A method for constructing a fluorescent sensor for dual parameter detection and applications thereof

Abstract

The application belongs to the field of sensors, and relates to the exploration of a double-parameter detection mode, in particular to a construction method and application of a fluorescent sensor for double-parameter detection. The application is a fluorescent sensor detection method for double-parameter simultaneous detection. Under optimized conditions, (PTEN, AD, HBV, P53-2) are taken as target 1, and five DNA sequences with different base lengths (BRCA2, DNA19, K-ras, P53, DNA36) are taken as target 2. The results show that the sensor can accurately measure different target 1 and target 2, proving that the method has the advantages of simple experimental design, convenient operation and strong universality, and can realize double-target simultaneous detection through single excitation. Meanwhile, the sensor can also be applied to the detection of related molecules other than nucleic acid sequences in biology, thereby providing a theoretical basis for the practical application of the double-parameter sensor.

Description

Technical Field

[0001] This invention belongs to the field of sensors and relates to the exploration of dual-parameter detection modes, specifically a method for constructing and applying a fluorescence sensor for dual-parameter detection. Background Technology

[0002] Currently, the main methods for simultaneous detection of multiple parameters include electrochemical methods, colorimetric methods, and fluorescence methods. In electrochemical methods, DNA probes immobilized on the electrode surface interact with the analyte, inducing changes in DNA structure and electrochemical properties, thereby generating an electrical signal to detect the analyte. For multi-parameter detection, the traditional method involves labeling the nucleic acid aptamer strand with ferrocene or methylene blue, using different redox potentials to detect different targets, as illustrated in Zheng Y, Liang W, Yuan Y, et al. Wavelength-resolved simultaneous photoelectrochemical bifunctional sensor on single interface: A newly in vitro approach for complexed DNA monitoring in cancer cells[J]. Biosensors and Bioelectronics. While electrochemical sensors offer advantages such as high sensitivity, good selectivity, and low detection limits, their electrode preparation process is cumbersome. Colorimetric methods typically obtain signals by observing color changes produced by DNA peroxidases catalyzing small molecules or by the color differences of metal nanorods in different dispersion states. In multi-parameter detection, the addition of different analytes produces different colors, and the type of analyte is determined by these color changes. Colorimetric sensors are low-cost, simple to operate, and highly practical, but they lack accuracy, and the levels of disease biomarker genes are often only trace in early diagnosis. Fluorescence methods rely on fluorescence signals for detection; common fluorescent molecules include fluorescent groups modified on DNA molecules, gold, silver, and copper nanoclusters. In addition, G-tetrachain molecules can specifically bind to some porphyrin molecules, such as N-methylporphyrin dipropionic acid IX and thiosulfate T, emitting fluorescence. For multi-parameter detection, detection is achieved by labeling the DNA strand or specifically binding to fluorescent dyes with different fluorescence emissions. Fluorescence sensors offer advantages such as good stability, high sensitivity, simple operation, and long lifespan, and are widely used in practical applications, showing broad prospects for future development.

[0003] To develop a biosensor method for simultaneous detection of two parameters that is simple to operate, widely applicable, and has a fast response speed. Summary of the Invention

[0004] This invention proposes a method for constructing and applying a fluorescence sensor for dual-parameter detection. It utilizes the luminescence properties of the fluorescent signal molecules thiamine T and FAM (6-carboxyfluorescein). Fluorescence conversion is achieved through molecular hairpin configuration changes. A fluorescence sensor for the simultaneous detection of two disease biomarker genes is established, and its versatility is studied.

[0005] The technical solution of this invention is implemented as follows:

[0006] The method for constructing a fluorescence sensor for dual-parameter detection includes the following steps:

[0007] (1) Prepare a Tris-HCl buffer solution containing hairpin probe HP1 and hairpin probe HP2, activate it by standing in the dark, and use it as a stock solution.

[0008] (2) Add buffer solution to the stock solution, then add ThT solution, and incubate at room temperature to obtain the working solution of the fluorescence sensor.

[0009] In step (1) above, hairpin probe HP1 is a labeled fluorescent probe and hairpin probe HP2 is an unlabeled fluorescent probe.

[0010] The 3' end of the hairpin probe HP1 is connected to -BHQ1, and the 5' end is connected to FAM-.

[0011] The hairpin probe HP1 is any one of HP(BRCA2), HP(DNA19), HP(K-ras), HP(P53-1), and HP(DNA36); wherein the HP(BRCA2) sequence is shown in SEQ ID No. 2, the HP(DNA19) sequence is shown in SEQ ID No. 3, the HP(K-ras) sequence is shown in SEQ ID No. 4, the HP(P53-1) sequence is shown in SEQ ID No. 5, and the HP(DNA36) sequence is shown in SEQ ID No. 6; the hairpin probe HP2 is any one of HP(AD), HP(P53-2), HP(PTEN), and HP(HBV); wherein the HP(AD) sequence is shown in SEQ ID No. 1, the HP(P53-2) sequence is shown in SEQ ID No. 7, the HP(PTEN) sequence is shown in SEQ ID No. 8, and the HP(HBV) sequence is shown in SEQ ID No. 9.

[0012] The concentration of the above Tris-HCl buffer was 20 mmol / L and the pH was 7.0; the concentrations of hairpin probe HP1 and hairpin probe HP2 in the stock solution were both 5 μmol / L.

