An electrochemical DNA synthesis method based on pulsed direct current voltage

Abstract

The application discloses an electrochemical DNA synthesis method based on pulse direct current voltage, and comprises the following steps: performing hydroxylation modification on an electrode surface to obtain a modified electrode; coupling an active intermediate, which is obtained by mixing a phosphoramidite synthesis monomer with an activating agent, with a hydroxyl active site on the modified electrode to form a phosphite triester bond; after flushing the modified electrode with anhydrous acetonitrile, adding an oxidizing agent on the modified electrode to perform oxidation, and the phosphite triester bond is oxidized into a stable phosphate ester bond by the oxidizing agent; adopting electrochemical deprotection to remove a DMT protective group to expose the hydroxyl active site, which is used for addition of a next base; and repeating the above steps to perform synthesis of an oligonucleotide sequence. In the electrochemical deprotection step, hydrogen ions are generated more uniformly, so that the synthesis quality of DNA can be guaranteed and improved while the gold layer of the gold electrode is slowed down in falling off and the service life of the gold electrode is prolonged.

Description

Technical Field

[0001] This invention relates to DNA synthesis methods, specifically an electrochemical DNA synthesis method based on pulsed DC voltage. Background Technology

[0002] Today, humanity lives in an age of information explosion. We receive tens of thousands of pieces of information and generate vast amounts of data every day. In this information age, storage demands are increasing daily, forcing an upgrade in storage capabilities. The contradiction between the exponentially rising daily demand for information storage and the inability of current information storage media, methods, and capabilities to meet this demand, coupled with the high costs of information preservation and equipment maintenance, is particularly prominent. Throughout human history, countless things have served as storage media, reflecting both human ingenuity and the necessity and importance of information storage. From 62,000 BC, when people used the extremely primitive and traditional skill of drawing in caves to record events and store information; to the invention of writing in 400 BC; to the invention of papermaking and printing in 170 BC; to the advent of computer memory chips in 1960; and now to the proposed and proven concept of DNA storage (Goldman, Bertone et al. 2013), storage media have undergone numerous updates and iterations. The replacement of storage media is an inevitable trend, but whether the exponentially increasing demand for massive data storage can be met depends on the development of emerging technologies and breakthroughs in cutting-edge science.

[0003] DNA storage is highly anticipated by the industry due to its numerous advantages over conventional storage media, including superior durability, ease of maintenance, format invariance, high density, high energy efficiency, high sustainability, and low cost. It is particularly suitable for storing cold data such as government documents (Xu, Zhao et al. 2021). DNA storage mainly consists of six key steps: encoding; synthesis; storage; retrieval; sequencing; and decoding. However, the complex and expensive DNA synthesis process significantly reduces the likelihood of practical applications for DNA storage.

[0004] To date, DNA synthesis technology has undergone three generations of evolution, from classic chemical synthesis (solid-phase phosphoramide synthesis) to microarray DNA synthesis and then to enzymatic synthesis. Because enzymatic synthesis is still immature, researchers are currently focusing on and the industry has high hopes for microarray DNA synthesis. Classical chemical synthesis involves four steps: deprotection; activation coupling; capping; and oxidation. Microarray DNA synthesis is an improvement on classical chemical synthesis, primarily in the deprotection step. The improvements are in two areas: in-situ synthesis and (multi-channel) parallel synthesis.

[0005] Currently, the main next-generation DNA synthesis technologies available globally include photolithography, inkjet printing, and electrochemical arrays. Photolithography can be further divided into masked photolithography synthesis and maskless photolithography synthesis. Agilent Technologies is a leading company in inkjet printing, while Custom Array primarily owns and holds a monopolistic position in electrochemical array technology. Currently, inkjet printing remains the mainstream technology in the market, mainly because the electrical stimulation in electrochemical array technology can affect the synthesis of bases (nucleotides), causing damage to the bases and producing side effects such as depurination. This, in turn, affects the accuracy and purity of the final product, i.e., the synthesis efficiency, leading to a further increase in synthesis costs.

