CN114324559B

CN114324559B - SiNW chip for biological sample detection and preparation method and application thereof

Info

Publication number: CN114324559B
Application number: CN202111668280.XA
Authority: CN
Inventors: 邬建敏; 曲雪彤; 张金银; 张秋美; 陈晓明; 李桐
Original assignee: Hangzhou Well Healthcare Technologies Co ltd; Zhejiang University ZJU
Current assignee: Hangzhou Well Healthcare Technologies Co ltd; Zhejiang University ZJU
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-12-12
Anticipated expiration: 2041-12-31
Also published as: CN114324559A

Abstract

The application discloses a SiNW chip for biological sample detection and a preparation method and application thereof, belonging to the technical field of mass spectrum detection, wherein the method comprises the following steps: preparing a SiNW chip with a vertical nanowire array; performing silanization modification on the SiNW chip by using a silanization reagent to obtain a silanized SiNW chip; the silanized SiNW chip can be subjected to gold modification, and the SiNW chip can be subjected to polydopamine modification before or after gold modification. The SiNW chip of the application is used for carrying out mass spectrometry analysis on biological samples, thereby not only reducing background interference in a detection range, but also having certain ultraviolet absorption characteristics, simultaneously improving the sensitivity of sample detection in a positive ion mode and/or a negative ion mode, and realizing multi-category, high-selectivity and high-coverage rate detection of lipids and metabolites in complex biological samples.

Description

SiNW chip for biological sample detection and preparation method and application thereof

Technical Field

The application belongs to the technical field of mass spectrometry detection, and particularly relates to a SiNW chip for biological sample detection and a preparation method and application thereof, and more particularly relates to a SiNW chip for biological sample detection based on a surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) platform and a preparation method and application thereof.

Background

In recent years, the developed lipidomics based on mass spectrometry technology are increasingly popular by researching the structure and the function of lipid molecules in organisms, revealing the relationship between lipid metabolism and physiological and pathological processes of the organisms, and further searching for potential biomarkers related to tumors. Lipid molecules can be classified into eight classes according to chemical composition and hydrophobicity, and furthermore, lipids can be simply classified into two classes, namely, polar lipids, which are mainly represented by phospholipids and sphingolipids, and neutral lipids, which include glycerides and sterol lipids. In the study of potential biomarkers associated with tumors, metabolic abnormalities of polar and neutral lipids have been reported in many literature. For example, lipid combinations of Lysophosphatidylethanolamine (LPE) (18:1), phosphatidylethanolamine (PE) (P-40:3), cholesterol Esters (CE) (18:2), sphingolipids (SM) (22:0) in serum successfully achieved good differentiation between lung cancer patients and healthy humans.

The platform for researching the lipidomic and metabonomics based on the mass spectrometry technology is mainly a liquid chromatography-mass spectrometer (Liquid Chromatograph Mass Spectrometer, LC-MS), and in a positive ion mode, a human plasma non-targeted lipid analysis crossing nine LC-MS platforms shows that 307 lipids in the human plasma can be annotated. However, the slower assay speed of this platform has been a limitation in its clinical large sample screening. Although those skilled in the art are working to improve the ability of LC-MS rapid analysis. However, it still takes a week to analyze 300-400 samples in practical serum sample clinical applications, which makes the analysis of large lipidomic samples very time consuming. Surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) developed in recent years based on matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been widely popular because it greatly reduces the background in the small molecular range (< 1000 Da) by replacing the conventional organic matrix with nanomaterials, while combining its ability to analyze at high speeds. Common nano materials of the SALDI-MS platform at the present stage comprise carbon materials, silicon-based materials, noble metal nano particles, metal oxide semiconductors and the like.

However, current research on lipid and metabolite detection based on SALDI-MS platform still faces two problems. Firstly, the problem of background interference in the lipid detection range, secondly, the selectivity of the matrix to lipid types in serum in a positive ion mode, and thirdly, the high coverage rate and the detection sensitivity of the lipid and the metabolite in the positive ion mode and the negative ion mode cannot be simultaneously considered. This is mainly because the lipids or metabolites in serum are rich in species, and existing materials are difficult to meet the requirement of simultaneously detecting multiple types of lipids or metabolites. For example, when LDI-MS analysis is performed using SiNW, positively charged polar lipids significantly suppress the signal of neutral lipids that are not charged. In order to solve the above problems, jingang et al detected lipids extracted from serum by PDA modified anti-reflective material, and detected 41 lipid signals in total in positive ion mode, which included five lipid classes (Yang, j.; zhang, w.; zhang, h.; zhong, m.; cao, w.; li, z.; huang, x.; nie, z.; liu, j.; li, p.; ma, x.; ouyang, z.acs Appl Mater Interfaces2019,11, 46140-46148). Kai Liang et al developed a method for direct organic solvent-free extraction of serum lipids using graphene oxide material polymers (AGO) (Liang, K.; gao, H.; gu, Y.; yang, S.; zhang, J.; li, J.; wang, Y.; li, Y.; anal.Chim. Acta2018,1035, 108-118.). Adsorption of TAGs in serum by AGO and in situ SALDI-MS detection was achieved without elution, which showed that it could detect lipid signals of 36 TAGs in positive ion mode.

