CN107142311B - Method for detecting DNA by chemiluminescence technology - Google Patents
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Abstract
The invention belongs to the field of analytical chemistry, and particularly relates to a method for detecting DNA by a chemiluminescence technology, which adopts the principle that luminol is used for reducing chloroauric acid to obtain luminol colloidal gold nanoparticles LumAuNPs, and probe DNA is used for modifying the LumAuNPs to obtain a chemiluminescent probe; then fixing the capture DNA of the hairpin structure by taking the magnetic bead as a carrier, opening the hairpin structure for capturing the DNA when the target DNA exists, and connecting the chemiluminescent probe to the surface of the magnetic bead by a catalytic hairpin self-assembly technology under the hybridization action of the probe DNA on the chemiluminescent probe; and after magnetic separation, adding hydroxylamine-O-sulfonic acid, taking LumAuNPs-hydroxylamine-O-sulfonic acid as a chemiluminescence system, performing chemiluminescence measurement, and realizing the measurement of target DNA according to the generated chemiluminescence. The method has the advantages of simplicity and high sensitivity.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a method for detecting DNA by a chemiluminescence technology.
Background
Luminol is a commonly used chemiluminescence reagent and is widely applied to various chemiluminescence analysis and detection. However, luminol chemiluminescence produces a high background signal and is therefore limited in its application. In recent years, functionalized colloidal gold has received attention from researchers, and gold can be synthesized by reducing chloroauric acid using ruthenium complex, luminol and its derivatives (Cui H, Wang W, Duan C F, et al Synthesis, chromatography, and electrochemical of luminescent-reduced gold nanoparticles and the reaction in a hydrogen peroxide sensor [ J ]. Chemistry,2007,13(24): 6984; Gao W, Qi W, Lai J, et al. thiourea as a crude-inorganic complex for luminescent in reaction [ J ], and gold by reducing the signal obtained by reacting with hydrochloric acid [ 7 ] and by reacting with hydrochloric acid [ 12 ] wherein the gold is available as luminescent in the luminescent in the luminescent in. Hydrogen peroxide is not selective because it is unstable and can react with many metal ions. In view of the defects of the prior art, the invention realizes the determination of DNA by utilizing a LumAuNPs-hydroxylamine-O-sulfonic acid chemiluminescence detection system, and has the characteristics of high sensitivity, simple method and the like.
Disclosure of Invention
The invention aims to invent a method for measuring DNA with simple method and high sensitivity.
In view of the shortcomings of the prior art, the present invention aims to provide a method for detecting DNA by chemiluminescence technology.
The technical scheme for realizing the aim of the invention is as follows:
a method for detecting DNA by a chemiluminescence technology is characterized in that luminol is used for reducing chloroauric acid to obtain luminol colloidal gold nanoparticles LumAuNPs, and probe DNA is used for modifying the LumAuNPs to obtain a chemiluminescence probe; then fixing the capture DNA of the hairpin structure by taking the magnetic bead as a carrier, opening the hairpin structure for capturing the DNA when the target DNA exists, and connecting the chemiluminescent probe to the surface of the magnetic bead by a catalytic hairpin self-assembly technology under the hybridization action of the probe DNA on the chemiluminescent probe; and after magnetic separation, adding hydroxylamine-O-sulfonic acid, taking LumAuNPs-hydroxylamine-O-sulfonic acid as a chemiluminescence system, performing chemiluminescence measurement, and realizing the measurement of target DNA according to the generated chemiluminescence.
The invention is realized by the following measures: a method for detecting DNA by a chemiluminescence technology is characterized by comprising the following steps:
(1) preparing luminol colloidal gold nanoparticles;
(2) preparing capture DNA modified magnetic beads;
(3) preparing probe DNA modified LumAuNPs;
(4) and (4) detecting the target DNA.
Preferably, the preparation of the luminol colloidal gold nanoparticles comprises the following steps:
before the start of the experiment, the used glass apparatus was subjected to HNO3Soaking in aqua regia of HCl (3:1, v/v) for 24h, washing with secondary distilled water, and oven drying. Diluting a certain amount of 1% chloroauric acid solution with deionized water to obtain a 0.02% chloroauric acid solution, placing the chloroauric acid solution in a three-neck flask, and heating, refluxing and boiling under magnetic stirring; and after the solution is boiled, quickly adding 0.01-5 mL of 0.01M luminol solution, continuously heating and boiling, changing the color of the solution from light yellow to black, finally changing the solution into wine red, stopping heating after 40min, continuously stirring and cooling to room temperature to obtain luminol colloidal gold nanoparticles, namely LumAuNPs, transferring the prepared LumAuNPs into a brown wide-mouth bottle, and storing at 4 ℃ for later use.
