CN1390955A - Nano-class amplifying detection method for biochip - Google Patents

Abstract

A nano amplifying detection method for biochip includes such steps as labelling nucleic acid or protein with nano metal particles, hybridizing said nucleic acid or protein with the probe or tissue on biochip, using a reactive metal reagent to react on said nano metal particle, amplifying said nano metal particle and optical detecting. Its advantages are high sensitivity and specificity, high detection speed, and low cost.

Description

A kind of nanometer amplification detecting process of biochip

Technical field:

The present invention relates to the nano metal amplification detecting process in biochip field, particularly a kind of biochip.

Technical background:

Biochip technology is a kind of new Measurement for Biotechnique that development in recent years is got up.It comprises gene chip, protein chip, organization chip etc.It combines the micro-fabrication technique and the molecular biology of semi-conductor industry, produces two-dimentional probe array on a substrate surface.Then with known probe " hybridization " on unknown object to be determined and the chip, to determine unknown molecular.Biochip technology is owing to incite somebody to action a large amount of different probe stationary simultaneously on upholder, so can disposable multiple different substances in the sample be detected and analyze, the traditional detection technological operation is numerous and diverse, level of automation is low, detect deficiencies such as target sequence quantity is few, detection efficiency is low thereby solved.Therefore this technology has at a high speed, high-throughput, high-level efficiency, detects the characteristics of the biological sample of hybridizing with it concurrently, compares with traditional diagnostic techniques, and chip technology has remarkable advantages.And, by designing different probe arrays, using specific analytical procedure can make this technology have multiple different using value.This technology is in a plurality of fields widespread uses such as gene sequencing, sudden change detection, polymorphism analysis, expression pattern analysis, drug development, medicals diagnosis on disease at present.

Yet up to the present, the domestic and international biochip product-feed market of all not having the acquisition certification is used for clinical, and the biochip and the detection system thereof that particularly can directly apply to common lab and field environment do not see that as yet the report of succeeding in developing is arranged.

The method that its major cause is to use exists various deficiencies.As on marking method, fluorescent method is maximum method of using at present, but its sensitivity not high, need lucifuge, want rapid detection after hybridization is finished in order to avoid fluorescent quenching, the result detects in addition needs with the price specific installations such as laser scanner of costliness extremely; Isotope-labelling method has the inconvenience of use owing to need isotropic substance and radioautograph, complicated operation, the shortcoming of growing, pollution being arranged consuming time.Because the restriction of labeling technique and hybridization signal amplifying technique, for improving detection sensitivity and specificity, common way is at mark with before analyzing sample to be carried out the amplification of appropriate procedure, and the most frequently used is round pcr.Yet, can influence pcr amplification efficient with fluorescent mark, isotopic labeling commonly used as ³²P and ³³The P transformation period is too short again, all is difficult for promoting the use of.And pcr amplification both needed to design primer, and the PCR instrument need be set up different amplification systems, amplification method and amplification condition to the target sequence of each detected object again, and this just increases workload and work difficulty greatly.In order to address these problems, many companies have carried out various explorations both at home and abroad, but method does not reach practical application as yet at present.

In a word,, obtain attracting attention of common people although chip technology has been obtained significant progress, the preparation of sample, probe is synthetic with fix, the reading and analyze etc. and still exist many insoluble problems aspect the several of mark of molecule, data.Particularly lower, the poor repeatability of technical costs costliness, complexity, detection sensitivity, analysis are general encloses problem such as narrower and has limited the application of this technology in biological detection.And these the very corn of a subject reasons are mainly aspect these two: being what improve that sensitivity uses is hybridization front signal amplifying technique, and there are various deficiencies in the signal reporter molecules that is used for mark hybridization sample.

The gold grain of nanometer diameter is used for the existing long history of life science tracer technique, stablizes because it has chemical property, can be marked on albumen and nucleic acid.Simultaneously, can form bigger by 10 after it reacts with silver than original volume ⁶Black particle doubly, can detect by an unaided eye or with common recording unit as take a picture, the characteristics of sweep record, being subjected to scientific research personnel's attention in recent years, then is a impressive progress during bio signal detects in recent years with not possessing the application of the discrete nanometer gold of adsorptive power as the signal reporter molecules particularly.At present, that what adopt is Radioactive colloidal gold is more as the nanometer gold of signal reporter molecules.Radioactive colloidal gold is owing to be not the single molecule with fixed sturcture, and its surface is positively charged under physiological pH, and therefore, it does not need to handle especially just can be with electronegative oligonucleotide combination.But this combination is illusive.The latter is the macromole complex body that the gold atom by different quantities constitutes, and gold atom is positioned at this macromolecular surface makes it constitute the stable and stereoscopic configuration by halide ions.The nm gold particles neutral that constitutes by gold atom, but because gold atom is positioned at the particulate surface, it is carried out specific modification just can make itself and nucleic acid form covalent attachment, so just with the nm gold particles mark on specific nucleic acid, albumen, can be used as the use of signal reporter molecules.Owing to be covalent attachment, therefore, it has more superiority than Radioactive colloidal gold.

NATURE magazine in 1996 has been delivered two pieces simultaneously makes nanometer gold be self-assembled into the report of nanocrystal as link molecule the DNA, Zehbe had reported with the hybridization in situ technique of nanometer gold as the signal reporter molecules in 1997, under the situation of developing in conjunction with silver, can make the detection sensitivity of viral nucleic acid in the tissue is brought up to 1 copy by 10～50 copies, the author thinks that so high sensitivity can make this method replace the original position round pcr in a lot of the application.But have not yet to see the report that this technology is used for biological chip testing technology.

Summary of the invention:

The objective of the invention is to set up a kind of with the bio-chip test method of nano metal as the signal reporter molecules.This detection method can be amplified the signal of results of hybridization, can reach to walk around sample is increased so that improve the requirement of detection sensitivity; And the signal that produces is promptly stable, is easy to again detect, and the equipment and the technology that make the result detect, analyze are greatly simplified.Thereby can utilize the high-throughput of biochip technology, high responsive, high special characteristic, overcome the main drawback that present detection chip exists.Because the biochip of this rapid detection that the present invention's research provides has hybridization, test set is simple, consistent being easy to grasped, highly sensitive, good reproducibility, the characteristic of applied range etc., it will become can be widely used in common lab, even the biochip of new generation of field environment.

The technical solution adopted in the present invention is such: i.e. a kind of nanometer amplification detecting process of biochip, and it is characterized in that: method comprises following three steps:

(1), uses the metallic particles labeling nucleic acid or the albumen of nanometer diameter;

(2), the nucleic acid of mark or albumen are combined with probe hybridization on the biochip;

(3), utilize can with the metal reagent of the metallic particles generation metallographic phase reaction of the nanometer diameter of this mark and the nano metal reaction of having hybridized mark on the nucleic acid that is combined on the biochip or the albumen, amplify nano-metal particle and also carry out ordinary optical and detect.

Term used in the present invention " biochip " is to make up the array that forms by biomacromolecule or tissue apposition on glass, pottery, tinsel or substrate materials such as nylon membrane, nitrocellulose filter.The example of biochip comprises gene (nucleic acid) chip, cell chip, protein chip, antibody chip or organization chip etc.

