Sox-hrp double enzyme conjugate, preparation method and application thereof

Abstract

The application belongs to the technical field of biochemical analysis, and more particularly relates to a SOX-HRP double-enzyme conjugate, a preparation method and application thereof. The SOX-HRP double-enzyme conjugate provided by the application can significantly shorten the substrate diffusion distance between enzymes, directly transfer reaction intermediates from the active center of the oxidase to the active center of the peroxidase in the nanometer scale, effectively reduce side reactions in the diffusion process, and thus realize strong anti-interference optical detection.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis technology, and more specifically, relates to SOX-HRP dual-enzyme conjugates, their preparation methods, and applications. Background Technology

[0002] The concentration levels of small molecule disease biomarkers such as creatine, uric acid, ascorbic acid, and glucose in biological samples such as blood, urine, saliva, and sweat are closely related to various metabolic diseases, kidney diseases, diabetes, and oxidative stress. Rapid, sensitive, and accurate quantitative analysis of these small molecules has significant clinical importance.

[0003] Currently, the commonly used detection method is based on a cascade reaction system of oxidase + peroxidase. For example, sarcosine oxidase (SOX) catalyzes the oxidation of sarcosine to produce hydrogen peroxide (H2O2), and then horseradish peroxidase (HRP) uses the generated H2O2 to oxidize chromogenic reagents (such as TMB, Amplex Red), and completes the quantitative analysis of sarcosine based on absorbance or fluorescence intensity.

[0004] However, reducing interfering substances are widely present in biological samples, including ascorbic acid, glutathione, uric acid, and dopamine. These substances react with and are consumed by H2O2 in the system, compete with HRP for substrates, and inhibit the normal amplification reaction of the colorimetric system, thus causing a significant negative bias in the detection results. Especially at low target concentrations, due to the limited amount of H2O2 generated, and the long diffusion distance and exposure time of the H2O2 generated by the oxidase in existing enzyme-catalyzed detection reagents, it is easily consumed by reducing substances such as ascorbic acid, making it susceptible to interference and resulting in reduced sensitivity and large detection errors in the low concentration range. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides SOX-HRP dual-enzyme conjugates, their preparation methods, and applications.

[0006] The present invention specifically adopts the following technical solution: The present invention provides a SOX-HRP dual-enzyme conjugate, which is obtained by covalently coupling a thiol-modified sarcosine oxidase and a maleimide-modified horseradish peroxidase.

[0007] This invention addresses the problems of low sensitivity and poor anti-interference ability in existing biodetection methods based on oxidase and peroxidase cascade reactions, where the intermediate product hydrogen peroxide (H2O2) has a long diffusion distance and is easily consumed by reducing substances in the sample (such as ascorbic acid). The invention creatively constructs an SOX-HRP dual-enzyme conjugate by chemically modifying and covalently coupling sarcosine oxidase (SOX) and horseradish peroxidase (HRP). This structure allows H2O2 generated by SOX catalysis to be directly transferred to the active site of adjacent HRP at the nanoscale, greatly shortening the substrate diffusion path and exposure time, thereby significantly reducing the consumption of interfering substances and achieving highly sensitive and highly interference-resistant optical detection of small molecule markers such as sarcosine.

[0008] The present invention also provides a method for preparing the SOX-HRP dual-enzyme conjugate, the method being as follows: a thiol-modified sarcosine oxidase solution, a maleimide-modified horseradish peroxidase solution, and a PBS-EDTA buffer are mixed and stirred at room temperature in the dark for 10-15 hours to covalently couple SOX and HRP. After covalent coupling, the mixture is washed twice by ultrafiltration and centrifugation with PBS-EDTA buffer. The volume ratio of the SOX solution, HRP solution, and PBS-EDTA buffer is 0.5-1.5:0.5-1.5:0.5-1.5.

[0009] Furthermore, the ultrafiltration molecular weight is 40~60 kDa.

[0010] The present invention also provides the application of the SOX-HRP dual-enzyme conjugate in the preparation of detection reagents or kits for detecting small molecule markers in biological samples.

[0011] Furthermore, the small molecule markers include sarcosine.

