CN105548108A - PSA ultrasensitive detection method based on fluorescent signal of gold nanorod core-quantum dot satellite-like nanostructure assembly - Google Patents
PSA ultrasensitive detection method based on fluorescent signal of gold nanorod core-quantum dot satellite-like nanostructure assembly Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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Abstract
A PSA ultrasensitive detection method based on a fluorescent signal of a gold nanorod core-quantum dot satellite-like nanostructure assembly, and belongs to the technical field of chemical analysis. The present invention includes the synthesis of gold nanorod; coupling of gold nanorod with a PSA aptamer; coupling of quantum dots with a partially complementary sequence of the PSA aptamer; assembly of a gold nanorod-quantum dot satellite nanostructure; and characterization and measurement of TEM and fluorescent signals. The different degrees of dissociation of different concentrations of PSA on the assembly affect the strength of the fluorescence signals, so a relationship curve between the PSA concentration and assembly fluorescence signal is established to achieve the quantitative detection of PSA. The method uses PSA aptamer and partially complementary sequence thereof to conduct satellite-like assembly of gold nanorod and quantum dots, and uses fluorescent signals to achieve ultrasensitive detection of PSA concentration.
Description
Technical field
The present invention relates to a kind of method based on gold nanorods core-quantum dot satellite shape nanostructured assembling body fluorescence signal super sensitivity detection PSA, belong to technical field of analytical chemistry.
Background technology
Prostate specific antigen PSA is a kind of proteolytic enzyme, mainly results from prostate epithelial cell, is to detect the important cancer markers of of prostate cancer.Although the normality threshold of PSA is lower than 4ng/mL in serum, when PSA concentration is lower, the probability suffering from prostate cancer is still very large.Therefore, in the period of prostate early diagnosis, be necessary to detect more exactly the concentration of PSA, to diagnosing tumor, Index for diagnosis and treat significant.
So the present invention proposes a kind of novel, easy, sensitive detection PSA method, its self-assembling technique based on nano material constructs the gold nanorods core-quantum dot satellite nanostructured with fluorescence spectrum, and in this, as detection substrate.By the concentration Criterion curve of fluorescence signal and object PSA, quantitatively detect PSA with this.
Summary of the invention
The object of this invention is to provide and a kind ofly utilize fluorescence spectrum to carry out the method for super sensitivity detection to PSA based on gold nanorods core-quantum dot satellite shape nanostructured assembling body.
Technical scheme of the present invention, a kind of method based on gold nanorods core-quantum dot satellite shape nanostructured assembling body fluorescence signal super sensitivity detection PSA, coupling there is the gold nanorods of PSA aptamers, the carboxylated quantum dot with PSA aptamers partial complementarity sequence is had with coupling, assembling obtains gold nanorods core-quantum dot satellite shape nanostructured assembling body, when there is determinand PSA, the structure of assembly changes, quantum dot dissociates from assembly, causes the detection of change realization to PSA of fluorescence signal; Concrete steps are as follows:
(1) structure of detecting sensor:
(1) synthesis of gold nanorods
Crystal seed synthesizes: under room temperature, be the three hydration tetra chlorauric acids of 10mM by 0.05mL concentration, join in the cetyl trimethyl ammonium bromide solution of the 0.2M of 1mL, solution colour becomes tawny by colourless, then the 0.01M sodium borohydride solution that 0.12mL newly prepares is added, rapid stirring 2min, namely solution colour becomes light brown from tawny;
Gold nanorods grows: the three hydration tetra chlorauric acids of the 1mM of 5mL join in 5mL, 0.2M cetyl trimethyl ammonium bromide solution, add the ultrapure water of 4mL, mixing, the 0.01M liquor argenti nitratis ophthalmicus of 0.125mL joins in above-mentioned mixed system, 70 μ L, 0.1M ascorbic acid solutions are added by mixing subsequently.Vigorous stirring, solution becomes colorless, and after 2min, adds the crystal seed of 12 μ L, stirs 20s, puts into 30 DEG C of water-baths, 2h.
