WO2023274367A1 - 纳米增强芯片的制备及其在小分子代谢物激光解离质谱检测中的应用 - Google Patents

纳米增强芯片的制备及其在小分子代谢物激光解离质谱检测中的应用 Download PDF

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WO2023274367A1
WO2023274367A1 PCT/CN2022/102857 CN2022102857W WO2023274367A1 WO 2023274367 A1 WO2023274367 A1 WO 2023274367A1 CN 2022102857 W CN2022102857 W CN 2022102857W WO 2023274367 A1 WO2023274367 A1 WO 2023274367A1
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detection
matrix
preparation
mass spectrometry
apf
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French (fr)
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钱昆
曹敬
李顺祥
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Shanghai Jiao Tong University
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Shanghai Jiao Tong University
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Priority claimed from CN202110751014.7A external-priority patent/CN113484404A/zh
Priority claimed from CN202110756337.5A external-priority patent/CN113484405B/zh
Priority claimed from CN202110780476.1A external-priority patent/CN113533491A/zh
Priority claimed from CN202110852545.5A external-priority patent/CN113588769A/zh
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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
    • H01J49/0459—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for solid samples
    • H01J49/0463—Desorption by laser or particle beam, followed by ionisation as a separate step
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material

Definitions

  • the invention relates to the field of composite nanomaterial chip synthesis, in particular to a nano-enhanced chip and its application in laser dissociation mass spectrometry detection of small molecular metabolites.
  • biomarkers proteins, nucleic acids, metabolites, etc.
  • metabolites are direct markers of biochemical activity and are more easily correlated with phenotypes.
  • Macromolecular substances such as nucleic acids, proteins, etc., can be extended to detectable levels through polymerase chain reaction, fluorescence enhancement and other technical support, so as to be used for disease diagnosis.
  • the signal amplification of small molecule metabolites still faces great challenges, and it is necessary to develop high-sensitivity detection methods for small molecule metabolites.
  • Mass spectrometry is considered the primary technique for the detection of small molecule metabolites due to inherent limitations in both sensitivity and molecular recognition of NMR spectroscopy.
  • gas chromatography/liquid chromatography-mass spectrometry is the most important mass spectrometry method for metabolite detection.
  • these methods require complex pretreatments such as desalting, protein removal, derivatization, and concentration of samples to achieve metabolite detection. , requires a high sample size and is time-consuming.
  • Matrix-assisted laser desorption ionization mass spectrometry promotes the conversion of the analyte from the solid phase to the gas phase by introducing a matrix material for photon absorption, and can directly achieve high-sensitivity and high-throughput detection of trace analyte substances.
  • the matrix material determines the detection performance of MALDI MS.
  • the traditional organic matrix tends to generate strong background signals at the small molecular weight end (m/z ⁇ 400), and these noises bring great interference to the detection of small molecules and affect the detection effect.
  • there are various biological macromolecules in complex biological samples and different pH and high salinity will hinder the detection of small molecules, so traditional organic matrices are difficult to meet the detection needs of small molecules.
  • traditional inorganic nanomaterials such as carbon-based, silicon-based, noble metal materials, etc.
  • the purpose of the invention is to develop a nano-enhanced chip and its application in the detection of small molecular metabolites by laser dissociation mass spectrometry.
  • the solutions involved in the present invention include:
  • the nano-reinforced chip involved in this application can be the following metal-organic framework materials, submicroreactors, polygonal star-shaped Au@ZnO nanocomposites, and porous alloy nanomaterials.
  • a method for preparing a metal organic framework material comprising the following steps:
  • Step 1 Mix dimethylformamide, ethanol, deionized water, triethylamine and terephthalic acid into a closed container, and add metal chloride to form a mixture;
  • Step 2 subjecting the mixture to an ultrasonic reaction in a water bath to obtain a reactant
  • Step 3 centrifuging and washing the reactant to remove the residual dimethylformamide to obtain the product
  • Step 4 Disperse the product described in step 3 in deionized water, and use low-speed centrifugation to separate the ultrathin metal organic framework material.
  • the metal chloride described in step 1 is one of ferrous chloride, cobalt chloride and nickel chloride, corresponding to step 4 to obtain ultra-thin iron metal organic framework materials, ultra-thin cobalt metal organic framework materials and ultra-thin cobalt metal organic framework materials and ultra-thin Thin nickel metal-organic framework materials.
  • step 2 the water-bath ultrasonic reaction condition in step 2 is to react at 0°C for 4 hours.
  • washing method described in step 3 is to use deionized water and isopropanol to wash five times respectively.
  • the ultra-thin iron metal organic framework material is further separated to obtain a multilayer iron metal organic framework material.
  • the ultra-thin nickel metal organic framework material is a single-layer nickel metal organic framework material.
  • the present invention also provides the application of a metal organic framework material in the detection of small molecules.
  • Step 1 Set the matrix-assisted laser desorption ionization mass spectrometry detection mode to positive ion reflection mode
  • Step 2 performing proportional dilution on the serum sample
  • Step 3 sample preparation is carried out on the mass spectrometer target plate, and the ultra-thin metal organic framework material is used as a matrix, and dried at room temperature;
  • Step 4 detecting small molecules in the serum sample
  • Step 5 analyze the original mass spectrum and obtain the detection result.
  • the molecular weight range of the small molecule in step 4 is less than 1000Da.
  • step 4 the small molecules described in step 4 include amino acids, nucleosides and the like.
  • a kind of preparation method of submicroreactor comprises the steps:
  • Step 2.1 Dissolving 3-aminophenol APF in deionized water, and adding formaldehyde solution and ammonia solution;
  • Step 2.2 react the mixture in step 2.1 at 30°C for 30 minutes;
  • Step 2.3 The reactants in step 2.2 are centrifuged and washed to obtain APF submicron materials.
  • step 2.2 the reactant in step 2.2 is directly centrifuged and washed with deionized water to obtain the spherical APF submicron material APF-sphere.
  • step 2.2 also includes adding acetone solution to the reactant in step 2.2, and reacting for 180 minutes at 30°C; centrifuging the reactant and washing it with deionized water to obtain a bowl-shaped APF submicron material APF- bowl.
  • APF-sphere is dispersed in deionized water, and a chloroauric acid solution is added to react to obtain APF-sphere&Au.
  • the addition amount of the chloroauric acid solution is 2ml.
  • APF-bowl was dispersed in deionized water to prepare three reaction solutions, which were respectively added to 1.5, 2 and 2.5ml of chloroauric acid solution, and reacted to obtain APF-bowl&Au-1, APF-bowl&Au-2 And APF-bowl&Au-3.
  • reaction condition is to react at 70° C. for 10 minutes.
  • the present invention also provides a method for detecting serum metabolites according to the above-mentioned submicroreactor, comprising the following steps:
  • the detected molecular weight range is less than 1000Da.
  • the detected substances include amino acids and sugar alcohols.
  • the invention provides an application of a multi-pointed star-shaped Au@ZnO nanocomposite material as a matrix material in matrix-assisted laser desorption ionization mass spectrometry detection of small molecule metabolites.
  • the small molecule in the small molecule metabolite is selected from more than one of proline, lysine, arginine, sucrose and glucose.
  • the invention provides an application of a multi-pointed star-shaped Au@ZnO nanocomposite material as a matrix material in matrix-assisted laser desorption ionization mass spectrometry detection of serum metabolites.
  • the present invention provides a preparation method according to the above-mentioned polygonal star-shaped Au@ZnO nanocomposite material, which includes the following steps:
  • the stirring speed is 800 ⁇ 10 rpm.
  • the rotational speed of centrifugation is 1000 ⁇ 10 rpm.
  • the present invention provides a polygonal star-shaped Au@ZnO nanocomposite material, which is obtained by the above preparation method.
  • the technical problem to be solved by the present invention is how to have a MALDI MS matrix with better LDI efficiency for the detection of complex biological samples (plasma) metabolites.
  • the present invention provides a kind of preparation method of porous alloy nanomaterial, described method comprises the following steps:
  • Step 1 Thoroughly mix Na2PdCl4, H2PtCl6 ⁇ 6H2O, hydrochloric acid and F127, and ultrasonically dissolve;
  • Step 2 After completely dissolving, add ascorbic acid solution, and immediately place it in a water bath for ultrasonic reaction;
  • Step 3 then add HAuCl4.4H2O solution to react;
  • Step 4 the porous alloy nanomaterial PdPtAu is obtained by centrifuging and washing with absolute ethanol and water, respectively, and then drying.
  • the dosages of Na2PdCl4, H2PtCl6 ⁇ 6H2O, hydrochloric acid and F127 described in step 1 were 0.6mL of 20Mm Na2PdCl4, 3mL of 20Mm H2PtCl6 ⁇ 6H2O, 60 ⁇ L of 6.0M hydrochloric acid, and 60mg of F127.
  • the ascorbic acid solution in step 2 is added in an amount of 0.1M ascorbic acid solution in 3 mL.
  • step 2 the water bath ultrasonic reaction in step 2 is to react at 45° C. for 3 hours.
  • the concentration of the HAuCl4 ⁇ 4H2O solution described in step 3 is 10Mm-40Mm, and the addition amount is 1.2mL.
  • the number of times of centrifugal washing in step 3 is 3 times, the rotation speed is 10,000 rpm, and the drying temperature is 50°C. °C.
  • Step 1 dispersing the porous alloy nanomaterial PdPtAu in deionized water
  • Step 2 Mix the plasma with an equal volume of methanol/acetonitrile mixture, the methanol/acetonitrile volume ratio is 1:1, shake on a shaker for 10 minutes, centrifuge for 10 minutes, and take the supernatant for mass spectrometry detection;
  • Step 3 the matrix-assisted laser desorption ionization mass spectrometry adopts the positive ion reflectance mode, and the sample preparation is carried out on the mass spectrometer target plate of the matrix-assisted laser desorption ionization mass spectrometry. Molecules are detected.
  • the molecular weight range of the small molecule is less than 1000Da.
  • the small molecules are carbohydrates or amino acids.
