Pinhole type solid-liquid binary SERS sensor and application thereof in rapid detection of bacterial substances

Abstract

The application discloses a preparation method of a pinhole type solid-liquid dual-state SERS (Surface Enhanced Raman Scattering) sensor and application of the pinhole type solid-liquid dual-state SERS sensor in rapid detection of bacterial substances. The preparation method of the pinhole type solid-liquid dual-state SERS sensor comprises the following steps: a small hole is formed on a base material, vanadium dioxide nanosol is dropped on the small hole position and dried, and then a sample to be detected, silver nanosheet sol and cysteine aqueous solution are sequentially dropped on the small hole position, and the SERS detection is performed on a semiconductor refrigeration hot table. The pinhole type solid-liquid dual-state SERS sensor can accurately position and enrich the bacterial substances near the pinhole, the solid-liquid two-state base material can generate a stronger resonance effect, and the base material can be combined with the bacterial substances closely, so that the overall Raman spectrum information of the bacterial bodies can be effectively captured.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of SERS detection technology, and particularly relates to a pinhole type solid-liquid dual-state SERS sensor and application thereof in rapid detection of bacterial substances. BACKGROUND

[0002] At present, bacterial infection events occur frequently worldwide. The main manifestations of bacterial infection of different degrees include primary inflammation, bacteremia, skin rash, septic shock, and even death. The development of rapid detection methods for pathogenic microorganisms is the key to cope with infection events, especially in the fields of medical examination and public health safety.

[0003] The existing technologies for bacterial detection include methods based on bacterial culture and counting, molecular detection technologies, immunological detection technologies, and biosensor detection technologies. The method based on bacterial culture and counting is relatively mature, but has limitations in terms of species, long cycle (more than 18 hours), and most bacteria cannot be artificially cultured. Common molecular detection technologies include polymerase chain reaction (PCR), and fluorescence quantitative PCR, multiplex PCR, and reverse transcription PCR based on PCR technology. Such methods are relatively accurate, but require professional personnel and the detection process is relatively complicated, and are not suitable for on-site detection. Immunological detection methods include enzyme-linked immunosorbent assay, immunochromatography, and immunofluorescence. Immunological detection methods have good specificity and high sensitivity, but it is difficult to obtain antibodies and the cost is high. Biosensor detection technologies include fluorescence method and electrochemical method, and sometimes there are false positive problems. In summary, the existing technologies cannot meet the actual needs of rapid detection of bacterial microorganisms.

[0004] With the progress of nanotechnology and spectroscopy technology, surface-enhanced Raman spectroscopy (SERS) detection technology brings opportunities for rapid detection of pathogenic bacterial microorganisms. SERS is very suitable for rapid detection of bacterial microorganisms due to its high sensitivity, inherent selectivity of spectral fingerprint, simple and rapid preparation, and non-destructive data acquisition in liquid environment. However, in traditional bacterial SERS detection, bacteria cannot fully contact the substrate material due to size, making it difficult to capture effective spectral information, and the obtained SERS spectral signal is only a local signal of the bacterial body, which cannot reflect the overall spectral information of the bacterial body. SUMMARY

[0005] The present application provides a preparation method of a pinhole type solid-liquid dual-state SERS sensor and application thereof in rapid detection of bacterial substances. In a sample containing bacteria, the spectral signal of the target bacteria is obtained to realize rapid detection of bacterial substances.

