A method for detecting sarcosine based on nucleic acid-mediated enzyme cascade

Abstract

The present invention belongs to the field of chemical detection, and relates to a detection method and kit for sarcosine based on a nucleic acid-mediated enzyme cascade. The method comprises: regulating the distance between sarcosine oxidase (SOX) and peroxidase (HRP) by nucleic acid to limit the enzyme cascade reaction to a specific area at the nanoscale; color development reaction, and determination of sarcosine content. In the method, nucleic acid molecules achieve precise regulation and high specificity of the distance. The method has strong specificity, high efficiency, significantly improves the sensitivity and accuracy of detection, and the required detection equipment and site are not harsh, low cost, and has important research significance and application value.

Description

Technical Field

[0001] The present invention belongs to the field of chemical detection, and relates to a detection method and a kit for sarcosine based on nucleic acid-mediated enzyme cascade, and more specifically, to a detection method and a kit for determining sarcosine, a specific target of prostate cancer. Background Art

[0002] As the second most common cancer in men, prostate cancer is attracting more and more attention. The current technology for detecting prostate cancer is mainly through the detection of prostate specific antigen (PSA), but the PSA detection has a gray range of 2ng / ml-10ng / ml. When the PSA content is in this range, its detection accuracy is not high, which is not conducive to the early diagnosis of prostate cancer. The sensitivity and accuracy of PSA detection need to be improved.

[0003] In 2009, Sreekumar et al. proposed that sarcosine is closely related to the development of prostate cancer, and the content of sarcosine is closely related to the development of the disease. Traditional methods for determining sarcosine include: electrochemical method (Chinese patent CN106596693A, invention name: an electrochemical detection method for sarcosine); ultraviolet spectroscopy: (Chinese patent CN110702670A, invention name: a sarcosine detection method based on metal organic framework materials); electrophoresis: (Chinese patent CN101718746A, invention name: a sarcosine detection method); chromatography mass spectrometry (literature: Areproducible and high-throughput HPLC / MS method to separate sarcosine fromα-andβ-alanine and to quantify sarcosine in human serum andurine. Anal. Chem. 2011, 83, 14, 5735–5740). However, most of these detection technologies require expensive detection instruments and require professional personnel to operate, which is not suitable for developing low-cost portable detection devices. Therefore, developing a highly sensitive, low-cost and rapid detection method has always been a need for those skilled in the art.

[0004] Furthermore, since sarcosine is a small amino acid molecule, there are difficulties in the specific and sensitive analysis of sarcosine small molecules. The detection of sarcosine small molecules by high performance liquid chromatography-mass spectrometry technology has the problem of being unable to effectively identify and distinguish sarcosine from other metabolic small molecules. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned prior art, the present invention aims to provide a method for detecting creatine with low operating difficulty, low cost, high efficiency, strong specificity and high sensitivity.

[0006] An object of the present invention is to provide a method for detecting sarcosine based on a nucleic acid-mediated enzyme cascade reaction, comprising the following steps:

[0007] Step 1: Use nucleic acids to regulate the distance between sarcosine oxidase SOX and peroxidase HRP to limit the enzyme cascade reaction to a specific area at the nanoscale;

[0008] Step 2: Color reaction and determination of creatine content.

[0009] Specifically, the step 1 includes:

[0010] Add a cross-linking agent to a SOX enzyme solution, incubate, and determine the SOX concentration; add a first nucleic acid, incubate so that the first nucleic acid is connected to the SOX enzyme molecule, and obtain a SOX enzyme molecule connected to the first nucleic acid; wherein each SOX enzyme molecule in the SOX enzyme connected to the first nucleic acid is connected to at least one first nucleic acid chain;

[0011] A cross-linking agent is added to the HRP enzyme solution, incubated, and the concentration of the HRP enzyme is determined; a second nucleic acid is added, incubated so that the second nucleic acid is connected to the HRP enzyme molecule, and an HRP enzyme molecule connected to the second nucleic acid is obtained; wherein each SOX enzyme molecule in the SOX enzyme connected to the second nucleic acid is connected to at least one second nucleic acid chain.

[0012] Furthermore, the step 1 further comprises: connecting the SOX enzyme connected to the first nucleic acid and the HRP enzyme connected to the second nucleic acid via a third nucleic acid, wherein the sequence of the third nucleic acid is different from the sequence of the first nucleic acid.

