentally different approach to detectbacteria and determine their antibiotic sensitivity,Boedicker and team used the method of stochasticconfinement of bacteria into nano-microliter plugs inchannels of a PDMS device [52]. In these plugs bac-teria (<1 cell/plug) were exposed to viability indica-tors which visualised the effect of also present anti-biotics. The method represents a highly interestingway to accelerate the assessment of antibiotic resist-ance. Although the limit of detection is theoreticallya single cell per plug, the authors used relativelyhigh numbers of cells (105 cfu/mL) and claimed 7 hfor a complete characterisation of a bacterial samplewhich is still too long to be competitive with stand-ard culture based methods. The idea of stochasticconfinement of bacteria or analytes, termed digitalPCR, was used in a molecular approach by Ottesento analyse communities of environmental bacteria[53] using Fluidigm’s BioMark technology. Rain-dance Technologies uses a similar method for tar-geted sequencing by sequence enrichment.

Figure 3 (online colour at: www.biophotonics-journal.org)Disposable Enterics Card (DEC), an example of a sample-to-answer device for molecular bacterial pathogen detec-tion. Reproduced from PATH website at www.path.org/projects/microfluidics_card.php [27. 02. 2009].(Image was kindly provided by Patrick McKern).

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3. mTAS for viral detection

3.1 Integrated reverse transcriptionPCR systems

As mentioned above reverse transcription PCR (RT-PCR) is a crucial part of many viral detection sys-tems. Several approaches combined the RT-PCReither with on chip sample preparation or with a de-tection module, but up to now there are only fewcomplete systems including sample preparation, RT-PCR, target capturing, and detection on a single de-vice. Lien et al. combined RT-PCR and sample pre-paration modules in an integrated device for the de-tection of dengue virus and enterovirus 71 [54, 55].Antibody coated paramagnetic beads were used toisolate and concentrate virus particles out of clinicalserum samples. The automated purification and en-richment process using magnetic fields generated byplanar microcoils reduced the sample preparationtime from 48 min needed with traditional methodsdown to 10 min [56]. The microtemperature controlmodule reduced the required time for PCR amplifi-cation by 50% requiring an overall time for samplepreparation and RT-PCR of 3 h 20 min. The detec-tion of the amplified viral specific PCR products wasperformed externally, applying slab gel analysis. Thismicrofluidic chip has also been applied for bacterialdetection [57, 58]. Streptococcus pneumoniae couldbe successfully detected out of whole blood samplewith a detection limit of 102 CFU/mL. The detectionlimit for viral detection was 102 PFU/mL. This sys-tem has a high degree of multiplexing capability asthe bead bound capture antibody provide specificityand the PCR can be multiplexed. Lee et al. combineda RT-PCR with chemiluminescence detection in alab-on-a-chip system for human immunodefiencyvirus (HIV) detection [59]. The reverse transcriptionand PCR was performed on chip with an integratedtemperature control module consisting of an infraredlamp and a cooling fan. This enabled a fast heatingrate of 3 �C/s and a cooling rate of 1.8 �C/s which re-duced the time for RT-PCR to 35 min including twominutes reverse transcription. During the PCR stepbiotin was introduced into the PCR products via bio-tinylated primer. Amplified HIV p24 and gp120genes were hybridised towards surface bound probesin an integrated detection chamber. The hybridiza-tion event was detected via a streptavidin-horse rad-ish peroxidise (HRP) conjugate and a HRP sub-strate. The emitted light was detected on chip with aportable optical detection system consisting of a lensand a photodiode. The authors describe an overallassay time of less than 1h, whereas the sample pre-paration was not included in this lab-on-a-chip sys-tem. This system with surface bound probes could

