One-Step Sensitive Detection of Salmonella typhimurium by Coupling

Article pubs.acs.org/ac

One-Step Sensitive Detection of Salmonella typhimurium by Coupling Magnetic Capture and Fluorescence Identification with Functional Nanospheres Cong-Ying Wen,† Jun Hu,† Zhi-Ling Zhang,† Zhi-Quan Tian,† Guo-Ping Ou,‡ Ya-Long Liao,‡ Yong Li,† Min Xie,† Zi-Yong Sun,‡ and Dai-Wen Pang*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, People’s Republic of China ‡ Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China S Supporting Information *

ABSTRACT: Sensitive, rapid, and reliable detection of bacteria has always been pursued due to the great threat of the bacteria to human health. In this study, a convenient one-step strategy for detecting Salmonella typhimurium was developed. Immunomagnetic nanospheres (IMNS) and immunofluorescent nanospheres (IFNS) were used to specifically capture and recognize S. typhimurium simultaneously. After magnetic separation, the sandwich immune complexes (IMNS−bacteria− IFNS) were detected under a fluorescence microscope with a detection limit as low as ca. 10 CFU/mL. When they were detected by fluorescence spectrometer, a linear range was exhibited at the concentration from 105 to 107 CFU/mL with R2 = 0.9994. Compared with the two-step detection strategy, in which the bacteria were first captured with the IMNS and subsequently identified with the IFNS, this one-step strategy simplified the detection process and improved the sensitivity. Escherichia coli and Shigella flexneri both showed negative results with this method, indicating that this method had excellent selectivity and specificity. Moreover, this method had strong anti-interference ability, and it had been successfully used to detect S. typhimurium in synthetic samples (milk, fetal bovine serum, and urine), showing the potential application in practice.

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als,15−27 silver nanoshells,28 gold nanorods,29,30 carbon nanotubes,31 and so on all have been used in the detection of bacteria to reduce detection time, improve sensitivity, or simplify the manipulation process. As the foodborne pathogens usually exist within complex biological matrixes at a very low concentration, separation and enrichment of the target pathogens are crucial steps for accurate detection. Magnetic nanomatarials, due to their unique properties, including being conveniently manipulated by a magnet, high surface-to-volume ratios, and fast kinetics in solution,7,21,32 have been widely applied to rapid, efficient, and specific capture and enrichment of target bacteria from the original samples. For example, Xu’s group22,27 and Simard’s group33 both had successfully used vancomycin-modified magnetic nanomaterials to capture vancomycin-resistant bacteria at ultralow concentration (∼10 CFU/mL). On the other hand, magnetic capture can easily be coupled with other detection methods, such as PCR,24 fluorescence observation,18−20,23,27 electrochemical detection,34

oodborne pathogenic bacteria have always been a major threat to human health and widely responsible for many foodborne diseases. In 2011, the U.S. Centers for Disease Control and Prevention (CDC) reported the damage caused by foodborne diseases each year in the United States: they caused 48 million people to fall sick, 128 000 to be hospitalized, and 3000 to die.1,2 The situation in developing countries is even more serious, so it is urgent to reduce foodborne illness. In consideration of the low infectious dose and high health risk of many bacterial pathogens (e.g., the doses for Escherichia coli O157:H7 and Salmonella are as low as 10 cells),3 a convenient, rapid, sensitive, and reliable detection method is essential for minimizing or eliminating potential infections. However, conventional methods, such as culture-based techniques and enzyme-linked immunosorbent assays (ELISA), suffer from the disadvantages of being time-consuming and having low sensitivity and a laborious process, and polymerase chain reaction (PCR) analyses often require intensive and careful sample prepurification and skilled technical staff.4−7 In recent years, the great superiority of nanomaterials in the pathogen detection has been reported. Dye-filled nanoparticles,8−11 quantum dots (QDs),12−20 magnetic nanomateri© 2012 American Chemical Society

