Intuitive Label-Free SERS Detection of Bacteria Using Aptamer-Based

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Intuitive Label-Free SERS Detection of Bacteria Using Aptamer-Based in Situ Ag Nanoparticles Synthesis Weicun Gao, Bo Li, Ruizhi Yao, Zhiping Li, Xiwen Wang, Xiaolin Dong, Han Qu, Qianxue Li, Nan Li, Hang Chi, Bo Zhou, and Zhiping Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01813 • Publication Date (Web): 13 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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Analytical Chemistry

Intuitive Label-Free SERS Detection of Bacteria Using Aptamer-Based in Situ Ag Nanoparticles Synthesis Weicun Gao1, 2‡, Bo Li1‡, Ruizhi Yao3, Zhiping Li1, Xiwen Wang1, Xiaolin Dong1, Han Qu1, Qianxue Li1, Nan Li1, Hang Chi1, Bo Zhou1* and Zhiping Xia1* 1 Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, AMMS Changchun 130122, China 2 College of Animal Sciences and Technology, Jilin Agricultural University, Changchun 130118, China 3 College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163000, Heilongjiang, China ‡

These authors contributed equally to this work.

*

Corresponding author

Tel.: +86 431 86985910; Fax: +86 43186985889 E-mail: [email protected] (Zhiping Xia); [email protected] (Bo Zhou)

1

ACS Paragon Plus Environment


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ABSTRACT The characteristic of an ideal bacteria-detection method should have high sensitivity, specificity, easy to operate and without time-consuming culture process. In this study, we report a new bacteria detection strategy that can recognize bacteria quickly and directly by surface-enhanced Raman scattering (SERS) with the formation of well-defined Bacteria-aptamer@AgNPs. SERS signals generated by Bacteria-aptamer@AgNPs exhibited a linear dependence on bacteria (R2=0.9671) concentration ranged from 101 to 107 cfu/mL. The detection limit is sensitive down to 1.5 cfu/mL. Meanwhile, the bacteria SERS signal was dramatically enhanced by its specifically recognized aptamer and the bacteria could be identified directly and visually through the SERS spectrum. This strategy eliminates the puzzling data analysis of previous studies and offers significant advantages over existing approaches, getting a critical step towards the creation of SERS-based biochips for rapid in situ bacteria detection in mixture samples.

Key words: label-free; SERS; aptamer; Staphylococcus aureus; Ag Nanoparticles

INTRODUCTION Direct bacteria detecting strategies without time-consuming culture process are urgently desired by clinical diagnosis and biosecurity area. The development of fast, sensitive and accurate diagnostic methods for bacteria thus appears as a major goal for public health. Traditionally, detection methods of bacteria include staining, light microscopic examination, microbial culture and amplification techniques such as immunoassays (enzyme-linked immunosorbent assay, ELISA)1, nucleic acid identification (polymerase chain reaction, PCR)2. However, staining culture only discriminates Gram+ (G+) and Gram-(G-). Microbial culture requires 24-72 hours to identify the pathogenic bacteria. Although the amplification strategies can achieve high specificity and sensitivity, they all need tedious sample preparation and the manual procedure is sophisticated. Although the next generation sequencing3 and microarray4 can achieve simultaneous detection of multiple pathogenic bacteria, these techniques still require prior isolation of bacterial DNA, preparation of enzyme reaction mixture and expensive instruments for nucleic acid amplification. The direct detection technique has been extensively developed. The bacteria can be bombarded by 2


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laser for the time-of-flight mass spectrometry detection, but the fingerprint difference is too little among different species and the instrument is too heavy for on-site detection5. Optical elastic scattering could also be used to analyze bacterial micro-colony on agar, but it could only discriminate microbe by clone morphology6. Vibrational spectroscopy including vibrational infrared and Raman

