CN113702462B - Preparation method of double-strand specific nuclease-assisted controlled release electrochemical DNA hydrogel composite material - Google Patents
Preparation method of double-strand specific nuclease-assisted controlled release electrochemical DNA hydrogel composite material Download PDFInfo
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Classifications
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
- C08F220/603—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen and containing oxygen in addition to the carbonamido oxygen and nitrogen
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a preparation method of a double-strand specific nuclease-assisted controlled release electrochemical DNA hydrogel composite material. The method is characterized in that: the base sequences of the DNA Strand A (SA) and the DNA Strand B (SB) in the DNA hydrogel were designed. So that the chlamydia trachomatis 16S rRNA (target RNA) can respond more obviously to the stimulation of a small amount of chlamydia trachomatis 16S rRNA with the help of double-strand specific nuclease (DSN). In most types of DNA hydrogels, each separation of a DNA probe requires hybridization of one strand of interest thereto. When the amount of target rRNA is small, this results in that it cannot produce an effective stimulus to the DNA hydrogel. The method successfully introduces DSN into the DNA hydrogel to obtain the controlled release DNA hydrogel which can be repeatedly cracked by a small amount of target objects. Therefore, it has higher reaction rate and sensitivity.
Description
Technical Field
The invention relates to a preparation method of a double-strand specific nuclease (DSN) -assisted controlled release electrochemical DNA hydrogel composite material and application thereof in electrochemical detection of chlamydia trachomatis 16S rRNA, belonging to the field of biological sensing.
Background
The controlled release system may be responsive to specific stimuli, such as nucleic acids, light, molecules, etc., to alter its physicochemical properties. Numerous controlled release systems based on hydrogel construction have been designed and widely used for smart drug delivery and targeted cancer therapy due to their excellent loading capacity and stimulus response capability. Recently, controlled release systems have been combined with a variety of detection techniques to develop bioanalytical sensors for different targets. The electrochemical detection technology is a qualitative and quantitative detection technology based on the electrochemical property of a substance, and can have high sensitivity only by simple equipment. To our knowledge, little research has been done to develop biosensors that combine the advantages of controlled release systems and electrochemical detection, although these sensors may have the advantages of simple structure, signal probe loading capability, high sensitivity and selectivity. Combining the two can integrate the advantages of hydrogel and an electrochemical system, and a novel biosensor is constructed for biological analysis.
Chlamydia Trachomatis (CT) is one of the most common pathogens for sexually transmitted diseases. A significant feature of CT infection is the absence of symptoms, especially in women with infection. More importantly, although there is no obvious symptom, serious sequelae such as pelvic inflammatory disease, infertility of fallopian tubes and the like can be caused. Thus, they need rapid point-of-care detection and extensive screening to facilitate identification of asymptomatic infections and enable early treatment, which requires rapid and sensitive biosensors as support. The DSN-assisted controlled release electrochemical DNA hydrogel composite material can respond to the stimulation of the 16S rRNA due to the complete pairing of the base sequence of SA and CT 16S rRNA. With this response, the convenient and rapid detection of CT 16S rRNA is of great importance for the identification and early treatment of asymptomatic infections.
The invention aims to provide a preparation method of a controllable-release electrochemical DNA hydrogel composite material, which is characterized in that the DNA probe sequence is carefully designed, and horseradish peroxidase (HRP) is uniformly embedded in the hydrogel, so that the composite material for CT 16S rRNA electrochemical detection is provided in the technical field of biosensing under the assistance of DSN.
Disclosure of Invention
It is an object of the present invention to provide a DSN-assisted controlled release electrochemical DNA hydrogel composite,
the chemical components of the material are acrylamide, ammonium Persulfate (APS), tetramethyl diethylamine (TEMED), and partially complementary acrylamide-modified DNA probes A and B (SA and SB), wherein HRP is filled in hydrogel to serve as an electrochemical signal molecule.
