CHIP SURFACE LINKER AND PREPARATION METHOD AND USE THEREFOR

Abstract

The present invention relates to the field of biochips, and provides a chip surface linker and a preparation method and a use therefor. The chip surface linker is obtained by means of applying a direct current voltage to an aromatic amine bonding molecule in the presence of an acid and a nitrite to cause a reaction with a chip surface to form a bonding molecular group connected the chip surface, and then using a functional molecular for reaction and modification to add a functional molecular group containing a hydroxyl group and an ester group. The chip surface linker obtained in the present invention is able to bond more stably with a chip surface, being stable in hot water and basic conditions, and features relatively good electrical conductivity, stability during energization, and resistance to organic solvents required for nucleic acid synthesis, and is thus very advantageous for subsequent nucleic acid synthesis and other uses.

Claims

1. A chip surface linker, wherein the linker is prepared by the following steps: step 1: applying a direct current voltage to cause an aromatic amine bonding molecule to react with a chip surface in the presence of an acid and a nitrite to form a bonding molecule group connected to the chip surface; and step 2: using a functional molecule for reaction and modification to obtain a linker containing a functional molecule group, wherein the functional molecule group contains a hydroxyl group and an ester group.

2. The chip surface linker according to claim 1, wherein the bonding molecule is an aniline substance.

3. The chip surface linker according to claim 1, wherein the bonding molecule is selected from p-aminophenylacetic acid, p-aminophenylethanol or p-aminophenylenediamine.

4. The chip surface linker according to claim 1, wherein the direct current voltage is a constant direct current voltage, and the direct current voltage applied is selected from 0.5 to 5.0 V, wherein the direct current voltage is applied for 10 to 50 min.

5. (canceled)

6. The chip surface linker according to of claim 1, wherein the nitrite is selected from sodium nitrite, potassium nitrite or calcium nitrite.

7-8. (canceled)

9. The chip surface linker according to claim 1, wherein the functional molecule is selected from a succinic anhydride modified base monomer, hydroxyethyl methacrylate, a succinic acid modified base monomer or an oxalic acid modified base monomer.

10. (canceled)

11. The chip surface linker according to of claim 1, wherein a connecting arm molecule group is further included between the bonding molecule group and the functional molecule group.

12. The chip surface linker according to claim 11, wherein the connecting arm molecule group is reacted with and connected to the bonding molecule group by a connecting arm molecule, and the functional molecule group is reacted with and connected to the connecting arm molecule group by a functional molecule.

13. The chip surface linker according to claim 12, wherein the connecting arm molecule is a diamine or diol substance.

14. The chip surface linker according to claim 12, wherein the connecting arm molecule is selected from ethylenediamine, hexamethylenediamine, decanediamine, 1,8-octanediamine, ethylene glycol, hexanediol, decanediol or 1,8-octanediol.

15-16. (canceled)

17. A preparation method of a chip surface linker, comprising the following steps: step 1: mixing an aromatic amine bonding molecule with an acid and a nitrite to obtain a mixed solution; step 2: bringing the mixed solution in step 1 into contact with a chip surface, and applying a direct current voltage for reaction to form a bonding molecule group connected to the chip surface; and step 3: reacting the reacted chip surface with a functional molecule to obtain a linker containing a functional molecule group, wherein the functional molecule group contains a hydroxyl group and an ester group.

18. The preparation method according to claim 17, wherein before step 3, the preparation method comprises bringing the reacted chip surface in step 2 into contact with a connecting arm molecule for reaction, so as to connect a connecting arm molecule group.

19. (canceled)

20. The preparation method according to claim 17, wherein the bonding molecule is selected from p-aminophenylacetic acid, p-aminophenylethanol or p-aminophenylenediamine.

21. The preparation method according to claim 17, wherein the direct current voltage is a constant direct current voltage, and the direct current voltage applied is selected from 0.5 to 5.0 V, wherein the direct current voltage is applied for 10 to 50 min.