  • the present invention has the following advantages and beneficial effects:
  • the metal ions (including iron, cobalt, nickel) and thickness (including ultra-thin and multi-layer) in the metal organic framework material can be detected Precise adjustment to overcome the defects of traditional matrix, low sample volume (0.1 microliter), fast (less than 1 minute), high throughput (about 120,000 data points, more than 300 metabolic characteristics), and high sensitivity for serum detection.
  • Metal-organic framework materials use transition metals, which are less expensive to prepare than noble metals, and have superior cost-effectiveness, and can be prepared by a one-step method, and the synthesis steps are simple.
  • the optimized ultra-thin iron metal-organic framework material provides a large number of active sites for molecular detection.
  • As a matrix material in MALDI-TOF-MS detection it can solve the problem of traditional organic matrix Inherent defects such as interference of low molecular weight segments (m/z ⁇ 1000) and hot spot effects enable efficient analysis of serum samples. Serum samples can be efficiently and quickly detected and analyzed small molecule metabolites in serum only after a simple process.
  • the preparation steps of the series submicroreactor chip materials are simple, the synthesis process is safe and the yield is large, and it has superior cost-effectiveness.
  • the optimized submicroreactor chip material is used as the substrate material of laser desorption ionization time-of-flight mass spectrometry. Compared with traditional organic matrix materials, sensitive detection of small molecular substances in serum metabolism can be realized in the low molecular weight range (m/z ⁇ 400) .
  • the optimized submicroreactor chip material can realize high-throughput (about 120,000 data points, more than 300 metabolic characteristics) and rapid (less than 1 minute) detection of serum with only 0.1 microliter of serum sample. Based on the above significant advantages, the submicroreactor chip material is expected to realize a wide range of clinical serum detection, and be applied to treatment detection and screening of corresponding therapeutic efficacy metabolic biomarkers.
  • Au@ZnO nanocomposite material can solve the hot spot effect of traditional organic matrix as the matrix of laser desorption ionization mass spectrometry and the problem of interference in the low molecular weight region.
  • the Au@ZnO nanocomposite material of the present invention can combine the advantages of both noble metals and semiconductor materials, improve the hot carrier generation effect through the Schottky effect, and have a high degree of controllability. The synergistic effect improves detection efficiency and reduces costs.
  • the special multi-pointed star-shaped composite nanoparticles of the present invention have enhanced electromagnetic fields at the tips and depressions, help to enhance surface plasmon resonance, improve the laser desorption ionization effect, and have It helps to increase the specific surface area and enhance the adsorption of small molecule metabolites, so the polygonal star-shaped Au@ZnO composite nanomaterials are used for the detection of small molecule metabolites and serum metabolites in LDI MS.
  • the porous PdPtAu alloy can be synthesized by a one-step method, the synthesis steps are simple, and the preparation cost is low.
  • the nanomaterial can solve the problems of hot spot effect of traditional organic matrix and interference in low molecular weight region.
  • the plasma sample only needs simple pretreatment, and only 1 ⁇ L of plasma extract is needed for each sample, so that small molecule metabolites in plasma can be detected rapidly and sensitively. This method has high accuracy, low cost, and high detection throughput, which meets the needs of clinical plasma detection.
  • Fig. 1 is a scanning electron microscope characterization picture of an ultra-thin iron metal organic framework prepared in a preferred embodiment of the metal organic framework material preparation method and its small molecule detection application;
  • Fig. 2 is the mass spectrogram of the proline standard molecule detected by the metal organic framework matrix-assisted laser desorption ionization mass spectrometry prepared in the preparation method of the metal organic framework material and its small molecule detection application;
  • Fig. 3 is the mass spectrogram of the metal organic framework matrix-assisted laser desorption ionization mass spectrometry detection creatinine standard molecule prepared by the preferred embodiment of the metal organic framework material and its small molecule detection application;
  • Fig. 4 is the mass spectrogram of the metal organic framework desorption matrix-assisted laser desorption ionization mass spectrometry to detect the low molecular weight part of serum prepared in the preferred embodiment of the metal organic framework material and its small molecule detection application;
  • Figure 5 shows the metal-organic framework material prepared in the preferred embodiment of the metal-organic framework material and its small molecule detection application.
  • the metal-organic framework desorption matrix-assisted laser desorption ionization mass spectrometry detects small molecules in different serum samples, and the disease group and the control group are carried out in MATLAB. Serum sample detection and identification.
  • Fig. 6 is a scanning electron micrograph of APF-sphere of a preferred embodiment in the preparation method of the submicroreactor and the serum metabolite detection method based on it;
  • Fig. 7 is the APF-bowl transmission electron microscope characterization diagram of a preferred embodiment in the preparation method of the submicroreactor and the serum metabolite detection method based on it;
  • Fig. 8 is a APF-sphere&Au scanning electron microscope characterization diagram of a preferred embodiment in the preparation method of the submicroreactor and the serum metabolite detection method based on it;
  • Fig. 9 is the APF-bowl&Au scanning electron microscope characterization diagram of a preferred embodiment in the preparation method of the submicroreactor and the serum metabolite detection method based on it;
  • Figure 10 is the preparation method of the submicroreactor and the serum metabolite detection method based on it.
  • Example 1 a series of submicroreactor chip materials (including APF-sphere&Au, APF-bowl&Au-1, APF-bowl&Au-2 and APF -bowl&Au-3) Statistical histogram results of five independent experiments for MALDI-TOF-MS detection of leucine standard molecules;
  • Figure 11 is the preparation method of the submicroreactor and the serum metabolite detection method based on it.
  • a series of submicroreactor chip materials including APF-sphere&Au, APF-bowl&Au-1, APF-bowl&Au-2 and APF -bowl&Au-3) Statistical histogram results of five independent experiments for MALDI-TOF-MS detection of mannitol standard molecules;
  • Fig. 12 is the mass spectrogram of the low molecular weight segment of serum detected by matrix-assisted laser desorption ionization mass spectrometry in Example 3 in the preparation method of the submicroreactor and the serum metabolite detection method based on it;
  • Fig. 13 is the submicroreactor preparation method and its serum metabolite detection method based on the matrix-assisted laser desorption ionization mass spectrometry detection of small molecules in different serum samples in Example 4, and the differential monitoring of patients before and after chemotherapy.
  • Fig. 14 is the characterization diagram of ZnO in the prior art and the polygonal star shape Au@ZnO nanocomposite material of the present invention (Fig. The scanning electron microscope picture of ZnO nanocomposite material, Fig. 14c is the line-scan energy dispersive x-ray (EDX) analysis picture of multipoint star shape Au@ZnO nanocomposite material of the present invention, Fig.
  • EDX line-scan energy dispersive x-ray
  • FIG. 14d is the multipoint star shape Au@ZnO nanometer of the present invention
  • the element distribution diagram of the composite material Figure 14e is the EDX result map of ZnO and the multi-pointed star-shaped Au@ZnO nanocomposite of the present invention
  • Figure 14f is the ultraviolet ray of Au, ZnO and the multi-pointed star-shaped Au@ZnO nanocomposite of the present invention Visible absorption spectrum
  • Figure 14g is the selected area electron diffraction pattern of the polygonal star-shaped Au@ZnO nanocomposite material of the present invention).
  • Fig. 15 is the mass spectrogram (Fig. 15a is glucose (Glucose), Fig. 15b is sucrose (Saccharose), Fig. 15c) in the MALDI MS detection of polygonal star shape Au@ZnO nanocomposite material in the embodiment of the present invention 4 kinds of standard small molecules is proline (Proline), Figure 15d is lysine (Lysine)).
  • Figure 16 is the mass spectrum of four kinds of standard small molecules and serum metabolites in the MALDI MS detection of multi-point star shape Au@ZnO nanocomposite in the embodiment of the present invention
  • Figure 16a is four kinds of metabolite mixed solutions containing sodium chloride
  • Figure 16b is a mixed solution of four metabolites containing bovine serum albumin
  • Figure 16c is a mixed solution of four metabolites containing CHCA
  • Figure 16d is a mixed solution of four metabolites containing CHCA
  • Figure 16e is the mass spectrum of standard serum detection map).
  • Figure 17 is a scanning electron microscope characterization picture of a preferred embodiment of the porous PdPtAu nanomaterial in the preparation method of the porous alloy nanomaterial and its application in the detection of plasma metabolites;
  • Figure 18 is a transmission electron microscope characterization picture of a preferred embodiment of the porous PdPtAu nanomaterial in the preparation method of the porous alloy nanomaterial and its application in the detection of plasma metabolites;
  • Figure 19 is a mass spectrogram of a porous PdPtAu nanomaterial used as a matrix-assisted laser desorption ionization mass spectrometry detection standard small molecule in a preferred embodiment of the preparation method of the porous alloy nanomaterial and its application in the detection of plasma metabolites;
  • Figure 20 is a preparation method of porous alloy nanomaterials and its application in the detection of plasma metabolites.
  • a preferred embodiment of porous PdPtAu nanomaterials is used as a matrix-assisted laser desorption ionization mass spectrometry detection containing a) NaCl and b) bovine serum albumin The mass spectrum of the small molecule;
  • Fig. 21 is a mass spectrogram of porous PdPtAu nanomaterials used as matrix-assisted laser desorption ionization mass spectrometry to detect small molecule metabolites in plasma samples in a preferred embodiment of the preparation method of porous alloy nanomaterials and its application in the detection of plasma metabolites.
  • Step 1 Preparation of instruments and reagents: set the matrix-assisted laser desorption ionization mass spectrometry detection mode to positive ion reflection mode;
  • Step 2 preparing metal-organic framework series substrates, including the following steps;
  • Step 2.1 Mix dimethylformamide, ethanol, deionized water, triethylamine, and terephthalic acid into a closed container, and add gold ferrous chloride or cobalt chloride or nickel chloride respectively;
  • Step 2.2 The mixture in step 2.1 was subjected to ultrasonic reaction in a water bath for 4 hours at 0°C;
  • Step 2.3 centrifuge the reactant in step 2.2, and wash with deionized water and isopropanol five times each to remove residual dimethylformamide;
  • Step 2.4 Disperse the product in step 2.3 in deionized water, and use low-speed centrifugation to separate the ultra-thin metal organic framework material (MOF-UL).
