CN115369153A - Smad4 gene detection biosensor and preparation method thereof - Google Patents
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Abstract
The invention belongs to the technical field of biosensors, and relates to a Smad4 gene detection biosensor and a preparation method thereof. The Smad4 gene detection biosensor provided by the invention comprises a Smad4 gene detection DNA probe; the nucleotide sequence of the Smad4 gene detection DNA probe is as follows: 5' -SH- (CH) 2 ） 6 -CATACCAGTCTAGACTTAT-3'. The invention combines electrochemistry, magnetic materials and nano molecules to detect the Smad4 sequence in the Smad protein pathway, and has excellent detection specificity, sensitivity, biocompatibility and detection limit performance.
Description
Technical Field
The invention belongs to the technical field of gene detection, and relates to a Smad4 gene detection biosensor and a preparation method thereof.
Background
The biosensor consists of a biomolecule recognition element and various physical and chemical transducers and is used for analyzing and detecting various living substances and chemical substances. An electrochemical DNA biosensor is an instrument which uses DNA as a recognition element and converts the concentration of the DNA into an electrochemical signal for detection. Electrochemical DNA biosensors can be divided into two categories: one type is an electrochemical biosensor based on DNA hybridization, which fixes single-stranded DNA on the surface of an electrode as a probe to realize the detection of probe complementary DNA. The other type is a non-gene-recognition electrochemical DNA sensor, which is a sensitive device with single-strand or double-strand DNA fixed on the surface of an electrode as a sensor, and realizes the detection and research of specific substances by using the action of other substances and the DNA or using the characteristics of the DNA. The electrochemical DNA hybridization biosensor is characterized in that SSDNA molecules are used as sensitive elements and fixed on the surface of an electrode, the SSDNA molecules are hybridized with target DNA by utilizing a molecular hybridization technology, and the sequence of the target DNA is determined by measuring the electrochemical signal change before and after the hybridization of an electroactive substance.
The nano gold refers to gold micro particles, the diameter of which is 1 to 100nm, the nano gold has high specific surface area and high surface energy, can be combined with various biological macromolecules, and does not influence the biological activity of the nano gold. Nano meterThe gold nanoparticles have the characteristics of common nano materials, namely surface effect, small-size effect, quantum size effect and tunnel quantum size effect, and meanwhile, the gold nanoparticles have unique optical properties and the capability of promoting electron transfer. Fe 3 O 4 Are one of the most important magnetic materials. Due to the multiple functions of the nano composite material, the biosensor can meet the requirements of quick response, high sensitivity and high selectivity. But also has the problems of complex preparation process, incapability of being reused, high cost and the like, which limits the clinical use of the traditional Chinese medicine. CN113584129A provides a p53 gene detection probe, which is composed of a porous hollow magnetic nanoparticle, an electrochemical redox active probe encapsulated inside the porous hollow magnetic nanoparticle, a cationic polymer functional layer coated on the surface of the porous hollow magnetic nanoparticle, and capture DNA adsorbed on the surface of the functional layer. However, the probe is packaged in a porous hollow magnetic nanoparticle mode, so that the preparation difficulty is high, and the success rate is low.
In mammals, 8 different Smad proteins, smad1 to Smad8, are found, and can be divided into 3 subfamilies: receptor-activated Smads (R-Smad), general-type Smads (Co-Smad), inhibitory Smads (I-Smad). Only one species of Co-Smad, smad4, is found in mammals. Binding of phosphorylated Smad3 to Smad4 may prevent binding of Smad4 to nuclear receptor chromosome maintenance region 1 (nuclear export receptor chromosome region 1) to localize Smad4 in the nucleus. Smad4 expression and mutations have significant effects on a variety of cancers, including colon cancer, and aberrant Smad4 expression (mutations or reduced numbers) correlates with the degree of tumor differentiation and Dukes staging. Therefore, the Smad4 gene detection biosensor is provided, detects the expression abnormality of Smad4, and has important significance for the related research of Smad protein channels.
Disclosure of Invention
The invention aims to develop a Smad4 gene detection biosensor.
In view of the above-mentioned objects, the present invention provides a Smad4 gene detection biosensor and a method for preparing the same to meet the needs in the art.
