CN1209466C - Nucleic acid exparding gene chip hybridization intellectualization and inspecting instrument thereof - Google Patents
Nucleic acid exparding gene chip hybridization intellectualization and inspecting instrument thereof Download PDFInfo
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- CN1209466C CN1209466C CN 01128122 CN01128122A CN1209466C CN 1209466 C CN1209466 C CN 1209466C CN 01128122 CN01128122 CN 01128122 CN 01128122 A CN01128122 A CN 01128122A CN 1209466 C CN1209466 C CN 1209466C
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Abstract
The present invention relates to a method of detecting the intellectualized parallel operation of nucleic acid amplification and gene chip hybridization, and a detecting instrument thereof, which belongs to the field of biochip detection. The detecting instrument is characterized in that the detecting instrument comprises a temperature cycler (3), a hybrid instrument (4), three-dimensional transmission mechanical hands (5), a detecting mechanism (7) and a computer (8), wherein a position sensor and a photoelectric transducer which is electrically connected with the computer (8) are arranged in the detecting mechanism. The detection is carried out by the following steps: introducing a PCR chip to a temperature controlled area, moving the PCR chip to a hybridizing area after the PCR circulation process is finished, cleaning the PCR chip after the hybridizing process is finished, carrying out detection after the cleaning process is finished, and showing the test result after the detecting data is processed by the computer.
Description
Technical field
The invention belongs to the biochip test field, particularly a kind of Nucteic acid amplification gene chip hybridization Intelligent and Parallel detection method and proofing unit thereof.
Background technology
Human genome (order-checking) plan (Humen genome project HGP) is one of greatest heroic undertaking on the human history of natural science.Along with the progressively enforcement of the Human Genome Project and the fast development of molecular biology related discipline, HGP has fulfiled the making of genetic map ahead of schedule, measure 800,000 cDNA fragments (ESTs), be equivalent to 450,000 genes, accounted for about 50% of human total gene.Estimate to finish complete sequence analysis in 2003.Increasing animals and plants, microbial genome sequence are measured, and the gene order data just increase rapidly with unprecedented speed.Yet, how to go to study the function that numerous gene like this undertaken and just become whole world life science worker common problem in vital process.For this reason, people have proposed post genome project, protein groups plan, disease gene batch total and draw to wait and further decode this book from heaven of Human genome.In order to study gene expression differences such as Different Individual genovariation, different tissues, different time, different physiological statuss, set up novel hybridization and sequence measurement and just seem especially important a large amount of genetic information is carried out efficiently, detect fast and analyze.
In recent years, biochip was international research focus always, and biochip mainly is meant and utilizes plane micro-processing technology and numerator self-assembly technique, makes up microanalysis unit and system on the solid chip.Biochip can integrated many difference in functionality devices, realize multiple function, as: the pre-treatment of biological sample, the extraction of nucleic acid, the amplification of gene fragment, the microfluid system that biological probe array and kapillary form can be realized screening or detection to accurate, quick, the large information capacity of compound, protein, nucleic acid, cell and other biological components.Along with going deep into and development of research, grow some new technology in genomics field, their all potential DNA of making (thymus nucleic acid) order-checkings and genetics mapping operations are accelerated greatly, and expense reduces.Wherein most promising is biochip technology.1996, the Ai Fei company (Affymetrix) of santa clara successfully produced first batch of practical DNA chip, and produced first batch of practical DNA chip, and produced " gene chip " system.Biochip claims gene chip, DNA chip, DNA microprobe array again, or the like.It the is integrated gene probe of a large amount of dense arrangement can be analyzed a large amount of genes at short notice, makes people can promptly read and analyze biological gene information.Biochip technology is that the micro-fabrication technique of semi-conductor industry technology and molecular biology are combined, on a substrate surface, produce two-dimentional dna probe array, its working process is known organic molecule in the dna probe and determined unknown dna molecular " hybridization ", therefrom obtain DNA information, to determine unknown dna molecular " hybridization ", therefrom obtain DNA information, to determine unknown dna molecular.
