CN116169005B

CN116169005B - Disposable hydrophobic nano-liter sample application needle head and sample application test method

Info

Publication number: CN116169005B
Application number: CN202310179779.7A
Authority: CN
Inventors: 沈杰晖; 黄昊; 林勤瑶; 张爱权
Original assignee: Xiamen Jinnuohua Scientific Instrument Co ltd
Current assignee: Xiamen Jinnuohua Scientific Instrument Co ltd
Priority date: 2023-02-28
Filing date: 2023-02-28
Publication date: 2023-07-28
Anticipated expiration: 2043-02-28
Also published as: CN116169005A

Abstract

The invention provides a disposable hydrophobic nano-liter sample application needle head and a sample application test method. The method comprises the following steps: s100, fixing the nanoliter sample application needle on a nanoliter sample application instrument, sucking 20-30nl diluted nucleic acid matrix, applying sample on a 96-hole silicon target, waiting for crystallization of the nucleic acid matrix, observing and recording the number of times N1 of moving and attaching the liquid beads to the side wall; s200, replacing the nanoliter sample application needle, sucking 20-30nl undiluted nucleic acid matrix, repeatedly applying sample on the crystallized 96-hole silicon target, waiting for secondary crystallization, observing and recording the number of times N2 of moving and attaching the liquid beads to the side wall; s300, replacing the nanoliter sample application needle, sucking 20-30nl undiluted nucleic acid matrix again, repeatedly applying sample on the crystallized 96-hole silicon target, waiting for the sample preparation of the three crystallization, observing and recording the number of times N3 of moving and attaching the liquid beads to the side wall; s400, let M= (N1+N2+N13)/3, judge whether M exceeds the setting value, if so, it is disqualified, otherwise, it is qualified.

Description

Disposable hydrophobic nano-liter sample application needle head and sample application test method

Technical Field

The invention relates to a disposable hydrophobic nano-liter sample application needle head and a sample application test method.

Background

In recent years, with the improvement of sensitivity, resolution and detection mass range of matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), they have been widely used in the field of biomolecule detection such as proteomics, genomics and microbiological detection. Compared with the traditional protein and nucleic acid detection means, the detection means based on MALDI-MS has the characteristics of high speed and high accuracy, and the MALDI-MS can detect biological macromolecules with the relative molecular mass of hundreds of thousands at the level of femtomoles to attomoles and only needs a few seconds at the same time. The MALDI-MS detection scheme has become a trend of detection of microorganisms and nucleic acid mass spectra in the future by virtue of its simple operation, high reproducibility and high accuracy.

The basic workflow of MALDI-MS can be divided into four parts, sample preparation and matrix selection, sample and matrix co-crystallization, mass spectrometry, data statistics and processing. The process of co-crystallization of the sample and the matrix directly determines the accuracy and sensitivity of the mass spectrum detection result, and the sample application of the trace sample is generally performed by a nanoliter sample application instrument. However, the condition that the liquid beads are attached to the side wall in a moving way cannot be avoided in the sample application process of the needle head of the existing nanoliter sample application instrument, so that accurate sample application of 20-30nl of nanoliter micro-sample cannot be realized.

Disclosure of Invention

The invention provides a disposable hydrophobic nano-liter sample application needle head and a sample application test method, which can effectively solve the problems.

The invention is realized in the following way:

the invention provides a disposable hydrophobic nano-liter sample application needle, which comprises the following components:

the inner diameter of the liquid drop end of the glass needle is 0.5-1.5 mm;

and the polydimethylsiloxane coating is formed on the inner surface and the outer surface of the glass needle.