[0006] In order to achieve the above technical purposes of the present application, the following technical solutions are adopted in the present application:

[0007] A preparation method of a pinhole type solid-liquid dual-state SERS sensor, comprising the steps of: drilling a small hole on a base material, dropping a vanadium dioxide nanosol at the small hole position after drying treatment, then dropping a sample to be detected, a silver nanosheet sol and a cysteine sulfinate aqueous solution at the small hole position in sequence, placing on a semiconductor refrigeration hot table, and performing SERS detection. The base material is preferably flax cloth, and the small hole diameter is preferably 0.01-0.1 mm. The vanadium dioxide nanosol can be obtained by a known preparation method or purchased from a professional manufacturer. One preparation method can be as follows: 3 m mol of ammonium metavanadate (NH4VO3), 6 m mol of oxalic acid (H2C2O4·2H2O) and 35 mL of deionized water are added to a beaker, and stirred vigorously at room temperature for 30 min. Then the solution is transferred to a 50 mL stainless steel autoclave and heated at 180°C, and after 24 hours, it is naturally cooled to room temperature. The obtained blue-black precipitate is filtered and washed with distilled water and anhydrous alcohol, and dried in air at 70°C for 12 h to obtain nanometer vanadium dioxide with a particle size of 100-300 nm, which is then dispersed in ultrapure water for standby use. The silver nanosheet sol has a particle size of 60-100 nm, which can be obtained by a known preparation method such as the sodium citrate method or purchased from a professional manufacturer.

[0008] In the present application, the vanadium dioxide nanosol is preferably concentrated 50-300 times to form a star-shaped vanadium dioxide nanosol, more preferably concentrated 60-220 times, and the particle number of the concentrated vanadium dioxide nanosol is 3.55×10 10 -1.3046×10 11 / μL; the silver nanosheet sol is preferably concentrated 50-300 times for use, more preferably concentrated 80-200 times, and the particle number of the concentrated silver nanosol is 1.896×10 10 -4.74×10 12 / μL; and the cysteine sulfinate solution has an antioxidation protection effect, and the aqueous solution concentration is preferably 10 -7 -10 -9 mol / L.

[0009] In the present application, flax cloth is used as the base material, which has good hygroscopicity and can adsorb the sample to be detected on the surface of the nanomaterial, so that the sample to be detected is in the hot spot area, which is conducive to the surface enhancement of the signal and further conducive to SERS detection. At the same time, due to the rough surface of the flax cloth, there are many small gaps after the nanomaterial is attached, and due to the electric field coupling effect, there are also a large number of "hot spot effects" in these gaps, which further enhances the Raman scattering signal of the sample to be detected, and reflects more detailed structure information of the sample to be detected.

[0010] The pinhole type solid-liquid dual-state SERS sensor of the application can accurately position and enrich the fungi near the pinhole, and the solid-liquid two-state substrate, i.e., the vanadium dioxide nanosol is solid after drying, and the silver nanosheet sol is liquid, can produce stronger resonance effect, and the substrate can be closely combined with the fungi, so that the overall Raman spectrum information of the fungi can be effectively captured.

[0011] In the application, the vanadium dioxide nanosol, the silver nanosheet sol and the aqueous cysteine sulfinate solution can be prepared in advance for on-site and timely application. Therefore, the pinhole type solid-liquid dual-state SERS sensor based on the application can be made into a detection kit set, which can include bottles or cans or cylinders respectively containing the vanadium dioxide nanosol, the silver nanosheet sol and the aqueous cysteine sulfinate solution, a substrate material linen cloth provided with small holes, a semiconductor refrigeration hot table, a handheld Raman detector and the like.

[0012] The application also proposes the application of the pinhole type solid-liquid dual-state SERS sensor or the detection kit set in rapid detection of fungi, using the following method steps:

[0013] (1) Take the substrate material, and wear small holes with a diameter of 0.03-0.08 mm, and then take out after ultrasonic cleaning with ultrapure water and air drying without excess water drops;

[0014] (2) Take 20-50 μL of star-shaped vanadium dioxide nanosol concentrated by 60-220 times, and drop it at the position of the small hole, and then ultrasonic standing and drying for use;

[0015] (3) Drop 30-80 μL of liquid sample of fungi at the small hole obtained in step (2), shake, then drop 10-30 μL of silver nanosheet sol concentrated by 80-200 times, then drop 10 -7 -10 -9 mol / L aqueous cysteine sulfinate solution 5-15 μL, mix uniformly, and place on a semiconductor refrigeration hot table at 38-42℃;

