Method for quantitatively detecting low-abundance protein biomarker by combining photosensitive nanoprobe with Cas12a

Abstract

The invention provides a method for quantitatively detecting a low-abundance protein biomarker by combining a photosensitive nanoprobe with Cas12a, which comprises the following steps: identifying and enriching a target to be detected in serum through the photosensitive nanoprobe DA / PL-coated Cu NPs, and converting a signal of the target to be detected into a plurality of PL signals through photolysis to generate first signal amplification; the released PL is specifically recognized through Cas12a, the Cas12a is activated after being combined with the PL and does not specifically cut a signal label containing a fluorescence quenching group, a fluorescence signal is released, secondary signal amplification is generated, and quantitative detection of a to-be-detected target in serum is achieved by detecting the fluorescence signal. According to the method disclosed by the invention, ultra-sensitive dual signal amplification is realized through combination of the photosensitive nanoprobe and Cas12a, and the method has the advantages of high reliability, high sensitivity and high specificity and can be used for rapidly and accurately detecting low-abundance protein biomarkers in serum.

Description

Technical Field

[0001] The present invention relates to the field of biology and medicine, and in particular to a method for quantitatively detecting low-abundance protein biomarkers using a photosensitive nanoprobe combined with Cas12a. Background Art

[0002] Proteins are the executors of life's functions and the primary source of molecular biomarkers. These protein biomarkers play a crucial role in the development and progression of tumors, and their highly sensitive and accurate quantitative analysis is crucial for early diagnosis and effective treatment of tumors. Currently, commonly used quantitative methods for protein markers include enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS). Although these methods have made significant progress in the quantitative study of serum proteins, they still suffer from issues such as low specificity, high false-negative and false-positive rates, and complex sample pretreatment. To address these issues, it is urgent to explore and establish a new, highly sensitive, specific, and convenient clinical molecular detection technology.

[0003] Currently, there are two common methods for enhancing detection signals: one is to use highly sensitive detection instruments to perform secondary amplification of the output signal; the other is to use functional nanomaterials and bio-amplification technologies to amplify the signal. Nanoparticle-based amplification technologies, such as functionalized copper nanoprobes (CuNPs), have been widely used in biochemical assays due to their ease of preparation, ease of modification and storage, high loading efficiency, and good biocompatibility. While these methods can improve detection sensitivity and specificity to a certain extent, they still cannot quickly and accurately detect some low-abundance protein biomarkers. Summary of the Invention

[0004] The purpose of the present invention is to address the problem that low-abundance protein biomarkers cannot be detected quickly and accurately in the existing field. A method for quantitatively detecting low-abundance protein biomarkers by combining a photosensitive nanoprobe with Cas12a is provided. Ultra-sensitive dual signal amplification is achieved by combining a photosensitive nanoprobe with Cas12a, which has the advantages of high reliability, high sensitivity, and high specificity, and can quickly and accurately detect low-abundance protein biomarkers in serum.

[0005] According to the purpose of the present invention, a method for quantitatively detecting low-abundance protein biomarkers by combining a photosensitive nanoprobe with Cas12a is provided, comprising the following steps:

[0006] The target to be detected in the serum is identified and enriched by a photosensitive nanoprobe (DA / PL@Cu NPs), and the signal of a target to be detected is converted into the signals of several photosensitive linker probes (PL) by photolysis, generating the first signal amplification; wherein the DA / PL@Cu NPs are composed of a detection antibody (DA) and a photosensitive linker probe (PL) modified on copper nanoparticles (Cu NPs), the DA can specifically recognize the target to be detected, and the PL is a DNA sequence modified with a disulfide bond and a photosensitive group, and the DNA sequence specifically binds to the crRNA required for Cas12a activation;

[0007] Cas12a specifically recognizes the released PL. After binding to PL, Cas12a is activated and non-specifically cuts the signal tag containing the fluorescent quenching group, releasing the fluorescent signal, generating a second signal amplification, and realizing quantitative detection of the target to be detected in the serum by detecting the fluorescent signal.

