Electrochemical DNA synthesis and control method based on updatable metal substrate

Abstract

The invention discloses an electrochemical DNA synthesis and control method based on an updatable metal substrate, which comprises the following steps: preparing a liquid metal microelectrode or an electrode array, and carrying out electrochemical DNA synthesis on the surface of the electrode; after the phase change of the obtained electrode, the endocytosis of the DNA molecules on the surface of the electrode is realized by manually or automatically mechanically extruding the liquid metal, so that electrochemical synthesis and in-situ packaging are completed; the liquid metal oxide layer is removed to release internal molecules; after the liquid metal is extruded out, new liquid metal flows to the original position, or after the liquid metal is taken out of the array, new liquid metal is added in the original position of the array, and electrode or electrode array updating is achieved and used for a new round of synthetic reaction and molecular manipulation. The method provided by the invention is simple and efficient, can realize updating of the electrode for DNA synthesis and integrated molecular control, has extremely high expandability, and provides a new thought for further development of a DNA synthesis platform and practical application in the field of DNA data storage.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an electrochemical DNA synthesis and manipulation method based on a renewable metal substrate. Background Art

[0002] The application of phosphoramidite chemistry has driven rapid development in the field of DNA synthesis and has become a common technique for commercial DNA synthesis. With technological advancements and innovations in various fields, a variety of array-based DNA synthesis technologies have emerged, such as inkjet printing, electrochemical synthesis, and photochemical synthesis. Among these, electrochemical methods stand out due to their ease of use and high sensitivity.

[0003] However, existing DNA synthesis methods typically use solid and rigid substrate arrays, including glass slides, silicon wafers, polymers, and metal electrodes that are complex to process and expensive. After DNA synthesis is completed on these substrate surfaces, it is difficult to update and reuse the substrates.

[0004] Although some studies have added a porous reaction layer to the electrode surface as a synthesis carrier, the removal and encapsulation of DNA molecules after synthesis is still very complicated. For example, chemical cutting agents can be used to remove DNA molecules, but this will also destroy the substrate performance. In addition, DNA encapsulation still requires the introduction of additional materials (such as silica, etc.). Not only are the steps cumbersome, but the release also requires additional chemical treatment (such as etching, etc.), and DNA synthesis has poor compatibility with subsequent molecular manipulation.

[0005] Therefore, the difficulty in updating and reusing synthetic substrates greatly reduces the scalability of DNA synthesis platforms and limits the further development of DNA synthesis technology. Summary of the Invention

[0006] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides an electrochemical DNA synthesis and manipulation method based on a renewable metal substrate. The present invention replaces traditional metal materials (gold, platinum, etc.) with low-temperature melting point liquid metal materials (such as gallium 29.8°C). The prepared electrode can realize electrochemical DNA synthesis after functional modification. The prepared electrode can be deformed, and the electrode can be renewed after solid-liquid phase change to complete a new round of synthesis and manipulation; at the same time, the present invention can realize rapid in-situ encapsulation of surface molecules through mechanical extrusion of liquid metal, and the internal molecules can be released by removing the oxide layer. In addition, the microfluidic chip prepared by the present invention can complete DNA encapsulation through automated mechanical extrusion, thereby improving the integration of DNA synthesis and manipulation and realizing integrated electrochemical DNA synthesis and molecular manipulation.

[0007] Technical Solution: To achieve the above objectives, the present invention provides a method for electrochemical DNA synthesis and manipulation based on a renewable metal substrate, comprising the following steps:

[0008] (1) preparing liquid metal microelectrodes or electrode arrays and performing electrochemical DNA synthesis on the electrode surface;

[0009] (2) After the electrode obtained in step (1) undergoes phase transformation, the liquid metal is squeezed manually or automatically to achieve internalization of the DNA molecules on its surface, thereby completing electrochemical synthesis and in situ encapsulation; and the liquid metal oxide layer is removed to achieve release of the internal molecules;

[0010] (3) After the liquid metal is squeezed out, new liquid metal flows to the original position, or the liquid metal is taken out of the array and new liquid metal is added to the original position of the array to update the electrode or electrode array and use it for a new round of synthesis reaction and molecular manipulation.

[0011] Among them, utilizing the properties of liquid metal, during the mechanical extrusion process, the liquid metal is continuously exposed to the air, the degree of oxidation continues to increase, the oxide layer infiltrates the surface molecules, and in the process of continuous expansion and contraction, the DNA molecules are internalized into the metal.

[0012] Among them, by utilizing the properties of liquid metal, adding NaOH solution and vortexing it at the same time can remove the oxide layer on the surface of the liquid metal, as well as the oxide layer internalized by molecular infiltration during the extrusion process, and release the DNA molecules from the inside of the metal.

[0013] Wherein, the liquid metal in step (1) is one or more of gallium, indium, tin, bismuth, zinc, and lead-based alloys, or one or more of binary, ternary, or quaternary alloys of gallium, indium, tin, and bismuth.

[0014] Preferably, the liquid metal material is gallium. The metals of the present invention are all low-temperature melting point liquid metals.