The DNA hybridization is the extremely important phenomenon that molecular biology research is found, it has become the means of a kind of extremely important dna sequencing and medical science genetic diagnosis.Hybridization is applied to dna sequencing, is called " sequencing by hybridization ", and gene chip is a core devices of dna sequencing and diagnositc system, it and read the instrument of DNA information and the software of decipher gained data is formed system or the platform that obtains genetics information together.The basic purposes of this technology is to carry out hybrid gene order-checking and hybrid gene diagnosis.
Utilize gene chip to realize that the identification of nucleotide sequence needs following steps:
1, extract the target nucleic acid sequence, carry out pcr amplification and target-marking sequence:
2, Kuo Zeng nucleotide sequence and gene chip carry out hybridization;
3, rinsing;
4, fluoroscopic examination:
5, analyzing and testing result provides the biological significance of results of hybridization.
At present, ordinary method need be finished said process respectively with pcr amplification instrument, hybridization instrument, chip scanner.Because complicated operating process, interfering factors is more, the difficult stdn that realizes analytical results, and required instrument is very expensive, has limited popularizing that biochip technology is used.
Round pcr is as the external nucleic acid amplification method of a kind of selectivity, can make template DNA increase 100,000 times after taking turns circulation through 25～35, for many years in molecular biological fundamental research and Clinical Laboratory to using widely.But, many drawbacks are arranged in clinical application because there are defectives such as false positive and complicated operation in PCR.Reason be because: 1, the labile factor of many test conditionss causes the mistake amplification of PCR in the PCR process.2, the judgement of PCR product is the nucleic acid fragment that judges whether to obtain length-specific with electrophoresis method, can't determine its concrete sequence.3, the PCR process operation is loaded down with trivial details, very easily pollutes.Therefore, for technical level of operators and quality very high requirement is arranged.
Summary of the invention
It is a kind of under microcomputer control that technical problem to be solved by this invention is to provide, and realizes controlled pattern hybridization; Automatically finish cleaning; Automatically the results of hybridization of gene chip probes array is detected and exports automatically the result's who detects Nucteic acid amplification gene chip hybridization Intelligent and Parallel detection method and detector thereof.
Technical solution of the present invention can realize according to following mode:
The intelligent detection method of Nucteic acid amplification gene chip hybridization is characterized in: carry out according to the following step successively:
(A) pcr chip is sent into temperature controlled region, finish sex change, annealing, the extension process of PCR process according to the temperature model of setting;
(B) after the PCR working cycle is finished, pcr chip is moved to hybridization region, and whole crossover process is carried out temperature control according to the temperature of setting:
(C) after crossover process is finished, by washing unit pcr chip is cleaned, after cleaning is finished, gene chip is sent into detection zone by transport unit and is detected, the position signal of proofing unit, the position signal of pcr chip hybridization point is defeated by computer by transmitter, photoelectric commutator, is numerary signal by the A/D card with analog signal conversion, and hybridization signal is transferred to computer by photoelectric conversion device.
(D) after computer is handled the data of accepting, provide the result of biological significance.
Detector according to the intelligent detection method of above-mentioned Nucteic acid amplification gene chip hybridization is adopted is characterized in: contain: frame, temperature cycler, hybridization instrument, three-dimensional transfer robots, the feeler mechanism that has three-dimensional transfer robots, cleaning system, computer; Pcr chip carriage in the described temperature cycler and pcr chip carriage and the frame in the hybridization instrument are coupling; Described cleaning system, temperature cycler and hybridization instrument are assigned on the frame; Described three-dimensional transfer robots is assigned between temperature cycler and the hybridization instrument; Corresponding P C R chip position is provided with position transducer and photoelectric commutator in described feeler mechanism; Described position transducer and photoelectric commutator are electrically connected with computer after the A/D conversion.
