WO2020103497A1 - 一种用于质谱分析的通用型纳米芯片及其制备方法与应用 - Google Patents

一种用于质谱分析的通用型纳米芯片及其制备方法与应用

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Publication number
WO2020103497A1
WO2020103497A1 PCT/CN2019/099604 CN2019099604W WO2020103497A1 WO 2020103497 A1 WO2020103497 A1 WO 2020103497A1 CN 2019099604 W CN2019099604 W CN 2019099604W WO 2020103497 A1 WO2020103497 A1 WO 2020103497A1
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Prior art keywords
nanochip
mass spectrometry
array
spotting
main material
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PCT/CN2019/099604
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English (en)
French (fr)
Inventor
邬建敏
陈晓明
陈锡胜
钟巧玲
栾春燕
余捷凯
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杭州汇健科技有限公司
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Priority to KR1020217016489A priority Critical patent/KR102533281B1/ko
Priority to JP2021529106A priority patent/JP7213595B2/ja
Priority to EP19886491.0A priority patent/EP3872486A4/en
Publication of WO2020103497A1 publication Critical patent/WO2020103497A1/zh
Priority to US17/303,218 priority patent/US11764047B2/en

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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0409—Sample holders or containers
    • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0009—Calibration of the apparatus

Definitions

  • the invention relates to the technical field of mass spectrometry, in particular to a general-purpose nanochip used for mass spectrometry and its preparation method and application.
  • MALDI-TOF-MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • the sample target plate is one of the most important consumables for the MALDI-TOF mass spectrometer.
  • the commercial common target plate is made of stainless steel.
  • the problems are: (1) After use, organic reagents such as acetone, acetonitrile, and ethanol are required Ultrasonic cleaning, the process is cumbersome; (2) It is easy to cause sample residues and scratches on the surface of the target plate, causing cross-contamination, affecting the flatness of the target plate and the effect of matrix crystallization, which in turn affects the accuracy of clinical sample identification; (3) Detection of small molecular samples It is susceptible to interference from matrix peaks, the matrix crystal is not uniform when detecting nucleic acid samples, the signal to noise is relatively low, tissue samples can not be directly imaged by mass spectrometry, and the scope of application is narrow; (4) the preparation cost is high and it is difficult to detect in high-throughput clinical mass spectrometry Large-scale promotion in the field.
  • Patent CN107907585 uses fibrous filter paper as the base material of the target board, and the wax layer on the surface of the filter paper is used to achieve hydrophobic performance;
  • patent CN202230053 uses conductive plastic as the main body of the target board, and the surface is covered with a hydrophobic film.
  • these two disposable target boards cost It is lower, but the surface of the target plate does not have a microstructure, and the sample holes on the surface of the target plate only serve as the sample carrier, which contributes less to the peak of the mass spectrum signal. Compared with the stainless steel target plate, the mass spectrum signal does not increase significantly.
  • Patent CN107515243 sinters the titanium dioxide nanocrystalline layer on the surface of the stainless steel target plate, which improves the signal intensity of the mass spectrum above 20K Da, and the molecular weight range of microbial identification, peptide and nucleic acid testing is below 20K Da;
  • Patent CN106884156 deposits a layer on the surface of the stainless steel target plate Titanium dioxide nano-films are only used in the purification of phosphorylated peptides.
  • Disposable target plates that use metals as conductive materials for doping or surface modification of nanomaterials have complex manufacturing processes, further increase in cost, single test samples and limited application fields.
  • the purpose of the present invention is to provide a general-purpose nanochip for mass spectrometry analysis and its preparation method and application, which improves energy absorption and utilization, improves ionization efficiency, enhances mass spectrometry signals, and can be widely used in the field of clinical testing.
  • the present invention provides the following technical solutions:
  • a general-purpose nanochip for mass spectrometric analysis characterized in that the main material of the nanochip is a silicon-based semiconductor material, the surface of the main material is distributed with array type spotting holes, and the inner surface of the spotting hole is a nanostructure; the body The surface of the material has a regional hydrophobic modification, and the hydrophobic region is the surface of the host material inside or outside the array spotting holes.
  • the thickness of the nanostructure is 0.2-5 ⁇ m.
  • the nanostructure includes any one or more of nanowires, nanofibers, nanopillars, nanopyramids, nanoparticles, and nanoporous.
  • the silicon-based semiconductor material includes any one or more of monocrystalline silicon, polycrystalline silicon, silicon-based epitaxial metal, non-metallic element, and oxide.
  • the surface modification of the hydrophobic region adopts chemical vapor deposition or liquid phase chemical modification
  • the reagents used include any one or more of silanes, siloxanes, thiols, and terminal olefins.
  • the shape of the array spotting holes is circular or square.
