A highly stable SERS substrate for rapid detection of pathogens and its application

Abstract

The present invention discloses a highly stable SERS substrate for rapid detection of pathogens. The present invention proposes a SERS detection substrate for identifying samples containing bacteria to achieve high-stability detection of bacteria. The bacteria detection method of the present invention is simple to operate, and the detection time is shortened from several days (or even longer) of traditional culture methods to less than 10 minutes, and the professional level requirements of the operator are low. The present invention has high stability in the detection of pathogens such as Escherichia coli and Staphylococcus aureus, with an RSD of less than 5%. The detection limit of Escherichia coli and Staphylococcus aureus can reach 10 4 cfu / mL, which meets the requirements for clinical pathogen testing.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a highly stable SERS substrate for rapid detection of pathogens and its application. Background Art

[0002] Escherichia coli, Staphylococcus aureus and other pathogenic bacteria pose a threat to human health. The main reason is that many serious or even fatal diseases are caused by bacterial infection or contamination, such as intestinal infections, urinary tract infections and other diseases. Rapid and sensitive bacterial detection methods can prevent and control pathogen-related diseases. Polymerase chain reaction, genomics and proteomics studies are used to reveal antibacterial activity at the molecular level. These technologies have some problems, including false negatives of polymerase chain reaction, high cost of genomics and susceptibility to interference of proteomics. Since the discovery that Raman signals can be greatly enhanced on the surface of rough silver electrodes, surface enhanced Raman spectroscopy (SERS) technology has become the focus of material detection, and the detection and identification of bacteria has also attracted attention. [1,2] However, the instability of conventional SERS substrates and their weak binding ability with bacteria result in weak spectral signal intensity and poor reproducibility. Here, we propose a new highly stable SERS substrate for bacterial detection, which can achieve rapid and reliable detection of bacteria.

[0003] References

[0004] [1] Li Wenshuai, Wu Guorui, Zhang Qianjing, Yue Aiqin, Du Weijun, Zhao Jinzhong, Liu Dingbin. Research progress of bacterial detection based on Raman spectroscopy. Journal of Chemistry in Universities. 2020, 41(5), 872-883.

[0005] [2] Yunfan Chen, Qi An,* Kaixuan Teng, Chao Liu, Fuwei Sun,* and Guangtao Li,* Application of SERS in in Vitro Biomedical Detection, Chemistry-An Asian Journal, 10.1002 / asia.202201194. Summary of the Invention

[0006] To address the current issues of weak spectral signal intensity and poor reproducibility due to the poor stability of SERS for bacterial detection, the present invention provides a substrate and preparation method for SERS detection of pathogenic bacteria. Specifically, the present invention utilizes the following technical solutions to achieve this:

[0007] A highly stable SERS substrate for rapid detection of pathogens is prepared by the following method:

[0008] (1) Add 5-20 μm polycaprolactone porous framework to urine containing target pathogens and disperse it evenly in distilled water;

[0009] (2) adding the dihydrochalcone solution to step (1) and shaking to mix;

[0010] (3) Centrifuge at 1000-5000 rpm for 1-5 min, remove the supernatant, and retain the material at the bottom of the centrifuge tube for later use;

[0011] (4) mixing the tungsten telluride (WTe2) nanoparticles with the material at the bottom of the centrifuge tube in step (3) and allowing to stand;

[0012] (5) Take the mixed solution from step (4) and drop it onto a glass slide until it dries to form a film.

[0013] In the present invention, preferably, 5-20 mg of a polycaprolactone porous skeleton is added to 1 mL of a sample solution containing target pathogens. The polycaprolactone porous skeleton serves to anchor bacteria and is a commonly used biological scaffold material that can be purchased from professional manufacturers such as Xi'an Ruixi Biotechnology Co., Ltd.; the role of dihydrochalcone is to act as an affinity agent to firmly couple the pathogens to the surface of the nanoparticles. The concentration of the dihydrochalcone solution is 1*10 -5 -1*10 -7 M; The function of WTe2 nanoparticles is to enhance the SERS signal of pathogens. The dosage is generally very small. They can be purchased from professional manufacturers such as Shenzhen Liutan Technology Co., Ltd., and can also be prepared by referring to relevant literature. The sample liquid containing the target pathogens includes but is not limited to any one or a mixture of urine, blood, saliva, and tissue fluid. The pathogens include but are not limited to one or more of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Candida albicans. There is no requirement for the standing time, generally 1-5 minutes is sufficient. The film can be dried and air-dried at room temperature.

