Lead ion visual detection method

A detection method and technology for lead ions are applied in the field of rapid visual detection of lead ions, which can solve the problems of interfering lead ion detection and low detection sensitivity, and achieve the effects of high sensitivity and rapid detection, high detection sensitivity, and improved use efficiency.

Inactive Publication Date: 2017-03-29
HUAZHONG UNIV OF SCI & TECH
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AI-Extracted Technical Summary

Problems solved by technology

[0005] Aiming at the above defects or improvement needs of the prior art, the present invention provides a rapid visual detection method for lead ions, the purpose of which is to treat DNase with graphene oxide, absorb unreacted free single-stranded nucleic acid, and combine double gold standard Probe detection technology improves the use efficiency of fragments prod...
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Method used

After two kinds of gold standard probes are sprayed on the gold standard pad, the utilization efficiency of fragment after GR-5 DNA enzyme digestion can be fully improved, and independent gold standard probe can only use the DNA fragment of part, on the other hand nucleic acid The sensitivity of hybridization also ensures the sensitivity of the system, so the method of using two gold standard probes improves the sensitivity of lead ion detection.
Because the concentration of the first gold standard probe that step (1) prepares is identical with the second gold standard probe, is 14nM, so the first gold standard probe and the second gold standard probe on the gold standard pad The molar ratio is 0.25-4:1, preferably 2:3. The bin...
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Abstract

The invention discloses a lead ion fast visual detection method. The method comprises: performing hybrid hybridization of fundamental chains and enzyme chains to form a GR-5 DNA enzyme; adding oxidized graphene for treatment to remove unreacted free single-stranded nucleic acid to eliminate the false positive effect; spraying two kinds of gold label probes on a gold label mat; and adding two chains which are obtained from the GR-5 DNA enzyme which is a pretreated to-be-detected sample through cutting at the rA position, wherein the two chains together with the gold label probes and nucleotide sequences located on a detection line form a sandwich structure through chromatography, so that the chains are fixed at the detection line for color development, and excessive free nucleic acid is fixed at a quality control line for color development. According to the intensity of color development on the detection line, the lead ion concentration is obtained. Lead ions can be detected in 15 min at the room temperature, and the minimum limit of detection of the lead ions can reach 0.05 nM. The method is high in sensitivity and good in specificity, is not affected by other divalent metal ions, is simple and easy to operate, and can be directly applied to detection of lead ions in the environment.

Application Domain

Material analysis

Technology Topic

Nucleic acid sequenceGraphene +12

Image

  • Lead ion visual detection method
  • Lead ion visual detection method
  • Lead ion visual detection method

Examples

  • Experimental program(5)