spectroscopy can differ the vibrational spectra of bacteria molecular fingerprints7. However, infrared spectroscopy is sensitive to water, which makes it cannot give full play to the advantages of sensitive analysis for biological samples. Raman spectra can provide complementary microbial structure information to infrared spectroscopy and is affected weakly by water, which makes Raman spectroscopy can be used for biological samples detection in water.The Raman spectroscopy of a whole-organism fingerprinting is commonly used to discriminate8 and identify microorganisms9, evaluate the response of microorganisms to abiotic10and biotic stress11. It has been used to classify different bacterial species at the colony and single cell levels. However, there are little differences among various bacteria species in Raman spectra. Generally, the specific spectral peaks are so weak that the recondite mathematical analysis must be used to discriminate the fingerprinting of different bacteria12. Aptamer is a group of single strand DNA that can bind to the target specifically by non-covalent bond. Aptamer is selected from a random oligonucleotides library by the SELEX technology. After several rounds selection, the oligonucleotides (aptamer) which bind to the target with high specificity and affinity will be selected13. Plenty of aptamer were identified to target ion14, organic molecules15, proteins16, bacteria17 and tumor cells18. Researchers have successfully detected Staphylococcusaureus19and Salmonella typhimurium20by aptamer and Raman markers based SERS. However, these strategies need tedious modification process or depend on Raman markers. Inspired by in situ coating with Ag nanoparticles around bacteria11,21, we developed a direct label-free SERS detection of bacteria (Figure 1). Aptamer interacted with its specifically recognized bacteria and folded into the bind configuration. The bound aptamer could be used as the template for Ag nanoparticles synthesis in situ. SERS signal could only be enhanced with the existence of aptamer bound bacteria. Because one bacterium can bind many aptamer molecules22, the SERS signal can be amplified in situ. Therefore, the sensitive detection is achieved. This label-free strategy method is simple, sensitive, rapid and accurate with lower costs. More importantly, it eliminates the puzzling data analysis of previous bacteria SERS studies. The results 3



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could be obtained from the SERS spectra directly. It has offered significant advantages over existing approaches, getting a critical step towards the creation of SERS-based biochips for rapid in situ bacteria detection in mixture samples.

EXPERIMENTAL SECTION Chemical and biochemical materials Sixty-five percent concentrated nitric acid (HNO3), 37% hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium choride (NaCl), sodium borohydride (NaBH4) and silver nitrate (AgNO3) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Luria Bertni (LB) medium, brain-heart infusion (BHI) medium and Glass slides (25.4 x 76.2 x 1.2mm) were purchased from Sangon Biotech (Shanghai,China). Milli-Q water (18.2 MΩ) was produced using a Millipore water purification system. Phosphate buffered saline (PBS, pH 7.4, 200 mM) was prepared by solving 3.40 g (25 mM) of KH2PO4, 30.5 g (175 mM) of K2HPO4 and 21.25 g (362.5 mM) of NaCl to 1 L of Milli-Q water. Different concentrations of PBS (0.1, 1, 10, 80 and 100 mM) were diluted from the bulk PBS buffer (200 mM). Binding buffer (pH 8.8) is prepared by solving 12.114 g (40 mM) of Tris-base, 0.931 g (5 mM) of KCl, 0.2775 g (1 mM) of CaCl2, 1.015 g (2 mM) of MgCl2 and 21.9375 g (150 mM) of NaCl to 1 L of Milli-Q water. All of the components were filtered using a 0.22µm filter column before use. Instrument Raman microscope (LabRAM HR Evolution, HORIBA Scientific, Japan); Specord plus spectrometer (UV-2501PC, Shimadzu, Japan); Light fluorescence spectrophotometer (LS-55, PerkinElmer, USA); Transmission electron microscope (JEM-1200EX, JEOL, Japan); Flow cytometer (FACSCalibur, Becton Dickinson, USA); Scanning electron microscope (JEOL, JSM-5500LV, Japan). Bacteria cultures ShigellaFlexneri

(CICC:

21534),

Escherichia

coli

O157:H7

(CICC:

21530),

Staphylococcusaureus(CICC: 21600) and Listeria monocytogenes (CICC: 21633) were purchased 4


