CN115165983B - Reverse stem-loop specific ratio type electrochemical DNA biosensor based on polyadenylation and application thereof - Google Patents

Reverse stem-loop specific ratio type electrochemical DNA biosensor based on polyadenylation and application thereof Download PDF

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CN115165983B
CN115165983B CN202210784859.0A CN202210784859A CN115165983B CN 115165983 B CN115165983 B CN 115165983B CN 202210784859 A CN202210784859 A CN 202210784859A CN 115165983 B CN115165983 B CN 115165983B
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李兰英
闻艳丽
刘刚
陶晴
王乐乐
李妍
罗明
丁敏
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Shanghai Institute of Measurement and Testing Technology
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    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract

The invention provides an inverted stem-loop specific ratio type electrochemical DNA biosensor based on polyadenine and application thereof, wherein the electrochemical DNA biosensor comprises a polyadenine inverted stem-loop probe, a methylene blue modified signal probe, a ferrocene modified internal standard probe and a gold electrode, and the polyadenine inverted stem-loop probe is of a three-block inverted stem-loop structure. The skeleton structure of the polyadenylation inverted stem-loop probe is as follows from 5 '-3': and a third binding region-the first binding region-the second binding region, the second binding region and the third binding region being hybridized complementarily paired to form an inverted stem-loop structure. The electrochemical DNA biosensor disclosed by the invention adopts the ratio of peak current of methylene blue to peak current of ferrocene as signal output, can effectively eliminate the influence of factors such as change of microenvironment, different effective areas of electrodes, different density of assembled capture probes and the like in the detection process, has excellent detection performance and has wide application prospect.

Description

Reverse stem-loop specific ratio type electrochemical DNA biosensor based on polyadenylation and application thereof
Technical Field
The invention belongs to the technical field of biomolecule detection, and particularly relates to an inverted stem-loop specific ratio type electrochemical DNA biosensor based on polyadenylation and application thereof.
Background
Electrochemical DNA biosensors are known to have many advantages such as simplicity, high sensitivity, good selectivity, and low detection cost. It has been widely used for detection of various analytes, from ions to small molecules to nucleic acids and even proteins. However, conventional electrochemical DNA biosensors are usually limited to single signal mode, which inevitably introduces interference factors from substrates and detection systems, including different surface areas of individual electrodes, different numbers of assembled DNA on the surfaces of electrodes, nonspecific adsorption of other biomolecules in complex biological samples on the sensing interface, slow electron transfer kinetics, etc., which results in poor reproducibility of experimental results, high background signals, and insufficient sensitivity.
To overcome the inherent drawbacks of the single signal mode, scientists have recently focused on developing electrochemical DNA biosensors in the dual signal mode, i.e., ratio-type electrochemical DNA biosensors, which can obtain a more accurate signal through self-calibration capability because the ratio strength of the dual signal is independent of the sensor or detection substrate reagent concentration.
In 2015, ellington et al construct a ratio electrochemical biosensor based on molecular beacons for the first time, two ends of a molecular signal are respectively marked with classical electroactive substances (ferrocene and methylene blue), methylene blue is taken as signal output of conformational change, ferrocene is taken as internal control, in the conformational change process, only the distance between a methylene blue signal probe and the electrode surface is obviously changed to cause the change of peak current on a square wave volt-ampere curve, the distance between a ferrocene control probe and the electrode surface is kept relatively constant, the ratio of the peak current of the methylene blue to the peak current of the ferrocene is taken as signal output, and the repeatability is 1 order of magnitude smaller than that of the classical electrochemical DNA biosensor, and the sensitivity or the selectivity is not affected. Subsequently, a series of ratiometric electrochemical biosensors were developed for detection of circulating tumor DNA, mercury ions, thrombin, kanamycin.
However, the existing ratio-type electrochemical biosensor for detecting DNA still has the problem of poor stability, so that the ratio-type electrochemical biosensor for detecting DNA, which has good repeatability and higher stability, has important application value.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide an inverted stem-loop specific ratio type electrochemical DNA biosensor based on polyadenine and application thereof, wherein the electrochemical DNA biosensor contains a polyadenine DNA probe with a three-block inverted stem-loop structure, and the compact and orderly assembly of a capture probe on the surface of a gold electrode can be realized only by virtue of covalent bonding of middle polyadenine and gold without special modification.
The electrochemical DNA biosensor adopts the ratio of peak current of methylene blue and ferrocene as signal output, can effectively eliminate the influence of micro-environment change, different electrode effective areas, different assembly capture probe density and other factors in the detection process, has reproducibility of less than 2.77%, and can achieve the excellent performance of 0.34pM without signal amplification.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides an inverted stem-loop specific electrochemical DNA biosensor based on polyadenine, which comprises a polyadenine inverted stem-loop probe, a methylene blue modified signal probe, a ferrocene modified internal standard probe and a gold electrode; the polyadenine inverted stem-loop probe is of a three-block inverted stem-loop structure.
Preferably, the framework structure of the polyadenylation inverted stem-loop probe is as follows in order from 5 '-3': and a third binding region-the first binding region-the second binding region, the second binding region and the third binding region being hybridized complementarily paired to form an inverted stem-loop structure.
Preferably, the first binding region is for covalent binding to a gold electrode to form a self-assembled monolayer film.
Preferably, the second binding region is used for specifically recognizing a captured target, and the captured target is combined with a methylene blue modified signaling probe by adopting a sandwich structure for recognizing signal output.
The nucleotide sequence of the second binding region in the present invention may be used for specific binding to a target substance which is a substance capable of specifically binding to the nucleotide sequence, and the target substance includes RNA, DNA, protein, polypeptide, antibiotic, or the like.
Preferably, the third binding region is for specific binding to a ferrocene modified internal standard probe for controlling signal output.
Preferably, the first binding region is a polyadenylation sequence unit, the nucleotide sequence of which comprises: (A) n, where n is a positive integer, n=10-40, n may be, for example, 10, 20, 30, 40, etc.
Preferably, the first binding region comprises polyA10, polyA20, polyA30 or polyA40.
Preferably, the second binding region is sequence 1 that specifically binds to the target, the nucleotide sequence of sequence 1 comprising 10-40 bases (e.g., 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, etc.); wherein 6-8 bases at the 3' -end of the nucleotide sequence of the sequence 1 are also complementarily paired with the third binding region, and the number of bases may be, for example, 6, 7 or 8.
After the 3' end of the second binding region, 3 arbitrary bases are also attached, the presence of which 3 bases helps to open the stem-loop structure after binding to the target.
Preferably, the third binding region is a nucleotide sequence complementary to and paired with the internal standard probe, and the nucleotide sequence of the third binding region comprises 16-28 bases, and the number of bases can be, for example, 16, 18, 20, 22, 24, 26 or 28, etc.
Preferably, the third binding region comprises, from 5' -3', a sequence 3 and a sequence 2, which are sequentially connected, wherein the sequence 3 is a nucleotide sequence complementary to and paired with 6-8 bases at the 3' -end of the sequence 1 in the second binding region, and the sequence 2 is a nucleotide sequence complementary and paired with a ferrocene modified internal standard probe.
