CN117686476A - Mixed bimetal plasma array, preparation method, plasma array assisted laser desorption ionization mass spectrum detection method and application - Google Patents

Mixed bimetal plasma array, preparation method, plasma array assisted laser desorption ionization mass spectrum detection method and application Download PDF

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CN117686476A
CN117686476A CN202311708780.0A CN202311708780A CN117686476A CN 117686476 A CN117686476 A CN 117686476A CN 202311708780 A CN202311708780 A CN 202311708780A CN 117686476 A CN117686476 A CN 117686476A
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noble metal
plasma array
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metal nanoparticles
nanoparticles
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黄琳
李雪联
陈伟
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Shanghai Chest Hospital
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Shanghai Chest Hospital
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Abstract

The invention relates to a quantitative detection technology, in particular to a mixed bimetal plasma array, a preparation method and a detection method and application of plasma array assisted laser desorption ionization mass spectrometry, wherein the plasma array is obtained by self-assembling positively charged noble metal nano particles and negatively charged noble metal nano particles, and the positively charged noble metal nano particles and the negatively charged noble metal nano particles are nano particles of different noble metals; or, at least the positively charged noble metal nanoparticles are double noble metal nanoparticles; or, at least the negatively charged noble metal nanoparticles are double noble metal nanoparticles. Compared with the prior art, the method solves the problems that in the prior art, the accurate quantitative detection of the nucleoside substances is difficult to carry out by a chromatography method and an enzyme-linked immunosorbent assay method, and detection inaccuracy is caused by strong background noise of MALDI MS in a low-quality area, realizes rapid and selective identification of specific molecules, and obtains the signal intensity of the specific molecules by a fingerprint.

Description

Mixed bimetal plasma array, preparation method, plasma array assisted laser desorption ionization mass spectrum detection method and application
Technical Field
The invention relates to a quantitative detection technology, in particular to a mixed bimetal plasma array and a preparation method thereof, and a detection method and application of plasma array assisted laser desorption ionization mass spectrometry.
Background
The metabolites are located at the end of the biochemical pathway, providing a functional fingerprint of the physiological and pathophysiological state of the body. Disease-related processes, such as tumor progression, drug resistance, immune response and maintenance of pluripotency, are regulated by coordinated changes in metabolic enzymes and transporters and their corresponding metabolites. Therefore, the detection of the metabolic molecules has important significance in the aspects of disease diagnosis, treatment and the like.
Preferential, quantitative detection of conventional metabolites relies primarily on gas/liquid chromatography and mass spectrometry (GC/LC-MS). Both of these methods require cumbersome sample pretreatment and additional chromatographic run times (40 minutes) to process the clinical sample to address the composition complexity of the clinical sample and trace abundance of the target metabolite. It remains a significant challenge to establish an accurate and rapid quantitative analysis method to monitor metabolites in a clinically useful biological sample.
Matrix-assisted laser desorption/ionization MS (MALDI MS) is capable of rapid, sensitive and high throughput detection of metabolite molecules with the aid of a matrix, without or with only simple sample pretreatment for small amounts of sample (-nL). However, conventional organic matrices such as α -cyano-4-hydroxycinnamic acid (CHCA) and 2, 5-dihydroxybenzoic acid (DHB) present strong background noise in the low mass region (< m/z 700) due to self-dissociation, which hinders their use in metabolite detection.
In summary, currently quantitative detection of metabolites mainly relies on chromatography and enzyme-linked immunosorbent assay: 1) For the chromatography, the complex sample needs to be pretreated, and the problems of long detection time, high price and the like exist, so that the low-cost and high-flux detection of the actual complex sample is difficult to realize, and the method is difficult to be used for clinic. 2) For the ELISA, the detection time is long, the antibody is expensive, and the high-throughput and low-cost detection of the clinical complex sample is difficult to realize. In addition, the nucleoside substance has the problems of short half-life (for example, the half-life of adenosine is extremely short to a few seconds) and the like, and cannot be accurately and quantitatively detected by means of conventional chromatography and enzyme-linked immunosorbent assay. Therefore, there is a need to propose a method for preferential, quantitative detection of nucleoside substances.
