CN115792231B - DNase I biosensor based on thrombin aptamer-regulated enzyme cascade reaction - Google Patents
DNase I biosensor based on thrombin aptamer-regulated enzyme cascade reaction Download PDFInfo
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Abstract
The invention relates to a DNase I biosensor based on thrombin aptamer-mediated enzyme cascade reaction. The construction comprises the following steps: (1) preparing a thrombin solution and an aptamer solution; (2) preparing a DNase I standard solution with gradient concentration; (3) drawing a DNase I standard curve; (5) detecting the DNase I content in the sample to be detected. The DNase I biosensor based on thrombin aptamer-regulated enzyme cascade reaction has the advantages of simple operation process, low cost, less reagent consumption and the like, and the detection limit of DNase I is 0.2U/mL, so that the problems of complex and high cost of the existing detection method are effectively solved, and the development prospect is provided for clinical diagnosis and application.
Description
Technical Field
The invention provides a DNase I biosensor based on thrombin aptamer-regulated enzyme cascade reaction, and belongs to the technical field of detection and analysis.
Background
DNase I is an enzyme capable of cleaving the phosphodiester bond of DNA molecules. DNase I is present in blood, urine, sweat and other body fluids and is involved in various physiological processes of the human body, including gene recombination, DNA repair and DNA digestion. Meanwhile, it is also an important tool enzyme for genome DNA probe, DNA template digestion and in vitro high throughput sequencing. DNase I activity is closely related to many diseases. Clinical studies show that DNase I can alleviate exacerbation of respiratory symptoms, inhibit proliferation and metastasis of tumor cells, and enhance apoptosis. Recent studies have shown that gastric and colorectal cancer are closely related to reduced DNase I activity. Meanwhile, DNase I is currently used clinically for the treatment of bronchodilators, lung abscesses, cystic lung fibrosis and oral diseases associated with mucosal effects. In addition, it is considered a promising predictor of systemic lupus erythematosus and myocardial infarction area. These studies indicate that DNase I detection platforms can be a powerful tool for early clinical diagnosis. The detection of DNase I is clinically significant.
An aptamer refers to a single-stranded nucleotide sequence capable of specifically binding to a target molecule, and is selected from a random single-stranded nucleic acid library by "exponential enrichment of ligand system evolution". The aptamer can form a specific three-dimensional structure after adaptive folding through nucleotide-base complementary pairing, hydrogen bonding, pi-pi stacking, electrostatic force and the like interactions. Such three-dimensional structures can specifically bind to target molecules by intermolecular forces. Due to the high specificity and affinity of aptamer binding to targets, and the advantages of easy large-scale synthesis and flexible chemical modification, aptamers have been widely used in recent years as a new molecular recognition element in the fields of analysis and sensing. Cascade reactions refer to a series of successive reactions, two or more of which may be integrated. The products of the previous reaction may be consumed in or affect the subsequent reaction. In the field of biosensors, the design and implementation of cascade reactions has attracted considerable interest to researchers due to their advantages of signal amplification and signal transmission. Among them, thrombin aptamers have great potential applications in regulating cascade reactions due to inhibition of thrombin activity.
In the past studies, many DNase I detection methods have been designed and successfully applied, such as classical fluorescence detection and gel electrophoresis methods, electrochemical methods, and chemiluminescent methods in combination with nanomaterials. Although these methods are highly sensitive and reliable, they are costly and time consuming, some of which require even toxic markers. Therefore, the development of a simple and rapid DNase I detection new technology has important significance.
The point-of-care testing (POCT) technology product reduces the requirements on the professional of matched equipment and operation, can be used for on-site rapid detection and early-stage screening diagnosis of diseases, and can be applied to basic medical units or remote areas with limited resources to realize self-checking of patients at home. The biosensor has great potential in the development of POCT devices because of the advantages of simple operation, high sensitivity, good specificity and the like. Biosensors have a variety of construction materials such as nanomaterials, electronic chips, and paper. Among them, paper is particularly attractive in the development of portable biosensors due to its low cost and ease of operation. Paper biosensors have been used to detect various analytes, such as enzymes, proteins, nucleic acids, and pathogens. In particular a distance-based paper sensor. It can intuitively quantify the detection target by simply measuring the change in distance. Paper-based biosensors have attracted considerable attention due to their good cost effectiveness, simple operation and rapid data analysis.
