In-situ synthesized polypeptide wafer for maldi-TOF quality control detection, and manufacturing method and quality control detection method
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-08-13
Smart Images
Figure CN2025148132_13082026_PF_FP_ABST
Abstract
Description
An in-situ synthesized polypeptide wafer for MALDI-TOF quality control detection, its fabrication method, and its quality control detection method. Technical Field
[0001] This invention relates to the field of biowafer technology, and more particularly to an in-situ synthesized polypeptide wafer for MALDI-TOF quality control detection, its fabrication method, and its quality control detection method. Background Technology
[0002] In-situ synthesis of peptide wafers is a high-throughput biowafer technology. Its advantage lies in its ability to prepare peptides with different sequences ranging from submicron, micron to millimeter scales using stepwise synthesis techniques such as photopatterning or droplet printing on substrates like silicon, glass, and plastic. This allows for peptide synthesis, analysis, and research on a single wafer. During in-situ peptide wafer synthesis, monitoring the coupling efficiency of the stepwise amino acids becomes crucial to ensuring wafer quality.
[0003] MALDI-TOF imaging, as a detection technique, can detect the coupling status and synthesis efficiency of each amino acid layer, thereby ensuring the accuracy and high quality of wafer synthesis. The principle of MALDI (matrix-assisted laser desorption / ionization) is to irradiate the co-crystallized film formed by the sample and matrix with a laser. The matrix transfers the energy absorbed from the laser to the biomolecules, causing the biomolecules to gain or lose protons and become ionized. Therefore, MALDI is a soft ionization technique, particularly suitable for analyzing mixtures and biomolecules or unstable molecules, such as peptides, proteins, nucleic acids, the molecular weight distribution of polymers, and oligomer analysis. MALDI mass spectrometry is characterized by high sensitivity, wide applicability, and simple operation, making it the most commonly used method for the detection of biomolecules. In MALDI-TOF (matrix-assisted laser desorption / ionization-time of flight) mass spectrometry, the matrix and sample are typically dropped separately or mixed onto a stainless steel target plate and thoroughly dried before detection, or biological samples are processed, thoroughly dried, and then attached to conductive ITO glass for detection.
[0004] Wafers with non-conductive substrates, such as glass or plastic wafers, are optically transparent and electrically insulating. They can be modified using specific chemical methods, exhibiting superior optical transmittance and high-voltage resistance, as well as chemical inertness and biocompatibility. Glass is compatible with most biological samples and is a common substrate. However, non-conductive substrates suffer from low specificity, low sensitivity, and inaccuracy when used for MALDI-TOF detection, thus requiring solutions. Summary of the Invention
[0005] To address the problems existing in the prior art, this application proposes the following technical solution:
[0006] In one aspect, this application proposes an in-situ synthesized peptide wafer for MALDI-TOF quality control detection, comprising: a non-conductive substrate; wherein the non-conductive substrate is divided into one or more quality control regions and an application region; wherein a metal grid layer is covered on the quality control regions, the metal grid layer comprising a plurality of metal grid patterns composed of conductive lines; the metal grid layer divides the non-conductive substrate into conductive and non-conductive regions; wherein the spacing between adjacent conductive lines of the metal grid pattern is less than 100 μm; wherein the surface of the non-conductive substrate is exposed between the conductive lines of the metal grid pattern; wherein the metal grid layer is used to eliminate electron accumulation in the non-conductive regions of the quality control regions.
[0007] Another aspect of this application discloses a method for fabricating in-situ synthesized peptide wafers for MALDI-TOF quality control detection, comprising the following steps: marking the front and back sides of a non-conductive substrate; dividing the non-conductive substrate into one or more quality control regions and application regions; covering the front side of the quality control region with a metal grid layer, the metal grid layer including multiple metal grid patterns composed of conductive lines; wherein the metal grid layer divides the non-conductive substrate into conductive and non-conductive regions; wherein the spacing between adjacent conductive lines of the metal grid pattern is less than 100 μm; wherein the surface of the non-conductive substrate is exposed between the conductive lines of the metal grid pattern, and coupled with arm molecules for peptide synthesis after chemical modification; wherein the metal grid layer is used to eliminate electron accumulation in the non-conductive regions of the quality control region.
