CN109884299B - One-step fluorescence detection system and thrombin detection method - Google Patents

Abstract

The invention provides a one-step fluorescence detection system and a thrombin detection method, and relates to the technical field of analytical chemistry. The detection system comprises the B-H2 functionalized nanofiber membrane, supporting DNA, outputting DNA, competitive DNA, buffer solution, thioflavin T (ThT), H1 sequence and thrombin, and when the detection system is used for detecting the thrombin, the corresponding detection system has a detection limit of 1.0pM and has a good linear relation between 50pM and 5nM, excellent selectivity and long-term stability based on the improvement of reaction thermodynamics and kinetics on the surface interface of the nanofiber membrane. When the detection system is used for sample measurement, the separation and elution processes required by ELISA are avoided by integrating the PiDSD process, the CHA amplification and the ThT binding reaction, so that the time is saved, and the steps are simplified.

Description

One-step fluorescence detection system and thrombin detection method

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a one-step fluorescence detection system and a thrombin detection method.

Background

Sensitive, convenient, reliable and low-cost detection of protein markers is increasingly important in point-of-care testing (POCT) and early diagnosis. Detection methods such as enzyme-linked immunosorbent assay (ELISA), electrochemical assay, optical method, etc. are now common. These methods involve multiple separation and elution steps, are complicated to operate, require sophisticated instrumentation, have relatively low sensitivity, and the like.

The electrostatic spinning nanofiber membrane has the advantages of controllable diameter, porous structure, high specific surface area, easiness in modification and the like, and is considered to be an ideal sensing interface material. Electro-spun nanofiber membrane based photochemical sensors have been reported for the detection of protein markers such as Prostate Specific Antigen (PSA), alpha-fetoprotein (AFP), cardiac troponin i (ctni), C-reactive protein (CRP), and thrombin.

Thrombin is a serine protease that, during metabolism, converts soluble fibrinogen into insoluble fibrin chains. The Xiang subject group reported an electrochemical aptamer sensor with a thrombin detection limit as low as 5.6pM that combines both proximity-binding-induced strand displacement reaction and ion-dependent DNAzyme cycle signal amplification (J.M.Yang, B.T.Dou, R.Yuan, Y.X.Proximative binding and metal-dependent DNAzyme cycle amplification-integrated aptamer for label-free and sensitive electrochemical detection and analysis, 2016,88, 8218-8223). The Gu subject group reported a sandwich-type thrombin sensor based on aptamer-functionalized electrospinning, with a detection limit of 10pM, 2500-fold lower than that of 96-well plates (s.j.lee, r.tavarty, m.b.gu.electrospun polystyrene-poly (styrene-co-maleic anhydride) nanofiber as a new aptamer sensor for biosensors & Bioelectronics,2012,38, 302-. The Liu project group reported an electrospun based fluorescent thrombin sensor with a detection limit of 42pM (H.M.Wang, W.Tang, H.J.Wei, Y.ZHao, S.C.Hu, Y.Guan, W.Pan, B.Xia, N.Li, F.Liu.integrating DNA drivers with electrospinun nanofillers: a new fluorescent sensing platform for nucleic acids, proteins, and cells. journal of Materials chemistry B2015, 3, 3541-. These methods have a high sensitivity but involve multiple reaction and elution steps, making the entire experiment time and labor consuming.

The Xing topic set is based on two fluorophore-labeled aptamer probes of different wavelengths and fluorescence correlation spectroscopy (FCCS) such that the detection limit of thrombin is 0.8nM (X.M.Zhou, Y.H.Tang, D.xing.one-step homology protein detection based on aptamer probe and fluorescence cross-correlation. These one-step methods reduce the number of experimental steps and save time. However, these detection methods require labeling of the probe and preparation of nanoparticles, and are also low in sensitivity.

At present, a method for detecting thrombin by a one-step method with simple operation and high sensitivity does not exist.

