Liquid crystal biosensor for high-sensitivity visual detection of protein marker based on functional nucleic acid regulation and control of CRISPR-Cas12a as well as preparation and detection method of liquid crystal biosensor

Abstract

The invention provides a liquid crystal biosensor for high-sensitivity visual detection of a protein marker based on functional nucleic acid regulated CRISPR-Cas12a and a preparation and detection method of the liquid crystal biosensor. The sensor comprises a ready-to-use detection reagent and a nucleic acid molecule functionalized liquid crystal detection interface, the ready-to-use detection reagent comprises the following elements or components: a Cas enzyme-crRNA compound with shearing activity and an activation sequence locking system based on a nucleic acid aptamer-activation sequence-two-dimensional material, the nucleic acid molecule functionalized liquid crystal detection interface is a liquid crystal interface modified by DNA molecules or RNA molecules, and the nucleic acid aptamer specifically recognizes target protein. The sensor is simple and convenient to operate, does not need too much manpower, is high in sensitivity, and can realize semi-quantitative or quantitative detection.

Description

Technical Field

[0001] The invention relates to a liquid crystal biosensor for simultaneously detecting multiple cancer markers and a preparation and detection method thereof, belonging to the technical field of analysis and detection. Background Art

[0002] Protein is the executor of all life functions and is closely related to the occurrence and development of diseases. At present, the diagnosis of many diseases, including cancer, COVID-19, neurological diseases, and immune inflammation, involves the detection of proteins. For example, cancer screening and recurrence monitoring require the detection of protein cancer markers such as CEA, AFP, and CA199. The diagnosis of COVID-19 can detect markers such as N protein. The commonly used methods for detecting protein markers that have been reported include electrochemiluminescence (ECL), chemiluminescence immunoassay (LIA), enzyme-linked immunosorbent assay (ELISA), surface enhanced Raman spectroscopy (SERS), single particle counting, single molecule immune array (Simoa), surface plasmon resonance (SPR), electrochemical immunosensor, etc. However, the detection methods of these markers are suitable for testing laboratories in large hospitals with complete large-scale equipment and experienced operators, but they cannot fully meet the early screening of diseases for a wider population (such as early cancer screening, COVID-19 screening, etc.), especially for grassroots areas with weak equipment foundations and home self-examination.

[0003] Therefore, many research groups have begun to develop methods for point-of-care testing (POCT) of protein markers, such as paper-based microfluidics, gold nanoparticle colorimetry, SERS-based POCT and photonic crystal colorimetry, etc.6-8. However, there are still three important challenges: 1. The sensitivity must meet the detection requirements of some low-abundance disease markers when both detection and data reading are completely independent of the interpretation of laboratory instruments; 2. The detection method does not require strict operating specifications and requires trained and experienced professionals to operate. 3. It does not require excessive manpower. It is difficult for existing POCT technology to overcome these three challenges at the same time. Therefore, there is still an urgent need to develop a detection method that combines the advantages of high sensitivity, simple operation, and independence from large instruments.

[0004] Since the CRISPR-Cas system-based point-of-care testing (POCT) technology was first reported by Professor Zhang Feng's laboratory in 2017, due to its high amplification efficiency, compatibility with constant-temperature nucleic acid amplification and paper-based flow chromatography technology, the detection limit of nucleic acid molecules can be as low as aM level without the need for complex instruments. This technology has attracted widespread attention from relevant researchers, and subsequent reports and products based on the CRISPR-Cas system for detecting cfDNA, viral DNA, RNA, bacterial DNA and other nucleic acid molecules have emerged in an endless stream. There have also been reports on the application of this technology to protein detection, but because proteins cannot be amplified, the detection sensitivity is limited, and its advantages are not obvious compared to the mature paper-based flow chromatography technology based on gold nanoparticle colorimetry. Detection of low-abundance proteins, such as the new crown N protein, still cannot meet the requirements.

[0005] Liquid crystal biosensors are also a type of biosensor technology suitable for constructing POCT devices. They have the advantages of simple structure, low cost, low energy consumption, no labeling, small sample dosage, and high sensitivity. Due to their unique advantages, liquid crystal biosensors have gradually attracted widespread attention from researchers at home and abroad, and have achieved certain results in the detection of protein markers, with broad market prospects. However, in order to ensure that the system has both specificity and sensitivity, it is often necessary to construct a sensitive monolayer membrane that can be destroyed by the target molecule for the target, such as designing and synthesizing an amphiphilic molecule containing the amide bond of the enzyme substrate for transpeptidase to construct a sensor. At present, the construction method lacks universality, and it is difficult to achieve the detection of a wide range of targets by simply replacing existing signal recognition molecules (such as antibodies, nucleic acid aptamers), which greatly limits the practical application of liquid crystal sensors. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a liquid crystal biosensor based on the visualization of functional nucleic acid regulation CRISPR-Cas12a activity, a highly sensitive protein marker, and its preparation and detection method. The present invention designs a CRISPR-Cas12a-liquid crystal cascade amplification system. When the target detection object exists, the activated CRISPR-Cas12a cuts the nucleic acid molecules at the detection interface. After the nucleic acid molecules are removed, the orientation of the liquid crystal film at the detection interface is changed, and the chemical signal is finally amplified and converted into a visual physical signal by transmission to the body phase. For ease of use, the present invention pre-freezes the required detection reagents. When used, it only needs to be added to the solution to be detected and mixed and then added to the pre-prepared detection chip. For the convenience of quantitative processing, MATLAB processing programs can be used to automatically identify and process images and obtain data; it is also possible to achieve visual colorimetric detection without relying on the processing program.

