Sensor, kit and application thereof for detecting target molecules based on changes in liquid motion state

Abstract

The present invention relates to a composition, a sensor, and a preparation method and application thereof for judging a target molecule based on a target molecule-driven liquid motion state change. Specifically, the present invention activates the CRISPR / Cas system through a target nucleic acid and then triggers the conversion of the hydrophilicity and hydrophobicity of the reaction area interface, thereby judging the presence or absence of the target nucleic acid by the change in the motion state of the sample liquid in the reaction area (mutual conversion between static and motion). The present invention can highly sensitively judge the presence or absence of the target nucleic acid. In addition, the present invention can even achieve rapid and highly sensitive detection of the target nucleic acid by naked eye observation without the need for a display and a chip, and the relevant device preparation is simple and low-cost.

Description

Technical Field

[0001] The present invention relates to the detection of target molecules, and specifically to a composition, a sensor, and a preparation method and application thereof for detecting target molecules based on a target molecule-driven change in the motion state of a liquid; more specifically, to a sensor that detects target molecules by activating a CRISPR / Cas system based on a target molecule to drive the sensor interface to change from hydrophilic to hydrophobic or vice versa, thereby causing the liquid motion state to change, as well as a preparation method and application thereof. Background Art

[0002] Nucleic acid testing is widely used in the diagnosis of a variety of diseases, including the novel coronavirus, monkeypox virus, hepatitis A, hepatitis B, tumors, AIDS, Alzheimer's disease, and Down syndrome (also known as trisomy syndrome). Optical and electrical signals are the two most common signals in nature, and a variety of analytical instruments based on these two signals are being continuously developed and optimized for nucleic acid analysis, such as common PCR instruments, UV spectrophotometers, Raman spectrometers, and electrochemical workstations. Although these instruments have been successfully applied to nucleic acid analysis to varying degrees, the conversion of natural signals into digital signals necessitates the use of chips and displays. The presence of chips and displays inevitably increases the size and cost of the instruments, making these sensors inaccessible to the public and, therefore, unable to become widely available point-of-care (POCT) devices.

[0003] Although test strip sensors based on lateral flow and colloidal gold technologies have been widely used as point-of-care (POCT) devices during the COVID-19 pandemic, these devices focus on immunoassays and are not suitable for nucleic acid testing. This means that these sensors often only provide a signal after patients have already shown clinical discomfort, often failing to achieve early diagnosis. Therefore, the development of a highly sensitive nucleic acid detection platform that can eliminate the need for expensive instrumentation and is inexpensive is particularly urgent, as it is of great significance for reducing social operating costs and improving social efficiency. Summary of the Invention

[0004] In order to solve the above problems, the inventors have developed a detection method that activates the CRISPR / Cas system by target nucleic acid and triggers the conversion of the hydrophilicity and hydrophobicity of the reaction area interface, thereby judging the presence or absence of the target nucleic acid by the change in the motion state of the sample liquid in the reaction area (conversion between static and motion). This method can highly sensitively judge the presence or absence of the target nucleic acid. In addition, this method does not require a display and a chip, and can achieve rapid and highly sensitive detection of the target nucleic acid by naked eye observation. The relevant device preparation is simple and low cost.

[0005] In one aspect, the present invention provides a composition for detecting nucleic acid, the composition comprising:

[0006] substrate;

[0007] a conversion reagent, wherein the conversion reagent is attached to the substrate, wherein the conversion reagent is a probe; and

[0008] Identification reagents,

[0009] Wherein, the conversion reagent has the following structure:

[0010] [Hydrophilic group]-[Switch]-[Hydrophobic group],

[0011] wherein the switch comprises a single-stranded nucleic acid,

[0012] The substrate and the conversion reagent are connected at the hydrophilic group end, thereby making the substrate hydrophobic; or the substrate and the conversion reagent are connected at the hydrophobic group end, thereby making the substrate hydrophilic.

[0013] In some embodiments, the recognition reagent is a CRISPR / Cas system. In a preferred embodiment, the recognition element can be selected from the CRISPR / Cas12a system and the CRISPR / Cas13a system.

[0014] In some embodiments, the compositions of the invention are in the form of a biosensor.

[0015] On the other hand, the present invention provides a method for detecting nucleic acids in a sample, comprising: contacting the sample with the composition described in the first aspect, and observing the movement state of the sample to detect whether the nucleic acid exists in the sample.

[0016] In another aspect, the present invention provides a kit for detecting nucleic acid, wherein the kit comprises the composition described above and instructions for using the composition for detection.

[0017] In another aspect, the present invention provides use of the composition and kit of the present invention in detecting nucleic acids.

[0018] Beneficial effects

[0019] The present invention provides a sensing platform that is independent of instruments (especially electronic instruments) and can detect nucleic acids with high sensitivity through direct observation. It is a POCT home detection instrument with great potential.

[0020] Furthermore, the sensor platform of the present invention utilizes inexpensive raw materials and has a commercial production base. For example, the present invention can utilize inexpensive glass as the sensor substrate (e.g., glass tube). Compared to other substrate materials that require specialized laboratory synthesis or currently lack the potential for large-scale factory production, glass (e.g., glass tube) is highly suitable for commercial applications of the sensors of the present invention due to its mature, large-scale factory production and low cost.

[0021] Moreover, the CRISPR / Cas system used in the present invention enables large-scale cutting of the probe (switch) in the presence of a very small amount of target nucleic acid (e.g., even a single target nucleic acid molecule), achieving a one-time large-scale release of the conversion group in the conversion element, resulting in a dramatic conversion of the hydrophilic and hydrophobic properties of the substrate surface, thereby achieving rapid and highly sensitive detection. In the present invention, combining the probe structure of the present invention with the cutting ability of the CRISPR / Cas system, effective detection of RNA and DNA nucleic acid samples with a concentration as low as 1aM can be achieved within 6-10 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure (1a) is a schematic diagram of the dimensions of a glass test tube nucleic acid sensor in an upright state with a conversion reagent (i.e., a functional probe) connected to the bottom. Figure (1b) is a schematic diagram of the movement of a liquid containing a CRISPR / Cas system in a glass test tube nucleic acid sensor in an upright state with a functional probe connected to the bottom. Figure (1c) is a schematic diagram of the movement of a liquid containing a CRISPR / Cas system in a glass test tube nucleic acid sensor in an inverted state with a functional probe connected to the bottom. Figure (1d) is a schematic diagram of the movement of a mixed solution of a sample and a CRISPR / Cas system in a glass test tube nucleic acid sensor in an inverted state with a functional probe connected to the bottom after reacting with a sample containing a target nucleic acid for 1 minute.

[0023] Figure (2a) is a schematic diagram of the dimensions of a glass test tube nucleic acid sensor in an upright state with a conversion reagent (i.e., a functional probe) connected to the bottom. Figure (2b) is a schematic diagram of the movement of a liquid containing a CRISPR / Cas system in a glass test tube nucleic acid sensor in an upright state with a functional probe connected to the bottom. Figure (2c) is a schematic diagram of the movement of a liquid containing a CRISPR / Cas system in a glass test tube nucleic acid sensor in an inverted state with a functional probe connected to the bottom. Figure (2d) is a schematic diagram of the movement of a mixed solution of a sample and a CRISPR / Cas system in a glass test tube nucleic acid sensor in an inverted state with a functional probe connected to the bottom after reacting with a sample containing a target nucleic acid for 1 minute.

[0024] Figure (3a) is a photograph showing the change in motion state of a liquid containing a CRISPR / Cas12a system in a glass tube nucleic acid sensor with a conversion reagent (i.e., a functional probe) connected to the bottom in an upright state. Figure (3b) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a non-target nucleic acid is reacted with a mixed solution of the CRISPR / Cas12a system for 1 minute in the upright state of the sensor shown in Figure (3a). Figure (3c) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a target nucleic acid (African swine fever virus P72 gene nucleic acid) is reacted with a mixed solution of the CRISPR / Cas12a system for 1 minute in the upright state of the sensor shown in Figure (3a). Figure (3d) is a photograph of a blank control group, in which the same operation as Figure (3c) is used, but only a solvent is used to react with the CRISPR / Cas12a system.

[0025] Figure (4a) is a photograph showing the change in motion state of a glass tube nucleic acid sensor in which a liquid containing a CRISPR / Cas13a system is connected to a conversion reagent (i.e., a functional probe) at the bottom in an upright state. Figure (4b) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a non-target nucleic acid is reacted with a mixed solution of the CRISPR / Cas13a system for 1 minute in the upright state of the sensor shown in Figure (4a). Figure (4c) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a target nucleic acid (new coronavirus N gene nucleic acid) is reacted with a mixed solution of the CRISPR / Cas13a system for 1 minute in the upright state of the sensor shown in Figure (4a). Figure (4d) is a photograph of a blank control group, in which the same operation as Figure (4c) is used, but only a solvent is used to react with the CRISPR / Cas13a system.

