A highly sensitive detection method for D-lactic acid

Abstract

L-nucleic acid aptamers are used for highly sensitive detection of D-lactic acid in biological systems. The present invention relates to a novel method that can be used for detection of D-lactic acid in biological systems, which establishes a novel method for directly detecting D-lactic acid in biological systems, solving the problem that previous solutions cannot directly detect D-lactic acid. The method comprises the following steps: (1) based on the reported D-nucleic acid aptamer sequence that specifically responds to L-lactic acid, designing and synthesizing L-nucleic acid aptamers adsorbed with graphene oxide and labeled with fluorescent molecules; (2) proving that the L-nucleic acid aptamer can specifically respond to D-lactic acid, and constructing a detection system for the L-nucleic acid aptamer to D-lactic acid in a buffer system; (3) extracting serum and constructing a detection system for D-lactic acid in a biological matrix. The method has the advantages of simple operation, rapid response, stable signal, high sensitivity, and no false positive signals, 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 naturally occurring organic acid present in living organisms. It exists in two configurations: L-lactic acid and D-lactic acid. Normal metabolism in higher animals primarily consists of L-lactic acid, while D-lactic acid is primarily produced by various intestinal bacteria or exogenously ingested. Diabetes, traumatic shock, acidosis, intestinal ischemia, and the metabolism and metastasis of malignant tumors can all lead to elevated D-lactic acid concentrations. Therefore, D-lactic acid is also a biomarker for these diseases. Currently, the detection of D-lactic acid content primarily relies on HPLC, gas chromatography, neutralization titration, capillary electrophoresis, and enzymatic methods. These methods require either large instrumentation, complex operation, or interference from the biological matrix, requiring sample pretreatment and hindering immediate detection of D-lactic acid.

[0003] Aptamers are oligonucleotide fragments that can specifically bind to their targets, obtained through artificial screening using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. Currently, there are reports that a D-aptamer that specifically binds to L-lactic acid has been screened. Based on the principle of mirror image recognition, we inferred that the mirror image molecule of this D-aptamer, the L-aptamer, can recognize the mirror image of the original target, D-lactic acid. In this way, we have obtained an oligonucleotide fragment that specifically binds to D-lactic acid without the need for rescreening. At the same time, because this fragment has a mirror image structure of natural nucleic acids, it can resist degradation by nucleases in organisms and is not interfered with by nonspecific proteins, thus stably existing in biological matrices. Summary of the Invention

[0004] The present invention provides a stable, sensitive, and specific method for quantitatively analyzing D-lactic acid in a biological system. The L-aptamer-based D-lactic acid quantitative analysis method of the present invention is characterized in that a D-aptamer sequence that specifically recognizes L-lactic acid is directly converted into an L-aptamer. Based on the principle of chiral substrate specificity, the L-aptamer can also recognize the mirror image of L-lactic acid: D-lactic acid. At the same time, the L-aptamer will not be interfered with 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: a method for highly sensitive detection of D-lactic acid, comprising the following steps:

[0006] Step 1. Based on the reported D-aptamer sequence that specifically responds to L-lactic acid, a fluorescent molecule-labeled L-aptamer fluorescent probe is designed and synthesized: First, the sequence information of the reported D-aptamer that specifically responds to L-lactic acid is obtained. Based on the sequence, a suitable fluorescent molecule (such as FAM) and a chemical synthesis method are selected to introduce a functional group modification that can react with the fluorescent molecule at a specific position of the 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 activating 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 with the aptamer, thereby synthesizing the fluorescent molecule-labeled L-aptamer fluorescent probe. After the reaction is completed, the synthetic product is purified by purification methods 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 methods such as mass spectrometry.