[0007] The present invention specifically provides the following scheme:

[0008] The first aspect of the present invention provides a liquid crystal biosensor for detecting a target protein, the sensor comprising a ready-to-use detection reagent and a nucleic acid molecule functionalized liquid crystal detection interface, the ready-to-use detection reagent comprising the following elements or components: a Cas enzyme-crRNA complex with cleavage activity and an activation sequence locking system based on a nucleic acid aptamer-activation sequence-two-dimensional material, the nucleic acid molecule functionalized liquid crystal detection interface being a liquid crystal interface modified by a DNA molecule or an RNA molecule, wherein the nucleic acid aptamer specifically recognizes the target protein.

[0009] In a specific embodiment of the present invention, the Cas enzyme is Cas12, and the liquid crystal interface is modified by ssDNA; or the Cas enzyme is Cas13, and the liquid crystal interface is modified by ssRNA.

[0010] In a specific embodiment of the present invention, the aptamer-based activation sequence locking system is a nucleic acid aptamer-activation sequence-graphene complex, whose structure is that the nucleic acid aptamer-activation sequence is coupled through a spacer sequence and then adsorbed on the graphene surface through a connecting sequence.

[0011] In a specific embodiment of the present invention, the target protein is a marker protein indicating a disease or health risk or a marker protein of an infectious pathogen.

[0012] In a specific embodiment of the present invention, the target protein is the new coronavirus N protein, and the corresponding nucleic acid aptamer sequence is: ACGA CAA TAT TCC TTA GGG GCA CCG CTA CAT.

[0013] In a specific embodiment of the present invention, the target protein is carcinoembryonic antigen, and the corresponding nucleic acid aptamer sequence is: TAG CTA TAG GGG GTG AAG GGA TAC CC.

[0014] In a specific embodiment of the present invention,

[0015] The Cas enzyme is Cas12a;

[0016] The activation dsDNA sequence is TTTG GAT GAA TGC TAG CCT GTG TC; GA CAC AGG CTA GCA TTCATC CAAA;

[0017] The connecting DNA sequence is ACA TCC AGC ATA AGT TTT TTT CCC CCC CCC CCC CC;

[0018] The crRNA sequence is UAA UUU CUA CUA AGU GUA GAU GAT GAA TGC TAG CCT GTG TC.

[0019] In a specific embodiment of the present invention, the liquid crystal interface is formed by 4-cyano-4'-pentylbiphenyl, the DNA molecule is TAG CGA TAG TAG, and the modification is covalent coupling or non-covalent coupling.

[0020] The present invention further provides use of the liquid crystal biosensor in preparing a diagnostic kit.

[0021] The present invention further provides a diagnostic kit comprising the above-mentioned liquid crystal biosensor.

[0022] Preferably, the ready-to-use detection reagent and the corresponding buffer components are in the form of mixed frozen powder.

[0023] Beneficial effects:

[0024] 1. The present invention designs a liquid crystal biosensor based on the visualization of functional nucleic acid regulation of CRISPR-Cas12a activity and highly sensitive protein markers. Cascade amplification of Cas12a enzyme catalysis and liquid crystal orientation transmission is used to achieve high-sensitivity detection; according to the principle of nucleic acid aptamer specific recognition of target protein, the target protein activates Cas12a enzyme to trigger cascade amplification through reasonable design to achieve specific detection. At the same time, nucleic acid aptamers can be replaced according to different targets, so that the invention has the universality of detecting other protein markers.

[0025] 2. The output signal of the liquid crystal sensor is a visual optical signal, and it does not require lasers or xenon lamps, but only requires a small LED light source powered by a button battery. It does not require the use of large instruments and has the potential to be used for instant and rapid detection of protein markers.

[0026] 3. The present invention can use image recognition program for quantitative or visual colorimetric qualitative detection. If direct visual colorimetric qualitative detection is used, the operation is similar to the colorimetric test strip based on gold nanoparticles. This method has higher sensitivity than the gold nanoparticle colorimetric test strip.