[0026] Figure (5a) is a photograph showing the change in motion state of a liquid containing a CRISPR / Cas12a system in a glass tube nucleic acid sensor with a conversion reagent (i.e., a functional probe) connected to the bottom in an upright state. Figure (5b) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a non-target nucleic acid is reacted with a mixed solution of the CRISPR / Cas12a system for 1 minute in the upright state of the sensor shown in Figure (5a). Figure (5c) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a target nucleic acid (African swine fever virus P72 gene nucleic acid) is reacted with a mixed solution of the CRISPR / Cas12a system for 1 minute in the upright state of the sensor shown in Figure (5a). Figure (5d) is a photograph of a blank control group, in which the same operation as Figure (5c) is used, but only a solvent is used to react with the CRISPR / Cas12a system.

[0027] Figure (6a) is a photograph showing the change in motion state of a glass tube nucleic acid sensor in which a conversion reagent (i.e., a functional probe) is connected to the bottom of a liquid containing CRISPR / Cas13a in an upright state. Figure (6b) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a non-target nucleic acid is reacted with a mixed solution of the CRISPR / Cas13a system for 1 minute in the upright state of the sensor shown in Figure (6a). Figure (6c) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a target nucleic acid (new coronavirus N gene nucleic acid) is reacted with a mixed solution of the CRISPR / Cas13a system for 1 minute in the upright state of the sensor shown in Figure (6a). Figure (6d) is a photograph of a blank control group, in which the same operation as Figure (6c) is used, but only a solvent is used to react with the CRISPR / Cas13a system.

[0028] Figure (7a) is a photograph showing the change in motion state of a liquid containing a CRISPR / Cas12a system in a glass tube nucleic acid sensor with a conversion reagent (i.e., a functional probe) connected to the bottom in an upright state. Figure (7b) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a non-target nucleic acid is reacted with a mixed solution of the CRISPR / Cas12a system for 1 minute in the upright state of the sensor shown in Figure (7a). Figure (7c) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a target nucleic acid (African swine fever virus nucleic acid) is reacted with a mixed solution of the CRISPR / Cas12a system for 1 minute in the upright state of the sensor in Figure (7a). Figure (7d) is a photograph of a blank control group, in which the same operation as Figure (7c) is used, but only a solvent is used to react with the CRISPR / Cas12a system.

[0029] Figure (8a) is a photograph showing the change in motion state of a liquid containing CRISPR / Cas13a in a glass tube nucleic acid sensor with a conversion reagent (i.e., a functional probe) connected to the bottom in an upright state. Figure (8b) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a non-target nucleic acid is reacted with a mixed solution of the CRISPR / Cas13a system for 1 minute in the upright state of the sensor shown in Figure (8a). Figure (8c) is a photograph showing the change in motion state of the mixed solution in the sensor in an inverted state after a sample containing a target nucleic acid (new coronavirus nucleic acid) is reacted with a mixed solution of the CRISPR / Cas13a system for 1 minute in the upright state of the sensor shown in Figure (8a). Figure (8d) is a photograph of the blank control group, in which the same operation as Figure (8c) is used, but only a solvent is used to react with the CRISPR / Cas13a system. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only for illustration and explanation of the present invention and are not intended to limit the present invention. The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include ranges or values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in this article.

[0031] The present invention utilizes the CRISPR / Cas system activated by the target nucleic acid to drive the opening of the conversion switch in the conversion reagent to convert the hydrophilic and hydrophobic properties of the reaction area interface, and then the presence or absence of the target nucleic acid is highly sensitively judged by the conversion of the motion state of the test liquid in the reaction area (such as static and motion conversion). In this detection method, the CRISPR / Cas system acts as a target recognition element. After being recognized and activated by the target nucleic acid, based on its ability to randomly cut, the conversion element (such as a probe) acting on the reaction area interface converts the hydrophilic and hydrophobic properties of the reaction area interface in a relatively short period of time (such as within 6-10 minutes), thereby changing the motion state of the test liquid in the reaction area. Even the method based on the present invention has achieved the successful detection of a target nucleic acid with a concentration of 1aM.

[0032] Composition

[0033] In one aspect, the present invention provides a composition for detecting nucleic acid, the composition comprising:

[0034] substrate;

[0035] a conversion reagent, wherein the conversion reagent is attached to the substrate, wherein the conversion reagent is a probe; and

[0036] Identification reagents,

[0037] Wherein, the conversion reagent has the following structure:

[0038] [Hydrophilic group]-[Switch]-[Hydrophobic group],

[0039] wherein the switch comprises a single-stranded nucleic acid,

[0040] The substrate and the conversion reagent are connected at the hydrophilic group end, thereby making the substrate hydrophobic; or the substrate and the conversion reagent are connected at the hydrophobic group end, thereby making the substrate hydrophilic.

[0041] In some embodiments, the nucleic acid may be DNA or RNA. In some embodiments, the nucleic acid may be DNA. In some embodiments, the nucleic acid may be RNA. In some embodiments, the nucleic acid may be DNA and RNA.

[0042] In some embodiments, the recognition reagent is a CRISPR / Cas system. In a preferred embodiment, the recognition element can be selected from the CRISPR / Cas12a system and the CRISPR / Cas13a system.

[0043] In an embodiment of the present invention, the composition of the present invention may be in the form of a biosensor.

[0044] biosensors

[0045] A biosensor is generally considered to be a type of device that analyzes biological materials (such as tissues, microbial cells, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.) or biologically derived materials or biomimetic materials. It closely combines or links biological materials with physical and chemical sensors or sensor microsystems, and can generate intermittent or continuous signals, thereby exercising analytical functions. In this art, biosensors have a general definition and are generally composed of two parts: a recognition element (also known as a molecular recognition element) and a transducer element (also known as a transducer). In this article, the term "biosensor" refers to a device or equipment comprising a recognition element and a conversion element, also referred to as a "biosensing platform". Without being bound by theory, the biosensor described herein may include an amplifying element that further improves the detection sensitivity and / or a detection element that directly performs detection. In addition, the biosensor described herein is not limited to a specific form, and any form that includes the recognition element and conversion element described below is included within the scope of the term "biosensor" herein.

[0046] base

[0047] In the present invention, a conversion reagent (eg, a probe) is attached to the surface of a substrate to render the surface hydrophilic or hydrophobic.

[0048] In some embodiments, the probes may be attached to the entire surface of the substrate. In some embodiments, the probes may be attached to one or more regions of the substrate surface.

[0049] In some embodiments, the substrate can be in various configurations, such as a tube, a flat plate, a bowl, a microfluidic channel, and the like. For example, in the case of a tube / bowl, the probe can be attached to the bottom region of the substrate. For example, in the case of a flat plate, the probe can be attached to one or more regions of the substrate. For example, on a flat plate substrate, the probe can be attached to multiple regions at specific intervals (e.g., 1 mm to 5 cm).

[0050] In some embodiments, the substrate can be selected from glass substrates, silicon substrates, silicon dioxide substrates, indium tin oxide substrates, gold substrates, silver substrates, copper substrates, polydimethylsiloxane substrates, polymethyl methacrylate substrates, polycarbonate substrates, polyamide fiber substrates, polyvinyl chloride substrates and polyethylene terephthalate substrates, as well as any composite substrates thereof. For ease of visual observation, a transparent, translucent or white substrate is preferably used.

[0051] In the present invention, the substrate is preferably of a size suitable for operation. For example, in some embodiments, when a tubular or bowl-shaped substrate is used, its length is preferably such that liquid dripping can be observed, for example, preferably 2 cm or more, such as 2-10 cm, more preferably 5-10 cm.

[0052] In some embodiments, the substrate may be a glass test tube or a glass plate. For example, the glass test tube may be a conventional glass test tube of various specifications, such as a test tube with a length of 7.5 cm, a diameter of 1 cm, and a wall thickness of 0.1 cm.

[0053] In the present invention, the portion of the substrate to which the probe is attached may be referred to as a reaction region. Thus, in some embodiments, the entire substrate may be a reaction region. In a preferred embodiment, a portion of the substrate is a reaction region.

[0054] In a preferred embodiment, the size of the reaction area is preferably capable of accommodating 50-500 μl of liquid, preferably 50-200 μl of liquid, more preferably 80-160 μl of liquid.

[0055] In some embodiments, the substrate further comprises a signal stabilization region, which is preferably a hydrophobic region. In a preferred embodiment, the signal stabilization region is arranged in a manner that completely or partially, preferably completely, surrounds the reaction region, and the size of the signal stabilization region is less than, equal to, or greater than the size of the reaction region extending outward from the boundary of the reaction region. For example, when the reaction region is circular or rectangular, the diameter or side length of the signal stabilization region is less than, equal to, or greater than the diameter or side length of the reaction region extending outward from the boundary of the reaction region; the same applies when the reaction region is an irregular shape.

