Fluorescence sequencing chip, cleaning method thereof and fluorescence sequencing method

Abstract

The invention relates to a fluorescent sequencing chip, a cleaning method thereof and a fluorescent sequencing method, and belongs to the technical field of gene sequencing. The fluorescent sequencing chip comprises an insulating layer, a conductive layer, a covering layer and a power supply, wherein the insulating layer, the conductive layer and the covering layer are sequentially stacked in the thickness direction; the covering layer is provided with a groove penetrating through the covering layer in the thickness direction; the interior of the groove and the surface of the covering layer are used for jointly containing an ion solution, so that the biomolecules to be sequenced enter the groove, and fluorescence information in the groove can be collected; an electrode at one end of the power supply is connected with the conductive layer, and an electrode at the other end extends into the ion solution. Certain electric field force can be applied to the surface of the fluorescent sequencing chip, so that the distance between the non-specific binding substance and the focal plane of the surface of the fluorescent sequencing chip can be controlled, and the situation that the non-specific binding substance is adsorbed to the surface of the sequencing chip to affect a sequencing signal is reduced.

Description

Technical Field

[0001] The present application relates to the field of gene sequencing technology, and in particular to a fluorescent sequencing chip and a cleaning method thereof and a fluorescent sequencing method. Background Art

[0002] Gene sequencing using fluorescence technology is of great significance in the field of life sciences. During the sequencing process, the sample to be tested needs to be fixed on the surface of the chip, and the polymerase chain reaction (PCR) is used to synthesize and sequence the gene sequence to be tested. The principle of sequencing is to pass bases labeled with different fluorescent groups into the sequencing chip for biochemical reaction, and then use the optical microscopy imaging system to collect and analyze the changes in fluorescence in the sequencing chip to determine the sequence of the gene to be tested.

[0003] However, during sequencing, non-specific binding substances remaining on the surface of the sequencing chip often affect the accuracy of the sequencing results. Before sequencing, the surface of the sequencing chip needs to be biochemically treated to obtain a better hydrophilic surface, thereby obtaining a high-density modification of the sample to be tested. During the sequencing process, fluorescently labeled deoxyribonucleoside triphosphate (dNTP) will be captured by DNA and synthesized into a double strand. The synthesis of each dNTP is accompanied by the generation of image data. At the same time, the fluorescent label on the dNTP will fall off and leave, and there are still some free samples to be tested and dNTPs; however, due to the influence of the hydrophilicity of the surface of the sequencing chip, free fluorescent molecules, samples to be tested, dNTPs and other non-specific binding substances are easily adsorbed to the surface of the sequencing chip and captured by the camera, thereby affecting the sequencing signal. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the embodiments of the present application is to provide a fluorescent sequencing chip and a cleaning method thereof and a fluorescent sequencing method, so as to improve the current problem that free fluorescent molecules, test samples, dNTPs and other non-specific binding substances are easily adsorbed to the surface of the sequencing chip and affect the sequencing signal.

[0005] In a first aspect, an embodiment of the present application provides a fluorescent sequencing chip, comprising: an insulating layer, a conductive layer, a covering layer and a power supply; wherein the insulating layer, the conductive layer and the covering layer are stacked in sequence along the thickness direction; the covering layer has a groove that penetrates the covering layer along the thickness direction; the interior of the groove and the surface of the covering layer are used to jointly accommodate an ionic solution, so that the biological molecules to be sequenced enter the groove, and the fluorescence information in the groove can be collected; one end electrode of the power supply is used to be connected to the conductive layer, and the other end electrode is used to extend into the ionic solution.

[0006] The present application is to stack an insulating layer, a conductive layer and a covering layer in sequence along the thickness direction, and the covering layer has a groove that penetrates the covering layer along the thickness direction, and at the same time, one end electrode of the power supply is connected to the conductive layer, and the other end electrode extends into the ion solution, so that a certain voltage can be applied to the electrode, thereby applying a certain electric field force to the surface of the fluorescent sequencing chip, so as to control the distance between free fluorescent molecules, non-specific binding substances such as test samples and dNTPs and the surface focal plane of the fluorescent sequencing chip, thereby reducing the free fluorescent molecules, non-specific binding substances such as test samples and dNTPs being adsorbed on the surface of the sequencing chip and affecting the sequencing signal.

[0007] In some embodiments of the present application, the cover layer has a plurality of grooves, and the plurality of grooves are distributed in the cover layer in an array manner.

[0008] The present application arranges and distributes a plurality of grooves in the covering layer, thereby facilitating the simultaneous sequencing of a large number of samples to be tested.

[0009] In some embodiments of the present application, the depth of the groove is 100 nm to 500 nm, the diameter of the groove is 50 nm to 500 nm, and the distance between two adjacent grooves is 100 nm to 900 nm.

[0010] The present application sets a groove with a suitable depth and diameter to facilitate the entry of the sample to be tested and dNTP into the groove to improve the accuracy of detection; it also facilitates the free fluorescent molecules, non-specific binding substances such as the sample to be tested and dNTP to leave the groove, thereby controlling the sequencing step to stop or reducing the impact on the sequencing signal.

[0011] In some embodiments of the present application, the diameter of the groove is consistent along the thickness direction.

[0012] The present application sets the diameter of the groove to be consistent along the thickness direction, which is more conducive to free fluorescent molecules, samples to be tested, dNTPs and other substances entering or leaving the groove.

[0013] In some embodiments of the present application, the cross-section of the groove is circular, square, diamond or gear-shaped.

[0014] In some embodiments of the present application, the material of the conductive layer includes at least one of iron oxide, aluminum oxide, zinc oxide, copper oxide, indium tin oxide, titanium oxide, tantalum oxide, niobium oxide, fluorine-doped tin oxide and silicon carbide.

