CN116814745A - Preparation method of ratio type biosensor based on structure-specific fluorescent dye - Google Patents
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Abstract
The invention belongs to the technical field of biosensing detection, and discloses a preparation method and application of a ratio type biosensor based on a structure-specific fluorescent dye. Double-chain fluorescent dye SGI and G-quadruplex fluorescent dye NMM with structural specificity are used as double-output signals, a cascade signal amplification strategy combining DNAzyme auxiliary target circulation strategy and Exo III auxiliary DNA circulation strategy is utilized, DNAzyme is used as a biological recognition element, and the biological recognition element and target Pb are applied to the double-chain fluorescent dye SGI and G-quadruplex fluorescent dye NMM 2+ Specific binding characteristics, realizing Pb in actual sample 2+ High sensitivity and high (V)Accuracy and low cost detection. Pb 2+ The presence of (2) may cause a decrease in the fluorescence intensity of SGI at 520nm and a significant increase in the fluorescence intensity of NMM at 610 nm. The ratio type fluorescent biosensor pair Pb constructed by the invention 2+ The linear response range of (2) is 0.5-500 nM, the detection limit is 26.4 pM, and the device has good anti-interference capability and practical application capability, and is used for measuring Pb in a practical sample 2+ A novel biosensing platform is provided.
Description
Technical Field
The invention relates to a preparation method and application of a ratio type biosensor based on a structure-specific fluorescent dye.
Background
Lead ion (Pb) 2+ ) As a serious environmental contaminant, it has been recognized that it adversely affects the ecosystem, food safety, and human health. Pb due to the non-degradability and high usage of lead in the paint, battery and ceramic industries 2+ Has become a major environmental contaminant in water, soil and air. Prolonged exposure of humans to lead from drinking water has been reported to have irreversible health consequences. Very small amounts of lead ions can cause serious damage to the brain, kidneys and nervous system. Pb according to the United states Environmental Protection Agency (EPA) report 2+ Can lead to the difficulty in focusing attention, hyperactivity and mental retardation of the infants. Thus, development has been conducted for detecting Pb in environments, foods or biological samples 2+ The method of (2) has important significance.
There are many methods for detecting lead, such as differential potential elution analysis (DPSA), graphite furnace atomic absorption spectrometry (GF-AAS), hydride generation atomic fluorescence spectrometry (HG-AFS), inductively coupled plasma mass spectrometry (ICP-MS), dithizone Colorimetry (DC), etc., which can generally realize Pb detection 2+ But most of the methods need complicated and expensive instruments or complex operation steps, and are not suitable for on-site real-time detection. Various chemical and biological sensing technologies have been explored as alternatives to rapid and economical detection of trace lead contaminants, such as fluorescence, colorimetry, electrochemistry, electrochemiluminescence, and the like. Although these detection techniques have made great progress, there are also problems that the design or synthesis process is relatively complicated and even the detection performance is not ideal. Thus, sensitive, simple, rapid, inexpensive methods or strategies are developed for Pb 2+ Ion detection remains a challenge.
In recent years, a biological recognition element, DNAzyme, has attracted a great deal of attention in the field of metal ion detection due to its ease of design and modification. DNAzyme is obtained by in vitro screening, has high catalytic activity on a specific substrate, has high thermal stability and chemical stability, and has been read out in combination with various signal transduction modes, such as colorimetry, electrochemistry and fluorescence. Among them, fluorescence sensors are focused on the advantages of high sensitivity, easy operation, no damage to samples, and capability of providing real-time information for various applications. In order to meet the requirement of higher detection sensitivity in complex samples, isothermal amplification strategies such as exonuclease-assisted cyclic amplification are often introduced to achieve signal amplification. In addition, most fluorescent biosensors use fluorescent donors and acceptors to modify the aptamer, which is complex and expensive and double labeling can impair the binding affinity of the aptamer to the target, affecting detection sensitivity. In contrast, label-free sensing strategies have received great attention for their simplicity and economy. However, most of these detection platforms use a single signal, which is often affected by complex substrate interference, instrument fluctuation, probe concentration, and other factors. The ratio fluorescence analysis method can provide a built-in self-calibration function by measuring the change of the ratio of the fluorescence intensities of the two dyes, thereby effectively improving the anti-interference capability, the robustness and the reliability of the detection method.
