Portable method for detecting content of adenosine triphosphate (ATP)

Abstract

The invention discloses a portable method for detecting content of adenosine triphosphate (ATP). The method comprises the following steps of: respectively modifying a magnetic bead and invertase on an ATP aptamer which is split into two sections, performing specific identification and combination on the ATP aptamer which is split into two sections and a target ATP so as to form a sandwich structure, so that the aptamer modified with the invertase is fixed on the magnetic bead; separating the magnetic bead from the solution, dissolving the cleaned magnetic bead in a sucrose solution for carrying out an enzymatic hydrolysis reaction, measuring the reaction solution subjected to enzymatic hydrolysis by employing a glucometer, and indirectly detecting the concentration of ATP. The method is successfully applied to detection of the ATP concentration in a human serum sample, and the method has the advantages of simple operation, low cost, high sensitivity, high specificity and the like.

Description

technical field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a portable method for detecting the content of adenosine triphosphate. Background technique

[0002] Adenosine triphosphate (ATP) is the direct and main source of energy required for all life activities of tissue cells in the body. It can store and transfer chemical energy, participate in the synthesis of proteins, fats, sugars and nucleotides, and can promote the repair of various cells in the body and regeneration, enhance cell metabolism activity, and have strong pertinence for the treatment of various diseases. Therefore, quantitative detection of ATP content has important practical significance for scientific research and clinical diagnosis.

[0003] Aptamers are a class of single-stranded DNA or RNA oligonuclei that can bind to target molecules with high affinity and recognize target molecules with high specificity through in vitro screening by exponential e...

Examples

Embodiment 1

[0031] The following examples illustrate the operation process of applying the method of the present invention to the detection of ATP concentration in human serum samples in conjunction with the accompanying drawings:

[0032] 1. Synthesis of DNA compounds modified with sucrose invertase. References (Hermanson, G. T. Bioconjugate Techniques; Elsevier: London, 2008). Specific steps are as follows:

[0033] A. Mix 30 μM sulfhydryl-modified DNA with 60 μM TCEP in PBS buffer solution and incubate at room temperature for 1 hour;

[0034] B. Mix 10.0 mg / mL sucrose invertase and 1 mg Sulfo-SMCC in a buffer solution (0.01 M PBS, pH=7.3, 0.1 M NaCl) for 5 min, place on a shaker at room temperature for 1 hour, and shake the mixture Centrifuge and take the supernatant;

[0035] C. Mix the substances prepared in A and B, and react at room temperature for 48 hours.

[0036] 2. The preparation of magnetic beads modified with sucrose invertase and the detection of ATP, the specific step...

Embodiment 2

[0042] 1. The detection of ATP concentration in human serum, the specific steps are as follows: add 10 dilutions to the 4.0 uM two modified DNA mixtures 3 Double human serum sample, incubated at room temperature for (1.5±0.05) hours, then separated the mixture with a magnet, washed the magnetic beads with 50 uL buffer solution three times and added 10 uL buffer solution, then added 20 uL 1 M sucrose solution, and reacted at 45°C After 20 minutes, pipette 2.5 μL to measure with a blood glucose meter and record the data. The test results show that the ATP concentration in the human serum sample is about 1.81×10 -10 M.

[0043] 2. The specificity of the method of the present invention to the detection of ATP, the specific steps are as follows: in order to detect the specificity of the method of the present invention, the ATP used in the embodiment 1-2 is replaced with other interfering substances, respectively UTP , GTP and CTP, the concentration of ATP is 1×10 -12 M, and th...

technical field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a portable method for detecting the content of adenosine triphosphate. Background technique

[0002] Adenosine triphosphate (ATP) is the direct and main source of energy required for all life activities of tissue cells in the body. It can store and transfer chemical energy, participate in the synthesis of proteins, fats, sugars and nucleotides, and can promote the repair of various cells in the body and regeneration, enhance cell metabolism activity, and have strong pertinence for the treatment of various diseases. Therefore, quantitative detection of ATP content has important practical significance for scientific research and clinical diagnosis.

[0003] Aptamers are a class of single-stranded DNA or RNA oligonuclei that can bind to target molecules with high affinity and recognize target molecules with high specificity through in vitro screening by exponential e...

Examples

Embodiment 1

[0031] The following examples illustrate the operation process of applying the method of the present invention to the detection of ATP concentration in human serum samples in conjunction with the accompanying drawings:

[0032] 1. Synthesis of DNA compounds modified with sucrose invertase. References (Hermanson, G. T. Bioconjugate Techniques; Elsevier: London, 2008). Specific steps are as follows:

[0033] A. Mix 30 μM sulfhydryl-modified DNA with 60 μM TCEP in PBS buffer solution and incubate at room temperature for 1 hour;

[0034] B. Mix 10.0 mg / mL sucrose invertase and 1 mg Sulfo-SMCC in a buffer solution (0.01 M PBS, pH=7.3, 0.1 M NaCl) for 5 min, place on a shaker at room temperature for 1 hour, and shake the mixture Centrifuge and take the supernatant;

[0035] C. Mix the substances prepared in A and B, and react at room temperature for 48 hours.

[0036] 2. The preparation of magnetic beads modified with sucrose invertase and the detection of ATP, the specific step...

Embodiment 2

[0042] 1. The detection of ATP concentration in human serum, the specific steps are as follows: add 10 dilutions to the 4.0 uM two modified DNA mixtures 3 Double human serum sample, incubated at room temperature for (1.5±0.05) hours, then separated the mixture with a magnet, washed the magnetic beads with 50 uL buffer solution three times and added 10 uL buffer solution, then added 20 uL 1 M sucrose solution, and reacted at 45°C After 20 minutes, pipette 2.5 μL to measure with a blood glucose meter and record the data. The test results show that the ATP concentration in the human serum sample is about 1.81×10 -10 M.

[0043] 2. The specificity of the method of the present invention to the detection of ATP, the specific steps are as follows: in order to detect the specificity of the method of the present invention, the ATP used in the embodiment 1-2 is replaced with other interfering substances, respectively UTP , GTP and CTP, the concentration of ATP is 1×10 -12 M, and th...

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