Preferably, the nucleotide sequence of the sequence 3 comprises 6-8 bases, the number of bases can be, for example, 6, 7 or 8, the nucleotide sequence of the sequence 2 comprises 10-20 bases, the number of bases can be, for example, 10, 12, 14, 16, 18 or 20, etc.
Preferably, the framework structure of the polyadenylation inverted stem-loop probe is as follows in order from 5 '-3': sequence 3-sequence 2-poly (A) n-sequence 1; wherein n is a positive integer, n=10-40, n may be, for example, 10, 20, 30, 40, etc.;
the sequence 1 is specifically combined with a target object, and 6-8 bases at the 3' -end of the nucleotide sequence of the sequence 1 are also complementarily paired with the sequence 3; the sequence 2 is specifically combined with an internal standard probe modified by ferrocene, and the poly (A) n is covalently combined with a gold electrode.
The invention provides a three-block inverted stem-loop structure polyadenylation DNA probe, which can realize compact and orderly assembly of a capture probe on the surface of a gold electrode by covalent bonding of the middle polyadenylation and gold without special modification. The two sides of the polyadenylation DNA sequence are designed into an inverted stem-loop structure, one side is used for specifically identifying and capturing a target object, and the inverted stem-loop structure is combined with a signal probe modified by methylene blue by adopting a classical sandwich method structure and used for identifying signal output, and even single base mismatch of DNA can be identified due to the existence of the stem-loop structure; the other side is hybridized with a ferrocene modified control probe, no matter whether a target object exists or not, the distance between the ferrocene control probe and the electrode surface is kept relatively constant, and the control signal is output.
During detection, the methylene blue modified signal probe and the ferrocene modified control probe participate in the reaction at the same time, the concept of an internal standard in isotope dilution mass spectrometry is introduced into the sensor, the ratio of peak current of the methylene blue to that of the ferrocene is used as signal output, the influence of factors such as the change of microenvironment, the difference of the effective area of an electrode, the difference of the density of an assembled capture probe and the like in the detection process can be effectively eliminated, the reproducibility is less than 2.77%, and the excellent performance of 0.34pM can be achieved without signal amplification.
In a second aspect, the present invention provides a method for using the polyadenylation-based inverted stem-loop specific electrochemical DNA biosensor of the first aspect, the method comprising the steps of:
(1) Assembling a polyadenylation reverse stem-loop probe on the gold electrode;
(2) Sealing the empty space on the surface of the gold electrode by using a sealing liquid;
(3) Pre-hybridizing the target, the signal probe and the internal standard probe, hybridizing with the inverted stem-loop probe assembled on the surface of the gold electrode, and flushing the gold electrode after hybridization;
(4) Square wave voltammetry scanning is carried out on the washed gold electrode to obtain SWV peak current ratio I of MB marked on the signal probe and Fc marked on the internal standard probe MB /I Fc The method comprises the steps of carrying out a first treatment on the surface of the According to I MB /I Fc The target is analyzed.
Preferably, in step (1), the assembling is performed by the following steps:
and (3) carrying out electrochemical cleaning on the physically polished gold electrode, washing the surface of the gold electrode with deionized water after cleaning, removing water on the surface of the gold electrode, and dripping the polyadenine inverted stem-loop probe solution on the surface of the gold electrode for reaction assembly.
Preferably, the electrochemical cleaning is performed in a sulfuric acid solution having a concentration of 0.02-1M, which may be, for example, 0.02M, 0.03M, 0.05M, 0.08M, 0.1M, 0.2M, 0.4M, 0.6M, 0.8M, 1M, or the like.
Preferably, nitrogen purging is used to remove water from the gold electrode surface.
Preferably, the concentration of the polyadenylation inverted stem-loop probe solution is 0.02-1. Mu.M, and may be, for example, 0.02M, 0.03M, 0.05M, 0.08M, 0.1M, 0.2M, 0.4M, 0.6M, 0.8M, or 1M, etc.
Preferably, the polyadenine inverted stem-loop probe solution is used in an amount ofThe electrode may be, for example +> Electrode, & gt>Electrode, & gt>Electrode, & gt>Electrode, & gt>Electrode, & gt>Electrode, & gt>Electrode or->Electrodes, etc.
The amount of the polyadenine inverted stem-loop probe solution used on the surface of the electrode with the diameter of 2mm is 3-10 mu L in the invention.
Preferably, the temperature of the reactive assembly is 20-25 ℃, for example, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃ or 25 ℃, and the like, and the time of the reactive assembly is 2-24 hours, for example, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours or 24 hours, and the like.
Preferably, in step (2), the blocking solution comprises a solution of 6-Mercaptohexanol (MCH) at a concentration of 0.1-1mM, e.g., 0.1mM, 0.2mM, 0.3mM, 0.5mM, 0.8mM, 1mM, etc.
Preferably, the 6-mercaptohexanol solution is used in an amount ofThe electrodes may be, for example Pole(s)>Electrode, & gt>Electrode, & gt>Electrodes orElectrodes, etc.
Preferably, the sealing temperature is 20-25deg.C, such as 20deg.C, 21deg.C, 22deg.C, 23deg.C, 24deg.C or 25deg.C, and the sealing time is 25-35min, such as 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min or 35 min.
Preferably, in step (3), the prehybridization treatment is performed by the following steps:
mixing the target, signaling probe and internal standard probe with buffer solution to obtain prehybridization solution, holding the prehybridization solution at 80-90deg.C (such as 80deg.C, 1608 deg.C, 85deg.C, 87deg.C, 89 deg.C or 90deg.C) for 5-10min (such as 5min, 6min, 7min, 8min, 9min or 10 min), and standing at 20-25deg.C (such as 20deg.C, 21deg.C, 22deg.C, 23deg.C, 24 deg.C or 25deg.C) for 20min or more, such as 20min, 25min, 30min, 35min or 40 min.
Preferably, the concentration of the signaling probe in the pre-hybridization solution is 30 to 100. Mu.M, which may be, for example, 30. Mu.M, 40. Mu.M, 50. Mu.M, 60. Mu.M, 70. Mu.M, 80. Mu.M, 90. Mu.M, 100. Mu.M, or the like.
Preferably, the concentration of the internal standard probe in the pre-hybridization solution is 15-50. Mu.M, for example, 15. Mu.M, 20. Mu.M, 25. Mu.M, 30. Mu.M, 35. Mu.M, 40. Mu.M, 45. Mu.M, 50. Mu.M, etc.
Preferably, the buffer solution contains 0.8-1.2M NaCl and 0.03-0.07M NaClO 4 And 8-12mMThe pH of the buffer solution is 7.0-8.0 with water as solvent.
The concentration of NaCl in the buffer solution of the present invention is 0.8 to 1.2M, and for example, it may be 0.8M, 0.9M, 1.0M, 1.1M or 1.2M; naClO 4 The concentration of (2) is 0.03-0.07M, for example, 0.03M, 0.04M, 0.05M, 0.06M or 0.07M; the concentration of PBS may be 8-12mM PBS, for example, 8mM, 9mM, 10mM, 11mM or 12mM, and the pH of the buffer solution may be 7.0, 7.2, 7.5, 7.7, 7.8 or 8.0, for example.
