A cleaning method for a spotting needle of a chip spotter

Abstract

The application discloses a cleaning method for a spotting needle of a chip spotting instrument, and comprises the following steps: preparing a cleaning solution, wherein the cleaning solution comprises A liquid, B liquid and n groups of mixed liquid; and sequentially using the B liquid, the n groups of mixed liquid and the A liquid to clean the spotting needle; wherein the A liquid is 2-4% (V / V) Hellmanex III aqueous solution, the B liquid is 70-80% (V / V) isopropyl alcohol aqueous solution, the n groups of mixed liquid are obtained by mixing the A liquid and the B liquid and have different isopropyl alcohol concentrations, and n is greater than or equal to 1. The Hellmanex III aqueous solution is used as the A liquid and the isopropyl alcohol aqueous solution is used as the B liquid, so that the residual protein in the spotting needle can be better removed; meanwhile, the residual protein can be cleaned while avoiding the influence of the isopropyl alcohol solution on the next protein sample spotting, and SDS is not needed to be used, so that the salt crystallization is avoided to block the spotting needle.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for cleaning a spotting needle used in a chip spotting instrument. Background Art

[0002] The chip spotter is the main equipment used to prepare biochips, and can be divided into two types: non-contact inkjet and contact spotting. The GeSiM Nano-Plotter series spotter equipment is a piezoelectric micro-inkjet (non-contact) biochip spotting system based on picoliter-scale piezoelectric valveless jet printing technology. Its spotting needle is made of glass and silicone, has high biocompatibility, and has no mechanical physical valves. The sound waves generated by the piezoelectric chip can eject the biological sample out of the nozzle to the surface of the solid phase carrier, thereby leaving the biological sample on the surface of the solid phase carrier, and realizing switching between different biological samples without the risk of cross-contamination. During the spotting process, substances such as proteins are absorbed, and it is easy to produce water-insoluble solid crystals for a long time, and adsorb on the inner and outer walls of the spotting needle, thereby causing the spotting needle to be blocked, resulting in uneven spray points, scattered points, or even no spraying, affecting the spotting state of the spotting needle.

[0003] When cleaning a spotting needle, using only a 2-4% (v / v) Hellmanex III aqueous solution is difficult to dissolve water-insoluble solid crystals. Therefore, a 70-80% (v / v) isopropyl alcohol aqueous solution is used in combination. For example, Chinese patent CN114308854 A discloses a method for cleaning a spotting needle for a chip spotting instrument. This patent combines a 2-4% (v / v) Hellmanex III aqueous solution with a 1-10% (w / v) SDS aqueous solution as Solution A, and a 70-80% (v / v) isopropyl alcohol aqueous solution as Solution B. The cleaning process is designed to immerse the needle in each of Solution A and Solution B at least twice, and the immersion sequence is alternating, i.e., Solution A-Solution B-Solution A-Solution B. This immersion cleaning method achieves excellent decontamination effectiveness. However, the inventors found in actual operation that, on the one hand, SDS as a salt solution will cause salt crystals to form after long-term use, thereby clogging the spotting needle; on the other hand, the isopropanol aqueous solution will cause protein samples to denature, thereby affecting the spotting of the next protein sample.

[0004] It is therefore of great significance to provide a cleaning method that effectively cleans the water-insoluble crystals remaining on the wall of the spotting needle without affecting the next protein sample spotting, thereby solving the problem of poor spotting or damage caused by spotting needle blockage. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above technical deficiencies and propose a method for cleaning a spotting needle for a chip spotting instrument to solve the technical problems in the prior art of the existing cleaning methods causing spotting needle blockage, organic solvent residue, and damage to the spotting needle.

[0006] The present invention provides a method for cleaning a spotting needle for a chip spotting instrument, comprising the following steps:

[0007] Prepare a cleaning solution, wherein the cleaning solution includes solution A, solution B, and n mixed solutions;

[0008] The sample spotting needle is cleaned using the B solution, the n-group mixed solution, and the A solution in sequence; wherein,

