Article pubs.acs.org/ac
In Vitro Isothermal Nucleic Acid Amplification Assisted SurfaceEnhanced Raman Spectroscopic for Ultrasensitive Detection of Vibrio parahaemolyticus Li Yao,† Yingwang Ye,† Jun Teng,† Feng Xue,‡ Daodong Pan,§ Baoguang Li,∥ and Wei Chen*,† †
School of Food Science & Engineering, Hefei University of Technology, Hefei 230009, China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China § Faculty of Marine Science, Ningbo University, Ningbo 315211, China ∥ Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, Maryland 20993, United States ‡
S Supporting Information *
ABSTRACT: Detection of pathogenic microorganisms is of great importance for public health and food safety. Traditional protocols can hardly meet the continuously increasing demand in sensitivity and specificity of pathogen detections. In this study, we adopted Vibrio parahaemolyticus (V. parahaemolyticus, Vp) as the model analyte, and developed an antibody-Vp-aptamer heterosandwich-based surface-enhanced Raman scattering (SERS) method in conjunction with in vitro isothermal amplification for sensitive detection of V. parahaemolyticus. The rolling circular amplification (RCA) products provided enormous sites for assembling the Au@Ag nanoparticles and forming excess “hot-spot” sites for Raman measurement. By using this enhanced Raman signal strategy in the detection, a limit of detection (LOD) as low as 1 cfu/mL was successfully achieved for ultrasensitive detection of V. parahaemolyticus. In addition, we have applied this method to artificially contaminated food samples. The detection data indicated that this method is able to determine the concentrations of V. parahaemolyticus in the spiked food samples with satisfactory sensitivity and specificity and, thus, this developed ultrasensitive SERS scheme is well suited for the urgent need in pathogen detection and demonstrated great potential in food safety, environment monitoring, and a clinical setting.
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well. For example, expensive instrumentation and tedious data analysis are highly required for NGS, which renders this technology not that suitable for rapid on-site detection. In general, most of the current rapid detection methods are based on the principle of either PCR or ELISA. But, the drawbacks with these methods limit their wide applications.13,14 For instance, there is great room for the improvement in sensitivity of assays associated with PCR and immune-based methods; and all PCR-based methods could not differentiate live or dead pathogens.14,15
owadays, the threat of pathogenic microorganisms in foods, especially Escherichia coli,1 Salmonella typhimu2 rium, Listeria monocytogenes,3 Vibrio parahaemolyticus,4 and Staphylococcus aureus,5 is one of the serious food safety issues worldwide. Development of methods for rapid screening, identification, and quantification of common pathogens is a public health and food safety issue of great importance. Traditional culture-based standard protocol cannot well satisfy the high detection demands due to lengthy incubation time and labor-intensive procedures.6 The presence of molecular and biological methods including the polymerase chain reaction (PCR),7 enzyme-linked immunosorbent assay (ELISA),8−10 and next-generation sequencing (NGS)11,12 have already partially addressed some of these issues in pathogen detection. However, there are limitations with these molecular assays as © XXXX American Chemical Society