Received: November 2, 2012 Accepted: December 20, 2012 Published: December 20, 2012 1223

dx.doi.org/10.1021/ac303204q | Anal. Chem. 2013, 85, 1223−1230

Analytical Chemistry

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CdSe/ZnS QDs with the emission peak at about 565 nm into Pst−AAm−COOH. Then, MNS and FNS were modified with NH2−PEG−CM (MW 3400) as spacers to be conjugated with antibodies. Approximately 5 mg of MNS or FNS were activated in 100 mM EDC and 100 mM NHS in l mL of 0.01 M PBS (pH 6.8) at room temperature with continuous shaking. After incubating for 30 min, MNS were separated by magnetic force, and FNS by centrifugation, and washed with 0.01 M PBS (pH 7.2) three times. Then, they were dispersed in 1 mL of 0.01 M pH 7.2 PBS to react with 2 mg of NH2−PEG−CM for about 4 h at room temperature with continuous shaking. Afterward, the resultant MNS−PEG−COOH and FNS−PEG−COOH were washed by 0.01 M PBS (pH 7.2) five times to remove any unreacted NH2−PEG−CM. Then, MNS−PEG−COOH and FNS−PEG−COOH were reacted with the antibodies to S. typhimurium (about 50 μg), and the conjugation procedure was similar to that above except that the NH2−PEG−CM were replaced by antibodies. At last, the IMNS and IFNS were blocked with 1 mL of 1% BSA−PBS at 37 °C with gentle agitation for 30 min, and then stored at 4 °C for use. The transmission electron microscopy (TEM) image of S. typhimurium captured and identified with IMNS and IFNS was obtained by an FEI Tecnai G2 20 TWIN electron microscope. Detection of S. typhimurium. As illustrated in Scheme 1, IMNS and IFNS were simultaneously added to 1 mL of sample

and so on. However, the detection strategies mentioned above usually consisted of two or more steps, which made the manipulation tedious. Moreover, multiple steps would lead to more interference and more target loss to affect the reliability of the detection. In our previous work, we developed a convenient method to construct fluorescent−magnetic nanospheres (FMNS) by embedding nano-γ-Fe2O3 and CdSe/ZnS QDs into the poly(styrene/acrylamide) copolymer nanospheres (Pst− AAm−COOH). The FMNS have high structural stability, low nonspecific adsorption, fast binding kinetics, and have been successfully modified with various targeting biomolecules (such as antibody, lectin, avidin, biotin, etc.) to isolate and recognize proteins and cancer cells.35−37 Herein, magnetic nanospheres (MNS) and fluorescent nanospheres (FNS) were similarly prepared and respectively modified with antibodies to construct immunomagnetic nanospheres (IMNS) and immunofluorescent nanospheres (IFNS). And with the IMNS and IFNS, a convenient detection strategy was developed to detect Salmonella typhimurium by coupling magnetic capture and fluorescence recognition in one step. The IMNS with excellent superparamagnetic property were used for separation and enrichment. The IFNS, which encapsulated hundreds of QDs into a single nanosphere, were employed as reporter probes to provide a highly amplified and stable signal. Magnetic capture and fluorescence identification could be accomplished simultaneously in 1 h, and ca. 10 CFU/mL of S. typhimurium could be effectively captured and reliably detected. Compared with the two-step detection strategy, which included capture and a subsequent identification process, the one-step strategy not only simplified the detection process but also improved the sensitivity. Moreover, this new approach had good specificity and strong anti-interference ability, which made it have potential application in practice.