  • S1 Scribing of the main material, using laser or grinding wheel to scribing the main material in a clean room;
  • S2 Cleaning the main material, placing the diced main material in a concentrated sulfuric acid / hydrogen peroxide mixed solution for ultrasonic cleaning, and then using deionized water to absorb the solution on the surface of the main material; placing the main material in ethanol, followed by Ultrasonic cleaning in isopropanol solution;
  • S3 The patterning of array type spotting holes, using any one or more methods of metal stamping method, photolithography method, blue film method and screen printing method to realize the graphic design on the surface of the host material;
  • S4 Construction of nanostructures on the inner surface of the array of spotting holes, according to the pattern of the surface of the host material, reactive ion etching, chemical vapor deposition, physical vapor deposition, atomic layer deposition, wet chemical etching, template method, hydrothermal Any one or more of the methods and the drip coating method are used to prepare the nanostructure at the position corresponding to the array spotting holes;
  • An application of a general-purpose nanochip for mass spectrometry which is characterized by the following aspects: rapid identification of clinical microorganisms, fungi and other samples; rapid detection of small molecule metabolites in biological samples such as sweat, saliva, fingerprints, cells, tissues, etc. ; Antibiotic susceptibility test; rapid mass spectrometry imaging of tissue samples; SNP gene detection; serum protein and peptide detection.
  • the inner surface of the array spotting hole of the present invention is a nanostructure.
  • the nanostructure has a top enhancement effect, and the pinpoint shape of the surface is prone to generate a high-energy electric field, which promotes the ionization of the analyte;
  • the peak signal-to-noise ratio of the mass spectrum is enhanced, and the mass spectrum signal is enhanced;
  • the traditional mass spectrometer metal target plate needs to rely on the matrix to transfer energy to the sample to be tested.
  • the matrix and the sample to be tested are co-crystallized, which cannot avoid the interference of the matrix with the test of low molecular weight substances.
  • the nanostructures in the spotting holes of the nanochips of the present invention have significant electromagnetic field enhancement effects and charge transfer capabilities, so no additional organic matrix is required, and small molecular weight substances ( ⁇ 1000 Da) such as antibiotics (small drug molecules) and lipids can be achieved Matrix-free detection of body, amino acids, vitamins, etc .;
  • the traditional commercial metal target board is generally reused, which is easy to cause the influence of residual samples, and it needs to be continuously cleaned for clinical testing, which is time-consuming and labor-intensive, and the detection flux is reduced, which cannot meet the clinical needs.
  • the raw materials and equipment used are very common, and the preparation method is simple, so the cost of labor and material resources is low, the disposable performance of the chip is realized, the tedious steps of cleaning the target board are eliminated, and the cross contamination of the sample test can be avoided.
  • the present invention has a wide range of clinical applications, and can be used for rapid identification of clinical microbiological and fungal samples, rapid detection of small molecular metabolites, drug susceptibility testing of low molecular weight antibiotics, rapid mass spectrometry imaging of tissue samples, and SNP gene detection.
  • FIG. 1 is a schematic structural diagram of a nanochip according to an embodiment of the invention.
  • FIG. 2 is a test result diagram of the nanochip of the present invention in clinical microbial identification.
  • FIG. 3 is a comparison diagram of the mass spectrometry of the nanowire target plate of the present invention and the traditional metal target plate against Acinetobacter baumannii.
  • FIG. 4 is a chart showing the identification score of E. coli in the 55-well nanochip with the same target.
  • 5 is a schematic diagram of the peak position variation rate of the nanochip.
  • FIG. 6 is a comparison diagram of mass spectra of nucleic acids of the same concentration on a nanochip and a stainless steel target plate.
  • Figure 7a is a comparison of the mass spectra of ciprofloxacin tested on a stainless steel target plate and a nanochip.
  • Fig. 7b is a comparison chart of mass spectra obtained by testing erythromycin on a stainless steel target plate and a nanochip.
  • Fig. 8a is a mass spectrum obtained by testing small molecule metabolites in finger sweat on a nanochip.
  • Fig. 8b is a mass spectrum of mouse kidney tissue liposomes tested on a nanochip.
  • Fig. 9a is a mass spectrum of mouse kidney tissue liposome negative ion mode obtained by nanochips.
  • Fig. 9b is an MSI imaging diagram of 6 mass spectrum peaks in mouse kidney tissue.
  • Figure 10 is the serum peptide spectrum measured by the nanochip.
  • the invention provides a general-purpose nanochip for mass spectrometry analysis.
  • the main material of the nanochip is a silicon-based semiconductor material, including single-crystal silicon, polycrystalline silicon, silicon-based epitaxial metal, non-metallic element, and oxide.
  • silicon-based epitaxial metals include iron, copper, aluminum, and gold; non-metallic elements include graphene and carbon nanostructure materials; oxides include SiO 2 , Al 2 O 3 , TiO 2 and ZnO.
  • the surface of the main body material is distributed with array type spotting holes, the shape of the spotting holes is circular or square, the sample to be tested is placed in the array type spotting hole, and a matrix needs to be added in some test applications.