[0006] Electrochemical array technology typically uses metallic materials (such as precious metals like gold and platinum) as electrode materials to act as conductors and supports. On one hand, gold-sulfur bonds can be used to connect the support and functional groups (White, Dorfman et al. 2015); on the other hand, the metal electrode facilitates electron transport through conductivity, catalyzing the in-situ acid production of the electrolyte around the metal electrode. Studies have found that during the electrolytic acid production process, the metal electrode undergoes significant corrosion in the electrolyte environment. On the one hand, the electrode needs to be reused and recycled to reduce synthesis costs; on the other hand, even very slight corrosion (damage) of the electrode can significantly impact the quality of DNA synthesis, leading to a substantial reduction in the purity of the synthesized product, affecting the experiment's progress or delivery to the customer. Summary of the Invention

[0007] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide an electrochemical DNA synthesis method based on pulsed DC voltage that extends electrode life, produces high-purity products, and has good working stability.

[0008] Technical solution: The present invention provides an electrochemical DNA synthesis method based on pulsed DC voltage, comprising the following steps:

[0009] Step 1: Hydroxylation modification is performed on the electrode surface to obtain a modified electrode;

[0010] Step 2, Coupling: The active intermediate formed by mixing the phosphorus amide synthetic monomer and the activator is coupled to the hydroxyl active site on the modified electrode to form a phosphite triester bond. The phosphorus amide synthetic monomer is dA-DMT, dT-DMT, dG-DMT or dC-DMT.

[0011] Step 3, oxidation: After rinsing the modified electrode with anhydrous acetonitrile, the oxidant is dropped onto the modified electrode to carry out oxidation. The phosphite triester bond is oxidized by the oxidant into a stable phosphate ester bond.

[0012] Step 4, Deprotection: Electrochemical deprotection is used to remove the DMT protecting group to expose the hydroxyl active site for the addition of the next base;

[0013] Step 5: Repeat the above steps to synthesize the oligonucleotide sequence.

[0014] Furthermore, in step one, the electrode is made of one or more materials selected from platinum, gold, silicon, silicon dioxide, silicon nitride, and titanium nitride. The specific steps for the electrode hydroxylation modification are as follows:

[0015] S1. Place the electrode in acetone for ultrasonic cleaning, then add ethanol for ultrasonic cleaning, and blow dry.

[0016] S2, Place the electrode obtained in S1 in the piranha solution and bathe it in a water bath at 75-85°C;

[0017] S3, the electrode obtained in S2 is placed in a mixed solution of ethanol and 6-mercaptohex-1-ol, left to stand, and undergoes a self-assembly reaction;

[0018] S4. Place the electrode obtained in S3 into a mixed solution of hydroxysilane, toluene, and n-butylamine, and let it stand in the dark to carry out the self-assembly reaction; then wash it with toluene and dry it at 100-110℃ for later use.

[0019] Further, in step two, the activator is any one of 1H-tetrazole, 5-methylthio-1H-tetrazole (MTT), 5-ethylthio-1H-tetrazole (ETT), 5-nitrophenyl-1H-tetrazole (NPT), 5-benzylthio-1H-tetrazole (BTT), and 4,5-dicyanimidazolium (DCI), preferably ETT. To improve the purity of the synthesized product and reduce the workload of subsequent purification steps, unreacted hydroxyl groups are blocked by acylation; this step is optional and does not affect the successful addition of the final base.

[0020] Furthermore, in step three, the oxidant is either an iodine solution or hydrogen peroxide, preferably an iodine solution. The concentration of the iodine solution is 0.1–0.2 M, and the solvent is a mixed solution of water, pyridine, and tetrahydrofuran.

[0021] Further, in step four, the electrochemical deprotection involves using a pulsed current as input under energized conditions to generate hydrogen ions in situ on the anode surface, thereby removing the DMT protecting group. The pulsed current is a current with a constant direction and continuously varying intensity. The waveform of the pulsed current can be any one or more of square waves, sine waves, triangular waves, and sawtooth waves, with a duty cycle of 1% to 99%, an applied potential of 1mV to 10V, and a potential application time of 1ms to 120s each time.