[0013] Further, the first nucleic acid and the second nucleic acid are deoxyribonucleic acids, and the sequence of the first nucleic acid is the same as or different from the sequence of the second nucleic acid. The first nucleic acid, the second nucleic acid and the third nucleic acid are oligonucleotides, and the sequence of the first nucleic acid is the same as or different from the sequence of the second nucleic acid. Preferably, the first nucleic acid and the second nucleic acid are deoxyribonucleic acids, and the sequence of the first nucleic acid is the same as or different from the sequence of the second nucleic acid.

[0014] Further, the molar ratio of the first nucleic acid to the SOX enzyme is at least 5:1, and the molar ratio of the second nucleic acid to the HRP enzyme is at least 5:1. Preferably, the molar ratio of the first nucleic acid to the SOX enzyme is at least 10:1.

[0015] Preferably, the first nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO.1, and the second nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO.2. Preferably, the third nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO.3.

[0016] SEQ ID NO.1(5'-3'):TTTTTTTTTTTTTTTTTTTTTT

[0017] SEQ ID NO.2(5'-3'):TTGGTGGTGGTGGTGGTGGT

[0018] SEQ ID NO.3(5'-3'):

[0019] ACCACCACCACCACCACCAAAAAAAAAAAAAAAAAAAAAAAA

[0020] Another object of the present invention is to provide a kit for detecting sarcosine, the kit comprising at least:

[0021] Sarcosine oxidase SOX solution and peroxidase HRP solution, the solution used for connecting and diluting sarcosine oxidase and peroxidase is DPBS;

[0022] A first nucleic acid for modifying the sarcosine oxidase SOX, and a second nucleic acid for modifying the peroxidase HRP;

[0023] A third nucleic acid, used for linking the modified sarcosine oxidase SOX and the modified peroxidase HRP;

[0024] A substrate for an enzyme cascade reaction; the substrate is a sarcosine solution, and the concentration of the sarcosine solution is 1-100 μM;

[0025] The molar ratio of the first nucleic acid to the SOX enzyme is at least 5:1, and the molar ratio of the second nucleic acid to the HRP enzyme is at least 5:1; the molar ratio of the third nucleic acid to the linked modified sarcosine oxidase SOX and the modified peroxide oxidase HRP is at least 1:1:1.

[0026] Further, (1) Preparation of sarcosine solutions of different concentrations: 100 mg of sarcosine was weighed to prepare a 1 mol / L stock solution, and gradient dilution was performed using DPBS (pH 8.0) buffer solution, with each step being 10-fold dilution until the final concentration reached 1 μM; (2) Preparation of TMB buffer solution: 400 μL TMB stock solution, 150 μL 10×PBS and 450 μL ultrapure water; (3) Preparation of reaction buffer: 100 μL reaction system contained: 40 μL TMB reaction buffer, 20 μL mixed free enzyme, and 40 μL sarcosine of different concentrations.

[0027] More specifically, the present invention also provides a detection method and kit for determining the prostate cancer-specific target sarcosine.

[0028] The invention realizes high specificity detection through the cascade reaction of sarcosine oxidase and horseradish oxidase. The detection principle is: sarcosine oxidase catalyzes sarcosine to produce glycine, hydrogen peroxide and formaldehyde under the condition of sarcosine, and hydrogen peroxide is used as a substrate of peroxidase reaction to produce a color reaction, thereby realizing the detection of sarcosine. SOX and HRP are in a chaotic and disordered state in the solution, and random collision makes the catalytic efficiency of the enzyme cascade reaction insufficient to meet the sensitivity and accuracy required for detection.

[0029] Moreover, based on the base-to-base distance rule and calculation of the double-stranded DNA, it can be concluded that the distance between the two enzymes connected by the third single strand of about 42 bases is 12.8nm, which is different from the free enzyme. Due to the reduction in the distance between the enzymes, the detection rate, accuracy and sensitivity are significantly improved. The distance between the two enzymes can be determined by the number of bases in the DNA chain.

[0030] In contrast, the deoxynucleic acid DNA in the present invention replaces the hetero-bifunctional reagent SPDP, which can not only achieve nano-level regulation of the enzyme spacing, but also can connect enzyme molecules with high specificity. The DNA-mediated connection of SOX and HRP can not only achieve directional transmission of the dual enzyme cascade reaction, but also significantly improve the catalytic efficiency and catalytic rate of the enzyme cascade reaction. Therefore, the use of DNA-mediated connection of SOX and HRP has important research significance and application value for the efficient and rapid detection of sarcosine.