However, coverage of the above detection results is still far from that of LC-MS in positive ion mode, where an average of 300 lipids can be easily detected. Therefore, developing materials based on the SALDI-MS platform for low background, high selectivity, high sensitivity, high coverage lipid and metabolite detection is a primary problem for large-scale lipidomic and metabonomic screening, as well as a technical difficulty.

Disclosure of Invention

In order to solve at least one of the above technical problems, the present application aims to provide a novel composite material, which can not only reduce the background interference in the detection range of lipids and metabolites, but also realize multi-class high coverage detection of lipids and metabolites, improve the detection sensitivity, and achieve the purpose of:

the first aspect of the present application provides a method for manufacturing a silicon nanowire (SiNW) chip for biological sample detection, comprising the steps of:

s1, preparing a SiNW chip with a vertical nanowire array;

s2, performing silanization modification on the SiNW chip obtained in the step S1 by using a silanization reagent to obtain a silanized modified SiNW chip;

and S3, performing gold modification on the SiNW chip subjected to silanization modification obtained in the step S2.

When gold is used for modifying SiNW, the signal of partial lipid (such as polar lipid and neutral lipid) and/or metabolite can be obviously improved, and the detection coverage of the lipid and/or metabolite can be enlarged when mass spectrum is carried out.

In some embodiments of the present application, step S1 specifically includes:

s11, cutting p-type monocrystalline silicon, and etching in a solution containing hydrofluoric acid and a silver catalyst;

and S12, washing the etched p-type monocrystalline silicon by deionized water, and immersing in dilute nitric acid to remove the silver catalyst, thereby obtaining the SiNW chip with the vertical nanowire array.

In some preferred embodiments of the present application, the etching time of step S11 is 5 to 15 minutes, and in some more preferred embodiments of the present application, the etching time of step S11 is 10 minutes.

In some preferred embodiments of the application, the silver catalyst is silver nitrate.

In some embodiments of the present application, step S2 specifically includes:

s21, treating the SiNW chip obtained in the step S1 by oxygen plasma;

s22, reacting the SiNW chip subjected to oxygen plasma treatment with a silylation reagent;

s23, sequentially washing with toluene and absolute ethyl alcohol, and stabilizing at the temperature of 40-80 ℃ to obtain the silanized modified SiNW chip.

In some preferred embodiments of the present application, the silylating agent is a toluene solution of 3-aminopropyl trimethoxysilane. In some more preferred embodiments of the present application, the silylating agent is a toluene solution containing 2% (v/v) 3-aminopropyl trimethoxysilane.

In some preferred embodiments of the present application, in step S23, the stabilization is performed at 60 ℃ for 60min.

In some embodiments of the application, prior to step S3, further comprising the step of polydopamine modification of the silylated modified SiNW chip. In other embodiments of the present application, after step S3, the step of polydopamine modification of the silanized and gold-modified SiNW chip is further included.

In some preferred embodiments of the application, the step of performing a polyamine modification is:

immersing the chip in Tris buffer solution containing dopamine, reacting for 15-55 min, washing with deionized water and using N ₂ And (5) blow-drying.

In some embodiments of the application, the dopamine-containing Tris buffer is a 50mM Tris buffer containing 0.1M dopamine (ph=8.5).

In some more preferred embodiments of the application, the chip is immersed in a Tris buffer solution containing dopamine for 15min.

In some embodiments of the application, the performing a gold modification step in step S3 is:

immersing the chip in gold nanoparticle colloid solution, reacting for 15-180min, washing with deionized water, and using N ₂ And (5) blow-drying.

In some preferred embodiments of the application the chip is immersed in the colloidal gold nanoparticle solution for a reaction of 150 minutes.

In some embodiments of the present application, the preparation method of the gold nanoparticle colloidal solution specifically includes:

boiling a tetrachloroauric acid solution with the concentration of 0.01 percent, adding a sodium citrate solution with the volume ratio of 0.8-2 percent and continuously boiling for 20-60 minutes to obtain a gold nanoparticle colloid solution.

In some more preferred embodiments of the application, the gold nanoparticles have a particle size of 21 to 69nm. In some most preferred embodiments of the application, the gold nanoparticles have a particle size of 21nm.

