CN100587076C - Preparation method of visual bio-chip - Google Patents
Preparation method of visual bio-chip Download PDFInfo
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- CN100587076C CN100587076C CN200510096269A CN200510096269A CN100587076C CN 100587076 C CN100587076 C CN 100587076C CN 200510096269 A CN200510096269 A CN 200510096269A CN 200510096269 A CN200510096269 A CN 200510096269A CN 100587076 C CN100587076 C CN 100587076C
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Abstract
This invention involves the preparation methods of a visual biochip. Steps include (1) Preparation of the markers which labeled by magnetic particles: 1) the magnetic particles pretreated 2) pretreatment markers, 3)fixed response of markers, 4) cleaning, 5) preserved; (2) Preparation of the visual Biochip : adding the prepared magnetic particles labeled DNA probes to centrifuge tube, dilution, then add on the surface of gene chip; (3) by the gene chip hybridization, washing, drying after centrifugation, Immersion of silver enhancement solutions, Single silver deposition of the magnetic particles on the surface.
Description
Technical field
The present invention is with the gold-magnetic particles reagent that serves as a mark, but realizes the biochip that visualizing detects.Be specifically related to a kind of preparation method of visual bio-chip.
Background technology
Biochip is a nearly new and high technology that developed rapidly in life science in 10 years.It mainly is meant by little processing and microelectronics and makes up the miniature organism chemical analysis system at the solid chip surface, with realize to tissue, cell, protein, nucleic acid, carbohydrate and the other biological component of viable organism carry out accurately, the detection of quick, large information capacity.At present common biochip is divided into three major types: i.e. gene chip, protein chip, sugar bio-chip.The principal feature of biochip is high-throughput, microminiaturization and automatization.The highly integrated molecule microarray of thousands of dense arrangement makes people obtain the bioinformation in the sample rapidly and accurately in a large amount of biomolecules of very short time inner analysis on the biochip, its detection efficiency be the traditional detection means can not compare.
Gene chip is based on the complementary hybridization technique of nucleic acid probe, and nucleic acid probe is the base sequence of one section synthetic, connects some detectable materials on nucleic acid probe, according to the principle of base complementrity, utilizes gene probe to discern specific gene in the gene mixture.Gene chip claims DNA chip, dna microarray again, on it integrated molecule microarray actual be the gene probe of latticed dense arrangement.By the dna fragmentation of conventional base base sequence, in conjunction with the single stranded DNA of complementary base sequences thereof, determine corresponding sequence, can discern aberrant gene or its product etc. in this way.
Biochip technology mainly comprises: the fluorescent mark of the preparation of chip microarray, the preparation of sample, sample and the detection and the analysis of signal.The principle of protein chip, sugar bio-chip is similar to gene chip, and difference mainly contains two: one, and the fixed molecule is a protein on the protein chip, as antigen or antibody etc.; The fixed molecule is a sugar on the sugar bio-chip, as monose, oligosaccharides, polysaccharide etc.Its two, detect principle and be interaction according to albumen and albumen, albumen and nucleic acid, albumen and sugar, albumen and other molecule.The most frequently used chip signal detection method is that chip is placed chip scanner at present, gathers the strong and weak and fluorescence position of fluorescence of each reflecting point, through the related software analysis image, obtains relevant bioinformation.
Though biochip technology has presented wide application prospect at numerous areas such as gene expression analysis, gene diagnosis, drug screening and sequential analyses.But still there is following shortcoming in the technology of preparing of existing biochip:
1 detection is gone up many based on fluoroscopic examination, use fluorescent labeling reagent and confocal laser to detect scanner.It is all expensive that fluorescent labeling reagent and confocal laser detect the scanner price, causes the use cost costliness.
2 technology stay in the experimental phase, are difficult to penetration and promotion and use, and whole cost is too high, poor practicability.
3 utilize the Radioactive colloidal gold reagent that serves as a mark, and carry out the research that simple substance deposition of silver signal behind the silver ion reduction amplifies the DNA chip technology and are in the starting stage, still immature before not.
