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    A microfluidic nano-biosensor for the detection of pathogenic Salmonella

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    内容提示: A microfluidic nano-biosensor for the detection of pathogenicSalmonellaGiyoung Kimn , Ji-Hea Moon, Chang-Yeon Moh, Jong-guk LimNational Academy of Agricultural Science, Department of Agricultural Engineering, RDA, 150 Suin-Ro, Kweonseonku, Suwon 441-100, Republic of Koreaa r t i c l e i n f oArticle history:Received 3 June 2014Received in revised form28 July 2014Accepted 15 August 2014Available online 19 August 2014Keywords:MicrofluidicsNano-biosensorFood safetySalmonella Typhimuriuma b s t r a c tRapid de...

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    A microfluidic nano-biosensor for the detection of pathogenicSalmonellaGiyoung Kimn , Ji-Hea Moon, Chang-Yeon Moh, Jong-guk LimNational Academy of Agricultural Science, Department of Agricultural Engineering, RDA, 150 Suin-Ro, Kweonseonku, Suwon 441-100, Republic of Koreaa r t i c l e i n f oArticle history:Received 3 June 2014Received in revised form28 July 2014Accepted 15 August 2014Available online 19 August 2014Keywords:MicrofluidicsNano-biosensorFood safetySalmonella Typhimuriuma b s t r a c tRapid detection of pathogenic Salmonella in food products is extremely important for protecting thepublic from salmonellosis. The objective of the present study was to explore the feasibility of using amicrofluidic nano-biosensor to rapidly detect pathogenic Salmonella. Quantum dot nanoparticles wereused to detect Salmonella cells. For selective detection of Salmonella, anti-Salmonella polyclonalantibodies were covalently immobilized onto the quantum dot surface. To separate and concentratethe cells from the sample, superparamagnetic particles and a microfluidic chip were used. A portablefluorometer was developed to measure the fluorescence signal from the quantum dot nanoparticlesattached to Salmonella in the samples. The sensitivity for detection of pathogenic Salmonella wasevaluated using serially diluted Salmonella Typhimurium in borate buffer and chicken extract. Thefluorescence response of the nano-biosensor increased with increasing cell concentration. The detectionlimit of the sensor was 10 3 CFU/mL Salmonella in both borate buffer and food extract.& 2014 Elsevier B.V. All rights reserved.1. IntroductionContamination of food with pathogens poses a significantthreat to public health. Recent foodborne pathogen outbreaksfrom various food sources have increased public awareness ofthese pathogens. Salmonella is a major foodborne pathogen andSalmonella Typhimurium is the one of the most commonlyreported serotypes. S. Typhimurium is a gram-negative rod thatcauses self-limiting gastroenteritis in humans, and infection isassociated with symptoms of fever, abdominal pain, nausea andvomiting, diarrhea, dehydration, weakness, and loss of appetite.The symptoms may appear 12–72 h after consumption of acontaminated food or beverage. The pathogen is associated withraw or undercooked eggs, poultry, beef, and unwashed fruit. S.Typhimurium outbreaks continue to occur, and outbreaks fromvarious food sources have increased the need for simple, rapid,and sensitive methods to detect foodborne pathogens.Conventional methods for Salmonella detection and identifica-tion involve multiple, prolonged enrichment steps. Although someimmunological rapid assays are available, these assays still requireenrichment steps and give results in 18–48 h. Enzyme-linkedimmunoassay (ELISA) and polymerase chain reaction (PCR) arerobust but require several lengthy steps, expensive laboratoryinstruments, and experienced operators (Hart et al., 2011;Hossain et al., 2012). Biosensing methods, such as optical(Kramer et al., 2007), impedimetric (Radke and Alocilja, 2004;Varshney and Li, 2007; Yang et al., 2004), electrochemical(Setterington and Alocilja, 2012), and piezoelectric (Campbelland Mutharasan, 2008) methods, have shown great potential forrapid detection of foodborne pathogens.Among biosensing methods, fluorescent dye-based opticalimmunoassays have been widely used because they can be highlysensitive and simple to operate (Geng et al., 2004; Kim et al., 2007;Kramer and Lim, 