CN1094520C - Automatic combined target gene detecting method and detection instrument using said method - Google Patents
Automatic combined target gene detecting method and detection instrument using said method Download PDFInfo
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Abstract
The present invention relates to a method for automatically detecting composite target genes and a detector which is made based on the method. The method is mainly applied to a quartz crystal microbalance QCM technique to detect DNA by combining a gene chip technique. In the method, an ultra thin quartz resonator array is directly etched on a piezoelectric quartz crystal by using a micro-machining technique; then, a great amount of probes are solidified. In this way, a great amount of DNA molecules or RNA molecules can be detected and analyzed once, and thus, the disadvantages of a traditional nucleic acid blot hybridization technique of complication, low automaticity, less goal molecule detecting quality, etc. are solved. The method and the detector of the present invention can detect in an original position without marks and obtain detecting information at any time. The present invention has the advantages of small volume, convenient carrying, easy use, low cost, etc.
Description
Technical field
What the present invention relates to is a kind of combination target gene automatic testing method.
Background technology
Along with the progressively enforcement of the Human Genome Project and the fast development of molecular biology related discipline, geneseq database just increases rapidly with unprecedented speed.
Yet, the problem that the function of how studying the bioinformation of numerous genes and being undertaken in vital process has become this area researchist to solve, this has just proposed requirement quickly and accurately to a large amount of thymus nucleic acid DNA, RNA (ribonucleic acid) sequencing and analysis thereof.
Gene chip claims appearing as of DNA chip or biochip to solve this type of problem again provides possible terms of settlement.So-called gene chip (Gene chip) is meant a large amount of probe molecules is fixed on the upholder, hybridizes with sample then, and the quantity that target molecule exists in the judgement sample by the power that detects hybridization signal how much.Owing to can be fixed in extremely a large amount of probes on the upholder simultaneously with this technology, so once can carry out check and analysis, thereby solve deficiencies such as traditional nucleic acid blot hybridization technique complexity, level of automation is low, the testing goal molecular amounts is few to a large amount of dna moleculars or RNA molecule.
But, quartz crystal sensor and chip are directly combined to reach real-time detection and detection of dynamic exist
Do not disclose in the prior art.
Summary of the invention
Main purpose of the present invention is to provide a kind of target gene automatic testing method that utilizes the outer-gene chip technology human inheritance's disease, tumour and communicable disease to be carried out gene diagnosis, gene type and medical jurisprudence and environmental analysis.
Detection method of the present invention is to utilize Micrometer-Nanometer Processing Technology directly to etch ultra-thin quartz resonator array on quartz crystal, a large amount of probes are fixed in accordingly on the quartz resonator array of metal-plated membrane layer and form miniature quartz resonance array gene sensor chip, described quartz resonance array sensor chip comprises the quartz crystal 3 that is etched to array, lower surface metal-plated membrane layer 4 at quartz crystal, upper surface at the quartz crystal array is coated with the metallic membrane 2 that is identical array, be array on the upper surface of metallic diaphragm 2 be solidified with probe array layer 1, corresponding probe place is fixed with silver electrode 6, following metallic diaphragm and pedestal 5 thermocompression bondings.Apply voltage at the chip two ends by silver electrode and obtain a fixed frequency, make probe complementary sample hybridization with it together in solution then, mass loading and the viscosity coupled measured in the crossover process change by causing the frequency values of chip, the quantity that can whether be hybridized and hybridize.
Be that detection method of the present invention is described in detail below.
Of the present inventionly piezoelectric gene sensor is used for the basic functional principle that target gene detects is: a large amount of probe molecules are fixed in the quartz crystal upholder (the crystal both sides apply certain voltage by silver electrode) that is coated with gold or silver film, in reaction tank, hybridize then with sample.Owing to whether hybridize the change that can cause quartz-crystal resonance frequency, how many changing values by detecting quartz-crystal resonance frequency can judgement sample has or not target molecule and quantity.
The characteristics of the method for the invention are that biochip technology is combined application with the piezoelectric transducer technology, constitute the unique detection method.
The present invention utilizes Micrometer-Nanometer Processing Technology, directly etches ultra-thin quartz resonator array on quartz crystal, and quartz-crystal resonance frequency is very responsive to the fine change of plane of crystal quality, thereby its mass detection limit can reach the pg level.And, can reduce cost greatly because Micrometer-Nanometer Processing Technology makes it be easy to preparation in quantity.
The basic technique principle that the method for the invention adopts is as follows:
1, the piezo-electricity and piezoelectric principle
Crystal is subjected to the effect of extraneous mechanical pressure, produces the phenomenon of electric charge in its surface, is called piezoelectric effect.
As far back as 1880, the researchist just found that quartz waits some crystalline piezo-electricity and points out, some dielectric substance when the effect distortion that is subjected to external force in a certain direction, innerly can produce polarization phenomena, produces electric charge simultaneously in its surface; After external force is removed, get back to uncharged state again; And formed electric charge of plane of crystal and impressed pressure are directly proportional, and prior art changes this mechanical energy the phenomenon of electric energy into, are called " along piezoelectric effect ".
On the contrary, apply electric field on the direction of dielectric polarization, it can produce mechanical deformation; When removing extra electric field, dielectric distortion disappears thereupon, this electric energy is transformed into the phenomenon of mechanical energy, is called as " inverse piezoelectric effect "; The dielectric substance that will have piezoelectric effect in the prior art is referred to as piezoelectric.
