L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid

Abstract

The L-nucleic acid aptamer is used for high-sensitivity detection of D lactic acid in a biological system. The invention relates to a novel method for detecting D-type lactic acid in a biological system, establishes a novel method for directly detecting D-type lactic acid in the biological system, and solves the problem that D-type lactic acid cannot be directly detected by a previous scheme. The method comprises the following steps: (1) designing and synthesizing an L-nucleic acid aptamer adsorbed by graphene oxide and marked by a fluorescent molecule according to a reported D-nucleic acid aptamer sequence for specific response to L-lactic acid; (2) proving that the L-nucleic acid aptamer can specifically respond to D-lactic acid, and constructing a detection system of the L-nucleic acid aptamer to the D-lactic acid in a buffer solution system; and (3) extracting serum, and constructing a detection system of D lactic acid in the biological matrix. The method has the advantages of simple operation, rapid reaction, stable signal, high sensitivity, no false positive signal and the like, and can effectively detect lactic acid in organisms.

Description

Technical Field

[0001] The invention relates to a sensor for detecting D-lactic acid in a biological system, develops a method for directly detecting D-lactic acid, and belongs to the technical field of D-lactic acid detection and analysis methods. Background Art

[0002] Lactic acid is a natural organic acid existing in living organisms. Lactic acid has two configurations, L-lactic acid and D-lactic acid. The normal metabolism of higher animals is basically L-lactic acid. D-lactic acid is mainly produced by the metabolism of various intestinal bacteria or exogenous intake. Diabetes, traumatic shock, acidosis, intestinal ischemia, metabolism and metastasis of malignant tumors will cause an increase in D-lactic acid concentration. Therefore, D-lactic acid is also a biomarker for such diseases. At present, the detection of D-lactic acid content mainly relies on HPLC, gas chromatography, neutralization titration, capillary electrophoresis, and enzyme catalysis. These methods either require the use of large instruments, or are complicated to operate, or are interfered by biological matrices, and require sample pretreatment, which is not conducive to the instant detection of D-lactic acid.

[0003] Aptamers are oligonucleotide fragments that can specifically bind to the target obtained by artificial screening through the systematic evolution of ligands by exponential enrichment (SELEX) technology. At present, there are literature reports that people have screened out D-aptamers that can specifically bind to L-lactic acid. Based on the mirror recognition principle, we inferred that the mirror molecule of the D-aptamer: L-aptamer can recognize the mirror image of the original target: D-lactic acid. In this way, we obtained an oligonucleotide fragment that can specifically bind to D-lactic acid without the need for re-screening. At the same time, because the fragment is a mirror structure of natural nucleic acid, it can resist the degradation of nucleases in organisms and will not be interfered by non-specific proteins, so it can stably exist in the biological matrix. Summary of the invention

[0004] The present invention provides a stable, sensitive and specific quantitative analysis method for D-lactic acid in a biological system. The L-nucleic acid aptamer-based D-lactic acid quantitative analysis method of the present invention is characterized in that the D-nucleic acid aptamer sequence that specifically recognizes L-lactic acid is directly converted into the L-nucleic acid aptamer. According to the principle related to chiral substrate specificity, the L-nucleic acid aptamer can also recognize the mirror image body corresponding to L-lactic acid: D-lactic acid. At the same time, the L-nucleic acid aptamer will not be interfered by the biological matrix, thereby realizing the quantitative analysis of D-lactic acid in the biological system.