The invention further provides a sample application test method of the disposable hydrophobic nano-liter sample application needle head, which comprises the following steps:

s100, fixing the nanoliter sample application needle on a nanoliter sample application instrument, sucking 20-30nl diluted nucleic acid matrix, applying sample on a 96-hole silicon target, waiting for crystallization of the nucleic acid matrix, observing and recording the number of times N1 of moving and attaching the liquid beads to the side wall;

s200, replacing the nanoliter sample application needle, sucking 20-30nl undiluted nucleic acid matrix, repeatedly applying sample on the crystallized 96-hole silicon target, waiting for secondary crystallization, observing and recording the number of times N2 of moving and attaching the liquid beads to the side wall;

s300, replacing the nanoliter sample application needle, sucking 20-30nl undiluted nucleic acid matrix again, repeatedly applying sample on the crystallized 96-hole silicon target, waiting for the sample preparation of the three crystallization, observing and recording the number of times N3 of moving and attaching the liquid beads to the side wall;

s400, let M= (N1+N2+N13)/3, judge whether M exceeds the setting value, if so, it is disqualified, otherwise, it is qualified.

The beneficial effects of the invention are as follows: the disposable nano-liter sample application needle head prepared by the invention can realize accurate sample application of nano-liter micro-sample with the minimum volume of 20-30 nl. Furthermore, the disposable nano-liter sample needle provided by the invention can prevent the liquid beads from moving and attaching to the side wall during the liquid dropping process by the treatment of the polydimethylsiloxane coating hydrophobic coating, and ensure that the liquid in the needle is balanced with the gravity thereof through the hydrophobicity. In addition, the disposable nano-liter sample needle provided by the invention has a large-aperture needle, so that the needle can be prevented from being blocked by saturated solution crystallization. Finally, the disposable nano-liter sample application needle provided by the invention is of disposable design, avoids repeated use, and ensures that samples are not cross-contaminated. Furthermore, according to the sample application test method provided by the invention, the diluted nucleic acid matrix and the sample application test of the nucleic acid matrix on the polydimethylsiloxane coating are adopted, so that whether the disposable nano liter sample application needle is qualified can be judged, and the daily test requirement is met.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of a method for preparing a disposable nanoliter sample needle according to an embodiment of the invention.

FIG. 2 is a photograph of a disposable nanoliter sample needle provided by an embodiment of the invention.

FIG. 3 is a flow chart of a sample application test method for a nucleic acid matrix using a disposable nanoliter sample application needle according to an embodiment of the invention.

FIG. 4 is a photograph of crystals after spotting of nucleic acid substrates according to examples of the present invention and prior art.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.

In the description of the present invention, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present invention, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.

Referring to fig. 1, a method for preparing a disposable nanoliter sample needle comprises the following steps:

s1, preparing a polydimethylsiloxane solution, wherein the polydimethylsiloxane solution is formed by dissolving polydimethylsiloxane in a volatile organic solvent;

s2, using the polydimethylsiloxane solution to treat the inner surface and the outer surface of the glass needle, wherein the inner diameter of the liquid drop end of the glass needle is 0.5-1.5 mm;

and S3, drying the treated glass needle to volatilize the organic solvent so as to form polydimethylsiloxane coatings on the inner surface and the outer surface of the glass needle and form the fully-hydrophobic disposable nano-liter sample application needle.

In step S1, the polydimethylsiloxane has a number average molecular weight of 10,000 to 20,000g/mol. This is because, when the number average molecular weight of the polydimethylsiloxane is too large, on the one hand, it is difficult to dissolve, and on the other hand, the higher viscosity easily results in clogging of the glass needle during the subsequent preparation process, or in thicker films affecting the liquid output of the nanoliter spotting needle. In addition, when the number average molecular weight of the polydimethylsiloxane is too small, it is difficult to form a continuous polydimethylsiloxane coating.