Preferably, the preparation of the capture DNA modified magnetic beads comprises the following steps:
putting 10-100 mu L of carboxylated magnetic bead solution into a 1.5mL centrifuge tube, washing the solution for three times by using 10-200 mu L of 0.1M imidazole buffer solution, dispersing the solution into 0.01-2 mL of 0.1M imidazole buffer solution containing 0.1M EDC and 0.05M NHS, carrying out oscillation reaction for 30min at 37 ℃, and then adding 10-200 mu L of 5.0 × 10- 8M capture DNA, at 37 ℃ overnight oscillation, get capture DNA modified magnetic beads, then use 2.0mL 0.1M PBS buffer solution to wash three times, finally disperse into 2.0mL PBS buffer solution, 4 ℃ storage.
Preferably, the preparation of the probe DNA modified LumAuNPs comprises the following steps:
adding 1-20 microliter TCEP to 10-200 microliter TCEP with concentration of 1.0 × 10-6M probe DNA solution, at 37 ℃ for 1 hour oscillation activation, then 100-1000 mul of synthesized LumAuNPs are added into the solution, oscillation is carried out at 37 ℃ for overnight, then 10 mul-200 mul of 10mM Tris-HCl buffer solution containing 0.3M NaCl and pH 8.2 is added; after further shaking for 48h, centrifuging at 12000rpm for 30min, washing the red precipitate with 1mL of 0.1M PBS buffer solution with pH7.4, centrifuging again, and repeating the steps three times to obtain probe DNA modified LumAUNPs, namely the chemiluminescent probe. Dispersing the obtained chemiluminescent probe into 1000 μ L of 0.1M PBS buffer solution with pH7.4, and storing at 4 deg.C。
Preferably, the detection of the target DNA comprises the steps of:
putting 10-200 mu L of capture DNA modified magnetic bead solution into a centrifuge tube, then adding 10-100 mu L of solution containing target DNA into the centrifuge tube, carrying out oscillation reaction for 40min at 37 ℃, then adding 10-100 mu L of probe DNA modified LumAuNPs solution, carrying out oscillation reaction for 40min at 37 ℃, and connecting a chemiluminescent probe on the surface of the magnetic bead through the action of the target DNA and the capture DNA and the action of the probe DNA and the target DNA and the capture DNA; after magnetic separation, the magnetic isolate was dispersed in 50 μ L of 0.1M PBS buffer solution at pH7.4, then hydroxylamine-O-sulfonic acid solution was added to generate chemiluminescence, and the target DNA was determined quantitatively according to the chemiluminescence intensity.
The DNA sequence is as follows:
capturing DNA: 5' -ATATACGCCATGTAGCATTCGGT TAGG CGTAT ATT TG C TT-NH2-3`；
Target DNA: 5 '-AATATACGCCTAACCG-3';
probe DNA: 5' -SH-TATA CGCCTAACCGAATGCT TACCACGCGTAT AG C A T CCGA-3 ″
The invention researches the relation between target DNA with different concentrations and chemiluminescence intensity, and obtains a standard curve, a linear range and a linear equation of the target DNA to be detected.
Advantages and effects of the invention
When the concentration of the target DNA is between 80pM and 10nM, there is a significant change in the chemiluminescence intensity with changes in the target DNA concentration. The nonlinear equation of the target DNA to be detected was calculated as y 3663.39648+332.34823x (y: chemiluminescence intensity; x: logarithm of target DNA concentration in M), the linear correlation coefficient was 0.9987, and the detection limit was 30pM (3 σ) (fig. 2). The precision of the determination method is calculated by 11 times of parallel determination of target DNA with the concentration of 500pM, the relative standard deviation is 3.6 percent, and the determination method has better reproducibility.
In addition, LumAuNPs-H is utilized2O2For detecting the system, the target DNA is subjected to the method of the present invention in the same manner as the other stepsThe detection is carried out, and the detection limit of the measurement is 500 pM. The method for detecting DNA by using the chemiluminescence technology has high sensitivity.
Drawings
FIG. 1 is a schematic diagram of the principle of detecting a target DNA.