[0008] As an optional embodiment, the identification and enrichment of the target to be detected in serum by DA / PL@Cu NPs, and the conversion of the signal of a target to be detected into several PL signals by photolysis, specifically includes:

[0009] Magnetic beads modified with capture antibodies identify and capture the target to be detected in the serum, and then incubate with DA / PL@Cu NPs, allowing the DA / PL@Cu NPs to recognize and enrich the target to be detected in the serum. Subsequently, under the irradiation of ultraviolet light, PL is released, thereby converting the signal of one target to be detected into the signals of several PLs; wherein, the capture antibody can specifically recognize the target to be detected and has a different recognition site from DA.

[0010] As an optional embodiment, the method of identifying and capturing the target to be detected in the serum by using magnetic beads modified with capture antibodies specifically includes:

[0011] After washing the magnetic beads with phosphate buffered saline (PBS), the capture antibody was added and the beads were shaken at 37°C in PBS. After the reaction, the beads were washed with PBS. Then, the serum to be tested was added and the beads were shaken at 37°C in PBS. After the reaction, the beads were washed with PBS and the supernatant was discarded to obtain magnetic beads that captured the target to be tested.

[0012] As an optional embodiment, the molar ratio between the magnetic beads and the capture antibody is (1:1) to (50:1).

[0013] As an optional embodiment, the co-incubation with DA / PL@Cu NPs allows the DA / PL@Cu NPs to recognize and enrich the target to be detected in the serum, and then the PL is released under ultraviolet light, specifically comprising:

[0014] The magnetic beads that captured the target to be detected were mixed with DA / PL@Cu NPs and incubated at 37°C with continuous shaking. After the incubation, they were washed with PBS, and the precipitate was added to PBS and released after ultraviolet photolysis.

[0015] As an optional embodiment, the preparation process of the DA / PL@Cu NPs includes:

[0016] PL was added to tris(2-carboxyethyl)phosphine (TCEP) and reduced at 37°C in the dark to reduce the disulfide bonds on PL to sulfhydryl groups, thereby obtaining a first solution;

[0017] Cu NPs were added to the first solution and continued to react at 37°C in the dark with shaking. Then, NaCl solution and DA were added, and the pH was adjusted to 9.0. After overnight in the dark at 4°C, the solution was washed to obtain DA / PL@Cu NPs.

[0018] As an optional embodiment, the molar ratio between PL and TCEP is (1:10) to (1:50), the molar ratio of Cu NPs to DA is (1:1) to (50:1), the molar ratio of Cu NPs to PL is (50:1) to (500:1), and the molar ratio of NaCl to Cu NPs is (5:1) to (50:1).

[0019] As an optional embodiment, the PL is specifically recognized by Cas12a, and Cas12a is activated after binding to PL and non-specifically cuts the signal tag containing the fluorescent quenching group to release the fluorescent signal, specifically including:

[0020] Cas12a and CrRNA were mixed and shaken at 37°C to form a complex to obtain a second solution;

[0021] The photolytically released PL solution and the signal tag solution containing a fluorescent quenching group were added to the second solution, and the reaction was shaken at 37 ° C. The complex of Cas12a and CrRNA combined with PL and activated Cas12a, thereby non-specifically cutting the signal tag containing the fluorescent quenching group and releasing the fluorescent signal.

[0022] As an optional embodiment, the molar ratio of Cas12a and CrRNA is (10: 1) to (1: 10), the concentration of the signal label solution containing the fluorescent quenching group is 500nM to 1500nM, and the volume ratio of the PL solution to the signal label solution containing the fluorescent quenching group is 1: 1.

[0023] As an optional embodiment, in the quantitative detection of the target to be detected in serum by detecting the fluorescence signal, the conditions of the fluorescence detection are determined by the fluorescent group of the signal tag containing a fluorescence quenching group.

[0024] It can be seen from the technical solution of the present invention above that the method for quantitatively detecting low-abundance protein biomarkers by combining photosensitive nanoprobes with Cas12a proposed in the present invention is that, first, the target to be detected is recognized and captured by magnetic beads modified with capture antibodies, and then a "sandwich" structure is formed with functionalized copper nanoprobes DA / PL@Cu NPs modified with DA and PL. After that, the PL of the end-modified photosensitive group is released under the irradiation of ultraviolet light, and the signal of a target can be converted into a large number of PL signals, thereby generating the first step of signal amplification; when Cas12a is activated after binding to the released PL, a large number of signal tags containing fluorescent quenching groups are non-specifically and rapidly cut, thereby generating the second step of signal amplification, thereby achieving high sensitivity and high specificity of rapid quantitative detection of low-abundance protein biomarkers in serum, and well avoiding the limitation that traditional Cas12a can only detect nucleic acid targets.