More common piezoelectric has quartz, pottery etc., and wherein quartz becomes piezoelectric sensing because of over-all propertieies such as its favorable mechanical, electrochemistry and temperature, particularly the main element of piezoelectricity chemistry and piezoelectric biological sensing.
Quartz is a kind of aeolotropic crystal, presses the different directions sliced crystal, and its physical properties (as elasticity, piezoelectric effect, temperature profile etc.) differs greatly.
Researchist of the present invention finds that when the alternation energizing voltage put on the electrode of piezoquartz both sides, crystal can produce the mechanical deformation vibration, when the alternating voltage frequency reaches the crystal natural frequency, amplitude strengthens, and forms piezoelectric resonator, and this CF is called resonant frequency.
According to the sensitive mechanism of piezoelectric sensing, be resonance structure with piezoquartz, can find that its output signal (resonant frequency) is closely related with crystalline physical size and character.
People also observe: after crystal was coated with coating substances, respective change can take place in its oscillation frequency.
Sauerbrey vibrates the relational expression (2-1) of at first derive contained material mass of relevant plane of crystal and resonance frequency displacement in gas phase by AT cutting quartz crystal, and propose on this basis piezoquartz is used as the little balance of sensitive, so formula often is called the Sauerbrey equation.
Δ F: the frequency change value (Hz) that causes by coating.
F
q: basic response frequency.
υ: the aerial velocity of propagation of sound wave.
t
q: quartzy thickness (cm).
μ
q: the shearing modulus (2.947 * 10 of AT-cut quartz
11G.cm
-1.s
-2).
ρ
q: quartzy density (2.648g.cm
-3).
Δ m: the coating quality on the surface of quartz wafer (g).
A: the surface-area (cm of quartz wafer
2). (note: Δ m is the coating total mass on quartz wafer two sides, and A is the surface-area of the one side of quartz wafer.) for the quartz crystal of AT cutting, its shearing modulus (μ
q=2.95 * 10
11Dyn/cm
2) and density (ρ
q=2.648g.cm
-3) all be definite value, with μ
qAnd ρ
qValue substitution (2-1) formula obtain:
More than the prerequisite that derives of two formulas be that the coating film of uniform deposition on the supposition quartz wafer is equivalent to one deck quartz that increases same quality.Promptly requiring film and quartz is the rigidity bonded, that is to say quality on the electrode surface when crystal oscillation without undergoing any shear deformation, so just can ignore rete for quartz crystal density and elastic difference.
From the Sauerbrey equation as can be seen, when quartz wafer vibrated in gas phase, Δ f and Δ m were simple linear relationship, so quartz wafer can be used to do highstrung mass detector, and its detectability can reach the ng level, even pg level level.But, the someone confirms: when the Sauerbrey equation was applied to the research of " rigidity " coating, Δ m/m should be smaller or equal to 2% (m be the quality that is not deposited preceding unit surface quartz).
2, crystalloid fluid phase oscillations theory
Because piezoelectric transducer all is to obtain required information by the variation of measuring oscillation frequency, thereby crystal in liquid phase, vibrating, two problems that at first run into and must understand are: which the factor that 1. influences crystal oscillation activity (vigor) has, and what the vibration interval of crystal in different properties solution determined by in other words; 2. how SOLUTION PROPERTIES influences crystal oscillation frequency.Have only to have solved this two problems, crystal oscillation (application) system is widened, also could change the information of obtaining mass sensitivity according to crystal oscillation frequency exactly.
In recent years, along with the vibration of quartz crystal liquid phase is succeedd, people are more and more deep to the understanding of quartz crystal liquid phase oscillating characteristic.The range of application of quartz crystal is also widened, particularly the application in biology.The researchist finds, in liquid phase, QCM (Quartz Crystal Microbalance) is not only to mass-sensitive, and can be subjected to outside temperature, air pressure, magnetic field and rise and fall, impact the influence that concussion and fluid density, viscosity, specific inductivity, electricity are led and flow through factor such as crystalline exciting current fluctuating.
Had a lot of people to carry out in liquid environment the research of piezoelectric quartz crystal responder as detector, these studies confirm that mass loading and viscosity coupling are two main mechanisms of action that cause the piezoelectric quartz crystal frequency change.
Prior art shows, after piezoelectric quartz crystal two ends are applied voltage, the piezoelectric quartz crystal frequency that is pressed is executed fixed, researchist of the present invention on this basis, stationary probe on piezoquartz, will cause the variation of overall frequency like this, still, the frequency after the variation remains fixed value; But, because extraneous factor can cause frequency drift, therefore, the present invention has taked following measure, one reference promptly is set to be detected, if only solidify a probe on the monolithic crystal, so just being equipped with a crystal that has solidified same probe detects as reference, if obtain ultra-thin quartz resonator array by lithographic method at same crystal, just one of them that select to solidify same probe at this array detects as reference, so just can obtain stabilizied reference frequency and true and reliable experimental result.
And the variation of the frequency of the quartz crystal that institute's solidified different probe brings be can be easy to by prior art detected.