[0013] In step (2) above, the buffer solution is 20mM Tris-HCl, 300mM KCl, pH=9; the concentration of the ThT solution is 2100μmol / L.

[0014] The volume ratio of the above stock solution, buffer solution and ThT solution was 1:46:0.5; the incubation time at room temperature was 5 min.

[0015] The fluorescent sensor was prepared using the method described above.

[0016] The above-mentioned fluorescence sensor is used to detect multiple targets simultaneously for purposes other than disease diagnosis. The steps are as follows: add the test solution to 588 μL of the working solution of the fluorescence sensor, incubate at 37°C in a shaker for 60 min, use a fluorescence spectrophotometer to detect the fluorescence values ​​F and F0 before and after adding the test solution, calculate the change in fluorescence intensity F-F0, and substitute it into the linear equation to calculate the concentration of the target. The excitation wavelength of the fluorescence spectrophotometer is set to 460 nm, the emission wavelength is 465-650 nm, the excitation slit is 2.5 nm, and the emission slit is 5.0 nm.

[0017] When the target objects are AD and BRCA2, the linear equation is y = 3.9897x - 6.5337, R1 2 =0.9886, y=4.0938x+0.3458, R2 2 =0.9939, with detection limits of 0.53 and 0.36 nmol / L, respectively; the linear equation for target analytes AD and DNA19 is y = 3.3290x - 0.8471, R1 2 =0.9966, y=4.3307x+2.8466, R2 2 =0.9914, detection limits are 0.67 and 0.34 nmol / L, respectively; the linear equation for target analytes AD and K-ras is y = 3.0940x - 6.5998, R1 2 =0.9795, y=4.1836x+12.1746, R2 2 =0.9744, detection limits are 0.72 and 0.31 nmol / L respectively; when the target substances are AD and P53-1, the linear equation is y = 3.1007x - 4.2411, R1 2 =0.9966, y=4.4243x+0.1620, R2 2 =0.9996, detection limits are 0.81 and 0.37 nmol / L, respectively; the linear equation for target analytes AD and DNA36 is y = 2.9428x - 3.7615, R1 2 =0.9981, y=3.0487x+0.6233, R2 2=0.9997, detection limits are 0.83 and 0.63 nmol / L, respectively. When the target compounds are P53-2 and P53-1, the linear equation is y = 3.270x - 0.715, R1 2 =0.993, y=3.562x+18.558, R2 2 =0.986, with detection limits of 0.63 and 0.99 nmol / L, respectively; the linear equation for target analytes PTEN and P53-1 is y = 3.306x + 28.879, R1 2 =0.988, y=3.788x-6.956, R2 2 =0.999, detection limits are 0.54 and 1.06 nmol / L, respectively; the linear equation for target analytes HBV and P53-1 is y = 3.482x + 10.104, R1 2 =0.988, y=2.708x+2.364, R2 2 =0.999, and the detection limits are 1.26 and 1.19 nmol / L, respectively.

[0018] The detection mechanism of the dual-parameter detection fluorescence sensor in this application is as follows: Two hairpin probes are designed. Hairpin probe HP1 has a loop portion containing a base sequence complementary to the target DNA, a G-rich sequence at its 5′ end, and a hairpin stem formed by complementary pairing of the 3′ end with a partially G-rich sequence. The other hairpin probe HP2 has a loop portion containing a base sequence complementary to the target DNA or a nucleic acid aptamer strand that recognizes the target analyte. Its 5′ end is labeled with the fluorescent group FAM, and its 3′ end is labeled with the quencher group BHQ1. Both hairpin probes are individually locked and set to the same excitation wavelength. In the absence of a target analyte... Both HP1 and HP2 are hairpin structures. In HP1, most of the G-rich sequence is locked in the stem and cannot bind to ThT, resulting in low fluorescence intensity. In HP2, when the FAM and BHQ1 are very close, fluorescence resonance energy transfer occurs, suppressing the fluorescence signal. Therefore, both HP1 and HP2 exhibit weak fluorescence. Upon the addition of target 1, the target DNA binds complementary to the loop of HP1, opening the molecular hairpin and releasing the G-rich sequence in the stem. Under K+ induction, a G-quadruplex structure is formed, which binds to ThT and produces a strong fluorescence signal at 487 nm. At this time, HP2 remains a hairpin structure and therefore does not produce a fluorescence signal at 514 nm. Upon the addition of target 2, HP1 does not react and exhibits a low fluorescence signal at 487 nm. The hairpin of HP2 opens, and the fluorophore moves away from the quencher, producing a sensitive fluorescence signal at 514 nm. Upon the simultaneous addition of both target 1 and target 2, both HP1 and HP2 hairpin probes open, producing sensitive fluorescence signals at both 487 nm and 514 nm. Therefore, the simultaneous detection of two parameters can be achieved by observing the changes in fluorescence values ​​before and after the addition of the target.