[0031] Beneficial Effects

[0032] Based on nucleic acid-mediated connection between sarcosine oxidase and peroxidase, the two enzymes are placed in a limited environment, and the quantification of sarcosine is achieved through the color development phenomenon of the enzyme cascade reaction. This method not only achieves the regulation of the nanoscale distance between the two enzymes, making the enzyme cascade reaction faster and more sensitive, but also its detection equipment and environment are not harsh and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the principle of the nucleic acid-mediated enzyme cascade reaction according to the present invention.

[0034] Figure 2 The figure shows the change of the ultraviolet absorption peak at 343 nm of the nucleic acid-modified SOX enzyme in the present invention.

[0035] Figure 3 The figure shows the change of the ultraviolet absorption peak at 343 nm of the nucleic acid-modified HRP enzyme in the present invention.

[0036] Figure 4This is an atomic force microscopic image of the nucleic acid after linking SOX and HRP in the present invention.

[0037] Figure 5 It is the kinetic curve of free enzyme cascade reaction without nucleic acid mediation and enzyme cascade reaction mediated by nucleic acid.

[0038] Figure 6 It is a standard curve for detecting sarcosine by nucleic acid-mediated enzyme cascade reaction, and the concentrations of sarcosine solutions are 0μM, 1μM, 10μM, 20μM, 40μM, 60μM and 80μM respectively. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto. Without departing from the technical concept and technical solution of the present invention, any modification, adjustment or modification, or equivalent replacement method that can be implemented by a person of ordinary skill in the art to which the present invention belongs will fall within the scope of the protection claimed in the present invention.

[0040] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0041] In one aspect, the present invention provides a method for detecting sarcosine based on a nucleic acid-mediated enzyme cascade reaction, comprising the following steps:

[0042] Step 1: Use nucleic acids to regulate the distance between sarcosine oxidase SOX and peroxidase HRP to limit the enzyme cascade reaction to a specific area at the nanoscale;

[0043] Step 2: Color reaction and determination of creatine content.

[0044] Specifically, the step 1 includes:

[0045] Add a cross-linking agent to a SOX enzyme solution, incubate, and determine the SOX concentration; add a first nucleic acid, incubate so that the first nucleic acid is connected to the SOX enzyme molecule, and obtain a SOX enzyme molecule connected to the first nucleic acid; wherein each SOX enzyme molecule in the SOX enzyme connected to the first nucleic acid is connected to at least one first nucleic acid chain;

[0046] A cross-linking agent is added to the HRP enzyme solution, incubated, and the concentration of the HRP enzyme is determined; a second nucleic acid is added, incubated so that the second nucleic acid is connected to the HRP enzyme molecule, and an HRP enzyme molecule connected to the second nucleic acid is obtained; wherein each SOX enzyme molecule in the SOX enzyme connected to the second nucleic acid is connected to at least one second nucleic acid chain.

[0047] Furthermore, the step 1 further comprises: connecting the SOX enzyme connected to the first nucleic acid and the HRP enzyme connected to the second nucleic acid via a third nucleic acid, wherein the sequence of the third nucleic acid is different from the sequence of the first nucleic acid.

[0048] Further, the first nucleic acid and the second nucleic acid are deoxyribonucleic acids, and the sequence of the first nucleic acid is the same as or different from the sequence of the second nucleic acid. The first nucleic acid, the second nucleic acid and the third nucleic acid are oligonucleotides, and the sequence of the first nucleic acid is the same as or different from the sequence of the second nucleic acid.

[0049] Further, the molar ratio of the first nucleic acid to the SOX enzyme is at least 5:1, and the molar ratio of the second nucleic acid to the HRP enzyme is at least 5:1. Preferably, the molar ratio of the first nucleic acid to the SOX enzyme is at least 10:1.

[0050] Preferably, the first nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO.1, and the second nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO.2. Preferably, the third nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO.3.