[0005] In order to solve the above technical problems, the present invention is solved by the following technical solution: an L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid, comprising the following steps:

[0006] Step 1. Design and synthesize a fluorescent molecule-labeled L-aptamer fluorescent probe based on the reported D-aptamer sequence that specifically responds to L-lactic acid: First, obtain the reported D-aptamer sequence information that specifically responds to L-lactic acid, select a suitable fluorescent molecule (such as FAM) and a chemical synthesis method based on the sequence, introduce a functional group modification that can react with the fluorescent molecule at a specific position of the aptamer, and generally use a chemical synthesizer for solid phase synthesis. For example, during the synthesis process, when a specific nucleotide position is synthesized, a fluorescent molecule with an activation group is added to react with the corresponding group on the aptamer chain. The reaction conditions must strictly control parameters such as temperature, reaction time, and reagent concentration to ensure that the fluorescent molecule is accurately and efficiently labeled on the aptamer, and the fluorescent molecule-labeled L-aptamer fluorescent probe is synthesized. After the reaction is completed, the synthetic product is purified by purification means such as high performance liquid chromatography (HPLC) to remove unreacted raw materials and impurities to obtain a high-purity L-aptamer fluorescent probe, and its structure is characterized and confirmed by means such as mass spectrometry.

[0007] Step 2, prove that the L-nucleic acid aptamer has a specific binding ability to D-lactic acid, and construct a detection system of L-nucleic acid aptamer for D-lactic acid in a buffer solution: To prove the specific binding ability, prepare a series of solution systems containing different concentrations of D-lactic acid and other possible interfering substances (such as L-lactic acid, common sugars, amino acids, etc.), the solution is prepared using the buffer system (phosphate buffer) (PBS) described in claim 3, 5mM MgCl2, pH 7.4), add an equal amount of L-nucleic acid aptamer fluorescent probe to each solution system, incubate at a suitable temperature (such as 25-37°C) for a certain time (such as 10 to 30 minutes), after the incubation is completed, A fluorescence spectrometer is used to detect the changes in the fluorescence emission spectra of each system. If the fluorescence intensity is significantly enhanced in the solution system containing D-lactic acid, while the fluorescence intensity has no obvious change or changes very little in the system containing other interfering substances, it is proved that the L-nucleic acid aptamer has a specific binding ability to D-lactic acid. The detection system is constructed, and the working concentration of the L-nucleic acid aptamer is determined to be the final concentration of 100 nM level described in claim 4. An appropriate amount of the L-nucleic acid aptamer fluorescent probe is added to a certain volume (such as 1 mL) of the buffer system described in claim 3 to make its final concentration reach 100 nM, and the mixture is fully mixed to prepare a buffer detection system containing the L-nucleic acid aptamer fluorescent probe.

[0008] Step 3, extract serum and construct a detection system for D-lactic acid in a biological matrix: serum extraction, collect blood samples, centrifuge at a low temperature (such as 4° C.) and a certain speed (such as 3000-5000 rpm) for 10 to 20 minutes, collect the upper serum, and then perform appropriate pretreatment on the serum, such as filtering through a 0.22 μm filter membrane to remove possible impurity particles, etc., to obtain a pure serum sample, and construct a biological matrix detection system. Take a certain amount (such as 100 μL, that is, the reaction volume described in claim 5) of serum sample and add it to a buffer detection system containing an L-nucleic acid aptamer fluorescent probe to make the serum concentration reach 1% as described in claim 6, and incubate for 20 minutes as described in claim 7 at a suitable temperature (such as 25-37° C.). After the incubation, use a fluorescence detection device such as an enzyme marker to detect the change in the fluorescence signal of the system, and calculate the content of D-lactic acid in the serum sample according to a pre-established standard curve (constructed by a serum sample with a known concentration of D-lactic acid), thereby constructing a detection system for D-lactic acid in a biological matrix.

[0009] Preferably, the signal molecules may be FITC and graphene oxide, which is characterized in that when D-lactic acid is not present, the L-nucleic acid aptamer labeled with FAM is adsorbed on the surface of graphene oxide, and the fluorescence emitted by the FAM group is quenched by graphene oxide. When D-lactic acid is present, the nucleic acid aptamer combines with D-lactic acid to form a secondary structure that detaches from the surface of graphene oxide and emits a fluorescent signal.

