CN116287133A - Ribonucleic acid biosensor, and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a ribonucleic acid biosensor, a preparation method and application thereof, wherein the preparation method comprises the following steps: 1) Culturing 10-200 parts by weight of an oligonucleotide carrier in 0.01-0.2 parts by weight of an oligonucleotide solution to obtain an oligonucleotide carrier A; 2) Mixing and reacting the oligonucleotide carrier A obtained in the step 1), 20-200 parts by weight of carbodiimide, 5-50 parts by weight of N-hydroxy thiosuccinimide and 50-500 parts by weight of oligonucleotide target for 2-24 hours to obtain an oligonucleotide carrier B; 3) Adding cas13a carrying a targeting sequence to the oligonucleotide carrier B obtained in the step 2). The invention develops the cas13 a-based high-sensitivity ribonucleic acid biosensor, which has high detection sensitivity and is convenient to use.
Description
Technical Field
The invention relates to a ribonucleic acid biosensor, a preparation method and application thereof.
Background
Riboviruses are the most widely distributed viruses in nature, and some of them are severely threatening to human health and life. To date, there are few specific drugs to treat viral infections, the primary means of combating viral infections being vaccination. Vaccination is the most effective method to combat the known and long-lived riboviruses. Unlike deoxyriboviruses, the extreme instability of riboviruses makes them often mutated, for example, common influenza viruses are classified into three types a, b, and c and several tens of subtypes, and each year influenza is epidemic, the kind is first determined and then the corresponding vaccine is inoculated for prevention.
There are two main novel coronavirus detection methods: antigen detection and real-time fluorescent quantitative PCR based on Polymerase Chain Reaction (PCR). The antigen detection is very suitable for home self-detection due to the characteristics of rapidness, convenience and the like, but can only be used as an auxiliary means at the present stage. The gold standard of the novel coronavirus detection is real-time fluorescence quantitative PCR, and the severe sample treatment and detection equipment requirements of the novel coronavirus detection can only be carried out in a central laboratory with perfect infrastructure.
Disclosure of Invention
In one aspect, the invention provides a method for preparing a ribonucleic acid biosensor, comprising the following steps:
1) Culturing 10-200 parts by weight of an oligonucleotide carrier in 0.01-0.2 parts by weight of an oligonucleotide solution to obtain an oligonucleotide carrier A; wherein the oligonucleotide carrier comprises a gold electrode or magnetic beads coated with avidin;
2) Mixing and reacting the oligonucleotide carrier A obtained in the step 1), 20-200 parts by weight of carbodiimide, 5-50 parts by weight of N-hydroxy thiosuccinimide and 50-500 parts by weight of oligonucleotide target for 2-24 hours to obtain an oligonucleotide carrier B; the oligonucleotide target comprises N6- (6-aminohexyl) flavin adenine dinucleotide, quantum dots with amino on the surface or Prussian blue nano particles with amino on the surface;
3) Adding cas13a carrying a targeting sequence to the oligonucleotide carrier B obtained in the step 2).
Preferably, the targeting sequence comprises 5'-UUGCUGCUGCUUGACAGAUU-3', 5' -AACUAUACAACCUACUACCUCA-3 or 5'-UUACUCCUUGGAGGCCAUGUAGG-3'.
Preferably, in step 1), 50 to 120 parts by weight of the oligonucleotide carrier; 0.02-0.1 parts by weight of the oligonucleotide solution.
Preferably, in the step 2), 50 to 120 parts by weight of the carbodiimide; 10-30 parts by weight of N-hydroxy thiosuccinimide; 100-300 parts by weight of the oligonucleotide target.
Preferably, in step 2), the reaction time is 6 to 15 hours.
On the other hand, the invention also provides the ribonucleic acid biosensor prepared by the preparation method of the ribonucleic acid biosensor.
On the other hand, the invention also provides application of the ribonucleic acid biosensor, when the oligonucleotide target is N6- (6-aminohexyl) flavin adenine dinucleotide, the target ribonucleic acid and the ribonucleic acid biosensor are mixed and cultured for 1-30 minutes, then the oligonucleotide carrier is taken out, and a chromogenic culture solution of aminoantipyrine, 2-hydroxy-3-m-toluidine propane sodium sulfonate and peroxidase is added, and spectrophotometry is carried out after the reaction is completed.
On the other hand, the invention also provides application of the ribonucleic acid biosensor, when the oligonucleotide target is a quantum dot with amino on the surface, the target ribonucleic acid and the ribonucleic acid biosensor are mixed and cultured for 1-30 minutes, then the oligonucleotide carrier is taken out, and the solution is measured under ultraviolet light.