[0025] The method of quantitatively detecting low-abundance protein biomarkers by combining photosensitive nanoprobes with Cas12a of the present invention is a reliable, sensitive and specific non-invasive method that detects early cancer in an accurate and cost-effective manner, opening up a new avenue for early detection of cancer and having important implications for the current development of accurate and affordable early cancer detection and management strategies in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the process principle of the method for quantitatively detecting low-abundance protein biomarkers by combining photosensitive nanoprobes with Cas12a of the present invention.

[0027] Figure 2 It is a PL probe sequence optimization diagram in the example of the present invention; wherein, 2A is a polyacrylamide gel electrophoresis diagram of hybridization of PL of different sequence lengths with crRNA, and 2B is a relative fluorescence intensity diagram of Cas12a cleavage signal tags of PL of different sequence lengths with crRNA.

[0028] Figure 3 Figure 2 is a secondary structure diagram of PL-28 and the corresponding crRNA-28 in the examples of the present invention.

[0029] Figure 4 Characterization diagram of DA1 / PL-28@Cu NPs in the examples of the present invention; among them, 4A-4C are scanning electron microscopy images of Cu NPs, PL-28@Cu NPs and DA1 / PL-28@Cu NPs, respectively; 4D is an infrared characterization image of DA1 / PL-28@Cu NPs; 4E is a particle size distribution diagram; 4F is a zeta potential result diagram.

[0030] Figure 5This is a photolysis expansion diagram of PL-28 in an example of the present invention.

[0031] Figure 6 It is a feasibility verification diagram of Cas12a in the example of the present invention; wherein, 6A is a graph showing the change of fluorescence intensity of Cas12a cutting over time; 6B is a fluorescence spectrum graph of Cas12a cutting fluorescent probe.

[0032] Figure 7 It is a reaction condition optimization diagram of Cas12a in the example of the present invention, including (A) reaction solvent of Cas12a, (B) reaction temperature, (C) whether to add RNase inhibitor, and (D) optimization of reporter probe concentration.

[0033] Figure 8 Figure 8 is a graph showing the detection of ANGPTL2 by DA1 / PL-28@Cu NPs combined with Cas12a in an example of the present invention; Figure 8A shows the sensitivity of the detection method, Figure 8B shows the specificity of the detection method, Figure 8C shows a standard curve with the concentration of the standard ANGPTL2 as the horizontal axis and the fluorescence intensity as the vertical axis, and Figure 8D shows the quantitative results of ANGPTL2 in the serum of 9 healthy subjects and 56 colorectal cancer patients. DETAILED DESCRIPTION

[0034] In order to better understand the technical content of the present invention, specific embodiments are given and described below with reference to the accompanying drawings.

[0035] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to be comprehensive. It should be understood that the various concepts and embodiments described above, as well as those described in more detail below, can be implemented in any of a number of ways.

[0036] In recent years, the CRISPR / Cas system has garnered widespread attention for its high specificity and signal amplification capabilities. Cas12a, an RNA-guided endonuclease, rapidly trans-cleaves large amounts of single-stranded DNA upon activation, generating numerous signal tags. It has garnered particular attention for its ease of use, low cost, and high stability. However, conventional Cas12a can only detect nucleic acid targets, limiting its application.

[0037] Based on this, the present invention designs a new photosensitive probe to convert the signal of a protein target into a large number of nucleic acid PL signals, and cleverly combines with Cas12a, which effectively avoids the limitation of traditional Cas12a that can only detect nucleic acid targets, and can ultra-sensitively quantify protein biomarkers in the serum of cancer patients, which is of great significance in the early diagnosis of cancer.

[0038] Angiopoietin-like protein 2 (ANGPTL2) is a key molecule mediating chronic inflammation and its associated metabolic abnormalities. Furthermore, increased ANGPTL2 expression has been detected in tumor cells in hypoxic zones, suggesting that the tumor microenvironment induces ANGPTL2 expression in colorectal cancer. Therefore, ANGPTL2 in cancer cells and / or serum is considered a key factor in inflammation-induced carcinogenesis and cancer progression. Its expression is low in the early stages of colorectal cancer and gradually increases with disease progression.

