Au-DNA probe, metal ion rapid detection test strip and preparation method and application of Au-DNA probe and metal ion rapid detection test strip

Abstract

The invention discloses an Au-DNA probe, a metal ion rapid detection test strip and a preparation method and application thereof.The probe comprises a hybrid double strand formed by hybridization of a recognition strand and a substrate strand and gold nanoparticles, the recognition strand comprises a DNAzyme strand, an extension strand and terminal sulfydryl, the extension strand is complementary with the sequence of C DNA, the DNAzyme strand is provided with a metal ion recognition structure, and the substrate strand is provided with a metal ion detection structure. The substrate chain is provided with a metal ion cutting site, one end of the substrate chain is modified with terminal sulfydryl, and the gold nanoparticles are connected with the terminal sulfydryl. C DNA (deoxyribonucleic acid) and T DNA for capturing a substrate chain are sprayed on a reaction film of the test strip. Red based on AuNPs is used as a signal output mode, sensitive and rapid detection of metal ions is achieved, detection has low detection limit, wide linear response range and good selectivity and stability, the preparation is simple, convenient and rapid, easy-to-obtain raw materials are adopted for synthesis, the cost is low, rapid detection of Na < + > can be achieved, and the sensor is small, portable and easy to popularize. The development potential and the application background are realized.

Description

Technical Field

[0001] The present invention belongs to the field of colorimetric biosensing, and particularly relates to an Au-DNA probe, a metal ion rapid detection test strip based on the Au-DNA probe, and a preparation method and application thereof. Background Art

[0002] In recent years, with the acceleration of the urbanization process and the rapid development of industry, serious pollution problems have emerged. Among various pollutants, metal ions in the environment are difficult to degrade and can accumulate in the human body through the food chain, which makes them more harmful to the human body. Therefore, detection methods for metal ions have received extensive attention. Currently, spectroscopic detection techniques (atomic emission spectroscopy, atomic absorption spectroscopy, atomic fluorescence spectroscopy, and inductively coupled plasma mass spectrometry) are widely used for the detection of metal ions. Although these techniques are sensitive and reliable, they require large and precise instruments, complex operation procedures, and high maintenance costs. In contrast, electrochemical methods are cost-effective, time-efficient, user-friendly, and reliable. Electrochemical techniques such as voltammetry, impedance method, potentiometry, conductivity method, amperometry, etc. can be used for the detection of heavy metal ions. However, compared with spectroscopic techniques and optical methods, these electrochemical techniques generally have lower sensitivity. In optical methods, colorimetry is widely used for the determination of metal ions due to its high sensitivity, fast analysis speed, low cost, and simple operation.

[0003] Lateral flow chromatography (LFA) is a powerful in vitro diagnostic tool developed in the 20th century. Due to its simple operation, rapid analysis results, small size, good portability, low cost, and long-term stability under various conditions, it has been found to be a powerful in vitro diagnostic tool. In recent years, it has been widely used in many fields such as food safety, environmental detection, and disease diagnosis, and is one of the main means of point-of-care testing (POCT), playing an important role in the rapid on-site detection of analytes. The main signal generation mode in lateral flow chromatography mainly relies on nanoparticle labeling, such as quantum dots (QDs), magnetic nanoparticles (MNP), etc., which largely determine the type and performance of LFA. So far, various LFA labeling systems have been developed based on different signal conversion principles, such as colorimetric method, fluorescence method, magnetic method, Raman method, and chemiluminescence labeling method. In the colorimetric method, LFTA has received extensive favor. However, the difficulties in the detection of test strips are concentrated in aspects such as selectivity, sensitivity, matrix effect, stability, reproducibility, response time, calibration and quantification, cost, multi-ion detection, and environmental factors. Solving these problems requires optimizing reagents, improving materials, and optimizing the production process. Summary of the Invention

[0004] In view of the problems of the above-mentioned prior art, the present invention provides an Au-DNA probe, a metal ion rapid detection test strip based on the Au-DNA probe, and a preparation method and application thereof. The Au-DNA probe and the rapid detection test strip have high sensitivity, a wide linear range, convenience and rapidness, strong anti-interference ability, and can be used for on-site detection.