On the other hand, the invention also provides application of the ribonucleic acid biosensor, when the oligonucleotide target is Prussian blue nano particles with amino groups on the surfaces, the target ribonucleic acid and the ribonucleic acid biosensor are mixed and cultured for 1-30 minutes, then the oligonucleotide carrier is taken out, and current is detected in PBS solution containing ascorbic acid.
Preferably, the target ribonucleic acid comprises novel coronavirus ribonucleic acid, microribonucleic acid let-7a or GAPDH messenger ribonucleic acid.
In summary, the invention has the following beneficial effects:
the invention successfully develops a cas13 a-based high-sensitivity ribonucleic acid biosensor, when target ribonucleic acid exists, for example, ribonucleic acid of novel coronavirus, the ribonucleic acid hybridizes with a targeting sequence of cas13a under the condition of room temperature, cas13a is activated, and the detection sensitivity is greatly improved through the cooperative amplification of cas13a and chemical/electrochemical catalysis, and the use is more convenient.
Drawings
FIG. 1 is a schematic diagram of the working principle of a ribonucleic acid biosensor containing cas13a;
FIG. 2 is a graph of UV-visible spectra of different concentrations of novel coronavirus ribonucleic acids (synthetic fragments);
FIG. 3 shows the fluorescence intensities of different concentrations of novel coronavirus ribonucleic acids (synthetic fragments);
FIG. 4 shows the current response of different concentrations of novel coronavirus ribonucleic acid (synthetic fragment) after incubation for different times.
Detailed Description
The invention is further described with reference to the accompanying drawings.
The embodiment discloses a preparation method of a ribonucleic acid biosensor, which comprises the following steps:
1) Culturing 10-200 parts by weight of an oligonucleotide carrier in 0.01-0.2 parts by weight of an oligonucleotide solution for 1-6 hours, and then cleaning the oligonucleotide carrier for 1-5 times to obtain an oligonucleotide carrier A; wherein the oligonucleotide carrier comprises a gold electrode or magnetic beads coated with avidin; preferably, 50-120 parts by weight of an oligonucleotide carrier; 0.02-0.1 parts by weight of an oligonucleotide solution;
2) Mixing the oligonucleotide carrier A obtained in the step 1), 20-200 parts by weight of carbodiimide, 5-50 parts by weight of N-hydroxysulfosuccinimide and 50-500 parts by weight of oligonucleotide target, reacting for 2-24 hours at 4 ℃ on a shaking table, and then cleaning the oligonucleotide carrier A to obtain an oligonucleotide carrier B; the oligonucleotide target comprises N6- (6-aminohexyl) flavin adenine dinucleotide, quantum dots with amino on the surface or Prussian blue nano particles with amino on the surface; preferably, 50-120 parts by weight of carbodiimide; 10-30 parts by weight of N-hydroxysulfosuccinimide; 100-300 parts by weight of an oligonucleotide target; further, the reaction time is 6-15 hours;
3) Adding cas13a carrying a targeting sequence to the oligonucleotide carrier B obtained in the step 2); preferably, the targeting sequence comprises 5'-UUGCUGCUGCUUGACAGAUU-3', 5' -AACUAUACAACCUACUACCUCA-3 or 5'-UUACUCCUUGGAGGCCAUGUAGG-3'.
Wherein, the sequence: UUGCUGCUGCUUGACAGAUU, SEQ ID NO 1; sequence: AACUAUACAACCUACUACCUCA, SEQ ID NO 2; sequence: UUACUCCUUGGAGGCCAUGUAGG, SEQ ID NO 3.
The ribonucleic acid biosensor prepared by the preparation method of the ribonucleic acid biosensor.
When the oligonucleotide target is N6- (6-amino hexyl) flavin adenine dinucleotide, mixing and culturing the target ribonucleic acid and the ribonucleic acid biosensor for 1-30 minutes, then taking out an oligonucleotide carrier, adding a chromogenic culture solution of aminoantipyrine, 2-hydroxy-3-m-toluidine propane sodium sulfonate and peroxidase, and carrying out spectrophotometry at 555nm after the reaction is completed.
When the oligonucleotide target is a quantum dot with amino on the surface, the target ribonucleic acid and the ribonucleic acid biosensor are mixed and cultured for 1-30 minutes, then the oligonucleotide carrier is taken out, and the solution is measured under ultraviolet light.