[0019] The present invention has the following beneficial effects:

[0020] This application presents a fluorescence sensing detection method for simultaneous detection of two parameters. Under optimized conditions, with (PTEN, AD, HBV, P53-2) as target 1 and five DNA sequences of different base lengths (BRCA2, DNA19, K-ras, P53, DNA36) as target 2, the results show that the sensor can achieve accurate measurements even after changing the target 1 and target 2. This demonstrates that the method is simple in experimental design, convenient in operation, and highly universal. It can achieve simultaneous detection of two targets with a single excitation, providing a simple and universal method for the simultaneous detection of genes of multiple disease biomarkers. Furthermore, this sensor can also be applied to detect biomolecules other than nucleic acid sequences; simply adding the complementary sequence of the nucleic acid aptamer of the target 2 biomolecule to the existing sensor provides a foundation for the practical application of two-parameter sensors.

[0021] Labeled fluorescence sensors are widely used and highly sensitive, but require multiple labeling processes for simultaneous multi-parameter detection, which is cumbersome and expensive. Label-free fluorescence sensors eliminate the cumbersome labeling process, but often use noble metal nanoclusters for multi-target detection, resulting in unstable performance and interference between different dyes. This application's fluorescence sensing method for simultaneous dual-parameter detection combines the advantages of both labeled and label-free sensors, achieving dual-parameter detection while ensuring sensor stability and avoiding interference between the two parameters. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 To assess the feasibility of label-free sensors and optimize their stem length, the following diagrams are presented: (A) Detection principle diagram; (B) PTEN; (C) AD; (D) HBV; (E) P53-2; (F) HP (PTEN) stem length optimization; (G) HP (AD) stem length optimization; (H) HP (P53-2) stem length optimization.

[0024] Figure 2 The detection range and linear interval of the label-free sensor for detecting different target DNAs and the fluorescence spectra of different concentrations of target DNA added; where (A) PTEN (C) AD (E) HBV (G) P53-2; the fluorescence difference before and after adding different concentrations of target DNA (B) PTEN (D) AD (F) HBV (H) P53-2.

[0025] Figure 3 The schematic diagram and feasibility of the labeled sensor are shown; (A) Detection principle diagram (B) BRCA2 (C) DNA19 (D) K-ras (E) P53-1 (F) DNA36.

[0026] Figure 4 To detect fluorescence spectra at different concentrations and to plot the linear relationship between the fluorescence intensity of the system without a target and the fluorescence intensity difference F-F0 of the system with different concentrations of dual targets, and the target DNA concentration; where (AB) BRCA2; (CD) DNA19; (EF) K-ras; (GH) P53-1; (IJ) DNA36.

[0027] Figure 5 To detect the specificity of the detection system for P53-1, (A) fluorescence spectrum and (B) fluorescence intensity change bar graph are shown.

[0028] Figure 6 This is a schematic diagram of a highly versatile fluorescence sensor used for simultaneous detection of two targets.

[0029] Figure 7 This diagram illustrates the feasibility of a dual-parameter fluorescence sensor, showing: (A) fluorescence spectra of the system simultaneously detecting AD and BRCA2; (B) fluorescence spectra of the system simultaneously detecting AD and DNA19; (C) fluorescence spectra of the system simultaneously detecting AD and K-ras; (D) fluorescence spectra of the system simultaneously detecting AD and P53-1; (E) fluorescence spectra of the system simultaneously detecting AD and DNA36; (F) fluorescence spectra before adding AD and P53-1 at different reaction temperatures; and (G) fluorescence spectra after adding AD and P53-1 at different reaction temperatures. Figure; (H) Fluorescence intensity changes of AD gene at different reaction temperatures; (I) Fluorescence intensity changes of P53-1 gene at different reaction temperatures; (J) Optimization of reaction time of detection system when AD gene and P53-1 are added; Fluorescence spectra of different concentrations detected simultaneously by dual-parameter fluorescence sensor and linear relationship between the fluorescence intensity difference F-F0 of the system before and after adding different concentrations of dual targets and the target DNA concentration (KL) AD and BRCA2; (MN) AD and DNA19; (OP) AD and K-ras; (QR) AD and P53; (ST) AD and DNA36.

[0030] Figure 8Feasibility diagram of a dual-parameter fluorescence sensor; (A) Fluorescence spectrum of the system simultaneously detecting P53-1 and P53-2; (B) Fluorescence spectrum of the system simultaneously detecting P53-1 and PTEN; (C) Fluorescence spectrum of the system simultaneously detecting P53-1 and HBV; (D) Fluorescence spectrum after adding P53-1 and P53-2 at different reaction temperatures; (E) Fluorescence spectrum after adding P53-1 and PTEN at different reaction temperatures; (F) Fluorescence spectrum after adding P53-1 and HBV at different reaction temperatures; Fluorescence spectrum of the dual-parameter fluorescence sensor simultaneously detecting different concentrations and linear relationship between the fluorescence intensity difference F-F0 of the system before and after adding different concentrations of dual targets and the target DNA concentration (GH) P53-1 and P53-2; (IJ) P53-1 and PTEN; (KL) P53-1 and HBV.

[0031] Figure 9 (A) Selectivity of this method for the AD gene under optimized conditions; (B) Selectivity of this method for P53-1 under optimized conditions. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments used in the embodiments of the present invention, obtained by those of ordinary skill in the art without creative effort, are within the scope of protection of the present invention.

[0033] Reagents: All oligonucleotides were purchased from Shanghai Sangon Biotech Co., Ltd., and the oligonucleotide sequences are detailed in Table 1; Tris-HCl, sodium hydroxide (NaOH), and potassium chloride (KCl); all reagents were of analytical grade and purchased from Zhengzhou Xinmaijie Biotechnology Co., Ltd., and the solutions were prepared with ultrapure water (18.2 MΩ·cm).

