Method for detecting uric acid

Abstract

The invention discloses a method for detecting uric acid, according to the UA detection method, detection signals a1 and a2 can be obtained through two sensors, the sensor a1 is composed of an aptamer, complementary DNA of the aptamer, Exo III, a DNA walker and gold nanoparticles marked with PolyA-DNA-6-carboxyl fluorescein, and the sensor a2 is composed of gold nanoparticles marked with PolyA-DNA-6-carboxyl fluorescein; and the sensor a2 is composed of Apt and AuNPs modified by cDNA-DNAzyme and PolyA-DNA-FAM, and the AuNPs modified by the cDNA-DNAzyme and the PolyA-DNA-FAM are used for detecting the Apt. When UA exists in a sample, Apt is combined with UA, Exo III in the sensor a1 or DNAzyme in the sensor a2 is activated, PolyA-DNA-FAM is cut, FAM molecules are released into a solution, and a fluorescence signal is generated. On the basis, an Apt sensing array technology and linear discriminant analysis are combined, a UA signal is extracted from a mixed signal of UA and xanthine, the purpose of quantifying UA is achieved, and the method has high anti-interference capacity; the method is simple, has no radioactivity, does not need expensive instruments or special reagents, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to a method for uric acid detection. Background Art

[0002] Hyperuricemia is a known precursor of gout, hyperuricemic nephropathy, and cardiovascular diseases. The current main drugs for reducing uric acid (UA) levels are associated with long-term liver and kidney toxicity. Therefore, it is of great significance to develop new low-toxicity and effective UA-lowering drugs. Since more than 90% of hyperuricemia cases are due to impaired UA excretion, the main target of new UA-lowering drugs is the UA transporter, and screening inhibitors through in vitro cells is a rapid and economical method suitable for early-stage UA-lowering drug discovery. However, there are currently two main challenges in accurately quantifying intracellular UA: (1) extremely low UA concentration, about 0.5 amol (about 0.59 μM) per renal tubular cell. In 96-well plate experiments, the UA concentration in the lysis solution is about 27 pM, and a detection method with pM sensitivity is required. (2) Strong interference from complex intracellular components, such as high levels of hydrogen peroxide (H 2 O 2 ), structural analogs such as xanthine (Xan), and abundant proteins. These complexities have led to a scarcity of intracellular UA detection methods.

[0003] Currently, the key method for detecting intracellular UA is the radioisotope method, which requires corresponding safety protection measures and conditions and is costly, so it cannot be used in conventional laboratories. Although multiple UA detection techniques have been reported, including phosphotungstic acid method, capillary electrophoresis, high-performance liquid chromatography (HPLC), HPLC-tandem mass spectrometry, potentiometry, spectrophotometry, colorimetry, fluorescence method, and electrochemical sensors, etc., the detection limits of these methods are in the nanomolar (nM) to micromolar (μM) range and do not reach the pM sensitivity required for drug screening in 96- or 384-well plates. Chromatography and HPLC-MS are accurate but require expensive instruments, and due to the high protein content in cell lysates, sample preparation is complex, and the detection limits are in the nM to μM range, making them unsuitable for high-throughput screening of UA-lowering drugs. In addition, due to the high endogenous H 2 O 2 levels in cells, UA detection methods mainly relying on uricase to oxidize UA to H 2 O 2 combined with colorimetric, chemiluminescent, or electrochemical techniques are usually insufficient to detect intracellular UA. Therefore, it is necessary to develop UA detection methods based on new mechanisms.

[0004] Currently, the main method for detecting UA in cells is the radioactive isotope method. This method requires corresponding safety protection measures and conditions, and is costly, so it cannot be used in conventional laboratories. Although multiple UA detection techniques have been reported, including phosphotungstic acid method, capillary electrophoresis, high-performance liquid chromatography (HPLC), HPLC-tandem mass spectrometry, potentiometry, chemiluminescence method, colorimetry, fluorescence method, and electrochemical sensors, etc., the detection limits of these methods are in the nM to μM range, and cannot meet the picomolar (pM) sensitivity required for detecting UA in cell lysates during the drug screening process in 96-well or 384-well plates. Chromatography and HPLC-MS, although accurate, are not suitable for high-throughput screening of in vitro UA-lowering drugs based on cells due to the high protein content in cell lysates, complex sample preparation, and the need for expensive instruments. In addition, due to the relatively high intracellular H 2 O 2 level, UA detection methods mainly relying on uricase to oxidize UA to generate H 2 O 2 cannot be applied to the detection of UA in cells. Therefore, it is necessary to develop a detection method based on a new mechanism. Summary of the Invention