Disclosure of Invention

In view of the above, the present invention aims to provide a one-step fluorescence detection system and a thrombin detection method, which are simple to operate and have high sensitivity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a detection system for fluorescence detection of thrombin by a one-step method, which comprises a B-H2 functionalized nanofiber membrane, a support DNA, an output DNA, a competitive DNA, a buffer solution, a ThT, H1 sequence and thrombin; the sequence of the support DNA is shown as SEQ ID NO. 2-7, the sequence of the output DNA is shown as SEQ ID NO.8, the sequence of the competitive DNA is shown as any one of SEQ ID NO. 9-14, and the sequence of H1 is shown as SEQ ID NO. 15; the buffer solution comprises the following components in concentration: 10 to 50mM of Tris in a concentration of 10 to 50mM,50～200mM NaCl，5～15mM MgCl₂and 10-30 mM KCl, and the pH value is 7.5.

Preferably, the preparation method of the B-H2 functionalized nanofiber membrane comprises the following steps: (1) mixing polystyrene and tetrabutylammonium bromide, and dissolving the mixture in N, N-dimethylformamide to obtain a PS/TBAB/DMF solution; the mass of the polystyrene is 15-25% of that of the PS/TBAB/DMF solution; the mass volume ratio of the tetrabutylammonium bromide in the PS/TBAB/DMF solution is 0.1-0.5%.

(2) Performing electrostatic spinning on the PS/TBAB/DMF solution, and drying to obtain a PS nanofiber membrane; the voltage during electrostatic spinning is 10-20 kV, the sample introduction speed is 2-8 mu L/min, the receiving distance is 7-15 cm, and the collection time is 1.5-3 h;

(3) processing the PS nanofiber membrane by using argon plasma generated by dielectric barrier discharge to obtain a processed PS nanofiber membrane; the processing voltage is 40-50V, the current is 1.2-2.5A, and the discharge processing time is 1-3 min;

(4) soaking the treated PS nano-fiber membrane in an avidin solution for 0.5-1.3H, taking out and cleaning, then reacting with a B-H2 solution for 0.6-1.5H, and cleaning to obtain a B-H2 functionalized nano-fiber membrane; the B-H2 is a nucleotide sequence marked by biotin, and the nucleotide sequence is shown as SEQID NO. 1.

Preferably, the mass volume ratio of the tetrabutylammonium bromide in the PS/TBAB/DMF solution in the step (1) is 0.1-0.5%.

Preferably, the dissolving in the step (1) is accompanied by stirring, and the stirring time is 20-28 h.

Preferably, the drying temperature in the step (2) is 75-85 ℃, and the drying time is 3.2-4.5 h.

Preferably, after the PS nanofiber membrane is obtained in step (2), the PS nanofiber membrane is trimmed.

Preferably, the concentration of the avidin solution in the step (4) is 1.2-2.5 μ M.

Preferably, the concentration of the B-H2 solution in the step (4) is 600-1000 nM.

The invention also provides a method for fluorescence detection of thrombin by one-step method, which comprises the following steps: mixing the support DNA, the output DNA and the buffer solution, and annealing at the temperature of 95 ℃ to obtain SO double strands; and mixing the B-H2 functionalized nanofiber membrane, the SO double strand, the competitive DNA, the H1 sequence, the ThT and a sample to be detected, reacting under a dark condition, and measuring the fluorescence of the membrane.

Preferably, the excitation wavelength of the fluorescence is 450nm, and the emission spectrum between 470nm and 700nm is collected.

The invention provides a detection system for fluorescence detection of thrombin by a one-step method, which comprises a B-H2 functionalized nanofiber membrane, a support DNA, an output DNA, a competitive DNA, a buffer solution, a ThT, H1 sequence and thrombin. The detection system integrates three processes of Proximity Induced DNA Strand Displacement (PiDSD), catalytic hairpin self-assembly amplification (CHA) and ThT binding reaction. The surface interface behavior of the nanofiber membrane is functionalized through the B-H2, and the kinetic and thermodynamic reaction rates on the surface interface are promoted. In the embodiment of the invention, the thrombin is taken as a detection object, the detection limit can reach 1.0pM, the linear range is as wide as 50 pM-5 nM, and the thrombin-based detection method has the advantages of high specificity and good stability.