Behind the 4 employing colloid gold label biomacromolecules, superfluous Radioactive colloidal gold can not remove and remove, and marker is difficult to purifying, and labelled amount is uncertain, and the result who make detection, analyzes is inaccurate.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of visual bio-chip, it has solved use cost height in the background technology, detection, the inaccurate technical problem of analytical results.
Design of the present invention is:
A kind of preparation method of visual bio-chip, its special character is: the performing step of this method comprises
1) preparation gold-magnetic particles mark is labeled thing
(1) pre-treatment of gold-magnetic particles: get gold-magnetic particles, place centrifuge tube, add coupling buffer, with hand even, magnetic resolution is abandoned supernatant;
(2) be labeled the pre-treatment of thing: get the nucleic acid probe that is labeled the thing sulfydryl modification, the coupling buffer dilution with pH6.0～7.0 adds in the centrifuge tube, and the nucleic acid probe that makes sulfydryl modification is 1: 3 with the ratio of the amount of substance of gold-magnetic particles;
(3) be labeled the thing immobilized reactant: centrifuge tube is placed Air oscillator,, reacted 1～2 hour at 30～40 ℃ Celsius; Magnetic resolution is abandoned supernatant, obtains the nucleic acid probe of gold-magnetic particles mark;
(4) clean: add the cleaning buffer solution of pH6.0～7.0 in centrifuge tube, shake up, magnetic resolution is abandoned supernatant; Add the cleaning buffer solution of pH6.0～7.0 again, shake up, magnetic resolution is abandoned supernatant;
(5) preserve: in centrifuge tube, add the preservation damping fluid of pH6.0～7.0, in 2-6 Celsius ℃ preservation;
2) but the preparation visualizing biochip
(1) nucleic acid probe of getting the gold-magnetic particles mark for preparing adds centrifuge tube, adds the preservation damping fluid of pH6.0～7.0 again, and the nucleic acid probe of dilution gold-magnetic particles mark is to 100nM;
(2) get the nucleic acid probe of the gold-magnetic particles mark of 15～60 μ L dilution, be added on the gene chip surface;
(3) this gene chip is behind hybridization, washing, centrifugal drying, is immersed in the silver enhancement solution 10～20 minutes, and the simple substance deposition of silver is to the gold-magnetic particles surface.
Above-mentioned coupling buffer is good with the second triethylenetetraminehexaacetic acid ammonium damping fluid that is adopted as 0.1M; Be advisable with the phosphoric acid salt of the NaCl, the 10mM that adopt 0.3M, the damping fluid of 0.01% sodiumazide; Also can adopt or carbonate buffer solution; Or acetate buffer etc.
Above-mentioned preservation damping fluid and cleaning buffer solution all are advisable with the phosphoric acid salt of the NaCl, the 10mM that adopt 0.3M, the damping fluid of sodiumazide; Also can adopt phosphate buffered saline buffer; Or carbonate buffer solution; Or acetate buffer etc.
In the above-mentioned immobilized reactant that is labeled thing, centrifuge tube places the temperature of reaction of Air oscillator to be advisable with 37 ℃ Celsius; The temperature of the nucleic acid probe of described preservation gold-magnetic particles mark is advisable with 4 ℃ Celsius.