In summary, developing a biosensing detection platform that can simply, rapidly and sensitively detect DNase I using a paper substrate has important research significance in practical application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a DNase I biosensor based on thrombin aptamer-regulated enzyme cascade reaction. The instant detection of DNase I is achieved without the need for complex instrumentation and procedures.
The invention is realized by the following technical scheme:
a DNase I biosensor based on thrombin aptamer-mediated enzyme cascade reaction is constructed by the following steps:
(1) Dissolving thrombin in a buffer solution to obtain a thrombin solution; dissolving thrombin aptamer dry powder in a buffer solution to obtain an aptamer solution;
(2) Adding 250 mu L of PBS buffer solution into 0.5 ml freeze-dried rabbit plasma, and uniformly dissolving to obtain a rabbit plasma solution;
(3) Dissolving DNase I in a buffer solution to obtain a DNase I standard solution with gradient concentration;
(4) mu.L of the aptamer solution was added to 5. Mu.L of DNase I solution, mixed well, and incubated at 37℃for 30 minutes. After incubation, 10. Mu.L of thrombin solution was added to the incubation solution to give 20. Mu.L of a mixed solution.
(5) The mixture solution was incubated at 37 ℃ for 30 minutes. After the incubation, 10. Mu.L of rabbit plasma solution was added to each of 20. Mu.L of the mixture solutions containing DNase I at different concentrations to obtain 30. Mu.L of the solution to be tested.
(6) Incubate at 25℃for 5 min. To better separate the mobile phase aqueous solution from the clot in the gel state in the test solution, the test solution is placed in a centrifuge for centrifugation 60 s. Finally, the upper mobile phase aqueous solution is sucked out and dropped onto the PVC substrate, and the pH indicator strip is rapidly placed on the drop. After the watermark area on the indicator strip is unchanged, the intelligent mobile phone is used for photographing and recording experimental results, recording the watermark crawling distance on the indicator strip, and drawing a DNase I detection standard curve.
(7) And (3) dissolving a sample to be detected containing DNase I in a buffer solution to obtain a sample solution to be detected, measuring the moving distance of the sample mixture to be detected on the pH test strip according to the method described in the step (4), and comparing the moving distance with a DNase I standard curve to obtain the DNase I content in the sample to be detected.
According to a preferred embodiment of the invention, in step (1), the thrombin buffer is formulated as 50 mM Na 2 HPO 4 , 150 mM NaCl, 10 mM MgCl 2 , pH=6.5。
According to a preferred embodiment of the invention, in step (1), the thrombin solution has a concentration of 1 mg/mL.
According to a preferred embodiment of the invention, in step (1), the aptamer buffer is formulated as 50 mM Na 2 HPO 4 , 150 mM NaCl, 10 mM MgCl 2 , pH=6.5。
According to a preferred embodiment of the invention, in step (1), the aptamer sequence is AGAAAGGAGAGAGAGGTTGGTGTGGTTGGWherein 15 bases of the italic portion represent thrombin inhibitor domains.
According to a preferred embodiment of the present invention, in step (1), the concentration of the aptamer solution is 20. Mu.M.
According to a preferred embodiment of the present invention, in step (3), the DNase I buffer is formulated as 10 mM Tris-HCl, 2.5 mM MgCl 2 , 0.5 mM CaCl 2 , pH=7.6。
According to a preferred embodiment of the invention, in step (6), the centrifugation speed is 4000 r/min.
According to a preferred embodiment of the present invention, in the step (6), the pH indicator strip is a broad-spectrum test strip, and the size is 4.5 mm ×60 mm.
According to the invention, the above-described DNase I biosensor based on thrombin aptamer-mediated enzyme cascade is used for detection of DNase I in non-disease diagnostic serum.
The detection principle of the invention is as follows:
in the solution to be tested, thrombin aptamer can specifically bind thrombin and effectively inhibit thrombin activity when DNase I is not present, so that the solution to be tested remains in a liquid state and cannot form blood clots in a gel state. In contrast, when DNase I is present, the aptamer chain is hydrolyzed by DNase I and cannot normally bind thrombin to inhibit thrombin activity, so that thrombin can correctly coagulate plasma and form a clot in a gel state, and the solution to be tested undergoes delamination. Accordingly, the volume of the mobile phase in the solution to be measured is reduced. Based on this principle, we developed a DNase I biosensor based on thrombin aptamer-mediated enzyme cascade for visual quantitative detection of DNase I. In both cases, DNase I was present and DNase I was not present, the relative volume of the mobile phase aqueous solution in the solution to be tested was significantly changed. The volume of the aqueous solution may be further determined by a pH indicator strip. The concentration of DNase I can be quantified by measuring the length of the watermark on the indicator strip.
