CN113584130A - Biosensor for detecting cadmium ions and method for detecting cadmium ions - Google Patents
Biosensor for detecting cadmium ions and method for detecting cadmium ions Download PDFInfo
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Abstract
The invention relates to a biosensor for detecting cadmium ions, which comprises magnetic bead probes Cd-MBs and Hg-MBs, wherein a cadmium ion deoxynuclease sequence and a substrate chain sequence which is in complementary pairing connection with the cadmium ion deoxynuclease sequence are modified and connected on the Cd-MBs; the Hg-MBs was modified by ligation with a cleaved G-quadruplex fragment AT11A with a Poly-T region. Compared with the existing biosensor taking deoxyribozyme as a recognition element, the biosensor can accurately detect Cd2+Content of Hg in the sample, and can eliminate Hg in the sample2+For Cd2+The influence of the detection result promotes Cd2+The specificity of detection, the sensor can also realize Hg in the sample2+Accurate detection of. The biosensor can be used for detecting Cd of 1.9nM at the lowest2+And 19.5nM Hg2+To carry outDetection provides a new way for the construction of the subsequent nucleic acid biosensor.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a biosensor for detecting cadmium ions and a method for detecting the cadmium ions.
Background
Cadmium (Cd) naturally occurs in minerals and can be extracted from zinc-copper ore or sulfur-cadmium ore. Cadmium has been widely used in the production of nickel-cadmium batteries as a stabilizer of battery performance. With the growing awareness of the importance of environmental protection, cadmium has received increasing attention as a major heavy metal contaminant. Since the 1990 s, the use of cadmium has been greatly reduced in developed countries such as the European Union, but in most developing countries, the production and use of cadmium and the amount of emission into the environment have increased dramatically. Cadmium is difficult to degrade after entering the environment, can be accumulated in organisms through the action of biological enrichment, and cadmium poisoning in food also happens occasionally, thus causing great harm to the health and public health of people.
The drinking water sanitary standard GB 5749 and 2006 in China stipulates that the upper limit of cadmium is 0.005 mg/L. At present, the traditional detection methods for heavy metal cadmium mainly include atomic absorption spectrometry, atomic fluorescence spectrometry, inductively coupled plasma emission spectrometry and the like. Although these methods have good accuracy and sensitivity, they have limited popularization and application due to the need for expensive large-scale instruments, specialized detection personnel, and complicated pretreatment processes. Functional nucleic acid has the advantages of high stability, strong specificity, low cost, easy modification and the like, so biosensors based on the functional nucleic acid are more and more concerned by people and become a current research hotspot.
Liu et al developed a new Cd2+A screening method of specific recognition deoxyribozyme uses a single Phosphorothioate (PS) modified ribonucleobase (rA) containing DNA as a substrate, and isolates a deoxyribozyme named BN-Cd16 in vitro, wherein the catalytic loop of the deoxyribozyme contains 12 nucleotides. The deoxyribozyme pair Cd2+Has high selectivity, and is suitable for Cd2+The specific recognition deoxyribozyme lays a good foundation as a recognition element in the sensor. However, BN-Cd16 can still bind Hg2+And activity is generated, so when BN-Cd16 is used as Cd2+When the identification element is constructed in the specific biosensor, interference signals can be generated, so thatThe detection result is inaccurate.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a biosensor for detecting cadmium ions, which can detect Cd2+High-sensitivity detection is realized, and the accuracy of the detection result is high.
The invention also aims to provide a method for detecting cadmium ions.
Technical scheme
A biosensor for detecting cadmium ions comprises magnetic bead probes Cd-MBs and magnetic bead probes Hg-MBs, solid phase carriers of the magnetic bead probes Cd-MBs and the magnetic bead probes Hg-MBs are streptavidin modified nanometer magnetic beads, and cadmium ion deoxynuclease sequences (Cd-MBs) are modified and connected on the magnetic bead probes Cd-MBs2+DNAzyme) and a substrate strand sequence (PS substrate) linked in complementary pairing with a cadmium ion dnase sequence; a split G-quadruplex fragment AT11A with a Poly-T region is modified and connected on the magnetic bead probe Hg-MBs;
the cadmium ion deoxynuclease sequence (Cd)2+DNAzyme) is: 5'-GTT CGC CAT CTT CCT TCGATA GTTAAAATAGTG CAT-3', respectively;
the substrate chain sequence is: 5 '-TTC GGATGC ACTATrA GGAAGATGG CGAAC-biontin-3'; in the sequence, rA is a special ribonucleotide, is a ribonucleotide with phosphorothioate modification;
the sequence of the AT11A is: 5 '-biotin-A GGG ACG GGA TTT TTT TTT TTA GGG ACG GGA-3'.