[0051] SEQ ID NO.1(5'-3'):TTTTTTTTTTTTTTTTTTTTTT

[0052] SEQ ID NO.2(5'-3'):TTGGTGGTGGTGGTGGTGGT

[0053] SEQ ID NO.3(5'-3'):

[0054] ACCACCACCACCACCACCAAAAAAAAAAAAAAAAAAAAAAAA

[0055] In another aspect, the present invention provides a kit for detecting sarcosine, the kit comprising at least:

[0056] Sarcosine oxidase SOX solution and peroxidase HRP solution, the solution used for connecting and diluting sarcosine oxidase and peroxidase is DPBS;

[0057] A first nucleic acid for modifying the sarcosine oxidase SOX, and a second nucleic acid for modifying the peroxidase HRP;

[0058] A third nucleic acid, used for linking the modified sarcosine oxidase SOX and the modified peroxidase HRP;

[0059] A substrate for an enzyme cascade reaction; the substrate is a sarcosine solution, and the concentration of the sarcosine solution is 1-100 μM;

[0060] The molar ratio of the first nucleic acid to the SOX enzyme is at least 5:1, and the molar ratio of the second nucleic acid to the HRP enzyme is at least 5:1; the molar ratio of the third nucleic acid to the linked modified sarcosine oxidase SOX and the modified peroxide oxidase HRP is at least 1:1:1.

[0061] In one embodiment, (1) Preparation of sarcosine solutions of different concentrations: 100 mg of sarcosine is weighed to prepare a 1 mol / L stock solution, and gradient dilution is performed using DPBS (pH 8.0) buffer solution, with each step being a 10-fold dilution until the final concentration is 1 μM; (2) Preparation of TMB buffer solution: 400 μL TMB stock solution, 150 μL 10×PBS and 450 μL ultrapure water; (3) Preparation of reaction buffer: 100 μL reaction system contains: 40 μL TMB reaction buffer, 20 μL mixed free enzyme, and 40 μL sarcosine of different concentrations.

[0062] More specifically, the above detection method and kit can be a detection method and kit for measuring the prostate cancer-specific target sarcosine.

[0063] Example 1

[0064] (1) Experimental reagents:

[0065] The first nucleic acid and the second nucleic acid are used to modify the oligonucleotide chains of sarcosine oxidase SOX and peroxidase HRP, respectively: the first nucleic acid chain L1 (22 bp, ssDNA, 5'-terminal thiol modification) and the second nucleic acid chain L2' (20 bp, ssDNA, 5'-terminal thiol modification), which are purchased from Shanghai Shenggong Biotechnology Co., Ltd. Sarcosine oxidase SOX was purchased from Shanxi Ruiyali Company, and peroxidase HRP was purchased from Sigma. Sarcosine was purchased from Sigma. DPBS was purchased from Beijing Wokai Biotechnology Co., Ltd. Tetramethylbenzidine TMB was purchased from Thermo scientific, and the TMB does not contain hydrogen peroxide. Sarcosine was prepared into different concentrations using PB buffer solution.

[0066] Sequence of L1 (5'-3'):TTTTTTTTTTTTTTTTTTTTTT

[0067] L2 sequence (5'-3'):TTGGTGGTGGTGGTGGTGGT

[0068] (2) The first nucleic acid single-stranded DNA L1 modifies SOX:

[0069] Weigh 0.8 mg of SOX and dissolve it in 100 μL of DPBS. Quantify the SOX solution by UV absorption to 20 μM. Take 500 μL of SOX solution and put it into a 30 kDa centrifuge tube. Centrifuge at 8000 rcf, 10 min, 4°C. Centrifuge again under the same conditions and resuspend the precipitate with DPBS buffer to obtain SOX solution.

[0070] Add 0.06 mg / mL of 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester to the SOX solution, incubate for 1 hour, wash the SOX by centrifugation with DPBS, wash 4 times, the washing conditions are the same as above, resuspend the precipitate with DPBS, and obtain the SOX solution for use. Add 10 times the amount of the first nucleic acid L1 to the solution and incubate for 5 hours. After incubation, determine the connection ratio of L1 by ultraviolet spectrophotometer, and each SOX molecule connects 1-2 L1 chains. After connecting the DNA chain, the SOX is centrifuged and washed with HEPES buffer. After washing, set aside.

[0071] (3) Second nucleic acid single-stranded DNA L2 modified with HRP:

[0072] Weigh 0.8 mg of HRP and dissolve it in 100 μL of DPBS. Quantify the HRP solution by UV absorption to 20 μM. Take 500 μL of HRP solution and put it into a 30 kDa centrifuge tube. Centrifuge at 8000 rcf, 10 min, 4°C. Centrifuge again under the same conditions and resuspend the precipitate with DPBS buffer to obtain HRP solution.