[0012] Furthermore, the biological sample includes at least one of blood, urine, saliva, sweat, cell culture medium, or tissue homogenate.

[0013] Furthermore, the detection reagent or kit is used to perform optical detection, including fluorescence detection or colorimetric detection.

[0014] Furthermore, the detection reagents or kits are used in point-of-care testing devices, microfluidic chips, or wearable biosensors.

[0015] The present invention has the following beneficial effects: It exhibits substrate localization effect: the intermediate product between SOX and HRP can be utilized immediately without diffusion, and the H2O2 exposure is extremely short and almost unaffected by interfering agents.

[0016] Significantly enhanced anti-interference ability: Compared with the traditional cascade enzyme method, it can resist interference from dozens of times the concentration of ascorbic acid, glutathione, uric acid, etc.

[0017] Significantly improved sensitivity at low concentrations: Due to minimal loss of H2O2, stable signals can still be obtained even with low concentrations of small molecule substrates.

[0018] No sample pretreatment required, simple to operate: suitable for rapid POCT, clinical testing or wearable biosensors. Attached Figure Description

[0019] Figure 1 This is a standard curve of sarcosine concentration versus fluorescence intensity.

[0020] Figure 2 The graph shows the results of the anti-interference performance test. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0022] Example 1: Preparation of a two-enzyme covalent conjugate.

[0023] Prepare a 1 mg / mL SOX solution, a 5 mg / mL HRP solution, a 10 mM 2-iminothione solution, a 10 mM sulfosuccinimide 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (Sulfo-SMCC) solution, and a 0.1 M phosphate-EDTA buffer solution at pH 8.0.

[0024] 1 mL of SOX solution was placed in a centrifuge tube and stirred at low speed. During stirring, 100 times the molar amount of 2-iminothion was added for modification. The mixture was stirred continuously at room temperature in the dark for 2 h to modify the SOX with thiol groups.

[0025] Place 1 mL of HRP solution in a centrifuge tube and stir at low speed. Add 100 times the molar amount of Sulfo-SMCC for modification while stirring. Continue stirring at room temperature in the dark for 1 h to modify HRP with maleimide groups.

[0026] After modification, both were washed three times by ultrafiltration and centrifugation with PBS-EDTA buffer (pH=7.2). The enzyme solution was poured into Millipore ultrafiltration tubes and centrifuged at 4000 rpm for 10 minutes. The supernatant (i.e., the modified SOX and HRP) was collected to remove excess Sulfo-SMCC and 2-iminothione; (Millipore ultrafiltration tube, inner tube filter column is 15 mL, outer tube is 50 mL, 30 kDa).

[0027] Take 1 mL of each centrifuged solution and mix them. Then add 1 mL of PBS-EDTA buffer (pH=7.2) and stir continuously at room temperature in the dark for 12 h. After covalent coupling, wash twice with PBS-EDTA buffer (pH=7.2) by ultrafiltration and centrifugation (ultrafiltration procedure: pour the mother solution into a 50 kDa Millipore ultrafiltration tube, set the centrifugation force to 4000 rpm, centrifuge for 10 minutes, repeat twice; the free enzyme with smaller molecular weight enters the filtrate, while the conjugate with larger molecular weight is retained on the ultrafiltration membrane) to remove unreacted free enzymes and obtain the SOX-HRP dual enzyme conjugate (Millipore ultrafiltration tube specifications: inner tube filter column is 15 mL, outer tube is 50 mL, 50 kDa).

[0028] Example 2: Detection of sarcosine based on coupling enzyme.

[0029] 0.1M PBS buffer (pH=7.2).

[0030] SOX-HRP dual enzyme conjugate (diluted with 0.1M PBS buffer to a concentration of 0.1U / mL).

[0031] Amplex Red (prepared to 50 μM using anhydrous dimethyl sulfoxide).

[0032] The creatine sample to be tested was prepared into a series of standard solutions (0µM, 2µM, 5µM, 10µM, 40µM, 80µM, 100µM) using PBS buffer.

[0033] Take 100 μL of the above solution and mix them thoroughly in a cuvette. Incubate at 37 °C for 20 min, and then detect the fluorescence intensity using a fluorescence spectrophotometer (Ex / Em = 530 / 590 nm). The creatine concentration data can be obtained from the fluorescence intensity value.