easily be extended to a multianalyte detection plat-form. Kaigala et al. reported the possibility to detecthuman polyoma BK virus in unprocessed urine sam-ples with an integrated PCR-capillary electrophor-esis (CE) chip [60]. The only sample preparationstep was a 100 times dilution of the urine samplewith buffer. A 293 bp fragment was amplified apply-ing fluorescence labelled primer with a dual thermo-electric module temperature cycling system for PCRtemperature control with heating rates of 5–6 �C/sand cooling rates 3–4 �C/s. A non-denaturing poly-mer was used as sieving matrix in an integrated CEchip to detect the amplified PCR product. Fluores-cence from VIC dye-labelled PCR products wasmeasured. The detection sensitivity of the PCR-CEchip was ten copies of the BK virus in the on-chipPCR reaction mixture (5�105 copies/mL). Recently,the authors reported a ‘shoe-box’-sized portableplatform which included all required microfluidics,optics, and electronics parts for RT-PCR and CE de-tection for component costs of around $1000 [61].Zaytseva et al. developed a microfluidic biosensormodule for the detection of dengue viruses [62]. Theoff-chip amplified target RNA was captured byprobe oligonucleotides which were bound to mag-netic beads. Liposomes bound to universal reporteroligonucleotide probes were used for fluorescencedetection. The applied liposomes caused significantsignal amplification as a single liposome entrappedabout 105 fluorophores. The total time required foran assay was only 15 min. Pal and co-workers devel-oped an integrated device for genetic analysis [63].The combination of a PCR module and a tempera-ture controlled restriction digestion chamber en-abled the discrimination of influenza A/LA/1/87strains from influenza A/Sydney/5/97. A 690 bp frag-ment of the hemmaggultinin gene (HA1) was ampli-fied and digested with HpaI endonuclease. The di-gestion products were electrophoretically separatedand detected by their fluorescence with an externalCCD camera. The use of thin film platinum resistiveheaters and the high thermal conductivity of siliconresulted in high heating and cooling rates of 20 �C/s.This resulted in a very fast PCR time of 22 min for35 cycles.

3.2 Sample-to-answer RT-PCR systems

A complete system to detect the avian influenzavirus H5N1 was developed by Pipper and colleagues[64]. Sample preparation, RT-PCR and fluorescencedetection were done in droplets on a Teflon coateddisc, which is placed on a microfabricated heaterwith an integrated optical detection system (Fig-ure 4). The droplet itself became to a solid-phase ex-tractor and real-time thermocycler. Silica-coated


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superparamagnetic beads were used to isolate, purifyand concentrate the viral RNA from throat swabsamples. The purified viral RNA was then directlyreleased into a droplet, containing the RT-PCR mix-ture at processing temperature. Sealing the dropletwith mineral oil prevented the aqueous phase fromevaporating [65]. Thermocycling was performed bymoving the droplet clockwise over different tem-perature zones. The heating and cooling ratesachieved with this system were 11.5 �C/s and 5.6 �C/s, respectively. SYBR Green was used to monitorthe product formation in real-time with an assay sen-sitivity comparable to commercially available tests.The whole process took only 28 min.

3.3 Integrated immunodetection systems

In addition to PCR based methods several groupsdeveloped immunoassays for detection of viruses.Reichmuth et al. have very recently reported a sys-tem combining open channel electrophoresis and la-ser-induced fluorescence detection with a labelledantibody applied to detect swine influenza virus [66].Antibodies bound to the influenza virus were con-centrated in the gel matrix and thus separated fromunbound antibodies. Introduction of a nonporousmembrane improved detection sensitivity by a factorof four relative to the open-channel electrophoresisassay. The total assay time, including device regen-eration, is six minutes utilizing less than 50 mL of ma-terial. Liu and co-workers used a flow-through mi-crofluidic device and antibody-coated microbeads todetect marine fish iridovirus [67]. Virus particle werecaptured on the antibody-coated beads followed byincubation with specific secondary rabbit antibody

and quantum dot conjugated anti-rabbit IgG. Theconjugates were trapped in a microbead-trapping fil-ter followed by washing and fluorescence detection.This relative simple method improved the detectionsensitivity compared to a conventional ELISA testfrom 360 ng/mL down to 22 ng/mL and shortenedthe time-to-result from 3.25 h to 30 min and substan-tially reduced the amount of antibodies required forthe test. However, this approach has limited multi-plexing capability. Wang and Lee integrated micro-pneumatic valves and peristaltic micro-pneumaticpumps in a diagnostic chip in order to flow serumsamples and test reagents through a reaction/detec-tion area with immobilized antigens [68]. Theauthors showed the multiplexing capability of thesystem by detecting hepatitis C virus and syphiliscausing bacteria on the same chip in parallel. Boundserum antibodies were detected with secondaryHRP-conjugated antibodies. The sensitivity of thesystem was comparable with that of conventional 96-well plate tests with a time-to-result of 20 min.

3.4 Sample-to-answerimmunodetection systems

A complete system with multiplexing potentialbased on a sandwich immunoassay was developedby Yang et al. [69]. They combined a sample pre-paration module based on antibody-coated mag-netic beads with a micro flow cytometer for detect-ing dengue viruses. The virus particles trapped onthe antibody-coated magnetic beads were incubatedwith a secondary fluorescence-labelled antibody.The system showed good specificity and a limit ofdetection of 103 PFU/mL. The overall process in-cluding sample preparation and virus detection tookonly 40 min.