The invention aims to provide a preparation method of a DSN-assisted controlled release electrochemical DNA hydrogel composite material. The object of the invention is achieved in that it comprises the following steps:
(1) SA or SB was mixed with acrylamide solution and dried in vacuo to remove oxygen. Freshly prepared Ammonium Persulfate (APS) and tetramethyl diethylamine (TEMED) are then added and heated to form polymer A (P-SA) or polymer B (P-SB).
(2) The P-SA, P-SB and horseradish peroxidase (HRP) from step (1) are mixed in specific proportions and heated with sufficient agitation to form a DSN-assisted controlled release electrochemical hydrogel.
(3) In the step (1), the concentration of the SA and SB DNA chains is 100 mu M, the mass-volume ratio of the acrylamide solution is 25%, the mass-volume ratio of the APS is 10%, and the mass-volume ratio of the TEMED is 10%. During the polymer synthesis, SA or SB, acrylamide solution, APS and TEMED were added in a volume ratio of 80. Mu.L: 16 μl:1 μl:1 mul. The heating temperature for the synthesis of P-SA or P-SB was 37℃for 60 minutes.
(4) The dosage of the P-SA and the P-SB in the step (2) is 2.5 mu L, the concentration of the HRP enzyme is 100 mU, the dosage is 1 mu L, the gel forming mixing temperature is 65 ℃ and the time is 10 minutes.
(5) The sequence of the DNA strand in the step (1) is Strand A (SA) Acrydite-AAAAACTTAGGGGCCGA C TAACCC and Strand B (SB) Acrydite-AAAAGGGTTA C TCGGCC.
(III) application of DSN-assisted-based controlled release electrochemical DNA hydrogel composite material
The controllable-release electrochemical hydrogel composite material is used as an electrochemical sensor for detecting CT 16S rRNA, and comprises the following steps:
(1) CT 16S rRNA with different concentrations is respectively added into the DNA hydrogel composite material together with DSN to perform competitive binding on SA-SB chains to form an SA-rRNA structure. The latter is recognized by DSN, where SA is digested and rRNA is dissociated to bind new SA, forming a cyclic amplification effect. Eventually, the hydrogel disintegrates and the embedded HRP is released. Collecting the supernatant, adsorbing onto gold electrode, and separating with TMB and H 2 0 2 The mixed solutions were used as substrates and the electrochemical signal of each mixed system was measured using the CHI660E electrochemical workstation.
(2) Drawing a standard curve according to the collected electrochemical signals of each mixed system;
(3) Adding a sample to be tested and DSN together on the prepared electrochemical hydrogel composite material, collecting supernatant obtained after the reaction, adsorbing the supernatant on a gold electrode, taking a TMB and H202 mixed solution as a substrate, and measuring electrochemical signals of each mixed system by using a CHI660E electrochemical workstation. Combining the electrochemical signal with a standard curve to obtain the CT concentration in the sample to be detected.
The standard sample and the sample volume to be measured, the DSN volume, the supernatant volume, TMB and H of the steps (1) and (3) 2 0 2 The dosage ratio of the mixed solution is 20 mu L:1 [ mu ] L: 10 [ mu ] L: 2 mL.
The invention has the beneficial effects that:
in a typical controlled release DNA hydrogel, each separation of the DNA probe requires a target strand to hybridize to, and when the amount of target RNA is small, it does not produce effective stimulation of the DNA hydrogel. The DNA hydrogel composite material provided by the invention has the advantages that the base sequences of the DNA probes A (SA) and B (SB) are specially designed, and the DNA probes A (SA) and B (SB) can be repeatedly stimulated by a small amount of target RNA under the assistance of double-strand specific nuclease (DSN) to form cyclic enzymolysis, so that more obvious response is generated, and more electrochemical signal molecules are released. Therefore, compared with the general controllable release DNA hydrogel material, the material has more excellent reaction rate and sensitivity.
Drawings
FIG. 1 is a schematic diagram of a method for preparing a DNA hydrogel composite material of the present invention;
FIG. 2 is a graph showing the analysis and detection of a 16S rRNA standard sample based on a DSN-assisted controlled release electrochemical DNA hydrogel composite material according to the present invention, wherein: a) Blank buffer; b) DNA hydrogel supernatant without target rRNA; c) DNA hydrogel supernatant with 10pM target reaction.