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from China Center of Industrial Culture Collection(CICC). S.aureus (CICC: 21600) was cultured with Luria Bertni (LB) medium and L.mono (CICC: 21633) was cultured with brain-heart infusion (BHI) medium in a gyratory shaker at 200 rpm for 10 hours at 37°C. The bacteria were washed two times with binding buffer by centrifugation (5,000rpm, 2min) and stored at 4°C before use. Bonding of bacteria and aptamer Aptamer was fold by denaturing at 95°C for two minutes then cooling to 37°C at a rate of 2°C per 40 seconds using a PCR machine. The folded aptamer was stored at -20°C. Next, 1 mL 107 cfu/mL of S.aureus cells and 1 mL 107 cfu/mL of L.mono were incubated with 300 nM of aptamer (Aptamer for S.aureus, aptamerS:TCCCT ACGGC GCTAA CCTCC CAACC GCTCC ACCCT GCCTC CGCCT CGCCA CCGTG CTACA AC; Aptamer for L.mono, aptamerL: TATGG CGGCG TCACC CGACG GGGAC TTGAC ATTAT GACAG23) for 20 minutes at 4°C and then washed two times with PBS (100 mM) by centrifugation (5,000rpm) for two minutes. The mixtures were stored at 4°C till ready for assay. Synthesis of silver nanoparticles (AgNPs) AgNPs is synthesized according to previous research with slight modification24. Concisely, 17 mg (0.1 mM) of AgNO3 was dissolved in 10 mL Milli-Q water (placed at 0°C temperature); 37.8 mg (0.1 mM) of NaBH4 was dissolved in 100 mL Milli-Q water. 1 mL of AgNO3 was added to 1 mL NaBH4 at a flow rate of 0.67 mL/s-1 without mixing. The centrifuge tube was inverted one or two times. The particle size distribution of colloidal suspensions was characterized by TEM and UV-vis spectroscopy. The green /yellow colloidal sols were stored at 4°C without light. Bacteria-AgNPs 1 mL bacteria liquid was centrifuged at 5,000 rpm for 2 minutes and then the supernatant was discarded. 1 mL prepared AgNPs was pipetted into the sediments and vortexed to ensure that the mixture is evenly mixed. Then, 10 µL of (1 M) NaCl was added to the prepared mixture as coagulant. The resulting sample is termed as “Bacteria-AgNPs”. Bacteria-aptamer@AgNPs 5



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100 µL aptamer bound bacteria mixture was centrifuged at 5,000 rpm for 2 minutes and then the supernatant was discarded. 100 µL AgNO3 solution (10 mM) was added and vortexed for 5 minutes. Then, 100 µL of NaBH4 solution was added to the prepared mixture and vortexed. Finally, the sample was stored at 4°C without light. The resulting sample is designated as “Bacteria-aptamer@AgNPs”. TEM sample detection The carbon film was coated with a little S.aureus-aptamerS@AgNPs and 0.2% phosphotungstic acid staining, dry fixed and stored at 4°C. For energy dispersive spectroscopy (EDS), the S.aureus-aptamerS@AgNPs was simply washed with Milli-Q water three times and then sputter-coated with gold. Specimens were examined with the Scanning electron microscopy. SERS measurements and date analysis 3 µL of sample suspension was dropped on a fluted glass slide. The samples were observed from the Raman microscope and focused under 20× objective. The SERS spectra were recorded using a He−Ne 632.8 nm laser with 14 mW. The SERS spectra were continuously recorded at 30-seconds intervals. Random selection was carried out 10 times (30s-15min). The number of accumulations was two, the exposure time was two seconds, the width of confocal slit was 100 µm and the detecting spectra range was 400-2,000 cm-1. All data analysis was performed using Origin software 8.0 version. Mixture sample detection 500 µL S. flexneri, E.coli O157:H7, S. aureus and L. mono (107 cfu/mL), respectively, was added into centrifuge tube and vortexed. The mixture sample was incubated with 300 nM of aptamers. As aforementioned, the Bacteria-aptamer@AgNPs were synthesized and the SERS spectra were recorded.