[0015] Wherein, the preparation of the liquid metal microelectrode or electrode array in step (1) adopts polymer microtubes, including silicone tubes, polytetrafluoroethylene PTFE, polyvinylidene fluoride PVDF, polyvinyl fluoride PVC, polyethylene PE, polypropylene PP, polyurethane PU or polycarbonate PC.

[0016] Preferably, the microtube material is silicone or PTFE.

[0017] Furthermore, the diameter of the microtubular liquid metal electrode is 0.5 to 2 mm.

[0018] Furthermore, the liquid metal electrode array is 4 x 1, the anode is metal gallium, the cathode is silver, and the electrode diameter is 1 to 1.5 mm.

[0019] The steps of electrochemical DNA synthesis on the electrode surface in step (1) include DNA coupling, oxidation, electrochemical deprotection and cleaning; the groups used for functional modification of the electrode surface before electrochemical DNA synthesis include any one or more of carboxyl (-COOH), amino (-NH2) or sulfhydryl (-SH).

[0020] Preferably, thiol groups are used for modification, and the solution used is mercaptohexanol solution (MCH).

[0021] Preferably, the coupling time of electrochemical DNA synthesis is 2 minutes, the oxidation time is 40 seconds, and the deprotection time is 14 seconds;

[0022] Wherein, in step (1), the surface of the liquid metal electrode is modified with DNA molecules by using a DNA chain with terminal linking groups, wherein the groups include carboxyl (-COOH), amino (-NH2), sulfhydryl (-SH), and hydroxyl (-OH).

[0023] Preferably, the DNA modification on the surface of the liquid metal electrode uses a DNA chain with a thiol group connected to the end.

[0024] In the present invention, DNA modification is to directly connect the entire commercially synthesized DNA chain to the metal surface. The DNA end generally has a thiol, carboxyl or amino group; and the DNA synthesis uses MCH, which has a thiol end and a hydroxyl end, and the phosphoramidite monomers are connected to the hydroxyl groups one by one.

[0025] Preferably, the applied voltage of the electrochemical reaction of the present invention is in the range of 0.48V to 1.7V, preferably 1.4V.

[0026] Furthermore, the specific application of the electrochemical synthesis can be polymers such as DNA and RNA that are composed of a finite number of monomers.

[0027] The metal phase change in step (2) is to transform the low-temperature melting point liquid metal from solid to liquid by heating.

[0028] Preferably, the temperature is raised to 30° C. to liquefy the metallic gallium.

[0029] The mechanical extrusion in step (2) is to manually and repeatedly squeeze the liquid metal in the microtube, and each squeezing ensures that the liquid metal breaks through the oxide layer and contacts the external air, and the number of squeezing times is not less than 10 times.

[0030] The mechanical extrusion in step (2) automatically squeezes the liquid metal in the channel through a microfluidic chip having an extrusion structure, transfers the liquid metal to the microfluidic chip through a microtube, and collects it at the chip outlet.

[0031] Preferably, a microfluidic chip with an extrusion channel structure is designed to automatically and mechanically squeeze the liquid metal in the channel.

[0032] Furthermore, the microfluidic chip is made of polydimethylsiloxane (PDMS), and the ratio of PDMS prepolymer to curing liquid is 10:1.

[0033] Furthermore, the microfluidic chip substrate is a glass slide or a silicon wafer, and the PDMS chip and the substrate are treated with a plasma cleaning machine (Plasma) for 1 minute, and then the bonding is completed.

[0034] Furthermore, the channel height of the microfluidic chip is 200 microns, the width is 800 microns, the extrusion channel width is 400 microns, the length is 1000 microns, and the number is 10.

[0035] The molecular endocytosis in step (2) refers to the transfer of DNA molecules from the surface to the interior of the metal after repeated manual mechanical squeezing of the liquid metal in the microtube or automated mechanical squeezing of the liquid metal by a chip.

[0036] In step (2), the molecular endocytosis refers to the transfer of DNA molecules from the surface to the interior of the metal after repeated mechanical squeezing of the liquid metal in the microtubule; the molecular release is the removal of the liquid metal electrode oxide layer by an acidic or alkaline solution, while achieving the denaturation of the DNA double strand; the acidic or alkaline solution includes low-concentration hydrochloric acid or sodium hydroxide.

[0037] In step (3), the electrode is updated so that after the liquid metal is squeezed out, the remaining liquid metal in the microtube is transferred to the tube mouth to become a new electrode; or the electrode array is updated so that after the liquid metal is taken out of the array, new liquid metal is re-added to the electrode array of the microfluidic chip to serve as a new electrode.

[0038] Wherein, the new liquid metal flowing to the in-situ in step (3) is the new liquid metal flowing to the orifice of the microtubular electrode, and the array in-situ is the surface of the chip electrode array.

[0039] In step (3), the electrode is updated as follows: after the liquid metal in the microtube is repeatedly squeezed and squeezed out, the remaining liquid metal in the microtube is transferred to the tube mouth to become a new electrode.

[0040] As a preference, Figure 1 As shown, the present invention provides an electrochemical DNA synthesis and manipulation method (single electrode) based on a renewable metal substrate, comprising the following steps:

[0041] (1) Preparation of liquid metal electrodes. Specifically, liquid gallium is injected into the microtube via a syringe and solidified for later use.