Disclosure of Invention
The invention aims to solve at least one of the problems, and provides a mixed bimetal plasma array and a preparation method thereof, and a detection method and application of plasma array assisted laser desorption ionization mass spectrometry, so as to solve the problems that in the prior art, the chromatography and the enzyme-linked immunosorbent assay are difficult to accurately and quantitatively detect nucleoside substances, and the MALDI MS has strong background noise in a low-quality area to cause inaccurate detection, realize rapid and selective identification of specific molecules, and can acquire the signal intensity of the specific molecules through fingerprint, and the method has the advantages of simplicity in operation, high sensitivity, low cost and high detection flux, and has the potential of being applied to complex clinical samples.
The aim of the invention is achieved by the following technical scheme:
the first aspect of the invention discloses a mixed bimetallic plasma array, which is obtained by self-assembling positively charged noble metal nanoparticles and negatively charged noble metal nanoparticles, wherein,
the positively charged noble metal nanoparticles and the negatively charged noble metal nanoparticles are nanoparticles of different noble metals; or alternatively, the first and second heat exchangers may be,
at least the positively charged noble metal nanoparticles are double noble metal nanoparticles; or alternatively, the first and second heat exchangers may be,
at least the negatively charged noble metal nanoparticles are double noble metal nanoparticles.
Preferably, the noble metal nanoparticles include Ag nanoparticles, pt nanoparticles, au nanoparticles, and Pt/Au nanoparticles.
The second aspect of the invention discloses a method for preparing the mixed bimetal plasma array, which comprises the following steps:
s1: preparation of positively charged noble metal nanoparticles:
stirring a mixed metal source and a first end capping agent at room temperature, adding a reducing agent into the mixed solution, and standing away from light to obtain a suspension of positively charged noble metal nanoparticles;
s2: preparation of negatively charged noble metal nanoparticles:
heating and stirring the metal source to boil, adding a second end-capping agent into the boiled metal source and keeping the boiling state, and then continuously stirring in the natural cooling process to obtain a suspension of negatively charged noble metal nano particles;
s3: preparation of a plasma array:
dispersing the negatively charged noble metal nano particles prepared in the step S2 in oil, adding the positively charged aqueous suspension of the noble metal nano particles prepared in the step S1, vibrating, and self-assembling at a water-oil two-phase or oil-water-oil three-phase interface to form the mixed bimetal plasma array.
Preferably, the metal source comprises HAuCl 4 And H 2 PtCl 6 One or two of the following components; the first end capping agent comprises a cysteine solution; the second end capping agent comprises sodium citrate; the oil liquid comprises 1, 2-dichloroethane and n-hexane.
Preferably, in step S1, the stirring time is 20min, and the light-shielding standing time is 10min; in the step S2, the time for keeping the boiling state is 10min, and the time for continuing stirring in the natural cooling process is 15min; in step S3, the time of the oscillation is 30S.
Preferably, the volume ratio of the oil, negatively charged noble metal nanoparticles, and positively charged noble metal nanoparticles is 500:400:20.
the third aspect of the invention discloses a detection method of plasma array assisted laser desorption ionization mass spectrometry, which adopts the mixed bimetal plasma array as a matrix, or adopts the mixed bimetal plasma array prepared by any one of the methods as the matrix;
the detection method comprises the following steps:
s4: sample pretreatment:
adding the reaction sample into an extracting agent, vortex oscillating, centrifuging after freezing treatment, and taking supernatant;
s5: sample preparation:
preheating a target plate, transferring the plasma array to the target plate and drying, and dripping the sample processed in the step S4 on the plasma array and drying;
s6: sample detection:
in the matrix-assisted laser desorption ionization mass spectrometry, the sample prepared in the step S5 is subjected to mass spectrometry detection by adopting a positive ion reflection mode.