The volatile organic solvent is selected from n-hexane, n-heptane, tetrahydrofuran, cyclohexane, chloroform, dichloromethane or toluene. In the practical test process, the contact angle between solvents such as n-hexane, n-heptane, tetrahydrofuran, cyclohexane, chloroform, dichloromethane or toluene and glassware (neutral borosilicate glass, medical grade pipe) is above 25 degrees. However, it was found experimentally that mixing the solvent portions can reduce the contact angle of the mixed solvent. Preferably, the volatile organic solvent is selected from mixed solvents of normal hexane and tetrahydrofuran, and because the mixed solvents of chloroform and tetrahydrofuran have better wettability with glass, continuous liquid films, especially inner surfaces, can be formed on the inner surface and the outer surface of the glass needle. Through tests, the contact angle between the mixed solvent of chloroform and tetrahydrofuran and glass can reach about 15 degrees at the minimum, and the mixed solvent has good wettability with the glass. This is probably because chloroform and tetrahydrofuran are polar solvents, and the solvents interpenetrate molecules of different polarities in the process of mutual dissolution to strengthen the polarities, thereby improving the attraction and the binding force with the glass wall surface. More preferably, the volatile organic solvent is selected from mixed solvents of chloroform and tetrahydrofuran according to a ratio of 1:1-5. In various embodiments, the volatile organic solvent is selected from the group consisting of mixed solvents of chloroform and tetrahydrofuran in a ratio of 1:1, 1:2, 1:2.5, 1:3, 1:4, 1:5, respectively. Please refer to the following table 1, table 1 shows the contact angle test table with glass test when chloroform and tetrahydrofuran are mixed according to different ratios. As can be seen from the following Table 1, when the ratio of chloroform to tetrahydrofuran is 1:2.5-1:3, the contact angle between chloroform and tetrahydrofuran and the glass wall surface can reach about 15 degrees which is the smallest, and the contact angle between the mixed solvent of chloroform and tetrahydrofuran and the glass wall surface is increased to a certain extent along with the increase or decrease of the ratio.

Table 1 shows contact angles of chloroform and tetrahydrofuran at different ratios with glass test

Proportion of

1:1

1:2

1:2.5

1:3

1:4

1:5

Contact angle

20°

16°

15°

18°

22°

In some embodiments, the concentration of the polydimethylsiloxane solution is from 1wt% to 10wt%. This is due to: when the concentration of the polydimethylsiloxane solution is too high, the viscosity of the polydimethylsiloxane solution is high, so that the glass needle is easily blocked in the subsequent preparation process, or the film forming is thick, and the liquid outlet of the nanoliter sample application needle is affected. In addition, when the concentration of the polydimethylsiloxane solution is too small, it is difficult to form a continuous polydimethylsiloxane coating. In other embodiments, the concentration of the polydimethylsiloxane solution (polydimethylsiloxane/tetrahydrofuran) is 1wt%, 2wt%, 4wt%, 5wt%, 6wt%, 8wt%, 10wt%, respectively.

In the step S2, the inner diameter of the liquid drop end of the glass needle is 0.5-1.5 mm. The outer diameter of the glass needle is not limited, and is preferably 0.8-1.8 mm. In other embodiments, the glass needle has a droplet end with an inner diameter of 0.8mm, 1.0mm, 1.2mm, respectively, and a corresponding outer diameter of 1.1mm, 1.3mm, 1.5mm, respectively. The length of the glass needle is not limited, and is generally about 5-10 cm.

As a further improvement, the step of treating the inner and outer surfaces of the glass needle with the polydimethylsiloxane solution includes:

s21, immersing the glass needle in the polydimethylsiloxane solution to enable the polydimethylsiloxane solution to fill the inner surface and the outer surface of the glass needle;

s22, taking out the immersed glass needle, and discharging redundant liquid in the glass needle.