4. Optical detection systems for integratedpathogen detection

Optical methods offer, in principle, the prospect todetect pathogens at high levels of specificity and sen-sitivity in media that have potentially complex orpoorly characterized environments. The varieties ofoptical techniques used in combination with micro-fluidic devices have been recently reviewed byMyers [26] and Kuswandi [70]. Among those meth-ods fluorescence techniques offer some advantagesin terms of sensitivity and multiplexing capabilities.In this section we review fluorescent based techni-ques that have been used for the detection of patho-gens on chip-based platforms.

Figure 4 (online colour at: www.biophotonics-journal.org)Example of a microsystem for influenza virus detection in-cluding sample preparation and RT-PCR on a disc devel-oped by Pipper and colleagues [64]. Reprinted from Ref.[64] with permission from the Nature Publishing Group(Copyright 2007).

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4.1 Laser induced fluorescence

A large number of pathogens have been detectedusing microfluidic genetic analysis on PCR-CE plat-form [42, 47, 60] with novel integration of the techni-que allowing for dozens of gene transcripts to be de-tected in a multiplex format [71]. In those caseselectrophoresis detection is conducted by laser in-duced fluorescence (LIF). Briefly, in this technique,the samples are labelled with a particular fluoro-phore, and the fluorescence signals induced by a la-ser source are then detected as the samples flowthrough a separation channel. Conventional LIFtechnique utilizes multiple optical components con-sisting of a light source (Laser), excitation filters, di-chroic mirrors for the separation of excitation andemission channels, emission filter, and a detectorwith electronics for signal processing. For practical,integrated systems the amplitude of the excitationlight has to be kept low to avoid photobleaching.Therefore, a high gain amplification of the fluores-cence signal is required. Appropriate detectorsfound in fluorescence detection systems are photomultiplier tubes (PMTs), avalanche photodiodes,and photon counting modules (PCMs). Light cou-pling and photon collection to and from the micro-fluidic device can be performed with a confocal mi-croscope [42], or directly through optic fibers [57].Miniaturization of the fluorescence detection appa-ratus has been achieved by combining chip-inte-grated photodiode detectors coupled to a lock-in-amplifier [72, 73]. Such systems have been tested ondifferent pathogens such as MRSA and E. coli sys-tems with sensitivity in the low nanomolar range.Multiplexing capabilities have also been integratedusing multichannel disposable chip with integratedphotodiode [74].

4.2 Evanescent wave excitation

Common to all fluorescence-based detection modal-ities is the requirement that the optical signal repre-senting a given recognition event must be separablefrom the potentially high autofluorescence back-ground characteristic of most biological samples. Theuse of so-called single mode planar optical wave-guides has emerged as a powerful tool in fluores-cence-based pathogen sensors [75–79]. These arestructurally simple devices comprising a high indexdielectric layer on a low-index substrate. Machineddiffraction gratings can be used to couple monochro-matic light into the device. The operational principlerelies on the fact that not all the optical radiation isconfined within the guide. A very small portion leaksout into the biological material. This evanescent field

is very rapidly attenuated over length scales of a fewhundred nanometers. This geometry provides a nat-ural spatial filtering of the excitation thereby sup-pressing the production of background fluorescencesignal. Despite this rapid decay, optical field intensi-ties at the sample interface can be very high and, forcertain waveguide materials, it has been demon-strated that detection sensitivities using single-modeevanescent excitation can exceed that of confocalmicroscopes [75]. This also implies that low dye con-centrations are detectable. In recent years, effortshave been made to integrate evanescent wave exci-tations by total internal reflection into microfluidicsdevices using micro optics [80], air mirrors [81] andultimately by integrating all miniaturized opticalcomponents into a monolithic chip [82–84]. Evanes-cent wave excitation is particularly relevant to sur-face based assay formats such as DNA microarraysthat possess huge multiplexing potential. Polymermicrofluidic chips with integrated waveguides havealso been designed for the reading of such microar-rays [79].

4.3 Foerster-resonance energy transfer

Waveguide techniques exploiting fluorescence de-tection have now also been extended to use Foer-ster-resonance energy transfer as a higher informa-tion content reporter of binding events. FRET(Foerster (or Fluorescence) Resonance EnergyTransfer) is a technique for measuring interactionsbetween two molecules. In this technique, two dif-ferent fluorophores are tethered to two moleculesof interest. FRET is a distance-dependent interac-tion between the electronic excited states of thetwo dye molecules in which excitation is transferredfrom a donor molecule to an acceptor moleculewithout emission of a photon. The efficiency ofFRET is dependent on the inverse sixth power ofthe intermolecular separation, rendering a powerfulstructural and binding probe sensitivity over dis-tances comparable with the dimensions of biologicalmacromolecules. When FRET is used as a contrastmechanism, colocalization of proteins and other mo-lecules can be imaged with spatial resolution be-yond the limits of conventional optical microscopy.Also non trivial, the multiplexing capabilities ofFRET detection have recently been demonstratedfor the detection of DNA hybridization [85] and in-tegration of the technique in microfluidics has alsobeen reported [86, 87]. Rudimentary multiplexedmembrane-based assays based on waveguidecoupled FRET binding events involving multiple re-ceptors have been achieved for glycolipid GM1 – acellular receptor for cholera [88].