[0056] In a preferred embodiment, a sealing member that matches the substrate may also be included. For example, a tubular substrate may have a matching cover or cap, or a membrane may be used for sealing so that it can be closed when needed. To facilitate viewing, the cover, cap, or membrane is preferably transparent, translucent, or light-colored (e.g., white, off-white, etc.).

[0057] Conversion reagent or conversion element

[0058] In the present invention, the conversion reagent or conversion element (eg, probe) has the following structure: [hydrophilic group]-[conversion switch]-[hydrophobic group].

[0059] In a specific embodiment, the switch comprises one or more single-stranded nucleic acid portions. The single-stranded nucleic acid portions may be single-stranded DNA or single-stranded RNA. The length of each single-stranded nucleic acid portion may be selected from 5-30 nt, preferably 5-20 nt, for example, preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nt. When the single-stranded nucleic acid is single-stranded DNA, its length may be selected from 6-20 nt, more preferably 14-18 nt, and most preferably 15 or 16 nt. When the single-stranded nucleic acid is single-stranded RNA, its length may be selected from 5-10 nt, more preferably 5-8 nt, and most preferably 5 or 6 nt.

[0060] The sequence of the single-stranded nucleic acid can be any sequence. For example, a single-stranded DNA can be a 5-30 nt deoxyribonucleic acid chain composed of thymidine deoxynucleotides (hereinafter referred to as T). For example, a single-stranded RNA can be a 6-30 nt ribonucleic acid chain composed of uracil ribonucleotides (hereinafter referred to as U).

[0061] For example, the single-stranded DNA can be selected from the following: 5'-TTTTT-3', 5'-TTTTTT-3', 5'-TTTTTTTT-3', 5'-TTTTTTTTTT-3', 5'-TTTTTTTTTTTT-3', 5'-TTTTTTTTTTTTTT-3', 5'-TTTTTTTTTTTTTTTT-3', or 5'-TTTTTTTTTTTTTTTT-3'.

[0062] For example, the single-stranded RNA can be selected from the following: 5'-UUUUU-3', 5'-UUUUUU-3', or 5'-UUUUUUU-3'.

[0063] In the present invention, the hydrophilic group can be formed by one or more selected from the following: a nucleic acid chain composed of deoxynucleotides (e.g., deoxyribonucleic acid of 12-100 bases in length), polyethylene glycol (hereinafter referred to as PEG, such as HO(CH2CH2O) m H, wherein m is an integer between 5 and 13, for example, m can be 5, 6, 7, 8, 9, 10, 11, 12 or 13) or PEG derivatives, polyacrylic acid, polyacrylic acid, polyacrylic acid, polyurethane, and polyamide. For the above polymers, there is no upper limit to their degree of polymerization in the present invention, as long as they can provide the required hydrophilicity and hydrophobicity.

[0064] In a specific embodiment, the hydrophilic group can be formed by a substance selected from the group consisting of: HO(CH2CH2O) mH, wherein m is an integer between 5 and 13, and derivatives thereof; and nucleic acid chains composed of deoxynucleotides (for example, the group can comprise 12 to 100 bases in length, such as 20 to 40 bases in length, such as 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 bases in length).

[0065] In a preferred embodiment, the hydrophilic group is preferably a single-stranded nucleic acid chain composed of thymidine deoxynucleotides.

[0066] In the present invention, the polyethylene glycol derivative may include a monophosphate derivative of PEG. In a preferred embodiment, the hydrophilic group may include one or more monophosphate-PEG.

[0067] Preferably, the hydrophilic group is selected from: HO(CH2CH2O) m H, wherein m is an integer between 5 and 13, preferably between 5 and 8, and most preferably 6, and its monophosphate derivatives; and a single-stranded nucleic acid chain composed of thymidine deoxynucleotides, preferably 20 to 40 bases in length.

[0068] It should be noted that when using the CRISPR / Cas12a system, single-stranded nucleic acid chains composed of thymidine deoxynucleotides are not selected as hydrophilic groups.

[0069] In the present invention, the hydrophobic group is selected from a group based on optionally substituted or unsubstituted alkyl groups. In the present invention, preferably a straight chain alkyl group is used. In some embodiments, the C chain length of the alkyl group may be between 10-100, preferably 10-20, more preferably 10-15 (e.g., 10, 11, 12, 13, 14 or 15), for example, -(CH2)n-1CH3, wherein n may be an integer selected from 10-100, preferably 10-15. In some embodiments, the alkyl group may be optionally substituted by halogen (e.g., fluorine, chlorine, bromine or iodine).

[0070] In some embodiments, the probe may be selected from: [CH3(CH2) n-1 ]-[Switch]-[Single-stranded nucleic acid chain composed of T], and [CH3(CH2) n-1 ]-[Switch]-[HO(CH2CH2O) m H or its derivatives].

[0071] In a specific embodiment, the probe may be selected from: [CH3(CH2) n-1 ]-[single-stranded RNA]-[single-stranded nucleic acid chain composed of T], [CH3(CH2) n-1 ]-[single-stranded RNA]-[HO(CH2CH2O)m H or its derivatives], [CH3(CH2) n-1 ]-[single-stranded DNA]-[HO(CH2CH2O) m H or its derivatives], and [single-stranded nucleic acid chain composed of T]-[single-stranded RNA]-[(CH2) n- 1CH3].

[0072] In a specific embodiment, the probe may be selected from: [CH3(CH2) 11 ]-[single-stranded RNA]-[single-stranded nucleic acid chain composed of 22 T], [CH3(CH2) 11 ]-[single-stranded RNA]-[single-stranded nucleic acid chain composed of 40 Ts], [CH3(CH2) n-1 ]-[single-stranded RNA]-[HO(CH2CH2O)6H or its derivatives]; and [CH3(CH2) n-1 ]-[single-stranded DNA]-[HO(CH2CH2O)6H or its derivatives].

[0073] The preparation and connection of the various groups in the probe according to the present invention can be performed as needed based on known procedures. For example, the hydrophilic group or the hydrophobic group can be connected to the nucleic acid of the switch via a phosphodiester bond. For example, when bound to the 5' end of the switch, the hydrophilic group or the hydrophobic group can be connected to the switch via a phosphate group on the first nucleotide molecule of the switch; when bound to the 3' end of the switch, the hydrophilic group or the hydrophobic group can be connected to the switch via an additional phosphate group. For example, when bound to the 3' end of the switch, the additional phosphate group can form an ester bond with the 3'-hydroxyl group on the last nucleotide molecule of the switch. To facilitate connection, the termini of the hydrophilic group or the hydrophobic group can be modified as needed using known procedures, including but not limited to modification with functionalized groups.

[0074] For example, when an alkyl group is used as the hydrophobic group (terminally modified), it can be connected to the 5' end or 3' end of the switch by the following formula (I) or formula (II):

[0075]

[0076]

[0077] Here, B or B1 refers to the base in the nucleotide.

[0078] In some embodiments, B or B1 is selected from cytosine, thymine, guanine, adenine, uracil, preferably thymine or uracil.

[0079] For example, for monophosphate-PEG, it can be connected to the 3' end of the switch through its own phosphate group. For example, without limitation, it can be connected in the manner shown in the following formula (III):

[0080]

[0081] Here, B1 refers to the base in the nucleotide.

[0082] In some embodiments, B1 is selected from cytosine, thymine, guanine, adenine, and uracil, preferably thymine or uracil.

[0083] Therefore, in some preferred embodiments, the probe may be selected from:

[0084] 5'-[single-stranded nucleic acid chain composed of T]-[single-stranded RNA]-[(CH2) n-1 CH3]-3'; 5'-[CH3(CH2) n-1 ]-[single-stranded RNA]-[single-stranded nucleic acid chain composed of T]-3'; 5'-[HO(CH2CH2O) m H or its derivatives]-[single-stranded RNA]-[(CH2) n-1 CH3]-3'; 5'-[CH3(CH2) n-1 ]-[single-stranded RNA]-[HO(CH2CH2O) m H or its derivatives]-3';5'-[CH3(CH2) n-1 ]-[single-stranded DNA]-[HO(CH2CH2O) m H or its derivatives]-3'; and 5'-[HO(CH2CH2O) m H or its derivatives]-[single-stranded DNA]-[(CH2) n-1 CH3]-3'. In a specific embodiment, the probe can be selected from: 5'-[single-stranded nucleic acid chain consisting of 22 or 40 T]-[single-stranded RNA]-[(CH2) 11 CH3]-3'; 5'-[CH3(CH2) 11 ]-[single-stranded RNA]-[single-stranded nucleic acid chain composed of 22 or 40 Ts]-3'; 5'-[HO(CH2CH2O)6H or its derivatives]-[single-stranded RNA]-[(CH2) 11 CH3]-3'; 5'-[CH3(CH2) 11 ]-[single-stranded RNA]-[HO(CH2CH2O)6H or its derivatives]-3';5'-[CH3(CH2) 11]-[single-stranded DNA]-[HO(CH2CH2O)6H or its derivatives]-3'; and 5'-[HO(CH2CH2O)6H or its derivatives]-[single-stranded DNA]-[(CH2) 11 CH3]-3'.