In one aspect, the present invention provides a Smad4 gene detecting biosensor comprising a Smad4 gene detecting DNA probe; the nucleotide sequence of the Smad4 gene detection DNA probe is as follows: 5' -SH- (CH) 2 ） 6 -CATACCAGTCTAGACTTAT-3’。
In another aspect, the present invention provides a method for preparing a Smad4 gene detection biosensor, comprising: dissolving the Smad4 gene detection DNA probe into a tris (2-carboxyethyl) phosphine solution to obtain an activated Smad4 gene detection DNA probe; mixing hydroxyethyl piperazine ethyl sulfonic acid and HAuCl 4 ·XH 2 Mixing O and pure water, heating and stirring to prepare a nano gold solution; and mixing the activated Smad4 gene detection DNA probe and the nano gold solution at normal temperature, dripping the mixture on the surface of the treated gold electrode, reacting at 37 ℃ for 1.5 hours, immersing the mixture in mercaptoethanol, and sealing at 37 ℃ for 0.5 hour.
Further, in the preparation method of the Smad4 gene detection biosensor provided by the invention, the processing method of the gold electrode comprises the following steps: method for preparing Fe by adopting in-situ chemical oxidative polymerization 3 O 4 Removing an oxide layer and impurities on the surface of the gold electrode by using a-PANI-AuNPs composite solution, immersing the gold electrode in an HDT ethanol solution, reacting for 1h at 4 ℃, sequentially cleaning the electrode by using absolute ethanol and ultrapure water, drying the electrode by using ultrapure nitrogen, and immersing the electrode in the Fe composite solution 3 O 4 And (3) placing the solution in the-PANI-AuNPs composite solution at 37 ℃ for 12h, washing with water and drying.
Furthermore, in the preparation method of the Smad4 gene detection biosensor, the Fe is calculated according to the quantitative ratio of substances 3 O 4 Fe in-PANI-AuNPs composite solution 3 O 4 The proportion of PANI to AuNPs is 1.
In another aspect, the present invention relates to a Smad4 gene detection method using the Smad4 gene detection biosensor described above.
Further, the Smad4 gene detection method provided by the invention comprises the following steps: the Smad4 gene detection biosensor was immersed in a sample solution to be detected, reacted at 37 ℃ for 1 hour, and then the surface of the electrode was washed with PBS buffer, 0.2% SDS, and ultrapure water in this order to remove non-hybridized target DNA, to obtain a modified gold electrode, which was subjected to electrochemical measurement.
Further, in the Smad4 gene detection method provided by the present invention, the electrochemical assay comprises: and stirring and immersing the modified gold electrode in 1mmol/L MB for 20min, then stirring in PBS and ultrapure water for 5min, and recording the DPV curve of the hybridized electrode in the PBS solution.
In another aspect, the present invention relates to the use of the foregoing Smad4 gene detection biosensor in the detection of Smad4 gene.
In another aspect, the invention relates to the use of the foregoing Smad4 gene detection method in Smad4 gene detection.
Compared with the prior art, the invention has the following beneficial effects or advantages:
the invention provides a Smad4 gene detection biosensor and a preparation method thereof, on one hand, the invention designs a Smad4 gene detection DNA probe, which can realize the detection and identification of Smad4 gene in a Smad protein channel; in another aspect, the invention provides a Smad4 gene detection biosensor, and Fe is designed 3 O 4 The PANI-AuNPs realize the combination of the probe and the electrode and can better detect the electric signal change of the Smad protein channel in the sample to be detected.
Drawings
FIG. 1 shows the variation of peak current after hybridization of probe sequences with different concentrations of target sequences.
FIG. 2 shows the peak current values after hybridization of probe sequences with different sequences at the same concentration.
Detailed Description
In order to make the technical solutions of the present invention better understood and enable one skilled in the art to practice the present invention, the present invention is further described below with reference to specific examples and drawings, but the examples are not intended to limit the present invention.
The experimental methods and the detection methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
Example 1
This example provides the preparation of a Smad4 gene detection biosensor.
And (3) probe:
chemically synthesizing a Smad4 gene detection DNA probe; the nucleotide sequence of the Smad4 gene detection DNA probe is as follows: 5' -SH- (CH) 2 ） 6 -CATACCAGTCTAGACTTAT-3’。
Fe 3 O 4 -PANI-AuNPs complex solution preparation:
the Fe is calculated according to the mass ratio 3 O 4 Fe in-PANI-AuNPs composite solution 3 O 4 The proportion of PANI to AuNPs is 1.
50mL of a 0.01% strength by mass/volume tetrachloroauric acid solution are brought to boiling with vigorous stirring. Then 2mL of trisodium citrate at 1% by mass/volume are added rapidly to the boiling solution. When the color of the solution is changed from light yellow to wine red, the AuNPs solution is prepared.