The principle of the invention is as follows: the invention constructs a magnetic bead probe Cd-MBs as the identification Cd2+Identification element of (Cd)2+DNAzymes (cadmium ion deoxynucleases) are hybridized with a substrate strand (PS substrate) immobilized on magnetic beads by streptavidin-biotin chemistry, in Cd2+In the presence of Cd2+The catalytic activity of DNAzyme, PS substrate in phosphorothioate modified ribose nucleobase (rA) site is cut, and release single-stranded DNA (OD-1, sequence 5'-TTC GGA TGC ACT ATA-3'), OD-2 is a DNA sequence rich in G base; OD-3 is a complementary sequence fragment of sequences OD-1 and OD-2, andand also has part of FokI recognition site, and after separation by magnetic frame, OD-1 in the supernatant hybridized with OD-2/OD3 complex to form double-stranded DNA, in which the FokI recognition site appeared. The double stranded DNA is then cleaved by FokI endonuclease resulting in release of the OD-2 sequence. The sequence can be assembled with heme to form a G quadruplex structure with similar peroxidase activity, can be used as a catalyst of color reaction in a biosensor to accelerate ABTS color development, and finally realizes the purpose of detecting the change of the color by direct naked eye observation and the change of the absorbance of a final solution by an iMark enzyme-linked immunosorbent assay (iMark ELISA) instrument to Cd2+And (6) carrying out quantitative detection.
Magnetic bead probes Hg-MBs can be used to exclude Hg from the probe2+False positive results from non-specific substrate cleavage in Hg-MB, in which the magnetic beads were modified with AT11A, AT11A is a split G-quadruplex fragment with a Poly-T region, and Hg-MB serves two functions. First, due to thymine-Hg2+Formation of thymine base pairing, which can be in Cd2+As Hg in the detection process2+An enrichment probe for removing Hg from a sample2+The interference caused. The second function is for Hg2+And (6) detecting. So that Cd is being detected2+Previously, it was necessary to first add Hg-MBs to the sample to remove Hg that may be present in the sample2+And (4) interference. After separation with a magnetic rack, if Hg2+Absent, AT11A on the beads would form a complete G quadruplex; if Hg2+The G quadruplex on the AT11A on the magnetic beads is due to Hg2+Exist and are destroyed. As the structure of the G quadruplex is damaged, the enzymatic activity is reduced, and finally the ABTS color reaction is reversely catalyzed. Finally, the color change is observed by direct naked eyes and the absorbance change of the final solution is detected by an iMark microplate reader, so that Hg is detected2+And (6) carrying out quantitative detection.
Further, the preparation method of the magnetic bead probe Cd-MBs comprises the following steps:
1) taking 40 mu L of streptavidin coupled Magnetic Beads (MBs) with the concentration of 10mg/mL, washing the MBs with 1X PBST buffer solution, and re-dissolving the MBs with 200 mu L of LPBST buffer solution to obtain an MB solution;
2) adding 10 mu L of substrate chain and 10 mu L of cadmium ion deoxynuclease into 80 mu L of PBS buffer solution, incubating for 2min in a metal bath at 90 ℃, then slowly cooling to room temperature for annealing to obtain a mixed solution, adding 20 mu L of the mixed solution into 100 mu L of MBs solution, and fully mixing at room temperature to obtain a magnetic bead probe Cd-MBs; washed with PBST buffer and reconstituted in PBST buffer.
Further, the preparation method of the magnetic bead probe Hg-MBs comprises the following steps:
1) taking 40 mu L of streptavidin coupled Magnetic Beads (MBs) with the concentration of 10mg/mL, washing the MBs with 1X PBST buffer solution, and re-dissolving the MBs with 200 mu L of LPBST buffer solution to obtain an MB solution;
2) AT11A was diluted to a concentration of 10. mu.M in CB1 buffer solution and annealed, and then 20. mu.L of the solution was added to 100. mu.L of LMB solution and mixed well AT room temperature to obtain magnetic bead probes Hg-MBs, which were washed with PBST buffer solution and then reconstituted in PBST buffer solution.