Example Embodiment

[0046] 1) Preparation of gold-labeled probe:
[0047] Preparation of the first gold-labeled probe: Add the sulfhydryl-modified first single-stranded DNA to the nano gold solution, react for 24 hours, then add phosphate buffer and sodium dodecyl sulfonate, mix at room temperature for 30 minutes, and add chlorination Sodium aging for 2 days at room temperature, centrifugation at 4°C for 25 min, discard the supernatant, and resuspend the pellet with resuspension buffer to obtain the first gold-labeled probe prepared;
[0048] Preparation of the second gold-labeled probe: Add the second single-stranded DNA modified with sulfhydryl group to the nano gold solution, react for 24 hours, then add phosphate buffer and sodium dodecyl sulfonate, mix at room temperature for 30 minutes, add chlorination Sodium aging for 2 days at room temperature, centrifugation at 4°C for 25 min, discard the supernatant, and resuspend the pellet with resuspension buffer to obtain the second gold-labeled probe prepared;
[0049] Among them, the nano-gold particle size in the nano-gold solution is 13-35nm, preferably 20nm. In general, the larger the nano-gold particle size, the higher the detection sensitivity, but non-specific adsorption will cause false positive results, and vice versa. , The smaller the particle size, the less likely to be false positives, but the sensitivity of detection will decrease. Preferably, 20nm nano-gold is used to prepare gold-labeled probes.
[0050] The resuspension buffer is 20mM Na, pH 7.4 3 PO 4 , 5% BSA, 0.25% Tween 20 and 10% sucrose mixed solution.
[0051] The concentration of the prepared first gold-labeled probe is 14 nmol/L, and the concentration of the prepared second gold-labeled probe is 14 nmol/L.
[0052] Spraying two gold-labeled probes on the gold-labeled pad can fully improve the use efficiency of GR-5 DNA enzymatic digestion fragments, while a single gold-labeled probe can only use part of the DNA fragments. On the other hand, the sensitivity of nucleic acid hybridization The sensitivity of the system is also guaranteed, so the method of using two gold standard probes improves the sensitivity of lead ion detection.
[0053] 2) Preparation of gold label pad: immerse the glass fiber membrane in gold label pad treatment buffer for 0.5h, then dry at 37°C for 1h, and then use a gold label sprayer to test the first gold label prepared in step 1) The needle and the second gold-labeled probe are fixed to the glass fiber membrane in a volume ratio of 0.25-4:1, preferably 2:3, and dried at 37°C for 1 hour to obtain a prepared gold-labeled pad;
[0054] Since the concentration of the first gold-labeled probe and the second gold-labeled probe prepared in step (1) are the same, both are 14 nM, the molar ratio of the first gold-labeled probe to the second gold-labeled probe on the gold-labeled pad is 0.25~4:1, preferably 2:3. The binding ability of the two gold-labeled probes and the single-stranded DNA digested by GR-5 DNA is different. Because the gold-labeled sequence is different, the binding ability is different. Optimize the ratio of the two gold-labeled probes to achieve the best binding efficiency, thereby improving sensitivity.
[0055] The spray gold concentration of the gold sprayer is preferably 4 μL/cm. The volume of the gold-labeled probe sprayed on the gold-labeled pad will affect the color depth of the test strip, and a too low probe concentration will reduce the sensitivity of the detection system. Too high a concentration will cause non-specific adsorption, which may bring a certain false positive.
[0056] The gold label pad treatment buffer is a mixed solution of pH 8.0 4% Triton, 1% BSA, 2% glucose, 2% PEG-4000, 100 mM boric acid and 0.1% SDS.
[0057] 3) Preparation of test line and quality control line: use a scribing instrument to scribble on the nitrocellulose membrane at an amount of 1μL/cm, fix the biotin probe 3 and biotin probe 4 on the test line, and the quality control line The biotin probe 5 and the biotin probe 6 are fixed, and the distance between the two lines is 3-6 mm, preferably 5 mm.