(2) gold nanorods is modified PSA aptamers:
Adopt the gold nanorods of seeded growth method synthesis, after its centrifugal concentrating 2.5 times, be resuspended in ultrapure water.Get 100 μ L concentrate after gold nanorods, in order to avoid the non-specific adsorption of gold nanorods and carboxylated quantum dot, SH-PEG-COOH is added in solution, the mol ratio of SH-PEG-COOH and gold rod is made to be 20 ︰ 1, after abundant mixing, add the PSA aptamers of 5 μ L50 μM sulfhydrylations, and final concentration is the NaCl solution of 50mM, 37 DEG C of overnight incubation.Obtain the gold nanorods solution of coupling PSA aptamers, stand-by.
The concrete sequence of PSA aptamers is as follows:
PSA-aptamer：5’-SH-ATTAAAGCTCGCCATCAAATAGC-3’；
(3) quantum point coupling PSA aptamers partial complementarity sequence PSACS(complementarysequences): the carboxylated CdSeZnS quantum dot ultrapure water bought in gal garden, Wuhan dilutes 200 times makes final concentration be 40mM.By mol ratio 1000 ︰ 1000 ︰ 1 of EDC, NHS and CdSeZnS quantum dot, add EDC and NHS, react 2h when lucifuge.Afterwards, filtering with the super filter tube of interception 30kD, 10mMPB(pH=7.5) damping fluid is resuspended.Add the PSACS of final concentration 2 μMs wherein, reaction 3h, obtains quantum dot-PSACS solution, stand-by.
The concrete sequence of PSACS is as follows:
PSA-complementarysequence：5’-NH
2-TTTTTTGCTATTTGATGG-3’；
(4) assembling of gold nanorods core-quantum dot satellite shape nanostructured
The centrifugal 10min of 100 μ L gold nanorods coupling PSA aptamers solution 7500rpm, removing supernatant, the 100 μ L quantum dot-PSACS solution super filter tubes of molecular cut off 30kD filter, and most latter two solution is mixed in 100 μ L and contains 50mMNaCl, 50mMMg (NO
3)
2with in 1 × tbe buffer liquid of 0.01%SDS, hybridization 12h.The centrifugal 15min of 3000rpm, removing supernatant, resuspended with ultrapure water, obtain the gold nanorods core-quantum dot satellite shape nanostructured assembling body of high yield.
(2) fluorescence spectrum detects, set up the typical curve between the fluorescence signal of gold nanorods core-quantum dot satellite shape nanostructured assembling body and PSA concentration, set up typical curve between the two according to the relation of PSA concentration and fluorescence signal intensity, thus application fluorescence signal detects to PSA.
Fluorescence spectrum detects specific as follows: be mixed in 100 μ L in gold nanorods coupling PSA aptamers and quantum dot-PSACS and contain 50mMNaCl, 50mMMg (NO
3)
2during 1 × tbe buffer liquid with 0.01%SDS, add the object PSA of variable concentrations respectively, incubated at room 12h, because PSA can react with PSAaptamer, along with the increase of PSA concentration, the assembling of gold nanorods core-quantum dot satellite shape nanostructured is known from experience dissociating in various degree, therefore obtains different fluorescence signal intensities; Under the emission wavelength of 600nm, set up typical curve between the two according to the relation of PSA concentration and fluorescence signal intensity, thus application fluorescence signal detects to PSA.
Beneficial effect of the present invention: the invention provides and a kind ofly utilize fluorescence spectrum to carry out the method for super sensitivity detection based on gold nanorods core-quantum dot satellite shape nanostructured assembling body to PSA, have highly sensitive, detectability is low, advantage easily and efficiently, has extraordinary application prospect.
Accompanying drawing explanation
The transmission electron microscope photo of Fig. 1 gold nanorods of the present invention, quantum dot and gold nanorods core-quantum dot satellite shape nanostructured assembling body.A figure is gold nanorods, and B figure is quantum dot, and C figure and D figure is all transmission electron microscope pictures of gold nanorods core-quantum dot satellite shape nanostructured assembling body.