In step S21, the glass needle is immersed in the polydimethylsiloxane solution for a period of 1 to 5 minutes. In one embodiment, the glass needle is immersed in the polydimethylsiloxane solution for a period of about 2 minutes. As a further improvement, since the diameter of the glass needle is small, it is difficult to completely fill the glass needle with the polydimethylsiloxane solution, and it is preferable that after immersing the glass needle in the polydimethylsiloxane solution, it is further possible to treat a negative pressure state above the polydimethylsiloxane solution by means of vacuum pumping, thereby evacuating air in the glass needle and allowing the polydimethylsiloxane solution to rapidly and completely fill the glass needle. Specifically, in one embodiment thereof, the polydimethylsiloxane solution is poured into a vacuum vessel, and then the glass needle is placed on a support and immersed in the polydimethylsiloxane solution, followed by vacuum evacuation, so that the polydimethylsiloxane solution and the air in the glass needle are discharged. By means of vacuumizing, part of air in the polydimethylsiloxane solution can be removed, so that the finally formed coating is more uniform. This is because the polydimethylsiloxane is difficult to dissolve, and thus stirring or the like is inevitably required during the dissolution, and thus bubbles are formed in the polydimethylsiloxane solution, thereby affecting the uniformity of the finally formed coating.

In step S22, the excess liquid inside the glass needle may be discharged by slow blowing or centrifugal separation. In one embodiment, the excess liquid inside the glass needle is expelled by blowing.

In step 3, the step of drying the treated glass needle to volatilize the organic solvent to form a polydimethylsiloxane coating on the inner and outer surfaces of the glass needle comprises the following steps:

and drying the treated glass needle at 50-100 ℃ for 10-30 minutes. In one embodiment, the treated glass needle is dried at 70 ℃ for 15 minutes.

After drying, the thickness of the polydimethylsiloxane coating formed on the inner surface and the outer surface of the glass needle is in the micron level, namely, the thickness is between 1 micron and 50 microns. Preferably, the polydimethylsiloxane coating has a thickness of between 5 microns and 25 microns. Since the formed polydimethylsiloxane coating is relatively easily dissolved in an organic solvent, the polydimethylsiloxane coating is easily dissolved and destroyed after use, and thus the formed polydimethylsiloxane coating is a disposable coating.

As a further improvement, in other embodiments, the method for preparing the disposable nanoliter sample application needle further comprises:

s4, assembling the disposable nano-liter sample application needle head and the plastic needle cap together, and packaging after disinfection. The plastic material can be a PP plastic material needle cap or other types of plastic materials, and is not limited herein, so long as the plastic material can play a role in fixing the needle.

Referring to fig. 2, an embodiment of the present invention further provides a disposable nano-liter sample application needle prepared by the above method, wherein the disposable nano-liter sample application needle comprises:

the inner diameter of the liquid drop end of the glass needle is 0.5-1.5 mm;

Wherein the inner diameter of the liquid drop end of the glass needle is 0.5-1.5 mm. The outer diameter of the glass needle is not limited, and is preferably 0.8-1.8 mm. The length of the glass needle is not limited, and is generally about 5-10 cm.

The thickness of the polydimethylsiloxane coating is between 1 and 50 microns. Preferably, the polydimethylsiloxane coating has a thickness of between 5 microns and 25 microns.

As a further improvement, the disposable nanoliter sample needle further comprises:

the plastic needle cap is arranged at the end of the glass needle head, which is far away from the liquid drop.

The disposable nano-liter sample application needle provided by the invention can realize nano-liter micro sample application with the minimum volume of 20-30 nl. Furthermore, the disposable nano-liter sample needle provided by the invention can prevent the liquid beads from moving and attaching to the side wall during the liquid dropping process by the treatment of the polydimethylsiloxane coating hydrophobic coating, and ensure that the liquid in the needle is balanced with the gravity thereof through the hydrophobicity. In addition, the disposable nano-liter sample needle provided by the invention has a large-aperture needle, so that the needle can be prevented from being blocked by saturated solution crystallization. Finally, the disposable nano-liter sample application needle provided by the invention is of disposable design, avoids repeated use, and ensures that samples are not cross-contaminated.

The embodiment of the invention further provides a sample application test method of the disposable nano-liter sample application needle for the nucleic acid matrix. The disposable nano-liter sample application needle head prepared by the invention is mainly used for sample application of nucleic acid matrix, so the invention mainly tests the performance of the disposable nano-liter sample application needle head by the sample application test method and judges whether partial liquid beads are moved and attached to the side wall in the sample application process.