RESULTS AND DISCUSSION SERS detection of bacteria by aptamer based in situ Ag nanoparticles synthesis 6


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As a proof-of-concept study, the aptamer targeting to Staphylococcus aureus (S.aureus) was selected. Approximately, 1mL 1×107cfu/mL S.aureus was washed three times with binding buffer, re-suspended in 40µL binding buffer. The re-suspended solution was mixed with 60µL (500nM) aptamerS25 at 4°C for 20 minutes. The aptamer bound bacteria were washed three times by PBS (100mM). The mixtures were re-suspended by 100µL AgNO3 (10mM) for 5 minutes at the room temperature and then 100µL NaBH4 (10mM) were appended followed by vortexes for several seconds to obtain the mixture of S.aureus-aptamerS@AgNPs. The S.aureus-aptamerS@AgNPs (3 µL) was dropped on a fluted glass slide. The SERS spectra were recorded. The results showed that the general spectra of aptamerS bound S.aureus were greatly enhanced compared to the no-aptamerS S.aureus (Figure2A) SERS spectra. There are several distinct emission peaks at 735cm-1, 1337cm-1 and 1458 cm-1 in the aptamerS bound S.aureus SERS spectra, especially the 735 cm-1 peak was dramatically increased. However, when the Ag nanoparticle was synthesized in mixture of L.mono along with the aptamerS, the SERS signal of this mixture was very weak. This means the aptamerS could specifically enhance the SERS spectra of non-targeting bacteria. In fact, this SERS spectra of aptamerS bound S.aureus has very high stability. The SERS spectra of one positive focal point were continuously recorded at 30-seconds intervals for 10 times (30 s-15 min). The SERS spectra were very stable until the sample dried (Figure 2B). This is different from the previous research26 which showed that the SERS spectra were increased in the course of the droplet drying. The aptamer can bind to the surface of targeting bacteria by van der Waals force, hydrogen bonding, hydrophobic and electrostatic interaction. One bacterium can bind many aptamer, and these aptamer can absorb the Ag+ on to nucleotide and the addition of sodium borohydride can form the colloid nanoparticles, which deposited on the surface of aptamer forming the S.aureus-aptamerS@AgNPs. So the specific amplified signal can be achieved. It can be clearly observed from Transmission Electron Microscope (TEM) image that AgNPs were synthesized around the surface of bacterial cell wall (Figure 3). The Ag nanoparticles on the surface of S.aureus increased with the aptamer amount. Especially, when the S.aureus was mixed with interfering bacteria (Escherichia coli O157:H7, Shigella Flexneri, Listeria monocytogenes, all the interfering bacteria with a rod shaped bacteria), little Ag nanoparticle could be observed around the rod-shaped bacteria (Figure 3E). The Energy Dispersive Spectrometer (EDS) also support this 7



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observation (Figure S6). There were very high silver elements around the S.aureus. This strategy was also used to synthesize Listeria monocytogenes-aptamerL.@AgNPs (L.mono-aptamerL@ AgNPs), the SERS spectra of L.mono-aptamerL@AgNPs were enhanced with peaks at 735 cm-1 and 1358 cm-1, comparing to the aptamerL free L.mono@AgNPs (Figure S1A). Interestingly, although they are different bacteria, the SERS spectra are similar, especially in the peaks at 735 cm-1. In detail, the SERS spectra without aptamer also have a weak peak at 735 cm-1. This means that the aptamer based Ag nanoparticles could specifically enhance the peak 735 cm-1 of bacteria SERS spectra. The SERS spectra of L.mono-aptamerL@AgNPs were also continuously recorded at 30-seconds intervals for 10 times (30s-15min) and the SERS spectra were very stable (Figure S1B). To evaluate the reproducibility of this dynamic SERS approach for bacteria characterization, five different batches of samples containing 1×107 cfu/mL of S.aureus were detected. The results showed that there were no distinctive difference among the batches (Figure S2A). Figure S2B showed that the SERS spectra were stable among five different batches of samples containing 1×107 cfu/mL of L.mono. Unequivocally, the SERS spectra of aptamer based bacteria are highly reproducible and reliable.