  • the inner surface of the spotted hole is a nanostructure, including nanowires, nanofibers, nanopillars, nanopyramids, nanoparticles and nanoporous, the thickness of the nanostructure is 0.2-5 ⁇ m; the pinpoint morphology of the surface of the nanostructure has enhanced The functions of electric field and electron transfer, after the absorption of laser energy, the charge is separated to generate a high-energy electric field, which promotes the ionization of the analyte, which greatly enhances the strength and sensitivity of the signal; the tip of the nanostructure can be regarded as a micro extraction head, The molecules on the surface of the analyte can be sampled when it comes into contact with the analyte.
  • the signal strength is better than that of the traditional stainless steel target. From the data of antibiotics and metabolic small molecules, it can be seen that without the matrix assistance, the direct ionization analysis under the laser, the extracted chemicals can be more effective Detected; the nanostructure also has a large specific surface area, and the sample and matrix solution are more volatile on the surface of the nanostructure, speeding up the drying process, and thereby increasing the detection flux.
  • the surface of the host material has a regional hydrophobic modification. As shown in FIG. 1, the surface of the host material 2 in the array area of the patterned pores 1 or outside the array patterned pores;
  • the surface modification adopts chemical vapor deposition or liquid phase chemical modification, and the reagents used are silanes, siloxanes, thiols or terminal olefins.
  • the main material of the nanochip is silicon-based epitaxial metal, specifically aluminum.
  • the surface of the host material is distributed with 8 ⁇ 12 array spotting holes, and the shape of the spotting holes is circular.
  • the thickness of the nanowire structure is 0.2 ⁇ m, and the surface of the host material 2 outside the array spotting holes is a hydrophobic region.
  • the modification method of the hydrophobic region adopts the chemical vapor deposition method.
  • the main material of the nanochip is a non-metallic element, specifically graphene.
  • the surface of the host material is distributed with 8 ⁇ 12 array spotting holes, and the shape of the spotting holes is circular.
  • the thickness of the nanowire structure is 1.5 ⁇ m
  • the inner surface of the array-type spotting hole 1 is a hydrophobic region
  • the modification method of the hydrophobic region adopts a liquid phase chemical modification method.
  • This embodiment also provides a method for preparing a general-purpose nanochip for mass spectrometry analysis, which includes the following steps:
  • S1 Scribing of the main material, using a laser or grinding wheel to scribble the main material in a clean room; the size of the scoring varies according to the size of the target holder of the mass spectrometer and the number of sample holes (96 or 384 holes), the general size is 54mm * 36mm.
  • S2 Cleaning of the main material. Place the diced main material in a concentrated sulfuric acid / hydrogen peroxide mixed solution for ultrasonic cleaning. In the solution, the ratio of concentrated sulfuric acid to hydrogen peroxide is between 1: 1-10: 1 ; Then use deionized water to absorb the solution on the surface of the main material; then, place the main material in ethanol and isopropyl alcohol solution for ultrasonic cleaning; remove organic matter and dust on the surface of the main material; finally, use nitrogen to the main material Dry the surface.
  • S3 Patterning of array spotting holes, using metal stamping method, photolithography method, blue film method, screen printing method to achieve graphical design on the surface of the main material; the diameter of the spotting hole is 20 microns as required Up to 3 mm.
  • a blue film with a dot pattern of a suitable size and shape can be customized according to requirements, and the blue film can be closely attached to the surface of the main body material when heated to a temperature of 60 ° C.
  • the concentration of HF solution is 3 ⁇ 5M.
  • S4 Construction of nanostructures on the inner surface of the array of spotting holes, according to the pattern of the surface of the host material, reactive ion etching, chemical vapor deposition, physical vapor deposition, atomic layer deposition, wet chemical etching, template method, hydrothermal
  • the method and the drop coating method prepare the nanostructures at the positions corresponding to the array spotting holes.
  • a silane gas is introduced at a temperature of 800-950 ° C in a tube furnace, and the reaction takes 5 min-1 h, and the nanowire structure is grown in the spotting holes of the host material.
  • Embodiment 4 Application of nanochips in clinical microbial identification
  • Bacterial coating method use a 10 ⁇ L pipette tip to pick a small amount of colonies from the plate, lightly coat the nanochip and the stainless steel target, add 2 ⁇ L 50% formic acid solution, add 1 ⁇ L CHCH substrate dropwise after drying, and place at room temperature Dry in a desiccator.
  • the MALDI-TOF mass spectrometer was used for the test.
  • the test used a linear positive ion mode with a molecular weight of 2-20KDa and delayed extraction.
  • the test results are shown in Figure 2.
  • the identification of 9 common clinical pathogens on commercial stainless steel target plates and nanochips shows that the accuracy of nanochip identification results is high and the score is higher than that of stainless steel targets.
  • the peak position variation rate between the nanochip holes is below 600 ppm. Although the variation rate of the four peak positions with a molecular weight greater than 5000 is slightly higher, in general, the variation rate of the molecular weight within and between the pores of the nanochips is below 600 ppm, which meets the needs of microbial identification.