Temperature cycler of the present invention contains: the pcr chip carriage, have pcr chip turnover window working bin, be arranged on warm area dividing plate A, warm area dividing plate B and warm area dividing plate C in the working bin; Corresponding pcr chip position is provided with chip channel 26 on described warm area dividing plate A, warm area dividing plate B, warm area dividing plate C; In sex change district 10 that described warm area dividing plate delimited, amplification region 11, renaturation area 12, be furnished with heater block, blower fan, temperature control sensor respectively; Described temperature control sensor is electrically connected with computer 8 after the A/D conversion; Described heater block, blower fan connect computer 8.
Hybridization instrument of the present invention contains: the hybridization working bin, the pcr chip carriage that have pcr chip turnover window; Be furnished with heater block, blower fan, temperature control sensor in described hybridization working bin: described temperature control sensor is electrically connected with computer after the A/D conversion; Described heater block, blower fan connect computer.
Temperature cycler of the present invention is provided with the air channel regulating mechanism between heater block and pcr chip.
Cleaning system of the present invention contains: clean liquid storage bottle, pipeline, peristaltic pump, pcr chip interface; The inlet of described pipeline communicates with scavenging solution liquid storage bottle inner chamber, and its outlet is corresponding with the port of pcr chip behind peristaltic pump; Near described pipe outlet, is furnished with pressure transmitter; Described peristaltic pump, pressure transmitter are electrically connected with computer.
Feeler mechanism of the present invention is contained: detection part, have the mechanical manipulator of three-dimensional transmission capacity; Described detection part is installed on the mechanical manipulator.
The present invention is under microcomputer control, and cleaning is finished in amplification, hybridization that can the controlled pattern of integrated realization automatically, automatically the results of hybridization of gene chip probes array is detected and the result that detects of output automatically; It can shorten the PCR time, increases work efficiency and reaction system is isolated from the outside, and can reduce false positive results.
Description of drawings
Fig. 1 is a process flow sheet of the present invention;
Fig. 2 forms for detector of the present invention system;
Fig. 3 is a detector structural representation of the present invention;
Fig. 4 is a detector temperature control system structural representation of the present invention;
Fig. 5 is a gene chip cleaning system structural representation of the present invention;
Embodiment
As shown in the figure, the intelligent detection method of Nucteic acid amplification gene chip hybridization is characterized in: carry out according to the following step successively:
(A) pcr chip is sent into temperature controlled region, finish sex change, annealing, the extension process of PCR process according to the temperature model of setting;
(B) after the PCR working cycle is finished, pcr chip is moved to hybridization region, and whole crossover process is carried out temperature control according to the temperature of setting:
(C) after crossover process is finished, by washing unit pcr chip is cleaned, after cleaning is finished, gene chip is sent into detection zone by transport unit and is detected, the position signal of proofing unit, the position signal of pcr chip hybridization point is defeated by computer by transmitter, photoelectric commutator, is numerary signal by the A/D card with analog signal conversion, and hybridization signal is transferred to computer by photoelectric conversion device.
(D) after computer is handled the data of accepting, provide the result of biological significance.