Scheme 1. Schematic Diagram for the One-Step Detection of Bacteria with the IMNS and IFNS

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EXPERIMENTAL SECTION Chemicals. Monoclonal antibody to S. typhimurium was purchased from Abcam. NH2−PEG−CM (MW 3400, and CM here refers to carboxymethyl) was obtained from Laysan Bio. Difco skim milk was received from Becton, Dickinson and Company. Hoechst 33342 and the primers for PCR were from Invitrogen Corp. The 2× Tag PCR master mix was bought from Tiangen Biotech (Beijing) Co., Ltd. Agar for bacteria culture was purchased from Oxoid Ltd. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS) were bought from Sigma-Aldrich. Ultrapure water (18.2 MΩ·cm) was produced by a Millipore Milli-Q system. Bacteria Culture. The pure culture of S. typhimurium, E. coli, or Shigella flexneri was grown on the MacConkey agar at 37 °C for 24 h. Colonies were picked to prepare bacteria suspension with physiological saline. The exact cell number of the bacteria was determined by plating 10 μL of proper dilution onto the Mueller−Hinton agar. After the plates were incubated at 37 °C for 24 h, the number of colony forming units per milliliter (CFU/mL) was determined by counting the colonies grown on the plates. Considering biological safety, all experiments utilizing viable pathogens were done in a Biosafety level 2 laboratory by trained personnel, and the bacteria were heat-inactive for further use. Fabrication of IMNS and IFNS. According to the published procedure,38,39 carboxyl-terminated MNS and FNS were prepared by embedding, respectively, nano-γ-Fe2O3 and

solution (bacteria in the solution of 0.1% skim milk−0.05% Tween 20−0.9% NaCl). The mixture was incubated at 37 °C for 60 min with gentle shaking. Then the sandwich immune complexes were separated with a magnetic scaffold and rinsed by 0.1% skim milk−0.05% Tween 20−0.9% NaCl four times and 0.9% NaCl once. Afterward, they were resuspended in 300 μL of 0.9% NaCl for fluorescence spectrum measurements by a Fluorolog-3 (Horiba Jobin Yvon) fluorescence spectrometer, or they were resuspended in 20 μL of 0.9% NaCl and attracted by a magnet to the bottom of a minor hole made by PDMS (ca. 3 mm in diameter) for fluorescence microscopy imaging with a CCD camera (Retiga 2000R, Qimaging Corp., Canada) mounted on an inverted Nikon microscope (TE2000-U, Nikon, Japan). The bacteria at different concentrations were obtained by serial dilution. 1224



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Figure 1. Confocal microscopic images of S. typhimurium (A), physiological saline (B), E. coli (C), and S. flexneri (D) captured and identified by IMNS and IFNS, and S. typhimurium treated with MNS and FNS (E). (Bright field; Hoechst 33342: excitation 405 nm, emission 447 ± 30 nm band pass. FNS: excitation 488 nm, emission 565 ± 10 nm band pass. Merge: merge of Hoechst 33342 and FNS).

Confocal Microscopy Assay. Amounts of 200 μL of S. typhimurium (107 CFU/mL), E. coli (107 CFU/mL), and S. flexneri (107 CFU/mL) were stained by 30 μg/mL Hoechst 33342 at 37 °C for 30 min, then the cell solutions were rinsed three times with PBS (0.01 M pH 7.2) by centrifugation and resuspended in 0.1% skim milk−0.05% Tween 20−0.9% NaCl. Afterward, IMNS and IFNS were added to capture and identify the bacteria. After washing by magnetic separation, the immune complexes were observed by a confocal fluorescence microscope [the spinning-disk confocal microscope (Andor Revolution XD) was equipped with an Olympus IX 81 microscope, a Nipkow disk-type confocal unit (CSU 22, Yokogawa), and an EMCCD (Andor iXon DV885K single photon detector)]. Another two control experiments were performed: IMNS and IFNS were used to detect physiological saline without bacteria added; stained S. typhimurium (107 CFU/mL) was treated with MNS and FNS without antibodies modified following the detection procedure. Two-Step Detection Strategy. The two-step approach for detecting S. typhimurium was done to make a comparison with our method. First, the IMNS were incubated with the sample for 1 h to capture the bacteria, then the IMNS−bacteria composites were separated with a magnetic scaffold to remove