  • Embodiment 5 Application of nanochips in SNP detection
  • the DNA fragment containing the SNP site is amplified by PCR and the DNA is purified to remove free dNTPs in the system. Then, a single base extension reaction is performed, and further, resin purification is performed to remove impurities such as salts. After the completion, the amplified DNA sample can be dropped on the spotting area of the target plate according to the present invention.
  • the test adopts linear positive ion or negative ion mode, the test molecular weight is 2-10KDa, and delayed extraction is used. As shown in FIG.
  • the tested nucleic acid (sequence: CTA, CAG, GTG, AAG, GTG; molecular weight: 4657.09 Da) in the negative ion mode
  • the intensity of the mass spectrum peak detected by the nanochip of the present invention is much higher than that of a commercial metal target It is confirmed that the detection sensitivity of the nanochip mass spectrometry target is significantly improved.
  • the antibiotics ciprofloxacin and erythromycin were mixed with LB liquid medium at a concentration of 0.05 mg / ml.
  • the mixed solution of antibiotics and LB was first spotted on the stainless steel metal target. After drying, the CHCA matrix was added dropwise. The antibiotic and LB mixed solution is directly added dropwise without the need for a matrix. After drying, mass spectrometry is performed. The test uses a linear positive ion mode with a molecular weight of ⁇ 1000Da and delayed extraction. As shown in Figures 7a and 7b, the relative signal of ciprofloxacin or erythromycin on the nanochip is stronger than the metal target, which reduces the interference of the culture medium, and does not require a matrix to obtain high-quality antibiotics. Atlas.
  • Example 7 Application of nanochips in small molecule detection
  • Metabolic information in sweat and tissue samples can be obtained through micro-extraction of nanowire tips on nanochips.
  • the fingertips were cleaned and dried with deionized water, and the fist was clenched for 5 minutes to obtain fingerprint sweat. Then, the fingertip was pressed lightly on the nanochip for 15s and then directly subjected to mass spectrometry test.
  • the test adopts the reflection negative ion mode, and the test molecular weight is ⁇ 1000Da.
  • Figure 8a information about the small molecules of metabolites in sweat can be obtained without the matrix.
  • the nanochip was pressed on the surface of mouse kidney tissue or slice for 30 seconds, then rinsed with deionized water to remove excess tissue entities. After drying, it was directly subjected to mass spectrometry test.
  • the test adopts the reflection negative ion mode, the test molecular weight is ⁇ 1000Da, and delayed extraction is used. As shown in Figure 8b, information on liposomes on the surface of mouse kidney tissue can be obtained without the matrix.
  • Example 8 Application of nanochips in mass spectrometry imaging
  • Nanochips can quickly obtain metabolite information on the surface of living tissue and realize mass spectrometry imaging.
  • the nanochip of the present invention can be pressed and sampled directly on the tissue, without the need to spend hours making complicated frozen sections, and the test process does not require a matrix.
  • the specific process is as follows: put the nude mouse kidney tissue on a glass slide, the front face of the nanochip is directly contacted and pressed on the surface of the kidney tissue for 30 seconds, then thoroughly clean the chip surface with pure water, and then directly perform mass spectrometry test after drying. Reflection negative ion mode, test molecular weight is ⁇ 1000Da, laser pulse 500shots, using delayed extraction.
  • the negative ion pattern mass spectrogram of mouse kidney tissue as shown in Figure 9a and the imaging diagram of the six mass peaks shown in Figure 9b were obtained.
  • the metabolites on the surface of the kidney tissue were effectively detected, the signal was clear, and the mass spectrum peak was 776.5 He and 778.5 are mainly distributed in the area of the renal cortex.
  • the mass spectrum peaks 856.5, 878.5, and 906.6 are mainly concentrated in the renal medulla layer. From the mass spectrometry image, the tissue distribution characteristics of the characteristic metabolites can be obtained.