[0027] 4. The present invention uses ready-to-use reagents, which are easy and convenient to operate, do not require too much manpower, and do not require professionals and training. The operation method is similar to that of the test strip, but the sensitivity is higher than that. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the detection principle;

[0029] Figure 2 Grayscale statistics and heat maps of different liquid crystal optical signals;

[0030] Figure 3 This is the working curve for detecting N protein. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto. The drugs involved in the examples are all common commercial products unless otherwise specified.

[0032] Technical terms and definitions

[0033] Cas enzymes with cleavage activity

[0034] Cas12 and Cas13 are enzymes with cleavage activity. Under the guidance of the guide RNA sequence, once Cas12 and 13 bind to the target sequence, the cleavage activity is activated. This activity will simultaneously cleave the surrounding reporter sequences, thereby amplifying the signal.

[0035] Activate the sequence lock system

[0036] The present invention uses the activation sequence locking system based on aptamer-activation sequence-two-dimensional adsorption material, and this structure is known to the prior art, for example, aptamer sequence and Cas12a enzyme activation sequence are connected by TTTTTTTTT interval (spacer sequence can be nucleic acid molecule fragment such as polyT or polyA sequence, or non-nucleic acid molecule fragment such as polyethylene glycol or alkyl chain, or a combination of nucleic acid molecule fragment and non-nucleic acid molecule fragment), adsorbed on graphene oxide by connecting sequence, now due to aptamer-activation sequence adsorbed on graphene, Cas12a enzyme cannot be activated.When target protein exists, due to target protein and aptamer specific recognition binding effect, aptamer-activation sequence is separated from graphene, so that it can be combined with Cas12a enzyme to activate Cas12a enzyme.This process utilizes aptamer to ensure the specificity of identification to achieve specific detection.

[0037] Any two-dimensional material with strong adsorption capacity for nucleic acid sequences can be used in the present invention, such as graphene, germanene, graphene nanoribbons (GNR), double-layer graphene (BLG), phosphorene, graphene oxide, reduced graphene, fluorinated graphene, MoS2, two-dimensional metal carbide MXenes, etc.

[0038] The connection sequence has the following parts: the first part is a fragment complementary to the aptamer, the second part is a random sequence that is not complementary to the aptamer, the third part is a homopolymer base region (preferably PolyC) that enhances graphene adsorption, and an optional spacer such as polyT. There are two purposes for designing a random sequence: first, in order to eliminate or reduce non-specificity, the melting temperature of the connection sequence must be high enough, so there are certain requirements for the sequence length; second, in order to ensure that the aptamer can undergo conformational changes induced by the target after complementary hybridization with the connection sequence, thereby opening the double strand, the key bases in the stable secondary conformation of the aptamer after binding to the target cannot participate in hybridization. After experiments and software simulations, the connection sequence length of more than 14nt can effectively reduce non-specificity, and the appropriate length of the fragment complementary to the aptamer is 8nt, so the design of a 6nt random sequence meets the above two purposes.

[0039] The preferred linking sequence of the present invention is: ACATCCAGCATAAGT TTT TTT CCC CCC CCC CCC CC

[0040] Target protein

[0041] The detection system of the present invention can be used for any marker protein indicating disease or risk, such as carcinoembryonic antigen, prostate-specific antigen or alpha-fetoprotein, or proteins of infectious pathogens such as hepatitis B virus surface antigen, hepatitis B virus core antigen or new coronavirus N protein.

[0042] Aptamer

[0043] The concept of nucleic acid aptamers is well known to those skilled in the art. Those skilled in the art can obtain aptamer sequences that recognize specific target proteins by conventional screening methods. For example, in the present invention:

[0044] The nucleotide sequence of the SARS-CoV-2 N protein aptamer is A CGA CAA TAT TCC TTA GGG GCA CCG CTACAT;

[0045] The nucleotide sequence of the carcinoembryonic antigen aptamer is TAG CTA TAG GGG GTG AAG GGA TAC CC.

[0046] DMOAP: N,N-dimethyl-N-[3-(trimethoxysilyl)propyl]octadecyl ammonium chloride, CAS: 27668-52-6;

[0047] OTAB: octadecyltrimethylammonium chloride;

[0048] CRISPR: Clustered Regularly Interspaced Palindromic Repeats / Clustered Regularly Interspaced Palindromic Repeats

[0049] Cas12a: CRISPR-associated protein 12a

[0050] POCT: Point-of-care testing

[0051] DEPC water: ultrapure water treated with diethyl pyrocarbonate and sterilized by high temperature and high pressure

[0052] Room temperature: has a meaning well known to those skilled in the art, generally referring to 25±2°C.

[0053] The technical solution of the present invention is as follows:

[0054] A liquid crystal biosensor based on a visualized, highly sensitive protein marker regulated by functional nucleic acid CRISPR-Cas12a, the liquid crystal biosensor comprising a ready-to-use detection reagent and a DNA functionalized liquid crystal detection interface.

[0055] The above-mentioned ready-to-use detection reagent is prepared by freezing the cas12a-crRNA complex, the nucleic acid aptamer-activation sequence-graphene complex and the corresponding buffer in an EP tube.