Nucleic acid of the present invention comprises DNA, RNA, cDNA or peptide nucleic acid(PNA).

Albumen of the present invention comprises various antibody, antigen.

Term used in the present invention " metallic particles of nanometer diameter " is meant the metallic particles of diameter between 1.0-1000nm.The example comprises nanometer gold, nanometer vanadium, nanometer lead or nanometer silver etc., also comprises the metallic particles of modifying the nanometer diameter that is combined with groups such as amido, sulfydryl, acyl group or vitamin H.The metallic particles of the preferred nanometer diameter that the present invention uses is the nanometer gold of diameter as single maleic diacetyl imine beautify of 1.4nm.

Of the present inventionly can comprise pure metal reagent and metallic compound reagent such as gold and silver, iron, as lithium silver, quinhydrones silver with the metal reagent of the metallic particles generation metallographic phase reaction of the nanometer diameter of this mark.The preferred of the present invention's use can be lithium silver with the metal reagent of nanometer gold generation particle intensified response.

In the above-mentioned steps of the present invention (), the method for the metallic particles labeling nucleic acid of nanometer diameter comprises:

(1) by the mark of oligonucleotide joint realization to nucleic acid;

Or (2) are by the mark of primer realization to product nucleic acid;

Or (3) direct mark that nucleic acid is realized.

Term used in the present invention " oligonucleotide joint " is meant one section nucleic acid substances by 1-2000 based composition, comprises and has carried out being modified with as groups such as sulfydryl, amino, acyl groups.The oligonucleotide joint of the nanometer gold reaction of single maleic diacetyl imine beautify of the preferred and 1.4nm that the present invention uses is: 5 '-AAA AAA AAA AA-(CH ₂) ₆-SH-3 '.

Term used in the present invention " primer ", " PCR ", " RT-PCR " define reference: " round pcr experiment guide ", the C.W. Dieffenbacher, G.S. gets Vicks VapoRub and reins in.Science Press, in August, 1998 first version.

Term used in the present invention " product nucleic acid " is meant the nucleic acid substances that is generated by PCR or RT-PCR reaction, as DNA, cDNA.

The oligonucleotide joint that passes through in the above-mentioned steps () is realized the step of the marking method of nucleic acid is comprised:

(1), preparation nano metal solution;

(2), nano metal solution is mixed with oligonucleotide, nano metal is 1-100 with the ratio of oligonucleotide molecules number: 1, and reaction obtains to contain the oligonucleotide joint of nano metal;

(3), extract specimen dna, remove the phosphate group of the 5 ' end of DNA by the dephosphorylation reaction, prevent that himself from connecting;

(4), under the ligase enzyme effect, the oligonucleotide joint of combining nano metal mark and the specimen dna of above-mentioned processing obtain the sample DNA of nano metal mark.

Realize in the product nucleic acid labeling methods by primer in the described step () that adopt by the mark of PCR reaction primer realization to product D NA, the step of method comprises:

(1), preparation nano metal solution;

(2), nano metal solution is mixed with the PCR primer, nano metal is 1-100 with the ratio of primer molecule number: 1, obtain the PCR primer of nano metal mark;

(3), utilizing this labeled primer to carry out PCR reacts, obtains the PCR product D NA of nano metal mark.

Realize in the product nucleic acid labeling methods by primer in the described step () that adopt by the mark of RT-PCR reaction primer realization to product cDNA, the step of method comprises:

(1), preparation nano metal solution;

(2), nano metal solution is mixed with the RT-PCR primer, nano metal is 1-100 with the ratio of primer molecule number: 1, obtain the RT-PCR primer of nano metal mark;

(3), utilizing this labeled primer to carry out RT-PCR reacts, obtains the RT-PCR product cDNA of nano metal mark.

The marking method that directly nucleic acid is realized that adopts in the above-mentioned steps () is: react by the end group with nucleic acid, thereby make the end modified compound that sulfur-bearing is arranged or contain the imide class of nucleic acid, this compounds can react with nano metal, realizes the mark of nano metal to nucleic acid.

The method steps of the metallic particles labelled protein of nanometer diameter is in the above-mentioned steps ():

(1), preparation nano metal solution;

(2), the albumen handled of nano metal solution and reduction mixes, nano metal is counted ratio with protein molecular and was not less than 5: 1, obtains the albumen of nano metal mark.

The nucleic acid of the nano metal mark in the described step (two) and the step of the probe hybridization on the biochip are:

(1), the DNA to the nano metal mark carries out denaturing treatment;

(2), the DNA of nano metal mark that drips denaturing treatment is in biochip surface, carries out hybridization;

(3), clean the DNA that bonded nano metal mark is not hybridized in removal;

(4), drying treatment is standby.

The albumen of the nano metal mark in the described step (two) and the probe bonded step on the biochip are:

(1), PBS handles biochip, to block non-specific proteic binding site and to reduce non-specific;

(2), the albumen that drips the nano metal mark on biochip, educate reaction altogether;

(3), rinsing, remove the albumen of unconjugated nano metal mark;

(4), dry standby.

Comprise in the described step (three):

(1), drip the metallographic phase reaction reagent in chip surface, carry out metallographic phase and amplify reaction;

(2), clean removal unreacted metal phase reaction reagent;

(3), observations.The advantage and the effect of present technique invention are as follows:

(1) the nanometer amplifying technique of the present invention's employing can be amplified to 10 to the signal of hybridization ^4-8Doubly, have high detection sensitivity and specificity.

(2) the nanometer amplifying technique of the present invention's employing can be avoided the PCR reaction, thereby has simplified the treating processes of sample, has shortened detection time.

(3) the nanometer amplifying technique of the present invention's employing does not need expensive detector, has reduced the use cost and the experiment condition of chip.

In a word, this technology succeeds in developing the high-throughput that will really realize chip technology, special, responsive, characteristics fast.Can carry out rapid and precise detection and evaluation to common infection pathogen and difficult inspection pathogenic bacteria, can carry out the research of a certain or a plurality of specific genes or relative expression product, can carry out the research of gene and protein and disease relationship, the checking of disease related gene and the exploitation and the screening of novel drugs, the molecular diagnosis of disease, aspects such as the tracking of therapeutic process and prognosis are for the prevention and the treatment of clinical disease provides important directive function; This technology also can be applied under field condition and implement the investigation of battlefield pathogen distribution, the early diagnosis that war wound infects by the reviewer of basic unit simultaneously, and aspects such as the early discovery of biological warfare agent will produce favorable economic benefit and social benefit.

Embodiment

Embodiment 1 is by the nano gold mark of oligonucleotide joint realization to nucleic acid

Step is as follows:

(1), dissolving nanometer gold 6nmol in 0.02ml Virahol or DMSO, deionized water is diluted to 0.2ml.

(2), nano-Au solution mixes the (0.01M that sodium phosphate buffer concentration is in the end reaction system with the oligonucleotide sodium phosphate buffer, contain 150mMNaCl, 1mMEDTA, pH5-8), nanometer gold is 1-100 with oligonucleotide molecules number ratio: 1, educated altogether 2-24 hour for 4-37 ℃.Be prepared into the oligonucleotide joint that contains nanometer gold.