[0073] Add 3 mg / mL of 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester molecules to the HRP solution and incubate for 4 hours. Wash the HRP by centrifugation with DPBS, wash 4 times, the washing conditions are the same as above, resuspend the precipitate with DPBS, and obtain the HRP solution for use. Add 5 times the amount of the second nucleic acid L2 chain to the solution and incubate for 10 hours. After incubation, determine the connection ratio of L2 by ultraviolet spectrophotometer, and each HRP molecule connects 1-2 L2 chains. After connecting the DNA chain, the HRP is centrifuged and washed with HEPES solution. After washing, it is set aside.

[0074] (4) Preparation of sarcosine solutions of different concentrations

[0075] 100 mg of sarcosine was weighed to prepare a 1 mol / L stock solution, and DPBS (pH 8.0) buffer solution was used to perform 10-fold dilutions at each step until the final concentration reached 1 μM.

[0076] (6) Preparation of TMB buffer solution

[0077] 400 μL TMB stock solution, 150 μL 10× PBS and 450 μL ultrapure water.

[0078] (7) Preparation of reaction buffer

[0079] The 100 μL reaction system contains: 40 μL TMB reaction buffer, 20 μL mixed free enzyme, and 40 μL sarcosine of different concentrations.

[0080] (8) Detection of creatine

[0081] Dilute sarcosine with DPBS buffer solution to prepare 1-1000μM sarcosine solution. Turn on the microplate reader, preheat and set the parameters. Set up three parallel samples in each group, and the reaction system is 100μL. The 100μL reaction system contains 40μL TMB reaction buffer, 20μL mixed free enzyme, and 40μL of different concentrations of sarcosine. Detect the change in the absorbance of TMB at 370nm, and draw the kinetic curve and standard curve of sarcosine concentration.

[0082] Example 2

[0083] (1) Experimental reagents:

[0084] Except for the following operating steps, other experimental steps are basically the same as those in Example 1.

[0085] Three oligonucleotide chains were used to connect SOX and HRP: L1 (22 bp, ssDNA, 5'-terminal thiol modification), L2 (20 bp, ssDNA, 5'-terminal thiol modification), L3 (41 bp, ssDNA), all of which were purchased from Shanghai Shenggong Biotechnology Co., Ltd. Sarcosine oxidase SOX was purchased from Shanxi Ruiyali Company, and peroxidase HRP was purchased from Sigma. Sarcosine was purchased from Sigma. DPBS was purchased from Beijing Wokai Biotechnology Co., Ltd. Tetramethylbenzidine TMB was purchased from Thermoscientific, and the TMB did not contain hydrogen peroxide. Sarcosine was prepared in different concentrations using PB buffer solution.

[0086] Sequence of L1 (5'-3'):TTTTTTTTTTTTTTTTTTTTTT

[0087] L2 sequence (5'-3'):TTGGTGGTGGTGGTGGTGGT

[0088] Sequence of L3 (5'-3'):ACCACCACCACCACCACCAAAAAAAAAAAAAAAAAAAAAAAA

[0089] (2) The first nucleic acid single-stranded DNA L1 modifies SOX:

[0090] Weigh 0.8 mg of SOX and dissolve it in 100 μL of DPBS. Quantify the SOX solution by UV absorption to 20 μM. Take 500 μL of SOX solution and put it into a 30 kDa centrifuge tube. Centrifuge at 8000 rcf, 10 min, 4°C. Centrifuge again under the same conditions and resuspend the precipitate with DPBS buffer to obtain SOX solution.

[0091] Add 0.06 mg / mL of 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester to the SOX solution, incubate for 1 hour, wash the SOX by centrifugation with DPBS, wash 4 times, the washing conditions are the same as above, resuspend the precipitate with DPBS, and obtain the SOX solution for use. Add 10 times the amount of the first nucleic acid L1 to the solution and incubate for 5 hours. After incubation, determine the connection ratio of L1 by ultraviolet spectrophotometer, and each SOX molecule connects 1-2 L1 chains. After connecting the DNA chain, the SOX is centrifuged and washed with HEPES buffer. After washing, set aside.