[0058] 4) Preparation of sample working solution: Dissolve the base strand GR-5S and GR-5E of GR-5 DNA enzyme in a buffer solution in a water bath at 95°C for 5 minutes, slowly cool to room temperature, add graphene oxide solution and mix well. The reaction is carried out for 0.5-2h, preferably 1h, the graphene oxide is removed by centrifugation, and the supernatant is taken as the sample working solution.
[0059] The sample working solution is prepared by using a single-stranded DNA adsorbent to adsorb the free single strands of the GR-5 DNA enzyme base chain GR-5S and the enzyme chain GR-5E after the reaction is complete, to eliminate the free single strands in the lead ion analysis process False positive interference generated in The single-stranded DNA adsorbent is nano gold or graphene oxide, preferably graphene oxide.
[0060] The mass concentration ratio of the base chain GR-5S, the enzyme chain GR-5E and the graphene oxide of the GR-5 DNA enzyme is 1:0.3-2: 16.6-83.2, preferably 1:1.5:52. The excess of one of the single-stranded DNA in the base strand and the enzyme strand can maximize the formation of double strands. Under the optimal adsorption time, the unbound free single strands will be preferentially bound by graphene oxide, GR-5 DNA enzyme The double strands are adsorbed to a minimum, so that the sensitivity of detection will not be reduced.
[0061] The amount of graphene oxide solution added should not be too large or too small, and the reaction time after adding graphene oxide should not be too long or too short. If the added amount is too small or the reaction time is too short, the single chain adsorption of graphene oxide will not be complete. If the amount is too large or the reaction time is too long, the extra single-stranded DNA of the GR-5 DNase base strand GR-5S will also be adsorbed by graphene oxide, resulting in a decrease in the concentration of GR-5 DNase, which will affect the subsequent detection. Sensitivity. Therefore, the present invention is optimized through repeated trials, and the mass concentration ratio of the base chain GR-5S, the enzyme chain GR-5E and graphene oxide of the GR-5 DNA enzyme is 1:0.3-2:16.6-83.2, preferably 1 : 1.5:52, and found that after adding graphene oxide, the adsorption single-stranded reaction time is 0.5-2h, preferably 0.5-1h, more preferably 1h.
[0062] The centrifugal rotation speed is not less than 10000 rpm, and the centrifugation time is not less than 10 min. The removal of graphene oxide is very important for the subsequent lead ion detection process, so it is necessary to ensure the rotation speed and time during centrifugal separation so that the graphene oxide can be completely removed and avoid affecting the subsequent detection work.
[0063] The buffer solution is PBS, HEPES, Tris-Ac or sodium citrate buffer solution, preferably sodium citrate buffer solution. The concentration of the sodium citrate buffer solution is preferably a 5X sodium citrate buffer solution. Sodium citrate buffer solution is often used as a buffer for molecular hybridization. The sodium citrate buffer contains a large amount of sodium and chloride ions, which can reduce the repulsion between single-stranded DNA molecules and make it easier for single-strands to hybridize into double-strands. Other buffers Although there are sodium ions and chloride ions (that are ionized by sodium chloride), experimental results show that sodium citrate buffer is better than other buffers.
[0064] 5) Analysis of the sample to be tested: Take the sample working solution obtained in step 4) and add the sample to be tested. The volume of the sample to be tested is one-tenth of the sample working solution to obtain the sample to be tested. Drop the test liquid on the sample pad, chromatographically react for 5 minutes, and add 5×SSC to the sample pad to clean the test strip. After 15 minutes, a red strip can be seen on the test line and quality control line of the test strip. The paper strip image analyzer analyzes the gray value and calculates the concentration of lead ions in the sample to be tested through the standard curve.