A method for detecting cadmium ions comprises the following steps:
(1) detecting by adopting the biosensor, taking 5 mu L of cadmium ion standard solutions with different concentrations and 20 mu L of magnetic bead probes Hg-MBs, mixing the solutions in CB1 buffer solution until the final volume is 30 mu L, shaking the solutions at room temperature for incubation reaction, and then carrying out magnetic separation to obtain the cadmium ion standard solution containing Cd with different concentrations2+The supernatant of (a);
(2) respectively adding 15 μ L of Cd with different concentrations2+Mixing the supernatant with 10 mu L of magnetic bead probe Cd-MBs in a reaction buffer solution until the final volume is 50 mu L, incubating and reacting at room temperature, and then carrying out magnetic separation on each solution to obtain a series of supernatants; the reaction buffer solution formula comprises: 50mM MES, 25mM NaCl, pH 6.0;
(3) 15. mu.L of each supernatant obtained in step (2) was added to 1 Xreaction buffer (50mM NaCl, 10mM Tris-HCl, 10mM MgCl) containing 200nM OD-2/OD-3 complex and 5U FokI endonuclease21mM dithiothreitol, pH 7.0) at 37 ℃ for 30min to obtain a FokI reaction mixture;
the OD-2/OD-3 complex is obtained by annealing OD-2 and OD-3 fragments in T-Na buffer (25mM tris-HCl, 100mM NaCl, pH 7.3); the oligonucleotide sequence of OD-2 is: 5'-AGA GGG ACG GGAAGG GAC GGGA-3', wherein the oligonucleotide sequence of OD-3 is: 5'-ATC CCG TCC CTC TTA TAG TGC ATC CGAA-3', respectively; the formula of the T-Na buffer solution comprises the following steps: 25mM tris-HCl, 100mM NaCl, pH 7.3.
(4) mu.L of FokI reaction mixture and 10. mu.L of heme (10. mu.M) in CB2 buffer (25mM HEPES, 200mM NaCl, 50mM KCl, 0.025% Triton X-100, 150mM NH4Cl, pH 5.3) to a final volume of 190. mu.L, left at room temperature in the dark for 30min, and then 5. mu.L of 40mM ATBTS and 5. mu.L of 40mM H2O2Adding the mixture to a final volume of 200 mu L, reacting at room temperature for 15min, testing by using an iMark absorbance microplate reader, scanning the wavelength range of 400-500nm, recording the absorbance at 415nm, and drawing a standard curve of the concentration of cadmium ions and the absorbance;
(5) mixing 5 mu L of sample to be detected and 20 mu L of magnetic bead probe Hg-MBs in CB1 buffer solution until the final volume is 30 mu L, shaking at room temperature for incubation reaction, then carrying out magnetic separation to obtain supernatant, then carrying out the same treatment according to the steps (2) to (4), testing to obtain the absorbance at 415nm, substituting the absorbance into the standard curve of the cadmium ion concentration and the absorbance drawn in the step (4), and calculating to obtain the cadmium ion concentration in the sample to be detected.
The invention can also realize the detection of mercury ions in the sample, and the method comprises the following steps:
step one, 5 mu L of Hg-containing solution containing different Hg2+The concentrated sample solution, 10. mu.L of magnetic bead probe Hg-MBs and 10. mu.L of hemoglobin (10. mu.M) were mixed in CB1 buffer to a final volume of 190. mu.L, incubated at room temperature in the dark, gently shaken for 30min, and then 5. mu.L of 40mM ATBTS and 5. mu.L of 40mM H were added2O2After reacting for 15min at room temperature, testing by using an iMark absorbance microplate reader, scanning the wavelength range of 400-500nm, recording the absorbance at 415nm, and drawing a standard curve of mercury ions and the absorbance;
step two, mixing 5 μ L of sample solution, 10 μ L of magnetic bead probe Hg-MBs and 10 μ L of heme in CB1 buffer solution to a final volume of 190 μ L, incubating at room temperature in the dark, gently shaking for 30min, and then adding 5 μ L of 40mM ATBTS and 5 μ L of 40mM H2O2And (3) after reacting for 15min at room temperature, testing the absorbance at the position of 415nm by using an iMark absorbance microplate reader, substituting the absorbance into the standard curve in the step one, and calculating to obtain the concentration of mercury ions in the sample.
Has the advantages that: the invention provides a biosensor for detecting cadmium ions, which comprises magnetic bead probes Cd-MBs and magnetic bead probes Hg-MBs2+And can eliminate Hg in the sample2+For Cd2+The influence of the detection result promotes Cd2+The specificity of detection, the biosensor of the invention can also realize Hg in a sample2+Accurate detection of. By adopting the biosensor and combining with an enzyme-labeling instrument, the color change can be detected by naked eyes, and the lowest Cd content of 1.9nM2+And 19.5nM Hg2+The detection provides a new way for the construction of the subsequent nucleic acid biosensor.
Drawings
FIG. 1 is a schematic diagram of a biosensor according to the present invention;
FIG. 2 is an absorbance curve for different cadmium ion concentrations;
FIG. 3 is a standard curve of cadmium ion concentration versus absorbance;
FIG. 4 is a graph of absorbance at different concentrations of mercury ions;
FIG. 5 is a standard curve of mercury ion concentration versus absorbance;
FIG. 6 is Hg-MBs vs Hg2+The enrichment capacity test result of (1);
FIG. 7 shows the detection of Cd by the biosensor of the present invention2+And Hg2+The result of the specificity test of (1).