[0034] Table 1 shows the oligonucleotide sequences used.

[0035]

[0036]

[0037] Example

[0038] Sensor fabrication and fluorescence measurement

[0039] 1. Fabrication of label-free sensors

[0040] The hairpin probe was centrifuged at 7000 rpm for 1 min, and then dissolved in 20 mmol / L Tris-HCl buffer (pH 7.0) to prepare a 5 μmol / L solution. The solution was then activated by incubating at room temperature in the dark for 2 h. 12 μL of the hairpin probe was transferred, and 6 μL of 2100 μmol / L ThT was added to 570 μL of buffer (20 mmol / L Tris-HCl, 300 mmol / L KCl, pH 8.0) to obtain the label-free sensor HP1, which was stored at 4 °C for later use.

[0041] 2. Fabrication of labeled sensors

[0042] First, centrifuge the hairpin probe and target DNA at 7000 rpm for 1 min, then add 20 mmol / L Tris-HCl buffer (pH 7.0) to prepare a 5 μmol / L solution and activate at room temperature in the dark for 2 h. Next, take 12 μL of the probe solution and add it to 570 μL of buffer (20 mM Tris-HCl, 50 M NaCl, 5 mM KCl, 5 mM MgCl2, pH = 9) to obtain the working solution, which should be stored at 4 °C for later use. The final concentration of HP2 is 100 nmol / L.

[0043] 3. Fabrication of a dual-parameter sensor

[0044] First, centrifuge all DNA probes at 7000 rpm for 1 min, then add 20 mmol / L Tris-HCl buffer (pH 7.0) to prepare a 5 μmol / L solution and incubate at room temperature in the dark for 2 h for activation. Next, add 12 μL of HP1 and HP2 to 552 μL of buffer (20 mM Tris-HCl, 300 mM KCl, pH = 9), followed by 6 μL of 2100 μmol / L ThT and incubate at room temperature for 5 min to obtain working solutions. Store at 4 °C for later use. The final concentration of HP1 and HP2 is 100 nmol / L.

[0045] 4. Fluorescence Measurement

[0046] Take 588 μL of the prepared working solution and add 12 μL of target DNA at different concentrations, then incubate at 37°C in a shaker for 60 min. Fluorescence measurements are performed using a Hitachi F-7000 fluorescence spectrophotometer, with the excitation wavelength set to 460 nm and the emission wavelength ranging from 465 to 650 nm. The excitation slit is 2.5 nm, and the emission slit is 5.0 nm.

[0047] Implementation effect

[0048] 1. Feasibility and optimization of the experimental principle of label-free sensors

[0049] The principle of label-free fluorescent sensor detection of target DNA is as follows: Figure 1 As shown in Figure A, the detection principle utilizes the specific binding of the fluorescent dye ThT to G-rich sequences to generate a strong fluorescent signal. The detection of the target DNA is achieved by detecting the change in fluorescence signal intensity before and after the addition of the target DNA. The HP1 loop is the recognition region, the 5′ end is a G-rich sequence, and the 3′ end forms a hairpin stem with complementary pairing with some G-rich sequences. When no target DNA is present, most of the G-rich sequences in the HP1 molecular hairpin are locked in the stem and cannot bind to ThT. At this time, most of thioflavin T is free in the solution, resulting in low fluorescence intensity. When the target DNA is added, the molecular hairpin opens, releasing the G-rich sequences in the stem, which then bind to the K+ cation. + Under the induction of [a specific substance], a G-quadruplex structure is formed, which binds to thioflavin T and produces a strong fluorescent signal. The concentration of the target DNA can be detected based on the degree of increase in the fluorescent signal. For example... Figure 1 As shown in Figures B-1E, by changing the recognition sequence of the target gene in the HP1 loop, the detection of the target genes PTEN, AD, HBV, and P53-2 was achieved. Through stem length optimization, such as... Figure 1 As shown in F to 1H, the hairpin stem lengths of sensors HP(PTEN), HP(AD), and HP(P53-2) reached their optimal values ​​at 7, 8, and 7 minutes, respectively, based on the fluorescence difference before and after the addition of the target gene.

[0050] This experiment investigated the fluorescence response of the sensor to different concentrations of target DNA (PTEN, AD, HBV, P53-2). The sensor concentration was 100 nmol / L for all samples. Figure 2 The concentrations of (AB)PTEN were 1, 5, 15, 30, 50, 75, 100, 150, 200, and 250 nmol / L. The fluorescence intensity at 519 nm without target DNA was compared with the fluorescence intensity at different target DNA concentrations, with the difference F-F0 (F0 being the fluorescence intensity of the solution before adding target DNA, and F being the fluorescence intensity of the solution after adding target DNA) plotted on the ordinate (y) and the target DNA concentration plotted on the abscissa (x). R0 2 This is the linear correlation coefficient. The linear equation is y = 3.052x - 43.850, R0 2 =0.999, detection limit is 0.22 nmol / L; Figure 2 The concentrations of (CD)AD were 0, 0.1, 1, 5, 15, 30, 50, 75, 100, 150, and 200 nmol / L, respectively. The linear equation was y = 4.57x + 1.61, R0 2 =0.996, detection limit is 0.04 nmol / L; if Figure 2The concentrations of HBV (EF) were 0, 0.01, 0.05, 0.1, 1, 5, 15, 30, 50, 75, 100, 150, 200, 250, and 300 nmol / L, respectively. The linear equation was y = 5.280x + 24.038, RF 2 =0.999, detection limit is 2.74 nmol / L; if Figure 2 The concentrations of (GH)P53-2 were 0, 0.05, 0.1, 1, 5, 15, 30, 50, 75, 100, 150, and 200 nmol / L, respectively. The linear equation was y = 3.980x + 2.059, R0 2 =0.992, detection limit is 0.16 nmol / L;