[0009] The liquid A is a 2-4% (V / V) Hellmanex III aqueous solution, the liquid B is a 70-80% (V / V) isopropanol aqueous solution, and the n groups of mixed solutions are obtained by mixing the liquid A and the liquid B and have different isopropanol concentrations, and n≥1.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention uses a 2-4% (V / V) Hellmanex III aqueous solution as solution A and a 70-80% (V / V) isopropyl alcohol aqueous solution as solution B, thereby better removing residual protein in a spotting needle. At the same time, the 70-80% (V / V) isopropyl alcohol is gradiently diluted, so that the spotting needle is sequentially cleaned from a full 70-80% (V / V) isopropyl alcohol solution to a solution without 70-80% (V / V) isopropyl alcohol, thereby ensuring that residual protein can be cleaned while preventing the 70-80% (V / V) isopropyl alcohol solution from affecting the next protein sample spotting. Furthermore, no SDS is needed, thereby preventing the spotting needle from being clogged by salt crystals. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a schematic diagram of a cleaning process of an embodiment of a method for cleaning a spotting needle for a chip spotting instrument of the present invention;

[0013] Figure 2 This is a cleaning flow chart for Examples 1-4 of the present invention. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0015] See also Figure 1 The present invention provides a method for cleaning a spotting needle for a chip spotting instrument, comprising the following steps:

[0016] S1. Prepare a cleaning solution, wherein the cleaning solution includes liquid A, liquid B, and n groups of mixed liquids;

[0017] S2, sequentially using the B solution, the n-group mixed solution, and the A solution to clean the spotting needle; wherein,

[0018] The liquid A is a 2-4% (V / V) Hellmanex III aqueous solution (a cleaning agent produced by HELLMA Analytics, Germany), the liquid B is a 70-80% (V / V) isopropyl alcohol aqueous solution, and the n groups of mixed solutions are obtained by mixing the liquid A and the liquid B and have different isopropyl alcohol concentrations, and n≥1.

[0019] Hellmanex III is a weakly alkaline cleaning agent primarily composed of a surfactant and other raw materials. Its saponification and emulsification properties complement each other, ensuring the removal of dust particles while preventing the redeposition of dispersed particles. The surfactant's penetrating and wetting properties further remove residual matter. Isopropyl alcohol is a common cleaning agent that dissolves water-insoluble protein crystals in the spotting needle, preventing residual protein crystals from clogging the needle cavity. Both cleaning agents are non-corrosive to the needle tip and effectively clean stains.

[0020] The present invention adopts a method of gradient dilution of a 2-4% (V / V) Hellmanex III aqueous solution against a 70-80% (V / V) isopropanol aqueous solution followed by gradual washing, thereby ensuring that residual proteins can be washed while preventing the 70-80% (V / V) isopropanol solution from affecting the next protein sample spotting. Furthermore, no SDS is required, thereby preventing the formation of salt crystals that may clog the spotting needle.

[0021] In the present invention, the n groups of mixed solutions are cleaned in the order of decreasing isopropyl alcohol concentration.

[0022] In the present invention, the process of sequentially cleaning the sample needle using Solution B, the n mixed solutions, and Solution A includes: allowing the sample needle to absorb a sufficient amount of cleaning solution and then immerse it in the remaining cleaning solution of the same type. Furthermore, the volume of solution absorbed by the sample needle is 25-30 μl, and the immersion time is 300 seconds or more.

[0023] In the present invention, when cleaning the sample needle with a cleaning solution, after the cleaning with the previous cleaning solution is completed, the inner and outer walls of the sample needle are cleaned with pure water, and then the sample needle is cleaned with the next cleaning solution. Furthermore, each pure water cleaning time is 60 seconds or more.

[0024] In the present application, after the spotting needle is cleaned with the A liquid, the inner and outer walls of the spotting needle are further cleaned with pure water, and the cleaning time of the pure water is 160 seconds or more.

[0025] In some embodiments of the present application, the cleaning method of the spotting needle of the chip spotter comprises the following steps:

[0026] (I) Preparation of A liquid and B liquid:

[0027] A liquid: 2-4% (V / V) Hellmanex III aqueous solution;

[0028] B liquid: 70-80% (V / V) isopropanol aqueous solution.

[0029] (II) Preparation of mixed liquid:

[0030] The mixed liquid of the mth group is denoted as W m , W m is prepared by mixing a m volume fraction of A liquid and b m volume fraction of B liquid, wherein a m+1 >a m >0, 0<b m+1 <b m , and m≥1.