22. (canceled)

23. The preparation method according to claim 17, wherein the nitrite is selected from sodium nitrite, potassium nitrite or calcium nitrite.

24. (canceled)

25. The preparation method according to claim 17, wherein the functional molecule is selected from a succinic anhydride modified base monomer, hydroxyethyl methacrylate, a succinic acid modified base monomer or an oxalic acid modified base monomer.

26. (canceled)

27. The preparation method according to claim 18, wherein the connecting arm molecule is a diamine or diol substance.

28. The preparation method according to claim 27, wherein the connecting arm molecule is selected from ethylenediamine, hexamethylenediamine, decanediamine, 1,8-octanediamine, ethylene glycol, hexanediol, decanediol or 1,8-octanediol.

29. The preparation method according to claim 17, wherein when the direct current voltage is applied for reaction in step 2, the temperature is 4° C. to 40° C.; and the reaction time is 10 to 50 min.

30. (canceled)

31. Use of the chip surface linker according to claim 1 in nucleic acid synthesis or chip kit preparation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 is a schematic diagram of the preparation of a chip surface linker and use thereof in DNA synthesis in Example 1 of the present invention, wherein (1) is a metal chip, (2) is the linker prepared by this method, and (3) is DNA synthesized on this linker;

[0049] FIG. 2 is a schematic diagram of the comparison of the stability of the chip surface linker prepared by the method in Example 1 of the present invention in hot water and that of a traditional linker;

[0050] FIG. 3 shows the stability of the chip surface linker prepared by the method in Example 1 of the present invention in nucleic acid synthesis;

[0051] FIG. 4 is a schematic diagram of hybridization experiment of the chip surface linker prepared by the method in Example 1 of the present invention after being used for DNA synthesis;

[0052] FIG. 5 is a schematic diagram of the cleavability of DNA synthesized by the chip surface linker prepared by the method in Example 1 of the present invention;

[0053] FIG. 6 is a schematic diagram of DNA synthesized by the chip surface linker prepared by the method in Example 1 of the present invention;

[0054] FIG. 7 is a gel electrophoresis diagram of DNA synthesized by the chip surface linker prepared by the method in Example 1 of the present invention, wherein the sample is DNA (120 nt) synthesized by the chip surface linker prepared in Example 1, and the control is DNA (120 nt) synthesized by the original linker chip disclosed in Example 1 of US 20060105355 A1, and ladder represents the standard (after electrophoresis, the positions of 50 nt, 75 nt, 150 nt, 200 nt and 300 nt nucleic acids can be displayed).

DETAILED DESCRIPTION OF EMBODIMENTS

[0055] The technical solution in the examples of the present disclosure will be clearly and completely described below: obviously, the examples described are merely some, rather than all, of the examples of the present disclosure. On the basis of the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without involving any inventive effort fall within the scope of protection of the present invention.

[0056] In order to further understand the present application, the chip surface linker and a preparation method and use thereof provided by the present application will be described in detail below in combination with examples. However, it should be understood that these examples are implemented on the premise of the technical solution of the present invention, and the detailed embodiments and specific operation processes are given only to further illustrate the features and advantages of the present invention, but not to limit the claims of the present invention. The protection scope of the present invention is also not limited to the examples described below.

Example 1: Preparation of Chip Linker and Use Thereof in DNA Synthesis

[0057] Specific Experimental Steps:

1. chip cleaning: as shown in FIG. 1a, a platinum chip is sequentially rinsed with distilled water for 5 times, ethanol for 5 times and distilled water for 3 times, and then the chip is soaked in prinaha solution (the volume ratio of H.sub.2SO.sub.4:H.sub.2O.sub.2=3:1), placed in an oven at 65° C. for 30 min, and the chip is rinsed with distilled water for 5 times and blow-dried with argon;
2. chip modification: 18.15 mg cold p-aminophenylacetic acid is taken and mixed with 15 mM hydrochloric acid (1820 uL H.sub.2O, 180 uL 0.5 M HCl) uniformly, 6.21 mg sodium nitrite is then added, and same is quickly shaken well;
3. this mixed liquid is quickly added onto the chip surface, 2.5 v of constant direct current voltage is applied to the chip, and the chip is left to stand for an electrically promoted reaction at room temperature for 20 min, and the chip modification reaction is as follows:

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4. the chip is taken out and rinsed with distilled water for 5 times, ethanol for 5 times, 15 mM hydrochloric acid for 5 times and distilled water for 3 times, and then blow-dried with argon;
5. the chip is immersed in 8.7 mg of a solution of 1,8-octanediamine in methanol (1 mL methanol, 1.53 mg NHS and 7.64 mg EDC dissolved in 100 uL water, wherein NHS is N-hydroxysuccinimide and EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and same is left to stand for reaction for 8 h, rinsed with distilled water for 5 times and blow-dried with nitrogen;
6. the chip is soaked in a succinic anhydride modified base monomer mixed solution (10.8 mg base monomer (adenine, thymine, guanine or cytosine), 1.53 mg NHS, 7.64 mg EDC dissolved in 100 uL water), and same is left to stand for reaction for 8 h;
7. the chip is sequentially rinsed with ethanol, acetone, ethanol and distilled water for 5 times and blow-dried with nitrogen to obtain this stable linker, as shown in FIG. 1b;
8. the linker is placed on a CustomArray chip synthesizer for DNA synthesis, as shown in FIG. 1c.

[0058] The schematic diagram of the linker prepared by the above steps is shown in FIG. 1d. The chip is sequentially connected to a bonding molecule, a connecting arm molecule and a functional molecule to obtain a chip linker, and the linker includes a bonding molecule group, a connecting arm molecule group and a functional molecule group. The prepared linker is then used for DNA synthesis (using the CustomArray chip synthesizer), the schematic diagram of the obtained effect is shown in FIG. 1c, and perfect DNA can be synthesized.

Example 2: Comparison of Stability of New and Old Linkers in Hot Water

[0059] After the linker is modified on the chip, it is placed in distilled water at 80° C. for 2 days, followed by DNA synthesis.

[0060] For an original linker, a chip is deposited with polyhydroxy micromolecules (see Example 1 in US 20060105355 A1), rinsed with distilled water and dried, and then placed in distilled water at 80° C. for 2 days and blow-dried with argon, then 33 nt DNA is synthesized on the chip by using a CustomArray chip synthesizer, and the chip is scanned on a chip scanner (CustomArray, GenePix4000B). As shown in FIG. 2a, DNA synthesis is impossible in most areas.

[0061] The new linker is prepared according to the method in Example 1 of the present invention. For the new linker, a chip is placed in distilled water at 80° C. for 2 days and blow-dried with argon, then 33 nt DNA is synthesized on the chip by using a CustomArray chip synthesizer, and the chip is scanned on a chip scanner (CustomArray, GenePix4000B). As shown in FIG. 2b, the DNA on the chip surface can still exist stably, indicating that the new linker has sufficient stability in hot water, which is of great value to the subsequent high-temperature hybridization of the chip surface or other detection applications.

Example 3: Stability of New Linker in Nucleic Acid Synthesis Application

[0062] After hot water treatment and DNA synthesis, the new linker is subjected to hot TE and basic solution treatment and then used for hybridization.