A kind of preparation method of visual bio-chip, its special character is: the performing step of this method comprises
1) preparation gold-magnetic particles mark is labeled thing
(1) pre-treatment of gold-magnetic particles: get gold-magnetic particles, place centrifuge tube, add coupling buffer, with hand even, magnetic resolution is abandoned supernatant;
(2) be labeled the pre-treatment of thing: get and be labeled thing antibody/albumen, be diluted to 0.1～0.4mg/mL, add in the centrifuge tube with the coupling buffer of pH6.0～7.0;
(3) be labeled the thing immobilized reactant: centrifuge tube is placed Air oscillator,, reacted 15～25 minutes at 30～40 ℃ Celsius; Magnetic resolution is got supernatant liquor, obtains the antibody/albumen of gold-magnetic particles mark;
(4) clean: add the cleaning buffer solution of pH6.0～7.0 in centrifuge tube, shake up, magnetic resolution is abandoned supernatant; Add the cleaning buffer solution of pH6.0～7.0 again, shake up, magnetic resolution is abandoned supernatant;
(5) sealing: add confining liquid in centrifuge tube, centrifuge tube is placed Air oscillator, at 30～40 ℃ Celsius, reacted 40～80 minutes, magnetic resolution is abandoned supernatant;
(6) clean: add cleaning buffer solution in centrifuge tube, shake up, magnetic resolution is abandoned supernatant, finishes once and cleans; Repeated washing is 2 times again;
(7) preserve: in centrifuge tube, add the preservation damping fluid of pH6.0～7.0, in 2-6 Celsius ℃ preservation;
2) but the preparation visualizing biochip
(1) gets the antibody of the gold-magnetic particles mark for preparing/albumen and add centrifuge tube, be added on antibody chip or protein chip surface;
(2) antibody chip or protein chip were hatched 40～80 minutes;
(3) again with behind the washing of antibody chip or protein chip, the centrifugal drying, be immersed in the silver enhancement solution 10～20 minutes, the simple substance deposition of silver is to the gold-magnetic particles surface.
In the above-mentioned pre-treatment that is labeled thing, get and be labeled thing antibody/albumen, be diluted to 0.25mg/mL, add in the centrifuge tube again and be advisable with the coupling buffer of pH7.0.
Above-mentioned coupling buffer is advisable with the NaHCO that adopts 0.1M; Also can adopt the NaCl of 0.5M, or the phosphoric acid salt of 0.5M, or the carbonate of 0.5M, or the acetate of 0.5M etc.
Above-mentioned confining liquid can adopt the skim-milk of adding 1mg/ml or phosphoric acid salt, carbonate or the acetate etc. of bovine serum albumin.
Above-mentioned cleaning buffer solution is advisable to adopt 0.5% the tween 20 1 * phosphoric acid salt that contains; Also can adopt carbonate or acetate etc.
Above-mentioned preservation damping fluid is advisable with the phosphoric acid salt of NaCl, the 10mM of employing 0.3M, the damping fluid of sodiumazide; Also can adopt phosphate buffered saline buffer, carbonate buffer solution or acetate buffer.
The present invention has the following advantages:
1. the detection sensitivity height detects quite with fluorescent method.
2. Zhi Bei biochip can prolonged preservation.
3. the expense that make, detection needs is all lower, is easy to popularize, apply.
4. with the gold-magnetic particles reagent that serves as a mark, but visual detection.
5. preparation, detecting operation is easy, fast, uses flexibly.
Description of drawings
Fig. 1 prepares the principle schematic of gene chip for the present invention.
Fig. 2 prepares the principle schematic of albumen/antibody chip for the present invention.
Fig. 3 prepares the principle schematic of sugar bio-chip for the present invention.
Fig. 4 is the result schematic diagram of gene chip of the present invention.
Embodiment
The present invention is with the gold-magnetic particles reagent that serves as a mark, nucleic acid probe, albumen and antibody that the difference labeling SH groups is modified, be respectively applied on the biochips such as gene chip, protein chip, antibody chip and carbohydrate chip, again biochip is immersed in the silver enhancement solution, behind the silver ion reduction, the simple substance deposition of silver is to the gold-magnetic particles surface, but the realization visualizing detects.Also available ordinary flat scanner scans biochip, obtains signal data, data is analyzed again.
With the gold-magnetic particles reagent that serves as a mark, mainly be the physical property of utilizing gold-magnetic particles gold surface electrostatic adhesion, with as the nucleic acid modified, protein, multiple biomacromolecule combination such as lipid forms gold-magnetic particles-nucleic acid complexes, gold-magnetic particles-albumen composition etc.Utilize the magnetic kernel of gold-magnetic particles to carry out magnetic resolution, obtain the mixture of purifying.Referring to Fig. 1-3.Play a part effective nuclear in the deposition of gold-magnetic particles simple substance silver behind silver ion reduction, have significant signal amplification, so, also available ordinary flat scanner scanning can with the naked eye be observed.