[0031] (III) Sample addition: sequentially add solution 1 to solution N in the 1th to Nth holes of the sample addition strip, respectively; wherein solution 1 is B liquid, solution 2 to N-1 are mixed liquids with decreasing isopropanol concentration, and solution N is A liquid.

[0032] (IV) Start the cleaning process, which is as follows:

[0033] (1) Clean the inner and outer walls of the spotting needle with pure water for 60 seconds;

[0034] (2) Move the spotting needle to the 1st hole of the sample addition strip to suck enough solution 1, and soak in the remaining solution 1 in the 1st hole for 300 seconds;

[0035] (3) Move the spotting needle to the Mth hole of the sample addition strip, and cyclically execute steps (1) and (2) until the A liquid cleaning is completed, then clean the inner and outer walls of the spotting needle with pure water, and terminate the cleaning.

[0036] Further, the volume of the solution sucked by the spotting needle is 30ul.

[0037] Further, the sample addition amount in each hole of the sample addition strip is 200ul.

[0038] In some more specific embodiments of the present application, n=2, and the mixed liquid is prepared as follows:

[0039] Prepare 4 solutions according to the following formula: full reagent B; 1 volume part reagent A + 2 volume parts reagent B; 2 volume parts reagent A + 1 volume part reagent B; full reagent A and mark them as solution 1, solution 2, solution 3, solution 4 in sequence.

[0040] In some more specific embodiments of the present invention, n=2, and the cleaning process is:

[0041] (1) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0042] (2) Move the sample needle to well 1 of the sample strip, draw 30 μl of solution 1, and immerse it in the remaining solution 1 in well 1 for 300 seconds;

[0043] (3) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0044] (4) Move the sample needle to well 2 of the sample strip, draw 30 μl of solution 2, and immerse it in the remaining solution 2 in well 2 for 300 s;

[0045] (5) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0046] (6) Move the sample needle to well 3 of the sample strip, draw 30 μl of solution 3, and immerse it in the remaining solution 3 in well 3 for 300 s;

[0047] (7) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0048] (8) Move the sample needle to well 4 of the sample strip, draw 30 μl of solution 4, and immerse it in the remaining solution 4 in well 4 for 300 s;

[0049] (9) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 160 seconds.

[0050] Example 1

[0051] This embodiment provides a method for cleaning a spotting needle for a chip spotting instrument, which is performed according to the following steps:

[0052] (1) Prepare two reagents in advance:

[0053] A: 2% (V / V) Hellmanex III aqueous solution (a cleaning agent produced by HELLMA Analytics, Germany).

[0054] B: 80% (V / V) isopropyl alcohol aqueous solution.

[0055] (2) Prepare the mixed solution of reagents A and B:

[0056] Reagent A and reagent B were prepared in the following order: full reagent B; 1 volume part reagent A + 2 volume parts reagent B; 2 volume parts reagent A + 1 volume part reagent B; full reagent A to prepare 4 solutions, and marked them as solution 1, solution 2, solution 3, solution 4 in sequence.

[0057] (3) Add sufficient amount of solution 1 to well 1 of the sample plate; add sufficient amount of solution 2 to well 2; add sufficient amount of solution 3 to well 3; and add sufficient amount of solution 4 to well 4.

[0058] (IV) The cleaning process is:

[0059] (1) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0060] (2) Move the sample needle to well 1 of the sample strip, draw 30 μl of solution 1, and immerse it in the remaining solution 1 in well 1 for 300 seconds;

[0061] (3) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0062] (4) Move the sample needle to well 2 of the sample strip, draw 30 μl of solution 2, and immerse it in the remaining solution 2 in well 2 for 300 s;

[0063] (5) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0064] (6) Move the sample needle to well 3 of the sample strip, draw 30 μl of solution 3, and immerse it in the remaining solution 3 in well 3 for 300 s;

[0065] (7) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0066] (8) Move the sample needle to well 4 of the sample strip, draw 30 μl of solution 4, and immerse it in the remaining solution 4 in well 4 for 300 s;

[0067] (9) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 160 seconds.

[0068] Example 2

[0069] This embodiment provides a method for cleaning a spotting needle for a chip spotting instrument, which is performed according to the following steps:

[0070] (1) Prepare two reagents in advance:

[0071] A: 3% (V / V) Hellmanex III aqueous solution (a cleaning agent produced by HELLMA Analytics, Germany).