In still other embodiments of the present application, the performing a gold modification step in step S3 is: and depositing a gold layer on the chip by utilizing a magnetron sputtering method, wherein the sputtering time is 5-10 s. In some preferred embodiments of the application, the sputtering time is 8s.

The second aspect of the present application provides a SiNW chip prepared by using the preparation method according to any one of the first aspect of the present application.

According to the first aspect of the present application, three kinds of SiNW chips are included:

first chip: a gold-modified SiNW chip only;

a second chip: a SiNW chip modified by gold and then modified by polydopamine; and

third chip: a SiNW chip modified by polydopamine and then gold.

In a third aspect, the present application provides an application of the silicon nano-chip according to the second aspect of the present application in preparing a kit for detecting lipids and/or metabolites in a biological sample.

In some embodiments of the application, the biological sample is selected from at least one of blood, urine, brain spinal cord, milk, cell lysate, cell culture fluid, ascites fluid, saliva, and sweat.

In some embodiments of the application, the biological sample is a saliva sample, with which detection of, for example, a metabolite can be performed. In this arrangement, the first chip according to the second aspect of the application is more suitable. The second chip of the second aspect of the application is used for detecting metabolites in saliva samples, and has the advantages of more peaks, less background interference and better detection effect.

In some embodiments of the application, the biological sample is a blood sample. In some preferred embodiments of the present application, the blood sample is selected from at least one of a whole blood sample, a serum sample, a plasma sample. In some embodiments of the application, the blood sample is a serum sample. With blood samples, detection of lipids and/or metabolites may be performed. In this scheme, the second chip and the third chip according to the second aspect of the present application can be completed, wherein the second chip is modified with Au first and then modified with PDA, and since Au is modified on the side wall of SiNW, and then a layer of PDA is covered on the surface, the chip surface is smoother, and more effective peaks can be obtained.

In a fourth aspect, the present application provides a kit for detecting lipids and/or metabolites in a biological sample, comprising a silicon nano-chip according to any one of the second aspects of the present application.

In some embodiments of the application, the kit is used to diagnose a tumor or predict the risk of tumorigenesis.

In some preferred embodiments of the application, the neoplasm is a malignancy, such as lung cancer, bowel cancer, and the like.

In some embodiments of the application, the kit further comprises a biological sample lipid and/or metabolite extraction reagent.

In some embodiments of the application, the kit further comprises a quality control and/or standard.

In some embodiments of the application, the kit further comprises a mass spectrometry detection reagent.

In a fifth aspect, the present application provides a method for detecting lipids and/or metabolites in a biological sample based on a mass spectrometry platform, comprising the steps of:

s101, obtaining a total lipid extract and/or a total metabolite extract in a biological sample;

s102, fixing the SiNW chip according to the second aspect of the application on a target holder matched with the mass spectrum platform, dropwise adding 1-5 mu L of the total lipid extract or the total metabolite extract obtained in the step S101, and performing positive ion mode mass spectrum and/or negative ion mode analysis after drying.

In some embodiments of the application, the mass spectrometry platform is MALDI-TOF-MS.

In some embodiments of the present application, the step of obtaining the total lipid extract in step S101 is specifically: a biological sample of 20-40. Mu.L was taken and added with a methyl tert-butyl ether/methanol (MTBE/MeOH) (v/v=10/3) solution, vortexed for 10-30 min, and then H was added ₂ O, continuing to vortex for 10-30 min, centrifuging for 10min, extracting supernatant, repeatedly extracting supernatant twice, and drying under nitrogen flow.

In some embodiments of the application, in step S102, mass spectrometry is performed in positive ion mode and negative ion mode.

In the present application, the lipid includes a polar lipid and a neutral lipid. In some embodiments of the application, the polar lipids include phospholipids and sphingolipids and the neutral lipids include glycerides and sterol lipids.

In the present application, the metabolites refer to metabolic intermediates and metabolic end product molecules having a molecular weight of less than 1000Da produced by metabolic processes of an organism. In some preferred embodiments of the application, the metabolite refers to small molecule substances with a molecular weight of less than 300Da, such as uric acid, taurine, glucose.

The beneficial effects of the application are that

Compared with the prior art, the application has the following beneficial effects:

the prior art typically analyzes serum lipids based on LC-MS platforms, which, although give good lipid coverage, have been limited in clinical large sample screening due to the slow analysis rate, which takes a week to analyze 300-400 samples in practical serum sample clinical applications. The SALDI-MS platform is used for analysis, and the high-speed analysis capability of the SALDI-MS platform allows 300 samples to be analyzed within 1h, so that the SALDI-MS platform can play a greater role in future clinical large-scale queue screening.

