CN115931820A

CN115931820A - Digital SERS chip for quantitatively detecting microorganisms and preparation method thereof

Info

Publication number: CN115931820A
Application number: CN202211589805.5A
Authority: CN
Inventors: 陈李; 文枰; 徐溢; 李顺波
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2023-04-07

Abstract

The invention discloses a digital SERS chip for quantitatively detecting microorganisms and a preparation method thereof. The preparation process is simple, and can realize single-bacterium capture and digital quantitative detection of microorganisms.

Description

Digital SERS chip for quantitatively detecting microorganisms and preparation method thereof

Technical Field

The invention relates to the technical field of surface enhanced Raman spectroscopy, in particular to a digital SERS chip for quantitatively detecting microorganisms and a preparation method thereof.

Background

The existing microorganism detection methods mainly comprise standard plate colony counting, polymerase Chain Reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and the like. The colony counting method is a standard method for detecting microorganisms, is simple to operate and low in cost, but has a long detection period and cannot meet the requirement of rapid detection. Furthermore, many microorganisms (including viable but non-culturable cells) cannot be cultured at all, making this standard method unusable. ELISA and PCR can be detected within hours at the fastest speed, but both methods need complex pretreatment procedures and precise instruments, can be operated only by professionals, and are greatly limited in practical application.

The Surface Enhanced Raman Spectroscopy (SERS) technology has many advantages such as high detection sensitivity, fast detection speed, no water-based interference, "fingerprint" information, non-contact and non-destructive detection, and is very advantageous in the fast detection of microorganisms. Most of the reported researches reflect the concentration of a sample to be measured by measuring the intensity of an SERS spectrum after enhancing a Raman signal of the sample by an SERS substrate. This method has an important drawback that is difficult to overcome, namely: the enhancement of the SERS substrate on the spectrum signal mainly depends on a 'hot spot' generated by a plasma nano structure to enhance a weak Raman scattering signal, so that an enhancement factor is extremely sensitive to factors such as the distribution state and uniformity of the nano structure and the relative position of a sample molecule to be detected and the 'hot spot', most of the existing physical and chemical methods are difficult to prepare the SERS substrate with excellent uniformity and consistency on a nano scale, and therefore, the mode of measuring the strength of the SERS signal to reflect the concentration of the sample to be detected is adopted, the accuracy is not high, and the repeatability is not good. Methods such as electron beam lithography and particle beam etching are reported to be adopted to prepare the high-uniformity SERS substrate so as to improve the uniformity and the repeatability of signals, but the methods are very high in manufacturing cost and difficult to popularize in a large scale. Particularly for microorganism measurement, because the cell structure is complex, the volume is large, and the fluorescence signal is strong, the measurement accuracy of the existing SERS intensity measurement method for microorganisms is lower.

In order to overcome the problem, the invention designs a digital SERS chip, which realizes the capture of microorganisms by designing a micro-column array structure, and realizes the capture of single microorganism by optimizing the volume of a groove on a micro-column aiming at microorganisms with different volumes. Then, active point positions (micro-columns on which microorganisms are successfully paved) of the SERS chip are counted in a scanning mode, and the concentration of the microorganisms is measured by a mathematical statistics method. The method is insensitive to SERS signal intensity change, greatly reduces the requirement on uniformity of the SERS substrate, and can effectively improve the repeatability and precision of detection compared with the existing method.

Disclosure of Invention

The invention aims to provide a digital SERS chip for quantitatively detecting microorganisms and a preparation method thereof, wherein the digital SERS chip is simple in manufacturing process and can realize single-bacterium capture and digital quantitative detection of the microorganisms.

The invention relates to a digital SERS chip for quantitatively detecting microorganisms, which comprises a substrate and a transparent cover plate connected to the substrate, wherein a gap is reserved between the transparent cover plate and the substrate, a sample inlet and a sample outlet are formed in the transparent cover plate, microcolumns distributed in an array form are distributed on the substrate, the array quantity scale is 100 x 100 to 1000 x 1000, a columnar groove is formed in the top surface of each microcolumn, a high-reflection film is deposited on the inner surface of each columnar groove, precious metal nanoparticles are deposited on the surface of the high-reflection film at the bottom of each columnar groove, and an adapter, an antibody or vancomycin for capturing microorganisms are modified on the surface of each precious metal nanoparticle.

