CN101071135B

CN101071135B - Method for preparing semiconductor nano crystal of bio marker utilizing Raman signal

Info

Publication number: CN101071135B
Application number: CN2007101031401A
Authority: CN
Inventors: 刘益春; 单桂晔; 邵长路
Original assignee: Northeast Normal University
Current assignee: Northeast Normal University
Priority date: 2005-09-26
Filing date: 2005-09-26
Publication date: 2011-11-16
Anticipated expiration: 2025-09-26
Also published as: CN101071135A

Abstract

This invention belongs to nano-materials and biotechnology fields, involving the preparation of nano-materials, nano-materials in the application of biomarkers. First synthesized in the ethanol the nanocrystals of different sizes, and secondly, reduced perchlorate material and metal were dissolved in ethanol, the solution will be prepared as a mother liquor by centrifugal separation, by adding organic allocation preparation of such a Raman signals in the semiconductor nanocrystals, and through electrostatic and biological molecular docking, the implementation of the markers of biological molecules. This invention produced plasma resonance, allowing semiconductor nanocrystals Resonance Raman signal enhancement. Because of the nanocrystalline metal surface with a group of organic molecules strong coordination role, the organisms can be stable, sound marker, and the further implementation of the use of fingerprint characteristics of semiconductor nanocrystals resonance Raman signal of multi-stage unknown molecular testing.

Description

Utilize the preparation method of Raman signal to the semiconductor nano of biomarker

Technical field

The invention belongs to nano material and biological technical field, relate to the preparation of nano material, the application of nano material aspect biomarker.

Background technology

Inorganic nano-particle and biomolecule assembling, and utilize the physical characteristics of inorganic nano-particle self to implement the mark of biomolecule has been become the advanced subject of research in the world.At present, the physical property of the inorganic nano material of utilization comprises that mainly the plasma of metal absorbs colour developing, the photoluminescence characteristic of semiconductor nano, the magnetic property of magnetic nanometer etc.These methods play an important role for mark and the macromolecular characteristic of detection of biological and activity aspect.Current many in the world research groups are devoted to replace the organic molecule fluorescence labeling with the nanocrystalline labeling method of inorganic fluorescence semiconductor, the mark of biomolecule has experienced from the mark of the radiomaterial of the first generation, the enzyme of the second generation connects immune labeled, the evolution of the fluorescence labeling method of the inorganic semiconductor fluorescence nano in the organic dye molecule mark of the third generation the 4th generation till now.Utilize inorganic nano-crystal to carry out its starting point of mark and be three aspects: at first, organic dye molecule has unsettled characteristics, the photobleaching phenomenon appears under the irradiation of light easily, and nanocrystalline this shortcoming that has overcome organic molecule of inorganic semiconductor, inorganic semiconductor is nanocrystalline to have strong anti-photobleaching; Secondly, with respect to the half-peak breadth of inorganic nano-crystal fluorescent emission, organic dye molecule has wide half-peak breadth, and the fluorescence spectrum between the different dyes molecule overlaps, and this is restricted application multi-pass mark.The 3rd, the excitation spectrum of organic dye molecule all is very narrow, different dye molecules can only excite with different excitation sources, and it is nanocrystalline for inorganic semiconductor, has very wide excitation spectrum, use a kind of light source and can excite the nanocrystalline of different size simultaneously, this has saved transmitting illuminant for the multi-pass mark.Just be based on above 3 points, the researchist of various countries has made number of research projects to the fluorescence labeling biomolecule of inorganic nano-crystal.But, also exist problem, first for the nanocrystalline fluorescence labeling of inorganic semiconductor for biomolecule, having the nanocrystalline of fluorescent characteristic, generally all is to contain Cd, Pd, HgII-VI semiconductor nanos etc., these materials generally all are that environment is disagreeableness, and biosome itself is harmful to.Second, when using these materials and biomolecule coupling, relate to nanocrystalline phase transfer, nanocrystalline and problems such as docking of biomolecule, these processes are loaded down with trivial details and complicated, it is very difficult accessing the fluorescence nano that can be applied to biomarker, and this has limited the application of inorganic nano-crystal.Can obtain eco-friendlyly, preparation is simple for this reason, and the highly sensitive new indicia means of marking signal is the problem that the various countries researchist is paid close attention to.2003, professor Mirkin of Northwestern Univ USA proposes to strengthen the Raman signal of dyestuff of gold surface to the idea of unknown biomolecule mark with Ag first, utilize the detection of the different Raman signals enforcements of dye molecule to biomolecule, it is a kind of novel indicia means, it has overcome fluorescence signal and has been subjected to the media environment characteristics of interference, the live width of Raman is narrower with respect to the live width of fluorescence simultaneously, and this sensitivity for detection signal has more advantage.But with dyestuff RamanSignal carries out mark to material and has the unsettled characteristics of dye molecule equally, and the present invention designs and adopted the mark of the nanocrystalline resonance raman signal enforcement of inorganic semiconductor to biomolecule.