The preparation method of the nucleic acid probe that embodiment one, gold-magnetic particles labeling SH groups are modified:
1) pre-treatment of gold-magnetic particles: get 200 μ L gold-magnetic particles 1mg, place centrifuge tube, add coupling buffer 400 μ L, with hand even, magnetic resolution 2 minutes is abandoned supernatant.
2) be labeled the pre-treatment of thing: get the nucleic acid probe that is labeled the thing sulfydryl modification,, add in the centrifuge tube with the coupling buffer dilution.The nucleic acid probe of sulfydryl modification and gold-magnetic particles are 1: 3 by the ratio of amount of substance.
3) be labeled the thing immobilized reactant: centrifuge tube is placed Air oscillator,, reacted 1-2 hour at 37 ℃ Celsius; Magnetic resolution 2 minutes is abandoned supernatant.Obtain the nucleic acid probe of gold-magnetic particles mark.
4) clean: add cleaning buffer solution 400 μ L in centrifuge tube, shake up, magnetic resolution 2 minutes is abandoned supernatant; Add 400 μ L cleaning buffer solutions again, shake up, magnetic resolution 2 minutes is abandoned supernatant.
5) preserve: 7.0, the 4 ℃ of preservations of preservation pH of buffer of phosphoric acid salt, 0.01% sodiumazide of NaCl, 10mM of 0.3M that add 1mL are standby.Approximately can preserve more than February.
But the biochip of preparation visualizing:
1) nucleic acid probe of getting the gold-magnetic particles mark for preparing adds centrifuge tube, adds the PBS of NaCl, the 10mM of 0.3M, the preservation pH of buffer 7.0 of 0.01% sodiumazide again, the nucleic acid probe concentration dilution of gold-magnetic particles mark to 100nM.
2) get the nucleic acid probe of the gold-magnetic particles mark of 15～60 μ L dilution, be added on the gene chip surface.
3) this gene chip is behind hybridization, washing, centrifugal drying, be immersed in the silver enhancement solution 10～20 minutes, and silver ion reduction, the simple substance deposition of silver is to the gold-magnetic particles surface.On gene chip, can see the spot of black, referring to Fig. 4.
Embodiment two, gold-magnetic particles traget antibody/proteic preparation method:
1) pre-treatment of gold-magnetic particles: get 200 μ L gold-magnetic particles 1mg, place centrifuge tube, add coupling buffer 400 μ L, with hand even, magnetic resolution 2 minutes is abandoned supernatant.
2) be labeled the pre-treatment of thing: with antibody/albumen, as bovine serum (BSA) antibody/bovine serum albumin, be diluted to 0.25mg/mL with coupling buffer, the antibody-solutions of getting after the 400 μ L dilution adds in the centrifuge tube.
3) be labeled the thing immobilized reactant: centrifuge tube is placed Air oscillator,, reacted 20 minutes at 37 ℃ Celsius; Magnetic resolution 2 minutes is got supernatant liquor.Obtain the antibody/albumen of gold-magnetic particles mark.
4) clean: add cleaning buffer solution 400 μ L in centrifuge tube, shake up, magnetic resolution 2 minutes is abandoned supernatant; Add 400 μ L, 1 * phosphate buffered saline buffer again, shake up, magnetic resolution 2 minutes is abandoned supernatant.
5) sealing: add the 1mL confining liquid in centrifuge tube, centrifuge tube is placed Air oscillator, at 37 ℃ Celsius, reacted 1 hour, magnetic resolution 2 minutes is abandoned supernatant.
6) clean: the tween 20 1 * phosphoric acid salt that contains that adds 800 μ L 0.5% in centrifuge tube cleans cleaning buffer solution, shakes up, and magnetic resolution 2 minutes is abandoned supernatant, finishes once and cleans; Repeated washing 3 times.