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
Example 1
A biosensor for detecting cadmium ions comprises magnetic bead probes Cd-MBs and magnetic bead probes Hg-MBs, solid phase carriers of the magnetic bead probes Cd-MBs and the magnetic bead probes Hg-MBs are streptavidin modified nanometer magnetic beads, and cadmium ion deoxynuclease sequences (Cd-MBs) are modified and connected on the magnetic bead probes Cd-MBs2+DNAzyme) and substrates linked to complementary pairs of cadmium ion deoxynuclease sequencesChain sequence (PS substrate); a split G-quadruplex fragment AT11A with a Poly-T region is modified and connected on the magnetic bead probe Hg-MBs;
the cadmium ion deoxynuclease sequence (Cd)2+DNAzyme) is: 5'-GTT CGC CAT CTT CCT TCG ATA GTT AAA ATA GTG CAT-3', respectively;
the substrate chain sequence is: 5 '-TTC GGA TGC ACT ATrA GGA AGA TGG CGA AC-biontin-3'; in the sequence, rA is a special ribonucleotide, is a ribonucleotide with phosphorothioate modification;
the sequence of the AT11A is: 5 '-biotin-A GGG ACG GGA TTT TTT TTT TTA GGG ACG GGA-3'.
The principle diagram of the biosensor for detecting cadmium ions of the present invention is shown in FIG. 1. The magnetic bead probe Cd-MBs constructed by the invention is used for identifying Cd2+Identification element of (Cd)2+DNAzymes (cadmium ion deoxynucleases) are hybridized with a substrate strand (PS substrate) immobilized on magnetic beads by streptavidin-biotin chemistry, in Cd2+In the presence of Cd2+The catalytic activity of DNAzyme, PS substrate in phosphorothioate modified ribose nucleobase (rA) site is cut, and release single-stranded DNA (OD-1, sequence 5'-TTC GGA TGC ACT ATA-3'), OD-2 is a DNA sequence rich in G base; OD-3 is a complementary sequence fragment of sequences OD-1 and OD-2, and also has a part of FokI recognition site, and after separation with a magnetic stand, OD-1 in the supernatant hybridizes with OD-2/OD3 complex to form double-stranded DNA in which the FokI recognition site appears. The double stranded DNA is then cleaved by FokI endonuclease resulting in release of the OD-2 sequence. The sequence can be assembled with heme to form a G quadruplex structure with similar peroxidase activity, can be used as a catalyst of color reaction in a biosensor to accelerate ABTS color development, and finally realizes the purpose of detecting the change of the color by direct naked eye observation and the change of the absorbance of a final solution by an iMark enzyme-linked immunosorbent assay (iMark ELISA) instrument to Cd2+And (6) carrying out quantitative detection. Magnetic bead probes Hg-MBs can be used to exclude Hg from the probe2+False positive results from non-specific substrate cleavage in Hg-MBs, magnetic beads were modified with AT11A, AT11A is a split G-quadruplex fragment with a Poly-T region due to thymine-Hg2+Formation of thymine base pairing, Hg-MBs can be in Cd2+As Hg in the detection process2+An enrichment probe for removing Hg from a sample2+The interference caused. The sequences of the oligonucleotides involved in the invention are shown in Table 1:
TABLE 1
The preparation method of the magnetic bead probe Cd-MBs comprises the following steps:
1) mu.L of 10mg/mL streptavidin-coupled Magnetic Beads (MBs) were loaded with 1 XPBST buffer (136.89mM NaCl, 2.67mM KCl, 8.1mM Na)2HPO4、1.76mM KH2PO4After washing 3 times with 0.05% (w/v) Tween-20 at pH 7.4, the resulting mixture was reconstituted with 200 μ LPBST buffer to obtain an MB solution;
2) mu.L of substrate strand (100. mu.M) and 10. mu.L of cadmium ion deoxynuclease were added to 80. mu.L of PBS buffer solution (136.89mM NaCl, 2.67mM KCl, 8.1mM Na)2HPO4、1.76mM KH2PO4pH 7.4), incubating in a metal bath at 90 ℃ for 2min, then slowly cooling to room temperature for annealing to obtain a mixed solution, adding 20 μ L of the mixed solution into 100 μ LMB solution, and fully mixing at room temperature to obtain a magnetic bead probe Cd-MB; after washing with PBST buffer, the solution was reconstituted in 50. mu.L of PBST buffer.
