CN110702662B - Method for detecting bacteria based on tag surface enhanced Raman scattering - Google Patents

Abstract

The invention discloses a method for detecting bacteria based on tag surface enhanced Raman scattering. The method adopts IgG modified magnetic beads to enrich and capture bacteria, and adopts borated SERS labels (Au @ Ag @ PDA) attached to the surface of target bacteria for signal amplification, so that Raman spectrum signals of the bacteria are enhanced by 10⁸And about twice, the unique fingerprint spectrum is displayed. The invention further adopts PCA and HCA to compare different Raman spectrum regions, and the result shows that the surface protein and glycan signals of the bacteria are the optimal regions for bacteria classification. The invention classifies 5 bacteria such as staphylococcus aureus, escherichia coli, shigella dysenteriae, pseudomonas aeruginosa, klebsiella pneumoniae and the like, and the detection sensitivity reaches 10¹CFU/mL, detection time less than 30 min. The method has the advantages of good stability, simple and convenient operation, low cost and wide applicability, and can be popularized to the detection or classification of bacteria in clinic and food safety.

Description

Method for detecting bacteria based on tag surface enhanced Raman scattering

Technical Field

The method is applied to the technical field of microorganism detection and analysis, and relates to a method for detecting bacteria based on tag surface enhanced Raman scattering.

Background

Bacteria exist in their enormous diversity and complexity in the living environment, classification of bacteria is the first step in bacterial identification, and development of rapid, sensitive and accurate bacterial diagnostic methods is a major goal in ensuring public as well as animal health. Traditionally, there are gram stain and serotyping methods for bacterial detection, however, both methods are complicated and time consuming to operate. Recently developed molecular biology methods such as Polymerase Chain Reaction (PCR), enzyme-linked immunosorbent assay (ELISA), second generation sequencing (NGS), etc., all require complicated procedures and are not suitable for field detection, although they can achieve high specificity and sensitivity. Matrix-assisted laser desorption time-of-flight mass spectrometry has been widely applied to clinical microorganism identification in the last five years, the main basis for identifying microorganisms is the difference of microbial ribosomal proteins, and ribosomal proteins with different masses present specific fingerprint spectra in the mass spectrum. Although low cost and ease of operation make MALDI-TOF mass spectrometry an attractive option for bacterial identification and classification, this assay requires expensive equipment and a time-consuming process that cannot be decoupled from bacterial isolation and culture. In contrast, molecular spectroscopy, such as infrared spectroscopy and raman spectroscopy, can be used to rapidly scan the fingerprint of the microorganism. However, the infrared spectrum is extremely sensitive to water, so that the application of the infrared spectrum in the microbial domain is limited, and the Raman spectrum can provide fingerprint information of microbes complementary to the infrared spectrum and is not influenced by water, so that the application prospect of the Raman spectrum in microbial detection is good.

Surface-Enhanced Raman Scattering (SERS) is a vibrational spectroscopy technique with single-molecule sensitivity. At present, the method for detecting bacteria based on SERS is divided into two categories, namely directly obtaining the Raman fingerprint of bacteria by taking gold, silver and other precious metals as an enhanced substrate and detecting the bacteria after marking by a Raman label (SERS tag). The former method directly obtains a bacterial fingerprint spectrogram by using surface enhanced Raman, and the latter method detects bacteria by indirectly detecting a Raman signal label with strong intensity. The invention combines the advantages of the two methods, and the bacterial fingerprint can be directly obtained while the signal of the bacteria is amplified by adopting the label.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a method for detecting bacteria by Surface Enhanced Raman Scattering (SERS), which is rapid, convenient, low in cost and high in sensitivity. The invention discloses a preparation method of IgG modified magnetic beads and a borated SERS label, and realizes the rapid capture and SERS detection of bacteria based on the combination of the two materials.

In the present invention, IgG-modified Fe is used₃O₄Magnetic bead IgG @ Fe₃O₄The bacteria are captured and separated, a borated SERS label is attached to the surface of target bacteria for amplifying bacterial signals, and IgG @ Fe is formed in the process₃O₄Sandwich of-bacteria-SERS tagsThe nano sensor further enriches and enhances the Raman signal of the bacteria, thereby displaying a unique fingerprint. The invention further carries out Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) on the obtained bacterial specific fingerprint spectra, and finds the difference of the SERS fingerprints of different bacteria by comparing different Raman spectrum regions, thereby realizing the detection of the bacteria.

The technical scheme of the invention is specifically introduced as follows.