[0008] Another aspect of this application provides a quality control method for in-situ synthesized peptide wafers for MALDI-TOF quality control detection, comprising: providing an in-situ synthesized peptide wafer,
[0009] The wafer includes a non-conductive substrate, which is divided into one or more quality control regions and application regions. A metal grid layer covers the quality control regions, and the metal grid layer includes multiple metal grid patterns composed of conductive lines, exposing the surface of the non-conductive substrate between the conductive lines of the metal grid patterns. The metal grid layer divides the non-conductive substrate into conductive and non-conductive regions. Surface chemical modification is performed on the surface of the non-conductive regions to couple arm molecules. Layer-by-layer in-situ solid-state synthesis using photolithography is employed, coupling corresponding amino acids at each synthesis step, with corresponding amino acids coupled to the quality control and application regions. Furthermore, each selected amino acid is coupled sequentially at each synthesis step, forming specific peptide sequences at corresponding positions in the quality control and application regions. The quality control regions undergo MALDI detection pretreatment. MALDI-TOF is used to detect the quality control regions. Attached Figure Description
[0010] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:
[0011] Figure 1A shows a polypeptide wafer according to one embodiment of this application;
[0012] Figure 1B is a partial schematic diagram of the wafer shown in Figure 1 in the quality control area;
[0013] Figure 2 shows the amino acid detection spectrum of the polypeptide wafer shown in Figure 1 under MALDI-TOF detection.
[0014] Figure 3 shows the quality control peptide detection spectrum of the polypeptide wafer shown in Figure 1 under MALDI-TOF detection.
[0015] Figure 4 shows the spectra of the five wafers in the first control group under MALDI-TOF detection;
[0016] Figure 5 shows the spectra of the five wafers in the second control group under MALDI-TOF detection. Embodiments of the present invention
[0017] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.
[0018] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.
[0019] In this application, the term "in-situ synthesis" refers to the process of directly synthesizing a polypeptide sequence at the desired application site on a matrix, substrate, or slab. In some exemplary embodiments, a linker with protecting groups is deposited on the substrate surface, which can be removed, followed by the addition of an amino acid layer, and polypeptide synthesis is achieved by chemically coupling amino acid monomers. In some exemplary embodiments, a specially designed photoliographic mask is used to protect the sites that do not need to be synthesized, while exposing the sites that need to be synthesized. A photoresist, under light, generates an alkaline substance that removes the protecting groups on amino groups, allowing for the coupling of free hydroxyl groups to synthesize the polypeptide.
[0020] In this application, the term "matrix, substrate, or substrate" refers to a carrier in the process of biopeptide synthesis that has various chemical groups and can immobilize biomolecules such as proteins, antibodies, enzymes, peptides, DNA, etc.
[0021] In this application, the term "linker molecule" refers to a linker molecule laid on a treated glass slide in in situ synthesis, and amino acids can be synthesized into peptides based on linker molecules.
[0022] In this application, the term "protecting group" is used to protect the active side chain of amino acids. The protection of amino acids in peptide chemical synthesis is crucial and directly determines the success of the synthesis. These protecting groups are stable during the synthesis process, have no side reactions, and can be completely and quantitatively removed after the synthesis is completed.
[0023] In this application, the term "BOC protecting group" refers to tert-butyloxycarbonyl, an amino protecting group used in organic synthesis, particularly in peptide synthesis.
[0024] In this application, the term "chemical modification" refers to the process of attaching active groups or catalytic substances to the electrode surface by means of adsorption, coating, polymerization, chemical reaction, etc., to protect the electrode or improve the electrode's characteristic functions.
[0025] In this application, the term "selected amino acid" refers to an amino acid whose specific amino acid monomer is selected for coupling with a matrix or substrate according to the purpose.
[0026] In this application, the term "metal grid pattern" refers to a pattern composed of metal lines of regular or irregular shapes, with a non-metallic conductive substrate arranged between adjacent metal lines.