[0016] (4) Immediately use a laser with a wavelength of 1064 nm to perform SERS detection around the edge range of the small hole; randomly select 2-3 small holes, randomly take points on the circular path around the edge of each small hole to collect 2-3 spectra, then compound 4-9 spectra into one spectrum in a Gaussian fitting manner, and import into the Raman spectrometer library;

[0017] (5) Perform comparison and analysis detection of spectral data in a matching manner of characteristic peak identification, set the matching coefficient to 85%, the laser power to 30-50 mW, and the detection time to 2-4 s; the obtained result can be directly presented in a visual manner, and determined as the name of the specific fungi.

[0018] As a preferred technical solution, the application of the pinhole type solid-liquid dual-state SERS sensor in rapid detection of bacterial substances can adopt the following method steps:

[0019] (1) A 0.8*0.8 cm square flax material cloth strip is randomly and uniformly perforated with a 0.06 mm diameter needle, and then washed with ultrapure water for 1 min, and taken out to dry without excess water dripping.

[0020] (2) 30 μL of concentrated (60-220 times) star-shaped vanadium dioxide nanosol is added to the cloth strip hole position, then ultrasonicated for 2 min, placed for 5 min, and dried in a 40-60°C oven, ready for use. -7 -10 -9 mol / L aqueous solution of cysteine sulfinate 10 μL, mixed uniformly, and placed on a semiconductor refrigeration hot table at 38-42°C.

[0021] (4) Immediately use a 1064 nm wavelength laser to perform SERS detection around the edge range of the hole. Randomly select two holes, randomly take points to collect three spectra around the edge circular path of each hole, then compound the six spectra into one spectrum in a Gaussian fitting manner, and import into the spectrum library of a handheld Raman spectrometer.

[0022] (5) Perform spectral data comparison and analysis detection in a characteristic peak recognition matching manner, with a matching coefficient of 85%, a laser power of 30-50 mW, and a detection time of 2-4 s. The obtained detection results can be presented in a visual manner, and determined as the name of a specific bacterial substance.

[0023] The beneficial effects of the present application are:

[0024] (1) The detection method of the present application for bacterial substances does not require cultivation, can utilize pinhole positioning to enrich bacteria, is simple and fast to operate, and in the case of solid-state part-vanadium dioxide nanosol drying, the preparation of the solid-state SERS sensor is completed, and the whole process of single sample detection is about 3 minutes, which can provide an important basis for the rapid clinical judgment of doctors.

[0025] (2) The present application can comprehensively reflect the spectral signal of bacterial substances by collecting multiple spectra and then performing Gaussian compounding, overcoming the limitations and misjudgment of single signal, and improving the accuracy of the detection results, which can reach more than 95% (based on the results obtained by the cultivation method).

[0026] (3) The present application is suitable for rapid detection and determination of Klebsiella pneumoniae, Candida albicans, Salmonella, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus, Cronobacter sakazakii, Enterococcus faecium, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii and other bacteria, has wide detection range and strong universality. At present, although there are reports of Raman detection of bacteria in the industry, a standard Raman detection method and detection spectrum for bacteria have not been formed, and the present application can provide a selection basis for establishing a more complete SERS detection of bacteria.

[0027] (4) The detection lower limit of the present application for bacteria is 10 CFU / mL, which has high sensitivity and can fully meet the actual detection requirements. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the SERS spectrum of Candida albicans detected in Example 1.

[0029] Figure 2 is the SERS spectrum of Klebsiella pneumoniae detected in Example 2.

[0030] Figure 3 is the SERS spectrum of Staphylococcus epidermidis detected in Example 3.

[0031] Figure 4 is the SERS spectrum of Pseudomonas aeruginosa detected in Example 4. DETAILED DESCRIPTION

[0032] The following examples are further illustrations of the present application and are set forth to aid in the understanding of the present application, the true scope of which is indicated by the appended claims. Those skilled in the art can and will realize changes and modifications to the preferred embodiments described herein, and it is intended to claim all such changes and modifications as fall within the true scope of the application.