[0051] 2. Feasibility and Optimization of the Experimental Principle of Labeled Sensors

[0052] The schematic diagram of the marker sensor detection principle is as follows: Figure 3 As shown in Figure A, a molecular beacon is designed with a fluorophore FAM at its 5′ end and a quencher BHQ1 at its 3′ end. The loop portion of the beacon is completely complementary to the target DNA, and the stem portion has 6 complementary base pairs. When FAM and BHQ1 are very close, fluorescence resonance energy transfer occurs, and the fluorescence signal of FAM is quenched by BHQ1. When FAM moves away from BHQ1, the fluorescence signal of FAM is restored. In the absence of target DNA, HP folds into a stem-loop secondary structure in solution due to the complementary base pairing at the stem. At this point, the fluorophore and quencher are close to each other, and the fluorescence of the fluorophore is quenched, resulting in weak fluorescence in the solution. After the target DNA is added, the target DNA binds to the complementary base pairing at the loop portion of the molecular beacon, forcing the HP stem to open. FAM moves away from BHQ1, preventing the fluorescence of the fluorophore from being absorbed by the quencher, resulting in a sensitive fluorescence signal and a significant increase in the fluorescence intensity of the solution. The concentration of target DNA added can be deduced from the magnitude of the increase in fluorescence signal. Furthermore, by inserting the hairpin loop of a molecular gene into the corresponding complementary sequence of different target genes, it is possible to detect different target DNAs. For example... Figure 3 As shown in (BF), by changing the recognition sequence of the target in the HP2 loop, the target genes BRCA2, DNA19, K-ras, P53-1, and DNA36 were detected.

[0053] This experiment investigated the fluorescence response of the sensor to different concentrations of target DNA (BRCA2, DNA19, K-ras, P53-1, and DNA36). The concentrations of the target DNA were 0, 1, 30, 75, 100, and 200 nmol / L, and the fluorescence spectra are shown below. Figure 4As shown, the fluorescence intensity of the detection system gradually increases with the increase of the target DNA concentration. The fluorescence intensity at 519 nm without target DNA was compared with the fluorescence intensity at different target DNA concentrations, with the difference F-F0 (F0 being the fluorescence intensity of the solution before adding target DNA, and F being the fluorescence intensity of the solution after adding target DNA) plotted as the ordinate (y) and the target DNA concentration as the abscissa (x). R0 2 The coefficient represents the linear correlation coefficient. When the target DNA is the BRCA2 gene, a good linear relationship is observed between F-F0 and BRCA2 concentration in the range of 1-200 nmol / L, with the linear equation being y = 2.7987x + 9.9146, R0. 2 =0.9939, detection limit is 0.43 nmol / L; when the target DNA is DNA19 gene, F-F0 and DNA19 concentration show a good linear relationship between DNA19 concentration and concentration in the range of 1-150 nmol / L, the linear equation is y = 7.7182x + 21.7833, R 2 =0.9721, detection limit is 0.19 nmol / L; when the target DNA is the K-ras gene, F-F0 and K-ras concentration show a good linear relationship between K-ras concentration and concentration in the range of 1-100 nmol / L, the linear equation is y = 11.1675x + 14.0681, R 2 =0.9969, detection limit is 0.12 nmol / L; when the target DNA is the P53-1 gene, F-F0 and P53-1 concentration show a good linear relationship between P53-1 concentration and concentration in the range of 1-100 nmol / L, the linear equation is y = 14.0941x + 24.9667, R 2 =0.9861, detection limit is 0.1 nmol / L; when the target DNA is the DNA36 gene, F-F0 and DNA36 concentration show a good linear relationship between DNA36 concentration and concentration in the range of 1-100 nmol / L, the linear equation is y = 5.4846x + 26.8733, R 2 =0.9823, detection limit is 0.25 nmol / L. This result indicates that as the number of bases in the target DNA sequence increases, the detection limit first decreases and then increases. When the target DNA sequence is the P53-1 gene (30 bases), the sensor sensitivity is highest, with a detection limit of 0.10 nmol / L. The detection range gradually narrows from 1-200 nmol / L to 1-100 nmol / L, and then remains unchanged. When the target DNA sequence is the K-ras gene (15 bases), the detection range is the largest, 1-200 nmol / L.

[0054] Application Example 1

[0055] The above sensors were used to detect P53-1 DNA (T), single-base mismatched DNA (MT1), triple-base mismatched DNA (MT3), and random sequence DNA (MTR) at a concentration of 100 nmol / L. The results are as follows: Figure 5 As shown, the fluorescence intensity corresponding to the mismatched sequences is lower than that of the target DNA, indicating that this method has good specificity.