[0039] Photosensitive nanoprobe DA / PL@Cu NPs

[0040] Combine Figure 1 As shown in the figure, DA / PL@Cu NPs are composed of copper nanoparticles (Cu NPs) modified with detection antibodies (DA) and photosensitive linker probes (PL).

[0041] Among them, DA can specifically recognize the target to be detected. It can be understood that DA, as a recognition element, can be determined according to the actual target to be detected. For example, if the target to be detected is ANGPTL2, then an antibody that can specifically recognize ANGPTL2 can be selected. In the following example, DA1 is used as an antibody that can specifically recognize ANGPTL2 for distinction.

[0042] Once the target to be detected is determined, detection antibodies and capture antibodies can be purchased directly from the market or customized as required.

[0043] PL is a DNA sequence modified with a disulfide bond and a photosensitive group. The DNA sequence specifically binds to the crRNA required for Cas12a activation. The specific structural formula of PL is shown in Formula I.

[0044] Among them, Oligo~ is a DNA sequence.

[0045] In one preferred example, the DNA sequence of PL is a nucleotide sequence corresponding to the target to be detected. For example, if the target to be detected is ANGPTL2, the DNA sequence is a nucleotide sequence converted according to ANGPTL2. Then, the crRNA can be designed accordingly according to the DNA sequence of PL to enable stable hybridization between PL and crRNA.

[0046] It is understandable that as the crRNA required for Cas12a activation, there is a part of the fixed sequence necessary for activating Cas12a. On the basis of this part of the fixed sequence, another sequence is further designed according to the designed PL DNA sequence, so that the PL DNA sequence crRNA is stably hybridized, effectively activating the nuclease activity of Cas12a and improving Cas12a enzyme cutting efficiency.

[0047] In one example, ANGPTL2 was detected. Based on this target, the corresponding PL DNA sequence and the corresponding CrRNA were designed. The base number is 28bp and the sequence is as follows:

[0048] PL-28: AGAGAT GTACAGGTAA AACAC ACAAA CC

[0049] CrRNA-28:UAAUU UCUAC UAAGU GUAGA U AAGG UUUGU GUGUU UUACC UGUAC AUCU

[0050] Among them, the fixed sequence required for activating Cas12a in crRNA-28 is UAAUU UCUAC UAAGU GUAGA U.

[0051] Preparation of DA / PL@Cu NPs

[0052] PL was added to tris(2-carboxyethyl)phosphine (TCEP) and reduced at 37° C. in the dark. The disulfide bonds on PL were reduced to sulfhydryl groups by TCPE to obtain a first solution.

[0053] Cu NPs were added to the first solution and continued to react at 37°C in the dark with shaking. Then, NaCl solution and DA were added, and the pH was adjusted to 9.0. After overnight in the dark at 4°C, the solution was washed to obtain DA1 / PL@Cu NPs.

[0054] Among them, the molar ratio between PL and TCEP is (1:10) to (1:50), the molar ratio of Cu NPs to DA is (1:1) to (50:1), the molar ratio of Cu NPs to PL is (50:1) to (500:1), and the molar ratio of NaCl to Cu NPs is (5:1) to (50:1).

[0055] A method for quantitative detection of low-abundance protein biomarkers using photosensitive nanoprobes combined with Cas12a

[0056] Combine Figure 1 As shown, using the aforementioned photosensitive nanoprobe, taking the quantification of ANGPTL2 levels in the serum of colorectal cancer patients as an example, the method of quantitatively detecting low-abundance protein biomarkers using an exemplary photosensitive nanoprobe combined with Cas12a of the present invention comprises the following steps:

[0057] ANGPTL2 in serum is recognized and captured by magnetic beads modified with capture antibodies and then incubated with DA1 / PL@Cu NPs. DA1 / PL@Cu NPs then recognize and enrich ANGPTL2 in serum. Subsequently, under UV irradiation, PL is released, converting the signal of one ANGPTL2 into signals of several PLs, resulting in the first signal amplification.