3, hybridization principle brief introduction
People such as Watson and Crick has proposed the notion of the double-spiral structure model of DNA in nineteen fifty-three.They point out:
(1) dna molecular is to be made of two opposite parallel polynucleotide chains of direction, and these two chains chemically have reverse direction.Promptly ... the structure of P-5 '-ribose-3 '-P...... with ... the structure of P-3 '-ribose-5 '-P...... is relative.
(2) base has certain rule in pairs: application chromatography such as Chargaff are analyzed the based composition of multiple biological DNA, find that VITAMIN B4 number (A) equates that with the number of thymus pyrimidine (T) number of cytosine(Cyt) (C) equates with guanine (G) number among the DNA.Therefore, four kinds of possible base pair: A-T, T-A, G-C, C-G are arranged in DNA.
Article (3) two, chain is mainly linked to each other by the hydrogen bond between base: screw shaft is passed on the plane of base pair, and is approximately vertical with screw shaft.Two hydrogen bonds can be formed between AT, three hydrogen bonds can be formed between GC.Simultaneously, for the Stability Analysis of Structures of dna double spiral, also need reactive force by hydrophobic bond.
(4) because four kinds of base pairs all are fit to this model, and every chain can have base sequence arbitrarily, but because the paired regularity of base, definite as the base sequence of a chain, then another chain must have corresponding base sequence.
Because the dna double spirane structure mainly maintains by hydrogen bond and hydrophobic bond, so the every factors that can destroy hydrogen bond and hydrophobic bond such as heating, soda acid, organic solvent can both cause sex change, make the double-spiral structure of DNA become a random ball of string.Between the different denaturation dna fragmentation, " renaturation " of being undertaken by the base complementrity pairing is called hybridization.Hybridization not only can occur in DNA and DNA interchain, also can carry out between the homologous sequence of DNA and RNA chain.Two complementary single stranded DNAs form two key heterozygotes in the non covalent bond mode in the crossover process.When the sequence of a chain wherein is known,, just can verifies whether to contain in the unknown DNA sample and exist with known array complementary DNA by detecting crossover process.
Though the long-chain dna molecular is not an inflexible, and each nucleotide residue all has 6 strands that can rotate freely, and the dna double spiral may be existed with different conformation forms; But the short chain dna molecular under the dry condition still has certain rigid, and it is very little to be fixed on the space length of the oligonucleotide on the electrode; Because the DNA after fixing is film forming, adds that the relative crystal sole mass of quality of fixed oligonucleotide probe is very little, therefore can satisfy requirement of the present invention on wafer.
4, the general situation of development of prior art
In recent years, the research of related gene transmitter (being also referred to as DNA and biological nucleic acid transmitter) is both at home and abroad just becoming the research focus of biosensor technology, gene sensor is with its simple and easy, quick, inexpensive unique superiority, be with a wide range of applications in fields such as molecular biology, medical test and environmental monitorings, except that gene sequencing, transgenation, gene test and diagnosis, also relate to the research of DNA and medicine, protein molecule interphase interaction etc.
The reversible hybridization of complementary DNA be duplicate, transcribe, the basis of bioprocess such as translation, nucleic acid hybridization is absolutely necessary to understand these important bioprocesss on molecular level.
Current, the genetic analysis method mainly is to detect the sequence of concrete DNA in nonhomogeneous system, relatively Chang Yong method is a nucleic acid hybridization, nucleic acid hybridization is two complementary single-stranded dnas form two key heterozygotes in the non covalent bond mode a process, when the sequence of a chain wherein is known, this is a kind of very useful analytical technology, by detecting crossover process, can verify whether to contain in the unknown DNA sample and exist with known array complementary DNA, the most frequently used method is to fix the gene of one section known array on certain solid support, utilize its same complementary oligonucleotide with it in solution to hybridize then, thereby realize the detection of concrete DNA in the liquid phase.
In recent years; people are more and more deep to the research that detects concrete dna sequence dna in the liquid phase by hybridizing method; detect dna sequence dna by hybrid method very important use value is arranged; be mainly used in: fields such as clinical gene diagnosis, medical jurisprudence, food, biological chemistry, environment protection; and the detection method of gene also becomes more convenient and safe because of non-radioactive markers' such as vitamin H, digoxin, fluorescence dye application; the particularly application of polymerase chain reaction,PCR round pcr makes gene test sensitive more.
Traditional DNA hybridization all requires the applying marking method to detect hybridization signal, and these methods allow in situ detection, and can sensitivity very high.As: the detectability of round pcr can reach nmol/l; The dna computer technology also provides the method that detects a certain concrete dna sequence dna from the macro-mixing system; Because having become, shortwave fluorescence and Laser Scanning Confocal Microscope The Application of Technology, fluorescent marker method detect very sensitive common method of minim DNA.But these methods also have certain deficiency: 1. probe mark and modification cost an arm and a leg, operate loaded down with trivial details; 2. the forward and backward processing of label probe is complicated; 3. be difficult to accurately obtain the absolute quantity in the crossover process and hybridize quantitative informations such as consuming time; 4. hybridization time is long, often needs several hrs, even several days; 5. often need special testing installation and condition; 6. very high to operator's technical requirements, be difficult for grasping.Also have many investigators use the SPR method realize liquid phase mark and unmarked DNA hybridization quantitatively and in-situ monitoring, but this method exists instrument costliness, shortcoming such as quantitatively inaccurate.