Preferably, the reaction sample comprises a blood sample, a biological sample, a food sample and an environmental sample; the extractant comprises the following components in percentage by volume: 2:1 methanol, acetonitrile and water.
Preferably, in step S4, the vortex shaking time is 30S, the freezing treatment is freezing for 2 hours at-20 ℃, and the centrifugation is centrifugation for 10 minutes at 12000rpm at 4 ℃; in the step S5, the preheating temperature is 0-100 ℃.
The fourth aspect of the invention discloses an application of the detection method of the plasma array assisted laser desorption ionization mass spectrum in quantitatively detecting nucleoside substances.
Noble metals are promising laser desorption/ionization mass spectrometry (ldims) matrix materials exhibiting superior surface plasmon effects and higher hot carrier effects, thereby improving desorption and ionization of analytes. According to the invention, the mixed double noble metal nano particles are self-assembled through a liquid-liquid interface or an oil-water-oil three-phase interface, so that a novel uniformly and densely arranged plasma array is prepared; the plasma array can preferentially detect specific metabolites (e.g., nucleosides) by enhancing the binding affinity between chemical groups (e.g., basic nitrogen heterocycles (base ring nitrogen), exocyclic ketone groups (exocyclic keto groups), imidazole groups, etc.) and noble metals.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a novel plasma array formed by self-assembly of mixed bimetal nano materials, which can be used as a matrix material of matrix-assisted laser desorption ionization mass spectrum and is used for quantitative detection of nucleoside substances.
The invention aims to utilize self-assembled plasma arrays to assist LDI MS to detect nucleoside substances preferentially and quantitatively. The mixed bimetal plasma array is combined with mass spectrum, and has extremely strong affinity to nucleoside substances.
The detection method can realize the rapid (several seconds) and accurate (R) of inosine and adenosine by only consuming 4 mu L of natural biological fluid 2 >0.99 Quantification).
Compared with the existing method, the method can rapidly and selectively identify the specific molecules and can acquire the signal intensity of the specific molecules through the fingerprint; the method has the advantages of simple operation, high sensitivity, low cost, high detection flux and potential of being applied to complex clinical samples.
Drawings
FIG. 1 is a schematic diagram of an experimental workflow for preferential metabolite detection in laser desorption/ionization mass spectrometry (LDI-MS) using a plasma array as a matrix;
FIG. 2 shows the results of detection of inosine and adenosine in clinical samples, wherein (a), (b) and (c) are respectively the standard curves of inosine detection by LDI MS, the results of detection of 23 samples by LDI-MS, and the consistency comparison of the results of detection of LDI-MS and ELSA kit; (d) (e) and (f) are respectively the standard curve of adenosine detected by LDI MS, 19 samples of LDI-MS detection results, and consistency comparison of LDI-MS and ELSA kit detection results;
FIG. 3 is a graph showing the characteristics of the plasma array (A2) obtained in example 1, the plasma array (A1) obtained in comparative example 1, and the plasma array (A3) obtained in comparative example 2,
(a) I: SEM image of A1, ii: SEM image of A2, iii: an SEM image of A3 was taken of the sample,
(b) I: when A1 is used as a matrix, inosine (1 mg mL) -1 ) Aspartic acid (1 mg mL) -1 ) Arginine (1 mg mL) -1 ) Mass spectrum intensity plot of ii):when A2 was used as a substrate, inosine (1 mg mL) -1 ) Aspartic acid (1 mg mL) -1 ) Arginine (1 mg mL) -1 ) Mass spectrum intensity profile of iii): when A3 is used as a matrix, inosine (1 mg mL) -1 ) Aspartic acid (1 mg mL) -1 ) Arginine (1 mg mL) -1 ) Is a mass spectrum intensity map of (2);
FIG. 4 shows the tendency of A2 as a matrix for detecting 37 small molecules by laser desorption/ionization mass spectrometry in application example 1;
fig. 5 is a mass spectrum of a plasma sample detected by A2-matrix assisted laser desorption/ionization mass spectrometry in application example 1.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
In the examples below, the reagents used may be conventional commercial products, using methods well known in the art, unless specifically indicated.
