CN111398241A - High-flux SERS detection method for food-borne pathogenic bacteria - Google Patents
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- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 title claims abstract description 33
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- 238000001069 Raman spectroscopy Methods 0.000 claims description 24
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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Abstract
The invention relates to the technical field of food-borne pathogenic bacteria detection, and provides a high-flux SERS (surface enhanced Raman scattering) detection method for food-borne pathogenic bacteria, which comprises the following steps: preparing a silent region function detection probe, preparing a capture probe, establishing a high-flux detection standard working curve of two food-borne pathogenic bacteria, and simultaneously performing SERS (surface enhanced Raman Scattering) determination on the contents of the two food-borne pathogenic bacteria in water. The high-flux Surface-enhanced Raman spectroscopy (SERS) detection method provided by the invention can specifically, sensitively and rapidly detect the contents of two food-borne pathogenic bacteria simultaneously.
Description
Technical Field
The invention belongs to the technical field of food-borne pathogenic bacteria detection, and particularly relates to a high-flux SERS (surface enhanced Raman scattering) detection method for food-borne pathogenic bacteria.
Background
Food-borne diseases not only seriously affect the life health of people, but also affect the development of global economy. According to the united states centers for disease control and prevention (CDC), it is estimated that about one sixth of americans are ill each year from eating contaminated food or water sources, and that 3000 of them die as a result, with economic losses of up to $ 156 billion. Meanwhile, world health organization statistics indicate that about 100 million people suffer from food-borne diseases in the united states each year. The food-borne pathogenic bacteria are pathogenic microorganisms which can cause disease reactions and exist in food, and the common food-borne pathogenic bacteria comprise seven kinds of pathogenic escherichia coli, salmonella, shigella, listeria monocytogenes, vibrio parahaemolyticus, streptococcus haemolyticus and staphylococcus aureus.
At present, the traditional bacteria detection comprises staining, optical microscopy, microorganism culture and the like, but only Gram + (G +) and Gram- (G-) can be distinguished, and the microorganism culture process needs 24-72 hours, which has little effect on evaluating the product sanitation condition of food with short shelf life and is difficult to meet the requirement of food manufacturers, particularly exporters, for short detection period, the novel amplification technology comprises immunoassay (enzyme-linked immunosorbent assay (E L ISA)), nucleic acid identification (polymerase chain reaction (PCR)) and the like, which all need operation processes, the detection result of the E L ISA is easy to be influenced by various factors and is easy to generate false positive and detect on site, the high-throughput sequencing and microarray can simultaneously detect various pathogenic bacteria, but the technologies need to separate the DNA of the bacteria in advance, except for needing professional operators, needing expensive nucleic acid amplification, being difficult to meet the requirement of rapid detection of the current pathogenic bacteria, urgently judging the food-borne pathogenic bacteria or accurately detecting the multiple pathogenic bacteria simultaneously, and needing to effectively control the multiple pathogenic bacteria and multiple food-borne diseases.
The Surface Enhanced Raman Scattering (SERS) technology has narrow spectral peak, short detection time (usually, detection can be completed within fractions of seconds to tens of seconds), good specificity (the spectral peak directly reflects the vibration and rotation information of molecules, and is a fingerprint spectrum), very high sensitivity (single-molecule detection can be realized at present), and is very suitable for rapid and highly sensitive detection of complex samples. At present, two methods for detecting food-borne pathogenic bacteria by SERS at home and abroad are summarized, and the methods are classified into label-free detection and label detection. The food-borne pathogenic bacteria are detected without marks, generally a nano metal sol or a solid substrate is adopted, the pathogenic bacteria are directly detected by utilizing the Raman signals of the pathogenic bacteria, the detection mode is simple, but the reproducibility is poor, the acquired spectral signals are distinguished by chemometric analysis (principal component analysis, chromatographic clustering analysis and the like), and the detection method is not suitable for on-site rapid detection; SERS marker detection generally adopts a fingerprint area Raman signal as a signal molecule, modifies the signal molecule on the surface of an enhanced substrate, and realizes the identification of pathogenic bacteria by connecting a specific antibody. However, this method is difficult to use for high throughput detection of multiple pathogens, mainly for the following reasons: (1) raman signal molecules of a plurality of fingerprint areas are easy to overlap with each other in Raman peaks; (2) the Raman signal of other coexisting materials in the fingerprint area of the biological system is easy to overlap with the Raman signal molecular signal of the selected fingerprint area. Therefore, the difficulty in identifying the spectrogram is greatly increased, and the accuracy of the detection result is seriously influenced.
Disclosure of Invention
Aiming at the situation, the invention provides a high-flux SERS detection method for food-borne pathogenic bacteria, which can effectively solve the problems in the prior art by using Surface-enhanced Raman spectroscopy (SERS) to measure the content of the pathogenic bacteria at high flux.