Received: May 7, 2017 Accepted: August 23, 2017 Published: August 23, 2017 A
DOI: 10.1021/acs.analchem.7b01717 Anal. Chem. XXXX, XXX, XXX−XXX
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characterized by a transmission electron microscope (TEM; JEOL 2100 HR, Japan). Vp-aptamer included the following: ssDNA probe, 5′-TCT AAA AAT GGG CAA AGA AAC AGT GAC TCG TTG AGA TAC TAA AAA AAA ACA GGG CTG GGC ATA GAA GTC AGG GCA GA-3′ (sequence with underline was the aptamer to V. parahaemolyticus); circular template, 5′-P-TAT GCC CAG CCC TGT AAG ATG AAG ATA GCG CAG AATG GTC GGA TTC TCA ACT CGT ATC TGC CCT GAC TTC-3′; Au@Ag labeled probe, 5′-SH-(CH2)6-GCG CAG AAT GGT3′; Vp, 5′-(CH2)6-SH-TCT AAA AAT GGG CAA AGA AAC AGT GAC TCG TTG AGA TAC T-3′. All other chemicals were all of analytical grade and used directly without any further purifications. Antibody solution was prepared by diluting in 0.1 M phosphate buffer solution (PBS). Double distilled water was used throughout the study. Vibrio parahaemolyticus 17802 was dissolved in 50 mM TrisHCl buffer (pH7.4), including 100 mM NaCl, 5 mM KCl, 1 mM MgCl2, and hybridization buffer 0.01 M PBS (10 mM Na2HPO4 and 10 mM NaH2PO4, pH7.4) containing 2.5 mM MgCl2 and 10 mM KCl. Preparation of Au@Ag Nanoparticles. Uniform Au nanoparticles were first synthesized in aqueous solution based on our previous research.32,33 Briefly, 850 μL of 5 g/L HAuCl4 was added into 60 mL of ultrapure water and then heated to boiling with magnetic stirring. Afterward, 850 μL of 1% trisodium citrate solution was quickly injected and the solution was stirred for 10 min until it became wine-red and stirring was continued for another 5 min. After gradual cooling to room temperature, 4.5 mL of 0.1 M ascorbic acid and 9 mL of 1 mM AgNO3 solution were added into 50 mL of the above prepared Au seed solution. The solution turned to bright golden yellow, indicating the coating of Au seeds with a Ag shell. A sequential procedure for the immobilization of Raman reporter molecules on the surface of Au@Ag nanoparticles was as follows. Briefly, 10 μL of 10−6 M R6G was added to 1 mL of Au@Ag nanoparticles; then the mixture was reacted for 30 min under gentle stirring. Immobilization of R6G on the surface of Au NPs was identified using SERS measurements. Preparation of Au@Ag Labeled ssDNA Probes. Initially, the disulfide bond of 15 μL of 5 μM ssDNA probe was reduced by 3 μL of 1 mM TCEP in 100 μL of PBS in the dark for 1 h. A 1 mL aliquot Au@Ag was added with 4 μL of Tween-80 (10%) and vortexed at room temperature for 30 min. The mixture was concentrated to 100 μL and kept in a water bath at 52 °C for 2 h. Afterward, Au@Ag labeled probes were centrifuged at 10000 rpm for 10 min and washed with DI water twice. The sediment was resuspended in 100 μL of 10 mM Tris-HCl with 1 mM EDTA solution (pH 8.0) at 4 °C for subsequent use. Preparation of Circularization Mixture for RCA. First, 20 μL of phosphorylated padlock DNA probe (5 μM) was mixed with 20 μL of Vp-aptamer (5 μM). Then, 4 μL of PEG [50% (w/v) poly(ethylene glycol) 4000] and 60 μL of 1× ligation buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 0.5 mM ATP (pH7.8 at 25 °C)) were added into the above solution, and the mixture was incubated at 37 °C for 1 h. A 4 μL aliquot of T4 DNA ligase solution (5 U/ μL) was added, and the ligation was performed at 22 °C for 1 h. After the ligation, T4 DNA ligase in the mixture was inactivated by heating the reaction mixture to 65 °C for 10 min. The resulting mixture can be used directly for amplification or stored at 4 °C.