[0005] To achieve the above object, the present invention adopts the following technical solutions: An Au-DNA probe includes a hybrid double strand formed by hybridization of an identification strand and a substrate strand, and gold nanoparticles. The identification strand includes a DNAzyme strand, an extension strand, and a terminal mercapto group. The extension strand and the terminal mercapto group are respectively connected to both ends of the DNAzyme strand. The extension strand is complementary to the sequence of the cDNA that captures the identification strand. The DNAzyme strand has a metal ion recognition structure. The substrate strand has a cleavage site for the metal ion. The DNAzyme strand hybridizes with the entire sequence of the substrate strand. One end of the substrate strand close to the extension strand is modified with a terminal mercapto group. The hybrid double strand is connected to the gold nanoparticles through the terminal mercapto group.

[0006] The specific sequences of the DNAzyme strand with a metal ion recognition structure and the substrate strand with a metal ion cleavage site are prior art.

[0007] When the cDNA is fixed on a fixed carrier, the identification strand can be captured in a specific area.

[0008] When the metal ion is not contained in the system, the cDNA captures the hybrid double strand and the gold nanoparticles connected thereto, and free substrate strands and the gold nanoparticles carried thereby cannot be detected or captured in the system.

[0009] When the metal ion is contained in the system, the substrate strand breaks and separates from the DNAzyme strand. The broken substrate strand carries the gold nanoparticles connected thereto and is free in the system, and is detected or captured, as Figure 2 shown.

[0010] The detection of the metal ion is achieved by the color output of different gold nanoparticles.

[0011] Preferably, the terminal mercapto group is connected to the DNAzyme strand through -(CH 2 ) 6 -.

[0012] Preferably, the molar ratio of the identification strand to the substrate strand is 1:3.5.

[0013] Preferably, the metal ion is Na + , Mg 2+ , Pb 2+ , Zn 2+ or Mn 2+ .

[0014] Preferably, the particle size of the gold nanoparticles is 15 ± 5 nm.

[0015] Preferably, the sequence of the DNAzyme strand is 5’- TTT TTT GG CGG AAC CAG GTC AAAG GTGGGT GAG GGG ACG CCA AGA GTC CCC GCG GTT AGG AGA TCG-3’; The sequence of the substrate strand is 5’- TTT TTT GAC TTA TGA CGA TCT CCT AT / rA / GGA AGTTCC GCC-3’; wherein, / rA / is the cleavage site of Na + .

[0016] Preferably, the sequence of the extension strand is 5’-GAT CAT TAC TAG GCA C-3’.

[0017] The second object of the present invention is to provide a metal ion rapid detection test strip based on the Au-DNA probe, including a reaction membrane, on which a T line and a C line are sprayed, The area of the C line is sprayed with a conjugate of biotin-modified C DNA and streptavidin, and the area of the T line is sprayed with a conjugate of biotin-modified T DNA and streptavidin, The C DNA is complementary to the extension strand in the Au-DNA probe, The T DNA is complementary to the cleavage fragment of the substrate strand in the Au-DNA probe, and the cleavage fragment of the substrate strand is the part between the cleavage site of the metal ion in the substrate strand and the terminal mercapto group.

[0018] Preferably, the sequence of the C DNA is 5’-Biotin - G TGC CTA GTA ATG ATC-3’.

[0019] Preferably, the sequence of the T DNA is 5’-Biotin- T AGG AGA TCG TCA TAA GTC-3’.

[0020] Preferably, the distance between the C line and the T line is 4 - 8 mm.

[0021] The third object of the present invention is to provide a method for preparing the Au-DNA probe, comprising the following steps: (1-1) Using the salt aging method, coupling the recognition strand to the surface of the gold nanoparticles through the terminal mercapto group to prepare DNA-AuNPs 1, and coupling the substrate strand to the surface of the gold nanoparticles through the terminal mercapto group to prepare DNA-AuNPs 2; (1-2) Hybridizing DNA-AuNPs 1 and DNA-AuNPs 2 to obtain the Au-DNA probe.

