Analytica Chimica Acta 556 (2006) 171–177

Microbial detection in microfluidic devices through dual staining ofquantum dots-labeled immunoassay and RNA hybridization

Qing Zhanga, Liang Zhua, Hanhua Fengc,Simon Angd, Fook Siong Chaub, Wen-Tso Liua,∗

a Division of Environmental Science and Engineering, National University of Singapore, Blk E1A,#07-03, Engineering Drive 2, Singapore 117576, Singapore

b Department of Mechanical Engineering, National University of Singapore, Singaporec Institute of Microelectronics, Singapore

d Department of Electrical Engineering, University of Arkansas, Fayetteville, USA

Received 10 March 2005; received in revised form 2 July 2005; accepted 6 July 2005Available online 15 August 2005

Abstract

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This paper reported the development of a microfludic device for the rapid detection of viable and nonviable microbial cells throabeling by fluorescent in situ hybridization (FISH) and quantum dots (QDs)-labeled immunofluorescent assay (IFA). The coin sizonsists of a microchannel and filtering pillars (gap = 1–2�m) and was demonstrated to effectively trap and concentrate microbial ceiardia lamblia). After sample injection, FISH probe solution and QDs-labeled antibody solution were sequentially pumped into th

o accelerate the fluorescent labeling reactions at optimized flow rates (i.e. 1 and 20�L/min, respectively). After 2 min washing for each asshe whole process could be finished within 30 min, with minimum consumption of labeling reagents and superior fluorescent signahe choice of QDs 525 for IFA resulted in bright and stable fluorescent signal, with minimum interference with the Cy3 signal froetection.2005 Elsevier B.V. All rights reserved.

eywords: Microfluidic device; FISH; Immunofluorescent assay; Quantum dots;Giardia lamblia

. Introduction

Combined immunofluorescent assay (IFA) and florescentn situ hybridization (FISH) have been successfully demon-trated for the detection of microbial cells[1]. IFA stainingrovides strong signal intensity, while FISH staining is highlypecific and could be used to differentiate viable and nonvi-ble cells[2]. However, normal practices performed on glasslide or in test tube often require multiple reactions and wash-ng steps, which are considered to be time consuming, reagentonsuming and labor intensive[3,4].

Alternatively, rapid IFA could be achieved by performinghis assay on microfluidic or lab-on-a-chip devices[6,7]. Oneimple approach is to create sample inlet and outlet, microflu-

∗ Corresponding author. Tel.: +65 68741315; fax: +65 67791635.E-mail address: cveliuwt@nus.edu.sg (W.-T. Liu).

idic channels and weir-type trapping region on a silicon-bdevice [7]. After injecting sample solution containing ttarget cells in the microchannel, these cells can be meically trapped at the weir region, and be labeled by flusthe microchannel with a solution containing fluorescconjugated antibody specific to the target cells. After bwashing, these fluorescently labeled target cells can bedetected under a fluorescent microscopy at single cellSince all the reactions were performed in micro-scales,ple and reagent consumption could be reduced significaBy employing a flow through format, assaying time coalso be reduced from hours to minutes. Further integraautomation and parallel processing of multiple samplealso possible based on these microfluidic platforms[8,9].

Based on the concept described above, this study hather demonstrated that IFA and FISH could be perforin sequence on a microfluidic filter-based device to ach

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.07.003

172 Q. Zhang et al. / Analytica Chimica Acta 556 (2006) 171–177

rapid detection of viable and nonviable microbial cells. Thedevice consisted of a microchannel and filtering pillars totrap and concentrate the model microbial cells (i.e.Giardialamblia). To further increase the signal intensity and repro-ducibility of IFA, semiconductor quantum dots (QDs) havebeen recently introduced as a novel inorganic fluorescencedye. QDs are small nano-particles (ca., 2–50 nm in size) withnarrow, symmetrical and tunable emission spectra and canbe excited by a wide spectrum of wavelength[10]. With theexcellent emission property, signal intensity and photostabil-ity, QD label can be easily differentiated from autofluores-cence particles present in environments, thereby appearing tobe a preferred fluorescence dye in the IFA detection of pro-tozoa cells[11]. It is expected that these microfluidic devicescan be potentially used in the rapid diagnostic of clinical sam-ples, and in the monitoring of water quality and public healthin environments.