Nowadays, aside from an antibody, aptamer, as the recognition probe of pathogens, has been widely adopted for pathogen detection due to its intrinsic properties including high affinity and specificity, easy batch preparation and chemical modifications, and high stability.16−18 A great deal of work has been done on pathogen detections using aptamer as the recognition probe.19−21 More importantly, a distinctive advantage of using aptamer as the recognition probe lies in its ability to further integrate an in vitro nucleic acid based signal amplification strategy for sensitivity improvement, which cannot be realized by using a classic antibody.22,23 Rolling circular amplification (RCA), a type of in vitro amplification model, has been widely adopted for signal enhancement due to its properties of isothermal and extra-high amplification efficiency.24,25 The amplified long ssDNA products with repetitive sequence units complementary to the circular DNA template enable signal reporting and enhancement. Surface-enhanced Raman scattering (SERS) spectroscopy has become one of the most widely pursued spectroscopic tools owing to its production of unique spectroscopic fingerprints, high sensitivity, and nondestructive data acquisition.26−29 The substrate for SERS measurement plays a critical role in facilitating molecule adsorption and optimizing SERS sensitivity through both electromagnetic and chemical mechanisms. Meanwhile, the Raman signal can be significantly enhanced by the hot-spot sites created by assembly of metallic nanoparticles; and this signal enhancement strategy has been successfully used for various sensing systems to detect a single molecule.30,31 Herein, we describe a very promising and fascinating protocol for specific and ultrasensitive detection of Vibrio parhaemolyticus (V. parahaemolyticus, Vp) in food samples by the immuno-RCA strategy. In the immuno-RCA system, the immobilized antibodies on the microplate capture the target V. parahaemolyticus followed by the recognition and binding of aptamer-included ssDNA probe, which also acts as the primer of the RCA process. The silver coated gold nanoparticles (Au@ Ag) labeled ssDNA probe hybridize with the RCA products and assemble Au@Ag along the single strand RCA products to form an Au@Ag/RCA product structure. This particular Au@Ag/ RCA product structure constitutes hot-spot sites for Raman measurement and thus extraordinarily enhances the Raman response. Consequentially, the sensitivity of the detection of V. parahaemolyticus is greatly improved by this designed immunoRCA strategy.
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EXPERIMENTAL SECTION Materials and Methods. Deoxy-oligonucleotides were synthesized and purified by Sangon Biotechnology Co. Ltd., (Shanghai, China). Gold(III) chloride trihydrate (HAuCl4· 3H2O) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium citrate dehydrate (Na3C6H5O7·2H2O) was obtained from Alfa Aesar. Phi29 DNA polymerase, T4 DNA ligase solution, and dNTPs were also ordered from Sangon. Anti-Vibrio parahaemolyticus and Vibrio parahaemolyticus 17802 were purchased from Prajna Biology Technique Ltd. (Shanghai, China), and the concentrations were determined with the classic culture-based method by the Animal-Plant-Food Center of Jiangsu Entry-Exit Inspection and Quarantine Bureau (Nanjing, China). LabRAM HR Evolution (HORRIBA JOBIN YVON, France) was adopted for SERS measurements. The morphology of porous Au@Ag was B
DOI: 10.1021/acs.analchem.7b01717 Anal. Chem. XXXX, XXX, XXX−XXX
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Preparation of the Sensing Platform for V. parahaemolyticus Detection. The heterosandwich sensing platform was developed based on a 96-well microplate. First, 100 μL of antibody (2 μg/mL) was diluted in 0.05 M pH 9.6 carbonate buffer in the wells of the microplate and incubated at 4 °C overnight according to the classic ELISA protocol.34 Then, 100 μL of BSA solution (1%) was added and incubated at room temperature for 1 h to prevent nonspecific binding. After blocking with BSA, wells of the plate were added with 20 μL of V. parahaemolyticus at different concentrations and incubated at 37 °C for a certain time (of note, the samples at the low concentration should be first centrifuged to the volume of 20 μL to ensure the accuracy of the detections). Then, 100 μL of aptamer-included ssDNA probe was distributed to each well and further interacted with the captured V. parahaemolyticus at room temperature for 45 min. The microplate was washed 5 times with PBST [phosphate buffered saline containing 0.05% (v/v) Tween 20, pH 7.4]. Of note, after adding and incubation of V. parahaemolyticus with antibody in the microplate, the washing steps were omitted before subsequent adding of ssDNA probes in order to decrease the whole detection time. The washing treatments were carried out before the final RCA and SERS measurements to remove all nonspecific adsorption molecules. For RCA, 2 μL of Phi29 DNA polymerase (10 U/μL), 10 μL of 10 mM dNTPs, 90 μL 1× Phi29 buffer [33 mM pH 7.9 TrisHCl, 10 mM MgCl2, 66 mM KCl, 0.1% (v/v) Tween 20, 1 mM DTT] and the above prepared circular template were successively added. The polymerization reaction was performed at 37 °C for 1 h, and the plate was rinsed with PBST. Finally, 100 μL of Au@Ag labeled detection probe (containing 0.5 μM R6G) was added into the RCA product and hybridized at 37 °C for 30 min. After rinsing with PBST twice, each well incubated
with different V. parahaemolyticus was measured with Raman (acquisition time of 2 s, accumulation 3, and excited at 633 nm), and the signals of R6G were collected for quantitative analysis. The confirmed V. parahaemolyticus negative drink waters were bought directly from the local supermarket and spiked with the concentration-known standard V. parahaemolyticus solution to different concentrations. After thorough mixing with vortex, the spiked water samples were centrifuged to 100 μL and then directly determined with this signal amplified SERS protocol.