The preparation method of the magnetic bead probe Hg-MBs comprises the following steps:
1) taking 40 mu L of 10mg/mL streptavidin coupled Magnetic Beads (MBs), washing with 1X PBST buffer solution, and then re-dissolving with 200 mu L of PBST buffer solution to obtain MB solution;
2) AT11A was diluted to a concentration of 10. mu.M in CB1 buffer (25mM HEPES, 20mM KCl, 200mM NaCl, 0.025% (w/v) Triton X-100, pH 5.3) and annealed, and then 20. mu.L was added to 100. mu.L of MB solution and mixed well AT room temperature to give magnetic bead probes Hg-MBs, which were washed with PBST buffer and then reconstituted in 50. mu.L of PBST buffer.
Example 2 plotting cadmium ion concentration versus absorbance
(1) Taking 5 mu L of cadmium ion standard solution (0, 1nM, 10nM, 30nM, 50nM, 70nM, 100nM, 200nM, 300nM, 500nM) with different concentrations and 20 mu L of magnetic bead probe Hg-MBs, mixing in CB1 buffer solution to reach a final volume of 30 mu L, shaking at room temperature for incubation reaction, and then carrying out magnetic separation to obtain the cadmium ion standard solution containing different concentrations of Cd2+The supernatant of (a);
(2) respectively adding 15 μ L of Cd with different concentrations2+Mixing the supernatant with 10. mu.L of magnetic bead probe Cd-MBs in a solution (50mM MES, 25mM NaCl, pH 6.0) to a final volume of 50. mu.L, incubating the mixture at room temperature for 20min, and magnetically separating each solution to obtain a series of supernatants;
(3) 15. mu.L of each supernatant obtained in step (2) was added to 1 Xreaction buffer (50mM NaCl, 10mM Tris-HCl, 10mM MgCl) containing 200nM OD-2/OD-3 complex and 5U FokI endonuclease21mM dithiothreitol, pH 7.0) at 37 ℃ for 30min to obtain a FokI reaction mixture;
the OD-2/OD-3 complex is obtained by annealing OD-2 and OD-3 fragments in T-Na buffer (25mM tris-HCl, 100mM NaCl, pH 7.3); the oligonucleotide sequence of OD-2 is: 5'-AGA GGGACG GGA AGG GAC GGGA-3', wherein the oligonucleotide sequence of OD-3 is: 5'-ATC CCG TCC CTC TTA TAG TGC ATC CGAA-3' are provided.
(4) mu.L of FokI reaction mixture and 10. mu.L of heme (10. mu.M) in CB2 buffer (25mM HEPES, 200mM NaCl, 50mM KCl, 0.025% Triton X-100, 150mM NH4Cl, pH 5.3) to a final volume of 190. mu.L, left at room temperature in the dark for 30min, and then 5. mu.L of 40mM ABTS and 5. mu.L of 40mM H2O2Adding the mixture to a final volume of 200 mu L, reacting at room temperature, testing by using an iMark absorbance microplate reader, scanning the wavelength range of 400-500nm, recording the absorbance at 415nm, and showing the absorbance curve under different cadmium ion concentrations in figure 2, wherein the absorbance intensity depends on the dose along with the increase of the cadmium ion concentration(ii) is increased sexually; the absorbance intensity (Abs) at 415nM was recorded after 16min reaction at room temperature, and a standard curve of cadmium ion concentration versus absorbance was plotted, as shown in fig. 3, and it can be seen that the absorbance response and cadmium ion concentration showed a good linear relationship in the range of 1 to 100nM, and the linear equation was that Abs is 0.221+0.00455CCd(CCdIs Cd2+Concentration). Correlation coefficient (R) of linear regression equation2) It was 0.996, and the limit of detection (LOD) was estimated to be 1.9nM (3 SNR). In addition, the oxidation of colorless ABTS to green ABTS due to the enhancement of G-quadruplex/hemin catalytic activity·+With Cd2+With increasing concentration, a noticeable color change was observed.
Example 3 Standard Curve plotting Mercury ion concentration vs. Absorbance
mu.L of different concentrations of mercury ion standard solutions (0, 100nM, 200nM, 300nM, 400nM, 500nM, 1000nM, 2000nM), 10. mu.L of magnetic bead probe Hg-MBs and 10. mu.L of heme (10. mu.M) mixed in CB1 buffer to a final volume of 190. mu.L, incubated at room temperature in the dark, shaken gently for 30min, then 5. mu.L of 40mM ATBTS and 5. mu.L of 40mM H were added2O2Reacting at room temperature, testing with iMark absorbance microplate reader, scanning wavelength range of 400-500nm, recording absorbance at 415nm, and recording absorbance curve at different mercury ion concentrations as shown in FIG. 4, which can be seen in the absence of Hg2+In this case, the intact G-quadruplex structure can bind heme, activate its peroxidase activity, catalyze the oxidation of colorless ABTS to green ABTS·+With Hg2+An increase in concentration, a decrease in peroxidase activity of G-quadruplexes/heme, results in a decrease in uv absorbance at 415 nm; reacting at room temperature for 15min, testing with iMark absorbance microplate reader, recording the absorbance at 415nm, and drawing standard curve of mercury ion concentration and absorbance, as shown in FIG. 5, showing the absorbance and Hg2+The concentration is linear in the range of 0 to 400nM, and the linear equation is Abs ═ 1.048-0.002CHg(CHgIs Hg2+Concentration). Correlation coefficient (R) of linear regression equation2) Was 0.983. The limit of detection (LOD) was estimated to be 19.5nM (3 signal to noise ratio).
