Chip for time-of-flight mass spectrometry detection and preparation method thereof

Abstract

The present invention discloses a chip for time-of-flight mass spectrometry detection and a preparation method thereof. The chip for time-of-flight mass spectrometry detection includes: a base layer, a conductive layer is provided on one side of the base layer, a hydrophobic layer is provided on the side of the base layer facing away from the conductive layer, and the hydrophobic layer has a plurality of through holes arranged in an array; the base layer includes a glass base, and the side of the glass base facing away from the conductive layer is hydroxylated to form a hydrophilic layer; or the base layer includes a stacked glass base and a hydrophilic layer, and the hydrophilic layer is located between the conductive layer and the glass base. In the present invention, the chip adopts a glass base, which is low in cost; wherein the conductive layer ensures that the chip can be used for mass spectrometry detection; a hydrophilic layer and a hydrophobic layer are sequentially provided on the side of the glass base facing away from the conductive layer, and the through holes provided on the hydrophobic layer expose the hydrophilic layer, which can stably carry the matrix solution and the sample to be tested. The chip has a simple structure, low cost and is disposable, which can effectively avoid the problem of sample contamination affecting the mass spectrometry results.

Description

Technical Field

[0001] The present invention generally relates to the technical field of mass spectrometry analysis, and in particular to a chip for time-of-flight mass spectrometry detection and a preparation method thereof. Background Art

[0002] MALDI-TOF-MS (Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry) is a novel soft ionization organic mass spectrometry technique developed in the 1980s. This technique boasts high sensitivity, accuracy, minimal sample usage, rapid detection, high resolution, and high throughput. It is currently widely used in numerous biochemical analysis fields, including molecular weight and purity determination of biomacromolecules such as nucleic acids, proteins, and polysaccharides, nucleic acid and protein sequencing, and molecular weight distribution of polymers. The MALDI-TOF time-of-flight mass spectrometry platform is an internationally recognized platform for single nucleotide polymorphism (SNP) research. Its scientific and accurate nature has set a new standard in this field.

[0003] MALDI-TOF time-of-flight mass spectrometry can be used to detect proteins or nucleic acid molecules, but most of the chips currently used for time-of-flight mass spectrometry detection are imported chips, and most of them use metal or silicon substrates, which are relatively expensive; the detection sites mostly use pit structures, which may cause problems such as sample residue and detection interference, affecting the mass spectrometry results; due to the high cost, a chip is used multiple times, and the cleaning process is complicated, which may cause sample residue to affect the mass spectrometry results. Summary of the Invention

[0004] In view of the above-mentioned defects or deficiencies in the prior art, it is desired to provide a chip for time-of-flight mass spectrometry detection and a preparation method thereof.

[0005] In a first aspect, an embodiment of the present invention provides a chip for time-of-flight mass spectrometry detection, comprising: a base layer, a conductive layer being provided on one side of the base layer, a hydrophobic layer being provided on a side of the base layer facing away from the conductive layer, the hydrophobic layer having a plurality of through holes arranged in an array;

[0006] The base layer comprises a glass substrate, and the side of the glass substrate facing away from the conductive layer is hydroxylated to form a hydrophilic layer; or

[0007] The base layer includes a glass substrate and a hydrophilic layer that are stacked, and the hydrophilic layer is located between the conductive layer and the glass substrate.

[0008] Optionally, the water drop angle of the hydrophilic layer is less than 60°, and the water drop angle of the hydrophobic layer is greater than 100°.

[0009] Optionally, the water drop angle of the hydrophilic layer is less than 20°, and the water drop angle of the hydrophobic layer is greater than 140°.

[0010] Optionally, the diameter of the through hole is 200um-1mm.

[0011] Optionally, the glass substrate is made of borosilicate glass, and the thickness of the glass substrate is 0.2 mm-1 mm.

[0012] Optionally, the thickness of the glass substrate is 0.5 mm.

[0013] Optionally, the conductive layer is made of indium tin oxide.