  • MOF-UL ultra-thin metal organic framework material
  • the ultra-thin iron metal organic framework material Fe -MOF-UL
  • ultrathin cobalt metal organic framework Co-MOF-UL
  • Ni-MOF-UL single-layer nickel metal organic framework
  • Step 2.5 separating the multilayer iron metal organic framework material (Fe-MOF-Bulk) from the deposit in step 2.4;
  • Step 2.6 Disperse Fe-MOF-UL, Co-MOF-UL or Ni-MOF-UL, and Fe-MOF-Bulk obtained in step 2.4 and step 2.5 in deionized water and use it as a matrix;
  • Step 3 Proportionally dilute the serum sample
  • Step 4 Sample preparation is carried out on the mass spectrometer target plate, the matrix is made of optimized Fe-MOF-UL, and dried at room temperature;
  • Step 5 detecting small molecules in serum samples
  • Step 6 Analyze the original mass spectrum and obtain the detection result.
  • the prepared ultra-thin Fe metal organic framework material has a layered structure, and the synthesized material has a thinner thickness and a uniform surface structure.
  • Step 1 Preparation of instruments and reagents: set the matrix-assisted laser desorption ionization mass spectrometry detection mode to positive ion reflection mode;
  • Step 2 preparing a submicroreactor, comprising the following steps;
  • Step 2.1 Dissolve 0.1 g of 3-aminophenol (APF) in 30 ml of deionized water, add 0.1 ml of formaldehyde solution and 0.1 ml of ammonia solution;
  • Step 2.2 react the mixture in step 2.1 at 30°C for 30 minutes;
  • Step 2.3 Centrifuge the reactant in step 2.2, and wash it five times with deionized water to obtain a spherical APF submicron material (APF-sphere);
  • Step 2.4 Add 40 ml of acetone solution to the reactant in step 2.2, and react for 180 minutes at 30°C;
  • Step 2.5 Centrifuge the reactant in step 2.4, and wash it five times with deionized water to obtain a bowl-shaped APF submicron material (APF-bowl);
  • Step 2.6 Disperse 10 mg of the APF-sphere obtained in step 2.3 in 10 ml of deionized water, and add 2 ml of 1% chloroauric acid solution;
  • Step 2.7 Disperse 10 mg of the APF-bowl obtained in Step 2.5 in 10 ml of deionized water to prepare three parts of the above reaction solution, and add 1.5, 2, and 2.5 ml of 1% chloroauric acid solution respectively;
  • Step 2.8 React the reactants obtained in Step 2.6 and Step 2.7 at 70°C for 10 minutes, centrifuge and wash with water to obtain APF-sphere&Au, APF-bowl&Au-1, APF-bowl&Au-2 and APF-bowl&Au- 3;
  • Step 2.9 Disperse the APF-sphere&Au, APF-bowl&Au-1, APF-bowl&Au-2, and APF-bowl&Au-3 obtained in step 2.8 in deionized water and use them as substrates;
  • Step 3 Proportional dilution of the serum sample, 10-fold proportional dilution
  • Step 4 Sample preparation is carried out on the mass spectrometer target plate, using the matrix in step 2.9, preferably APF-bowl&Au-2, and drying at room temperature;
  • Step 5 detecting small molecules in serum samples
  • Step 6 Analyze the original mass spectrum and obtain the detection result.
  • the APF-bowl&Au-2 mass spectrometry detection performance obtained by using a bowl-shaped structure and reacting with 2 ml of chloroauric acid is the best.
  • the detection molecular weight range is less than 1000Da.
  • Substances tested include amino acids and sugar alcohols.
  • the prepared APF-sphere and APF-bowl have smooth surfaces and uniform shapes, while the surfaces of APF-sphere&Au and APF-bowl&Au modified by chloroauric acid are rough, and obvious gold particles can be seen .
  • Embodiment one detection of leucine standard substance
  • Preparation of instruments and reagents set the matrix-assisted laser desorption ionization mass spectrometry detection mode to positive ion reflection mode; the prepared submicroreactor matrix materials include APF-sphere&Au, APF-bowl&Au-1, APF-bowl&Au-2 and APF- bowl&Au-3; prepare the leucine standard solution; prepare the sample on the mass spectrometer target plate and dry it at room temperature; perform detection under the mass spectrometer and analyze the mass spectrometer image, as shown in Figure 10.
  • Embodiment two the detection of mannitol standard substance
  • Preparation of instruments and reagents set the matrix-assisted laser desorption ionization mass spectrometry detection mode to positive ion reflection mode; the prepared submicroreactor matrix materials include APF-sphere&Au, APF-bowl&Au-1, APF-bowl&Au-2, and APF -bowl&Au-3; Prepare the mannitol standard solution; prepare the sample on the mass spectrometer target plate and dry it at room temperature; perform detection under the mass spectrometer and analyze the mass spectrometer image, as shown in Figure 11;
  • Example 4 Detection and monitoring of serum samples before and after chemotherapy
  • a novel submicroreactor chip provided in the present invention is used as a matrix to assist matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and improve its detection performance, especially for complex serum samples.
  • MALDI-TOF-MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • the defects of traditional substrates can be overcome, and the rapid, high-throughput, and high-sensitivity serum metabolic small molecules can be realized. detection.
  • the submicroreactor chip material is easy to prepare, the synthesis process is safe and the yield is high.
  • the optimized APF-bowl&Au loaded with gold nanoparticles can be used as the matrix material in MALDI-TOF-MS detection, which can solve the inherent defects such as interference and hot spot effect of traditional organic matrix in the low molecular weight section , to achieve efficient analysis of serum samples.
  • the submicroreactor chip material can assist mass spectrometry to detect small molecule metabolites in serum samples, and this method has high detection sensitivity and high throughput, and has the potential for clinical application.
  • This embodiment provides a multi-pointed star-shaped Au@ZnO nanocomposite material and its preparation method and application.
  • the multi-pointed star-shaped Au@ZnO nanocomposite material of the present invention can be used as a matrix material in MALDI MS detection. Furthermore, the multi-pointed star-shaped Au@ZnO nanocomposite material of the present invention is used as a matrix material in the MALDI MS detection of small molecule metabolites.
  • the small molecule in the small molecule metabolite is selected from more than one of proline, lysine, arginine, sucrose and glucose.
  • the multi-pointed star-shaped Au@ZnO nanocomposite material of the present invention is used as a matrix material in the MALDI MS detection of serum metabolites.
  • the preparation method of the polygonal star shape Au@ZnO nanocomposite material of the present invention comprises the following steps:
  • step (1) the stirring speed is 800 ⁇ 10 rpm.
  • step (2) and step (3) the rotational speed of centrifugation is 1000 ⁇ 10 rpm.
  • the polygonal star-shaped Au@ZnO nanocomposite material of the present invention is obtained by the above-mentioned preparation method.
  • the inventive multipoint star-shaped Au@ZnO nanocomposite was used as matrix-assisted laser desorption ionization mass spectrometry for the detection of small molecule metabolites and serum metabolites.
  • the photoelectric effect and hot carrier generation efficiency are enhanced through the Schottky effect, which is helpful for the ionization of the analyte;
  • the special polygonal star shape in The electromagnetic field is enhanced at the tip and the depression, which helps to enhance the surface plasmon resonance, and at the same time increases the specific surface area, which helps to adsorb small molecules of metabolites to be detected;
  • the nanocomposite has a strong absorption at a wavelength of 355nm, which is compatible with The light source wavelength of matrix-assisted laser desorption ionization mass spectrometry matches, so through simple pretreatment, the sample processing steps are simplified, the efficiency of LDI is improved, and the process of traditional GC/LC MS
  • the preparation method of the multi-pointed star-shaped Au@ZnO nanocomposite material of the present invention is simple and uniform in shape.
  • the nanoparticle is used as the matrix of LDI MS, which can solve the problems existing in the traditional matrix, such as the background interference of the low molecular weight region.
  • only 1.5 ⁇ L of serum extract is needed for the serum sample, and the small molecular metabolites in the serum can be detected efficiently and rapidly.
  • This detection method has high sensitivity, low cost, and high detection throughput, which meets the needs of clinical serum detection and has the potential for clinical application.
  • the extinction spectra and surface charge values of the nanomaterials were obtained using an AuCy UV1900 spectrophotometer and a Malvern Zetasizer NanZS90.
  • Hitachi SU8100 was used to obtain scanning electron microscope pictures, and JEOL JEM-2100F was used to obtain transmission electron microscope pictures, line-scan energy dispersive X-spectrum and selected electron diffraction patterns.
  • the preparation method of the polygonal star-shaped Au@ZnO nanocomposite material of the present embodiment comprises the following steps:
  • Zinc acetate solution 39mL, 0.01mol/L was added to sodium hydroxide solution (65mL, 0.03mol/L), heated to 60°C and kept for 1h, then centrifuged at 10000rpm for 10min, The precipitate was collected, the supernatant was discarded, and then washed three times with ethanol and water respectively, and the obtained precipitate was dried in an oven at 50°C for future use.
  • the polygonal star-shaped Au@ZnO nanocomposite material obtained in the embodiment was subjected to the following experiments.
  • Standard small molecules proline, lysine, sucrose and glucose
  • standard small molecules proline, lysine, sucrose and glucose
  • the salt-tolerance test sample is to add sodium chloride to the mixed solution of four standard small molecules (proline, lysine, arginine and glucose), and the final concentrations of sodium chloride and small molecules are 0.2mg respectively /mL and 1mg/mL.
  • the test sample is a mixture of albumin and standard small molecules (proline, lysine, arginine and glucose), and the final concentration of albumin and small molecules is 1 mg/mL, so that Explore the detection performance of different matrices at high salt concentrations and proteins.
  • Au@ZnO nanocomposites were dispersed in water at a concentration of 1 mg/mL as a matrix.
  • Au nanoparticles and ZnO nanoparticles as the matrix, they were also dispersed in water at a concentration of 1 mg/mL.
