CN104931478A - Method for super-sensitively detecting alpha fetalprotein based on surface enhanced raman scattering effect of silver nanoparticle tripolymer - Google Patents
Method for super-sensitively detecting alpha fetalprotein based on surface enhanced raman scattering effect of silver nanoparticle tripolymer Download PDFInfo
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Abstract
The invention provides a method for super-sensitively detecting alpha fetalprotein based on the surface enhanced raman scattering effect of a silver nanoparticle tripolymer and belongs to the technical field of analytical chemistry. The method comprises the following steps: synthetizing silver nanoparticles with the grain sizes of 15 nm, modifying DNA by the silver nanoparticles, assembling the silver nanoparticle tripolymer, and conducting TEM characterizing and raman signal testing on the silver nanoparticle tripolymer. The silver nanoparticle tripolymer is assembled for the first time, and has a strong raman signal, the raman signal changes after the alpha fetoprotein is added, and the quick super-sensitive detection of the alpha fetalprotein is achieved by the change of the raman signal.
Description
Technical field
The present invention relates to the method based on Nano silver grain trimerical surface enhanced Raman scattering effect super sensitivity detection alpha-fetoprotein, belong to technical field of analytical chemistry.
Background technology
Alpha-fetoprotein (α-fetoprotein, AFP) is a kind of carcinogenic glycoprotein, as a kind of become " molecular labeling " of person's physiological state, and through being commonly used for the suitable biomarker of diagnosing hepatocellular carcinoma, yolk sac tumor, germinoma, cancer of the stomach.The method of current detection AFP mainly contains radio immunoassay and euzymelinked immunosorbent assay (ELISA) (ELISA), but all there are certain relative merits, and the scope of application is also had nothing in common with each other, so need in clinical practice to consider that these two kinds of methods all also exist some shortcomings part.When ELISA method detects tumor markers, because of the easy thermal denaturation of protein, so be difficult to detect protide tumor markers, the sensitivity of another ELISA method is lower, and effort is consuming time; There is cross reaction, false positive reaction in radio immunoassay, tissue sample process rapid not sometimes, can not deactivation digestive enzyme, and salt and pH affect the shortcomings such as result sometimes.The method how setting up sensitive and special detection tumor markers is also the difficulties needing to be badly in need of solving, and nano material is the study hotspot of society, can using nano material as a kind of novel probe, the rapid sensitive detection carrying out AFP is a spotlight.
Due to own dimensions size, nano structural material has unique physicochemical property, becomes present material and studies one of most active field.Nearest decades, Nano silver grain is due to the physicochemical property (as small-size effect, surface effect, quantum tunneling effect, Dielectric confinement effect, macro quanta tunnel effect etc.) of its uniqueness, especially silver nano material also has good antibacterial activity, has become the object that many fundamental researchs and Applied Research Laboratory are paid close attention to.The self assembly of nano material is a study hotspot of nanometer technology, reason is that self-assembled nanometer material can show the optics of the uniqueness being different from mono-dispersed nano particle, electricity, magnetics and chemical property, can be understood the physics and chemistry character of macroscopic material by the character studying self-assembled material better.Surface enhanced raman spectroscopy (SERS) is a kind of analytical approach being widely used in chemistry and bio-sensing, detect compared to fluorescent technique, due to Surface enhanced raman spectroscopy be a kind of by nonspecific wavelength laser excite the finger-print that obtains specific molecular and it keep the stability of its light by Raman beacon around metallics and chemical bond information be provided, therefore SERS can collect the peak width of narrower Raman spectrum, has larger potential source biomolecule analysis of markers application power.
First the present invention by the aptamers of the finishing alpha-fetoprotein of Nano silver grain and complementary series thereof, carries out tripolymer assembling, and is studied its Raman character and inquires into.Add the alpha-fetoprotein solution of variable concentrations, test trimerical Raman signal, to reach the sensing detection object to alpha-fetoprotein, detection line can reach 0.097aM.
Summary of the invention
The object of this invention is to provide the method based on Nano silver grain trimerical surface enhanced Raman scattering effect super sensitivity detection alpha-fetoprotein.
Technical scheme of the present invention, based on the method for Nano silver grain trimerical surface enhanced Raman scattering effect super sensitivity detection alpha-fetoprotein, comprise the synthesis of Nano silver grain, the trimerical assembling of Nano silver grain and structural characterization, Raman signal test, detect alpha-fetoprotein with Nano silver grain tripolymer.Concrete steps are as follows:
(1) synthesis of Nano silver grain: content carries out the synthesis of 15 nm Nano silver grains disclosed in application number 201110274220.X; Get 20mL ultrapure water to mix with the polyvinylpyrrolidone of 5mL massfraction 1%, then add the sodium borohydride aqueous solution of 0.6 mL 0.1mol/L, this reaction is carried out in ice bath.Then, by 5 mL massfractions be 1% aqueous povidone solution and 5mL massfraction be 0.15% silver nitrate aqueous solution simultaneously join in above-mentioned solution with the speed syringe pump of 30mL/h, note adding while stirring, after end to be added, put into 80 DEG C of water-baths immediately and grow 2h, namely obtain 15nm Nano silver grain.