[0027] In this application, the term "conductive line" refers to a metal line on a metal grid pattern.
[0028] In this application, the term "comprising" means including, but not limited to, the following elements, that is, it does not exclude other elements.
[0029] In this application, the terms "about" and "approximately" represent a range of accuracy that a person skilled in the art would understand while still ensuring the accuracy of the technical effects of the features in question. This term typically indicates a deviation of 10% from the numerical values shown, preferably 5%.
[0030] Non-conductive substrates, including glass wafers, conductive substrates with insulating layers, and plastic wafers, are non-conductive. Their non-conductive surfaces lead to charge accumulation during ionization, reducing ionization efficiency and resulting in defects in the synthesis quality when directly used for MALDI-TOF detection and monitoring. Existing technologies involve attaching copper adhesive tape to the back of a non-conductive glass slide for protein MALDI imaging on an FFPE tissue sectioner [Reference 1: "Copper adhesive tape attached to the reverse side of a non-conductive glass slide to achieve protein MALDI-imaging in FFPE-tissue sections"]. Experimental results from this article show that non-conductive glass with conductive copper tape acquires more protein ion signals and intensities than ITO glass sections. However, manually attaching the copper tape can cause air bubbles or uneven adhesion, leading to signal shifts in the MALDI detection of the glass slide. There is still significant room for improvement in this area. According to JEOL's research, "Mass spectrometry imaging on mixed conductive / non-conductive substrate using JMS-S3000 Spiral TOF™," [Reference 2: "Mass spectrometry imaging on mixed conductive / non-conductive substrate using JMS-S3000 Spiral TOF™"], non-conductive glass substrates have a significant impact on the MALDI detection of present substances, potentially causing "present" to appear as "absent." When the conductive metal portions are spaced 800 μm apart, the conductive metal regions can detect target signals in MALDI, while the non-conductive regions cannot. Forming a uniform, dense metal film on the glass substrate surface using gold deposition achieves good conductivity and detection, with improved resolution and signal intensity within the same conductive region. Existing technologies also include solutions using metal coatings or ITO conductive glass, but these methods alter the properties of the glass surface, affecting the feasibility of modifying the glass surface in in-situ synthesis to introduce appropriate functional molecules and the sequential coupling of amino acid monomers.
[0031] To date, there is still a lack of highly specific and sensitive detection methods for quality control of in-situ synthesized MALDI.
[0032] To address this issue, one aspect of this application proposes an in-situ synthesized peptide wafer for MALDI-TOF quality control detection, comprising:
[0033] A non-conductive substrate is used, which is divided into one or more quality control regions and application regions. A metal grid layer covers the quality control regions, and the metal grid layer includes multiple metal grid patterns composed of conductive lines. The metal grid layer divides the non-conductive substrate into conductive and non-conductive regions. The spacing between adjacent conductive lines of the metal grid pattern is less than 100 μm. The surface of the non-conductive substrate is exposed between the conductive lines of the metal grid pattern. The metal grid layer is used to eliminate electron accumulation in the non-conductive regions of the quality control regions. The structure of the conductive metal grid exposes the surface of the non-conductive substrate, providing feasibility for chemical modification and linking molecular arms. Furthermore, it ensures that accumulated charge is unloaded through the metal grid during MALDI-TOF laser ionization, guaranteeing the accuracy and reliability of the MALDI-TOF signal.
[0034] In at least one embodiment, the spacing between adjacent conductive lines of the metal grid pattern is greater than or equal to 10 μm and less than 100 μm.
[0035] In at least one embodiment, the quality control area includes more than one detection area, wherein each detection area includes a plurality of metal grid patterns arranged in an array; the distance between each detection area is greater than 200 μm.