the suspension and resuspended in 0.1% skim milk−0.05% Tween 20−0.9% NaCl. Afterward, the IFNS were added and the solution was incubated for another 1 h. At last, the immune complexes were washed by magnetic separation and detected with a fluorescence spectrometer. For comparison, the operation and detection conditions were the same with the one-step approach. Detection of S. typhimurium in the Synthetic Samples. Milk, fetal bovine serum, and urine were tested by both selective plating and PCR methods to certify they were not contaminated by S. typhimurium. Then they were mixed with the bacteria suspension (9:1 by volume) to get the synthetic samples. IMNS and IFNS were added to the samples to detect S. typhimurium following the procedure, and control experiments were done the same except no bacterium was added.

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RESULTS AND DISCUSSION

Characterization of the IMNS and IFNS. The Pst− AAm−COOH have hydrophilic surfaces and hydrophobic hollow cavities, and hydrophobic nano-γ-Fe2O3 or QDs can be embedded into the cavities in weakly polar organic solvent via hydrophobic interactions.38,39 As shown in the TEM images 1225



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Figure 2. TEM characterization, fluorescence, and PCR analyses. (A and B) TEM images of S. typhimurium captured and identified with IMNS and IFNS. (C) Histogram of fluorescence intensity for the specificity test. (The bacteria concentrations were 107 CFU/mL. “Blank” was the detection of physiological saline, and “MNS/FNS” was the treatment of S. typhimurium with MNS and FNS. Corresponding fluorescence spectra are shown in the inset.) (D) Agarose gel electrophoresis of PCR products from the bacteria captured with the one-step method. [Left part is the result of S. typhimurium: lane 1, 5 kbp DNA ladder; lane 2, positive control of S. typhimurium (107 CFU/mL); lane 3, positive control of S. typhimurium (107 CFU/mL) mixed with the nanospheres; lanes 4−9, S. typhimurium captured at the concentration of 107, 106, 105, 104, 103, 0 CFU/mL, respectively; lane 10, S. typhimurium (107 CFU/mL) treated with the nanospheres without antibodies modified; lane 11, negative control using sterile water as template. Middle part is the result of E. coli: lane 1, 5 kbp DNA ladder; lane 2, positive control of E. coli (107 CFU/mL); lane 3, positive control of E. coli (107 CFU/mL) mixed with the nanospheres; lane 4, E. coli (107 CFU/mL) captured; lane 5, negative control using sterile water as template. Right part is the result of S. flexneri.]

scaffold and detected with a fluorescence microscope and a fluorescence spectrometer. Optimization of the Detection Conditions. The detection signal is determined by two factors: the number of the bacteria captured by IMNS and the number of IFNS associated with each bacterium. To get the best signal, the dosages of IMNS and IFNS and the incubation time were optimized with the orthogonal experimental method according to the orthogonal layout L9 (33). Capture efficiency and fluorescence intensity [signal-to-noise ratio (S/N)] were selected as the evaluating indexes. The experiment program, results, and analysis are described in detail in Supporting Information section S.4 (Tables S1−S3, Supporting Information). From the analysis of the results (Table S3, Supporting Information), it can be seen that the optimal condition for capture efficiency was “A3B1C1” (0.64 mg IMNS, 0.87 mg IFNS, and 30 min incubation), while the optimal condition for S/N was “A1B3C2” (0.32 mg IMNS, 2.61 mg IFNS, and 60 min incubation). The inconsistency between the results could be ascribed to the mutual competition between IMNS and IFNS. Because the antibodies conjugated to them were identical, they competitively bound with the bacteria in the solution. Under the optimized condition for fluorescence signal (A1B3C2), the S/N was tested to be 34.32 ± 0.32, which was higher than all experimental groups in the orthogonal experiment. This verified that “A1B3C2” was indeed the optimal condition for the fluorescence signal. We also tested the capture efficiencies in this condition, and they reached 88.9%, 82.3%, and 91.4% at the bacteria concentration of 107, 105, and 103 CFU/mL, which were acceptable. Considering that the fluorescence signal is the signal