Drawings

FIG. 1 is a schematic diagram of the thrombin detection according to the present invention;

FIG. 2 is a graph showing the result of the PiDSD process and a graph showing the change in fluorescence in an example of the present invention;

FIG. 3 is a graph showing the results of performance tests for detecting thrombin at different concentrations in example 3 of the present invention;

FIG. 4 is a graph showing the results of the specificity and stability of the assay in example 4 of the present invention.

Detailed Description

The invention provides a detection system for fluorescence detection of thrombin by a one-step method, which comprises a B-H2 functionalized nanofiber membrane, a support DNA, an output DNA, a competitive DNA, a buffer solution, a ThT, H1 sequence and thrombin; the sequence of the support DNA is shown as SEQ ID NO. 2-7, the sequence of the output DNA is shown as SEQ ID NO.8, the sequence of the competitive DNA is shown as any one of SEQ ID NO. 9-14, and the sequence of H1 is shown as SEQ ID NO. 15; the buffer solutionThe liquid comprises the following components in concentration: 10 to 50mM Tris, 50 to 200mM NaCl, 5 to 15mM MgCl₂And 10-30 mM KCl, and the pH value is 7.5. In the PiDSD process of the present invention, the competitive dna (c) and the supporting dna(s) may include a T base sequence as a connecting strand for the supporting dna(s) and the export dna (o) strands, so as to reduce steric hindrance and promote strand displacement efficiency.

The sequences applicable to the detection system of the present invention are shown in table 1:

TABLE 1 sequence names and sequences

In the detection system, the preparation method of the B-H2 functionalized nanofiber membrane comprises the following steps: (1) mixing polystyrene and tetrabutylammonium bromide, and dissolving the mixture in N, N-dimethylformamide to obtain a PS/TBAB/DMF solution; the mass of the mixture of the polystyrene and the tetrabutylammonium bromide is 15-25% of that of the PS/TBAB/DMF solution;

(2) performing electrostatic spinning on the PS/TBAB/DMF solution, and drying to obtain a PS nanofiber membrane; the voltage during electrostatic spinning is 10-20 kV, the sample introduction speed is 2-8 mu L/min, the receiving distance is 7-15 cm, and the collection time is 1.5-3 h;

(3) processing the PS nanofiber membrane by using argon plasma generated by dielectric barrier discharge to obtain a processed PS nanofiber membrane; the processing voltage is 40-50V, the current is 1.2-2.5A, and the discharge processing time is 1-3 min;

(4) soaking the treated PS nano-fiber membrane in an avidin solution for 0.5-1.3H, taking out and cleaning, then reacting with a B-H2 solution for 0.6-1.5H, and cleaning to obtain a B-H2 functionalized nano-fiber membrane; the B-H2 is a nucleotide sequence marked by biotin, and the nucleotide sequence is shown as SEQID NO. 1.

When the B-H2 functionalized nanofiber membrane is prepared, polystyrene and tetrabutylammonium bromide are mixed and then dissolved in N, N-dimethylformamide to obtain a PS/TBAB/DMF solution; the mass of the polystyrene is 15-25% of that of the PS/TBAB/DMF solution; the mass volume ratio of the tetrabutylammonium bromide in the PS/TBAB/DMF solution is 0.1-0.5%. . According to the invention, the N, N-Dimethylformamide (DMF) is used as a solvent, Polystyrene (PS) and tetrabutylammonium bromide (TBAB) are dissolved, and a PS/TBAB/DMF solution is finally formed, wherein in the PS/TBAB/DMF solution, the PS concentration is preferably 16-24%, more preferably 18-22%, and most preferably 20%. In the PS/TBAB/DMF solution, the mass-to-volume ratio of the TBAB is preferably 0.1-0.5%, more preferably 0.2-0.4%, and most preferably 0.3%. In the invention, when the PS/TBAB/DMF solution is prepared, stirring is preferably carried out, and the stirring time is preferably 20-28 h, more preferably 22-25 h, and most preferably 24 h. The stirring speed of the invention is preferably 30-40 r/s. In the present invention, the stirring temperature is not particularly limited, and the stirring may be performed at room temperature, and is preferably 18 to 25 ℃.