When the oligonucleotide target is Prussian blue nano particles with amino groups on the surface, mixing and culturing the target ribonucleic acid and the ribonucleic acid biosensor for 1-30 minutes, taking out the oligonucleotide carrier, washing the oligonucleotide carrier for 1-5 times by using 0.05M phosphate buffer solution with pH of 7.0, and detecting current in PBS solution containing 1-100 mmol/L ascorbic acid at 0-0.3V. Specific examples are shown below:
preparation of colorimetric ribonucleic acid biosensor: 100mg of the avidin-coated magnetic beads were incubated in a solution of 0.05mg of biotin oligonucleotide (also carrying terminal carboxyl groups) for 4 hours, then washed 3 times with water, then 100mg of carbodiimide and 25mg of N-hydroxysulfosuccinimide were put in, and 200mg of N6- (6-aminohexyl) flavin adenine dinucleotide was added, reacted on a shaker at 4℃for 8 hours, and after completion of the reaction, the magnetic beads were washed 3 times with a 0.05M phosphate buffer solution at pH 7.0. After the treatment, N6- (6-amino hexyl) flavin adenine dinucleotide is coupled on the oligonucleotide, and finally, the flavin adenine dinucleotide prosthetic group at the tail end of the oligonucleotide and the apo-glucose oxidase are recombined and embedded into the apo-glucose oxidase to reduce the apo-glucose oxidase into the original glucose oxidase to be used as a marker of a target sequence. Then 5'-UUGCUGCUGCUUGACAGAUU-3' is used as a cas13a targeting sequence to prepare a ribonucleic acid biosensor, and the biosensor becomes a colorimetric type biosensor with high selectivity and sensitivity to novel coronavirus ribonucleic acid.
When there is a target ribonucleic acid in a nucleic acid sample, such as ribonucleic acid of a novel coronavirus, after cas13a carrying a targeting sequence is added, the target ribonucleic acid hybridizes with the targeting sequence, cas13a is activated, the activated cas13a cleaves an oligonucleotide with glucose oxidase at the end, and the number of cleaves increases with the increase of the culture time, and in order to rapidly detect the novel coronavirus gene while maintaining higher sensitivity, the culture time is usually 10 minutes. After the completion of the culture, the beads were removed by a magnet, and then a chromogenic medium of aminoantipyrine, sodium 2-hydroxy-3-m-toluidine propane sulfonate and peroxidase was added thereto, and after the completion of the reaction, spectrophotometry was performed at 555nm (see FIG. 2).
Preparation of a fluorescence ribonucleic acid biosensor: 100mg of the avidin-coated magnetic beads were incubated in 0.05mg of biotin oligonucleotide (also carrying terminal carboxyl groups) for 4 hours, then washed 3 times with water, then 100mg of carbodiimide and 25mg of N-hydroxysulfosuccinimide were put in, 200mg of quantum dots having amino groups on the surface were added, reacted on a shaker at 4℃for 8 hours, and after completion of the reaction, the magnetic beads were washed 3 times with a 0.05M phosphate buffer solution at pH 7.0. After the above treatment, the quantum dot is attached to the end of the oligonucleotide. Then 5'-UUGCUGCUGCUUGACAGAUU-3' is used as a targeting sequence of cas13a to prepare a ribonucleic acid biosensor, and the biosensor becomes a fluorescent biosensor with high selectivity and sensitivity to ribonucleic acid of novel coronaviruses. When there is a target ribonucleic acid in a nucleic acid sample, such as ribonucleic acid of a novel coronavirus, after cas13a carrying a targeting sequence is added, the target ribonucleic acid hybridizes with the targeting sequence, cas13a is activated, the activated cas13a cleaves oligonucleotides with quantum dots at the end, and the number of cleaves increases with the increase of culture time, the culture time is usually 10 minutes in order to rapidly detect the novel coronavirus while maintaining higher sensitivity. After the culture is completed, the magnetic beads are taken out by a magnet, and the fluorescence intensity of the quantum dots in the solution is measured under the excitation of ultraviolet light with the wavelength of 365 nm. As shown in fig. 3, the fluorescence intensity of the quantum dots is directly related to the concentration of the novel coronavirus ribonucleotides.
Preparation of electrochemical ribonucleic acid biosensor: nanosstrip is combined with TM After 100mg of the washed gold electrode was incubated in 0.05mg of a thiol oligonucleotide (also carrying a terminal carboxyl group) solution for 4 hours, washed 3 times with water, then 100mg of carbodiimide and 25mg of N-hydroxysulfosuccinimide were put in, 200mg of Prussian blue nanoparticles having amino groups on the surface were added, and reacted on a shaker at 4℃for 4 hours, after the completion of the reaction, the gold electrode was washed 3 times with a 0.05M phosphate buffer solution of pH 7.0. After the treatment, prussian blue nano-particles are connected to the tail end of the oligonucleotide. Then 5'-UUGCUGCUGCUUGACAGAUU-3' is used as a cas13a targeting sequence to prepare a ribonucleic acid biosensor, and the biosensor becomes an electrochemical biosensor with high selectivity and sensitivity to novel coronavirus ribonucleic acid. When there is a target ribonucleic acid in a nucleic acid sample, such as ribonucleic acid of a novel coronavirus, after Cas13a carrying a targeting sequence is added, the target ribonucleic acid hybridizes with the targeting sequence, cas13a is activated, cas13a after activation cleaves an oligonucleotide with Prussian blue nanoparticles at the end, and the number of cleaves increases with the increase of culture time, and in order to rapidly detect a novel coronavirus gene while maintaining higher sensitivity, the culture time is usually 10 minutes. After completion of the culture, the electrode was taken out and dissolved in 0.05M phosphate buffer pH7.0The solution was washed 3 times and then the current was measured at 0.2V in a PBS solution containing 25 mmol/l ascorbic acid. FIG. 4 shows the current response of different concentrations of novel coronavirus ribonucleic acid (synthetic fragment) after incubation for different times. As shown in FIG. 4, the longer the incubation time or the higher the concentration of novel coronavirus ribonucleic acid, the smaller the detected current. This is because the more Prussian blue nanoparticles as electrochemical catalysts are cut from the electrode, the less is left on the gold electrode, and the lower is the electrochemical catalytic oxidation current of ascorbic acid.
