CN1424405A - Gene chip molecular probe and related technology - Google Patents

Abstract

A complete set of specific amplification and label techniques for the cross signals of gene chip is disclosed, which can amplify the cross signals by several orders of magnitude, so improving the power for detecting nucleic acid to single-molecule level. A technique for increasing the control efficiency to solution and molecular movement and a technique for exciting the surface plasma resonance for shielding background light or exciting optical noise are also disclosed. A molecular probe of gene chip is composed of HCL instrument, HCL reagent kit, gene chip cross chamber, and SRR-CCD signal detecting and data processing system. Its advantages are low cost, high processing power and easy operation.

Description

Gene chip molecular probe and correlation technique

The invention belongs to the gene chip applied technical field, particularly relate to the hybridization of gene chip, mark, wash-out, signal detection and data analysis technique.

Gene chip is the gene analysis technique that had just risen in recent years, it is to utilize parallel analysis principle not homotactic nucleic acid molecule in a large number, gene probe as target gene in the analytic sample, mode with high density arrays is solidificated in silicon chip, glass on the matrix such as nylon membrane, thereby is made into gene chip on the ordinary meaning, or be called gene microarray, microarray ^(1-2)Through the fast development of a few years, in order to satisfy different market demands, under the guidance of parallel analysis thought, gene chip has derived various manifestation, as U.S. Affymetrix company ⁽³⁾The oligonucleotide chip ⁽⁴⁾, Genomic Solutions ⁽⁵⁾Company, Incyte ⁽⁶⁾Company, Shanghai associating Gene science group ⁽⁷⁾Deng the glass gene chip of producing ⁽⁸⁾, be used for the research of gene expression profile ⁽⁹⁾, medical diagnosis on disease and treatment, new drug research and environment protection ^(4,8)Nanogen by the U.S. ⁽¹⁰⁾, Aclara ⁽¹¹⁾, Motorola ⁽¹²⁾Lab-on-a-chip of producing Deng company (Lab-on-a-chips) and gene film square formation etc.Above-mentioned various chip is in integrated level, specificity, and sensitivity, all many-sides such as process automation are each has something to recommend him.

The gene chip utilisation technology develops into now; as the most practical; most convenient a kind of; glass DNA chip is with its specificity; manufacturing cost; the handiness and the production efficiency of scale or short run gene chip content arrangement, thus adopted by the most vast scientific research and industry member, become the main flow in the application.It is ripe that the manufacturing technology of this gene chip has become, and domestic manufacturing cost can be reduced to the universalness degree.China has grasped advanced high-quality gene chip dot matrix techniques, and the gene chip integrated level of some system can reach every gene chip (16800 gene points of 18mm * 36mm), number of chemical finishing ⁽⁷⁾And the standardized production of this gene chip establishes in international community just gradually, its product batch between stability and batch in repeatability can obtain fine control.Gene chip is to its maximum market--and as if the carrying out on a large scale of practical field one popularization and application such as clinical diagnosis drive ons that Everything is ready boldly, just save up strength to start out.Yet, from global range, the application facet of gene chip in practical field not the picture anticipation optimism with develop rapidly.Take a broad view of various forms of gene chip application systems, can find they always following certain or some aspect expose defective:

(1) the biochemical operation steps of gene chip is numerous and diverse, experimental technique to operator requires high: in gene chip uses, gene isolation, purifying, mark, PCR, hybridization, wash-out, many complex technology processes such as signal detection and data processing make the professional scientific researcher that gene chip is still only done some training very often and theoretical tray is higher to grasp.And in wide practical field, as medical diagnosis, food, health quarantines such as medicine, science of law evaluation etc., general practitioner is difficult to reach professional scientific researcher's state of the art;

(2) instrument and operation (referring to test kit, consumptive material prices such as gene chip) cost height;

(3) detection sensitivity is low, can not satisfy and detect micro-virus and a large amount of actual requirements of hanging down the abundance genes of research etc.;

(4) system integration, level of automation is low, how to occur with the individual feature product form, as detector, interpretoscope, hybridization instruments etc. are difficult to improve user's timeliness and comfort level;

(5) there is problem separately in the gene chip that adopted: oligonucleotide chip gene density height but false positive is also high; Chip lab functional integration height, specificity is good and density is too low, but also is not suitable for scale standard production; Glass-based is because of chip gene density height, and specificity is good, is fit to scale standard production, and the function course level of automation of hardware support kit system is low or the like.

Analysis-by-synthesis once, gene chip promoteing the wider application in practical field requires each gene chip application system all prerequisites below must satisfying simultaneously:

(1) the necessary specificity of the gene chip that system adopted is good, gene density height, and the production efficiency height, low cost of manufacture can realize the stdn scale production, and can reach sufficiently high stability and repeatability,

(2) the own household function integrated level of system height, the level of automation height is operated simple and easyly, and system hardware and test kit cost are low, and the interface close friend is powerful, and technical feature is stable,

(3) system's detection sensitivity must reach enough high levels, can satisfy exhausted 8 most of users' the requirement to sensitivity for analysis,

(4) last, competent genetic resources is all gene chips and the prerequisite (except the Gene Chip system of scientific research and pharmaceutical industry) of drive oing respective market towards the supporting treatment system in each practical field boldly thereof.Except last point depended on progressively finishing of the Human Genome Project and the back Human Genome Project, other three aspects all were the requirements to gene chip and treatment system itself thereof.With regard to my understanding, in the world, still do not have any gene chip system for handling at present and can meet above-mentioned requirements simultaneously.Therefore, the gene chip treatment system that satisfies above-mentioned all requirements simultaneously becomes the primary affair in present international biochip technology field, also is the bottleneck place of gene chip career development simultaneously.

Purpose of the present invention is said technically, just provides one and overlaps lifting glass gene chip detection sensitivity significantly, and economic and practical and can realize the technological method system of automatic operating, and a kind of equipment and test kit of realizing above-mentioned functions in the end is provided.Its aim just is to provide gene chip in the needed base molecule biological processes of each field popularization and application of society, signal detection and interpretation of result system, matched reagent box and practical gene chip hybridization chamber 2.

The present invention takes the lead in the gene chip treatment technology adopting optics particulate (high molecular fluorescent microballoons or contain the polymeric particles of a large amount of fluorescence dyes, silica optics magnetic bead, or pass through any other means with fluorescence dye, chemistry or noclilucence material etc. are fixed in the surface of particulate or are embedded in the high lotus signal vehicle of various ways such as its inside) thing that serves as a mark carries out molecular signal and amplifies, and concrete grammar can be as follows:

(1) the optics particulate is connected on the double chain acid molecule that mark specifically hybridized to the gene chip surface by covalent linkage:

(2) or by nucleic acid to be analyzed in the sample is connected on the optics particulate, straight with the optics particulate that carries target gene

Connect with gene chip on probe hybridize, at last through carrying out optical excitation and signal detection after the process such as wash-out.This signal amplification technique by the optics particulate can improve the detection sensitivity of gene chip greatly and reduce its requirement to the high-precision optical detection system, and even spends the level that subzero 15 degree cooling CCD can realize Single Molecule Detection by subzero 10.Moreover, owing to passed through the signal amplification, simplified the molecular biological technical finesse step of gene chip, especially saved the PCR process of DNA cloning, made whole process can pass through temperature, light and various fluidic sequencing control realize automatization, removed the uncertainty that each step manual operation brings, the experimenter is got fed up from miscellaneous, procedural, free in the operation of machinery.The more important thing is that the employing of this technology has also been avoided by the round pcr process to the poor specificity that gene chip analysis itself is brought, be difficult to quantitatively, fatal problem such as the technological operation difficulty is big.