Aiming at the problems of poor selectivity, low sensitivity, unstable detection and the like existing in the prior detection technology, the invention constructs a novel label-free ratio fluorescent biosensor. First, target Pb was treated with DNAzyme 2+ Carrying out specific recognition and enhancing detection specificity; secondly, a DNAzyme and Exo III auxiliary cascade signal amplification strategy is adopted to realize signal amplification, so that the detection sensitivity is improved; finally, the ratio fluorescent sensing platform is formed by skillfully utilizing two structure-specific fluorescent dyes, so that the interference of the external environment can be effectively reduced, false positive and false negative signals are avoided, and the reliability of the sensor is improved. The fluorescence sensing strategy based on the invention overcomes the problems in the prior art, has the characteristics of higher sensitivity, economy, simplicity and stability, and is beneficial to popularization and application of the invention.
Disclosure of Invention
The preparation method of the ratio type biosensor based on the structure-specific fluorescent dye comprises the following steps:
(1) Construction of a target recognition unit: E-DNA and S-DNA are subjected to base complementation to form a DNAzyme secondary structure, and the E-DNA/S-DNA@MBs system is formed by immobilizing the E-DNA and the S-DNA on MBs through the combination effect of biotin modified at the tail end of the S-DNA and streptavidin coated on the surface of the MBs, performing magnetic separation and washing, and removing supernatant.
(2) Sensing process without target: when Pb-free 2+ In the system, DNAzyme structure on the surface of MBs is in a stable state and is removed through a magnetic separation process, dsDNA formed by hybridization of S1 and G1 and exonuclease are added into supernatant, and the exonuclease cannot exert shearing activity due to the fact that 3' -blunt ends or concave ends are not present in the double-stranded structure, so that G1 rich in guanine bases is blocked in double chains, double-chain and G-quadruplex specific fluorescent dyes are added, the double-chain fluorescent dyes are embedded in a large amount of dsDNA, and the G-quadruplex fluorescent dyes are free in solution.
(3) Sensing process in the presence of target: when Pb exists in the system 2+ When DNAzyme is excited, thereby inducing rA site on S-DNA to shear off ssDNA and simultaneously releasing Pb 2+ And E-DNA, start DNAzyme assisted target circulation system. Subsequently, the E-DNA continues to complementarily pair with another S-DNA on the surface of MBs, at Pb 2+ The shearing reaction is continued under the action of the catalyst, and more ssDNA is obtained in the supernatant after magnetic separation. Continuing to add dsDNA and exonuclease, ssDNA will complementarily pair with the partial sequence exposed by S1 to form a 3' blunt end, which initiates the exonuclease-assisted DNA circulation system. The enzyme digests S1 into mononucleotide from 3' end, released ssDNA is combined with new dsDNA, the next cleavage reaction is started, dsDNA is reduced in the final system, double-chain fluorescent dye effect is reduced, and exposed G1 is in K + Is folded under the stability to form a G-quadruplex structure, and the G-quadruplex fluorescent dye is embedded in a large amount.
(4) Establishment of linear relationship between the ratio fluorescent signal and the target: pb-free 2+ In the case of (2), there is little in the supernatantThe ssDNA is contained, the continuous introduction of the dsDNA and the exonuclease do not play a role, and finally, the double-stranded fluorescent dye is combined with a large amount of dsDNA and emits strong fluorescence, and the G1 is blocked so that the fluorescent dye of the G-quadruplex is free and has weaker fluorescence intensity in the solution; pb 2+ The existence of the double-stranded fluorescent dye activates DNAzyme, and simultaneously triggers the DNAzyme and an exonuclease auxiliary cascade signal amplification system, so that dsDNA is sheared and digested, the fluorescence intensity of the double-stranded fluorescent dye is obviously reduced, and the exposed large amount of G1 and G-quadruplex fluorescent dye change in action conformation, so that the fluorescence is obviously enhanced. According to addition of Pb 2+ Then, quantitative analysis is realized by the change of the fluorescence intensity ratio of the two signal probes. The invention combines the dual signal amplification strategy with the label-free ratio type sensing platform, can further improve the detection sensitivity, and further enhances the reliability compared with a single signal sensor.