[0085] In some cases, to increase hydrophilicity, multiple [HO(CH2CH2O)6H or its derivatives] may be used, for example, 2, 3, or 4.

[0086] In an embodiment of the present invention, in the case where the hydrophilic surface is converted to the hydrophobic surface during the reaction, when the nucleic acid to be detected is RNA, [CH3(CH2) 11 ]-[single-stranded RNA composed of 6 U]-[single-stranded nucleic acid chain composed of 22 or 40 T]; when the nucleic acid to be detected is DNA, [CH3(CH2) 11 ]-[single-stranded DNA composed of 15 Ts]-[HO(CH2CH2O)6H or its derivatives].

[0087] In an embodiment of the present invention, in the case where the hydrophobic surface is converted to a hydrophilic surface during the reaction, when the nucleic acid to be detected is RNA, [CH3(CH2) 11 ]-[single-stranded RNA composed of 6 U]-[single-stranded nucleic acid chain composed of 22 or 40 T]; when the nucleic acid to be detected is DNA, [CH3(CH2) 11 ]-[single-stranded DNA composed of 15 Ts]-1 or 2 [HO(CH2CH2O)6H or its derivatives].

[0088] Connection of probe to substrate

[0089] For the connection between the probe and the substrate, the connection can be carried out in a covalent or non-covalent manner. In the present invention, the two can be connected using methods known in the art. In some embodiments, the connection method and the bridge reagent used are determined based on the substrate material. A common method is generally to perform hydrophilic treatment on the substrate surface, and then the bridge reagent modifies the substrate surface and / or the probe end with functional groups. The functional groups that can be modified include but are not limited to the following modifications such as amino, carboxyl, hydroxyl, sulfonic acid, sulfhydryl, biotin and streptavidin, and then covalently linked through the above group interactions. Based on methods known in the art, the amount of the connected probe can be adjusted as needed, for example, by adjusting the concentration of the added probe, as well as other reaction conditions.

[0090] For example, the probe may be modified with an amino group. 1-6Alkylamino. 1-6 The alkylamino group may be CH3(CH2)5NH2. In some embodiments, for a hydrophobic group, an amino group may be directly connected to the end thereof as a linker group; for a hydrophilic group, a C 1-6 The alkylamino group is linked to the hydrophilic group.

[0091] Optional substrate hydrophilic treatment methods include, but are not limited to: plasma treatment, sodium hydroxide solution treatment, piranha solution treatment, and concentrated hydrochloric acid treatment.

[0092] Functional group bridging agents that can be used on the substrate surface include, but are not limited to, mercaptopropyltriethoxysilane, aminopropyltriethoxysilane (eg, APTES), triethoxysilane, and trimethoxysilane.

[0093] For example, in a specific embodiment, APTES can be used to modify a glass substrate, and then glutaraldehyde can be used as a cross-linking agent to attach amino-modified probes.

[0094] In a specific embodiment, for the method of changing the reaction area of ​​the substrate from hydrophilic to hydrophobic and from hydrophilic to hydrophobic, the connection between the substrate and the probe may include the following steps: hydrophilic treatment of the substrate and preparation of a probe binding area; and fixing the probe to the probe binding area.

[0095] Optionally, before fixing the probe, a hydrophobic signal stabilization region is set in the area adjacent to the reaction area. Preferably, the signal stabilization region is set in a manner that completely or partially, preferably completely surrounds the reaction area, and the size of the signal stabilization region is less than, equal to or greater than the size of the reaction area extending outward from the boundary of the reaction area, preferably equal to or greater than the size of the reaction area. For example, when the reaction area is circular or rectangular, the diameter or side length of the signal stabilization region is less than, equal to or greater than the diameter or side length of the reaction area extending outward from the boundary of the reaction area. The setting of the signal stabilization region can be carried out based on methods known in the art, such as using perfluorosilane for hydrophobic modification.

[0096] In some embodiments, for the method of converting the reaction area of ​​the substrate from hydrophilic to hydrophobic and from hydrophobic to hydrophilic, the connection between the substrate and the probe may include the following steps: hydrophilizing the substrate and functionalizing the reaction area of ​​the substrate (such as a glass tube) by adding an amino modifier and an amino cross-linking agent to prepare a probe binding area; providing a signal stabilization area with hydrophobicity; and fixing the probe to the probe binding area.

[0097] In a specific embodiment, the connection between the substrate and the probe may include the following steps:

[0098] The substrate is hydrophilized by sodium hydroxide, and then the reaction area of ​​the substrate (such as a glass tube) is functionalized by adding 3-aminopropyltriethoxysilane as an amino modifier and glutaraldehyde as an amino cross-linker to prepare a probe binding area; the signal stabilization area is set by hydrophobic modification using perfluorosilane; and the probe is fixed to the probe binding area by adding a probe.

[0099] In a specific embodiment, the connection between the substrate and the probe may include the following steps:

[0100] Immersing a substrate (e.g., a glass tube) in a sodium hydroxide aqueous solution (e.g., a concentration of 3 M) for 3-6 hours (e.g., 4 hours), washing and drying, then adding a 3-aminopropyltriethoxysilane solution to the reaction region for reaction, washing and drying, and then adding glutaraldehyde as an amino cross-linking agent to the reaction region for reaction, washing and drying;

[0101] The region adjacent to the reaction region is hydrophobically modified by perfluorosilane to provide a hydrophobic signal stabilization region;

[0102] And, by adding the probe to the reaction area, the probe is fixed to the probe binding region.

[0103] During the step of setting up the signal stabilization region, the reaction region needs to be protected by physical means, for example, by filling or coating the region with a substance (e.g., an aqueous solution such as water) or a masking layer / membrane that does not participate in the hydrophobic modification reaction of the signal stabilization region.

[0104] In some embodiments, in a manner where the target-activated CRISPR-Cas nucleic acid cleavage system drives the sensor interface to change from hydrophilic to hydrophobic and / or from hydrophobic to hydrophilic, the connection between the substrate and the probe may include the following steps:

[0105] Hydrophilic treatment of the substrate and preparation of the probe binding region: The substrate (e.g., a glass tube) is hydrophilicized, then cleaned and dried; an amino modifier and an amino cross-linker are sequentially added to the reaction region (e.g., corresponding to a volume of 110 μL of liquid) of the hydrophilic treated substrate (e.g., within the glass tube) to functionalize the substrate (e.g., glass tube);

[0106] Setting a signal stabilization domain (e.g., corresponding to a volume of 110 μl-165 μl of liquid): adding water (e.g., 110 μl) to a substrate (e.g., a glass tube) to protect the reaction area, and hydrophobically modifying it with 10-55 μl of perfluorosilane (e.g., 0.1%-10% perfluorosilane in toluene); then adding a polymethyl methacrylate (PMMA) solution (e.g., 120-165 μl, 1-30 mg / mL in acetone) to protect the hydrophobic signal stabilization domain; after the PMMA solution dries to form a film, bombarding the substrate with a plasma cleaning technique to hydrophilize the area above 165 μl; then removing the PMMA film with acetone;

[0107] Immobilizing the probe: adding an amino-modified functional probe solution to the probe binding region and reacting, then washing and drying, and storing at -80°C for future use.

[0108] In a specific embodiment, for a method in which the target activates the CRISPR / Cas nucleic acid cleavage system to drive the sensor interface from a hydrophilic property to a hydrophobic property, the connection between the substrate (e.g., a glass tube) and the probe may include the following steps:

[0109] Hydrophilic treatment of the substrate and preparation of the probe binding region: The substrate (e.g., a glass tube) is hydrophilic treated, then cleaned and dried. A 3-aminopropyltriethoxysilane solution (e.g., an ethanol solution, 5 wt%-30 wt%) is then added to the reaction region (e.g., corresponding to a volume of 110 μL of liquid) of the substrate (e.g., within the glass tube) and allowed to react for one hour, followed by washing and drying. A glutaraldehyde solution is then added and allowed to react for one hour, followed by washing and drying.