After a PS/TBAB/DMF solution is obtained, the PS/TBAB/DMF solution is subjected to electrostatic spinning and dried to obtain a PS nano-fiber membrane; the voltage during electrostatic spinning is 10-20 kV, the sample injection speed is 2-8 mu L/min, the receiving distance is 7-15 cm, and the collection time is 1.5-3 h. The voltage of the electrostatic spinning is preferably 12-18 kV, more preferably 14-16 kV, and most preferably 15 kV. The sample injection speed in the electrostatic spinning process is preferably 3-7 mu L/min, more preferably 4-6 mu L/min, and most preferably 5 mu L/min. According to the invention, the fiber membrane after electrostatic spinning is preferably received on the aluminum foil, and the receiving distance is preferably 8-14 cm, more preferably 9-12 cm, and most preferably 10 cm. The collection time is preferably 1.6-2.5 h, more preferably 1.8-2.2 h, and most preferably 2 h. In the electrostatic spinning process, the preferred environmental humidity is 45-55%, and the more preferred environmental humidity is 49%. According to the invention, the nanofiber membrane received on the aluminum foil is preferably subjected to drying treatment, wherein the drying temperature is preferably 75-85 ℃, more preferably 78-82 ℃ and most preferably 80 ℃. The drying time is preferably 3.2-4.5 h, more preferably 3.6-4.2 h, and most preferably 4 h. After the PS nanofiber membrane is obtained, the PS nanofiber membrane is preferably trimmed, and the trimming is preferably performed to form a circular sheet with a diameter d equal to 5 mm.

After the PS nano fiber film is obtained, the PS nano fiber film is processed by argon plasma generated by dielectric barrier discharge to obtain the processed PS nano fiber film; the processing voltage is 40-50V, the current is 1.2-2.5A, and the discharge processing time is 1-3 min. The voltage of the treatment is preferably 42-48V, more preferably 44-46V, and most preferably 45V. The current for the treatment of the invention is preferably 1.5-2.4A, more preferably 1.8-2.2A, and most preferably 2A. The time of the discharge treatment is preferably 1.2-2.8 min, more preferably 1.6-2.5 min, and most preferably 2 min. The plasma treatment of the invention can improve the surface hydrophilicity of the PS film.

After the treated PS nano-fiber membrane is obtained, the treated PS nano-fiber membrane is soaked in an avidin solution for 0.5-1.3H, taken out and cleaned, and then reacts with a B-H2 solution for 0.6-1.5H, and the B-H2 functionalized nano-fiber membrane is obtained after cleaning; the B-H2 is a nucleotide sequence marked by biotin, and the nucleotide sequence is shown as SEQ ID NO. 1. In the present invention, the temperature for the soaking is not particularly limited, and is preferably room temperature, and more preferably 18 to 25 ℃. The concentration of the avidin solution is preferably 1.2-2.5 μ M, more preferably 1.8-2.2 μ M, and most preferably 2 μ M. The soaking time is preferably 0.6-1.2 hours, and more preferably 0.8-1 hour. The avidin is soaked in the solution to be immobilized on the surface interface of the membrane. In the present invention, both of the two cleaning operations are preferably performed by ultrapure water cleaning, and the number of the cleaning operations is preferably three. The concentration of the B-H2 solution is preferably 600-1000 nM, more preferably 700-900 nM, and most preferably 800 nM. The reaction temperature is preferably 35-40 ℃, and more preferably 37 ℃. The reaction time is preferably 0.8-1.2 h, and more preferably 1 h. The sequence of B-H2 is preferably as follows: Biotin-CTGACTAAAACCCAAAACCCGCTAGAGAAGTCAGTGTGGAAAATCTCTAGCGGGTTTTGGGTTTTGGGTTTTGGG (SEQ ID NO. 1). The B-H2 solution reaction can obtain the B-H2 functionalized nanofiber membrane and has the best optimal signal-to-back ratio.

The invention also provides a method for fluorescence detection of thrombin by one-step method, which comprises the following steps: mixing the support DNA, the output DNA and the buffer solution, and annealing at the temperature of 95 ℃ to obtain SO double strands; and mixing the B-H2 functionalized nanofiber membrane, the SO double strand, the competitive DNA, the H1 sequence, the ThT and a sample to be detected, reacting under a dark condition, and measuring the fluorescence of the membrane.