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4.4 Fluorescence cross-correlationspectroscopy

Emerging technologies based on fluorescence detec-tion include the family of microscopy-based methodsmaking use of dynamic transport and mobility meas-urements with the view to detect changes related tobinding events. In fluorescence cross-correlationspectroscopy (FCCS) one measures the temporalfluorescence fluctuations due to differently labelledmolecule pairs diffusing through a small sample vo-lume. Cross-correlation optical signals from separatedetection channels extract information of the dy-namics of the dual-labelled molecules. In the case ofpathogen detection for example, FCCS is implemen-ted by labelling both reactants (ligand and virus)with fluorophores of separate colors. Both fluoro-phores are excited, and the fluorescence intensityfluctuations from both reactants are monitored si-multaneously. The cross-correlation amplitude be-tween the two reactants is derived as a function ofdelay time. When the two reactants with separatecolors bind, they will diffuse in and out of the obser-vation volume together, leaving (perfectly) positivelycorrelated fluorescence fluctuation traces. Zhanget al. have used FCCS to probe dengue virus in a na-nomolar bulk solution by following the specific asso-ciation of dengue antibody [89]. This was performedin a microfluidic chamber. Moreover, they differen-tiated the compartments containing the dengue virusand the virus-free compartments. They suggest thatby expanding the throughput using microfluidic de-vices integrated with FCCS, there are realistic pro-spects for detecting single viral particles in humanbody fluids in the near future.

4.5 Single molecule detection

Pathogen detection required ever increasing sensitiv-ities and thus, the ultimate achievement would bethe detection of individual pathogen entities in frontof a sample background. Enumeration of bacterialpathogen Vibrio cholerae biomolecules has beenachieved by combining Single Molecule Detection(SMD) with confined nucleic acid amplificationwhich converts nanometer scale target molecules tofluorescent micrometer size DNA molecules [90].During the amplification stage two separate probesare brought to proximity with the target (DNA orprotein) and joined by enzymatic ligation, resultingin the formation of a unique circular DNA moleculewhich is used as a template in a rolling circle amplifi-cation (RCA) reaction. After hybridization withfluorescent-tagged probes, the RCA product is de-tectable as a bright object of approximately 1 mm in

diameter. The method allows for separate circulartemplates to be formed corresponding to uniqueanalyte entities and hybridization of detectionprobes of distinct colors would allow multiplexedsignal readout. Individual DNA molecules were de-tected and quantified by pumping a sample througha thermoplastic microchannel mounted in a standardconfocal fluorescence microscope operating in line-scan mode. In their work, Jarvius et al. were able todemonstrate analyte number measurements overseven orders of magnitude.

5. Conclusion

The detection of viral and bacterial pathogens withintegrated devices is a particular challenge for thedesign and realisation of lab-on-a-chip devices. Overthe last decade substantial achievements drove astep change from solutions for individual assay mod-ules towards systems which integrate a completesample-to-answer process. Examples have been giv-en for bacterial and viral targets. For the latter im-munoassay based schemes are still very frequentnext to molecular assays based on RT-PCR. At thesame time transduction methods rely heavily on op-tical techniques, which offer a number of modalitiesallowing combining multiplex detection capabilitiesand high sensitivity. This combination of optical de-tection and molecular diagnostics has still a great po-tential for exploitation in lab-on-a-chip devices. Itcan be anticipated that future studies will increas-ingly target this field to arrive at the next level ofdiagnostic tools to pave the way towards a systemsapproach to diagnostics and therapy.

Till Bachmann is Head ofBiochip Research and ChiefOperating Officer at the Di-vision of Pathway Medicineof the University of Edin-burgh. His scientific interestlocates at the interface ofmedicine and biochip re-search with a key focus onmolecular diagnostics of in-fectious diseases with multi-

parametric detection technologies. He has a Ph. D inbiosensors obtained from research conducted atUniversity of Stuttgart and the University of Tokyo anda German Habilitation in Analytical Biotechnology. Hehas extensive experience in research and scientificmanagement in particular of interdisciplinary diagnos-tic projects with a strong focus on translational activ-ities.

J. Biophoton. 2, No. 4 (2009) 207



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