Recently, more and more researchers begins to use non-marked method detection gene order.Studying more is the DNA bio-sensor system, and it is by hybridizing method detection and Identification dna sequence dna, and can carry out the DNA quantitative examination, and this detection method is simple to operate, time-consuming less, and the undesired signal molecule just can directly carry out quantitative analysis.It not only can be used for the mensuration of dna sequence dna, point mutation, the more important thing is the carrying out that can monitor hybridization dynamically, quantitatively, and does not need cleaning electrode, does not need drying, just can directly obtain hybridization information under liquid phase state.The DNA biosensor is fixed on the converting unit through the dna probe of modifying one, and this converting unit is transformed into electrical signal with physics or chemical signal again.According to selecting the different of medium and transverter for use, the DNA biosensor can be divided into several big classes such as electrochemistry, optics and piezoeletric quartz sensor.
In the present invention, the researchist has mainly used QCM (Quartz Crystal Microbalance) QCM (Quartz CrystalMicrobalance) technology to be the piezoelectric transducer technology and to detect DNA in conjunction with biochip technology, and has compared the influence of different fixing method to situations such as transmitter time of response, hybridization efficiencies.
In order to be illustrated more clearly in creative place of the present invention, the applicant makes following description in this new development to gene sensor research:
A, optics gene sensor
Mainly contain fluorescence optical fiber gene sensor, surface-enhanced Raman gene probe and surface plasma body resonant vibration gene sensor three classes at present.
Henke etc. measure the hybridization of optical fiber surface by bromination second pyridine hybridization indicator and total internal reflection fluorescent method, studied the preparation of fluorescence optical fiber DNA transmitter, and two kinds of process for fixation of optical fiber surface single stranded DNA have been compared by scattering method and UV-UIS spectrography, the result show oligonucleotide be difficult to directly to be fixed on the surface hydrophobicity linking agent aminoterminal on, but then can successfully fix with the acid amides coupling reaction.Uddion etc. adopt dna synthesizer, and directly at the silica fibre surface synthetic oligonucleotide after the linking agent processing, the optical fiber DNA transmitter of fluoroscopic examination has been developed in the dsDNA embedding bromination second pyridine that utilizes hybridization to form, and is used to detect the formation of triple helical DNA.Abel etc. [6] have developed automated optical DNA sensing system, its principle is with avidin or Streptavidin biotinylated probe to be fixed on optical fiber surface, utilize the fluorescence excitation and the detection of silica fibre loss field, realize the field monitoring of probe and fluorescein-labelled complementary strand hybridization, sensitivity can reach 132pmol.Compare with fluoroscopic examination, surface-enhanced Raman detects has higher sensitivity.Therefore utilize surface-enhanced Raman (SERS) reagent to modify the gene probe of preparation, can need not amplification and be directly used in gene test.Graham etc. have reported that sensitivity reaches the SERS gene probe of 0.8pmol.Isola etc. will have spectral selectivity and highly sensitive SERS technology combines with round pcr, are applied to the detection of HIV-Gag gene.Though SERS technology required equipment is expensive complicated, but based on its minimum spectral bandwidth (peak width at half height＜1nm), be expected to be implemented on the chip and detect a plurality of target genes simultaneously with a plurality of SERS gene probes, and this to be general optical detective technology (peak width at half height as fluoroscopic examination is 50-100nm) be difficult to accomplish.
To the sensitivity that sensor surface changes, need not hybridization indicator also need not be to dna marker, direct field monitoring crossover process based on SPR for surface plasma body resonant vibration (SPR) gene sensor.Peterlinz etc. report realizes that with dichromatism SPR spectrum the non-marked in situ quantitation of DNA hybridization detects, and thus follow-up study the kinetics of hybridization and thermotropic denaturalization process.Corn and Smith research department report will scan SPR to be measured and the coupling of micro-SPR technology, in order to characterize the DNA hybridization absorption of golden film surface and with the combining of Streptavidin, and, come the spr signal of amplifying probe hybridization by vitamin H-Streptavidin effect formation Streptavidin/DNA polymolecular layer.Simultaneously, this research department has made up the array of 2 * 2 oligonucleotide probes (the about 2.0mm of probe spot diameter) on golden film surface, detects many DNA hybridization simultaneously with on-the-spot micro-SPR technology. and this research has tentatively shown the feasibility of carrying out the DNA chip detection with the SPR technology.
B, electrochemical gene sensor
Usually the electrochemical gene sensor of making is because precision and poor reproducibility are difficult to practical the popularization.Therefore, research at present mainly is to utilize the Micrometer-Nanometer Processing Technology that is easy to make in enormous quantities, and development is expected to realize disposable electrochemical gene sensor.The Wang laboratory utilizes screen printing technique, developed series mini DNA thick film carbon dioxide process carbon electrode, its application comprises based on hybrid method and detects nucleotide sequence, embeds dsDNA or the direct adsorptive [stripping that these small molecules and ultramicron nucleic acid are measured in the influence of nuclear base oxidation signal is measured by medicine and agricultural thing, and this laboratory hybridization of having proposed to detect with high-sensitive continuous current stripping chronopotentiometry DNA is simultaneously discerned; The dna sequence dna of measuring has M.Tuberculosis, HIV-I, E.Coli, Protozoancrypyosporidium parvum etc.Hashimoto etc. utilize the photoetching Micrometer-Nanometer Processing Technology, etch little gold film electrode of the fixing DNA probe of diameter 0.3mm, can disposablely use, and make hybridization indicator with electric active matter Hoechst 33258, have detected the concentration of patients serum HBV-DNA.Singhal and Kuhr utilize at copper surface ribose and amino catalytic oxidation, have proposed to detect different DNA electrochemical sensors with utilizing VITAMIN B4 and the oxidation of guanosint base at present.Because all Nucleotide and dna molecular all contain ribose and amino, therefore, such transmitter can be suitable for the mensuration of all kinds of Nucleotide.