7) preserve: 7.0, the 4 ℃ of preservations of preservation pH of buffer of phosphoric acid salt, 0.01% sodiumazide of NaCl, 10mM of 0.3M that add 1mL are standby.Approximately can preserve for 2 weeks.
But the biochip of preparation visualizing:
1) antibody/albumen of getting the gold-magnetic particles mark that 15～60 μ L prepare adds centrifuge tube, is added on antibody chip or the protein chip surface.
2) antibody chip or protein chip were hatched 1 hour; Washing, behind the centrifugal drying, be immersed in the silver enhancement solution 10～20 minutes, silver ion reduction, the simple substance deposition of silver is to the gold-magnetic particles surface.Can be visual on antibody chip or protein chip to the spot of black, referring to Fig. 4.
Claims (10)
1. preparation method of visual bio-chip, it is characterized in that: the performing step of this method comprises
1) preparation gold-magnetic particles mark is labeled thing
(1) pre-treatment of gold-magnetic particles: get gold-magnetic particles, place centrifuge tube, add coupling buffer, with hand even, magnetic resolution is abandoned supernatant;
(2) be labeled the pre-treatment of thing: get the nucleic acid probe that is labeled the thing sulfydryl modification, the coupling buffer dilution with pH6.0～7.0 adds in the centrifuge tube, and the nucleic acid probe that makes sulfydryl modification is 1: 3 with the ratio of the amount of substance of gold-magnetic particles;
(3) be labeled the thing immobilized reactant: centrifuge tube is placed Air oscillator,, reacted 1～2 hour at 30～40 ℃ Celsius; Magnetic resolution is abandoned supernatant, obtains the nucleic acid probe of gold-magnetic particles mark;
(4) clean: add the cleaning buffer solution of pH6.0～7.0 in centrifuge tube, shake up, magnetic resolution is abandoned supernatant; Add the cleaning buffer solution of pH6.0～7.0 again, shake up, magnetic resolution is abandoned supernatant;
(5) preserve: in centrifuge tube, add the preservation damping fluid of pH6.0～7.0, in 2-6 Celsius ℃ preservation;
2) but the preparation visualizing biochip
(1) nucleic acid probe of getting the gold-magnetic particles mark for preparing adds centrifuge tube, adds the preservation damping fluid of pH6.0～7.0 again, and the nucleic acid probe of dilution gold-magnetic particles mark is to 100nM;
(2) get the nucleic acid probe of the gold-magnetic particles mark of 15～60 μ L dilution, be added on the gene chip surface;
(3) this gene chip is behind hybridization, washing, centrifugal drying, is immersed in the silver enhancement solution 10～20 minutes, and the simple substance deposition of silver is to the gold-magnetic particles surface.
2. preparation method of visual bio-chip according to claim 1 is characterized in that: described coupling buffer is the second triethylenetetraminehexaacetic acid ammonium damping fluid of 0.1M; Or the damping fluid of the phosphoric acid salt of the NaCl of 0.3M, 10mM, 0.01% sodiumazide; Or carbonate buffer solution; Or acetate buffer.
3. preparation method of visual bio-chip according to claim 1 and 2 is characterized in that: described preservation damping fluid and cleaning buffer solution are the phosphoric acid salt of NaCl, the 10mM of 0.3M, the damping fluid of sodiumazide; Or phosphate buffered saline buffer; Or carbonate buffer solution; Or acetate buffer.
4. preparation method of visual bio-chip according to claim 3 is characterized in that: in the described immobilized reactant that is labeled thing, it is 37 ℃ Celsius that centrifuge tube places the temperature of reaction of Air oscillator; The temperature of the nucleic acid probe of described preservation gold-magnetic particles mark is 4 ℃ Celsius.