The invention provides a method for detecting bacteria based on tag surface enhanced Raman scattering, which comprises the following steps:

1) IgG-modified Fe₃O₄Magnetic bead IgG @ Fe ₃0₄Adding the test bacteria sample to incubate for capturing and separating bacteria;

2) adding a borated SERS label to the separated sample, and attaching the label to the surface of the bacteria to enhance a Raman signal by utilizing the combination of boric acid and glycopeptide on the surface of the bacteria;

3) separating IgG @ Fe generated in the reaction in the step 2) by external magnetism₃O₄-performing SERS detection on the bacteria-SERS tag nanocomplex.

Preferably, in step 1), the bacteria include staphylococcus aureus, escherichia coli, staphylococcus dysentery, pseudomonas aeruginosa and klebsiella pneumoniae.

Preferably, in step 1), IgG-modified Fe ₃0₄Magnetic bead IgG @ Fe₃O₄The preparation method comprises the following steps: commercial carboxyl modified magnetic beads and immunoglobulin I_gG is coupled for 1-3 h at room temperature_，After magnetic separation, the cells were blocked with 200 μ L PBST buffer containing 1% BSA, and finally washed three times with PBS to remove impurities, resuspended in PBST buffer, and stored at 4 ℃ for future use.

Preferably, in step 2), the preparation method of the borated SERS tag is as follows:

firstly preparing 18 nm gold nanoparticle AuNPs, then adding 600 muL AuNPs into a clean EP tube, and then sequentially adding 20 muL 0.1 mol/L ascorbic acid, 80 muL 5 mmol/L silver nitrate and 20 muL 5X 10^-5 mol/L pATP, 500 mu L50 mmol/L Tris-HCl buffer area, 100 mu L5 mg/mL dopamine, and then centrifuging at 6000 rpm for 15min and re-suspending in 500 mu L PBS; and adding 100 muL EDC with the concentration of 10mg/mL and 100 muL NHS with the concentration of 10mg/mL, incubating for 20-40 min, adding 4-carboxyphenylboronic acid, coupling for 20-40 min, and storing the final solution at 4 ℃ for later use.

Further preferably, the method for detecting bacteria based on tag surface enhanced raman scattering of the present invention comprises the following specific steps:

1) respectively adding 50 muL of IgG modified Fe into 1 mL of different bacterial liquids₃O₄Magnetic bead IgG @ Fe ₃0₄Are combined together

Incubating for 10-30 min to generate IgG @ Fe₃O₄-bacterial complexes, followed by magnetic collection, washing 2-4 times with PBS buffer at pH 7.4, concentration 10mmol/L, and resuspension with PBST;

2) adding 100 mu L of borated SERS label into the mixed solution obtained in the step 1), and incubating for 5-15 min to generate IgG @ Fe₃O₄-bacteria-SERS tag complex, followed by magnetic collection, washing 2-4 times with PBS buffer at pH 7.4, concentration 10 mmol/L;

3）IgG@Fe₃O₄measurement of SERS spectra with capillary tubes for bacteria-SERS tag complexes.

Preferably, the method further comprises a step 4) of comparing different Raman spectrum regions through Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) to find the difference of the SERS fingerprints of different bacteria, so as to realize the detection of the bacteria.

Further preferably, the results of Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) show bacterial surface protein and glycan signals (1200-^-1) Is the optimal area for bacterial classification.

Compared with the prior art, the invention has the following beneficial effects:

IgG-modified Fe in the invention₃O₄The magnetic bead can catch and separate the bacterium and catch efficiently from solution fast, and the SERS label also can directly acquire bacterium fingerprint map when enlargeing the signal of bacterium. Sensitivity of detectionThe degree can reach 10¹CFU/mL, detection time less than 30 minutes. The method has good stability, simple operation and low cost. Therefore, the method can be popularized and used for bacteria detection in clinical and food safety.

Drawings

FIG. 1 is a representation of a SERS nanosensor; wherein A is TEM image of 18 nm AuNPs, B is TEM image of Au (core) @ Ag (shell), C is TEM image of SERS tag, and D is IgG @ Fe₃O₄TEM image of magnetic beads, E is IgG @ Fe₃O₄TEM image of the bacterial complex, F is IgG @ Fe₃O₄TEM image of bacteria-SERS tag complex.

FIG. 2 illustrates optimization of SERS tag synthesis conditions; wherein, when A is synthesized Au @ Ag, 5 mM AgNO with different volumes is added₃Effect on SERS intensity, B is 5 mM AgNO in different volumes₃To 1081cm^-1SERS intensity curve, C is 5X 10 of different volumes^-5Effect of M pATP on SERS intensity, D is 5X 10 of different volumes^-5M pATP to 1081cm^-1And (3) processing an SERS intensity curve graph, wherein E is an ultraviolet absorption spectrogram of AuNPs (black curve) and an SERS label (red curve), F is the research on the stability of the SERS label, and the SERS label is stored for 0, 1,7 and 30 days of SERS intensity change at 4 ℃.

