Ultra-sensitive biosensor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biosensing, and discloses an ultra-sensitive biosensor as well as a preparation method and application thereof. The ultra-sensitive biosensor comprises a substrate, a two-dimensional material loaded on the substrate, DNA origami loaded on the two-dimensional material and quantum dots modified on the DNA origami, wherein the DNA origami contains an exposed single-stranded DNA fragment, and the single-stranded DNA fragment can be cut by an activated Cas protein; the quantum dots are connected with the tail ends of the single-stranded DNA fragments. The ultrasensitive biosensor is excellent in performance, and compared with a conventional pure optical biosensor combining surface plasma resonance and DNA origami, the sensitivity of the ultrasensitive biosensor is improved by four orders of magnitude and can reach the aM level.

Description

Technical Field

[0001] The present invention relates to the field of biosensing technology, and particularly to an ultrasensitive biosensor and its preparation method and application. Background Art

[0002] As a product of the intersection of multiple disciplines such as chemistry, physics, medicine, and engineering, biosensors have the advantages of high specificity, high sensitivity, and low cost. They can quickly analyze and monitor target substances and are widely used in fields such as clinical diagnosis, food and drug analysis, healthcare, immunology, and environmental monitoring. Its core consists of two parts: an identification unit (for identifying target substances) and a sensor unit (converting the interaction between the identification unit and the target substance into a measurable physical signal, such as optical, electrochemical, piezoelectric, thermal, mechanical, acoustic, or magnetic signals). The main biosensing materials include enzymes, proteins, DNA, antibodies, antigens, cells, and tissues, etc., which act as sensitive elements for identifying analytes. According to the different recognition materials, biosensors can be divided into sensors based on enzymes, cells, DNA, tissues, etc. and immunosensors.

[0003] In recent years, DNA origami has shown extensive application potential in aspects such as precisely controlling chemical and enzymatic reactions, assembling plasmonic antennas, drug delivery, bio-computation, three-dimensional lattice engineering of nanoparticles (NPs), and nanomachining in surface engineering. For biosensors based on DNA origami, a variety of signal reading strategies have been developed, and the sensing mechanisms, potential, and challenges in their practical applications have been deeply explored. However, the sensitivity of these biosensors can only reach fM, which is not conducive to the detection of some biomarkers with extremely low contents.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above deficiencies of the existing technology, the purpose of the present invention is to provide an ultrasensitive biosensor and its preparation method and application, aiming to solve the problem of low sensitivity of existing biosensors.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect, an ultrasensitive biosensor is provided, including: a substrate, a two-dimensional material loaded on the substrate, a DNA origami loaded on the two-dimensional material, and quantum dots modified on the DNA origami;

[0008] Wherein, the DNA origami contains exposed single-stranded DNA fragments that can be cleaved by an activatable Cas protein; the quantum dots are connected to the ends of the single-stranded DNA fragments.

[0009] In a preferred technical solution, the two-dimensional material is modified with a carboxyl group or an N-hydroxysuccinimide ester group, the DNA origami is modified with an amino group, and the DNA origami is loaded on the two-dimensional material through an amide bond.

[0010] In a preferred technical solution, the quantum dot is modified with a carboxyl group or an N-hydroxysuccinimide ester group, the end of the single-stranded DNA fragment is modified with an amino group, and the quantum dot is connected to the end of the single-stranded DNA fragment through an amide bond.

[0011] In a preferred technical solution, the two-dimensional material is selected from one or more of molybdenum disulfide, tungsten disulfide, molybdenum diselenide, and tungsten diselenide.

[0012] In a preferred technical solution, the quantum dot is selected from one or more of CdS, CdSe, CdTe, and ZnSe.

[0013] In a preferred technical solution, the Cas protein is selected from one or more of Cas12a, Cas13a, and Cas9.

[0014] In a preferred technical solution, the DNA origami is composed of splicing of 4 single-stranded DNAs with nucleotide sequences shown in SEQ ID NO.1-4.