1.25g of dodecylbenzenesulfonic acid (DBSA) and 0.5mL of aniline monomer were dissolved in 50mL of distilled water, and after stirring for 20min, 25mL of an aqueous solution containing 1.25g of (NH) S20S was added dropwise, and the reaction was carried out at a constant temperature for 8 hours. After the reaction is completed, CHC1 is used 3 Extracting to obtain PANI (DBSA)/CHC 1 3 And (3) solution. Mixing the solution with a certain amount of FeCl 3 ·6H 2 O and FeC1 2 ·4H 2 O solution (FeCl) 3 ·6H 2 O and FeC1 2 ·4H 2 The mass ratio of O is 2: 1) And uniformly mixing, heating under reflux and stirring for reaction, adding a certain amount of uncooled AuNPs solution, uniformly and slowly dripping the heated NaOH solution after 30min until the pH is =7.4, and continuously reacting for 8h. After the reaction was completed, CHC1 was used again 3 Extracting, washing with distilled water to obtain the Fe 3 O 4 -PANI-AuNPs complex solution.
And (3) treating the treated gold electrode:
polishing aluminum oxide powder to remove an oxide layer on the surface of the gold electrode, removing impurities on the surface of the gold electrode by ultrasonic waves, immersing the gold electrode into a 2.5mmol/L HDT ethanol solution, reacting for 1 hour at 4 ℃, cleaning the electrode by absolute ethyl alcohol and ultrapure water in sequence, and drying the electrode by blowing with ultrapure nitrogen. Immersing in 1mol/L Fe 3 O 4 And (3) placing the mixture in the PANI-AuNPs compound solution for 12 hours at 37 ℃, washing with water and drying in the air.
Preparation of Smad4 gene detection biosensor:
and dissolving the Smad4 gene detection DNA probe into a tris (2-carboxyethyl) phosphine solution to obtain 50 mu L of 3 mu mol/L activated Smad4 gene detection DNA probe. Reacting hydroxyethyl piperazine ethyl sulfonic acid with HAuCl 4 ·XH 2 And mixing O and pure water, heating and stirring to prepare the nano gold solution. Mixing 50 mu L of 3 mu mol/L activated Smad4 gene detection DNA probe and 50mL of 5mmol/L nano-gold solution at normal temperature, dripping the mixture on the surface of a treated gold electrode, reacting at 37 ℃ for 1.5h, immersing the mixture in 1mmol/L mercaptoethanol, and sealing at 37 ℃ for 0.5h.
Example 2
This example provides detection by a Smad4 gene detection biosensor.
Target DNA sequence: 5 'ATAAGTCTAGACTGGTATG-3';
single base mismatches: 5 'ATAACTCTAGACTGGTATG-3';
non-complementary sequences: 5 'GCGGTCTCGAGTCAACACA-3'.
Adding TE buffer solution with pH 7.4 in the amount required by the preparation concentration into each sequence DNA, then slightly covering the tube cover, shaking uniformly, and preparing into 1.0 × 10 5 And (5) freezing and storing the solution in mol/L.
The assembled Smad4 gene detection biosensor was immersed in 100. Mu.L of target DNA solutions of different concentrations, reacted at 37 ℃ for 1 hour, and then the surface of the gold electrode was washed with PBS buffer, 0.2% SDS, and ultrapure water in this order to remove non-hybridized target DNA, i.e., hybridized gold electrode, and then electrochemically measured.
The test adopts a three-electrode system, and takes a hybridized gold electrode as a working electrode, a saturated calomel electrode as a reference electrode and a platinum electrode as a counter electrode. In the presence of 1mmol/L [ Fe (CN) ] containing 0.1mol/L KCl 6 ] 3-/4- In the solution, an electrochemical workstation records the electrochemical impedance spectrum of each modified gold electrode, and the frequency range is 10 5 Hz-0.01Hz。
The Smad4 gene detection biosensor is stirred and immersed in 1mmol/L MB for 20min, then stirred in PBS and ultrapure water for 5min in sequence to remove non-specifically adsorbed MB, and the DPV curve of the electrode after hybridization is recorded in the PBS solution. The PBS buffer was flushed with nitrogen for 20min prior to the assay to remove oxygen.