[0010] Preferably, the buffer system used in the method comprises the following components when used for D-lactic acid sensing: phosphate buffer PBS, 5mM MgCl2, pH 7.4.

[0011] Preferably, the final concentration of the L-aptamer in the method is at a level of 100 nM.

[0012] Preferably, the reaction volume is 100 μL when the quantitative detection is performed by an ELISA instrument.

[0013] Preferably, in serum, the serum concentration is 1%.

[0014] Preferably, the reaction time is 20 min in both the standard buffer and serum.

[0015] Preferably, the detection sensitivity of D-lactic acid in standard buffer is 0.1 mM and in serum is 0.25 mM.

[0016] The advantages of the present invention are: first, the method is an innovation in the principle of chiral substrate specificity, proving that while chiral substance A specifically binds to substance B, the mirror image A' of chiral substance A also specifically binds to the mirror image B' of B; second, the method does not require screening of nucleic acid sequences, and only needs to synthesize the screened D-nucleic acid aptamer sequence into an L-nucleic acid aptamer sequence, which is simple and fast to design; third, with the help of the excellent performance of L-DNA in resisting interference from biological matrices, the method can be used for the detection of D-lactic acid in biological systems. In view of the advantages of simple design, good stability, high sensitivity, etc., the L-nucleic acid aptamer proposed by us may develop into a popular D-lactic acid quantitative detection tool.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid of the present invention has a short detection cycle from sample processing to final result acquisition, which can generally be completed within 2-3 hours. This is due to the rapid binding kinetics of the nucleic acid aptamer and D-lactic acid and the efficient hybridization reaction between the signal amplification probe and the nucleic acid aptamer. The short detection time enables the method to meet the needs of clinical rapid diagnosis, such as rapid screening of patients suspected of having D-lactic acid-related diseases in the emergency room, and timely providing doctors with diagnostic evidence to formulate corresponding treatment plans. In the food industry production process, it can also monitor the changes in the content of D-lactic acid in the fermented product in real time, adjust the fermentation process parameters in time, ensure the stability and consistency of product quality, improve production efficiency, reduce production costs, and respond quickly;

[0019] 2. The L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid of the present invention has good chemical stability and thermal stability in the L-nucleic acid aptamer. Compared with some biological enzyme recognition elements, it is not easily affected by environmental factors such as temperature, pH value, etc. to be inactivated or denatured. In multiple repeated detection experiments, the sensor can maintain stable performance, and the detection results have good repeatability. Whether in the preparation process of different batches of sensors or in multiple repeated detections of the same sample, the deviation of the obtained D-lactic acid detection results is within an acceptable range. This stability and repeatability make the detection method of the present invention more reliable, which is conducive to the establishment of a standardized detection process and quality control system. It has important application value in large-scale clinical detection and industrial production detection, and the signal is stable without false positive signals;

[0020] 3. The entire detection process design of the L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid of the present invention is simple and efficient. The operator only needs to set the corresponding parameters according to the instrument operating procedures to quickly obtain the detection data. The method is simple and easy to operate and does not require long-term and complicated training for professionals to master, which greatly reduces the labor cost and time cost of detection, and is conducive to promotion and application in different laboratories and on-site detection scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the overall design idea of ​​the patent. The L-nucleic acid sequence is labeled with a fluorescent molecule and adsorbed on the surface of graphene oxide (GO). At this time, the fluorescence emitted by the fluorescent molecule labeled with the nucleic acid fragment is quenched by GO. After adding D-lactic acid, D-lactic acid binds to the nucleic acid fragment, detaches from the GO surface, and the fluorescent molecule recovers the signal. The intensity of the fluorescent signal is linearly related to the concentration of D-lactic acid, so it can be used for the detection of D-lactic acid.

[0022] Figure 2 is the nucleic acid aptamer sequence.

[0023] Figure 3 The responsiveness of the aptamer to D-lactic acid. Compared with the blank control group, the fluorescence signal increased by 3.9 times after adding 50 mM lactic acid.