2. Experimental

2.1. Design, fabrication and simulation of microfluidicdevice

Fig. 1 illustrates the design and features of the microflu-idic device used. The device (20 mm× 10 mm) consists ofa sc nd,

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MI). The base plate contains a reaction chamber (50�m indepth), an inlet (1 mm in width), an outlet (1 mm in width), acoarse filter region and a trapping filter region for microbialcells. The coarse screen which is designed for pre-filtratinglarge impurities from the sample contains six rows of pillar-type filters 500�m apart (Fig. 1b). The gaps between any twopillars gradually decrease from 50�m in the first two rows,to 30�m in the third and fourth rows, and 20�m in the lasttwo rows. The microbial cell-trapping region consists of twosymmetric fine pillar-type filters. Both filters are made of pil-lars with a gap of 1–2�m between any two pillars (Fig. 1c).Its symmetric design is for the purpose of further reducingthe chance of clogging both trapping filters. The device wasfabricated using semiconductor processing technology[12]at the Institute of Microelectronics, Singapore. The reactionchamber and pillars were fabricated by photolithographyand deep reactive ion etching (DRIE). The inlet and outlet(at the bottom of the plate) were etched by anisotropicSi etching, with a patterned nitride layer as the etchingmask. A surface-polished Pyrex glass cover was anodicallybonded to the etched silicon prior to dicing into individualchips. Chip holder and tubing connection were similar tothose described previously[7]. A heating plate (Instec Inc.,Boulder, CO) with a thermocouple attached onto the surfaceof the microchip was used to control the temperature of themicrochip.

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650�m thick silicon base and a 500�m thick Pyrex glasover (corning 7740, Dow corning corporation, Midla

ig. 1. Illustration of the microfluidic device design. (a) Top view ofesign showing the four main regions: inlet, cell-trapping filter region, colter region and outlet. (b) Part of the coarse filter region. The spacing las d1 between each pillar was varied for each two rows as 20, 30, 5�m,

espectively. (c) Part of the microbial cell-trapping filter region. The spacingabeled as d2 between each pillar was 1–2�m. The size of each pillar was0�m× 10�m.

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The fluidic flow in the microchannels and the coarse fiegion was simulated using Fluent 6.1 (Fluent India, Pndia). The width of the channel is 50�m, and the gaetween any two given diamond-shaped pillars in the cha

s 2�m. The inlet velocity was set 2 mm/s and the fluid wssumed to be water.

.2. Testing materials and reagents

Concentrated water sample was prepared by filteL of tapped water through 1�m hollow fiber filter andluting the concentrate into 1 mL of mQ water. Fixed via. lamblia cysts in 5% formalin (∼106 cells/mL) andiotinylated monoclonal antibody A300BIOT specific toG.

amblia were purchased from Waterborne Inc. (New OrleA). Prior to the experiments in the microfluidic devi. lamblia cysts were pretreated in 50% ethanol/PBS0◦C for 20 min to increase the cell wall permeability[13].D-conjugated antibody solution was prepared accordi

he manufacturer’s protocol (Quantum Dot Corporationrief, 1�L of streptavidin-coated QD525 (2 mM, Quantot Corporation, Hayward, CA), 5�L of antibody solutionnd 94�L of QDs dilution buffer (supplied with QDs) weixed and incubated at 37◦C for 1 h. No further purificatio

tep was performed because the quantity of QDs can sall the binding sites of antibodies in the solution. Cy3 labligonucleotide probe (5′-CGGCGGGGGGCCAACTAC-3′

13]) targeting the 18S rRNA ofG. lamblia was pur-hased from Qiagen Operon GmbH (Cologne, Germall the solutions were filtered through a 0.2�m mem-

Q. Zhang et al. / Analytica Chimica Acta 556 (2006) 171–177 173

brane filter immediately prior to use in the microfluidicdevice.

2.3. Principle and procedures of combined FISH andIFA in microfluidic device

The concept for trapping and dual labeling of the micro-bial cells within the microfluidic device was illustrated inFig. 2. Initially, the procedures for performing FISH andIFA were individually optimized. For FISH assay, a newmicrofluidic chip was washed with 1× PBS for 2 min ata constant flow rate using a syringe pump (KDS100, KDScientific, Boston, MA). Then, 1–5�L of solution contain-ing approximately 1–5× 103 Giardia cysts were deliveredinto the device using a micro-syringe. Simultaneously, FISHprobe solution (0.5 ng/�L of oligonucleotide probes whichis 10 times diluted compared with the probe concentrationof FISH assay on conventional platform, 0.9 M of NaCl,20 mM of Tris/HCl, and 0.01% SDS) was pumped into thedevice at a constant flow rate up to 40 min at 48◦C. Becausethe size ofGiardia cysts (7–10�m in width and 8–13�min length) was much larger than the gap between any twogiven pillars (1–2�m), Giardia cysts were trapped in frontof the pillars and retained in the chamber by the flow pressuretoward the pillars. After hybridization, the chip was washedw

