CN102517202A - Method for modifying DNA micro-fluidic chip micro-channel based on nano gold particles - Google Patents
Method for modifying DNA micro-fluidic chip micro-channel based on nano gold particles Download PDFInfo
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- CN102517202A CN102517202A CN2011103843532A CN201110384353A CN102517202A CN 102517202 A CN102517202 A CN 102517202A CN 2011103843532 A CN2011103843532 A CN 2011103843532A CN 201110384353 A CN201110384353 A CN 201110384353A CN 102517202 A CN102517202 A CN 102517202A
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Abstract
The invention discloses a method for modifying a DNA micro-fluidic chip micro-channel based on nano gold particles. The method comprises the following steps of: pre-treating a DNA micro-fluidic chip, preparing a composite screening medium containing the nano gold particles, and modifying the DNA micro-fluidic chip micro-channel. The method has the advantages of simplicity in operation, difficulty in blocking the channel and the like; and the DNA micro-fluidic chip modified by the method has a good screening effect and short analysis time.
Description
Technical field
The invention belongs to the technical field that the DNA micro-fluid control chip electrophoretic is analyzed, be specifically related to the method that a kind of utilization contains the compound sieving medium modifying DNA micro-fluidic chip microchannel of nm gold particles.
Background technology
In recent years, micro-total analysis system receives very big concern, but outstanding advantage such as analysis speed is fast because it has, separator column is imitated high and flexible combination and scale be integrated, and be widely used in the research of biomacromolecules such as DNA.But DNA micro-fluidic chip passage has absorption to a certain degree to biological samples such as DNA in the substantial sepn process, causes that peak hangover and resolving power are low, has limited the application of DNA micro-fluidic chip analytical technology.Therefore, on the basis that guarantees chip analysis safety, repeatability, the microchannel of DNA micro-fluidic chip is modified, become one of critical path of promoting DNA micro-fluidic chip analytical technology and industrialization.
The method of directly utilizing multipolymer that DNA micro-fluidic chip microchannel is modified is arranged in the prior art, but the preparation process of multipolymer is complicated, productive rate is low, and the easy blocking microchannel.
The present invention overcome the process that exists in the method for in the prior art DNA micro-fluidic chip passage being modified complicated, be prone to the microchannel caused defective such as obstruction; A kind of method based on nm gold particles modifying DNA micro-fluidic chip microchannel has been proposed; Have simple to operate, be difficult for beneficial effects such as blocking channel, sieving medium are stable, the life-span is long; The screening effect of the DNA micro-fluidic chip of modifying through the inventive method is good, and analysis time is short.
Summary of the invention
The present invention proposes a kind of method, may further comprise the steps based on nm gold particles modifying DNA micro-fluidic chip microchannel:
The pre-treatment of the first step DNA micro-fluidic chip and microchannel thereof
The DNA micro-fluidic chip is put into H
2SO
4Solution soaks, and behind deionized water, hydrochloric acid soln, deionized water rinsing, dries up subsequent use successively;
Second step preparation nm gold particles
With HAuCl
4Under intense agitation, be heated to boiling, add Trisodium Citrate, after reaction obtained nm gold particles, is cooled to room temperature, it was subsequent use under 4 ℃ of temperature, to keep in Dark Place;
The compound sieving medium of the 3rd step preparation nano-golden particle-containing
Said nm gold particles is added in the mixing solutions of the PVP K120 of supersound process and Natvosol and mixes; Under agitation splash into acetone; Obtain flocculent precipitate, after deposition, washing, drying, obtain the compound sieving medium of said nano-golden particle-containing; The compound sieving medium of said nano-golden particle-containing is stored in the tbe buffer solution subsequent use;
The 4th step modification step
The compound sieving medium of the 3rd nano-golden particle-containing that obtain of step is poured in the microchannel of the said DNA micro-fluidic chip that the first step is handled; Make it to be full of; Leave standstill up to the compound sieving medium and the microchannel of said nano-golden particle-containing and fully interact the completion modification.
In the inventive method, H in the said the first step
2SO
4Mass concentration be 98%, concentration of hydrochloric acid solution is 1mol/L.
In the inventive method, HAuCl in said second step
4Concentration is 2.5 * 10
-4Mol/L, said sodium citrate concentration are 2.5 * 10
-4Mol/L.
In the inventive method, acetone is anhydrous propanone in said the 3rd step.
In the inventive method, the lip-deep negative charge of said nm gold particles is partly replaced by the neutral polymer molecular chain in the compound sieving medium or shields, and nm gold particles stably is scattered in the compound sieving medium.