[0022] Compared to DC or AC voltages of equal amplitude, the space charge behavior under pulsed voltage is drastically different. The accumulation and aggregation of surface charge increases the probability of initial electron generation, accelerating the formation and development of electron avalanches, while the charge steady-state characteristics under pulsed voltage can effectively mitigate this accumulation. As the pulse frequency and duty cycle increase, surface charge accumulation intensifies. Changes in pulsed voltage parameters alter the movement and accumulation characteristics of both space and surface charges, thereby affecting DNA synthesis at the electrodes.

[0023] Preparation principle: Utilizing the charge steady-state characteristics under pulsed voltage, the accumulation and aggregation of surface charge can be effectively slowed down, preventing the formation and development of electron avalanches. This avoids the release of enormous energy, which could cause the metal layer, i.e., the solid-phase support for DNA synthesis, to detach, thus significantly improving the efficiency of DNA synthesis on the solid-phase support. Furthermore, by adjusting pulsed voltage parameters such as pulse frequency and duty cycle, the movement and accumulation characteristics of space charge and surface charge can be altered to obtain optimal space charge behavior, providing the best conditions for efficient and stable DNA synthesis on the electrode.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0025] 1. Using pulsed DC voltage as input for electro-acid desorption protection can significantly slow down gold layer detachment from gold electrodes and extend the working life of gold electrodes;

[0026] 2. Using pulsed DC voltage as input for electro-acid deprotection can generate hydrogen ions more uniformly. This method can slow down the gold layer peeling off the gold electrode, extend the working life of the gold electrode, and at the same time ensure and improve the quality of DNA synthesis.

[0027] 3. Suitable for large-scale industrial production, multiple electrodes work together, enabling "high throughput" of DNA synthesis; on the other hand, the electrodes can be recycled multiple times, which helps to save costs and enhance the stability of electrode operation.

[0028] 4. The electrode operating parameters are easy to control. By adjusting the pulse voltage parameters such as pulse frequency and duty cycle, the movement and accumulation characteristics of space charge and surface charge can be changed to obtain the optimal space charge behavior, providing the best conditions for the efficient and stable synthesis of DNA on the electrode.

[0029] 5. It facilitates the construction of an integrated electrochemical DNA synthesis platform, where all four steps—deprotection, activation coupling, capping, and oxidation—can be efficiently and sensitively regulated through electrochemical methods. Attached Figure Description

[0030] Figure 1This is the current density-time curve of Embodiment 1 of the present invention;

[0031] Figure 2 This is the current density-time curve for Comparative Example 1;

[0032] Figure 3 This is a comparison of the average fluorescence intensity during electrochemical DNA synthesis on a gold electrode.

[0033] Figure 4 This is a line graph showing the gold layer peeling rate of gold-plated silicon wafers under different applied voltage conditions;

[0034] Figure 5 This is an X-ray diffraction pattern of a partially electrochemically etched gold-plated silicon wafer. Detailed Implementation

[0035] In the following embodiments, the volume ratio of concentrated sulfuric acid to hydrogen peroxide in the piranha solution is 3:1. A pulse current is a current whose direction remains constant but whose intensity continuously changes.

[0036] Example 1

[0037] An electrochemical DNA synthesis method based on pulsed DC voltage includes the following steps:

[0038] (1) Hydroxylation modification of the electrode:

[0039] S1, the electrode is a gold-plated silicon wafer. The electrode is placed in 5 ml of acetone and ultrasonically cleaned for 10 minutes. Then, 5 ml of ethanol is added and ultrasonically cleaned for 10 minutes. Then, it is dried.

[0040] S2, Place the electrode obtained in S1 in a freshly prepared piranha solution and incubate it in a water bath at 80°C for 30 minutes;

[0041] S3. Place the electrode obtained in S2 in a mixed solution of 10 mL ethanol and 15 μL 6-mercaptohex-1-ol, let it stand for 24 hours, and carry out the self-assembly reaction.