[0014] In a second aspect, an embodiment of the present invention provides a method for preparing a chip for time-of-flight mass spectrometry detection, comprising:

[0015] providing a glass substrate;

[0016] forming a conductive layer on one side of the glass substrate;

[0017] forming a hydrophilic layer on a side of the glass substrate facing away from the conductive layer;

[0018] A hydrophobic layer is formed on a side of the hydrophilic layer facing away from the conductive layer.

[0019] Optionally, forming a hydrophilic layer on a side of the glass substrate facing away from the conductive layer includes:

[0020] A first plasma is used to bombard and modify the side of the glass substrate facing away from the conductive layer to form a hydrophilic layer.

[0021] Optionally, forming a hydrophobic layer on a side of the hydrophilic layer facing away from the conductive layer includes:

[0022] Coating a hydrophobic material on the side of the hydrophilic layer facing away from the conductive layer to form a hydrophobic film layer;

[0023] A mask is used to cover a portion of the hydrophobic film layer, and a second plasma is used to bombard a portion of the hydrophobic film layer not covered by the mask, so as to form a hydrophobic layer with a plurality of through holes.

[0024] The technical solutions provided by the embodiments of the present invention may have the following beneficial effects:

[0025] The chip for time-of-flight mass spectrometry detection and the preparation method thereof provided in an embodiment of the present invention adopt a glass substrate and are low in cost. A conductive layer is provided on one side of the glass substrate to meet the conductivity requirements of the chip and ensure that the chip can be used for mass spectrometry detection. A hydrophilic layer is provided on the side of the glass substrate facing away from the conductive layer, and a hydrophobic layer with a through hole is provided on the side of the hydrophilic layer facing away from the glass substrate, through which the hydrophilic layer is exposed. By utilizing the difference in hydrophilic and hydrophobic properties, the through hole can stably carry a matrix solution and a sample to be tested for mass spectrometry analysis. The chip has a simple structure, is low in cost, and is disposable, which can effectively avoid the problem of sample contamination affecting mass spectrometry results. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0027] Figure 1 A schematic structural diagram of a chip for time-of-flight mass spectrometry detection provided by an embodiment of the present invention;

[0028] Figure 2 A flowchart of a method for preparing a chip for time-of-flight mass spectrometry detection provided by an embodiment of the present invention;

[0029] Figures 3 to 7 A schematic structural diagram of a method for preparing a chip for time-of-flight mass spectrometry detection provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are intended only to illustrate the relevant invention and are not intended to limit the invention. It should also be noted that, for ease of description, only portions relevant to the invention are shown in the accompanying drawings.

[0031] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0032] like Figure 1 As shown, an embodiment of the present invention provides a chip for time-of-flight mass spectrometry detection, comprising: a base layer 1, a conductive layer 2 is provided on one side of the base layer 1, a hydrophobic layer 3 is provided on the side of the base layer 1 facing away from the conductive layer 2, and the hydrophobic layer 3 has a plurality of through holes 31 arranged in an array;

[0033] The base layer 1 includes a glass substrate 11, and the side of the glass substrate 11 facing away from the conductive layer 2 is hydroxylated to form a hydrophilic layer 12; or,

[0034] The base layer 1 includes a glass base 11 and a hydrophilic layer 12 which are stacked together. The hydrophilic layer 12 is located between the conductive layer 2 and the glass base 11 .

[0035] The chip for time-of-flight mass spectrometry provided in this embodiment comprises a glass substrate 11 and multiple functional film layers, including a conductive layer 2, a hydrophilic layer 12, and a hydrophobic layer 3. The hydrophilic layer 12 may be formed by modifying the surface of the glass substrate 11 facing away from the conductive layer 2, or by coating the surface of the glass substrate 11 facing away from the conductive layer 2 with a hydrophilic material.