Detector according to the intelligent detection method of above-mentioned Nucteic acid amplification gene chip hybridization is adopted is characterized in: contain: frame 1, temperature cycler 3, hybridization instrument 4, three-dimensional transfer robots 5, the feeler mechanism 7 that has three-dimensional transfer robots, cleaning system 15, computer 8; Pcr chip carriage 21 in the described temperature cycler 3 and the pcr chip carriage 22 in the hybridization instrument 4 are coupling with frame 1 respectively: described cleaning system 15, temperature cycler 3 and hybridization instrument 4 are assigned on the frame 1; Described three-dimensional transfer robots 5 is assigned between temperature cycler 3 and the hybridization instrument 4; Can finish the alternation procedure of pcr amplification and hybridization smoothly to guarantee three-dimensional transfer robots 5.Corresponding pcr chip position is provided with position transducer and the photoelectric commutator that is used to detect in described feeler mechanism 7; Described position transducer and photoelectric commutator are electrically connected with computer 8 after the A/D conversion.Described temperature cycler 3 contains: pcr chip carriage 21, have pcr chip turnover window 30 working bin 31, be arranged on warm area dividing plate 23A, warm area dividing plate 24B and warm area dividing plate 25C in the working bin 31; Corresponding pcr chip position is provided with chip channel 26 on described warm area dividing plate 23A, warm area dividing plate 24B, warm area dividing plate 25C: the size of chip channel 26 is corresponding with pcr chip; In sex change district 10 that described warm area dividing plate delimited, amplification region 11, renaturation area 12, be furnished with heater block, blower fan, temperature control sensor respectively; Three cover heater blocks, blower fan, temperature control sensor are controlled sex change district 10, amplification region 11 and renaturation area 12 respectively: described temperature control sensor is electrically connected with computer 8 after the A/D conversion; Described heater block, blower fan connect computer 8.Described hybridization instrument 4 contains: the hybridization working bin 33, the pcr chip carriage 22 that have pcr chip turnover window 32: be furnished with heater block, blower fan, temperature control sensor in described hybridization working bin 33; Described temperature control sensor is electrically connected with computer 8 after the A/D conversion; Described heater block, blower fan connect computer 8.Described temperature cycler 3 is provided with the air channel regulating mechanism between heater block and pcr chip, be used for regulating the thermal radiation dosage of temperature field; The air channel regulating mechanism can be designed to venetian blind type; Cleaning system of the present invention contains: clean liquid storage bottle 16, pipeline 17, peristaltic pump 18, pcr chip interface 28; The inlet of described pipeline 17 communicates with scavenging solution liquid storage bottle 16 inner chambers, and its outlet is corresponding with the port of pcr chip behind peristaltic pump 18; Near described pipe outlet, is furnished with pressure transmitter 19; Described peristaltic pump 18, pressure transmitter 19 are electrically connected with computer 8.Described feeler mechanism 7 is contained: detection part, have the mechanical manipulator 29 of three-dimensional transmission capacity; Described detection part is installed on the mechanical manipulator 29.
The axis of pcr chip carriage 21 is driven by stepper-motor 34D in the temperature cycler 3 of the present invention, and the axis of pcr chip carriage 22 is driven by stepper-motor 35F in the hybridization instrument 4.36 is ultraviolet source, and 37 is CCD.Three-dimensional transfer robots 5, the mechanical manipulator 29 that has a three-dimensional transmission capacity are the prior art part.As shown in Figure 5,38 are the pcr chip inlet, and 20 is level sensor; 39 is the waste liquid chamber.
Present embodiment constitutes " multi-functional Nucteic acid amplification gene chip hybridization detecting instrument " jointly by triple channel PCR air heating thermostat (temperature cycler 3), chip transfer apparatus, instrument self checking warning device, computer and peripherals.
This instrument has many warm areas temperature control system, can walk abreast simultaneously a plurality of gene chips are handled.Temperature cycler 3 comprises heating thermal source (a plurality of 220V, the temperature control sensor of 300VA～500VA), blower fan, air channel, highly sensitive, little plastisied dispersion, temperature control software/hardware etc., the temperature of sensor detecting warm area and with design temperature relatively, temperature control software control phase shift silicon controlled rectifier is regulated the heating power of heater block, make air themperature be controlled at ± 0.3 ℃, in the chip temperature be controlled at ± 0.1 ℃, a temperature temperature difference at whole warm area is ± 0.3 ℃.In addition, this temperature control unit has automatic warning, self-checking function.The temperature control software system can be carried out log management work such as temperature control time, temperature control mode, temperature control user simultaneously.The user can set the pattern of temperature control, the curve of temperature control as required, can be in the temperature control process variation of monitoring temperature in real time, can interrupt, suspend the temperature control process and revise temperature control curve: the user can only make amendment other people parameter of no weight update and data to the temperature control parameter that oneself is provided with.