Referring to fig. 3, the spotting test method includes the following steps:

In steps S100 and S200, the preparation method of the diluted nucleic acid matrix includes:

s101, dissolving fructose (fructose) and a nucleic acid matrix in a first solvent to form an undiluted nucleic acid matrix;

s102, mixing and diluting the undiluted nucleic acid matrix with a second solvent in proportion to form a diluted nucleic acid matrix.

In step S101, the main function of the fructose is to optimize crystallization to prevent the laser energy from being excessively large. The nucleic acid matrix is: the citric acid diamine is mixed with 3HPA (trihydroxy picolinic acid) to form. The first solvent is a mixed solvent of acetonitrile and water. The ratio of acetonitrile to water is 1:2-5, and in a plurality of embodiments, the ratio of acetonitrile to water in the mixed solvent is 1:2, 1:3, 1:4, 1:5 respectively. The concentration of the fructose is 1 g-1.5 g/100ml of the mixed solvent, namely, the 100ml of the mixed solvent contains 1 g-1.5 g of fructose, and in one embodiment, the concentration of the fructose is 1.15g/100ml of the mixed solvent. The concentration of the diamine citrate is 1 g-1.5 g/100ml mixed solvent, namely, the mixed solvent of 100ml contains 1 g-1.5 g of diamine citrate, and in one embodiment, the concentration of the diamine citrate is 1.24g/100ml mixed solvent. The concentration of the trihydroxy picolinic acid is 5 g-6 g/100ml, namely, the 100ml mixed solvent contains 5 g-6 g of the trihydroxy picolinic acid, and in one embodiment, the concentration of the trihydroxy picolinic acid is 5.84g/100 ml. Specifically, in one of the preferred embodiments I, the undiluted nucleic acid matrix comprises: acetonitrile (200 ul), water (600 ul), fructise (0.0092 g), diamine citrate (0.0099 g), 3HPA (0.0467 g). In one preferred embodiment, the undiluted nucleic acid matrix comprises: acetonitrile (160 ul), water (640 ul), fructise (0.0092 g), diamine citrate (0.0099 g), 3HPA (0.0467 g).

Experiments show that by selecting a mixture of acetonitrile and water as primary dilution solvent, a primary crystal layer with larger size, density and thickness can be produced. Referring to fig. 4, a is a photograph of a prior art after performing spotting primary crystallization using water as a primary dilution solvent, and as can be seen from fig. 4a, the size, density and thickness of crystals produced by performing spotting primary crystallization using water as a dilution solvent are small. FIG. 4c is a photograph of example I of the present invention after spotting and once crystallization using a mixed solvent of acetonitrile and water as a primary dilution solvent. As is apparent from fig. 4c, the size, density and thickness of crystals produced by spotting one-time crystallization using a mixed solvent of acetonitrile and water as a dilution solvent are significantly larger than those of the prior art. FIG. 4e is a photograph of example II of the present invention after spotting and once crystallization using a mixed solvent of acetonitrile and water as a primary dilution solvent. As is apparent from fig. 4e, the size, density and thickness of crystals produced by the spotting primary crystallization using a mixed solvent of acetonitrile and water as a dilution solvent are better than those of fig. 4 c.

In step S102, the second solvent is selected from acetonitrile solvents, or a mixture of acetonitrile and acetone, and experiments show that by selecting acetonitrile or a mixture of acetonitrile and acetone as the secondary dilution solvent, a secondary crystal layer with higher density and thickness can be produced. The ratio of the mixture of acetonitrile and acetone is not limited. Preferably, the undiluted nucleic acid matrix and the second solvent are mixed according to 1: mixing in the volume ratio of 5-10. In one preferred embodiment I, the undiluted nucleic acid matrix and the second solvent are mixed according to 1:9, i.e., 180ul acetonitrile at a 9:1 ratio: 20ul of nucleic acid matrix. In one preferred embodiment II, the undiluted nucleic acid matrix and the second solvent are mixed according to 1:9, i.e., 180ul (acetonitrile to acetone 1:1) diluted in a 9:1 ratio: 20ul of nucleic acid matrix.