of MNS and FNS (Figures S1A and S2A, Supporting Information), nano-γ-Fe2O3 or QDs were well-distributed in the nanospheres. The magnetic hysteresis loop of MNS (Figure S1B, Supporting Information) showed that they had excellent superparamagnetic property at room temperature. It can be seen that FNS showed good dispersibility and strong fluorescence from the fluorescence microscopic image (Figure S2B, Supporting Information). The fluorescence spectra of the QDs and the FNS (Figure S2C, Supporting Information) showed that the photoluminescence properties of the QDs were essentially unchanged in the nanospheres, in spite of a slight red-shift because of the change of QD environments from n-hexane to the copolymer nanospheres. Furthermore, the fluorescence intensity was proportional to the FNS concentration over a range from 0.004 to 0.875 mg/mL (Figure S2D, Supporting Information), which confirmed the homogeneity of the FNS and made quantitative detection possible. In the preparation of IMNS and IFNS, NH2−PEG−CM (MW 3400) was introduced as a spacer between the nanosphere and the antibody, which could partly reduce the strong steric hindrance between the rigid nanosphere and the bacterial surface to improve the binding efficiency.9,33 The rough value of antibodies on each nanosphere was evaluated to be about 40 by the fluorescence intensity of FITC-labeled IgG conjugated to the nanospheres (section S.3, Supporting Information).32,40 Detection of S. typhimurium. The entire immunoassay procedure for S. typhimurium detection was only one step. As illustrated in Scheme 1, IMNS and IFNS were simultaneously added to the sample solution. After incubation, the sandwich immune complexes were separated and washed with a magnetic 1226



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Figure 3. Detection results of S. typhimurium. (A−F) Fluorescence microscopic images of S. typhimurium captured and identified by IMNS and IFNS. (Bacteria concentrations of the samples corresponding to panels A−E were 0, 10, 100, 1000, 10 000 CFU/mL. Panel F is the empty magnification of a single typical bacterium.) (G) Table of the capture efficiencies at ultralow bacteria concentrations. (H) Linear relationship of fluorescence intensity vs bacteria concentration from 105 to 107 CFU/mL.

detected finally, “A1B3C2”, which were 0.32 mg IMNS, 2.61 mg IFNS, and 60 min incubation, was selected as the condition for following experiments. Reliability and Specificity. Colocalization analysis was done to prove the capture and identification of S. typhimurium by IMNS and IFNS. The bacteria were stained by a nuclear dye Hoechst 33342, whose fluorescence can be separated from IFNS’s by different filters. The confocal fluorescence micro-

scopic images (Figure 1) showed that the fluorescence of Hoechst 33342 from the bacteria (green in Figure 1A) and the fluorescence of IFNS (red in Figure 1A) were overlapped in the positive samples, while no fluorescence was found in the controls (Figure 1B−E). Thus, it could be concluded that IMNS and IFNS specifically captured and identified S. typhimurium. The TEM images (Figure 2, parts A and B) showed that many immunonanospheres bound to the S. 1227