  • 1.5 ⁇ L of analyte solution standard small molecule solution or standard serum
  • 1.5 ⁇ L of matrix suspension was dropped on a polished target plate, dried at room temperature, covered with 1.5 ⁇ L of matrix suspension, and then analyzed by LDI mass spectrometry after drying.
  • Mass spectrometry was performed using an AutoFlex TOF/TOF mass spectrometer (Bruker, Germany) equipped with a Nd:YAG laser (2 kHz, 355 nm).
  • sample preparation is carried out on the mass spectrometer target plate, and dried at room temperature;
  • the polygonal star-shaped Au@ZnO nanocomposite material of this embodiment that is, four typical small molecule metabolites of p-ZnO (Glucose, sucrose, proline and lysine) are better detected than pure zinc oxide (ZnO) nanoparticles, especially sucrose, the mass spectrometry signals differ by more than 17 times, so it can be said that the polygonal star shape Au@ZnO
  • the nanocomposite material (p-ZnO) is more suitable for LDI mass spectrometry than ZnO.
  • sample preparation is carried out on the mass spectrometer target plate, and dried at room temperature;
  • Figure 16a is the mass spectrometry detection result of four metabolite mixed solutions (proline, lysine, arginine and glucose are all 1mg/mL) containing 0.2mg/mL sodium chloride, as can be seen from the figure, this
  • the multi-pointed star-shaped Au@ZnO nanocomposite material in the embodiment can detect all four metabolites under the condition of high salt concentration, and only detects the hydrogenation peak of arginine when CHCA is used as the matrix in Figure 16c, so the four metabolites Metabolite mixed solution (1 mg/mL for proline, lysine, arginine and glucose) had better salt tolerance.
  • Figure 16b is a mixed solution of four metabolites (proline, lysine, arginine and glucose are all 1 mg/mL) added with 1 mg/mL bovine serum albumin, as can be seen from Figure 16b, the four metabolites can also be detected by the multi-pointed star-shaped Au@ZnO nanomatrix in this example, and CHCA in Figure 16d only detects the hydrogenation peak of arginine, so the multi-pointed star-shaped Au@ZnO nanocomposite material has better protein-resistant properties.
  • four metabolites proline, lysine, arginine and glucose are all 1 mg/mL
  • bovine serum albumin 1 mg/mL bovine serum albumin
  • CHCA has a poor detection effect on metabolites in high-salt and high-protein mixed solutions, and only detects the hydrogenation peak of arginine, while the polygonal star-shaped Au@ZnO nanocomposite material of the present invention can combine four All kinds of amino acids are detected, which proves that the multi-pointed star-shaped Au@ZnO nanocomposite material of the present invention is more suitable for the detection of complex biological samples than the traditional organic matrix CHCA, and has lower background interference.
  • Step 1 Preparation of instruments and reagents: matrix-assisted laser desorption ionization mass spectrometry, using positive ion reflectance mode;
  • Step 2 The preparation of the porous PdPtAu alloy comprises the following steps;
  • Step 2.1 Mix 0.6mL Na2PdCl4 (20mM), 3mL H2PtCl6 ⁇ 6H2O (20mM), 60 ⁇ L hydrochloric acid (6.0M) and 60mg F127, and dissolve them by ultrasonic. After F127 is completely dissolved, add 3mL ascorbic acid solution (0.1M), immediately Place in a water bath at 45°C for ultrasonic reaction for 3 h, add 1.2 mL of HAuCl4 ⁇ 4H2O solution (10 mM, 20 mM, 30 mM, 40 mM respectively) and react for 1 h. After the reaction, wash with absolute ethanol and water centrifuge (10,000 rpm) three times, and dry at 50°C for later use.
  • Step 2.2 Disperse the above porous PdPtAu alloy in deionized water and use it as a matrix;
  • Step 3 The ratio (glucose, phenylalanine, lysine) is dissolved in deionized water, and the mass spectrometry performance of PdPtAu with different pore sizes and gold contents is detected;
  • Step 5 Sample preparation is carried out on the mass spectrometer target plate, the matrix is made of optimized PdPtAu material, and dried at room temperature;
  • Step 6 Detect small molecules in plasma samples
  • Step 7 Analyze the mass spectrometry results and draw conclusions.
  • the synthesized porous PdPtAu alloy has a particle size of about 170nm, uniform particle size, and a porous structure.
  • Transmission electron microscopy results were obtained using a JEOL JEM-2100F. As shown in Figure 18, the nanomaterial has a porous structure, which is consistent with the results of scanning electron microscopy.
  • Figure 20a is the mass spectrum that detects glucose, phenylalanine and lysine in the solution containing NaCl
  • Figure 20b shows Mass spectra for the detection of glucose, phenylalanine, and lysine in solution of serum albumin.
  • FIG. 21 and FIG. 21a, 21b are the mass spectrograms of the healthy plasma sample and the cancer plasma sample respectively.

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Abstract

一种金属有机框架材料的制备方法及其小分子检测应用，制备方法包括将二甲基甲酰胺、乙醇、去离子水、三乙胺、对苯二甲酸混合加入一密闭容器中，并加入金属氯化物形成混合物；水浴超声后离心并洗涤；产物分散在去离子水中，采用低速离心分离出超薄金属有机框架材料。可对金属有机框架材料中金属离子和厚度进行精准调节，克服传统基质的缺陷，实现低样本量、快速、高通量、高灵敏度地对血清小分子进行检测。

Description

纳米增强芯片的制备及其在小分子代谢物激光解离质谱检测中的应用 技术领域
本发明涉及复合纳米材料芯片合成领域，尤其是涉及纳米增强芯片及其在小分子代谢物激光解离质谱检测中的应用。
背景技术
生物标志物(蛋白质、核酸、代谢物等)检测因其无创等特点，在体外诊断中发挥越来越重要的作用。与功能受表观遗传调控的基因和翻译后修饰的蛋白质不同，代谢物是生化活性的直接标志，更易与表型相关联。大分子物质如核酸、蛋白等，可经由聚合酶链式反应、荧光增强等技术支持下，将微量物质扩展至可检测水平，从而用于疾病诊断。相比于大分子物质，针对小分子代谢物的信号扩增仍然面临巨大挑战，需要开发用于小分子代谢物的高灵敏度检测方法。
目前，小分子代谢物的检测主要依托于两大技术：质谱技术和核磁共振波谱技术。由于核磁共振波谱技术的灵敏度和分子识别方面均存在固有的局限性，因此质谱被认为是小分子代谢物的首要检测技术。其中，气相/液相色谱-质谱联用技术是代谢物检测最主要的质谱技术手段，然而这些方法需要对样品进行脱盐、除蛋白、衍生化、浓缩等复杂的预处理，才能实现代谢物检测，对样本量需求高且耗时。基质辅助激光解吸电离质谱(MALDI MS)通过引入基质材料用于光子吸收，促进待测物质从固相至气相转换，可以直接实现微量待测物质的高灵敏度、高通量检测。
基质材料决定MALDI MS检测性能，传统的有机基质容易在小分子量端(m/z<400)产生强的背景信号，而这些噪声对于小分子的检测带来极大的干扰，影响检测效果。此外，复杂生物样品中存在着各种的生物大分子，且不同的酸碱度以及高盐度都会对小分子的检测带来阻碍，因此传统的有机基质难以满足小分子的检测需求。传统的无机纳米材料(如碳基、硅基、贵金属材料等)虽然可以用于小分子代谢物的检测，但在对复杂生物样本检测时，仍然存在局限性。
发明内容
本发明的目的在于开发纳米增强芯片及其在小分子代谢物激光解离质谱检测中的应用。
具体而言，本发明涉及的方案包括：
(一)提供一种金属有机框架材料的制备方法及其小分子检测应用；
(二)提供一种亚微反应器的制备方法及基于其的血清代谢物检测方法；
(三)提供一种多角星形状Au@ZnO纳米复合材料及其制备方法和应用；
(四)提供一种多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用。
也就是本申请中涉及的纳米增强芯片可以为下述的金属有机框架材料、亚微反应器、多角星形状Au@ZnO纳米复合材料以及多孔合金纳米材料。
本发明的目的可以通过以下技术方案来实现：
(一)提供一种金属有机框架材料的制备方法及其小分子检测应用
首先提供了一种金属有机框架材料的制备方法，所述方法包括以下步骤：
步骤1、将二甲基甲酰胺，乙醇，去离子水，三乙胺，对苯二甲酸混合加入一密闭容器中，并加入金属氯化物形成混合物；
步骤2、将所述混合物进行水浴超声反应，得到反应物；
步骤3、将所述反应物进行离心并洗涤，除去残留的所述二甲基甲酰胺，得到产物；
步骤4、将步骤3中所述产物分散在去离子水中，采用低速离心分离出超薄金属有机框架材料。
进一步地，步骤1所述金属氯化物为氯化亚铁、氯化钴和氯化镍中的一种，对应步骤4分别得到超薄铁金属有机框架材料、超薄钴金属有机框架材料和超薄镍金属有机框架材料。
进一步地，步骤2所述水浴超声反应条件为0℃条件下反应4小时。
进一步地，步骤3所述洗涤方法为采用去离子水和异丙醇各洗涤五次。
进一步地，所述超薄铁金属有机框架材料进一步分离得到多层铁金属有机框架材料。
进一步地，所述超薄镍金属有机框架材料为单层镍金属有机框架材料。
进一步，本发明还提供一种金属有机框架材料在小分子检测中的应用。
进一步地，包括以下步骤：
步骤1、将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；
步骤2、对血清样品进行比例稀释；
步骤3、在质谱靶板上进行样品制备，所述超薄金属有机框架材料作为基质，室温下干燥；
步骤4、对所述血清样品中的小分子进行检测；
步骤5、分析原始质谱图谱，并获得检测结果.