In gold nanorods core-quantum dot satellite shape nanostructured assembling body, the PSA of variable concentrations is added, the variation diagram of the fluorescence spectrum caused in Fig. 2 the present invention.
Fluorescence spectrum in Fig. 3 the present invention detects the canonical plotting of PSA.
Embodiment
Embodiment 1
(1) structure of detecting sensor
The synthesis of a, gold nanorods
Crystal seed synthesizes: under room temperature, be the three hydration tetra chlorauric acids of 10mM by 0.05mL concentration, join in the cetyl trimethyl ammonium bromide solution of the 0.2M of 1mL, solution colour becomes tawny by colourless, then the 0.01M sodium borohydride solution that 0.12mL newly prepares is added, rapid stirring 2min, namely solution colour becomes light brown from tawny;
Gold nanorods grows: the three hydration tetra chlorauric acids of the 1mM of 5mL join in 5mL, 0.2M cetyl trimethyl ammonium bromide solution, add the ultrapure water of 4mL, mixing, the 0.01M liquor argenti nitratis ophthalmicus of 0.125mL joins in above-mentioned mixed system, 70 μ L, 0.1M ascorbic acid solutions are added by mixing subsequently.Vigorous stirring, solution becomes colorless, and after 2min, adds the crystal seed of 12 μ L, stirs 20s, puts into 30 DEG C of water-baths, 2h.
B, gold nanorods are modified PSA aptamers:
Adopt the gold nanorods of seeded growth method synthesis, after its centrifugal concentrating 2.5 times, be resuspended in ultrapure water.Get 100 μ L concentrate after gold nanorods, in order to avoid the non-specific adsorption of gold nanorods and carboxylated quantum dot, SH-PEG-COOH is added in solution, the mol ratio of SH-PEG-COOH and gold rod is made to be 20 ︰ 1, after abundant mixing, add the PSA aptamers of 5 μ L50 μM sulfhydrylations, and final concentration is the NaCl solution of 50mM, 37 DEG C of overnight incubation.Obtain the gold nanorods solution of coupling PSA aptamers, stand-by.
PSA-aptamer：5’-SH-ATTAAAGCTCGCCATCAAATAGC-3’；
C, quantum point coupling PSA aptamers partial complementarity sequence PSACS(complementarysequences): the carboxylated CdSeZnS quantum dot ultrapure water bought in gal garden, Wuhan dilutes 200 times makes final concentration be 40mM.By mol ratio 1000 ︰ 1000 ︰ 1 of EDC, NHS and CdSeZnS quantum dot, add EDC and NHS, react 2h when lucifuge.Afterwards, filtering with the super filter tube of interception 30kD, 10mMPB(pH=7.5) damping fluid is resuspended.Add the PSACS of final concentration 2 μMs wherein, reaction 3h, obtains quantum dot-PSACS solution, stand-by.
PSA-complementarysequence：5’-NH
2-TTTTTTGCTATTTGATGG-3’；
The assembling of d, gold nanorods core-quantum dot satellite shape nanostructured
The centrifugal 10min of 100 μ L gold nanorods coupling PSA aptamers solution 7500rpm, removing supernatant, the 100 μ L quantum dot-PSACS solution super filter tubes of molecular cut off 30kD filter, and most latter two solution is mixed in 100 μ L and contains 50mMNaCl, 50mMMg (NO
3)
2with in 1 × tbe buffer liquid of 0.01%SDS, hybridization 12h.The centrifugal 15min of 3000rpm, removing supernatant, resuspended with ultrapure water, obtain gold nanorods core-quantum dot satellite shape nanostructured assembling body (as shown in Figure 1) of high yield.
(2) fluorescence spectrum detects, set up the typical curve between the fluorescence signal of gold nanorods core-quantum dot satellite shape nanostructured assembling body and PSA concentration, set up typical curve between the two according to the relation of PSA concentration and fluorescence signal intensity, thus application fluorescence signal detects to PSA.