Except that said gene was surveyed, another important application of electrochemical gene sensor was the detection to transgenation and damage.Domestic Sun Xing inflammation etc. utilizes the ssDNA Covalent Immobilization at graphite electrode surface, with daunomycin (DRN) is hybridization indicator, make that complementary ssDNA hybridization forms dsDNA in the ssDNA of electrode surface and the solution, DRN with electrochemical activity is in crossover process in the intercalation of DNA double-spiral structure, form the DNA electrochemical sensor, according to the difference that under the effect of UV-irradiation, nitrous acid, can hybridize and hybridize degree, detect the variation of dna structure, and tentatively inquired into above-mentioned two kinds of factors and cause the degree of dna damage and possible mutation mechanism.Before this, Wang etc. then directly use the fixedly miniature electrochemical of dsDNA, have inquired into the dna damage that ultraviolet radiation causes based on the oxidation signal variation of guanine among the DNA, comprise the conformational change of DNA and the induced chemical reaction of guanine thereof.
C, piezoelectric gene sensor
Piezoelectric gene sensor is based on the sensitivity (mass sensitivity can reach the ng level) that bulk acoustic wave device-piezoelectric quartz resonator changes its surface quality, DNA hybridization that need not the direct detecting sensor of hybridization indicator surface, this type of transmitter also have the bulk acoustic wave of being called and quartz (crystal) little/receive balance DNA transmitter.In the research of transmitter dna probe process for fixation, usefulness LB membrane techniquies such as Nicolili deposit the unimolecular layer that obtains with aliphatic amide blended ssDNA on quartz resonator, and good hybridization activity is arranged; Fawcett etc. use hydrophobic polyphenyl alkene, polyethylene and acrylic acid crosslinking copolymers stationary probe respectively at sensor surface, have tentatively inquired into the application of polymer in development DNA transmitter.Because biomacromolecule is easily adsorbed at hydrophilic surface, for making the probe activity reach maximum, reduce nonspecific absorption simultaneously, should weaken the bioaffinity of sensor surface except that probe site as far as possible, connect the ideal surfaced that reaction site may become hybridization type DNA transmitter on the hydrophobic polymer surface.
The quality change of transmitter desiccated surface detected before and after mostly the past piezoelectric gene sensor was relatively to hybridize, and along with the maturation of liquid phase piezoelectric transducer technology, by the field monitoring crossover process, made piezoelectric gene sensor more easy and quick; Simultaneously also can carry out the dynamic (dynamical) research of surperficial crossover process, for the optimization of gene sensor provides foundation.What this respect took the lead in conducting a research is the Okahato laboratory, and they have measured 10 to kinetic parameter and surperficial hybridization binding capacities such as the double-stranded complementary bonded equilibrium constant of 30-mer oligonucleotide, combination and dissociative velocity constants; And factors such as the length of fixing means, probe and target gene by changing probe, mismatched bases number, hybridization temperature, hybridization solution ionic strength, studied sensor surface crossover process dynamics in great detail.
Compare with the hybridization detection technique of material labeling nucleic acid such as radio isotope, the sensitivity of gene sensor also has big gap.Except that the detection sensitivity of further raising senser element itself, domestic old reputation China etc. is example with hsv and people's endocytoplasmic reticulum molecular chaperones Grp94 genome, has attempted the development of primer strand extension piezoelectric gene sensor first.Its principle is to utilize the extension of the primer-template hybridization back primer strand in the dna sequence analysis, and making hybridization back short chain probe is that template is progressively extended with the target gene, thereby causes the bigger quality in surface to increase, and sensitivity is improved.In addition, Wang etc. utilize peptide nucleic acid(PNA) (PNA) probe, have realized the on-the-spot discrimination of single base mismatch, and the specificity of transmitter is significantly improved, and hybridization time also can be reduced to 3-5 minute.
Gene probe--single stranded DNA fragment (oligonucleotide) is the primary and primary condition of gene sensor in the immobilization of sensor surface.
The method that the present invention adopts includes absorption method, covalent linkage is legal and three kinds of probe-immobilized methods of combined method, adopt back of the prior art two class methods, though can obtain firm probe modification layer, its surperficial hybridization avtive spot is few, and the method complexity.
Researchist of the present invention notices, utilization possesses the self-assembly effect of the molecule of some structure, prepare end modified probe, make it form unimolecular layer (SAMs) stable, high-sequential naturally on the surface, has the ideal reactive behavior undoubtedly, it is end modified that the present invention preferably adopts sulfydryl to carry out, and promptly preferably adopts sulfydryl covalent bonds method.
In the aforementioned range gene transmitter, the SPR and the piezoelectric transducer that need not mark or hybridization indicator are easy to preparation in enormous quantities, and technology is simple and efficient, can carry out real-time field monitoring, and required equipment is also than simple cheap.This two classes transmitter has become the main flow of gene sensor research.