[0024] Figure 4 The linear relationship of the nucleic acid aptamer probe in response to D-lactic acid in buffer solution is 0.1 mM.

[0025] Figure 5 The selectivity of the nucleic acid aptamer probe in the buffer solution can effectively distinguish D-lactic acid from L-lactic acid.

[0026] Figure 6 The linear relationship of the nucleic acid aptamer probe in response to D-lactic acid in serum samples is shown in Figure 1, and the detection sensitivity is 0.25 mM. DETAILED DESCRIPTION

[0027] Unless the context clearly dictates otherwise, unmodified nouns and nouns modified by "the" include singular and plural referents.

[0028] As used in the specification and claims, the terms "comprises," "comprising," "having," "may," "containing," and variations thereof as used herein refer to open transitional phrases, terms, or words that require the presence of specified ingredients / steps and allow for the presence of other ingredients / steps. However, such descriptions should be interpreted as also describing compositions or methods as "consisting of" and "consisting essentially of" the recited ingredients / steps, which allows for the presence of only the specified ingredients / steps and any unavoidable impurities that may result therefrom, and excludes other ingredients / steps.

[0029] Numerical values ​​in the specification and claims of this application should be understood to include the same numerical values ​​when reduced to the same number of significant figures and numerical values ​​that differ from the stated value by less than the experimental error of ordinary measurement techniques of the type described in this application for determining the stated value.

[0030] All ranges disclosed herein are inclusive of the indicated endpoints and are independently combinable (eg, the range of "2 grams to 10 grams" includes the endpoints 2 grams and 10 grams, and all intermediate values).

[0031] The terms "about" and "approximately" can be used to include any numerical value that can be varied without changing the basic function of the value. When used with a range, "about" and "approximately" also disclose the range defined by the absolute values ​​of the two endpoints, for example, "about 2 to about 4" also discloses a range of "2 to 4". Generally, the terms "about" and "approximately" can refer to ±10% of the indicated number. However, for temperature, the term "approximately" refers to ±1°C.

[0032] Unless expressly stated otherwise, the percentages of elements are to be considered as percentages by weight of the alloy in question.

[0033] The present disclosure may refer to the temperature of certain method steps. It should be noted that these specifications generally refer to the temperature set by the heat source (such as a furnace), and not necessarily the temperature that the heated material must reach.

[0034] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be used for other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0035] Example 1. Design, synthesis and purification of L-aptamers

[0036] The D-type nucleic acid aptamer sequence reported in the literature for detecting L-lactic acid was directly converted into an L-type nucleic acid aptamer sequence. The L-DNA synthetic monomers were purchased from ChemGene. The monomers were dissolved in acetonitrile to a concentration of 0.05 g / ml. The sequence was synthesized on a DNA automatic synthesizer using CPG labeled with FITC at the 3 ends. After the synthesis, the CPG was transferred to a centrifuge tube, and 0.5 mL of saturated ammonia was added to perform aminolysis at 65°C for 8 h to cut the DNA from the CPG. After the aminolysis was completed, the ammonia gas was removed, the supernatant was taken at a speed of 10000 rpm for 10 min, and then 0.5 mL of ultrapure water was added to the CPG to elute the sample adsorbed on the CPG. The supernatant was taken at a speed of 10000 rpm again for 10 min. The supernatant obtained by combining the obtained supernatants was the crude product. The obtained crude product was concentrated and dissolved in 0.1 M triethylamine acetate (TEAA), and purified using a reversed-phase high performance liquid chromatography.

[0037] The product purified by reverse phase-HPLC was dried under vacuum and dissolved in ultrapure water, desalted using a gel filtration column, and concentrated under vacuum after desalting. The absorbance of nucleic acid at 260 nm was measured using a UV-visible spectrophotometer to quantify DNA.