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RESULTS AND DISCUSSION Design of Electrochemical Biosensor. Effective detection of pathogens in food products is the first control point for the prevention of foodborne diseases. Improvement the sensitivity of the rapid detection methods is of great importance for accurate detection of the target pathogenic bacterium. The schematic diagram of the SERS sensing protocol of V. parahaemolyticus with in vitro isothermal nucleic acid amplification as the signal enhancement strategy is shown in Scheme 1. First, the antibody of V. parahaemolyticus is immobilized on each well of the microplate. In the presence of target V. parahaemolyticus, the added aptamer-included ssDNA can form the heterosandwich structure (HSS) of antibody-target-aptamer. After the isothermal RCA, the elongated ssDNA product is labeled with Au@Ag nanoparticles, which act as the substrate for SERS measurement. Due to the repeat sequence in the RCA product, tremendous Au@Ag nanoparticles are assembled together along the ssDNA, which greatly enhance the SERS signal and sensing performance of the detection of V. parahaemolyticus. This in vitro nucleic acid amplification based signal enhancement SERS C
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Figure 1. Results of RCA-based signal amplification. Agarose gel results of RCA products in (a) dark field and (b) bright field; TEM images of (c) prepared Au@Ag NPs and (d) assembled Au@Ag NPs; (e) Raman results of traditional and amplified signals (in panels a and b: lane 1, marker; lane 2, aptamer-included ssDNA probe modified Au@Ag; lane 3, RCA product; lane 4, RCA product-Au@Ag composites).
Figure 2. (a) Typical Raman results for detection of V. parahaemolyticus at different concentrations by the signal amplified method; (b) standard curve of the Raman intensity versus the concentration of V. parahaemolyticus.
Figure 1e), while the Raman signal of 2.2 × 104 cfu/mL V. parahaemolyticus can be easily distinguished from that of blank (curve of traditional process in Figure 1e). Of more significance, the Raman signal of the measurement with RCA treatment is dramatically enhanced as designed (curve of amplified signal in Figure 1e), indicating the effectiveness of our designed protocol for ultrasensitive detection of V. parahaemolyticus. Sensing Performance of the Designed in Vitro Nucleic Acid Amplification Based Protocol for Ultrasensitive Detection of V. parahaemolyticus. Under the optimized conditions including the concentration of the immobilized antibody (2 μg/mL), concentration of aptamer-included ssDNA probe (1.5 μM), incubation time of antibody (15 min) or aptamer (45 min) and RCA amplification time (1 h) for sensing of V. parahaemolyticus (see detailed optimization results in the Supporting Information), the practical sensing performance was investigated by standard V. parahaemolyticus solution at different concentrations. As the results show in Figure 2, the characteristic Raman peak of R6G is increased with the increase of V. parahaemolyticus concentration. To our surprise, V. parahaemolyticus at the concentration of 3 cfu/mL in the sensing system can be easily and well distinguished against the blank group, demonstrating the extraordinary sensing performance of our protocol. Based on the intensity of the characteristic peak of R6G (at 1651 cm−1) at different concentrations, a calibration curve was constructed for quantitative analysis of V. parahaemolyticus. Each sample was repeatedly measured five times, and the average values were adopted for the quantitative analysis. The quantitative assay exhibited a well and wide linear range from 3 × 108 to 2.2 × 108
strategy provides a novel sensing platform for ultrasensitive detection of V. parahaemolyticus. Results of RCA Characterization and Signal Amplification Confirmation. As the critical step for signal enhancement in the sensing performance, RCA and sequentially assembled of Au@Ag nanoparticles were carefully studied beforehand. The agarose gel electrophoresis results in Figure 1a,b indicated that (i) the aptamer-included ssDNA template was successfully amplified with product of high molecular weight (lane 3 in the dark field of the gel); and (ii) the Au@Ag nanoparticles labeling the RCA product further increased the molecular weight and steric hindrance, which