[0056] The steps of using the above-mentioned liquid crystal biosensor are as follows: adding the object to be detected into the EP tube, mixing and dissolving it, and then adding the DNA-modified liquid crystal biointerface.

[0057] Preferably, according to the present invention, the protein marker includes but is not limited to the novel coronavirus N protein, wherein the nucleotide sequence for detecting the novel coronavirus N protein is A CGA CAA TAT TCC TTA GGG GCA CCG CTA CAT,

[0058] The preferred CRISPR-Cas12a activation dsDNA sequence of the present invention is TTTG GAT GAA TGC TAG CCTGTG TC; GA CAC AGG CTA GCA TTC ATC CAAA

[0059] The crRNA sequence optimized by the present invention is UAA UUU CUA CUA AGU GUA GAU GAT GAA TGCTAGCCT GTG TC

[0060] The preferred connecting DNA sequence of the present invention is ACA TCC AGC ATA AGT TTT TTT CCC CCC CCCCCCCC

[0061] The preferred sample buffer of the present invention is: 10 mM Tri-HCl, 150 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, pH=7.5, prepared with DEPC water.

[0062] The more specific implementation steps of the present invention are as follows:

[0063] The preparation method of the above-mentioned nucleic acid aptamer-activation sequence-graphene complex in the present invention comprises the following steps:

[0064] The connection sequence and graphene oxide were mixed and incubated in HEPES buffer (10mM HEPES, 300mM NaCl, pH = 7.5) for 3-6h, then incubated at 95°C for 3-15min, and then slowly cooled to room temperature. The ratio of the connection sequence to graphene oxide was 1nmol / 10ug to ensure saturated adsorption of the connection DNA on graphene oxide. The nucleic acid aptamer containing the CRISPR-Cas12a activation dsDNA sequence and the complementary DNA sequence were denatured and annealed at a ratio of 1:1, and the activation sequence base-specific complementary pairing was formed into double-stranded DNA. The annealed aptamer-activation sequence was added to the graphene oxide adsorbed with the connection sequence and incubated at room temperature for 10-120min to ensure that the nucleic acid aptamer-activation sequence was bound to the graphene oxide through base complementary pairing. BSA (1-20mg / mL) was added and incubated overnight to block the graphene oxide. Centrifuge at 10,000 rpm for 10 minutes, remove the supernatant, wash the precipitate, repeat 3-5 times to remove free DNA. Store the prepared complex at 4°C for future use.

[0065] The preparation method of the above-mentioned cas12a-crRNA complex in the present invention comprises the following steps:

[0066] The Cas12a enzyme and crRNA were mixed and incubated for 1 hour to obtain a Cas12a-crRNA complex, and the preferred ratio was 2:1-1.5:1.

[0067] Preferably, the crRNA needs to be prepared with DEPC water.

[0068] The preparation method of the above-mentioned ready-to-use detection reagent in the present invention comprises the following steps:

[0069] The prepared nucleic acid aptamer-activation sequence-graphene complex is mixed with the cas12a-crRNA complex. The concentration of the nucleic acid aptamer-activation sequence-graphene complex in the preferred mixed solution is 10ug / mL, and the concentration of the cas12a-crRNA complex is 50-500pM. 5U / uL nuclease inhibitor. The freeze-drying method is to place the prepared reaction system without the sample to be tested in a -80°C refrigerator for 2 hours, and then vacuum dry it at -50°C for 12 hours; the method of using the freeze-dried reaction system is to dissolve the freeze-dried reaction system with 18-19.6μL RNAase-free water, and add 0.4-2μL of the nucleic acid extraction product to be tested for reaction.

[0070] In the present invention, after a solution containing the target (such as saliva, urine, PBS solution, etc.) is mixed with a sample buffer, 50-300uL is added to an EP tube containing a ready-to-use reaction reagent. The preferred mixing ratio of the target solution to the sample buffer is 1:1 to 1:10.

[0071] The preparation method of the above-mentioned liquid crystal biosensor for detecting protein markers comprises the following steps:

[0072] (1) Soaking the glass sheet in a washing solution at 70-90° C. for 30-60 min, then washing with water, anhydrous ethanol, and methanol for 3-6 times, drying with nitrogen, and drying at 100-120° C. for 10-20 h, placing the glass sheet in a DMOAP solution with a mass concentration of 0.1%-3% for 15-30 min at room temperature, washing with three distilled water for 3-6 times, drying with nitrogen, and curing at 100-110° C. for 3-5 h, and cooling to obtain a glass substrate;

[0073] (2) placing a copper mesh on the glass substrate of step (1), injecting OTAB-doped 4-cyano-4'-pentylbiphenyl into the copper mesh while hot at 35-45° C. to construct a liquid crystal biosensor interface, wherein the thickness of the liquid crystal film is 20-60 μm. The preferred OTAB doping concentration of this patent is:

[0074] (3) Inject the DNA solution into the liquid crystal biosensor interface obtained in step (2), let it stand at room temperature for 10 to 20 minutes, and wash away the free DNA with PBS buffer to obtain a DNA-modified liquid crystal biosensor.