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Abstract

一种用于质谱分析的通用型纳米芯片及该纳米芯片的制备方法与临床应用，涉及质谱分析技术领域。纳米芯片的主体材料（2）为硅基半导体材料，主体材料（2）的表面分布有阵列式点样孔（1），点样孔（1）的内表面为纳米结构；主体材料（2）的表面具有区域性疏水修饰，阵列式点样孔（1）内为亲水区，点样孔（1）外为疏水区；或者点样孔（1）内为疏水区，点样孔（1）外为亲水区；该纳米结构可萃取待测生物组织样本表面的分子，并提高了激光能量吸收和利用率，进而提高离子化效率，增强质谱信号。该纳米芯片可广泛应用于临床检验领域。

Description

一种用于质谱分析的通用型纳米芯片及其制备方法与应用 技术领域
本发明涉及质谱分析技术领域，尤其涉及一种用于质谱分析的通用型纳米芯片及其制备方法与应用。
背景技术
基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF-MS)的原理是将能吸收激光能量的基质化合物与待测样品混合形成共结晶，基质吸收能量传递给待测样品，使待测样品进行离子化，离子在电场中加速飞过飞行管道，根据到达检测器的飞行时间不同而被检测出不同的质荷比(m/z)。MALDI-TOF作为一种软电离技术，是一种在药物筛选及临床诊断中重要的检测手段，可应用于蛋白、多肽、微生物、SNP基因检测等。
样品靶板是MALDI-TOF质谱仪最重要的耗材之一，商业化普通靶板采用不锈钢材质，虽然可以重复使用，但存在的问题是：(1)使用后需要丙酮、乙腈、乙醇等有机试剂超声清洗，过程繁琐；(2)易在靶板表面造成样品残留和划痕，产生交叉污染，影响靶板平整度和基质结晶效果，进而影响临床样本鉴定准确度；(3)检测小分子样本时易受基质峰干扰，检测核酸样本时基质结晶不均匀，信噪比较低、无法直接进行组织样本质谱成像，应用范围窄；(4)制备成本较高，难以在高通量临床质谱检测领域进行大规模推广。目前一次性靶板大多采用不导电的塑料、纤维制成，不利于样本离子化和质谱出峰。专利CN107907585以纤维性滤纸为靶板基体材料，滤纸表面的涂蜡层以实现疏水性能；专利CN202230053以导电塑料为靶板主体，表面覆一层疏水性膜，这两种一次性靶板虽成本较低，但靶板表面不具备微结构，靶板表面样品孔只起到样品载体的作用，对质谱信号的出峰贡献较小，与不锈钢靶板相比，质谱信号没有明显的提高。专利CN107515243在不锈钢靶板表面烧结二氧化钛纳米结晶层，对20K Da以上的质谱信号强度有所提高，而微生 物鉴定、多肽及核酸测试分子量区域为20K Da以下；专利CN106884156在不锈钢靶板表面沉积一层二氧化钛纳米薄膜，仅应用于纯化磷酸化肽领域。以金属为导电材料进行掺杂或表面修饰纳米材料的一次性靶板，制作工艺复杂，成本进一步增加，检测样本单一且应用领域有限。
发明内容
本发明的目的在于提供一种用于质谱分析的通用型纳米芯片及其制备方法与应用，提高了能量吸收和利用率，提高了离子化效率，增强质谱信号且可广泛应用于临床检验领域。
为实现上述目的，本发明提供如下技术方案：
一种用于质谱分析的通用型纳米芯片，其特征在于，纳米芯片的主体材料为硅基半导体材料，主体材料的表面分布有阵列式点样孔，点样孔的内表面为纳米结构；主体材料的表面具有区域性疏水修饰，疏水区为阵列式点样孔内或阵列式点样孔外的主体材料表面。
进一步的，纳米结构的厚度为0.2-5μm。
进一步的，纳米结构包括纳米线、纳米纤维、纳米柱、纳米金字塔、纳米颗粒及纳米多孔中的任意一种或多种。
进一步的，硅基半导体材料包括单晶硅、多晶硅、硅基底外延金属、非金属单质及氧化物中的任意一种或多种。
进一步的，疏水区的表面修饰采用化学气相沉积或液相化学修饰，采用的试剂包括硅烷类、硅氧烷类、硫醇类及末端烯烃类中的任意一种或多种。
进一步的，阵列式点样孔的形状为圆形或方形。
一种用于质谱分析的通用型纳米芯片的制备方法，其特征在于，包括以下步骤：
S1：主体材料的划片，在无尘室中利用激光或砂轮对主体材料进行划片；
S2：主体材料的清洗，将划片后的主体材料放置在浓硫酸/过氧化氢混合 溶液中进行超声清洗，再用去离子水冲吸主体材料表面的溶液；将主体材料依次放置在乙醇、异丙醇溶液中进行超声清洗；
S3：阵列式点样孔的图形化，采用金属印章法、光刻法、蓝膜法及丝网印刷法中的任意一种或多种方法在主体材料的表面实现图形化设计；