[0033] Example 1

[0034] (1) A 0.8x0.8cm square linen material cloth strip is randomly and uniformly perforated with a 0.06mm diameter needle, and then washed with ultrapure water for 1min, and taken out to dry without dripping excess water.

[0035] (2) 30μL of star-shaped vanadium dioxide nanosol concentrated by about 60 times is added to the cloth hole position, and then ultrasonicated for 2min, placed for 5min, and placed in a 40℃ oven until dry.

[0036] (3) The cloth strip prepared in (2) was added with 50 μL of Candida albicans liquid sample (CMCC 98001, 10 CFU / mL) at the small hole, and shaken for 1 min. Then, 20 μL of concentrated (80 times) silver nanosheet sol was added, and 10 μL of 0.1 mol / L aqueous cysteine solution was added, and mixed uniformly, and placed on a semiconductor refrigeration hot table at 38°C. -7 mol / L aqueous cysteine solution 10 μL, and mixed uniformly, and placed on a semiconductor refrigeration hot table at 42°C.

[0037] (4) Immediately, SERS detection was performed around the edge of the small hole using a laser with a wavelength of 1064 nm. Two small holes were randomly selected, and three spectra were collected at random points around the circular path of the edge of each small hole, and then six spectra were compounded into one spectrum in a Gaussian fitting manner, and introduced into the spectrum library of a handheld Raman spectrometer.

[0038] (5) Spectral data were compared and analyzed in a matching manner of characteristic peak identification, the matching coefficient was set to 85%, the laser power was 30 mW, and the detection time was 2 s. The detection result was determined as Candida albicans. For example, Figure 1 738, 997, 1024, 1241, 1440, 1601 cm -1 are characteristic peaks of Candida albicans.

[0039] Example 2

[0040] (1) A 0.8*0.8 cm square linen material cloth strip was randomly and uniformly perforated with a needle with a diameter of 0.06 mm, and then washed with ultrapure water for 1 min, and taken out to dry without excess water dripping.

[0041] (2) 30 μL of star-shaped vanadium dioxide nanosol concentrated by 220 times was added to the small hole position of the cloth strip, and then ultrasonicated for 2 min, and left to stand for 5 min, and placed in a 60°C oven until dry.

[0042] (3) The cloth strip prepared in (2) was added with 50 μL of Candida albicans liquid sample (CMCC 98001, 10 CFU / mL) at the small hole, and shaken for 1 min. Then, 20 μL of concentrated (80 times) silver nanosheet sol was added, and 10 μL of 0.1 mol / L aqueous cysteine solution was added, and mixed uniformly, and placed on a semiconductor refrigeration hot table at 38°C. -9 mol / L aqueous cysteine solution 10 μL, and mixed uniformly, and placed on a semiconductor refrigeration hot table at 42°C.

[0043] (4) Immediately, SERS detection was performed around the edge of the small hole using a laser with a wavelength of 1064 nm. Two small holes were randomly selected, and three spectra were collected at random points around the circular path of the edge of each small hole, and then six spectra were compounded into one spectrum in a Gaussian fitting manner, and introduced into the spectrum library of a handheld Raman spectrometer.

[0044] (5) The spectral data was compared and analyzed using a characteristic peak identification matching method, with a matching coefficient set to 85%, laser power of 50mW, and detection time of 4s. The detection result identified it as Klebsiella pneumoniae. Figure 2 At 647, 725, 751, 952, 1318, and 1437 cm -1 The peak at this point is characteristic of Klebsiella pneumoniae.

[0045] Example 3

[0046] Step (3) Add 50 μL of Staphylococcus epidermidis liquid sample (CMCC26069, 10 CFU / mL) to the small well. Everything else is the same as in Example 1. The detection result indicates Staphylococcus epidermidis. Figure 3 At 736, 1010, 1250, 1331, 1448, and 1610 cm -1 The peak at this location is characteristic of Staphylococcus epidermidis.