FIG. 3 shows the utilization of IgG @ Fe₃O₄bacteria/SERS tag nanosensor at 10³-10¹In the bacterial concentration range of CFU/mL, SERS detection is carried out on the bacteria; a, B, C, D and E are respectively staphylococcus aureus, escherichia coli, Shigella dysenteriae, pseudomonas aeruginosa and klebsiella pneumoniae, F is the bacteria with different concentrations and a control sample (without bacteria) at 1081cm^-1The SERS intensity.

FIG. 4 shows the concentration of bacteria at 10³Average spectra of Raman spectra of bacteria collected 10 times per mL CFU, wherein A is SERS spectra comparison of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Streptococcus dysenteriae, and R is₁And R₂Respectively 700-1050 cm^-1And 1200 ion 1550 cm^-1(ii) a raman spectral region of; b is R₂Region (1200 ion 1550 cm)^-1) SER ofAnd (4) S spectrum.

FIG. 5 shows data analysis of five strains of Staphylococcus aureus (cyan), Escherichia coli (red), Shigella dysenteriae (pink), Pseudomonas aeruginosa (blue) and Klebsiella pneumoniae (green), the left graph shows the result of Principal Component Analysis (PCA), and the right graph shows the result of Hierarchical Cluster Analysis (HCA).

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the method of the present invention are not limited thereto.

The reagents and instrumentation used in the examples were as follows:

1. reagent

Carboxylated magnetic beads (10mg/mL, 200 nm) were purchased from Nanjing Donna, Inc.; tetrachloroauric acid (HAuCl)₄•3H₂O), human immunoglobulin IgG (. gtoreq.95%), 4-aminobenzenethiol (pATP), 4-carboxyphenylboronic acid, Bovine Serum Albumin (BSA), N- (3-methylenepropyl) -N' -ethylcarbadiimide hydrochloride (EDC) (. gtoreq.98.0%), N-hydroxysuccinimide (NHS) (98%), dopamine, Tween 20, sodium citrate, silver nitrate from Sigma;

PBS buffer (pH 7.4), Tris-HCl (pH 8.0), 4-morpholinoethanesulfofonic acid (MES, ≧ 99%), MEST solution was 50 μ L0.05% Tween 20 by 100 mM MES, PBST solution (19990 μ L10 μ L Tween 20 by PBS buffer).

Staphylococcus aureus (ATCC 25923), Escherichia coli (CICC 21530), Shigella dysenteriae (CMCC B51105), Pseudomonas aeruginosa (CICC 21636), Klebsiella pneumoniae (CICC 21106) were purchased from China center for culture Collection (CICC) and American center for culture Collection (ATCC).

2. Instrument for measuring the position of a moving object

A transmission electron microscope 200KV (model: JEM 2011, manufacturer: JEOL company, Japan) and Horiba Jobin Yvon XPloRA confocal micro-Raman spectrometer excite a sample by using a laser with the power of 785 nm of 0.5mw, and the acquisition time of each SERS spectrum of the excited sample is 10 seconds. Ten spectra were collected for each sample and averaged to ensure reproducibility of the signal.

Example 1

A method for detecting bacteria based on tag surface enhanced raman scattering, the method comprising the steps of:

1. preparation of microbial Standard samples

Taking bacterial strain to be detected, carrying out streak culture on LB solid culture medium at 37 ℃ for 24 h, selecting a single colony, inoculating the single colony in 20mLLB liquid culture medium, placing the single colony in 160rpm, carrying out constant temperature shaking culture at 37 ℃ for 14 h, and then using distilled water to adjust the bacterial concentration to OD₆₀₀=1.0, a strain standard sample was prepared. The bacteria to be detected are staphylococcus aureus, escherichia coli, shigella dysenteriae, pseudomonas aeruginosa and klebsiella pneumoniae.

2. Preparation of IgG-modified magnetic beads

1) After commercial carboxyl modified magnetic beads (200 nm, 5 mg/mL) are uniformly mixed by ultrasound for 3min, 200 muL of the magnetic beads are placed in a clean EP tube, the mixture is washed for 3 times by PBST solution, and then 100 muL of EDC (10mg/mL) and 100 muL of NHS (10mg/mL) solution are added for shake incubation for 30min at 25 ℃ and then are subjected to magnetic separation;

2) 100 μ L of immunoglobulin IgG (2mg/mL) was added, magnetically separated after 2 h of 25 ℃ concussion coupling, and blocked with 200 μ L of PBST buffer (containing 1% BSA) for 1 h, where BSA was used to reduce non-specific adsorption. Finally, three washes with PBS were performed to remove impurities, resuspended in PBST buffer, and stored at 4 ℃ until the final IgG concentration was 0.4 mg/mL.