[0036] The through holes 31 provided on the hydrophobic layer 3 of the chip of this embodiment are used for dripping the matrix solution and the sample to be tested. The location of each through hole 31 is the detection site. The chip adopts a glass substrate 11, which can effectively reduce costs; the conductive layer 2 on one side of the glass substrate 11 serves as a common electrode for the detection site, ensuring that the chip with the glass substrate 11 can be used for mass spectrometry analysis; the side of the glass substrate 11 facing away from the conductive layer 2 is provided with a hydrophilic layer 12 and a hydrophobic layer 3 having through holes 31, and the through holes 31 expose the hydrophilic layer 12. By utilizing the difference in water drop angle between the hydrophilic layer 12 and the hydrophobic layer 3, the matrix solution and the sample to be tested can be stably maintained in the through holes 31 for mass spectrometry analysis. The chip has a simple structure, low cost and is disposable, which can effectively avoid the problem of sample contamination affecting the mass spectrometry results.

[0037] The chip provided in this embodiment has a detection area and an information area. The through holes 31 are distributed in the detection area, and the chip number is set in the information area.

[0038] In an embodiment of the present invention, the water drop angle of the hydrophilic layer 12 is less than 60°, and the water drop angle of the hydrophobic layer 3 is greater than 100°. Thus, based on the fact that the difference in the water drop angles of the hydrophilic layer 12 and the hydrophobic layer 3 is greater than 40°, the hydrophilic layer 12 and the hydrophobic layer 3 are both highly hydrophilic. Since the hydrophobic layer 3 is further away from the glass substrate 11 than the hydrophilic layer 12, the through holes 31 in the hydrophobic layer 3 expose the hydrophilic layer 12. Due to the difference in hydrophilicity and hydrophobicity between the hydrophilic layer 12 and the hydrophobic layer 3, each through hole 31 can stably support the matrix solution and the sample to be tested.

[0039] Furthermore, the water drop angle of the hydrophilic layer 12 is less than 20°, and the water drop angle of the hydrophobic layer 3 is greater than 140°. This further improves the stability of the matrix solution and the sample to be tested in the through hole 31.

[0040] In some embodiments, the diameter of the through-holes 31 is 200 μm to 1 mm. The size of the chip is limited by the size and number of the through-holes 31 and the spacing between the through-holes 31. In this embodiment, the diameter of the through-holes 31 is 200 μm to 1 mm. This ensures that each detection site can accommodate the volume of the matrix solution and the sample to be tested. It also limits the size of the chip and improves its compatibility with different mass spectrometers.

[0041] In some embodiments, the glass substrate 11 is made of borosilicate glass, and the thickness of the glass substrate 11 is 0.2 mm to 1 mm. In this embodiment, the glass substrate 11 is made of relatively hard borosilicate glass. The functional film layers (conductive layer 2, hydrophilic layer 12, and hydrophobic layer 3) are fabricated on the surface of the glass substrate 11, making it less prone to breakage and ensuring the smoothness of each functional film layer.

[0042] If the thickness of the glass substrate 11 is less than 0.2 μm, the glass substrate 11 may be easily broken during processing of the functional film layers. If the thickness of the glass substrate 11 is greater than 1 mm, the excessive thickness of the substrate may also result in excessive waste of materials.

[0043] Preferably, the thickness of the glass substrate 11 is 0.5 mm, which has good hardness and can ensure that it is not easily broken during processing of each functional film layer; the thickness is moderate and no material is wasted.

[0044] In some embodiments, the conductive layer 2 is made of indium tin oxide.

[0045] Mass spectrometry analysis requires a common electrode to ionize the sample in the mass spectrometer's electric field. Since the glass substrate 11 is non-conductive, adding a conductive layer 2 allows the chip to meet mass spectrometry requirements. Conductive layer 2 is formed on one side of the glass substrate 11 using a magnetron sputtering process. The resistance of conductive layer 2 can range from tens to hundreds of ohms.

[0046] like Figure 2 As shown, an embodiment of the present invention provides a method for preparing a chip for time-of-flight mass spectrometry detection, comprising the following steps:

[0047] S10, providing a glass substrate;

[0048] S20, forming a conductive layer on one side of the glass substrate;

[0049] S30, forming a hydrophilic layer on a side of the glass substrate facing away from the conductive layer 2;

[0050] S40 , forming a hydrophobic layer on the side of the hydrophilic layer facing away from the conductive layer 2 .