The prior art research on detection of lipids in serum based on the SALDI-MS platform faces two problems. Firstly, the problem of background interference in the lipid detection range, and secondly, the selectivity of the matrix to the lipid types in serum in a positive ion mode, so that the existing material is difficult to meet the requirement of simultaneously detecting multiple types of lipids. By utilizing the silicon nano chip provided by the application, not only can the background interference in the lipid detection range be reduced, but also the detection sensitivity can be improved, the multi-class high coverage rate detection of serum lipid can be realized, the number of detected effective peaks can be increased to approximately 300, and the silicon nano chip simultaneously comprises neutral lipid and polar lipid.

The preparation method of the silicon nano chip is simple, siNW is prepared only by a metal-assisted chemical etching method, gold modification is performed by a self-assembly method, the gold-modified SiNW chip is obtained, polydopamine modification can be performed before or after gold modification, the gold-modified and polydopamine-modified SiNW chip is prepared, and the SiNW chip can be used as a disposable consumable material to replace a traditional target plate by matching with a mass spectrum target holder, so that the detection pollution risk is reduced.

The silicon nano chip and the kit are widely applied, can analyze lipid and metabolites of various biological samples, and can be finally used for diagnosing diseases or predicting the occurrence risk of the diseases.

Drawings

Fig. 1 shows a flow chart of the preparation of SiNW-Au-PDA in an embodiment of the present application.

Fig. 2 shows a vertical nanowire array of SiNW prepared in an embodiment of the application.

Fig. 3 shows the effect of etching time of a SINW chip on a mass spectrum detection signal.

Fig. 4 shows ultraviolet absorbance spectra (a) of aunps of different particle sizes, differences (b) in total intensities of lipid peak signals obtained by aunps of different particle sizes, and TEM images (c) of aunps of particle sizes of 21nm, when serum lipid extracts were detected.

FIG. 5 shows the effect of modification time of AuNP on total intensity of lipid peaks and number of peaks S/N.gtoreq.5 in serum (a) and the effect of modification time of PDA on total intensity of lipid peaks and number of peaks S/N.gtoreq.5 in serum (b).

Fig. 6 shows a top view (a), a cross-sectional view (b) and a 45 ° oblique view (c) of a SiNW-Au-PDA SEM image.

Fig. 7 shows background spectra of the LDI-MS analysis performed by SiNW-Au and SiNW-Au-PDA.

Fig. 8 shows mass spectra of LDI-MS analysis of serum lipids of human body for different materials, a: auNP, b: siNW, c: siNW-Au, d: siNW-PDA, e: siNW-Au-PDA, f: siNW-PDA-Au.

FIG. 9 shows the total ionic strength (a) and peak number (b) of the SiNW-PDa-Au and SiNW-Au-PDA LDI-MS analysis of serum lipids; in-batch stability of serum lipids analyzed by SiNW-Au-PDA (c); in-batch stability of serum lipids analyzed by SiNW-PDA-Au (d); metallographic microscope image (e) of SiNW-Au-PDA magnification 500 times; and a metallographic microscope image (f) at 500 Xmagnification of SiNW-PDA-Au.

Fig. 10 shows a mass spectrum (a) of the LDI-MS analysis of serum lipids by SiNW-Ag-PDA and a mass spectrum (b) of the LDI-MS analysis of serum lipids by SiNW-Au-PDA.

FIG. 11 shows the effect of SiNW-Au-PDA chip on the relative intensity of TAG/DSPC (a), the total intensity of TAG+DSPC (b) and the number of detected peaks of serum lipid extract (S/N.gtoreq.5) (c) at different sputtering times when gold modification is performed using magnetron sputtering.

Fig. 12 shows a mass spectrum (a) of LDI-MS analysis of human serum lipids and intra-batch stability (b) and inter-batch stability (c) of the analysis using a SiNW-Au-PDA chip prepared by gold modification using magnetron sputtering.

Fig. 13 shows spectra of saliva metabolites in negative ion mode on SiNW chip, siNW-Au chip and SiNW-Au-PDA chip substrates.

Fig. 14 shows background spectra in negative ion mode on SiNW chip, siNW-Au chip and SiNW-Au-PDA chip substrates.

Fig. 15 shows spectra of saliva metabolites in positive ion mode on SiNW chip, siNW-Au chip and SiNW-Au-PDA chip substrates.

Detailed Description

Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this disclosure is incorporated herein by reference in its entirety, and the equivalent patents are incorporated herein by reference, especially with respect to the definitions of synthetic techniques, product and process designs, polymers, comonomers, initiators or catalysts, etc. in the art, as disclosed in these documents. If the definition of a particular term disclosed in the prior art is inconsistent with any definition provided in the present application, the definition of the term provided in the present application controls.