Characterization of bacteria surface-enhanced Raman scattering by Ag nanoparticles To explore the characteristic of the Bacteria-aptamer@AgNPs based SERS, the SERS spectra and absorbance spectra of different bacterial samples were analyzed. The SERS spectra of S.aureus and aptamerS bound S.aureus were very weak .For the S.aureus@AgNPs sample, there were little enhancement in all spectra, but the typical peak of 735cm-1, 1337 cm-1could be observed. The spectra of S.aureus-AgNPs showed that there were general enhancements from 1000 cm-1

to 1600 cm-1, except for the peak 735 cm-1. Interestingly, for the

S.aureus-aptamerS@AgNPs samples, the peak at 735 cm-1 was significantly increased (Figure 4A) and the amplification factor is over 18 times (Figure 4B). UV−vis spectroscopy (Figure S3) showed that no distinct peaks could be observed from the S.aureus and aptamerS bound S.aureus solution from 300 nm to 800 nm. The conventional AgNPs were displayed as yellow suspensions with a narrow absorption peak at 391 nm, which indicated that mono-disperse particles had a diameter

of

approximately

4-5nm

(FigureS4).

The

UV−vis

8


spectroscopy

of

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S.aureus-aptamerS-AgNPs has a main peak at 413 nm, with a red-shift compared to the counterpart of AgNPs. The peak width of S.aureus-aptamerS-AgNPs was larger than the conventional AgNPs. This phenomenon is similar to the aggregation of AgNPs induced by 0.01 M of

NaCl.

The

S.aureus-aptamerS@AgNPs

owned

a

spectrum

similar

to

the

S.aureus-aptamerS-AgNPs, with a subtle red shift. This red shift may be caused by decreased inter-particle distance and the size of AgNPs increased. The results mentioned above stated that AgNPs in the S.aureus-aptamerS@AgNPs owning a different morphology, and exhibiting different aggregations or assembly behaviors. Optimization of the S.aureus detection condition In this detection strategy, the aptamer concentration and ionic strength are the main factors. Different concentrations of PBS buffer (0, 1.0, 10, 50 and 100 mM) were used to wash the aptamerS bound S.aureus compound to assess the influence of ionic strength. 100 µL 1×107 cfu/mL aptamerS bound S.aureus compound were washed with different concentrations of PBS solution for twice. S.aureus-aptamerS@AgNPs was prepared as described above respectively. The corresponding SERS spectra showed that the SERS signals of S.aureus-aptamerS@AgNPs decreased along with the reduction of PBS concentrations (Figure S5A), simultaneously the signal of 735 cm-1 peak was attenuated too (Figure S5B). These results are completely opposed to previous research results26. To explore the reasons for this phenomenon, we used FAM-labeled aptamerS to form aptamer bound S.aureus compound and then the compound was washed twice with PBS (100 mM) and Milli-Q water(18.2Ω) respectively. The fluorescence of the washing solutions supernatant was detected to reflect the binding status of bacteria and aptamer. The fluorescence of supernatant of water washing solution is significantly higher than the counterpart of PBS washing solution (Figure S5C). This result indicated that the binding of aptamer and S.aureus was mediated by ionic strength. Another important factor is the amounts of aptamer. Different amounts of aptamer (0, 50, 100, 150, 200 and 300 nM) were used in the same sample (107 cfu/mL S. aureus) to acquire the optimized dosage of aptamer. Figure 5A revealed that the main SERS spectra peak at 735cm-1 increased as the dosage of aptamer increased. A linear dependence on aptamer concentration (Y=5.5*X+80.4, R2=0.9968) was proved by the 735 cm-1 SERS signals generated by S. aureus-aptamerS@AgNPs from 0 to 200 nM (Figure5B). The TEM images result of 9



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S.aureus-aptamerS@AgNPs showed that the number of AgNPs that conjugated outside S.aureus increased as the amount of aptamer increased from 0 nM to 300 nM. With the FAM-labeled aptamer, the flow cytometer analysis showed that the number of FAM-labeled S.aureus increased with the aptamer concentration in a linear relationship from 0 nM to 200 nM and the growth slowed down at 300 nM aptamer (Figure 5C). In order to guarantee the stability of our results, this study used the aptamer at 300 nM.