(3), behind the ultrasonic fracture specimen dna long-chain, reaction solution below in Eppendorf tube, adding respectively, full dose be 50 μ l:DNA fragments at TE buffer 1-20pmol, 10 * Alkaline Phosphatasebuffer, 5 μ l, CIAP (10-30U/ul) 1-2ul, aqua sterilisa adds to 50 μ l.37-50 ℃, 15-120 minute insulation.Phenol/chloroform (1: 1) extracting 1-3 time.Add the NaCL 2.5 μ l (final concentration 150mM) of 3M.Add the cold ethanol of 125 μ l (2.5 times), at-20 ℃ of cold insulation 30-60 minutes.Centrifugal recovery precipitates, after cleaning with 200 μ l, 70% cold ethanol, and drying under reduced pressure.With the TE buffer dissolution precipitation below the 20 μ l.

(4), specimen dna is heated to 90-100 ℃, after 5-20 minute, it is cooled to rapidly below 4 ℃.

(5), in reaction Tube pipe, modulate following reaction solution, full dose 50 μ l.The sample DNA 1-2 μ l of strand, the oligonucleotide joint 5-10 μ l of nano gold mark, 10 * T4 RNA Ligase Buffer, 10 μ l, 0.1%BSA 3 μ l, T4 RNA Ligase 40-100U, PEG#6000 final concentration 25%, aqua sterilisa add to 50 μ l.5 ℃ of reactions 12-24 hour.The 0.5M EDTA termination reaction that adds 2 μ l.The oligonucleotide joint concentration of described nano gold mark was not less than 5: 1 with the ratio of the molecule number of the sample DNA of strand.

Embodiment 2 is by the nano gold mark of PCR reaction primer realization to product D NA

Step is as follows:

(1), dissolving nanometer gold 6nmol in 0.02ml Virahol or DMSO, deionized water is diluted to 0.2ml.

(2), nano-Au solution mixes with the sodium phosphate buffer of the special PCR primer 1 of DNA to be checked and 2 respectively that (concentration of sodium phosphate buffer is 0.01M in the end reaction system, contain 150mMNaCl, 1mMEDTA, pH5-8), nanometer gold is 1-100 with primer molecule number ratio: 1, educated altogether 2-24 hour for 4-37 ℃.Preparation contains the special PCR primer 1 and 2 of DNA to be checked of nano gold mark.

(3), add following anti-composition (100 μ l reaction system) in the reaction Tube pipe: Taq enzyme (5U/ μ l) 0.5 μ l, 10 * Taq enzyme Buffer, 10 μ l, MgCl ₂(25mM) 8 μ l, dNTP (every kind of each 2.5mM) 8 μ l, sample DNA 1 μ g, golden labeled primer 1 (20 μ M) 1-5 μ l, golden marking primer 2 (20 μ M) 1-5 μ l adds sterilization ddH ₂O to 100 μ l.

(4), be put in the PCR instrument of preheating, program is as follows: 95 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ of 45-60 seconds.Carry out 30-40 circulation.Last 72 ℃ were extended 10 minutes.

Embodiment 3 is by the nano gold mark of RT-PCR reaction primer realization to product cDNA

Step is as follows:

(1), dissolving nanometer gold 6nmol in 0.02ml Virahol or DMSO, deionized water is diluted to 0.2ml.

(2), nano-Au solution mixes the (0.01M that sodium phosphate buffer concentration is in the end reaction system with poly-T primer sodium phosphate buffer, contain 150mMNaCl, 1mMEDTA, pH5-8), nanometer gold is 1-100 with random primer molecule number ratio: 1, educated altogether 2-24 hour for 4-37 ℃.Be prepared into the poly-T primer that contains nano gold mark.

(3), in reaction Tube pipe, add following reaction solution: dNTP (every kind of each 2.5mmol/L) 2.5 μ l, 0.1mol/L mercaptoethanol 2.0 μ l, RNasin (40,000U/ml) 0.5 μ l, poly-T primer (20-40U/ml) the 2.0 μ l of nano gold mark, 3.0 μ g RNA (deionized water dilution), 18.8 μ l.Heated mixt 5 minutes to 70 ℃, cooled on ice.Brief centrifugal sample.

(4), add again in the reaction Tube pipe: ThermoScript II damping fluid 5.0 μ l, ThermoScript II 200U1.2 μ l.Causing the reaction final volume is 2.5 μ l.37 ℃ of insulations are after 60 minutes, 90 ℃ of sex change 5 minutes, cooled on ice 5 minutes.

Embodiment 4 nano gold mark albumen

Step is:

(1), soluble protein 0.15mg in the 0.1M sodium phosphate buffer, pH6.0 contains 5mM EDTA (1ml), adds MEA (8mg).Incubated at room 1 hour;

(2), gel-filtration chromatography separates reductive albumen.Elutriant uses 0.02M sodium phosphate, pH6.5,150mMNaCl, 1mM EDTA.Reductive albumen is in first peak elutriant;

(3), dissolving nanometer gold reagent 6nmol in the 0.02ml Virahol, deionized water is diluted to 0.2ml.

(4), active nano-Au solution adds in the reductive albumen, wherein nanometer gold is 10: 1 with the ratio of the proteic molecule number of reduction, educates altogether 2-12 hour for 4-37 ℃;

(5), utilize gel-filtration chromatography to separate unlabelled nm gold particles from antibody complex, elutriant 0.02M sodium phosphate pH7.4,150mMNaCl wash-out.First incarnadine peak is a labeled complex, and second dark-coloured band is unconjugated nano metal.Repeat once can further obtain purification effect.

The nucleic acid of embodiment 5 nano gold marks and the probe hybridization on the DNA chip

Step is:

(1), the DNA chip of preparation is placed in the hybridization chamber of preheating, add 5.0 μ l, 5 * SSC-2g/LSDS in hybridization chamber to keep humidity.

(2), 1.0 μ l nano gold mark DNA add in the 9.0 μ l hybridization buffers, in 95 ℃ of sex change 5-10 minutes, ice bath immediately.

(3), drip DNA sample hybridization buffer in chip surface, add a cover the cover glass of one 2cm * cm, sealing hybridization cell immerses in the 40-60 ℃ of water-bath, hybridizes 1-10 hour.

(4), take out chip and immerse immediately in 1 * SSC-0.1%SDS scavenging solution of room temperature, cleaned 5 minutes.Move in 40-60 ℃ of 0.1 * SSC-0.1%SDS scavenging solution and washed 5-10 minute.Cleaned 5 minutes with fresh 0.1 * SSC-0.1%SDS more immediately, the back moves into 0.1 * SSC and cleaned 5 minutes, to remove SDS.

(5), lucifuge drying.

The albumen of embodiment 6 nano gold marks combines with the probe on the protein chip

Step is:

(1), the protein chip with preparation places in 37 ℃ the wet box, drip 10.0 μ l, 1% PBS-BSA damping fluid (20mM phosphoric acid buffer pH7.40, contain 150mM NaCl, 0.5%BSA, 0.1% glycerine, 0.05%Tween 20) on the chip, to block nonspecific protein binding site and to reduce nonspecific antibodies.

(2), discard the PBS-BSA on the chip, do not wash, drip the nano metal binding substances of 10 times of PBS-BSA damping fluids dilution, the reaction solution of educating altogether 0.5-3 hour and gently shook frequently.