Polymerase chain reaction,PCR (PCR) technology has been widely used in the medical science every field, but it is used and need therefore carry out field monitoring to polymerase chain reaction,PCR again to the product compartment analysis behind pcr amplification at present, and promptly real time pcr has important use value.
Some sample and probe are fainter in conjunction with the signal that is produced, researchist of the present invention is an example with the detection of human papillomavirus oncogene transcript, attempted first but the piezoelectric gene sensor of field monitoring is combined with round pcr, made up novel real time pcr; In other words, in order to improve strength of signal, the present invention has adopted the advanced performing PCR amplification of sample, or adopts the method for PCR in real time amplification, has improved the intensity and the detection sensitivity of signal.
A significant trend of gene sensor development is that the miniature gene sensor array of body plan is a gene sensor chip.So far except that the preceding trial of having mentioned of carrying out with the SPR microtechnique, Arlinghaus etc. are with the hybridization of sputter excitation resonance ionization microprobe technology for detection with isotopic labeling DNA and gene sensor chip, and the present invention's target gene group automatic checkout system of the present invention that adopted the miniature piezoelectric quartz resonator array body plan that is different from above-mentioned prior art.
The detection step of target gene group automatic testing method of the present invention is as follows:
At first carry out collection of specimens and handle, by sampling system sample is added the hybridization system then, hybridization system described here is made of pedestal, chip, lead; The hybridization system feeds back to data gathering and treatment system with sample with the frequency change parameter after probe contacts and the data of comparison and detection parameter, described data gathering and treatment system comprised that multiple tracks test is gathered and in real time central signal handle, described multiple tracks test gather be actually to a plurality of probes on the probe on a plurality of piezoelectric quartz crystals or the piezoelectric quartz crystal array because of and sample gather in regular turn in conjunction with the variation of the parameter that produces; Central signal is handled finally and is showed with shunting data presentation, aggregation of data processing, pictorial display in real time.
In fact, miniature piezoelectric quartz resonator of the present invention can be that the monolithic quartz crystal solidifies a kind of probe, also can adopt miniature piezoelectric quartz resonator array, so just can on a large amount of fritters that are etched on the quartz crystal, be provided with a large amount of probes, be typically provided with at least 9 kinds of probes.
The present invention can adopt the different types of probe of mark on a chip (being multiple special gene fragment--nucleotide sequence).The difference of probe kind, the gene information of detection are just different; If a kind of probe of mark only can carry out the detection of gene information of the same race and analysis to a plurality of samples of different sources (as certain pathogenic agent: hepatitis B virus etc.); As the multiple probe of mark, can diagnose and analyze (as: when detecting hepatitis, can fix multiple probe in detecting first, second, third gradegrade C hepatitis) to the several genes information of a sample.
Miniature quartz resonance array gene sensor chip of the present invention is to utilize Micrometer-Nanometer Processing Technology directly to etch ultra-thin quartz resonator array on quartz crystal, a large amount of probe molecules are fixed on the quartz crystal upholder that is coated with gold or silver film then, the crystal both sides apply certain voltage by silver electrode, probe and sample are hybridized, owing to whether hybridize the change that can cause quartz-crystal resonance frequency, how many changing values by detecting quartz-crystal resonance frequency can judgement sample has or not target molecule and quantity.
Of the present inventionly the piezoelectric gene sensor chip is used for the basic functional principle that target gene detects is: on certain solid support, fix a fragment gene, specifically, described fixedly upholder can be a piezoelectric quartz crystal, two ends at piezoelectric quartz crystal apply voltage by silver electrode, thereby obtain a fixed frequency, utilize its same complementary oligonucleotide with it in solution to hybridize then, the mass loading of crossover process and viscosity coupled change by causing the frequency change of quartz piezoelectric crystal, the changing value of this frequency just can be drawn the quantity of whether hybridizing and hybridizing by analyzing, thereby realized the detection of concrete DNA in the liquid phase.
Be that described miniature piezoelectric quartz resonator array gene sensor chip of the present invention is concluded below:
Wherein, miniature piezoelectric quartz resonator array gene sensor chip of the present invention comprises that the AT that is etched to array cuts quartz crystal 3, lower surface metal-plated membrane layer 4 at quartz crystal, upper surface in the quartz crystal battle array is coated with the metallic diaphragm 2 that is identical array, be array on the upper surface of metallic diaphragm 2 be solidified with probe array layer 1, corresponding probe place is fixed with electrode 6, following metallic diaphragm and pedestal 5 thermocompression bondings; Described number of arrays is at least one, preferably is at least six, preferably is at least nine; Wherein, the piece number of described array is identical with the quantity of the probe layer 1 that is cured; Be provided with groove 9 between the piece that described array presented; Described metallic diaphragm can be golden membranous layer or silver film; Described electrode is a silver electrode; Described pedestal is a glass pedestal; In order to reduce the influence of other factors, one that is solidified with in described array in the same probe is used as the reference detection; The curing that the present invention adopts can be an absorption method, covalent linkage is legal and combined method, but preferably adopts the end modified covalent bonds method of sulfydryl.