Problem to prior art exists the objective of the invention is to release a kind of method based on nm gold particles modifying DNA micro-fluidic chip microchannel.This method has been inherited whole advantages of prior art modifying method, and has overcome the weak point of its existence.
For realizing above-mentioned purpose, the present invention adopts following technical scheme.
The pre-treatment of the first step DNA micro-fluidic chip and microchannel thereof
Earlier mass concentration being put in DNA micro-fluidic chip and microchannel thereof is 98% H
2SO
4Solution soaking 20 minutes, deionized water are cleaned, and use concentration to be the hydrochloric acid soln flushing of 1mol/L 10 minutes again, and deionized water rinsing 10 minutes dries up subsequent use.
Second step preparation nm gold particles (GNPs)
Before the preparation gold sol, all glassware are all used chloroazotic acid and are soaked, and water is rinsed well then, and complete drying.Use the different sodium citrate aqueous solutions of measuring to prepare the GNPs of different-grain diameter.In three round-bottomed flasks that reflux condensing tube is housed, add 50 ml respectively, HAuCl
4The aqueous solution is heated to boiling under intense agitation.In this solution, add 0.6 ml, the sodium citrate aqueous solution of 0.3 ml and 0.05 ml respectively fast.The color of solution becomes red-purple then from the faint yellow mazarine that becomes, and shows to have generated nm gold particles.Behind the constantly boiling 10-15 min, remove thermal source.Gold sol continue to stir, and is cooled to room temperature, puts into 4 ℃ refrigerator and keeps in Dark Place.According to size difference called after GNPs3, GNPs30, GNPs70.
The compound sieving medium of the 3rd step preparation nano-golden particle-containing
With the above-mentioned gold size solution of 2 ml (GNPs3; GNPs30 and GNPs70) add respectively treat to mix fully in the mixing solutions of simple supersound process blended PVP K120 (PVP) and Natvosol (HEC) after; Under agitation splash into above-mentioned 3 parts of aqueous solution in the acetone soln of handling well respectively; Obtain flocculent precipitate, take out filtering-depositing and use washing with acetone, obtain the compound sieving medium of nano-golden particle-containing after the drying.The compound sieving medium of the above-mentioned nano-golden particle-containing for preparing is added in the buffered soln of 1 * TBE, for use.
The 4th step modification step
The modifier of the compound sieving medium of the 3rd nano-golden particle-containing that obtain of step is sucked injector for medical purpose; Syringe needle through injector for medical purpose pours into the modifier of the compound sieving medium of described nano-golden particle-containing in the microchannel of the DNA micro-fluidic chip that the first step is handled; Make it to be full of; Left standstill 20 minutes, this modifier and microchannel are fully interacted, so far modification is all accomplished.
Compare with background technology, the present invention has the following advantages:
1, simple operating steps is prone to row.
2, the microchannel is when the DNA micro-fluidic chip of method modification of the present invention is done the analysis of dna fragmentation; Screening effect to biological sample DNA is good, and analysis time is short, and the work-ing life of modifier is longer; Use continuously and do not see that its performance obviously changes a week, data see the following form 1.
Technical scheme of the present invention is further characterized in that, described HAuCl
4Concentration is 2.5 * 10-4 mol/L, and sodium citrate concentration is 2.5 * 10-4 mol/L.
Technical scheme of the present invention is further characterized in that described acetone is anhydrous propanone.
The inventive method preparation earlier comprises the compound sieving medium of nm gold particles, and the microchannel inwall is being modified.The present invention has the following advantages: the preparation process operation of modifier is simple, and the inventive method is difficult for blocking channel; The DNA micro-fluidic chip of modifying through the inventive method is good to the screening effect of biological sample DNA, and analysis time is short, and the work-ing life of modifier is longer.The microchannel that the present invention is particularly suitable for being used for to the DNA micro-fluidic chip is modified, and has expanded the application of nano material in micro-fluid control chip electrophoretic.
Description of drawings
Shown in Figure 1 is the microfluidic chip structure synoptic diagram.The 1st, sample pool, the 2nd, buffer pool, the 3rd, sample waste liquid pool, the 4th, damping fluid waste liquid pool, the 5th, sample channel, the 6th, sample intake passage, the 7th, microchannel, the 8th, microchannel.
Shown in Figure 2 is the structural representation of the compound sieving medium of nano-golden particle-containing.
