CN116930502A - Serum amino acid rapid detection method based on paper-based electric sample processing device and mass spectrometry - Google Patents
Serum amino acid rapid detection method based on paper-based electric sample processing device and mass spectrometry Download PDFInfo
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 description 1
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- 238000002156 mixing Methods 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
Abstract
The invention discloses a rapid detection method of serum amino acid based on a paper-based electric sample processing device and mass spectrometry, which belongs to the technical field of serum amino acid detection, and aims of separating and purifying with other coexisting media are fulfilled based on the characteristic that a target component has an isoelectric point.
Description
Technical Field
The invention belongs to the technical field of serum amino acid detection, and particularly relates to a rapid detection method of 11 amino acids in serum based on a paper-based electric sample processing device and high-sensitivity mass spectrometry.
Background
Amino acids are the basic building blocks of proteins, whose metabolic levels are closely related to health. The amino acid levels in blood samples can be used for clinical diagnosis of a variety of diseases. For example, amino acid levels in neonatal heel blood can be diagnostic of a variety of genetic metabolic disorders. Rapid determination of amino acids in blood samples can be achieved using isotope labeling and high sensitivity Mass Spectrometry (MS). In order to achieve accurate detection, the isotopic dilution method for determining amino acids has very high requirements for sample processing, for example, the isotopic internal standard and the native measured components must be sufficiently balanced. The current commercial kit method is ensured by extracting the dried blood slices with a labeled extraction solution at a higher temperature (50 ℃) and under high mechanical strength conditions (700 rmp) and for a long time (50 min). The method without power-up in the commercialized kit method has low diffusion balance speed, and the movement speed of charged components under the drive of an electric field can be effectively enhanced. The amino acid can be aggregated on the basis of isoelectric point characteristics of the amino acid on the established pH gradient so as to achieve the purposes of enrichment and separation. Paper-based analysis device (Paper-based analytical devices, PAD) is a newer microfluidic technology, and can realize functions of on-site rapid and instant detection and sample processing based on cheap Paper-based materials and simple processing means.
Disclosure of Invention
The invention aims to provide a rapid detection method of 11 amino acids in serum based on a paper-based microfluidic electric sample processing device and high-sensitivity mass spectrum. An isoelectric focusing (Isoelectric focusing, IEF) sample electrokinetic treatment method without the aid of free carrier ampholytes and without the addition of inhibitors achieves separation and purification from other coexisting media based on the characteristic of the target component having an isoelectric point.
The invention aims to separate amino acid components in a sample from a complex matrix on a paper-based microfluidic device based on an electric method, and then dissolve the purified components with an organic solvent and detect the components with ESI MS. The invention provides a new strategy and more choices for developing a paper-based biomarker detection method, and meets the urgent need of a low-cost and high-efficiency sample pretreatment method in clinic.
The invention realizes the aim through the following technical scheme:
as shown in fig. 1, the amino acids and proteins form a purification band with a length of 2-10 mm on a dynamic double gradient (pH/electric field) established on a paper-based fluid channel (1-10 mm wide, 10-100 mm long) under the drive of a direct current voltage of 1-300V based on isoelectric point characteristics, thereby being separated from other medium components in a sample; the extraction liquid is used for dissolving the amino acid in the purification band on the paper-based channel and then directly carrying out ESI MS detection.
In the present invention, the anode end of the paper-based fluid channel is used with an acidic solution, the cathode end is used with an alkaline solution, and the alkaline solution and the acidic solution are respectively 10mM NH 3 ·H 2 O and HAc solutions, the volume of which is between 10 and 500. Mu.L, are loaded with a reservoir or directly onto the paper-based fluid channel.
In the invention, the serum sample is directly loaded in a liquid (1-30 mu L) mode; or the sample is loaded in a form of being preloaded to form a dry spot on the paper-based channel; or in the form of dry paper sample pieces having a diameter smaller than the width on the paper-based fluid channel, preferably a diameter of 1-8 mm.
The paper-based fluid channel in the invention is a hydrophilic glass fiber material.
The electrodes to which the voltage is applied in the present invention are applied to the paper-based fluid channels either through the reservoir or directly by contact with the paper-based fluid channels.
In the invention, the dry paper sample is moistened with water for 1-2 min before treatment.
The invention has the following beneficial effects:
the invention provides an isoelectric focusing sample electrokinetic treatment method based on a paper-based analysis device, which is free from carrier ampholyte and does not need to add inhibitors, and is used for measuring 11 amino acids in serum. The method achieves the aim of separating and purifying with other coexisting media based on the characteristic that the target component has an isoelectric point, and 11 amino acids in serum can be detected in a highly reproducible manner by means of an isotope internal standard method through the isoelectric focusing treatment of a carrier-free ampholyte of a paper-based analysis device by optimizing paper-based materials, operation steps and experimental conditions. Compared with the commercial kit method, the method has certain comparability, but has low price and high analysis speed compared with the commercial kit. By establishing a stable high pH gradient on the paper-based fluid channel, the amino acid component can be migrated from the sample medium and focused, thereby achieving the purpose of separating from the complex medium, providing favorable conditions for offline MS rapid detection of amino acid and protein biomarker molecules, and providing a premise for online MS detection.