[0005] The object of the present invention is to provide a UA detection method, which can be used for the accurate quantification of intracellular UA and is suitable for the screening of UA-lowering drugs. The new method established in this study has high sensitivity and can detect UA at a level as low as picomolar; by adopting the aptamer (Apt) sensing array technology and combining linear discriminant analysis (LDA), it can effectively quantify the UA concentration from the mixed signals of UA and Xan, and has strong anti-interference ability; it is simple, non-radioactive, does not require expensive instruments or special reagents, and has the advantages of high sensitivity, high specificity, low cost, and wide applicability compared with the existing technology.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] A method for UA detection. Based on the Apt of UA, two sensors a1 and a2 were respectively constructed for obtaining UA signals, and were used in combination with LDA to achieve accurate quantification of UA. Sensor a1: In the Exo III-driven DNA walker system, the DNA walking track was constructed by modifying PolyA-DNA-FAM labeled with 6-carboxyfluorescein onto the surface of gold nanoparticles. When UA exists in the sample, Apt binds to UA and releases the complementary DNA (cDNA) of Apt into the solution. Sensor a2: cDNA hybridizes with PolyA-DNA-FAM on the surface of AuNPs, activates Exo III to cleave PolyA-DNA-FAM in the double strand, releases cDNA, and moves along the walking track unit through the burnt-bridge mechanism, releasing FAM molecules from the surface of AuNPs into the solution.

[0008] Preferably, the length of PolyA in sensor a1 is 10 to 40 nucleotides, the molar ratio of PolyA-DNA to AuNPs is 50:1 to 250:1, and the concentration of FAM-Sub@AuNPs is 0.025 to 0.5 nM.

[0009] Preferably, the Exo III enzymatic digestion time in sensor a1 is 20 to 60 minutes, and the enzyme concentration is 0.25 to 1.25 U / mL.

[0010] Preferably, sensor a2 is modified with FAM-Sub and DNAzyme locked by the complement of Apt on AuNPs to construct a signal probe for UA. When there is no UA in the sample, Apt locks DNAzyme to prevent it from cleaving FAM-Sub; when UA exists, Apt binds to UA, releases DNAzyme, and activates the DNA walker to cleave FAM-Sub to generate a fluorescence signal.

[0011] Preferably, the concentration of the UA probe in sensor a2 is 0.25 to 2 nM, and the molar ratio of DNAzyme to FAM-Sub is 6:01 to 1:2.

[0012] Preferably, the three Apt sequences of sensors a1 and a2 have different affinities and are respectively used for distinguishing UA and Xan signals.

[0013] Preferably, LDA is used. By setting different mixing ratios of UA and Xan and constructing a training set with different total concentrations, combined with the LDA two-dimensional score map, quantitative detection of UA in the mixture is carried out.

[0014] Preferably, the LDA two-dimensional score map is used to screen drugs for reducing UA.

[0015] The beneficial effects of the present invention are:

[0016] High sensitivity: The developed method realizes the detection of UA at the picomolar (pM) level through the combination of Apt and Exo III or DNAzyme-driven DNA walker.

[0017] High specificity: Based on LDA, accurate quantification of UA in mixed signals is achieved.