[0007] Step 2: Prove that the L-aptamer has a specific binding ability to D-lactic acid, and construct a detection system for the L-aptamer to 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 potentially interfering substances (such as L-lactic acid, common sugars, amino acids, etc.). The solution is prepared using the buffer system described in claim 3 (phosphate buffer solution) (PBS), 5mM MgCl2, pH 7.4). An equal amount of L-aptamer fluorescent probe is added to each solution system, and the solution is incubated 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 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 does not change significantly or changes very little in the system containing other interfering substances, it is demonstrated that the L-aptamer has specific binding ability to D-lactic acid. The detection system is constructed, and the working concentration of the L-aptamer is determined to be the final concentration of 100 nM as described in claim 4. An appropriate amount of the L-aptamer fluorescent probe is added to a certain volume (e.g., 1 mL) of the buffer system according to claim 3 to a final concentration of 100 nM. The mixture is thoroughly mixed to prepare a buffer detection system containing the L-aptamer fluorescent probe.

[0008] Step 3, extract serum and construct a detection system for D-lactic acid in a biological matrix: serum extraction, collect a blood sample, 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 of 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. Incubate at a suitable temperature (such as 25-37 ° C) for 20 minutes as described in claim 7. After the incubation, use a fluorescence detection device such as an enzyme marker to detect the change in the fluorescence signal of the detection system. According to the pre-established standard curve (constructed by serum samples with known concentrations of D-lactic acid), the content of D-lactic acid in the serum sample is calculated, thereby constructing a detection system for D-lactic acid in the biological matrix.

[0009] Preferably, the signal molecules can 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 using an enzyme-labeled instrument.

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

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

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

[0016] The advantages of this invention are: First, this method is an innovation based on the principle of chiral substrate specificity, demonstrating that while chiral substance A specifically binds to substance B, its mirror image A' also specifically binds to B'; second, this method does not require nucleic acid sequence screening; it simply synthesizes L-aptamer sequences from pre-screened D-aptamer sequences, making the design simple and rapid; third, leveraging L-DNA's excellent resistance to biological matrix interference, this method can be used to detect D-lactic acid in biological systems. Given its advantages such as simple design, good stability, and high sensitivity, our proposed L-aptamer-based method has the potential to become a widespread tool for quantitative detection of D-lactic acid.

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

[0018] 1. The highly sensitive detection method for D-lactic acid of the present invention has a short detection cycle, from sample processing to final result acquisition, and can generally be completed within 2-3 hours. This is due to the rapid binding kinetics of the nucleic acid aptamer to 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 rapid clinical diagnosis. For example, in the emergency room, patients suspected of having D-lactic acid-related diseases can be quickly screened, providing doctors with timely diagnostic evidence to formulate appropriate treatment plans. In the food industry production process, it can also monitor the changes in the D-lactic acid content of fermented products in real time, and adjust the fermentation process parameters in a timely manner to ensure the stability and consistency of product quality, improve production efficiency, reduce production costs, and achieve rapid response.

[0019] 2. The highly sensitive detection method for D-lactic acid of the present invention has good chemical and thermal stability. Compared with some biological enzyme recognition elements, the L-aptamer is not easily affected by environmental factors such as temperature and pH value and inactivated or denatured. In 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 repeated detection of the same sample, the deviation of the D-lactic acid detection results obtained is within an acceptable range. This stability and repeatability make the detection method of the present invention more reliable, conducive to the establishment of standardized detection processes and quality control systems, and has important application value in large-scale clinical testing and industrial production detection. The signal is stable and there are no false positive signals.

[0020] 3. The entire detection process design of the high-sensitivity detection method for 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 complex training for professionals to master it. It 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 concept of the patent. The L-nucleic acid sequence is labeled with a fluorescent molecule and adsorbed onto the graphene oxide (GO) surface. The fluorescence emitted by the fluorescent molecule labeled with the nucleic acid fragment is quenched by the GO. Upon addition of D-lactic acid, the D-lactic acid binds to the nucleic acid fragment, detaches from the GO surface, and the fluorescent molecule resumes its signal. The intensity of the fluorescence signal is linearly correlated with the D-lactic acid concentration, making it suitable for D-lactic acid detection.