Preferably, the hybridization temperature is 20-25deg.C, such as 20deg.C, 21deg.C, 22deg.C, 23deg.C, 24deg.C or 25deg.C, and the hybridization time is 0.5-4h, such as 0.5h, 1.0h, 1.5h, 2h, 2.5h, 3h, 3.5h or 4 h.
Preferably, the solution used for flushing the gold electrode contains 0.08-0.12M NaCl and 0.03-0.07M NaClO 4 And 8-12mM PBS, the pH of the solution is 7.0-8.0, and the solvent is water.
The concentration of NaCl in the solution used for flushing the gold electrode is 0.08-0.12M, for example, 0.08M, 0.09M, 0.1M, 0.11M or 0.12M, etc.; naClO 4 The concentration of (2) is 0.03-0.07M, for example, 0.03M, 0.04M, 0.05M, 0.06M or 0.07M; the concentration of PBS may be 8-12mM, for example, 8mM, 9mM, 10mM, 11mM or 12mM, and the pH of the solution may be 7.0, 7.2, 7.5, 7.7, 7.8 or 8.0, for example.
Preferably, in the step (4), the electrolyte system used for square wave voltammetry scanning contains 0.01-0.10M NaClO 4 And 8-12mM PBS, the pH of the electrolyte system is 7.0-8.0, and the solvent is water.
NaClO in the electrolyte system of the invention 4 The concentration of (2) is 0.01-0.10M, for example, 0.01M, 0.03M, 0.05M, 0.08M or 0.10M, etc.; the concentration of PBS may be 8-12mM, for example, 8mM, 9mM, 10mM, 11mM, or 12mM, and the pH of the electrolyte system may be 7.0, 7.2, 7.5, 7.7, 7.8, or 8.0, for example.
Preferably, in step (4), the method according to I MB /I Fc The method for quantitatively analyzing the target comprises the following steps: square wave is carried out on the DNA standard substance according to the processing method of the target objectVoltammetric scanning, construction of DNA Standard I MB /I Fc And calculating the concentration of the target object according to the linear relation equation.
As a preferred technical scheme of the invention, the using method of the multi-adenine-based inverted stem-loop specific ratio type electrochemical DNA biosensor comprises the following steps:
(1) Electrochemical cleaning of the physically polished gold electrode in sulfuric acid solution with concentration of 0.02-1 mu M, flushing the surface of the gold electrode with deionized water, removing water on the surface of the gold electrode by nitrogen purging, and adding the polyadenine inverted stem-loop probe solution with concentration of 0.02-1 mu M according to the usage amountThe electrode is dripped on the surface of the gold electrode, and the electrode is assembled for 8 to 12 hours in a reaction way at the temperature of 20 to 25 ℃;
(2) The vacancy of the gold electrode surface is closed by using MCH solution with the concentration of 0.1-1mM, the temperature of 20-25 ℃ is closed for 25-35min, and the usage amount of the MCH solution is thatAn electrode;
(3) Mixing a target, a signal probe and an internal standard probe with a buffer solution to obtain a prehybridization solution, wherein the concentration of the signal probe in the prehybridization solution is 30-100 mu M, the concentration of the internal standard probe is 15-50 mu M, and the buffer solution contains 0.8-1.2M NaCl and 0.03-0.07M NaClO 4 And 8-12mM PBS, ph=7.0-8.0 of the buffer solution, the solvent being water; maintaining the prehybridization solution at 80-90deg.C for 5-10min, and standing at 20-25deg.C for more than 20 min; after prehybridization treatment, hybridization is carried out for 0.5 to 4 hours with an inverted stem-loop probe assembled on the surface of the gold electrode, the gold electrode is washed after hybridization, and the solution adopted for washing the gold electrode contains 0.08 to 0.12M NaCl and 0.03 to 0.07M NaClO 4 And 8-12mM PBS, ph=7.0-8.0 of the solution, the solvent being water;
(4) Square wave voltammetry scanning is carried out on the washed gold electrode, wherein an electrolyte system adopted by the square wave voltammetry scanning contains 0.01-0.1M NaClO 4 And 8-12mM PBS, pH=7.0-8.0 of electrolyte system, water as solvent, to obtain SWV peak current ratio I of MB marked on signal probe and Fc marked on internal standard probe MB /I Fc The method comprises the steps of carrying out a first treatment on the surface of the Square wave voltammetry scanning is carried out on the DNA standard substance by referring to the treatment method of the target substance, and I of the DNA standard substance is constructed MB /I Fc And calculating the concentration of the target object according to the linear relation equation.
In a third aspect, the present invention provides a polyadenine-based inverted stem-loop ratio type DNA detection kit comprising the polyadenine-based inverted stem-loop ratio type electrochemical DNA biosensor of the first aspect.
In a fourth aspect, the invention provides the application of any one or the combination of at least two of the polyadenine-based inverted stem-loop ratio type electrochemical DNA biosensor in the first aspect, the polyadenine-based inverted stem-loop ratio type electrochemical DNA biosensor in the second aspect, or the polyadenine-based inverted stem-loop ratio type DNA detection kit in the third aspect in DNA quantitative detection.
The numerical ranges recited herein include not only the above-listed point values, but also any point values between the above-listed numerical ranges that are not listed, and are limited in space and for the sake of brevity, the present invention is not intended to be exhaustive of the specific point values that the stated ranges include.
Compared with the prior art, the invention has the following beneficial effects:
(1) The covalent combination of the polyadenylation sequence unit and gold can realize the compact and orderly assembly of the capture probe on the surface of the gold electrode, the two sides of the polyadenylation sequence unit are designed into an inverted stem-loop structure, one side is used for specifically identifying the capture target, and the capture target is combined with a signal probe modified by methylene blue by adopting a classical sandwich method structure for identifying signal output, and even single base mismatch of DNA can be identified due to the existence of the stem-loop structure; the other side is hybridized with a ferrocene modified control probe, no matter whether a target object exists or not, the distance between the ferrocene control probe and the electrode surface is kept relatively constant, and the control signal is output.
(2) The methylene blue modified signal probe and the ferrocene modified control probe participate in the reaction at the same time, the concept of 'internal standard' in isotope dilution mass spectrometry is introduced into the sensor, the ratio of peak current of the methylene blue and ferrocene is adopted as signal output, the influence of factors such as the change of microenvironment, the difference of the effective area of electrodes and the difference of the density of the assembled capture probe in the detection process can be effectively eliminated, the reproducibility is less than 2.77%, and the excellent performance of 0.34pM can be achieved without signal amplification and the detection limit.
(3) The reverse stem-loop specific ratio type electrochemical DNA biosensor based on the polyadenylation has high selectivity and can distinguish single base mismatch.
Drawings
FIG. 1 is a schematic representation of the design and detection principle of an inverted stem-loop specific electrochemical DNA biosensor based on polyadenylation in example 1.