[0056] To further investigate the applicability of this method to complex samples, it was applied to the detection of P53-1 in human serum samples. Fluorescence signals were detected by adding 15, 50, and 100 nmol / L P53-1 to 10% of the serum sample, and the results are shown in Table 2. The recoveries ranged from 98% to 100.5%, and the relative errors ranged from 0.48% to 6.4%. This demonstrates that this method has good applicability for the detection of P53-1 in serum samples.

[0057] Table 2. Detection of target DNA P53-1 in serum samples.

[0058]

[0059] Application Example 2

[0060] The detection principle diagram of the fluorescence sensor used for dual-parameter detection is as follows: Figure 6As shown, two hairpin probes were designed. Hairpin probe HP1 has a loop containing a base sequence complementary to the target DNA, a G-rich sequence at its 5' end, and a hairpin stem formed by complementary pairing of the 3' end with a partially G-rich sequence. Hairpin probe HP2 has a loop containing a base sequence complementary to the target DNA or a nucleic acid aptamer strand that recognizes the target analyte, a fluorophore FAM at its 5' end, and a quencher BHQ1 at its 3' end. Both hairpin probes are individually locked and set to the same excitation wavelength. In the absence of a target analyte, both HP1 and HP2 emit light. In HP1, the G-rich sequence is mostly locked in the stem, preventing it from binding to ThT and resulting in low fluorescence intensity. In HP2, when the FAM and BHQ1 are very close, fluorescence resonance energy transfer occurs, suppressing the fluorescence signal. Therefore, both HP1 and HP2 exhibit weak fluorescence. Upon the addition of target 1, the target DNA binds complementary to the loop of HP1, opening the molecular hairpin and releasing the G-rich sequence in the stem. Under K+ induction, a G-quadruplex structure is formed, which binds to ThT and produces a strong fluorescence signal at 487 nm. At this time, HP2 remains a hairpin structure and therefore does not produce a fluorescence signal at 514 nm. Upon the addition of target 2, HP1 does not react and exhibits a low fluorescence signal at 487 nm. The HP2 hairpin opens, and the fluorophore moves away from the quencher, producing a sensitive fluorescence signal at 514 nm. Simultaneously, upon the addition of both target 1 and target 2, both HP1 and HP2 hairpin probes open, producing sensitive fluorescence signals at both 487 nm and 514 nm. Therefore, the simultaneous detection of two parameters can be achieved by observing the change in fluorescence values ​​before and after the addition of the target. This single-emission dual-detection strategy is simpler than dual-emission and dual-emission detection in simultaneous detection processes.

[0061] Objective 1 remains unchanged, Objective 2 changes: To verify the sensor's universality, the ring recognition region sequence of the HP1 sensor probe is first changed to accommodate various labeled and unlabeled analytes, and the fluorescence spectra are then observed. For example... Figure 7 As shown in (AE), the constructed dual-parameter sensor can effectively detect target 1 (AD) and target 2 (BRCA2, DNA19, K-ras, P53-1, DNA36) simultaneously.

[0062] To achieve better detection results, the temperature of the HP(AD) and HP(P53-1) detectors was optimized, such as... Figure 7(FJ) Within the range of 0–50℃, before the addition of the two target genes, the fluorescence intensity of ThT gradually decreased with increasing test temperature, while FAM showed no obvious peak (7F). After the addition of the two target genes, within the range of 0–25℃, only ThT showed a peak, while FAM showed no obvious peak. This is because the molecular hairpin HP2 and FAM are more affected by temperature, resulting in a large difference in fluorescence signal intensity between ThT and FAM, thus preventing the appearance of a FAM peak. Within the range of 25–50℃, both ThT and FAM showed obvious peaks. Therefore, for simultaneous detection of two parameters, the temperature range of 25–50℃, where ThT and FAM show obvious peaks, should be selected for subsequent experiments (7G). The relationship between the change in fluorescence signal F-F0 and temperature is as follows: Figure 7 As shown in H and 7I, the F-F0 of ThT decreased with increasing temperature before and after the addition of the two target genes. This is because increasing temperature makes the G-quadruplex structure unstable and unable to bind to more ThT. The fluorescence change of FAM first increased and then decreased with increasing temperature, and F-F0 reached its highest value at 30℃. Figure 7 (I) This is because increased temperature affects the molecular hairpin structure and the luminescence efficiency of FAM. To achieve optimal detection results, 30℃ was chosen for subsequent experiments. To obtain the optimal reaction time, the fluorescence spectra of the AD gene and P53-1 gene were measured every 3 minutes, and the fluorescence signal change curves over time are shown below. Figure 7 As shown in Figure J, within 0-60 min, the fluorescence signals of both the fluorescent dyes ThT and FAM showed a significant increase in the initial stage of the reaction. When the reaction time was increased to 60 min, the fluorescence intensity reached its maximum value, and thereafter, the fluorescence intensity remained basically unchanged. Therefore, 60 min was chosen as the reaction time.