Referring to fig. 4, b is a photograph of a prior art sample obtained by performing the sample application secondary crystallization using water as a secondary dilution solvent, and as can be seen from fig. 4b, the size, density and thickness of crystals produced by performing the sample application secondary crystallization using water as a dilution solvent are small. FIG. 4d is a photograph of example I of the present invention obtained by spotting and secondary crystallization using acetonitrile as a secondary dilution solvent. As is evident from fig. 4d, the size, density, thickness of crystals produced by spotting secondary crystallization using acetonitrile solvent as dilution solvent are significantly larger than in the prior art. FIG. 4f is a photograph of example II of the present invention obtained by spotting the sample using a mixed solvent of acetonitrile and acetone as a secondary dilution solvent. As is apparent from fig. 4f, the size, density, and thickness of crystals produced by spotting secondary crystallization using a mixed solvent of acetonitrile and acetone as a dilution solvent are better than those of fig. 4 f.

In step S400, the value of M is preferably 0 to 5. In one embodiment, M is 5, i.e., more than 5 is unacceptable, otherwise acceptable.

Example 1:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:2.5) to form a polydimethylsiloxane solution with the concentration of about 4 weight percent, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample A1.

Example 2:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:1) to form a polydimethylsiloxane solution with the concentration of about 4 weight percent, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample A2.

Example 3:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:2) to form a polydimethylsiloxane solution with the concentration of about 4 weight percent, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample A3.

Example 4:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:3) to form a polydimethylsiloxane solution with the concentration of about 4 weight percent, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample A4.

Example 5:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:4) to form a polydimethylsiloxane solution with the concentration of about 4 weight percent, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample A5.

Example 6:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:5) to form a polydimethylsiloxane solution with the concentration of about 4 weight percent, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample A6.

Test example 1:

fixing the nanoliter sample application needle head samples A1-A6 on a nanoliter sample application instrument, sucking 20-30nl diluted nucleic acid matrix, and applying sample on a 96-hole silicon target; replacing the nanoliter sample application needle, sucking 20-30nl undiluted nucleic acid matrix, repeatedly applying sample on the crystallized 96-hole silicon target, and waiting for secondary crystallization; and replacing the nanoliter sample application needle, sucking 20-30nl undiluted nucleic acid matrix again, repeatedly applying sample on the crystallized 96-hole silicon target, and waiting for the sample preparation to be completed after three times of crystallization. During spotting, observations were made to obtain the number of times the beads were moved to attach to the sidewall, 3 needles were taken per sample and the average recorded after the number of times was recorded as shown in table 2.

Table 2 is a sample application record of nanoliter sample application needle samples A1-A6

Sample of	A1	A2	A3	A4	A5	A6
							Number of times	0	3	2	1	2	4

As can be seen from table 2, in the spotting process performed on the 96-well silicon target, the situation where no bead moves to adhere to the sidewall occurred when the sample in which chloroform and tetrahydrofuran were mixed in a volume ratio of 1:2.5 was the best in the spotting process. With the ratio of chloroform to tetrahydrofuran, some of the beads were removed and attached to the sidewall during spotting on 96-well silicon targets.

Example 7:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:2.5) to form a polydimethylsiloxane solution with the concentration of about 1wt%, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample B1.

Example 8:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:2.5) to form a polydimethylsiloxane solution with the concentration of about 6 weight percent, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample B2.

Example 9:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:2.5) to form a polydimethylsiloxane solution with the concentration of about 10 weight percent, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample B3.