FIG. 2A is a schematic illustration of the different electrodes of example 4 at 5mM K with 0.1M KCl 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]Cyclic voltammogram in solution.
FIG. 2B is a schematic illustration of the different electrodes of example 4 at 5mM K with 0.1M KCl 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]Electrochemical ac impedance plot in solution.
FIG. 3A is a graph showing SWV response results for 100nM target DNA using three separate electrodes in example 5.
Fig. 3B is a graph showing the reproducibility of the non-ratio type sensor and the ratio type sensor in example 5.
FIG. 4A is a plot of inverted stem-loop probe concentration versus signal I for example 6 MB /I Fc Is a graph of the relationship of (1).
FIG. 4B shows hybridization time and signal I in example 6 MB /I Fc Is a graph of the relationship of (1).
FIG. 5A shows signals I of target DNA at different concentrations in example 7 MB /I Fc Graph of concentration.
FIG. 5B is the log of target DNA at different concentrations and signal I in example 7 MB /I Fc Is a fit of the curve.
FIG. 6 shows the results of analysis of a base mismatch sample in example 8.
FIG. 7 is the results of analysis of a simulated biological sample in example 9.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
The nucleotide sequences of the nucleic acid molecules used in the design of the polyadenylation stem-loop probe, the signaling probe and the internal standard probe are shown in Table 1:
TABLE 1
Abbreviations: a polyA10 inverted stem-loop (IMB-PA 10), a polyA20 inverted stem-loop (IMB-PA 20), a polyA30 inverted stem-loop (IMB-PA 30), a polyA40 inverted stem-loop (IMB-PA 40), the single underlined portions of IMB-PA10, IMB-PA20, IMB-PA30, IMB-PA40 representing the complementary pair of sequence 1 and sequence 3; methylene Blue (MB), ferrocene (Fc), single base mismatches (Sm-target), two base mismatches (Twm-target), three base mismatches (Thm-target), four base mismatches (Fm-target), mismatched bases are bolded in italics and double-underlined.
In Table 1, IMB-PA10 is taken as an example:
TGAACCTATCGCATCGTATCGTA 10 GTATATAGCCTCCGGTTCATGC；
wherein'TGAACCTATCGCATCGTATCGT "is a third binding region, where"TGAACC"is sequence 3 (SEQ ID NO: 3)," TATCGCATCGTATCGT "is sequence 2 (SEQ ID NO: 2).
A 10 Is the first binding region (polyadenylation sequence unit).
“GTATATAGCCTCCGGTTCATGC "is the second binding region, wherein" GTATATAGCCTCCGGTTCATGC "is sequence 1 (SEQ ID NO: 1)," at the 3' -end of the nucleotide sequence of said sequence 1 "GGTTCA"complementary pairing to sequence 3 of the third binding region".
Wherein:
SEQ ID NO:1：GTATATAGCCTCCGGTTCATGC。
SEQ ID NO:2：TATCGCATCGTATCGT。
SEQ ID NO:3：TGAACC。
SEQ ID NO:4：ACGATACGATGCGATA。
example 1
The embodiment provides an inverted stem-loop specific electrochemical DNA biosensor based on polyadenine and a use method thereof. The design and detection principle of the reverse stem-loop specific ratio type electrochemical DNA biosensor based on the polyadenylation is shown in figure 1. The polyadenylation inverted stem-loop probe used in this example was IMB-PA20 in Table 1, the signaling probe was Reporter probe (MB), the internal standard probe was Control probe (Fc), and the Target probe was Target.
(1) Electrochemical cleaning of physically polished gold electrode in sulfuric acid solution of 0.5 mu M concentration, flushing the surface of the gold electrode with deionized water (Milli-Q water), nitrogen purging to eliminate water on the surface of the gold electrode, and adding 0.5 mu M concentration of polyadenylation inverted stem-loop probe solution in the following amount The electrode is dripped on the surface of the gold electrode, and the reaction and the assembly are carried out for 10 hours at 20 ℃.
(2) The vacancy on the surface of the gold electrode is closed by using MCH solution with the concentration of 0.1mM, the temperature is 20 ℃ for 30min, and the usage amount of the MCH solution is thatAn electrode.
(3) Mixing target DNA, signal probe and internal standard probe with buffer solution to obtain prehybridization solution, in which the concentration of target DNA is 100nM, the concentration of signal probe is 40. Mu.M and the concentration of internal standard probe is 20. Mu.M, and the buffer solution contains 1M NaCl and 0.05M NaClO 4 And 10mM PBS, ph=7.4 of buffer solution, water as solvent; maintaining the prehybridization solution at 80deg.C for 5min, and standing at 20deg.C for 25min; after prehybridization treatment, the hybridization is carried out for 2 hours with an inverted stem-loop probe assembled on the surface of the gold electrode, the gold electrode is washed after hybridization, and the solution adopted for washing the gold electrode contains 0.1M NaCl and 0.05M NaClO 4 And 10mM PBS, ph=7.4 of the solution, and the solvent is water.
(4) Square wave voltammetry scanning is carried out on the washed gold electrode, wherein an electrolyte system adopted by the square wave voltammetry scanning contains 0.05M NaClO 4 And 10mM PBS, pH=7.4 of the electrolyte system, water as solvent, to obtain SWV peak current ratio I of MB labeled on the signaling probe and Fc labeled on the internal standard probe MB /I Fc The method comprises the steps of carrying out a first treatment on the surface of the Square wave voltammetry scanning is carried out on a DNA standard substance according to a treatment method of target DNA, and I of the DNA standard substance is constructed MB /I Fc And calculating the concentration of the target DNA according to the linear relation equation.
Example 2
The embodiment provides an inverted stem-loop specific electrochemical DNA biosensor based on polyadenine and a use method thereof. The using method is as follows:
the polyadenylation inverted stem-loop probe used in this example was the polyadenylation inverted stem-loop probe (IMB-PA 10) shown in Table 1, the signaling probe was the Reporter probe (MB), the internal standard probe was the Control probe (Fc), and the Target probe was the Target.
(1) The physically polished gold electrode is subjected to electrochemical cleaning in sulfuric acid solution with the concentration of 0.02 mu M, and the surface of the gold electrode is washed by deionized water (Milli-Q water) after the cleaning, and is purged by nitrogenRemoving water on the surface of the gold electrode, and adding 0.02 mu M of polyadenylation inverted stem-loop probe solution into the solution according to the usage amountThe electrode is dripped on the surface of the gold electrode, and the electrode is assembled for 12 hours at 25 ℃.