[0018] Low cost and wide applicability: This method is simple, non-radioactive, does not require expensive instruments or special reagents, and is applicable to a variety of occasions. Description of the Drawings

[0019] Figure 1 For the influence of the length of PolyA (a), the influence of the molar ratio of PolyA20 to AuNPs (b), and the influence of the concentration of FAM-Sub@AuNPs (c);

[0020] Figure 2 For the influence of ExoⅢ digestion time and concentration on fluorescence intensity (a) Influence of different Exo-III digestion times (b) Influence of different concentrations of Exo-III;

[0021] Figure 3 For the quantitative performance of the ASA-Exo III-DW-LDA method for UA;

[0022] Figure 4 For the quantitative performance of the ASA-DNAzyme-DW-LDA method for UA. Specific Implementation Method

[0024] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0026] Example 1

[0027] Synthesis of AuNPs and Modification of PolyA-DNA-FAM

[0028] Synthesis of AuNPs:

[0029] Synthesize 13nm gold nanoparticles (AuNPs) according to the sodium citrate reduction method. First, add 1.7 mL of 1% HAuCl4 The solution was added to a three-necked flask containing 48.3 mL of ultrapure water, and then stirred and heated in an oil bath until boiling. Subsequently, 5 mL of 38.8 mM sodium citrate solution was rapidly added to the boiling solution. When the color of the solution changed from yellow to dark red (about 15 minutes), heating was stopped, and stirring was continued while allowing it to cool naturally to room temperature to obtain the AuNPs solution.

[0030] Modification of AuNPs with PolyA-DNA-FAM:

[0031] The 100 nM PolyA-DNA-FAM solution was mixed with the prepared AuNPs solution, gently shaken, and frozen overnight. After thawing to room temperature after freezing, the mixture was centrifuged at 13,000 rpm for 10 minutes, the supernatant was discarded, and it was resuspended with 80 mM phosphate buffer (pH 5.4, 0.1 M NaCl) and stored for later use.

[0032] Example 2

[0033] UA detection experiment

[0034] Hybridization of Apt and cDNA:

[0035] 2 μL of 1 nM Apt was mixed with 2 μL of 1 nM cDNA, and 175 μL of Tris-HCl buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 800 mM NaCl, pH 7.9) was added, and incubated in a 96-well plate for 30 minutes to form a double-stranded Apt-cDNA complex.

[0036] Addition of sample and PolyA-DNA-FAM@AuNPs:

[0037] After adding the sample to be detected, it was mixed thoroughly to allow Apt to bind to UA and release cDNA. Then, 5 μL of PolyA-DNA-FAM@AuNPs solution and 1 μL of Exo III enzyme were added, and incubation was continued for 30 minutes to activate Exo III to cleave the PolyA-DNA-FAM strand and release FAM fluorescent molecules through the walking track.

[0038] Fluorescence signal measurement:

[0039] At an excitation wavelength of 470 nm, the fluorescence intensity at 520 nm was measured, and the fluorescence signal of the sample was recorded.

[0040] Example 3

[0041] Quantitative detection of UA

[0042] Training set design:

[0043] Set 7 different mixing ratios of UA and Xan, and set 6 different concentrations for each mixing ratio to construct the training set data. Use SPSS 27.0 software to perform linear discriminant analysis (LDA) on the fluorescence intensity data to generate the LDA two-dimensional score plot.

[0044] Calculation of the relationship between fluorescence signal and concentration:

[0045] According to the position of the sample on the LDA two-dimensional score plot generated from the training set, calculate the molar ratio and total concentration of UA in the mixture sample.

[0046] Example 4:

[0047] Detection of UA in the lysate of HEK293 cells with high expression of URAT1

[0048] UA uptake experiment of hURAT1-HEK293 cells:

[0049] Wash the cells once with 100 μL of UA uptake buffer, and add the UA uptake buffer containing Lesinurad or benzbromarone, and incubate at 37 °C. After aspirating the liquid in the well, add the UA uptake buffer for the UA uptake experiment. After 15 minutes, lyse the cells with 1 M NaOH and take the cell lysate. Then use the ASA-Exo III-DW-LDA method to detect the UA concentration in the lysate.

[0050] 14C-labeled UA uptake experiment:

[0051] Wash the cells with 100 μL of UA uptake buffer, and add the uptake buffer containing 50 mM 14C-labeled UA for the UA uptake experiment. After 15 minutes, add the scintillation fluid, and use a liquid scintillation counter to measure the radioactivity of 14C-labeled UA. Repeat the measurement three times and take the average value.