[0022] Preferably, in step (1-1), the concentrations of the recognition strand and the substrate strand are 100 μM respectively, the incubation temperature for the coupling is room temperature, and the incubation time is 10-16 h, preferably 16 h; the concentration of the salt solution in the salt aging method is 1 M, the incubation temperature is room temperature, and the incubation time is 12-24 h, preferably 24 h.

[0023] Preferably, in step (1-2), the hybridization temperature is 90-95 °C, the hybridization time is 5 min, and the hybridization method is natural cooling to room temperature.

[0024] Preferably, the gold nanoparticles are prepared by the following method: using chloroauric acid (HAuCl 4 ), a sodium citrate solution as a raw material, and preparing AuNPs by the sodium citrate reduction method. The average particle size of the obtained AuNPs is 15 nm.

[0025] Preferably, the concentration of chloroauric acid is 24 mM, and the concentration of sodium citrate is 38.8 mM.

[0026] The fourth object of the present invention is to provide a method for preparing the metal ion rapid detection test strip based on the Au-DNA probe, comprising: (2-1) Assembling the substrate of the reaction membrane with the sample pad and the absorbent pad into a test strip; (2-2) Spraying the C-region solution on the substrate of the reaction membrane and then drying to form a C line, and spraying the T-region solution on the substrate of the reaction membrane and then drying to form a T line; The C-region solution contains a conjugate of C DNA, biotin and streptavidin; The T-region solution contains a conjugate of T DNA, biotin and streptavidin.

[0027] The components of the test strip are assembled on the PVC bottom plate with an overlap of 2 mm to ensure smooth flow between the components, and a portable test strip colorimetric sensor is prepared.

[0028] Preferably, the conjugate of cDNA, biotin, and streptavidin is obtained by the following method: co-incubating cDNA, biotin, and streptavidin, and that's it. The conjugate of T DNA, biotin, and streptavidin is obtained by the following method: co-incubating T DNA, biotin, and streptavidin, and that's it.

[0029] The concentration of the cDNA or the T DNA is 50 - 100 μM, preferably 100 μM; the concentration of the streptavidin is 1 - 5 mg / mL, preferably 1 mg / mL; the temperature of the incubation is 37 °C, and the time of the incubation is 1 - 3 h, preferably 1 h.

[0030] Preferably, the temperature of the drying is 37 °C, and the time of the drying is 1 - 2 h, preferably 2 h.

[0031] The fifth object of the present invention is to provide the application of the Au-DNA probe and the metal ion rapid detection test strip based on the Au-DNA probe, and the application is: detecting metal ions. The detection principle is as Figure 3 shown.

[0032] The beneficial effects of the Au-DNA probe provided by the present invention and the colorimetric biosensor of the metal ion rapid detection test strip based on the Au-DNA probe are as follows: AuNPs have the advantages of simple preparation, short labeling time, good stability, low cost, etc. At the same time, combining nanoparticles with DNAzyme in the detection of metal ions can directly obtain the detection results in a visually observable way.

[0033] The Au-DNA probe provided by the present invention and the metal ion rapid detection test strip based on the Au-DNA probe detect metal ions with high sensitivity, a wide linear range of 1 nM - 120 mM, and a detection limit as low as 0.05 nM (S / N≥3).

[0034] The constructed lateral flow test strip can complete the detection of metal ions within 12 min, which is convenient, fast, and can be used for on-site detection.

[0035] The construction of the portable lateral flow test strip reduces the operation cost, is simple to operate, has good selectivity, strong anti-interference ability, is used for sensitive detection of metal ions, and can be applied and popularized. Description of the Drawings

[0036] Figure 1 It is the preparation flow chart of the Au-DNA probe.

[0037] Figure 2Flow chart of the change of Au-DNA probe in a system containing metal ions.

[0038] Figure 3 Schematic diagram of the principle of the detection process of the present invention.

[0039] Figure 4 Transmission electron microscopy (TEM) images of the materials prepared in Example 1, from left to right are AuNPs, DNA-AuNPs1, and DNA-AuNPs 2.

[0040] Figure 5 Dynamic light scattering (DLS) diagrams of the materials prepared in Example 1, from left to right are AuNPs, DNA-AuNPs 1, and DNA-AuNPs 2.