Example Embodiment

[0066] Example 1
[0067] A rapid visual detection method for lead ions includes the following steps:
[0068] (1) Preparation of nano gold solution:
[0069] Put 100mL HAuCl 4 The aqueous solution (0.01%) was poured into a round bottom flask equipped with a reflux condenser and heated to boiling, and then 2 mL of trisodium citrate solution (1%) was added to the flask. Continue heating to reflux under strong magnetic stirring. After the color of the solution changes from colorless and black to dark red, continue heating for 10 minutes, stop heating, cool to room temperature, filter with 0.22μm nylon membrane to remove large particles, and store it at temperature Keep in the refrigerator at 4°C.
[0070] (2) Preparation of gold-labeled probe:
[0071] Add 45μL of sulfhydryl modified first single-stranded DNA and second single-stranded DNA (100μM, 1OD) into 14mL nano-gold solution, react for 24h, add 100mmol/L PB (phosphate buffer) and 10% SDS ( Sodium dodecyl sulfate) to make the final concentration of PB and SDS be 9mmol/L and 0.1%, respectively, the shaker was oscillated at room temperature for 30min, 2M sodium chloride was added to make the final concentration 0.2M, and the final concentration was at room temperature for 2 days. Centrifuge at 4°C for 25 min (12000 rpm), discard the supernatant to remove the unmodified DNA probe, and resuspend the red precipitate with 1 mL of resuspension buffer. This process was repeated three times. Finally, it was resuspended with 700μL of resuspension buffer to obtain the first gold-labeled probe with a concentration of 14nM and the second gold-labeled probe with a concentration of 14nM, which were stored at 4°C for later use.
[0072] (3) Preparation of gold label pad:
[0073] Cut the glass fiber membrane of appropriate size, and then use gold label pad to process the buffer (4% Triton, 1% BSA, 2% glucose, 2% PEG-4000, 100 mM boric acid, 0.1% SDS, pH 8.0) soaking treatment, 37 ℃ Dry for 1h. Take the gold-labeled probe 1 with a concentration of 14nM and the gold-labeled probe 2 with a concentration of 14nM, spray gold with a film sprayer at a volume of 4μL/cm, dry at 37℃ for 1h, store at 4℃, the first gold The volume ratio of the standard probe to the second gold standard probe is 2:3.
[0074] (4) Preparation of test line and quality control line:
[0075] Take 20μL of streptavidin (1mg/ml), 15μL of biotin probe 3 (100μM) and 15μL of biotin probe 4 (100μM), mix the three evenly, react for 2h, and use for streaking to the detection line; Also take 20μL of streptavidin (1mg/ml), 15μL of biotin probe 5 (100μM) and 15μL of biotin probe 6 (100μM), mix the three evenly, react for 2h, and use for marking to the quality control line on. Then use a gold spray scribing machine to scribe and fix the nitrocellulose membrane at an amount of 1 μL/cm, with a distance of 5 mm between the two lines, dry at room temperature for 12 hours, and store at 4°C.
[0076] (5) Preparation of sample loading working solution:
[0077] The basal chain GR-5S and the enzyme chain GR-5E of GR-5 DNase are dissolved in 5×SSC (pH 7.4, sodium citrate buffer) to make the final concentration of 100nM (1.2μg/mL) and 150nM (1.8μg) respectively. /mL), 95℃ water bath for 5min, slowly cooling to room temperature, 3h in total. Add 2mg/mL graphene oxide solution to the above solution to make the final concentration 62.5μg/mL, react for 1h, adsorb unreacted free single-stranded nucleic acid, centrifuge at 10500rpm for 10min to remove graphene oxide, take the supernatant as the upper Sample working fluid.
[0078] (6) Drawing of standard curve:
[0079] Prepare 13 standard lead ion concentration gradient standard solutions, the concentrations are 0mol/L, 5pmol/L, 10pmol/L, 50pmol/L, 100pmol/L, 500pmol/L, 1nmol/L, 5nmol/L, 10nmol/ L, 100nmol/L, 1μmol/L, 10μmol/L, 100μmol/L.
[0080] Add 13 parts of lead ion standard solutions of different concentrations to the sample working solution, mix well and react for 1 hour, drop 100 μL of the sample solution on the sample pad, chromatographically react for 5 minutes, add 5×SSC to the sample pad to clean the remaining gold standard Free gold-labeled probes and single-stranded DNA on the pad and nitrocellulose membrane. After 15 minutes, red bands can be seen on the detection line and quality control line of the test strip. The intensity of the strip is analyzed by the test strip image analyzer Gray value (normalized intensity of the band).
[0081] image 3 A shows that as the lead ion concentration increases, the normalized intensity of the band increases. And when the lead ion concentration is 10nmol/L~100μmol/L, the lead ion concentration has a good linear relationship with the normalized intensity of the band; image 3 B shows the physical picture when the lead ion concentration is 10nmol/L~100μmol/L; image 3 C is the detection signal intensity of low-concentration lead ions. The lowest detection limit (LOD) of the test strip is obtained by adding 3 times the standard deviation of the normalized intensity of the blank response to the average value of the normalized intensity of the blank response. The minimum detection limit of lead ion is 0.05nM.
[0082] (7) Detection of samples to be tested
[0083] Add an excessive amount of concentrated nitric acid solution to the soil sample to be tested, stir and heat to above 90°C without boiling, so that all the sample to be tested is dissolved and oxidized by concentrated nitric acid to obtain a nitrated solution; then, let the solution cool to 60°C Next, add excess hydrogen peroxide slowly, stir and heat to above 90℃ but boiling until no bubbles are generated; then cool the resulting solution to below 60℃, add excess hydrochloric acid solution, stir and heat without boiling, keep it for 10 minutes ~1h; After cooling to room temperature, filter the obtained solution, evaporate and concentrate, and then make the volume with buffer to obtain the sample to be tested after pretreatment. After the sample to be tested is diluted 100 times, the concentration of lead ion is measured according to the above method 19.1 nM.