The numerical ranges in the present application are approximations, so that it may include the numerical values outside the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For example, if a component, physical or other property (e.g., molecular weight, melt index, etc.) is recited as being 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

As used with respect to chemical compounds, the singular includes all isomeric forms and vice versa unless explicitly stated otherwise (e.g., "hexane" includes all isomers of hexane, either individually or collectively). In addition, unless explicitly stated otherwise, the use of the terms "a," "an," or "the" include plural referents.

The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or process, and are not related to whether or not such other component, step or process is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising", "including" or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant or compound. Rather, the term "consisting essentially of … …" excludes any other component, step or process from the scope of any of the terms recited below, as those out of necessity for operability. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.

In order to make the technical problems, technical schemes and beneficial effects solved by the application more clear, the application is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present application. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the application, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the claims.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1 preparation of gold nanoparticles and polydopamine-modified silicon nanowire chips (SiNW-Au-PDA)

The preparation of the SiNW-Au-PDA is divided into 3 steps, as shown in FIG. 1, firstly, a silicon nanowire chip (SiNW) with a vertical nanowire array is prepared, then a gold nanoparticle (AuNP) solution with uniform particle size is prepared, and modification is carried out by using the AuNP solution and a Dopamine (DA) solution respectively, so that the SiNW-Au-PDA chip is finally obtained. The detailed steps are as follows:

step one, preparing SiNW chip with vertical nanowire array

The inventor prepares a SiNW chip by using a one-step metal-assisted chemical etching method, and the preparation process is as follows:

the p-type silicon single crystal was cut into square with a size of 2X 2cm by a diamond knife, and then a silicon single crystal was grown in a solution containing 4.8M hydrofluoric acid (HF) and 0.02M silver nitrate (AgNO) ₃ ) And (5) performing reactive etching in the solution for 5-15min. After etching, the etching solution was washed three times with deionized water and then immersed in dilute nitric acid (HNO) ₃ ) Soaking for 1h to dissolve silver (Ag) catalyst.

Through this step a SiNW chip with a vertical nanowire array (as shown in fig. 2) can be produced.

In the detection of a mass spectrometry lipid standard solution (PC (36:0)), it was found that when the etching time was 10min, the obtained SiNW chip had a better mass spectrometry signal (as shown in FIG. 3), and thus 10min was selected as the optimal etching time.

Step two, preparing gold nanoparticle (AuNP) colloidal solution with uniform particle size

The inventors utilized conventional sodium citrate (Na ₃ Cit) reduction method, the specific preparation process is as follows:

when 50ml of 0.01% tetrachloroauric acid (HAuCl) ₄ ) After the solution is boiled, 400 to 1000 mu L of 1 percent Na is added ₃ The Cit solution was boiled for a further 30min, eventually forming a homogeneous AuNP colloidal solution.

The inventors found that different volumes of Na were added ₃ Cit solution, auNP solutions prepared were not uniform in particle size (as shown in Table 1), and also differed in peak intensity effect upon serum lipid detection (as shown in panels a and b of FIG. 4).

TABLE 1 Experimental data for preparing gold nanoparticle (AuNP) colloidal solutions having uniform particle size

Add volume (μL)	Aunp diameter (nm)
		600	7
415	21
		300	32
160	69

For the stability of the chip and the stability of the AuNP colloidal solution, the inventors finally selected an AuNP colloidal solution with a particle size of about 21nm for the preparation of SiNW-Au-PDA chips. FIG. 4, panel c, is a Transmission Electron Microscope (TEM) image of AuNP having a particle size of 21nm.

Step three, preparing SiNW-Au-PDA chip

(1) The SiNW chip treated by oxygen plasma is reacted with toluene solution (v/v) containing 2% 3-aminopropyl trimethoxysilane (APTES) for 10-15min at room temperature;

(2) Washing the reaction product with toluene and absolute ethyl alcohol in sequence, and stabilizing the reaction product at 60 ℃ for 60min to obtain SiNW-APTES;

(3) Immersing the SiNW-APTES in AuNP colloid solution for 15-180min, washing with deionized water, and adding N ₂ Drying to obtain the gold modified SiNW chip (SiNW-Au).

(4) Soaking SiNW-Au in 50mM Tris buffer solution (pH=8.5) containing 0.1M Dopamine (DA), reacting for 15-55 min, washing with deionized water and using N ₂ And drying to finally prepare the SiNW-Au-PDA chip.

The modification time of AuNP and PDA had an effect on both the number and intensity of serum lipid peak signals detected, as shown in fig. 5. The inventors selected AuNP modification time as 150min and PDA modification time as 15min as the optimal etching conditions, and the SEM diagram of the SiNW-Au-PDA chip thus prepared is shown in FIG. 6.