The method has the characteristics of low cost, simple operation, high speed and the like, can be used for not only liquid samples, but also dry spots, and has wide application prospect in clinical disease screening and diagnosis.
Drawings
FIG. 1 is a schematic flow diagram of the operation.
FIG. 2 (a) shows a pH gradient profile and three protein stacking effect profiles with different isoelectric points: phycocyanin (blue, pI-4.5), bovine hemoglobin (brown, pI-6.8), cytochrome C (reddish brown, pI-10.0); class (b) separation schematic and actual effect diagram: the initial state (up), class separation of components on the dual gradient interface after electrokinetic treatment and focusing effect of target components (down) are respectively anionic probes (brilliant blue, blue), electrically neutral probes (rhodamine B, pink), ampholytic substance probes (bovine hemoglobin, brown), cationic probes (methyl green, blue green).
FIG. 3 (a) is a graph of the parallel packing effect of amino acids in serum after electrokinetic treatment based on PAD (ninhydrin solution specific chromogenic amino acids); (b) Typical results are based on mass spectrometric detection of amino acids in serum after PAD electrokinetic treatment.
Detailed Description
The following further describes the technical solution of the present invention by way of examples with reference to fig. 1 to 3, but the invention is not limited to the examples.
Examples
A rapid detection method of serum amino acid based on paper-based electric sample processing device and mass spectrometry comprises the following steps:
(1) Preparation of isotope internal standard, standard solution and standard-added sample
Isotopic internal standard solution: the standard dry powder of the stable isotope of amino acids (NeoBase) was reconstituted with 1.0mL of extract (from Perkinelmer or self-formulated methanol solution containing 0.01% formic acid V/V) according to Kit instructions, and the solution was thoroughly mixed until completely dissolved to give a standard stock solution of the isotope (concentration level was 0.5. Mu. Mol/mL except Gly) for the remaining 10 amino acids. Diluting the isotope internal standard stock solution by 1 time by using the extract to obtain isotope internal standard PAD working solution, and placing the working solution in a refrigerator (4 ℃) for standby. Amino acid standard solution: the 11 amino acid pure products are dissolved in methanol to prepare an amino acid standard stock solution with the concentration of 0.5 mu mol/mL (Gly 2.5 mu mol/mL) and the stock solution is placed in a refrigerator (4 ℃) for standby. And (3) preparing a marked sample: the 40. Mu.L of the isotope internal standard working solution and 40. Mu.L of the extract are added into 60. Mu.L of serum samples, and the mixture is placed in a refrigerator (4 ℃) for storage after vortex mixing.
(2) Sample loading
The loading mode of the labeled serum sample on the PAD is divided into three types: (1) directly loading the sample liquid on the PAD, and then carrying out electric treatment. Directly loading the labeled serum sample liquid onto a paper-based fluid channel (within the range of 5-10 mm from the edge of the anode liquid storage tank), and immediately performing electric treatment; (2) And loading the sample liquid onto a protein card sample sheet, drying, and then performing electric treatment. Whatman 903 TM Preparing a sample wafer with the diameter of 6mm by using a puncher, loading a labeled serum sample liquid on the sample wafer, and drying for 48 hours at the temperature of 4 ℃; (3) And loading the sample liquid on the PAD channel, drying and then carrying out electric treatment. The labeled serum sample solution is preloaded on a paper-based fluid channel (within 5-10 mm from the edge of the anode liquid storage tank), and is dried for 48 hours at 4 ℃. Each test sample contained 3. Mu.L of serum stock in each of the above three loading modes.
(3) Sample processing
PAD laboratory treatment: the glass fiber filter was cut into 3X 35mm paper-based fluid channels, and the samples were loaded onto the paper-based fluid channels. For sample loading or PAD preloading of dry spots, 5. Mu.L deionized water was added dropwise to the spot to wet it for 1min before treatment, and 400. Mu.L of 10mM NH was then added to the cathode cell 3 ·H 2 O (pH-10.7) solution, 400 mu L of 10mM HOC (pH-2.6) solution is loaded in the anode pool. When (when)The solution in the cathode cell and the anode cell wets the paper-based fluid channel and is then treated for 2min by applying 200V voltage. After the treatment, the packed strip (about 5 mm) was cut off and placed in a 0.5mL centrifuge tube, 150. Mu.L of the extract was added, and the mixture was shaken on a shaker at 120rpm for 5min, and after standing, the supernatant was taken for MS detection.
DBS solvent treatment method: based on Kit instructions, DBS was placed in 96-well plates, 150. Mu.L of extract was added, the 96-well plates were placed in incubators, and the plates were shaken at 45℃and 600rpm for 45min, and after returning to room temperature, the supernatants were removed for MS detection.