Fluorescence spectrum detects specific as follows: be mixed in 100 μ L in gold nanorods coupling PSA aptamers and quantum dot-PSACS and contain 50mMNaCl, 50mMMg (NO
3)
2during 1 × tbe buffer liquid with 0.01%SDS, add the object PSA of variable concentrations respectively, incubated at room 12h, because PSA can react with PSAaptamer, along with the increase of PSA concentration, the assembling of gold nanorods core-quantum dot satellite shape nanostructured is known from experience dissociating in various degree, therefore obtains different fluorescence signal intensities.The change of fluorescence spectrum figure as shown in Figure 2.
Under the excitation wavelength of 600nm, set up typical curve between the two according to the relation of PSA concentration and fluorescence signal intensity, thus application fluorescence signal detects to PSA.Fluorescence spectrum detects the canonical plotting of PSA as shown in Figure 3.
Claims (2)
1. the method based on gold nanorods core-quantum dot satellite shape nanostructured assembling body fluorescence signal super sensitivity detection PSA, it is characterized in that: coupling had the gold nanorods of PSA aptamers and coupling to have the carboxylated quantum dot with PSA aptamers partial complementarity sequence, assembling obtains gold nanorods core-quantum dot satellite shape nanostructured assembling body, when there is determinand PSA, the structure of assembly changes, quantum dot dissociates from assembly, and then causes the detection of change realization to PSA of fluorescence signal; Concrete steps are as follows:
(1) structure of detecting sensor:
A, gold nanorods are modified PSA aptamers: the gold nanorods adopting the synthesis of seeded growth method, after its centrifugal concentrating 2.5 times, is resuspended in ultrapure water; Get 100 μ L concentrate after gold nanorods, SH-PEG-COOH is added in solution, the mol ratio of SH-PEG-COOH and gold nanorods is made to be 20 ︰ 1, after abundant mixing, add the PSA aptamers of 5 μ L50 μM sulfhydrylations, and final concentration is the NaCl solution of 50mM, 37 DEG C of overnight incubation, obtain the gold nanorods solution of coupling PSA aptamers, stand-by;
PSA aptamers sequence is as follows:
PSA-aptamer：5’-SH-ATTAAAGCTCGCCATCAAATAGC-3’；
B, quantum point coupling PSA aptamers partial complementarity sequence PSACS: get CdSeZnS quantum dot ultrapure water and dilute 200 times and make final concentration be 40mM; Be 1000 ︰ 1000 ︰ 1 by the mol ratio of EDC, NHS and CdSeZnS quantum dot, add EDC and NHS, react 2h when lucifuge; Afterwards, filter with the super filter tube of interception 30kD, pH is 7.5, the PB damping fluid of 10mM is resuspended; Add the PSACS of final concentration 2 μMs wherein, reaction 3h, obtains quantum dot-PSACS solution, stand-by;
PSACS sequence is as follows:
PSA-complementarysequence：5’-NH
2-TTTTTTGCTATTTGATGG-3’；
The assembling of c, gold nanorods core-quantum dot satellite shape nanostructured: the centrifugal 10min of 100 μ L gold nanorods coupling PSA aptamers solution 7500rpm, removing supernatant, 100 μ L quantum dot-PSACS solution molecular cut offs are that the super filter tube of 30kD filters, and most latter two solution is mixed in 100 μ L and contains 50mMNaCl, 50mMMg (NO
3)
2with in 1 × tbe buffer liquid of 0.01%SDS, hybridization 12h; The centrifugal 15min of 3000rpm, removing supernatant, resuspended with ultrapure water, obtain the gold nanorods core-quantum dot satellite shape nanostructured assembling body of high yield;
(2) fluorescence spectrum detects: the typical curve setting up gold nanorods core-quantum dot satellite shape nanostructured assembling body fluorescence signal and PSA concentration, set up typical curve between the two according to the relation of PSA concentration and fluorescence signal intensity, thus application fluorescence signal detects to PSA.