[0042] S4. The electrode obtained in S3 was placed in a mixed solution of 62.5 μL hydroxysilane, 5 mL toluene, and 6.25 μL n-butylamine and left to stand in the dark for 4 hours to carry out the self-assembly reaction. Then, it was washed three times with toluene and dried in an oven at 110 °C for 40 min to obtain the modified electrode.

[0043] (2) Electrochemical synthesis of DNA:

[0044] S1, Coupling: An active intermediate prepared by mixing an equal volume of 0.10M phosphorous amide monomer dT-DMT and 0.50M activator 5-ethylthio-1H-tetrazole (ETT) was dropped onto the modified electrode for coupling. The reaction lasted 120s to form a phosphite triester bond.

[0045] S2, Oxidation: After rinsing the modified electrode with anhydrous acetonitrile, 0.1 M oxidant was dropped onto the modified electrode for oxidation. The reaction lasted for 40 s, during which the unstable phosphite triester bonds were oxidized to stable phosphate ester bonds by the oxidant. The oxidant was an iodine solution, and the solvent was a mixed solution of 10 vol% water, 20 vol% pyridine, and 70 vol% tetrahydrofuran;

[0046] S3, Deprotection: After rinsing the electrode with anhydrous acetonitrile, the electrode was immersed in an anhydrous acetonitrile solution containing 25 mM hydroquinone, 25 mM benzoquinone, and 25 mM tetrabutylammonium hexafluorophosphate. Electrochemical deprotection was carried out using a three - electrode system of an electrochemical workstation. The working electrode was the modified electrode clamped by a glassy carbon electrode. The modified electrode was suspended like the character "Fu", with the lower half along the diagonal immersed in the electrolyte and the upper half not in contact with the electrolyte; the counter electrode was a platinum wire; the reference electrode was a saturated calomel electrode. As Figure 1 , a pulsed DC voltage was used, with the voltage set to 2 V and 0 V alternating in turn, each cycle lasting 2 s, for a total of 60 cycles, to remove the DMT protecting group to expose the hydroxyl active sites for the addition of the next base. The waveform of the pulsed current was a sine wave, and its duty cycle was 50%.

[0047] S4, Repeat the above steps to synthesize the oligonucleotide sequence TTTTT.

[0048] Comparative Example 1

[0049] The rest of the steps in this comparative example were the same as those in Example 1, except for the deprotection step, where the pulsed DC voltage was replaced with a DC voltage, the voltage was set to 2 V, and the effective time was 60 s.

[0050] To compare the effects of generating hydrogen ions by the two methods in Example 1 and Comparative Example 1, an average fluorescence intensity comparison of electrochemical DNA synthesis was carried out. As <000011l>, the experimental results of fluorescence detection showed that the average fluorescence intensity of Comparative Example 1 was 147.909, and the average fluorescence intensity of Example 1 was 142.064, with the values being comparable. Additionally, by analyzing the fluorescence images, it was found that in Example 1, a pulsed DC voltage was used as the input for electro - acid deprotection, and the generation of hydrogen ions was more uniform. This indicates that this method can slow down the shedding of the gold layer on the gold electrode, extend the service life of the gold electrode, while ensuring and improving the quality of DNA synthesis.

[0051] Example 2

[0052] An electrochemical DNA synthesis method based on pulsed DC voltage, comprising the following steps: ​​​​S1, the electrode is a gold-plated silicon wafer. The electrode is placed in 5 ml of acetone and ultrasonically cleaned for 10 minutes. Then, 5 ml of ethanol is added and ultrasonically cleaned for 10 minutes. Then, it is dried.

[0055] S2, Place the electrode obtained in S1 in a freshly prepared piranha solution and incubate it in a water bath at 80°C for 30 minutes;

[0056] S3. Place the electrode obtained in S2 in a mixed solution of 10 mL ethanol and 15 μL 6-mercaptohex-1-ol, let it stand for 24 hours, and carry out the self-assembly reaction.