Example 1
Step 1: preparation of instruments and reagents: matrix-assisted laser desorption ionization mass spectrometry adopts a positive ion reflection mode;
step 2: the preparation of the plasma array material comprises the following steps of;
step 2.1: pt/Au NPs (-) with negative surface charge were synthesized using sodium citrate as a capping agent.
In detail, HAuCl 4 And H 2 PtCl 6 The (1/2, v/v) mixed aqueous solution (50 mL,1 mM) was heated to boiling with vigorous stirring. Then, sodium citrate solution (5 ml,38.8 mm) was added rapidly to the mixture. After boiling for 10 minutes, the mixture solution is stirred for 15 minutes under the condition of no heating, and then Pt/Au NPs (-) can be obtained.
Step 2.2: by NaBH 4 As a reducing agent, cysteamine solution is used as a blocking agent to prepare Au NPs (+) with positive charges.
In detail, will 40mL 1.42mM HAuCl 4 Mix with 400. Mu.L 213mM cysteamine solution and stir at room temperature for 20 minutes. Subsequently, 10. Mu.L of 10mM NaBH was added 4 Adding the solution into the mixture, and standing the solution in dark placeAnd obtaining Au NPs (+)' after 10 min.
Step 2.3: the plasma array is prepared by adopting an improved water-oil interface self-assembly method, and through simple electrostatic interaction, self-assembling noble metal nano particles with opposite charges on a liquid-liquid interface.
First, 400. Mu.L of negatively charged metal NPs (Pt/Au NPs (-)) were dispersed in 500. Mu.L of 1, 2-dichloroethane. Then, 20. Mu.L of positively charged metal NP (Au NPs (+) aqueous suspension) was added thereto. The mixture was vigorously shaken for 30 seconds to form a bright nanofilm (i.e., a plasma array) at the two-phase interface.
Step 2.4: the above-mentioned plasma array, denoted as A2, was used as a substrate;
step 3: sample pretreatment: adding 150 μl of extracting agent (methanol: acetonitrile: water=2:2:1 (v: v)) into 50 μl of plasma, vortex shaking for 30s, placing in a refrigerator at-20deg.C for 2 hr, centrifuging (12000 rpm,10min, 4deg.C), and collecting supernatant;
step 4: sample preparation is performed on a target plate, comprising the steps of:
step 4.1: heating the target plate and controlling the temperature to 30 ℃;
step 4.2: taking the plasma subarray prepared in the step 2 as a matrix, transferring the matrix onto a target plate, and drying for later use;
step 4.3: taking the sample pretreated in the step 3, dripping the sample on a plasma array, and drying the sample for later use;
step 5: mass spectrum detection is carried out on the sample points dripped on the target plate;
step 6: and analyzing the mass spectrum detection result to obtain a conclusion.
Comparative example 1
This comparative example is substantially identical to the protocol of example 1, except that an Au plasma array, designated A1, was prepared and synthesized from Au NPs (+) and Au NP (-).
Comparative example 2
This comparative example is essentially identical to the protocol of example 1, except that a Pt plasma array, designated A3, was prepared, synthesized from Pt NPs (+) and Pt NPs (-).
Characterization results:
from the scanning electron microscope results (fig. 3 (a)), the A2 array was more uniformly and tightly arranged than the A1 and A3 arrays, and showed the highest intensity among the 3 indicator molecules of inosine, aspartic acid, and arginine (fig. 3 (b)).
Fig. 1 shows a schematic diagram of an experimental workflow of preferential metabolite detection using a plasma array as a matrix in laser desorption/ionization mass spectrometry (ldims), wherein fig. 1 (a) is a schematic diagram of a preparation process of self-assembly of the matrix (plasma array) through a liquid-liquid interface, fig. 1 (b) shows a schematic diagram of preferential detection of specific metabolites (such as nucleosides) by the plasma array, and fig. 1 (c) shows a schematic diagram of accurate quantification of nucleoside substances in a trace serum sample by the plasma array assisted ldims.