Further defined, in step (1), the volume of the MBs is 1-10 mu L, and the constant temperature oscillation temperature is 20-50 ℃.
Further defined, in steps (1) (2) (3), the sequence of the E-DNA is: 5'-CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG GAAGCA CAC TA-3'; the sequence of the S-DNA is as follows: 5' -GGT GAG TGC TTC CAC TAT rA GGA AGA GAT GAA AAA A-3', wherein the 3' end is modified with biotin; the sequence of S1 is as follows: 5'-ATC ACT CTG GCA CAA AAC ACC CAT CCC GCC CAA CCC GTG GAA GCA CTC ACC-3'; the sequence of G1 is as follows: 5'-GGG TTG GGC GGG ATG GGT GTT TTG TGC CAG TAC TCC-3'.
Further defined, in the steps (1), (2) and (3), the concentration of the DNA strand is 0.5-2. Mu. Mol/L, the volume is 5-10. Mu.L, and the incubation time is 0.5-2 h.
Further defined, in step (2) (3), the double-stranded fluorescent dye is one or more of DAPI, 7-AAD and SGI, and SGI is preferably selected; the G-quadruplex fluorescent dye is one or more of thioflavin T, crystal violet and NMM, and NMM is preferably selected.
Further defined, in the step (2) and the step (3), the concentration of the double-chain fluorescent dye is 1-100X, and the volume is 5-20 mu L; the concentration of the G-quadruplex fluorescent dye is 1-100 mu mol/L, and the volume is 5-20 mu L.
Further defined, in the step (2) and (3), the exonuclease is one or more of Exo I, exo III and RecJF Exo, and preferably Exo III is selected in an amount of 1-10U.
Further defined, in step (2) (3), the K + The solution is one or more of potassium chloride, potassium nitrate and potassium sulfate solution.
Compared with the prior art, the invention has the following remarkable advantages:
1. the invention uses DNAzyme to make Pb 2+ The specificity recognition function of the fluorescent biosensor is improved, and simultaneously, the magnetic separation technology is combined, so that the interference of background signals is reduced, and the detection accuracy is improved.
2. The invention adopts DNAzyme and exonuclease auxiliary cascade signal amplification strategy to realize signal amplification and enhance the sensitivity of the fluorescent biosensor.
3. The invention utilizes two non-marked fluorescent probes to output signals, and skillfully designs the sensor into a ratio type sensor, thereby reducing the experimental cost, reducing the detection environment interference and improving the reliability of the fluorescent sensor.
The foregoing description is only an overview of the technical solution of the present invention, and the following detailed description of the preferred embodiments of the present invention is provided for the purpose of making the technical means of the present invention more clearly understood and may be implemented according to the content of the specification.
Drawings
FIG. 1 is a schematic illustration of the preparation of a structure-specific fluorescent dye-based ratiometric biosensor.
FIG. 2 shows Pb addition of the sensor constructed in example 1 of the present invention 2+ Front (solid line), rear (dashed line).
FIG. 3 is a calibration curve of the sensor constructed in example 1 of the present invention for detecting lead ions.
Fig. 4 shows the selectivity of the sensor constructed in example 1 of the present invention for lead ions in the presence of other interfering ions.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1
A preparation method and application of a ratio type biosensor based on structure specific fluorescent dye are shown in figure 1.
A method for preparing a ratio-type biosensor based on a structure-specific fluorescent dye, comprising the following steps:
(1) Equal volumes of equal concentrations of S1 and G1 were mixed evenly in a centrifuge tube, run in a PCR apparatus at 95℃for 10min, and cooled slowly to 25℃to form a stable double-stranded structure. mu.L of 1. Mu. Mol/L biotin-modified S-DNA was mixed with 10. Mu.L of 0.5. Mu. Mol/L E-DNA and incubated at 37℃for 1 hour. Adding 5 mu L of 10mg/mL MBs into a 200 mu L centrifuge tube, uniformly mixing, washing 3 times by using 100 mu L of Buffer I Buffer solution, adding 20 mu L of the mixture of the S-DNA and the E-DNA into the centrifuge tube, standing at 37 ℃ for shaking incubation for 1h, magnetically separating, washing 3 times by using the Buffer solution, and removing the supernatant, thereby successfully constructing the E-DNA/S-DNA@MBs system.