The present invention has also adopted the fully shielding of surface plasma body resonant vibration (SPR) signal excitation technology realization to bias light for the first time, improves signal to noise ratio, has further improved the signal detection effect.

Also in the hybridization of gene chip, mark has been introduced sequencing alternating electromagnetism field action comprehensively in the processes such as wash-out, be used to accelerate the efficient of said process in the present invention, shortens the process required time.

Advantage of the present invention not only is to have improved the gene chip detection sensitivity, handle timeliness, reduce cost, also make non-specialized-technical personnel simultaneously, can utilize this supermatic equipment to carry out high level and high efficiency genetic analysis through brief training on operation or guidance.Everything is all used for the universalness of gene chip the technology impellent is provided.

The present invention is divided on the whole:

1. biochip technology is handled (hybridization, mark, wash-out, photochmeical reaction etc.) system-HCL instrument and HCL test kit 19 parts,

2. gene chip optical detection (containing the novel gene chip) and data analysis system,

3. the gene chip hybridization chamber 2,

The automatization total system integrated-the GMPS gene chip molecular probe,

5. intelligent chip diagnosis user interface utilizes multicomponent signal data fusion technology or multifactor data regression technique in the clinical diagnosis

The principle of work of gene chip molecular probe

The present invention carries out specific marker as high lotus signal vehicle to the double-strandednucleic acid of having hybridized on the gene chip by the optics particulate, thereby realizes that signal amplifies.The HCL instrument is the hybridization that will pass through before gene chip is detected, and mark, pre-treatment steps such as wash-out are realized the equipment of operation automatically and control, and all biochemical reagents and the various solution that move in the HCL instrument are referred to as HCL test kit 19.Gene chip will could finally obtain our desired detected result by signal detection and data analysis system after handling through the HCL instrument.Add that latter two part is collectively referred to as gene chip molecular probe (GMPS).Carry out division with regard to each subsystem below.HCL test kit 19

HCL test kit 19 is cores of whole GMPS gene chip molecular probe.According to biological process, can be divided into following program:

One, gene isolation, purifying: in that we utilize commercially available various DNA, RNA are arranged at present aspect this, isogeneity test kits such as mRNA, since isogeneity, non-patent abilities such as hybridization buffer, and different samples has different biological processes processes, thereby provides in the mode of principle.General procedure is as follows:

1. the extracting of the DNA of biological sample or RNA (comprising mRNA)

2.DNA or the purifying of RNA

3. detect as RNA, then at first reverse transcription becomes DNA, and carries out the purifying (suitable temperature and time) of product

4. the DNA behind DNA or the reverse transcription is carried out alkaline denaturation or high-temperature denatured

5. the template after the sex change is hybridized (suitable temperature and time) at hybridization solution and chip probe

6. carry out wash-out after the hybridization, residual to remove hybridization solution.

We will add following technology in the HCL instrument, to improve the processing efficiency of HCL instrument:

(1) quick hybridization is oozed in swimming: impel nucleic acid molecule (carrying negative charge) to do the touch opportunity that well-regulated electrophoresis motion increases target gene and probe gene in gene chip hybridization chamber 2 by the electric field that gene chip dot matrix zone (being effective hybridization system of gene chip hybridization chamber 2) is applied alternation, thereby quicken crossover process.In the design of hybridization buffered soln, the stronger electroosmotic effect (direction of motion is identical with the direction of nucleic acid in solution) of utilizing high ionic strength citric acid or sodium phosphate salt damping fluid to be produced under electric field further improves nucleic acid molecule and follows the sport efficiency of solution integral body at solid phase surface.Impel nucleic acid molecule to move on the gene chip surface, will improve the touch opportunity of probe and target gene effectively, thereby improve hybridization efficiency with proper speed and mode.

(2) swimming to be oozed quick mark and to be carried out specific marker in order reaching at the dna double chain of having hybridized on the gene chip, we select can specificity intercalation of DNA two strands organic reagent such as psoralene or derivatives thereof 4 '-amine methyl-4,5 ', 8-trimethylpsoralen (AMT) etc. embed " probe " part of the complementary heteroduplex of nucleic acid (DNA or RNA) as specificity.

Under the illumination of 330-380 nano-ultraviolet light, take place and the covalently cross-linked photochemical reaction of dna double chain interchain.After the reaction, it is connected with optics particulate (LB) with connecting arm (L), realizes the signal mark.Perhaps, with the AMT or derivatives thereof directly and connecting arm synthesize one.Program is as follows: $\left(1\right)\mathrm{AMT},\mathrm{Suc}-L^{n^{+}/n^{-}-}\mathrm{SH},$ $\left(2\right)\mathrm{AMT},\mathrm{Suc}-L^{n^{+}/n^{-}}-B$ $\left(3\right)\mathrm{AMT}-L^{n^{+}/n^{-}}-\mathrm{SH},$ $\left(4\right)\mathrm{AMT}-L^{n^{+}/n-}B,$ $\left(5\right){\mathrm{LB}}^{n^{+}/n^{-}-}{L}^{n^{+}/n^{-}-}\mathrm{MAL}$ (tensio-active agent)

Annotate: Suc-is a succinimide ester, the specific efficient reactive group of amido; B is a vitamin H, and Mal-is a maleimide, or the functional group of iodo-acid amide modification.

(3) optics particulate optical particulate can be a silica optics magnetic bead, high polymer micro balloons etc.But its optimal selection form is the superpolymer fluorescent microsphere, as polystyrene fluorescent microsphere etc.Existing at present commercially available various surface functional groups are modified (as amido, carboxyl, hydroxyl, sulfonic group etc.), contain the fluorescent microsphere of the fluorescence dye of various excitation wavelengths (350-750nm) and emission wavelength (400-800nm).Particle diameter also can be chosen arbitrarily at 20nm to tens micron as required, is equivalent to contain 10 respectively in each microballoon ²-10 ¹⁰Individual above fluorescein(e) dye molecule.We select the concrete parameter attribute of fluorescent microsphere will be according to the finishing of gene chip slide 26, and detection sensitivity and linearity range need, and the electrical requirement of ion of hybridization and labeling process is oozed in swimming, and excitation light source etc. are all multifactor.Such as, for fear of non-specific mark, when considering the surface functional group of optics particulate, the function of surface group that need fully take into account gene chip slide 26 modifies: if the surface of glass slide modification group is aldehyde group modified, behind the gene chip sample applying, the aldehyde radical of uncured nucleic acid sealed make it be converted into hydroxyl.So, the finishing group of optics particulate is preferably avoided carboxyl, and can select amido or hydroxyl.It is directly related that the particle diameter of optics particulate and the detection sensitivity of system and linear detection range actual requires.The big more detection sensitivity of particle diameter is high more and linear detection range is narrow more.Ooze operation for satisfying swimming, electric charge such as ammonium ion are carried in the surface of optics particulate, imidazoles, and carboxylate radical, azochlorosulfonate acid anion, but inside embedding magnetic core are so that magnetic field operation down.

Tensio-active agent in the labelled reagent composition, adds tensio-active agent, and with ultrasonic oscillation the optics particulate is dispersed in the solution effectively for eliminating the assembly that the optics particulate produces because of the surface hydrophobicity effect.For the functional group that makes the optics microparticle surfaces " is not flooded " by the molecular chain of tensio-active agent, influence labeling effciency, preferably select the low fluorine series of surfactants of short molecular chain and micelle-forming concentration, as the FC-143 that 3M company produces, FC-129 etc.HCL instrument HCL instrument designs in order to help HCL test kit 19 to finish required molecular biology process.It provides HCL test kit 19 needed temperature control in each process, photochemical reaction, alternating electromagnetic field, the hardware supported of ambient conditionss such as the sequencing conveying of various solution.Under the control of the automatic control system of being formed by computer 20 (upper computer and software) and micro-chip 14 (lower computer CPU and software thereof), by the performed task division in gene chip hybridization chamber 2 (control terminal) is had following several executive system, the control principle synoptic diagram as shown in Figure 1.