[0051] Reference Figure 3, perform step S10 to provide a glass substrate 11. The glass substrate 11 is preferably made of borosilicate glass and has a thickness of 0.2 μm to 1 mm, preferably 0.5 μm.

[0052] Reference Figure 4 , performing step S20 to form a conductive layer 2 on one side of the glass substrate 11. Exemplarily, this step specifically includes: forming the conductive layer 2 on one side of the glass substrate 11 by a magnetron sputtering process, wherein the conductive layer 2 is an indium tin oxide layer.

[0053] The magnetron sputtering power for forming the conductive layer 2 is 80W to 120W, the pressure is 0.5Pa-0.9Pa, and the sputtering time is 15min to 25min, which can make the resistance of the conductive layer 2 tens or hundreds of ohms, meeting the conductivity requirements of the chip.

[0054] Preferably, the magnetron sputtering power is 100 watts, the pressure is 0.7 Pa, and the sputtering time is 20 minutes.

[0055] Reference Figure 5 , performing step S30 to form a hydrophilic layer 12 on the side of the glass substrate 11 facing away from the conductive layer 2 .

[0056] Exemplarily, the first plasma is used to bombard and modify the side of the glass substrate facing away from the conductive layer 2 to form the hydrophilic layer 12 .

[0057] This embodiment can use a plasma cleaning machine to treat the surface of the glass substrate 11 through a first plasma. By utilizing the bombardment of energetic particles in the plasma, foreign matter adsorbed on the glass surface can be completely removed, the surface energy of the glass can be increased, and the glass surface can be cleaned and activated, so that a hydrophilic layer 12 is formed on the side of the glass substrate 11 facing away from the conductive layer 2.

[0058] Alternatively, a hydrophilic material may be coated on the side of the glass substrate 11 facing away from the conductive layer 2 to form a hydrophilic layer.

[0059] Then, step S40 is performed to form a hydrophobic layer 3 on the side of the hydrophilic layer 12 facing away from the conductive layer 2 .

[0060] Reference Figure 6 A layer of hydrophobic material, such as Teflon or Cytop, is spin-coated or spray-coated on the side of the hydrophilic layer 12 facing away from the conductive layer 2. After heating and curing, a dense hydrophobic film layer 32 is formed. Cytop is a trade name for perfluoro (1-butenyl vinyl ether) polymer, which is a non-crystalline, highly transparent fluorine-containing polymer.

[0061] Reference Figure 7A mask (not shown) is used to cover a portion of the hydrophobic film layer 32 , and a second plasma is used to bombard the portion of the hydrophobic film layer 32 not covered by the mask, thereby forming a hydrophobic layer 3 having a plurality of through holes 31 .

[0062] In this embodiment, a plasma etcher may be used to process the hydrophobic film layer 32 with a second plasma to obtain a hydrophobic layer 3 having a plurality of through holes 31 arranged in an array.

[0063] Embodiments of the present invention also provide a detection method for a chip used for time-of-flight mass spectrometry. Each through-hole 31 of the chip can also be referred to as a detection site. First, a certain amount of matrix solution is dripped onto the detection site. Then, using a spotter, 1-500 nL of sample is dripped onto the detection site, dispersing the sample macromolecules within the small molecule matrix. The chip, with the matrix solution and sample, is then fed into a mass spectrometer. When a pulsed laser irradiates the detection site, the matrix absorbs energy and transfers it to the sample molecules, ionizing them and accelerating them under the action of a strong electric field. The mass spectrometer calculates the sample's mass spectrum based on the ions' time of flight in the tube, enabling accurate identification of biomacromolecules.

[0064] The chip for time-of-flight mass spectrometry detection provided by the embodiment of the present invention is suitable for detecting specific proteins or nucleic acid molecules.