[0057] S4. The electrode obtained in S3 was placed in a mixed solution of 62.5 μL hydroxysilane, 5 mL toluene, and 6.25 μL n-butylamine and left to stand in the dark for 4 hours to carry out the self-assembly reaction. Then, it was washed three times with toluene and dried in an oven at 110 °C for 40 min to obtain the modified electrode.

[0058] (2) Electrochemical synthesis of DNA:

[0059] S1, Coupling: An active intermediate prepared by mixing an equal volume of 0.10M phosphorous amide monomer dA-DMT and 0.50M activator 5-benzylthio-1H-tetrazole (BTT) was dropped onto the modified electrode for coupling. The reaction lasted 120s to form a phosphite triester bond.

[0060] S2, Oxidation: After rinsing the modified electrode with anhydrous acetonitrile, 0.1M oxidant is dropped onto the modified electrode for oxidation. The reaction lasts for 40 seconds, and the unstable phosphite triester bond is oxidized to a stable phosphate ester bond by the oxidant. The oxidant is an iodine solution, and the solvent is a mixed solution of 10 vol% water, 20 vol% pyridine, and 70 vol% tetrahydrofuran.

[0061] S3, Deprotection: After rinsing the electrode with anhydrous acetonitrile, the electrode was immersed in anhydrous acetonitrile solution containing 25 mM hydroquinone, 25 mM benzoquinone, and 25 mM tetrabutylammonium hexafluorophosphate. Electrochemical deprotection was performed using a three-electrode system of an electrochemical workstation. The working electrode was a modified electrode clamped between glassy carbon electrodes, suspended like a Chinese character for "good fortune," with the lower half immersed in the electrolyte and the upper half not in contact with the electrolyte. The counter electrode was a platinum wire; the reference electrode was a saturated calomel electrode. A pulsed DC voltage was used, alternating between 2V and 0V, with each cycle lasting 1 second for a total of 60 cycles. This removed the DMT protecting group to expose the hydroxyl active site for the addition of the next base. The pulsed current waveform was a sine wave with a 50% duty cycle.

[0062] S4. Repeat the above steps to synthesize the oligonucleotide sequence AAAAA.

[0063] Example 3

[0064] An electrochemical DNA synthesis method based on pulsed DC voltage includes the following steps:

[0065] (1) Hydroxylation modification of the electrode:

[0066] S1, the electrode is platinum-plated titanium nitride. The electrode is placed in 5 ml of acetone and ultrasonically cleaned for 10 minutes. Then 5 ml of ethanol is added and ultrasonically cleaned for 10 minutes. Then it is dried.

[0067] S2, Place the electrode obtained in S1 in a freshly prepared piranha solution and incubate it in a water bath at 75°C for 30 minutes;

[0068] S3. Place the electrode obtained in S2 in a mixed solution of 10 mL ethanol and 15 μL 6-mercaptohex-1-ol, let it stand for 24 hours, and carry out the self-assembly reaction.

[0069] S4. The electrode obtained in S3 was placed in a mixed solution of 62.5 μL hydroxysilane, 5 mL toluene, and 6.25 μL n-butylamine and allowed to stand in the dark for 4 hours to carry out the self-assembly reaction. Then, it was washed three times with toluene and dried in an oven at 100 °C for 40 min to obtain the modified electrode.

[0070] (2) Electrochemical synthesis of DNA:

[0071] S1, Coupling: An active intermediate, prepared by mixing 0.10M phosphorous amide monomer dT-DMT with 0.50M activator 1H-tetrazole and 5-methylthio-1H-tetrazole (MTT) in equal volumes, is dropped onto a modified electrode for coupling. The reaction lasts for 120s to form a phosphite triester bond.

[0072] S2, Oxidation: After rinsing the modified electrode with anhydrous acetonitrile, 0.2M oxidant is added dropwise to the modified electrode for oxidation. The reaction lasts for 40 seconds, and the unstable phosphite triester bond is oxidized to a stable phosphate ester bond by hydrogen peroxide.