Each numbering is corresponding as follows among the figure: ultraviolet lamp 1, and gene chip hybridization chamber 2 connects key 3 soon, electronic circuit in the gene chip hybridization chamber is printed version Wiring port 4, thermoelectric heat pump 5 is arranged at the temperature probe 6 between gene chip hybridization chamber 2 and the thermoelectric heat pump 5, is used for the temperature of testing end; Be used to detect the temperature probe 7 of the envrionment temperature (medium temperature during the cold or water-cooled of air) of thermoelectric heat pump 5, threeway 8, peristaltic pump pump head 9 can be accepted the peristaltic pump motor 10 that micro-chip 14 instructs, switch power supply 12, unicircuit 13 is accepted the air cooling or the water-cooled of micro-chip 14 instructions and is carried out terminal 11, micro-chip 14, hyperchannel solenoid valve 15, i=1,2,3; Step motor 16, step motor servomechanism 17, laser apparatus 18, HCL test kit 19, computer 20.

One, temperature controlling system: this system is by thermoelectric heat pump 5 and air is cold or water-cooled device 11, switch power supply 12, temperature probe 6,7 compositions such as grade.Utilizing thermoelectric heat pump 5 to carry out temperature control is principle according to peltier effect, and cold junction becomes with electric current (I) necessarily to concern with temperature head (Δ T) between the hot junction.The temperature of temperature probe 7 and 6 difference testing ends (towards gene chip hybridization chamber 22) and environment (towards working medium water that is used to dispel the heat or air).

Two, alternating electric field Controlling System: there is power amplification circuit 13/ triode in (seeing shown in Figure 1) this system, and connection terminal, gene chip hybridization chamber 2 electronics are printed version PCB core 23 devices such as grade, and the structure design of PCB core 23 as shown in Figure 3; Several groups of magnetic core coils of settling along different angles and orientation etc.;

Annotate: hybridization buffer aperture 29, printed circuit board PCB core 23.

Three, light control system: this system is by ultraviolet lamp 1, and switching value output is controlled;

Four, fluid delivery system: this system is by HCL test kit 19, multiple valve, and computer 20 compatible variable-speed pumps, pipeline connects key 3 soon, threeway 8, compositions such as four-way.

Five, the gene chip hybridization chamber 2: its structure design as shown in Figure 2.

Gene chip hybridization chamber 2, by upper plastic cover, the opaque plastics pedestal, gene chip, device formations such as PCB core are oozed in swimming, it is characterized in that, pedestal is the shaped as frame structure, gene chip slide 26 is embedded in pedestal central authorities with facing up, pedestal bottom shaped as frame opening greater than the gene dot matrix area in gene chip slide 26 fronts less than gene chip itself, make excitation beam gene dot matrix area in covering gene chip slide 26 fronts fully still after refraction, simultaneously can hold gene chip slide 26 again, on gene chip slide 26, place the shaped as frame swimming and ooze the PCB core, latter's frame inwall is shown discrete metalized surface, by the plated through-hole that forms in the PCB course of processing by after tailor technology and form, the discrete metalized surface of the metal plating circuit that comes out by etching in the metallization of these frame inwalls (gold or silver etc.) surface and frame outer wall links to each other, the latter is as the external tapping of PCB core, oozing ion manipulation with the swimming of HCL instrument partly links to each other, the PCB core is oozed in whole swimming, gene chip and base part are covered by a upper plastic cover, the liquid access way is located on upper plastic cover or the pedestal, and gene chip hybridization chamber 2 humidity keep damping fluid storage aperture to ooze on the PCB core in swimming, and lead to extraneous by the aperture of upper plastic cover corresponding position, this aperture is by its outlet of the little plug envelope of a rubber degree, gene chip is from bottom to top respectively by slide, chromium metal (1～100 sodium rice, optimum thickness can be 3 sodium rice), silver (or gold 10～1000 sodium rice, optimum thickness can be 40,50,75 sodium rice etc.), quartzy (1～100 sodium rice, optimum thickness can be 3 sodium rice or 5 sodium rice) etc. coating constitute, at quartzy coating surface process silanization, finishing such as amido or aldehyde radical technology forms the gene chip slide 26 that can directly solidify gene molecule, forms gene chip through gene chip point system technology again.Annotate: the numbering among Fig. 2 is described as follows: electronics track plate 23 (detailed structure is seen Fig. 2), PCB Wiring port 24, gene chip hybridization chamber base 25, gene chip slide 26, gene chip hybridization chamber window lid 27, gangway, gene chip hybridization chamber 28, damping fluid sulculus 29, the glass window 30 that gene chip hybridization chamber window covers, the liquid of gene chip hybridization chamber is drilled inner chamber 31.

The structure design purpose of gene chip hybridization chamber 2 is in order to satisfy light, electromagnetism, and heat, extraneous various factors such as logistics is to the programming of gene chip.

The HCL instrument will be used for being achieved as follows control and require (following procedural order can not change):

1. quick hybridization process: quick hybridization is oozed in swimming.Needed hybridization buffer of this process and dna solution are seen through in the aperture at 2 two ends, gene chip hybridization chamber by means of micropipette by the technician and are added drop-wise on the gene chip.And damping fluid is added drop-wise in the damping fluid sulculus 29 of gene chip hybridization chamber 2.Subsequently, by connecing gene chip hybridization chamber 2 that the top-notch player is mounted with the DNA hybridization solution HCL instrument gene chip processing enter of packing into soon, enter following automatic hybridization program.

Procedural order	Flow (ml/min)	Process duration (s)	Temperature (℃)	Remarks
					2.No.1 solution	0～2000	?60～600000	?-10～100	Collaborative processes
3.NO.2 solution	10～10000	?1～5000	?0～100	This strength of solution changes sees embodiment
					4.NO.3 solution	0.006～3000	?1～120000	?-30～30	(a)
5.No.4 solution	10～10000	?1～5000	?0～100	Elute soln, rinsing solution
					6.NO.5 solution	0.1～5000	?1～300000	?0～60	Label solution
7.NO.6 solution	3000～5400	?2～1000	?0～60	Elute soln
					8.NO.7 solution	10～10000	?2～10000	?0～60	Rinsing liquid
9.No.8 ventilate	1～100000	?1～100000	?0～120	Wind drenches (b)

Annotate: provide parameter variation range often require be designed to the laboratory can be convenient adjustable by computer 20, to be convenient to finding more optimal experimental data by the automation process of HCL model machine.Each solution composition is as follows respectively in the HCL test kit 19: No.1 solution: hybridization buffer, and as 5 * SSC, dna solution, pH2.5-10.0; No.2 solution: hybridization elution liquid, select traditional ripe SDS wash-out prescription for use; Crossover process is according to concrete needs adjustment and controlled temperature.Collaborative processes: (1) is by closed electromagnetic valve gene chip hybridization chamber 2; (2) start the alternating electric field system and accelerate the hybridization kinetics process; No.3 solution: probe molecule solution, contain general formula as be shown in the examples (I) compounds, be dissolved in the Na that is lower than 10mM ₂HPO ₄Damping fluid.The pH value of final solution should be in the 2.5-10.0 scope, and salt concn must guarantee to be lower than 20mM.This process can at room temperature be carried out, and the time length is not waited from 5 minutes by 1 hour; No.4 solution: the non-specific or elute soln of bonding probes molecule not on the gene chip.Concrete composition is looked probe molecule and different; No.5 solution: label solution, in the finishing of optics particulate molecular recognition group that a large amount of and probe molecule can specific reaction or immune specific reaction molecule etc. are arranged, constructional feature sees embodiment for details.Simultaneously, in order to overcome the adhesive attraction between the optics particulate, in the preparation of label solution, add the short chain surfactants (as fluorine surfactant etc.) of capacity, and make it to form uniform system by ultrasonic concussion.No.6 solution: elute soln, use the above-mentioned fluorine surfactant or the SDS solution of same concentration.No.7 solution: rinsing liquid, be triple distillation water, or deionized water.