Furthermore, the diameter of the single microcolumn is 1 to 30 μm, and the height is 20 to 200 μm.

Further, the inner diameter of the columnar groove is 0.5-20 μm, and the depth is 0.5-20 μm.

Furthermore, the material of the high-reflection film is gold, and the thickness of the high-reflection film is 100 to 150nm.

Further, the noble metal nanoparticles are gold nanoparticles, silver nanoparticles, gold-coated silver nanoparticles, or silver-coated gold nanoparticles.

A preparation method of a digital SERS chip for quantitatively detecting microorganisms comprises the following steps:

s1, preparing a plurality of micro-columns distributed in an array manner and columnar grooves on the top surfaces of the micro-columns on a substrate by adopting an ICP (inductively coupled plasma) etching method;

s2, depositing a high-reflection film on the surface of the columnar groove;

s3, depositing precious metal nanoparticles on the surface of the high-reflection film at the bottom of the columnar groove, and modifying an aptamer, an antibody or vancomycin for capturing microorganisms on the surface of the precious metal nanoparticles;

and S4, connecting the transparent cover plate to the substrate to obtain the SERS chip for quantitatively detecting the microorganisms.

The invention has the beneficial effects that:

1. according to the invention, the columnar groove is designed on the microcolumn to capture microorganisms, the size of the columnar groove can be optimized according to the sizes of different microorganisms, the single microorganism is spread, and the counting accuracy is improved.

2. The microcolumn structure designed by the invention can focus a focus on the groove structure in the vertical direction during scanning measurement, thereby avoiding interference caused by bacteria attached to other positions on the substrate, such as the bottom of the substrate, and further improving the measurement accuracy.

3. The invention designs the digital SERS chip of the micro-column array, the number of micro-columns which successfully capture microorganisms is obtained by counting active points on the chip during measurement, and the microorganism concentration is measured by a mathematical statistics method, so that the problems of low accuracy, poor consistency and the like of SERS intensity measurement are avoided, and the anti-interference capability is effectively improved by a digital measurement mode. The method is insensitive to SERS signal intensity change, greatly reduces the requirement on uniformity of the SERS substrate, and can effectively improve the repeatability and precision of detection compared with the existing method.

Drawings

FIG. 1 is a top view of a digital SERS chip for quantitatively detecting microorganisms according to the present invention;

fig. 2 is a side view of a digital SERS chip for quantitatively detecting microorganisms according to the present invention.

In the figure, 1 is a substrate, 2 is a microcolumn, 3 is a columnar groove, 4 is a high-reflection film, 5 is a noble metal nanoparticle, 6 is a transparent cover plate, 7 is a sample inlet, and 8 is a sample outlet.

Detailed Description

In order to make the objects, technical solutions and advantages 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 accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Referring to fig. 1 and 2, the digital SERS chip for quantitatively detecting microorganisms comprises a substrate 1 and a transparent cover plate 6 connected to the substrate, wherein a gap is reserved between the transparent cover plate 6 and the substrate 1, a sample inlet 7 and a sample outlet 8 are arranged on the transparent cover plate 6, a plurality of microcolumns 2 distributed in an array are distributed on the substrate 1, a columnar groove 3 is arranged on the top surface of each microcolumn 2, a high-reflection film 4 is deposited on the surface of each columnar groove 3, and a noble metal nanoparticle 5 is deposited on the surface of each high-reflection film 4 at the bottom of each columnar groove 3.

The diameter D of each single microcolumn 2 is 1 to 30 mu m, and the height H is 20 to 200 mu m. The inner diameter d of the columnar groove 3 is 0.5-20 mu m, and the depth h is 0.5-20 mu m. The high-reflection film is made of gold, and the thickness of the high-reflection film is 100 to 150nm. The noble metal nanoparticles are gold nanoparticles, silver nanoparticles, gold-coated silver nanoparticles or silver-coated gold nanoparticles.

The preparation method of the digital SERS chip for quantitatively detecting the microorganisms comprises the following steps:

s1, preparing a plurality of micro-columns 2 distributed in an array manner and columnar grooves 3 on the top surfaces of the micro-columns 2 on a substrate 1 by adopting an ICP etching method. Specifically, (100) monocrystalline polished silicon wafers are selected as substrates, and the thickness of the substrates is 0.2 to 1mm. Depositing a silicon nitride layer on the surface of the monocrystalline silicon piece, wherein the thickness of the silicon nitride layer is 500nm;

the silicon wafer is patterned on one side by standard photolithography (200 x 200 array structure) and the exposed silicon nitride layer is removed by wet etching process.