induced slower mobility and a more concentrated band in the gel (lane 4 in both the dark and bright gels). These results demonstrate that the labeling of RCA product of Au@Ag nanoparticles was successful. Furthermore, this labeling step was also characterized by transmission electron microscopy (TEM). On the one hand, pure Au@Ag nanoparticles with the diameter of about 20 nm (Figure 1c) were dispersed randomly while the assembled Au@Ag nanoparticles dispersed along the linear structure even to micrometer scale (Figure 1d); on the other hand, the assembly density of Au@Ag nanoparticles was also greatly increased due to multiple complicated RCA products as the templates for assembly, which was better for subsequent enhancement of Raman signals. In short, all these results clearly confirmed the successful labeling of RCA product with Au@Ag nanoparticles for SERS measurement. Meanwhile, the signal amplification effect was also assessed before the measurement of V. parahaemolyticus. There was almost no Raman signal in the absence of target V. parahaemolyticus in the sensing system (curve of blank in D
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Figure 3. (a) Typical Raman spectrum of the specificity against other control bacteria; (b) corresponding intensity results of the specificity studies.
cfu/mL toward V. parahaemolyticus. The LOD was based on three times the standard deviation of the blank/slope and treated as 1 cfu/mL. This LOD was better than any other current methods for V. parahaemolyticus detection8,15 and even our previously reported heterosandwich-based electrochemical method.35 This excellent sensitivity should be attributed to both the RCA-based amplification for Au@Ag nanoparticles labeling and the powerful measurement ability of SERS. In addition, we also assessed other important parameters of the scheme including the specificity, reproducibility, and stability of the method. For specificity confirmations, control samples of the common potential interferents including Staphylococcus aureus (S. aureus), Shigella, Escherichia coli (E. coli), Salmonella, and Campylobacter jejuni (C. jejuni) were tested together with the target V. parahaemolyticus and V. parahaemolyticus-included mixture. As the results show in Figure 3, it was easily to observe that only the V. parahaemolyticus and the mixture groups (at the concentration of 2.2 × 104 cfu/mL) induced the obvious Raman response of R6G while the signals of those control samples can be ignored even at 100-fold higher concentrations (2.2 × 106 cfu/mL). This excellent specificity could be attributed to the design of simultaneous application of both antibody and aptamer as the recognition probes in this detection method. As mentioned above, taking advantage of the in vitro nucleic acid amplification for signal enhancement, aptamer was adopted as the replacement of traditional antibody and as the second recognition probe. The superb recognition ability of both antibody and aptamer applied in the sensing system contributed to the excellent specificity of the developed method for ultrasensitive detection of V. parahaemolyticus. The reproducibility of the developed method was examined by measuring the same V. parahaemolyticus samples (at the concentration of 2.2 × 104 cfu/mL) with the parallel prepared sensing systems for five times. The results indicated that the RSD of Raman signals of different sensing batches was in an acceptable range from 4.2% to 6.0%, demonstrating the good reproducibility of the developed method for V. parahaemolyticus detections. The stability of the proposed Raman detection protocol was also evaluated by the long-term storage assay. After 4 weeks of storage at 4 °C, the response of Raman measurement retained 94.8% of its initial response level (see detailed results in Supporting Information), suggesting the acceptable stability and precision of the proposed signalenhanced Raman sensing protocol for V. parahaemolyticus detection. Besides, another important parameter of the new
