A digital SERS chip for quantitatively detecting microorganisms and a preparation method thereof

Abstract

The application discloses a digital SERS chip for quantitatively detecting microorganisms and a preparation method thereof. The chip comprises a substrate and a transparent cover plate connected to the substrate, a gap is left between the transparent cover plate and the substrate, the transparent cover plate is provided with a sample inlet and a sample outlet, the substrate is distributed with micro columns in an array, each micro column is provided with a columnar groove on the top surface, a high-reflection film is deposited on the inner surface of the columnar groove, and noble metal nanoparticles are deposited on the surface of the high-reflection film at the groove bottom. The manufacturing process is simple, and single-bacterium capturing and digital quantitative detection of microorganisms can be realized.

Description

Technical Field

[0001] This invention relates to the field of surface-enhanced Raman spectroscopy, specifically to a digital SERS chip for quantitative detection of microorganisms and its preparation method. Background Technology

[0002] Existing microbial detection methods mainly include standard plate colony counting, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA). Colony counting is the standard method for microbial detection; it is simple to operate and inexpensive, but its detection cycle is long, failing to meet the needs of rapid detection. Furthermore, many microorganisms (including viable but unculturable cells) cannot be cultured at all, rendering this standard method unusable. ELISA and PCR can complete detection in as little as a few hours, but both methods require complex pretreatment procedures and sophisticated instruments, requiring specialized personnel to operate, which greatly limits their practical application.

[0003] Surface-enhanced Raman spectroscopy (SERS) technology boasts numerous advantages, including high detection sensitivity, fast detection speed, immunity to water-based interference, fingerprint information, non-contact, and non-destructive testing, making it highly advantageous for rapid microbial detection. Most reported studies enhance the Raman signal of a sample using a SERS substrate, then measure the intensity of the SERS spectrum to reflect the sample concentration. However, this method suffers from a significant drawback: the enhancement of the spectral signal by the SERS substrate relies heavily on "hot spots" generated by the plasmonic nanostructure to amplify the weak Raman scattering signal. Therefore, the enhancement factor is extremely sensitive to factors such as the distribution and uniformity of the nanostructure itself, as well as the relative position of the sample molecules to the "hot spots." Most existing physical and chemical methods struggle to fabricate SERS substrates with excellent uniformity and consistency at the nanoscale. Consequently, the method of measuring SERS signal intensity to reflect sample concentration suffers from low accuracy and poor repeatability. Reports have described the use of electron beam lithography and particle beam etching to prepare highly uniform SERS substrates, thereby improving signal uniformity and repeatability. However, these methods are very expensive to manufacture, making large-scale deployment difficult. This is particularly true for microbial measurements, where the complex structure, large size, and strong fluorescence signals of cells result in lower accuracy for existing SERS intensity measurement methods.

[0004] To overcome this challenge, this invention designs a digital SERS chip. It achieves microbial capture through a micropillar array structure, and optimizes the volume of grooves on the micropillars to capture individual microorganisms of different sizes. Then, the active sites (micropillars successfully deposited with microorganisms) on the SERS chip are counted using a scanning method, and the microbial concentration is measured through mathematical statistical methods. This method is insensitive to changes in SERS signal intensity and significantly reduces the requirement for uniformity of the SERS substrate. Compared to existing methods, it can effectively improve the repeatability and accuracy of detection. Summary of the Invention

[0005] The purpose of this invention is to provide a digital SERS chip for quantitative detection of microorganisms and its preparation method. The manufacturing process is simple and can realize single-cell capture and digital quantitative detection of microorganisms.

[0006] The digital SERS chip for quantitative detection of microorganisms according to the present invention includes a substrate and a transparent cover plate connected to the substrate. A gap is left between the transparent cover plate and the substrate. The transparent cover plate is provided with an inlet and an outlet. Micropillars are distributed in an array on the substrate, with the array size ranging from 100×100 to 1000×1000. Each micropillar has a columnar groove on its top surface. A high-reflectivity film is deposited on the inner surface of the columnar groove. Noble metal nanoparticles are deposited on the surface of the high-reflectivity film at the bottom of the columnar groove. The surface of the noble metal nanoparticles is modified with aptamers, antibodies, or vancomycin for capturing microorganisms.