(3), rinsing, (3 * 1min), PBS (3 * 1min) for PBS-BSA.

(4), dry.Embodiment 7 signals amplify reaction test

Utilize lithium argentum reagent and the nanometer gold reaction of having hybridized mark on the nucleic acid that is combined on the biochip or the albumen, amplify nm gold particles and with the naked eye or the step of ordinary optical instrument detecting comprise:

(1), lithium argentum reagent 2ml adds chip surface, room temperature reaction 3-7 minute, 0.1 * SSC cleaned 5 minutes.Dry.

(2), ordinary optical instrument (as microscope, scanner etc.) or naked eyes direct viewing result, black particle is promptly represented the hybridization positive findings.Embodiment 8 implements the detection reagent of gene chip by the oligonucleotide joint labeling nucleic acid: the nanometer gold reagent of single maleic diacetyl imine beautify (diameter 1.4nm), the lithium silvering solution is available from Nanoprobes company.

The two hexyl deoxy-oligonucleotides of 3 ' two sulphur: 5 '-AAA AAA AAA AA-(CH ₂) ₆-SH-3 ' and verdigris

The pseudomonas specific probe: 5 '-TCC TAT GGT AAA GAG CGT CCG-3 ' is by Shanghai

Sangon company provides.

The DNA extraction test kit is available from Shanghai Shenergy Biocolor BioScience ﹠ Technology Company

CIAP and 10 * T4 RNA Ligase are provided by precious biotech firm.Step:

(1), DNA chip self-control (reference: Linda A.Chrisey, Gil U Lee and C.ElizabethO ' Ferrall.Covalent attachment of synthetic DNA to self-assembled monolayerfilms.Nucleic Acids Research, 1996,24 (15): 3031-3039), the DNA chip of preparation places in the hybridization chamber of preheating, add 5.0 μ l, 5 * SSC-2g/LSDS in hybridization chamber to keep humidity.

(2), oligonucleotide 9.6nmol and TECP-HCl 6.2mmol, 0.1M sodium phosphate buffer (pH8.0 contains 51mMEDTA) mixes.Carry out reduction reaction under 37 ℃ of the temperature.

(3), dissolving nanometer gold 6nmol in the 0.02ml Virahol, deionized water is diluted to 0.2ml.

(4), nano-Au solution mixes the (0.01M that sodium phosphate buffer concentration is in the end reaction system with the oligonucleotide sodium phosphate buffer that as-reduced SH modifies, contain 150mMNaCl, 1mMEDTA, pH6.5), it is 10: 1 that nanometer gold is counted ratio with thiol molecule, educated altogether 6 hours, and be prepared into the oligonucleotide joint that contains nanometer gold for 4 ℃.Reaction product HPLC separation and purification, first incarnadine peak is a labeled complex, repeats once can further obtain purification effect.Labeled complex is stored in 0.02M sodium phosphate, 150mM NaCl, 0.05% sodiumazide (pH7.5).

(5), extract genomic dna in the patient blood.(reference reagent box specification sheets)

(6), behind the ultrasonic fracture specimen dna long-chain, reaction solution below in Eppendorf tube, adding respectively, full dose is 50 μ l:DNA fragment 1-20pmol in the TE damping fluid, 10 * Alkaline phosphoric acid buffer, 5 μ l, CIAP (10-30U/ul) 1-2ul, aqua sterilisa adds to 50 μ l.Insulation in 37 ℃, 15 minutes.Phenol/chloroform (1: 1) extracting 1 time.Add the NaCL 2.5 μ l (final concentration 150mM) of 3M.Add the cold ethanol of 125 μ l (2.5 times) ,-20 ℃ of cold insulations 30 minutes.Centrifugal recovery precipitates, after cleaning with 200 μ l, 70% cold ethanol, and drying under reduced pressure.With the TE damping fluid dissolution precipitation below the 20 μ l.

(7), specimen dna is heated to 98 ℃, after 10 minutes, it is cooled to rapidly below 4 ℃.

(8), in reaction Tube pipe, modulate following reaction solution, full dose 50 μ l.The sample DNA 1-2 μ l of strand, the oligonucleotide joint 5-10 μ l of nano gold mark, 10 * T4 RNA Ligase Buffer, 10 μ l, 0.1%BSA 3 μ l, T4 RNA Ligase 40-100U, PEG#6000 final concentration 25%, aqua sterilisa add to 50 μ l.5 ℃ of reactions 12 hours.The 0.5M EDTA termination reaction that adds 2 μ l.

(9), 1.0 μ l gold marker DNA adds in the 9.0 μ l hybridization buffers, in 95 ℃ of sex change 10 minutes, ice bath immediately.

(10), drip this DNA sample hybridization buffer in chip surface, add a cover the cover glass of one 2cm * cm, sealing hybridization cell immerses in 42 ℃ of water-baths, hybridizes 1 hour.

(11), take out chip and immerse immediately in 1 * SSC-0.1%SDS scavenging solution of room temperature, cleaned 5 minutes.Move in 40 ℃ of 0.1 * SSC-0.1%SDS scavenging solutions and washed 5 minutes.Cleaned 5 minutes with fresh 0.1 * SSC-0.1%SDS more immediately, the back moves into 0.1 * SSC and cleaned 5 minutes, to remove SDS.The lucifuge drying.

(12), lithium silvering solution 2ml adds chip surface, room temperature reaction 5 minutes, 0.1 * SSC cleaning 5 minutes.Dry.

(13), ordinary optical instrument (as microscope, scanner etc.) or naked eyes direct viewing result, black particle is promptly represented the hybridization positive findings.Embodiment 9 implements the detection reagent of gene chip by reverse transcription PCR (RT-PCR) marked product cDNA: the nanometer gold reagent of single maleic diacetyl imine beautify (diameter 1.4nm), the lithium silvering solution is available from Nanoprobes company.

Two hexyl random primers of 5 ' two sulphur and hepatitis C virus specific probe: 5 '-GGG AGT GAT

CTA TGG TGG AG-3 ' is provided by Shanghai Sangon company.

RNA extracts test kit, RT-PCR test kit available from Shanghai Shenergy Biocolor BioScience ﹠ Technology Company's step:

(1), DNA chip self-control (reference: Linda A.Chrisey, Gil U Lee and C.ElizabethO ' Ferrall.Covalent attachment of synthetic DNA to self-assembled monolayerfilms.Nucleic Acids Research, 1996,24 (15): 3031-3039), the DNA chip of preparation places in the hybridization chamber of preheating, add 5.0 μ l, 5 * SSC-2g/LSDS in hybridization chamber to keep humidity.

(2), two hexyl random primer 9.6nmol of 5 ' two sulphur and TECP-HCl 6.2mmol, 0.1M sodium phosphate buffer (pH8.0 contains 51mMEDTA) mixing.Carry out reduction reaction under 37 ℃ of the temperature.

(3), dissolving nanometer gold 6nmol in 0.02ml Virahol or DMSO, deionized water is diluted to 0.2ml.