[0073] S3, Deprotection: After rinsing the electrode with anhydrous acetonitrile, the electrode was immersed in anhydrous acetonitrile solution containing 25 mM hydroquinone, 25 mM benzoquinone, and 25 mM tetrabutylammonium hexafluorophosphate. Electrochemical deprotection was performed using a three-electrode system on an electrochemical workstation. The working electrode was a modified electrode clamped between glassy carbon electrodes, suspended diagonally with the lower half immersed in the electrolyte and the upper half not in contact with the electrolyte. The counter electrode was a platinum wire; the reference electrode was a saturated calomel electrode. A pulsed DC voltage was used, alternating between 1 mV and 0 V, for 120 s per cycle, for a total of 60 cycles. This removed the DMT protecting group to expose the hydroxyl active site for the addition of the next base. The pulsed current waveform was a square wave with a duty cycle of 1%.

[0074] S4. Repeat the above steps to synthesize the oligonucleotide sequence TTTTT.

[0075] In this embodiment, the square wave waveform of the pulsed current can be replaced by any one of a sine wave, a triangle wave, and a sawtooth wave.

[0076] Example 4

[0077] An electrochemical DNA synthesis method based on pulsed DC voltage includes the following steps:

[0078] (1) Hydroxylation modification of the electrode:

[0079] S1, the electrode is gold-plated silicon dioxide. The electrode is placed in 5 ml of acetone and ultrasonically cleaned for 10 minutes. Then 5 ml of ethanol is added and ultrasonically cleaned for 10 minutes. Then it is dried.

[0080] S2, Place the electrode obtained in S1 in a freshly prepared piranha solution and incubate it in a water bath at 85°C for 30 minutes;

[0081] S3. Place the electrode obtained in S2 in a mixed solution of 10 mL ethanol and 15 μL 6-mercaptohex-1-ol, let it stand for 24 hours, and carry out the self-assembly reaction.

[0082] S4. The electrode obtained in S3 was placed in a mixed solution of 62.5 μL hydroxysilane, 5 mL toluene, and 6.25 μL n-butylamine and allowed to stand in the dark for 4 hours to carry out the self-assembly reaction. Then, it was washed three times with toluene and dried in an oven at 102 °C for 40 min to obtain the modified electrode.

[0083] (2) Electrochemical synthesis of DNA:

[0084] S1, Coupling: An active intermediate prepared by mixing 0.10M phosphorous amide monomer dG-DMT with 0.50M activator 5-nitrophenyl-1H-tetrazole (NPT) in equal volumes was dropped onto the modified electrode for coupling. The reaction was carried out for 120s to form a phosphite triester bond.

[0085] S2, Oxidation: After rinsing the modified electrode with anhydrous acetonitrile, 0.15M oxidant is dropped onto the modified electrode for oxidation. The reaction lasts for 40 seconds, and the unstable phosphite triester bond is oxidized to a stable phosphate ester bond by the oxidant. The oxidant is an iodine solution, and the solvent is a mixed solution of 10 vol% water, 20 vol% pyridine, and 70 vol% tetrahydrofuran.

[0086] S3, Deprotection: After rinsing the electrode with anhydrous acetonitrile, the electrode was immersed in anhydrous acetonitrile solution containing 25 mM hydroquinone, 25 mM benzoquinone, and 25 mM tetrabutylammonium hexafluorophosphate. Electrochemical deprotection was performed using a three-electrode system on an electrochemical workstation. The working electrode was a modified electrode clamped between glassy carbon electrodes, suspended diagonally with the lower half immersed in the electrolyte and the upper half not in contact with the electrolyte. The counter electrode was a platinum wire; the reference electrode was a saturated calomel electrode. A pulsed DC voltage was used, alternating between 10V and 0V, for 90 seconds per cycle, for a total of 60 cycles. This removed the DMT protecting group to expose the hydroxyl active site for the addition of the next base. The pulsed current waveform was a triangular wave with a duty cycle of 99%.

[0087] S4. Repeat the above steps to synthesize the oligonucleotide sequence GGGGG.