In fig. 2, the detection results of ldims (using plasma array A2 as a substrate) are taken as examples of inosine and adenosine, and it can be seen that the detection results of the method are basically consistent with the detection results of a commercial kit (ELISA kit), and the comparison of the detection results of the two results shows that the consistency is more than 98%, which indicates that the method has excellent accuracy.
Application example 1: affinity detection of 37 Small molecules
Step 1: preparation of instruments and reagents: matrix-assisted laser desorption ionization mass spectrometry, which adopts a cation reflection mode for detection;
step 2: preparation of plasma array materials: in accordance with the protocol in example 1, A2;
step 3: configuration of 37 small molecule solutions: at 1mg mL -1 Sodium chloride solution of (2) is used as solvent, and 1mM solution is prepared respectively;
among them, 37 kinds of small molecules are mainly classified into 6 major categories: including nucleosides (inosine, guanosine, adenosine, cytidine, thymidine), pyrimidines (cytosine, thymine), amino acids (aspartic acid, glutamic acid, histidine, arginine, lysine, asparagine, glutamine, serine, alanine, glycine, cysteine, methionine, tyrosine, isoleucine, phenylalanine, proline, valine, leucine), sugars (raffinose, glucose, cellobiose), hormones (estradiol, androstenedione, testosterone, estrone), other classes (vitamin B1, nicotinamide, lactic acid, ascorbic acid, cholesterol);
step 4: the mass spectrum signal intensity of 37 small molecules (1 mM) was detected under a mass spectrometer using A2 as a matrix, and data analysis was performed, and the results are shown in FIG. 4. Shows a stronger tendency to nucleoside.
Application example 2: detection of authentic plasma samples
Step 1: preparation of instruments and reagents: matrix-assisted laser desorption ionization mass spectrometry, which adopts a cation reflection mode for detection;
step 2: preparation of plasma array materials: in accordance with the protocol in example 1, A2;
step 3: pretreatment of clinical samples: taking 50. Mu.L of plasma sample, adding 150. Mu.L of extractant (methanol: acetonitrile: water=2:2:1, v/v/v), swirling for 30s, standing in a refrigerator at-20deg.C for 2h, centrifuging (12000 rpm,15min, 4deg.C), and collecting supernatant for use;
step 4: taking A2 as a matrix, transferring to a target plate, drying, dripping 2 mu L of the sample in the step 3, drying at room temperature, detecting a clinical plasma sample under a mass spectrometer, and observing fragment peaks of guanosine, adenosine and inosine when m/z is 174.01, 180.11 and 181.01 respectively when the mass spectrometer is shown in a result of a mass spectrometer shown in FIG. 5.
In other embodiments, the matrix material is not limited to Pt/Au alloy materials, but extends to matrix materials that are commonly used with laser desorption ionization mass spectrometry, such as Au/Ag alloy materials, pt/Ag alloy materials, and the like. And the matrix material can be prepared from a combination of different noble metal nanoparticles, such as: the Pt/Au alloy material can also be prepared from Au NPs (+) and Pt NPs (-), pt/Au NPs (+) and Au NPs (-), and the like; the Au/Ag alloy material can be prepared from Au NPs (+) and Ag NPs (-), au/Ag NPs (+) and Ag NPs (-), and the like; the main difference from example 1 is that the ratio of the configuration is different, and the optimal ratio or the preferable ratio range is selected according to experimental optimization.
In other embodiments, the reaction system sample may be extended to other biological samples (e.g., urine, sweat, saliva, pericardial fluid, etc.), food samples (e.g., milk), environmental samples (waste, river water), etc.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims (10)

1. A mixed bimetal plasma array is characterized by being obtained by self-assembling positively charged noble metal nano particles and negatively charged noble metal nano particles,
the positively charged noble metal nanoparticles and the negatively charged noble metal nanoparticles are nanoparticles of different noble metals; or alternatively, the first and second heat exchangers may be,
at least the positively charged noble metal nanoparticles are double noble metal nanoparticles; or alternatively, the first and second heat exchangers may be,
at least the negatively charged noble metal nanoparticles are double noble metal nanoparticles.