[0015] In some embodiments of the present application, the thickness of the conductive layer is 10 nm to 500 nm, and includes one or more layers of the above materials.

[0016] In some embodiments of the present application, the material of the insulating layer includes glass.

[0017] In some embodiments of the present application, the material of the electrode includes at least one of platinum, gold and silver.

[0018] In some embodiments of the present application, the material of the covering layer includes polymer.

[0019] In the second aspect, an embodiment of the present application provides a cleaning method for the fluorescent sequencing chip provided in the first aspect, the cleaning method is used to remove residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing, and the cleaning method includes: placing an ionic solution on the fluorescent sequencing chip, and making the liquid level of the ionic solution higher than the top of the groove; electrically connecting one electrode of the power supply to the conductive layer, and extending the other electrode into the ionic solution, and making the power supply output an AC voltage; when the fluorescent information in the groove decreases, continuously outputting the voltage in the direction of the voltage output by the power supply; and then removing the liquid phase on the fluorescent sequencing chip.

[0020] The present application outputs an AC voltage through a power supply, and when the fluorescence information in the groove decreases, the voltage is continuously output in the direction of the voltage output by the power supply to control free fluorescent molecules, test samples, dNTPs and other non-specific binding substances away from the surface focal plane of the fluorescent sequencing chip, that is, it is beneficial to clean and remove free fluorescent molecules, test samples, dNTPs and other residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing, thereby reducing the free fluorescent molecules, test samples, dNTPs and other non-specific binding substances being adsorbed to the surface of the sequencing chip and affecting the sequencing signal.

[0021] In some embodiments of the present application, the ionic solution contains at least one of Na2SO3, Na2SO4, Na2HPO4, KH2PO4, NaCl, KCl, MgCl2, glucose oxidase, catalase, glucose and cysteine.

[0022] In some embodiments of the present application, the ionic solution contains 2-4 M Na2SO3, 1-2 M Na2SO4, 1-2 M Na2HPO4, 1-2 M KH2PO4, 1-2 M NaCl, 1-2 M KCl, 1-2 M MgCl2, 100-200 μg / mL glucose oxidase, 100-200 μg / mL catalase, 10%-15% glucose, and 100-200 mM cysteine.

[0023] In some embodiments of the present application, the residual molecules include at least one of DNA, RNA, protein, polymerase, helicase, transcriptase, dNTP, biotin and streptavidin; during cleaning, the voltage continuously output in the voltage direction of the power supply is ≥0.1V.

[0024] In some embodiments of the present application, during cleaning, the frequency of the voltage continuously output in the voltage direction of the power supply is ≤100 Hz.

[0025] In some embodiments of the present application, the cleaning method includes: adding a cleaning agent to the ion solution on the fluorescent sequencing chip before the power supply outputs the AC voltage.

[0026] The present application adds a cleaning agent to the ion solution on the fluorescent sequencing chip, and after controlling the free fluorescent molecules, the sample to be tested and non-specific binding substances such as dNTP to be away from the surface focal plane of the fluorescent sequencing chip, it is helpful to clean and remove these residual molecules.

[0027] In some embodiments of the present application, the cleaning agent includes at least one of sodium dodecyl sulfate, Triton-100, ethanol, propanol, and polysorbate-20.

[0028] In a third aspect, an embodiment of the present application provides a fluorescent sequencing method, which adopts the fluorescent sequencing chip provided in the first aspect, and the sequencing method includes a sequencing step and a sequencing stop step; the sequencing step includes: placing an ion solution on the fluorescent sequencing chip, and making the liquid level of the ion solution higher than the top of the groove, so that the biological molecules to be sequenced enter the groove, and by obtaining the fluorescence information corresponding to the biological molecules to be sequenced in the groove, the sequence of the biological molecules to be sequenced is determined; the sequencing stop step includes: electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ion solution, and making the power supply output an AC voltage; when the fluorescence information in the groove decreases, continuously outputting the voltage in the direction of the voltage output by the power supply.

[0029] The present application outputs an AC voltage through a power supply, and when the fluorescence information in the groove decreases, the voltage is continuously output in the direction of the voltage output by the power supply to control the dNTP to be away from the surface focal plane of the fluorescent sequencing chip, so that the dNTP is separated from the sample to be tested, thereby controlling the sequencing step to stop.

[0030] In some embodiments of the present application, the sequencing method also includes a cleaning step; the cleaning step is used to remove residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing; the cleaning step includes: placing an ionic solution on the fluorescent sequencing chip, and making the liquid level of the ionic solution higher than the top of the groove; electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ionic solution, and making the power supply output an AC voltage; when the fluorescence information in the groove decreases, continuously outputting the voltage in the direction of the voltage output by the power supply; then removing the liquid phase on the fluorescent sequencing chip; or / and, the sequencing step includes: electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ionic solution, and making the power supply output an AC voltage; when the fluorescence information in the groove increases, continuously outputting the voltage in the direction of the voltage output by the power supply; determining the sequence of the biological molecule to be sequenced by obtaining the fluorescence information corresponding to the biological molecule to be sequenced in the groove.