S4：阵列式点样孔内表面纳米结构的构建，根据主体材料表面的图形，采用反应离子刻蚀、化学气相沉积、物理气相沉积、原子层沉积、湿法化学刻蚀、模板法、水热法及滴涂法中的任意一种或多种方法在对应阵列式点样孔的位置制备纳米结构；
S5：主体材料表面的疏水修饰。
一种用于质谱分析的通用型纳米芯片的应用，其特征在于，包括以下方面：临床微生物、真菌等样本的快速鉴定；汗液、唾液、指纹、细胞、组织等生物样本中小分子代谢物快速检测；抗生素的药敏试验；组织样本快速质谱成像；SNP基因检测；血清中蛋白、多肽检测。
与现有技术相比，本发明的有益效果是：
1、本发明的阵列式点样孔内表面为纳米结构，纳米结构具备顶端增强效应，其表面的针尖形貌容易产生高能电场，促进被分析物离子化；因此点样孔内的纳米结构提高了质谱出峰信噪比，增强了质谱信号；
2、传统的质谱金属靶板需要依赖基质将能量传递给待测样品，基质与待测样品共结晶，无法避免基质对低分子量物质测试的干扰。本发明的纳米芯片点样孔内的纳米结构具有显著的电磁场增强效应及电荷转移能力，因而无需额外添加有机基质，可实现对小分子量物质(＜1000Da)如抗生素(药物小分子)、脂质体、氨基酸、维生素等的免基质检测；
3、传统的商业金属靶板一般重复利用，易引起残留的样本影响，且对临床测试来说需要不断清洗，这样耗时耗力，检测通量下降，不能满足临床的需求，而本发明所用的原材料及使用的设备都很常见，且制备方法简单，因 此人力物力成本较低，实现了芯片的一次性可抛性能，省去了清洗靶板的繁琐步骤，还能避免样品测试的交叉污染，提高质谱检测简便性与通量；
4、本发明的临床应用广，可用于临床微生物、真菌等样本的快速鉴定、小分子代谢物快速检测、低分子量抗生素的药敏试验、组织样本快速质谱成像和SNP基因检测等。
附图说明
图1为本发明一实施例的纳米芯片的结构示意图。
图2为本发明的纳米芯片在临床微生物鉴定中的测试结果图。
图3为本发明的纳米线靶板与传统金属靶板对鲍曼不动杆菌质谱对比图。
图4为大肠杆菌在纳米芯片同靶55孔内的鉴定得分图。
图5为纳米芯片峰位置变异率示意图。
图6为相同浓度核酸在纳米芯片和不锈钢靶板上的质谱对比图。
图7a为环丙沙星在不锈钢靶板和纳米芯片上测试得到的质谱对比图。
图7b为红霉素在不锈钢靶板和纳米芯片上测试得到的质谱对比图。
图8a为手指汗液中小分子代谢物在纳米芯片上测试得到的质谱图。
图8b为小鼠肾脏组织脂质体在纳米芯片上测试得到的质谱图。
图9a为纳米芯片获得的小鼠肾组织脂质体负离子模式质谱图。
图9b为小鼠肾组织的6个质谱峰的MSI成像图。
图10为纳米芯片测得的血清肽谱。
图中：1-阵列式点样孔；2-主体材料。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供一种用于质谱分析的通用型纳米芯片，纳米芯片的主体材料为硅基半导体材料，包括单晶硅、多晶硅、硅基底外延金属、非金属单质和氧化物。进一步的，硅基底外延金属又包括铁、铜、铝和金等；非金属单质又包括石墨烯、碳纳米结构材料；氧化物又包括SiO 2、Al 2O 3、TiO 2和ZnO等。所述主体材料的表面分布有阵列式点样孔，点样孔的形状为圆形或方形，阵列式点样孔内放置待测样品，某些测试应用中需添加基质。
所述点样孔的内表面为纳米结构，包括纳米线、纳米纤维、纳米柱、纳米金字塔、纳米颗粒和纳米多孔，纳米结构的厚度为0.2-5μm；纳米结构表面的针尖形貌具有增强的电场和电子转移的功能，在吸收激光能量后电荷分离而产生高能电场，促进被分析物离子化，极大地增强了信号的强度和敏感度；纳米结构的尖端可以被视为微萃提取头，当其与分析物接触时可对分析物表面的分子进行取样。因此从测试微生物数据看，信号强度要优于传统的不锈钢靶，从抗生素和代谢小分子数据可以看出在没有基质辅助的情况下在激光下直接电离解析，所提取的化学物质可以更加有效地被检测到；纳米结构还具有较大的比表面积，样品和基质溶液在纳米结构的表面更易挥发，加速干燥过程，进而提高检测通量。
为了实现限域，所述主体材料的表面具有区域性疏水修饰，如图1所示，疏水区阵列式点样孔1内或阵列式点样孔外的主体材料2表面；所述疏水区的表面修饰采用化学气相沉积或液相化学修饰，采用的试剂为硅烷类、硅氧烷类、硫醇类或末端烯烃类。
实施例一：
本实施例中，纳米芯片的主体材料采用硅基底外延金属，具体采用铝。主体材料的表面分布有8×12的阵列式点样孔，点样孔的形状为圆形。该纳米线结构的厚度为0.2μm，阵列式点样孔外的主体材料2表面为疏水区。疏水区的修饰方法采用化学气相沉积法。
实施例二：