IFA, target cells (∼1–5× 103 cells) were injected into a newmicrofluidic device. The chip was flushed with a labelingsolution containing QD-conjugated antibody at a flow rate of20�L/min up to 15 min, and subsequently washed with PBSat a flow rate of 20�L/min for 2 min. Detection of FISH andIFA signal was performed using a microscopy-based imagingsystem. Real-time monitoring of FISH and IFA were per-formed and monitored. After separately optimizing the FISHand IFA procedures, these two procedures were performed insequence as shown inFig. 2.

2.4. FISH and IFA in solution

For FISH analysis, 1–5�L of the pretreatedG. lambliacysts and 10�L of FISH probe solution containing 5 ng/�Lof fluorescently labeled probes were mixed in a 1.5 mL cen-trifuge tube and incubated at 48◦C for 20 min. The hybridiza-tion was stopped by adding 40�L of PBS (pH 7.4) solutioninto the tube. For IFA, 1–5�L of G. lamblia cysts was mixedwith 10�L of 1× QD-conjugated antibody solution in a1.5 mL tube, and incubated at 37◦C for at least 1 h. Bothassays were subjected to a final wash by centrifugation. Thelabeled cells were then dissolved in PBS solution, spottedonto a glass slide, and examined using an epifluorescencem

ith PBS for 2 min at a flow rate of 20�L/min. To perform

Fig. 2. Schematic illustration of the principle to conduct QDs-la

icroscopy.

beled immunoassay with FISH assay in the microfluidic device.

174 Q. Zhang et al. / Analytica Chimica Acta 556 (2006) 171–177

2.5. Imaging system and signal quantification

An Olympus BX51 epifluorescence microscope equippedwith a cooled CCD camera SPOT-RT Slider (DiagnosticInstruments Inc.), a 100 W HBO bulb, and fluorescence fil-ter sets (U-MWU2, U-MWB2, and U-MF2) was used forthe FISH analysis. To observe QD signals, Chroma QDs fil-ter sets (32003 and 32005) (Chroma Inc., USA) were used.The image analysis software, MetaMorph (Universal Imag-ine Corporation, USA), was used to control the camera and toperform image analysis. Fluorescence images were taken atan exposure time of 500 and 100 ms for FISH and IFA, respec-tively. Based on the captured images, the signal was quanti-fied. Signal-to-noise ratio (S/N) of fluorescently labeled cellswas calculated by dividing the whole-cell signal intensitywith its nearby background signal intensity.

3. Results

3.1. Microfluidic device

Fig. 3a and c show the scanning electron microscope(SEM) images for the cell-trapping filter region and the coarsefilter region. The two pillar-type trapping regions were fab-ricated toward to the end of the microchannels (Fig. 3a). The

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Fig. 3. SEM images of the fabricated pillar-type microfilter on silicon. (a)The microbial cell-trapping filter region. (b) The zoom view of the fine filterregion. (c) The coarse filter region. (d) The zoom view of the coarse filterregion.

distance between any two given pillars (Fig. 3b) was approximately 1.5�m and the depth of the pillars was 50�m. Thecoarse filter region (Fig. 3c) was made of arrays of diamonshape pillars (30�m× 30�m) with a space of 20, 30 o50�m between any two given pillars (Fig. 3d).

The diamond-shape coarse filters in the device maserved as two functions. The first function was to trap lparticles in the fluidic sample to avoid large impurities reaing the fine pillar region and clogging the device. To validthis, concentrated water sample was injected into the dto fully saturate the blocking region provided by the coafilter, and followed by flushing with PBS for 5 min.Fig. 4ashows an image at the coarse filter region after sample ition under visible light. Large debris or particles were cleblocked by those coarse pillars. As the result, only few lparticles were observed in the fine pillar region, and thewas not affected by the trapping of the large particles incoarse filter region (Fig. 4b). However, it should be noted ththis would be an extreme case to demonstrate the functithe coarse filter. The second function of the coarse filterto facilitate the distribution of fluid inside the microchanel. This was validated through the simulation of fluid flinside the microchannel as shown inFig. 4c. Prior to therow of coarse filter, the flow velocity at a cross sectionobserved to be higher in the middle of the channel than aedge. By introducing a row of coarse filters, the uniformitthe flow velocity at the edge and the center between anygiven filter was greatly improved. This improvement cofurther facilitate mixing and movements of particles inflow before reaching the fine cell-trapping region.