[0031] The present application outputs an AC voltage through a power supply, and when the fluorescence information in the groove decreases, the voltage is continuously output in the direction of the voltage output by the power supply to control the free fluorescent molecules, the sample to be tested and the non-specific binding substances such as dNTP to be away from the surface focal plane of the fluorescent sequencing chip, that is, it is conducive to cleaning and removing the free fluorescent molecules, the sample to be tested and the residual molecules such as dNTP on the surface of the fluorescent sequencing chip after the fluorescent sequencing, thereby reducing the free fluorescent molecules, the sample to be tested and the non-specific binding substances such as dNTP to be adsorbed on the surface of the sequencing chip and affecting the sequencing signal. Or / and, the power supply outputs an AC voltage, and when the fluorescence information in the groove increases, the voltage is continuously output in the direction of the voltage output by the power supply to control the dNTP to be close to the surface focal plane of the fluorescent sequencing chip, improve the penetration rate of the dNTP into the groove, and improve the binding efficiency of the dNTP with the biological molecules to be sequenced in the groove, thereby improving the detection accuracy of the fluorescent sequencing.

[0032] In some embodiments of the present application, in the sequencing step, when the voltage is continuously output in the voltage direction of the power supply output, the voltage value of the power supply output is increased.

[0033] In some embodiments of the present application, in the sequencing step, when the voltage is continuously output in the voltage direction of the power supply output, the voltage value of the power supply output is increased to ≥0.1V.

[0034] In some embodiments of the present application, the ionic solution contains at least one of Na2SO3, Na2SO4, Na2HPO4, KH2PO4, NaCl, KCl, MgCl2, glucose oxidase, catalase, glucose and cysteine.

[0035] In some embodiments of the present application, the ionic solution contains 2-4 M Na2SO3, 1-2 M Na2SO4, 1-2 M Na2HPO4, 1-2 M KH2PO4, 1-2 M NaCl, 1-2 M KCl, 1-2 M MgCl2, 100-200 μg / mL glucose oxidase, 100-200 μg / mL catalase, 10%-15% glucose, and 100-200 mM cysteine.

[0036] In some embodiments of the present application, the sequencing steps include: connecting a modifier to the bottom wall of the groove; placing an ionic solution on the chip and making the liquid level of the ionic solution higher than the top of the groove; placing the biological molecule to be sequenced in the ionic solution so that the biological molecule to be sequenced enters the groove and combines with the modifier in the groove; placing a fluorescently labeled biological molecule in the ionic solution so that the fluorescently labeled biological molecule enters the groove and combines with the biological molecule to be sequenced in the groove; determining the sequence of the biological molecule to be sequenced by obtaining the fluorescent information corresponding to the biological molecule to be sequenced in the groove; wherein the biological molecule to be sequenced is DNA, and a DNA polymerase that specifically binds to DNA is bound to the molecule to be sequenced; the fluorescently labeled biological molecule is a fluorescently labeled dNTP; the modifier is biotin and streptavidin; when the sequencing stop step, the voltage continuously output in the voltage direction of the power supply output is ≥0.1V.

[0037] In some embodiments of the present application, during the sequencing stop step, the frequency of the voltage continuously output in the voltage direction of the power supply is ≤100 Hz.

[0038] In a fourth aspect, an embodiment of the present application provides a method for improving the accuracy of fluorescent sequencing. The method for improving the accuracy of sequencing adopts the fluorescent sequencing chip provided in the first aspect. The method for improving the accuracy of sequencing includes: placing an ionic solution on the chip, and making the liquid level of the ionic solution higher than the top of the groove; electrically connecting one electrode of the power supply to the conductive layer, and extending the other electrode into the ionic solution, and making the power supply output an AC voltage; when the fluorescence information in the groove increases, continuously outputting the voltage in the direction of the voltage output of the power supply and increasing the voltage value of the power supply output; determining the sequence of the biological molecule to be sequenced by obtaining the fluorescence information corresponding to the biological molecule to be sequenced in the groove.

[0039] The present application outputs an AC voltage through a power supply, and when the fluorescence information in the groove increases, the voltage is continuously output in the direction of the voltage output by the power supply to control the dNTP to be close to the surface focal plane of the fluorescent sequencing chip, thereby increasing the penetration rate of the dNTP into the groove, and increasing the binding efficiency of the dNTP with the biological molecules to be sequenced in the groove, thereby improving the detection accuracy of the fluorescent sequencing.

[0040] In some embodiments of the present application, the voltage is continuously output in the voltage direction of the power supply output and the voltage value of the power supply output is increased to ≥0.1V. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 A side view of the fluorescent sequencing chip provided in an embodiment of the present application.

[0043] Icon: 1-insulating layer; 2-conductive layer; 3-covering layer; 31-groove; 4-power supply.

[0044] Figure 2 A top view of the fluorescent sequencing chip provided in an embodiment of the present application.

[0045] Figure 3 This is a fluorescence event result diagram provided in Example 1 of the present application.

[0046] Figure 4 This is a fluorescence event result diagram provided in Example 2 of the present application. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0048] In order to improve the problem that free fluorescent molecules, test samples, dNTPs and other non-specific binding substances are easily adsorbed to the surface of the sequencing chip and affect the sequencing signal in the current technology, the present application embodiment provides a fluorescent sequencing chip, such as Figure 1 As shown, it includes: an insulating layer 1, a conductive layer 2, a covering layer 3 and a power supply 4; wherein the insulating layer 1, the conductive layer 2 and the covering layer 3 are stacked in sequence along the thickness direction; the covering layer 3 has a groove 31 that penetrates the covering layer 3 along the thickness direction; the inside of the groove 31 and the surface of the covering layer 3 are used to jointly accommodate an ion solution, so that the biological molecules to be sequenced enter the groove 31, and the fluorescence information in the groove 31 can be collected; one end electrode of the power supply 4 is used to be connected to the conductive layer 2, and the other end electrode is used to extend into the ion solution.