(2) The vacancy on the surface of the gold electrode is closed by using MCH solution with the concentration of 0.1mM, the temperature is 25 ℃ for 25min, and the usage amount of the MCH solution is thatAn electrode;
(3) Mixing target DNA, signal probe and internal standard probe with buffer solution to obtain prehybridization solution, in which the concentration of target DNA is 0.001nM, the concentration of signal probe is 30 μM and the concentration of internal standard probe is 15 μM, and the buffer solution contains 0.8M NaCl and 0.03M NaClO 4 And 8mM PBS, ph=7.0 of buffer solution, water as solvent; maintaining the prehybridization solution at 80deg.C for 5min, and standing at 25deg.C for 30min; after prehybridization treatment, the hybridization is carried out for 0.5h with an inverted stem-loop probe assembled on the surface of the gold electrode, the gold electrode is washed after hybridization, and the solution adopted for washing the gold electrode contains 0.08M NaCl and 0.03M NaClO 4 And 8mM PBS, ph=7.0 of the solution, and the solvent is water.
(4) Square wave voltammetry scanning is carried out on the washed gold electrode, wherein an electrolyte system adopted by the square wave voltammetry scanning contains 0.01M NaClO 4 And 8mM PBS, the pH=7.0 solvent of the electrolyte system being water, to obtain the SWV peak current ratio I of the MB marked on the signal probe and the Fc marked on the internal standard probe MB /I Fc The method comprises the steps of carrying out a first treatment on the surface of the Square wave voltammetry scanning is carried out on a DNA standard substance according to a treatment method of target DNA, and I of the DNA standard substance is constructed MB /I Fc Linear relation equation with logarithm of concentration, R of linear relation equation 2 The concentration of target DNA was calculated from a linear relationship equation at 0.9968, where the target DNA recovery was 85-96%.
Example 3
The embodiment provides an inverted stem-loop specific electrochemical DNA biosensor based on polyadenine and a use method thereof. The using method is as follows:
the polyadenylation inverted stem-loop probe used in this example was the polyadenylation inverted stem-loop probe (IMB-PA 40) shown in Table 1, the signaling probe was the Reporter probe (MB), the internal standard probe was the Control probe (Fc), and the Target probe was the Target.
(1) Electrochemical cleaning of physically polished gold electrode in sulfuric acid solution with concentration of 1 μm, washing the surface of gold electrode with deionized water (Milli-Q water), removing water on the surface of gold electrode by nitrogen purging, and adding 1 μm concentration of polyadenylation inverted stem-loop probe solution according to the usage amountThe electrode is dripped on the surface of the gold electrode, and the electrode is assembled for 24 hours in a reaction way at 25 ℃;
(2) The vacancy on the surface of the gold electrode is closed by using MCH solution with the concentration of 1mM, the room temperature is closed for 35min, and the usage amount of the MCH solution is thatAn electrode;
(3) Mixing target DNA, signal probe and internal standard probe with buffer solution to obtain prehybridization solution, in which the concentration of target DNA is 100nM, the concentration of signal probe is 100 μM and the concentration of internal standard probe is 50 μM, and the buffer solution contains 1.2M NaCl and 0.07M NaClO 4 And 12mM PBS, ph=8.0 of buffer solution, water as solvent; maintaining the prehybridization solution at 90 ℃ for 10min, and then standing at 25 ℃ for more than 20 min; after prehybridization treatment, the hybridization is carried out for 4 hours with an inverted stem-loop probe assembled on the surface of the gold electrode, the gold electrode is washed after hybridization, and the solution adopted for washing the gold electrode contains 0.12M NaCl and 0.07M NaClO 4 And 12mM PBS, ph=8.0 of the solution, the solvent being water;
(4) Square wave voltammetry scanning is carried out on the washed gold electrode, wherein an electrolyte system adopted by the square wave voltammetry scanning contains 0.1M NaClO 4 And 12mM PBS, ph=8.0, solvent of the electrolyte systemFor water, SWV peak current ratio I of MB labeled on the signaling probe and Fc labeled on the internal standard probe was obtained MB /I Fc The method comprises the steps of carrying out a first treatment on the surface of the Square wave voltammetry scanning is carried out on a DNA standard substance according to a treatment method of target DNA, and I of the DNA standard substance is constructed MB /I Fc Linear relation equation with logarithm of concentration, R of linear relation equation 2 The concentration of the target DNA was calculated from a linear relationship equation at 0.9971, where the detection accuracy of the target DNA was 84-95%.
Example 4
This example provides a polyadenine-based inverted stem-loop ratio type DNA detection kit containing the electrochemical DNA biosensor obtained in example 1. The electrochemical DNA biosensor was characterized using cyclic voltammetry and electrochemical ac impedance.
The cyclic voltammetric scan conditions are as follows:
electrolyte system: 5mM K containing 0.1M KCl 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]A solution;
scanning potential interval: (-0.2 to 0.6) V;
sweep speed: 0.1V/s.
The conditions of the electrochemical ac impedance are as follows:
electrolyte system: 5mM K containing 0.1M KCl 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]A solution;
frequency range: 0.01Hz-100kHz.
The characterization results are shown in fig. 2A and 2B. FIG. 2A shows the different electrodes at 5mM K with 0.1M KCl 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]Cyclic voltammograms in solution; FIG. 2B shows the different electrodes at 5mM K with 0.1M KCl 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]Electrochemical ac impedance diagram in solution, wherein in fig. 2B, the abscissa is impedance and the ordinate is reactance; the electrode includes: a: bare gold electrode (Au); b: IMB-PA/Au; c: IMB-PA/MCH/Au; d: IMB-PA/MCH/Au hybridizes to the target DNA, signaling probes, and control probes.
FIG. 2A is a schematic view ofDifferent electrodes at K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]According to the cyclic voltammogram of [ Fe (CN) ] 6 ] 3-/4- The change of the volt-ampere signal verifies the formation of a self-assembled monolayer film on the gold electrode, and as can be seen from the figure, the bare gold electrode has good reversibility, and the peak potential difference is 85mV (curve a); when the inverted stem-loop probe is assembled on the gold electrode surface (curve b), the negative charge on the DNA phosphate backbone causes [ Fe (CN) 6 ] 3-/4- The electron transfer process on the gold electrode was suppressed, the peak potential difference increased to 388mV, and the peak current was greatly reduced. MCH (6-mercapto hexanol) seals the free unbound sites of the modified electrode (curve c), and the inverted stem-loop probes lodged on the gold electrode surface are arranged in order, [ Fe (CN) 6 ] 3-/4- The electron transfer process on the gold electrode is smooth, the peak potential difference is reduced to 300mV, and the peak current is slightly increased; when the inverted stem-loop probe is hybridized with the target DNA, the signal probe and the control probe, the two ends of the polyadenine respectively form a rigid double-stranded DNA structure, the arrangement is more ordered, and the methylene blue of the signal probe and the ferrocene on the control probe also promote [ Fe (CN) 6 ] 3-/4- The electron transfer on the gold electrode reduced the peak potential difference to 280mV and the peak current increased slightly.
The result shows that the DNA of the inverted stem-loop probe is self-assembled and fixed on the surface of the gold electrode through the inherent binding force of the polyadenylation and the gold electrode, a better self-assembled monolayer film is formed, and the constructed inverted stem-loop DNA sensor is hybridized with the target DNA, the signal probe and the control probe.