(4), nano-Au solution mixes the (0.01M that sodium phosphate buffer concentration is in the end reaction system with the random primer sodium phosphate buffer that as-reduced SH modifies, contain 150mMNaCl, 1mMEDTA, pH6.5), it is 10: 1 that nanometer gold is counted ratio with thiol molecule, educated altogether 6 hours, and be prepared into the random primer that contains nanometer gold for 4 ℃.Reaction product HPLC separation and purification, first incarnadine peak is a labeled complex, repeats once can further obtain purification effect.Labeled complex is stored in 0.02M sodium phosphate, 150mMNaCl, 0.05% sodiumazide (pH7.5).

(5), extract geneome RNA in the patient blood.(reference reagent box specification sheets)

(6), in reaction Tube pipe, add following reaction solution: dNTP (every kind of each 2.5mmol/L) 2.5 μ l, 0.1mol/L mercaptoethanol 2.0 μ l, RNasin (40,000U/ml) 0.5 μ l, the random primer of nano gold mark (20-40U/ml) 2.0 μ l, 3.0 μ g RNA (deionized water dilution), 18.8 μ l.Heated mixt 5 minutes to 70 ℃, cooled on ice.Brief centrifugal sample.

(7), add again in the reaction Tube pipe: ThermoScript II damping fluid 5.0 μ l, ThermoScript II 200U1.2 μ l.Causing the reaction final volume is 2.5 μ l.37 ℃ of insulations are after 60 minutes, 90 ℃ of sex change 5 minutes, cooled on ice 5 minutes.

(8), 1.0 μ l gold mark cDNA adds in the 9.0 μ l hybridization buffers.Drip this cDNA sample hybridization buffer in chip surface, add a cover the cover glass of one 2cm * cm, sealing hybridization cell immerses in 42 ℃ of water-baths, hybridizes 1 hour.

(9), take out chip and immerse immediately in 1 * SSC-0.1%SDS scavenging solution of room temperature, cleaned 5 minutes.Move in 40 ℃ of 0.1 * SSC-0.1%SDS scavenging solutions and washed 5 minutes.Cleaned 5 minutes with fresh 0.1 * SSC-0.1%SDS more immediately, the back moves into 0.1 * SSC and cleaned 5 minutes, to remove SDS.The lucifuge drying.

(10), lithium silvering solution 2ml adds chip surface, room temperature reaction 4 minutes, 0.1 * SSC cleaning 5 minutes.Dry.

(11), ordinary optical instrument (as microscope, scanner etc.) or naked eyes direct viewing result, black particle is promptly represented the hybridization positive findings.Embodiment 10 implements the detection reagent of gene chip by PCR reaction marking product D NA: the nanometer gold reagent of single maleic diacetyl imine beautify (diameter 1.4nm), the lithium silvering solution available from

Nanoprobes company.The two hexyl deoxy-oligonucleotide primer 1:5 ' of colon bacillus 5 ' two sulphur

SH-(CH ₂) ₆-TAT GAA CTG TGC GTC ACA GCC-3 ', the two hexyls of 5 ' two sulphur take off

Oxygen Oligonucleolide primers 2:5 ' SH-(CH ₂) ₆-CAT CAG CAC GTT ATC GAA

TCC-3 ' and colon bacillus specific probe: 5 '-TTC TAC TTT ACT GGC TTT

GGT CG-3 ' is provided by Shanghai Sangon company.

DNA extraction test kit, pcr amplification test kit are available from Shanghai Shenergy Biocolor BioScience ﹠ Technology Company's step:

(1), DNA chip self-control (reference: Linda A.Chrisey, Gil U Lee and C.ElizabethO ' Ferrall.Covalent attachment of synthetic DNA to self-assembled monolayerfilms.Nucleic Acids Research, 1996,24 (15): 3031-3039), the DNA chip of preparation places in the hybridization chamber of preheating, add 5.0 μ l, 5 * SSC-2g/LSDS in hybridization chamber to keep humidity.

(2), Oligonucleolide primers 1 and 2 each 9.6nmol respectively with TECP-HCl 6.2mmol, 0.1M sodium phosphate buffer (pH8.0 contains 51mMEDTA) mixes.Carry out reduction reaction under 37 ℃ of the temperature.

(3), dissolving nanometer gold 6nmol in 0.02ml Virahol or DMSO, deionized water is diluted to 0.2ml.

(4), nano-Au solution mixes the (0.01M that sodium phosphate buffer concentration is in the end reaction system with the Oligonucleolide primers 1 of as-reduced SH modification and 2 sodium phosphate buffer respectively, contain 150mMNaCl, 1mMEDTA, pH6.5), it is 10: 1 that nanometer gold is counted ratio with thiol molecule, educated altogether 6 hours for 4 ℃, be prepared into the Oligonucleolide primers 1 and 2 that contains nanometer gold respectively.Reaction product is used the HPLC separation and purification respectively, and first incarnadine peak is a labeled complex, repeats once can further obtain purification effect.Labeled complex is stored in 0.02M sodium phosphate, 150mM NaCl, 0.05% sodiumazide (pH7.5).

(5), extract genomic dna in the patient blood.(reference reagent box specification sheets)

(6), add following reaction in the reaction Tube pipe and form (100 μ l reaction system): Taq enzyme (5U/ μ l) 0.5 μ l, 10 * Taq enzyme Buffer, 10 μ l, MgCl ₂(25mM) 8 μ l, dNTP (every kind of each 2.5mM) 8 μ l, sample DNA 1 μ g, golden labeled primer 1 (20 μ M) 1-5 μ l, golden marking primer 2 (20 μ M) 1-5 μ l adds sterilization deionization H ₂O to 100 μ l.

(7), be put in the PCR instrument of preheating, program is as follows: 95 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 60 seconds.Carry out 32 circulations.Last 72 ℃ were extended 10 minutes.

(8), 1.0 μ l gold mark PCR product D NA adds in the 9.0 μ l hybridization buffers.In 95 ℃ of sex change 10 minutes, ice bath immediately.

(9), drip this dna hybridization buffer liquid in chip surface, add a cover the cover glass of one 2cm * cm, sealing hybridization cell immerses in 42 ℃ of water-baths, hybridizes 1 hour.

(10), take out chip and immerse immediately in 1 * SSC-0.1%SDS scavenging solution of room temperature, cleaned 5 minutes.Move in 40 ℃ of 0.1 * SSC-0.1%SDS scavenging solutions and washed 5 minutes.Cleaned 5 minutes with fresh 0.1 * SSC-0.1%SDS more immediately, the back moves into 0.1 * SSC and cleaned 5 minutes, to remove SDS.The lucifuge drying.

(11), lithium silvering solution 2ml adds chip surface, room temperature reaction 5 minutes, 0.1 * SSC cleaning 5 minutes.Dry.

(12), ordinary optical instrument (as microscope, scanner etc.) or naked eyes direct viewing result, black particle is promptly represented the hybridization positive findings.Embodiment 11 implements the detection reagent of gene chip by the direct reaction labeling nucleic acid: the nanometer gold reagent (diameter 1.4nm) that monoamine is modified, the lithium silvering solution is available from Nanoprobes company.

Streptococcus pneumoniae specific probe: 5 '-TTT CGA GTG TTG CTT TTTATG GGC-3 '

Provide by Shanghai Sangon company.