本实施例中，纳米芯片的主体材料采用非金属单质，具体采用石墨烯。主体材料的表面分布有8×12的阵列式点样孔，点样孔的形状为圆形。该纳米线结构的厚度为1.5μm，阵列式点样孔1的内表面为疏水区，疏水区的修饰方法采用液相化学修饰法。
实施例三：
本实施例还提供一种用于质谱分析的通用型纳米芯片的制备方法，包括以下步骤：
S1：主体材料的划片，在无尘室中利用激光或砂轮对主体材料进行划片；划片尺寸根据质谱仪靶托大小及样品孔数(96孔或384孔)不定，一般尺寸为54mm*36mm。
S2：主体材料的清洗，将划片后的主体材料放置在浓硫酸/过氧化氢混合溶液中进行超声清洗，溶液中，浓硫酸与过氧化氢的比例在1:1-10:1之间；再用去离子水冲吸主体材料表面的溶液；然后，将主体材料依次放置在乙醇、异丙醇溶液中进行超声清洗；除去主体材料表面的有机物、灰尘等；最后用氮气对主体材料的表面进行吹干。
S3：阵列式点样孔的图形化，采用金属印章法、光刻法、蓝膜法、丝网印刷法在主体材料的表面实现图形化设计；其中点样孔的直径尺寸根据需要在20微米至3毫米范围。如蓝膜法，按照需求定制带有合适尺寸、形状的点样孔图案的蓝膜，在加热至60℃温度下将蓝膜紧密贴在主体材料的表面即可。再称取0.01-0.2g的AgNO 3固体溶于10-50ml的HF溶液中，HF溶液的浓度为3～5M，将图形化完毕的单晶硅片置于上述溶液中反应10～60min。刻蚀结束后将硅片转移至硝酸溶液中除银，反应时间为30～60min。完毕后用去离子水冲洗硅片并用氮气吹干即得到阵列式点样孔。
S4：阵列式点样孔内表面纳米结构的构建，根据主体材料表面的图形，采 用反应离子刻蚀、化学气相沉积、物理气相沉积、原子层沉积、湿法化学刻蚀、模板法、水热法、滴涂法在对应阵列式点样孔的位置制备纳米结构。如化学气相沉积法，在管式炉内800-950℃温度下通入硅烷气体，反应5min-1h，主体材料的点样孔内即生长纳米线结构。
S5：主体材料表面的疏水修饰；修饰方法采用液相化学修饰法，采用甲苯或丙酮作为溶剂，十一碳烯酸的浓度为1-20％，加热回流5-30min。
实施例四：纳米芯片在临床微生物鉴定中的应用
涂菌法，用10μL枪头从平板中挑取少量菌落，分别轻轻涂在纳米芯片和不锈钢靶上，加入2μL 50％的甲酸溶液，待其干燥后滴加1μL CHCA基质，室温下置于干燥器中干燥。使用MALDI-TOF质谱仪进行测试，测试采用线性正离子模式，测试分子量为2-20KDa，采用延迟提取。测试结果如附图2所示，9株常见临床致病菌在商业化不锈钢靶板和纳米芯片上的鉴定情况，可以看到纳米芯片鉴定结果准确率高，且得分比不锈钢靶高，体现了纳米芯片在微生物鉴定上的优势。
从附图3可以看到同为鲍曼不动杆菌的质谱图，纳米芯片上的出峰数目明显多于不锈钢靶，较高的出峰效率使得纳米芯片的微生物鉴定得分与准确率高于不锈钢靶。利用大肠杆菌测试了纳米芯片点样孔间的鉴定重复性，结果如附图4所示，纳米芯片上55孔，鉴定分均在2.0以上，孔间重复性非常好。如图5所示，进一步利用大肠杆菌的5个标准峰位置评估了纳米芯片孔内与孔间的分子量变异率，与金属靶相比，纳米芯片孔内峰位置变异率更低，在300ppm以下。纳米芯片孔间峰位置变异率在600ppm以下。虽然分子量大于5000的四个峰位置变异率略高，但总体上看纳米芯片孔内与孔间的分子量变异率均在600ppm以下，满足微生物鉴定需求。
实施例五：纳米芯片在SNP检测中的应用
通过PCR扩增含有SNP位点的DNA片段后纯化DNA以除去体系中游离的 dNTP。然后进行单碱基延伸反应，进一步地，进行树脂纯化去除盐等杂质。完毕后即可进行将扩增后的DNA试样滴于本发明所述的靶板点样区，测试采用线性正离子或负离子模式，测试分子量为2-10KDa，采用延迟提取。如附图6所示，受试核酸(序列：CTA CAG GTG AAG GTG；分子量：4657.09Da)在负离子模式下采用本发明所述的纳米芯片检测到的质谱峰强度远远高于商业金属靶，证实了纳米芯片质谱靶的检测灵敏度显著提高。
实施例六：纳米芯片在抗生素药敏实验中的应用
首先把抗生素环丙沙星和红霉素分别与LB液体培养基混合，浓度为0.05mg/ml，在不锈钢金属靶上先点抗生素与LB混合液，干燥后再滴加CHCA基质，在纳米芯片上直接滴加抗生素与LB混合液，无需基质，干燥后进行质谱测试，测试采用线性正离子模式，测试分子量为<1000Da，采用延迟提取。如附图7a和图7b所示,无论是环丙沙星还是红霉素在纳米芯片上的相对信号强于金属靶，降低了培养基的干扰，且不需要基质就可以获得高质量的抗生素图谱。
实施例七：纳米芯片在小分子检测中的应用