[0075] Preferably, according to the present invention, the washing solution in step (1) is prepared by mixing concentrated sulfuric acid with a mass concentration of 98% and hydrogen peroxide with a mass concentration of 30% in a volume ratio of 7:3.

[0076] Preferably, according to the present invention, the preparation method of OTAB-doped 4-cyano-4'-pentylbiphenyl in step (2) is as follows: OTAB powder is dissolved in chloroform to prepare a 0.1 mM OTAB chloroform solution, the OTAB chloroform solution and 4-cyano-4'-pentylbiphenyl are mixed in a volume ratio of 1:1, vortexed at 2500 rpm for 1 to 5 min to fully mix, and the chloroform is dried with nitrogen to obtain OTAB-doped 4-cyano-4'-pentylbiphenyl.

[0077] In the present invention, the liquid crystal biosensor modified with DNA in step (3) has an optical response to the addition of the activated Cas12a enzyme.

[0078] In the present invention, in step (3), the DNA at the liquid crystal biosensor interface reaches a saturated state; preferably, in the present invention, the concentration of the DNA solution in step (4) is ≥ 0.01 μM; further preferably, the concentration of the encoding DNA solution is 0.01 to 100 μM.

[0079] Preferably according to the present invention, the pH of the Tris buffer in step (4) is 7-8.

[0080] Preferably according to the present invention, the solvent used for the DNA solution in step (4) is a buffer solution of 20 mM Tri-HCl, 150 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, pH = 7.5.

[0081] The above-mentioned detection method of the liquid crystal biosensor for detecting protein markers comprises the following steps:

[0082] S1. Mix the ready-to-use reagent with the sample: Mix the pre-selected diluted sample with the ready-to-use reagent frozen in the EP tube at room temperature, shake well and set aside;

[0083] S2. Adding DNA-modified liquid crystal sensing substrate: adding the solution obtained in S1 to the DNA-modified liquid crystal sensing substrate, and observing the change of liquid crystal optical signal using a polarizing microscope or a homemade small device based on a mobile phone;

[0084] S3. Visual signal collection and processing: Quantitative or semi-quantitative detection: Collect liquid crystal optical morphology images every 5 minutes, process the liquid crystal images to obtain the brightness ratio, and use the dynamic relationship between the brightness ratio and time to obtain quantitative or semi-quantitative data. Qualitative detection: After the liquid crystal morphology is stable for 30-90 minutes, visually observe or collect liquid crystal morphology images and analyze them using data processing software.

[0085] S4. For actual samples, such as saliva samples, since they contain a high concentration of salivary mucin, the nonspecific adsorption capacity of the protein is strong and has a strong matrix effect. Therefore, saliva samples need to be pretreated. The pretreatment method steps are as follows: take a saliva sample and add an equal volume of polystyrene microspheres, shake well, dilute with loading buffer, shake gently for 3-10 minutes, centrifuge for 1-5 minutes in a handheld centrifuge to remove large particles and polystyrene microspheres, pass through a 0.45μm filter membrane, further remove polystyrene microspheres and small particles, take 50-300μL and add it to the EP tube containing the ready-to-use reagent, mix well, and drop it onto the liquid crystal detection chip.

[0086] Preferably according to the present invention, the protein markers in step S1 include but are not limited to carcinoembryonic antigen and SARS-CoV-2N protein, wherein the nucleotide sequence of the carcinoembryonic antigen aptamer is TAG CTA TAG GGG GTG AAG GGA TACCC.

[0087] Preferably according to the present invention, the pre-dilution ratio in step S1 is 5-10 times.

[0088] According to the preferred embodiment of the present invention, the loading buffer composition in steps S1 and S4 is a buffer solution of 20 mM Tri-HCl, 150 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, pH = 7.5

[0089] According to the preferred embodiment of the present invention, the preferred polystyrene properties in step S4 are single particle, water-soluble, highly stable, positively charged, with a particle size of 5 μm, and a high specific surface area.

[0090] According to the preferred embodiment of the present invention, the preferred final concentration of polystyrene in step S4 is 0.1-5 mg / mL

[0091] Preferably according to the present invention, the detection method further comprises: establishing a working curve according to changes in the cancer marker standard solution and its liquid crystal optical morphology to achieve quantitative detection of multiple cancer markers in the sample to be detected.