[0042] (2) The solid-state electrode obtained in step (1) is exposed to different reaction reagents to complete functional modification and electrochemical DNA synthesis. The synthesis reaction steps are: coupling reaction, oxidation reaction, and deprotection reaction. The above steps are repeated until all bases on the electrode are synthesized. The electrode is washed with anhydrous acetonitrile between reaction steps.

[0043] (3) heating the electrode after the reaction in step (2) to change it from a solid phase to a liquid phase, and manually and repeatedly mechanically squeezing it to cause it to continuously expand and contract in the air, so that the surface DNA molecules are internalized into the metal;

[0044] (4) After completing step (3), the liquid metal containing the DNA molecules is squeezed out, and the liquid metal in the tube is transferred to the tube mouth, generating a new electrode surface for a new round of DNA synthesis and manipulation.

[0045] As a preference, Figure 2 As shown, the electrochemical DNA synthesis and manipulation method (electrode array) based on a renewable metal substrate of the present invention includes the following steps:

[0046] (1) Prepare the glass substrate electrode array, PDMS chip and slider separately to complete the assembly of the microfluidic chip; gallium and silver paste are added to the electrode array of the microfluidic chip as the anode and cathode respectively.

[0047] (2) Slide the slider to move the coupling gel to the electrode surface to complete the coupling reaction. Specifically, the coupling reagent is adsorbed inside the gel and diffuses to the electrode surface after contact with the electrode. After the reaction, the electrode area is cleaned.

[0048] (3) Sliding the slider to move the oxidizing reagent to the electrode surface to complete the oxidation reaction, and then cleaning the electrode area and the slider channel after the reaction;

[0049] (4) Slide the slider to move the deprotection reagent to the electrode surface, apply voltage to complete the electrochemical deprotection reaction, and clean the electrode area and slider channel after the reaction;

[0050] (5) Cycle steps (2) to (4) to complete the synthesis of all bases;

[0051] (6) Heating the electrode array to liquefy the gallium electrode into liquid metal, and transferring the liquid metal to a channel with an extrusion structure through the upper slider to complete automated mechanical extrusion, so that the surface DNA molecules are internalized into the metal;

[0052] (7) Gallium is added back into the microfluidic chip electrode array as a new electrode for a new round of DNA synthesis and manipulation.

[0053] Furthermore, when a coupling reaction is carried out on the surface of the gallium electrode array of the microfluidic chip, the coupling reagent is adsorbed by the gel, and the gel contacts the electrode to complete the coupling reaction;

[0054] Preferably, the gel monomer used is N-isopropylacrylamide, the crosslinking agent is N,N-methylenebisacrylamide, the photoinitiator is 2-hydroxy-2-methylacetophenone, and the polymerization solution is a mixed solution of dimethyl sulfoxide and water;

[0055] This invention, based on a renewable metal substrate, involves the preparation of a liquid metal electrode, electrochemical DNA synthesis on the surface of the liquid metal electrode, and mechanical extrusion of the metal after a solid-liquid phase transition. The invention achieves electrochemical DNA synthesis using the liquid metal electrode as the substrate, and then mechanically extrudes the liquid metal manually or automatically to internalize the DNA molecules attached to the metal surface, achieving integrated DNA synthesis and packaging.

[0056] The present invention realizes electrochemical DNA synthesis based on a renewable liquid metal substrate. Compared with traditional rigid substrates, liquid metal has unique solid-liquid phase transition properties, is deformable, renewable, and reusable, and provides new ideas for further development in the field of DNA synthesis. The microfluidic chip designed in the present invention has a high degree of integration and can improve the efficiency of DNA synthesis. The designed liquid metal electrode array is scalable and can further reduce synthesis costs by increasing electrode flux.

[0057] The present invention realizes chemical DNA synthesis and encapsulation based on liquid metal. Traditional encapsulation steps are cumbersome and incompatible with synthesis reactions. For example, silica encapsulation takes three or four days, and release requires highly corrosive hydrofluoric acid, which poses certain risks to both DNA molecules and operators themselves. The present invention can achieve encapsulation through mechanical extrusion of liquid metal without the introduction of additional materials. In addition, the molecule can be released by removing the oxide layer with a low-concentration alkaline solution, which is simpler, more efficient and low-risk.

[0058] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0059] (1) The present invention proposes an electrochemical DNA synthesis and manipulation method based on a renewable metal substrate. By utilizing liquid metal with solid-liquid phase transition properties, the electrode can be renewed and reused. Compared with electrodes such as gold and platinum that are complex to process and difficult to reuse, the processing cost of the synthesis platform and the cost of DNA synthesis are effectively reduced, and the scalability of the DNA synthesis platform is improved.

[0060] (2) The present invention proposes an electrochemical DNA synthesis and manipulation method based on a renewable metal substrate, which uses liquid metal as a material to achieve integrated electrochemical DNA synthesis and in situ packaging without the need to introduce other reagents or materials, thereby achieving compatibility between synthesis and packaging operations.