FIG. 2B shows the different electrodes at K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]The electrochemical alternating current impedance diagram of the electrode is very small, the impedance of the bare gold electrode (curve a) is only 130kΩ, and after the inverted stem-loop probe is assembled (curve b), the impedance is greatly increased to 4571kΩ, which indicates that the inverted stem-loop capture probe is assembled on the gold electrode surface, thereby preventing the electron transfer on the gold electrode surface and causing the impedance increase; when the MCH of 0.1mM is used for 30min (curve c), the impedance becomes lower to 2384kΩ again, because the inverted stem-loop probe on the electrode surface becomes ordered after the MCH is used for the spacer The electron transfer is facilitated; after the inverted stem-loop probe is hybridized with the target DNA, the signal probe and the control probe (curve d), the two ends of the polyadenine respectively form a rigid double-stranded DNA structure, the arrangement is more ordered, and the methylene blue of the signal probe and the ferrocene on the control probe can generate electron transfer on a gold electrode, the electrochemical alternating current impedance is continuously reduced to 1766kΩ, and the results are consistent with the results of the cyclic voltammetry characterization, so that the inverted stem-loop DNA sensor is successfully prepared and can be used for detecting the target DNA.
Example 5
This example uses the polyadenylation-based inverted stem-loop specific electrochemical DNA biosensor constructed in example 1 to detect 100nM target DNA, taking 50 separate measurements, each with separate electrodes, provided that: length of multiple polyadenines in inverted stem-loop probe: 10nt, 20nt, 30nt, 40nt respectively; different concentrations of IMB-PA20 were used: 0.02. Mu.M, 0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, respectively.
Results of random extraction three times (I MB /I FC Length of polyadenine corresponding to =3.02: 10nt; IMB-PA20 concentration: 0.02 μm; i MB /I FC Length of polyadenine corresponding to =2.97: 40nt; IMB-PA20 concentration: 1. Mu.M; i MB /I FC Length of polyadenine corresponding to =2.86: 20nt; IMB-PA20 concentration: 0.5 μm) is shown in fig. 3A. FIG. 3A shows SWV response results when 100nM target DNA was detected using three separate electrodes. Characteristic peaks of the MB labeled on the signaling probe appeared at-0.23V, three times of peak current values were 131.5nA, 89.4nA, 117.3nA, respectively, the repeatability was poor, characteristic peaks of the Fc labeled on the control probe appeared at 0.26V, and also varied correspondingly with the peak current values of the MB, but when the peak current ratio (I MB /I Fc ) The signal ratios of the three times are 3.02, 2.97 and 2.86 respectively, and the repeatability is good.
More data statistics are shown in FIG. 3B, and FIG. 3B is a graph comparing reproducibility of non-ratio sensors and ratio sensors. The signal of the non-ratiometric sensor is a signaling probe-labeled MB (I MB ) SWV peak of (2)The signal of the current, ratio sensor is the SWV peak current ratio (I MB /I Fc ) The signals are subjected to normalization treatment, the reproducibility of the non-ratio type biosensor is very poor, and the relative standard deviation of the experimental result is 20.29%; the reproducibility of the experimental result of the ratio type biosensor is improved to 2.77%, which proves that the ratio type electrochemical biosensor constructed by us has the advantage of good reproducibility.
Example 6
This example optimizes the use condition parameters of the polyadenine-based inverted stem-loop specific electrochemical DNA biosensor of example 1. In order to optimize the performance of the reversed stem-loop specific ratio type electrochemical DNA biosensor based on the polyadenine, two parameters of signal probe concentration and hybridization time in experimental conditions are optimized in sequence.
The optimization results are shown in FIG. 4A and FIG. 4B, and FIG. 4A shows the concentration of the inverted stem-loop probe and the signal I MB /I Fc FIG. 4B is a graph showing hybridization time and signal I MB /I Fc Is a graph of the relationship of (1).
As shown in FIG. 4A, with increasing signal concentration, signal I was detected at 100nM target DNA MB /I Fc The signal increases gradually but also when detecting a blank, and when using a signaling probe of 40. Mu.M, the signal-to-noise ratio (S/N) is maximum, and therefore, the optimal concentration of the signaling probe is selected to be 40. Mu.M.
Hybridization time is an important parameter affecting sensor performance, and as shown in FIG. 4B, signal I is detected at 100nM target DNA with increasing hybridization time MB /I Fc The method firstly shows an increasing trend, the hybridization time reaches 2.0h, I MB /I Fc When the maximum value is reached, the signal when detecting the blank shows a decreasing trend, the hybridization time reaches 2.0h, the blank signal is minimum, and the blank signal is increased along with the continuous increase of the hybridization time. When the hybridization time was 2.0 hours, the signal to noise ratio (S/N) was maximum, and therefore, the hybridization time was selected to be 2.0 hours.
Example 7
This example evaluates the analytical performance of an inverted stem-loop specific electrochemical DNA biosensor based on polyadenine. Under the optimal experimental conditions, the reversed stem-loop specific ratio type electrochemical DNA biosensor based on the polyadenine constructed in the example 1 is adopted to detect target DNA with different concentrations.
The evaluation results are shown in FIG. 5A and FIG. 5B, FIG. 5A shows signals I of target DNA of different concentrations MB /I Fc FIG. 5B is a graph showing the logarithmic relationship of target DNA at different concentrations and signal I MB /I Fc Is a fit of the curve.
As shown in FIG. 5A, signal I increases with increasing target concentration in the range of 1pM-100nM MB /I Fc Gradually increasing. When the target concentration is in the range of 10pM-1nM, as shown in FIG. 5B, signal I MB /I Fc The linear regression equation y=0.00196 lgx+0.0084 shows a linear relationship with the logarithm of the target DNA concentration, the linear correlation coefficient being 0.997, where x represents the target DNA concentration and y represents the signal I MB /I Fc The limit of detection was 0.34pM (S/n=3). The result shows that the sensor can be used for quantitative detection of DNA, and is more sensitive and accurate.
Example 8
This example uses the multiple adenine-based inverted stem-loop specific electrochemical DNA biosensor described in example 1 to distinguish single base mismatches. The multi-adenine inverted stem-loop probe used in the detection experiment is IMB-PA20, the signal probe is Reporter probe (MB), the internal standard probe is Control probe (Fc), and the target probes are Sm-target-1, sm-target-2, sm-target-3, tlm-target and Fm-target.
It is well known that distinguishing single base mismatches has important applications in single nucleotide polymorphism detection. The reverse stem-loop specific electrochemical DNA biosensor based on the polyadenine constructed in the embodiment 1 is used for respectively detecting a complete complementary sequence, a single base mismatch sequence, a two base mismatch sequence, a three base mismatch sequence and a four base mismatch sequence, the concentration of all sequences is 50nM, the analysis result is shown in the figure 6, the signal intensity obtained by detecting the single base mismatch DNA sequence is about half of the complete complementary signal intensity, and the signal intensity obtained by detecting the two base mismatch sequence, the three base mismatch sequence and the four base mismatch sequence is close to the background signal of a system, so that the reverse stem-loop specific electrochemical DNA biosensor based on the polyadenine has high selectivity.