An ICP etching method is adopted to manufacture a micro-column array and a columnar groove at the top of the micro-column, the diameter D of a single micro-column 2 is 5 mu m, and the height H is 100 mu m. The inner diameter d of the columnar groove 3 is 2 μm, and the depth h is 2 μm.

And S2, photoetching, adopting a stripping method, depositing a high-reflection film 4 on the surface of the columnar groove 3, and depositing the high-reflection film 4 with the thickness of 100nm on the surface of the columnar groove by utilizing a magnetron sputtering technology, wherein the high-reflection film 4 is made of gold.

And S3, reducing silver nitrate by using sodium citrate to prepare silver nanoparticle sol, uniformly assembling silver nanoparticles on the surface of a high-reflection film at the bottom of the columnar groove 3 to serve as a Raman enhancement medium, and modifying a layer of vancomycin on the surface of the silver nanoparticles.

And S4, bonding and connecting the transparent cover plate 6 to the substrate 1 to obtain the SERS chip for quantitatively detecting the microorganisms.

During the specific use, inject into foretell digital SERS chip with bacterium sample solution through introduction port 7 on the transparent cover 6, the bacterium is staphylococcus aureus or escherichia coli, ensures that all column recesses 3 all are filled by sample solution. Standing for 30 to 60 minutes, and capturing bacteria by the vancomycin at the bottom of the columnar groove 3. The redundant liquid outside the cavity is discharged through a sample outlet 8 on the transparent cover plate 6; and then collecting the SERS spectrum of the bacteria by utilizing the SERS mapping function of a Raman instrument. When SERS mapping is tested, the scanning step length is set to be consistent with the distance between the micro-column arrays, each scanning point is located at the center of the columnar groove, and meanwhile exciting light covers the whole columnar groove. And judging whether bacteria are contained in the pits or not through the characteristic Raman peak, and calculating the concentration of the bacteria to be detected according to the occurrence frequency of the characteristic signal.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A digital SERS chip for quantitatively detecting microorganisms, comprising: including substrate (1) and connect transparent cover (6) on substrate (1), leave the clearance between transparent cover (6) and substrate (1), be equipped with on transparent cover (6) and advance appearance mouth (7) and go out appearance mouth (8), it has microcolumn (2) that are array distribution to distribute on substrate (1), and every microcolumn (2) top surface all is equipped with column recess (3), column recess (3) internal surface deposit has high reflectance coating (4), and the high reflectance coating surface deposit of the tank bottom of column recess (3) has noble metal nanoparticle (5), noble metal nanoparticle (5) surface modification has aptamer, antibody or vancomycin that are used for catching the microorganism.

2. The digital SERS chip according to claim 1, wherein: the diameter D of each single microcolumn (2) is 1 to 30 mu m, the height H is 20 to 200 mu m, and the microcolumn array scale is 100 x 100 to 1000 x 1000.

3. The digital SERS chip according to claim 1, wherein: the inner diameter d of the columnar groove (3) is 0.5 to 20 mu m, and the depth h is 0.5 to 20 mu m.

4. The digital SERS chip according to claim 1, wherein: the high-reflection film (4) is made of gold, and the thickness of the high-reflection film is 100-150nm.

5. The digital SERS chip according to claim 1, wherein: the noble metal nanoparticles are gold nanoparticles, silver nanoparticles, gold-coated silver nanoparticles or silver-coated gold nanoparticles.

6. A preparation method of an SERS chip for quantitatively detecting microorganisms is characterized by comprising the following steps:

s1, photoetching, and preparing microcolumns (2) distributed in an array manner and columnar grooves (3) on the top surfaces of the microcolumns (2) on a substrate (1) by adopting a dry etching technology or a wet etching technology;

s2, photoetching, and depositing a high-reflection film (4) on the inner surface of the columnar groove (3) by adopting a stripping method;

s3, depositing the noble metal nanoparticles (5) on the surface of the high-reflection film (4) at the bottom of the columnar groove, and modifying the surface of the noble metal nanoparticles (5) with aptamers, antibodies or vancomycin for capturing microorganisms;

and S4, bonding the transparent cover plate (6) with the substrate (1) to obtain the SERS chip for quantitatively detecting the microorganisms.