[0007] Furthermore, the diameter of a single micropillar is 1~30μm and the height is 20~200μm.

[0008] Furthermore, the inner diameter of the columnar groove is 0.5~20μm, and the depth is 0.5~20μm.

[0009] Furthermore, the high-reflectivity film is made of gold, and its thickness is 100~150nm.

[0010] Furthermore, the precious metal nanoparticles are gold nanoparticles, silver nanoparticles, gold-coated silver nanoparticles, or silver-coated gold nanoparticles.

[0011] A method for preparing a digital SERS chip for quantitative detection of microorganisms includes the following steps: S1, Multiple micropillars arranged in an array and columnar grooves on the top surface of the micropillars are fabricated on the substrate using ICP etching. S2, depositing a high-reflectivity film on the surface of the columnar groove; S3 involves depositing noble metal nanoparticles on the surface of a high-reflectivity film at the bottom of a columnar groove, and then modifying the surface of the noble metal nanoparticles with aptamers, antibodies, or vancomycin for capturing microorganisms. S4. Connect the transparent cover plate to the substrate to obtain the SERS chip for quantitative detection of microorganisms.

[0012] The beneficial effects of this invention are: 1. This invention uses columnar grooves designed on micropillars to capture microorganisms. The volume of the columnar grooves can be optimized according to the size of different microorganisms, so as to achieve the capture of individual microorganisms and improve the accuracy of counting.

[0013] 2. The micropillar structure designed in this invention can focus the focal point on the groove structure in the vertical direction during scanning measurement, avoiding interference from other locations on the substrate, such as bacteria attached to the bottom of the substrate, and further improving the measurement accuracy.

[0014] 3. This invention designs a micropillar array digital SERS chip. During measurement, the number of micropillars successfully capturing microorganisms is determined by counting the active points on the chip. Microbial concentration is measured using mathematical statistical methods, avoiding problems such as low accuracy and poor consistency in SERS intensity measurements. The digital measurement method effectively improves anti-interference capability. This method is insensitive to changes in SERS signal intensity and significantly reduces the requirement for uniformity of the SERS substrate. Compared to existing methods, it can effectively improve the repeatability and accuracy of detection. Attached Figure Description

[0015] Figure 1 This is a top view of the digital SERS chip for quantitative detection of microorganisms as described in this invention; Figure 2 This is a side view of the digital SERS chip for quantitative detection of microorganisms as described in this invention.

[0016] In the figure, 1—substrate, 2—micropillar, 3—columnar groove, 4—high reflectivity film, 5—noble metal nanoparticles, 6—transparent cover plate, 7—sample inlet, 8—sample outlet. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0018] See Figure 1 and Figure 2 The digital SERS chip shown includes a substrate 1 and a transparent cover plate 6 connected to the substrate. A gap is left between the transparent cover plate 6 and the substrate 1. The transparent cover plate 6 is provided with an inlet 7 and an outlet 8. Multiple micropillars 2 are distributed in an array on the substrate 1. Each micropillar 2 has a columnar groove 3 on its top surface. A high-reflectivity film 4 is deposited on the surface of the columnar groove 3. Noble metal nanoparticles 5 are deposited on the surface of the high-reflectivity film 4 at the bottom of the columnar groove 3.

[0019] The diameter D of a single micropillar 2 is 1~30μm, and the height H is 20~200μm. The inner diameter d of the columnar groove 3 is 0.5~20μm, and the depth h is 0.5~20μm. The high-reflectivity film is made of gold, and its thickness is 100~150nm. The noble metal nanoparticles are gold nanoparticles, silver nanoparticles, gold-coated silver nanoparticles, or silver-coated gold nanoparticles.

[0020] The method for preparing the digital SERS chip for quantitative detection of microorganisms includes the following steps: S1, multiple arrayed micropillars 2 and columnar grooves 3 on the top surface of the micropillars 2 are fabricated on the substrate 1 using ICP etching. Specifically, a (100) single-crystal polished silicon wafer is selected as the substrate, with a substrate thickness of 0.2~1mm. A silicon nitride layer with a thickness of 500nm is deposited on the surface of the single-crystal silicon wafer; The silicon wafer was patterned on one side using standard photolithography (a 200×200 array structure), and the exposed silicon nitride layer was removed using a wet etching process.