In order to achieve the purpose, the technical scheme provided by the invention is as follows: the food-borne pathogenic bacteria high-flux SERS detection method is characterized by comprising the following steps:
(1) preparation of probes for detecting the functionality of silent region
Firstly, selecting gold, silver or core-shell materials with the particle size of more than 30nm as an enhanced substrate of SERS signals, self-assembling Raman signal molecules of a silence region containing sulfydryl on the surface of the enhanced substrate, optimizing the signal output intensity of the enhanced substrate, then selecting molecules which are good in biocompatibility, hydrophilic and rich in active reaction groups for wrapping, and finally connecting specific targeting functional molecules on the outer layer of a wrapping material to obtain the silence region function detection probe without optical interference;
(2) preparation of functional Capture probes
Connecting a specific targeting functional molecule on the surface of a carboxyl magnetic bead with a specific particle size based on EDC-NHS reaction to obtain a functional capture probe;
(3) establishment of high-flux simultaneous SERS detection standard working curve for two food-borne pathogenic bacteria
When two food-borne pathogenic bacteria exist simultaneously, under different concentrations, two functional capture probes are respectively added to respectively capture the two food-borne pathogenic bacteria, then two functional detection probes are added to respectively target the surfaces of the corresponding food-borne pathogenic bacteria to form a sandwich structure of magnetic beads, food-borne pathogenic bacteria and detection probes, then the sandwich structure is separated under the action of magnetic force and focused on the laser of a Raman spectrometer to carry out Raman test, and then mercaptophenylacetylene molecules in the two detection probes are respectively established to be 2106cm-1Para-mercaptobenzonitrile molecule at 2228cm-1The standard working curve of the peak intensity y and the corresponding pathogenic bacteria concentration x;
(4) determination of contents of two food-borne pathogenic bacteria
Adding two food-borne pathogenic bacteria with different concentrations into a sample to be tested, adding a functional capture probe and a functional detection probe respectively, incubating, and performing Raman test to obtain p-mercaptophenylacetylene molecules at 2106cm-1The neutralized p-mercaptobenzonitrile molecule is 2228cm-1And (4) comparing the peak intensity with the standard working curve established in the step (3) to determine the contents of the two food-borne pathogenic bacteria in the sample to be detected.
Preferably, the SERS enhancing substrate in step (1) includes gold, silver or core-shell material structured nanomaterials with different shapes and sizes.
Preferably, the Raman signal molecule in the silent region in step (1) comprises a molecule which has all thiol groups and Raman shifts in the biological silent region.
Preferably, the wrapping material in the step (1) is all polymers capable of providing naked amino groups.
Preferably, the particle size of the carboxyl magnetic beads in the step (2) is 380nm-1 μm.
Preferably, the target functional molecule in step (1) or (2) is an antibody, ligand, peptide chain or other chemical or biological molecule related to the detection object.
The invention has the following beneficial effects:
(1) the silence area functional detection probe constructed by the invention emits a narrow-band (1-2nm) single-peak Raman scattering signal in a biological silence area, can effectively avoid the interference of intrinsic Raman signals and fluorescence signals of other substances, and has high accuracy;
(2) according to the invention, the capture probe is constructed based on the carboxyl magnetic beads, and the magnetic separation performance of the magnetic beads greatly simplifies the experimental operation process and shortens the time required by detection;
(3) the detection probe and the capture probe constructed by the invention have strong specificity;
(4) the invention provides a high-flux SERS detection method capable of simultaneously detecting two food-borne pathogenic bacteria.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a standard operating curve for the present invention;
FIG. 3 is a transmission electron microscope image of a sandwich structure formed by the coexistence of the detection probe, the capture probe and two food-borne pathogenic bacteria of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
The specific embodiment of the invention is as follows:
standard working curve making
(1) Instruments and reagents
785nm of excitation light wavelength;
gold nano sol: the self-synthesis is carried out by adopting a sodium citrate reduction chloroauric acid method;
two functional detection probes: two kinds of signal molecules which contain sulfydryl and have different Raman shifts of the biological silence region are anchored on the surface of the nanogold through forming an S-Au bond, the nanogold is coated by polylysine and provides an amino group, and the two kinds of signal molecules are respectively connected with monoclonal antibodies of salmonella and staphylococcus aureus through EDC-NHS reaction to construct two kinds of different functional detection probes for autonomous preparation;
two functional capture probes: respectively modifying monoclonal antibodies of salmonella and staphylococcus aureus on the surfaces of the carboxyl magnetic beads, and independently preparing;
the transmission electron microscope with the sandwich structure formed by the coexistence of the detection probe, the capture probe and the two food-borne pathogenic bacteria is shown in figure 3;
and (4) autonomously culturing salmonella and staphylococcus aureus liquid with different concentrations.