[0052] Example 5

[0053] Preparation of UA signal probe

[0054] Preparation of FAM-Sub and DNAzyme:

[0055] Mix FAM-Sub, DNAzyme and AuNPs, heat in a glass beaker to 95 °C and continue heating for 50 minutes. After heating, dry the mixture and redissolve it in 50 mM Tris-HCl buffer (pH 5.5). Add Apt to hybridize with the DNAzyme on the surface of AuNPs, and resuspend it in 20 mM Tris-HCl (pH 5.5) solution for storage.

[0056] Example 6

[0057] Detection of UA in HEK293 cell lysate with high expression of URAT1 by Apt-DNAzyme-DW method

[0058] Incubation of UA probe with sample:

[0059] Add the UA probe into Tris-HCl buffer, add the analyte, with a final volume of 200 μL, and let it react with the probe to activate the DNA walker. Detect the fluorescence signal with a microplate reader.

[0060] Example 7

[0061] Detection of in-situ UA in hGLUT9-HEK293 cells by ASA-DNAzyme-DW-LDA method

[0062] Incubation of UA probe:

[0063] Add the UA probe into a 96-well plate, and add UA uptake buffer for UA uptake of cells. After incubation for 10 minutes, aspirate the liquid in the wells, and add UA uptake buffer containing Isobavachin for continued incubation. After 15 minutes, add 1 M NaOH to lyse the cells, and detect the fluorescence signal with a microplate reader.

[0064] Experimental example

[0065] Application of ASA-Exo III-DW-LDA / ASA-DNAzyme-DW-LDA method in quantitative analysis of UA and drug screening

[0066] 1. Experimental purpose

[0067] Verify the accuracy of the ASA-Exo III-DW-LDA / ASA-DNAzyme-DW-LDA method in the mixture of UA and Xan, and the potential of this method in screening inhibitors of URAT1 and GLUT9 transporters.

[0068] 2. Experimental materials and reagents

[0069] Cell lines: hURAT1-HEK293, hGLUT9-HEK293

[0070] Drugs: Lesinurad, Benzbromarone, Isobavachi

[0071] Chemical reagents:

[0072] 1 M NaOH, cell culture medium, UA uptake buffer

[0073] 14 C radioactive labeling reagent

[0074] Various RNA sequences (Apt1, cDNA1, PloyA-DNA, etc., see Tables 1 and 2)

[0075] Table 1 Sequences used in the experiments of Method 1

[0076]

[0077]

[0078] Table 2 Sequences used in the experiments of Method 2

[0079]

[0080] Equipment:

[0081] PCR instrument, flow cytometer, fluorescence spectrophotometer, patch clamp, 14 C liquid scintillation counter

[0082] 3. Experimental procedures

[0083] 3.1 Sample preparation for the ASA-Exo III-DW-LDA / ASA-DNAzyme-DW-LDA method

[0084] Preparation of mixed samples: Prepare samples containing mixtures of UA and Xan at 7 different concentrations, with 7 mixing ratios set for each concentration (such as 15:0, 14:1, 11:4, 7.5:7.5, 4:11, 1:14, 0:15).

[0085] Establishment of standard curves: Use SPSS 27.0 software for linear discriminant analysis (LDA) to generate an LDA two-dimensional score plot. According to the linear relationship between the value of ΔFactor 2 / ΔFactor 1 (ΔFactor 2 = Factor 2 sample -Factor2 blank , ΔFactor 1 = Factor 1 sample -Factor 1 blank ) and the percentage of UA, plot the standard curve. Group all samples with the same total concentration in the training samples into the same category, and then perform LDA analysis. According to the linear relationship between ΔFactor 1 (ΔFactor 1 = Factor 1 sample -Factor 1 blank ) and the natural logarithm of the total concentration of the mixture, plot the standard curve.

[0086] 3.2 Drug action experiment

[0087] Drug treatment:

[0088] Incubate hURAT1-HEK293 cells with Lesinurad and Benzbromarone at different concentration gradients.

[0089] Set different concentration gradients of Isobavachi to incubate hGLUT9-HEK293 cells.