EDC (1-ethyl-3,3 '-dimethylaminopropyl carbodiimide) available from Sigma company.Step:

(1), DNA chip self-control (reference: Linda A.Chrisey, Gil U Lee and C.ElizabethO ' Ferrall.Covalent attachment of synthetic DNA to self-assembled monolayerfilms.Nucleic Acids Research, 1996,24 (15): 3031-3039), the DNA chip of preparation places in the hybridization chamber of preheating, add 5.0 μ l, 5 * SSC-2g/LSDS in hybridization chamber to keep humidity.

(2), extract genomic dna in the patient blood.(reference reagent box specification sheets)

(3), specimen dna is heated to 98 ℃, after 10 minutes, it is cooled to rapidly below 4 ℃.

(4), in reaction Tube pipe, add following reaction and form: the DNA-phosphoric acid buffer 20pmol of sex change, EDC 2nmol, 1nmol EDTA, pH6.5, the μ l of deionized water man to 50,4 ℃ of reactions 6 hours.Gel-filtration chromatography separates removes unreacted EDC.

(5), dissolving nanometer gold 6nmol in 0.02ml Virahol or DMSO, deionized water is diluted to 0.2ml.

(6), nano-Au solution and the DNA-phosphoric acid buffer of modification reaction (0.01M that phosphate buffer density is contains 150 mMNaCl, 1mMEDTA, pH7.5) mixes, and it is 10: 1 that nanometer gold is counted ratio with dna molecular, educates altogether 6 hours for 4 ℃.Reaction product HPLC separation and purification, first incarnadine peak is a labeled complex, repeats once can further obtain purification effect.Labeled complex is stored in 0.02M sodium phosphate, 150mM NaCl, 0.05% sodiumazide (pH7.5).

(7), 1.0 μ l gold marker DNA adds in the 9.0 μ l hybridization buffers.In 95 ℃ of sex change 10 minutes, ice bath immediately.

(8), drip this dna hybridization buffer liquid in chip surface, add a cover the cover glass of one 2cm * cm, sealing hybridization cell immerses in 42 ℃ of water-baths, hybridizes 1 hour.

(9), take out chip and immerse immediately in 1 * SSC-0.1%SDS scavenging solution of room temperature, cleaned 5 minutes.Move in 40 ℃ of 0.1 * SSC-0.1%SDS scavenging solutions and washed 5 minutes.Cleaned 5 minutes with fresh 0.1 * SSC-0.1%SDS more immediately, the back moves into 0.1 * SSC and cleaned 5 minutes, to remove SDS.The lucifuge drying.

(10), lithium silvering solution 2ml adds chip surface, room temperature reaction 5 minutes, 0.1 * SSC cleaning 5 minutes.Dry.

(11), ordinary optical instrument (as microscope, scanner etc.) or naked eyes direct viewing result, black particle is promptly represented the hybridization positive findings.Embodiment 12 utilizes nanometer gold-Yin to amplify the detection reagent that protein chip is implemented in reaction: the nanometer gold reagent (diameter 1.4nm) that monoamine is modified, the lithium silvering solution is available from Nanoprobes company.

Hepatitis E antigen is available from 3V diagnostic techniques company

DTT is available from Sigma company.Step:

(1), protein chip self-control.(reference: Gavin Mac Beath and Stuart L.Schreiber.Printing proteins as microarrys for high-throughput function deternination.Science, 289:1760-1763.)

(2), patients serum 0.5ml in the 0.1M sodium phosphate buffer, pH6.0 contains 5mM EDTA (1ml), adds MEA (8mg).Incubated at room 1 hour.

(3), gel-filtration chromatography separates reductive albumen.Elutriant uses 0.02M sodium phosphate, pH6.5,150mMNaCl, 1mM EDTA.Reductive albumen is in first peak elutriant.

(4), dissolving nano metal reagent 6nmol in the 0.02ml Virahol, deionized water is diluted to 0.2ml.

(5), active nano metal solution adds in the reductive albumen, educated altogether 2-12 hour for 4-37 ℃.

(6), utilize gel-filtration chromatography to separate unlabelled nano-metal particle from antibody complex.Elutriant 0.02M sodium phosphate pH7.4,150mMNaCl wash-out.First incarnadine peak is a labeled complex, and second dark-coloured band is unconjugated nano metal.Repeat once can further obtain purification effect.

(7), the protein chip with preparation places in 37 ℃ the wet box, drip 10.0 μ l, 1% PBS-BSA damping fluid (20mM phosphoric acid buffer pH7.40, contain 150mM NaCl, 0.5%BSA, 0.1% glycerine, 0.05%Tween 20) on the chip, to block nonspecific protein binding site and to reduce nonspecific antibodies.

(8), discard the PBS-BSA on the chip, do not wash, drip the nano metal binding substances of 10 times of PBS-BSA damping fluids dilution, the reaction solution of educating altogether 0.5-3 hour and gently shook frequently.

(9), rinsing, (3 * 1min), PBS (3 * 1min) for PBS-BSA.Dry.

(10), lithium silvering solution 2ml adds chip surface, room temperature reaction 5 minutes, 0.1 * SSC cleaning 5 minutes.Dry.

(11), ordinary optical instrument (as microscope, scanner etc.) or naked eyes direct viewing result, black particle is promptly represented the hybridization positive findings.Embodiment 13 utilizes nanometer gold-Yin to amplify the detection reagent that organization chip is implemented in reaction: the nanometer gold reagent (diameter 1.4nm) that monoamine is modified, the lithium silvering solution is available from Nanoprobes company.

Anti-ICAM-1 antibody is available from crystalline substance U.S. company.Step:

(1), organization chip self-control.(reference: Nocito A, Kononen J, Kallioniemi OP.Tissuemicroarrays (TMAs) for high-throughput molecular pathology research.Int JCancer.2001 Oct 1; 94 (1): 1-5.).

(2), lytic antibody (0.15mg) in the 0.1M sodium phosphate buffer, pH6.0 contains 5mMEDTA (1ml), adds MEA (8mg).Incubated at room 1 hour.

(3), gel-filtration chromatography separates reductive albumen.Elutriant uses 0.02M sodium phosphate, pH6.5,150mMNaCl, 1mM EDTA.Reductive albumen is in first peak elutriant.

(4), dissolving nano metal reagent 6nmol in the 0.02ml Virahol, deionized water is diluted to 0.2ml.

(5), active nano-Au solution adds in the reductive antibody, educated altogether 2-12 hour for 4-37 ℃.

(6), gel-filtration chromatography separates unlabelled nano-metal particle from antibody complex.Elutriant 0.02 M sodium phosphate pH7.4,150mMNaCl wash-out.First incarnadine peak is a labeled complex, and second dark-coloured band is unconjugated nano metal.Repeat once can further obtain purification effect.

(7), the protein chip with preparation places in 37 ℃ the wet box, drip 10.0 μ l, 1% PBS-BSA damping fluid (20mM phosphoric acid buffer pH7.40, contain 150mM NaCl, 0.5%BSA, 0.1% glycerine, 0.05%Tween 20) on the chip, hatched 5 minutes, to block nonspecific protein binding site and to reduce nonspecific antibodies.

(8), the PBS-BSA rinsing is 1 minute.

(9), room temperature and nano-Au composite were hatched 30 minutes.Nano-Au composite PBS-BSA dilutes 50 times, wherein contains the homologous serum of 1% normal nano gold mark antibody.