At present, utilize the biological fluorescent labelling method of dyestuff and quantum dot owing to have half-peak breadth, shortcoming such as the big and environment of background interference is unfriendly, the present invention mainly is that the shortcoming at above mark has proposed the new method of the albumen or the nucleic acid of the unknown being carried out mark with the characteristic fingerprint Raman signal of inorganic nano-crystal.

Broad-band gaps such as ZnO are nanocrystalline as a kind of nontoxic, and harmless biological friendly oxide is widely used in the ultraviolet emission optoelectronic device.Simultaneously we to be also noted that ZnO etc. has under He-Cd laser excitation multistage RamanThe characteristic signal peak, and can strengthen by metal (Au, Ag, etc.).For this reason, we propose to utilize semiconductor materials such as ZnO first Draw GracefulSignal carries out mark to biomolecule.Simultaneously in order to obtain stronger Raman signal, we have adopted metallic nano crystal (Au, Ag, Deng) semiconductor nano is carried out surperficial coating, the effect of two aspects has been played in this coating, at first, semiconductor nanos such as metal and ZnO produce the resonance effect of plasma, and it can make the resonance raman signal of semiconductor nano strengthen.In addition, because very strong coordination can take place with organic molecule group (sulfydryl etc.) in the surface of metallic nano crystal, this has played the effect of a bridge for inorganic nano material and with the biomolecule coupling, and further utilizes the detection of the multistage Raman fingerprint signal enforcement of the resonance with fingerprint characteristic semiconductor nano to unknown molecular.

The content of invention

One of purpose of the present invention provides a kind of inorganic semiconductor preparation of nano crystal of utilizing Raman signal to biomarker

Two of purpose of the present invention is to utilize the nanocrystalline biomarker that carries out of inorganic semiconductor.

1. preparation strengthens the ZnO/Au core-shell nano crystalline substance of Raman signal

The ZnO that at first synthesizes different size in absolute ethyl alcohol is nanocrystalline, and the preparation of ZnO is adopted is the salt (zinc acetate) that will contain zinc ion and the reaction of alkali (lithium hydroxide, tetramethyl oxyammonia etc.).The ZnO of different size can obtain by the control temperature.The ZnO that obtains is nanocrystalline to be stable existence in ethanolic solution.

Secondly, utilize perchloric acid gold salt and metallic reducing material (p-dihydroxy-benzene, sodium citrate etc.) are dissolved in respectively in the absolute ethyl alcohol, then with the ZnO solution of above-mentioned preparation as mother liquor, stir the solution of configuration to such an extent that splash in the mother liquor respectively under the condition more fast.Result's solution is come out by centrifuging, and the centrifugal material that goes out cleans, and is added in the ethanolic solution again.

Adopt at last in the solution of ZnO/Au of preparation to add organic ligand (mercaptoacetic acid, mercaptoethylmaine etc.), adopt centrifugation method that solution is separated again once more, and with sample dispersion in the buffer solution between the PH=7-10.This probe with Raman signal can dock with biomolecule by static, thereby implements the mark to biomolecule.