[0063] The new linker is prepared according to the method in Example 1 of the present invention. For the new linker, a chip is placed in distilled water at 80° C. for 2 days and blow-dried with argon, then 33 nt DNA is synthesized on the chip by using a CustomArray chip synthesizer, and the chip is scanned on a chip scanner. The results are shown in FIGS. 3a and 3b. Two DNA primers with fluorescence (100 pM each) are taken and hybridized with the DNA synthesized on the chip (at room temperature, hybridization reaction for 2 h), and the chip is rinsed with PBS buffer and scanned on a chip scanner. As shown in FIG. 3c, different fluorescent spots are displayed, which indicates that the two DNA primers can be hybridized with the DNA synthesized on the chip. The chip is then rinsed with 1 M NaOH solution to remove the hybridized primer, placed in TE buffer (a mixed solution of 5 mL of 1 M Tris-HCl Buffer with pH 8.0 and 1 mL of 0.5 M EDTA with pH 8.0) at 80° C. for 2 days, blow-dried with argon, and then scanned on a chip scanner. The result is shown in FIG. 3d. Two DNA primers with fluorescence (100 pM each) are taken and hybridized with the DNA synthesized on the chip (at room temperature, hybridization reaction for 2 h), and the chip is rinsed with PBS buffer and scanned on a chip scanner. As shown in FIG. 3e, different fluorescent spots are displayed, which indicates that the two DNA primers can be hybridized with the DNA synthesized on the chip. It indicates that the new linker has sufficient stability in hot water and is resistant to alkali and hot TE, which is of great value for the subsequent high-temperature hybridization of the chip surface or other detection applications.

Example 4: Stability of New Linker in Repeated Hybridization-Elution-Hybridization Applications

[0064] The new linker is prepared according to the method in Example 1 of the present invention. As shown in FIG. 4, after a chip is blow-dried with argon, 33 nt DNA is synthesized on the chip by using a CustomArray chip synthesizer, and the chip is scanned on a chip scanner. The result is shown in FIG. 4b. Two DNA primers with fluorescence (100 pM each) are taken and hybridized with the DNA synthesized on the chip (at room temperature, hybridization reaction for 2 h), and the chip is rinsed with PBS buffer and scanned on a chip scanner. As shown in FIG. 4c, different fluorescent spots are displayed. The chip is then rinsed with 100 mM NaOH solution to remove the hybridized primer, blow-dried with argon and scanned on a chip scanner. The result is shown in FIG. 4d. Two DNA primers with fluorescence (100 pM each) are taken and hybridized with the DNA synthesized on the chip (at room temperature, hybridization reaction for 2 h), and the chip is rinsed with PBS buffer and scanned on a chip scanner. As shown in FIG. 4e on the right, after multiple cycles, the chip can still be used for hybridization and has obvious fluorescence signals, which indicates that this linker is stable enough to withstand repeated hybridization and alkali-solution elution.

Example 5: Cleavability of Synthesized DNA from the Chip

[0065] The linker (FIG. 5a) is prepared by the method in Example 1 of the present invention, and then 120 nt DNA is synthesized on the chip by a CustomArray chip synthesizer (FIG. 5b). The formed linker contains a cleavage group (an ester group), which can cleave oligonucleotide (oligo) from the chip (16 h) under basic (ammonia) and heating condition (65° C.) to form free oligo. The chip is rinsed with water and then scanned by using a chip scanner (FIG. 5c), which indicates that the whole DNA is basically cleaved.

Example 6: DNA Quality Characterization of New Linker for DNA Synthesis

[0066] The linker is prepared by the method in Example 1 of the present invention, and then 120 nt DNA is synthesized on the chip by using a CustomArray chip synthesizer (as shown in FIG. 6). The formed linker contains a cleavage group (an ester group), which can cleave oligonucleotide (oligo) from the chip (16 h) under basic (ammonia) and heating condition (at 65° C.) to form a free oligonucleotide pool (oligo pool). The concentrations of the two chips determined by Thermo Scientific™ NanoDrop™ One Microvolume UV-Vis Spectrophotometers are 21.4 ng/uL and 19.4 ng/uL, which meets the requirements. Next, PCR amplification is carried out, and the resulting product is subjected to gel electrophoresis diagram analysis. As shown in FIG. 7, the size of the resulting product is similar to that of the standard product, and they are located at the same position, which indicates that the product synthesized by such linker is correct.

REFERENCES

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