[0092] (3) Second nucleic acid single-stranded DNA L2 modified with HRP:

[0093] Weigh 0.8 mg of HRP and dissolve it in 100 μL of DPBS. Quantify the HRP solution by UV absorption to 20 μM. Take 500 μL of HRP solution and put it into a 30 kDa centrifuge tube. Centrifuge at 8000 rcf, 10 min, 4°C. Centrifuge again under the same conditions and resuspend the precipitate with DPBS buffer to obtain HRP solution.

[0094] Add 3 mg / mL of 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester molecules to the HRP solution and incubate for 4 hours. Wash the HRP by centrifugation with DPBS, wash 4 times, the washing conditions are the same as above, resuspend the precipitate with DPBS, and obtain the HRP solution for use. Add 5 times the amount of the second nucleic acid L2 chain to the solution and incubate for 10 hours. After incubation, determine the connection ratio of L2 by ultraviolet spectrophotometer, and each HRP molecule connects 1-2 L2 chains. After connecting the DNA chain, the HRP is centrifuged and washed with HEPES solution. After washing, it is set aside.

[0095] (4) Third nucleic acid single-stranded DNA L3 mediated SOX and HRP connection:

[0096] The third nucleic acid single-strand L3 was added to the SOX and HRP enzyme solution in a volume ratio of 1:1:1, incubated for 3 hours at a temperature of 37°C, and then placed at 4°C for 30 minutes.

[0097] (5) Preparation of sarcosine solutions of different concentrations

[0098] 100 mg of sarcosine was weighed to prepare a 1 mol / L stock solution, and DPBS (pH 8.0) buffer solution was used to perform 10-fold dilutions at each step until the final concentration reached 1 μM.

[0099] (6) Preparation of TMB buffer solution

[0100] 400 μL TMB stock solution, 150 μL 10× PBS and 450 μL ultrapure water.

[0101] (7) Preparation of reaction buffer

[0102] The 100 μL reaction system contains: 40 μL TMB reaction buffer, 20 μL mixed free enzyme, and 40 μL sarcosine of different concentrations.

[0103] (8) Detection of creatine

[0104] Dilute sarcosine with DPBS buffer solution to prepare 1-1000μM sarcosine solution. Turn on the microplate reader, preheat and set the parameters. Set up three parallel samples in each group, and the reaction system is 100μL. The 100μL reaction system contains 40μL TMB reaction buffer, 20μL mixed free enzyme, and 40μL of different concentrations of sarcosine. Detect the change in the absorbance of TMB at 370nm, and draw the kinetic curve and standard curve of sarcosine concentration.

[0105] The test results are shown in the attached figure.

[0106] Figure 1 The schematic diagram is a schematic diagram of the principle of the enzyme cascade reaction mediated by nucleic acid according to the present invention. The schematic diagram shows that after DNA connection, the distance between SOX and HRP is shortened. This clearly demonstrates the principle of double-stranded mediated enzyme cascade.

[0107] Figure 2 The change of the ultraviolet absorption peak of the nucleic acid-modified SOX enzyme at 343nm in the present invention. The single-chain L1 replaces the aromatic heterocyclic ring on the SPDP molecule on the SOX molecule, and the directional heterocyclic ring after the substitution has characteristic absorption at 350nm. Figure 2 This indicates that the single-stranded DNA L1 was successfully modified onto the SOX molecule.

[0108] Figure 3 The figure shows the change of the ultraviolet absorption peak of the nucleic acid-modified HRP enzyme at 343 nm. The single-chain L2' replaces the aromatic heterocycle on the SPDP molecule on the HRP molecule, and the substituted directional heterocycle has characteristic absorption at 350 nm. Figure 3 This indicates that the DNA single-strand L2' was successfully modified onto the HRP molecule.

[0109] Figure 4The atomic force microscopy image of the nucleic acid after linking SOX and HRP in the present invention shows that DNA shortens the enzyme distance between the two enzymes.

[0110] Figure 5 It is the kinetic curve of free enzyme cascade reaction without nucleic acid mediation and enzyme cascade reaction mediated by nucleic acid. The kinetic curve shows that under the same concentration conditions, the enzyme cascade reaction mediated by nucleic acid quickly reaches equilibrium.