fabricated signal-enhanced SERS protocol for target bacteria detection, the whole detection time, is also compared with current main methods. Without considering the sample pretreatments, it usually takes 3−7 days or longer to get the results of the classical culture-based methods while the molecular biological methods of 2−4 h depend on the adopted category and designed amplification programs. For the signalenhanced SERS protocol in this study, it requires 3−4 h to complete the whole detection including the 0.25 h for antibody recognition, 0.75 h for aptamer recognition, 1 h for RCA, 0.5 h for hybridization of AuNPs labeled probes, and 5−10 min for SERS measurements, which is comparable to the molecular biological methods. Research in the future can focus on further simplification of the detection protocol and improvement of the detection efficiency. Detection of V. parahaemolyticus in Food Samples. The developed nucleic acid amplification based signal enhancement SERS protocol was adopted for the detection of target V. parahaemolyticus in spiked water samples. Water samples spiked with different concentrations of Vp were measured with this protocol. The detection results were used to assess the accuracy of the protocol. The data in Table 1 clearly showed that the Table 1. Detection of V. parahaemolyticus in Spiked Water Samples by the Proposed Method (n = 5) sample spiked concn (cfu/mL) 1 2 3 4 5
1 1 1 1 1
× × × × ×
102 105 104 106 103
measured concn (cfu/mL) (0.95 (1.02 (0.95 (1.07 (1.03
± ± ± ± ±
0.021) 0.035) 0.058) 0.042) 0.087)
× × × × ×
102 105 104 106 103
recovery (%) 95 ± 2.1 102 ± 3.5 95 ± 5.8 107 ± 4.2 103 ± 8.7
measured concentrations were in proportion to the spiked concentrations and recoveries of the spiked samples achieved between 95% and 107% for V. parahaemolyticus detection, indicating a good accuracy for the developed protocol for V. parahaemolyticus detection. The key aspect of superb performance of this detection protocol is the synergy of the new factors, i.e., the usage of antibody, aptamer, Au@Ag, and RCA in the system. In this research, antibody was adopted as the capture recognition probe and aptamer was used as the signal reporting and amplification probe. However, in future studies, this strategy can be modified to widen its application in foodborne pathogen detection. For instance, an antibody against a specific target pathogen can be used, while a universal aptamer probe E
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can be adopted for the detection of a broader range of pathogens such as different strains from the same species or genus.
4. CONCLUSIONS In summary, a signal-enhanced SERS protocol was developed for ultrasensitive detection of target Vibrio parahaemolyticus by using in vitro nucleic acid amplification to assemble Au@Ag nanoparticles for Raman measurements. The RCA product acted as the template for Au@Ag assembly, and the resulting assembly structure was used as the substrate for Raman measurement. The Au@Ag in the assembled structure can form the hot-spot for Raman measurement, and the Raman response was dramatically increased even at trace amounts of target V. parahaemolyticus. The LOD of V. parahaemolyticus detection was achieved as low as 1 cfu/mL. In addition, the spiked food samples were well determined with our developed method. Both the sensing model and the signal-enhancement strategy in this research are attributed to the superb performance of the protocol for ultrasensitive detection of V. parahaemolyticus. Practically, this strategy may be used as well in the detection of other pathogens in clinical diagnosis, food safety, biothreat detection, and environmental monitoring.
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ASSOCIATED CONTENT
S Supporting Information *
Additional figures as noted in text and experimental details for optimization of method, different batch detection results and stability results. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.analchem.7b01717. SERS optimization results, batch detection results, and SERS protocol results after storage, (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mails:
[email protected];
[email protected]. ORCID
Wei Chen: 0000-0003-3763-1183 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is financially supported by the Science and Technology Ministry of the People’s Republic of China (Grant 2015BAD17B02-3), the NSFC Grant of 21475030, the S&T Research Project of Anhui Province Grant 15czz03109, the National 10000 Talents-Youth Top-notch Talent Program, and the KC Wong Magna Fund in Ningbo University.
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