[0015] In a preferred technical solution, the DNA origami is composed of splicing of 8 single-stranded DNAs with nucleotide sequences shown in SEQ ID NO.5-12.

[0016] In a preferred technical solution, the DNA origami is composed of splicing of 22 single-stranded DNAs with nucleotide sequences shown in SEQ ID NO.13-34.

[0017] In a preferred technical solution, the DNA origami is composed of splicing of 31 single-stranded DNAs with nucleotide sequences shown in SEQ ID NO.35-65.

[0018] In a further preferred technical solution, the 5'-end of the single-stranded DNA is modified with an amino group.

[0019] In a second aspect, a kit is provided, including the ultrasensitive biosensor as described in the first aspect.

[0020] In a third aspect, a preparation method of the ultrasensitive biosensor as described in the first aspect is provided, including:

[0021] providing a substrate;

[0022] loading a two-dimensional material on the substrate to obtain a two-dimensional material / substrate structure;

[0023] Load DNA origami on the two-dimensional material to obtain a DNA origami / two-dimensional material / substrate structure;

[0024] Modify quantum dots on the DNA origami to obtain the ultra-sensitive biosensor described above.

[0025] Preferred technical solution, the preparation method of the DNA origami includes:

[0026] Provide single-stranded DNA required for assembling DNA origami, and sequentially add TE buffer and TM buffer to obtain a DNA mixture;

[0027] Self-assemble the single-stranded DNA in the DNA mixture by heating to obtain the DNA origami;

[0028] Among them, the TE buffer is prepared with water and includes: tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid; the TM buffer is prepared with water and includes: tris(hydroxymethyl)aminomethane and magnesium sulfate.

[0029] Fourthly, provide the application of the ultra-sensitive biosensor described in the first aspect, the kit described in the second aspect or the ultra-sensitive biosensor prepared by the preparation method described in the third aspect in the preparation of products for nucleic acid detection.

[0030] Beneficial effects: The present invention provides an ultra-sensitive biosensor, which combines a two-dimensional material and quantum dots, and uses DNA origami to precisely control the distance between the two, thereby significantly enhancing the second harmonic generation effect. Quantum dots are linked to the single-stranded DNA fragments exposed in the DNA origami. Combining with the CRISPR / Cas technology, when the target nucleic acid is present, the single-stranded DNA fragments can be cleaved to release quantum dots, reducing the second harmonic generation effect and thus emitting a detection signal. This ultra-sensitive biosensor has excellent performance. Compared with the conventional surface plasmon resonance and the pure optical biosensor combined with DNA origami, the sensitivity is increased by four orders of magnitude and can reach the aM level. Brief Description of the Drawings

[0031] Figure 1 It is a schematic diagram of the structure and detection of the ultra-sensitive biosensor of the present invention.

[0032] Figure 2 It is the detection effect diagram of the ultra-sensitive biosensor of Example 1. Detailed Embodiments

[0033] The present invention provides an ultra-sensitive biosensor and its preparation method and application. To make the purpose, technical solution and effect of the present invention clearer and more definite, the present invention is further described in detail below.

[0034] An embodiment of the present invention provides a highly sensitive biosensor, comprising: a substrate, a two-dimensional material loaded on the substrate, a DNA origami loaded on the two-dimensional material, and quantum dots modified on the DNA origami;

[0035] Wherein, the DNA origami contains exposed single-stranded DNA fragments, and the single-stranded DNA fragments can be cleaved by an activatable Cas protein; the quantum dots are connected to the ends of the single-stranded DNA fragments.