(10), rinsing, PBS-BSA 1 minute, totally 3 times, PBS rinsing 1 minute, totally 3 times.

(11), the PBS of 1% glutaraldehyde fixes 10 minutes.

(12), rinsed with deionized water 5 minutes, 2 times.

(13), drip an amount of lithium argentum reagent dyeing 3 minutes.

(14), rinsed with deionized water 5 minutes, 2 times.

(15), use low-temperature resins ordinary method dehydration embedding.

(16), microscopically observations.Embodiment 14 utilizes nano gold mark to adopt hybridization in situ technique to implement the detection reagent of organization chip: the nanometer gold reagent of single maleic diacetyl imine beautify (diameter 1.4nm), the lithium silvering solution available from

Nanoprobes company.

The IL-8 oligonucleotide probe that SH modifies: 5 '-GTT GGC GCA GTG TGG TCC ACT

CTC AAT CAT-(CH ₂) ₆-SH-3 ' is provided by Shanghai Sangon company.

Proteinase K is available from Po Lam Man.

Buffer I (Ph7.4) autogamy: toxilic acid 0.1M, NaCl 0.15M.Step:

(1), organization chip self-control.(reference: Nocito A, Kononen J, Kallioniemi OP.Tissuemicroarrays (TMAs) for high-throughput molecular pathology research.Int JCancer.2001 Oct 1; 94 (1): 1-5.).

(2), oligonucleotide 9.6nmol and TECP-HCl 6.2mmol, 0.1M sodium phosphate buffer (pH8.0 contains 51mMEDTA) mixes.Carry out reduction reaction under 37 ℃ of the temperature.

(3), dissolving nanometer gold 6nmol in the 0.02ml Virahol, deionized water is diluted to 0.2ml.

(4), nano-Au solution mixes the (0.01M that sodium phosphate buffer concentration is in the end reaction system with the oligonucleotide sodium phosphate buffer that as-reduced SH modifies, contain 150mMNaCl, 1mMEDTA, pH6.5), it is 10: 1 that nanometer gold is counted ratio with thiol molecule, educated altogether 6 hours, and be prepared into the oligonucleotide probe that contains nanometer gold for 4 ℃.Reaction product HPLC separation and purification, first incarnadine peak is a labeled complex, repeats once can further obtain purification effect.Labeled complex is stored in 0.02M sodium phosphate, 150mM NaCl, 0.05% sodiumazide (pH7.5).

(5), the protein chip with preparation places in 37 ℃ the wet box 0.1M PBS rinsing 5 minutes 3 times.

(6), 5 minutes permeabilities of 3%Triton X-100/0.1M PBS rinsing with the increase tissue.

(7), 2 * SSC rinsing is 5 minutes.

(8), Proteinase K (20 μ g/m1) digestion, hatched 5 minutes for 37 ℃.

(9), 0.1M glycine/0.1MPBS rinsing is 5 minutes.

(10), 4% Paraformaldehyde 96 rinsing is 5 minutes.

(11), 1M PBS rinsing 5 minutes * 3 times, used 2 * SSC rinsing again 10 minutes.

(12), prehybridization: get prehybridization solution and drip on the cell sheet in right amount, put 42 ℃ of water-baths and hatched 2 hours.

(13), hybridization: in 1: 50 (the probe liquid of mark: ratio preparing hybrid liquid prehybridization solution).Hybridization solution is dripped on organization chip, and the Parafilm film covering with the DEPC water treatment places in the wet box again, and 42 ℃ of water-baths were hatched 20 hours.

(14), take out chip, striping, each rinsing 5 minutes * 3 times in 4 * SSC, 2 * SSC, 1 * SSC, 0.5 * SSC successively under 37 ℃ of conditions.

(15), the rinsing of 0.1M PBS room temperature is 5 minutes.

(16), Buffer I room temperature was embathed 5 minutes.

(17), BufferII (the Buffer I that contains 1% blocking agent) was hatched 1 hour for 37 ℃.

(18), the rinsing of Buffer I room temperature is 5 minutes.

(19), drip an amount of lithium argentum reagent dyeing 3 minutes.

(20), the abundant rinsing of 0.1M PBS, termination reaction.

(21), the conventional dehydration of chip, transparent, neutral gum mounting.

(22), microscopically observations.Simultaneous test 1 fluorescent mark detection method experimental procedure

(1), DNA chip self-control (reference: Linda A.Chrisey, Gil U Lee and C.ElizabethO ' Ferrall.Covalent attachment of synthetic DNA to self-assembled monolayerfilms.Nucleic Acids Research, 1996,24 (15): 3031-3039), the DNA chip of preparation places in the hybridization chamber of preheating, add 5.0 μ l, 5 * SSC-2g/LSDS in hybridization chamber to keep humidity.

(2), in infection of staphylococcus aureus septic patient 50 μ l serum, extract DNA with reference to Shanghai Shenergy Biocolor BioScience ﹠ Technology Company DNA extraction test kit specification sheets, getting this DNA extraction liquid of 2 μ l is that template is carried out the PCR reaction.Reaction system is as follows: cumulative volume is 25 μ l.50mM KCl, 10mM Tis-HCl (pH9.0), 5mM MgCl ₂, 200 μ M dATP, 200 μ M dGTP, 200 μ M dCTP, 80 μ M dTTP, 40 μ M CY5 mark dUTP, 1.25U archaeal dna polymerase, upstream and the downstream primer of each 0.2 μ M.Primer sequence is: upstream 5 '-gTC ggT ACA CgA TAT TCT TCA Cg-3 ', downstream 5 '-CTC TCg TAT gAC Cag CTT Cgg TAC-3 '.Amplification condition is: 94 ℃ 5 minutes, with 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ of circulations in 1 minute 40 times, 72 ℃ were extended 10 minutes.Electrophoresis detection PCR product.

(3), 1.0 μ l fluorescent label DNAs add in the 9.0 μ l hybridization buffers, in 95 ℃ of sex change 10 minutes, ice bath immediately.

(4), drip this DNA sample hybridization buffer in chip surface, add a cover the cover glass of one 2cm * cm, sealing hybridization cell immerses in 42 ℃ of water-baths, hybridizes 1 hour.

(5), take out chip and immerse immediately in 1 * SSC-0.1%SDS scavenging solution of room temperature, cleaned 5 minutes.Move in 40 ℃ of 0.1 * SSC-0.1%SDS scavenging solutions and washed 5 minutes.Cleaned 5 minutes with fresh 0.1 * SSC-0.1%SDS more immediately, the back moves into 0.1 * SSC and cleaned 5 minutes, to remove SDS.The lucifuge drying.

(6), the chip that hybridization is finished scans the detection hybridization signal under proper condition with the laser confocal scanning instrument.Simultaneous test 2 isotopic labeling detection method experimental procedures

(1), DNA chip self-control (reference: Linda A.Chrisey, Gil U Lee and C.ElizabethO ' Ferrall.Covalent attachment of synthetic DNA to self-assembled monolayerfilms.Nucleic Acids Research, 1996,24 (15): 3031-3039), the DNA chip of preparation places in the hybridization chamber of preheating, add 5.0 μ l, 5 * SSC-2g/LSDS in hybridization chamber to keep humidity.