In addition, if 5' -AACUAUACAACCUACUACCUCA-3 is used as the cas13a targeting sequence, a ribonucleic acid biosensor is produced, and the biosensor becomes a biosensor with high selectivity and sensitivity to the microribonucleic acid let-7 a. If 5'-UUACUCCUUGGAGGCCAUGUAGG-3' is used as a cas13a targeting sequence, the ribonucleic acid biosensor is prepared, and the GAPDH messenger ribonucleic acid biosensor is obtained.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (10)
1. The preparation method of the ribonucleic acid biosensor is characterized by comprising the following steps: the method comprises the following steps:
1) Culturing 10-200 parts by weight of an oligonucleotide carrier in 0.01-0.2 parts by weight of an oligonucleotide solution to obtain an oligonucleotide carrier A; wherein the oligonucleotide carrier comprises a gold electrode or magnetic beads coated with avidin;
2) Mixing and reacting the oligonucleotide carrier A obtained in the step 1), 20-200 parts by weight of carbodiimide, 5-50 parts by weight of N-hydroxy thiosuccinimide and 50-500 parts by weight of oligonucleotide target for 2-24 hours to obtain an oligonucleotide carrier B; the oligonucleotide target comprises N6- (6-aminohexyl) flavin adenine dinucleotide, quantum dots with amino on the surface or Prussian blue nano particles with amino on the surface;
3) Adding cas13a carrying a targeting sequence to the oligonucleotide carrier B obtained in the step 2).
2. The method for preparing ribonucleic acid biosensor according to claim 1, where: the targeting sequence includes 5'-UUGCUGCUGCUUGACAGAUU-3', 5' -AACUAUACAACCUACUACCUCA-3 or 5'-UUACUCCUUGGAGGCCAUGUAGG-3'.
3. The method for preparing ribonucleic acid biosensor according to claim 1, where: in the step 1), 50-120 parts by weight of the oligonucleotide carrier; 0.02-0.1 parts by weight of the oligonucleotide solution.
4. The method for preparing ribonucleic acid biosensor according to claim 3, where: in the step 2), 50-120 parts by weight of carbodiimide; 10-30 parts by weight of N-hydroxy thiosuccinimide; 100-300 parts by weight of the oligonucleotide target.
5. The method for preparing ribonucleic acid biosensor according to claim 4, where: in the step 2), the reaction time is 6-15 hours.
6. A ribonucleic acid biosensor prepared by the method for preparing a ribonucleic acid biosensor according to any one of claims 1 to 5.
7. The use of the ribonucleic acid biosensor of claim 6, where: when the oligonucleotide target is N6- (6-aminohexyl) flavin adenine dinucleotide, mixing and culturing the target ribonucleic acid and a ribonucleic acid biosensor for 1-30 minutes, then taking out an oligonucleotide carrier, adding an amino antipyrine, 2-hydroxy-3-m-toluidine propane sodium sulfonate and a chromogenic culture solution of peroxidase, and carrying out spectrophotometry after the reaction is completed.
8. The use of the ribonucleic acid biosensor of claim 6, where: when the oligonucleotide target is a quantum dot with amino on the surface, mixing and culturing the target ribonucleic acid and ribonucleic acid biosensor for 1-30 minutes, then taking out the oligonucleotide carrier, and measuring the solution under ultraviolet light.
9. The use of the ribonucleic acid biosensor of claim 6, where: when the oligonucleotide target is Prussian blue nano particles with amino groups on the surface, mixing and culturing the target ribonucleic acid and ribonucleic acid biosensor for 1-30 minutes, taking out the oligonucleotide carrier, and detecting current in PBS solution containing ascorbic acid.
10. The use of the ribonucleic acid biosensor according to any one of claims 7 to 9, characterised in that: the target ribonucleic acid comprises novel coronavirus ribonucleic acid, microribonucleic acid let-7a or GAPDH messenger ribonucleic acid.
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