Example Embodiment

[0084] Example 2
[0085] The invention provides a lead ion gold standard chromatography test strip, such as figure 2 As shown, it includes a sample pad, a gold label pad, a nitrocellulose membrane and absorbent paper which are sequentially mounted on a PVC bottom plate. The gold label pad is fixed with the first gold label probe ( figure 2 The gold-labeled probe 1) and the second gold-labeled probe ( figure 2 The gold standard probe 2); there are detection lines and quality control lines on the nitrocellulose membrane, and the third biotin probe ( figure 2 Biotin probe 3) and the fourth biotin probe ( figure 2 The biotin probe 4), the fifth biotin probe ( figure 2 Biotin probe 5) and the sixth biotin probe ( figure 2 The biotin probe in 6). The base chain GR-5S and the enzyme chain GR-5E undergo complementary pairing, the excess free single chains are adsorbed by graphene oxide, and lead ions are added to the sample pad as a loading solution.
[0086] The basal strand of GR-5 DNA enzyme (GR-5S), we call it sequence 1: 5'-GGTCTCACTATrAGGAAGAGATGATGTCTGTCAGATGTAG-3', part of this sequence is complementary to the sequence of gold label probe 1 and the other is complementary to the sequence of biotin probe 3 , To form a sandwich structure for detecting color development on the line.
[0087] The enzyme chain of GR5-DNA enzyme (GR-5E), we call it sequence 2: 5'-GACATCATCTCTGAAGTAGCGCCGCCGTATAGTGAGACC-3', part of this sequence is complementary to the sequence of gold label probe 2 and part of it is complementary to the sequence of biotin probe 4. A sandwich structure is formed to detect color development on the wire.
[0088] The first single-stranded DNA used to prepare the first gold-labeled probe: 5'-GACATCATCTCT-SH-3' (SH is the 3'end of the DNA sequence modified with a sulfhydryl group), which is 5'-AGAGATGATGTC-3' of the base strand This part of the sequence is complementary.
[0089] The second single-stranded DNA used to prepare the second gold-labeled probe: 5'-SH-GGTCTCACTATA-3' (SH is the 5'end of the DNA sequence is modified with a sulfhydryl group), which is the same as the 5'-TATAGTGAGACC-3' of the enzyme chain This part of the sequence is complementary.
[0090] Biotin probe 3: 5'-biotin-AAAAAACTACATCTGACA-3' (biotin is the 5'end of the DNA sequence is modified with biotin), which is complementary to this part of the 5'-TGTCAGATGTAG-3' sequence of the base strand.
[0091] Biotin probe 4: 5'-CGGCGCTACTTCAAAAAA-biotin-3' (biotin is the 3'end of the DNA sequence modified with a sulfhydryl group), which is complementary to this part of the 5'-GAAGTAGCGCCG-3' sequence of the enzyme chain.
[0092] Biotin probe 5: 5'-AGAGATGATGTCAAAAAA-biotin-3' (biotin is the 3'end of the DNA sequence modified with biotin), which is complementary to the sequence of the gold label probe 1.
[0093] Biotin probe 6: 5'-biotin-AAAAAATATAGTGAGACC-3' (biotin is the 5'end of the DNA sequence modified with biotin), which is complementary to the sequence of the gold label probe 2.

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Size13.0 ~ 35.0nm

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