[0111] Figure 6 This is a standard curve for detecting sarcosine by nucleic acid-mediated enzyme cascade reaction, with sarcosine solutions at concentrations of 0μM, 1μM, 10μM, 20μM, 40μM, 60μM and 80μM. The correlation coefficient between the data points constituting the standard curve is 0.99, which is conducive to obtaining more accurate detection results.

[0112] The above are only specific embodiments of the present invention, but the present invention is not limited thereto. All variations that can be directly derived or associated with the contents disclosed by a person skilled in the art should be considered to be included in the protection scope of the present invention. <110> Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine <120> A method for detecting sarcosine based on nucleic acid-mediated enzyme cascade <160> 2 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <400> ttttttttttt ttttttttttt 22 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> ttggtggtgg tggtggtggt 20 <210> 3 <211> 42 <212> DNA <213> Artificial sequence <400> accaccacca ccaccaccaa aaaaaaaaaa aaaaaaaaaa aa 42

Claims

1. A method for detecting sarcosine based on a nucleic acid-mediated enzyme cascade reaction, characterized in that: The method comprises the following steps: Step 1: regulating the distance between sarcosine oxidase SOX and peroxidase HRP through nucleic acid to limit the enzyme cascade reaction to a specific area at the nanoscale; Step 2: color reaction and determination of sarcosine content; The step 1 comprises: Adding a cross-linking agent to a SOX enzyme solution, incubating, and determining the SOX concentration; adding a first nucleic acid, incubating so that the first nucleic acid is linked to a SOX enzyme molecule, and obtaining a SOX enzyme molecule linked to the first nucleic acid; wherein each SOX enzyme molecule in the SOX enzyme linked to the first nucleic acid is linked to at least one first nucleic acid chain; and Adding a crosslinking agent to an HRP enzyme solution, incubating, and determining the concentration of the HRP enzyme; adding a second nucleic acid, incubating so that the second nucleic acid is connected to the HRP enzyme molecule, and obtaining an HRP enzyme molecule connected to the second nucleic acid; wherein each SOX enzyme molecule in the SOX enzyme connected to the second nucleic acid is connected to at least one second nucleic acid chain; Connecting the SOX enzyme connected to the first nucleic acid and the HRP enzyme connected to the second nucleic acid through a third nucleic acid; The detection method is not directly used for diagnosis or treatment of diseases.

2. The detection method according to claim 1, characterized in that: The first nucleic acid and the second nucleic acid are deoxyribonucleic acids, and the sequence of the first nucleic acid is the same as or different from the sequence of the second nucleic acid.

3. The detection method according to claim 1, characterized in that: The first nucleic acid, the second nucleic acid and the third nucleic acid are oligonucleotides, and the sequence of the first nucleic acid is the same as or different from the sequence of the second nucleic acid.

4. The detection method according to claim 1, characterized in that: The molar ratio of the first nucleic acid to the SOX enzyme is at least 5:1, and the molar ratio of the second nucleic acid to the HRP enzyme is at least 5:

1.

5. The detection method according to claim 1, characterized in that: The sequence of the third nucleic acid is different from the sequence of the first nucleic acid.

6. The detection method according to any one of claims 1 to 4, characterized in that: The sequence of the first nucleic acid is the nucleotide sequence shown in SEQ ID NO.1, and the sequence of the second nucleic acid is the nucleotide sequence shown in SEQ ID NO.

2.

7. The detection method according to claim 6, characterized in that: The sequence of the third nucleic acid is the nucleotide sequence shown in SEQ ID NO.

3.

8. A kit for detecting sarcosine, characterized in that: The kit at least comprises: Sarcosine oxidase SOX solution and peroxidase HRP solution, the solution used for connecting and diluting sarcosine oxidase and peroxidase is DPBS; A first nucleic acid for modifying the sarcosine oxidase SOX, and a second nucleic acid for modifying the peroxidase HRP; A third nucleic acid, used for linking the modified sarcosine oxidase SOX and the modified peroxidase HRP; A substrate for an enzyme cascade reaction; the substrate is a sarcosine solution, and the concentration of the substrate solution is 1-100 uM; Among them, the molar ratio of the first nucleic acid to the SOX enzyme is at least 5:1, and the molar ratio of the second nucleic acid to the HRP enzyme is at least 5:1; the molar ratio of the third nucleic acid to the modified sarcosine oxidase SOX and the modified peroxide oxidase HRP is 1:1:1.

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