[0058] Among them, the capture antibody can specifically recognize ANGPTL2, and its recognition site is different from that of DA1. The capture antibody is modified with biotin and can specifically bind to the streptavidin-modified magnetic beads, so that the magnetic beads can specifically recognize and capture ANGPTL2. Afterwards, when the magnetic beads that captured ANGPTL2 were incubated with DA1 / PL@Cu NPs, ANGPTL2 was recognized and captured by DA1, thus forming a "sandwich" structure of magnetic beads, ANGPTL2, and DA / PL@Cu NPs, completing the identification and enrichment of ANGPTL2.

[0059] It is understood that the capture antibody is also determined according to the actual target to be detected.

[0060] Cas12a specifically recognizes the released PL. After binding to the PL, Cas12a is activated and non-specifically cuts the signal tag containing the fluorescent quenching group, releasing the fluorescent signal, generating a second signal amplification, and realizing the quantitative detection of ANGPTL2 in serum by detecting the fluorescent signal. In this way, a large number of signal tags containing fluorescent quenching groups are cut by a very small amount of protein biomarkers, greatly improving the sensitivity of detection.

[0061] Among them, the signal tag containing a fluorescence quenching group is a short DNA chain with quenched fluorescent groups connected at both ends. The fluorescence is quenched when the chain is intact, and the fluorescence is restored when the DNA chain is cut. Generally, a DNA chain of 5 bases is selected to ensure that the fluorescent group is quenched. For example, 5'-FAM-TTATT-BHQ-3' can be preferably used as a signal tag.

[0062] As an optional example, ANGPTL2 in serum is recognized and captured by magnetic beads modified with a capture antibody, specifically comprising:

[0063] After washing the magnetic beads with phosphate buffered saline (PBS), the capture antibody was added and the beads were shaken at 37°C in PBS. After the reaction, they were washed with PBS. Then, the serum to be tested was added and shaken at 37°C in PBS. After the reaction, they were washed with PBS and the supernatant was discarded to obtain magnetic beads that captured ANGPTL2.

[0064] As an optional example, the molar ratio between the magnetic beads and the capture antibody is (1:1) to (50:1). The magnetic beads are modified with streptavidin and can specifically bind to the capture antibody modified with biotin. The magnetic beads modified with streptavidin can be purchased directly.

[0065] As an optional example, DA / PL@Cu NPs are co-incubated to allow DA / PL@Cu NPs to recognize and enrich ANGPTL2 in serum. Subsequently, the PL probe is released under UV irradiation, specifically including:

[0066] The magnetic beads that captured ANGPTL2 were mixed with DA / PL@Cu NPs and incubated at 37°C with continuous shaking. After the incubation, they were washed with PBS, and the precipitate was added to PBS and released after ultraviolet photolysis.

[0067] As an optional example, Cas12a specifically recognizes the released PL. After binding to the PL, Cas12a is activated and non-specifically cuts the signal tag containing the fluorescent quenching group to release the fluorescent signal, specifically including:

[0068] Cas12a and CrRNA were mixed and shaken at 37°C to form a complex to obtain a second solution;

[0069] The photolytically released PL solution and signal tag solution were added to the second solution, and the reaction was shaken at 37°C. The complex of Cas12a and CrRNA combined with PL and activated Cas12a, thereby non-specifically cutting the signal tag containing the fluorescent quenching group and releasing the fluorescent signal.

[0070] As an optional example, the molar ratio of Cas12a and CrRNA is (10:1) to (1:10), the concentration of the signal label solution containing the fluorescent quenching group is 500nM to 1500nM, and the volume ratio of the PL solution to the signal label solution containing the fluorescent quenching group is 1:1.

[0071] As an optional example, in the quantitative detection of ANGPTL2 in serum by detecting fluorescence signals, the conditions for fluorescence detection are determined by the fluorescent gene of the signal tag containing a fluorescence quenching group; for example, when 5'-FAM-TTATT-BHQ-3' is used, the detection conditions are: excitation wavelength 492 nm, emission wavelength 518 nm.

[0072] It can be understood that in the method of the present invention, the quantification of low-abundance protein biomarkers of various cancers can be achieved by simply replacing the recognition elements (i.e., the capture antibody and the detection antibody, which recognize different sites of the target protein marker and can form a "sandwich" structure).