FIG. 2 is a graph showing the relationship between the concentration of a target DNA and the intensity of chemiluminescence.
Detailed Description
The following examples further illustrate the method of operation of the present invention, but are not to be construed as further limiting the invention.
Example 1: method for detecting DNA by chemiluminescence technology
1. Experimental part
1.1 instruments and reagents
1.1.1 instrumentation
DHG forced air drying ovens (shanghai, charm instruments ltd); model AR224CN aohaus analytical balance (qingdao and zengxin electronics limited, qingdao); THZ type constant temperature oscillating box (haoyuxin technologies ltd., beijing); RFL-1 type ultra-weak chemiluminescence detector (Raymei analytical instruments, Inc., Xian); an ke-TGL-16C Fei Weng brand high speed centrifuge (an pavilion scientific instruments factory, Shanghai).
1.1.2 reagents
1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl)4) Purchased from Sigma company; the carboxyl magnetic beads with the particle size of 0.5 mu m and the concentration of 10mg/mL are purchased from the Tianjin BeisLe chromatographic technology development center; luminol (Luminol), hydroxylamine-O-sulfonic acid (HOSA), and TCEP (tris (2-carboxyethyl) phosphine hydrochloride) were purchased from Aladdin; 0.01M luminol was dissolved in 0.1M NaOH and stored in a brown bottle in a refrigerator at 4 ℃; 1g of chloroauric acid is added with 100mL of water to prepare a 1% chloroauric acid solution, the chloroauric acid solution is stored in a brown bottle, and the chloroauric acid solution is diluted by secondary distilled water before use.
PBS buffer solution is 0.10M, pH7.4, and is prepared by weighing 0.1g KH2PO4、4.0g NaCl、1.45gNa2HPO4·12H2Dissolving O and 0.1g KCl in 1L water to obtain the final product.
The sequences of the single-stranded DNA and hairpin DNA (synthesized by Shanghai Biotechnology, Ltd.) used in this experiment were as follows:
capturing DNA: 5' -ATATACGCCATGTAGCATTCGGT TAGG CGTAT ATT TG C TT-NH2-3`；
Target DNA: 5 '-AATATACGCCTAACCG-3';
probe DNA: 5 '-SH-TATA CGCCTAACCGAATGCT TACCACGCGTAT AG C A T CCGA-3'.
The DNA of the hairpin structure is used after incubation treatment.
1.2 Synthesis of LumAuNPs
Before the experiment, all glassware used was HNO3Soaking in aqua regia of HCl (3:1, v/v) for 24h, washing with secondary distilled water, and oven drying. 100 mu.L of 1% chloroauric acid solution is diluted with deionized water to 50mL of 0.02% chloroauric acid solution and placed in a three-neck flask, a magneton is added in the three-neck flask and placed in a magnetic stirrer, and the mixture is heated under reflux and boiled under magnetic stirring. After the solution is boiled, 1mL of 0.01M luminol solution is rapidly added, the solution is continuously heated and boiled for 40min, the color of the solution is changed from light yellow to black and finally to wine red, the heating is stopped after 40min, and the solution is cooled to room temperature under the condition of continuous stirring. The LumAuNPs obtained were transferred to a brown jar and stored at 4 ℃ for further use.
1.3 preparation of magnetic beads modified with Capture DNA
Adding 50 μ L of carboxylated magnetic bead solution into 1.5mL centrifuge tube, washing with 100 μ L of 0.1M imidazole buffer solution for three times, dispersing into 1mL of 0.1M imidazole buffer solution containing 0.1M EDC and 0.05M NHS, reacting at 37 deg.C for 30min with shaking, adding 100 μ L of 5.0 × 10-8M capture DNA, at 37 ℃ oscillation overnight, get capture DNA modified magnetic beads, then use 2.0mL 0.1M PBS buffer solution to wash three times, finally disperse into 2.0mLPBS buffer solution, 4 ℃ storage.
1.4 preparation of Probe DNA modified LumAuNPs
5 μ L of TCEP was added to 100 μ L of 1.0 × 10-6M in the probe DNA solution, activated by shaking at 37 ℃ for 1 hour, andadding 600 μ L of the synthesized LumAuNPs into the solution, shaking overnight at 37 ℃, and then adding 50 μ L of 10mM Tris-HCl buffer solution containing 0.3M NaCl and pH 8.2; after further shaking for 48h, centrifuging at 12000rpm for 30min, washing the red precipitate with 1mL of 0.1M PBS buffer solution with pH7.4, centrifuging again, and repeating the steps three times to obtain probe DNA modified LumAUNPs, namely the chemiluminescent probe. The resulting chemiluminescent probe was dispersed in 1000. mu.L of 0.1M PBS buffer pH7.4 and stored at 4 ℃ until needed.