[0036] The technical principle of the embodiment of the present invention is as follows: The second harmonic generation effect of two-dimensional materials is a common nonlinear physical phenomenon. Second harmonic generation refers to that when the material is irradiated by strong laser light, the material doubles the frequency of the incident light, thereby generating a new light wave with a frequency twice that of the incident light. Quantum dots (QDs) are nanostructures with three-dimensional quantum confinement effects, having unique optical and electronic properties, and can be combined with two-dimensional materials to significantly enhance the nonlinear optical effect, and enhance the second harmonic generation through methods such as electric field localization, energy resonance transfer, and charge carrier coupling. DNA origami can precisely control the distance between two-dimensional materials and QDs, thereby precisely controlling the enhancement of the second harmonic generation of two-dimensional materials by QDs, and avoiding the entanglement problem between conventional single-stranded DNA probes and the non-specific adsorption problem caused by the adsorption of QDs on the surface of two-dimensional materials. At the same time, combined with the CRISPR / Cas technology, it can ensure the recognition ability for target nucleic acids. As Figure 1 shown, the presence of QDs significantly enhances the conversion efficiency of the second harmonic generation of two-dimensional materials. When the target nucleic acid is recognized, the Cas protein is activated, thereby cleaving the single-stranded DNA fragment linking the QDs in the DNA origami, releasing the QDs, reducing the conversion efficiency of the second harmonic generation of two-dimensional materials, and then emitting a detection signal.

[0037] The embodiment of the present invention constructs a highly sensitive biosensor combining QD, CRISPR / Cas, and DNA origami technologies, and detects the target nucleic acid by detecting the change in the second harmonic generation before and after adding samples to the highly sensitive biosensor, and can identify low-abundance nucleic acid targets in complex biological samples, especially in the case of no large-scale sample preparation or amplification. Compared with the optical biosensor combined with conventional surface plasmon resonance (SPR) and CRISPR / Cas technologies, the highly sensitive biosensor of the embodiment of the present invention has significantly improved detection sensitivity and can reach the aM level.

[0038] In one embodiment, the two-dimensional material is modified with a carboxyl group or an N-hydroxysuccinimide ester group, the DNA origami is modified with an amino group, and the DNA origami is loaded on the two-dimensional material through an amide bond.

[0039] In one embodiment, the quantum dots are modified with carboxyl or N-hydroxysuccinimide ester groups, the ends of the single-stranded DNA fragments are modified with amino groups, and the quantum dots are connected to the ends of the single-stranded DNA fragments through amide bonds.

[0040] In one embodiment, the two-dimensional material is selected from one or more of molybdenum disulfide (MoS2), tungsten disulfide (WS2), molybdenum diselenide (MoSe2), and tungsten diselenide (WSe2).

[0041] In one embodiment, the quantum dots are selected from one or more of CdS, CdSe, CdTe, and ZnSe.

[0042] In one embodiment, the Cas protein is selected from one or more of Cas12a, Cas13a, and Cas9.

[0043] In one embodiment, the DNA origami is composed of splicing of 4 single-stranded DNAs with nucleotide sequences shown in SEQ ID NO.1-4. Specifically, the 4 single-stranded DNAs with nucleotide sequences shown in SEQ ID NO.1-4 can be spliced into a "tetrahedron" DNA origami with a height of 5 nm, providing a suitable distance between the quantum dots and the two-dimensional material, so as to enhance the second harmonic of the two-dimensional material by the quantum dots. Among them, the 5' end of the single-stranded DNA can be modified with an amino group for fixing the DNA origami on the two-dimensional material or linking the quantum dots. SEQ ID NO.1-4 can be as follows:

[0044]

[0045] In one embodiment, the DNA origami is composed of splicing of 8 single-stranded DNAs with nucleotide sequences shown in SEQ ID NO.5-12. Specifically, the 8 single-stranded DNAs with nucleotide sequences shown in SEQ ID NO.5-12 can be spliced into a "tetrahedron" DNA origami with a height of 10 nm, providing a suitable distance between the quantum dots and the two-dimensional material, so as to enhance the second harmonic of the two-dimensional material by the quantum dots. Among them, the 5' end of the single-stranded DNA can be modified with an amino group for fixing the DNA origami on the two-dimensional material or linking the quantum dots. SEQ ID NO.5-12 can be as follows:

[0046]

[0047] In one embodiment, the DNA origami is composed of 22 single-stranded DNAs with nucleotide sequences shown in SEQ ID NOs. 13 to 34 spliced together. Specifically, the 22 single-stranded DNAs with nucleotide sequences shown in SEQ ID NOs. 13 to 34 can be spliced together to form a "tetrahedron" DNA origami with a height of 15 nm, providing a suitable distance between the quantum dots and the two-dimensional material, thereby enhancing the second harmonic of the two-dimensional material by the quantum dots. Among them, the 5'-end of the single-stranded DNA can be modified with an amino group for immobilizing the DNA origami on the two-dimensional material or linking quantum dots. SEQ ID NOs. 13 to 34 can be as follows:

[0048]

[0049]

[0050] In one embodiment, the DNA origami is composed of 31 single-stranded DNAs with nucleotide sequences shown in SEQ ID NOs. 35 to 65 spliced together. Specifically, the 31 single-stranded DNAs with nucleotide sequences shown in SEQ ID NOs. 35 to 65 can be spliced together to form a "tetrahedron" DNA origami with a height of 20 nm, providing a suitable distance between the quantum dots and the two-dimensional material, thereby enhancing the second harmonic of the two-dimensional material by the quantum dots. Among them, the 5'-end of some single-stranded DNAs can be modified with an amino group for immobilizing the DNA origami on the two-dimensional material or linking quantum dots. SEQ ID NOs. 35 to 65 can be as follows:

[0051]

[0052]

[0053]

[0054] In one embodiment, the substrate is selected from one or more of a silica / silicon (SiO2 / Si) substrate, a sapphire substrate, a fused silica glass substrate, and a silicon nitride substrate. Specifically, the substrate only provides support and its material is not limited.

[0055] In one embodiment, the height of the DNA origami is 5 to 20 nm.

[0056] In one embodiment, the detection signal of the ultrasensitive biosensor is the second harmonic. Specifically, by detecting the change in the second harmonic before and after adding samples to the ultrasensitive biosensor, the concentration of the target nucleic acid is calculated.

[0057] The embodiment of the present invention provides a kit, including the ultrasensitive biosensor as described above.

[0058] In one embodiment, the kit further includes a CRISPR / Cas reaction system; the CRISPR / Cas reaction system includes: a CRISPR / Cas system and a buffer for the reaction of the CRISPR / Cas system; the CRISPR / Cas system includes: a Cas protein and a crRNA or gRNA that is complementary to the target nucleic acid.

[0059] In one embodiment, the kit further includes an instruction manual; the instruction manual records the second harmonic value I1 of the ultrasensitive biosensor before sample addition, and / or the instruction manual records the second harmonic value I0 of the ultrasensitive biosensor before modification with quantum dots; and / or the instruction manual records the upper limit value of the second harmonic change (I0 - I1) after sample addition to the ultrasensitive biosensor.

[0060] In one embodiment, the method of using the kit for nucleic acid detection includes the steps:

[0061] Detect the second harmonic of the ultrasensitive biosensor before sample addition to obtain the value I1;

[0062] After mixing the sample to be tested with the CRISPR / Cas reaction system, add the mixture to the ultrasensitive biosensor and incubate.

[0063] After the incubation is completed, wash the ultrasensitive biosensor and remove non-specific adsorption, and detect the second harmonic of the ultrasensitive biosensor to obtain the value I2;

[0064] Analyze the change in the second harmonic of the ultrasensitive biosensor before and after sample addition to obtain the concentration of the target nucleic acid in the sample to be tested.

[0065] The method of use is for non-diagnostic and non-therapeutic purposes.