(2) Subsequently, 10. Mu.L of Pb-free powder was added to the above system 2+ And Pb-containing 2+ After magnetic separation of the mixture, the supernatant was aspirated, 10. Mu.L of a double-stranded complex formed by hybridization of 1. Mu. Mol/L S1-G1 was added to the supernatant, incubated at 37℃for 1 hour, 5U of Exo III was further added to the system, and incubation at 37℃was continued for 1 hour, finally 10. Mu.L of 1 XSGI and 10. Mu.L of 30. Mu. Mol/L NMM fluorescent dye were added, and 10. Mu.L of 1mmol/L KCl solution were added, incubated at 37℃for 30 minutes, 10mmol/L Tris-HCl buffer was supplemented to 200. Mu.L, excitation wavelengths of 399nm were set to obtain fluorescence intensity of NMM at 610nm, and excitation wavelengths of 485nm were set to obtain fluorescence intensity of SGI at 520 nm.
(3) Establishment of a standard curve: 10 mu L of Pb with different concentrations 2+ Adding standard solution into the step (2) to obtain sample detection solutions with different gradients, incubating to obtain different fluorescence signals, and taking the logarithmic value of the lead ion concentration as the abscissa, wherein the difference F of the fluorescence intensity of SGI and NMM is SGI /F NMM And (5) performing linear fitting on the ordinate, and establishing a standard curve of the sensor to lead ions.
As shown in FIG. 2, pb was added to the sensor constructed in example 1 of the present invention 2+ Front (solid line), rear (dashed line).
As shown in FIG. 3, a standard curve for detecting lead ions by the sensor constructed in example 1 of the present invention is shown.
Example 2:
a preparation method and application of a ratio type biosensor based on a structure-specific fluorescent dye, and the practical application thereof, comprises the following steps:
in order to verify that the prepared novel label-free ratio fluorescent biosensor has a specific recognition effect on lead ions, a lead ion standard substance is added into a Tris buffer solution to lead Pb in a sample 2+ Is 50nM; preparing other 7 standard solutions of interference metal ions with concentration of 5 μm and Pb respectively by adopting Tris buffer solution 2+ 100 times the concentration. The detection system constructed according to example 1 was used for the above 7 different interfering metal ion standard solutions and their combination with Pb 2+ The mixed sample of (2) is detected, the detection result is shown in figure 4, and the method of the invention is illustrated for Pb 2+ Has good selectivity.
Example 3:
a preparation method and application of a ratio type biosensor based on a structure-specific fluorescent dye, and the practical application thereof, comprises the following steps:
(1) Food sample treatment: pretreating sample by microwave digestion, firstly weighing 0.5g food standard-added sample (oatmeal and salted vegetable) and adding into a tank of a microwave digestion oven, and simultaneously adding 8mL HNO 3 、2mL H 2 O 2 The reaction temperature, pressure, heating time and holding time were set at 180deg.C, 400psi, 12min, 15min, respectively. After digestion, the tank containing the digestion solution is heated on a heating plate to remove the acid from the digestion solution, and finally cooled at room temperature, using standard addition methods, to obtain a food extract.
(2) Sample detection: taking 10 mu L of food extract according to the following stepsStep (1) (2) of example 1 to obtain a fluorescent signal, and substituting the fluorescent signal into a standard curve to obtain Pb in the sample 2+ Is a concentration of (3).