[0049] In the present application, an insulating layer 1, a conductive layer 2 and a covering layer 3 are stacked in sequence along the thickness direction, and the covering layer 3 has a groove 31 that penetrates the covering layer 3 along the thickness direction. At the same time, one end electrode of a power supply 4 is connected to the conductive layer 2, and the other end electrode extends into the ion solution. A certain voltage can be applied to the electrode, thereby applying a certain electric field force to the surface of the fluorescent sequencing chip, so as to control the distance between free fluorescent molecules, non-specific binding substances such as test samples and dNTPs and the surface focal plane of the fluorescent sequencing chip, thereby reducing the free fluorescent molecules, non-specific binding substances such as test samples and dNTPs being adsorbed on the surface of the sequencing chip and affecting the sequencing signal.

[0050] In some embodiments of the present application, Figure 2 As shown, the cover layer 3 has a plurality of grooves 31, and the plurality of grooves 31 are arranged in an array in the cover layer 3. Arranging and distributing the plurality of grooves 31 in the cover layer 3 is conducive to sequencing a large number of samples to be tested at the same time.

[0051] In some embodiments of the present application, the depth of the groove 31 is 100nm-500nm, the diameter of the groove 31 is 50nm-500nm, and the distance between two adjacent grooves 31 is 100nm-900nm. As an example, the depth of the groove 31 can be, but is not limited to, 100nm, 120nm, 150nm, 160nm, 180nm, 200nm, 220nm, 250nm, 260nm, 280nm, 300nm, 320nm, 350nm, 360nm, 380nm, 400nm, 420nm, 450nm, 460nm, 480nm, 500nm. The diameter of the groove 31 may be, but is not limited to, 50nm, 80nm, 100nm, 150nm, 180nm, 200nm, 250nm, 280nm, 300nm, 350nm, 380nm, 400nm, 450nm, 480nm, 500nm. The spacing between two adjacent grooves 31 may be, but is not limited to, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm. The groove 31 is set with a suitable depth and diameter to facilitate the test sample and dNTP to enter the groove 31, so as to improve the accuracy of detection; it is also conducive to the free fluorescent molecules, test samples, dNTP and other non-specific binding substances to leave the groove 31, so as to control the sequencing step to stop or reduce the impact on the sequencing signal.

[0052] In some embodiments of the present application, the diameter of the groove 31 is consistent along the thickness direction, which is more conducive to free fluorescent molecules, samples to be tested, dNTPs and other substances entering or leaving the groove 31.

[0053] In some embodiments of the present application, the cross-section of the groove 31 may be, but is not limited to, circular, square, diamond or gear-shaped, as long as it facilitates the entry of the sample to be tested and dNTPs for sequencing.

[0054] In some embodiments of the present application, the material of the conductive layer 2 may include but is not limited to at least one of iron oxide, aluminum oxide, zinc oxide, copper oxide, indium tin oxide, titanium oxide, tantalum oxide, niobium oxide, fluorine-doped tin oxide and silicon carbide, etc. As long as a certain voltage is applied to the electrode, a certain electric field force is applied to the surface of the fluorescent sequencing chip.

[0055] In some embodiments of the present application, the thickness of the conductive layer 2 is 10nm to 500nm, and includes one or more layers of the above materials. As an example, the thickness of the conductive layer 2 can be, but is not limited to, 10nm, 30nm, 50nm, 80nm, 100nm, 150nm, 180nm, 200nm, 250nm, 280nm, 300nm, 350nm, 380nm, 400nm, 450nm, 480nm, 500nm.

[0056] In some embodiments of the present application, the material of the insulating layer 1 may include, but is not limited to, glass. The material of the insulating layer 1 may also be other insulating materials (eg, silicon dioxide).

[0057] In some embodiments of the present application, the material of the electrode may include but is not limited to at least one of platinum, gold and silver. The material of the electrode can be conductive.

[0058] In some embodiments of the present application, the material of the cover layer 3 includes a polymer, which is conducive to embossing suitable grooves 31 along the thickness direction thereof.

[0059] An embodiment of the present application provides a method for cleaning the above-mentioned fluorescent sequencing chip, which is used to remove residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing. The cleaning method includes: placing an ionic solution on the fluorescent sequencing chip, and making the liquid level of the ionic solution higher than the top of the groove; electrically connecting one electrode of the power supply to the conductive layer, and extending the other electrode into the ionic solution, and making the power supply output an AC voltage; when the fluorescent information in the groove decreases, continuously outputting the voltage in the direction of the voltage output by the power supply; and then removing the liquid phase on the fluorescent sequencing chip.

[0060] The present application outputs an AC voltage through a power supply, and when the fluorescence information in the groove decreases, the voltage is continuously output in the direction of the voltage output by the power supply to control free fluorescent molecules, test samples, dNTPs and other non-specific binding substances away from the surface focal plane of the fluorescent sequencing chip, that is, it is beneficial to clean and remove free fluorescent molecules, test samples, dNTPs and other residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing, thereby reducing the free fluorescent molecules, test samples, dNTPs and other non-specific binding substances being adsorbed to the surface of the sequencing chip and affecting the sequencing signal.

[0061] In some embodiments of the present application, the ionic solution may contain, but is not limited to, at least one of Na2SO3, Na2SO4, Na2HPO4, KH2PO4, NaCl, KCl, MgCl2, glucose oxidase, catalase, glucose and cysteine.

[0062] In some embodiments of the present application, the ionic solution may contain, but is not limited to, 2-4M Na2SO3, 1-2M Na2SO4, 1-2M Na2HPO4, 1-2M KH2PO4, 1-2M NaCl, 1-2M KCl, 1-2M MgCl2, 100-200μg / mL glucose oxidase, 100-200μg / mL catalase, 10%-15% glucose, and 100-200mM cysteine. The use of an ionic solution with a suitable component and a suitable concentration is conducive to applying a suitable electric field force to the surface of the fluorescent sequencing chip to control free fluorescent molecules, samples to be tested, dNTPs and other non-specific binding substances away from the surface focal plane of the fluorescent sequencing chip.