Example 9
This example uses the polyadenylation-based inverted stem-loop specific electrochemical DNA biosensor described in example 1 to detect biological samples.
To further evaluate the practicality of the constructed inverted stem-loop ratio electrochemical biosensor, in this example, 50nM of synthetic Target DNA (Target) was added to undiluted human serum and human saliva, respectively, to simulate a biological positive sample, undiluted human serum and human saliva were used as simulated biological negative samples, and the poly-adenine-based inverted stem-loop ratio electrochemical DNA biosensor described in example 1 was used for detection, respectively, and the results are shown in fig. 7, and fig. 7 shows the results of analysis of the simulated biological samples, and the sensor can distinguish the simulated biological positive sample from the simulated biological negative sample well, and the signal intensity obtained by detecting the simulated biological positive sample is substantially identical to the signal intensity obtained by detecting in the hybridization solution, indicating that the sensor can be used for analysis of complex biological samples.
Example 10
This example provides a series of polyadenine-based inverted stem-loop specific electrochemical DNA biosensors with the sequences shown in table 2.
TABLE 2
In the sequences of PDGF-BB-IMB-PA20 and KAN-IMB-PA20 in Table 2, the single underlined parts indicate the sequence A portion of column 1 complementarily paired with sequence 3;
PDGF-BB-IMB-PA 20:
GATCATGGTGATTATCGCATCGTATCGTAAAAAAAAAAAAAAAAAAAACAGGCTACGGCACGTAGAGCATCACCATGATCCTG
wherein "GATCATGGTGATTATCGCATCGTATCGT "is a third binding region, where"GATCAT"is sequence 3 (SEQ ID NO: 5)," GGTGATTATCGCATCGTATCGT "is sequence 2 (SEQ ID NO: 23).
"AAAAAAAAAAAAAAAAAAAA" is the first binding region (polyadenylation sequence unit).
“CAGGCTACGGCACGTAGAGCATCACCATGATCCTG "is a second binding region, capable of specifically binding to platelet derived growth factor (PDGF-BB), wherein" CAGGCTACGGCACGTAGAGCATCACCATGATCCTG "is sequence 1 (SEQ ID NO: 24), the 3' -end of the nucleotide sequence of said sequence 1"ATGATC"complementary pairing to sequence 3 of the third binding region".
KAN-IMB-PA 20:
GCTTAGCCTATCGCATCGTATCGTAAAAAAAAAAAAAAAAAAAAAGATGGGGGTTGAGGCTAAGCCGA
wherein "GCTTAGCCTATCGCATCGTATCGT "is a third binding region, where"GCTTAG"is sequence 3 (SEQ ID NO: 25) and" CCTATCGCATCGTATCGT "is sequence 2 (SEQ ID NO: 26).
"AAAAAAAAAAAAAAAAAAAAA" is the first binding region (polyadenylation sequence unit).
“GATGGGGGTTGAGGCTAAGCCGA "is a second binding region that binds Kanamycin (KAN), wherein" GATGGGGGTTGAGGCTAAGCCGA "is sequence 1 (SEQ ID NO: 27), the 3' -end of the nucleotide sequence of said sequence 1"CTAAGC"complementary pairing to sequence 3 of the third binding region".
The PDGF-BB-IMB-PA20 is an electrochemical DNA biosensor directed to platelet-derived growth factor; the KAN-IMB-PA20 is an electrochemical DNA biosensor for kanamycin; the method of example 1 was used to detect the target. The test results are shown in Table 3.
TABLE 3 Table 3
Sample of RSD(％) Detection limit
Platelet derived growth factor 2.75 0.1pM
Kanamycin 2.58 2pM
As can be seen from the results in Table 3, the reverse stem-loop ratio electrochemical DNA biosensor based on the polyadenine designed by the invention has excellent repeatability and lower detection limit when detecting platelet-derived growth factor and kanamycin, and proves that the invention has good universality.
In conclusion, the reverse stem-loop specific ratio type electrochemical DNA biosensor based on the polyadenine has the advantages of good detection effect, high sensitivity, high accuracy and good selectivity. The electrochemical DNA biosensor disclosed by the invention adopts the ratio of peak current of methylene blue to peak current of ferrocene as signal output, can effectively eliminate the influence of factors such as the change of microenvironment, the difference of effective areas of electrodes, the difference of density of assembled capture probes and the like in the detection process, has the repeatability of detection results of less than 2.77%, can achieve the excellent detection performance of 0.34pM without signal amplification, and has wide application prospect.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Sequence listing
<110> Shanghai market metering test technology institute
<120> an inverted stem-loop specific electrochemical DNA biosensor based on polyadenylation and application thereof
<130> 2022
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<170> PatentIn version 3.3
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Claims (28)

1. An inverted stem-loop specific ratio type electrochemical DNA biosensor based on polyadenine is characterized by comprising a polyadenine inverted stem-loop probe, a methylene blue modified signal probe, a ferrocene modified internal standard probe and a gold electrode; the polyadenine inverted stem-loop probe is of a three-block inverted stem-loop structure;
The skeleton structure of the polyadenylation inverted stem-loop probe is as follows from 5 '-3': a third binding region-a first binding region-a second binding region, the second binding region and the third binding region being hybridized complementarily paired to form an inverted stem-loop structure;
the first binding region is used for being covalently bound with the gold electrode to form a self-assembled monolayer film;
the second binding region is used for specifically identifying and capturing a target object, and is combined with the captured target object and the methylene blue modified signal probe by adopting a sandwich method structure and used for identifying and outputting signals;
the third binding region is used for specifically binding with the ferrocene modified internal standard probe and controlling signal output;
the nucleotide sequence of the methylene blue modified signaling probe is complementarily paired with a part of the nucleotide sequence of the target object;
the nucleotide sequence of the ferrocene modified internal standard probe is complementarily paired with a part of the nucleotide sequence of the third binding region.
2. The polyadenylation-based inverted stem-loop specific electrochemical DNA biosensor of claim 1, wherein the first binding region is a polyadenylation sequence unit, the nucleotide sequence of the sequence unit comprising: (a) n, wherein n is a positive integer, n=10-40.
3. The polyadenylation-based inverted stem-loop ratio electrochemical DNA biosensor of claim 2, wherein the first binding region comprises polyA10, polyA20, polyA30 or polyA40.
4. The polyadenylation based inverted stem-loop specific electrochemical DNA biosensor of claim 1, wherein the second binding region is sequence 1 that specifically binds to a target, the nucleotide sequence of sequence 1 comprising 10-40 bases; wherein 6-8 bases at the 3' -end of the nucleotide sequence of sequence 1 are also complementarily paired with the third binding region.
5. The polyadenylation based inverted stem-loop specific electrochemical DNA biosensor of claim 1, wherein the third binding region is a nucleotide sequence complementary to the internal standard probe, and the nucleotide sequence of the third binding region comprises 16-28 bases.