2004). However, methods that rely on organicfluorescent dyes are limited by their sensitivity to photobleaching,which limits long-term analysis. They also have a narrow excita-tion bandwidth and overlapping emission profiles from differentfluorophores, which limits multiplexed applications (Kampaniet al., 2007). Recently developed nanotechnology has the potentialto overcome the limitations of organic fluorophores. Fluorescentnanoparticles and quantum dots (QDs) have several advantagesover conventional organic dyes, such as high quantum yield andbrightness, photostability, and resistance to chemical degradation.Semiconductor QDs exhibit size- and composition-dependentfluorescence properties that are suitable for multi-target andhighly sensitive imaging using single excitation wavelengths(Tallury et al., 2010). The water solubility of QDs makes themsuitable for biological applications, including imaging, detection,and biomolecular conjugation. Several groups have reported theapplication of QDs for detection of microorganisms, includingEscherichia coli O157:H7 (Su and Li, 2004; Wang et al., 2012; Zhuet al., 2012), Campylobacter jejuni (Bruno et al., 2009), ListeriaContents lists available at ScienceDirectjournal homepage: www.elsevier.com/locate/biosBiosensors and Bioelectronicshttp://dx.doi.org/10.1016/j.bios.2014.08.0230956-5663/& 2014 Elsevier B.V. All rights reserved.n Corresponding author. Tel.: þ82 31 290 1899; fax: þ82 31 290 1900.E-mail address: giyoung@korea.kr (G. Kim).Biosensors and Bioelectronics 67 (2015) 243–247 monocytogenes (Wang et al., 2007), and avian influenza viruses(Chou and Huang, 2012).To increase the sensitivity of detection, many researchers haveused immunomagnetic separation (IMS) methods. In this techni-que, antibodies conjugated to superparamagnetic beads captureanalytes and are collected from the sample solution to concentratethe target analytes. Rotariu et al. (2005) designed a flow-throughimmunomagnetic separator that consists of a ferromagnetic wireand a silicone tube to improve the recovery of E. coli O157 fromlarge-volume samples. Yang and Li (2006) separated E. coli O157:H7 and S. Typhimurium from samples by using specific antibody-coated magnetic beads. Quantum dots were used as fluorescencelabels in immunoassays for simultaneous detection of two speciesof bacteria.Several studies also coupled immunomagnetic separation withQD labeling (Dudak and Boyaci, 2008; Liandris et al., 2011; Zhaoet al., 2009). The IMS method effectively separates target analytesfrom the sample; however, many steps (such as reaction, washing,collection with external magnetic force, and dispersion in buffersolution) are involved in the separation process, and analytes maybe lost between these steps.An increasing research effort has focused on the use of micro-fluidic devices to further improve the sensitivity of detection.Microfluidic devices are mainly used to enhance analytical perfor-mance by miniaturization. Reduction of the size of the devicesprovides additional advantages, such as reduced consumption ofreagents and the ability to integrate several analytical stepstogether (Mairhofer et al., 2009). Microfluidic systems are able toefficiently and rapidly obtain measurements from small volumesof complex fluids, and they can be used to concentrate pathogensinto a small volume and remove interfering foreign materials fromthe sample (Ramadan and Gijs, 2012). Many reports have indicatedthat microfluidic devices enhance the sensitivity of bacterialdetection. Liu et al. (2005) improved the minimal detectableconcentration of a virus by using antibody-coated microbeadsand a pillar-type filter-equipped microfluidic system. Zaytsevaet al. (2005) collected dengue virus RNA by using superparamag-netic beads inside a microfluidic device. Chen et al. (2010)generated a highly enriched solution of viral products concen-trated 40–80-fold relative to the initial sample by using micro-fluidic immunomagnetic separation. Beyor et al. (2008) developeda microfluidic concentration device based on a polydimethylsiloxane (PDMS) valve membrane, a glass microfluidic wafer, anda glass manifold for immunomagnetic E. coli isolation from a dilutesample.In this study, S. Typhimurium was detected using a microfluidicdevice to demonstrate