[0063] In some embodiments of the present application, the residual molecules include at least one of DNA, RNA, protein, polymerase, helicase, transcriptase, dNTP, biotin and streptavidin; during cleaning, the voltage continuously output in the voltage direction of the power supply output is ≥ 0.1 V. Continuously outputting a suitable voltage in the voltage direction of the power supply output is conducive to applying a suitable electric field force to the surface of the fluorescent sequencing chip, so as to control the distance between the above-mentioned residual molecules and the surface focal plane of the fluorescent sequencing chip.

[0064] In some embodiments of the present application, during cleaning, the frequency of the voltage continuously output in the voltage direction of the power supply is ≤100 Hz.

[0065] In some embodiments of the present application, the cleaning method includes: before the power supply outputs the AC voltage, a cleaning agent is first added to the ion solution on the fluorescent sequencing chip. After the free fluorescent molecules, the sample to be tested, and non-specific binding substances such as dNTP are controlled to be away from the surface focal plane of the fluorescent sequencing chip, the cleaning agent is conducive to cleaning and removing these residual molecules.

[0066] In some embodiments of the present application, the cleaning agent may include, but is not limited to, at least one of sodium dodecyl sulfate, Triton-100, ethanol, propanol, and polysorbate-20. Selecting a suitable cleaning agent can efficiently clean and remove residual molecules.

[0067] An embodiment of the present application provides a fluorescence sequencing method, which uses the fluorescence sequencing chip as described above, and the sequencing method includes a sequencing step and a sequencing stop step; the sequencing step includes: placing an ion solution on the fluorescence sequencing chip, and making the liquid level of the ion solution higher than the top of the groove, so that the biological molecules to be sequenced enter the groove, and by obtaining the fluorescence information corresponding to the biological molecules to be sequenced in the groove, the sequence of the biological molecules to be sequenced is determined; the sequencing stop step includes: electrically connecting one end electrode of a power supply to the conductive layer, extending the other end electrode into the ion solution, and making the power supply output an AC voltage; when the fluorescence information in the groove decreases, continuously outputting the voltage in the direction of the voltage output by the power supply.

[0068] The present application outputs an AC voltage through a power supply, and when the fluorescence information in the groove decreases, the voltage is continuously output in the direction of the voltage output by the power supply to control the dNTP to be away from the surface focal plane of the fluorescent sequencing chip, so that the dNTP is separated from the sample to be tested, thereby controlling the sequencing step to stop.

[0069] In some embodiments of the present application, the sequencing method also includes a cleaning step; the cleaning step is used to remove residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing; the cleaning step includes: placing an ionic solution on the fluorescent sequencing chip, and making the liquid level of the ionic solution higher than the top of the groove; electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ionic solution, and making the power supply output an AC voltage; when the fluorescence information in the groove decreases, continuously outputting the voltage in the direction of the voltage output by the power supply; then removing the liquid phase on the fluorescent sequencing chip; or / and, the sequencing step includes: electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ionic solution, and making the power supply output an AC voltage; when the fluorescence information in the groove increases, continuously outputting the voltage in the direction of the voltage output by the power supply; determining the sequence of the biological molecule to be sequenced by obtaining the fluorescence information corresponding to the biological molecule to be sequenced in the groove.

[0070] The present application outputs an AC voltage through a power supply, and when the fluorescence information in the groove decreases, the voltage is continuously output in the direction of the voltage output by the power supply to control free fluorescent molecules, samples to be tested, dNTPs and other non-specific binding substances to be away from the surface focal plane of the fluorescent sequencing chip, that is, it is beneficial to clean and remove the free fluorescent molecules, samples to be tested, dNTPs and other residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing, thereby reducing the free fluorescent molecules, samples to be tested, dNTPs and other non-specific binding substances adsorbed on the surface of the sequencing chip and affecting the sequencing signal; or / and, outputs an AC voltage through a power supply, and when the fluorescence information in the groove increases, the voltage is continuously output in the direction of the voltage output by the power supply to control the dNTP to be close to the surface focal plane of the fluorescent sequencing chip, thereby increasing the penetration rate of the dNTP into the groove, and increasing the binding efficiency of the dNTP with the biological molecules to be sequenced in the groove, thereby improving the detection accuracy of the fluorescent sequencing.

[0071] In some embodiments of the present application, in the sequencing step, when the voltage is continuously output in the voltage direction of the power supply output, the voltage value of the power supply output is increased. Continuously outputting a suitable voltage in the voltage direction of the power supply output and increasing the voltage value of the power supply output are beneficial to increase the electric field force applied to the surface of the fluorescent sequencing chip, so as to increase the penetration rate of dNTP into the groove and improve the binding efficiency of dNTP and the biological molecule to be sequenced in the groove.

[0072] In some embodiments of the present application, in the sequencing step, when the voltage is continuously output in the voltage direction of the power supply output, the voltage value of the power supply output is increased to ≥ 0.1 V. Continuously outputting a suitable voltage in the voltage direction of the power supply output is conducive to applying a suitable electric field force to the surface of the fluorescent sequencing chip, so as to control the dNTP to be close to the surface focal plane of the fluorescent sequencing chip, increase the penetration rate of the dNTP into the groove, and improve the binding efficiency of the dNTP with the biological molecule to be sequenced in the groove.

[0073] In some embodiments of the present application, the ionic solution may contain, but is not limited to, at least one of Na2SO3, Na2SO4, Na2HPO4, KH2PO4, NaCl, KCl, MgCl2, glucose oxidase, catalase, glucose and cysteine.