[0061] (3) The present invention proposes an electrochemical DNA synthesis and manipulation method based on a renewable metal substrate, which uses a microfluidic chip with an extrusion structure to achieve automated mechanical extrusion DNA packaging. Compared with traditional packaging methods, this method is simpler, more efficient, and more automated, thus achieving integrated DNA synthesis and manipulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the electrochemical DNA synthesis and manipulation method based on a renewable metal substrate of the present invention (single electrode);

[0063] Figure 2 Schematic diagram of the electrochemical DNA synthesis and manipulation method based on a renewable metal substrate (electrode array) of the present invention;

[0064] Figure 3 The actual images of the microtubular gallium electrode and microfluidic chip gallium electrode array prepared by the present invention, as well as the extruded structure chip parameters;

[0065] Figure 4 This is the fluorescence image after the surface of the gallium electrode is modified with DNA and hybridized with the complementary fluorescent chain;

[0066] Figure 5 The relationship between the amount of DNA synthesised on the gallium electrode surface and the square root of time, as well as the difference in the amount of DNA synthesised on the gallium electrode array surface;

[0067] Figure 6 Fluorescence images after mechanical extrusion in air environment and oxygen-free environment

[0068] Figure 7 Fluorescence images of the liquid metal surface before and after manual and automated mechanical extrusion;

[0069] Figure 8 Schematic diagram of DNA molecule release after mechanical extrusion, and comparison of surface and internal DNA concentrations after manual and automated mechanical extrusion. DETAILED DESCRIPTION

[0070] The present invention will be further described below with reference to the accompanying drawings and examples.

[0071] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0072] Example 1

[0073] like Figure 3 As shown in a, the preparation steps of the microtubular gallium electrode used in the present invention are as follows:

[0074] (1) Take a 1 mm diameter silicone tube and clean it with deionized water and ethanol respectively;

[0075] (2) taking the microtube obtained in step (1), and injecting liquid metal gallium into the microtube;

[0076] (3) Connect the copper wire to the end of the microtube and place it at -20°C. Wait for the liquid metal electrode to solidify before use. The diameter of the microtubular liquid metal electrode is about 1 mm.

[0077] like Figure 3 As shown in Figures 3b and 3c, the preparation steps of the microfluidic chip used in the present invention are as follows:

[0078] (1) Design the electrode array pattern and process it into a carbon electrode array on a glass substrate ( Figure 3 the lowermost layer in b);

[0079] (2) Design a microfluidic chip and process it into a mold. Mix polydimethylsiloxane (PDMS) prepolymer and curing agent in a ratio of 10:1, remove bubbles by vacuum, pour into the mold, and place in an oven at 60°C for two hours. The inlet and outlet radius of the microfluidic chip is 500 μm, the channel height is 200 μm, and the width is 800 μm. The channel with an extruded structure is 400 μm wide and 1000 μm long, and the number is 10.

[0080] (3) Chip assembly: Bond the PDMS obtained in step (2) to the glass electrode obtained in step (1). The plasma cleaning time is 1 min.

[0081] (4) Electrode filling: Add metal gallium and silver paste to the chip electrodes, which serve as the anode and cathode of the reaction respectively;

[0082] (5) Superhydrophobic treatment of the extrusion channel: A superhydrophobic reagent (NC319, Changzhou Naroko New Material Technology Co., Ltd.) was injected into the PDMS chip obtained in step (2). The superhydrophobic reagent completely covered the chip and placed it in an oven at 60°C for half an hour. The liquid metal electrode array was 4 x 1, the anode was metal gallium, the cathode was silver, and the electrode diameter was about 1 mm.

[0083] Example 2

[0084] The steps for DNA modification and characterization on the surface of liquid metal electrodes are as follows:

[0085] (1) Take 10 μL of 10 mM Tris-HCl buffer (pH 8.5) and dissolve 1 μM DNA single strand with thiol groups at the end (5'-CACTTTGTACATTTTTT-SH-3');

[0086] (2) Add 1 μL of 5 μM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution to the solution obtained in step (1) and react at room temperature in the dark for 1 hour;

[0087] (3) placing the microtubular gallium electrode prepared in Example 1 into the solution after the reaction in step (2) and incubating at room temperature for 12 hours;

[0088] (4) The electrode obtained in step (3) was washed with 10 mM Tris-HCl buffer, and then placed in 10 μL of 20 mM MCH solution and incubated at room temperature for 1 hour;

[0089] (5) The electrode obtained in step (4) was washed with 10mM Tris-HCl buffer and then placed in 10μL of a 1μM fluorescent single chain (5'-ATGTACAAAGTG-Cy3-3', 5'-ATGTACAATGTG-Cy3-3') solution with a Cy3 molecule attached to the end. The reaction was carried out at room temperature in the dark for 1 hour. The electrode was then observed under a fluorescence microscope and the fluorescence intensity was calculated. The control group was DNA-unmodified; the matched group was DNA-modified and then hybridized with a complementary fluorescent chain; the mismatched group was DNA-modified and then hybridized with a base-mismatched fluorescent chain (not completely complementary, with a single base mismatch).

[0090] like Figure 4 As shown in the figure, compared with the control group, the matched group showed obvious fluorescence, indicating the successful modification and hybridization of thiol DNA; at the same time, the weakened fluorescence of the single-base mismatch group further confirmed the result.