the advantages of using magnetic beadsand QDs as a fluorescent label. S. Typhimurium cells wereseparated and concentrated into small-volume samples by usinganti-Salmonella antibody-coated magnetic beads. The cells werelabeled with antibody-conjugated QDs to form “sandwich” com-plexes. The fluorescence signals from the complexes were mea-sured using a custom-made fluorometer to provide a quantitativedetection method for S. Typhimurium.2. Materials and methods2.1. Bacteria and mediaS. Typhimurium (ATCC 14028) and E. coli (ATCC 25922) wereobtained from American Type Culture Collection (Manassas, VA,USA). Fresh cultures of S. Typhimurium and E. coli were preparedby incubation in brain heart infusion (BHI; Difco Laboratories,USA) broth at 37 °C for 14 h. The cell-containing buffer waschanged to 50 mM borate buffer (pH 7.4) by the followingprocedure: 1 mL of the enriched cell suspensions was centrifugedfor 10 min at 5000g, and the supernatant was discarded. Thecollected cell pellet was resuspended in 1 mL of borate buffer andwashed two more times with the same procedure. The cells werediluted to appropriate numbers (10 3 –10 6 CFU/mL) with boratebuffer for the experiment. Borate buffer without inoculation of S.Typhimurium cells was used as a control. The enriched S. Typhi-murium was enumerated using the standard plate count (SPC)method.Food samples were prepared by inoculating 100 mL of the cellsuspension into homogenized food extracts. The cells were alsodiluted to appropriate numbers (10 3 –10 6 CFU/mL) with the foodextract. Borate buffer or plain food extract that did not containSalmonella cells was used as a negative control. For food samplepreparation, packages of chicken breast were purchased from alocal grocery store.2.2. Reagents and antibodiesBacTrace affinity-purified goat anti-Salmonella antibodies werepurchased from Kirkegaard & Perry Laboratories (Gaithersburg,MD, USA). Anti-Salmonella antibody-conjugated superparamag-netic beads (Dynabeads s anti-Salmonella, 2.8 μ m in diameter)and CdSe/ZnS core/shell QDs (Qdot s 605 antibody conjugationkit, 20 nm in diameter) were purchased from Life Technologies(Carlsbad, CA, USA). Bovine serum albumin (BSA) was purchasedfrom Pierce (Rockford, IL, USA), and borate buffer was purchasedfrom Sigma (St. Louis, MO, USA). The Sylgard s 184 siliconeelastomer kit containing PDMS prepolymer and catalyst waspurchased from Dow Corning Corp. (Midland, MI, USA). Syringeswere purchased from BD Biosciences (San Jose, CA, USA), andtubing was obtained from Thermo Fisher Scientific Inc. (Waltham,MA, USA).2.3. Conjugation of antibodies to QDsAntibodies were conjugated to QDs prior to the assay. Goatanti-Salmonella antibodies were coupled directly to the QD surfaceusing the Qdot antibody conjugation kit. Antibody conjugationwith QDs was performed according to the manufacturer's instruc-tions. Briefly, 10 mM succinimidyl 4-[N-maleimidomethyl] cyclo-hexane-1-carboxylate (SMCC) was first added to the QD nanocrys-tals and incubated at room temperature for 1 h to activate the QDs.Second, 1 M dithiothreitol (DTT) was added to the purified anti-body (1 mg/mL) and incubated at room temperature for 30 min toreduce it. Then, the activated QDs and the reduced antibodies weremixed and incubated at room temperature for 1 h. After theconjugation reaction, the antibody-QD conjugates were concen-trated via ultrafiltration (centrifugation at 7000 rpm for 15 min).Finally, the concentrated QD-antibody conjugate was purifiedusing size exclusion chromatography and dispersed in 200 μ L of50 mM borate buffer, pH 7.4, containing 1% BSA.2.4. Fabrication of microfluidic channelsA silicon microfluidic channel design (width, 400 mm; height,50 mm) was created using a CFD program (Coventorware 2010,Coventor Inc., Cary, NC, USA). The microfluidic design is shown inFig. 