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

[0023] Figure 3 The fluorescence signal of the aptamer increased by 3.9 times when 50 mM lactic acid was added to the blank control group.

[0024] Figure 4 The linear relationship of the nucleic acid aptamer probe's response to D-lactic acid in buffer solution is shown, and the detection sensitivity 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's response to D-lactic acid in serum samples is shown in FIG. 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 permit 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 permits the presence of only the specified ingredients / steps and any unavoidable impurities that may result therefrom, and excludes other ingredients / steps.

[0029] The numerical values ​​in the specification and claims of this application should be understood to include the same value when reduced to the same number of significant figures and 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 stated 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 value that can vary without changing the basic function of the value. When used in conjunction 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". In general, 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, percentages of elements are to be considered as percentages by weight of the alloy in question.

[0033] The present disclosure may refer to temperatures for 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 intended to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are for illustrative purposes only, and those skilled in the art will readily appreciate other obvious variations. The basic principles of the present invention defined in the following description may be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the present invention.

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

[0036] The D-type aptamer sequence for detecting L-lactic acid reported in the literature was directly converted to an L-type aptamer sequence. The L-DNA synthetic monomers were purchased from ChemGene and dissolved in acetonitrile to a concentration of 0.05 g / ml. The sequence was synthesized using CPG labeled with FITC at the three ends on an automatic DNA synthesizer. After synthesis, the CPG was transferred to a centrifuge tube and aminolyzed with 0.5 mL of saturated ammonia at 65°C for 8 h to cleave the DNA from the CPG. After aminolysis, the ammonia was removed and the supernatant was collected by centrifugation at 10,000 rpm for 10 min. 0.5 mL of ultrapure water was then added to the CPG to elute the sample adsorbed on the CPG. The supernatant was collected by centrifugation at 10,000 rpm for 10 min. The resulting supernatants were combined to form the crude product. The crude product was concentrated, dissolved in 0.1 M triethylamine acetate (TEAA), and purified using reversed-phase high-performance liquid chromatography.

[0037] The product purified by reverse phase-HPLC was vacuum dried and dissolved in ultrapure water. It was desalted using a gel filtration column and concentrated in 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] Using PBS buffer as the standard buffer, for each sample to be tested, 100 mL of PBS buffer, 100 nM L-aptamer probe, and 20 mg / mL GO were prepared and 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 a microplate reader using a fixed-point detection mode with an excitation wavelength of 485 nm and a detection emission wavelength of 520 nm. The measured fluorescence signal was 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 Using L-aptamer / GO Sensor

[0041] 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.

[0042] Example 4: A highly sensitive method for detecting D-lactic acid, comprising the following steps:

[0043] Step 1: Based on the reported D-aptamer sequence that specifically responds to L-lactic acid, a fluorescent aptamer probe labeled with a fluorescent molecule was designed and synthesized:

[0044] 1. First, obtain the sequence information of the reported D-aptamer that specifically responds to L-lactic acid.

[0045] 2. Based on the sequence, a suitable fluorescent molecule (such as FAM) and chemical synthesis method are selected to introduce functional groups that react with the fluorescent molecule 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 activating group is added to cause 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 with the nucleic acid aptamer to synthesize the fluorescent molecule-labeled L-nucleic acid aptamer fluorescent probe. After the reaction is completed, the synthetic product is purified by purification methods 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 its structure is characterized and confirmed by methods such as mass spectrometry.

[0046] Step 2: Prove that the L-aptamer has the specific binding ability to D-lactic acid, and construct a detection system for the L-aptamer to D-lactic acid in a buffer solution:

[0047] 1. Demonstration of specific binding ability:

[0048] Prepare 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.). The solutions are prepared using the buffer system described in claim 3 (phosphate buffered saline) (PBS), 5mM MgCl2, pH 7.4).

[0049] An equal amount of L-aptamer fluorescent probe is added to each solution system, and the mixture is incubated at a suitable temperature (eg, 25-37° C.) for a certain period of time (eg, 10-30 minutes).