[0036] In at least one embodiment, a metal grid layer is deposited onto a non-conductive substrate, and the material of the metal grid layer is selected from any one of gold, silver, copper, chromium, platinum, titanium, and their alloys. In a further embodiment, multiple periodically repeating metal grid patterns are formed on the metal grid layer using photolithography. In at least one embodiment, the metal grid pattern includes any one or more of square, rectangular, circular, linear, spiral, hexagonal, and mesh shapes. In at least one embodiment, the minimum linewidth of the conductive line is from 1 μm to 500 μm. In at least one embodiment, the minimum linewidth of the conductive line is from 2 μm to 100 μm. In at least one embodiment, the minimum linewidth of the conductive line is from 5 μm to 50 μm. In at least one embodiment, the minimum linewidth of the conductive line is from 10 μm to 20 μm. The minimum linewidth of the conductive line should be preferably selected based on the minimum linewidth of the laser focusing point of the mass spectrometer.
[0037] In at least one embodiment, the spacing between adjacent conductive lines of the metal grid pattern is any one of 10μm, 20μm, 30μm, or 40μm.
[0038] In at least one embodiment, the non-conductive substrate is configured to perform peptide synthesis thereon in an in-situ synthetic manner.
[0039] In at least one embodiment, the non-conductive substrate surface exposed between the conductive lines of the metal grid pattern is chemically modified and coupled with arm molecules for peptide synthesis.
[0040] Another aspect of this application discloses a method for fabricating in-situ synthesized peptide wafers for MALDI-TOF quality control detection, comprising the following steps: marking the front and back sides of a non-conductive substrate; dividing the non-conductive substrate into one or more quality control regions and application regions; covering the front side of the quality control region with a metal grid layer, the metal grid layer including multiple metal grid patterns composed of conductive lines; wherein the metal grid layer divides the non-conductive substrate into conductive and non-conductive regions; wherein the spacing between adjacent conductive lines of the metal grid pattern is less than 100 μm; wherein the surface of the non-conductive substrate is exposed between the conductive lines of the metal grid pattern, and coupled with arm molecules for peptide synthesis after chemical modification; wherein the metal grid layer is used to eliminate electron accumulation in the non-conductive regions of the quality control region.
[0041] Another aspect of this application discloses a quality control detection method for in-situ synthesized peptide wafers for MALDI-TOF quality control detection, comprising: providing an in-situ synthesized peptide wafer, the wafer including a non-conductive substrate; wherein the non-conductive substrate is divided into one or more quality control regions and application regions; a metal grid layer is covered on the quality control regions, the metal grid layer including multiple metal grid patterns composed of conductive lines, the conductive lines of the metal grid patterns exposing the surface of the non-conductive substrate; the metal grid layer divides the non-conductive substrate into metal conductive regions and non-conductive regions; surface chemical modification is performed on the surface of the non-conductive regions to couple arm molecules; layer-by-layer in-situ solid-state synthesis technology using photolithography, coupling corresponding amino acids in each synthesis step, and correspondingly coupling amino acids on the quality control regions and application regions; and coupling the selected amino acids one by one in each synthesis step, forming specific peptide sequences at corresponding positions on the quality control regions and application regions; performing MALDI detection pretreatment on the quality control regions; and using MALDI-TOF to detect the quality control regions.
[0042] In a further embodiment, the amino acid is an amino acid with a protecting group, and in yet another embodiment, the protecting group is a BOC protecting group.
[0043] The specific implementation process and technical effects of this application will be described in detail below with reference to the accompanying drawings:
[0044] One embodiment relates to a method for fabricating an in-situ synthesized peptide wafer for MALDI-TOF quality control detection. First, a non-conductive substrate of appropriate size is cut, and the front and back sides of the non-conductive substrate are marked. Then, the application area and quality control area of the peptide wafer are divided. The quality control area includes a corresponding metal grid conductive area. The remaining non-conductive area on the peptide wafer is divided into multiple application areas. A metal grid layer is formed by metal deposition on the non-conductive substrate front side in the quality control area. Specific methods include: first, forming a metal layer using methods such as physical vapor deposition, chemical vapor deposition, electrochemical deposition, atomic layer deposition, electron beam evaporation, magnetron sputtering, and laser deposition; then, using photolithography to create a zero layer on the metal layer, i.e., forming multiple periodically repeating metal grid patterns on the metal layer. The conductive lines of the metal grid pattern expose the surface of the non-conductive substrate, thus forming the metal grid layer. The shape of the metal grid can be rectangular, circular, linear, spiral, hexagonal, or mesh-like. The function of the metal grid is to connect with the entire metal area and sample holder through line connections, preserving the original surface groups and functional arm modifications of the substrate in a certain proportion of the area, while also unloading the accumulated charge during the laser ionization process of MALDI-TOF detection, ensuring ionization efficiency and achieving effective ionization and mass spectrometry analysis of the target analyte.