[0034] like Figure 1 As shown, a standard curve of creatine concentration (x) versus fluorescence intensity (y) was plotted. The linear equation of the fitted curve is as follows: y = 276.1x - 326.7 , R 2 =0.9980The linear relationship is good.

[0035] Detection of sarcosine in actual serum: Sarcosine standard samples were added to serum, and the fluorescence intensity value was obtained by fluorescence detection. The value was then substituted into the linear equation of the curve for calculation. The recovery rate and RSD (relative standard deviation) of sarcosine were obtained and are shown in Table 1. The results show that this method can be used for the detection of sarcosine in actual samples.

[0036] Table 1: Detection of creatine in actual samples Example 3: Anti-interference performance test.

[0037] Add 200 μM ascorbic acid, 200 μM glutathione, and 200 μM uric acid to the experimental system (0.1 U / mL SOX-HRP dual-enzyme conjugate + 1 mM Amplex Red + 200 μM sarcosine + 0.1 M PBS buffer) and the control system (conventional mixed cascade enzyme or co-immobilized cascade enzyme + 1 mM Amplex Red + 200 μM sarcosine + 0.1 M PBS buffer).

[0038] Traditional mixed cascade enzymes refer to free dual-enzyme solutions of 0.1 U / mL SOX and 0.1 U / mL HRP, while co-immobilized cascade enzymes refer to 0.1 U / mL MOF-immobilized SOX-HRP dual-enzyme solutions. The MOF-immobilized dual enzymes were purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.

[0039] Mix the above solution in a cuvette and incubate at 37°C for 60 minutes. Detect the fluorescence intensity value (Ex / Em=530 / 590nm) every 10 minutes using a fluorescence spectrophotometer. The results can be obtained by comparing the changes in fluorescence intensity.

[0040] The results are as follows Figure 2 The results showed that the signal of traditional mixed-cascade enzyme or co-immobilized cascade enzyme systems decreased by more than 90%, while the signal of the dual-enzyme coupled system of this invention remained above 95%. When detecting substrates using traditional mixed-cascade enzyme or co-immobilized cascade enzyme systems, the system showed almost no fluorescence signal within 30 minutes when the system contained the reducing substance ascorbic acid; however, using the dual-enzyme covalently coupled system, the fluorescence signal increased rapidly with reaction time, almost unaffected by ascorbic acid. This demonstrates its significant anti-interference ability.

[0041] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0042] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0043] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A SOX-HRP dual-enzyme conjugate, characterized in that, The SOX-HRP dual-enzyme conjugate is obtained by covalently coupling a thiol-modified sarcosine oxidase and a maleimide-modified horseradish peroxidase.

2. The method for preparing the SOX-HRP dual-enzyme conjugate according to claim 1, characterized in that, The preparation method is as follows: A thiol-modified sarcosine oxidase solution, a maleimide-modified horseradish peroxidase solution, and a PBS-EDTA buffer are mixed and stirred at room temperature in the dark for 10-15 hours to covalently couple the sarcosine oxidase and horseradish peroxidase. After covalent coupling, the mixture is washed twice by ultrafiltration and centrifugation with PBS-EDTA buffer. The volume ratio of the sarcosine oxidase solution, horseradish peroxidase solution, and PBS-EDTA buffer is 0.5-1.5:0.5-1.5:0.5-1.

5.

3. The preparation method according to claim 2, characterized in that, The ultrafiltration molecular weight is 40~60 kDa.

4. The use of the SOX-HRP dual-enzyme conjugate according to claim 1 in the preparation of detection reagents or kits for detecting small molecule markers in biological samples.

5. The application according to claim 4, characterized in that, The small molecule markers include sarcosine.

6. The application according to claim 4, characterized in that, The biological sample includes at least one of blood, urine, saliva, sweat, cell culture medium, or tissue homogenate.

7. The application according to claim 4, characterized in that, The detection reagent or kit is used to perform optical detection, including fluorescence detection or colorimetric detection.

8. The application according to claim 4, characterized in that, The detection reagents or kits are used in point-of-care testing devices, microfluidic chips, or wearable biosensors.

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