Sensitive detection of S.aureus. using aptamer-based Ag nanoparticles in situ synthesis A series dilution of S. aureus were washed for three times with binding buffer and re-suspended in 100 µL binding buffer containing 300 nM aptamerS. The mixtures were kept at 4 °C for 20 minutes to form the aptamerS bound S. aureus compound. The compound was washed for three times with PBS (100 mM). The S.aureus-aptamerS@AgNPs was synthesized as described above. The SERS spectra of S.aureus-aptamerS@AgNPs compound were decreased along with the reduction of the amount of S.aureus (Figure 6A). The intensity of 735 cm−1 was plotted against the S.aureus concentration and a linear relationship was obtained. The linear regression equation is Y=2.2*X-1.14 (R2=0.967) (Figure 6B). This detection strategy is highly sensitive and the peak at 735 cm−1 can be easily discriminated just only 10cfu/mL S.aureus were present. The linearity range is very wide from 101 to 107 cfu/mL. Based on the 3x standard error, the detection limit of 1.5 cfu/mL was obtained, which is almost 1000 times of Triton X-100 pre-treated in situ synthesis Ag nanoparticles strategy11. If the S.aureus was directly used as the template of Ag nanoparticles, no characteristic SERS spectra could be obtained. The SERS signal will disappear when the concentration of S.aureus was less than 105 cfu/mL (Figure 6C). This phenomenon should be the results from the bacteria washed with PBS, which could decrease the zeta potential of cell wall26. The Supplementary table1 shows the typical SERS spectra peaks that can be observed in bacteria. 10


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Recently, many studies have confirmed that bacteria SERS signals might come from molecules involved in the bio-chemical activity of cells, such as metabolite or species specific cell signaling molecule27, rather than from the outer cell wall. For these typical peaks, 1,128 cm-1and 1,631 cm-1 bands were assigned to the Amide III and Amide I of proteins, respectively, 735 cm-1 (adenine), 788 cm-1 (cytosine, uracil) and 1,337 cm-1 (guanine) were from purine and pyrimidine metabolites. Moreover, 628cm-1,648cm-1and 1,337 cm-1 were assigned to phenylalanine (skeletal), tyrosine (skeletal) and tryptophan. The 1,458 cm-1 band was assigned to the CH2 deformation of lipid28. With the help of SERS mapping technique, single-cell detection can be easily achieved. The emissions from 720 to 740 cm-1 were used as the standard for SERS mapping. Figure 7A showed that the SERS map of S.aureus-aptamerS@AgNPs was intensively enhanced around the bacteria clone shape. However, there were few enhancements in the S.aureus@AgNPs group (Figure7B), even the bacteria clones were visible under the light microscope in both samples. Moreover, the single cell could be easily detected with S.aureus-aptamerS@AgNPs (Figure7C). It implied this technique could recognize single bacterium specifically.

Discrimination of S. aureus in the complex sample To test the potential clinical applications of Bacteria-aptamer@AgNPs based bacteria discrimination in the complex sample, four irrelevant bacteria including Shigella flexneri (S.flexneri), Escherichia coli O157:H7 (E.coli O157:H7), L.mono and S. aureus were mixed with aptamerS (300 nM) respectively. Based on the Bacteria-aptamer@AgNPs stratgey, the SERS spectra were recorded. The results showed that all bacteria have no enhanced emission signal, except for the S.aureus having a dramatic enhancement with peaks at 735cm-1, 1337cm-1 and 1458 cm-1 (Figure 8). The S.aureus containing mixed sample also presented the special S.aureus SERS 11



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spectra with slight reduction compare to the equally S.aureus alone (Figure 8E). Nevertheless, the S.aureus could be easily discriminated in the complex sample. In our strategy, all complex samples have to undergo the filtration and concentration to enrich the bacteria and then the sample will be washed with PBS, which could purify the different bacteria samples. Based on the specific reorganization of aptamer and bacteria, the aptamer bound bacteria could be formed. And the Bacteria-aptamer@AgNPs could give a specifically enhanced SERS spectra.Another important factor should be clarified is that the SERS spectra of Bacteria-aptamer@AgNPs strategy were similar, even though they are completely different bacteria (L.mono and S.aureus). The specific peak 735cm-1was specifically enhanced. This property maybe is a characteristic of the Bacteri@AgNPs strategy. However based the specific reorganization of aptamer and its targeting bacteria, the specific result still could be obtained.