[0041] Figure 6 Zeta potential diagrams (ζ) of AuNPs, DNA-AuNPs 1, and DNA-AuNPs 2 prepared in Example 1.

[0042] Figure 7 Ultraviolet-visible spectroscopy (UV-vis) diagrams of AuNPs, DNA-AuNPs 1, and DNA-AuNPs 2 prepared in Example 1.

[0043] Figure 8 Transmission electron microscopy (TEM) and dynamic light scattering (DLS) diagrams of the Au-DNA probe prepared in Example 1.

[0044] Figure 9 Circular dichroism (CD) diagrams of AuNPs, DNA-AuNPs 1, DNA-AuNPs 2, and Au-DNA probe in Example 1.

[0045] Figure 10 Ultraviolet-visible spectroscopy (UV-vis) diagrams of AuNPs, DNA-AuNPs 1, DNA-AuNPs 2, and Au-DNA probe in Example 1.

[0046] Figure 11 Schematic diagram and polyacrylamide gel electrophoresis diagram of the process of DNA, strand displacement reaction, and metal particle cleavage in Example 1 (3-1-1). Among them, lane 1 is the substrate strand; lane 2 is the recognition strand; lane 3 is the cleavage strand; lane 4 is the control group; lane 5 is the hybrid double strand; lane 6 is the experimental group.

[0047] Figure 12Schematic diagram of the process of DNA, strand displacement reaction and metal particle cleavage in Example 1 (3-1-2), and polyacrylamide gel electrophoresis pattern. Among them: Lane 1 is the substrate strand; Lane 2 is the recognition strand; Lane 3 is the cleavage strand; Lane 4 is the T-captured cleavage strand; Lane 5 is the hybrid double strand; Lane 6 is the T-captured substrate strand; Lane 7 is the C-captured recognition strand; Lane 8 is the C-captured hybrid double strand.

[0048] Figure 13 Schematic diagram of the process of DNA, strand displacement reaction and metal particle cleavage in Example 1 (3-1-3), and polyacrylamide gel electrophoresis pattern. Among them: Lane 1 is the T DNA; Lane 2 is the C DNA; Lane 3 is the cleavage strand; Lane 4 is the hybrid double strand; Lane 5 is the experimental group; Lane 6 is the T-captured cleavage strand; Lane 7 is the C-captured recognition strand; Lane 8 is the C-captured hybrid double strand; Lane 9 is the verification group.

[0049] Figure 14 Graph showing the color intensity analysis of the C line and T line after each sample passes through the test strip in Example 1 (3-2).

[0050] Figure 15 For each concentration of Na + solution and Au-DNA probe solution after being mixed separately and passing through the test strip, photos and relative color intensity analysis graphs.

[0051] Figure 16 Graph showing the photos and relative color intensity after each ionic solution and Au-DNA probe solution are mixed separately and pass through the test strip in Example 1 (5).

[0052] Figure 17 For Na + and the relative color intensity comparison graph of the Au-DNA probe mixture after passing through each test strip in Example 1 (6).

[0053] Figure 18 For the detection of Na 2 O 2 after hydrolysis, photos of the test strip and relative color intensity graphs obtained in Example 1 (7). + Detailed implementation mode

[0054] The present invention will be described in detail below in conjunction with examples and drawings. Example 1

[0055] The target metal ion in this example is Na + as an example.

[0056] (1) Preparation of Au-DNA probe: Using 24 mM chloroauric acid (HAuCl 4Using ultrapure water and 38.8 mM sodium citrate solution as raw materials, 15 nm AuNPs were prepared by the sodium citrate reduction method.

[0057] According to the target Na + The designed DNAzyme strand is 5'-TTT TTT GG CGG AAC CAG GTC AAAG GTG GGTGAG GGG ACG CCA AGA GTC CCC GCG GTT AGG AGA TCG-3'; the corresponding sequence with the Na + cleavage site is 5'-TTT TTT GAC TTA TGA CGA TCT CCT AT / rA / GGA AGT TCC GCC-3'.