[0066] Specifically, when detecting whether the sample to be tested contains the target nucleic acid, it can be determined whether the target nucleic acid is contained by whether there is a difference between I2 and I1. If I2 = I1, the sample to be tested does not contain the target nucleic acid; if I2 < I1, the sample to be tested contains the target nucleic acid.

[0067] Specifically, when detecting the concentration of the target nucleic acid in the sample to be detected, different concentrations of the target nucleic acid standard solution can be prepared first. Detect the corresponding I2 for different concentrations, calculate the percentage of the change in the second harmonic (I2 - I1) corresponding to different concentrations to the total change in the second harmonic (I0 - I1) of the ultrasensitive biosensor. The formula is Δ(%) = (I2 - I1) / (I0 - I1). Then, use the negative logarithm of the target nucleic acid concentration as the abscissa and Δ(%) as the ordinate to plot the standard curve of the target nucleic acid concentration. Then detect the I2 of the sample to be detected, substitute it into the formula to calculate its Δ(%), and then substitute Δ(%) into the standard curve to obtain the concentration of the target nucleic acid in the sample to be detected.

[0068] In one embodiment, in the step of adding the sample to react on the ultrasensitive biosensor, the incubation temperature is 35 - 42 °C and the time is 20 - 40 minutes.

[0069] In one embodiment, the removal of non-specific adsorption is completed by incubating with proteinase K; preferably, the incubation temperature is 35 - 42 °C and the time is 20 - 40 minutes.

[0070] The embodiment of the present invention provides a preparation method of the ultrasensitive biosensor as described above, including:

[0071] Providing a substrate;

[0072] Loading a two-dimensional material on the substrate to obtain a two-dimensional material / substrate structure;

[0073] Loading DNA origami on the two-dimensional material to obtain a DNA origami / two-dimensional material / substrate structure;

[0074] Modifying quantum dots on the DNA origami to obtain the ultrasensitive biosensor.

[0075] In one embodiment, the preparation method of the DNA origami includes:

[0076] Providing single-stranded DNA required for assembling DNA origami, and sequentially adding TE buffer and TM buffer to obtain a DNA mixture;

[0077] Making the single-stranded DNA in the DNA mixture self-assemble by heating to obtain the DNA origami;

[0078] Among them, the TE buffer is prepared with water and includes: tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid; the TM buffer is prepared with water and includes: tris(hydroxymethyl)aminomethane and magnesium sulfate.

[0079] In one embodiment, the two-dimensional material is loaded on the substrate by mechanical exfoliation or chemical vapor deposition (CVD). The deposition thickness requirement of the two-dimensional material is not strict, and bulk, few-layer, and single-layer are all acceptable; preferably few-layer or single-layer, and the prepared ultrasensitive biosensor will have better detection effect.

[0080] The embodiments of the present invention provide the application of the ultrasensitive biosensor as described above, the kit as described above, or the ultrasensitive biosensor prepared by the preparation method as described above in the preparation of products for nucleic acid detection.

[0081] In one embodiment, the nucleic acid is derived from a virus, and the virus includes one or more of SARS-COV-2, HPV16, Monkeypox, but is not limited thereto; or, the nucleic acid is a biomarker of a disease such as miRNA, and the disease includes one or more of lung cancer, breast cancer, but is not limited thereto.

[0082] In one embodiment, when it is necessary to detect whether a patient is carrying a certain virus, only a sample of the patient's body fluid needs to be taken. After mixing the Cas12a protein, the protein reaction solution (i.e., the buffer for the CRISPR reaction), the sampling solution, and the crRNA designed according to different viruses, it is dropped into the ultrasensitive biosensor. The ultrasensitive biosensor is placed in a flow cell and incubated at 37 °C for 30 minutes for the CRISPR reaction. After rinsing with TM buffer, non-specific adsorption is removed by incubating with proteinase K for 10 minutes, followed by one rinse. By detecting the second harmonic change before and after the ultrasensitive biosensor, the diagnosis of a specific virus can be made.