1 and consists of a serpentine channel and detection well. Asilicon wafer master mold containing the microfluidic channeldesign was fabricated by a third-party service company (BuysemiInc., Suwon, Korea) by standard photolithography techniques. Thesurface of the master mold was passivated with fluorosilane toprevent it from permanently adhering to the PDMS during themolding. To create a PDMS mold, a 10:1 mixture of PDMSprepolymer and coring agent was stirred thoroughly and thenG. Kim et al. / Biosensors and Bioelectronics 67 (2015) 243–247 244 degassed under vacuum. The prepolymer-coring agent mixturewas poured onto the silicon master, which was placed inside acontainer formed by an aluminum foil. The PDMS mold was curedin an oven for 2 h at 65 °C. After curing, the PDMS mold waspeeled from the master. The final thickness of the PDMS mold was?3 mm. To provide inlets and outlets, connecting holes to thechannels were fabricated in the PDMS mold using a puncher. Awide-gauge (23 G) needle with a blunt end was inserted into eachhole to connect the tubing. The microfluidic channels containedtwo inlet ports and one outlet port (Fig. 1). Inlet port 1 was usedfor injections of magnetic beads mixed with sample solutions,followed by a rinse buffer to wash away unbound components. Anantibody-conjugated QD-containing solution was injected throughinlet port 2. The patterned PDMS mold was bonded to a glass slideby air plasma treatment to form closed channels and wells.2.5. Portable fluorometerFluorescence signals from QD-labeled S. Typhimurium cellswere measured using a custom-built portable fluorometer devel-oped in a previous study (Kim et al., 2013). As shown in Fig. 2, thefluorometer used a LED unit as the excitation light source andhighly sensitive silicon PMT (PCDmini, SenSL, Santa Clara, CA, USA)as a detector for signals with very low fluorescence. The lightsource unit comprised a 415 nm LED module connected to a fibercoupler module. The excitation light was delivered to the samplingunit via a bifurcated fiber optic bundle that was connected to thefiber coupler module of the light source unit. The fiber probe wasplaced at the bottom of the sampling unit to reduce the signalvariation caused by gravity-induced falling of target cells. Thesampling unit contained a tray to mount a microfluidic chipcontaining the concentrated sample liquid and a housing to blockambient light. The collected fluorescence signal entered thedetection unit from the collection fibers to the fiber couplermodule, which collimated the light and transmitted it to theC-mount adaptor module. In the C-mount adaptor module, anedge filter with a 515 nm cut-on wavelength was installed to passonly the fluorescence signal from the QDs. Finally, the fluorescencesignals acquired by PMT were digitized and transferred to a PC forstorage by the digital to analog converter of the silicon PMT.2.6. Detection procedureBacteria were detected using a sandwich assay. First, 20 μ L ofthe antibody-coated magnetic beads were mixed with 1 mL ofsample solution containing 10 3 –10 6 CFU/mL of S. Typhimuriumand vortexed on a Genie-2 mixer (Daigger, Vernon Hills, IL, USA)for several seconds. The mixtures were incubated at room tem-perature for 30 min on an RKVSD mixer at a speed of 10 rpm. Themagnetic bead–cell conjugates were separated from the solutionby loading into MPC-S magnetic particle concentrators (DynalBiotech) for 3 min. The liquid portion was carefully removed usinga pipette. The bead–cell conjugates were rinsed with 0.5 mL of 1%PBS–BSA, followed by immunomagnetic separation, which wasrepeated twice. Then, the captured and washed cells were dis-persed in 200 μ L of 50 mM borate buffer, pH 7.4, containing 1%BSA. Finally, the captured cells and antibody-conjugated QDs wereintroduced to inlet ports 1 and 2 of the microfluidic chip,respectively.The solutions containing the captured cells and the antibody-conjugated QDs were withdrawn by negative pressure created by aperistaltic pump at a flow rate of 40 mL/min. The two solutionswere mixed in the meandering channel, and the cells were labeledwith the antibody-conjugated QDs. The QD-labeled cells werecaptured in the detection zone using an external magnetic fieldfrom a permanent magnet placed under the detection zone (Fig.S1). Immediately after the sample introduction, 0.5 mL of boratebuffer was introduced into the two inlet ports to remove theFig.1. Layout of the microfluidic channel. The well adjacent to the outlet hole is thedetection zone.Fig. 2. Schematic diagram of the portable fluorometer.G. Kim et al. / Biosensors and Bioelectronics 67 (2015) 243–247 245 unbound QDs. The microfluidic chip was then inserted into theportable fluorometer