Detection method step apportion of the present invention is as follows:
1. the extraction of sample disposal sample amplifying nucleic acid, restriction endonuclease are handled (different specimens with different enzymes) and are carried out the segmental separation purification of purpose etc. according to different samples;
2. if the use of chip non-once is cleaned at sample introduction test advancing column electrode, detector of the present invention preferably uses disposable chip;
3. start the computation software program, content comprises sample introduction, monitoring in real time, signal processing, image display system and interpretation of result;
4. make it to produce hybridization by sample being closed the probe contact through the sample holes sample introduction;
5. obtain test result according to computer data and image;
6. clean sample inlet pool and reaction tank;
7. cyclical operation.
Multiple tracks of the present invention (hole) reaction unit is produced and public sale by the applicant.
Target gene group automatic testing method of the present invention has adopted physiotechnology, molecular biology, transmitter, Micrometer-Nanometer Processing Technology etc., and its main application comprises the diagnosis (communicable disease, inherited disease, tumour, cardiovascular disorder etc.) to disease, the detection of transgenation, the development and the environmental monitoring of novel drugs.
Adopt the detection performance of detection method of the present invention as shown in table 1 below:
Table 1
Main economic and technical indices limit of identification: 5pg, suitable detection time with isotopic labeling: 10min sensor chip size: 10mm * 10mm transmitter and array size: 1.0mm * 1.0mm, 7 * 7 chips can be surveyed the target gene number: 49 sensor chip work-ing lifes: 10 times, and preferred disposable use detection system work-ing life: more than ten thousand times
Adopt the advantage of the method for the invention as follows:
The sensitivity of the chip of miniature piezoelectric quartz resonator array of the present invention body plan can reach the pg level; Specificity is identical with present used mark detection technique.
But adopt automatic testing method of the present invention to possess in-site detecting, need not mark, can obtain detection information at any time, volume is less, be easy to carry, advantage such as easy to use, with low cost, be suitable for clinical and field environment detects.
Property/the price ratio of table 2 detection method of the present invention and ordinary method
Annotate: ELISA and culture method are the methods that present wide clinical application detects pathogenic agent
| Detection time | The operation difficulty or ease | Price (RMB) | Support equipment | ||
| Conventional | ELISA | 3hour | Complicated | ￥50.0 | Need |
| Cultivate | ＞1week | Complicated | ￥150.0 | Need | |
| Sensor chip of the present invention | ＜10min | Simple and easy | ￥30.0 | Do not need | |
But the protection domain see claim of detection method of the present invention, but be not limited only to this all should be within protection scope of the present invention to the change of the order of detection method of the present invention and combination under the present invention's design.
Table 5 is circuit signals of automatic testing method of the present invention.
Automatic testing method of the present invention, except chip, the each several part of circuit can be adopted circuit of the prior art, one of contribution of the present invention be with the combination of each testing circuit and in conjunction with chip of the present invention to reach purpose of the present invention.
Description of drawings
Be description of drawings of the present invention below,, can more be expressly understood principle of the present invention and structure by following explanation and in conjunction with the following detailed description, wherein:
Accompanying drawing 1 is the DNA chip synoptic diagram that adopts in the combination target gene automatic testing method of the present invention;
Accompanying drawing 2 is that the A-A of accompanying drawing 1 described chip is to sectional view;
Accompanying drawing 3 is electrode surface probe stationary situation synoptic diagram in the combination target gene automatic testing method of the present invention;
Accompanying drawing 4 combination target gene detection methods of the present invention are to HPV and LT detected result synoptic diagram, and wherein, Y-axis is a frequency minimizing value, and X-axis is the time;
Accompanying drawing 5 is schematic flow sheets of combination target gene automatic testing method of the present invention;
Accompanying drawing 6 is circuit and each parts mode of connection synoptic diagram of combination target gene automatic testing method of the present invention.
Be the label of each parts of the above-mentioned accompanying drawing of the present invention below, wherein, 1 probe layer, the 2nd, last gold (or silver) rete, the 3rd, quartz crystal, the 4th, following gold (or silver) rete, 5 pedestals, the 6th, silver electrode, the 9th, groove.
Embodiment
Be embodiment below, described embodiment just is used for illustrating the present invention, rather than is used for limiting of the present invention.
Embodiment 1
Adopt the automatic testing method of the invention described above, heat-labile toxin (LT) fragment that produces with the toxin originality intestinal bacteria (ETEC) of Hpv18 (7021-7050) fragment of 30bp and 20bp is fixed on the chip, sample is detected, see Fig. 4, as shown in Figure 4, when reaction reaches balance in the time of about 5 minutes, chip concussion this moment frequency reduces about 40-50HZ, and wherein, the ■ line is the sample negative control, ▲ line is the test result to HPV, and the X line is the test result to LT.
Table 3 and table 4 are respectively the result that tubercule bacillus, gonococcus (having known the sample that has or not) detect to be compared with conventional cultural method, PCR method and the method for the invention.
Negative sample is 1,8,9,10,11,14,15,18,19,20,21,22,23,26,27 in 30 parts of gonococcus samples shown in the table 3, and all the other are all positive;
Negative sample is 1,6,7,9,11,18,19,21,22,27,28 in 30 parts of tubercule bacillus samples shown in the table 4, and all the other are all positive.