[0110] Setting a signal stabilization domain (e.g., corresponding to a volume of 110 μL-165 μL of liquid): adding water (e.g., 110 μL) to a substrate (e.g., a glass tube) to protect the reaction area, and adding 10-55 μL of perfluorosilane (e.g., 0.1%-10% perfluorosilane in toluene) for hydrophobic modification, followed by washing and drying after the reaction; then adding 120-165 μL of polymethyl methacrylate (PMMA) solution (e.g., the solvent can be acetone, with a concentration of 1-30 mg / 10 mL), heating to dry the PMMA solution to form a film, bombarding and cleaning the glass tube area not protected by PMMA with oxygen plasma; and finally, removing the PMMA film with acetone;

[0111] Immobilize the probe: add an appropriate amount of functional probe solution (e.g., a volume corresponding to 110 μL of liquid) to the reaction area of ​​the substrate (e.g., in a glass tube) and incubate (e.g., incubate for 5-20 minutes), rinse and dry, and then store at -80°C for future use.

[0112] In some embodiments, during the hydrophilic treatment of the substrate and the preparation of the probe binding region, the hydrophilic treatment is performed by soaking in a 3M sodium hydroxide aqueous solution for 3-6 hours, for example, 4 hours.

[0113] In some embodiments, in the step of fixing the probe, the functional probe structure is [hydrophobic group]-[switch]-[hydrophilic group]. In some embodiments, for RNA targets, NH2+(CH2) 12 + a switch consisting of 6 Us + a hydrophilic group consisting of 22 Ts; for DNA targets, NH2+(CH2) is used 12 + A transfer switch consisting of 15 Ts + HO(CH2CH2O)6H.

[0114] In some embodiments, in a method where the target activates the CRISPR-Cas nucleic acid cleavage system to drive the sensor interface from hydrophobic to hydrophilic, the connection between the substrate and the probe may include the following steps:

[0115] Hydrophilic treatment of the substrate and preparation of the probe binding region: The substrate (e.g., a glass tube) is hydrophilicized, then cleaned and dried; an amino modifier and an amino cross-linker are sequentially added to the reaction region (e.g., corresponding to a volume of 110 μL of liquid) of the hydrophilic treated substrate (e.g., within the glass tube) to functionalize the substrate (e.g., glass tube);

[0116] To create a signal-stabilizing domain (e.g., corresponding to a volume of 110 μL to 165 μL of liquid), add 110 μL of water and 15 μL of a 0.5% perfluorosilane solution in toluene to a substrate (e.g., a glass tube) for hydrophobic modification. Then, add 165 μL of a 1 mg / mL polymethyl methacrylate (PMMA) solution in acetone to localize and protect the hydrophobic region. After the PMMA solution dries to form a film, use plasma cleaning technology to bombard the inner wall of the tube to remove the hydrophobic modification. Then, remove the PMMA film with acetone.

[0117] Immobilize the probe: add the amino-modified functional probe solution to the reaction area of ​​the substrate and react for 50 minutes, then rinse and dry, and store at -80°C for future use.

[0118] In some embodiments, in a method where the target activates the CRISPR-Cas nucleic acid cleavage system to drive the sensor interface from hydrophobic to hydrophilic, the connection between the substrate and the probe may include the following steps:

[0119] Hydrophilic treatment of the substrate and preparation of the probe binding region: The substrate (e.g., a glass tube) is hydrophilic treated, then cleaned and dried. A 3-aminopropyltriethoxysilane solution is then added to the reaction region (e.g., a volume corresponding to 110 μL of liquid) of the substrate (e.g., a glass tube) and allowed to react for one hour, followed by washing and drying. A glutaraldehyde solution is then added and allowed to react for one hour, followed by washing and drying.

[0120] Setting a signal stabilization domain (e.g., corresponding to a volume of 110 μL-165 μL of liquid): adding water (e.g., 110 μL) to a substrate (e.g., a glass tube) to protect the reaction area, and adding 10-55 μL of perfluorosilane (e.g., 0.1%-10% perfluorosilane in toluene) for hydrophobic modification, followed by washing and drying after the reaction; then adding 120-165 μL of polymethyl methacrylate (PMMA) solution (e.g., the solvent can be acetone, with a concentration of 1-30 mg / 10 mL), heating to dry the PMMA solution to form a film, bombarding and cleaning the glass tube area not protected by PMMA with oxygen plasma; and finally, removing the PMMA film with acetone;

[0121] Immobilize the probe: add an appropriate amount of functional probe solution (e.g., a volume corresponding to 110 μL of liquid) to the reaction area of ​​the substrate (e.g., in a glass tube) and incubate (e.g., incubate for 5-20 minutes), rinse and dry, and then store at -80°C for future use.

[0122] In some embodiments, during the step of preparing the probe binding region, the hydrophilic treatment is performed with a 3M sodium hydroxide aqueous solution (3 mL to 6 mL) for 3 to 6 hours, for example, 4 hours. The amino-modifying agent is 3-aminopropyltriethoxysilane. The amino-crosslinking agent is glutaraldehyde.

[0123] In some embodiments, in the step of fixing the probe, the functional probe structure is [hydrophilic group]-[switch]-[hydrophobic group]. For DNA targets, NH2+HO(CH2CH2O)6H+HO(CH2CH2O)6H+switch composed of 15 T+(CH2) 12 For RNA targets, a hydrophilic composition consisting of NH2+40 T+6 RNA+(CH2) was selected. 12 .

[0124] In some embodiments, the probe solution is an aqueous solvent, such as water or ethanol, including RNase- and DNase-free water. The probe concentration is 10 nM-10 μM, and the reaction time is 1 min-2 h, for example, 5 min-60 min, at room temperature.

[0125] In some embodiments, for a method in which the target activates the CRISPR / Cas nucleic acid cleavage system to drive the sensor interface from hydrophilic to hydrophobic, when the target nucleic acid is RNA, the probe is C 12 Alkyl + 6U + 22T, the concentration is 100nM, the solvent is RNase-free water, the reaction time is 8 minutes at room temperature (eg 25°C); when the target nucleic acid is DNA, the probe is C 12 Alkyl + 15T + polyethylene glycol (6-mer), the concentration is 100 nM, the solvent is RNase-free water, and the reaction time is 8 minutes at room temperature (e.g., 25°C).

[0126] In some embodiments, for a method in which the target activates the CRISPR / Cas nucleic acid cleavage system to drive the sensor interface from hydrophobic to hydrophilic, when the target nucleic acid is RNA, the probe is 40T+6U+C 12 Alkyl, the concentration is 100nM, the solvent is RNase-free water, the reaction time is 50 minutes at room temperature (such as 25°C); when the target nucleic acid is DNA, the probe is 2 polyethylene glycol (6-mer) + 15T + C 12 The concentration of alkyl is 1 μM, the solvent is ethanol, and the reaction time is 50 minutes at room temperature (eg, 25° C.).

[0127] In a specific embodiment, the specific probes used are as follows:

[0128] Probes for hydrophilic-to-hydrophobic conversion based on the CRISPR / Cas12a system:

[0129]

[0130] Probes for hydrophilic-to-hydrophobic conversion based on the CRISPR / Cas13a system:

[0131]

[0132] Probes for hydrophobic to hydrophilic conversion based on the CRISPR / Cas12a system:

[0133]

[0134] Probes for hydrophobic to hydrophilic conversion based on the CRISPR / Cas13a system:

[0135]

[0136] In the present invention, the term "hydrophilic" refers to a substance having a strong affinity for water, attracting water molecules. In the present invention, "exhibiting hydrophilicity" at the substrate surface / reaction region interface means that the substrate surface / reaction region interface is easily wetted by water and exhibits an affinity for water.

[0137] In the present invention, the term "hydrophobic" refers to a substance that repels water. In the present invention, the substrate surface / reaction region interface "exhibits hydrophobicity" means that the substrate surface / reaction region interface repels water.

[0138] Recognition reagent or recognition element

[0139] The CRISPR / Cas system, present in most bacteria, is an immune system designed to identify and destroy invading pathogens. In the CRISPR / Cas system, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) consists of DNA fragments that repeat short base sequences in the bacterial genome, as well as stored pathogen DNA fragments that allow the cell to recognize any pathogens attempting to invade again. CRISPR is transcribed into an RNA sequence (crRNA).

[0140] In this article, the “CRISPR / Cas system” refers to two types of CRISPR / Cas systems, which use a single effector protein to resist the invasion of exogenous nucleic acids and can perform gene editing and nucleic acid detection simply and efficiently. Among them, the most typical two types of CRISPR / Cas systems are CRISPR / Cas12a (Cpf1) and CRISPR / Cas13a (C2C2) systems. In this article, Cas (CRISPR-associated protein) is a nuclease guided by crRNA. crRNA is a specific nucleic acid sequence that guides CRISPR / Cas protein to recognize and cut target nucleic acid molecules, which can be transcribed in vitro or artificially chemically synthesized.