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typhimurium cell surfaces, which further confirmed the successful association between the immunonanospheres and the bacteria. Fluorescence intensity of the immune complex was collected by a fluorescence spectrometer, which is shown in Figure 2C, and typical fluorescence spectra are in the inset. The IFNS fluorescence was easily detected in the positive sample, while no obvious IFNS fluorescence was found in the negative samples of E. coli, S. flexneri, and physiological saline. Similarly, when MNS and FNS without antibodies modified were incubated with S. typhimurium, no obvious fluorescence signal of FNS was found either, which proved that the nonspecific binding was minimal. The reliability and specificity of the method were further confirmed from the perspective of molecular biology by PCR (section S.5, Supporting Information). The results (Figure 2D) showed that the reaction of the bacteria mixed with the nanospheres all got corresponding gene fragments (lane 3 in the left, middle, and right parts of Figure 2D), which suggested the influence of nanospheres on PCR was insignificant. The 605 bp fragment of the iroB gene of S. typhimurium was found in the positive samples (left part of Figure 2D, lanes 4−8), while no 622 bp fragment of the phoA gene of E. coli or 435 bp fragment of the ipaH gene of S. flexneri was found when this method was used to capture E. coli or S. flexneri (middle and right parts of Figure 2D, lane 4), indicating that S. typhimurium was successfully and specifically captured. All these results suggested that our method was able to capture and identify S. typhimurium with high reliability and specificity. Qualitative Detection and Quantitative Analysis. As shown in the diagram (Figure S4, Supporting Information), a small PDMS device with a hole (diameter ∼3 mm) stuck on the surface of a coverglass, which had been successfully used in the tumor cell detection, 41 was used for fluorescence microscopy detection. A sample would be considered positive when the bacteria identified with IFNS are found. [Fluorescence spots that match the size of a single bacterium (∼2 μm)27]. Figure 3A−E shows the detection results of serially diluted solutions of S. typhimurium. Only one picture is presented as a representative of each concentration, and Figure 3A−E corresponds to 0, 10, 100, 1000, 10 000 CFU/mL. The objects marked by circles were the bacteria associated with IFNS, while the other few fluorescence spots may be caused by fragmentations of the bacteria which might have occurred when they were heat-killed or due to the slight nonspecific adsorption.12 The empty magnification of a typical single bacterium captured and identified by IMNS and IFNS (Figure 3F) showed that the IFNS bind the bacterium around, just as in the results of TEM images (Figure 2B). The replicated experiments (five times) of each concentration (Table S4, Supporting Information) showed that S. typhimurium could be reliably detected with a limit of ca. 10 CFU/mL. (When the bacteria suspension is diluted to very low concentrations such as ∼10 CFU/mL, the concentration calculated according to the dilution is usually untruthful. So we just say “ca. 10 CFU/mL”.) To further confirm the ability of this strategy to detect bacteria at ultralow concentration (∼10 CFU/mL), the corresponding capture efficiencies were investigated and are shown in Figure 3G. The capture efficiencies were calculated by the equation capture efficiency = Nc/(Ns + Nc) × 100%

controls, nanospheres without antibodies modified were used to evaluate the nonspecific interaction (Table S5, Supporting Information). As the results showed, S. typhimurium could be efficiently captured even at very low bacteria concentration, while the nonspecific interaction was slight and can be ignored. When the fluorescence intensities at different bacteria concentrations were collected by the fluorescence spectrometer, a linear range was exhibited at the concentration from 105 to 107 CFU/mL with R2 = 0.9994 (Figure 3H). The intra-assay CV (coefficient of variation) and interassay CV, which were calculated to be 5.2% and 7.2%, respectively, suggested that this method had good reproducibility.3,32 Superiority of the One-Step Strategy over the TwoStep Strategy. It is obvious that the one-step strategy is more convenient and more time-saving than the two-step strategy. Besides, did the two have some differences in detection signal? The samples with different concentrations of S. typhimurium were tested by the two strategies, respectively, and the fluorescence signal obtained is shown in Figure 4. It can be

Figure 4. Linear relationship of the fluorescence intensity vs the bacteria concentration with the one-step strategy and the two-step strategy.