[0014] As a preferred embodiment, the present invention provides a highly stable SERS substrate, which is prepared by the following method:

[0015] (1) Add 8-15 mg of a 5-20 μm polycaprolactone porous framework to a sample solution containing target pathogens and disperse it evenly in 1-3 mL of distilled water;

[0016] (2) Add 10% ethanol to step (1). -5 -10 -7 Add 1-3 mL of dihydrochalcone solution of M and shake to mix;

[0017] (3) Centrifuge at 3000-4000 rpm for 1-3 min, remove the supernatant, and retain the material at the bottom of the centrifuge tube for later use;

[0018] (4) mixing the tungsten telluride (WTe2) nanoparticles with the material at the bottom of the centrifuge tube in step (3) and allowing to stand;

[0019] (5) Take 8-12 μL of the mixed solution after standing in step (4) and drop it onto a glass slide until it dries to form a film.

[0020] The highly stable SERS substrate described in this invention can be used for rapid pathogen detection. By irradiating the substrate with a 785nm laser, the characteristic SERS spectrum of the target pathogen can be obtained in 1-5 seconds. The laser power is 20-120mW, preferably 50-100mW. A 10-20μL WTe2 nanoparticle sol is used.

[0021] The key problem of the present invention when using a substrate prepared from ordinary nanoparticles for detection in biological samples containing pathogenic bacteria is that the stability of the detection is defective. When ordinary gold or silver nanoparticles are used to detect bacteria, the volume of the bacteria is relatively large relative to the nanoparticles, about 1-5μm, and the binding strength between the bacteria and the nanoparticles is relatively weak, which usually leads to weak and unstable spectral signals. Furthermore, bacteria are usually in a relatively complex matrix. The influence of this complex matrix on the detection effect is undoubtedly huge, and it also directly affects the stability of the detection signal. Centrifugation and washing can remove most of the influence of the complex matrix, but it is still difficult to ensure the stability of the detection signal. It is based on these problems that the present invention proposes a SERS detection substrate to identify samples containing bacteria and achieve high-stability detection of bacteria.

[0022] The beneficial effects of the present invention are:

[0023] (1) The bacteria detection method of the present invention is simple to operate, and the detection time is shortened from several days (or even longer) of the traditional culture method to less than 10 minutes, and the professional level requirement of the operator is low.

[0024] (2) The present invention has high stability in detecting pathogens such as Escherichia coli and Staphylococcus aureus, with a relative standard deviation (RSD) of less than 5%. The detection limit for Escherichia coli and Staphylococcus aureus can reach 10 4 cfu / mL, which meets the requirements for clinical pathogen testing.

[0025] (3) The present invention can detect pathogens including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Candida albicans, etc. It has a wide range of applications and is universal. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the SERS spectrum of E. coli detection.

[0027] Figure 2 This is the SERS spectrum of Staphylococcus aureus detection.

[0028] Figure 3 This is a diagram of the SERS detection results of Comparative Example 1.

[0029] Figure 4 This is a diagram of the SERS detection results of Comparative Example 2.

[0030] Figure 5 This is a diagram of the SERS detection results of Comparative Example 3.

[0031] Figure 6 This is a diagram of the SERS detection results of Comparative Example 4.

[0032] Figure 7 The vertical axis in the figure represents the relative Raman intensity, that is, the difference between the maximum and minimum Raman intensity in Example 1.

[0033] Figure 8 The vertical axis in the figure represents the relative Raman intensity, that is, the difference between the maximum and minimum Raman intensity in Example 2. DETAILED DESCRIPTION

[0034] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0035] Example 1

[0036] (1) 0.01 g of 5 μm polycaprolactone porous framework was added to 1 mL of urine containing Escherichia coli pathogens and evenly dispersed in 2 mL of distilled water; the urine sample was obtained from the Second Affiliated Hospital of Anhui Medical University and was previously confirmed by culture method to be a urine sample from a patient infected with Escherichia coli.