for measurement.In addition to labeling with QDs and concentration of the cellsinside the microfluidic device, a typical QD labeling method wasused for comparison with the microfluidic concentration method.The cells captured with the antibody-coated magnetic beads wereincubated with 200 μ L of 10 μ g/mL antibody-QD conjugates atroom temperature for 30 min. The magnetic bead–cell–QD con-jugates were separated from the solution by loading into themagnetic particle concentrators for 3 min. The liquid portion wascarefully removed, and the remaining bead–cell–QD conjugateswere rinsed three times with 0.5 mL of borate buffer containing 1%BSA. The captured and washed bead–cell–QD conjugates werethen dispersed in 200 μ L of borate buffer containing 1% BSA.Finally, the bead–cell–QD conjugate solution was loaded into an8-well chamber slide (Lab-Tek II, Thermo Fisher Scientific Inc., MA,USA) and inserted into the fluorometer for measurement.The fluorescence signal was recorded for 5 s. For each experi-ment, the standard deviation (SD) for signals from 3 samples wascalculated. The error bars in each graph indicate 7SD. The limit ofdetection was designated as the number of cells producingfluorescence values higher than three times the SD plus the meanof the control signals. A sample was considered to test positive ifthe signal difference was higher than the limit of detection.3. Results and discussionThe feasibility of detecting S. Typhimurium using a microflui-dic-based nano-biosensor for immunomagnetic separation and QDlabeling was investigated. The responses of the microfluidic-basednano-biosensor to increasing concentrations of S. Typhimuriumspiked into borate buffer are shown in Fig. 3, which shows that theintensity of the fluorescence signals linearly increasing withincreasing cell numbers. When the concentration of S. Typhimur-ium was increased from 0 to 10 6 CFU/mL, the fluorescence in-tensity increased from 686,689 counts to 1,177,415 counts. Thesandwich assay with the microfluidic nano-biosensor producedpositive results at a S. Typhimurium concentration of 10 3 CFU/mLin the borate buffer sample within 30 min.The detection performance of the microfluidic nano-biosensorwas compared to that of a typical QD labeling method in whichbead–cell–QD conjugates were separated and applied to an 8-wellchamber slide for signal measurement. The responses of the8-well chamber slide biosensor to increasing concentrations of S.Typhimurium spiked into borate buffer are shown in Fig. 4. Theintensity of the fluorescence signals also increased with increasingcell numbers. When the concentration of S. Typhimurium in-creased from 0 to 10 6 CFU/mL, the fluorescence intensity increasedfrom 49,117 counts to 64,022 counts. The 8-well chamber slidemethod produced a positive result at an S. Typhimurium concen-tration of 4?10 3 CFU/mL of in the borate buffer sample. The signalincrease from 0 to 10 6 CFU/mL for the microfluidic nano-biosensor,490,926, was more than 33 times larger than that for the 8-wellchamber slide, 14,905. This enhanced signal increase could beexplained by the concentration effect of the reduced samplevolume, as the sample volume of the detection well in themicrofluidic chip, 0.7 mL, was 142 times lower than that of the8-well chamber slide, 100 mL. In addition to the concentrationeffect, the signal increase may have resulted from reduced loss ofthe analyte. Ramadan and Gijs (2012) suggested that samplepreparation should be performed inside a single confined channelto minimize analyte loss.The microfluidic nano-biosensor specificity for S. Typhimuriumwas evaluated with E. coli. Fig. S4 shows fluorescence signalsobtained from samples containing 10 3 –10 6 CFU/mL E. coli and 10 3CFU/mL S. Typhimurium in borate buffer. The sample consisting of10 3 CFU/mL S. Typhimurium produced 17,983 counts more thanthe 10 6 CFU/mL E. coli sample. The results indicate that the anti-Salmonella antibody conjugated QD and magnetic bead selectivelybind to S. Typhimurium and produce a specific signal for S.Typhimurium.The effectiveness of S. Typhimurium detection using themicrofluidic-based nano-biosensor was also tested in complexfood samples. Chicken extracts were used as a sample matrix,and S. Typhimurium cells were inoculated to