[0045] The quality control area is defined as a 1000μm square detection zone, containing multiple metal grid patterns arranged in an array. Each detection zone is spaced 200μm apart and marked with edges for easy positioning. The non-conductive substrate between the conductive lines of the metal grid patterns is then chemically modified. Following this modification, silanol groups on the substrate surface are used to couple arm molecules, and then a layer-by-layer in-situ solid-state synthesis technique using photolithography is employed. In each synthesis step, corresponding amino acids are coupled, and these amino acids are coupled onto the quality control area and the application area. Furthermore, the selected amino acids are coupled one by one in each synthesis step, forming specific peptide sequences at corresponding positions in the quality control area and the application area.
[0046] One embodiment provides a wafer 100 for synthesizing a peptide chip, the structure of which is shown in Figures 1A and 1B. The wafer 100 includes four quality control regions, namely the first quality control region A, the second quality control region B, the third quality control region C, and the fourth quality control region D as shown in Figure 1A. The other regions outside the quality control regions are application regions, which include 13 application detection wafers, namely application detection wafers 1-13 as shown in Figure 1A.
[0047] As shown in Figure 1B, each quality control area contains 213 square detection areas 200, each 1000 μm in size, with adjacent detection areas spaced 200 μm apart. Each of the 213 detection areas 200 in the quality control area has multiple metal grid patterns arranged in an array, with the spacing between adjacent conductive lines of the metal grid patterns being 20 μm.
[0048] Following the principles of in-situ peptide synthesis, 18 amino acids are synthesized in an ordered manner within a customized automated system. During the synthesis of each amino acid, one or more amino acids are coupled by exposure at corresponding positions in the quality control area, ultimately used for quality control of each coupling step. The coupling reaction of amino acids or peptides on the peptide wafer is completed by designing exposure at different layers and sites, and the peptide wafer is synthesized according to the designed amino acid sequence. The completed application area can be cut into 13 application detection wafers (detection wafers 1-13 in Figure 1A) for medical or specific-purpose chips. Four small wafers in the quality control area are used for MALDI detection to test the synthesis effect and quality.
[0049] In another embodiment, the number of quality control zones is other numbers that need to be set, such as 2, 6, or 8.
[0050] Pretreatment before quality control testing:
[0051] The cut quality control slides underwent MALDI pretreatment, including removing the surface protective adhesive, removing the Boc protecting group with TFA, linking TMPP, activating with iodoacetonitrile, and ammonolysis to bring the peptides and amino acids to be detected into a deprotected and free state. Then, a saturated CHCA solution was sprayed onto the surface to ensure thorough mixing of the matrix and sample, ready for analysis. The matrix-coated glass quality control slides (any one of the quality control slides A / B / C / D in Figure 1A) were placed on a Bruker standard stainless steel target plate, and the MALDI-TOF mass spectrometry method parameters were adjusted to suit the peptides and amino acids to be detected, thus completing the quality control detection.
[0052] MALDI-TOF testing was performed on the above quality control areas:
[0053] The test results show:
[0054] As shown in Figure 2, the target peaks of the detection spectrum in the corresponding detection region match the designed amino acid / peptide target peaks, and the resolution is high.
[0055] As shown in Figure 3, each quality control peptide was correctly and significantly detected. This example demonstrates that amino acid monolayers and quality control peptides can be accurately represented in the MALDI detection spectrum. Based on the goal of controlling the wafer synthesis effect, the synthesis status of each layer and the final synthesis effect can be determined by intuitively comparing the peak shape and mass number in the MALDI spectrum, thus achieving quality control. The metal grid in this invention enables MALDI detection on a non-conductive substrate, playing a crucial role in product export quality control.
