SURFACE LINKER OF SEMICONDUCTOR CHIP, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF

Abstract

The present invention relates to the field of biochips, and provides a surface linker for a semiconductor chip, a preparation method therefor and an application thereof. The chip surface linker reacts with a chip surface by means of using silanized molecules as a solute and toluene as a solvent so as to form bonding molecules connected to the chip surface, and is prepared by reacting with functionalized molecules to modify a hydroxyl group and an ester group. The chip surface linker obtained by the present invention may be stably bonded to the chip surface, is stable under acidic and alkaline conditions, has good electrical conductivity, electrical stability and resistance to organic solvents required for nucleic acid synthesis, and is extremely advantageous for subsequent nucleic acid.

Claims

1. A chip surface linker, wherein the linker is prepared by using a silanized molecule to react with a surface of a chip to form a bonding molecule linked to the surface of the chip, and further causing the bonding molecule to react with a functional molecule and be modified with a hydroxyl group and an ester group.

2. The chip surface linker according to claim 1, wherein the silanized molecule is selected from one or more of 3-aminopropyltriethoxysilane, dodecyltrimethoxysilane, vinyltriethoxysilane, bis(γ-trimethylsilylpropyl)amine, or γ-(2,3-epoxypropoxy)propyltrimethoxysilane.

3. The chip surface linker according to claim 1, wherein the silanized molecule is reacted with the surface of the chip in a suitable solvent, and the solvent is selected from one or more of toluene, ethanol or methanol.

4. The chip surface linker according to claim 3, wherein a volume ratio of the solvent to the silanized molecule is 2:1 to 40:1.

5. The chip surface linker according to claim 1, wherein the silanized molecule is reacted with the surface of the chip at a temperature of 50° C. to 100° C.

6. The chip surface linker according to claim 1, wherein the silanized molecule is reacted with the surface of the chip for 1 to 6 h.

7. The chip surface linker according to claim 1, wherein the functional molecule is a hydroxyl-containing compound containing a long carbon chain and an ester group.

8. The chip surface linker according to claim 7, wherein the functional molecule is selected from one or more of a succinic anhydride-modified base monomer, hydroxyethyl methacrylate, a succinic acid modified base monomer, or an oxalic acid-modified base monomer, and is preferably a succinic anhydride-modified base monomer, wherein the base monomer moiety is selected from one or more of an adenine nucleoside, a guanine nucleoside, a cytosine nucleoside, a thymine nucleoside, or an uracil nucleoside.

9. The chip surface linker according to claim 1, wherein before the bonding molecule is reacted with and modified by the functional molecule, a step of placing in a drying device at 60° C. to 100° C. for 1 to 6 h to reinforce the bonding molecule linked to the surface of the chip is further comprised.

10. (canceled)

11. The chip surface linker according to claim 1, wherein the chip is a semiconductor chip, and wherein the surface of the semiconductor chip comprises TiN or TiW.

12. (canceled)

13. A method for preparing a chip surface linker, comprising following steps: step 1: mixing a silanized molecule with a suitable solvent in a volume ratio to obtain a mixed solution; step 2: contacting and reacting a chip with the mixed solution in the step 1 to form a bonding molecule linked to a surface of the chip; and step 3: contacting and reacting the surface of the chip linked to the bonding molecule after the reaction with a functional molecule to modify with a hydroxyl group and an ester group.

14. The method according to claim 13, wherein the silanized molecule is selected from one or more of 3-aminopropyltriethoxysilane, dodecyltrimethoxysilane, vinyltriethoxysilane, bis(γ-trimethylsilylpropyl)amine, or γ-(2,3-epoxypropoxy)propyltrimethoxysilane.

15. The method according to claim 13, wherein the solvent in the step 1 is selected from one or more of toluene, ethanol or methanol.