prepare sample sets.The responses of the nano-biosensors to increasing concentrationsof S. Typhimurium spiked into chicken extracts are shown in Fig. 5.The intensity of the fluorescence signals increased with increasingcell numbers, as observed for the test with the borate buffersample. When the concentration of S. Typhimurium increasedfrom 0 to 10 6 CFU/mL, the fluorescence intensity increased from755,485 counts to 1,184,996 counts. The detection limit of the QD-based nano-biosensor was also 10 3 CFU/mL S. Typhimurium in thefood sample.The microfluidic nano-biosensor had good linearity with thenumber of S. Typhimurium cells at concentrations of 0–10 6CFU/mL for both borate and chicken extract samples. The standardcurve showed a linear relationship between the cell concentra-tions (C) and the fluorescence intensity (FI) values of the micro-fluidic nano-biosensor. The regression model can be expressed asFI¼1,23,791Cþ574,052 with R 2 ¼0.995 and FI¼103,114Cþ625,349 with R 2 ¼0.949 for the borate buffer and chicken extract,respectively.Fig. 3. Response of the microfluidic nano-biosensor for different concentrations ofS. Typhimurium in borate buffer.Fig. 4. Response of a nano-biosensor in an 8-well chamber slide for differentconcentrations of S. Typhimurium in borate buffer.G. Kim et al. / Biosensors and Bioelectronics 67 (2015) 243–247 246 The detection limit of this method is comparable to or betterthan that of other studies: 10 4 CFU/mL (Yang and Li, 2006) and6?10 3 CFU/mL (Zhao et al., 2009). Similarly, a lateral flow stripwas only able to detect 10 6 CFU/mL Salmonella cells in 30 min (Kimet al., 2011). PCR-based analyses (real-time PCR or real-time RT-PCR) were able to increase the detection limit from 10 3 CFU/mL to10 CFU/mL, but required approximately 24 h (Miller et al., 2011).When using an interdigitated microelectrode-based impedancebiosensor, Liébana et al. (2009) detected 7.5?10 3 CFU/mL Salmo-nella cells in milk using a magneto-electrode. However, they pre-concentrated the cells prior to the measurement and required anadditional electrochemical signal enhancement method. For E. coliO157:H7, the limit of detection was 8?10 5 CFU/mL in beefsamples when using a similar magnetic enhancement strategywith an impedimetric biosensor.At least 10 5 Salmonella cells are required to cause salmonellosisin healthy adults (Kothary and Babup); thus, the detection limit ofour method should be suitable for outbreak prevention.4. ConclusionsConventional methods for pathogen detection and identifica-tion are labor-intensive and take days to complete. Some rapidimmunological assays have been developed, but these assaysrequire several lengthy steps, expensive laboratory instruments,and experienced operators. Recently developed nano-biosensorsprovide the potential for rapid detection of foodborne pathogens.This study showed that a microfluidic nano-biosensor enablessensitive detection of S. Typhimurium in borate buffer and chickensamples. For selective detection, an anti-Salmonella polyclonalantibody was used to capture and label Salmonella. Semiconduc-tive fluorescent QDs were attached to Salmonella in the samplesand produced fluorescent light. Magnetic separation and a micro-fluidic chip were used to separate and concentrate the cells fromthe sample. The fluorescence response of the nano-biosensor wasmeasured using a custom-built fluorometer. The sensitivity of thesensor was 10 3 CFU/mL Salmonella in both borate buffer and foodextracts.AcknowledgmentThis study was conducted with the support of “ResearchProgram for Agricultural Science & Technology Development(Project no. PJ009987)”, National Academy of Agricultural Science,Rural Development Administration, Republic of Korea.Appendix A. Supplementary informationSupplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2014.08.023.ReferencesBeyor, N., Seo, T.S., Liu, P., Mathies, R.A., 2008. Biomed. 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Response of the microfluidic nano-biosensor for different concentrations ofS. Typhimurium in chicken extracts.G. Kim et al. / Biosensors and Bioelectronics 67 (2015) 243–247 247

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  • 免费下载A microfluidic nano-biosensor for the detection of pathogenic Salmonella.XDF