(a) can in the static system under the zero degrees celsius, carry out during irradiation, not wait from 5 minutes by 60 minutes according to the irradiation dose time length.Light source: high voltage mercury lamp or RUL3500 Jupiter, the distance of itself and gene chip hybridization chamber is less than 2 centimetres.350～380nm, preferably the about 8.3-40kJ/m of radiation dose is carried out in the UV radiation of 365nm synchronously ²

(b) employed air-flow process air filterability equipment in the wind pouring process.The spr signal detection system

This system utilizes surface plasma body resonant vibration signal excitation technology, at the metal and the quartzy plated film that are used for plating on the slide material of producer gene chip one deck ultra-thin (thickness that has only tens nanometers), and on the quartz surfaces that is plated, solidify nucleic acid molecule, realize hybridization, mark, the process that wash-out and signal detection etc. are necessary.Its principle schematic as shown in Figure 3.

1. total system is excited by spr signal, and three parts of ccd signal collection and gene chip constitute.Preceding two systems are designed to strict light and isolate, and promptly are designed to be mutual light and isolate and isolate with the light in the external world separately.Position and angle are fixed in whole instrument to the angle of excitation light source 18, collimator tube, beam expanding lens 37 etc. in design with when making.When the user places gene chip hybridization chamber 2, itself and exciting light collimated beam angulation are not critical angle usually, main dependence critical angle this moment servosystem 42 is adjusted the angle of gene chip hybridization chamber 2 automatically by the mode of asking extreme value by step motor (microstepper motor) 16 performers such as grade, make input angle reach the SPR critical angle, thereby at positive surface phasmon TM ripple, i.e. the surface plasma body resonant vibration ripple of producing of gene chip.In the gene chip front, we are gene marker---Gao He signal vehicle on the secondary excitation light source activation gene chip with this surface plasma body resonant vibration ripple, thereby produce the exciting field that the gene chip surface closely suddenly dies.The fluorescent signal that signal vehicle in this exciting field is subjected to exciting the back to produce, by optical imaging system with these signal focus and be projected on the CCD chip, thereby realize by the conversion of optical image to digital code information.At last, by computer these digital code informations are handled and analyzed again.Because as the surface phasmon of excitation light source is the evanscent field that acts in the micron-scale from nano surface, can't effectively enter becomes background noise in the imaging system, and it excites the fluorescent signal of generation to arrive imaging system away from the gene chip surface, so the exciting light noise can effectively shield.

2.SPR the two is all the optics closed system mutually and separately signal generation system and ccd signal acquisition system.

The key points in design of above-mentioned three parts is set forth respectively below.

3.SPR signal excitation part: as shown in Figure 3,, with the parallel expansion bundle of light beam that laser apparatus 18 sends, make it to shine abreast the back side of gene chip by beam expanding lens in the spr signal excitation portion.Plate chromium, silver and quartzy respectively on gene chip slide 26 surfaces that are used for some the system gene, afterwards through silanization, functional group finishing, in order to make a gene chip.During signal detection, the parallel polarization exciting light is through plane prism 47 and the back surface incident of optical match liquid 48 from gene chip, adjust the angle of carrying genes chip hybridization chamber 2 Movable bracket platforms 49 (plane prism 47 is fixed wherein) when luminous energy 38 detected energy of reflection light reach mnm. by critical angle servosystem 42, critical angle servosystem 42 angular adjustment stop and make fixed angle in this angle, thereby make the incident excitation beam become SPR critical angle incident with gene chip, cause at the positive surface plasma resonance ripple that produces of gene chip, excite the gene chip surface signal vehicle of mark by this resonance wave, thereby produce fluorescence or corresponding optical signalling, utilize lens imaging system 33 to transmit signals to CCD32 and be converted into the digital code information that can be write down and analyze by computer 20.

Numbering explanation among Fig. 3: charge coupled device ccd 32 (including CCD driving circuit and data acquisition circuit plate), imaging system 33, gene chip hybridization chamber 34, platform bracket 49, rotating shaft (fulcrum) 35,36; Beam expanding lens 37, luminous energy 38, laser apparatus 18 (built-in collimation head), step motor 16, recliner 39, switch power supply 40,41; Critical angle servosystem 42, micro-chip 43, computer 20, spr signal excitation chamber 44 and ccd signal are gathered chamber 45, polarizer 46, plane prism 47 (specific refractory power 1.70), optical match liquid 48 (glycerine is the Cargill product, and specific refractory power is 1.70).

2.CCI) the signals collecting part:

The optical design of gene chip: 26 preparations of gene chip slide and cleaning step are as follows:

1. be 1.70 with specific refractory power, surface finish is wiped surface blot and dust at the slide of 0.5 Newton's rings with lens paper.

2. put into Virahol and chloroform and respectively soaked 30 minutes, then, insert in the vitriol oil and hydrogen peroxide (30%) mixed solution and spend the night in (V/V, 1: 1).

3. take out the back with the ultrapure water flushing, put into about 20 minutes of the alcohol saturated solution of KOH again.

4. again through the ultrapure water flushing, place the ethanolic soln of 10%HCL to preserve.

5. before the depositing metallic films, with the ultrapure water flushing, nitrogen dries up, and places EDWARDS E306A vacuum plating unit sputtering sedimentation metallic membrane: deposit 3 nanometer titaniums successively, 50 nanometer silvers, 10 nanometer titanium dioxide silicon films.

6. the cleaning step of this substrate by semiconductor material cleaned, put into the HF dilute solution again and handled 2 minutes, take out the back and wash repeatedly with distilled water, dry under nitrogen afterwards.The finishing step of gene chip slide 26 is as follows:

1. substrate is immersed in the benzole soln of APTES (APTES is a γ-An Jibingjisanyiyangjiguiwan, and Tokyo changes into) (10 ^-1Mmol/l) reaction is 6 hours, takes out back toluene, and acetone cleans, and dries up with nitrogen simultaneously.

2. substrate is immersed and contain 1% glutaraldehyde (glutaraldehyde, Shanghai chemical reagent station packing.The all homemade analytical pure of all the other reagent) (pH7.2,0.05M) reaction is 2 hours in the PBS damping fluid.

3. activatory carrier acetone, ethanol, and distilled water flushing, and dry under nitrogen.

Because the technology at glass or quartz surfaces curing nucleic acid is a lot ^(18-24), and very ripe, can ensure the firm connection of nucleic acid, adjustable density, breviary herein.The point system of gene chip slide 26: this part technology belongs to routine techniques, thereby repeats no more because of non-patent ability.

Except that metallic membranes such as chrome-faced, gold and silver, plate again outside the quartz thin film, also can remove from gold or silver surface and plate quartz again, but the method for direct fixed nucleic acid.Because in the technology of gold surface fixed nucleic acid comparative maturity ^(13-17), thereby, repeat no more.Kind is a lot of on the present gene chip data analysis software market of data analysis system, and therefore, this respect is based on purchase.Intelligent chip diagnosis (ICD) necessity: because biochip test has the high-throughput characteristic, its detected result provides magnanimity information, and biomedical development causes the division of labor more and more thinner, and the clinician is difficult to the biochip test result is comprehensively analyzed.Thereby be necessary to adopt biometrical method, utilize the detection quantity of information that disease is differentiated diagnosis to greatest extent, and will differentiate diagnostic result and be arranged at biochip customer analysis interface purpose: by the detection of biochip magnanimity information, utilize biometrical method easily to suffer from the diagnosis of physique, disease is differentiated diagnosis.Method:

1. the foundation of discriminant function formula:, and collect clinical data according to the detection of biochip.At first each factor is selected by biological significance and statistics multiplicity method, utilized biometrical method to set up the discriminant function formula according to the factor after selecting then.