[0141] For CRISPR / Cas12a, CRISPR / Cas12a can not only efficiently identify and cut the DNA double-stranded of specific sequence (dsDNA, which contains a sequence complementary to crRNA and is recognized by crRNA) when relying on PAM sequence, but also can cut any single-stranded DNA (ssDNA) when activated to form CRISPR / Cas / dsDNA ternary complex by the DNA double-stranded of specific sequence (such as the target nucleic acid sequence herein). These characteristics enable CRISPR / Cas12a to improve the sensitivity, specificity and speed of detection. Therefore, in some embodiments, CRISPR / Cas12a system is preferred.

[0142] The CRISPR / Cas12a protein recognition site PAM described herein is present in the complementary sequence of the crRNA target sequence, guiding the activation of the cleavage activity of the CRISPR / Cas12a system. This sequence is called the protospacer adjacent motif (PAM) and is generally T-rich (for CRISPR / Cas12a).

[0143] In a specific embodiment, the Cas12a may be LbuCas12a.

[0144] In the present invention, the length of the PAM is preferably 3-8 bp, more preferably 3-6 bp, and most preferably 4 bp. The PAM sequence is not limited to the PAM present in the natural system. It is a technique known in the art to select several candidate sequences by random or directed mutagenesis of the active site sequence, or by combining computer simulation and verifying the binding ability of the PAM to CRISPR / Cas12a.

[0145] The most recently discovered Cas protein is Cas13. The Cas13 protein family consists of two subtypes: (1) Cas13a protein (LshCas13a) from Leptotrichia shahii, formally known as C2c2, belongs to type VI; (2) Cas13b protein (PspCas13b) from Prevotella spp. belongs to type III. This system only targets and cuts single-stranded RNA, not single-stranded or double-stranded DNA. The CRISPR / Cas13 system acts as an "adaptive" immune system in archaea and bacteria to defend against invading RNA. The CRISPR-Cas13a system consists of two parts: the Cas13a protein and the guide RNA.

[0146] In terms of working mechanism, the Cas13a protein forms a complex with a specific guide RNA, which interacts with RNA molecules in the cell. Once it finds a target RNA sequence complementary to the guide RNA, Cas13a will produce double-stranded cuts in that region. More importantly, once this cutting activity of Cas13a is activated, it will degrade cellular RNA in a more extensive manner, which is called the RNA damage diffusion effect.

[0147] The mechanism of action of the Cas13a protein. After recognizing its target, the CRISPR / Cas13 system cleaves single-stranded RNA at a sequence complementary to the crRNA spacer. The target sequence has a protospacer flanking site (PFS) at its 3' end, typically consisting of adenosine (A), uracil (U), and cytosine (C).

[0148] Another special feature of Cas13a is that once Cas13a recognizes and cuts the RNA target specified by the crRNA sequence, it enters an enzymatic "activated" state, at which time it will bind to and cut other RNAs, regardless of whether they are homologous to the crRNA or whether PFS exists.

[0149] In a specific embodiment, the Cas13a may be LwaCas13a.

[0150] In the present invention, the length of crRNA is preferably 60-70 bp, more preferably 62-67 bp, and most preferably 64 bp. The crRNA can be designed based on the needs of the user. Preferably, it is designed in combination with bioinformatics software.

[0151] In the present application, commercially available Cas12a and Cas13a and their reaction solutions can be used as recognition reagents or recognition elements. For example, LbuCas12a and LwaCas13a purchased from Shanghai Huicheng Biotechnology Co., Ltd. can be used.

[0152] In recent years, the new coronavirus and African swine fever have caused great damage to humans and pigs respectively, brought many inconveniences to society, and seriously hindered the normal production and life of human society. In the context of the current lack of effective drugs and effective vaccines, the rapid identification of new coronavirus and African swine fever cases through nucleic acid testing and timely isolation is still the preferred method to control their spread. In view of the above background, the new coronavirus nucleic acid is selected as a preferred example of the application of this application in the detection of RNA nucleic acid-related diseases, and the African swine fever virus nucleic acid is selected as a preferred example of the application of this application in the detection of DNA nucleic acid-related diseases. As an example, for the target sequence, the N gene of the new coronavirus and the P72 gene of the African swine fever virus can be selected respectively. For example, the N gene sequence of the new coronavirus can be NCBI: NC_045512.2; the P72 gene sequence of the African swine fever virus can be GenBank: MN886939.1.

[0153] For the above genes, the available gRNAs are as follows:

[0154] For the new coronavirus N gene

[0155] crRNA1

[0156] GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACACGCGGGUCCACCAAACGUAAUGCGUGC (SEQID NO: 1)

[0157] crRNA2

[0158] GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACACGCAUUUGCGGCCAAUGUUUGUAAUGC (SEQID NO: 2)

[0159] For African swine fever virus P72 gene

[0160] crRNA1

[0161] UAAUUUCUACUAAGUGUAGAUUGGAAGAGCUGUAUCUCUAUCCUGAAA (SEQ ID NO: 3)

[0162] crRNA2

[0163] UAAUUUCUACUAAGUGUAGAUUUCCAUGAUUUGCACAAGCCGCACCAAA (SEQ ID NO: 4)

[0164] Additional reagents or components

[0165] In a preferred embodiment, the composition may further contain a pigment, etc., for displaying the detection results.

[0166] Biological samples

[0167] The biosensors, compositions, kits, and methods of the present invention can be used for diagnostic or non-diagnostic or non-clinical applications, such as for environmental pollution monitoring, food and cosmetic quality control, and disease diagnosis. In the present invention, the nucleic acids used as detection targets can be naturally derived, biosynthesized, or chemically synthesized. In some embodiments, the nucleic acids are derived from biological samples.

[0168] In the present invention, the nucleic acid to be detected can be present in any liquid sample or a solid sample that can be converted into a liquid sample by appropriate operation. The sample can be an environmental sample, for example, a sample of groundwater, reclaimed water, seawater, wastewater, mining waste. Alternatively, the sample can be a biological sample, in particular a sample from a subject, for example one or more of the following samples: blood, serum, plasma, sputum, cerebrospinal fluid, urine, tears, alveolar isolate, pleural fluid, cystic fluid, tissue, saliva. The sample can also come from food, drinking water, cosmetics or feed.

[0169] In some embodiments, the sample can be pretreated to enrich and extract the nucleic acid to be detected, or to remove impurities that may interfere with the detection. For example, the sample can be pretreated by centrifugation, filtration, ultrasound, homogenization, heating, freezing, thawing, mechanical treatment or a combination of various operating methods, and / or adding pretreated reagents. Those skilled in the art are aware of common pretreatment methods and pretreatment reagents for specific samples. For example, conventional pretreatment reagents include surfactants and detergents, salts, cell lysis agents, anticoagulants, degradative enzymes (such as proteases, lipases, nucleases, lipases, collagenases, cellulases, amylases, etc.) and solutions (such as buffers), etc.

[0170] Detection method

[0171] In an embodiment of the present invention, the nucleic acid in a sample is detected using the biosensor of the present invention or the composition of the present invention.

[0172] The present invention provides a method for detecting nucleic acid in a sample to be tested, the method comprising:

[0173] In the reaction area of ​​the substrate, the sample to be tested is mixed with the recognition reagent and incubated to react; the movement change of the sample to be tested is observed; and the presence of the target nucleic acid is detected based on whether the movement change occurs.

[0174] The specific conditions of mixing and incubation can be selected by those skilled in the art according to actual needs, as long as the sample and the recognition reagent can be fully combined to form a complex, for example, reacting at room temperature for more than 1 minute, for example, 1-10 minutes (but not limited thereto).

[0175] In some embodiments, the method further comprises a step of pre-treating the sample to be tested, for example, a step of lysing the viruses or cells in the sample to be tested.

[0176] In the present application, the method further includes using a reference. For example, a reaction solution without the addition of a nucleic acid sample can be used as a reference to observe changes in the motion state of the liquid.

[0177] For CRISPR-Cas12a, the crRNA concentration is 10nM-1μM, preferably 40nM. The crRNA to Cas protein ratio is 2:1.

[0178] The ratio of crRNA to Cas protein ranges from 0.5 to 4:1; the optimal ratio is 2:1.

[0179] For CRISPR-Cas13a, the crRNA concentration is 10nM-1μM; preferably 40nM.

[0180] The ratio of crRNA to Cas protein ranges from 0.25 to 4:1; the optimal ratio is 1:2.

[0181] In this application, a change in motion state can refer to a liquid changing from a static state to a dynamic state, or it can refer to a liquid moving from one region to another without external influence (e.g., without the application of an external force). For example, for a tubular or bowl-shaped substrate, the liquid can change from being static in an inverted state to falling; for a plate-shaped substrate, the liquid can move from a reactive region to a non-reactive region.