Example 2 application of SiNW-Au-PDA chip in mass spectrometric detection of serum lipids

1. Extraction of serum lipid metabolites

The total lipid extract in the serum samples was prepared by a modified Matyash method (Matyash, v.; liebisch, g.; kurzchalia, t.v.; shevchenko, a.; schwucke, d.j. Lipid res.2008,49, 1137-1146.), as follows:

that is, 20 to 40. Mu.L of serum was taken and 335. Mu.L of methyl tert-butyl ether/methanol (MTBE/MeOH) (v/v=10/3) solution was added theretoVortex for 10-30 min, then add 65 μL H ₂ O, continuing to vortex for 10-30 min, centrifuging for 10min, extracting supernatant, repeatedly extracting supernatant twice, and drying under nitrogen flow. The precipitate was resuspended in isopropyl alcohol (IPA) and stored at-20deg.C prior to analysis.

2. Preparation of different chips

To verify the effect of Au modification and PDA modification on the chip on serum lipid mass spectrometry detection, the inventors additionally prepared Au-alone modified SiNW chip (SiNW-Au) and PDA-alone modified SiNW chip (SiNW-PDA), and a PDA-followed by Au-modified SiNW chip (SiNW-PDA-Au).

The preparation method of SiNW-Au comprises the following steps:

the preparation method of SiNW-Au is the same as that of example 1, i.e. SiNW-Au obtained in step three (3).

The preparation method of SiNW-PDA comprises the following steps:

step one and step two are the same as in example 1, and step three is replaced by:

(1) The oxygen plasma treated SiNW was reacted with toluene solution (v/v) containing 2% APTES at room temperature for 10-15min.

(2) The reaction was washed with toluene and absolute ethanol in this order and stabilized at 60℃for 60min.

(3) The SiNW-APTES preparation was first immersed in 50mM Tris buffer (pH=8.5) containing 0.1M DA, reacted for 15min, rinsed with deionized water and washed with N ₂ Drying to obtain a SiNW chip modified by the PDA (SiNW-PDA).

Preparation of SiNW-PDA-Au:

the SiNW-PDA is prepared by the method, then is immersed into an AuNP colloid solution and kept for 150min, and is cleaned by deionized water and then is treated by N ₂ Drying to obtain SiNW-PDA-Au.

3. Different chips for serum lipid detection

The chip was cut to a small chip size of 3×3mm, fixed with a carbon conductive gel on an aluminum target plate matched with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and then 2 μl of serum lipid extraction solution was added dropwise, and after drying, positive ion mode mass spectrometry was performed.

Before serum lipids were detected using a SiNW-Au-PDA chip, the inventors first studied the background peaks during laser desorption ionization mass spectrometry (LDI-MS) of the material.

As shown in fig. 7, comparing, the serum lipid was detected by LDI-MS using SiNW-Au, and a clear peak was observed; the serum lipid is detected by utilizing SiNW-Au-PDA, and almost no background peak exists in the m/z range of 400-1000, so that the method shows that the PDA has a remarkable inhibition effect on the Au cluster ion background, and further shows that the SiNW-Au-PDA is a good substrate for LDI-MS analysis.

The serum lipids were analyzed by LDI-MS using different chips in positive ion mode, and as shown in FIG. 8, it was found that SiNW-Au (c), siNW-PDA (d), siNW-Au-PDA (e) and SiNW-PDa-Au (f) each had higher detection coverage than the current-stage pure AuNP (a) and SiNW (b). Further, au modification and PDA modification (SiNW-Au-PDA or SiNW-PDA-Au) are performed simultaneously, and more effective peaks can be detected than Au modification or PDA modification alone.

Specifically, when serum lipids were subjected to LDI-MS analysis using SiNW-Au, the enhancement of neutral lipid peak-emitting ability by means of AuNP was found in the mass spectrum, although part of neutral lipids (CE (18:2) -m/z 671.6, TG (52:3) -m/z 879.7, TG (54:4) -905.8, etc.) could be detected, [ Au ₃ ] ⁺ Such Au clusters interfere with the peaks, while the signal intensity of the mass spectrum is not very high. When the SiNW-PDA is used for analyzing serum lipid, the number of effective lipid peaks is improved to a certain extent compared with that of the SiNW, but the effective lipid peaks lack neutral lipid signals in the range of m/z 850-1000, so that the mass spectrum analysis performance of the effective lipid peaks is lower than that of the SiNW-Au-PDA or SiNW-PDa-Au, for example, the effective lipid peaks 236 in the range of 400-1000Da (S/N is more than or equal to 5) are detected in a positive ion mode by using the SiNW-Au-PDA for LDI-MS analysis of serum lipid. Therefore, the single modification of SiNW can not achieve ideal effect, and the synergistic effect of SiNW, au and PDA is the key of the application for detecting the high coverage rate of serum lipid.