[0047] Example 4

[0048] Step (3) Add 50 μL of Pseudomonas aeruginosa (CMCC10104, 10 CFU / mL) liquid sample to the well. Everything else is the same as in Example 1. The test result indicates Pseudomonas aeruginosa. Figure 4 At 677, 1006, 1365, 1566, and 1625 cm -1 The peak at this location is characteristic of Pseudomonas aeruginosa.

[0049] It should be noted that the above-described technical content of this invention is merely an explanation and clarification to enable those skilled in the art to understand the technical essence of this invention, and therefore is not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the claims. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the essential spirit of this invention should be within the scope of protection of this invention.

Claims

1. A method for preparing a pinhole type solid-liquid dual-state SERS sensor, comprising the steps of: drilling a small hole on a base material, dropping vanadium dioxide nanosol at the position of the small hole and drying, then dropping the sample to be detected, silver nanoplate sol, and aqueous cysteine sulfinate solution at the position of the small hole in sequence, placing on a semiconductor refrigeration hot table, and performing SERS detection.

2. The production method according to claim 1, wherein The base material is linen, and the small hole has a diameter of 0.01-0.1 mm.

3. The production method according to claim 1, wherein The vanadium dioxide nanosol is concentrated by 50-300 times to form a star-shaped vanadium dioxide nanosol.

4. The production method according to claim 1, wherein The silver nanoplate sol is concentrated by 50-300 times.

5. The production method according to claim 1, wherein The aqueous cysteine sulfinate solution has a concentration of 10 -7 -10 -9 mol / L.

6. The pinhole type solid-liquid dual-state SERS sensor obtained by the preparation method of any one of claims 1-5.

7. A detection kit set based on the pinhole type solid-liquid dual-state SERS sensor of claim 6, comprising bottles or cans or cylinders respectively containing vanadium dioxide nanosol, silver nanoplate sol, and aqueous cysteine sulfinate solution, and further comprising linen base material provided with small holes, a semiconductor refrigeration hot table, and a handheld Raman detector.

8. Use of the pinhole type solid-liquid dual-state SERS sensor of claim 6 or the detection kit set of claim 7 in rapid detection of bacterial substances, wherein the bacterial substances include any one of Klebsiella pneumoniae, Candida albicans, Salmonella, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus, Cronobacter sakazakii, Enterococcus faecium, Proteus mirabilis, Pseudomonas aeruginosa, and Acinetobacter baumannii.

9. Use according to claim 8, wherein the compound is ###0002### The following method steps are used: (1) Take the base material, drill a small hole with a diameter of 0.03-0.08 mm, wash with ultrapure water, and then take out and air dry until no excess water droplets are present; (2) Take 20-50 μL of star-shaped vanadium dioxide nanosol concentrated by 60-220 times, drop it at the position of the small hole, then ultrasonically stand, and dry for use; (3) At the small hole of step (2), 30-80 μL of liquid sample of the bacteria is added, after oscillation, 10-30 μL of silver nanosheet sol with concentration of 80-200 times is added, then 10 -7 -10 -9 μL of 5-15 μL of aqueous solution of 10 mol / L cysteine sulfinate is added, and mixed uniformly, and placed on a semiconductor refrigeration hot table at 38-42 °C; (4) Immediately use a 1064 nm wavelength laser to perform SERS detection around the edge of the small hole; randomly select 2-3 small holes, randomly take points along a circular path around the edge of each small hole to collect 2-3 spectra, then compound 4-9 spectra into one spectrum in a Gaussian fitting manner, and import into the Raman spectrometer library; (5) Perform spectral data comparison and analysis detection in a characteristic peak recognition matching manner, with a matching coefficient of 80%-90%, a laser power of 30-50 mW, and a detection time of 2-4 s; the obtained results can be directly presented in a visual manner, and are determined as the name of a specific bacterial substance.

10. Use according to claim 9, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The detection lower limit of the bacterial substance liquid sample in step (3) is 10 CFU / mL.

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