[0058] The designed extension strand is 5'-GAT CAT TAC TAG GCA C-3'.

[0059] According to the design, the recognition strand was customized as 5'-(CH 2 ) 6 -TTT TTT GG CGG AAC CAG GTC AAAGGTG GGT GAG GGG ACG CCA AGA GTC CCC GCG GTT AGG AGA TCG GAT CAT TAC TAG GCA C-3'; the substrate strand is 5'-(CH 2 ) 6 -TTT TTT GAC TTA TGA CGA TCT CCT AT / rA / GGA AGT TCC GCC-3'.

[0060] 15 μL of 100 μM substrate strand and recognition strand were added to 1.5 μL of 100 mM TCEP (dissolved in pure water), and reacted at room temperature for 1 h to reduce disulfide bonds. The above-activated substrate strand and recognition strand were respectively mixed with 500 μL of AuNPs, incubated at room temperature for 16 h, then 50 μL of 1 M LiCl was added to increase the final salt concentration in the solution to 0.1 M, and incubated at room temperature for 24 h. Centrifuge at 12,000 rpm for 30 min to remove the excess DNA, and DNA-AuNPs 1 was obtained with the substrate strand, and DNA-AuNPs 2 was obtained with the recognition strand.

[0061] Finally, DNA-AuNPs 1 and DNA-AuNPs 2 were respectively dispersed in pure water and stored at 4°C for later use.

[0062] Mix the prepared DNA-AuNPs 1 and DNA-AuNPs 2 in a hybridization buffer solution at a molar ratio of 1:3, hybridize at 95 °C for 5 min, and then cool naturally to room temperature to obtain the Au-DNA probe, which is stored at 4 °C for future use.

[0063] (2)Structures and characterizations of AuNPs, DNA-AuNPs 1, and DNA-AuNPs 2: Characterize AuNPs, DNA-AuNPs 1, and DNA-AuNPs 2 using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis (ζ). The results are as Figures 4 - 7 shown.

[0064] As can be seen from the TEM image ( Figure 4 ), the surface of AuNPs is spherical and smooth, and the DNA layer after DNA is connected to the surface of AuNPs can be seen after uranyl acetate staining.

[0065] As can be seen from the DLS graph ( Figure 5 ), the hydrodynamic diameter of AuNPs is 16.44 ± 0.04 mm, while the hydrodynamic diameters of DNA-AuNPs 1 and DNA-AuNPs 2 are 27.29 ± 0.03 mm and 24.75 ± 2.25 mm, respectively, which are significantly larger than those of bare AuNPs, preliminarily indicating the successful coupling of DNA and AuNPs.

[0066] In addition, characterize DNA-AuNPs 1, DNA-AuNPs 2, and AuNPs using ultraviolet-visible absorption spectroscopy ( Figure 7 ). Compared with bare AuNPs, obvious red shifts appear in the DNA-modified AuNPs at 520 nm to 526 nm. The zeta potential ( Figure 6 ) shows that the negative potentials of DNA-AuNPs 1 and DNA-AuNPs 2 are significantly higher than that of AuNPs. The zeta potential of AuNPs encapsulated with sodium citrate is measured to be -2.24 mV. When the zeta potential is -6.74 mV and -4.30 mV, the zeta potential after DNA molecules are replicated on the surface of AuNPs is significantly negative, which is due to the electronegativity of DNA molecules themselves. These results indicate the successful preparation of AuNPs, DNA-AuNPs 1, and DNA-AuNPs 2.

[0067] As Figure 8 shown, the morphology of the Au-DNA probe is similar to the morphologies and TEM images of DNA-AuNPs 1 and DNA-AuNPs 2, preliminarily indicating the successful assembly of the Au-DNA probe.