进一步地，步骤4所述小分子的分子量范围为小于1000Da。
进一步地，步骤4所述小分子包括氨基酸、核苷等。
(二)提供一种亚微反应器的制备方法及基于其的血清代谢物检测方法；
本发明中提供了一种亚微反应器的制备方法，包括如下步骤：
步骤2.1：将3-氨基苯酚APF溶解于去离子水中，并加入甲醛溶液和氨水溶液；
步骤2.2：将步骤2.1的混合物在30℃条件下，反应30分钟；
步骤2.3：将步骤2.2中的反应物进行离心洗涤，得到APF亚微材料。
进一步地，直接将步骤2.2中的反应物进行离心，并采用去离子水洗涤，得到球形的APF亚微材料APF-sphere。
进一步地，还包括向步骤2.2中的反应物中加入丙酮溶液，在30℃条件下，反应180分钟；将反应物进行离心，并采用去离子水洗涤，得到碗形的APF亚微材料APF-bowl。
进一步地，将得到的所述APF-sphere分散在去离子水中，并加入氯金酸溶液，反应得到APF-sphere&Au。
进一步地，所述氯金酸溶液的加入量为2ml。
进一步地，将得到的所述APF-bowl分散在去离子水中，制备三份反应液，分别加入1.5、2和2.5ml氯金酸溶液中，反应得到APF-bowl&Au-1、APF-bowl&Au-2以及APF-bowl&Au-3。
进一步地，反应条件为在70℃条件下，反应10分钟。
本发明中还提供一种根据上述的亚微反应器的血清代谢物检测方法，包括如下步骤：
(1)仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；
(2)将得到的APF-sphere&Au、APF-bowl&Au-1、APF-bowl&Au-2以及APF-bowl&Au-3分散在去离子水中，作为基质使用
(3)对血清样品进行比例稀释；
(4)在质谱靶板上进行样品制备，采用(2)中的所述基质，室温下干燥；
(5)对血清样品中的小分子进行检测；
(6)分析原始质谱图谱，并获得检测结果。
进一步地，检测分子量范围为小于1000Da。
进一步地，检测的物质包括氨基酸和糖醇。
(三)提供一种多角星形状Au@ZnO纳米复合材料及其制备方法和应用
本发明提供一种多角星形状Au@ZnO纳米复合材料作为基质材料在小分子代谢物的基质辅助激光解吸电离质谱检测中的应用。
优选地，小分子代谢物中的小分子选自脯氨酸、赖氨酸、精氨酸、蔗糖和葡萄糖中的一种以上。
本发明提供一种多角星形状Au@ZnO纳米复合材料作为基质材料在血清代谢物的基质辅助激光解吸电离质谱检测中的应用。
本发明提供一种根据上述的多角星形状Au@ZnO纳米复合材料的制备方法，其包括如下步骤：
(1)、纳米金溶液的制备：水合四氯金酸溶液搅拌加热至120±1℃后，加入二水合柠檬酸钠，恒温下搅拌30±0.1min，冷却至室温备用；
(2)、氧化锌的制备：将醋酸锌溶液加入氢氧化钠溶液中，加热至60±1℃且保温1±0.1h，离心收集沉淀，洗涤，在50±1℃下干燥备用；
(3)、多角星形状Au@ZnO纳米复合材料的制备：醋酸锌溶液和纳米金溶液混合，得到混合液，在混合液内加入氢氧化钠溶液，加热至60±1℃且保温1±0.1h，离心收集沉淀，洗涤，在50±1℃下干燥备用。
优选地，步骤(1)中，搅拌的转速为800±10rpm。
优选地，步骤(2)和步骤(3)中，离心的转速为1000±10rpm。
本发明提供一种多角星形状Au@ZnO纳米复合材料，其由上述的制备方法得到。
(四)提供一种多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用
本发明所要解决的技术问题是如何具有更好的LDI效率的MALDI MS基质，用于复杂生物样本(血浆)代谢物的检测。基于此，本发明提供一种多孔合金纳米材料的制备方法，所述方法包括以下步骤：
步骤1、将Na2PdCl4、H2PtCl6·6H2O、盐酸和F127充分混合，超声溶解；
步骤2、待完全溶解后，加入抗坏血酸溶液，并立刻置于水浴超声反应；
步骤3、随后加入HAuCl4·4H2O溶液反应；
步骤4、最后分别用无水乙醇和水离心洗涤后干燥得到所述多孔合金纳米材料PdPtAu。
进一步地，步骤1所述Na2PdCl4、H2PtCl6·6H2O、盐酸和F127用量分别为20Mm的Na2PdCl4加入0.6mL，20Mm的H2PtCl6·6H2O加入3mL，6.0M的盐酸加入60μL，F127加入60mg。
进一步地，步骤2所述抗坏血酸溶液加入量为0.1M的抗坏血酸溶液加入3mL。
进一步地，步骤2所述水浴超声反应为在45℃反应3h。
进一步地，步骤3所述HAuCl4·4H2O溶液的浓度为10Mm-40Mm，加入量为1.2mL。
进一步地，步骤3所述离心洗涤次数为3次，转速为10000rpm，干燥温度为50。℃上述多孔合金纳米材料在检测血浆代谢物中的应用，所述多孔合金纳米材料作为基质辅助激光解吸电离质谱的基质。
进一步地，包括以下步骤：
步骤1、将所述多孔合金纳米材料PdPtAu分散在去离子水中；
步骤2、将血浆与等体积甲醇/乙腈混合液混合，所述甲醇/乙腈体积比为1:1，摇床震荡10min，离心10min，取上清液用于质谱检测；
步骤3、所述基质辅助激光解吸电离质谱采用正离子反射模式，在所述基质辅助激光解吸电离质谱的质谱靶板上进行样品制备，所述样品为1μL血浆提取液，对血浆样品中的小分子进行检测。
进一步地，所述小分子的分子量范围为小于1000Da。
进一步地，所述小分子为糖类或氨基酸。
与现有技术相比，本发明具有以下优点及有益效果：
(一)提供的一种金属有机框架材料的制备方法及其小分子检测应用中，可以对金属有机框架材料中金属离子(包括铁、钴、镍)和厚度(包括超薄和多层)进行精准调节，实现了克服传统基质的缺陷，样本量低(0.1微升)、快速(小于1分钟)、高通量(约120000个数据点，300多个代谢特征)、高灵敏度地对血清进行检测。金属有机框架材料采用过渡金属，相较于贵金属制备成本低，具有优越的成本效益， 且可以通过一步法制备，合成步骤简易。通过对系列金属有机框架材料进行优化，优化后的超薄铁金属有机框架材料，为分子检测提供了大量的活性位点，作为MALDI-TOF-MS检测中的基质材料，可以解决传统有机基质在低分子量段(m/z<1000)的干扰以及热点效应等固有缺陷，实现血清样本的高效分析。血清样本仅需经过简单的，就可高效、快速的检测分析血清中的小分子代谢物。
(二)提供的一种亚微反应器的制备方法及基于其的血清代谢物检测方法中，系列亚微反应器芯片材料制备步骤简单，合成过程安全且产量较大，具有优越的成本效益。将优化的亚微反应器芯片材料作为激光解吸电离飞行时间质谱的基底材料，相较于传统的有机基质材料，在低分子量段(m/z<400)可实现血清代谢小分子物质的灵敏检测。优化后的亚微反应器芯片材料仅0.1微升的血清样本，即可实现血清的高通量(约120000个数据点，300多个代谢特征)、快速(小于1分钟)检测。基于以上显著优势，亚微反应器芯片材料有望实现临床大范围的血清检测，并应用于治疗检测中并筛选出相应的治疗疗效代谢生物标志物。
(三)提供的多角星形状Au@ZnO纳米复合材料及其制备方法和应用中，与有机基质相比，Au@ZnO纳米复合材料作为激光解吸电离质谱的基质，可以解决传统有机基质的热点效应以及在低分子量区有干扰的问题。与无机基质相比，本发明的Au@ZnO纳米复合材料却能结合贵金属和半导体材料两者的优势，通过肖特基效应提高热载流子产生效应，具有高度的可调控性，通过两者的协同效应提高检测效率并降低成本。本发明特殊多角星形状的复合纳米颗粒，与传统核壳纳米材料或者球状纳米材料相比，在尖端和凹陷处增强了电磁场，有助于增强表面等离子共振，提高了激光解吸电离效应，同时有助于提高比表面积增强小分子代谢物的吸附性，故多角星形的Au@ZnO复合纳米材料用于LDI MS中的小分子代谢物和血清代谢物的检测。
(四)提供的多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用中，多孔PdPtAu合金，通过一步法即可合成，合成步骤简单，制备成本低。该纳米材料作为激光解吸电离质谱的基质，可以解决传统有机基质的热点效应以及在低分子量区有干扰的问题。本发明中，血浆样本只需经过简单的预处理，并且每份样本只需要1μL血浆提取液，就可快速、灵敏的检测血浆中的小分子代谢物。这种方法准确率高、成本低、检测通量高，满足了临床血浆检测的需求。
附图说明
图1是金属有机框架材料的制备方法及其小分子检测应用中一个较佳实施例制备得到的超薄铁金属有机框架的扫描电子显微镜表征图片；
图2是金属有机框架材料的制备方法及其小分子检测应用中制备得到的金属有机框架基质辅助激光解吸电离质谱检测脯氨酸标准分子的质谱图；
图3是金属有机框架材料的制备方法及其小分子检测应用中较佳实施例制备得到的金属有机框架基质辅助激光解吸电离质谱检测肌酐标准分子的质谱图；
图4是金属有机框架材料的制备方法及其小分子检测应用中较佳实施例制备得到的金属有机框架解吸基质辅助激光解吸电离质谱检测血清低分子量段的质谱图；
图5是金属有机框架材料的制备方法及其小分子检测应用中较佳实施例制备得到的金属有机框架解吸基质辅助激光解吸电离质谱检测不同血清样本的小分子，在MATLAB进行疾病组和对照组的血清样本检测及鉴别。
图6是亚微反应器的制备方法及基于其的血清代谢物检测方法中一个较佳实施例的APF-sphere的扫描电子显微镜图；
图7是亚微反应器的制备方法及基于其的血清代谢物检测方法中一个较佳实施例的APF-bowl透射电子显微镜表征图；
图8是亚微反应器的制备方法及基于其的血清代谢物检测方法中一个较佳实施例的APF-sphere&Au扫描电子显微镜表征图；