5. preparation method of visual bio-chip, it is characterized in that: the performing step of this method comprises
1) preparation gold-magnetic particles mark is labeled thing
(1) pre-treatment of gold-magnetic particles: get gold-magnetic particles, place centrifuge tube, add coupling buffer, with hand even, magnetic resolution is abandoned supernatant;
(2) be labeled the pre-treatment of thing: get and be labeled thing albumen, be diluted to 0.1～0.4mg/mL, add in the centrifuge tube with the coupling buffer of pH6.0～7.0;
(3) be labeled the thing immobilized reactant: centrifuge tube is placed Air oscillator,, reacted 15～25 minutes at 30～40 ℃ Celsius; Magnetic resolution is got supernatant liquor, obtains the albumen of gold-magnetic particles mark;
(4) clean: add the cleaning buffer solution of pH6.0～7.0 in centrifuge tube, shake up, magnetic resolution is abandoned supernatant; Add the cleaning buffer solution of pH6.0～7.0 again, shake up, magnetic resolution is abandoned supernatant;
(5) sealing: add confining liquid in centrifuge tube, centrifuge tube is placed Air oscillator, at 30～40 ℃ Celsius, reacted 40～80 minutes, magnetic resolution is abandoned supernatant;
(6) clean: add cleaning buffer solution in centrifuge tube, shake up, magnetic resolution is abandoned supernatant, finishes once and cleans; Repeated washing is 2 times again;
(7) preserve: in centrifuge tube, add the preservation damping fluid of pH6.0～7.0, in 2-6 Celsius ℃ preservation;
2) but the preparation visualizing biochip
(1) albumen of getting the gold-magnetic particles mark for preparing adds centrifuge tube, is added on antibody chip or protein chip surface;
(2) antibody chip or protein chip were hatched 40～80 minutes;
(3) again with behind the washing of antibody chip or protein chip, the centrifugal drying, be immersed in the silver enhancement solution 10～20 minutes, the simple substance deposition of silver is to the gold-magnetic particles surface.
6. preparation method of visual bio-chip according to claim 5 is characterized in that: in the described pre-treatment that is labeled thing, get and be labeled thing albumen, be diluted to 0.25mg/mL with the coupling buffer of pH7.0, add in the centrifuge tube.
7. preparation method of visual bio-chip according to claim 6 is characterized in that: described coupling buffer is the NaHCO of 0.1M; Or the NaCl of 0.5M; Or 0.5M phosphoric acid salt; Or 0.5M carbonate; Or 0.5M acetate.
8. preparation method of visual bio-chip according to claim 7 is characterized in that: described confining liquid is the skim-milk of adding 1mg/ml or phosphoric acid salt, carbonate or the acetate of bovine serum albumin.
9. preparation method of visual bio-chip according to claim 8 is characterized in that: described cleaning buffer solution be 0.5% contain tween 20 1 * phosphoric acid salt; Or carbonate or acetate.
10. preparation method of visual bio-chip according to claim 9 is characterized in that: the phosphoric acid salt of NaCl, 10mM that described preservation damping fluid is 0.3M, the damping fluid of sodiumazide; Or phosphate buffered saline buffer; Carbonate buffer solution; Or acetate buffer.
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| CN103257233B (en) * | 2013-05-31 | 2015-08-05 | 南京祥中生物科技有限公司 | The biochip of Visual retrieval Multiple Classes of Antibiotics while of a kind of, illegal adjuvant and biotoxin and method |
| CN105002566A (en) * | 2015-07-30 | 2015-10-28 | 江苏猎阵生物科技有限公司 | Visual chip and preparation method thereof and method for chip visualization |
| CN105733842B (en) * | 2016-03-21 | 2018-10-19 | 珠海国际旅行卫生保健中心 | A kind of gene chip probes stripper |
| CN109821134A (en) * | 2019-01-18 | 2019-05-31 | 武汉大学 | A kind of three-dimensional network bioprobe and the preparation method and application thereof for living body |
| CN116953224B (en) * | 2023-05-31 | 2024-07-19 | 复旦大学附属华山医院 | Luminous reagent for detecting FGF21 and application thereof |
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