[0092] The technical features of the present invention are as follows:

[0093] The present invention is by chemical synthesis, and nucleic acid aptamer sequence and Cas12a enzyme activation sequence are connected together by TTTTTTTTT intervals. Adsorbed on graphene oxide by connecting sequence, this method is document reported method, can ensure that subsequent experiments have better signal-to-noise ratio, i.e., have smaller matrix effect. At this time, due to nucleic acid aptamer-activation sequence adsorbed on graphene, Cas12a enzyme cannot be activated. When target protein exists, due to target protein and nucleic acid aptamer specific recognition binding effect, nucleic acid aptamer-activation sequence is separated from graphene, so that it can be combined with Cas12a enzyme to activate Cas12a enzyme. This process uses nucleic acid aptamer to ensure the specificity of identification so as to achieve specific detection.

[0094] In the case of the presence of target protein, Cas12a is activated, and the ability to cut any DNA sequence is generated, and there is a one-to-many relationship between the target protein and the cut DNA, and the signal is amplified; After the DNA modified at the liquid crystal interface is cut, the interface liquid crystal molecule orientation will be affected, and the molecular orientation will be further transferred from the interface to the body phase (transmission depth of about 50 μm) so as to further amplify the signal. The present invention ensures the sensitivity of detection by the cascade amplification method.

[0095] The liquid crystal detection chip affects the birefringence of light through the change of molecular orientation, and generates an optical morphology signal visible to the naked eye. It only needs a button battery-powered LED as a light source, and the signal can be obtained by direct visual observation or mobile phone shooting. The data acquisition method is similar to the currently commonly used gold particle color test strips. It does not rely on large instruments, which is convenient for real-time detection. The present invention can also be used for semi-quantitative and quantitative detection, that is, after obtaining the signal image, it relies on the software program to automatically identify and process, obtain the brightness ratio (Br), and establish a standard curve through the Br of the standard sample to obtain semi-quantitative and quantitative data.

[0096] The required reagents can be frozen in EP tubes in advance. When using, just mix the sample with the frozen powder in the EP tube and then add it to the detection chip. No complicated operation is required, and no experienced personnel are required. The detection process is similar to the currently commonly used gold particle colorimetric test strips.

[0097] The method of identifying target proteins based on nucleic acid aptamers is universal, that is, other target proteins can be detected by replacing nucleic acid aptamers. This overcomes the problem that liquid crystal sensors currently need to design and synthesize specific sensing films based on the characteristics of target proteins. It is easy to promote and utilize the detection of other protein markers.

[0098] The present invention is further illustrated by taking the novel coronavirus N protein as an example.

[0099] The detection principle of the novel coronavirus N protein in the embodiment is as follows Figure 1 As shown, when the SARS-CoV-2N protein is present, the corresponding aptamer recognizes the target and detaches from the graphene due to phase changes. After entering the bulk phase, the Cas12a activation sequence pre-connected to the aptamer can bind to the crRNA in the Cas12a enzyme to activate the Cas12a ribozyme activity. The activated Cas12a enzyme affects the orientation of the liquid crystal molecules at the interface by cutting the DNA pre-modified on the liquid crystal interface, and the molecular orientation is further transmitted to the bulk phase to amplify the signal. And the birefringence of the liquid crystal film is affected by the change in molecular orientation to obtain a visible optical signal change.

[0100] Example 1: Preparation of ready-to-use reagents

[0101] A method for preparing a ready-to-use reagent for detecting the novel coronavirus N protein, the steps are as follows:

[0102] The connection sequence and graphene oxide were mixed and incubated in HEPES buffer (10mM HEPES, 300mM NaCl, pH=7.5) for 3h, then incubated at 95℃ for 5min, and then slowly cooled to room temperature. The ratio of the connection sequence to graphene oxide was 1nmol / 10ug to ensure saturated adsorption of the connection DNA on graphene oxide. The aptamer containing the CRISPR-Cas12a activation dsDNA sequence and the complementary DNA sequence were denatured and annealed at a ratio of 1:1, and the activation sequence base-specific complementary pairing was formed into double-stranded DNA. The annealed aptamer-activation sequence was added to the graphene oxide adsorbed with the connection sequence and incubated at room temperature for 30min to ensure that the aptamer-activation sequence was bound to the graphene oxide through base complementary pairing. The ratio of the aptamer-activation sequence to graphene oxide was 1nmol / 10ug. BSA (1mg / mL) was added and incubated overnight to block the graphene oxide. Centrifuge at 25°C and 10,000 rpm for ten minutes, remove the supernatant, wash the precipitate, and repeat three times to remove free DNA. The prepared complex is stored at 4°C for later use. Cas12a enzyme and crRNA are mixed and incubated at a ratio of 2:1 for 1 hour to obtain the cas12a-crRNA complex.

[0103] The prepared nucleic acid aptamer-activation sequence-graphene complex is mixed with the cas12a-crRNA complex, and the nuclease inhibitor is mixed. The concentration of the nucleic acid aptamer-activation sequence-graphene complex in the mixed solution is 10ug / mL, and the concentration of the cas12a-crRNA complex is 50pM. The concentration of the nuclease inhibitor is 5U / uL. The total volume is 100μL. The prepared mixed solution without the sample to be tested is frozen in a -80℃ refrigerator for 2h, and then vacuum dried at -50℃ for 12h; the freeze-dried powder is stored at 4℃ for later use

[0104] The nucleotide sequence of the SARS-CoV-2 N protein detected is ACT TAT GCT GGA TGT CGC TTA CGA CAATAT TCC TTA GGG GCA CCG CTA CAT TGA CAC ATC CAG C.