[0073] For better understanding, the present invention is further described below with reference to several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0074] Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0075] The detection antibodies and capture antibodies used in the following examples are shown in Table 1. The general structural formula of PL is shown in Formula I, and was customized from Sangon Biotech (Shanghai) Co., Ltd. based on the provided nucleic acid sequence; crRNA was also customized from Sangon Biotech (Shanghai) Co., Ltd. based on the provided nucleic acid sequence; the nucleic acid sequence on PL hybridized with the corresponding nucleic acid sequence of crRNA, and the nucleic acid sequence on PL was identical to the nucleic acid sequence converted by ANGPTL2.

[0076] Table 1

[0077]

[0078] Example 1

[0079] [Synthesis and Characterization of Photosensitive Nanoprobes]

[0080] (1) Synthesis

[0081] Take 100 μL, 10 μM photosensitive ligation probe (PL), add 1 mg of tris(2-carboxyethyl)phosphine and reduce at 37°C in the dark for 2 hours. Weigh 40 mg of Cu NPs and add them to the above solution for reaction. Continue to shake and react at 37°C in the dark for 30 minutes to obtain PL@CuNPs. Then add 400 μL, 500 nM NaCl solution and 4 μL, 100 μg / mL ANGPTL2 detection antibody (DA1,) and adjust the pH to 9.0. After overnight at 4°C in the dark, wash three times at 8000 rpm for 5 minutes to synthesize DA1 / PL@Cu NPs.

[0082] {Sequence Optimization of PL}

[0083] According to the above method, photosensitive nanoprobes synthesized with different base PLs were prepared, and the corresponding Cas12a enzyme cleavage efficiency was investigated. The results are as follows Figure 2 shown.

[0084] As can be seen from the figure, when the number of PL bases is ≥24bp, PL and CrRNA can stably hybridize; when the number of PL bases reaches 28bp, the Cas12a enzyme cleavage efficiency is basically saturated. Therefore, PL-28 and CrRNA-28 were selected for subsequent experiments. The secondary structures of PL-28 and CrRNA-28 are as follows. Figure 3 The nucleic acid sequences related to the experiment are shown in Table 2.

[0085] Table 2. Sequences of PL, crRNA, and signal tags

[0086]

[0087] (2) Characterization

[0088] DA1 / PL-28@Cu NPs were synthesized using PL-28 according to the method in (1) and characterized. Figure 4 shown.

[0089] Figure 4 A-4C are the scanning electron microscopy results of Cu NPs, PL-28@Cu NPs, and DA1 / PL-28@Cu NPs, respectively. The results show that the photosensitive nanoprobe DA1 / PL-28@Cu NPs are spherical and evenly distributed; Figure 4 The infrared characterization of DA1 / PL-28@Cu NPs in D showed that the characteristic group PO4 3- As well as the appearance of the characteristic group amide of DA1, it preliminarily proved the successful synthesis of DA1 / PL-28@CuNPs; Figure 4 The particle size distribution results of Figure E show that the particle sizes of Cu NPs, PL-28@Cu NPs, and DA1 / PL-28@Cu NPs are 258 nm, 281 nm, and 313 nm, respectively. The gradual increase in particle size further supports the successful synthesis of DA1 / PL-28@CuNPs. Figure 4 The zeta potential results of F showed that the potential flipped from positive to negative, which further proved the successful synthesis of DA1 / PL-28@Cu NPs.

[0090] (3) Photolysis investigation of PL probe

[0091] 1 mg of the synthesized DA1 / PL-28@Cu NPs was redissolved in 200 μL of PBS and irradiated with UV light (wavelength 365 nm, power 16 W). 3 μL of the supernatant was taken every 5 min to measure the PL-28 concentration and investigate the optimal photolysis time of PL-28. The results are as follows: Figure 5 As shown in the figure, PL-28 was almost completely released at 25 min (concentration was 37.545 ng / μL).

[0092] Example 2

[0093] [Feasibility verification and reaction condition optimization based on CRISPR / Cas12a fluorescence detection system]

[0094] (1) Feasibility verification of CRISPR / Cas12a fluorescence detection system

[0095] Take 40 μL, 100 nM Cas 12a, 40 μL, 100 nM CrRNA-28, shake at 37 ° C for 30 minutes, and store in a -20 ° C refrigerator until use.