3. Preparation of borated SERS tags

1) 18 nm AuNPs were prepared according to the method of Frens: adding 100 mL of deionized water into a 250 mL three-necked bottle, heating the three-necked bottle in an oil bath until the water is boiled, adding 0.5 mL of 0.5% chloroauric acid solution, then adding 1.8 mL of 1% sodium citrate solution, gradually changing the color of the solution from light yellow to purple, heating the solution for 20 min, stopping the reaction when the color of the solution is changed into wine red, cooling the solution to room temperature, and keeping the solution at 4 ℃ away from light for later use;

2) preparation of the SERS label: adding the prepared 600 mu LAuNPs into a 1.5 mL EP tube, slightly shaking the mixture for several times by hand after adding 20 mu L ascorbic acid, then adding 80 mu L5 mM silver nitrate, reacting the mixture for 3min after shaking the mixture by hand, and then adding 20 mu L5 × 10^-5 M pATP signal molecule solution, wherein the solution gradually changes into gray, then 500 muL of 50 mM Tris-HCl buffer solution (pH 8.5) and 100 muL of 5mg/mL dopamine are added for reaction for 30min, and after centrifugation at 6000 rpm for 15min, the solution is resuspended in 500 muL PBS solution and mixed uniformly by ultrasound for 1 min;

3) boration: and adding 100 muL EDC (10mg/mL) and 100 muL NHS (10mg/mL) into the prepared SERS tag, oscillating and incubating for 30min at normal temperature, then adding 500 muL 1mg/mL 4-carboxyphenylboronic acid, oscillating and incubating for 30min, carrying out boration, centrifuging at 6000 rpm for 15min, then re-suspending in 500 muL PBS solution, carrying out ultrasonic mixing for 1min, and storing for later use at 4 ℃ in a dark place.

4. IgG@Fe₃O₄Catch bacteria in 1 mL 10³Adding 50 mu L IgG @ Fe into CFU/mL bacterial liquid₃O₄Performing shake incubation on magnetic beads at 37 ℃ for 20 min, and performing magnetic separation to obtain IgG @ Fe₃O₄Bacterial complexes, washed three times with PBS.

5. In the above IgG @ Fe₃O₄Adding 100 mu L SERS tag into the bacterial complex, incubating for 10 min, and passing

Magnetic separation to obtain IgG_@F_e3O₄-bacteria-SERS tag sandwich complexes washed three times with PBS.

FIG. 1 is a representation of a SERS nanosensor; wherein A is TEM image of 18 nm AuNPs, B is TEM image of Au (core) @ Ag (shell), C is TEM image of SERS tag, and D is IgG @ Fe₃O₄TEM image of magnetic beads, E is IgG @ Fe₃O₄TEM image of the bacterial complex, F is IgG @ Fe₃O₄TEM image of bacteria-SERS tag complex.

6. IgG@Fe₃O₄Bacteria-_SE_RThe S-tag complex was measured by SERS spectroscopy using a capillary, and the spectra of each bacterium were collected 10 times.

7. And comparing different Raman spectrum regions by adopting Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) to determine the optimal region for bacteria classification.

Example 2 optimization of SERS tags

Taking silver nitrate, p in the synthesis of the SERS tag in the step 3Study of the effect of the amount of ATP on SERS intensity and SERS tag stability (fig. 2). Wherein FIG. 2A shows the addition of different volumes of 5 mM AgNO₃Effect on SERS tag Synthesis, FIG. 2B 5 mM AgNO in different volumes₃To 1081cm^-1Graph of SERS intensity effect, FIG. 2C is 5X 10 of different volumes^-5Effect of M pATP on SERS tag Synthesis, FIG. 2D 5X 10 for different volumes^-5M pATP to 1081cm^-1The graph of the influence of SERS intensity is shown in FIG. 2E, which is the graph of the ultraviolet absorption spectrum of AuNPs (black curve) and SERS tag (red curve), and FIG. 2F is the research of the stability of SERS tag, which is stored for 0, 1,7,30 days at 4 deg.C.

Example 3 SERS detection of bacteria

Using IgG @ Fe₃O₄bacteria/SERS tag nanosensor at 10³-10¹The detection limit of the SES nano-sensor (figure 3) is shown in figures 3A-E, which are the detection limit of Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Pseudomonas aeruginosa and Klebsiella pneumoniae respectively, and figure 3F, which is the detection limit of 1081cm of five bacteria at three different concentrations^-1A comparison of raman intensities.