Advantageous effects
1. The DNase I biosensor based on thrombin aptamer-regulated enzyme cascade reaction has the advantages of simple operation process, low cost, less reagent consumption and the like, and the detection limit of DNase I is 0.2U/mL, so that the problems of complex and high cost of the existing detection method are effectively solved, and the development prospect is provided for clinical diagnosis and application.
2. The DNase I biosensor based on thrombin aptamer-mediated enzyme cascade reaction provided by the invention can realize the quantification of DNase I by visually reading the flowing distance of the DNase I-containing mixture on a test strip. And establishing an indirect relation between the concentration of DNase I and the crawling distance of the mixture on the test strip, so that the DNase I can be rapidly and quantitatively detected simply and immediately by only relying on the paper-based test strip under the condition of not depending on a complex instrument.
Drawings
FIG. 1 is a schematic diagram of DNase I biosensor principle based on thrombin aptamer-mediated enzyme cascade.
FIG. 2 is a photograph showing the response of the paper-based biosensor of example 1 to thrombin solutions of different concentrations.
FIG. 3 is a photograph of the response of the paper-based biosensor of example 2 to different concentrations of aptamer solutions.
FIG. 4 is a photograph showing the response of the paper-based biosensor in example 3 to DNase I solutions of different concentrations.
FIG. 5 shows DNase I concentration versus gray scale in example 3Cr) calibration curve of values.
FIG. 6 is a graph showing the results of detecting DNase I in serum in example 4.
Detailed Description
The technical scheme of the invention is further described below with reference to the examples and the drawings in the specification, but the protection scope of the invention is not limited thereto.
In the following examples, thrombin Shanghai microphone Lin Shenghua, inc., DNase I Beijing Soy Bao technology, inc., and aptamers were synthesized by Shanghai Biotechnology, inc.
Unless otherwise specified, the drugs and reagents involved in this example are all commercially available products.
The principle of the detection method is shown in fig. 1, in the solution to be detected, when DNase I does not exist, thrombin aptamer can specifically bind thrombin and effectively inhibit thrombin activity, so that the solution to be detected keeps in a liquid state and can not form blood clots in a gel state. In contrast, when DNase I is present, the aptamer chain is hydrolyzed by DNase I and cannot normally bind thrombin to inhibit thrombin activity, so that thrombin can correctly coagulate plasma and form a clot in a gel state, and the solution to be tested undergoes delamination. Accordingly, the volume of the mobile phase in the solution to be measured is reduced. Based on this principle, we developed a DNase I biosensor based on thrombin aptamer-mediated enzyme cascade for visual quantitative detection of DNase I. In both cases, DNase I was present and DNase I was not present, the relative volume of the mobile phase aqueous solution in the solution to be tested was significantly changed. The volume of the aqueous solution may be further determined by a pH indicator strip. The concentration of DNase I can be quantified by measuring the length of the watermark on the indicator strip. Therefore, the DNase I can be detected rapidly, quantitatively, simply and immediately under the condition of not depending on complex instruments.
EXAMPLE 1 determination of thrombin concentration
We studied the optimal concentration of thrombin to coagulate rabbit plasma in buffer (50 mM Na 2 HPO 4 、150 mM NaCl、10 mM MgCl 2 Ph=6.5) to prepare a range of thrombin solutions at concentrations of 0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 1.25 mg/mL and 2 mg/mL. Then, 10. Mu.L of the rabbit plasma solution was added to 20. Mu.L of the solution containing thrombin at various concentrations, to obtain 30. Mu.L of the solution to be tested. To better separate the mobile phase solution from the clot in the gel state in the solution to be treated, the solution to be tested was placed in a centrifuge 60 s (4000 r/min). Finally, the upper mobile phase solution is sucked out and dropped onto the PVC substrate, and the pH indicator strip is quickly placed on the drop. After the watermark area on the indicator strip is unchanged, the intelligent mobile phone is used for photographing and recording experimental results.