2. differentiate diagnosis: with the discriminant function formula that each factor value substitution of biont has been set up, obtain its functional value, and draw the probability that it suffers from various diseases, it is carried out disease differentiate diagnosis.

3. the optimization of discriminant function formula: because the exactness of discriminant function formula is along with the number positive correlation of observing routine number and detection index thereof, thereby we will set up the biochip test database, list the detection individuality that each example increases newly in database, regularly data of database is examined, and in conjunction with the individual discriminant function formula (promptly enlarge the routine number of observation and rebulid the discriminant function formula) of optimizing of the detection that increases newly.Along with being extensive use of of biochip, our database enlarges gradually, and the discriminant function formula of You Huaing will have high differentiation diagnostic value gradually.Characteristics:

1. unite two into one: the detected result interface of common biochip only provides the detected result of each index (as each gene) that is detected, we dissolve in gene chip detected result customer analysis interface with the discriminant function formula, thereby when each index is detected, and further comprehensive all indexs are carried out the differentiation diagnosis of disease to biont.

2. dynamic: when biont being detected and disease differentiates diagnosis, the result feeds back to database with its last diagnostic, and in conjunction with ICD check and analysis result the discriminant function formula is optimized processing.The simple example explanation: the discriminant function formula is found the solution very complicated, and existing two classes differentiation with two detection indexs illustrates

1. the foundation of discriminant function formula: the diastolic pressure (x that obtains n example coronary heart disease and m example normal people ₁) and plasma cholesterol (x ₂) value, then utilize the biostatistics discriminatory analysis can be in the hope of discriminant function formula Z=b ₁x ₁+ b ₂x ₂, b wherein ₁, b ₂Be constant term.Then with patient and normal people's x ₁And x ₂Substitution discriminant can obtain the average discriminant value Z of patient _{The patient}, the average discriminant value z of normal people _{The normal people}, then can set up discriminant critical value Z ₀=(Z _{The patient}+ Z _{The normal people})/2.

2. differentiate diagnosis: to some new prescription on individual diagnosis persons according to its diastolic pressure (x ₁) and plasma cholesterol (x ₂) be worth, can try to achieve the value Z of its discriminant function formula _{Go to a doctor}, then with Z _{Go to a doctor}With Z ₀Relatively, illustrate greatly then that as the former this prescription on individual diagnosis case is the coronary heart disease patient, show then that less than the latter this prescription on individual diagnosis case is non-coronary heart disease patient as the former, and can be according to Z _{Go to a doctor}With Z ₀Difference try to achieve the probable value of this prescription on individual diagnosis patient's trouble coronary heart disease possibility.

3. the optimization of discriminant function formula: above-mentioned a certain new case's diagnostic result is fed back to database, so the case last diagnostic is a coronary heart disease, and then we reach the n example coronary heart disease and the m example normal people (n+1 example patient altogether) that set up the discriminant function formula first in conjunction with this routine patient and rebulid discriminant function.Along with the increase of database, the discriminant function model is optimized gradually.

The beneficial effect that invention is compared with background technology

The beneficial effect that the present invention compares with background technology has following several aspect:

1, system is to the detection sensitivity height of gene chip, and the signal to noise ratio height can satisfy the high requirement to the sensitivity aspect of most scientific researches and practical field, helps promoting the popularization and application of gene chip at aspects such as clinical diagnosis, industrial and commercial quarantine and environment measurings,

2, system handles process integration degree height, collection hybridization, mark, wash-out, signal detection and data processing etc. improve professional and technical personnel's processing efficiency in one, have also reduced the professional technique requirement to non-specialized-technical personnel simultaneously, improved gene chip use in result's repeatability and stable

3, system is to the efficient height of gene chip processing, because in hybridization, mark has adopted (alternation) electrophoresis in the processes such as wash-out, electroosmosis technology improves the timeliness of the main process in the processing of said gene chip greatly,

4, system cost and running cost are cheap, are suitable for the popularization and application of gene chip,

5, system is applicable to the glass gene chip, and helps the application of this kind gene chip universalness, becomes the main product in the parallel detection technique of range gene.

Most preferred embodiment is about the explanation of HCL test kit composition

In the gene chip hybridization signal magnifying tags technology, there are two class reagent to mention.They are respectively through after the crossover process at gene chip, as with gene chip on the nucleic acid hybridization two strands of complementary pairing under certain condition can specificity bonded probe molecule general formula (I) compound, and general formula (II) compound that the heteroduplex nucleic acid that is connected with probe molecule is carried out the fluorescent signal mark as high lotus signal vehicle.Their constitutional features is respectively:

General formula (I) compound: Ps-L ₁-A,

General formula (II) compound: FB (L ₂-A ') _n, wherein,

One, Ps-represents any derivative of psoralene (Psoralen), it can be a water-soluble 4 '-methylol-4,5 ', 8 '-trimethylpsoralen (4 '-Hydroxymethyl-4,5 ', 8-trimethylpsoralen or 4 '-hydroxymethyltrioxalen, write a Chinese character in simplified form HMT), 4 '-amine methyl-4,5 ', 8-trimethylpsoralen (4 '-hydroxymethyl-4,5 ', 8-trimethylpsoralen or 4 '-aminomethyltrioxalen, write a Chinese character in simplified form AMT), trimethylpsoralen (trimethylpsoralen writes a Chinese character in simplified form TMP), 8-methoxypsoralen (8-methoxypsoralen is abbreviated as 8-MOP), 4,7,4 '-and 4,7,5 '-trimethylammonium isopsoralen (4,7,4 ' and4,7,5 '-trimethylallopsoralen) and psoralene itself etc.;

Two, A-and A '-be meant any a pair of have certain chemical reaction or interaction specific probe molecule joint, they can be one of following situations: (1) special reaction functional group is right; (2) immune response is right; (3) specific molecular of other form identification interaction partners;

Three ,-L ₁-and-L ₂-expression solvent compatibility connecting arm, promptly when solvent was hydrophobic systems, connecting arm also was hydrophobic connecting arm; When solvent was hydrophilic system, connecting arm also was hydrophilic connecting arm, as PEG, peptide chain, carbohydrate chain etc.;

Four, FB represents the high lotus signal vehicle that exists with various fine particle forms, it can be that superpolymer fluorescent microsphere, silica magnetic bead and other contain the carrier of high-content fluorescence dye, its finishing have can with the functional group of probe molecule joint A-specific reaction in general formula (I) compound or effect to A '-.

Below set forth with regard to various different sights combinations by some embodiment.

The labelling method of the fluorescent microsphere of the surperficial avidin 9 white marker of example one usefulness

By means of the specific molecular recognition capability of vitamin H, nucleic acid double chains such as vitamin H and psoralene or derivatives thereof AMT are embedded molecule synthesize biotinylated molecule by connecting arm, biotinylation psoralene or derivatives thereof AMT is called for short BP.Thereby reach the purpose of having hybridized to become double-stranded nucleic acid molecule with biotin labeling, the bonding strength of utilizing psoralene or derivatives thereof AMT to reinforce heteroduplex again simultaneously.The process of synthetic BP is from Tetraglycol 99, successively at p-methyl benzene sulfonic chloride (in pyridine), to generate the disulfonic acid ester through affine substitution reaction; Nitrogen salt and reductive agent react generation two repeatedly nitrogen salt and diamine compounds respectively with repeatedly again.Generating BP with step reactions such as two-(tertiary butyl)-dicarboxylic ester and AMT.