[0068] As Figure 9As shown, single AuNPs, as well as DNA-AuNPs 1 and DNA-AuNPs 2, did not exhibit a strong CD response, while in the Au-DNA probe, a strong CD signal was observed at 283 nm. The CD signal was caused by the deviation of the NPs from sphericity. From the DLS graph ( Figure 8 ), it can be seen that the hydrodynamic diameter after assembling into the Au-DNA probe was significantly larger than the hydrodynamic diameter of the AuNPs in DNA-AuNPs 1 and DNA-AuNPs 2, being 75.96 ± 0.28 nm. In addition, the prepared probe was characterized using ultraviolet-visible absorption spectroscopy ( Figure 10 ), and the experimental results showed that there was an obvious red shift in the ultraviolet absorption spectrum. These results indicate that the Au-DNA probe was successfully prepared.

[0069] (2)Preparation of the Na + rapid detection test strip based on the Au-DNA probe: The sample pad, reaction membrane (NC membrane), and absorbent pad components were assembled on a PVC bottom plate with an overlap of 2 mm, enabling smooth flow between the components to prepare a portable lateral flow test strip colorimetric sensor.

[0070] 100 μM of T DNA and C DNA were respectively incubated with 1 mg / mL streptavidin at 37 °C for 1 h, and then centrifuged at 8,000 rpm for 10 min in a 30KDa ultrafiltration tube to remove the unbound DNA. The prepared T DNA-streptavidin conjugate and C DNA-streptavidin conjugate were respectively sprayed on the nitrocellulose membrane to form a test line and a control line. The distance between the two lines was approximately 8 mm. Then the nitrocellulose membrane was dried at 37 °C for 2 h for immobilization.

[0071] (3)Feasibility analysis of the biosensor composed of the constructed Au-DNA probe and the Na + rapid detection test strip based on the Au-DNA probe: (3-1)The characteristics of the hybridization and cleavage processes of the recognition strand and the substrate strand were analyzed by PAGE after natural cooling at 95 °C for 5 min to prove the feasibility of the proposed method: (3-1-1)Polyacrylamide gel electrophoresis was respectively performed on the substrate strand, recognition strand, cleavage strand, control group, hybrid double strand, and experimental group.

[0072] Among them, the cleavage strand was separately prepared according to the sequence after the substrate strand was cleaved into fragments.

[0073] The control group was obtained by the following method: The part of the substrate strand excluding the cleavage strand was separately prepared and hybridized with the recognition strand to obtain the control group.

[0074] The experimental group was obtained by the following method: Hybrid double strands were mixed and incubated with Na + ions at 37 °C for 3 h.

[0075] The results were as Figure 13 shown. The substrate strand, recognition strand, and cleavage strand showed distinct single bands in lanes 1-3 respectively. Compared with lanes 1 and 2, the hybrid double strands in lane 5 had a slower migration rate, indicating successful hybridization of the substrate strand and the recognition strand.

[0076] In the experimental group in lane 6, after adding Na + ions, the lane shifted significantly downward, showing the same downward-shifted band as in lane 4, proving that Na + could successfully cleave the hybrid double strands.

[0077] (3-1-2) Polyacrylamide gel electrophoresis was performed on the substrate strand, recognition strand, cleavage strand, T-capture cleavage strand, hybrid double strands, T-capture substrate strand, C-capture recognition strand, and C-capture hybrid double strands respectively.

[0078] Among them, the cleavage strand was the same as in (3-1-1). The T-capture cleavage strand was obtained by hybridizing the cleavage strand with T DNA. The T-capture substrate strand was obtained by hybridizing the substrate strand with T DNA. The C-capture recognition strand was obtained by hybridizing C DNA with the recognition strand through an extension strand. The C-capture hybrid double strands were obtained by hybridizing C DNA with the hybrid double strands through an extension strand.

[0079] The results were as Figure 12 shown. The band formed by the hybridization of the hybrid double strands and C DNA was verified in lane 8, indicating the feasibility of the capture of the hybrid double strands by the C region of the test strip. The position of the band of the C-capture recognition strand in lane 7 was higher than that of the hybrid double strands in lane 5, indicating successful hybridization of C DNA and the hybrid double strands.

[0080] (3-1-3) Polyacrylamide gel electrophoresis was performed on T DNA, C DNA, cleavage strand, hybrid double strands, experimental group, T-capture cleavage strand, C-capture recognition strand, C-capture hybrid double strands, and verification group respectively.