We studied the optimal concentration of thrombin-coagulated rabbit plasma. As shown in fig. 2, as the thrombin concentration increases, the amount of upper mobile phase solution in the solution to be treated gradually decreases, and the watermark coverage of the corresponding indicator strip decreases significantly. However, when the thrombin concentration is greater than 1 mg/mL, the volume of the mobile phase solution formed stops changing. And it was found that the change in the volume of the blood clot stopped when the concentration of thrombin was greater than 1 mg/mL. Therefore, we used 1 mg/mL thrombin solution as the optimal condition for paper-based biosensor preparation.
Example 2 determination of aptamer concentration
The aptamer can specifically bind thrombin and effectively inhibit thrombin activity, so that the selection of a suitable aptamer concentration is critical to the experimental system. Will adapt toThe dry powder was dissolved in buffer (50 mM Na 2 HPO 4 、150 mM NaCl、10 mM MgCl 2 Ph=6.5) to prepare a series of aptamer solutions of different concentrations. mu.L of thrombin solution was mixed with 10. Mu.L of aptamer solution of different concentrations to give 20. Mu.L of mixed solution. The series of mixed solutions contained 1 mg/mL thrombin and different concentrations (0. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 30. Mu.M) of aptamer. The mixture was incubated at 37℃for 30 minutes. After incubation, 10. Mu.L of the rabbit plasma solution was added to 20. Mu.L of the mixed solution containing the different concentrations of the aptamer, yielding 30. Mu.L of the solution to be tested. The subsequent treatment of the solution to be measured was the same as in example 1.
Fig. 2 shows a picture of the indicator strip corresponding to a variation in aptamer concentration between 0 and 30 μm. As the concentration of the aptamer increases, the volume of the upper mobile phase solution in the solution to be treated gradually increases, and the watermark coverage of the corresponding indicator strip increases significantly. This indicates that more thrombin activity is inhibited. To increase the sensitivity of the assay, we selected the smaller value of the aptamer concentration (20 μm) that effectively inhibited thrombin activity as the optimal condition for the next experiment.
Example 3 detection of DNase I
We studied the response of DNase I at different concentrations in a paper flow sensor. DNase I was dissolved in buffer 2 (10 mM Tris HCl,2.5 mM MgCl 2 ，0.5 mM CaCl 2 Ph=7.6) to obtain a series of DNase i solutions of different concentrations. Then, 5. Mu.L of the aptamer solution was added to 5. Mu.L of DNase I solution, mixed well, and incubated at 37℃for 30 minutes. After incubation, 10. Mu.L of thrombin solution was added to the incubation solution to give 20. Mu.L of a mixed solution. At this time, the resulting mixed solution contained 1 mg/mL thrombin, 20. Mu.M aptamer and DNase I at various concentrations (0U/mL, 0.01U/mL, 0.1U/mL and 1U/mL). The mixture solution was incubated at 37 ℃ for 30 minutes. After the incubation, 10. Mu.L of rabbit plasma solution was added to each of 20. Mu.L of the mixture solutions containing DNase I at different concentrations to obtain 30. Mu.L of the solution to be tested. The subsequent treatment of the solution to be measured is the same as described above.
FIG. 4 shows representative photographs of the response of paper-based biosensors to DNase I at concentrations ranging from 0 to 100U/mL. As shown in fig. 4, an increase in DNase I concentration resulted in a decrease in the water flow distance on the paper web, and a significant decrease in watermark coverage. For DNase I concentrations in the range of 0-100U/mL, a linear relationship between watermark coverage on the indicator strip and CDNase I was obtained (fig. 5). The final calculation gave a DNase I detection limit of 0.2U/mL (based on 3σ/slope).
EXAMPLE 4 the method of the invention is used for detection of DNse I in serum
Normal human serum was diluted 40-fold with PBS buffer, and DNase I was added thereto to give gradient serum standards with concentrations of 0U/mL, 0.2U/mL, 2U/mL, 20U/mL, and then tested as described in example 3, with the results shown in fig. 6.
As shown in fig. 6, as DNase I concentration increases, the length of the watermark on the test strip becomes shorter, which proves that DNase I with different concentrations can be clearly distinguished by different water flow distances on the paper-based biosensor.