[0091] Example 3

[0092] The synthesis and characterization of DNA on the gallium electrode surface are as follows:

[0093] (1) Mercaptohexanol modification: The microtubular gallium electrode prepared in Example 1 was placed in a 20 mM 6-mercapto-1-hexanol solution and incubated at room temperature for 12 h;

[0094] (2) Coupling reaction: The gallium electrode was placed in a coupling reagent (0.1 M different phosphoramidite monomers (ATGC, all T was used in this experiment) and 0.5 M activator 5-ethylthiotetrazolyl) and reacted at room temperature for 2 minutes. Anhydrous acetonitrile was used to clean the electrode surface;

[0095] (3) Oxidation reaction: The electrode obtained in step (2) was placed in an oxidizing agent (0.1 M trichloroperbenzoic acid) and reacted at room temperature for 40 seconds. Anhydrous acetonitrile was used to clean the electrode surface;

[0096] (4) Deprotection reaction: The electrode obtained in step (2) was placed in a deprotection reagent (25 mM hydroquinone, 25 mM p-benzoquinone, and 200 mM tetrabutylamine hexafluorophosphate), a voltage of 1.4 V was applied, and the reaction was carried out at room temperature for 14 seconds. Anhydrous acetonitrile was then used to clean the electrode surface;

[0097] (5) Repeat the above steps (2) to (4) 8 times until all bases on the electrode are synthesized (5'-TTTTTTTT-3');

[0098] (6) Weigh 0.012 g of hexaaminoruthenium trichloride (RuHex), dissolve it in 10 mM Tris-HCl (pH 8.3) to a final concentration of 20 mM in a volume of 2 mL, and further dilute it to 50 μM;

[0099] (7) Blowing nitrogen gas through the Tris-HCl buffer solution without hexaaminoruthenium trichloride added in step (6) and the 50 μM hexaaminoruthenium trichloride Tris-HCl buffer solution for 10 minutes respectively;

[0100] (8) The electrode obtained in step (5) was placed in a deoxygenated Tris-HCl buffer solution without hexaaminoruthenium trichloride and a Tris-HCl solution containing 50 μM hexaaminoruthenium trichloride, respectively. The chronocoulometry method was used for measurement. The starting voltage was 0.2 V, the ending voltage was -0.6 V, the number of steps was 2, the pulse duration was 0.25 s, and the measurement sensitivity was 1×10 - 5 A / V;

[0101] (9) The measurement results obtained in the above step (8) are used to make a curve of charge versus square root of time, and a fitting straight line is drawn to the curve to obtain the intercept difference, that is, the charge difference.

[0102] like Figure 5 As shown in a, the chrono-Coulomb curve shows a significant change in the Tris solution containing 50 μM RuHex (red curve), and the intercept of the extended lines of the two curves is greater than 0, and the charge difference is about 17.8 nC. This indicates that the added RuHex is partially adsorbed on the electrode surface through electrostatic interaction, generating a redox signal in addition to the double-layer capacitance charge, proving the successful synthesis of DNA on the gallium electrode surface.

[0103] Example 4

[0104] The synthesis and characterization of DNA on the surface of the gallium electrode array of the microfluidic chip are as follows:

[0105] (1) Mercaptohexanol modification: Prepare a 20 mM 6-mercapto-1-hexanol solution, add the solution dropwise onto the surface of the gallium electrode array prepared in Example 1, and incubate at room temperature for 12 hours;

[0106] (2) Gel preparation: Weigh 0.4 g of monomer N-isopropylacrylamide, 0.04 g of N,N-methylenebisacrylamide, 0.5 mL of deionized water, and 1.5 mL of dimethyl sulfoxide. Vortex at room temperature until completely dissolved to obtain a gel prepolymer solution. Then add 20 μL of photoinitiator 2-hydroxy-2-methylacetophenone and irradiate with a UV lamp (5W) for 45 seconds to obtain a gel. Then transfer the gel to a hot plate or oven and heat it at 80°C for more than 30 minutes to completely dry the gel.

[0107] (3) Slider assembly: Prepare a PDMS slider with a groove structure for storing gels and reagents. After the slider surface contacts silicone oil, it is assembled with the chip;

[0108] (4) Coupling reaction: Prepare coupling reagents (0.1 M different phosphoramidite monomers (ATGC) and 0.5 M activator 5-ethylthiotetrazolyl) and inject them into the microfluidic chip channel. The reagents come into contact with the dry gel at the outlet, and the gel gradually becomes saturated. Move the gel to the electrode array surface using a slider. React at room temperature for 2 minutes. Transfer anhydrous acetonitrile to the electrode array surface using the slider to complete the cleaning.

[0109] (5) Oxidation reaction: Prepare an oxidizing reagent (0.1 M trichloroperbenzoic acid) and inject it into the microfluidic channel. Move the oxidizing reagent to the electrode array surface via the slider. Allow the reaction to proceed at room temperature for 40 seconds. Then, transfer anhydrous acetonitrile to the electrode array surface via the slider to complete the cleaning.