Embodiment
In conjunction with following specific embodiment and accompanying drawing, the present invention is done further detailed description, protection content of the present invention is not limited to following examples.Under spirit that does not deviate from inventive concept and scope, variation and advantage that those skilled in the art can expect all are included among the present invention, and are protection domain with the appending claims.
The present invention is based on the method for nm gold particles modifying DNA micro-fluidic chip microchannel, may further comprise the steps:
The pre-treatment of the first step DNA micro-fluidic chip and microchannel thereof
Earlier mass concentration being put in DNA micro-fluidic chip and microchannel thereof is 98% H
2SO
4Solution soaking 20 minutes, deionized water are cleaned, and use concentration to be the hydrochloric acid soln flushing of 1mol/L 10 minutes again, and deionized water rinsing 10 minutes dries up subsequent use.
Second step preparation nm gold particles (GNPs)
Before the preparation gold sol, all glassware are all used chloroazotic acid and are soaked, and water is rinsed well then, and complete drying.Use the different sodium citrate aqueous solutions of measuring to prepare the GNPs of different-grain diameter.In three round-bottomed flasks that reflux condensing tube is housed, add 50 ml respectively, HAuCl
4The aqueous solution is heated to boiling under intense agitation.In this solution, add 0.6 ml, the sodium citrate aqueous solution of 0.3 ml and 0.05 ml respectively fast.The color of solution becomes red-purple then from the faint yellow mazarine that becomes, and shows to have generated nm gold particles.Behind the constantly boiling 10-15 min, remove thermal source.Gold sol continue to stir, and is cooled to room temperature, puts into 4 ℃ refrigerator and keeps in Dark Place.According to size difference called after GNPs3, GNPs30, GNPs70.
The compound sieving medium of the 3rd step preparation nano-golden particle-containing
With the above-mentioned gold size solution of 2 ml (GNPs3; GNPs30 and GNPs70) add respectively treat to mix fully in the mixing solutions of simple supersound process blended PVP K120 (PVP) and Natvosol (HEC) after; Under agitation splash into above-mentioned 3 parts of aqueous solution in the acetone soln of handling well respectively; Obtain flocculent precipitate, take out filtering-depositing and use washing with acetone, obtain the compound sieving medium of nano-golden particle-containing after the drying.The compound sieving medium of the above-mentioned nano-golden particle-containing for preparing is added in the buffered soln of 1 * TBE, for use.
The 4th step modification step
The modifier of the compound sieving medium of the 3rd nano-golden particle-containing that obtain of step is sucked injector for medical purpose; Syringe needle through injector for medical purpose pours into the modifier of the compound sieving medium of described nano-golden particle-containing in the microchannel of the DNA micro-fluidic chip that the first step is handled; Make it to be full of; Left standstill 20 minutes, this modifier and microchannel are fully interacted, so far modification is all accomplished.
Embodiment
Present embodiment concrete operations step is fully according to the method described above operated, and same steps as repeats no more.
In the first step, H
2SO
4Mass concentration be 98%, concentration of hydrochloric acid solution is 1mol/L; In second step, HAuCl
4Concentration is 2.5 * 10
-4Mol/L, sodium citrate concentration are 2.5 * 10
-4Mol/L; In the 3rd step, acetone is anhydrous propanone.
In the present embodiment; The compound screening dielectric structure of the nano-golden particle-containing that the 3rd step prepared is as shown in Figure 2; The black bead is depicted as the GNPs nm gold particles in the compound sieving medium among the figure; Because its lip-deep negative charge is partly replaced by the neutral polymer molecular chain or shields, the electric density meeting of GNPs obviously descends, and GNPs also stably is scattered in the polymers soln of compound sieving medium near neutral.
Utilize DNA micro-fluidic chip that present embodiment modifies that the electrophoresis process of DNA sample analysis may further comprise the steps:
Before analyzing experiment, the Φ X174-Hae Ш digest DNA maker that gets 5 μ L is dissolved in the deionized water of 50 μ L, as the DNA sample, adds the optical dye TO-PRO-3 of 1 μ L, and the refrigerator of putting into 4 ℃ is subsequent use.