(2) Experimental procedure
Taking salmonella and staphylococcus aureus bacteria liquid with different concentration gradients in a certain volume, respectively adding a 10 mu L salmonella functional capture probe and a 10 mu L staphylococcus aureus functional capture probe, respectively adding a 100 mu L salmonella functional detection probe and a 100 mu L staphylococcus aureus functional detection probe, respectively incubating for 30min in a shaking table at room temperature, separating under the action of magnetic force, washing for 3 times, calibrating a raman spectrometer, performing raman test, simultaneously collecting two SERS signals with different raman shifts in a biological silence area, and establishing that the p-mercaptophenylacetylene molecules in the two detection probes are 2106cm-1The peak intensity y at (a) is plotted against the standard working curve for salmonella and staphylococcus aureus concentration x, respectively, as shown in fig. 2.
Example 1
500ml of ultrapure water was taken, and salmonella and staphylococcus aureus were added thereto in different concentrations, respectively, according to the procedures and detection methods described above, and the results are shown in Table 1.
Example 2
5000ml of tap water was taken, and salmonella and staphylococcus aureus with different concentrations were added thereto, respectively, and the procedure and detection method were performed as described above, and the results are shown in table 1.
Table 1 results of rapid SERS measurements with ultra pure water and tap water with different amounts of added e.
| Examples | Added value (cfu/m L) | Detected value (cfu/m L) | Recovery (%) |
| Ultrapure water | 500/500 | 523/484 | 1.05/0.97 |
| Tap water | 5000/5000 | 5204/5166 | 1.04/1.03 |
Although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. The food-borne pathogenic bacteria high-flux SERS detection method is characterized by comprising the following steps:
(1) preparation of probes for detecting the functionality of silent region
Firstly, selecting gold, silver or core-shell materials with the particle size of more than 30nm as an enhanced substrate of SERS signals, self-assembling Raman signal molecules of a silence region containing sulfydryl on the surface of the enhanced substrate, optimizing the signal output intensity of the enhanced substrate, then selecting molecules which are good in biocompatibility, hydrophilic and rich in active reaction groups for wrapping, and finally connecting specific targeting functional molecules on the outer layer of a wrapping material to obtain the silence region function detection probe without optical interference;
(2) preparation of functional Capture probes
Connecting a specific targeting functional molecule on the surface of a carboxyl magnetic bead with a specific particle size based on EDC-NHS reaction to obtain a functional capture probe;
(3) establishment of high-flux simultaneous SERS detection standard working curve for two food-borne pathogenic bacteria
When two food-borne pathogenic bacteria exist simultaneously, under different concentrations, two functional capture probes are respectively added to respectively capture the two food-borne pathogenic bacteria, then two functional detection probes are added to respectively target the surfaces of the corresponding food-borne pathogenic bacteria to form a sandwich structure of magnetic beads, food-borne pathogenic bacteria and detection probes, then the sandwich structure is separated under the action of magnetic force and focused on the laser of a Raman spectrometer to carry out Raman test, and then mercaptophenylacetylene molecules in the two detection probes are respectively established to be 2106cm-1Para-mercaptobenzonitrile molecule at 2228cm-1The standard working curve of the peak intensity y and the corresponding pathogenic bacteria concentration x;
(4) determination of contents of two food-borne pathogenic bacteria
Adding two food-borne pathogenic bacteria with different concentrations into a sample to be tested, adding a functional capture probe and a functional detection probe respectively, incubating, and performing Raman test to obtain p-mercaptophenylacetylene molecules at 2106cm-1The neutralized p-mercaptobenzonitrile molecule is 2228cm-1And (4) comparing the peak intensity with the standard working curve established in the step (3) to determine the contents of the two food-borne pathogenic bacteria in the sample to be detected.
2. The high-flux SERS detection method for food-borne pathogenic bacteria according to claim 1, wherein the SERS enhanced substrate in the step (1) comprises gold, silver or core-shell structured nano materials with different shapes and sizes.
3. The high-throughput SERS detection method for food-borne pathogenic bacteria according to claim 1, wherein in the step (1), Raman signal molecules in the silent region comprise all molecules with sulfydryl and Raman shifts in a biological silent region.
4. The high-flux SERS detection method for food-borne pathogenic bacteria according to claim 1, wherein the coating material in the step (1) is all polymers capable of providing naked amino groups.
5. The high-flux SERS detection method for food-borne pathogenic bacteria according to claim 1, wherein the particle size of the carboxyl magnetic beads in the step (2) is 380nm-1 μm.
6. The high-throughput SERS detection method for food-borne pathogenic bacteria according to claim 1, wherein the targeting functional molecule in step (1) or (2) is an antibody, a ligand, a peptide chain or other chemical or biological molecule related to a detection object.
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| CN112098389A (en) * | 2020-08-31 | 2020-12-18 | 华南理工大学 | Detection method of Listeria monocytogenes |
| CN112098389B (en) * | 2020-08-31 | 2022-04-22 | 华南理工大学 | Detection method of Listeria monocytogenes |
| CN113092440A (en) * | 2021-04-06 | 2021-07-09 | 江苏大学 | Hollow gold-silver core-shell nanoflower SERS (surface enhanced Raman scattering) nanoprobe HAu/AgSNFs-ATP (adenosine triphosphate) |
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