[0105] The CRISPR-Cas12a activation dsDNA sequence is TTTG GAT GAA TGC TAG CCT GTG TC; GA CACAGG CTA GCA TTC ATC CAAA.

[0106] The crRNA sequence is UAA UUU CUA CUA AGU GUA GAU GAT GAA TGC TAG CCT GTG TC.

[0107] The connecting DNA sequence is ACA TCC AGC ATA AGT TTT TTT CCC CCC CCC CCC CC.

[0108] The sample buffer was: 10 mM Tri-HCl, 150 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, pH = 7.5.

[0109] Example 2: Preparation of liquid crystal detection chip

[0110] A method for preparing a liquid crystal detection chip for detecting the novel coronavirus N protein, the steps are as follows

[0111] (1) Preparation of glass substrate: a glass slide was immersed in 60 mL of a washing solution prepared by mixing 98% concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3 at 75°C for 30 min, then washed with water, anhydrous ethanol, and methanol for 3 times, dried with nitrogen, dried at 110°C for 12 h, placed in a 0.5% DMOAP solution at room temperature for 15 min, washed with three distilled water for 5 times, dried with nitrogen, and cured at 105°C for 3 h. After cooling, a glass substrate was obtained.

[0112] (2) A G75 copper mesh was placed at the bottom of a separate channel, and 1.5 μL of OTAB-doped 4-cyano-4'-pentylbiphenyl heated to 35-45°C was injected into the copper mesh while hot. Then, a capillary with an inner diameter of 0.5 mm was used to remove excess OTAB-doped 4-cyano-4'-pentylbiphenyl to construct a liquid crystal biosensor interface. The thickness of the liquid crystal biosensor interface was consistent with the thickness of the copper mesh, which was approximately 20 μm.

[0113] The preparation method of the OTAB-doped 4-cyano-4'-pentylbiphenyl is as follows: OTAB powder is dissolved in chloroform to prepare a 0.1 mM OTAB chloroform solution, the OTAB chloroform solution and 4-cyano-4'-pentylbiphenyl are mixed in a volume ratio of 1:1, vortexed at 2500 rpm for 3 min to fully mix, and the chloroform is dried with nitrogen to obtain OTAB-doped 4-cyano-4'-pentylbiphenyl;

[0114] (3) The reporter DNA was prepared into a solution with a concentration of 0.01 μM, and the solvent used was 10 mM Tri-HCl, 150 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, pH = 7.5 buffer, and the liquid crystal biosensor interface was modified. The solution was allowed to stand at room temperature for 60 min, and then the solution was exchanged with Tris buffer (pH 7-8). The free reporter DNA was washed away by exchanging the solution ten times to obtain a liquid crystal biosensor;

[0115] The nucleotide sequence of the reporter DNA is TAG CGA TAG TAG

[0116] Example 3: Method for detecting SARS-CoV-2N protein

[0117] A method for detecting the novel coronavirus N protein, which method requires the use of a ready-to-use reagent in combination with a detection chip, and the steps are as follows:

[0118] S1. Mix 20 μL of PBS solution of SARS-CoV-2 N protein of different concentrations with 80 μL of loading buffer and shake well, then add to the EP tube containing the ready-to-use reagent, shake well and set aside;

[0119] S2. Add the mixed reagent and sample mixture obtained in S1 to the DNA-modified liquid crystal sensing substrate, and observe the change of the liquid crystal optical signal using a polarizing microscope or a self-made small device based on a mobile phone. Quantitative or semi-quantitative detection requires collecting liquid crystal optical morphology images every 5 minutes. This operation can be completed by setting automatic timing photography using mobile phone software;

[0120] S3. Visual signal collection and processing: Quantitative or semi-quantitative detection: Collect liquid crystal optical morphology images every 5 minutes, process the liquid crystal images to obtain the brightness ratio, and use the slope of brightness change and time to obtain quantitative or semi-quantitative data. Qualitative detection: After the liquid crystal morphology is stable for 30-90 minutes, visually observe or collect liquid crystal morphology images and analyze them with data processing software to obtain the brightness ratio, thereby qualitatively estimating the concentration.