图9是亚微反应器的制备方法及基于其的血清代谢物检测方法中一个较佳实施例的APF-bowl&Au扫描电子显微镜表征图；
图10是亚微反应器的制备方法及基于其的血清代谢物检测方法实施例一中采用系列亚微反应器芯片材料(包括APF-sphere&Au、APF-bowl&Au-1、APF-bowl&Au-2以及APF-bowl&Au-3)用于MALDI-TOF-MS检测亮氨酸标准分子的五次独立实验的统计柱状图结果；
图11是亚微反应器的制备方法及基于其的血清代谢物检测方法实施例二中采用系列亚微反应器芯片材料(包括APF-sphere&Au、APF-bowl&Au-1、APF-bowl&Au-2以及APF-bowl&Au-3)用于MALDI-TOF-MS检测甘露醇标准分子的五次独立实验的统计柱状图结果；
图12是亚微反应器的制备方法及基于其的血清代谢物检测方法中实施例三中 基质辅助激光解吸电离质谱检测血清低分子量段的质谱图；
图13是亚微反应器的制备方法及基于其的血清代谢物检测方法中实施例四中基质辅助激光解吸电离质谱检测不同血清样本的小分子，在患者进行化疗前和化疗后的差异监测。
图14为现有技术中ZnO和本发明的多角星形状Au@ZnO纳米复合材料的表征图(图14a为现有技术中纳米ZnO的扫描电镜图，图14b为本发明的多角星形状Au@ZnO纳米复合材料的扫描电镜图，图14c为本发明的多角星形状Au@ZnO纳米复合材料的线扫能量色散x射线(EDX)分析图，图14d为本发明的多角星形状Au@ZnO纳米复合材料的元素分布图，图14e为ZnO和本发明的多角星形状Au@ZnO纳米复合材料的EDX结果图，图14f为Au、ZnO和本发明的多角星形状Au@ZnO纳米复合材料的紫外可见吸收光谱图，图14g为本发明的多角星形状Au@ZnO纳米复合材料的选区电子衍射图)。
图15为本发明的实施例中多角星形状Au@ZnO纳米复合材料的MALDI MS检测中4种标准小分子的质谱图(图15a为葡萄糖(Glucose)，图15b为蔗糖(Saccharose)，图15c为脯氨酸(Proline)，图15d为赖氨酸(Lysine))。
图16为本发明的实施例中多角星形状Au@ZnO纳米复合材料的MALDI MS检测中4种标准小分子和血清代谢物的质谱图(图16a为含有氯化钠的四种代谢物混合溶液，图16b为含有牛血清白蛋白的四种代谢物混合溶液，图16c为含有CHCA的四种代谢物混合溶液，图16d为含有CHCA的四种代谢物混合溶液，图16e是标准血清的质谱检测图)。
图17是多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用中一个较佳实施例的多孔PdPtAu纳米材料的扫描电镜表征图片；
图18是多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用中一个较佳实施例的多孔PdPtAu纳米材料的透射电镜表征图片；
图19是多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用中一个较佳实施例的多孔PdPtAu纳米材料作为基质辅助激光解吸电离质谱检测标准小分子的质谱图；
图20是多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用中一个较佳实施例的多孔PdPtAu纳米材料作为基质辅助激光解吸电离质谱检测含有a)NaCl和b)牛血清白蛋白的小分子的质谱图；
图21是多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用中一个较佳实施例的多孔PdPtAu纳米材料作为基质辅助激光解吸电离质谱检测血浆样本中小分子代谢物的质谱图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。
(一)提供关于金属有机框架材料的制备方法及其小分子检测应用的实施例
制备方法实施例1
步骤1：仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；
步骤2：制备金属有机框架系列基质，包括以下步骤；
步骤2.1：将二甲基甲酰胺，乙醇，去离子水，三乙胺，对苯二甲酸混合加入一密闭容器中，并分别加入金氯化亚铁或氯化钴或氯化镍；
步骤2.2：在步骤2.1的混合物在0℃条件下，水浴超声反应4小时；
步骤2.3：将步骤2.2中的反应物进行离心，并采用去离子水和异丙醇各洗涤五次，除去残留的二甲基甲酰胺；
步骤2.4：将步骤2.3中的产物分散在去离子水中，采用低速离心分离出超薄金属有机框架材料(MOF-UL)，根据金属氯化物的差异，分别得到超薄铁金属有机框架材料(Fe-MOF-UL)、超薄钴金属有机框架材料(Co-MOF-UL)和单层镍金属有机框架材料(Ni-MOF-UL)；
步骤2.5：将步骤2.4中的沉积物中分离出多层铁金属有机框架材料(Fe-MOF-Bulk)；
步骤2.6：将步骤2.4和步骤2.5中得到的Fe-MOF-UL，Co-MOF-UL或Ni-MOF-UL，以及Fe-MOF-Bulk，分散在去离子水中，作为基质使用；
步骤3：对血清样品进行比例稀释；
步骤4：在质谱靶板上进行样品制备，基质采用优化的Fe-MOF-UL，室温下干燥；
步骤5：对血清样品中的小分子进行检测；
步骤6：分析原始质谱图谱，并获得检测结果。
如图1所示，采用Hitachi S-4800扫描电子显微镜获取透射电镜结果。所制备的超薄铁金属有机框架材料呈层状结构，合成的材料厚度较薄，表面结构均一。
应用实施例1
对脯氨酸标准品的检测：
(1)仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；所制备MOF材料；配置好的脯氨酸标准溶液；
(2)在质谱靶板上进行样本制备，室温下干燥；
(3)在质谱仪下进行检测，并对质谱图像进行分析，结果如图2所示；
应用实施例2
对肌酐标准品的检测：
(1)仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；所制备MOF材料；配置好的肌酐标准溶液；
(2)在质谱靶板上进行样本制备，室温下干燥；
(3)在质谱仪下进行检测，并对质谱图像进行分析，结果如图3所示；
应用实施例3
对血清样本小分子的检测：
(1)仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；所制备的系列金属有机框架材料，包括Fe-MOF-UL,Co-MOF-UL，Ni-MOF-UL，以及Fe-MOF-Bulk；
(2)按一定比例稀释血清样本；
(3)在质谱靶板上进行样本制备，室温下干燥；
(4)在质谱仪下进行检测，并对质谱图像进行分析，结果如图4所示。
应用实施例4
对疾病组和对照组血清样本检测及鉴别，，其中疾病组为23例病人血清样本，对照组为23个健康志愿者血清样本：
(1)仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；所制备MOF材料；MATLAB分析软件；
(2)按一定比例稀释血清样本；
(3)在质谱靶板上进行样本制备，室温下干燥；
(4)在质谱仪下进行检测，收集质谱数据；
(5)对质谱数据进行预处理，并采用MATLAB分析软件进行分析，结果如图5所示。
(二)提供一种亚微反应器的制备方法及基于其的血清代谢物检测方法的实施例；
技术方案如下：
步骤1：仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；
步骤2：制备亚微反应器，包括以下步骤；
步骤2.1：将0.1克3-氨基苯酚(APF)溶解于30毫升去离子水中，加入0.1毫升甲醛溶液和0.1毫升氨水溶液；
步骤2.2：步骤2.1的混合物在30℃条件下，反应30分钟；
步骤2.3：将步骤2.2中的反应物进行离心，并采用去离子水洗涤五次，得到球形的APF亚微材料(APF-sphere)；
步骤2.4：向步骤2.2中的反应物中加入40毫升丙酮溶液，在30℃条件下，反应180分钟；
步骤2.5：将步骤2.4中的反应物进行离心，并采用去离子水洗涤五次，得到碗形的APF亚微材料(APF-bowl)；
步骤2.6：将步骤2.3得到的APF-sphere，取10毫克分散在10毫升去离子水中，并加入2毫升质量浓度为1％的氯金酸溶液；
步骤2.7：将步骤2.5得到的APF-bowl，取10毫克分散在10毫升去离子水中，制备三份上述反应液，并分别加入1.5、2、2.5毫升质量浓度为1％的氯金酸溶液；
步骤2.8：将步骤2.6和步骤2.7中分别得到的反应物在70℃条件下，反应10分钟，离心水洗，依次得到APF-sphere&Au、APF-bowl&Au-1、APF-bowl&Au-2以及APF-bowl&Au-3；
步骤2.9：将步骤2.8中得到的APF-sphere&Au、APF-bowl&Au-1、APF-bowl&Au-2、以及APF-bowl&Au-3分散在去离子水中，作为基质使用；
步骤3：对血清样品进行比例稀释，10倍比例稀释；
步骤4：在质谱靶板上进行样品制备，采用步骤2.9中基质，优选采用APF-bowl&Au-2，室温下干燥；
步骤5：对血清样品中的小分子进行检测；
步骤6：分析原始质谱图谱，并获得检测结果。
进一步地，采用碗形结构、以及2毫升氯金酸反应所得的APF-bowl&Au-2质谱检测性能最佳。检测分子量范围为小于1000Da。检测的物质包括氨基酸和糖醇。
表征所用仪器