Synthetic BP carries out mark as the nucleus in the No.3 solution by photochemical crosslinking reaction pair double-strandednucleic acid.Afterwards, polymer (as the polystyrene etc.) fluorescent microsphere (SIGMA company, the FluoroSpheres that molecular probe (Molecular Probes) company etc. produce) of selecting commercially available avidin 9 albefaction for use is as the nucleus in high lotus signal vehicle and the No.4 solution.

Also can directly buy commercially available BP product (Molecular Biosciences company):

(+)-vitamin H-PEO ₄-psoralene C ₃₃H ₄₄N ₄SO ₁₀Mr=688.79; Mp 98-100 (+)-vitamin H-PEO that also can the said firm ₄-amine [(+)-Biotin-PEO ₄-amine] be raw material,

With it add vinyl alcohol two-(succsinic acid)-two (succinates) [ethylene glycol bis-(succinicacid), bis-(succinimidyl ester), write a Chinese character in simplified form EGS] (Molecular Probes company Cat.#E-6306) can synthesize PB at normal temperatures with AMT.

In addition, the production of vitamin H-psoralene (biotinylated psoralen) also can be produced (USPatent No:4,868,311 according to the United States Patent (USP) technology; 9/19/89).Example dual-purpose psoralene-vitamin H carries out mark

Utilize Schleicher ﹠amp; Psoralene-vitamin H (United States Patent (USP) U.S.Patent#4,599,303) that Scheull company produces carries out mark.Process is as follows:

(1) with the gene chip hybridization chamber 90 degrees centigrade of following incubations 10 minutes,

(2) after the hybridization of gene chip hybridization chamber, fast cooling to-20～0 degree centigrade with iced it, after 2～5 minutes, flow velocity with 3ml/min during beginning pumps into psoralene-biotinylated probes solution, flow velocity with 0.06～1ml/min after about 30 seconds pumps into, and continues to accept irradiation 1～60 minute under iced condition;

(3) temperature is set to room temperature, stops irradiation, pumps into the dH of propyl carbinol with the flow velocity of 1～10000ml/min ₂O saturated solution 10 seconds to 30 minutes pumps into the dH of ether afterwards ₂The O saturated solution;

(4) under the room temperature, pump into the label solution 10～30000 seconds of the fluorescent microsphere (SIGMA company or Molecular Probes company product) that surperficial avidin modifies with the flow velocity of 0.1～5000ml/min;

(5) flow velocity with ml/min pumped into dH in lasting 1～1000 second under the room temperature ₂The O elutriant.The biotinylated fluorescent microsphere labelling method of example avometer face

(FluoroSpheres of Molecular Probes company) carries out mark with the fluorescent microsphere that commercially available surface biological elementization is arranged.At this moment, an end of synthetic connecting arm is an avidin, and the other end is AMT.Example four marking sulfhydryl methods

Sulfydryl specific reaction functional group has maleimide, iodo-acid amide.During the nucleic acid probe, with (Ionized) hydrophilic connecting arm (as the artificial synthetic polypeptide chain, polyoxyethylene glycol (PEG) etc.) with psoralene or its following derivative AMT, 4 '-methylol-4,5 ', 8-trimethylpsoralen (HMT) etc. couple together with sulfydryl or maleimide, form: the chain molecule of " probe-connecting arm-joint A " pattern.Structure as AMT-PEG-Cys:

Or as the structure of HMT-polypeptide-halfcystine (APC):

AMT-Peptide-Cys is abbreviated as: APC or as the structure of AMT-PP-MAL: AMT-PP-MAL is abbreviated as APM.R-, R '-for non-sulfydryl or contain the group of disulfide linkage or utilize AMT and succinimido-instead-4-(maleimide ylmethyl)-cyclohexyl-1-carboxylicesters ^(25,26)(SMCC, Molecular Probes, Cat.#S-1534) synthetic AMT-CH-MAL, thus with the amino on the AMT be converted into can with the dimaleoyl imino of sulfydryl specific reaction [other reagent such as succinimido-own ester of 6-dimaleoyl imino (and EMCS, Cat.#S-1563) also can]:

AMT-CH-MAL is abbreviated as ACM HPM-PEG-MAL is abbreviated as HPM or as the structure of HMT-PP-MAL:

And fluorescent tag molecule synthetic followed following structural pattern: " fluorescent microsphere (connecting arm-joint A ') _n".Above-mentioned joint A and joint A ' they are a pair of specific reaction functional groups, as

1) LB (PEG-SH) _nWith the MAL-PEG-AMT reaction, or

2) LB (PP-SH) _nWith the MAL-PP-AMT reaction, or

3) LB (PEG-SH) n and APM, or

4) LB (PP-SH) _nWith HPM, or

5) LB (PP-SH) _nWith ACM reaction etc.

With the 4th) or the 5th) mark chemical equation that reaction pair is carried out is as follows:

1) under iced cold condition, HPM forms double-stranded embedded type mixture with the nucleic acid hybridization two strands (dsDNA) of surface of glass slide specifically:

2) under 5-10 minute 330-380 sodium rice UV-irradiation, the psoralene of HPM part is reacted with double-strandednucleic acid generation interchain covalent cross-linking:

3) under the normal temperature, the mercapto-functionalized superpolymer fluorescent microsphere (as the polystyrene fluorescent microsphere) in surface just can form covalently bound reaction in the aqueous solution (add in the solution fluorine surfactant etc. as stablizer):

The fluorescent microsphere that example five usefulness surface is modified by amido carries out mark

Generally, because the gene chip slide often forms one deck polylysine film behind silylation modification, thereby for avoiding the caused background of non-specific binding too high, general polylysine slide gene chip system is not used the fluorescent microsphere mark of surface amino groups modification.Unless the gene chip slide is when gold-plated processing is solidified gene by sulfydryl, the fluorescent microsphere that can adopt surperficial amido to modify.This fluorescent microsphere can directly buy from Molecular Probes company and Sigma company.

As the probe molecule of specific double-strand nucleic acid, and succinimido-[4-(psoralene-8-oxygen base) butyl ester] of purchase Molecular Biosciences company (Succinimidyl-[4-(psoralen-8-yloxy)] butyrate, be abbreviated as SPB) ^(26-30)Effective constituent as No. three solution in the HCL test kit.

Index

1.Ramsay?G，et?al.DNA?Chip：State-of-the-art.Nature?Biotechnology?16(1)：40-44，1998

2.Chee?M，Yang?R，Hubbel，et?al.Accessing?genetic?information?with?high-density?DNA?arrays.Science?274(5287)：610-613，1996

3.http：// www.affymetrix.com

4.Yershov?K，et?al.DNA?Analysis?and?Diagnosis?on?Oligonucleotide?Chips.Proc.Natl.Acad.Sci.USA?93(10)：4913-4918，1996

5.http：// www.genomicsolutions.com

6. http：//www.incyte.com

7. http：//www.chinagenenet.com

8.Christine?D，et?al.DNA?Microarray?in?drug?discovery?and?development.?Nature?GeneticsSupplement.21(1)：48-50，1999

9.Duggan?D?J.Nature?Genetics?Supplement，21(1)，42-47，1999.