[0037] (2) Add 1×10 -6 M dihydrochalcone solution 1mL, and shake to mix;

[0038] (3) Centrifuge for 3 min (at 4000 rpm), remove the supernatant with a pipette, and retain the material at the bottom of the centrifuge tube for later use;

[0039] (4) Mix 20 μL of tungsten telluride (WTe2) nanoparticle sol with the material at the bottom of the centrifuge tube obtained in step (3) and let it stand for 1 min;

[0040] (5) Using a dropper, take 10 μL of the mixed solution obtained in step (4) and drop it onto a glass slide until it dries to form a film;

[0041] (6) Irradiate the substrate prepared in step (5) with a 785 nm laser at a laser power of 50 mW. After 3 seconds, the characteristic SERS spectrum of the E. coli pathogen can be obtained. The results of the parallel detection of this embodiment are compared as follows: Figure 7 As shown, the detection of Escherichia coli by the present invention has high stability, with RSD less than 5%.

[0042] Example 2

[0043] (1) 0.015 g of a 20 μm polycaprolactone porous framework was added to 1 mL of urine containing Staphylococcus aureus pathogens and dispersed evenly in 2 mL of distilled water. The urine sample was obtained from the Second Affiliated Hospital of Anhui Medical University and was previously confirmed by culture to be a urine sample from a patient infected with Staphylococcus aureus.

[0044] (2) Add 5×10 -7 M dihydrochalcone solution 1mL, and shake to mix;

[0045] (3) Centrifuge for 3 min (at 4000 rpm), remove the supernatant with a pipette, and retain the material at the bottom of the centrifuge tube for later use;

[0046] (4) Mix 20 μL of tungsten telluride (WTe2) nanoparticle sol with the material at the bottom of the centrifuge tube obtained in step (3) and let it stand for 1 min;

[0047] (5) Using a dropper, take 10 μL of the mixed solution obtained in step (4) and drop it onto a glass slide until it dries to form a film;

[0048] (6) Irradiate the substrate prepared in step (5) with a 785 nm laser at a laser power of 100 mW. After 2 seconds, the characteristic SERS spectrum of Staphylococcus aureus pathogen can be obtained. The results of the parallel detection of this embodiment are compared as follows: Figure 8 The detection of Staphylococcus aureus by the present invention has high stability, with an RSD of less than 5%.

[0049] Example 3

[0050] (1) 0.008 g of a 10 μm polycaprolactone porous framework was added to 1 mL of urine containing Staphylococcus aureus pathogens and evenly dispersed in 2 mL of distilled water. The urine sample was obtained from the Second Affiliated Hospital of Anhui Medical University and was previously confirmed by culture to be a urine sample from a patient infected with Staphylococcus aureus.

[0051] (2) Add 3×10 -6 M dihydrochalcone solution 1mL, and shake to mix;

[0052] (3) Centrifuge for 2 min (at 4000 rpm), remove the supernatant with a pipette, and retain the material at the bottom of the centrifuge tube for later use;

[0053] (4) Mix 15 μL of tungsten telluride (WTe2) nanoparticle sol with the material at the bottom of the centrifuge tube obtained in step (3) and let it stand for 1 min;

[0054] (5) Using a dropper, take 10 μL of the mixed solution obtained in step (4) and drop it onto a glass slide until it dries to form a film;

[0055] (6) Irradiating the substrate prepared in step (5) with a 785 nm laser at a laser power of 80 mW, a characteristic SERS spectrum of the Staphylococcus aureus pathogen can be obtained after 4 seconds.

[0056] Comparative Example 1

[0057] Compared with Example 3, conventional gold nanoparticles were used instead of tungsten telluride (WTe2) nanoparticles, and no signal of Staphylococcus aureus was detected. Experimental process:

[0058] (1) Add 0.008 g of a 10 μm polycaprolactone porous framework to 1 mL of urine containing Staphylococcus aureus pathogens and disperse evenly in 2 mL of distilled water;

[0059] (2) Add 3×10 -6 M dihydrochalcone solution 1mL, and shake to mix;

[0060] (3) Centrifuge for 2 min (at 4000 rpm), remove the supernatant with a pipette, and retain the material at the bottom of the centrifuge tube for later use;

[0061] (4) Mix 15 μL of the gold nanoparticle sol with the material at the bottom of the centrifuge tube obtained in step (3) and let it stand for 1 min;

[0062] (5) Using a dropper, take 10 μL of the mixed solution obtained in step (4) and drop it onto a glass slide until it dries to form a film;

[0063] (6) Irradiate the substrate prepared in step (5) with a 785 nm laser at a laser power of 80 mW. The result can be obtained after 4 seconds ( Figure 3 ).