2. preparation strengthens the ZnO/Ag core-shell nano crystalline substance of Raman signal.

Under ultrasonic condition, freshly prepd ZnO ethanolic solution is joined in the aqueous solution of silver nitrate, simultaneously in this mixed solution, dropping p-dihydroxy-benzene aqueous solution gradually.P-dihydroxy-benzene can reduce silver nitrate and form argent bunch, silver-colored bunch can be that crystal seed forms the nanocrystalline shell of Ag at its surface deposition with ZnO, Ag has humidification to the Raman signal of ZnO, because Ag can have strong coordination ability with organic group, the ZnO/Ag nano-crystal with core-shell structure of preparation can with the biomolecule coupling.

The present invention produces the resonance effect of plasma, and the resonance raman signal of semiconductor nano is strengthened.Because very strong coordination can take place with organic molecule group (sulfydryl etc.) in the surface of metallic nano crystal, played the effect of bridge for inorganic nano material and biomolecule coupling, can stablize biosome, harmless mark, and further implement to utilize to have of the detection of the multistage Raman signal of resonance of fingerprint characteristic semiconductor nano to unknown molecular.

Embodiment

1. prepare the core-shell nano crystalline substance of ZnO/Au and select the precursor material of its phase transfer gold for use HAuCl ₄, the reductive agent of reduction gold can be selected p-dihydroxy-benzene or sodium citrate for use.ZnO is as crystal seed, and Au is nanocrystalline in its superficial growth.The process of concrete test is as follows: get 5～10ml0.1M ZnO ethanolic solution, put it in the volume and with absolute absolute ethyl alcohol and dilute 10 times, other takes by weighing HAuCl ₄, the ratio of the amount of substance of its amount of substance and ZnO between 1:5～1:10, both HAuCl of the 0.05～0.2mM that will take by weighing ₄Be dissolved in the 10ml ethanolic solution, take by weighing the reductive agent p-dihydroxy-benzene simultaneously, its amount of substance should with HAuCl ₄Amount of substance identical, reductive agent also is dissolved in the ethanolic solution.HAuCl then ₄Ethanolic solution and p-dihydroxy-benzene ethanolic solution dropwise join in the ZnO ethanolic solution of quick stirring, allow solution left standstill ageing 10～12 hours after dripping off, and its objective is that to make crystal growth complete.Solution after the ageing fully also is distributed to nanocrystalline separation in the ethanol solution again by centrifugation method.Further in order to be applied to biomarker, adopt the Au coordination of sulfhydryl compound (as mercaptoaniline, mercaptoacetic acid etc.) as part and ZnO surface, the ratio of the amount of ZnO and sulfhydryl compound should be between 1:10～1:20.The sulfhydryl compound of specified quantitative is joined in the ZnO/Au ethanolic solution, stirring reaction 24-30h, after reaction finishes, come out and be distributed in the buffer solution of PH between 7～10 particle is centrifugal, prepared ZnO/Au solution can be used for docking with biomolecule coupled.

2. the core-shell nano of preparation ZnO/Ag is brilliant and with its phase transfer

The precursor material of silver is selected AgNO for use ₃, the reductive agent of reduction gold can be selected p-dihydroxy-benzene or sodium citrate for use.Its preparation method is similar to the method for preparing ZnO/Au core-shell nano crystalline substance.Take by weighing 5～10ml0.1M ZnO ethanolic solution equally, put it in the volume and and dilute 10 times with absolute absolute ethyl alcohol, and AgNO ₃Amount of substance and the ratio of the amount of substance of amount and ZnO between 1:5～1:10, both with the AgNO of respective amount ₃Be dissolved in the ethanolic solution, simultaneously corresponding reductive agent p-dihydroxy-benzene also be dissolved in the ethanolic solution, then AgNO ₃Ethanolic solution and p-dihydroxy-benzene ethanolic solution dropwise join in the ZnO ethanolic solution of quick stirring, allow solution left standstill ageing 10～12 hours after dripping off, and its objective is that to make crystal growth complete.Solution after the ageing fully also is distributed to nanocrystalline separation in the ethanol solution again by centrifugation method.Further in order to be applied to biomarker, we adopt the Ag coordination of sulfhydryl compound (as mercaptoaniline, mercaptoacetic acid etc.) as part and ZnO surface, and the ratio of the amount of ZnO and sulfhydryl compound should be between 1:10～1:20.The sulfhydryl compound of specified quantitative is joined in the ZnO/Au ethanolic solution, stirring reaction 24-30h, after reaction finishes, come out and be distributed in the buffer solution of PH between 7～10 particle is centrifugal, prepared ZnO/Ag solution can be used for docking with biomolecule coupled.