2. The hybrid bimetallic plasmonic array of claim 1, wherein the noble metal nanoparticles include Ag nanoparticles, pt nanoparticles, au/Ag nanoparticles, and Pt/Au nanoparticles.
3. A method of making a hybrid bimetallic plasma array as claimed in claim 1 or 2, comprising the steps of:
s1: preparation of positively charged noble metal nanoparticles:
stirring a mixed metal source and a first end capping agent at room temperature, adding a reducing agent into the mixed solution, and standing away from light to obtain a suspension of positively charged noble metal nanoparticles;
s2: preparation of negatively charged noble metal nanoparticles:
heating and stirring the metal source to boil, adding a second end-capping agent into the boiled metal source and keeping the boiling state, and then continuously stirring in the natural cooling process to obtain a suspension of negatively charged noble metal nano particles;
s3: preparation of a plasma array:
dispersing the suspension of the noble metal nano particles with negative charges prepared in the step S2 in oil, adding the suspension of the noble metal nano particles with positive charges prepared in the step S1, vibrating, and self-assembling at a water-oil two-phase interface or an oil-water-oil three-phase interface to form the mixed bimetal plasma array.
4. The method of claim 3, wherein the metal source comprises HAuCl 4 And H 2 PtCl 6 One or two of the following components; the first end capping agent comprises a cysteine solution; the second end capping agent comprises sodium citrate; the oil liquid comprises 1, 2-dichloroethane and n-hexane.
5. The method for preparing a mixed bimetal plasma array according to claim 3, wherein in the step S1, the stirring time is 20min, and the light-shielding standing time is 10min; in the step S2, the time for keeping the boiling state is 10min, and the time for continuing stirring in the natural cooling process is 15min; in step S3, the time of the oscillation is 30S.
6. A method of preparing a hybrid bimetallic plasma array as claimed in claim 3, wherein the volume ratio of oil, negatively charged precious metal nanoparticles to positively charged precious metal nanoparticles is 500:400:20.
7. a method for detecting plasma array assisted laser desorption ionization mass spectrometry, which is characterized in that the mixed bimetal plasma array according to claim 1 or 2 is adopted as a matrix, or the mixed bimetal plasma array prepared by the method according to any one of claims 3 to 6 is adopted as a matrix;
the detection method comprises the following steps:
s4: sample pretreatment:
adding the reaction sample into an extracting agent, vortex oscillating, centrifuging after freezing treatment, and taking supernatant;
s5: sample preparation:
preheating a target plate, transferring the plasma array to the target plate and drying, and dripping the sample processed in the step S4 on the plasma array and drying;
s6: sample detection:
in the matrix-assisted laser desorption ionization mass spectrometry, the sample prepared in the step S5 is subjected to mass spectrometry detection by adopting a positive ion reflection mode.
8. The method for detecting plasma-array assisted laser desorption ionization mass spectrometry of claim 7 wherein the reaction sample comprises a blood sample, a biological sample, a food sample and an environmental sample; the extractant comprises the following components in percentage by volume: 2:1 methanol, acetonitrile and water.
9. The method for detecting plasma array assisted laser desorption ionization mass spectrometry according to claim 7, wherein in the step S4, the vortex oscillation time is 30S, the freezing treatment is freezing for 2h at-20 ℃, and the centrifugation is centrifugation for 10min at 12000rpm at 4 ℃; in the step S5, the preheating temperature is 0-100 ℃.
10. Use of a detection method of a plasma array assisted laser desorption ionization mass spectrum according to any one of claims 7 to 9 for quantitative detection of nucleoside substances.
CN202311708780.0A 2023-12-13 2023-12-13 Mixed bimetal plasma array, preparation method, plasma array assisted laser desorption ionization mass spectrum detection method and application Pending CN117686476A (en)

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