[0088] Example 5

[0089] An electrochemical DNA synthesis method based on pulsed DC voltage includes the following steps:

[0090] (1) Hydroxylation modification of the electrode:

[0091] S1, the electrode is silicon nitride. The electrode is placed in 5 ml of acetone and ultrasonically cleaned for 10 minutes. Then 5 ml of ethanol is added and ultrasonically cleaned for 10 minutes. Then it is dried.

[0092] S2, Place the electrode obtained in S1 in a freshly prepared piranha solution and incubate it in a water bath at 82°C for 30 minutes;

[0093] S3. Place the electrode obtained in S2 in a mixed solution of 10 mL ethanol and 15 μL 6-mercaptohex-1-ol, let it stand for 24 hours, and carry out the self-assembly reaction.

[0094] S4. The electrode obtained in S3 was placed in a mixed solution of 62.5 μL hydroxysilane, 5 mL toluene, and 6.25 μL n-butylamine and allowed to stand in the dark for 4 hours to carry out the self-assembly reaction. Then, it was washed three times with toluene and dried in an oven at 108 °C for 40 min to obtain the modified electrode.

[0095] (2) Electrochemical synthesis of DNA:

[0096] S1, Coupling: An active intermediate prepared by mixing 0.10M phosphorous amide monomer dC-DMT with 0.50M activator 4,5-dicyanimidazolium (DCI) in equal volumes was dropped onto the modified electrode for coupling. The reaction was carried out for 120s to form a phosphite triester bond.

[0097] S2, Closed:

[0098] S3, Oxidation: After rinsing the modified electrode with anhydrous acetonitrile, 0.2M oxidant is added dropwise to the modified electrode for oxidation. The reaction lasts for 40 seconds, and the unstable phosphite triester bond is oxidized to a stable phosphate ester bond by hydrogen peroxide.

[0099] S4, Deprotection: After rinsing the electrode with anhydrous acetonitrile, the electrode was immersed in anhydrous acetonitrile solution containing 25 mM hydroquinone, 25 mM benzoquinone, and 25 mM tetrabutylammonium hexafluorophosphate. Electrochemical deprotection was performed using a three-electrode system on an electrochemical workstation. The working electrode was a modified electrode clamped between glassy carbon electrodes, suspended diagonally with the lower half immersed in the electrolyte and the upper half not in contact with the electrolyte. The counter electrode was a platinum wire; the reference electrode was a saturated calomel electrode. A pulsed DC voltage was used, alternating between 5V and 0V, with each cycle lasting 1 ms for a total of 60 cycles. This removed the DMT protecting group to expose the hydroxyl active site for the addition of the next base. The pulsed current waveform was a sine wave with a 50% duty cycle.

[0100] S6. Repeat the above steps to synthesize the oligonucleotide sequence CCCCC.

[0101] Example 6

[0102] The remaining steps of this embodiment are the same as in Embodiment 1, except that the voltage for deprotection is different. The initial point is the gold layer peeling ratio corresponding to a DC voltage of 2V and an application time of 300s. The four points after Group I correspond to the gold layer peeling ratios of (A) to (D); the four points after Group II correspond to the gold layer peeling ratios of (E) to (H). DC voltage is used for (A) to (B), with a voltage of 2V. The effective application time for potential (A) is 600s, and the effective application time for potential (B) is 300s. Pulsed DC voltage is used for (C) to (H), and the effective application time for potential is 300s for all. (C) Alternate between 2V and 0V, 20 seconds per cycle, for a total of 30 cycles; (D) Alternate between 2V and 0V, 10 seconds per cycle, for a total of 60 cycles; (E) Alternate between 2V and 0V, 5 seconds per cycle, for a total of 120 cycles; (F) Alternate between 2V and 0V, 2.5 seconds per cycle, for a total of 240 cycles; (G) Alternate between 0V and 2V, 20 seconds per cycle, for a total of 30 cycles; (H) Alternate between 0V and 2V, 10 seconds per cycle, for a total of 60 cycles.