[0038] Example 2: Quantitative detection of D-lactic acid by L-aptamer / GO sensor in standard buffer

[0039] PBS buffer was used as the standard buffer. For each sample to be tested, 100 mL of PBS buffer and 100 nM L-aptamer probe were prepared, and GO with a concentration of 20 mg / ml was added. The samples were incubated at room temperature for 10 minutes. After incubation, samples with different concentrations of D-lactic acid were added and incubated at room temperature for 20 minutes. The samples were analyzed by fluorescence titration on an ELISA instrument. The fixed-point detection mode was adopted, with an excitation wavelength of 485 nm and a detection emission wavelength of 520 nm. The measured fluorescence signals were recorded for quantitative detection of D-lactic acid. In the selective experiment, L-lactic acid was used as a control to examine the responses of 20 mM D-lactic acid and 20 mM L-lactic acid to the sensor.

[0040] Example 3. Quantitative detection of D-lactic acid in serum by L-aptamer / GO sensor Human blood was obtained from the hospital and centrifuged at 6000 rpm for 5 min to obtain serum, which was diluted with PBS to 1% serum buffer and quantified using the method in Example 2.

[0041] Example 4: An L-nucleic acid aptamer sensor for highly sensitive detection of D-lactic acid, comprising the following steps:

[0042] Step 1: Based on the reported D-aptamer sequence that specifically responds to L-lactic acid, a fluorescent aptamer fluorescent probe labeled with a fluorescent molecule was designed and synthesized:

[0043] 1. First, obtain the sequence information of the D-aptamer that has been reported to specifically respond to L-lactic acid.

[0044] 2. According to the sequence, select a suitable fluorescent molecule (such as FAM) and a chemical synthesis method, introduce a functional group modification that can react with the fluorescent molecule at a specific position of the nucleic acid aptamer, and generally use a chemical synthesizer for solid phase synthesis. For example, during the synthesis process, when synthesizing to a specific nucleotide position, add a fluorescent molecule with an activation group to make it undergo a coupling reaction with the corresponding group on the nucleic acid aptamer chain. The reaction conditions must strictly control parameters such as temperature, reaction time, and reagent concentration to ensure that the fluorescent molecule is accurately and efficiently labeled on the nucleic acid aptamer, and synthesize the L-nucleic acid aptamer fluorescent probe labeled with the fluorescent molecule. After the reaction is completed, purify the synthetic product by purification means such as high performance liquid chromatography (HPLC) to remove unreacted raw materials and impurities to obtain a high-purity L-nucleic acid aptamer fluorescent probe, and characterize its structure by means such as mass spectrometry to confirm its structure.

[0045] Step 2: Prove that the L-nucleic acid aptamer has the specific binding ability to D-lactic acid, and construct the detection system of the L-nucleic acid aptamer to D-lactic acid in the buffer solution:

[0046] 1. Proof of specific binding ability:

[0047] A series of solution systems containing different concentrations of D-lactic acid and other potentially interfering substances (such as L-lactic acid, common sugars, amino acids, etc.) were prepared. The solutions were prepared using the buffer system (phosphate buffer) (PBS) described in claim 3, 5 mM MgCl2, pH 7.4).

[0048] An equal amount of L-nucleic acid aptamer fluorescent probe is added to each solution system, and incubated at a suitable temperature (such as 25-37° C.) for a certain time (such as 10 to 30 minutes).

[0049] After the incubation, a fluorescence spectrometer is used to detect the changes in the fluorescence emission spectra of each system. If the fluorescence intensity is significantly enhanced in the solution system containing D-lactic acid, while the fluorescence intensity has no obvious change or changes very little in the system containing other interfering substances, it proves that the L-nucleic acid aptamer has the ability to bind specifically to D-lactic acid.

[0050] 2. Construction of detection system:

[0051] The working concentration of the L-aptamer is determined to be the final concentration of 100 nM as described in claim 4, and an appropriate amount of the L-aptamer fluorescent probe is added to a certain volume (such as 1 mL) of the buffer system as described in claim 3 to make its final concentration reach 100 nM, and the mixture is fully mixed to prepare a buffer detection system containing the L-aptamer fluorescent probe.