FIG. 1 is a regression curve of MB after hybridization of probe sequences with different concentrations of target sequences. The change in peak current (. DELTA.I) and the logarithm of DNA concentration (lgc) DNA ) In a good linear relation, the regression equation delta I =0.7642 lgc DNA +11.084, linear correlation coefficient of 0.9992, detection limit of 1.3X 10 -13 mol/L（σ=3）。
To further verify the selectivity of the biosensor prepared according to the present invention, 1X 10 was used -5 Change in detection signal of different DNA sequences of target DNA sequence (TD), single base mismatch target (SM) and non-complementary DNA (NC) in mol/L. Smad4 gene detection biosensor at 1mmol/L [ Ru (NH) ] 3 ) 6 ] 3+ After immersing for 20min under stirring, stirring in PBS and ultrapure water for 5min to remove non-specifically adsorbed [ Ru (NH) 3 ) 6 ] 3+ The DPV curves of the hybridized electrodes were recorded in PBS solution. The PBS buffer was flushed with nitrogen for 20min prior to the assay to remove oxygen.
As can be seen from FIG. 2, the electrochemical signal of the non-complementary DNA is very low for different DNA sequences at the same concentration as the target, and the electrochemical signal of the single base mismatch target (SM) is lower than that of the target DNA sequence (TD) for the single base mismatch target DNA sequence at the same concentration as the target DNA sequence. As can be seen from comparison of FIG. 1, [ Ru (NH) ]was used 3 ) 6 ] 3+ As an indicator, it is less effective than MB. The result shows that the biosensor constructed by the strategy of the invention has good selectivity and discrimination capability of base mutation analysis on the target DNA, and has huge application potential.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.
Claims (10)
1. A Smad4 gene detection biosensor, comprising a Smad4 gene detection DNA probe; the nucleotide sequence of the Smad4 gene detection DNA probe is as follows: 5' -SH- (CH) 2 ） 6 -CATACCAGTCTAGACTTAT-3’。
2. The Smad4 gene detection biosensor according to claim 1, wherein the preparation method comprises: dissolving the Smad4 gene detection DNA probe into a tris (2-carboxyethyl) phosphine solution to obtain an activated Smad4 gene detection DNA probe; mixing hydroxyethyl piperazine ethyl sulfonic acid and HAuCl 4 ·XH 2 Mixing O and pure water, heating and stirring to prepare a nano gold solution; and mixing the activated Smad4 gene detection DNA probe and the nano-gold solution at normal temperature, dripping the mixture on the surface of the treated gold electrode, reacting at 37 ℃ for 1.5h, immersing the mixture in mercaptoethanol, and sealing at 37 ℃ for 0.5h.
3. The Smad4 gene detection biosensor of claim 2, wherein the gold electrode treatment method comprises: method for preparing Fe by adopting in-situ chemical oxidative polymerization 3 O 4 Removing an oxide layer and impurities on the surface of the gold electrode by using a-PANI-AuNPs composite solution, immersing the gold electrode in an HDT ethanol solution, reacting for 1h at 4 ℃, sequentially cleaning the electrode by using absolute ethanol and ultrapure water, drying the electrode by using ultrapure nitrogen, and immersing the electrode in the Fe composite solution 3 O 4 And (3) placing the solution in the-PANI-AuNPs composite solution at 37 ℃ for 12h, washing with water and drying.
4. The Smad4 gene detection biosensor according to claim 3, wherein the Fe is in a mass ratio of substances 3 O 4 Fe in-PANI-AuNPs composite solution 3 O 4 The proportion of PANI to AuNPs is 1.
5. A Smad4 gene detection method using the Smad4 gene detection biosensor according to any one of claims 1 to 4.
6. The method for detecting a Smad4 gene of claim 5, comprising: the Smad4 gene detection biosensor was immersed in a sample solution to be detected, reacted at 37 ℃ for 1 hour, and then the surface of the electrode was washed with PBS buffer, 0.2% SDS, and ultrapure water in this order to remove non-hybridized target DNA, to obtain a modified gold electrode, which was subjected to electrochemical measurement.
7. The Smad4 gene detection method of claim 5, wherein the electrochemical assay comprises: and stirring and immersing the modified gold electrode in 1mmol/L MB for 20min, then stirring in PBS and ultrapure water for 5min in sequence, and recording the DPV curve of the hybridized electrode in the PBS solution.
8. Use of the Smad4 gene detection biosensor according to any one of claims 1 to 4 for Smad4 gene detection.
9. Use of the detection method using the Smad4 gene detection biosensor according to claim 8 in Smad4 gene detection.
10. The use of claim 9, wherein the Smad4 gene assay comprises assaying a sample containing a human Smad4 gene.
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