The inventors further verified the effect of the modification sequence of Au and PDA on the accuracy and sensitivity of the detection. When serum lipids were analyzed by LDI-MS using SiNW-Au-PDA or SiNW-PDa-Au, respectively, the two mass spectra were found to have a certain similarity (e-plot and f-plot in FIG. 8). To verify further which modification sequence has a better effect, the inventors performed a more in-depth analysis, as shown in fig. 8.

The results showed that there was no significant difference in total ionic strength of LDI-MS analysis of serum lipids using SiNW-Au-PDA or SiNW-PDa-Au (panel a in FIG. 9), but SiNW-Au-PDA was significantly higher than SiNW-PDa-Au in the number of effective lipid peaks (panel b in FIG. 9). Furthermore, siNW-Au-PDA was found to have better stability in the batch (i.e. nine measurements in parallel with three samples in the same batch) than SiNW-PDA-Au in terms of stability (fig. 9, panels c and d). The result is also reflected on the surface of the material, as shown in e and f of fig. 9, since the surface of the SiNW-Au-PDA is covered with a layer of PDA, and AuNP can be modified on the sidewall of the SiNW, the surface is uniform. While SiNW-PDa-Au has a certain aggregation on the surface due to the fact that a layer of PDA film is coated first and then Au is deposited.

Therefore, the SiNW-Au-PDA has better characterization and detection performance relative to the SiNW-PDa-Au by combining various factors.

Example 3 Effect of different Metal modifications on serum lipid Mass Spectrometry detection

Regarding the choice of metal materials, au and Ag are the most commonly used at this stage. Accordingly, the inventors have made a selection of these two metals. Under the same 15min modification time, the inventors prepared a SiNW chip (SiNW-Ag-PDA) modified with PDA after selecting Ag modification.

The preparation method of the SiNW-Ag-PDA comprises the following steps:

(1) Preparation of SiNW-Ag:

immersing the etched SiNW into 0.01M AgNO ₃ And (5) the solution is in the state of 15min.

(2) Preparation of SiNW-Ag-PDA:

immersing the prepared SiNW-Ag in 50mM Tris buffer solution (pH=8.5) containing 0.1M DA, reacting for 15min, washing with deionized water and washing with N ₂ Drying to obtain a SiNW-Ag chip modified by PDA (SiNW-Ag-PDA).

The inventors performed LDI-MS analysis on serum lipids using SiNW-Au-PDA and SiNW-Ag-PDA, respectively, and as shown in FIG. 10, it was found that SiNW-Au-PDA has better detection intensity and detection coverage than SiNW-Ag-PDA, indicating that Au modification can better act synergistically with PDA.

Example 4 Effect of different gold modification methods on detection

In order to realize the mass production of the chip and further improve the stability, the inventor further improves the gold modification method, namely, adopts a physical method, namely, magnetron sputtering Au plating to implement gold modification, and the specific steps are as follows.

Step one, a SiNW chip with a vertical nanowire array was prepared, and the specific steps are the same as in example 1.

Step two, preparing gold nanoparticle (AuNP) colloidal solution with uniform particle size, and the specific steps are the same as in example 1.

Step three in example 1 was modified as follows:

(1) The SiNW chip treated with oxygen plasma was reacted with toluene solution (v/v) containing 2% APTES at room temperature for 10-15min.

(2) The SiNW chip was washed successively with toluene and absolute ethanol at the end of the reaction and stabilized at 60℃for 60min.

(3) And (3) performing magnetron sputtering processing on the SiNW-APTES chip prepared in the steps (1) - (2): an Au layer was deposited on the SiNW-APTES chip using AuNP in an argon atmosphere at a flow rate of 37sccm using a discover-635 magnetron sputtering system (DENTON, usa). Sputtering for 5s-10s in DC mode under 200W power to obtain SiNW-Au.

The inventors have evaluated by varying the sputtering time and using lipid standards (TAG and DSPC) and serum lipid extract to obtain optimal sputtering conditions, the results are shown in fig. 11. The results show that under the sputtering conditions of 9s and 10s, the neutral lipid peaks are too obvious, but the peak of the polar lipid is inhibited; whereas under sputtering conditions of 5s and 6s, neutral lipids did not show a significant peak. Under the sputtering conditions of 7s and 8s, the stability of mass spectrum detection and the number of peaks are ideal. Finally, the inventors selected a sputtering time of 8s as the optimal sputtering time for preparing SiNW-Au-PDA.

The serum lipid extract prepared in example 2 was also mass-detected using a SiNW-Au-PDA chip prepared with a sputtering time of 8s, and the mass spectrum is shown in fig. 12, panel a. The results show that the SiNW-Au-PDA chip prepared by the sputtering method is equivalent to the SiNW-Au-PDA chip prepared by the chemical modification method in the detection effect of serum lipid.