In the method, S and O are preferably added into the buffer solution for mixing, and the concentration of S is preferably 80-120 mu M, more preferably 90-110 mu M, and most preferably 100 mu M. The concentration of O in the invention is preferably 80-120 mu M, more preferably 90-110 mu M, and most preferably 100 mu M. The volume ratio of S to O in the invention is preferably (1-1.5): 1, more preferably (1.1 to 1.3): 1, most preferably 1.2: 1. In the present example, 2.4. mu.L of 100. mu. M S and 2.0. mu.L of 100. mu.MO were added to 20.0. mu.L of the buffer solution, annealed at 95 ℃ for 5min and cooled to room temperature to prepare an SO duplex with a final concentration of 10.0. mu.M.

After the SO double strand of the present invention, the B-H2 functionalized nanofiber membrane is preferably immersed in a mixed solution of 20nM SO, 20nM C, 200nM H1, 6. mu.M ThT and sample for reaction. The reaction is preferably carried out at room temperature in a dark condition, and the reaction time is preferably 80-120 min, more preferably 90-110 min, and most preferably 100 min. In the present invention, after the reaction is completed, it is preferable to wash three times with a buffer solution and measure the fluorescence of the membrane. In the present invention, the excitation wavelength of the fluorescence is preferably 450nm, the emission spectrum between 470nm and 700nm is collected, and the fluorescence at 492nm is recorded. The excitation and emission slit widths were 10nm and 10nm, respectively.

In the present invention, the detection principle of the method is shown in fig. 1, during the soaking process, thrombin specifically promotes the PiDSD process, and the output DNA will initiate CHA amplification reaction and ThT binding reaction on the nanofiber membrane. The PiDSD process comprises 3 DNA strands: support DNA (S), export DNA (O), and competitor DNA (C). Two aptamers are contained in S and C chains respectively (Apt29 in S and Apt15 in C), and a T base sequence is designed to be used as a connecting chain of the S and C chains so as to reduce steric hindrance and promote the chain replacement efficiency. Thrombin binds to aptamers in SO, C, closing the distance between C and SO. This process promotes the rate of strand displacement between SO and C. Subsequent CHA amplification reactions lead to the formation of G-quadruplex-ThT complexes on the nanofiber membrane, resulting in significant fluorescence signals. In general, thrombin is able to specifically trigger the generation of the G-quadruplex-ThT complex in a single step by a functionalized nanofiber membrane. Thus, the nanofiber membrane produces significantly enhanced fluorescence intensity and thus allows convenient and sensitive detection of thrombin.

The reaction system and the detection method for fluorescence detection of thrombin by one-step method provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

The PS nanofiber membrane is prepared by adopting an electrostatic spinning technology, and the specific operation is as follows: taking DMF as a solvent, preparing a PS/TBAB/DMF solution with the mass fraction of 20%, wherein the TBAB is 0.3% (w/v), stirring for 24h at room temperature, and carrying out electrostatic spinning after uniform mixing. Spinning conditions are as follows: the voltage is 15kV, the sample introduction speed is 5 mu L/min, the receiving distance is 10cm, the collection time is 2h, and the environmental humidity is 49 percent. The nanofiber membrane received on the aluminum foil was placed in an oven at 80 ℃ for 4h and then cut into disks with a diameter d of 5mm for use.

The PS nanofiber membrane is treated by argon plasma generated by dielectric barrier discharge, the voltage is set to be 45V, the current is 2.0A, and the discharge treatment time is 2 min. And immediately soaking the treated PS nano-fiber membrane in 100 mu L of 2.0 mu M avidin solution for 1H at room temperature, taking out the PS nano-fiber membrane, soaking and cleaning the PS nano-fiber membrane for three times by using ultrapure water, soaking the PS nano-fiber membrane in 100 mu L of 800nM B-H2 solution for reaction at 37 ℃ for 1H, taking out the PS nano-fiber membrane, and cleaning the PS nano-fiber membrane for three times by using the ultrapure water to prepare the B-H2 functionalized nano-fiber membrane. The B-H2 functionalized nanofiber membrane was observed under a scanning electron microscope, and as a result, as shown in FIG. 2-A, the B-H2 functionalized nanofiber membrane was composed of uniform, randomly distributed nanofibers, and had a diameter of about 350 nm.