[0083] In one embodiment, when it is necessary to detect whether a patient is carrying a certain cancer, only a sample of the patient's blood needs to be taken. After mixing the Cas12a protein, the protein reaction solution (i.e., the buffer for the CRISPR reaction), the reverse transcribed DNA (miRNA in the blood), and the crRNA designed according to different cancer markers, it is dropped into the ultrasensitive biosensor. The ultrasensitive biosensor is placed in a flow cell and incubated at 37 °C for 30 minutes for the CRISPR reaction. After rinsing with TM buffer, non-specific adsorption is removed by incubating with proteinase K for 10 minutes, followed by one rinse. By detecting the second harmonic change before and after the ultrasensitive biosensor, the diagnosis of a specific cancer can be made.

[0084] The present invention will be further described below through specific examples.

[0085] Example 1

[0086] This example provides an ultrasensitive biosensor, which is specifically as follows:

[0087] (1) Substrate cleaning: The SiO2 / Si substrate was ultrasonically cleaned with acetone and alcohol for 30 minutes respectively, and then dried with nitrogen to obtain a clean SiO2 / Si substrate.

[0088] (2) Construction of MoS2 / SiO2 / Si structure: A monolayer of MoS2 was deposited on the SiO2 / Si substrate by chemical vapor deposition to form a MoS2 / SiO2 / Si structure.

[0089] (3) Coupling agent modification: The MoS2 / SiO2 / Si structure was immersed in a 5 mM solution of 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) and treated at room temperature for 2 hours to form free N-hydroxysuccinimide ester groups on the surface of MoS2. Then it was rinsed with acetone and deionized water, and finally dried with nitrogen.

[0090] (4) DNA origami assembly: Single-stranded DNAs with nucleotide sequences customized as shown in SEQ ID NO.1 - 4 (the 5'-ends of the single-stranded DNAs were modified with amino groups) were concentrated by centrifugation (7000 rpm, 4 °C, 5 minutes) respectively, and then diluted to 100 μM with TE buffer to obtain single-stranded DNA solutions, which were stored frozen at -20 °C to -80 °C. 2 μL of each single-stranded DNA solution was mixed (16 μL in total), and 184 μL of TM buffer was added to obtain a DNA mixture, so that the final concentration of each single-stranded DNA in the DNA mixture was 1 μM. The DNA mixture was heated to 95 °C for 10 minutes and then immediately cooled to room temperature in ice to form a DNA origami with a tetrahedral structure (height: 5 nm). The solution containing the DNA origami (1 μM) was dropped onto the MoS2 / SiO2 / Si structure and incubated at 37 °C for 2 h, and then rinsed three times with water. Since there are free amino groups (-NH2) at the bottom of the DNA origami, the amino groups and the free N-hydroxysuccinimide ester groups on the surface of MoS2 underwent dehydration condensation to form amide bonds (-CONH-), enabling the DNA origami to be fixed on the surface of the MoS2 / SiO2 / Si structure, completing the fixation of the DNA origami and obtaining a DNAorigami / MoS2 / SiO2 / Si structure.

[0091] (5) Second harmonic benchmark test: The DNA origami / MoS2 / SiO2 / Si structure was benchmark measured using a second harmonic test system, and the initial value I0 was recorded.

[0092] (6) Quantum dot modification: Prepare a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 20 mM) and N-hydroxysuccinimide (NHS, 10 mM), and add it to MES buffer (0.1 M, pH 6.0) at a volume ratio of 1:9 and mix for 1 hour. Drop the CdTe quantum dot (CdTe QD, 10 μM) solution onto the DNA origami / MoS2 / SiO2 / Si structure and incubate at 37 °C for 2 h. Since the surface of CdTe QD is modified with carboxyl groups (-COOH), the carboxyl groups and the free amino groups (-NH2) at the top of the DNA origami undergo dehydration condensation to form amide bonds (-CONH-), thus fixing the CdTe QD on the DNA origami and completing the construction of the CdTe QD / DNA origami / MoS2 / SiO2 / Si sensor, that is, a ultrasensitive biosensor is obtained.