[0096] When detecting the target, add 10 μL of photolytically released PL-28 and 10 μL of 750 nM signal tag containing a fluorescence quenching group to the above reaction solution, shake the reaction at 37°C for 6 minutes, and detect fluorescence under the conditions of excitation wavelength 492 nm and emission wavelength 518 nm.

[0097] The reaction solvent for all the above steps was Cas reaction solution containing 5 U / μL RNase inhibitor.

[0098] like Figure 6 As shown in the figure, the fluorescence intensity when all components of Cas12a, crRNA-28, and PL-28 were present was significantly higher than that of the control group; at 6 minutes, Cas12a basically completely cut the signal tag containing the fluorescence quenching group.

[0099] (2) Optimization of reaction conditions based on CRISPR / Cas12a fluorescence detection system

[0100] like Figure 7 As shown in the figure, the Cas12a reaction solvent, reaction temperature, whether to add RNase inhibitors, and the concentration of the signal tag containing the fluorescent quenching group were optimized. The results showed that when the reaction solvent was Cas reaction buffer, the reaction temperature was 37°C, RNase inhibitors were added, and the concentration of the signal tag containing the fluorescent quenching group was 750nM, the best detection results were achieved.

[0101] Example 3

[0102] [Quantitative detection of ANGPTL2 in serum by photosensitive nanoprobe combined with Cas12a]

[0103] Take 20 μL of streptavidin-modified magnetic beads (50 mg beads / mL, 0.5 μm, product number: D1 10557-0001, manufacturer: Sangon Biotech (Shanghai) Co., Ltd.) and wash three times with PBS. Then add 30 μL of 100 μg / mL ANGPTL2 capture antibody in 200 μL PBS and shake at 37°C for 30 minutes. After the reaction is completed, wash three times with PBS, add 100 μL of the serum to be tested, shake at 37°C for 30 minutes in 200 μL PBS, wash three times with PBS, discard the supernatant, and collect the precipitate for later use.

[0104] The collected precipitate was incubated with 200 μL of 1 mg / mL DA1 / PL-28@Cu NPs at 37°C for 30 min with continuous shaking. After washing three times with PBS, the precipitate was added with 100 μL of PBS and subjected to UV photolysis (wavelength 365 nm, power 16 W) for 25 min to release PL-28.

[0105] Take 40 μL, 100 nM Cas 12a, 40 μL, 100 nM CrRNA-28 and shake at 37 ° C for 30 minutes, then add 10 μL of the above-mentioned photolyzed PL-28 and 10 μL of 750 nM signal tag containing a fluorescence quencher group, shake at 37 ° C for 6 minutes, and detect fluorescence at an excitation wavelength of 492 nm and an emission wavelength of 518 nm. The reaction solvent for all the above steps is the Cas reaction solution containing 5 U / μL RNase inhibitor.

[0106] like Figure 8 As shown in A, the detection limit of ANGPTL2 is 50 pg / mL, and the detection method has high sensitivity; Figure 8 As shown in B, the interfering protein detection signal is basically consistent with the blank matrix signal, indicating that the detection method has good specificity; Figure 8 As shown in Figure C, as the concentration of ANGPTL2 increases, the fluorescence signal intensity also gradually increases, and has a good linear relationship; Figure 8 As shown in Figure D, the concentration of ANGPTL2 in the serum of colorectal cancer patients (CRC) was significantly higher than that in the serum of normal controls (NC) (P<0.05); thus, it can be seen that the detection method of the present invention can successfully and quantitatively detect ANGPTL2 in serum samples, revealing that the method of the present invention is of great significance in the early diagnosis of colorectal cancer.

[0107] While the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Persons skilled in the art will readily appreciate that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for quantitatively detecting low-abundance protein biomarkers using a photosensitive nanoprobe combined with Cas12a, characterized in that The following steps are involved: The target to be detected in the serum is identified and enriched by a photosensitive nanoprobe (DA / PL@Cu NPs), and the signal of a target to be detected is converted into the signals of several photosensitive linker probes (PL) by photolysis, generating the first signal amplification; wherein the DA / PL@Cu NPs are composed of a detection antibody (DA) and a photosensitive linker probe (PL) modified on copper nanoparticles (Cu NPs), the DA can specifically recognize the target to be detected, and the PL is a DNA sequence modified with a disulfide bond and a photosensitive group, and the DNA sequence specifically binds to the crRNA required for Cas12a activation; Cas12a specifically recognizes the released PL. After binding to PL, Cas12a is activated and non-specifically cuts the signal tag containing the fluorescent quenching group, releasing the fluorescent signal, generating a second signal amplification, and realizing quantitative detection of the target to be detected in the serum by detecting the fluorescent signal.