CONCLUSION Direct discrimination of bacteria by SERS spectra is a promising strategy since it quickly gives us confirmed results. However, the chemical composition of bacteria were very complicated, including lipid, polysaccharide, nucleic acid, protein and the metabolic intermediate. The SERS spectroscopy is vibrational spectra of the molecule. So it is very difficult to detect the special emission peaks of the complex bacterium. The more excruciating trouble is that the composition of different bacteria is extremely similar. Therefore, it is almost impossible to detect bacteria by special emission peaks of SERS spectra directly. The utilization of whole spectroscopy fingerprint analysis could discriminate different bacteria. However, it is indirect results and needs confusing mathematical calculation. Although several groups have reported the capability of in situ Ag nanoparticles synthesis for bacteria detection8, 29, it still needs principal component analysis (PCA)30 or hierarchy cluster analysis (HCA)31 to discriminate different bacteria. To obtain the direct special SERS spectra, Raman marker molecules have to intricately modify for the bacteria detection. To clear these obstructers, we used the aptamer as the template for nanoparticle synthesis. We can get special SERS spectra for bacteria dependent on the special recognition of aptamer and bacteria. This is the first report about SERS spectra based bacteria identification in absence of Raman markers and PCA/HCA. Furthermore, in experimental conditions, bacteria can be identified directly through SERS spectra changes. It takes less than 40 12


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minutes to detect S.aureus with a good linear relationship ranged from 101 to 107 cfu/mL. With the help of the SERS mapping technique, the single-cell detection of S.aureus is easily achieved. This provides a foundation for the SERS technique to be widely used in areas of rapid bacteria detection. This SERS bioassay can be widely employed in the detection of various pathogenic bacteria or even tumor cells using corresponding aptamer. We expect that this bioassay would contribute remarkable significance for pathogenic bacteria detection in clinical diagnosis and biomedical research in the future.

AUTHOR INFORMATION Corresponding author: E-mail (Zhiping Xia): [email protected]; E-mail(Bo Zhou):[email protected] Zhiping Xia &Bo Zhou , Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China Notes The authors declare no competing financial interest.

ACKNOLEDGMENTS This work was funded in part by The National Key Research and Development Program of China (2016YFD0501001)

and

the

“3551 Schema for Talents”

in China OpticsValley (No.J (Q)

H20140716-006).The authors thanks Wenxue Cao for her help and computer technical support, and thanks to strong support from chemical instrument center of Jilin University.

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Figure legend Figure 1 The SERS bacterial detection based on aptamer dependent in situ formations of AgNPs Figure 2 Detection of S.aureus by S.aureus-aptamerS@AgNPs based SERS (A) SERS spectra of S.aureus-aptamerS@AgNPs (greenline), L.mono-aptamerS@AgNPs (blue line)and S.aureus@AgNPs (red line); (B) The SERS spectra of one positive focal point of S.aureus-aptamerS@AgNPs were continuously recorded at 30-second intervals for 10 times(30 s-15 min) until the samples were dried. Figure 3 TEM images of S.aureus binding to different aptamer The S. aureus-aptamerS@AgNPs results by (A)50nM, (B)100nM, (C) 200nM and (D) 300nM of aptamerS to capture the same S.aureus sample (107cfu/mL), respectively.(E)TEM images of bacteria-aptamerS@AgNPs take from the mixed bacteria samples (S.aureus,L.mono, S.Flexneri, E. coliO157:H7). Figure 4 SERS spectra of S.aureus by different detection method (A) SERS spectra of 1 × 107cfu/mL S.aureus-aptamerS@AgNPs (green line), S.aureus@AgNPs (black line), S.aureus-AgNPs (blue line), S.aureus (pink line) and S.aureus-aptamerS (red line). (B) SERS spectra intensity at 735cm-1, 1: S.aureus-aptamerS@AgNPs; 2: S.aureus-AgNPs; 3:S.aureus@AgNPs (P

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