Example 2

The proximity-induced DNA strand displacement (PiDSD) process was demonstrated by polyacrylamide gel electrophoresis (PAGE)

A12% polyacrylamide gel was prepared, and then 8. mu.L of the DNA sample was mixed well with 2. mu.L of 6 Xgel loading buffer (0.25% bromophenol blue, 0.25% xylene cyan, 40% (w/v) sucrose solution), added to the gel loading well, and subjected to electrophoresis. Electrophoresis was performed for 1h at constant voltage of 110V in 1 × TBE buffer (89mM Tris, 89mM boric acid, 2mM EDTA, pH 8.3). At this point the gel was stained with EB for 30 minutes, followed by photographing with a UV imaging system (Clinx GenoSens, China).

The PiDSD results are shown in FIG. 2B, where the 1, 2, 5 bands represent SO, C, O chains, respectively. The SO and C chains do not react for 100min in the absence of thrombin to produce the export chain O (lane 3). When thrombin is present, a band may be formed at the bottom of band 4 that exports the O chain. In addition, a new S-thrombin-C band with a slow migration rate is generated at the top of the band 4, and the mixed band of SO and C chains becomes shallow, which further proves that the output O is replaced.

And detecting different test solutions by using the functionalized nanofiber membrane, and measuring the fluorescence intensity of the nanofiber membrane. Respectively immersing the B-H2 functionalized nanofiber membrane into different test solutions, wherein each test solution respectively comprises the following components: (a) h1, SO, C, thrombin; (b) ThT, (c) H1, ThT; (d) h1, SO, ThT; (e) h1, C, ThT; (f) h1, SO, C, ThT; (g) h1, SO, C, thrombin, ThT. The concentrations of H1, SO, C, thrombin and ThT used in (a) - (g) were 200nM, 20nM, 20nM, 10nM and 6. mu.M, respectively.

As shown in FIG. 2C, when the B-H2 functionalized nanofibers were immersed in the test solutions (a) - (f), the fluorescence intensities were very weak (FIG. 2C a-f). This is because neither CHA amplification nor G-quadruplex-ThT complexes occur. However, once the B-H2 functionalized nanofibers were immersed in the test solutions containing thrombin, SO, C, H1, and ThT, the fluorescence intensity was significantly enhanced (fig. 2C g). The fluorescence intensity at 492nm of test solution (g) was enhanced by 3.3-fold relative to that of test solution (f) containing no thrombin. The increase in fluorescence intensity is due to the formation of the G-quadruplex-ThT complex by the test solution (G). The detection result proves the feasibility of the thrombin one-step fluorescence detection strategy based on the nanofiber sensing platform.

Example 3

Setting the concentration of H1 as 200nM, the concentration of ThT as 6 μ M, the reaction time as 100min, and the staining time as 15min to carry out thrombin detection performance tests with different concentrations, and marking the test solution as: (a)0pM, (b)1.0pM, (c)10pM, (d)50pM, (e)100pM, (f)200pM, (g)500pM, (h)1nM, (i)5nM, (j)10nM, and (k)20 nM.

As shown in FIG. 3-A, the concentration of thrombin was increased from 0pM to 20nM, and the fluorescence intensity of the nanofibers at 492nM was gradually increased. When the concentration of thrombin is 0pM to 20nM, the nanofiber membrane fluorescence intensity shows a good linear correlation with the logarithm of thrombin concentration, as shown in FIG. 3-B, the linear equation is: FL 58.7+213.4lgC, and r 0.9910, the detection limit of 1.0pM can be calculated according to the 3 σ rule.

Example 4

The specificity and stability of the assay system were tested by substituting thrombin at different concentrations with common proteins and amino acids (100nM), including Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), Glucose Oxidase (GOD) threonine (Thr), serine (Ser) and alanine (Ala), respectively, by the experimental conditions in example 3, and the results are shown in fig. 4: when detecting proteins or amino acids, the fluorescence intensity of the nanofiber membrane was not significantly increased compared to the blank. However, when 10nM thrombin (Tb) was added, the fluorescence intensity increased significantly. The detection system of the invention is proved to have good selectivity.