[0074] In some embodiments of the present application, the ionic solution may contain, but is not limited to, 2-4 M Na2SO3, 1-2 M Na2SO4, 1-2 M Na2HPO4, 1-2 M KH2PO4, 1-2 M NaCl, 1-2 M KCl, 1-2 M MgCl2, 100-200 μg / mL glucose oxidase, 100-200 μg / mL catalase, 10%-15% glucose, and 100-200 mM cysteine.

[0075] In some embodiments of the present application, the sequencing steps include: connecting a modifier to the bottom wall of the groove; placing an ionic solution on the chip and making the liquid level of the ionic solution higher than the top of the groove; placing the biological molecule to be sequenced in the ionic solution so that the biological molecule to be sequenced enters the groove and combines with the modifier in the groove; placing a fluorescently labeled biological molecule in the ionic solution so that the fluorescently labeled biological molecule enters the groove and combines with the biological molecule to be sequenced in the groove; determining the sequence of the biological molecule to be sequenced by obtaining the fluorescent information corresponding to the biological molecule to be sequenced in the groove; wherein the biological molecule to be sequenced is DNA, and a DNA polymerase that specifically binds to DNA is bound to the molecule to be sequenced; the fluorescently labeled biological molecule is a fluorescently labeled dNTP; the modifier is biotin and streptavidin; when the sequencing stop step, the voltage continuously output in the voltage direction of the power supply output is ≥0.1V.

[0076] The present application connects biotin and streptavidin modifiers on the bottom wall of the groove, allows the biomolecule DNA to be sequenced to enter the groove and bind to the modifiers in the groove, and allows the fluorescently labeled dNTP to enter the groove and bind to the biomolecule DNA to be sequenced in the groove, so as to determine the sequence of the biomolecule DNA to be sequenced; at the same time, when the sequencing stops, the appropriate voltage is continuously output in the voltage direction of the power supply output to control the dNTP to stay away from the surface focal plane of the fluorescent sequencing chip, so that the dNTP is separated from the sample to be tested, thereby achieving the control of stopping the sequencing step.

[0077] In some embodiments of the present application, during the sequencing stop step, the frequency of the voltage continuously output in the voltage direction of the power supply is ≤100 Hz.

[0078] The embodiment of the present application provides a method for improving the accuracy of fluorescent sequencing. The method for improving the accuracy of sequencing uses the fluorescent sequencing chip as described above. The method for improving the accuracy of sequencing includes: placing an ion solution on the chip, and making the liquid level of the ion solution higher than the top of the groove; electrically connecting one electrode of a power supply to the conductive layer, and extending the other electrode into the ion solution, and making the power supply output an AC voltage; when the fluorescence information in the groove increases, continuously outputting the voltage in the direction of the voltage output of the power supply and increasing the voltage value of the power supply output; and determining the sequence of the biological molecule to be sequenced by obtaining the fluorescence information corresponding to the biological molecule to be sequenced in the groove.

[0079] The present application outputs an AC voltage through a power supply, and when the fluorescence information in the groove increases, the voltage is continuously output in the direction of the voltage output by the power supply to control the dNTP to be close to the surface focal plane of the fluorescent sequencing chip, thereby increasing the penetration rate of the dNTP into the groove, and increasing the binding efficiency of the dNTP with the biological molecules to be sequenced in the groove, thereby improving the detection accuracy of the fluorescent sequencing.

[0080] In some embodiments of the present application, the voltage is continuously output in the voltage direction of the power supply output and the voltage value of the power supply output is increased to ≥ 0.1 V. Continuously outputting a suitable voltage in the voltage direction of the power supply output is conducive to applying a suitable electric field force to the surface of the fluorescent sequencing chip, so as to control the dNTP to be close to the surface focal plane of the fluorescent sequencing chip, increase the penetration rate of the dNTP into the groove, and increase the binding efficiency of the dNTP with the biological molecules to be sequenced in the groove, thereby improving the detection accuracy of the fluorescent sequencing.

[0081] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0082] Example 1

[0083] The present embodiment provides a fluorescence sequencing method, which uses a fluorescence sequencing chip. The fluorescence sequencing chip includes: an insulating layer, a conductive layer, a covering layer and a power supply; wherein the insulating layer, the conductive layer and the covering layer are stacked in sequence along the thickness direction; the covering layer has a groove that penetrates the covering layer along the thickness direction; the insulating layer is made of glass; the conductive layer is indium tin oxide, and the thickness of the conductive layer is 200nm; the depth of the groove on the covering layer is 200nm, the diameter of the groove is 300nm, and the distance between two adjacent grooves is 500nm.

[0084] The sequencing method includes a sequencing step and a sequencing stop step; the sequencing step includes: connecting the modifiers biotin and streptavidin and a DNA polymerase specifically binding to the DNA to be sequenced on the bottom wall of the groove, then adding fluorescently labeled dNTP and an ion solution containing 1M Na2HPO4, 1M KH2PO4, 1M NaCl, 1M KCl, and 1M MgCl2, and making the liquid level of the ion solution higher than the top of the groove, electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ion solution, and making the power supply output ±1.5V AC voltage, when the fluorescence information in the groove increases, the voltage direction of the power supply output continuously outputs +1.5V voltage, and the sequence of the DNA to be sequenced is determined by obtaining the fluorescence information corresponding to the DNA to be sequenced in the groove; the sequencing stop step includes: electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ion solution, and making the power supply output ±1.5V AC voltage; when the fluorescence information in the groove decreases, continuously outputting -1.5V voltage in the voltage direction of the power supply output;

[0085] The sequencing method also includes a cleaning step; the cleaning step is used to remove residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing; the cleaning step includes: electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ion solution, and making the power supply output ±1.5V AC voltage, when the fluorescent information in the groove decreases, continuously outputting -1.5V voltage in the direction of the voltage output of the power supply; then removing the liquid phase on the fluorescent sequencing chip.