Q. Zhang et al. / Analytica Chimica Acta 556 (2006) 171–177 175

Fig. 4. (a) Part of the coarse filter region under normal visible light aftersample injection. (b) Part of the fine filter region under normal visible lightafter sample injection. (c) CFD simulation showing the contours of velocitymagnitude (m/s) in a microchannel.

3.2. FISH assay in microfluidic device and in tube

The labeling efficiency of FISH inside the microfluidicdevice was evaluated at different pumping rates (i.e. 0.5, 1,2, 5, 10 and 20�L/min) using a Cy3-labeled oligonucleotideprobe at a concentration of 0.5 ng/�L. Fig. 5a shows thata flow rate of 1�L/min gave the highest end-point signal-to-noise ratio (S/N) ratio, 11.4, for whole cell FISH. TheS/N ratio decreased when the flow rate was decreased to0.5�L/min or increased gradually to 20�L/min. The S/Nratios of FISH obtained in tube were about 2.0 and 7.4 usinga hybridization solution containing 0.5 or 5 ng/�L of fluores-cently labeled oligonucleotide probe, respectively (Fig. 5b).Thus, the S/N ratio obtained in microfluidic device with10 times diluted probe concentration under a flow rate of1�L/min was 1.5–5.7-fold higher than that obtained in tube.

Fig. 5. Effect of hybridization flow rate in FISH assay on the labelingefficiency. (a) Effect of hybridization flow rate on the FISH signal. (b) Com-parison of FISH signal in the microfluidic device with that in tube. The flowrate used in the microfludic device is 1�L/min. For experiments evolved inboth figures, the hybridization time was the same (20 min). (c) Real-timemonitoring of the FISH signal at hybridization temperature of 37, 48 and60◦C. The two dashed arrow lines showed the S/N ratio after 2 min washing.

The effect of hybridization temperature on labeling effi-ciency was further evaluated at a flow rate of 1�L/min underdifferent hybridization temperatures (i.e. 37, 48 and 60◦C).Fig. 5c indicated that the S/N ratio obtained at 48◦C wasalways higher than that obtained at 37 and 60◦C. Real-timemonitoring those fluorescence labeled cells at an interval of5 min for 40 min revealed that the S/N ratio rapidly increasedfrom 1 to 3.9 within the first 5 min, and leveled off at a plateauaround 5 from 10 min onward (Fig. 5c). Subsequent washingat a flow rate of 20�L/min for 2 min resulted in a signif-icant increase in the S/N ratio from 5.8 to 11.5, because ofthe reduction of background noise attributed by the unlabeledfluorescence probe in the bulk solution. We also stopped thehybridization process after 10 min, and observed an increasein S/N ratio from 4 to 7.9 after 2 min of washing. This sug-gested that a hybridization time of 10 min was sufficient forFISH in the microfluidic device.

176 Q. Zhang et al. / Analytica Chimica Acta 556 (2006) 171–177

3.3. QD-based IFA in microfluidic device

For QDs-based IFA, QDs with emission peak at 525 wasselected as the label to minimize the possible overlappedspectrum of emission from Cy3-labled FISH assay. The label-ing efficiency of IFA inside the microfluidic device wasevaluated using 0.1× QDs conjugated antibody solution atdifferent pumping rates. We initially tried different flow ratesfrom 20 up to 100�L/min. However, the variation of theobserved S/N values was high. The microfluidic chips wereoften broken during the reagent injection process due to highflow resistance in the filter chamber. Cell-like debris wassometimes observed on the other side of the cell-trappingpillars after the labeling process, indicating that some of thecells were broken and squeezed through the filter region.Thus, flow rates at or below 20�L/min were tested togetherwith different labeling antibody concentrations to obtain opti-mized conditions for IFA in microfluidic device.

Fig. 6 shows the S/N ratio of those QDs-labeled cells asdetermined during the real-time monitoring of the labelingprocess. Using a labeling solution containing 0.1× antibody,the highest S/N ratio at any given time was observed under aflow rate of 20�L/min. The S/N ratio gradually increasedfrom 1.1 to 19.8 in the first 8 min and remained almostunchanged for 7 min. The labeling efficiency reduced alongwith the decrease in the flow rate or labeling antibody con-ce oodd .

tion( r-m att eh inedf e oft h the0 -i utt rate

F n thei

of 20�L/min with 0.1× labeling antibody concentration wasselected for IFA in the combined analysis of FISH and IFA.