[0021] The micropillar array and the columnar grooves on the top of the micropillars were fabricated using ICP etching. The diameter D of each micropillar 2 is 5 μm and the height H is 100 μm. The inner diameter d of the columnar groove 3 is 2 μm and the depth h is 2 μm.

[0022] S2, photolithography, using the lift-off method, and then depositing a high-reflectivity film 4 on the surface of the columnar groove 3. The high-reflectivity film 4 with a thickness of 100nm is deposited on the surface of the columnar groove using magnetron sputtering technology. The material of the high-reflectivity film 4 is gold.

[0023] S3, silver nanoparticle sol was prepared by reducing silver nitrate with sodium citrate, and the silver nanoparticles were uniformly assembled on the surface of a high-reflectivity film at the bottom of the columnar groove 3 as a Raman enhancement medium, and a layer of vancomycin was modified on the surface of the silver nanoparticles.

[0024] S4, the transparent cover plate 6 is bonded to the substrate 1 to obtain the SERS chip for quantitative detection of microorganisms.

[0025] In practical use, the bacterial sample solution, either Staphylococcus aureus or Escherichia coli, is injected into the digital SERS chip through the inlet 7 on the transparent cover 6, ensuring that all columnar grooves 3 are filled with the sample solution. After standing for 30-60 minutes, the bacteria are captured by vancomycin at the bottom of the columnar grooves 3. Excess liquid outside the cavity is removed through the outlet 8 on the transparent cover 6; then, the SERS spectrum of the bacteria is collected using the SERS mapping function of a Raman spectrometer. During the SERS mapping test, the scanning step size is set to match the spacing of the microcolumn array, ensuring that each scanning point is located at the center of the columnar groove, while simultaneously ensuring that the excitation light covers the entire columnar groove. The presence of bacteria in the groove is determined by the characteristic Raman peaks, and the concentration of the bacteria to be tested is calculated based on the frequency of the characteristic signals.

[0026] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A digital SERS chip for quantitative detection of microorganisms, characterized in that: The device includes a substrate (1) and a transparent cover plate (6) connected to the substrate (1). There is a gap between the transparent cover plate (6) and the substrate (1). The transparent cover plate (6) is provided with an inlet (7) and an outlet (8). Micropillars (2) are distributed in an array on the substrate (1). Each micropillar (2) has a columnar groove (3) on its top surface. A high-reflectivity film (4) is deposited on the inner surface of the columnar groove (3). Noble metal nanoparticles (5) are deposited on the surface of the high-reflectivity film at the bottom of the columnar groove (3). The surface of the noble metal nanoparticles (5) is modified with aptamers, antibodies or vancomycin for capturing microorganisms. The high-reflectivity film (4) is made of gold and has a thickness of 100~150nm.

2. The digital SERS chip for quantitative detection of microorganisms according to claim 1, characterized in that: The diameter D of a single micropillar (2) is 1~30μm, the height H is 20~200μm, and the size of the micropillar array is 100*100 to 1000*1000.

3. The digital SERS chip for quantitative detection of microorganisms according to claim 1, characterized in that: The inner diameter d of the columnar groove (3) is 0.5~20μm, and the depth h is 0.5~20μm.

4. The digital SERS chip for quantitative detection of microorganisms according to claim 1, characterized in that: The precious metal nanoparticles are gold nanoparticles, silver nanoparticles, gold-coated silver nanoparticles, or silver-coated gold nanoparticles.

5. A method for preparing a SERS chip for quantitative detection of microorganisms, characterized in that, Includes the following steps: S1, photolithography, and then dry or wet etching techniques are used to fabricate micropillars (2) arranged in an array and columnar grooves (3) on the top surface of the micropillars (2) on the substrate (1). S2, photolithography, using the lift-off method, deposit a high-reflectivity film (4) on the inner surface of the columnar groove (3). S3, deposit noble metal nanoparticles (5) on the surface of a high-reflectivity film (4) at the bottom of a columnar groove, and modify the surface of the noble metal nanoparticles (5) with aptamers, antibodies or vancomycin for capturing microorganisms. S4, the transparent cover plate (6) is bonded to the substrate (1) to obtain the SERS chip for quantitative detection of microorganisms.

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