[0086] Example 2

[0087] This embodiment provides a fluorescence sequencing method, which is different from Embodiment 1 in that: the sequencing step includes: making the power supply output ±3V AC voltage, and when the fluorescence information in the groove increases, the power supply outputs a voltage direction that continuously outputs a +3V voltage; the sequencing stop step includes: making the power supply output ±3V AC voltage, and when the fluorescence information in the groove decreases, the power supply outputs a voltage direction that continuously outputs a -3V voltage; the cleaning step includes: making the power supply output ±3V AC voltage, and when the fluorescence information in the groove decreases, the power supply outputs a voltage direction that continuously outputs a -3V voltage.

[0088] Test Example 1

[0089] This test example detects and counts the number of fluorescence events that occur in the groove of the fluorescent sequencing chip when an AC voltage is applied to the fluorescent sequencing chip in Example 1-2. Figure 3-Figure 4 shown.

[0090] Depend on Figure 3-Figure 4The results show that when a +1.5V voltage or a +3V voltage is applied to the fluorescent sequencing chip, the fluorescence events occurring in the groove of the fluorescent sequencing chip increase, and when a -1.5V voltage or a -3V voltage is applied to the fluorescent sequencing chip, the fluorescence events occurring in the groove of the fluorescent sequencing chip decrease; this indicates that when the present application applies a positive voltage to the fluorescent sequencing chip, free fluorescent molecules, dNTPs, etc. can be controlled to approach the surface focal plane of the fluorescent sequencing chip, and a large number of fluorescent molecules move near the surface focal plane of the fluorescent sequencing chip to be collected and detected, thereby facilitating the improvement of the penetration rate of dNTPs into the grooves and the improvement of the binding of dNTPs with the biological molecules to be sequenced in the grooves. Efficiency, improve the detection accuracy of fluorescent sequencing; when a negative voltage is applied to the fluorescent sequencing chip, the free fluorescent molecules, dNTPs, etc. can be controlled to be away from the surface focal plane of the fluorescent sequencing chip, so that the number of fluorescent molecules moving near the surface focal plane of the fluorescent sequencing chip is reduced, thereby facilitating the separation of dNTPs from the sample to be tested, achieving the control of stopping the sequencing step, and facilitating the control of the distance of free fluorescent molecules, the sample to be tested, dNTPs and other non-specific binding substances away from the surface focal plane of the fluorescent sequencing chip, making them easy to wash away, thereby reducing the free fluorescent molecules, the sample to be tested, dNTPs and other non-specific binding substances being adsorbed to the surface of the sequencing chip and affecting the sequencing signal;

[0091] At the same time, when a voltage of ±3V is applied to the fluorescent sequencing chip, the difference in fluorescence events occurring in the groove of the fluorescent sequencing chip is greater than when a voltage of ±1.5V is applied, which indicates that increasing the voltage value of the power supply output can better control the distance between free fluorescent molecules, test samples, dNTPs and other non-specific binding substances and the surface focal plane of the fluorescent sequencing chip.

[0092] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A fluorescent sequencing chip, characterized in that: include: Insulating layer, conductive layer, covering layer and power supply; The insulating layer, the conductive layer and the covering layer are stacked in sequence along the thickness direction; the covering layer has a groove penetrating the covering layer along the thickness direction; the inside of the groove and the surface of the covering layer are used to jointly contain an ion solution, so that the biological molecules to be sequenced enter the groove, and the fluorescence information in the groove can be collected; One end electrode of the power supply is used to be connected to the conductive layer, and the other end electrode is used to extend into the ion solution.

2. The fluorescent sequencing chip according to claim 1, characterized in that: The covering layer has a plurality of the grooves, and the plurality of the grooves are distributed in the covering layer in an array manner; Optionally, the depth of the groove is 100nm to 500nm, the diameter of the groove is 50nm to 500nm, and the distance between two adjacent grooves is 100nm to 900nm; Optionally, along the thickness direction, the diameter of the groove is consistent; Optionally, the cross-section of the groove is circular, square, diamond or gear-shaped.

3. The fluorescent sequencing chip according to claim 1, characterized in that: The material of the conductive layer includes at least one of iron oxide, aluminum oxide, zinc oxide, copper oxide, indium tin oxide, titanium oxide, tantalum oxide, niobium oxide, fluorine-doped tin oxide and silicon carbide; or / and, the conductive layer has a thickness of 10 nm to 500 nm and comprises one or more layers of the above materials; Or / and, the material of the insulating layer includes glass; Or / and, the material of the electrode includes at least one of platinum, gold and silver; Or / and, the material of the covering layer includes polymer.

4. A method for cleaning the fluorescent sequencing chip according to any one of claims 1 to 3, characterized in that: The cleaning method is used to remove residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing, and the cleaning method comprises: Placing an ion solution on the fluorescent sequencing chip, and making the liquid level of the ion solution higher than the top of the groove; One end electrode of the power supply is electrically connected to the conductive layer, and the other end electrode is extended into the ion solution, so that the power supply outputs an AC voltage; when the fluorescent information in the groove decreases, the voltage is continuously output in the direction of the voltage output by the power supply; Then, the liquid phase on the fluorescent sequencing chip is removed.