(2) the trimerical assembling of Nano silver grain: get the 15nm particle diameter Nano silver grain colloidal sol that three pipes have concentrated, ensure each nano grain of silver sub-surface in every pipe respectively a controlled modification alpha-fetoprotein aptamers and and its complementary series, carry out hybridizing Nano silver grain three is concrete;
By Electronic Speculum TEM and Raman spectrometer, the trimerical structure of Nano silver grain and Raman signal are characterized and tested;
Aptamers 5 '-SH-GGCAGGAAGA CAAACAGGAC CGGGTTGTGT
GGGGTTTTAA GAGCGTCGCC TGTGTGTGGT CTGTGGTGCT GT-3’；
Complementary series 1:5 '-SH-CCGGTCCTGT TTGTCTTCCT-3 ',
Complementary series 2:5 '-SH-GGCGACGCTC TTAAAACCCCA-3 ';
(3) detection of alpha-fetoprotein: alpha-fetoprotein is added in the Nano silver grain tripolymer assembled, reaction 10min, carries out Raman test;
The trimerical assembling of described Nano silver grain: get Nano silver grain that three pipe 50mL have concentrated and carry out coupling with the aptamers of the alpha-fetoprotein of 50mL sulfydryl modification and complementary series thereof respectively, at room temperature reaction overnight, respectively at centrifugal 15 min of 13000r/min, abandon supernatant, afterwards by the ultrapure water dispersion precipitation of 50mL, stand-by;
By 50mL silver-aptamers, 50 mL silver-complementary seriess 1,50mL silver-complementary series 2 in the 5 mM PB damping fluids of 150mL, hybridization reaction 12h, centrifugal 15 min of assembling product, abandon supernatant, and the ultrapure water dispersion of rear 100mL, carries out TEM and Raman characterizes.
The detection of described alpha-fetoprotein: the Nano silver grain tripolymer getting 100mL, adds the alpha-fetoprotein of 1mL variable concentrations, makes its final concentration be specially 0,0.2,0.5,1,2,5,10,20 aM at 0 ~ 20aM(), react 10 min, carry out Raman test.
Beneficial effect of the present invention: the present invention is based on Raman signal as detection signal, not only stable but also detectability is low, and Nano silver grain has the strongest plasma resonance peak in infrared region.The present invention assembles Nano silver grain tripolymer and shows stronger Raman signal, carries out the detection of alpha-fetoprotein, greatly can improve the sensitivity of detection based on strong raman scattering intensity.
Accompanying drawing explanation
The TEM figure of Fig. 1 Nano silver grain tripolymer package assembly.
The trimerical Raman signal of Nano silver grain under Fig. 2 variable concentrations alpha-fetoprotein.
Fig. 3 carries out alpha-fetoprotein examination criteria curve based on Nano silver grain tripolymer package assembly.
Embodiment
Embodiment 1 is based on the method for Nano silver grain trimerical surface enhanced Raman scattering effect super sensitivity detection alpha-fetoprotein
(1) the trimerical synthesis of Nano silver grain: content carries out the synthesis of 15 nm Nano silver grains disclosed in application number 201110274220.X; Get 20 mL ultrapure waters to mix with the polyvinylpyrrolidone of 5 mL massfractions 1%, then add the sodium borohydride aqueous solution of 0.6 mL 0.1mol/L, this reaction is carried out in ice bath.Then, by 5 mL massfractions be 1% aqueous povidone solution and 5 mL massfractions be 0.15% silver nitrate aqueous solution simultaneously join in above-mentioned solution with the speed syringe pump of 30mL/h, note adding while stirring, after end to be added, put into 80 DEG C of water-baths immediately and grow 2h, namely obtain 15nm Nano silver grain.
By the Nano silver grain of synthetic 100mL 15 nm at the centrifugal 15min of 13000r/min, abandon supernatant, the ultrapure water dispersion precipitation of rear 200mL, stand-by.
(2) the trimerical assembling of Nano silver grain: get Nano silver grain that three pipe 50mL have concentrated and carry out coupling with the aptamers of the alpha-fetoprotein of 50mL sulfydryl modification and complementary series thereof respectively, at room temperature reaction overnight, respectively at the centrifugal 15min of 13000r/min, abandon supernatant, afterwards by the ultrapure water dispersion precipitation of 50mL, stand-by;
By 50mL silver-aptamers, 50mL silver-complementary series 1,50mL silver-complementary series 2 in the 5 mM PB damping fluids of 150mL, hybridization reaction 12h, centrifugal 15 min of assembling product, abandon supernatant, and the ultrapure water dispersion of rear 100mL, carries out TEM and Raman characterizes.
(3) detection of alpha-fetoprotein: the Nano silver grain tripolymer assembly getting 100mL, add 1mL alpha-fetoprotein solution, final concentration scope is 0,0.2,0.5,1,2,5,10,20aM, reacts 10 min, carries out Raman test.Lowest detectable limit reaches 0.097aM.