16. The method according to claim 13, wherein a volume ratio of the solvent to the silanized molecule is 2:1 to 40:1.

17. The method according to claim 13, wherein in the step 2, the surface of the chip is contacted and reacted with the mixed solution at 50° C. to 100° C. for 1 to 8 h.

18. The method according to claim 13, wherein the functional molecule in the step 3 is a hydroxyl-containing compound containing a long carbon chain and an ester group.

19. The method according to claim 18, wherein the functional molecule is selected from one or more of a succinic anhydride-modified base monomer, hydroxyethyl methacrylate, a succinic acid modified base monomer, or an oxalic acid-modified base monomer, and is preferably a succinic anhydride-modified base monomer, wherein the base monomer moiety is selected from one or more of an adenine nucleoside, a guanine nucleoside, a cytosine nucleoside, a thymine nucleoside, or an uracil nucleoside.

20. The method according to claim 13, wherein before the step 3, the method further comprises a step of placing in a drying device at 60° C. to 100° C. for 1 to 6 h to reinforce the bonding molecule linked to the surface of the chip.

21. (canceled)

22. The method according to claim 13, wherein the chip is a semiconductor chip-, wherein the surface of the semiconductor chip comprises TiN or TiW.

23. (canceled)

24. Use of the chip surface linker according to claim 1 for nucleic acid synthesis or preparation of chip kits.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 is a flowchart showing preparation of a TiN chip linker;

[0046] FIG. 2 is a diagram of a chromogenic test for feasibility of the TiN chip linker;

[0047] FIG. 3 is a diagram of a stability test against organic solvents of the TiN chip linker;

[0048] FIG. 4 is a diagram of a stability test against ammonia of the TiN chip linker;

[0049] FIG. 5 is a diagram of a stability test against acids and bases of the TiN chip linker; and

[0050] FIG. 6 is a diagram showing the results of application of the TiN chip linker in DNA synthesis and hybridization.

DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present invention are described clearly and completely below. Apparently, the embodiments described are merely some embodiments, rather than all of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention shall fall within the protection scope of the present invention.

[0052] In order to provide a further understanding of the present invention, the chip surface linker and the preparation method and use thereof provided in the present invention are described in detail below in conjunction with examples. However, it should be understood that these examples are implemented on the premise of the technical solutions of the present invention, detailed implementations and specific operating procedures are set forth only for the purpose of further explaining the features and advantages of the present invention rather than limiting the claims of the present invention, and the scope of protection of the present invention is also not limited to the following examples.

Example 1: Linker Preparation Test

[0053] Specific experimental steps for linker preparation are as follows.

[0054] 1. A TiN chip (from an IC factory) was rinsed 5 times with distilled water, 5 times with ethanol, 5 times with methanol, 5 times with distilled water, and blow dried with nitrogen.

[0055] 2. A mixture of toluene and APTES (3-aminopropyltriethoxysilane) (30:1, volume ratio) was formulated. The TiN chip was immersed in this mixture, and reacted at 65° C. for 4 h after sealing. The specific reaction is as follows:

##STR00002##

[0056] 3. The chip was taken out, placed in an oven at 90° C., cured at high temperature for 1 h, then washed with ethanol 3 times, washed with distilled water 3 times, and blow dried with nitrogen.

[0057] 4. The chip was immersed in a mixed solution of 10.8 mg of a succinic anhydride-modified base monomer (where the base monomer was adenine nucleoside, thymine nucleoside, guanine nucleoside or cytosine nucleoside), 1.53 mg of NHS, and 7.64 mg of EDC dissolved in 100 μL of water), and allowed to stand for 8 h.

[0058] 5. The chip was rinsed with ethanol, acetone, ethanol and distilled water 5 times in sequence, and blow dried with nitrogen to obtain this stable linker, as shown in FIG. 1. The linker includes a bonding molecule and a functional molecule, where the bonding molecule is a silanized molecule and the functional molecule is a molecule containing a hydroxyl (or protected hydroxyl) group and an ester group.