3. the nanocrystalline biomarker that carries out of inorganic semiconductor

Lamivudine is the new drug of the anti-hepatitis B virus (HBV) of ucleosides, it is the strong inhibitor of hbv replication, its effect target is a HBV DNA polymerase, by suppressing the pregenome RNA reverse transcription is to play chain termination in minus-strand dna and the DNA chain extension process to suppress hbv replication. inside and outside research all shows, in case lamivudine resistance is caused by HBVP gene C district YMDD variation. the YMDD variant appears, no longer responsive to Lamivudine. resistance may be with HBV DNA quantitatively knock-on and changes of liver function, even causes that sb.'s illness took a turn for the worse.The present invention mainly is to use common template and ZnO Raman microprobe to implement to detect. and universal primer is made of common template (UT) sequence of 5 ' end and 3 ' special primer sequence, the common template sequence with any natural acid sequence be not complementary can only with general Raman probe coupling. this indicia means has highly sensitive, high special, pinpoint accuracy and high flux characteristics, has broad application prospects in clinical pathogen detection, drug effect monitoring, mutation analysis and related science research field.

Claims

1. utilize the preparation method of Raman signal to the semiconductor ZnO/Ag core-shell nano crystalline substance of biomarker, it is characterized in that: under ultrasonic condition, freshly prepd ZnO ethanolic solution is joined in the aqueous solution of silver nitrate, simultaneously in this mixed solution, dropping p-dihydroxy-benzene aqueous solution gradually, p-dihydroxy-benzene forms argent bunch with the reduction silver nitrate, silver-colored bunch can be that crystal seed forms the nanocrystalline shell of Ag at its surface deposition with ZnO, Ag has humidification to the Raman signal of ZnO, because Ag can have strong coordination ability with organic group, the ZnO/Ag nano-crystal with core-shell structure of preparation can with the biomolecule coupling;

The precursor material of silver is selected AgNO for use ₃, the reductive agent p-dihydroxy-benzene or the sodium citrate of reduction silver, concrete grammar is: takes by weighing 5～10ml 0.1M ZnO ethanolic solution, puts it in the container and and dilute 10 times with absolute absolute ethyl alcohol, and AgNO ₃Amount of substance and the ratio of the amount of substance of ZnO between 1: 5～1: 10, both with the AgNO of respective amount ₃Be dissolved in the ethanolic solution, simultaneously corresponding reductive agent p-dihydroxy-benzene also be dissolved in the ethanolic solution, then AgNO ₃Ethanolic solution and p-dihydroxy-benzene ethanolic solution dropwise join in the ZnO ethanolic solution of quick stirring, allow solution left standstill ageing 10～12 hours after dripping off, solution after the ageing fully also is distributed in the ethanol solution nanocrystalline separation by centrifugation method again, adopt the Ag coordination of sulfhydryl compound mercaptoaniline or mercaptoacetic acid as part and ZnO surface, the ratio of the amount of ZnO and sulfhydryl compound should be between 1: 10～1: 20, quantitative sulfhydryl compound is joined in the ZnO/Ag ethanolic solution, stirring reaction 24-30h, after reaction finishes, come out and be distributed in the buffer solution of PH between 7～10 particle is centrifugal.

2. utilize semiconductor nano the application in biomarker of Raman signal by claim 1 preparation to biomarker.