3.4. Combined FISH and IFA assay in microfluidicdevice

We finally performed the FISH and IFA in sequence in themicrofluidic device. The assay was completed within 30 min.Fig. 7 shows that viableG. lamblia cysts were labeled byFISH (panel a) and IFA (panel b).

4. Discussion

This study has clearly demonstrated that the use ofmicrofluidic device as a platform could achieve rapid bio-chemical reactions or microbial cells detection. For IFA, rapiddetection was likely achieved by improving the mass trans-fer rate for the labeling reagent (antibodies) in the solutionto bind onto the target sites (i.e. antigens) on the surface ofmicrobial cells. Thus, the time required to achieve a high S/Nratio could be shortened by increasing either the concentra-tions of the labeling reagent or the flow rates of the labelingsolution (Fig. 6). In contrast, for FISH assay, the highestlabeling efficiency was not obtained with the highest flowrate tested (Fig. 5a). Our results suggested that the labelinge etedR an

F gpillars after the combined QDs-labeled IFA and FISH in the microfluidicdevice. The images were taken under fluorescent filter sets for QD 525 andCy3, respectively: (a) IFA signal and (b) FISH signal.

entration. When there was no flow (0�L/min), the labelingfficiency based on S/N ratio was too low to provide gistinction between background noise and labeled cells

The effect of the QDs-conjugated antibody concentrai.e. 0.01×, 0.1×, 0.5×) on the S/N ratio was further deteined under a flow rate of 10�L/min. Results indicated th

he S/N ratio obtained from a 0.5× antibody solution was thighest in the first 5 min, but was lower than that obta

rom 0.1× labeling solution after then. This was becaushe comparatively high background signal associated wit.5× antibody labeling solution. For 0.01× antibody label

ng solution, poor S/N ratio (∼1.0) was observed throughohe labeling process. Based on this observation, a flow

ig. 6. Effect of flow rate and concentration of the antibody solution ommunoassay signal.

fficiency was possibly affected by the presence of targNA molecules inside the cell wall. At a flow rate higher th

ig. 7. Fluorescent image of labeledG. lamblia cysts in front of the trappin

Q. Zhang et al. / Analytica Chimica Acta 556 (2006) 171–177 177

1�L/min, most of the oligonucleotide probes could possiblybe flushed away before they could diffuse into the cell walland hybridize with the rRNA targets inside. So, probe diffu-sion into the cell wall could be a rate-limiting step in additionto the temperature effect (Fig. 5c).

It was further demonstrated that not only the labeling pro-cess could be accelerated, but the washing steps could also begreatly simplified and shortened. Standard washing practicescarried out manually in tube or on glass slide require lengthyand repetitive incubation, centrifugation and re-suspendingsteps, which may take more than 20 min for a complete wash-ing step[4,5], whereas a one-step washing for only 2 min wasmore than enough to effectively remove the background noisein the microfluidic device (Fig. 5c).

Performance of the microfluidice device was seldomobserved to be affected by clogging of the trapping regionduring experiments. This was because the volume of sam-ple solutions (1–5�L) or the total number of microbial cellsintroduced into the microchannel was relative low. We alsoobserved that after sample injection, microbial cells wereusually evenly distributed and trapped in front of the cell-trapping pillars instead of accumulating at specific locationsof the pillar region. The incorporation of coarse filters beforethe cell-trapping region could also prevent large impuritiesreaching the trapping region and blocking the fluid flow. Inaddition, the pillar height (50�m) was almost five timesl , theo hosep ghth fine-p

horesc ingd mis-s QDsh ave-ld anicfl ever,

QDs are usually larger than organic fluorescent dye in size,and cannot easily penetrate or diffuse through cell membraneinto cell. Thus, Cy3 was selected as the fluorescent dye forFISH, and combined with QDs 525 for IFA to minimize pos-sible signal interference between IFA and FISH detection ina dual labeling assay.

In conclusion, this paper presented a lab-on-a-chip methodfor combined assay of FISH and QDs-labeled IFA for thedetection of microbial cells. Unlike normal methods thatare performed in tube or glass slide[2–4,11,13], the cur-rent method could provide advantages on (i) rapid labelingefficiency and detection time; (ii) low volume of reagentsneed (low cost); (iii) strong fluorescence signal intensity.Portable, automated and high throughput instruments couldbe ultimately fabricated based on the proposed microfluidicfilter-based device, with the integration of power system anddetection system.

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