[0050] After the incubation, a fluorescence spectrometer is used to detect 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 in the system containing other interfering substances has no obvious change or changes very little, it proves that the L-nucleic acid aptamer has the specific binding ability to D-lactic acid.

[0051] 2. Construction of detection system:

[0052] The working concentration of the L-aptamer is determined to be the final concentration of 100 nM as described in claim 4. An appropriate amount of the L-aptamer fluorescent probe is added to a certain volume (e.g., 1 mL) of the buffer system described in claim 3 to a final concentration of 100 nM. The mixture is thoroughly mixed to prepare a buffer detection system containing the L-aptamer fluorescent probe.

[0053] Step 3: Extract serum and construct a detection system for D-lactic acid in biological matrix:

[0054] 1. Serum extraction:

[0055] Collect a blood sample and use centrifugation technology to centrifuge 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.

[0056] 2. Construction of biological matrix detection system:

[0057] A certain amount of serum sample (such as 100 μL, i.e., the reaction volume according to claim 5) 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. The sample is 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 using a fluorescence detection device such as a microplate reader. The content of D-lactic acid in the serum sample is calculated according to a pre-established standard curve (constructed using serum samples with known D-lactic acid concentrations), thereby constructing a detection system for D-lactic acid in a biological matrix.

[0058] The specific steps are as follows: the staff designed and synthesized an L-nucleic acid aptamer fluorescent probe labeled with a fluorescent molecule. First, the D-nucleic acid aptamer sequence information that has been reported to specifically respond 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 a functional group modification that can react with the fluorescent molecule at a specific position 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 to synthesize the L-nucleic acid aptamer fluorescent probe labeled with a fluorescent molecule. 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, 5mMMgCl2, pH7.4. 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 a solution system containing D-lactic acid, while the fluorescence intensity does not change significantly or changes very little in a 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 at the final concentration of 100 nM level as described in claim 4, adds an appropriate amount of the 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 centrifugation separation technology, and centrifuge at a low temperature, such as 4°C, and a certain speed, such as 3 The sample is centrifuged at 1000-5000 rpm for 10-20 minutes, and the upper serum layer is collected. The serum is then appropriately pretreated, such as filtering through a 0.22 μm filter membrane to remove any impurities, to obtain a pure serum sample. At this point, the staff constructs a biological matrix detection system: a certain amount of serum sample, such as 100 μL, i.e., the reaction volume, is added to a buffer detection system containing an L-aptamer fluorescent probe to a serum concentration of 1%. The sample is incubated at an appropriate temperature, such as 25-37°C, for 20 minutes. After the incubation, the fluorescence signal change of the system is detected using a fluorescence detection device such as a microplate reader. The D-lactic acid content in the serum sample is calculated based on a pre-established standard curve using serum samples with known D-lactic acid concentrations, thereby constructing a detection system for D-lactic acid in the biological matrix.

[0059] 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, replacements 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 appended claims and their equivalents. This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment as needed without creative contribution, but as long as they are within the scope of the claims of the present application, they are protected by patent law.

Claims

1. A highly sensitive method for detecting D-lactic acid, characterized in that: The following steps are involved: Step 1: Based on the reported D-aptamer sequence that specifically responds to L-lactic acid, a fluorescent aptamer probe labeled with a fluorescent molecule was designed and synthesized; Step 2: Prove that the L-aptamer has a specific binding ability to D-lactic acid, and construct a detection system for the L-aptamer to D-lactic acid in a buffer solution; Step 3: extracting serum and constructing a detection system for D-lactic acid in biological matrix; The signal molecules are FITC and graphene oxide. In the absence of D-lactic acid, the L-aptamer labeled with FAM is adsorbed on the graphene oxide surface, and the fluorescence emitted by the FAM group is quenched by graphene oxide. In the presence of D-lactic acid, the aptamer combines with D-lactic acid to form a secondary structure that detaches from the graphene oxide surface and emits a fluorescent signal.

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