(2), in infection of staphylococcus aureus septic patient 50 μ l serum, extract DNA with reference to Shanghai Shenergy Biocolor BioScience ﹠ Technology Company DNA extraction test kit specification sheets, getting this DNA extraction liquid of 2 μ l is that template is carried out the PCR reaction.Reaction system is as follows: cumulative volume is 25 μ l.50mM KCl, 10mM Tis-HCl (pH9.0), 5mM MgCl ₂, 200 μ M dATP, 200 μ M dGTP, 200 μ M dCTP, 200 μ M dTTP, 1.25U archaeal dna polymerase, upstream and the downstream primer of each 0.2 μ M.Primer sequence is: upstream 5 '-gTC ggT ACA CgA TAT TCT TCA Cg-3 ', downstream 5 '-CTC TCg TATgAC Cag CTT Cgg TAC-3 '.Amplification condition is: 94 ℃ 5 minutes, with 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ of circulations in 1 minute 40 times, 72 ℃ were extended 10 minutes.Electrophoresis detection PCR product.

(3), reclaim purification kit with vast Tyke dna fragmentation and return the PCR product, and adopt random primering to carry out isotopic labeling.

(4), 1.0 μ l isotopic labeling DNA add in the 9.0 μ l hybridization buffers, in 95 ℃ of sex change 10 minutes, ice bath immediately.

(5), drip this DNA sample hybridization buffer in chip surface, add a cover the cover glass of one 2cm * cm, sealing hybridization cell immerses in 42 ℃ of water-baths, hybridizes 1 hour.

(6), take out chip and immerse immediately in 1 * SSC-0.1%SDS scavenging solution of room temperature, cleaned 5 minutes.Move in 40 ℃ of 0.1 * SSC-0.1%SDS scavenging solutions and washed 5 minutes.Cleaned 5 minutes with fresh 0.1 * SSC-0.1%SDS more immediately, the back moves into 0.1 * SSC and cleaned 5 minutes, to remove SDS.The lucifuge drying.

(7), the chip that hybridization is finished carries out radioautograph detection hybridization signal.

According to specific embodiment 8 methods, adopt the streptococcus aureus of pure culture to extract the different concns detected result that its DNA and two kinds of simultaneous tests carry out and be compared as follows: (the notes result is expressed as x: y, x represent to detect positive number of times, and y represents to detect number of times)

Comparison by above-mentioned simultaneous test can be found out:

(1) the nanometer amplifying technique of the present invention's employing can be amplified to 10 to the signal of hybridization ^4-8Doubly, have high detection sensitivity and specificity.

(2) the nanometer amplifying technique of the present invention's employing can be avoided the PCR reaction, thereby has simplified the treating processes of sample, has shortened detection time.

(3) the nanometer amplifying technique of the present invention's employing is pollution-free, does not need expensive detector, has reduced the use cost and the experiment condition of chip.

Claims (9)

1, a kind of nanometer amplification detecting process of biochip, it is characterized in that: method may further comprise the steps:

(1), uses the metallic particles labeling nucleic acid or the albumen of nanometer diameter;

(2), the nucleic acid of mark or albumen are combined with probe or tissue hybridization on the biochip;

(3), utilize can with the metal reagent of the metallic particles generation metallographic phase reaction of the nanometer diameter of this mark and the nano metal reaction of having hybridized mark on the nucleic acid that is combined on the biochip or the albumen, amplify nano-metal particle and also carry out ordinary optical and detect.

2, the nanometer amplification detecting process of biochip according to claim 1 is characterized in that: the method for the metallic particles labeling nucleic acid of nanometer diameter comprises in the described step ():

(1) by the mark of oligonucleotide joint realization to nucleic acid;

Or (2) are by the mark of primer realization to product nucleic acid;

Or (3) direct mark that nucleic acid is realized.

3, according to the nanometer amplification detecting process of claim 1,2 described biochips, it is characterized in that: by the mark of oligonucleotide joint realization to nucleic acid, the step of method comprises:

(1), preparation nano metal solution;

(2), nano metal solution is mixed with oligonucleotide, nano metal is 1-100 with the ratio of oligonucleotide molecules number: 1, and reaction obtains to contain the oligonucleotide joint of nano metal;

(3), extract specimen dna, remove the phosphate group of the 5 ' end of DNA by the dephosphorylation reaction, prevent that himself from connecting;

(4), under the ligase enzyme effect, the oligonucleotide joint of combining nano metal mark and the specimen dna of above-mentioned processing obtain the sample DNA of nano metal mark.

4, according to the nanometer amplification detecting process of claim 1,2 described biochips, it is characterized in that: realize in the product nucleic acid labeling methods that by primer adopt by the mark of PCR reaction primer realization to product D NA, the step of method comprises:

(1), preparation nano metal solution;

(2), nano metal solution is mixed with the PCR primer, nano metal is 1-100 with the ratio of primer molecule number: 1, obtain the PCR primer of nano metal mark;

(3), utilizing this labeled primer to carry out PCR reacts, obtains the PCR product D NA of nano metal mark.

5, according to the nanometer amplification detecting process of claim 1,2 described biochips, it is characterized in that: in the marking method of primer realization to product nucleic acid, adopt by the mark of RT-PCR reaction primer realization to product cDNA, the step of method comprises:

(1), preparation nano metal solution;

(2), nano metal solution is mixed with the RT-PCR primer, nano metal is 1-100 with the ratio of primer molecule number: 1, obtain the RT-PCR primer of nano metal mark;

(3), utilizing this labeled primer to carry out RT-PCR reacts, obtains the RT-PCR product cDNA of nano metal mark.

6, according to the nanometer amplification detecting process of claim 1,2 described biochips, it is characterized in that: directly the method for the mark that nucleic acid is realized is: by with the end group reaction of nucleic acid, thereby make the end modified compound that sulfur-bearing is arranged or contain the imide class of nucleic acid, this compounds can react with nano metal, realizes the mark of nano metal to nucleic acid.

7, the nanometer amplification detecting process of biochip according to claim 1 is characterized in that: the method steps of the metallic particles labelled protein of nanometer diameter is in the described step ():

(1), preparation nano metal solution;

(2), the albumen handled of nano metal solution and reduction mixes, the ratio of nano metal and protein molecular number obtains the albumen of nano metal mark for being not less than 5: 1.

8, the nanometer amplification detecting process of biochip according to claim 1 is characterized in that: the nucleic acid of the nano metal mark in the described step (two) and the step of the probe hybridization on the biochip are:

(1), the DNA to the nano metal mark carries out denaturing treatment;

(2), the DNA of nano metal mark that drips denaturing treatment is in biochip surface, carries out hybridization;

(3), clean the DNA that bonded nano metal mark is not hybridized in removal;

(4), drying treatment is standby.

9, the nanometer amplification detecting process of biochip according to claim 1 is characterized in that: the albumen of the nano metal mark in the described step (two) and the probe bonded step on the biochip are:

(1), PBS handles biochip, to block non-specific proteic binding site and to reduce non-specific;

(2), the albumen that drips the nano metal mark on biochip, educate reaction altogether;

(3), rinsing, remove the albumen of unconjugated nano metal mark;

(4), dry standby.