1.5 detection of target DNA
Putting 50 mu L of capture DNA modified magnetic bead solution into a centrifuge tube, then adding 50 mu L of solution containing target DNA into the centrifuge tube, carrying out oscillation reaction at 37 ℃ for 40min, then adding 50 mu L of probe DNA modified LumAUNPs solution, carrying out oscillation reaction at 37 ℃ for 40min, and connecting a chemiluminescent probe to the surface of the magnetic bead through the action of the target DNA and the capture DNA and the action of the probe DNA and the target DNA and the capture DNA; after magnetic separation, the magnetic isolate was dispersed in 50 μ L of 0.1MPBS buffer solution at pH7.4, followed by addition of hydroxylamine-O-sulfonic acid solution to produce chemiluminescence. And (4) drawing a standard curve according to the relation between the concentration of the standard solution and the chemiluminescence intensity.
Example 2: sample analysis
The sample solution containing the target DNA was subjected to the experiment according to the method of step 1.5 in example 1, and the target DNA content was obtained from the chemiluminescence intensity and the standard curve obtained in step 1.5 in example 1.
The content of the target DNA is measured according to the method, the method is evaluated by adopting a standard addition method, the measured recovery rate of the sample is 96.00-102.2%, the measurement result is shown in table 1, and the method has the characteristic of high precision in the detection of the target DNA.
TABLE 1 analysis of samples
| Numbering | Content (wt.)a,b | Standard addition of | Measured quantity | Recovery rate |
| 1 | 1.22 | 1.00 | 2.18 | 96.0％ |
| 2 | 3.59 | 5.00 | 8.67 | 103.4％ |
| 3 | 4.70 | 5.00 | 9.81 | 102.2％ |
aResults of 7 measurements
bUnit: nM.
SEQUENCE LISTING
<110> Qingdao university of science and technology
<120> a method for detecting DNA by chemiluminescence technology
<160>3
<170>PatentIn version 3.3
<210>1
<211>40
<212>DNA
<213> Artificial sequence
<400>1
ATATACGCCA TGTAGCATTC GGTTAGGCGT ATATTTGCTT 40
<210>2
<211>16
<212>DNA
<213> Artificial series
<400>2
AATATACGCC TAACCG 16
<210>3
<211>41
<212>DNA
<213> Artificial sequence
<400>1
TATACGCCTA ACCGAATGCT TACCACGCGT ATAGCATCCG A 41
Claims (3)
1. A method for detecting DNA by a chemiluminescence technology is characterized in that luminol is used for reducing chloroauric acid to obtain luminol colloidal gold nanoparticles LumAuNPs, and probe DNA is used for modifying the LumAuNPs to obtain a chemiluminescence probe; then fixing the capture DNA of the hairpin structure by taking the magnetic bead as a carrier, opening the hairpin structure for capturing the DNA when the target DNA exists, and connecting the chemiluminescent probe to the surface of the magnetic bead by a catalytic hairpin self-assembly technology under the hybridization action of the probe DNA on the chemiluminescent probe; after magnetic separation, adding hydroxylamine-O-sulfonic acid, taking LumAuNPs-hydroxylamine-O-sulfonic acid as a chemiluminescence system to perform chemiluminescence measurement, and realizing the measurement of target DNA according to the generated chemiluminescence, wherein the method comprises the following specific steps:
(1) preparing luminol colloidal gold nanoparticles: before the experiment is started, soaking a used glass instrument in aqua regia for 24 hours, washing with secondary distilled water, and putting into an oven for drying; diluting a certain amount of 1% chloroauric acid solution with deionized water to obtain a 0.02% chloroauric acid solution, placing the chloroauric acid solution in a three-neck flask, and heating, refluxing and boiling under magnetic stirring; after the solution is boiled, 0.01-5 mL of 0.01M luminol solution is quickly added, the solution is continuously heated and boiled until the color of the solution is changed from light yellow to black and finally to wine red, the heating is stopped after 40min, the solution is continuously stirred and cooled to room temperature to obtain luminol colloidal gold nanoparticles, namely LumAuNPs, the prepared LumAuNPs are transferred into a brown wide-mouth bottle, and the solution is stored for later use at 4 ℃;