[0121] Data processing: The MATLAB processing program is used to automatically identify the image and automatically calculate the value. The calculation principle is that the grayscale values ​​of the brightness conversion of the bright area and the dark area are different. According to the threshold set by machine learning, the bright area and the dark area are distinguished, and the proportion of the bright area pixels to the entire liquid crystal image area is calculated to obtain the proportion of the bright area of ​​the initial liquid crystal morphology. Then, this method is used to obtain the proportion of the liquid crystal bright area after the sample is added. The difference between the two is calculated as the change in the bright area. The standard concentration is used as the horizontal axis and the change in the bright area is used as the vertical axis to draw a graph to obtain the working curve. The working curve is as follows: Figure 2 As shown, the standard solutions with known concentrations were subsequently tested, and the yields were determined to be between 92.1% and 110.6%;

[0122] Among them, the working curve of the new crown N protein is y=44.02x-1.91, R 2 =0.985; detection limit is 0.31 ng / mL.

[0123] Example 4: Detection of SARS-CoV-2N protein in actual samples (saliva)

[0124] Take the saliva of healthy people, add different concentrations of SARS-CoV-2N protein to simulate the saliva samples of new crown patients, and take the saliva of healthy people and add the same volume of PBS solution as the healthy sample control. Then add positively charged polystyrene microspheres with a particle size of 5μm and high specific surface area to the saliva sample, shake it well, dilute it with loading buffer (the total dilution of saliva is 10 times), shake it gently for 2-3 minutes, centrifuge it with a handheld centrifuge to remove large particles and polystyrene microspheres, pass it through a 0.45μm filter membrane, further remove polystyrene microspheres and small particles, take 100μL and add it to the EP tube containing the ready-to-use reagent, mix it well, and drop it onto the liquid crystal detection chip.

[0125] Data processing: The algorithm software was used to identify and process, calculate the bright area, and bring it into the working curve to obtain the SARS-CoV-2N protein concentration. The yield of N protein detected in saliva is shown in Table 1:

[0126] Table 1 Detection rate of N protein in saliva

[0127]

[0128] The above is only a specific embodiment of the present invention, and the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined by the claims.

Claims

1. A liquid crystal biosensor for detecting a target protein, the sensor comprising a ready-to-use detection reagent and a nucleic acid molecule functionalized liquid crystal detection interface, the ready-to-use detection reagent comprising the following elements or components: a Cas enzyme-crRNA complex with cleavage activity and an activation sequence locking system based on a nucleic acid aptamer-activation sequence-two-dimensional material, the nucleic acid molecule functionalized liquid crystal detection interface is a liquid crystal interface modified by a DNA molecule or an RNA molecule, wherein the nucleic acid aptamer specifically recognizes the target protein to be detected.

2. A liquid crystal biosensor for detecting a target protein as described in claim 1, wherein the Cas enzyme is Cas12, and the liquid crystal interface is modified with ssDNA or dsDNA; or the Cas enzyme is Cas13, and the liquid crystal interface is modified with ssRNA.

3. The liquid crystal biosensor for detecting target protein according to any one of claims 1 to 2, wherein the aptamer-based activation sequence locking system is a nucleic acid aptamer-activation sequence-graphene complex, whose structure is that the nucleic acid aptamer-activation sequence is coupled through a spacer sequence and then adsorbed on the graphene surface through a connecting sequence.

4. A liquid crystal biosensor for detecting a target protein as claimed in claim 1, wherein the target protein is a marker protein indicating a disease or health risk or a marker protein of an infectious pathogen.

5. A liquid crystal biosensor for detecting a target protein as claimed in claim 4, wherein the target protein is the new crown N protein, and the corresponding nucleic acid aptamer sequence is: ACT TAT GCT GGA TGT CGC TTA CGA CAA TAT TCC TTA GGGGCA CCG CTA CAT TGA CAC ATC CAG C.

6. A liquid crystal biosensor for detecting a target protein according to claim 4, wherein the target protein is carcinoembryonic antigen, and the corresponding nucleic acid aptamer sequence is: ACT TAT GCT GGA TGT CGC TTA CGA CAA TAT TCC TTA GGGGCA CCG CTA CAT TGA CAC ATC CAG C.

7. The liquid crystal biosensor for detecting a target protein according to claim 3, The Cas enzyme is Cas12a; The activation dsDNA sequence is TTTG GAT GAA TGC TAG CCT GTG TC; GA CAC AGG CTA GCA TTC ATCCAAA; The connecting DNA sequence is ACA TCC AGC ATA AGT TTT TTT CCC CCC CCC CCC CC; The crRNA sequence is UAA UUU CUA CUA AGU GUA GAU GAT GAA TGC TAG CCT GTG TC.

8. The liquid crystal biosensor for detecting target protein as claimed in claim 1, wherein the liquid crystal interface is formed by nematic liquid crystal molecules such as 4-cyano-4'-pentylbiphenyl, the DNA molecule is TAG CGA TAG TAG, and the modification is covalent coupling or non-covalent coupling.

9. Use of the liquid crystal biosensor according to any one of claims 1 to 8 in preparing a diagnostic kit.

10. A diagnostic kit comprising the liquid crystal biosensor according to any one of claims 1 to 8; Preferably, the ready-to-use detection reagent and the corresponding buffer components are in the form of mixed frozen powder.

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