Example 4 testing of the enrichment Capacity of Hg-MBs
Different Hg is added2+Samples of concentration (samples 1 to 6, Hg)2+The concentrations are respectively: 0.2. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M) with Hg-MBs and separated with a magnetic stand to obtain a supernatant; mu.L of the supernatant was transferred to a new centrifuge tube, 15. mu.L of the supernatant and 10. mu.L of Cd-MBs solution were mixed in a solution (50mM MES, 25mM NaCl, pH 6.0) to a final volume of 50. mu.L and incubated at room temperature for 20min, then each solution was separated with a magnetic rack, 15. mu.L of the supernatant was transferred to a new centrifuge tube, after which the supernatant was added to a 1 Xreaction buffer (50mM NaCl, 10mM Tris-HCl, 10mM MgCl. sub.10U) containing 200nM OD-2/OD-3 complex (annealing in 25mM Tris-HCl, 100mM NaCl, pH 7.3), 5U FokI endonuclease21mM dithiothreitol, pH 7.0), and incubating the reaction solution at 37 ℃ for 30 min. Then, 70. mu.L of LFokI reaction mixture and 10. mu.L of hemoglobin (10. mu.M) were added in CB2 buffer (25mM HEPES, 200mM NaCl, 50mM KCl, 0.025% Triton X-100, 150mM NH4Cl, pH 5.3) to a final volume of 190. mu.L, and left at room temperature in the dark for 30 min. Finally, 5. mu.L of 40mM ABTS and 5. mu.L of 40mM H2O2Adding the mixture to a final volume of 200 mu L, reacting at room temperature for 15min, testing by an iMark absorbance microplate reader, scanning the wavelength range of 400-500nm, and recording the absorbance at 415nm for analysis.
The test results are shown in FIG. 6, in which samples 1 to 6, Hg are present2+The concentrations are respectively: 0.2. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, sample C without added Hg2+The control group of (1). It can be seen that all samples except sample C are in Hg2+All showed lower absorbance before separation. However, Hg is being carried out2+After the enrichment step, the absorbance intensity of samples 1, 2 and 3 was almost the same as sample C, indicating that almost all Hg was present2+Trapped by Hg-MBs, Hg in the supernatant2+The concentration is close to zero; the absorbance of samples 4 and 5 showed a significant increase compared to the samples not treated with Hg-MBs. Sample 6, which has a lower absorbance through Hg2+No significant change was found after the accumulation step. These results suggest that Hg-MBs do have Hg2+Accumulation capacity ofSo as to contain Hg at a concentration of not more than 5 μ M2+Used as a separation probe in solution.
Example 5 specific detection of biosensors
(1) Biosensor and detection of Cd Using the invention2+The method of (1), detecting a series of divalent metal cations, including Mg2+、Ba2+、Ca2+、Mn2+、Ni2+、Co2+And Zn2+Absorbance intensity at 415nm, three gradients were set for each metal ion: 100nM, 1. mu.M and 10. mu.M;
(2) biosensor and detection of Hg using the present invention2+The method of (1), detecting a series of divalent metal cations, including Mg2+、Ba2+、Ca2+、Mn2+、Ni2+、Co2+And Zn2+Absorbance intensity at 415nm, three gradients were set for each metal ion: 0nM, 200nM, 1000 nM.
The results of the specificity test are shown in FIG. 7, where FIG. 7A shows the application of the biosensor of the present invention and the detection of Cd2+FIG. 7B shows the measurement of the absorbance intensity at 415nm of other divalent metal ions, using the biosensor of the present invention and the detection of Hg2+As can be seen from the results of measuring the absorbance intensity at 415nm of other divalent metal ions, when Hg-MBs were used as a separation probe and Cd-MBs were used as a discrimination probe, Cd was contained2+The response of the reaction solution was evident at a concentration of 100 nM. In addition, the concentration of other metal ions was increased to 10. mu.M, and no significant increase in absorbance was observed. In the direction of Hg2+During detection, Hg is contained2+The absorbance of the reaction solution is obviously reduced along with the increase of the concentration, and other metal ions do not influence the result even if the concentration is increased to 1000 nM.