2., according to claim 1 based on the method for gold nanorods core-quantum dot satellite shape nanostructured assembling body fluorescence signal super sensitivity detection PSA, it is characterized in that step (2) is specific as follows: be mixed in 100 μ L in gold nanorods coupling PSA aptamers and quantum dot-PSACS and contain 50mMNaCl, 50mMMg (NO
3)
2during 1 × tbe buffer liquid with 0.01%SDS, add the object PSA of variable concentrations respectively, incubated at room 12h, because PSA can react with PSAaptamer, along with the increase of PSA concentration, the assembling of gold nanorods core-quantum dot satellite shape nanostructured is known from experience dissociating in various degree, therefore obtains different fluorescence signal intensities; Under the emission wavelength of 600nm, set up typical curve between the two according to the relation of PSA concentration and fluorescence signal intensity, thus application fluorescence signal detects to PSA.
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| CN106367510A (en) * | 2016-09-20 | 2017-02-01 | 江南大学 | Preparation method and application of satellite-shaped nanometer assembling body used for duplex detection of intracellular cancer biomarker |
| CN106525814A (en) * | 2016-11-07 | 2017-03-22 | 华南师范大学 | PSA detection method based on magnetic core-gold satellite assembly body |
| CN106771204A (en) * | 2017-01-18 | 2017-05-31 | 中南大学 | A kind of detection method of carcinomebryonic antigen concentration |
| CN108059955A (en) * | 2016-11-08 | 2018-05-22 | 财团法人工业技术研究院 | Quantum dot and preparation method thereof |
| CN109239064A (en) * | 2018-10-26 | 2019-01-18 | 湖南科技大学 | A kind of preparation method and application of cupric nanometer rods compound quick detection kit |
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| CN113960003A (en) * | 2021-10-20 | 2022-01-21 | 吉林大学 | Aptamer sensor for detecting tetracycline based on DNA silver nanoclusters and gold nanorods |
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Cited By (16)
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| CN106290873B (en) * | 2016-07-28 | 2018-03-30 | 江南大学 | A kind of preparation and application based on transformed space tetrahedral structure on the gold with Raman and fluorescent dual signal |
| CN106290873A (en) * | 2016-07-28 | 2017-01-04 | 江南大学 | A kind of based on preparation and the application with transformed space tetrahedral structure on the gold of Raman and fluorescent dual signal |
| CN106367510A (en) * | 2016-09-20 | 2017-02-01 | 江南大学 | Preparation method and application of satellite-shaped nanometer assembling body used for duplex detection of intracellular cancer biomarker |
| CN106367510B (en) * | 2016-09-20 | 2019-06-14 | 江南大学 | A kind of preparation method and application of the satellite shape Nanoscale assemblies for cancer markers double check intracellular |
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| CN106525814B (en) * | 2016-11-07 | 2018-12-25 | 华南师范大学 | It is a kind of based on magnetic core-gold satellite assembly PSA detection method |
| CN106525814A (en) * | 2016-11-07 | 2017-03-22 | 华南师范大学 | PSA detection method based on magnetic core-gold satellite assembly body |
| CN108059955A (en) * | 2016-11-08 | 2018-05-22 | 财团法人工业技术研究院 | Quantum dot and preparation method thereof |
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| CN108059955B (en) * | 2016-11-08 | 2021-03-05 | 财团法人工业技术研究院 | Quantum dot and preparation method thereof |
| CN106771204B (en) * | 2017-01-18 | 2018-12-14 | 中南大学 | A kind of detection method of carcinomebryonic antigen concentration |
| CN106771204A (en) * | 2017-01-18 | 2017-05-31 | 中南大学 | A kind of detection method of carcinomebryonic antigen concentration |
| CN109239064A (en) * | 2018-10-26 | 2019-01-18 | 湖南科技大学 | A kind of preparation method and application of cupric nanometer rods compound quick detection kit |
| CN110082530A (en) * | 2019-04-09 | 2019-08-02 | 天津大学 | Aqueogel based on quantum dot and gold nanorods and preparation method thereof, application |
| CN113960003A (en) * | 2021-10-20 | 2022-01-21 | 吉林大学 | Aptamer sensor for detecting tetracycline based on DNA silver nanoclusters and gold nanorods |
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Application publication date: 20160504 |