In addition, the inter-batch stability of the SiNW-Au-PDA chip prepared by the sputtering method was further improved by using the physical deposition method, and the average RSD was 10.7% and 12.6%, respectively (fig. 12, panels b and c).

Example 5 application of SiNW-Au chip in saliva Metabolic substance Spectrum detection

1. Chip preparation

A SiNW chip and a SiNW-Au chip were prepared separately using the method of example 1, and a SiNW-Au-PDA chip was prepared using the method of example 4 (i.e., au plating using magnetron sputtering).

2. Saliva metabolite extraction

Saliva metabolite extraction procedure:

(1) Taking 30-50 saliva samples of individuals, filtering and uniformly mixing, and then extracting saliva metabolites;

(2) Methanol and acetonitrile were mixed in volume 1:1, as a protein precipitant, precooling for 10min before use.

(3) Adding a protein precipitant into the saliva sample, wherein the volume ratio of the protein precipitant to the biological sample is 2:1-10:1, and the pretreatment process comprises the steps of oscillating for 5-30min, centrifuging and taking supernatant;

(4) Adding ultrapure water into the supernatant obtained in the step (1) in a ratio of 1:5-1:1 to obtain saliva metabolite extraction solution.

3. Saliva metabolic profile detection

And fixing the SiNW chip, the SiNW-Au chip and the SiNW-Au-PDA chip on an aluminum target plate matched with a matrix assisted laser desorption ionization time-of-flight mass spectrum (MALDI-TOF-MS) respectively by using carbon conductive adhesive, then dropwise adding 2 mu L of saliva metabolite extract, and performing mass spectrometry analysis of a negative ion mode and a positive ion mode after the saliva metabolite extract is dried, wherein the detection range is 20-400Da.

The spectrogram of saliva metabolite extract under the negative ion mode on three chips is shown in fig. 13, when SiNWS-Au is taken as a substrate chip, the peak intensity of saliva metabolite extract is strongest, the accumulated peak number of SiNWS-Au-PDA is largest, but because the interference of polymer peaks in metabolic molecule areas is more (shown in fig. 14), siNWS-Au is taken as a metabolism detection substrate under the optimal negative ion mode.

The spectrogram of saliva metabolite extract under the positive ion mode on three chips is shown in figure 15, when SiNWS-Au is taken as a substrate chip, the peak intensity of saliva metabolite extract is strongest, siNWS-Au-PDA is inferior, and the desorption ionization efficiency of SiNWS as a substrate chip under the positive ion detection mode is lowest. The result shows that the Au modified chip has an obvious improvement effect on improving the desorption ionization efficiency of the metabolic molecules, and further can improve the peak-out performance and coverage rate of the metabolic molecules.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A method for preparing a SiNW chip for biological sample detection, comprising the steps of:

s1, preparing a SiNW chip with a vertical nanowire array: cutting p-type monocrystalline silicon into squares with the size of 2X 2cm by using a diamond knife, performing reactive etching for 10min in a solution containing 4.8M hydrofluoric acid and 0.02M silver nitrate, washing three times by using deionized water after etching, and soaking in dilute nitric acid for 1h to dissolve a silver catalyst to obtain a SiNW chip with a vertical nanowire array;

s3, carrying out gold modification on the SiNW chip subjected to silanization modification obtained in the step S2: immersing the chip in gold nanoparticle colloid solution with particle diameter of 21nm, reacting for 150min, washing with deionized water, and using N ₂ Blow-drying the materials,

further comprises the steps of poly-dopamine modification of the silanized and gold-modified SiNW chip by immersing the chip in Tris buffer solution containing dopamine, reacting for 15min, washing with deionized water and washing with N ₂ And (5) blow-drying.

2. The method according to claim 1, wherein the gold modification in step S3 is further performed by:

and depositing a gold layer on the chip by using a magnetron sputtering method, wherein the sputtering time is 5-10 s.

3. The SiNW chip prepared by the preparation method of any one of claims 1 to 2.

4. Use of the SiNW chip of claim 3 for the preparation of a kit for the detection of lipids and/or metabolites in a biological sample.

5. A kit for detecting lipids and/or metabolites in a biological sample, comprising the SiNW chip of claim 3.

6. A method for detecting lipids and/or metabolites in a biological sample based on a mass spectrometry platform, comprising the steps of:

s102, fixing the SiNW chip in claim 3 on a target holder matched with the mass spectrum platform, dropwise adding 1-5 mu L of the total lipid extract or the total metabolite extract obtained in the step S101, and performing mass spectrum analysis in a positive ion mode and/or a negative ion mode after drying.