[0064] Comparative Example 2

[0065] Compared with Example 3, only the polycaprolactone porous skeleton is not used, and other processes remain unchanged. When Staphylococcus aureus is detected, 730cm -1 The SERS peaks at 1324, 1456 cm -1 The SERS peak signal-to-noise ratio at Figure 4 ).

[0066] Comparative Example 3

[0067] Compared with Example 1, only dihydrochalcone was not used, and other processes remained unchanged. The signal-to-noise ratio of the spectrum obtained when detecting Escherichia coli was very poor, and most of the signals at the main peak positions did not appear ( Figure 5 ).

[0068] Comparative Example 4

[0069] Compared with Example 1, the polycaprolactone porous framework and dihydrochalcone were not used, and other processes remained unchanged. The spectrum obtained when detecting Escherichia coli did not have the main SERS signal of Escherichia coli ( Figure 6 ).

[0070] It should be noted that the present invention claims protection for a highly stable SERS substrate for rapid detection of pathogens. The highly stable SERS substrate is a special material different from conventional SERS substrates. It has a stabilization mechanism that can be used for pathogen detection and can ensure the stability of the detection signal for common pathogens, so that it can obviously be used for common pathogens including Escherichia coli, Staphylococcus aureus, etc. involved in the embodiments of the present invention. This is obviously easy to imitate and verify for technicians who know the main purpose of the present invention, so the actual protection scope of the present invention is not limited by the embodiments of the present invention. The actual protection scope of the present invention shall be based on the claims. Those skilled in the art should know that any modifications, equivalent substitutions and improvements based on the essential spirit of the present invention should be within the actual protection scope of the present invention.

Claims

1. A highly stable SERS substrate for rapid detection of pathogens, prepared by the following method: (1) Add 5-20 μm polycaprolactone porous framework to the sample solution containing target pathogens and disperse it evenly in distilled water; (2) Add the dihydrochalcone solution to step (1) and shake to mix; (3) Centrifuge at 1000-5000 rpm for 1-5 min, remove the supernatant, and retain the material at the bottom of the centrifuge tube for later use; (4) Mixing the tungsten telluride nanoparticles with the material at the bottom of the centrifuge tube in step (3) and allowing to stand; (5) Take the mixed solution after standing in step (4) and drop it onto a glass slide until it dries to form a film.

2. The highly stable SERS substrate according to claim 1, wherein Add 5-20 mg of polycaprolactone porous framework to 1 mL of sample solution containing target pathogens.

3. The highly stable SERS substrate according to claim 1, wherein The particle size of the tungsten telluride nanoparticles is 60-90 nm.

4. The highly stable SERS substrate according to claim 1, wherein The dihydrochalcone solution has a concentration of 10 -5 -10 -7 M aqueous solution.

5. The highly stable SERS substrate according to claim 1, wherein Step (3) Centrifuge at 3000-4000 rpm for 1-5 minutes.

6. The highly stable SERS substrate according to claim 1, wherein The sample liquid containing the target pathogen includes but is not limited to any one of urine, blood, saliva, and tissue fluid, or a mixture of several of them.

7. The highly stable SERS substrate according to claim 1, wherein: The pathogens include but are not limited to one or more of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Candida albicans.

8. The highly stable SERS substrate according to any one of claims 1 to 7, wherein: The following preparation method is used to obtain: (1) Add 8-15 mg of 5-20 μm polycaprolactone porous framework to 1 mL of sample solution containing target pathogens and disperse evenly in 1-3 mL of distilled water; (2) Add concentration 10 to step (1) -5 -10 -7 Add 1-3 mL of dihydrochalcone solution of M and shake to mix; (3) Centrifuge at 3000-4000 rpm for 1-3 minutes, remove the supernatant, and retain the material at the bottom of the centrifuge tube for later use; (4) Mixing the tungsten telluride nanoparticles with the material at the bottom of the centrifuge tube in step (3) and allowing to stand; (5) Take 8-12 μL of the mixed solution after standing in step (4) and drop it onto a glass slide until it dries to form a film.

9. Use of the highly stable SERS substrate according to any one of claims 1 to 8 in rapid detection of pathogens, characterized in that: By irradiating a highly stable SERS substrate with a 785 nm laser, the characteristic SERS spectrum of the target pathogens can be obtained after 1-5 seconds; the target pathogens include but are not limited to one or more of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Candida albicans.

10. The use according to claim 9, characterized in that Laser power 20-120mW.

Images