[0063] When the objectives are AD and BRCA2, the linear equation is y = 3.9897x - 6.5337, R1. 2 =0.9886, y=4.0938x+0.3458, R2 2 =0.9939, with detection limits of 0.53 and 0.36 nmol / L, respectively; when the target is AD and DNA19, the linear equation is y = 3.3290x - 0.8471, R1 2 =0.9966, y=4.3307x+2.8466, R2 2 =0.9914, with detection limits of 0.67 and 0.34 nmol / L, respectively; when the target is AD and K-ras, the linear equation is y = 3.0940x - 6.5998, R1 2 =0.9795, y=4.1836x+12.1746, R2 2=0.9744, detection limits are 0.72 and 0.31 nmol / L respectively; when the target is AD and P53-1, the linear equation is y = 3.1007x - 4.2411, R1 2 =0.9966, y=4.4243x+0.1620, R2 2 =0.9996, with detection limits of 0.81 and 0.37 nmol / L, respectively; when the target is AD and DNA36, the linear equation is y = 2.9428x - 3.7615, R1 2 =0.9981, y=3.0487x+0.6233, R2 2 =0.9997, with detection limits of 0.83 and 0.63 nmol / L, respectively. These results indicate that the method has good universality, and the sensor performs optimally when the target gene is AD or K-ras (25 bases), with detection limits of 0.72 and 0.31 nmol / L, respectively.

[0064] Objective 1 changed, Objective 2 unchanged: Further replace the ring recognition region sequence of the HP2 sensor probe, and simultaneously observe the fluorescence spectra of various labeled and unlabeled analytes. Figure 8 (AC) shows that after replacing the ring recognition sequence of the unmarked hairpin HP2 with the inverse complementary sequence of P53-2, HBV, and PTEN, the sensor still exhibits good universality. Within the range of 20–30℃, as the temperature increases, the fluorescence value of FAM gradually increases, while the fluorescence value of ThT gradually decreases. Figure 8 To ensure that both DF and DF exhibit significant peak values, 20℃ was chosen as the reaction temperature.

[0065] The fluorescence intensity at 487 nm and 514 nm without target DNA and the fluorescence intensity corresponding to the concentrations of target DNA and target DNA were respectively compared. The difference F-F0 (F0 is the fluorescence intensity of the solution before the addition of target DNA, and F is the fluorescence intensity of the solution after the addition of target DNA) was plotted on the y-axis, and the concentrations of target DNA and target DNA were plotted on the x-axis. R 2 This is the correlation coefficient. For example... Figure 8 As shown in H, J, and L, when the objectives are P53-2 and P53-1, the linear equation is y = 3.270x - 0.715, R1 2 =0.993, y=3.562x+18.558, R2 2 =0.986, with detection limits of 0.63 and 0.99 nmol / L, respectively; when the targets are PTEN and P53-1, the linear equation is y = 3.306x + 28.879, R1 2 =0.988, y=3.788x-6.956, R2 2=0.999, with detection limits of 0.54 and 1.06 nmol / L, respectively; when the targets are HBV and P53-1, the linear equation is y = 3.482x + 10.104, R1 2 =0.988, y=2.708x+2.364, R2 2 =0.999, with detection limits of 1.26 and 1.19 nmol / L, respectively;

[0066] Application Example 3

[0067] To further verify the feasibility of the two methods, this experiment simultaneously detected different concentrations of AD and P53-1 genes, as well as PTEN and P53-1 genes in human serum samples to validate the practicality of the methods. The human serum was diluted 10-fold with ultrapure water for spiked recovery experiments. The results are shown in Tables 3 and 4. The sensor recoveries ranged from 101.01% to 119.3%, and the relative standard deviations (RSDs) of the recoveries for both sensors (n=5) were below 5.62%, indicating that the sensors have good practicality for detecting AD and P53-1 genes, as well as PTEN and P53-1 genes in human serum.

[0068] Table 3. Detection of AD and P53-1 in serum samples.

[0069]

[0070] Table 4. Detection of PTEN and P53-1 in serum samples

[0071]

[0072]

[0073] Implementation Result Analysis

[0074] This paper combines the above-mentioned label-free and labeled fluorescence sensing detection methods to design a fluorescence sensing detection method for simultaneous detection of two parameters. Under optimized conditions, (PTEN, AD, HBV, P53-2) were used as target 1, and five DNA sequences of different base lengths (BRCA2, DNA19, K-ras, P53, DNA36) were used as target 2. The results show that the sensor can achieve accurate measurement after changing different targets 1 and target 2, proving that this method has the advantages of simple experimental design, convenient operation and strong universality. It can achieve simultaneous detection of two targets with a single excitation, providing a simple and universal method for the simultaneous detection of genes of multiple disease biomarkers. At the same time, this sensor can also be applied to the multi-parameter detection of related molecules other than nucleic acid sequences in biology, providing a theoretical basis for the practical application of dual-parameter sensors.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a fluorescence sensor for dual-parameter detection, characterized in that, The steps are as follows: (1) Prepare Tris-HCl buffer containing hairpin probe HP1 and hairpin probe HP2, activate it by standing in the dark, and use it as a stock solution; (2) Add buffer solution to the stock solution, then add ThT solution, and incubate at room temperature to obtain the working solution of the fluorescence sensor; The hairpin probe HP1 is HP-BRCA2 and the hairpin probe HP2 is HP-AD; the HP-BRCA2 sequence is shown in SEQ ID No.2 and the HP-AD sequence is shown in SEQ ID No.

1.