Detailed Description
The invention is described in further detail below with reference to the drawings and the detailed description.
As shown in FIG. 1, acrylate modified DNA Strand A (SA) and DNA Strand B (SB) were copolymerized with acrylamide monomers to obtain corresponding DNA-polyacrylamide complexes, i.e., polymer strand A (P-SA) and polymer strand B (P-SB), respectively. The two were mixed and hybridized P-SA and P-SB to form a 16s-rRNA responsive hydrogel in which HRP was encapsulated. Since SA-SB hybrid double-stranded (dsDNA) has a set of mismatched base pairs, the DSN will not be able to cleave it without the target rRNA. However, the SA therein is fully complementary to the target sequence, and thus when presentAt the target sequence, the SA-SB dsDNA will be replaced with B-target rRNA to form an SA-rRNA hybridization duplex. The DNA-rRNA hybrid duplex is then specifically recognized by DSN. SA is digested by DSN, target rRNA is released and recovered for another round of hybridization and digestion. Thus, in this strategy, only a very small amount of target rRNA is required for collapse of the hydrogel and release of HRP, as the target rRNA is recovered by the DSN. The released HRP is then adsorbed to a working electrode and placed in a reaction chamber containing the substrates TMB and H 2 O 2 In the measurement solution of (2), TMB can be subjected to catalytic amplification oxidation-reduction reaction of a plurality of horseradish peroxidase on the surface of the sensor, and the reduction current signal of the catalytic oxidation product of TMB is measured at a fixed potential, so that the current signal is amplified, and the signal size is related to the concentration of target DNA.
Eventually, a significantly enhanced electrochemical signal may be detected. The electrochemical signal is positively correlated with the target concentration, thereby realizing quantitative detection of chlamydia trachomatis.
Example 1:
the preparation method of the DSN-assisted controlled release electrochemical DNA hydrogel composite material comprises the following steps:
(1) SA or SB was mixed with 25% by mass of acrylamide solution and vacuum dried for 15 minutes to remove oxygen. Then 10% mass fraction of freshly prepared APS and 10% mass fraction of TEMED were added and dried at 37 ℃ for 60 minutes to form P-SA or P-SB. Then, P-SA, P-SB, NE buffer and HRP were mixed and heated at 65℃for 10 minutes with sufficient shaking to form a DNA hydrogel.
(2) The DNA sequence SA in the step (1) is Acrydite-AAAAACTTAGGGGCCGA C TAACCC, SB, acrydite-AAAAGGGTTA C TCGGCC. During the synthesis of the polymer P-SA or P-SB, SA or SB, acrylamide solution, APS and TEMED were added in a volume ratio of 80. Mu.L: 16 μl:1 μl:1 mul. In the process of heating and gelling the DNA hydrogel composite material, the volume ratio of the P-SA, the P-SB, the NE buffer solution and the HRP is 2.5 mu L: 2.5 μl:1 μl:1 mul. The HRP enzyme activity unit is 100M U.
Example 2:
the DSN-assisted controlled release electrochemical DNA hydrogel composite prepared based on example 1 was tested for target RNA as follows:
(1) Mu.l of target rRNA sequence or 20. Mu.l of blank buffer were mixed with 1. Mu.l of DSN, respectively, and then added to the DNA hydrogel prepared in step (1) of example 1, respectively, to form a mixture. After incubating the mixture at 35℃for 60 minutes, 10. Mu.l of the supernatant was removed by gentle shaking, and each of them was dropped onto the activated gold electrode and allowed to stand for drying. And 10 μl of blank buffer was also added dropwise to the activated gold electrode, and left to stand for drying as a blank control.
(2) In step (1), the target rRNA sequence was GGGUUAGUCGGCCCCUAAGU at a concentration of 10pM and the DSN enzyme activity unit was 10M U. The blank buffer was 1 XPBS buffer.
(3) Immersing the gold electrode obtained in the step (1) into 1 ml of TMB substrate solution (K-blue, neogen corporation, H-containing) 2 O 2 ) In the above, a current-time curve (initial potential: 0 volts; sampling interval: 0.1 seconds; sampling time: 100 seconds). The measurement results are shown in FIG. 2.