[0182] Reagent test kit

[0183] The present invention also provides a kit for detecting nucleic acid, wherein the kit comprises the composition or biosensor of the present invention.

[0184] In some embodiments, the kit of the present invention further comprises a delivery tool or device (e.g., a pipette) for detecting nucleic acids using the biosensor and / or reagent of the present invention, one or more containers of wash buffer, a data carrier containing instructions for use (e.g., an instruction manual or a computer-readable medium), a standard (e.g., a sample containing a known amount of nucleic acid), and combinations thereof, etc.

[0185] Example

[0186] Example 1 Preparation of nucleic acid samples

[0187] Regarding sample pretreatment and sample extraction: For the N gene of the new coronavirus: pUC57-2019-nCoV-N plasmid from GenScript was used for preparation through amplification and transcription. The specific amplification and transcription process is as follows:

[0188] Amplification reactions were performed using a 50 μL reaction mixture containing 100 ng of plasmid DNA, 500 nM forward primer, 500 nM reverse primer, 750 μM dNTPs, 5 U Taq DNA polymerase, and 1× PCR buffer. PCR reactions were performed using the Step One Real-Time PCR System (Applied Biosystems, USA) with a 4-minute hot start at 95°C, followed by 40 cycles of 95°C for 20 seconds, 55°C for 30 seconds, and 72°C for 60 seconds. The forward primer contained a T7 promoter, and the amplified product served as a transcription template for generating the corresponding RNA transcripts via a T7 transcription reaction. The 50 μL reaction mixture contained 8 μL of PCR product, 1 mM NTP, 250 U T7 RNA polymerase, and 1× RNA Pol reaction buffer (40 mM Tris-HCl, 2 mM spermidine, 1 mM DTT, 6 mM MgCl2, pH 7.9). The mixture was incubated at 37°C for 5 hours, and then 5 U of DNase I was added to the mixture to digest the DNA template. Finally, the transcripts were purified using an RNA Clean Kit and quantified by Nanodrop 2000 (ThermoFisher Scientific).

[0189] The primers used are as follows:

[0190] Forward primer sequence (SEQ ID NO: 5): AATTCTAATACGACTCACTATAGGGCCAAATTGGCTACTACCGAAGAGCTAC

[0191] Reverse primer sequence (SEQ ID NO: 6): CACAGTTTGCTGTTTCTTCTGTCTCTGCGG

[0192] For the African swine fever P72 gene: The full-length African swine fever DNA provided by Shandong Academy of Agricultural Sciences was used for amplification. The specific full-length DNA extraction and P72 gene amplification process are as follows:

[0193] Add an equal volume of a phenol:chloroform (1:1) mixture to the blood sample collected from an African swine fever infected pig. Mix thoroughly by inverting the tube. Centrifuge at 12,000 g / min for 5 minutes at room temperature. If the sample contains a lot of protein or the protein boundary is unclear, centrifuge for 10 minutes. Remove the upper (aqueous) layer and transfer it to a new centrifuge tube. Then, add an equal volume of a phenol:chloroform:isovaleric acid (25:24:1) mixture to the sample. Mix thoroughly by inverting the tube. Centrifuge at 12,000 g / min for 5 minutes at room temperature. Remove the upper (aqueous) layer and transfer it to a new centrifuge tube. Add another equal volume of a chloroform:isovaleric acid (24:1) mixture to the sample. Mix thoroughly by inverting the tube. Centrifuge at 12,000 g / min for 5 minutes at room temperature. Remove the upper (aqueous) layer and transfer it to a new centrifuge tube. Finally, centrifuge at 8000 g / min at 4°C for 10 minutes, discard the supernatant, wash the precipitate with 75% ethanol, and centrifuge at 8000 g / min for 5 minutes. Discard the supernatant completely, dry the precipitate under natural conditions, dissolve it in 50 μL of DEPC-treated sterile water, and store it at -20°C until further use. This completes the extraction of full-length African swine fever DNA.

[0194] For amplification, a 50 μL reaction mixture containing 100 ng of full-length African swine fever DNA, 500 nM forward primer, 500 nM reverse primer, 750 μM dNTPs, 5 U Taq DNA polymerase, and 1× PCR buffer was used. PCR was performed using the StepOne Real-Time PCR System (Applied Biosystems, USA) with a 4-minute hot start at 95°C, followed by 40 cycles of 95°C for 20 seconds, 55°C for 30 seconds, and 72°C for 60 seconds. Quantification was performed using a Nanodrop 2000 (Thermo Fisher Scientific).

[0195] Forward primer sequence (SEQ ID NO: 7): TTAGGTACTGTAACGCAGCACAGC

[0196] Reverse primer sequence (SEQ ID NO: 8): ATGGCATCAGGAGGAGCTTTTTGT

[0197] Example 2 Detection of nucleic acids by converting hydrophilic properties to hydrophobic properties

[0198] Preparation of a sensor with a hydrophobic substrate. A method for preparing a sensor for nucleic acid-related disease detection by activating a CRISPR-Cas nucleic acid cleavage system based on a target nucleic acid to drive the bottom interface of a glass tube from hydrophilic to hydrophobic, causing the liquid to shift from being stable and static at the bottom of the glass tube to sliding down from the bottom of the glass tube when the glass tube is inverted. Figure 1 As shown), including the following steps:

[0199] Soak the glass tube in 3M sodium hydroxide aqueous solution for 4 hours, rinse with deionized water and dry

[0200] Prepare a 10 wt% 3-aminopropyltriethoxysilane ethanol solution, and drip 110 μL of the solution into the bottom of a glass test tube to react for 1 hour, then wash with ethanol and dry;

[0201] 110 μL of glutaraldehyde stock solution was dropped into the bottom of a glass test tube to react for 1 hour, then washed with deionized water and ethanol and dried;

[0202] 110 μL of water and 10 (RNA) / 15 (DNA) μL of 8 wt% perfluorosilane toluene solution were added sequentially, reacted for 1 hour, washed with ethanol and dried;

[0203] Add 165 μL of 10 mg / mL PMMA acetone solution and dry at 85 °C for 2 h;

[0204] The perfluorosilane not protected by PMMA was bombarded and removed using oxygen plasma. The oxygen plasma treatment mode was High mode, the pressure was 100 Pa, and the treatment time was 20 minutes.

[0205] The PMMA film was removed by immersing in acetone for 30 minutes each time, repeated three times, then rinsed with ethanol and dried naturally.

[0206] Prepare probe solution; for DNA diseases, prepare NH2+(CH2) with RNase-free water 12 + Single-stranded DNA consisting of 15 Ts + HO(CH2CH2O)6H type probe solution 110μL, concentration is 100nM. After the solution is prepared, add it to the bottom of the glass test tube and incubate at room temperature for 8 minutes; for RNA diseases, use RNase-free water to prepare NH2+(CH2) 12 110 μL of a single-stranded RNA probe consisting of 6 Us and a single-stranded DNA probe consisting of 22 Ts, with a concentration of 100 nM, was added to the bottom of a glass test tube after the solution was prepared and incubated at room temperature for 8 minutes.

[0207] For DNA diseases, 110 μL of CRISPR-Cas12a solution (purchased from Shanghai Huicheng Biotechnology Co., Ltd.) was prepared. The ratio of crRNA to Cas in the CRISPR / Cas solution was 2 to 1, the crRNA concentration was 80 nM, and the Cas concentration was 40 nM. The CRISPR / Cas solution was mixed with the 1aM target DNA obtained in Example 1 and incubated at 37°C for 5 minutes. The mixture was then added to the glass tube and reacted for 1 minute. If the liquid stably aggregates at the top of the glass tube when the glass tube is inverted, as shown in the following example, Figure 3 a and Figure 3b, it proves that the sample is negative; if the liquid slides down the wall of the glass tube from the top of the glass tube when the glass tube is inverted, as shown in Figure 3 a and Figure 3 As shown in c, the sample nucleic acid is positive.

[0208] For RNA diseases, prepare 110 μL of CRISPR-Cas13a solution. The ratio of crRNA to Cas in the CRISPR / Cas solution is 1:2, the crRNA concentration is 5 nM, and the Cas concentration is 10 nM. Mix the CRISPR / Cas solution with 1 aM target RNA and incubate at 37°C for 5 minutes. Then add the mixture to the glass tube and react for 1 minute. If the liquid accumulates stably at the top of the glass tube when the glass tube is inverted, such as Figure 4 a and Figure 4 b, it proves that the sample is negative; if the liquid slides down the wall of the glass tube from the top of the glass tube when the glass tube is inverted, as shown in Figure 4 a and Figure 4 As shown in c, the sample nucleic acid is positive.

[0209] In the experiments, reaction reagents without nucleic acid sample addition were used as controls.