[0052] Step 3: Extract serum and construct a detection system for D-lactic acid in biological matrix:

[0053] 1. Serum extraction:

[0054] Collect blood samples and centrifuge them at a low temperature (such as 4°C) and a certain speed (such as 3000-5000rpm) for 10 to 20 minutes to collect the upper serum. Then, the serum is appropriately pretreated, such as filtering through a 0.22μm filter membrane to remove possible impurity particles, to obtain a pure serum sample.

[0055] 2. Construction of biological matrix detection system:

[0056] A certain amount (such as 100 μL, i.e., the reaction volume described in claim 5) of serum sample is taken and added to a buffer detection system containing an L-nucleic acid aptamer fluorescent probe to make the serum concentration reach 1% as described in claim 6, and incubated for 20 minutes as described in claim 7 at a suitable temperature (such as 25-37° C.). After the incubation, the fluorescence signal change of the detection system is detected by a fluorescence detection device such as an enzyme marker, and the content of D-lactic acid in the serum sample is calculated according to a pre-established standard curve (constructed by serum samples with known concentrations of D-lactic acid), thereby constructing a detection system for D-lactic acid in a biological matrix.

[0057] The specific steps are as follows: the staff designs and synthesizes L-nucleic acid aptamer fluorescent probes labeled with fluorescent molecules. First, the reported D-nucleic acid aptamer sequence information that specifically responds to L-lactic acid is obtained. Then, based on the sequence, a suitable fluorescent molecule such as FAM and a chemical synthesis method are selected to introduce functional group modifications that can react with fluorescent molecules at specific positions of the nucleic acid aptamer. Generally, a chemical synthesizer is used for solid phase synthesis. For example, during the synthesis process, when a specific nucleotide position is synthesized, a fluorescent molecule with an activated group is added to allow it to undergo a coupling reaction with the corresponding group on the nucleic acid aptamer chain. The reaction conditions must strictly control parameters such as temperature, reaction time, and reagent concentration to ensure that the fluorescent molecule is accurately and efficiently labeled on the nucleic acid aptamer, and the L-nucleic acid aptamer fluorescent probe labeled with fluorescent molecules is synthesized. After the reaction is completed, the purified product is purified by high performance liquid chromatography (HPLC). The synthetic product is purified by chemical means to remove unreacted raw materials and impurities to obtain a high-purity L-nucleic acid aptamer fluorescent probe, and its structure is characterized and confirmed by mass spectrometry and other means. At this time, the staff proved the specific binding ability of the L-nucleic acid aptamer to D-lactic acid and constructed a detection system in a buffer solution. First, the specific binding ability was proved: first, a series of solution systems containing different concentrations of D-lactic acid and other possible interfering substances such as L-lactic acid, common sugars, amino acids, etc. were prepared. The solution was prepared using a buffer system phosphate buffer PBS, 5mM MgCl2, pH 7.4. Secondly, an equal amount of L-nucleic acid aptamer fluorescent probe was added to each solution system, and incubated at a suitable temperature such as 25-37°C for a certain time such as 10 to 30 minutes. Finally, after the incubation, a fluorescence spectrometer was used to detect the changes in the fluorescence emission spectrum of each system.If the fluorescence intensity is significantly enhanced in the solution system containing D-lactic acid, while the fluorescence intensity has no obvious change or changes very little in the system containing other interfering substances, it proves that the L-nucleic acid aptamer has a specific binding ability to D-lactic acid. At this time, the staff constructs a detection system, determines that the working concentration of the L-nucleic acid aptamer is the final concentration of 100 nM level as described in claim 4, adds an appropriate amount of L-nucleic acid aptamer fluorescent probe to a certain volume such as 1 mL of the buffer system as described in claim 3, so that its final concentration reaches 100 nM, and mixes thoroughly to prepare a buffer detection system containing the L-nucleic acid aptamer fluorescent probe. Finally, the staff extracts serum and constructs a detection system for D-lactic acid in a biological matrix. For serum extraction: collect blood samples, use centrifugal separation technology, and centrifuge at a low temperature such as 4°C and a certain speed such as 3 The mixture is centrifuged at 000-5000rpm for 10-20 minutes, the upper serum is collected, and then the serum is appropriately pretreated, such as filtering through a 0.22μm filter membrane to remove possible impurity particles, etc., to obtain a pure serum sample. At this time, the staff constructs a biological matrix detection system: a certain amount of serum sample, such as 100μL, i.e., the reaction volume, is taken, and added to a buffer detection system containing an L-nucleic acid aptamer fluorescent probe to make the serum concentration reach 1%, and incubated at a suitable temperature such as 25-37°C for 20 minutes. After the incubation, the fluorescence signal change of the detection system is detected by a fluorescence detection device such as an enzyme marker, and the content of D-lactic acid in the serum sample is calculated according to the pre-established standard curve through a serum sample with a known concentration of D-lactic acid, thereby constructing a detection system for D-lactic acid in the biological matrix.