(4) PAD electric purification and separation of amphoteric components
Blood samples are typically highly complex biological samples containing proteins and amino acids that are important endogenous metabolic and biomarker small molecules with pI characteristics. As a sample pretreatment means, the invention does not emphasize the separation between AA, but expects to separate the AA component to be detected from other complex media by utilizing the established pH gradient so as to achieve the purpose of sample purification, thereby creating conditions for MS direct detection. For this reason, it is desirable to establish a broad pH gradient over a small scale so that the target component is focused while achieving spatial separation from particulate and salt and neutral components. In view of the possible impact of the supporting electrolyte on the MS system, we have chosen a volatile acid-base system. The pH gradient characterization result of the established system using a glass fiber paper base with good hydrophilicity and low cost as a paper base fluid channel substrate is shown in FIG. 2 a. With HAc and NH 3 ·H 2 The O solution is used for supporting the dynamic pH gradient established by the electrolyte, and establishes a stable pH gradient between 3 and 10.5 within the range of 5mm under the given conditions. The gradient may be established within 2 minutes of applying the voltage and may last for at least 10 minutes. After three proteins with isoelectric spans of 4.5-10.0 were loaded from the anode end, they were focused to a pH gradient interval while being moved away from the loading position within 2min and the order was consistent with their respective pI (fig. 2 a). To further demonstrate the nature of the class separation on the paper-based fluid channels, a colored probe of four classes of components, namely a mixture of anions and cations (representing salt), neutral components and amphoteric components, is loaded onto the anode end and then subjected to an electrical treatment, resulting in the followingShown in figure 2 b. Also after 2min, the neutral probe (pink) therein was slightly shifted towards the cathode end relative to the initial loading position by EOF, the anionic probe (blue) was shifted towards the anode end, the cationic probe continued to be shifted towards the cathode end by the pH gradient zone, while the amphoteric probe (brown) was piled up in the form of a focal zone in the pH gradient zone. Thus, anions and cations (salts) can be removed from the pH gradient region for desalting. AA is a small amphoteric component relative to protein molecules. To further demonstrate the behavior of the small ampholytic component AA on this PAD system, we stained AA in serum samples after the same PAD electrical treatment, the results are shown in fig. 3 a. The expected repeatable AA color bands are also seen in the gradient zone. The MS detection is carried out, and the method has better precision as shown in figure 3 b.
TABLE 1 labeled recovery of 11 amino acids in serum after two different methods of treatment
Note that: the data in the table are based on the same batch of experiments performed on standard-added serum samples prepared from direct dilution stock, preloaded dry spot samples were wetted with 5 μl pure water for 1min and power-down for 2min at 200v voltage prior to treatment. N=5
In the above process:
sample loading position:
the sample may be loaded anywhere on the channel.
Sample loading mode:
the blood sample loading modes are mainly three types: liquid and paper carry dry spots and coupons.
ESI MRM MSMS off-line determination:
in order to carry out the MS detection of the AA in the sample by the isotope dilution method, the invention adopts a method of adding an isotope internal standard before sample treatment to prepare the sample. The stacked belt subjected to PAD treatment is directly subjected to ESI MRM MSMS detection after being extracted by extraction liquid.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A rapid detection method of serum amino acid based on paper-based electric sample processing device and mass spectrometry is characterized in that amino acid and protein form a purification band on a dynamic double gradient established on a paper-based fluid channel under the drive of direct-current voltage based on isoelectric point characteristics, so that the purification band is separated from other medium components in a sample; the extraction liquid is used for dissolving the amino acid in the purification band on the paper-based channel and then directly carrying out ESIMS detection.
2. The rapid detection method of serum amino acids based on paper-based electric sample processing device and mass spectrometry according to claim 1, wherein the paper-based fluid channel uses an acidic solution at the anode end and an alkaline solution at the cathode end, the alkaline solution and the acidic solution being NH respectively 3 ·H 2 O and HAc solutions are loaded with a reservoir or directly onto the paper-based fluid channel.
3. The rapid detection method of serum amino acids based on paper-based electric sample processing device and mass spectrometry according to claim 1, wherein the paper-based fluid channel is 1-10 mm wide and 10-100 mm long; the dynamic double gradient refers to a pH gradient and an electric field gradient; the length of the formed purifying belt is 2-10 mm.
4. The rapid detection method of serum amino acids based on paper-based electric sample processing device and mass spectrometry according to claim 1, characterized in that the serum sample is directly loaded in liquid form; or the sample is loaded in a form of being preloaded to form a dry spot on the paper-based channel; or in the form of a dry paper sample.
5. The rapid detection method of serum amino acids based on paper-based electronic sample processing device and mass spectrometry according to claim 1, wherein the paper-based fluid channel is a hydrophilic glass fiber material.
6. The rapid detection method of serum amino acids based on paper-based electronic sample processing device and mass spectrometry according to claim 1, wherein the electrode to which the voltage is applied to the paper-based fluid channel through a liquid reservoir or directly by contact with the paper-based fluid channel.
7. The rapid detection method of serum amino acids based on paper-based electric sample processing device and mass spectrometry according to claim 4, wherein the dry paper sample sheet has a diameter smaller than the width of the paper-based fluid channel and is wetted with water before processing.
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