(2) preparing the capture DNA modified magnetic beads, namely putting 10 mu L-100 mu L of carboxylated magnetic bead solution into a 1.5mL centrifuge tube, washing the solution for three times by using 10 mu L-200 mu L of 0.1M imidazole buffer solution, then dispersing the solution into 0.01 mL-2 mL of 0.1M imidazole buffer solution containing 0.1M EDC and 0.05M NHS, carrying out oscillation reaction for 30min at the temperature of 37 ℃, and then adding 10 mu L-200 mu L of 5.0 × 10-8M captures DNA, oscillates overnight at 37 ℃ to obtain captured DNA modified magnetic beads, then is washed three times by 2.0mL of 0.1M PBS buffer solution, and finally is dispersed into 2.0mL of PBS buffer solution and is stored at 4 ℃;
(3) the preparation of probe DNA modified LumAuNPs is carried out by adding 1-20 microliter TCEP to 10 microliter-200 microliter concentration of 1.0 × 10-6M probe DNA solution, at 37 ℃ for 1 hour of oscillation activation, then 100 to 1000 mul of synthesized LumAuNPs are added into the solution, oscillation is carried out at 37 ℃ for overnight, then 10 to 200 mul of 10mM Tris-HCl buffer solution containing 0.3M NaCl and pH 8.2 is added; continuing shaking for 48h, centrifuging for 30min at 12000rpm, washing the red precipitate with 1mL of 0.1M PBS buffer solution with pH7.4, centrifuging again, and repeating the steps for three times to obtain probe DNA modified LumAUNPs, namely a chemiluminescent probe; dispersing the obtained chemiluminescence probe into 1000 μ L of 0.1M PBS buffer solution with pH7.4, and storing at 4 deg.C for use;
(4) detection of target DNA: putting 10-200 mu L of capture DNA modified magnetic bead solution into a centrifuge tube, then adding 10-100 mu L of solution containing target DNA into the centrifuge tube, carrying out oscillation reaction for 40min at 37 ℃, then adding 10-100 mu L of probe DNA modified LumAuNPs solution, carrying out oscillation reaction for 40min at 37 ℃, and connecting a chemiluminescent probe on the surface of the magnetic bead through the action of the target DNA and the capture DNA and the action of the probe DNA and the target DNA and the capture DNA; after magnetic separation, the magnetic isolate is dispersed in 50 μ L of PBS buffer solution with pH 7.40.1M, then hydroxylamine-O-sulfonic acid solution is added to generate chemiluminescence, and the determination of target DNA is realized according to the quantification of chemiluminescence intensity.
2. A method for detecting DNA by chemiluminescence according to claim 1, wherein the DNA sequence is as follows:
the DNA sequence is as follows:
capturing DNA: 5' -ATATACGCCATGTAGCATTCGGT TAGG CGTAT ATT TG C TT-NH2-3`；
Target DNA: 5 '-AATATACGCCTAACCG-3';
probe DNA: 5 '-SH-TATA CGCCTAACCGAATGCT TACCACGCGTAT AG C A T CCGA-3'.
3. The method for detecting DNA according to claim 1, wherein the DNA is synthesized by Shanghai Bioengineering Co., Ltd.
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| CN103592291A (en) * | 2013-10-09 | 2014-02-19 | 青岛科技大学 | Method for measuring abscisic acid based on nano-gold marking and tyramine signal amplifying technology |
| CN103674935A (en) * | 2013-12-05 | 2014-03-26 | 青岛科技大学 | Method for determining gibberellin based on hybridization chain-reaction signal amplification technology |
| CN103954611A (en) * | 2014-04-09 | 2014-07-30 | 南昌大学 | Method for gold nanoparticle chemiluminiscence amplified detection of adenosine based on aptamer technology |
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| CN103592291A (en) * | 2013-10-09 | 2014-02-19 | 青岛科技大学 | Method for measuring abscisic acid based on nano-gold marking and tyramine signal amplifying technology |
| CN103674935A (en) * | 2013-12-05 | 2014-03-26 | 青岛科技大学 | Method for determining gibberellin based on hybridization chain-reaction signal amplification technology |
| CN103954611A (en) * | 2014-04-09 | 2014-07-30 | 南昌大学 | Method for gold nanoparticle chemiluminiscence amplified detection of adenosine based on aptamer technology |
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