2. The method for constructing a fluorescence sensor for dual-parameter detection according to claim 1, characterized in that: In step (1), hairpin probe HP1 is a labeled fluorescent probe and hairpin probe HP2 is an unlabeled fluorescent probe.

3. The method for constructing a fluorescence sensor for dual-parameter detection according to claim 2, characterized in that: The 3' end of the hairpin probe HP1 is connected to -BHQ1, and the 5' end is connected to FAM-.

4. The method for constructing a fluorescence sensor for dual-parameter detection according to claim 3, characterized in that: The hairpin probes HP1 and HP2 further include any of the following combinations: hairpin probe HP1 is HP-DNA19 and hairpin probe HP2 is HP-AD; hairpin probe HP1 is HP-K-ras and hairpin probe HP2 is HP-AD; hairpin probe HP1 is HP-P53-1 and hairpin probe HP2 is HP-AD; hairpin probe HP1 is HP-DNA36 and hairpin probe HP2 is HP-AD; hairpin probe HP1 is HP-P53-1 and hairpin probe HP2 is HP-P53-2; hairpin probe HP1 is HP-P53-1 and hairpin probe HP2 is HP-PTEN; hairpin probe HP1 is HP-P53-1 and hairpin probe HP2 is HP-HBV. The HP-DNA19 sequence is shown in SEQ ID No. 3, the HP-K-ras sequence is shown in SEQ ID No. 4, the HP-P53-1 sequence is shown in SEQ ID No. 5, the HP-DNA36 sequence is shown in SEQ ID No. 6, the HP-P53-2 sequence is shown in SEQ ID No. 7, the HP-PTEN sequence is shown in SEQ ID No. 8, and the HP-HBV sequence is shown in SEQ ID No.

9.

5. The method for constructing a fluorescence sensor for dual-parameter detection according to any one of claims 1-4, characterized in that: The Tris-HCl buffer solution had a concentration of 20 mmol / L and a pH of 7.0; the hairpin probes HP1 and HP2 in the stock solution each had a concentration of 5 μmol / L.

6. The method for constructing a fluorescence sensor for dual-parameter detection according to claim 5, characterized in that: In step (2), the buffer solution is 20 mM Tris-HCl, 300 mM KCl, pH=9; the concentration of the ThT solution is 2100 μmol / L.

7. The method for constructing a fluorescence sensor for dual-parameter detection according to claim 5, characterized in that: The volume ratio of the stock solution, buffer solution and ThT solution is 1:46:0.5; the incubation time at room temperature is 5 min.

8. A fluorescent sensor prepared by the construction method according to any one of claims 1-4 and 6-7.

9. The application of the fluorescence sensor of claim 8 in detecting multiple targets simultaneously for purposes other than disease diagnosis, characterized in that, The procedure is as follows: Add the test solution to 588 μL of the working solution of the fluorescence sensor, incubate at 37°C in a shaker for 60 min, and use a fluorescence spectrophotometer to detect the fluorescence values ​​before and after adding the test solution. F and F 0 Calculate the change in fluorescence intensity. FF 0 The concentration of the target analyte was calculated by substituting it into the linear equation; the excitation wavelength of the fluorescence spectrophotometer was set to 460 nm, the emission wavelength to 465~650 nm, the excitation slit to 2.5 nm, and the emission slit to 5.0 nm.

10. The application according to claim 9, characterized in that: When the target objects are AD and BRCA2, the linear equation is: y =3.9897 x -6.5337, R1 2 =0.9886, y =4.0938 x +0.3458, R2 2 =0.9939, detection limits were 0.53 and 0.36 nmol / L, respectively; the linear equations for target analytes AD and DNA19 were as follows: y =3.3290 x -0.8471, R1 2 =0.9966, y =4.3307 x +2.8466, R2 2 =0.9914, detection limits were 0.67 and 0.34 nmol / L, respectively; the linear equations for target analytes AD and K-ras were... y =3.0940 x -6.5998, R1 2 =0.9795, y =4.1836 x +12.1746, R2 2 =0.9744, detection limits are 0.72 and 0.31 nmol / L, respectively; the linear equations for target analytes AD and P53-1 are as follows: y =3.1007 x -4.2411, R1 2 =0.9966, y =4.4243 x +0.1620, R2 2 =0.9996, detection limits were 0.81 and 0.37 nmol / L, respectively; the linear equations for target analytes AD and DNA36 were as follows: y =2.9428 x -3.7615, R1 2 =0.9981, y =3.0487 x +0.6233, R2 2 =0.9997, detection limits were 0.83 and 0.63 nmol / L, respectively; the linear equations for target compounds P53-2 and P53-1 were as follows: y =3.270 x -0.715, R1 2 =0.993, y =3.562 x +18.558, R2 2 =0.986, detection limits are 0.63 and 0.99 nmol / L, respectively; the linear equations for target analytes PTEN and P53-1 are as follows: y =3.306 x +28.879, R1 2 =0.988, y =3.788 x -6.956, R2 2 =0.999, detection limits were 0.54 and 1.06 nmol / L, respectively; the linear equations for target analytes HBV and P53-1 were as follows: y =3.482 x +10.104, R1 2 =0.988, y =2.708 x +2.364, R2 2 =0.999, and the detection limits were 1.26 and 1.19 nmol / L, respectively.

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