[0081] Among them, the setting methods of the cleavage strand and the experimental group were the same as in (3-1-1). The setting methods of the T-capture cleavage strand, C-capture recognition strand, and C-capture hybrid double strands were the same as in (3-1-2).

[0082] The verification group was obtained by mixing and incubating the hybrid double strands with Na + ions at 37 °C for 3 h and then mixing and incubating with T DNA and C DNA.

[0083] The results were as Figure 13 shown. Na +In the presence of [substance], the recognition strand can achieve catalytic cleavage. Therefore, after adding an excessive amount of Na + , lane 5 shows the Ci-strand product generated by cleavage, releasing H DNA, which lays the foundation for the subsequent successful color development of the test strip.

[0084] The results in lane 9 show that the product of the interaction between the hybrid double-strand and Na + ions is at the same position as the control bands of the T-captured cleavage strand in lane 6 and the C-captured recognition strand in lane 7, indicating that the H DNA after cleaving the hybrid double-strand can be captured by T DNA, and the recognition strand can be captured by C DNA. +

[0085] (3-2) Using a colorimetric detection method in combination with Image J software, the Au-DNA probe solution (negetive), DNA-AuNPs 1 solution, DNA-AuNPs 2 solution, the mixed solution (Positive) obtained by reacting the saturated standard Na + and the Au-DNA probe at 37 °C for 3 h, and the mixed solution (Sample) obtained by reacting 500 ng / mL Na 2 O 2 hydrolyzed Na + and the Au-DNA probe at 37 °C for 3 h were respectively added to the sample pad, and Image J software was used to analyze and observe the results. As Figure 14 shown.

[0086] (4) Linear detection of the constructed biosensor: Target Na + with concentrations of 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, and 120 mM were respectively added to 30 nM, 50 μL of the Au-DNA probe solution. After incubation at 33 °C for 2 h, the mixtures were respectively added to the sample pad, and the results were observed after 12 min for linear detection.

[0087] The relative color intensity is the ratio of the color intensity of the T line to the color intensity of the C line obtained by Image J software for each group.

[0088] As Figure 15 shown, the detection range of this biosensor is 1 nM - 120 mM, and the detection limit is 0.05 nM.

[0089] (5) Selectivity detection of the constructed biosensor: 100 mM Na + , 120 mM K + , 120 mM NH​4 + 、 120 mM Li + 、 120 mM Ca 2+ 、 120 mM Mn 2+ 、 120 mM Fe 2+ 、 120 mM Cu 2+ 、 120 mM Mg 2+ 、 120 mM Zn 2+ 、 120 mM Fe 3+ and 500 ng / mL Na 2 O 2 solutions were separately added to the 30 nM Au-DNA probe solution. After incubation at 33 °C for 2 h, each mixture was separately added to the sample pad. After 12 min, the results were observed. Each group was detected in parallel three times to observe the signal change.

[0090] As Figure 16 shown, in the presence of the target Na + , the signal was significantly enhanced, indicating that the sensor has good selectivity.

[0091] (6) Detection of the stability of the constructed biosensor for Na + detection: Take the test strips and store them separately at room temperature under dry conditions for 1, 3, 7, 12, 35, 60 days before Na + detection. After that, the saturated standard Na + solution was mixed with the Au-DNA probe solution and reacted. After incubation at 37 °C for 3 h, the mixture was separately added to the sample pads of each test strip. The results were as Figure 17 shown. The color intensity and sensitivity of the test strips had no significant difference from those of the freshly prepared test strips, and the signal remained 93.9% of the original signal. This indicates that the biosensor based on this remains stable within 2 months.

[0092] (7) Detection of the Na + content after the hydrolysis of sodium peroxide by the constructed biosensor: To verify the amount of Na + after the hydrolysis of sodium peroxide, Na 2 O 2 was formulated into different concentrations: 5, 20, 50, 80, 100, 200, 500 ng / mL, and separately added to 30 nM, 50 μL of the Au-DNA probe solution. After incubation at 33 °C for 2 h, the mixture was separately added to the sample pads. After 12 min, the results were observed.