Each curve in fig. 2 shows the response of the time-current curve under different conditions. The drop of blank buffer on the gold electrode modified with capture probe gave a lower current signal (curve a), probably due to TMB in the absence of HRP catalysis, and H 2 O 2 Weak interactions between them. When the DNA hydrogel supernatant that was not reacted by the target rRNA was added dropwise to the gold electrode, a very slight increase in the curve signal was observed (curve b), probably due to a small portion of unintentional leakage of HRP embedded with the DNA hydrogel. Whereas when the DNA hydrogel supernatant with target rRNA reaction was added dropwise to the gold electrode modified with capture probe, a very pronounced enhancement of the i-t curve signal occurred (curve c). The results indicate that electrochemical sensors constructed using DSN-assisted controlled release electrochemical DNA hydrogel composites can be used to recognize target RNA sequences.
Claims (2)
1. A method for detecting chlamydia trachomatis 16S rRNA based on a double-strand specific nuclease-assisted controlled release electrochemical DNA hydrogel composite material is characterized by comprising the following steps:
sequence of DNA strand: SA, acrydite-AAAAACTTAGGGGCCGA C TAACCC;
SB：Acrydite-AAAAGGGTTA C TCGGCC；
sequence of 16S rRNA: GGGUUAGUCGGCCCCUAAGU;
the preparation method of the DNA hydrogel composite material comprises the following steps:
(1) Mixing DNA strand SA and DNA strand SB with acrylamide solution, and vacuum drying to remove oxygen; then adding freshly prepared ammonium persulfate and tetramethyl diethylamine, and heating to form polymer P-SA and polymer P-SB;
(2) Then, mixing polymer P-SA, polymer P-SB and horseradish peroxidase, and heating under sufficient shaking to form a DNA hydrogel composite;
the method for detecting 16S rRNA comprises the following steps:
1) Respectively adding 16S rRNA with different concentrations and double-strand specific nuclease into the DNA hydrogel composite material, wherein the 16S rRNA performs competitive binding on the SA-SB chain to form an SA-rRNA structure; SA-rRNA is recognized by double-strand specific nucleases, wherein DNA strand SA is digested, rRNA is dissociated to bind to new DNA strand SA, forming a cyclic amplification effect; finally, the hydrogel is cracked, and the embedded horseradish peroxidase is released; collecting the supernatant, adsorbing onto gold electrode, and separating with TMB and H 2 0 2 The mixed solution is used as a substrate, and electrochemical signals of each mixed system are measured by a CHI660E electrochemical workstation;
2) Drawing a standard curve according to the collected electrochemical signals of each mixed system;
3) Adding a sample to be detected and double-chain specific nuclease together on the prepared electrochemical hydrogel composite material, collecting supernatant obtained after the reaction, adsorbing the supernatant on a gold electrode, and using TMB and H 2 0 2 The mixed solution is used as a substrate, and electrochemical signals of each mixed system are measured by a CHI660E electrochemical workstation; combining the electrochemical signal with a standard curve to obtain16S rRNA concentration in the sample to be tested.
2. The method according to claim 1, characterized in that: in the step (1), the concentration of the DNA chain SA and the DNA chain SB is 100 mu M, the mass-volume ratio of the acrylamide solution is 25%, the mass fraction of ammonium persulfate is 10%, and the mass fraction of tetramethyl diethylamine is 10%; in the polymer synthesis process, DNA chain SA or DNA chain SB, acrylamide solution, ammonium persulfate and tetramethyl diethylamine are added in the volume ratio of 80 mu L:16 μl:1 μl:1 μl; the synthetic heating temperature of the polymer P-SA and the polymer P-SB is 37 ℃ and the time is 60 minutes; the dosage of the polymer P-SA in the step (2) is 2.5 mu L, the activity unit of horseradish peroxidase is 100 MU, the dosage is 1 mu L, the gel forming mixing temperature is 65 ℃ and the time is 10 minutes.
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