[0072] B: 75% (V / V) isopropyl alcohol aqueous solution.

[0073] (2) Prepare the mixed solution of reagents A and B:

[0074] Reagent A and reagent B were prepared in the following order: full reagent B; 1 volume part reagent A + 2 volume parts reagent B; 2 volume parts reagent A + 1 volume part reagent B; full reagent A to prepare 4 solutions, and marked them as solution 1, solution 2, solution 3, solution 4 in sequence.

[0075] (3) Add sufficient amount of solution 1 to well 1 of the sample plate; add sufficient amount of solution 2 to well 2; add sufficient amount of solution 3 to well 3; and add sufficient amount of solution 4 to well 4.

[0076] (IV) The cleaning process is:

[0077] (1) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0078] (2) Move the sample needle to well 1 of the sample strip, draw 30 μl of solution 1, and immerse it in the remaining solution 1 in well 1 for 300 seconds;

[0079] (3) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0080] (4) Move the sample needle to well 2 of the sample strip, draw 30 μl of solution 2, and immerse it in the remaining solution 2 in well 2 for 300 s;

[0081] (5) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0082] (6) Move the sample needle to well 3 of the sample strip, draw 30 μl of solution 3, and immerse it in the remaining solution 3 in well 3 for 300 s;

[0083] (7) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0084] (8) Move the sample needle to well 4 of the sample strip, draw 30 μl of solution 4, and immerse it in the remaining solution 4 in well 4 for 300 s;

[0085] (9) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 160 seconds.

[0086] Example 3

[0087] This embodiment provides a method for cleaning a spotting needle for a chip spotting instrument, which is performed according to the following steps:

[0088] (1) Prepare two reagents in advance:

[0089] A: 4% (V / V) Hellmanex III aqueous solution (a cleaning agent produced by HELLMA Analytics, Germany).

[0090] B: 70% (V / V) isopropyl alcohol aqueous solution.

[0091] (2) Prepare the mixed solution of reagents A and B:

[0092] Reagent A and reagent B were prepared in the following order: full reagent B; 1 volume part reagent A + 2 volume parts reagent B; 2 volume parts reagent A + 1 volume part reagent B; full reagent A to prepare 4 solutions, and marked them as solution 1, solution 2, solution 3, solution 4 in sequence.

[0093] (3) Add sufficient amount of solution 1 to well 1 of the sample plate; add sufficient amount of solution 2 to well 2; add sufficient amount of solution 3 to well 3; and add sufficient amount of solution 4 to well 4.

[0094] (IV) The cleaning process is:

[0095] (1) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0096] (2) Move the sample needle to well 1 of the sample strip, draw 30 μl of solution 1, and immerse it in the remaining solution 1 in well 1 for 300 seconds;

[0097] (3) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0098] (4) Move the sample needle to well 2 of the sample strip, draw 30 μl of solution 2, and immerse it in the remaining solution 2 in well 2 for 300 s;

[0099] (5) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0100] (6) Move the sample needle to well 3 of the sample strip, draw 30 μl of solution 3, and immerse it in the remaining solution 3 in well 3 for 300 s;

[0101] (7) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds;

[0102] (8) Move the sample needle to well 4 of the sample strip, draw 30 μl of solution 4, and immerse it in the remaining solution 4 in well 4 for 300 s;

[0103] (9) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 160 seconds.

[0104] like Figure 2 As shown, a method for cleaning a spotting needle for a chip spotter designed in Examples 1 to 4 of the present invention has a cleaning process designed as follows: start the cycle - (purified water) cleans the spotting needle - aspirates (solution 1 / solution 2 / solution 3 / solution 4) - wait - end the cycle, after the cycle ends, clean the spotting needle (purified water) - return to a safe position.

[0105] The present invention performs gradient dilution of 70-80% (V / V) isopropyl alcohol, so that the spotting needle is cleaned in sequence from a full 70-80% (V / V) isopropyl alcohol solution to a solution without 70-80% (V / V) isopropyl alcohol. This ensures that residual proteins are cleaned while preventing the 70-80% (V / V) isopropyl alcohol solution from affecting the next protein sample spotting. Furthermore, no SDS is required, thereby preventing the spotting needle from being clogged by salt crystals.