[0110] (6) Deprotection reaction: Prepare deprotection reagent (25 mM hydroquinone, 25 mM p-benzoquinone, and 200 mM tetrabutylamine hexafluorophosphate), move the deprotection reagent to the electrode array surface via a slider, apply a voltage of 1.4 V, react at room temperature for 14 seconds, and then transfer anhydrous acetonitrile to the electrode array surface via a slider to complete the cleaning;

[0111] (7) Repeat the above steps (4) to (6) until all bases on the electrode are synthesized (the specific sequence is 5'-ACATACAGACACACAA-3', 5'-TCAGTGTATGATTCTC-3', 5'-TGCGTGCGTGGTTGGT-3', 5'-TCTGTGTTTCAGTGAT-3';

[0112] (8) Weigh 0.012 g of hexaaminoruthenium trichloride (RuHex), dissolve it in 10 mM Tris-HCl (pH 8.3) to a final concentration of 20 mM in a volume of 2 mL, and further dilute it to 50 μM.

[0113] (9) Blowing nitrogen gas through the Tris-HCl buffer solution without hexaaminoruthenium trichloride added in step (7) and the 50 μM hexaaminoruthenium trichloride Tris-HCl buffer solution for 10 minutes respectively;

[0114] (10) The deoxygenated Tris-HCl buffer solution without hexaaminoruthenium trichloride and the Tris-HCl solution containing 50 μM hexaaminoruthenium trichloride were successively added dropwise to the surface of the electrode array obtained in step (7). The chronocoulometry method was used for measurement, with a starting voltage of 0.2 V, an ending voltage of -0.6 V, two steps, a pulse duration of 0.25 s, and a measurement sensitivity of 1×10 -5 A / V;

[0115] (11) The measurement results obtained in the above step (10) are used to make a curve of charge versus square root of time, and a fitting straight line is drawn to the curve to obtain the intercept difference, that is, the charge difference.

[0116] like Figure 5 As shown in Figure 3b, the charge differences on the electrode array surface are approximately 65.1nC, 66.0nC, 60.7nC, and 64.7nC, respectively. This first indicates that the added RuHex is partially adsorbed on the electrode surface through electrostatic action, generating a redox signal in addition to the double-layer capacitance charge, that is, DNA is successfully synthesized on the gallium electrode array surface; secondly, the charge differences detected on the four electrode surfaces are close, proving the stability and reliability of parallel synthesis on the electrode array surface through the microfluidic chip.

[0117] Example 5

[0118] The mechanical extrusion effect in air environment and oxygen-free environment is characterized by the following steps:

[0119] (1) Take 10 μL of 10 mM Tris-HCl buffer (pH 8.5) and dissolve 1 μM DNA single strand with thiol groups at the end (5'-CACTTTGTACATTTTTT-SH-3');

[0120] (2) Add 1 μL of 5 μM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution to the solution obtained in step (1) and react at room temperature in the dark for 1 hour;

[0121] (3) placing the microtubular gallium electrode prepared in Example 1 into the solution after the reaction in step (2) and incubating at room temperature for 12 hours;

[0122] (4) The electrode obtained in step (3) was washed with 10 mM Tris-HCl buffer, and then placed in 10 μL of 20 mM MCH solution and incubated at room temperature for 1 hour;

[0123] (5) The electrode obtained in step (4) was washed with 10 mM Tris-HCl buffer and then placed in 10 μL of a 1 μM fluorescent single-chain (5'-ATGTACAATGTG-Cy3-3') solution with a Cy3 molecule attached to the end, and reacted in a dark room for 1 hour;

[0124] (6) Take the electrode obtained in step (5) and place it on a hot plate at 30°C for 30 seconds to complete the phase transition from solid to liquid;

[0125] (7) Take the electrode obtained in step (6) and manually and repeatedly squeeze the liquid metal in the microtube in an air environment. Ensure that the liquid metal breaks through the surface oxide layer each time. This can be observed with the naked eye to ensure that bright metal breaks through the dark oxide layer each time. Keep in contact with the outside air. Squeeze 10 times.

[0126] (8) Take the electrode obtained in step (6) and manually and repeatedly mechanically squeeze the liquid metal in the microtube in an oxygen-free environment (glove box, oxygen content less than 0.01 ppm), as in step (7);

[0127] (9) Take the electrodes obtained in steps (7) and (8) and observe them using a fluorescence microscope.

[0128] like Figure 6 As shown, after manually and repeatedly mechanically squeezing the liquid metal in the microtube in an air environment, the fluorescence intensity on the electrode surface was significantly reduced compared to the initial state. In contrast, after squeezing in an oxygen-free environment, the fluorescence intensity remained essentially unchanged. The results indicate that the oxide layer during the squeezing process plays a key role in the molecular endocytosis process, wetting the molecules and assisting in their endocytosis. This further demonstrates that the present invention primarily utilizes the properties of the liquid metal itself. During the continuous squeezing process, the degree of oxidation will continue to increase, enabling the endocytosis of surface molecules.