The compound sieving medium that in four liquid baths of DNA micro-fluidic chip, adds the nano-golden particle-containing of equivalent respectively is as modifier; Carried out prerunning earlier 20 minutes; After treating baseline stability, the DNA sample of the compound sieving medium in the sample pool 1 with equivalent replaced, carry out sample introduction.As shown in Figure 1, sample gets into the sample waste liquid pools by liquid bath 1 through sample channel 5, sample intake passage 6 and converts separation voltage to after 3,40 seconds and separate, sample in microchannel 7 separated after, 8 places are to be detected in the microchannel, the sample after the separation gets into damping fluid waste liquid pool 4.Concrete dna fragmentation electrophoresis sample introduction and isolating voltage conditions such as following table 2 are provided with:
Laser-induced fluorescence detection system: it is 635 nm that light source adopts emission wavelength, the semiconductor laser of power 4 mW.Laser beam is perpendicular to the plane of DNA micro-fluidic chip, and (635 ± 10 nm, rich optics limited-liability company converges in Shenyang) filter through preposition bandpass filter, are converged at 8 places, microchannel of DNA micro-fluidic chip by condenser lens.Laser apparatus wherein, preposition bandpass filter, the condenser lens three constitutes integrated module.Fluorescence detecting system is by collecting lens, aperture, and bandpass filter (670 ± 10 nm, rich optics limited-liability company converges in Shenyang), PM (R928 type, Binsong Photon Technology Co., Ltd. Beijing) four parts are formed.On the inclined to one side 45 ° of directions of DNA micro-fluidic chip quadrature, detect the fluorescence of outgoing.
High voltage power supply: the output of four contact high pressure can be provided, every contact voltage output (scope 0-6000 V) but independent control.System comprises hardware and software two portions.The fluorescent signal that PM is collected is handled through signals collecting and amplification module, is input to external computingmachine, carries out data processing and demonstration by computingmachine through Chip Microcomputer A/D conversion.All parameter settings, control and the signals collecting of instrument are all through RS232 Serial Port Line and computer realization communication.
DNA micro-fluidic chip of the present invention microchannel modifying method has been simplified operating process, has avoided the obstruction of chip microchannel, has expanded the application of nano material in micro-fluid control chip electrophoretic.The DNA micro-fluidic chip that present embodiment utilizes the inventive method to modify can be applied to biological sample analyses such as DNA sample.
Claims (5)
1. the method based on nm gold particles modifying DNA micro-fluidic chip microchannel is characterized in that, said method comprising the steps of:
The pre-treatment of the first step DNA micro-fluidic chip and microchannel thereof
The DNA micro-fluidic chip is put into H
2SO
4Solution soaks, and behind deionized water, hydrochloric acid soln, deionized water rinsing, dries up subsequent use successively;
Second step preparation nm gold particles
With HAuCl
4Under intense agitation, be heated to boiling, add Trisodium Citrate, after reaction obtained nm gold particles, is cooled to room temperature, it was subsequent use under 4 ℃ of temperature, to keep in Dark Place;
The compound sieving medium of the 3rd step preparation nano-golden particle-containing
Said nm gold particles is added in the mixing solutions of the PVP K120 of supersound process and Natvosol and mixes; Under agitation splash into acetone; Obtain flocculent precipitate, after deposition, washing, drying, obtain the compound sieving medium of said nano-golden particle-containing; The compound sieving medium of said nano-golden particle-containing is stored in the tbe buffer solution subsequent use;
The 4th step modification step
The compound sieving medium of the 3rd nano-golden particle-containing that obtain of step is poured in the microchannel of the said DNA micro-fluidic chip that the first step is handled; Make it to be full of; Leave standstill up to the compound sieving medium and the microchannel of said nano-golden particle-containing and fully interact the completion modification.
2. the method based on nm gold particles modifying DNA micro-fluidic chip microchannel according to claim 1 is characterized in that H in the said the first step
2SO
4Mass concentration be 98%, concentration of hydrochloric acid solution is 1mol/L.
3. the method based on nm gold particles modifying DNA micro-fluidic chip microchannel according to claim 1 is characterized in that, HAuCl in said second step
4Concentration is 2.5 * 10
-4Mol/L, said sodium citrate concentration are 2.5 * 10
-4Mol/L.
4. the method based on nm gold particles modifying DNA micro-fluidic chip microchannel according to claim 1 is characterized in that, acetone is anhydrous propanone in said the 3rd step.
5. the method based on nm gold particles modifying DNA micro-fluidic chip microchannel according to claim 1; It is characterized in that; The lip-deep negative charge of said nm gold particles is partly replaced by the neutral polymer molecular chain in the compound sieving medium or shields, and nm gold particles stably is scattered in the compound sieving medium.
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| CN103115949A (en) * | 2013-01-22 | 2013-05-22 | 山东理工大学 | Preparation method of enzyme biosensor for detecting pesticide residues |
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Application publication date: 20120627 |