The foregoing is only a specific embodiment of the invention. The scope of the present invention is not limited thereto, and any changes or substitutions that would be easily recognized by those skilled in the art within the scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims (8)
1. An application of DNase I biosensor based on thrombin aptamer-mediated enzyme cascade reaction in detection of DNase I in non-disease diagnosis serum is provided, which comprises the following construction steps:
(1) Dissolving thrombin in a buffer solution to obtain a thrombin solution; dissolving thrombin aptamer in a buffer solution to obtain an aptamer solution;
(2) Adding 250 mu L of PBS buffer solution into 0.5 ml freeze-dried rabbit plasma, and uniformly dissolving to obtain a rabbit plasma solution;
(3) Dissolving DNase I in a buffer solution to obtain a DNase I standard solution with gradient concentration;
(4) mu.L of the aptamer solution was added to 5. Mu.L of DNase I solution, mixed well, and incubated at 37℃for 30 minutes; after incubation, 10 μl of thrombin solution was added to the incubation to give 20 μl of mixed solution;
(5) The mixture solution was incubated at 37 ℃ for 30 minutes; after the cultivation, 10. Mu.L of a rabbit plasma solution was added to each of 20. Mu.L of the mixture solutions containing DNase I at different concentrations to obtain 30. Mu.L of a solution to be tested;
(6) Incubate at 25℃for 5 min; placing the solution to be tested in a centrifuge for centrifugation 60 s; finally, sucking out the upper mobile phase aqueous solution and dripping the upper mobile phase aqueous solution on the PVC substrate, and rapidly placing the pH indicator strip on the liquid drop; after the watermark area on the indicator strip is unchanged, photographing and recording an experimental result, recording the watermark crawling distance on the indicator strip, and drawing a DNase I detection standard curve;
(7) And (3) dissolving a sample to be detected containing DNase I in a buffer solution to obtain a sample solution to be detected, measuring the moving distance of the sample mixture to be detected on the pH test strip according to the method described in the step (4), and comparing the moving distance with a DNase I standard curve to obtain the DNase I content in the sample to be detected.
2. The use according to claim 1, wherein in step (1) the thrombin buffer is formulated as 50 mM Na 2 HPO 4 , 150 mM NaCl, 10 mM MgCl 2 , pH=6.5。
3. The use according to claim 1, wherein in step (1) the thrombin solution has a concentration of 1 mg/mL.
4. The use according to claim 1, wherein in step (1) the aptamer buffer formulation is 50 mM Na 2 HPO 4 , 150 mM NaCl, 10 mM MgCl 2 , pH=6.5。
5. The use according to claim 1, wherein in step (1) the aptamer solution is at a concentration of 20 μm.
6. The use according to claim 1, wherein in step (3) the DNase I buffer is formulated as 10 mM Tris-HCl, 2.5 mM MgCl 2 , 0.5 mM CaCl 2 , pH=7.6。
7. The use according to claim 1, wherein in step (6) the centrifugation speed is 4000 r/min.
8. The use of claim 1, wherein in step (6) the pH indicator strip is a broad-spectrum test strip having a size of 4.5 mm x 60 mm.
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| WO2005072456A2 (en) * | 2004-01-28 | 2005-08-11 | Gerard Biotech, Llc | Single use lyophilized enzymatic reagents, and kits and methods for using same |
| NZ598557A (en) * | 2009-08-18 | 2013-08-30 | Baxter Int | Aptamers to tissue factor pathway inhibitor and their use as bleeding disorder therapeutics |
| CN106520913B (en) * | 2016-09-22 | 2020-02-21 | 江苏大学 | Preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection |
| CN107603981A (en) * | 2017-09-08 | 2018-01-19 | 中国科学院生态环境研究中心 | Detect the enzyme-linked aptamer of blood coagulation and detection method of aflatoxin B1 |
| CN108562633B (en) * | 2018-04-19 | 2020-06-02 | 山东农业大学 | Photoelectrochemical sensor for detecting sulfadimethoxine and detection method thereof |
| CN110220893B (en) * | 2019-06-11 | 2021-01-22 | 上海交通大学 | Visual method for measuring thrombin in urine sample based on interface moving distance |
| CN110423756B (en) * | 2019-08-05 | 2023-03-28 | 江南大学 | Short-chain DNA aptamer sequence for specifically recognizing streptomycin and application thereof |
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