[0059] The linker is prepared on the surface of the TiN chip by the above method, rinsed with a large amount of ethanol, then rinsed with distilled water, and blow dried with nitrogen. 20 μL of trichloroacetic acid was added to the linker-modified chip surface. After 1 second, the trichloroacetic acid was quickly collected to a centrifuge tube. As shown in FIG. 2, a significant color change (slight red) was observed compared with the trichloroacetic acid stock solution. This is due to the chromogenic reaction of the ODMT group (4,4′-bimethoxytrityl, ODMT on the base molecule monomer) at the top of the linker with the trichloroacetic acid, indicating that the linker was successfully modified on the surface.

Example 2: Linker Stability Test

[0060] 2.1 Linker Stability Against Organic Solvents

[0061] In order to test the stability of the linker against organic solvents, particularly the stability of the silanized molecule that react with the TiN surface, the silanized molecule was first reacted with the surface of the TiN chip according to the method of Example 1 (not including the functional molecule modification step). Then the chip was immersed in an organic solvent (including 4,5-dicyanoimidazole, trichloroacetic acid, acetic anhydride, 1-methylimidazole, and iodine solution, in a volume ratio of 1:1:1:1:1) used for synthesizing an oligonucleotide, and allowed to stand at room temperature for two days. Then, after rinsing with ethanol and distilled water, a probe DNA-H1 (2 μM, Nanjing GenScript Biotech Co., Ltd.) was linked to the silanized molecule through the reaction of carboxyl and amino groups, and then probes DNA-H2 modified with cy5 and cy3 fluorescent molecules (10 nM, Nanjing GenScript Biotech Co., Ltd.) were hybridized with the DNA-H1, as shown in FIG. 3a. As shown in FIG. 3b, the chip hybridization design was divided into four parts, where the upper two parts were hybridizations, and the lower two parts were for comparison. After hybridization, it was scanned under a chip scanner (CustomArray, GenePix 4000B) (532 nm/635 nm). It can be found that obvious fluorescence was only observed in the hybridized portion, as shown in FIG. 3c, which also indicates that the silanized molecule in the first modification step was still stable on the surface of the TiN chip after being soaked with organic solvents, and can be used for subsequent reactions and applications.

TABLE-US-00001 The sequence of the DNA-H1 probe was: (SEQ ID NO: 1) 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTTTT-NH2-3′ The sequence of the DNA-H2-cy5 probe was: (SEQ ID NO: 2) 5′-TAGGGAAAGAGTGT-Cy5-3′ The sequence of the DNA-H2-cy3 probe was: (SEQ ID NO: 3) 5′-AGATCGGAAGAGCG-Cy3-3′ 2.2 Linker stability against ammonia

[0062] In order to test the stability of the linker against ammonia, particularly the stability of the silanized molecule that react with the TiN surface, the silanized molecule was first reacted with the TiN surface according to the method of Example 1 (not including the functional molecule modification step). Then the chip was immersed in 28% ammonia water, and allowed to stand at 65° C. for 16 h. Then, after rinsing with ethanol and distilled water, a probe DNA-H1 (2 μM, Nanjing GenScript Biotech Co., Ltd., the sequence of which was the same as that in Example 2.1) was linked to the silanized molecule through the reaction of carboxyl and amino groups, and then probes DNA-H2 modified with cy5 and cy3 fluorescent molecules (10 nM, Nanjing GenScript Biotech Co., Ltd., the sequences of the two probes being the same as those of DNA-H2 in Example 2.1) were hybridized with the DNA-H1, as shown in FIG. 4a. As shown in FIG. 4b, the chip hybridization design was divided into four parts, where the upper two parts were hybridizations, and the lower two parts were for comparison. After hybridization, it was scanned under a chip scanner (532 nm/635 nm). It can be found that obvious fluorescence was only observed in the hybridized portion, as shown in FIG. 4c, which also indicates that the silanized molecule in the first modification step was still stable on the surface of the TiN chip after being soaked with the ammonia water, and can be used for subsequent reactions and applications.