10. http：//www.nanogen.com

11. http：//www.aclara.com

12. http：//www.motorola.com/lifesciences/

13.Journal?of?American?Chemical?Society?109(8)：1987

14.Journal?of?American?Chemical?Society?119(38)：8916，1997

15.Chemical?Letters?863，1996

16.Analytic?Chemistry?69,4948，1997

17.Analytic?Chemistry?69,4939，1997

18.Histochemistry?101,325，1994

19.Biosensors?and?Bioelectronics?7：487，1992

20.Nuclear?Acid?Research?22(24)，5456，1994

21.Analytical?Biochemistry?247，961997

22.Analytical?Biochemistry?187(1)，56，1990

23.Nuclear?Acid?Research?24(15)，3031，1996

24.Analytical?Chimica?Acta?344,201，1997

25.European?J.?Biochemistry?101,395，1979

26.Analytical?Biochemistry?198,75，1991

27.Laskin，J.D.，et.al.PNAS?USA?83,8211.1986

28.Inman，R.B.，et.al.J.Molecules.?Biol.193，377.1987

29.Hartley，J.A.，et.al.Anti-Cancer?Drug?Des.9,181.1994

30.Elsner，H.I.，et.al.Bioconjugate?Chemistry.?5,463-467.1994

Description of drawings

Accompanying drawing 1, gene chip molecular probe Automatic Control Theory synoptic diagram

1 ultraviolet lamp,

Gene chip hybridization chambers 2,

3 connect key soon,

Electronic circuit in the gene chip hybridization chambers 4 is printed version Wiring port 4,

5 thermoelectric heat pumps,

6 are arranged at the temperature probe 6 between gene chip hybridization chamber 2 and the thermoelectric heat pump 5, are used for the temperature of testing end,

The temperature probe 7 of 7 envrionment temperatures that are used to detect thermoelectric heat pump 5 (medium temperature during the cold or water-cooled of air),

8 threeways,

9 peristaltic pump pump heads,

10 can accept the peristaltic pump motor of micro-chip 14 instructions,

11 air cooling or the water-cooleds of accepting micro-chip 14 instructions are carried out terminal,

12 switch power supplies,

13 unicircuit,

14 micro-chips,

15 hyperchannel solenoid valves, i=1,2,3,

16 step motor,

17 step motor servomechanisms,

18 laser apparatus,

19 HCL test kits,

20 computers.

Accompanying drawing 2, gene chip hybridization cell structure schema

23 electronics track plates (detailed structure is seen accompanying drawing 2),

24 PCB Wiring ports,

25 gene chip hybridization chamber bases,

26 gene chip slides,

27 gene chip hybridization chamber window lids,

Gangway, gene chip hybridization chamber 28,

29 damping fluid sulculuses,

The glass window that 30 gene chip hybridization chamber windows cover,

The liquid of gene chip hybridization chambers 31 is drilled inner chamber 31.

Accompanying drawing 3, SPR-CCD signal detection and data handling system synoptic diagram

32 charge coupled device ccds (including CCD driving circuit and data acquisition circuit plate),

33 imaging systems,

Gene chip hybridization chambers 34,

35 rotating shafts (fulcrum),

36 rotating shafts (fulcrum),

37 beam expanding lenss,

38 luminous energies,

39 recliner,

40 switch power supplies,

41 switch power supplies,

42 critical angle servosystems,

43 micro-chips,

44 spr signal excitation chamber,

45 ccd signals are gathered the chamber,

46 polarizers,

47 plane prisms (specific refractory power 1.70),

48 optical match liquid 48 (glycerine is the Cargill product, and specific refractory power is 1.70),

49 platform brackets 49.

Claims (10)

1. gene chip hybridization signal magnifying tags technology, it is through after the crossover process at gene chip, utilize the nucleic acid hybridization double-stranded specific probe molecule of the compound of general formula (I) as complementary pairing, and utilize the compound of general formula (II) the heteroduplex nucleic acid that is connected with probe molecule to be carried out mark as high lotus signal vehicle

The compound structure of general formula (I) is characterized as: Ps-L ₁-A,

The compound structure of general formula (II) is characterized as: FB (L ₂-A ') _n, wherein,

Ps-represents psoralene (Psoralen), isopsoralen and their any derivative form, it can be 4 '-methylol-4,5 ', 8-trimethylpsoralen (4 '-hydroxymethyl-4,5 ', 8-trioxalen, write a Chinese character in simplified form HMT), 4 '-amine methyl-4,5 ', 8-trimethylpsoralen (4 '-aminomethyl-4,5 ', 8-trioxalen writes a Chinese character in simplified form AMT), trimethylpsoralen (trimethylpsoralen writes a Chinese character in simplified form TMP), 8-methoxypsoralen (8-methoxypsoralen, be abbreviated as 8-MOP), 4,7,4 '-and 4,7,5 '-trimethylammonium isopsoralen (4,7,4 '-and 4,7,5 '-trimethylallopsoralen) and psoralene itself etc.

A-and A '-be meant any a pair of have certain chemical reaction or interaction specific probe molecule joint, they can be one of following situations: (1) special reaction functional group is right; (2) immune response is right; (3) specific molecular of other form identification interaction partners,

-L ₁-and-L ₂-expression solvent compatibility connecting arm, promptly when solvent was hydrophobic systems, connecting arm also was hydrophobic connecting arm; When solvent was hydrophilic system, connecting arm also was hydrophilic connecting arm, can be PEG, synthetic polypeptide etc.,

FB represents the high lotus signal vehicle that exists with various molecule forms, it can be that superpolymer fluorescent microsphere, finishing have the silica magnetic bead of fluorescence dye and other to contain the carrier of high-content fluorescence dye, its finishing have can with the functional group of probe molecule joint A-specific reaction in general formula (1) compound or effect to A '-

Utilize the method feature of heteroduplex nucleic acid on general formula (I) the compound treatment gene chip to be: after crossover process is finished, under-50～100 degrees centigrade temperature condition, will contain general formula (I) compound concentration and be that the time length is in 20 hours in the solution quiding gene chip hybridization chamber 2 of 0.001mM～10M, the interchain covalent cross-linking reaction of this compound in nucleic acid double chain takes place under ultraviolet lighting, thereby the specificity that realizes itself and double-strandednucleic acid is covalently bound

The method feature that utilizes general formula (II) compound to carry out the signal mark is: general formula (I) compound and double-strandednucleic acid realize covalently bound after, importing contains the solution of general formula (II) compound, continue 1～100000 second time, then non-specific adsorption is carried out wash-out.

2. a gene chip signal mark of making according to claim 1 and amplify test kit, promptly the HCL test kit 19, comprised gene chip and handled the needed whole biochemical reagents of each process, contain following component:

General formula (I) compound, general formula (II) compound.

3. hybridization of mainly being engaged in gene chip according to claim 1 and claim 2 making, mark, the HCL instrument that wash-out equimolecular biological procedures is handled, mainly constitute by micro-chip 14 automatic control systems and each performer, wherein temperature is controlled performer formations such as the analog quantity control that relies on micro-chip 14 and semiconductor cooler 5 automatically, convection cell is given each pump in the heat-extraction system automatically, the control of valve etc. and ultraviolet lamp 1 relies on switching value control, its principle be according to gene chip must process hybridization, photo-crosslinking, mark, processes such as wash-out, gene chip in the gene chip hybridization chamber is heated to a certain temperature procedurally, and thermostatic control time length, refrigeration is to a certain temperature and constant temperature, carry different solutions, ultra violet lamp etc.