Example 6
Verification of the biosensor of the invention in the presence of Hg2+Detection of Cd in Presence2+Accuracy and reliability of (2): all samples were divided into two groups. Group A is Cd of different concentrations2+Solution, group B, 50nM Hg was added to each sample in group A2+. All samples were assayed with Hg-MBs before testingTreated to remove Hg2+And (4) pollution. And then based on the calibration curve plotted in example 2 (fig. 3). Calculating Cd in each sample2+And (4) concentration. The results are shown in table 2:
TABLE 2
Theoretical t(5,95%)=2.571
The t-value did not exceed the theoretical value within the 95% confidence interval, which means that Cd was detected from both sets of samples, as compared by student-t test2+There were no statistically significant differences in concentration. The experimental results show that the biosensor designed by the invention can accurately detect Cd2+And can also be used for detecting Hg2+A contaminated sample.
Example 7
Application test:
tap water, pond water (the pond water is taken from Nanjing forestry university) and lake water (the lake water is taken from Nanjing basalt lake water sample) are taken as actual samples, and the biosensor provided by the invention is used for detecting and analyzing the actual samples. Filtering the water sample by using a 0.22 mu m filter membrane, and then adding Cd with different concentrations into the sample2+And Hg2+Finally, the biosensor designed by the invention is used for actual detection.
The experimental results show table 3:
TABLE 3
Remarking: in the table, a is tap water, b is pond water, c is basalt lake water, and the recovery rate is the ratio of the detected amount to the additive amount.
From Table 3 can be seenFor Cd out2+The average recovery rate of the three samples is 99.75-101.39%, and the relative standard deviation is lower than 2.60%; hg is a mercury vapor2+The average recovery rate of the three samples is 101.60-102.85%, and the relative standard deviation is lower than 3.16%. The experimental results show that whether the method is applied to Cd2+Also Hg2+In each water sample, the error of the adding standard recovery rate of the actual sample is within 10% through the designed biosensor, and the RSD is less than 5%, so that the method can be effectively applied to accurate detection of the actual sample.
Claims (4)
1. A biosensor for detecting cadmium ions is characterized by comprising magnetic bead probes Cd-MBs and magnetic bead probes Hg-MBs, wherein solid phase carriers of the magnetic bead probes Cd-MBs and the magnetic bead probes Hg-MBs are streptavidin modified nano magnetic beads, and the magnetic bead probes Cd-MBs are modified and connected with cadmium ion deoxynuclease sequences and substrate chain sequences which are in complementary pairing connection with the cadmium ion deoxynuclease sequences; a split G-quadruplex fragment AT11A with a Poly-T region is modified and connected on the magnetic bead probe Hg-MBs;
the cadmium ion deoxynuclease sequence is as follows: 5'-GTT CGC CAT CTT CCT TCG ATA GTT AAA ATA GTG CAT-3', respectively;
the substrate chain sequence is: 5 '-TTC GGA TGC ACT ATrA GGA AGA TGG CGA AC-biontin-3';
the sequence of the AT11A is: 5 '-biotin-A GGG ACG GGA TTT TTT TTT TTA GGG ACG GGA-3'.
2. The biosensor for detecting cadmium ions according to claim 1, wherein the magnetic bead probes Cd-MBs are prepared by a method comprising the steps of:
1) taking 40 mu L of 10mg/mL streptavidin coupled magnetic beads, washing with 1X PBST buffer solution, and then re-dissolving with 200 mu L PBST buffer solution to obtain MB solution;
2) adding 10 mu L of substrate chain and 10 mu L of cadmium ion deoxynuclease into 80 mu L of PBS buffer solution, incubating for 2min in a metal bath at 90 ℃, then slowly cooling to room temperature for annealing to obtain a mixed solution, adding 20 mu L of the mixed solution into 100 mu L of MB solution, and fully mixing at room temperature to obtain the magnetic bead probe Cd-MBs.
3. The biosensor for detecting cadmium ions according to claim 1 or 2, wherein the magnetic bead probes Hg-MBs are prepared by a method comprising:
1) taking 40 mu L of 10mg/mL streptavidin coupled magnetic beads, washing with 1X PBST buffer solution, and then re-dissolving with 200 mu L PBST buffer solution to obtain MB solution;
2) AT11A was diluted to a concentration of 10. mu.M in CB1 buffer solution and annealed, and then 20. mu.L of the solution was added to 100. mu.L of LMB solution and mixed well AT room temperature to obtain magnetic bead probes Hg-MBs.