[0129] Example 6

[0130] Manual and automated mechanical extrusion of liquid metal to achieve molecular endocytosis, the steps are as follows:

[0131] (1) Take 10 μL of 10 mM Tris-HCl buffer (pH 8.5) and dissolve 1 μM DNA single strand with thiol groups at the end (5'-CACTTTGTACATTTTTT-SH-3');

[0132] (2) Add 1 μL of 5 μM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution to the solution obtained in step (1) and react at room temperature in the dark for 1 hour;

[0133] (3) placing the microtubular gallium electrode prepared in Example 1 in the solution after the reaction in step (2), or adding the solution dropwise to the surface of the electrode array prepared in Example 1, and incubating at room temperature for 12 hours;

[0134] (4) The electrode obtained in step (3) was washed with 10 mM Tris-HCl buffer, and then placed in 10 μL of 20 mM MCH solution and incubated at room temperature for 1 hour;

[0135] (5) The electrode obtained in step (4) was washed with 10 mM Tris-HCl buffer and then placed in 10 μL of a 1 μM fluorescent single-chain (5'-ATGTACAATGTG-Cy3-3') solution with a Cy3 molecule attached to the end, and reacted in a dark room for 1 hour;

[0136] (6) Take the electrode obtained in step (5) and place it on a hot plate at 30°C for 30 seconds to complete the phase transition from solid to liquid;

[0137] (7) Take the electrode obtained in step (6) and manually and repeatedly squeeze the liquid metal in the microtube. Each squeeze ensures that the liquid metal breaks through the surface oxide layer. This can be observed with the naked eye to ensure that bright metal breaks through the dark oxide layer each time. The electrode is continuously exposed to the outside air and squeezed 10 times. The degree of oxidation gradually increases, and the oxide layer infiltrates the surface molecules. The molecules are internalized during the repeated squeezing and contraction process.

[0138] (8) Alternatively, the electrode array obtained in step (6) is taken, and the microtube is connected to the inlet of the microfluidic chip of Example 1. The injection pump parameter is set to 500 μm / min. The liquid metal enters the chip through the inlet and undergoes continuous automated extrusion under the action of the injection pump to achieve endocytosis. Finally, the liquid metal is collected at the outlet.

[0139] like Figure 7 As shown in a, the electrode surface without squeezing operation showed obvious fluorescence, but after repeated mechanical squeezing, the surface fluorescence intensity was significantly weakened, indicating that the mechanical squeezing operation promoted the internalization of surface DNA molecules; Figure 7 As shown in b, the liquid metal surface before chip extrusion showed obvious fluorescence. After the chip automated extrusion, the fluorescence intensity was significantly weakened, indicating that the chip automated mechanical extrusion achieved effective internalization of surface DNA molecules.

[0140] Example 7

[0141] The steps of endocytosis and release of DNA molecules are as follows (e.g. Figure 8 a):

[0142] (1) Take 10 μL of 10 mM Tris-HCl buffer (pH 8.5) and dissolve 1 μM DNA single strand with thiol groups at the end (5'-CACTTTGTACATTTTTT-SH-3');

[0143] (2) Add 1 μL of 5 μM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution to the solution obtained in step (1) and react at room temperature in the dark for 1 hour;

[0144] (3) placing the microtubular gallium electrode prepared in Example 1 into the solution after the reaction in step (2) and incubating at room temperature for 12 hours;

[0145] (4) Add the solution from step (2) dropwise to the surface of the electrode array prepared in Example 1 and incubate at room temperature for 12 hours;

[0146] (5) washing the electrodes or electrode arrays obtained in steps (3) and (4) with 10 mM Tris-HCl buffer, and then placing them in 10 μL of 20 mM MCH solution and incubating at room temperature for 1 hour;

[0147] (6) washing the electrode or electrode array obtained in step (5) with 10 mM Tris-HCl buffer, then adding 1 μM complementary DNA strand (5'-ATGTACAAAGTG-3') solution and reacting at room temperature for 1 hour;

[0148] (7) The electrode or electrode array obtained in step (6) was washed with Tris-HCl buffer for more than three times, and then the electrode was placed on a 30°C hot plate and heated for 30 seconds to complete the phase transition from solid to liquid;

[0149] (8) Take the single electrode obtained in step (7), and manually and repeatedly squeeze the liquid metal in the microtube mechanically. The squeezing force ensures that the liquid metal breaks through the surface oxide layer and contacts the external air. The squeezing frequency is 10 times, and the DNA molecule is internalized during the continuous squeezing process.

[0150] (9) Taking the electrode array obtained in step (7), transferring the liquid metal to a microfluidic chip with an extrusion structure, completing automated mechanical extrusion, and achieving DNA molecule endocytosis;

[0151] Further, release of endocytosed DNA:

[0152] (10) Take the electrode or electrode array obtained in step (8) or (9), solidify it and place it in a 0.2 M NaOH solution for 5 minutes. The obtained supernatant is quantified as the surface DNA concentration; after washing the electrode at least three times with Tris-HCl buffer, place it on a 30°C hot plate to liquefy it, and then place it in a 0.2 M NaOH solution and vortex it for 5 minutes. The obtained supernatant is quantified as the internal DNA concentration;

[0153] (11) Take the supernatant obtained in step (10) and use Nanodrop 1000 TM Perform quantitative detection.