Stability was also verified and when after 51 days the fluorescence of the membrane decreased only 7.3% relative to the initial value.

Example 5

In order to verify the feasibility of the detection system in a biological complex system, thrombin with different concentrations is added into a human serum sample diluted by 100 times, detection is carried out by using the detection system, and the recovery rate is tested, and the results are shown in table 2, wherein the recovery rate and the RSD are respectively between 98.8% and 102.5% and 2.3% and 3.7%. The result shows that the detection system can be used for accurately and precisely detecting the thrombin in the biological complex system.

Table 2 detection and recovery experiments of detection systems in human serum samples

The invention provides a one-step fluorescence detection system and a thrombin detection method, based on the improvement of reaction thermodynamics and kinetics on a nanofiber membrane surface interface, the detection system has a detection limit of 1.0pM, and has a good linear relation between 50pM and 5nM, excellent selectivity and long-term stability. The detection method disclosed by the invention integrates the PiDSD process, the CHA amplification and the ThT binding reaction, and the one-step detection method avoids the separation and elution processes required by ELISA, saves the time and simplifies the experimental steps. The sensitive, simple and label-free sensing platform has potential application in the field of POCT and other protein detection.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims (7)

1. The one-step fluorescence detection system is characterized by comprising a B-H2 functionalized nanofiber membrane, a support DNA, an output DNA, a competitive DNA, a buffer solution, a ThT, H1 sequence and thrombin; the sequence of the support DNA is shown as SEQ ID NO. 2-7, the sequence of the output DNA is shown as SEQ ID NO.8, the sequence of the competitive DNA is shown as any one of SEQ ID NO. 9-14, and the sequence of H1 is shown as SEQ ID NO. 15; the buffer solution comprises the following components in concentration: 10 to 50mM Tris, 50 to 200mM NaCl, 5 to 15mM MgCl₂And 10-30 mM KCl, the pH value is 7.5;

the preparation method of the B-H2 functionalized nanofiber membrane comprises the following steps:

(1) mixing polystyrene and tetrabutylammonium bromide, and dissolving the mixture in N, N-dimethylformamide to obtain a PS/TBAB/DMF solution; the mass of the polystyrene is 15-25% of that of the PS/TBAB/DMF solution; the mass volume ratio of the tetrabutylammonium bromide in the PS/TBAB/DMF solution is 0.1-0.5%;

(2) performing electrostatic spinning on the PS/TBAB/DMF solution, and drying to obtain a PS nanofiber membrane; the voltage during electrostatic spinning is 10-20 kV, the sample introduction speed is 2-8 mu L/min, the receiving distance is 7-15 cm, and the collection time is 1.5-3 h;

(3) processing the PS nanofiber membrane by using argon plasma generated by dielectric barrier discharge to obtain a processed PS nanofiber membrane; the processing voltage is 40-50V, the current is 1.2-2.5A, and the discharge processing time is 1-3 min;

(4) soaking the treated PS nano-fiber membrane in an avidin solution for 0.5-1.3H, taking out and cleaning, then reacting with a B-H2 solution for 0.6-1.5H, and cleaning to obtain a B-H2 functionalized nano-fiber membrane; the B-H2 is a nucleotide sequence marked by biotin, and the nucleotide sequence is shown as SEQ ID NO. 1.

2. The detection system according to claim 1, wherein the mass-to-volume ratio of tetrabutylammonium bromide in the PS/TBAB/DMF solution in the step (1) is 0.1-0.5%.

3. The detection system according to claim 1, wherein the dissolution in the step (1) is accompanied by stirring, and the stirring time is 20-28 h.

4. The detection system according to claim 1, wherein the drying temperature in the step (2) is 75-85 ℃, and the drying time is 3.2-4.5 h.

5. The detection system according to claim 1, wherein after the PS nanofiber membrane is obtained in step (2), the method further comprises trimming the PS nanofiber membrane.

6. The detection system according to claim 1, wherein the concentration of the avidin solution in the step (4) is 1.2-2.5. mu.M.

7. The detection system according to claim 1, wherein the concentration of the B-H2 solution in the step (4) is 600-1000 nM.

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