采用Hitachi S-4800扫描电子显微镜获取SEM结果。采用JEOL JEM-2100F透射电子显微镜获取TEM结果。
表征结果为：
如图6至图9所示，所制备的APF-sphere、APF-bowl表面光滑，形态均一，而经过氯金酸修饰后的APF-sphere&Au和APF-bowl&Au表面粗糙，可以看到明显的金颗粒。
实施例一：亮氨酸标准品的检测
仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；所制备亚微反应器基质材料，包括APF-sphere&Au、APF-bowl&Au-1、APF-bowl&Au-2以及APF-bowl&Au-3；配置好亮氨酸标准溶液；在质谱靶板上进行样本制备，室温下干燥；在质谱仪下进行检测，并对质谱图像进行分析，如图10所示。
实施例二：甘露醇标准品的检测
仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；所制备亚微反应器基质材料，包括APF-sphere&Au、APF-bowl&Au-1、APF-bowl&Au-2、以及APF-bowl&Au-3；配置好甘露醇标准溶液；在质谱靶板上进行样本制备，室温下干燥；在质谱仪下进行检测，并对质谱图像进行分析，如图11所示；
实施例三：血清样本小分子的检测
仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；所制备优化的亚微反应器APF-bowl&Au-2作为基质材料；按一定比例稀释血清样本；在质谱靶板上进行样本制备，室温下干燥；在质谱仪下进行检测，并对质谱图像进行分析，如图12所示。
实施例四：化疗前后血清样本的检测及监测
仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；所制备优化的亚微反应器APF-bowl&Au-2作为基质材料；MATLAB和 Metaboanalyst分析软件；按一定比例稀释血清样本；在质谱靶板上进行样本制备，室温下干燥；在质谱仪下进行检测，收集质谱数据；对质谱数据进行预处理，并采用MATLAB分析软件进行分析，如图13。
本发明中提供的一种新型的亚微反应器芯片作为基质，用于辅助将基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)，提高其检测性能，尤其是针对复杂的血清样本。通过对亚微反应器芯片材料的形态结构(球性、碗形)和组成成分(金负载含量)进行调控，从而克服传统基质的缺陷，实现快速、高通量、高灵敏度的血清代谢小分子检测。
亚微反应器芯片材料制备简易，合成过程安全且产量较高。通过对亚微反应器芯片材料进行优化，优化后负载金纳米颗粒的APF-bowl&Au作为MALDI-TOF-MS检测中的基质材料，可以解决传统有机基质在低分子量段的干扰以及热点效应等固有缺陷，实现血清样本的高效分析。本发明中，亚微反应器芯片材料可以辅助质谱实现血清样本中的小分子代谢物检测，且此方法检测灵敏度高、通量高，有应用于临床的潜力。
(三)提供多角星形状Au@ZnO纳米复合材料及其制备方法和应用的实施例
本实施例中提供了一种多角星形状Au@ZnO纳米复合材料及其制备方法和应用。
本发明的多角星形状Au@ZnO纳米复合材料作为基质材料可以在MALDI MS检测中得以应用。进一步地，本发明的多角星形状Au@ZnO纳米复合材料作为基质材料在小分子代谢物的MALDI MS检测中得以应用。其中，小分子代谢物中的小分子选自脯氨酸、赖氨酸、精氨酸、蔗糖和葡萄糖中的一种以上。
进一步地，本发明的多角星形状Au@ZnO纳米复合材料作为基质材料在血清代谢物的MALDI MS检测中得以应用。
本发明的多角星形状Au@ZnO纳米复合材料的制备方法包括如下步骤：
(1)、采用柠檬酸还原法合成纳米金溶液：水合四氯金酸溶液搅拌加热至120±1℃后，加入二水合柠檬酸钠，恒温下搅拌30±0.1min，冷却至室温备用；
(2)、氧化锌的制备：将醋酸锌溶液加入氢氧化钠溶液中，搅拌加热至60±1℃且保温1±0.1h，弃去上清液，再用乙醇和水分别洗涤三次，在50±1℃下干燥备用；
(3)、多角星形状Au@ZnO纳米复合材料的制备：醋酸锌溶液和纳米金溶液 混合，得到混合液，在混合液内加入氢氧化钠溶液，继续搅拌加热至60±1℃且保温1±0.1h，弃去上清液，再用乙醇和水分别洗涤三次，在50±1℃下干燥备用。
其中，在步骤(1)中，搅拌的转速为800±10rpm。在步骤(2)和步骤(3)中，离心的转速为1000±10rpm。
由上述的制备方法得到本发明的多角星形状Au@ZnO纳米复合材料。
总之，本发明的多角星形状Au@ZnO纳米复合材料作为基质辅助激光解吸电离质谱用于小分子代谢物和血清代谢物的检测。首先，由于其复合了半导体氧化锌和金属金，通过肖特基效应增强了光电效应和热载流子生成效率，有助于待测物的电离；其次，由于其特殊的多角星形状，在尖端和凹陷处增强了电磁场，有助于增强表面等离子共振，同时增加了比表面积，有助于吸附待检测的代谢物小分子；最后该纳米复合材料在355nm波长处有很强的吸收，与基质辅助激光解吸电离质谱的光源波长相匹配，因此通过简单的预处理，简化了样本处理步骤，提高了LDI效率，克服了传统GC/LC MS需要复杂样本预处理的过程，也克服了传统LDI MS基质的缺陷，实现了小分子代谢物和血清代谢物的快速、灵敏和高通量检测。
本发明的多角星形状Au@ZnO纳米复合材料制备方法简单，形貌均一，该纳米粒子作为LDI MS的基质，可以解决传统基质存在的问题，如低分子量区的背景干扰。本发明中血清样本只需1.5μL血清提取液，就可高效、快速地检测血清中的小分子代谢物。这种检测方法灵敏度高、成本低、检测通量高，满足了临床血清检测的需求，有应用于临床的潜力。
表征所用仪器：
利用AuCy UV1900分光光度计和马尔文Zetasizer NanZS90获得了纳米材料的消光光谱和表面电荷值。
采用Hitachi SU8100获取扫描电子显微镜图片，用JEOL JEM-2100F获得透射电子显微镜图片、线扫能量色散X光谱和选取电子衍射图谱。
表征结果为：
从图14a和图14b可以看出纳米ZnO和纳米Au@ZnO都是多角星形结构，Au@ZnO(图14b)明显角更多，形貌也更均一，从线扫分析(图14c，从上至下依次为Zn、O和Au)和元素分布分析(图14d)来看，得到的Au@ZnO包含Zn、O和Au三种元素，证明了纳米金对氧化锌的形貌影响，由于金属种子粒径较小(30nm)，形成了像花瓣一样的多角星形结构。图14e所示的能量色散x射线光谱进一步记录了 金属含量从0％增加到1.51％(按重量计算)，表明结果与其他表征一致。紫外-可见光谱分析显示Au@ZnO在355nm处的吸收最高，与质谱的Nd:YAG激光器的波长一致，有利于质谱检测(图14f)。进一步表征了p-ZnO复合材料的晶体结构，与锌铁矿的[1,0,3]、[1,0,2]和[0,0,2]的晶面相关(图14g)。综上所述，等离子体ZnO复合材料具有独特的结构和成分，有LDI MS检测的潜力。
本实施例的多角星形状Au@ZnO纳米复合材料的制备方法包括如下步骤：
(1)、纳米金溶液的制备：将10.45mg水合四氯金酸溶解在100mL烧瓶的去离子水中，剧烈搅拌下(800rpm)加热至120℃后，加入2.0mL、1wt％二水合柠檬酸钠，保持120℃搅拌30min，冷却至室温备用。
(2)、ZnO的制备：将醋酸锌溶液(39mL、0.01mol/L)加入氢氧化钠溶液(65mL、0.03mol/L)中，加热至60℃且保温1h，然后以10000rpm下离心10min，收集沉淀，弃去上清液，再用乙醇和水分别洗涤三次，得到的沉淀在50℃烘箱中干燥备用。
(3)、多角星形状Au@ZnO纳米复合材料的制备：醋酸锌溶液(39mL、0.01mol/L)和39mL纳米金溶液混合搅拌2min(600rpm)，得到混合液，在混合液内加入氢氧化钠溶液(65mL、0.03mol/L)，继续搅拌并加热至60℃且保温1h，然后以10000rpm下离心10min，收集沉淀，弃去上清液，再用乙醇和水分别洗涤三次，得到的沉淀在50℃烘箱中干燥备用。
将实施例得到的多角星形状Au@ZnO纳米复合材料进行如下实验。
LDI MS检测：
小分子代谢物的检测：将标准品小分子(脯氨酸、赖氨酸、蔗糖和葡萄糖)用超纯水配成1mg/mL的溶液。
耐盐检测样本是在四种标准品小分子(脯氨酸、赖氨酸、精氨酸和葡萄糖)的混和溶液中加入氯化钠，最终氯化钠以及小分子的终浓度分别是0.2mg/mL和1mg/mL。对于耐蛋白检测，检测样本则是在白蛋白与标准品小分子(脯氨酸、赖氨酸、精氨酸和葡萄糖)的混合物，白蛋白和小分子的终浓度都是1mg/mL，从而探索不同基质在高盐浓度和蛋白质中的检测效果。
在一个典型的LDI MS实验中，将Au@ZnO纳米复合材料以1mg/mL的浓度分散在水中作为基质。对于Au纳米颗粒和ZnO纳米颗粒作为基质的情况下，同样以1mg/mL的浓度分散在水中。将1.5μL的分析物溶液(标准小分子溶液或标准血清)滴在抛光型的靶板上，室温干燥后覆盖上1.5μL的基质悬浮液，干燥后进行 LDI质谱分析。质谱检测采用AutoFlex TOF/TOF质谱仪(Bruker，德国)，装有Nd:YAG激光器(2kHz，355nm)。采集在正反射离子模式下进行，采用延迟提取，重复频率为1000Hz，加速电压为20千伏。本实验的延迟时间优化为250ns。在所有LDI MS实验中，每次分析的激光射击数为2000次。
小分子物质的质谱检测及耐盐耐蛋白测试：