6. The polyadenylation-based inverted stem-loop specific electrochemical DNA biosensor according to claim 1, wherein the third binding region comprises, from 5' -3', sequence 3 and sequence 2 connected in sequence, the sequence 3 being a 6-8 base complementary pairing nucleotide sequence to the 3' end of the sequence 1 in the second binding region, the sequence 2 being a complementary pairing nucleotide sequence to the ferrocene modified internal standard probe.
7. The polyadenylation-based inverted stem-loop specific electrochemical DNA biosensor of claim 6, wherein the nucleotide sequence of sequence 3 comprises 6-8 bases and the nucleotide sequence of sequence 2 comprises 10-20 bases.
8. The polyadenylation-based inverted stem-loop specific electrochemical DNA biosensor of claim 1, wherein the skeletal structure of the polyadenine inverted stem-loop probe is, in order from 5 '-3': sequence 3-sequence 2-poly (a) n-sequence 1, wherein n is a positive integer, n=10-40;
the sequence 1 is specifically combined with a target, 6-8 bases at the 3' -end of the nucleotide sequence of the sequence 1 are also complementarily paired with the sequence 3, the sequence 2 is specifically combined with a ferrocene modified internal standard probe, and the poly (A) n is covalently combined with a gold electrode.
9. A method of using the polyadenylation-based inverted stem-loop specific electrochemical DNA biosensor of any one of claims 1-8, comprising the steps of:
(1) Assembling a polyadenylation reverse stem-loop probe on the gold electrode;
(2) Sealing the empty space on the surface of the gold electrode by using a sealing liquid;
(3) Pre-hybridizing the target, the signal probe and the internal standard probe, hybridizing with the inverted stem-loop probe assembled on the surface of the gold electrode, and flushing the gold electrode after hybridization;
(4) Square wave voltammetry scanning is carried out on the washed gold electrode to obtain the SWV peak current ratio of MB marked on the signal probe and Fc marked on the internal standard probeI MB /I Fc The method comprises the steps of carrying out a first treatment on the surface of the According toI MB /I Fc The target is analyzed.
10. The method of using a multi-adenine based inverted stem-loop specific electrochemical DNA biosensor as in claim 9, wherein in step (1), the assembling is performed by:
and (3) carrying out electrochemical cleaning on the physically polished gold electrode, washing the surface of the gold electrode with deionized water after cleaning, removing water on the surface of the gold electrode, and dripping the polyadenine inverted stem-loop probe solution on the surface of the gold electrode for reaction assembly.
11. The method of using a multi-adenine based inverted stem-loop ratio electrochemical DNA biosensor as claimed in claim 10 wherein said electrochemical washing is performed in sulfuric acid solution at a concentration of 0.02-1 μm.
12. The method of using a multi-adenine based inverted stem-loop specific electrochemical DNA biosensor as claimed in claim 10 wherein said method of removing water from the surface of the gold electrode is a nitrogen purge.
13. The method of using a polyadenine-based inverted stem-loop specific electrochemical DNA biosensor according to claim 10, wherein the concentration of the polyadenine inverted stem-loop probe solution is 0.02-1 μΜ.
14. The method of using a polyadenine-based inverted stem-loop specific electrochemical DNA biosensor according to claim 10, wherein the polyadenine inverted stem-loop probe solution is used in an amount of 3-10 μl/Φ mm electrode.
15. The method of using a multi-adenine based inverted stem-loop specific electrochemical DNA biosensor according to claim 10, wherein the temperature of the reactive assembly is 20-25 ℃ and the time of the reactive assembly is 2-24 h.
16. The method of using a multi-adenine based inverted stem-loop ratio electrochemical DNA biosensor of claim 9, wherein in step (2), the blocking solution comprises a solution of 6-mercaptohexanol at a concentration of 0.1-1 mM.
17. The method of using a multi-adenine based inverted stem-loop ratio electrochemical DNA biosensor of claim 16, wherein the 6-mercaptohexanol solution is used in an amount of 100-300 μl/Φ mm electrode.
18. The method of using a multi-adenine based inverted stem-loop ratio electrochemical DNA biosensor of claim 9, wherein the blocking temperature is 20-25 ℃ and the blocking time is 25-35 min.
19. The method of using a multi-adenine based inverted stem-loop specific electrochemical DNA biosensor as claimed in claim 9 wherein in step (3), the pre-hybridization treatment is performed by:
mixing a target, a signal probe and an internal standard probe with a buffer solution to obtain a prehybridization solution, keeping the prehybridization solution at 80-90 ℃ for 5-10 min, and then standing at 20-25 ℃ for more than 20 min.
20. The method of using a multi-adenine based inverted stem-loop specific electrochemical DNA biosensor as claimed in claim 19 wherein the concentration of signaling probe in the pre-hybridization solution is 30-100 μm.
21. The method of using a multi-adenine based inverted stem-loop specific electrochemical DNA biosensor as claimed in claim 19 wherein the concentration of internal standard probe in the pre-hybridization solution is 15-50 μm.
22. The polyadenylation-based inverted stem-loop specific electrochemical of claim 19 The method for using the DNA biosensor is characterized in that the buffer solution contains 0.8-1.2M NaCl and 0.03-0.07M NaClO 4 And 8-12 mM PBS, the pH of the buffer solution is 7.0-8.0, and the solvent is water.
23. The method of using a multi-adenine based inverted stem-loop specific electrochemical DNA biosensor according to claim 9, wherein the hybridization is performed at a temperature of 20-25 ℃ for a time of 0.5-4 h.
24. The method for using the reverse stem-loop specific electrochemical DNA biosensor based on polyadenylation according to claim 9, wherein the solution used for washing the gold electrode contains 0.08-0.12M NaCl and 0.03-0.07M NaClO 4 And 8-12 mM PBS, the pH of the solution is 7.0-8.0, and the solvent is water.
25. The method of using a multi-adenine based inverted stem-loop specific electrochemical DNA biosensor as claimed in claim 9, wherein in step (4), the square wave voltammetry scanning electrolyte solution system contains 0.01-0.10M NaClO 4 And 8-12 mM PBS, the pH of the electrolyte system is 7.0-8.0, and the solvent is water.
26. The method of using a multi-adenine based inverted stem-loop specific electrochemical DNA biosensor as in claim 9, wherein in step (4), said method is based on I MB /I Fc The method for analyzing the target comprises the following steps: square wave voltammetry scanning is carried out on the DNA standard substance by referring to the treatment method of the target substance, and the DNA standard substance is constructedI MB /I Fc And calculating the concentration of the target object according to the linear relation equation.
27. A polyadenine-based inverted stem-loop ratio type DNA detection kit, characterized in that the DNA detection kit comprises the polyadenine-based inverted stem-loop ratio type electrochemical DNA biosensor of any one of claims 1 to 8.
28. Use of any one or a combination of at least two of the polyadenine-based inverted stem-loop ratio electrochemical DNA biosensor of any one of claims 1-8, the polyadenine-based inverted stem-loop ratio electrochemical DNA biosensor of any one of claims 9-26, the method of use of the polyadenine-based inverted stem-loop ratio electrochemical DNA biosensor, or the polyadenine-based inverted stem-loop ratio DNA detection kit of claim 27 in DNA quantitative detection.
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