[0093] Example 2

[0094] This example provides a ultrasensitive biosensor, the difference from Example 1 is only that: single-stranded DNA with nucleotide sequences shown in SEQ ID NO.5-12 is used to assemble DNA origami (height is 10 nm).

[0095] Example 3

[0096] This example provides a ultrasensitive biosensor, the difference from Example 1 is only that: single-stranded DNA with nucleotide sequences shown in SEQ ID NO.13-34 is used to assemble DNA origami (height is 15 nm).

[0097] Example 4

[0098] This example provides a ultrasensitive biosensor, the difference from Example 1 is only that: single-stranded DNA with nucleotide sequences shown in SEQ ID NO.35-65 is used to assemble DNA origami (height is 20 nm).

[0099] Application Example

[0100] This application example provides the application of the ultrasensitive biosensor of Example 1 in miRNA detection, specifically as follows:

[0101] (1) Second harmonic test before sample addition: Measure the ultrasensitive biosensor of Example 1 through a second harmonic test system and record the value I1.

[0102] (2) Preparation of the sample to be measured: Collect the patient's blood, centrifuge to separate the serum, extract miRNA in the serum using a miRNA extraction kit, and reverse transcribe miRNA into a DNA solution using a reverse transcription kit.

[0103] (3) CRISPR / Cas Reaction Configuration and Incubation: Using a commercially available Cas protein kit, 20 μL of Cas12a protein, 20 μL of protein reaction solution, 10 μL of reverse-transcribed DNA solution, and 10 μL of crRNA (1 μM, designed according to the target DNA) were mixed and made up to 200 μL to obtain a mixed solution. The mixed solution was dropped onto the CdTe QD / DNA origami / MoS2 / SiO2 / Si sensor and incubated at 37 °C for 30 minutes. After incubation, the CdTe QD / DNA origami / MoS2 / SiO2 / Si sensor was rinsed with TM buffer, and then 30 μL of proteinase K was dropped onto the CdTe QD / DNA origami / MoS2 / SiO2 / Si sensor and incubated at 37 °C for 30 minutes to remove non-specific adsorption, and finally rinsed.

[0104] (4) Second-Harmonic Generation Test after Sample Loading: The CdTe QD / DNA origami / MoS2 / SiO2 / Si sensor after sample loading was measured by a second-harmonic generation test system, and the value I2 was recorded.

[0105] By comparing the changes in the measurement results of I0, I1, and I2, analyze whether there is an abnormality in the expression of the target miRNA in the patient sample.

[0106] Test

[0107] The specificity of the ultrasensitive biosensor of Example 1 was detected by adding different miRNAs (miRNA-21, miRNA-155, miRNA-10b) and crRNAs (crRNA-21, crRNA-155, crRNA-10b) to the CRISPR / Cas reaction system, and the results are as shown in Figure 2 A. As can be seen from Figure 2 A, a detection signal can only be generated when the miRNA and crRNA match.

[0108] The assembly of the DNA origami in Examples 1 to 4 was verified by polyacrylamide gel electrophoresis (PAGE), and the results are as shown in Figure 2 B, where L1 is the single-stranded DNA that was not assembled in Example 1, L2 is the DNA origami after assembly in Example 1, L3 is the single-stranded DNA that was not assembled in Example 2, L4 is the DNA origami after assembly in Example 2, L5 is the single-stranded DNA that was not assembled in Example 3, L6 is the DNA origami after assembly in Example 3, L7 is the single-stranded DNA that was not assembled in Example 4, and L8 is the DNA origami after assembly in Example 4. As can be seen from Figure 2 B, the DNA origami in Examples 1 to 4 was successfully assembled.