2. The method according to claim 1, characterized in that The method of identifying and enriching the target to be detected in serum by DA / PL@Cu NPs and converting the signal of a target to be detected into several PL signals by photolysis specifically includes: Magnetic beads modified with capture antibodies identify and capture the target to be detected in the serum, and then incubate with DA / PL@Cu NPs, allowing the DA / PL@Cu NPs to recognize and enrich the target to be detected in the serum. Subsequently, under the irradiation of ultraviolet light, PL is released, thereby converting the signal of one target to be detected into the signals of several PLs; wherein, the capture antibody can specifically recognize the target to be detected and has a different recognition site from DA.

3. The method according to claim 2, characterized in that The method of identifying and capturing the target to be detected in the serum by using magnetic beads modified with capture antibodies specifically includes: After washing the magnetic beads with phosphate buffered saline (PBS), the capture antibody was added and the beads were shaken at 37°C in PBS. After the reaction, the beads were washed with PBS. Then, the serum to be tested was added and the beads were shaken at 37°C in PBS. After the reaction, the beads were washed with PBS and the supernatant was discarded to obtain magnetic beads that captured the target to be tested.

4. The method according to claim 3, characterized in that The molar ratio between the magnetic beads and the capture antibody is (1:1) to (50:1).

5. The method according to claim 2, characterized in that The co-incubation with DA / PL@Cu NPs enables DA / PL@Cu NPs to recognize and enrich the target to be detected in the serum. Thereafter, PL is released under the irradiation of ultraviolet light, specifically including: The magnetic beads that captured the target to be detected were mixed with DA / PL@Cu NPs and incubated at 37°C with continuous shaking. After the incubation, they were washed with PBS, and the precipitate was added to PBS and released after ultraviolet photolysis.

6. The method according to claim 1, characterized in that The preparation process of the DA / PL@Cu NPs includes: PL was added to tris(2-carboxyethyl)phosphine (TCEP) and reduced at 37°C in the dark to reduce the disulfide bonds on PL to sulfhydryl groups, thereby obtaining a first solution; Cu NPs were added to the first solution and continued to react at 37°C in the dark with shaking. Then, NaCl solution and DA were added, and the pH was adjusted to 9.

0. After overnight in the dark at 4°C, the solution was washed to obtain DA / PL@Cu NPs.

7. The method according to claim 6, characterized in that The molar ratio between PL and TCEP was (1:10) to (1:50), the molar ratio between Cu NPs and DA was (1:1) to (50:1), the molar ratio between Cu NPs and PL was (50:1) to (500:1), and the molar ratio between NaCl and Cu NPs was (5:1) to (50:1).

8. The method according to claim 1, characterized in that The PL is specifically recognized by Cas12a, and Cas12a is activated after binding to the PL and non-specifically cuts the signal tag containing the fluorescent quenching group to release the fluorescent signal, specifically including: Cas12a and CrRNA were mixed and shaken at 37°C to form a complex to obtain a second solution; The photolytically released PL solution and the signal tag solution containing a fluorescent quenching group were added to the second solution, and the reaction was shaken at 37 ° C. The complex of Cas12a and CrRNA combined with PL and activated Cas12a, thereby non-specifically cutting the signal tag containing the fluorescent quenching group and releasing the fluorescent signal.

9. The method according to claim 8, characterized in that The molar ratio of Cas12a and CrRNA is (10:1) to (1:10), the concentration of the signal label solution containing the fluorescent quenching group is 500nM to 1500nM, and the volume ratio of the PL solution to the signal label solution containing the fluorescent quenching group is 1:

1.

10. The method according to claim 1, characterized in that In the quantitative detection of the target to be detected in the serum by detecting the fluorescent signal, the conditions of the fluorescent detection are determined by the fluorescent group of the signal tag containing the fluorescent quenching group.

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