[0103] Using the image recognition function on the online website (http: / / mkweb.bcgsc.ca / color-summarizer / ?analyze), a line graph showing the gold layer peeling rate of gold-plated silicon wafers under the same applied voltage was obtained. For example... Figure 4Under the same conditions, the method of using pulsed DC voltage can significantly reduce the rate of gold layer detachment. The gold layer detachment rate before and after using this method was measured by image recognition technology, and it was found that the gold layer detachment rate was significantly reduced from the original 54.61% to 17.02%.

[0104] Figure 5 This is an X-ray diffraction pattern of the gold-plated silicon wafer partially electrochemically etched in Example 1. (As shown...) Figure 5 As shown, the X-ray diffraction pattern mainly consists of two peaks: one is the gold layer that has not yet detached, with its characteristic peak position at 37°; the other is the silicon (substrate) layer exposed after the gold layer has detached, with its characteristic peak position at 69°.

Claims

1. An electrochemical DNA synthesis method based on pulsed DC voltage, characterized in that, Includes the following steps: Step 1: Hydroxylation modification is performed on the electrode surface to obtain a modified electrode; Step 2: The active intermediate, which is a mixture of phosphorus amide monomer and activator, is coupled to the hydroxyl active site on the modified electrode to form a phosphite trimer bond. The phosphorus amide monomer is dA-DMT, dT-DMT, dG-DMT or dC-DMT. Step 3: After rinsing the modified electrode with anhydrous acetonitrile, the oxidant is dropped onto the modified electrode for oxidation. The phosphite triester bond is oxidized by the oxidant into a stable phosphate ester bond. Step four: Electrochemical deprotection is used to remove the DMT protecting group to expose the hydroxyl active site for the addition of the next base; Step 5: Repeat the above steps to synthesize the oligonucleotide sequence; In step four, electrochemical deprotection involves using a pulsed current as input under energized conditions to generate hydrogen ions in situ on the anode surface, thereby removing the DMT protecting group. The pulse current is a current whose direction remains constant but whose intensity changes continuously; The waveform of the pulsed current is any one or more of square wave, sine wave, triangle wave and sawtooth wave, with a duty cycle of 1% to 99%, an applied potential of 1 mV to 10 V, and a potential application time of 1 ms to 120 s each time.

2. The electrochemical DNA synthesis method based on pulsed DC voltage according to claim 1, characterized in that: In step one, the electrode is made of one or more materials selected from platinum, gold, silicon, silicon dioxide, silicon nitride, and titanium nitride.

3. The electrochemical DNA synthesis method based on pulsed DC voltage according to claim 1, characterized in that: In step one, the specific steps of electrode hydroxylation modification are as follows: S1. Place the electrode in acetone for ultrasonic cleaning, then add ethanol for ultrasonic cleaning, and blow dry. S2, place the electrode obtained in S1 in the piranha solution and in a water bath at 75~85 ℃; S3, the electrode obtained in S2 is placed in a mixed solution of ethanol and 6-mercaptohex-1-ol, left to stand, and undergoes a self-assembly reaction; S4. Place the electrode obtained in S3 into a mixed solution of hydroxysilane, toluene, and n-butylamine, and let it stand in the dark to carry out the self-assembly reaction; then, wash it with toluene and dry it at 100~110 ℃ for later use.

4. The electrochemical DNA synthesis method based on pulsed DC voltage according to claim 1, characterized in that: In step two, the activator is any one of 1H-tetrazole, 5-methylthio-1H-tetrazole, 5-ethylthio-1H-tetrazole, 5-nitrophenyl-1H-tetrazole, 5-benzylthio-1H-tetrazole, and 4,5-dicyanimidazolium.

5. The electrochemical DNA synthesis method based on pulsed DC voltage according to claim 1, characterized in that: The unreacted hydroxyl groups in step two are blocked by an acylation reaction.

6. The electrochemical DNA synthesis method based on pulsed DC voltage according to claim 1, characterized in that: In step three, the oxidant is either iodine solution or hydrogen peroxide.

7. The electrochemical DNA synthesis method based on pulsed DC voltage according to claim 6, characterized in that: The concentration of the iodine solution is 0.1~0.2 M, and the solvent is a mixed solution of water, pyridine, and tetrahydrofuran.

Images