Relatively detected result as can be seen, conventional cultural method is relatively accurately with objective, but sensitivity is limited, PCR method is highly sensitive but be prone to false positive, can get no matter accuracy or sensitivity by experimental data, the detection effect of automatic target gene detection method of the present invention is better than this two kinds of methods.
The characteristics of target gene automatic testing method of the present invention are DNA chip and sensor technology combination mutually, have following advantage:
1, cheap, be easy to carry;
2, easy to use, but original position and monitoring in real time;
3, need not mark, but prolonged preservation is reacted the 3rd susceptibility height;
4, can detect many group samples simultaneously.
Table 3 gonococcus Clinical Laboratory result report
| Sample number | 1 2 3 4 5 6 7 8 9 10 |
| Cultivate | - + + + + + + - - - |
| PCR | - + + + + + + - - + |
| MGI | - + + + + + + + - - |
| Sample number | 11 12 13 14 15 16 17 18 19 20 |
| Cultivate | - + + - - + + - - - |
| PCR | - + + - + + + - - + |
| MGI | - + + - - + + - - - |
| Sample number | 21 22 23 24 25 26 27 28 29 30 |
| Cultivate | - - - + + - - - + + |
| PCR | - - + + + + - + + + |
| MGI | - - - + + - - + + + |
Table 4 tubercule bacillus Clinical Laboratory result report
Table 5 is circuit signals of detection method of the present invention
| Sample number | 1 2 3 4 5 6 7 8 9 10 |
| Cultivate | - + + + + - - + - - |
| PCR | - + + + + - + + - + |
| MGI | - + + + + - - + - + |
| Sample number | 11 12 13 14 15 16 17 18 19 20 |
| Cultivate | - + + + + + + - - + |
| PCR | - + + + + + + - - + |
| MGI | - + + + + + + - - - |
| Sample number | 21 22 23 24 25 26 27 28 29 30 |
| Cultivate | - - + + + + - - - + |
| PCR | - + + + + + + + + + |
| MGI | - - + + + + - - + + |
Claims (10)
1. combination target gene automatic testing method, utilize Micrometer-Nanometer Processing Technology directly on quartz crystal, to etch ultra-thin quartz resonator array, a large amount of probes are fixed in accordingly on the quartz resonator array of metal-plated membrane layer and form miniature quartz resonance array gene sensor chip, described quartz resonance array sensor chip comprises the quartz crystal (3) that is etched to array, lower surface metal-plated membrane layer (4) at quartz crystal, upper surface at the quartz crystal array is coated with the metallic membrane (2) that is identical array, be array on the upper surface of metallic diaphragm (2) be solidified with probe array layer (1), corresponding probe place is fixed with silver electrode (6), following metallic diaphragm and pedestal (5) thermocompression bonding, apply voltage at the chip two ends by silver electrode and obtain a fixed frequency, make probe complementary sample hybridization with it together in solution then, mass loading and the viscosity coupled measured in the crossover process change by causing the frequency values of chip, the quantity that can whether be hybridized and hybridize.
2. detection method according to claim 1 is characterized in that described array forms one at least.
3. detection method according to claim 1 is characterized in that described array forms six at least.
4. detection method according to claim 1 is characterized in that described array forms nine at least.
5. detection method according to claim 1 is characterized in that described metallic diaphragm can be golden membranous layer or silver film, and described pedestal is a glass pedestal.
6. detection method according to claim 1 is characterized in that the piece number of described array and the quantity of the probe that is cured are identical; Be provided with groove (9) between the piece that described array presented.
7. detection method according to claim 1, one that it is characterized in that being solidified with in the described array in the same probe is used as the reference detection, and probe is the known segment, but the of the same race or different types of probe of mark on the chip array.
8. detection method according to claim 1 is characterized in that described probe curing comprises absorption method, covalent bonds method and combined method.
9. detection method according to claim 1, it is end modified to it is characterized in that described probe curing adopts sulfydryl to carry out, i.e. sulfydryl covalent bonds method.
10. detection method according to claim 1 is characterized in that described sample is advanced performing PCR amplification, or adopts the PCR in real time amplification.
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| CN 99123578 CN1094520C (en) | 1998-11-10 | 1999-11-10 | Automatic combined target gene detecting method and detection instrument using said method |
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| CN 99123578 CN1094520C (en) | 1998-11-10 | 1999-11-10 | Automatic combined target gene detecting method and detection instrument using said method |
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Cited By (1)
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| CN102094079B (en) * | 2009-12-11 | 2012-12-12 | 崔学晨 | Fast detection method of loop-mediated isothermal nucleic acid amplification of in-situ QCM (Quartz Crystal Microbalance) |
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| CN1395103A (en) * | 2002-07-31 | 2003-02-05 | 中华人民共和国北京出入境检验检疫局 | Piezoelectric biological chip for detecting disease cause of mad cattle disease and its preparation method |
| CN101545898B (en) * | 2008-03-25 | 2012-09-05 | 中国科学院化学研究所 | Micro-fluidic chip detecting system using quartz crystal microbalance as transducer |
| CN106253875B (en) * | 2016-10-09 | 2019-12-27 | 湖南农业大学 | High-flux piezoelectric resonance chip and measuring system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102094079B (en) * | 2009-12-11 | 2012-12-12 | 崔学晨 | Fast detection method of loop-mediated isothermal nucleic acid amplification of in-situ QCM (Quartz Crystal Microbalance) |
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