(3) When the oatmeal is used as a food sample for measurement, pb with the reference amount of 0.1 times and 10 times is respectively added into the rice flour by taking the 10nmol/L of the added quantity as the reference 2+ And (5) a standard substance. Taking 10. Mu.L of sample solution, measuring fluorescent signals according to the steps (1) and (2) of the example 1, and carrying out the standard curve detected in the example 1 to obtain Pb in the sample 2+ Concentration, 3 replicates per sample were averaged and recovery and RSD calculated as shown in the following table:
the label-free ratio type fluorescence sensor prepared through verification has the advantages of high sensitivity, good selectivity, good reliability and stability and the like for detecting lead ions. The detection of the actual sample shows that the prepared sensor has very good practical application value.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (8)
1. A method for preparing a ratio-type biosensor based on a structure-specific fluorescent dye, comprising the following steps:
(1) Target Pb 2+ Is characterized by comprising the following steps: the E-DNA and the S-DNA are complementary and paired through base to form a DNAzyme secondary structure, and the DNAzyme secondary structure is immobilized on the surface of MBs through the combination effect of biotin and streptavidin, namely the E-DNA/S-DNA@MBs system, pb is formed 2+ Specifically recognized by DNAzyme, activates cleavage of the substrate strand, cleaves the next ssDNA and releases E-DNA and Pb 2+ Further starting DNAzyme auxiliary target circulation system, and finally magnetically separating to obtain supernatant containing a large amount of ssDNA in common homogeneous Pb 2+ On the basis of DNAzyme recognition mechanism, the method is further combined with a magnetic separation technology, so that the target circulation reaction is better realized;
(2) Construction of a label-free ratio-type fluorescence sensor: sequentially adding dsDNA and exonuclease into the supernatant after magnetic separation, wherein the existence of the ssDNA triggers an exonuclease-assisted DNA circulation system, so that the dsDNA is sheared and digested by the exonuclease, the fluorescence intensity of double-stranded dye is obviously reduced, and the exposed G-rich sequence G1 is in K + Under the stabilization of the dye, the dye reacts with G-quadruplex dye to generate conformational change and generate strong fluorescence, and the fluorescence signal change of the two dyes is opposite to the fluorescence signal change of the two dyes in the absence of ssDNA, and Pb is realized according to the change of the fluorescence intensity ratio of the two signaling probes before and after the target is added 2+ Compared with the common single signal fluorescence sensor construction strategy, the quantitative analysis of the fluorescent dye takes two label-free fluorescent dyes as the ratio type signal output, and further introduces an exonuclease-assisted isothermal signal amplification strategy.
2. The method for preparing a structure-specific fluorescent dye-based ratiometric biosensor according to claim 1, wherein in the step (1), the volume of the MBs is 1-10 μl, and the constant-temperature oscillation temperature is 20-50deg.C.
3. The method for preparing a structure-specific fluorescent dye-based ratiometric biosensor according to claim 1, wherein in the step (1) (2), the sequence of the E-DNA is: 5'-CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG GAA GCA CAC TA-3'; the sequence of the S-DNA is as follows: 5' -GGT GAG TGC TTC CAC TAT rA GGA AGA GAT GAA AAA A-3', wherein the 3' end is modified with biotin; the sequence of S1 is as follows: 5'-ATC ACT CTG GCA CAA AAC ACC CAT CCC GCC CAA CCC GTG GAA GCA CTC ACC-3'; the sequence of G1 is as follows: 5'-GGG TTG GGC GGG ATG GGT GTT TTG TGC CAG TAC TCC-3'.
4. The method for preparing a structure-specific fluorescent dye-based ratiometric biosensor according to claim 1, wherein in the step (1) (2), the concentration of the DNA strand is 0.5-2. Mu. Mol/L, the volume is 5-10. Mu.L, and the incubation time is 0.5-2 h.
5. The method for preparing a structure-specific fluorochrome-based ratiometric biosensor according to claim 1, wherein in step (2), the fluorochrome is one or more of DAPI, 7-AAD, SGI, preferably SGI; the G-quadruplex fluorescent dye is one or more of thioflavin T, crystal violet and NMM, and NMM is preferably selected.
6. The method for preparing a structure-specific fluorescent dye-based ratiometric biosensor according to claim 1, wherein in the step (2), the concentration of the double-stranded fluorescent dye is 1-100X and the volume is 5-20 μl; the concentration of the G-quadruplex fluorescent dye is 1-100 mu mol/L, and the volume is 5-20 mu L.
7. The method for preparing a structure-specific fluorescent dye-based ratiometric biosensor according to claim 1, wherein in the step (2), the exonuclease is one or more of Exo I, exo III and RecJf Exo, and Exo III is preferably selected in an amount of 1 to 10U.
8. The method for preparing a structure-specific fluorochrome-based ratiometric biosensor according to claim 1, wherein in step (2), the K is selected from the group consisting of + The solution is one or more of potassium chloride, potassium nitrate and potassium sulfate solution.
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