[0058] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the accompanying drawings are only examples and do not limit the present invention. The purpose of the present invention has been fully and effectively achieved. The functions and structural principles of the present invention have been demonstrated and explained in the embodiments. Without departing from the principles, the embodiments of the present invention may be subject to any changes or modifications. Although the embodiments of the present invention have been shown and described, it is understood by those of ordinary skill in the art that various changes, modifications, substitutions and deformations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the attached claims and their equivalents. This specific embodiment is merely an explanation of the present application, and it is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without creative contributions as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. An L-nucleic acid aptamer sensor for highly sensitive detection of D-lactic acid, characterized in that: The following steps are involved: Step 1: Based on the reported D-nucleic acid aptamer sequence that specifically responds to L-lactic acid, a L-nucleic acid aptamer fluorescent probe labeled with a fluorescent molecule is designed and synthesized; Step 2, proving that the L-nucleic acid aptamer has a specific binding ability to D-lactic acid, and constructing a detection system of the L-nucleic acid aptamer to D-lactic acid in a buffer solution; Step 3: extract serum and construct a detection system for D-lactic acid in biological matrix.

2. The L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid according to claim 1, characterized in that: The signal molecules as described in claim 1 can be FITC and graphene oxide, and are characterized in that when D-lactic acid is not present, the L-nucleic acid aptamer labeled with FAM is adsorbed on the surface of graphene oxide, and the fluorescence emitted by the FAM group is quenched by graphene oxide. When D-lactic acid is present, the nucleic acid aptamer combines with D-lactic acid to form a secondary structure that detaches from the surface of graphene oxide and emits a fluorescent signal.

3. The L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid according to claim 1, characterized in that: The buffer system used in the method of claim 1, comprising the following components when used for D-lactic acid sensing: Phosphate buffered saline (PBS), 5 mM MgCl2, pH 7.

4.

4. The L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid according to claim 1, characterized in that: The final concentration of the L-nucleic acid aptamer in the method of claim 1 is at a level of 100 nM.

5. The L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid according to claim 1, characterized in that: The method according to claim 1 has a reaction volume of 100 μL when quantitative detection is performed using an ELISA instrument.

6. The L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid according to claim 1, characterized in that: The serum according to claim 1, wherein the serum concentration is 1%.

7. The L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid according to claim 1, characterized in that: In the reaction time as described in claim 1, the standard buffer and serum are both 20 minutes.

8. The L-nucleic acid aptamer sensor for high-sensitivity detection of D-lactic acid according to claim 1, characterized in that: In the method of claim 1, the detection sensitivity of D-lactic acid in standard buffer is 0.1 mM and in serum is 0.25 mM.

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