通过纳米芯片上纳米线顶端微萃提取作用可以获取汗液和组织样本中的代谢信息。手指尖用去离子水清洁并干燥，紧握拳头5min以得到指纹汗液，然后指尖轻轻按压纳米芯片15s后直接进行质谱测试，测试采用反射负离子模式，测试分子量为<1000Da，采用延迟提取。如图8a，免基质的情况可获得汗液中的代谢物小分子信息。
纳米芯片按压在小鼠肾组织实体或切片表面30s后用去离子水冲洗以去掉多余的组织实体，干燥后直接进行质谱测试，测试采用反射负离子模式，测试分子量为<1000Da，采用延迟提取。如图8b，免基质的情况可获得小鼠肾脏组织表面的脂质体信息。
实施例八：纳米芯片在质谱成像中的应用
纳米芯片可以快速获取活体组织表面的代谢物信息并实现质谱成像。本 发明的纳米芯片直接在组织上按压取样即可，无需花数小时制作复杂的冰冻切片，测试过程不需要基质。例举的具体过程如下：将裸鼠肾组织置于玻璃片上，纳米芯片的正面直接接触并按压在肾组织表面30秒，然后用纯水彻底清洗芯片表面，干燥后直接进行质谱测试，测试采用反射负离子模式，测试分子量为<1000Da，激光脉冲500shots，采用延迟提取。得到如图9a所示的小鼠肾组织脂质体负离子模式质谱图及图9b所示的六个质谱峰的成像图，肾组织表面的代谢物被有效地检测到，信号清晰，质谱峰776.5和778.5主要分布局于肾皮质层区域，质谱峰856.5,878.5,和906.6主要聚集在肾髓质层，从质谱成像图可以得到特征代谢物的组织分布特征。
实施例九：纳米芯片在血清肽谱检测中的应用
将血清样本用缓冲溶液稀释10倍后，取适量滴加至纳米芯片上，待自然风干后覆盖1μL CHCA(α-氰基-4-羟基肉桂酸)质谱基质进行共结晶，自然晾干后直接进行MALDI-TOF质谱检测，测试采用线性正离子模式，测试分子量为1-10KDa，激光脉冲1000shots，采用延迟提取。图10是在纳米芯片测得的血清肽谱，信号强度强，信噪比高。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。

Claims (8)

  1. 一种用于质谱分析的通用型纳米芯片，其特征在于，纳米芯片的主体材料为硅基半导体材料，所述主体材料的表面分布有阵列式点样孔，所述点样孔的内表面为纳米结构；所述主体材料的表面具有区域性疏水修饰，疏水区为阵列式点样孔内或阵列式点样孔外的主体材料表面。
  2. 根据权利要求1所述的用于质谱分析的通用型纳米芯片，其特征在于，所述纳米结构的厚度为0.2-5μm。
  3. 根据权利要求1所述的用于质谱分析的通用型纳米芯片，其特征在于，所述纳米结构包括纳米线、纳米纤维、纳米柱、纳米金字塔、纳米颗粒及纳米多孔中的任意一种或多种。
  4. 根据权利要求1所述的用于质谱分析的通用型纳米芯片，其特征在于，所述硅基半导体材料包括单晶硅、多晶硅、硅基底外延金属、非金属单质及氧化物中的任意一种或多种。
  5. 根据权利要求1所述的用于质谱分析的通用型纳米芯片，其特征在于，所述疏水区的表面修饰采用化学气相沉积或液相化学修饰，采用的试剂包括硅烷类、硅氧烷类、硫醇类及末端烯烃类中的任意一种或多种。
  6. 根据权利要求1所述的用于质谱分析的通用型纳米芯片，其特征在于，所述阵列式点样孔的形状为圆形或方形。
  7. 一种基于权利要求1所述的用于质谱分析的通用型纳米芯片的制备方法，其特征在于，包括以下步骤：
    S1：主体材料的划片，在无尘室中利用激光或砂轮对主体材料进行划片；
    S2：主体材料的清洗，将划片后的主体材料放置在浓硫酸/过氧化氢混合溶液中进行超声清洗，再用去离子水冲吸主体材料表面的溶液；将主体材料依次放置在乙醇、异丙醇溶液中进行超声清洗；
    S3：阵列式点样孔的图形化，采用金属印章法、光刻法、蓝膜法及丝网印刷法中的任意一种或多种方法在主体材料的表面实现图形化设计；
    S4：阵列式点样孔内表面纳米结构的构建，根据主体材料表面的图形，采用反应离子刻蚀、化学气相沉积、物理气相沉积、原子层沉积、湿法化学刻蚀、模板法、水热法及滴涂法中的任意一种或多种方法在对应阵列式点样孔的位置制备纳米结构；
    S5：主体材料表面的疏水修饰。
  8. 一种基于权利要求1所述的用于质谱分析的通用型纳米芯片的应用，其特征在于，包括以下方面：临床微生物样本的快速鉴定；生物样本中小分子代谢物快速检测；抗生素的药敏试验；组织样本快速质谱成像；SNP基因检测；血清中蛋白、多肽检测。
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