[0065] In any of the above embodiments of the present invention, if the hydrophilic layer 12 of the chip is formed by bombarding the surface of the glass substrate 11 facing away from the conductive layer 2 with the first plasma, the hydrophilic layer 12 is actually the surface of the glass substrate 11. In order to clearly illustrate the structure of the hydrophilic layer 12 relative to other film layers, Figure 1 、 Figures 3 to 7 The hydrophilic layer 12 is schematically distinguished from the glass substrate 11. Therefore, it can be understood that the drawings in the present invention are used to illustrate the structure of the chip and are not completely equivalent to the actual structure of the chip.

[0066] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0067] The present invention uses terms such as "first," "second," and so on to describe various types of information, but such information should not be limited to these terms. These terms are used solely to distinguish information of the same type from one another. For example, without departing from the scope of the present invention, first information could also be referred to as second information, and similarly, second information could also be referred to as first information.

[0068] The above description is merely an illustration of the preferred embodiments of the present invention and the technical principles employed. Those skilled in the art should understand that the scope of the invention is not limited to the technical solutions formed by the specific combination of the above-mentioned technical features, but also encompasses other technical solutions formed by any combination of the above-mentioned technical features or their equivalents without departing from the inventive concept. For example, a technical solution formed by replacing the above-mentioned features with (but not limited to) technical features with similar functions disclosed in the present invention.

Claims

1. A chip for time-of-flight mass spectrometry detection, characterized in that: The invention comprises: a base layer, a conductive layer is provided on one side of the base layer, a hydrophobic layer is provided on the side of the base layer away from the conductive layer, and the hydrophobic layer has a plurality of through holes arranged in an array; The base layer comprises a glass substrate, and a side of the glass substrate facing away from the conductive layer is hydroxylated to form a hydrophilic layer; The conductive layer is made of indium tin oxide and is formed on one side of the glass substrate by a magnetron sputtering process. The magnetron sputtering power of the magnetron sputtering process is 80 watts to 120 watts, the pressure is 0.5 Pa-0.9 Pa, and the sputtering time is 15 min to 25 min.

2. The chip for time-of-flight mass spectrometry detection according to claim 1, characterized in that: The water drop angle of the hydrophilic layer is less than 60°, and the water drop angle of the hydrophobic layer is greater than 100°.

3. The chip for time-of-flight mass spectrometry detection according to claim 2, characterized in that: The water drop angle of the hydrophilic layer is less than 20°, and the water drop angle of the hydrophobic layer is greater than 140°.

4. The chip for time-of-flight mass spectrometry detection according to claim 1, characterized in that: The diameter of the through hole is 200um-1mm.

5. The chip for time-of-flight mass spectrometry detection according to claim 1, characterized in that: The glass substrate is made of high borosilicate glass, and the thickness of the glass substrate is 0.2 mm to 1 mm.

6. The chip for time-of-flight mass spectrometry detection according to claim 5, characterized in that: The thickness of the glass substrate is 0.5 mm.

7. A method for preparing a chip for time-of-flight mass spectrometry detection according to any one of claims 1 to 6, characterized in that: include: providing a glass substrate; forming a conductive layer on one side of the glass substrate; forming a hydrophilic layer on a side of the glass substrate facing away from the conductive layer; A hydrophobic layer is formed on a side of the hydrophilic layer facing away from the conductive layer.

8. The method for preparing a chip for time-of-flight mass spectrometry detection according to claim 7, characterized in that: The forming of a hydrophilic layer on a side of the glass substrate facing away from the conductive layer comprises: A first plasma is used to bombard and modify the side of the glass substrate facing away from the conductive layer to form a hydrophilic layer.

9. The method for preparing a chip for time-of-flight mass spectrometry detection according to claim 7, characterized in that: The forming of a hydrophobic layer on a side of the hydrophilic layer facing away from the conductive layer comprises: Coating a hydrophobic material on the side of the hydrophilic layer facing away from the conductive layer to form a hydrophobic film layer; A mask is used to cover a portion of the hydrophobic film layer, and a second plasma is used to bombard a portion of the hydrophobic film layer not covered by the mask, thereby forming a hydrophobic layer with a plurality of through holes.

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