[0063] 2.3 Linker Stability Against Acids and Bases

[0064] In order to test the stability of the linker against acids and bases, particularly the stability of the silanized molecule that react with the TiN surface, the silanized molecule was first reacted with the TiN surface according to the method of Example 1 (not including the functional molecule modification step). Then the chip was immersed in 0.5 M sodium hydroxide solution for 0.5 h, rinsed with distilled water, and then placed in 0.5 M hydrochloric acid solution for 0.5 h. Then, after rinsing with distilled water, a probe DNA-H1 (2 μM, Nanjing GenScript Biotech Co., Ltd., the sequence of which was the same as SEQ ID NO:1 in Example 2.1) was linked to the silanized molecule through the reaction of carboxyl and amino groups, and then probes DNA-H2 modified with cy5 and cy3 fluorescent molecules (10 nM, Nanjing GenScript Biotech Co., Ltd., the sequences of the two probes being the same as those of DNA-H2 in Example 2.1, i.e., SEQ ID NO:2 and SEQ ID NO:3) were hybridized with the DNA-H1, as shown in FIG. 5a. As shown in FIG. 5b, the chip hybridization design was divided into four parts, where the upper two parts were hybridizations, and the lower two parts were for comparison. After hybridization, it was scanned under a chip scanner. It can be found that obvious fluorescence was only observed in the hybridized portion, as shown in FIG. 5c, which also indicates that the silanized molecule in the first modification step was still stable on the surface of the TiN chip after being soaked with strong bases and strong acids, and can be used for subsequent reactions and applications.

Example 3: Use of Linker in DNA Synthesis and Hybridization

[0065] DNA was synthesized on a TiN chip modified with this linker. The TiN chip was divided into two parts, where the upper part is modified with the linker, and the lower part is not modified with the linker, as a blank control, as shown in FIG. 6a. This chip was placed on a CustomArray chip synthesizer. Reagents for DNA synthesis were injected one by one into the surface of the chip by using, for example, a method of column synthesis (see Reference [4]), to synthesize 38 nt DNA (the sequence of which was the same as SEQ ID NO: 1 of the DNA-H1 in Example 2.1). After the synthesis, the chip was scanned on a chip scanner. As shown in FIG. 6b, the results show that a significant color change was observed in the linker-modified part and no color change was observed in the blank part, indicating that the DNA synthesis was successful. In order to verify whether the synthesized DNA is the desired DNA, a DNA probe modified with a cy3 fluorescent molecule (100 μM, Nanjing GenScript Biotech Co., Ltd., the sequence of the probe being the same as SEQ ID NO: 3 of the DNA-H2-cy3 in Example 2.1) was hybridized with the synthesized DNA. After hybridization, the chip was scanned on a chip scanner. As shown in FIG. 6c, the results show that a significant red light was observed, indicating that the hybridization was successful, and the synthesized sequence on the chip was the target sequence. This further proves that this linker can be used for in situ synthesis of DNA.

REFERENCES

[0066] 1. Malki, Maayan, et al. “Thin electroless Co (W, P) film growth on titanium-nitride layer modified by self-assembled monolayer.” Surface and Coatings Technology 252 (2014): 1-7. [0067] 2. Zeb, Gul, et al. “On the chemical grafting of titanium nitride by diazonium chemistry.” RSC Advances 5.62 (2015): 50298-50305. [0068] 3. Qiu, Guangyu, Siu Pang Ng, and Chi-Man Lawrence Wu. “Label-free surface plasmon resonance biosensing with titanium nitride thin film.” Biosensors and Bioelectronics 106 (2018): 129-135. [0069] 4. Lönnberg H. Synthesis of oligonucleotides on a soluble support[J]. Beilstein journal of organic chemistry, 2017, 13(1): 1368-1387.