[0106] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for cleaning a spotting needle for a chip spotting instrument, characterized in that: The following steps are involved: Preparation of Liquid A and Liquid B: Prepare mixed solution: The mixed solution of group m is recorded as W m , W m By a m Volume parts of liquid A and b m Volume parts of liquid B are mixed, where a m+1 >a m >0,0 m+1 m , m≥1;​​ Prepare a cleaning solution, wherein the cleaning solution includes solution A, solution B, and n mixed solutions; The sample spotting needle is cleaned using the B solution, the n-group mixed solution, and the A solution in sequence; wherein, The solution A is a 2-4% (V / V) Hellmanex III aqueous solution, the solution B is a 70-80% (V / V) isopropanol aqueous solution, and the n groups of mixed solutions are obtained by mixing the solution A and the solution B and have different isopropanol concentrations, where n ≥ 1; The n groups of mixed solutions are cleaned in the order of decreasing isopropyl alcohol concentration; Sample addition: Add solutions 1 to N into wells 1 to N of the sample plate, respectively; where solution 1 is solution B, solutions 2 to solution N-1 are mixtures with decreasing isopropanol concentrations, and solution N is solution A. Cleaning process: (1) Clean the inner and outer walls of the spotting needle with pure water; (2) The sample needle moves to well 1 of the sample plate to absorb solution 1 and then immerses in the remaining solution 1 in well 1; (3) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water; (4) The sample needle moves to well 2 of the sample strip to absorb solution 2 and immerse in the remaining solution 2 in well 2; (5) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water; (6) Repeat the following process: the sample needle moves to wells 3 to N-1 of the sample strip in sequence, absorbs the solution in the corresponding wells, and immerses it in the remaining solution in the corresponding wells; then the sample needle moves to the cleaning station and cleans the inner and outer walls of the sample needle with pure water; (7) The sample needle moves to the Nth hole of the sample plate to absorb solution N and immerse in the remaining solution N in the Nth hole; the sample needle moves to the cleaning station, and the inner and outer walls of the sample needle are cleaned with pure water to terminate the cleaning.

2. The method for cleaning a spotting needle for a chip spotting instrument according to claim 1, characterized in that: The process of sequentially using the solution B, the n mixed solutions, and the solution A to clean the spotting needle includes: allowing the spotting needle to absorb a sufficient amount of cleaning solution and then soaking it in the remaining cleaning solution of the same kind.

3. The method for cleaning a spotting needle for a chip spotting instrument according to claim 2, wherein: The volume of solution absorbed by the spotting needle is 25-30ul, and the immersion time is 300s or more.

4. The method for cleaning a spotting needle for a chip spotting instrument according to claim 1, wherein: In the process of cleaning the sample needle with the cleaning solution, after the cleaning with the previous cleaning solution is completed, the inner and outer walls of the sample needle are cleaned with pure water, and then the sample needle is cleaned with the next cleaning solution.

5. The method for cleaning a spotting needle for a chip spotting instrument according to claim 4, characterized in that: Each pure water cleaning time is 60s or more.

6. The method for cleaning a spotting needle for a chip spotting instrument according to claim 1, characterized in that: After cleaning the sample needle with solution A, the inner and outer walls of the sample needle need to be cleaned with pure water; the pure water cleaning time should be 160s or more.

7. The method for cleaning a spotting needle for a chip spotting instrument according to claim 1, characterized in that: The amount of sample added to each well of the sample strip is 200ul.

8. The method for cleaning a spotting needle for a chip spotting instrument according to claim 1, characterized in that: The n=2, and the cleaning process is: (1) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds; (2) Move the sample needle to well 1 of the sample strip, absorb 30 μl of solution 1, and immerse it in the remaining solution 1 in well 1 for 300 seconds; (3) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds; (4) Move the sample needle to well 2 of the sample strip, absorb 30 μl of solution 2, and immerse it in the remaining solution 2 in well 2 for 300 seconds; (5) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds; (6) Move the sample needle to well 3 of the sample strip, absorb 30 μl of solution 3, and immerse it in the remaining solution 3 in well 3 for 300 seconds; (7) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 60 seconds; (8) Move the sample needle to well 4 of the sample plate, absorb 30 μl of solution 4, and immerse it in the remaining solution 4 in well 4 for 300 seconds; (9) The sample needle moves to the cleaning station and the inner and outer walls of the sample needle are cleaned with pure water for 160 seconds.

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