[0210] Example 3 Detection of nucleic acids by converting hydrophobic properties to hydrophilic properties

[0211] A method for preparing a sensor that activates a CRISPR-Cas nucleic acid cleavage system based on a target nucleic acid to drive the bottom interface of a glass tube to change from a hydrophobic property to a hydrophilic property, so that when the glass tube is inverted, the liquid slides down the tube wall from the top of the glass tube to the liquid remains stable at the top of the glass tube when the glass tube is inverted, thereby realizing nucleic acid-related disease detection (such as Figure 2 As shown), including the following steps:

[0212] Soak the glass tube in 3M sodium hydroxide aqueous solution for 4 hours, rinse with deionized water and dry

[0213] A 10 wt % 3-aminopropyltriethoxysilane ethanol solution was prepared, and 110 μL of the solution was dropped into the bottom of a glass test tube to react for 1 hour, and then washed with ethanol and dried.

[0214] 115 μL of glutaraldehyde stock solution was dropped into the bottom of a glass test tube for reaction for 1 hour, then washed with deionized water and ethanol and dried.

[0215] 110 μL of water and 40 μL of a 0.1 wt % perfluorosilane / toluene solution were added in sequence, and the mixture was reacted for 1 hour, washed with ethanol, and dried.

[0216] Add 165 μL of 10 mg / mL PMMA acetone solution and dry at 85°C for 2 hours.

[0217] The perfluorosilane not protected by PMMA was bombarded and removed using oxygen plasma. The oxygen plasma treatment mode was High mode, the pressure was 100 Pa, and the treatment time was 20 minutes.

[0218] The PMMA film was removed by immersing in acetone for 30 minutes each time, repeated three times, then rinsed with ethanol and dried naturally.

[0219] For DNA diseases, use ethanol to prepare a single-stranded DNA consisting of NH2+2 HO(CH2CH2O)6H+15 T+(CH2) 12 110 μL of the probe was added to the bottom of the glass test tube at a concentration of 1 μM. After the solution was prepared, it was added to the bottom of the glass test tube and incubated at room temperature for 50 minutes. For RNA diseases, RNase-free water was used to prepare a single-stranded RNA composed of NH2+40 DNA+6 U+(CH2) 12 110 μL of the type probe with a concentration of 100 nM was added to the bottom of the glass test tube after the solution was prepared and incubated at room temperature for 50 minutes.

[0220] For DNA diseases, prepare 110μL CRISPR-Cas12a solution. The ratio of crRNA to Cas in the CRISPR / Cas solution is 2:1, the crRNA concentration is 80nM, and the Cas concentration is 40nM. Mix the CRISPR / Cas solution with 1aM target DNA and incubate at 37°C for 5 minutes. Then add the mixture to a glass tube and react for 1 minute. Invert the test tube. If the liquid slides down the tube wall from the top of the glass tube when the glass tube is inverted, Figure 5 a and Figure 5 b, it proves that the sample is negative; if it is stably gathered at the top of the glass tube when the glass tube is inverted, as shown in Figure 5 a and Figure 5 As shown in c, the sample nucleic acid is positive.

[0221] For RNA diseases, prepare 110μL of CRISPR-Cas13a solution. The ratio of crRNA to Cas in the CRISPR / Cas solution is 1:2, the crRNA concentration is 20nM, and the Cas concentration is 40nM. Mix the CRISPR / Cas solution with 1aM target DNA and incubate at 37°C for 5 minutes. Then add the mixture to the glass tube and react for 1 minute. After 1 minute, invert the test tube. If the liquid slides down the tube wall from the top of the glass tube when the glass tube is inverted, Figure 6 a and Figure 6 b, it proves that the sample is negative; if it is stably gathered at the top of the glass tube when the glass tube is inverted, as shown in Figure 6 a and Figure 6 As shown in c, the sample nucleic acid is positive.

[0222] In the experiments, reaction reagents without nucleic acid sample addition were used as controls.

[0223] Example 4: Actual sample detection

[0224] The close-to-close sensor of Example 2 was used to detect a real sample of African swine fever with a Ct value of 27; the close-to-close sensor of Example 3 was used to detect a real sample of the new coronavirus with a Ct value of 24.

[0225] The HUDSON method was used to extract nucleic acid from real samples of African swine fever virus and real samples of new coronavirus. The specific steps were to add tris(2-carboxyethyl) phosphate hydrochloride (TCEP) with a final concentration of 100mM and ethylenediaminetetraacetic acid (EDTA) with a final concentration of 1mM to the sample, then heat at 50°C for 20 minutes and finally heat at 95°C for 5 minutes. Then, take an appropriate amount of solution and add it to the phobic-affinity and affinity-phobic test tubes respectively, repeat Example 2 and Example 3, and the steps are as follows: Figure 7 He Ru Figure 8 Results shown.

Claims

1. A biosensor for detecting nucleic acid, the biosensor comprising: substrate; Conversion reagents, wherein The conversion reagent is connected to the substrate, wherein the conversion reagent is a probe; and Identification reagents, Wherein, the conversion reagent has the following structure: [Hydrophilic group]-[Switch]-[Hydrophobic group], Wherein, the probe is selected from: [CH3(CH2) n-1 ]-[Switch]-[Single-stranded nucleic acid chain composed of T], and [CH3(CH2) n-1 ]-[Switch]-[HO(CH2CH2O) m H], wherein m is an integer between 5 and 13, and n is an integer between 10 and 15, wherein the single-stranded nucleic acid chain composed of T as a hydrophilic group has a length of 20 to 40 bases, wherein the switch comprises a single-stranded nucleic acid, The substrate and the conversion reagent are connected at the hydrophilic group end, thereby making the substrate hydrophobic; or the substrate and the conversion reagent are connected at the hydrophobic group end, thereby making the substrate hydrophilic, Wherein, the recognition reagent is selected from the CRISPR / Cas12a system and the CRISPR / Cas13a system, wherein the CRISPR / Cas12a system and the CRISPR / Cas13a system use LbuCas12a and LwaCas13a, Wherein, the substrate is a glass tube, The substrate comprises a reaction area connected to a probe and a hydrophobic signal stabilization area, and the size of the reaction area is capable of accommodating 50-110 microliters of liquid. Among them, in the case of converting the hydrophilic surface to the hydrophobic surface during the reaction, when the nucleic acid to be detected is RNA, [CH3(CH2) 11 ]-[single-stranded RNA composed of 6 U]-[single-stranded nucleic acid chain composed of 22 or 40 T] as a probe, and CRISPR / Cas13a system; when the nucleic acid to be detected is DNA, [CH3(CH2) 11 ]-[single-stranded DNA composed of 15 Ts]-[HO(CH2CH2O)6H] as a probe, and the CRISPR / Cas12a system; Alternatively, in the case where the surface is converted from hydrophobic to hydrophilic during the reaction, when the nucleic acid to be detected is RNA, [CH3(CH2) 11 ]-[single-stranded RNA composed of 6 U]-[single-stranded nucleic acid chain composed of 22 or 40 T] as a probe, and CRISPR / Cas13a system; when the nucleic acid to be detected is DNA, [CH3(CH2) 11 ]-[single-stranded DNA composed of 15 Ts]-1 or 2 [HO(CH2CH2O)6H] as probes, and the CRISPR / Cas12a system; The CRISPR-Cas12a system comprises crRNA and Cas12a protein, wherein the concentration of crRNA is 10nM-1μM, and the ratio of crRNA to Cas12a protein is in the range of 0.5-4:1; The CRISPR-Cas13a system comprises crRNA and Cas13a protein, the crRNA concentration is 10nM-1μM, and the crRNA to Cas13a protein ratio ranges from 0.25-4:

1.

2. The biosensor according to claim 1, wherein The size of the reaction area is such that it can accommodate 80-110 μl of liquid.

3. The biosensor according to claim 1 or 2, wherein The ratio of crRNA to Cas12a protein is in the range of 2:

1.

4. The biosensor according to claim 1 or 2, wherein The ratio of crRNA to Cas13a protein is in the range of 1:

2.

5. The biosensor according to any one of claims 1 or 2, wherein The biosensor comprises the following crRNA for detecting the novel coronavirus: GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACACGCGGGUCCACCAAACGUAAUGCGUGC GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACACGCAUUUGCGGCCAAUGUUUGUAAUGC; Alternatively, the biosensor comprises the following crRNA for African swine fever virus detection: UAAUUUCUACUAAGUGUAGAUUGGAAGAGCUGUAUCUCUAUCCUGAAA UAAUUUCUACUAAGUGUAGAUUUCCAUGAUUUGCACAAGCCGCACCAAA.

6. A kit comprising the biosensor according to any one of claims 1 to 5, and instructions for use.

7. Use of the biosensor according to any one of claims 1 to 5 in preparing a kit for detecting viruses.

8. The use according to claim 7, wherein The virus is selected from the new coronavirus or African swine fever virus.

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