Claims (3)
1. based on the method for Nano silver grain trimerical surface enhanced Raman scattering effect super sensitivity detection alpha-fetoprotein, it is characterized in that the synthesis comprising Nano silver grain, the trimerical assembling of Nano silver grain and structural characterization, Raman signal test, detect alpha-fetoprotein with Nano silver grain tripolymer; Concrete steps are as follows:
(1) synthesis of Nano silver grain: the synthesis carrying out 15nm particle diameter Nano silver grain;
(2) the trimerical assembling of Nano silver grain: get the 15nm particle diameter Nano silver grain colloidal sol that three pipes have concentrated, ensure each nano grain of silver sub-surface in every pipe respectively a controlled modification alpha-fetoprotein aptamers and and its complementary series, carry out hybridizing to obtain Nano silver grain tripolymer, by Electronic Speculum TEM and Raman spectrometer, the trimerical structure of Nano silver grain and Raman signal are characterized and tested;
Aptamers: 5 '-SH-GGCAGGAAGA CAAACAGGAC CGGGTTGTGT GGGGTTTTAA GAGCGTCGCC TGTGTGTGGT CTGTGGTGCT GT-3 ';
Complementary series 1:5 '-SH-CCGGTCCTGT TTGTCTTCCT-3 ',
Complementary series 2:5 '-SH-GGCGACGCTC TTAAAACCCC A-3 ';
(3) detection of alpha-fetoprotein: added by alpha-fetoprotein in the Nano silver grain tripolymer assembled, reaction 10min, carries out Raman test.
2., according to claim 1 based on the method for Nano silver grain trimerical surface enhanced Raman scattering effect super sensitivity detection alpha-fetoprotein, it is characterized in that the trimerical number of assembling steps of described Nano silver grain is as follows:
Get 15nm particle diameter Nano silver grain that three pipe 50mL have concentrated and carry out coupling with the aptamers of the alpha-fetoprotein of 50 mL sulfydryl modifications and complementary series thereof respectively, at room temperature reaction overnight, respectively at the centrifugal 15min of 13000r/min, abandon supernatant, afterwards by the ultrapure water dispersion precipitation of 50 mL, stand-by;
By 50 mL silver-aptamers, 50 mL silver-complementary series 1,50 mL silver-complementary seriess 2 in the 5 mM PB damping fluids of 150 mL, hybridization reaction 12h, assembling product is Nano silver grain tripolymer, centrifugal 15min, abandon supernatant, the ultrapure water dispersion of rear 100mL, carries out TEM and Raman characterizes.
3. according to claim 1 based on the method for Nano silver grain trimerical surface enhanced Raman scattering effect super sensitivity detection alpha-fetoprotein, it is characterized in that the detecting step of alpha-fetoprotein is as follows: the Nano silver grain tripolymer getting 100mL, add the alpha-fetoprotein of 1 mL variable concentrations, make its final concentration at 0-20aM, reaction 10min, carries out Raman test.
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| CN105606582A (en) * | 2016-03-25 | 2016-05-25 | 徐大鹏 | Alpha fetoprotein kit based on aptamer fluorescent probe AFP4 and detection method thereof |
| CN105651754A (en) * | 2016-03-25 | 2016-06-08 | 徐大鹏 | Nucleic acid aptamer fluorescent probe based alpha-fetoprotein AFP2 kit and detection method |
| CN105651755A (en) * | 2016-03-25 | 2016-06-08 | 徐大鹏 | Nucleic acid aptamer fluorescent probe based alpha-fetoprotein AFP1 kit and detection method |
| CN105785034A (en) * | 2016-03-25 | 2016-07-20 | 徐大鹏 | Alpha fetal protein kit based on aptamer fluorescent probe AFP3 and detection method thereof |
| CN105785033A (en) * | 2016-03-25 | 2016-07-20 | 徐大鹏 | Alpha fetal protein kit based on aptamer fluorescent probe AFP5 and detection method thereof |
| CN106290898A (en) * | 2016-07-29 | 2017-01-04 | 江南大学 | A kind of gold up-conversion nanoparticles trimer preparation method and application thereof |
| CN111912827A (en) * | 2020-06-23 | 2020-11-10 | 河南工程学院 | Surface enhanced monomolecular super-Raman scattering substrate based on Fano resonance type silver nano trimer |
| CN113008864A (en) * | 2021-03-04 | 2021-06-22 | 天津中医药大学 | Method for detecting food-borne pathogenic bacteria by using surface-enhanced Raman spectrum sensor |
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| CN111912827A (en) * | 2020-06-23 | 2020-11-10 | 河南工程学院 | Surface enhanced monomolecular super-Raman scattering substrate based on Fano resonance type silver nano trimer |
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| CN113008864A (en) * | 2021-03-04 | 2021-06-22 | 天津中医药大学 | Method for detecting food-borne pathogenic bacteria by using surface-enhanced Raman spectrum sensor |
| CN113008864B (en) * | 2021-03-04 | 2022-10-25 | 天津中医药大学 | Method for detecting food-borne pathogenic bacteria by using surface-enhanced Raman spectrum sensor |
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