(1)、仪器与试剂的准备：基质辅助激光解吸电离质谱，采用阳离子反射模式检测；将Au@ZnO纳米复合材料配制成悬浮液，并配制单一的标准品小分子(葡萄糖、蔗糖、脯氨酸、赖氨酸)溶液以及混和标准品(脯氨酸、赖氨酸、精氨酸和葡萄糖)溶液和高浓度盐和蛋白的混合溶液；
(2)、在质谱靶板上进行样本制备，室温下干燥；
(3)、在质谱仪下进行检测，并对质谱图像进行分析，如图15所示，本实施例的多角星形状Au@ZnO纳米复合材料，即p-ZnO的四种典型小分子代谢物(葡萄糖、蔗糖、脯氨酸和赖氨酸)的检测效果比纯的氧化锌(ZnO)纳米颗粒效果更好，尤其是蔗糖，质谱信号相差17倍多，因此可以说多角星形状Au@ZnO纳米复合材料(p-ZnO)比氧化锌更适用于LDI质谱检测。
在耐盐耐蛋白实验中，如图16a和16b所示，p-ZnO在高盐和高蛋白浓度下仍能很好地检测这四种代谢物的混合物。
血清代谢物的质谱检测：
(1)、仪器与试剂的准备：基质辅助激光解吸电离质谱，采用阳离子反射模式检测；将Au@ZnO纳米复合材料配制成悬浮液检测标准血清；
(2)、在质谱靶板上进行样本制备，室温下干燥；
(3)、在质谱仪下进行检测，并对质谱图像进行分析，生理条件下的血浆含有各种无机盐和蛋白质，因此要考虑基质在质谱检测中的耐盐耐蛋白性质，如图16所示，图16a是含有0.2mg/mL氯化钠的四种代谢物混合溶液(脯氨酸、赖氨酸、精氨酸和葡萄糖均为1mg/mL)的质谱检测结果，由图可知，本实施例的多角星形状Au@ZnO纳米复合材料能在高盐浓度条件下将四种代谢物全部检测出来，与图16c中CHCA作为基质只检测出了精氨酸的加氢峰，因此四种代谢物混合溶液(脯氨酸、赖氨酸、精氨酸和葡萄糖均为1mg/mL)具有较好耐盐性。图16b则是加入了1mg/mL的牛血清白蛋白的四种代谢物混合溶液(脯氨酸、赖氨酸、精氨酸和葡萄糖均为1mg/mL)，由图16b可知，四种代谢物也能被本实施例中的多角星形状Au@ZnO 纳米基质检测出来，图16d中CHCA也只检测出了精氨酸的加氢峰，因此多角星形状Au@ZnO纳米复合材料具有较好的耐蛋白性质。
由此可知，CHCA对于高盐和高蛋白混合溶液中的代谢物检测效果较差，只检测出了精氨酸的加氢峰，而本发明的多角星形状Au@ZnO纳米复合材料能将四种氨基酸都检测出来，证明本发明的多角星形状Au@ZnO纳米复合材料与传统的有机基质CHCA相比更适用于复杂生物样本的检测，背景干扰更低。
(四)提供的多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用
实施例1多孔合金纳米材料的制备
步骤1：仪器与试剂的准备：基质辅助激光解吸电离质谱，采用正离子反射模式；
步骤2：多孔PdPtAu合金的制备，包括以下步骤；
步骤2.1：将0.6mL Na2PdCl4(20mM)，3mL H2PtCl6·6H2O(20mM)，60μL盐酸(6.0M)和60mg F127充分混合，超声溶解，待F127完全溶解后，加入3mL抗坏血酸溶液(0.1M)，立刻置于45℃水浴超声反应3h，分别加入1.2mL HAuCl4·4H2O溶液(浓度分别为10mM、20mM、30mM、40mM)，反应1h。反应结束后，分别用无水乙醇和水离心(10000rpm)洗涤3次，50干℃燥备用。
步骤2.2：将上述多孔PdPtAu合金分散在去离子水中，作为基质使用；
步骤3：配比(葡萄糖、苯丙氨酸、赖氨酸)溶解在去离子水中，检测不同孔径和金含量的PdPtAu质谱性能；
步骤4：将血浆与等体积甲醇/乙腈混合液(甲醇/乙腈，v/v＝1:1)混合，摇床震荡10min，离心10min，取上清液用于质谱检测；
步骤5：在质谱靶板上进行样品制备，基质采用优化的PdPtAu材料，室温下干燥；
步骤6：对血浆样品中的小分子进行检测；
步骤7：对质谱检测结果进行分析，得出结论。
采用Hitachi S-4800获取扫描电子显微镜与能量色散X射线光谱结果，如图17所示，合成的多孔PdPtAu合金，粒径约为170nm，粒径均匀，存在多孔结构。
采用JEOL JEM-2100F获得透射电子显微镜结果。如图18所示，纳米材料存在多孔结构，与扫描电镜结果一致。
实施例2标准小分子物质检测
(1)仪器与试剂的准备：基质辅助激光解吸电离质谱，采用阳离子反射模式检测；将多孔合金配制成悬浮液，并配制标准品小分子(葡萄糖、苯丙氨酸、赖氨酸)溶液；
(2)在质谱靶板上进行样本制备，室温下干燥；
(3)在质谱仪下进行检测，并对质谱图像进行分析，结果如图19所示，图19a、19b、19c分别是葡萄糖、苯丙氨酸、赖氨酸标准小分子溶液。
实施例3含有NaCl和牛血清白蛋白的小分子物质检测
(1)仪器与试剂的准备：基质辅助激光解吸电离质谱，采用阳离子反射模式检测；将多孔合金配制成悬浮液，并配制高浓度盐或蛋白的混合小分子(葡萄糖、苯丙氨酸、赖氨酸)溶液；
(2)在质谱靶板上进行样本制备，室温下干燥；
(3)在质谱仪下进行检测，并对质谱图像进行分析，结果如图20所示，图20a是含有NaCl的溶液中检测葡萄糖、苯丙氨酸和赖氨酸的质谱图，图20b牛血清白蛋白的溶液中检测葡萄糖、苯丙氨酸和赖氨酸的质谱图。
实施例4血浆代谢物的检测
(1)仪器与试剂的准备：基质辅助激光解吸电离质谱，采用阳离子反射模式检测；将多孔合金配制成悬浮液；对血浆用有机溶剂进行简单的预处理；
(2)在质谱靶板上进行样本制备，室温下干燥；
(3)在质谱仪下进行检测，并对质谱图像进行分析，如图21所示，图21a，21b分别为健康血浆样本和癌症血浆样本的质谱图。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。

Claims (10)

  1. 一种金属有机框架材料的制备方法，其特征在于，所述方法包括以下步骤：步骤1、将二甲基甲酰胺，乙醇，去离子水，三乙胺，对苯二甲酸混合加入一密闭容器中，并加入金属氯化物形成混合物；步骤2、将所述混合物进行水浴超声反应，得到反应物；步骤3、将所述反应物进行离心并洗涤，除去残留的所述二甲基甲酰胺，得到产物；步骤4、将步骤3中所述产物分散在去离子水中，采用低速离心分离出超薄金属有机框架材料。
  2. 如权利要求1所述金属有机框架材料在小分子检测中的应用。
  3. 根据权利要求2所述的金属有机框架材料在小分子检测中的应用，其特征在于，包括以下步骤：步骤1、将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；步骤2、对血清样品进行比例稀释；步骤3、在质谱靶板上进行样品制备，所述超薄金属有机框架材料作为基质，室温下干燥；步骤4、对所述血清样品中的小分子进行检测；步骤5、分析原始质谱图谱，并获得检测结果。
  4. 一种亚微反应器的制备方法，其特征在于，包括如下步骤：步骤2.1：将3-氨基苯酚APF溶解于去离子水中，并加入甲醛溶液和氨水溶液；步骤2.2：将步骤2.1的混合物在30℃条件下，反应30分钟；步骤2.3：将步骤2.2中的反应物进行离心洗涤，得到APF亚微材料。
  5. 权利要求4所述亚微反应器的血清代谢物检测方法，其特征在于，包括如下步骤：(1)仪器与试剂的准备：将基质辅助激光解吸电离质谱检测模式设置为正离子反射模式；(2)将得到的APF-sphere&Au、APF-bowl&Au-1、APF-bowl&Au-2以及APF-bowl&Au-3分散在去离子水中，作为基质使用(3)对血清样品进行比例稀释；(4)在质谱靶板上进行样品制备，采用(2)中的所述基质，室温下干燥；(5)对血清样品中的小分子进行检测；(6)分析原始质谱图谱，并获得检测结果。
  6. 一种多角星形状Au@ZnO纳米复合材料的制备方法，其特征在于：其包括如下步骤：(1)、纳米金溶液的制备：水合四氯金酸溶液搅拌加热至120±1℃后，加入二水合柠檬酸钠，恒温下搅拌30±0.1min，冷却至室温备用；(2)、氧化锌的制备：将醋酸锌溶液加入氢氧化钠溶液中，加热至60±1℃且保温1±0.1h，离心收集沉淀，洗涤，在50±1℃下干燥备用；(3)、多角星形状Au@ZnO纳米复合材料的制备：醋酸锌溶液和所述纳米金溶液混合，得到混合液，在所述混合液内加入 氢氧化钠溶液，加热至60±1℃且保温1±0.1h，离心收集沉淀，洗涤，在50±1℃下干燥备用。
  7. 一种多角星形状Au@ZnO纳米复合材料，其特征在于：其由权利要求6所述的制备方法得到。
  8. 权利要求7所述多角星形状Au@ZnO纳米复合材料作为基质材料在基质辅助激光解吸电离质谱检测、小分子代谢物的基质辅助激光解吸电离质谱检测或血清代谢物的基质辅助激光解吸电离质谱检测中的应用。
  9. 一种多孔合金纳米材料的制备方法，其特征在于，所述方法包括以下步骤：步骤1、将Na2PdCl4、H2PtCl6·6H2O、盐酸和F127充分混合，超声溶解；步骤2、待完全溶解后，加入抗坏血酸溶液，并立刻置于水浴超声反应；步骤3、随后加入HAuCl4·4H2O溶液反应；步骤4、最后分别用无水乙醇和水离心洗涤后干燥得到所述多孔合金纳米材料PdPtAu。
  10. 一种如权利要求9所述多孔合金纳米材料在检测血浆代谢物中的应用，其特征在于，包括以下步骤：步骤1、将所述多孔合金纳米材料PdPtAu分散在去离子水中；步骤2、将血浆与等体积甲醇/乙腈混合液混合，所述甲醇/乙腈体积比为1:1，摇床震荡10min，离心10min，取上清液用于质谱检测；步骤3、所述基质辅助激光解吸电离质谱采用正离子反射模式，在所述基质辅助激光解吸电离质谱的质谱靶板上进行样品制备，所述样品为1μL血浆提取液，对血浆样品中的小分子进行检测。
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