[0090] UA uptake experiment:

[0091] Cell culture: Cultivate the cells in a medium containing different concentrations of drugs for 24 hours. After cell treatment, use the ASA-Exo III-DW-LDA / ASA-DNAzyme-DW-LDA method to quantitatively analyze the UA concentration in the cells.

[0092] 3.3 Calculation of drug inhibitory effect

[0093] Use the following formula to calculate the inhibitory effect of the drug on the URAT1 or GLUT9 transporter:

[0094] Uptake of control(%)=((UA drug -UA control ) / (UA model –UA control ))×100%

[0095] Where UA_drug is the UA concentration in the drug treatment group, UA_model is the UA concentration in the model group, and UA_control is the UA concentration in the control group.

[0096] 3.4 Comparative experiment:

[0097] Use 14 C radioactive labeling / patch clamp method: Compare the IC of Lesinurad and Benzbromarone measured by the ASA-Exo III-DW-LDA / ASA-DNAzyme-DW-LDA method with the traditional 14 C radioactive labeling / patch clamp method. 50 value determination.

[0098] Pearson correlation analysis: Use the Pearson correlation analysis method to compare the correlation of UA concentrations obtained by the new method and the traditional method.

[0099] 4. Experimental data analysis

[0100] 4.1 Heat map analysis of UA recovery rate

[0101] According to the experimental results, Figure 3a shows the UA recovery rates in samples with 9 different concentrations and mixing ratios. The recovery rates range from 82.9% to 120.0%, indicating the high accuracy of the ASA-Exo III-DW-LDA method in complex samples.

[0102] According to the experimental results, Figure 3 a and 4a show the UA recovery rates in samples with 9 different concentrations and mixing ratios. The recovery rates range from 85.0% to 118.8%, indicating the high accuracy of the ASA-Exo III-DW-LDA and ASA-DNAzyme-DW-LDA methods in detecting UA in complex samples.

[0103] 4.2 Analysis of drug inhibitory effects

[0104] Figure 3 b shows the results of detecting the inhibitory effects of Lesinurad and Benzbromarone on the URAT1 transporter based on the ASA-Exo III-DW-LD method. As the drug concentration increases, the absorption rate of UA by cells gradually decreases; the IC 50 values of Lesinurad and Benzbromarone are 9.71 μM and 0.81 μM, respectively. Figure 3 c shows 14 the results of parallel experiments by the 14C radiolabeling method. The IC 50 values of Lesinurad and Benzbromarone are measured to be 7.52 μM and 1.18 μM, respectively, indicating good consistency between the results of the ASA-Exo III-DW-LDA method and the 14C radiolabeling method.

[0105] Figure 4 b shows the results of detecting the inhibitory effect of Isobavachin on the GLUT9 transporter based on ASA-DNAzyme-DW-LDA. As the drug concentration increases, the inhibitory effect on the absorption of UA by cells is enhanced; the IC 50 value of Isobavachin is 1.57 μM. Figure 4 c shows the results of parallel experiments by the patch clamp method. The IC 50 value of Isobavachin is measured to be 1.92 μM, indicating good consistency between the results of the ASA-DNAzyme-DW-LDA method and the patch clamp technique.

[0106] 4.3 Correlation analysis

[0107] Figure 3d and 3e show the results of Pearson bivariate correlation analysis between the ASA-Exo III-DW-LDA method and the UA concentrations measured by the 14C radiolabeling method. The Pearson correlation coefficients of Lesinurad and Benzbromarone are 0.9880 and 0.9777 respectively, indicating a very high consistency between the two methods.

[0108] Figure 4 d shows the results of Pearson bivariate correlation analysis between the ASA-DNAzyme-DW-LDA method and the UA concentrations measured by the patch clamp method. The Pearson correlation coefficient of Isobavachi is 0.8817, indicating a good consistency between the two methods.

[0109] 5. Summary of Results

[0110] This experiment verified the accuracy and reliability of the ASA-Exo III-DW-LDA and ASA-DNAzyme-DW-LDA methods in the quantitative analysis of UA, especially in the application of complex samples. Through the screening of URAT1 and GLUT9 transporter inhibitors, it was proved that this method has good drug screening potential and is highly correlated with the results of traditional 14 C radiolabeling method and patch clamp technique. Therefore, the ASA-Exo III-DW-LDA and ASA-DNAzyme-DW-LDA methods provide a sensitive, specific, economical and widely applicable new technology platform for UA detection and screening of UA-lowering drugs.