5. The cleaning method according to claim 4, characterized in that: The ion solution contains at least one of Na2SO3, Na2SO4, Na2HPO4, KH2PO4, NaCl, KCl, MgCl2, glucose oxidase, catalase, glucose and cysteine; Optionally, the ionic solution contains 2-4M Na2SO3, 1-2M Na2SO4, 1-2M Na2HPO4, 1-2M KH2PO4, 1-2M NaCl, 1-2M KCl, 1-2M MgCl2, 100-200 μg / mL glucose oxidase, 100-200 μg / mL catalase, 10%-15% glucose, and 100-200 mM cysteine; and / or, the residual molecules include at least one of DNA, RNA, protein, polymerase, helicase, transcriptase, dNTP, biotin and streptavidin; During the cleaning, the voltage continuously output in the voltage direction of the power supply is ≥ 0.1V; Optionally, during the cleaning, the frequency of the voltage continuously output in the voltage direction of the power supply is ≤100 Hz; Optionally, the cleaning method includes: before the power supply outputs the AC voltage, adding a cleaning agent to the ion solution on the fluorescent sequencing chip; Optionally, the cleaning agent includes at least one of sodium dodecyl sulfate, Triton-100, ethanol, propanol, and polysorbate-20.

6. A fluorescence sequencing method, characterized in that: The sequencing method uses the fluorescent sequencing chip according to any one of claims 1 to 3, and the sequencing method includes a sequencing proceeding step and a sequencing stopping step; The sequencing step includes: placing an ion solution on the fluorescent sequencing chip, and making the liquid level of the ion solution higher than the top of the groove, so that the biological molecule to be sequenced enters the groove, and determining the sequence of the biological molecule to be sequenced by acquiring the fluorescence information corresponding to the biological molecule to be sequenced in the groove; The sequencing stop step includes: electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ion solution, and making the power supply output an AC voltage; when the fluorescence information in the groove decreases, continuously outputting voltage in the direction of the voltage output by the power supply.

7. The sequencing method according to claim 6, characterized in that: The sequencing method further comprises a washing step; The cleaning step is used to remove residual molecules on the surface of the fluorescent sequencing chip after fluorescent sequencing; the cleaning step includes: placing an ion solution on the fluorescent sequencing chip, and making the liquid level of the ion solution higher than the top of the groove; electrically connecting one end electrode of the power supply to the conductive layer, and extending the other end electrode into the ion solution, and making the power supply output an AC voltage; when the fluorescent information in the groove decreases, continuously outputting the voltage in the direction of the voltage output by the power supply; and then removing the liquid phase on the fluorescent sequencing chip; Or / and, the sequencing step includes: electrically connecting one end electrode of the power supply to the conductive layer, extending the other end electrode into the ion solution, and making the power supply output an AC voltage; when the fluorescence information in the groove increases, continuously outputting the voltage in the direction of the voltage output by the power supply; determining the sequence of the biomolecule to be sequenced by acquiring the fluorescence information corresponding to the biomolecule to be sequenced in the groove; Optionally, in the sequencing step, when the voltage is continuously output in the voltage direction of the power supply output, the voltage value of the power supply output is increased; Optionally, in the sequencing step, when the voltage is continuously output in the voltage direction of the power supply output, the voltage value of the power supply output is increased to ≥0.1V.

8. The sequencing method according to claim 6 or 7, characterized in that: The ion solution contains at least one of Na2SO3, Na2SO4, Na2HPO4, KH2PO4, NaCl, KCl, MgCl2, glucose oxidase, catalase, glucose and cysteine; Optionally, the ionic solution contains 2-4M Na2SO3, 1-2M Na2SO4, 1-2M Na2HPO4, 1-2M KH2PO4, 1-2M NaCl, 1-2M KCl, 1-2M MgCl2, 100-200 μg / mL glucose oxidase, 100-200 μg / mL catalase, 10%-15% glucose and 100-200 mM cysteine.

9. The sequencing method according to claim 8, characterized in that The sequencing step comprises: connecting a decoration to the bottom wall of the groove; Placing an ion solution on the chip, and making the liquid level of the ion solution higher than the top of the groove; Placing the biomolecule to be sequenced in the ionic solution, so that the biomolecule to be sequenced enters the groove and binds to the modifier in the groove; Placing a fluorescently labeled biomolecule in the ionic solution, so that the fluorescently labeled biomolecule enters the groove and combines with the biomolecule to be sequenced in the groove; Determine the sequence of the biomolecule to be sequenced by acquiring fluorescence information corresponding to the biomolecule to be sequenced in the groove; Wherein, the biological molecule to be sequenced is DNA, and the molecule to be sequenced is bound to a DNA polymerase that specifically binds to the DNA; the fluorescently labeled biological molecule is a fluorescently labeled dNTP; and the modifier is biotin and streptavidin; When the sequencing stops, the voltage continuously output in the voltage direction of the power supply is ≥ 0.1V; Optionally, during the sequencing stop step, a voltage frequency continuously output in the voltage direction of the power supply is ≤100 Hz.

10. A method for improving the accuracy of fluorescent sequencing, characterized in that: The method for improving the accuracy of sequencing uses the fluorescent sequencing chip according to any one of claims 1 to 3, and the method for improving the accuracy of sequencing comprises: Placing an ion solution on the chip, and making the liquid level of the ion solution higher than the top of the groove; An electrode at one end of the power supply is electrically connected to the conductive layer, and an electrode at the other end is extended into the ion solution, so that the power supply outputs an AC voltage; when the fluorescence information in the groove increases, the voltage is continuously output in the direction of the voltage output by the power supply and the voltage value output by the power supply is increased; by obtaining the fluorescence information corresponding to the biological molecule to be sequenced in the groove, the sequence of the biological molecule to be sequenced is determined; Optionally, the voltage is continuously output in the voltage direction of the power supply output and the voltage value of the power supply output is increased to ≥0.1V.

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