[0056] In a further embodiment, a control experiment was conducted to fabricate multiple wafers using the above method for quality control testing, as detailed below:
[0057] Two control groups were set up, each containing five non-conductive glass substrates. MALDI-TOF detection was performed on both control groups to compare and analyze the effects on different objects. The non-conductive substrates included in the control groups are as follows:
[0058] Non-conductive glass substrate without a metal layer;
[0059] Chromium-coated non-conductive glass substrate;
[0060] Non-conductive glass substrate with a square chromium metal grid with a 40μm spacing between adjacent conductive lines;
[0061] A non-conductive glass substrate with a square chromium metal grid with a 20μm spacing between adjacent conductive lines;
[0062] A non-conductive glass substrate with a square chromium metal grid with a spacing of 10 μm between adjacent conductive lines;
[0063] All prepared non-conductive glass substrates were cleaned with IPA to remove surface dust and dried with nitrogen. Surface chemical modification was performed on the front side of the glass to couple arm molecules. Boc-Gly-OH was then deprotected and coupled onto the arm molecules via exposure to form single amino acid wafers.
[0064] The first control group was reserved for future testing;
[0065] For the second control group, Boc-Leu-OH amino acids were further synthesized and coupled to form detection zones for amino acids and peptides. The in-situ synthesized non-conductive glass substrate was deprotected with TFA (trifluoroacetic acid) to remove the Boc protecting group (tert-butyloxycarbonyl, abbreviated as "Boc"). It was then ultrasonically cleaned with NMP and IPA for 2 minutes each, followed by nitrogen drying. A 40 mg / ml TMPP solution was added to the surface and reacted for 2 hours. After ultrasonic cleaning with IPA for 2 minutes, it was dried with nitrogen. An iodoacetonitrile mixture was added and reacted overnight. The substrate was then ultrasonically cleaned with NMP and IPA for 2 minutes each, followed by nitrogen drying. It was then placed in an ammonolysis apparatus and subjected to ammonolysis with ammonia for 2 hours. Finally, a saturated CHCA solution was sprayed onto the surface to ensure thorough mixing of the matrix and sample, ready for analysis.
[0066] Turn on the Bruker AUTO-FLEX mass spectrometer, fix the non-conductive glass substrate of each control group onto the target plate with carbon ribbon, select positive ion reflection mode, molecular weight 900-2000 Da, laser pulse frequency 50 Hz, laser focus point "ULTRE", frequency 2000, shot 500 to test the detection area results.
[0067] Summary results:
[0068] The results of the first control group are shown in Figure 4. The figure shows that the non-conductive glass substrate without a metal grid layer does indeed cause the "present" to "absent" phenomenon in MALDI detection, with no signal value appearing. However, the surface spectrum of the non-conductive glass with chromium metal deposition shows that there is a signal, with differences in signal strength: among them, the non-conductive glass with full metal coverage can detect the target object, but the signal is very weak; the non-conductive glass with a square metal grid has clearly visible target peaks of Gly, Leu and dipeptide composed of two amino acids, with strong signals. The square metal grid is more suitable for the signal detection of amino acids and peptides, and the signals of 20μm and 10μm squares are significantly higher than those of 40μm squares.
[0069] The spectrum of the second control group, as shown in Figure 5, is consistent with that of single amino acids. The non-conductive glass substrate with square metal grid deposition can achieve the original surface groups and functional arm modification, and at the same time, it can achieve charge unloading during the laser ionization process of MALDI-TOF detection, so as to achieve effective ionization and mass spectrometry detection of the target analyte.
[0070] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.
[0071] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.