[0093] As Figure 17 shown, as the Na +With the increase in concentration, the signal intensity of the T line gradually increases, proving that the colorimetric side test strip can detect the Na after the hydrolysis of sodium peroxide + quantity, with relatively high sensitivity.

[0094] It can be seen that the biosensor of the present invention exhibits a relatively wide linear range, a low detection limit, simple preparation of the test strip, low cost, small size and portability, and can realize the on-site detection of Na + , having development potential and application background.

Claims

1. An Au-DNA probe, characterized in that: It includes a hybrid double strand formed by hybridization of a recognition strand and a substrate strand, and gold nanoparticles, The recognition chain includes a DNAzyme chain, an extension chain and a terminal thiol group, wherein the extension chain and the terminal thiol group are connected to both ends of the DNAzyme chain respectively. The extended strand is complementary to the sequence of the C DNA that captures the recognition strand, The DNAzyme chain has a metal ion recognition structure, The substrate chain has a cleavage site for the metal ion, The DNAzyme chain is hybridized with the entire sequence of the substrate chain. The end of the substrate chain close to the extension chain is modified with a terminal thiol group, The hybrid double strand is connected to the gold nanoparticle via a terminal thiol group.

2. The Au-DNA probe according to claim 1, characterized in that The metal ion is Na + Mg 2+ , Pb 2+ 、Zn 2+ or Mn 2+ .

3. The Au-DNA probe according to claim 1, characterized in that The sequence of the DNAzyme chain is 5'- TTTTTT GG CGG AAC CAG GTC AAAG GTG GGT GAG GGG ACG CCA AGA GTC CCC GCG GTT AGGAGA TCG-3'; The sequence of the substrate strand is 5'- TTT TTT GAC TTA TGA CGA TCT CCT AT / rA / GGA AGT TCCGCC-3'; Among them, / rA / is Na + cleavage site.

4. The Au-DNA probe according to claim 1, characterized in that The sequence of the extended chain is 5'-GAT CATTAC TAG GCA C-3'.

5. The method for preparing the Au-DNA probe according to any one of claims 1 to 4, characterized in that: The steps include: (1-1) using a salt aging method, coupling the recognition chain to the surface of the gold nanoparticles via the terminal thiol group to prepare DNA-AuNPs 1, and coupling the substrate chain to the surface of the gold nanoparticles via the terminal thiol group to prepare DNA-AuNPs 2; (1-2) Hybridizing DNA-AuNPs 1 and DNA-AuNPs 2 to obtain the Au-DNA probe.

6. A metal ion rapid detection test strip based on the Au-DNA probe according to any one of claims 1 to 4, characterized in that: It includes a reaction film, on which T lines and C lines are sprayed, The C line region is sprayed with a conjugate of biotin-modified C DNA and streptavidin, and the T line region is sprayed with a conjugate of biotin-modified T DNA and streptavidin. The C DNA is complementary to the extended strand in the Au-DNA probe, The T DNA is complementary to the cleavage fragment of the substrate chain in the Au-DNA probe, and the cleavage fragment of the substrate chain is the portion between the cleavage site of the metal ion and the terminal thiol group in the substrate chain.

7. The metal ion rapid detection test strip according to claim 6, characterized in that: The sequence of the C DNA is 5'-Biotin-GTGCCTAGTAATGATG ATC-3'.

8. The metal ion rapid test strip according to claim 6, characterized in that: The sequence of the T DNA is 5'-Biotin-TAGG AGA TCG TCA TAA GTC-3'.

9. The method for preparing the metal ion rapid detection test strip based on the Au-DNA probe according to any one of claims 6 to 8, characterized in that: include: (2-1) Assembling the reaction membrane substrate, the sample pad and the absorbent pad into a test strip; (2-2) spraying the C zone solution on the substrate of the reaction film and drying it to form a C line, and spraying the T zone solution on the substrate of the reaction film and drying it to form a T line; The C zone solution contains a conjugate of C DNA, biotin and streptavidin; The T region solution contains a conjugate of T DNA, biotin and streptavidin.

10. Use of the Au-DNA probe according to any one of claims 1 to 4 and the metal ion rapid detection test strip based on the Au-DNA probe according to any one of claims 6 to 8.

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