[0154] like Figure 8 As shown in Table b, the quantitative results after manual mechanical squeezing showed that the DNA concentration inside the liquid metal was 3.60 ng / μL, the DNA concentration on the surface was 0.62 ng / μL, and the blank control group was about 0.01 ng / μL. The internal DNA concentration was higher than that in the table, indicating that the DNA molecules were transferred to the liquid metal through manual mechanical squeezing and successfully released. Figure 8 As shown in Figure c, the ideal endocytosis effect was achieved and the DNA was successfully released through automated extrusion on the chip, with the internal DNA concentration being higher than that on the surface (3.86 ng / μL>0.80 ng / μL). In addition, the control group showed that the detection results of unmodified DNA and modified single-stranded DNA were significantly lower than those of the normal modification and hybridization experimental groups, indicating that the released chain was the complementary chain of the hybridization, rather than the single chain originally fixed on the surface, eliminating interference.

Claims

1. A method for electrochemical DNA synthesis and manipulation based on a renewable metal substrate, characterized in that: The steps include: (1) preparing liquid metal microelectrodes or electrode arrays and performing electrochemical DNA synthesis on the electrode surface; (2) After the electrode obtained in step (1) undergoes phase transformation, the liquid metal is squeezed manually or automatically to achieve internalization of the DNA molecules on its surface, thereby completing electrochemical synthesis and in situ encapsulation; and the liquid metal oxide layer is removed to achieve release of the internal molecules; (3) After the liquid metal is squeezed out, new liquid metal flows to the original position, or the liquid metal is taken out of the array and new liquid metal is added to the original position of the array to update the electrode or electrode array and use it for a new round of synthesis reaction and molecular manipulation.

2. The electrochemical DNA synthesis and manipulation method based on a renewable metal substrate according to claim 1, characterized in that: The liquid metal in step (1) is preferably one or more of gallium, indium, tin, bismuth, zinc, and lead-based alloys, or one or more of binary, ternary, or quaternary alloys of gallium, indium, tin, and bismuth.

3. The electrochemical DNA synthesis and manipulation method based on a renewable metal substrate according to claim 1, characterized in that: The preparation of the liquid metal microelectrode or electrode array in step (1) uses polymer microtubes, including silicone tubes, polytetrafluoroethylene PTFE, polyvinylidene fluoride PVDF, polyvinyl fluoride PVC, polyethylene PE, polypropylene PP, polyurethane PU or polycarbonate PC.

4. The electrochemical DNA synthesis and manipulation method based on a renewable metal substrate according to claim 1, characterized in that: The steps of electrochemical DNA synthesis on the electrode surface in step (1) include DNA coupling, oxidation, electrochemical deprotection and cleaning; the groups used for functional modification of the electrode surface before electrochemical DNA synthesis include any one or more of carboxyl (-COOH), amino (-NH2) or sulfhydryl (-SH).

5. The electrochemical DNA synthesis and manipulation method based on a renewable metal substrate according to claim 1, characterized in that: The phase change in step (2) is to transform the low-temperature melting point liquid metal from solid to liquid by heating.

6. The electrochemical DNA synthesis and manipulation method based on a renewable metal substrate according to claim 1, characterized in that: The manual mechanical extrusion in step (2) is to manually and repeatedly squeeze the liquid metal in the microtube, each squeezing ensuring that the liquid metal breaks through the oxide layer and contacts the external air, and the number of squeezing is not less than 10 times; the automated mechanical extrusion utilizes a microfluidic chip with an extrusion structure to automatically squeeze the liquid metal in the channel, transfer the liquid metal to the microfluidic chip through the microtube, and collect it at the chip outlet.

7. The electrochemical DNA synthesis and manipulation method based on a renewable metal substrate according to claim 1, characterized in that: The molecular endocytosis in step (2) refers to the transfer of DNA molecules from the surface to the interior of the metal after repeated mechanical squeezing of the liquid metal in the microtubule; the molecular release is the removal of the liquid metal electrode oxide layer by an acidic or alkaline solution, while achieving the denaturation of the DNA double strand; the acidic or alkaline solution includes low concentration hydrochloric acid or sodium hydroxide.

8. The electrochemical DNA synthesis and manipulation method based on a renewable metal substrate according to claim 1, characterized in that: In step (3), the electrode is updated to the liquid metal and after it is squeezed out, the remaining liquid metal in the microtube is transferred to the tube mouth to become a new electrode; or the electrode array is updated to the liquid metal and after it is taken out of the array, new liquid metal is re-added to the electrode array of the microfluidic chip to serve as a new electrode.

9. The electrochemical DNA synthesis and manipulation method based on a renewable metal substrate according to claim 1, characterized in that: In step (3), the new liquid metal flowing to the in-situ is the new liquid metal flowing to the tube mouth of the microtubular electrode, and the array in-situ is the surface of the chip electrode array.

10. The electrochemical DNA synthesis and manipulation method based on a renewable metal substrate according to claim 1 can be used in the fields of DNA synthesis and DNA data storage, thereby improving the scalability of the DNA synthesis platform, reducing the cost of electrochemical DNA synthesis, simplifying the packaging steps, and realizing integrated DNA synthesis and manipulation.

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