Claims
1. An in-situ synthesized polypeptide wafer for MALDI-TOF quality control detection, characterized in that... include: Non-conductive substrate; The non-conductive substrate is divided into one or more quality control areas and application areas; The quality control area is covered with a metal grid layer, which includes multiple metal grid patterns composed of conductive lines; the metal grid layer divides the non-conductive substrate into conductive and non-conductive regions. Wherein, the spacing between adjacent conductive lines of the metal grid pattern is less than 100 μm; The conductive lines of the metal grid pattern expose the surface of a non-conductive substrate. The metal grid layer is used to eliminate the accumulation of electrons in the non-conductive areas of the quality control region.
2. The in-situ synthesized polypeptide wafer according to claim 1, characterized in that: The spacing between adjacent conductive lines in a metal grid pattern is greater than or equal to 10 μm and less than 100 μm.
3. The in-situ synthesized polypeptide wafer according to claim 1, characterized in that: The quality control area includes more than one detection area, wherein each detection area includes multiple metal grid patterns arranged in an array; the distance between each detection area is greater than 200 μm.
4. The in-situ synthesized polypeptide wafer according to claim 1, characterized in that: The metal grid pattern includes any one or more of the following: square, rectangular, circular, linear, spiral, hexagonal, and mesh.
5. The in-situ synthesized polypeptide wafer according to claim 1, characterized in that: The metal grid layer is deposited onto the quality control area, and the material of the metal grid layer is selected from any one of gold, silver, copper, chromium, platinum, titanium and their alloys.
6. The in-situ synthesized polypeptide wafer according to claim 1, characterized in that: The minimum linewidth of the conductive lines is 1 μm to 500 μm.
7. The in-situ synthesized polypeptide wafer according to claim 6, characterized in that: The minimum line width of the conductive lines is 2 μm to 100 μm.
8. The in-situ synthesized polypeptide wafer according to claim 6, characterized in that: The minimum linewidth of the conductive lines is 5 μm to 50 μm.
9. The in-situ synthesized polypeptide wafer according to claim 6, characterized in that: The minimum linewidth of the conductive lines is 10 μm to 20 μm.
10. The in-situ synthesized polypeptide wafer according to claim 1, characterized in that: The spacing between adjacent conductive lines of the metal grid pattern is any one of 10μm, 20μm, 30μm, or 40μm.
11. The in-situ synthesized polypeptide wafer according to claim 1, characterized in that: The non-conductive substrate surface exposed between the conductive lines of the metal grid pattern is chemically modified and coupled with arm molecules used for peptide synthesis.
12. A method for fabricating in-situ synthesized peptide wafers for MALDI-TOF quality control detection, comprising the following steps: The non-conductive substrate is marked with its front and back sides, and the non-conductive substrate is divided into one or more quality control areas and application areas; A metal grid layer is covered on the front of the quality control area, and the metal grid layer includes a plurality of metal grid patterns composed of conductive lines; The metal grid layer divides the non-conductive substrate into conductive and non-conductive regions. Wherein, the spacing between adjacent conductive lines of the metal grid pattern is less than 100 μm; The conductive lines of the metal grid pattern expose the surface of a non-conductive substrate, which is then chemically modified and coupled with arm molecules used for peptide synthesis. The metal grid layer is used to eliminate the accumulation of electrons in the non-conductive areas of the quality control region.
13. A quality control detection method for in-situ synthesized peptide wafers for MALDI-TOF quality control detection, comprising: An in-situ synthesized peptide wafer is provided, the wafer comprising a non-conductive substrate; wherein the non-conductive substrate is divided into one or more quality control regions and application regions; a metal grid layer is covered on the quality control regions, the metal grid layer comprising a plurality of metal grid patterns composed of conductive lines, the conductive lines of the metal grid patterns exposing the surface of the non-conductive substrate; the metal grid layer divides the non-conductive substrate into conductive and non-conductive regions. Surface chemical modification of the surface in non-conductive regions to couple arm molecules; The in-situ solid-phase synthesis technique using photolithography is used layer by layer. In each synthesis step, the corresponding amino acids are coupled and linked to the quality control region and the application region. In each synthesis step, the selected amino acids are coupled one by one, forming a specific polypeptide sequence at the corresponding position in the quality control region and the application region. The quality control area undergoes MALDI testing pretreatment. MALDI-TOF was used to test the quality control area.