Processing of Raman spectral data

Smoothing, baseline calibration and spectrogram normalization are carried out on the collected spectrogram data, and the Raman spectrum range is intercepted to be 200-2000cm^-1And (6) carrying out clustering analysis. Dividing the Raman spectrum of the bacteria into R₁ (700-1050 cm^-1) And R₂ (1200-1550 cm^-1) The results of the clustering analysis based on the different regions are shown in table 1 for two regions (fig. 4):

TABLE 1

The results show that R₂(1200-1550 cm^-1) Is the optimal area for bacterial classification. Because different bacteria have different cell biochemical components, when the SERS tag is attachedObtaining a Raman spectrum with bacterial specificity when attached to the surface of the bacteria based on R₂The classification effect of the region is best because R₂The region represents the bacterial surface protein and glycan signals, closest to the SERS enhancing substrate (Ag).

The bacterial surface protein and glycan signal area (1200-^-1) The five bacteria of staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, escherichia coli and streptococcus dysenteriae are subjected to cluster analysis, and the results show the effectiveness of the detection method disclosed by the invention as shown in fig. 5.

Claims (4)

1. A method for detecting bacteria based on tag Surface Enhanced Raman Scattering (SERS), comprising the steps of:

1) IgG-modified Fe₃O₄Magnetic bead IgG @ Fe₃0₄Adding the test bacteria sample to incubate for capturing and separating bacteria;

2) adding a borated SERS label to the separated sample, and attaching the label to the surface of the bacteria to enhance a Raman signal by utilizing the combination of boric acid and glycopeptide on the surface of the bacteria;

3) separating IgG @ Fe generated in the reaction in the step 2) by external magnetism₃O₄-performing SERS detection on the bacteria-SERS tag nanocomplex;

4) comparing different Raman spectrum regions through Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA), finding out the difference of fingerprint SERS spectra of different bacteria, and realizing the detection of the bacteria; the method comprises the following specific steps: the Raman spectrogram of the bacteria is divided into two regions of R1 and R2, and the wave number of the R1 region is 700-1050 cm^-1The wave number of the R2 region is 1200-^-1Finally, by comparing the differences of the specific fingerprints of the bacteria in the R2 region, the classified detection of various bacteria is realized; wherein:

in the step 2), the preparation method of the borated SERS tag comprises the following steps:

firstly, preparing 18 nm gold nanoparticle AuNPs, then adding 600 muL AuNPs into a clean EP tube, then sequentially adding ascorbic acid, silver nitrate, pATP, 500 muL 50 mmol/L Tris-HCl buffer area, 100 muL 5mg/mL dopamine, and then centrifugally resuspending in 500 muL PBS; and adding 100 muL EDC with the concentration of 10mg/mL and 100 muL NHS with the concentration of 10mg/mL, incubating for 20-40 min, adding 4-carboxyphenylboronic acid, coupling for 20-40 min, and storing the final solution at 4 ℃ for later use.

2. The method according to claim 1, wherein in step 1), the bacteria comprise staphylococcus aureus, escherichia coli, staphylococcus dysentery, pseudomonas aeruginosa and klebsiella pneumoniae.

3. The method of claim 1, wherein in step 1), the IgG-modified Fe is₃0₄Magnetic bead IgG @ Fe₃O₄The preparation method comprises the following steps: commercial carboxyl modified magnetic beads and immunoglobulin I_gG is coupled for 1-3 h at room temperature_，After magnetic separation, the column was blocked with PBST buffer containing 1% BSA, and finally washed three times with PBS to remove impurities, resuspended in PBST buffer, and stored at 4 ℃ until use.

4. The method according to claim 1, characterized by the following specific steps:

1) respectively adding 50 muL of IgG modified Fe into 1 mL of different bacterial liquids₃O₄Magnetic bead IgG @ Fe₃0₄Incubate together

Culturing for 10-30 min to generate IgG @ Fe₃O₄-bacterial complexes, followed by magnetic collection, washing 2-4 times with PBS buffer at pH 7.4, concentration 10mmol/L, and resuspension with PBST;

2) adding 100 mu L of borated SERS label into the mixed solution obtained in the step 1), and incubating for 5-15 min to generate IgG @ Fe₃O₄-bacteria-SERS tag complex, followed by magnetic collection, washing 2-4 times with PBS buffer at pH 7.4, concentration 10 mmol/L;

3) IgG@Fe₃O₄measurement of SERS spectra with capillary tubes for bacteria-SERS tag complexes.

Images