4. A method for detecting cadmium ions is characterized by comprising the following steps:
(1) the biosensor of claim 1, 2 or 3, wherein 5 μ L of standard solution of cadmium ions with different concentrations and 20 μ L of magnetic bead probe Hg-MBs are mixed in CB1 buffer solution to a final volume of 30 μ L, the mixture is shaken at room temperature for incubation reaction, and then magnetic separation is carried out to obtain the product containing Cd with different concentrations2+The supernatant of (a);
(2) respectively adding 15 μ L of Cd with different concentrations2+Mixing the supernatant with 10 mu L of magnetic bead probe Cd-MBs in a reaction buffer solution until the final volume is 50 mu L, incubating and reacting at room temperature, and then carrying out magnetic separation on each solution to obtain a series of supernatants; the reaction buffer solution formula comprises: 50mM MES, 25mM NaCl, pH 6.0;
(3) taking 15 mu L of the supernatant in the step (2), respectively adding the supernatant into a 1X reaction buffer solution containing 200nM OD-2/OD-3 compound and 5U FokI endonuclease, and incubating the reaction solution at 37 ℃ for 30min to obtain a FokI reaction mixture;
the OD-2/OD-3 compound is obtained by annealing OD-2 and OD-3 fragments in a T-Na buffer solution; the oligonucleotide sequence of OD-2 is: 5'-AGA GGG ACG GGA AGG GAC GGGA-3', wherein the oligonucleotide sequence of OD-3 is: 5'-ATC CCG TCC CTC TTA TAG TGC ATC CGAA-3', respectively;
(4) will 7mu.L of LFokI reaction mixture and 10. mu.L of heme were mixed in CB2 buffer to a final volume of 190. mu.L, left at room temperature in the dark for 30min, and 5. mu.L of 40mM ABTS and 5. mu.L of 40mM H were added2O2Adding the mixture to a final volume of 200 mu L, reacting at room temperature for 15min, testing by using an iMark absorbance microplate reader, scanning the wavelength range of 400-500nm, recording the absorbance at 415nm, and drawing a standard curve of the concentration of cadmium ions and the absorbance;
(5) mixing 5 mu L of sample to be detected and 20 mu L of magnetic bead probe Hg-MBs in CB1 buffer solution until the final volume is 30 mu L, shaking at room temperature for incubation reaction, then carrying out magnetic separation to obtain supernatant, then carrying out the same treatment according to the steps (2) to (4), testing to obtain the absorbance at 415nm, substituting the absorbance into the standard curve of the cadmium ion concentration and the absorbance drawn in the step (4), and calculating to obtain the cadmium ion concentration in the sample to be detected.
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150031014A1 (en) * | 2013-07-25 | 2015-01-29 | The Board Of Trustees Of The University Of Illinois | Detecting analytes with a ph meter |
CN107966436A (en) * | 2017-10-27 | 2018-04-27 | 中国农业大学 | A kind of visible sensor of functional nucleic acid based on cadmium and its application |
CN108949934A (en) * | 2018-06-20 | 2018-12-07 | 中国农业大学 | A kind of general ultrafast amplification visible sensor of partition of chromium ion cutting-type |
CN110684789A (en) * | 2019-10-24 | 2020-01-14 | 南京林业大学 | Fusion gene, recombinant vector and preparation method thereof, cadmium ion whole-cell biosensor and preparation method and application thereof |
-
2021
- 2021-07-16 CN CN202110807990.XA patent/CN113584130A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150031014A1 (en) * | 2013-07-25 | 2015-01-29 | The Board Of Trustees Of The University Of Illinois | Detecting analytes with a ph meter |
CN107966436A (en) * | 2017-10-27 | 2018-04-27 | 中国农业大学 | A kind of visible sensor of functional nucleic acid based on cadmium and its application |
CN108949934A (en) * | 2018-06-20 | 2018-12-07 | 中国农业大学 | A kind of general ultrafast amplification visible sensor of partition of chromium ion cutting-type |
CN110684789A (en) * | 2019-10-24 | 2020-01-14 | 南京林业大学 | Fusion gene, recombinant vector and preparation method thereof, cadmium ion whole-cell biosensor and preparation method and application thereof |
Non-Patent Citations (3)
Title |
---|
LI D, ET AL: "Development of a DNAzyme-based colorimetric biosensor assay for dual detection of Cd2+ and Hg2", 《ANAL BIOANAL CHEM》, vol. 413, no. 28, XP037614950, DOI: 10.1007/s00216-021-03677-x * |
LI T, ET AL: "G-quadruplex-based DNAzyme for sensitive mercury detection with the naked eye", 《CHEM COMMUN (CAMB)》, no. 24, pages 1 - 3 * |
LU N ET AL: "Colorimetric Hg2+ detection with a label-free and fully DNA-structured sensor assembly incorporating G-quadruplex halves", 《ANALYST》, vol. 134, no. 9 * |
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