Comparative example 1:

dissolving polydimethylsiloxane with the number average molecular weight of 10,000-20,000 g/mol in a mixed solvent of chloroform and tetrahydrofuran (the chloroform and the tetrahydrofuran are mixed according to the volume ratio of 1:2.5) to form a polydimethylsiloxane solution with the concentration of about 12 weight percent, and pouring the polydimethylsiloxane solution into a vacuum container; placing a glass needle (neutral borosilicate glass with an inner diameter of 1.0mm, an outer diameter of 1.3mm and a length of 8 cm) on a bracket and vertically immersing in a polydimethylsiloxane solution; vacuumizing to exhaust the air of the glass needle head and soaking for 2 minutes; after the glass needle is taken out, discharging redundant liquid in the glass needle by a slow blowing mode; the final treated glass needle was dried at 70℃for 15 minutes and designated as sample C1.

Test example 2:

fixing the nanoliter sample application needle head samples A1, B1-A3 and C1 on a nanoliter sample application instrument, sucking 20-30nl diluted nucleic acid matrix, and applying sample on a 96-hole silicon target; replacing the nanoliter sample application needle, sucking 20-30nl undiluted nucleic acid matrix, repeatedly applying sample on the crystallized 96-hole silicon target, and waiting for secondary crystallization; and replacing the nanoliter sample application needle, sucking 20-30nl undiluted nucleic acid matrix again, repeatedly applying sample on the crystallized 96-hole silicon target, and waiting for the sample preparation to be completed after three times of crystallization. During spotting, observations were made to obtain the number of times the beads were moved to attach to the sidewall, 3 needles were taken for each sample and the average was taken after the number of times was recorded as shown in table 3.

Table 3 is a sample application record of nanoliter sample application needle samples A1, B1-A3

Sample of	A1	B1	B2	B3	C1
						Number of times	0	5	2	4	10

As can be seen from table 3, in performing spotting on 96-well silicon targets, when the polydimethylsiloxane solution was increased to 4wt% or more, the condition in which the beads were moved to be attached to the sidewalls began to increase. However, in the preparation of sample C1, since the polydimethylsiloxane was unevenly dispersed, the glass needle was easily clogged during the preparation, making it impossible to sufficiently feed the liquid into the inside of the needle tube or easily aggregate into clusters inside the needle tube to affect the effect, so that when the concentration was more than 10wt%, the movement of the liquid beads attached to the side wall was significantly increased.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations may 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

1. A method of sample application testing a disposable hydrophobic nano-liter sample application needle, the disposable hydrophobic nano-liter sample application needle comprising: the inner diameter of the liquid drop end of the glass needle is 0.5-1.5 mm; polydimethylsiloxane coatings formed on the inner and outer surfaces of the glass needle; characterized in that the method comprises the steps of:

2. The method for spotting a disposable hydrophobic nanoliter spotting needle of claim 1, wherein the method for preparing the diluted nucleic acid matrix comprises:

3. The method for testing the sample application of the disposable hydrophobic nano-liter sample application needle according to claim 1, wherein in the step S400, the M value is 0 to 5.

4. The sample application test method of the disposable hydrophobic nano-liter sample application needle head according to claim 1, wherein the outer diameter of the glass needle head is 0.8-1.8 mm; the length of the glass needle is 5-10 cm.

5. The method for spotting a disposable hydrophobic nanoliter spotting needle of claim 1 wherein the polydimethylsiloxane coating has a thickness of between 1 micron and 50 microns.

6. The method for spotting a disposable hydrophobic nano-liter spotting needle of claim 1 wherein the polydimethylsiloxane coating has a thickness of between 5 microns and 25 microns.

7. The method of spotting a disposable hydrophobic nanoliter spotting needle of claim 1, wherein the disposable nanoliter spotting needle further comprises:

8. The method for testing the sample application of the disposable hydrophobic nano-liter sample application needle according to claim 1, wherein the glass needle is made of neutral borosilicate glass.