4. gene chip signal detection technique, it is to utilize laser surface plasmon resonance (SPR) technology, shielding exciting light and bias light be to the interferential of signal,

Plate chromium, silver and quartzy respectively on gene chip slide 26 surfaces that are used for some the system gene, afterwards through silanization, functional group finishing, in order to making a gene chip,

When signal detection, the parallel polarization exciting light is through plane prism 47 and the back surface incident of optical match liquid 48 from gene chip, the angle of holding in the palm gene chip hybridization chamber 2 and being fixed with the carriage 49 of plane prism 47 by critical angle servosystem 42 adjustment reaches mnm. and is fixed in this angle until energy meter 38 detected reflected light, thereby make excitation beam become SPR critical angle incident with gene chip, cause at the positive surface phasmon TM ripple that produces of gene chip, excite the gene chip surface signal vehicle of mark by this ripple, thereby produce fluorescence or corresponding optical signalling, utilize lens imaging system 33 to transmit signals to CCD32 and be converted into the digital code information that to be analyzed by computer 20.

5. a SPR-CCD signal detection and data handling system of making according to claim 4, this system has spr signal to produce system and parts such as ccd signal collection and data analysis system, wherein spr signal produces system by laser 18 equal excitation light sources, collimator tube or collimate first-class, polariscope 46, beam expanding lens 37, critical angle servosystem 42, step motor 16, gene chip hybridization chamber 2, micro-chip 43 devices such as grade are formed, it is characterized in that, excitation light source 18, collimator tube, position and angle are fixed in whole instrument to the angle of beam expanding lens 37 grades in design with when making, when the user places gene chip hybridization chamber 2, itself and exciting light collimated beam angulation are not critical angle usually, main dependence critical angle this moment servosystem 42 is adjusted the angle of gene chip hybridization chamber 2 automatically by the mode of asking extreme value by step motor 16 performers such as grade, make input angle reach the SPR critical angle, thereby at the positive surface phasmon TM ripple that produces of gene chip, it is the surface plasma body resonant vibration ripple, in the gene chip front, we are gene marker---Gao He signal vehicle on the secondary excitation light source activation gene chip with this surface plasma body resonant vibration ripple, thereby produce the exciting field that the gene chip surface closely suddenly dies, signal vehicle in this exciting field is subjected to exciting the fluorescent signal of back generation, by optical imaging system with these signal focus and be projected on the CCD chip, thereby realize by the conversion of optical image to digital code information, at last, by computer these digital code informations are handled and analyzed again, because as the surface phasmon of excitation light source is the evanscent field that acts in the micron-scale from nano surface, can't effectively enter becomes background noise in the imaging system, and it excites the fluorescent signal of generation to arrive imaging system away from the gene chip surface, so the exciting light noise can effectively shield, the two is all the optics closed system mutually and separately spr signal generation system and ccd signal acquisition system, promptly, (be in gene chip reverse side one side with gene chip hybridization chamber 2 and platform bracket thereof 49 for marginal spr signal excitation chamber 44, be the side that gene chip is not put system gene square formation) and ccd signal collection chamber 45 (the positive side of gene chip, be a side of gene chip point system gene square formation) be optoisolated between the two on the physical space, promptly in instruments design with in making, the two airtight and insulating space of optics of respectively doing for oneself, bias light in the spr signal excitation chamber 44 and exciting light can't be by scatterings, reflection, various approach such as refraction are leaked to ccd signal and gather in the chamber 45, thereby do not excite the signal that is produced to cause noise jamming to the positive SPR of gene chip.

6. gene chip alternating electric field swimming osmometer solution system manipulation technology, it is the principle that can carry out orientation movement according to charged ion and high inonic strength solution in electric field, thereby impel wherein solution or ion to carry out desired orientation movement by applying alternating electric field on every side, help improving the efficient of hybridization, mark even elution process in the gene chip hybridization chamber.

7. the gene chip swimming osmometer solution system maneuvering system that fabrication techniques according to claim 6 becomes, it contains the integral parts such as PCB core 23, alternating electric field driving circuit----micro-chip, power amplification circuit and connection terminal in the gene chip hybridization chamber 2, it is characterized in that

The gene chip hybridization district arranges a shaped as frame PCB core 23 in gene chip hybridization chamber 2; Its thickness is determined by the size in hybridization chamber; Along being provided with vertically discrete gold-plated electrode of some numbers on the inwall of frame-shaped construction; They lead to two row's interface electrodes of both sides outer wall by up and down two lip-deep glodclad wires of PCB core 23; Gold-plated electrode on PCB core 23 inwalls is corresponding one by one with interface electrode on the outer wall; Link to each other by the glodclad wire on the upper and lower surface of PCB core 23; Electrode number depends on the needs of alternating electric field regulation and control

PCB core 23 is made special-purpose Protel design software design and the etching production process production according to electronic circuit printed board PCB,

The alternating electric field of hybridization region is produced the alternating voltage signal and is amplified generation through electron devices such as power amplification circuits by micro-chip in the PCB core 23 shaped as frame structures, be sent to PCB core 23 by connection terminal at last, and alternating electric field is applied to the hybridization zone on gene chip surface the most at last.

8. gene chip hybridization chamber 2 that described requirement designs according to claim 1-7, by upper plastic cover 27, opaque plastics pedestal 25, gene chip 26, swimming is oozed PCB core 23 devices such as grade and is constituted, wherein pedestal 25 is the shaped as frame structure, the gene chip slide is embedded in pedestal 25 central authorities with facing up, pedestal 25 bottom shaped as frame openings are own less than gene chip 25 greater than the gene dot matrix area in gene chip slide front, make excitation beam gene dot matrix area in covering gene chip slide front fully still after refraction, simultaneously can hold the gene chip slide again, on the gene chip slide, place the shaped as frame swimming and ooze PCB core 23, latter's frame inwall is shown discrete metalized surface, by the plated through-hole that forms in the PCB course of processing by after tailor technology and form, the discrete metalized surface of the metal plating circuit that metalized surface such as the gold of these frame inwalls or silver are come out by etching and frame outer wall links to each other, the latter is oozed the external tapping of PCB core 23 as swimming, oozing ion manipulation with the swimming of HCL instrument partly links to each other, PCB core 23 is oozed in whole swimming, gene chip 26 and pedestal 25 parts are covered by a upper plastic cover 27, the liquid access way is located on upper plastic cover 27 or the pedestal 25, and gene chip hybridization chamber 2 humidity keep damping fluid storage aperture 29 to ooze on the PCB core 23 in swimming, and lead to extraneous by the aperture of upper plastic cover 27 corresponding positions, this aperture is by little its outlet of plug shutoff of rubber

Gene chip is from bottom to top respectively by slide, chromium metal (1～100 sodium rice, can be 3 sodium rice), silver (or the gold 10～1000 sodium rice, can be 40,50,75 sodium rice etc.), quartzy (1～100 sodium rice, can be 3 sodium rice or 5 sodium rice) etc. coating constitute, form the gene chip slide that can directly solidify gene molecule at quartzy coating surface through finishing technologies such as silanization, amido or aldehyde radicals, form gene chip 26 through gene chip point system technology again

The gene chip surface also can only be plated one deck chromium and gold respectively, need not plate one deck quartz film, afterwards directly at the gold surface immobilized nucleic acid molecule, and some system gene chip.

9. gene chip molecular probe, this system have respectively be engaged in mainly that gene chip is hybridized under the effect of swimming osmometer solution system maneuvering system, HCL test kit 19, the gene chip hybridization chamber 2 of mark, the efficient HCL instrument of handling of wash-out equimolecular biological procedures, the special use of HCL instrument and the SPR-CCD information of mainly being engaged in signal excitation, generation, collection, data processing and the interpretation of result of gene chip detects and several sections such as data handling system.

10. claim of using about the gene chip molecular probe in the claim 9, it can be applied to the genetic analysis in following field: the biological defence agent in the research of range gene group, teaching, drug screening genome research, various clinical diagnosis genetic analysis, various industry and commerce, medical and hygiene article and environment quarantine, the investigation of epidemic situation or generaI investigation, various science of law gene identity authentication, blood safety inspection, national defence field etc.