[0109] The detection limit of the ultrasensitive biosensor of Example 1 was tested by preparing miRNA-21 with different concentrations, and the results are as Figure 2 shown in C and D in Figure 2 , where C is the percentage change in the second harmonic signal tested with miRNA-21 at different concentrations, and D is an entire SHG mapping test corresponding to the change in C. As can be seen from

[0110] C and D in , the detection limit of the ultrasensitive biosensor of Example 1 can reach the aM level. It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or changes can be made according to the above description, and all such improvements and changes should fall within the protection scope of the appended claims of the present invention.

Claims

1. An ultrasensitive biosensor, characterized in that: include: A substrate, a two-dimensional material loaded on the substrate, a DNA origami loaded on the two-dimensional material, and quantum dots modified on the DNA origami; The DNA origami contains exposed single-stranded DNA fragments, which can be cut by activated Cas proteins; and the quantum dots are connected to the ends of the single-stranded DNA fragments.

2. The ultrasensitive biosensor according to claim 1, characterized in that: The two-dimensional material is modified with a carboxyl group or an N-hydroxysuccinimide ester group, the DNA origami is modified with an amino group, and the DNA origami is loaded on the two-dimensional material via an amide bond.

3. The ultrasensitive biosensor according to claim 1, characterized in that: The quantum dot is modified with a carboxyl group or an N-hydroxysuccinimide ester group, the end of the single-stranded DNA fragment is modified with an amino group, and the quantum dot is connected to the end of the single-stranded DNA fragment via an amide bond.

4. The ultrasensitive biosensor according to claim 1, characterized in that: The two-dimensional material is selected from one or more of molybdenum disulfide, tungsten disulfide, molybdenum diselenide, and tungsten diselenide; And / or, the quantum dots are selected from one or more of CdS, CdSe, CdTe, and ZnSe; And / or, the Cas protein is selected from one or more of Cas12a, Cas13a, and Cas9.

5. The ultrasensitive biosensor according to claim 1, characterized in that: The DNA origami is composed of four single-stranded DNA sequences as shown in SEQ ID NO.1 to 4; Or, the DNA origami is composed of 8 single-stranded DNAs whose nucleotide sequences are shown in SEQ ID NOs. 5 to 12; Or, the DNA origami is composed of 22 single-stranded DNAs whose nucleotide sequences are shown in SEQ ID NOs. 13 to 34; Alternatively, the DNA origami is composed of 31 single-stranded DNAs whose nucleotide sequences are shown in SEQ ID NOs. 35 to 65.

6. The ultrasensitive biosensor according to claim 5, characterized in that: The 5' end of the single-stranded DNA is modified with an amino group.

7. A kit for nucleic acid detection, characterized in that: The invention comprises the ultra-sensitive biosensor as claimed in any one of claims 1 to 6.

8. A method for preparing an ultrasensitive biosensor according to any one of claims 1 to 6, characterized in that: include: providing a substrate; Loading a two-dimensional material on the substrate to obtain a two-dimensional material / substrate structure; Loading DNA origami on the two-dimensional material to obtain a DNA origami / two-dimensional material / substrate structure; The ultra-sensitive biosensor is obtained by modifying quantum dots on the DNA origami.

9. The preparation method according to claim 8, characterized in that: The preparation method of the DNA origami comprises: Providing single-stranded DNA required for assembling DNA origami, and sequentially adding TE buffer and TM buffer to obtain a DNA mixed solution; The single-stranded DNA in the DNA mixture is self-assembled by heating to obtain the DNA origami; The TE buffer is prepared with water and includes tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid; the TM buffer is prepared with water and includes tris(hydroxymethyl)aminomethane and magnesium sulfate.

10. Use of the ultrasensitive biosensor according to any one of claims 1 to 6, the kit according to claim 7, or the ultrasensitive biosensor prepared by the preparation method according to claims 8 to 9 in preparing products for nucleic acid detection.

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