seen that the one-step strategy got stronger signal and better linear relationship (R2 = 0.9957) compared with the two-step strategy (R2 = 0.9315). It is not difficult to understand that the IMNS and IFNS competitively bind to the same sites of the bacteria because of the same antibodies coated on their surfaces. In the two-step detection, IMNS were first added and preferentially occupied the binding sites, which made less IFNS bind to the bacteria and lower signal be obtained. On the other hand, the same amounts of IMNS were used to detect the bacteria at different concentrations, and they occupied different proportions of the binding sites. As a result, the remaining sites for IFNS were not proportional, and the linear relationship between the signal and the bacteria concentration was not as good as that of the one-step strategy. Though two kinds of antibodies to different antigens of the bacteria would minimize the competition, only one kind of antibody to the lipopolysaccharides of S. typhimurium was picked in consideration of the cost and the limitation of the kinds of antigens on the surface of S. typhimurium for choice. By optimizing the detection conditions, favorable dosages of IMNS and IFNS and incubation time were found to achieve the best fluorescence signal and acceptable capture efficiency. Application to the Synthetic Samples. As the pathogens usually exist in complex biological matrixes, synthetic samples were detected to further assess potential applications of this

(1)

where Nc is the number of captured bacteria, Ns is the number of bacteria in the supernatant, and Ns + Nc is consistent with the total number of bacteria employed in the experiments.33 Ns and Nc were determined by conventional plate counting. As 1228



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Figure 5. Detection results of the synthetic samples. (A) Fluorescence microscopic images. (B) Histogram for fluorescence intensities. (C) Linear relationship of fluorescence intensity vs bacteria concentration from 105 to 107 CFU/mL in the synthetic milk sample.

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method in practice. Milk, fetal bovine serum, and urine, which contained a lot of different species, were used to mimic real samples to be detected with IMNS and IFNS. As shown in the fluorescence microscopic images (Figure 5A), all positive samples presented IFNS identified bacteria, while no bacterium was found in the negative samples. The corresponding fluorescence signal (Figure 5B) was slightly lower than that of the ideal sample (bacteria in NaCl solution), which was due to the complexity of the matrixes. Besides, a similar linear range was exhibited at the concentration from 105 to 107 CFU/mL with R2 = 0.9985, and the error bars of signal were acceptable to be around 5% (Figure 5C). The results suggested that this method might be used in real, complex samples.

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ASSOCIATED CONTENT

S Supporting Information *

Sections S.1−S.8, Figures S1−S4, and Tables S1−S5. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 0086-27-68756759. Fax: 0086-27-68754067. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, no. 2011CB933600), the Science Fund for Creative Research Groups of NSFC (no. 20921062), the National Natural Science Foundation of China (Grants 20833006, 21175100, and 21005056), the Program for New Century Excellent Talents in University (NCET-100656), and the “3551 Talent Program” of the Administrative Committee of East Lake Hi-Tech Development Zone (Grant [2011]137).

CONCLUSIONS

In summary, we reported a convenient, highly sensitive, and reliable strategy for S. typhimurium detection without pretreatment of the sample. The process could be accomplished with only one step, in which IMNS and IFNS were used to capture and identify the target bacteria simultaneously. With this method, S. typhimurium could be detected specifically and reproducibly with a limit of ca. 10 CFU/mL. Moreover, this method was easy to manipulate and time-saving, and its successful application to synthetic samples had validated its potentiality in the real, complex sample detection. The concept described herein can be expanded to other pathogen detection and can be modified for simultaneous detection of multiple pathogens with multiantibody-coated IMNS and multicolor IFNS.

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REFERENCES

(1) Scallan, E.; Hoekstra, R. M.; Angulo, F. J.; Tauxe, R. V.; Widdowson, M. A.; Roy, S. L.; Jones, J. L.; Griffin, P. M. Emerging Infect. Dis. 2011, 17, 7−15. (2) Scallan, E.; Griffin, P. M.; Angulo, F. J.; Tauxe, R. V.; Hoekstra, R. M. Emerging Infect. Dis. 2011, 17, 16−22. (3) Wolter, A.; Niessner, R.; Seidel, M. Anal. Chem. 2008, 80, 5854− 5863.

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