[0111] 6. Beneficial Effects of Experimental Results

[0112] This experiment provides a sensitive, specific, simple and economical analytical method for the quantitative detection of UA, and solves the technical bottleneck problem of traditional methods in drug screening, especially in the screening of UA-lowering drugs.

[0113] The above embodiments of the present invention do not limit the protection scope of the present invention. The implementation manners of the present invention are not limited thereto. All kinds of modifications, substitutions or changes made to the above structure of the present invention according to the above content of the present invention, in accordance with the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, shall fall within the protection scope of the present invention.

Claims

1. A method for detecting uric acid, characterized in that: Based on the aptamer of uric acid, two sensors a1 and a2 were constructed for capturing uric acid signals, and combined with linear discriminant analysis to achieve accurate quantification of uric acid. Sensor a1: In the exonuclease III-driven DNA walker system, the DNA walking track is composed of PolyA-DNA-FAM labeled with 6-carboxyfluorescein modified on the surface of gold nanoparticles. When uric acid exists in the sample, the aptamer binds to uric acid and releases the complementary DNA of the aptamer into the solution, that is, cDNA into the solution; Sensor a2: cDNA hybridizes with PolyA-DNA-FAM on the surface of gold nanoparticles, activates exonuclease III to cut PolyA-DNA-FAM in the double-stranded chain, releases cDNA, and moves along the walking track unit through the burnt-bridge mechanism, releasing FAM molecules from the gold nanoparticle surface into the solution.

2. The method for detecting uric acid according to claim 1, characterized in that: In sensor a1, the length of PolyA is 10 to 40 nucleotides, and the nucleotide sequences of PolyA-DNA, aptamer and cDNA are shown in Table 1; the molar ratio of PolyA-DNA to gold nanoparticles is 50:1 to 250:1, and the concentration of FAM-Sub@AuNPs is 0.025 to 0.5 nM.

3. The method for detecting uric acid according to claim 1, characterized in that: In sensor a1, the exonuclease cleavage time is 20 to 60 minutes, and the enzyme concentration is 0.25 to 1.25 U / mL.

4. The method for detecting uric acid according to claim 1, characterized in that: The three Apt sequences have different affinities, and an Apt array sensor is constructed to distinguish between uric acid and xanthine signals.

5. The method for detecting uric acid according to claim 1, characterized in that: Using linear discriminant analysis, by setting different mixing ratios of uric acid and xanthine and constructing training sets with different total concentrations, combined with linear discriminant analysis two-dimensional score graph, quantitative detection of uric acid in the mixture was performed; The linear discriminant analysis two-dimensional score plot was used to screen uric acid-lowering drugs that inhibit URAT1.

6. The method for detecting uric acid according to claim 1, characterized in that: In sensor a2, the nucleotide sequences of the aptamer, the FAM-Sub modified on the gold nanoparticles, and the DNAzyme complementary locked by the aptamer are shown in Table 2.

7. The method for detecting uric acid according to claim 6, characterized in that: In sensor a2, the concentration of the uric acid probe is 0.25 to 2 nM, and the molar ratio of DNAzyme to FAM-Sub is 6:1 to 1:

2.

8. The method for detecting uric acid according to claim 6, characterized in that: The three uric acid aptamer sequences have different affinities for binding to uric acid, and an aptamer array sensor is constructed to distinguish between uric acid and xanthine signals.

9. The method for detecting uric acid according to claim 6, characterized in that: Using line discriminant analysis, by setting different mixing ratios of uric acid and xanthine and constructing training sets with different total concentrations, combined with the two-dimensional score graph of line discriminant analysis, quantitative detection of uric acid in the mixture was performed.

10. The method for detecting uric acid according to claim 9, characterized in that: Line discriminant analysis two-dimensional score plot is used to screen uric acid-lowering drugs.

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