Feeler mechanism comprises detection part, has the mechanical manipulator of three-dimensional transmission capacity; Described detection part is installed on the mechanical manipulator.Detection part mainly is made up of photoelectric commutator and optical system, and detection part detects hybridization signal under the driving of three-dimensional precision transmission system, and machine provides detection, analytical results as calculated simultaneously.Three-dimensional precision drive mechanism precision requires: directions X: ± 0.1mm; Y direction: ± 0.05mm; Z direction: ± 0.01mm.Three-dimensional precision drive mechanism has position transducer and department's clothes motor of three directions, shows the coordinate of detection position with respect to design origin in real time, and the signal of transmitter is by RS232 interface and external communication.Data collecting system mainly is signal digitalized with collection optical system, and analytical system is analyzed simultaneously, and 3 D positioning system can accurately, promptly navigate under software control needs the position detected.Carry out quantification treatment detecting signal, and show and write down detected strength of signal, positional information.The automatic form deposit on demand of detected result.
The transport unit of gene chip is by X, Y, the three-dimensional driving device hand of Z, the transport unit function be with gene chip automatically slave be transported to amplification region, hybridization region and detection zone outward, wherein, comprise clamping device and locating mechanism, move to temperature controlled region outside by machine at pcr chip, the mechanical manipulator clamping device finish to chip from dynamic seal, guarantee that the liquid in the chip is limited in the effective temperature controlled region.Transport unit comprises stepper-motor and position transducer, and position transducer provides the positional information of chip, the motion of control step motor.
The function of gene chip cleaning system 15 is after finishing amplification and hybridization, cleans chip automatically.This system comprises: peristaltic pump, pressure transmitter, magnetic valve, level sensor and tubing system.When amplification, after hybridization finishes, by mechanical manipulator 29 pcr chip is taken out from hybridization instrument 4, and dock at pcr chip interface 28 places that are transplanted on cleaning system 15; After butt joint finishes, provide by computer that scavenging pump is opened in instruction and magnetic valve cleans chip, clean pressure, flow and scavenging period that software control is cleaned.Simultaneously, the pressure of the real-time display pipes of system is regulated pressure, the time of cleaning automatically.Report to the police in case of necessity.
The instrument self checking warning device enters self check together after the power-on, check whether transmission, location, heating, blower fan and temperature control system be normal, as undesired, reports to the police automatically.In the working process, various controlled variable surpass threshold values or the location is unpunctual, send guard signal together.
Chip stops certain hour in warm area in crossover process, control valve does not stop to carry out break-make, and controlled liq is shaken in chip, can improve hybridization efficiency, shortens hybridization time.
Realize automatic control processes such as this detector transports, PCR temperature control, liquid transfer, hybridization, cleaning, detection by independently developed computer software, finish that chip transports, pcr amplification, cleaning, hybridization, detect, provide detected result according to the program of setting.
Claims (7)
1, Nucteic acid amplification gene chip hybridization Intelligent and Parallel detection method is characterized in that: carry out according to the following step successively:
(A) pcr chip is sent into temperature controlled region, finish sex change, annealing, the extension process of PCR process according to the temperature model of setting;
(B) after the PCR working cycle is finished, pcr chip is moved to hybridization region, and whole crossover process is carried out temperature control according to the temperature of setting;
(C) after crossover process is finished, white washing unit cleans pcr chip; After cleaning is finished, gene chip is sent into detection zone by transport unit and is detected, the position signal of proofing unit, the position signal of pcr chip hybridization point is defeated by computer by transmitter, photoelectric commutator, is numerary signal by the A/D card with analog signal conversion, and hybridization signal is transferred to computer by photoelectric conversion device.
(D) after computer is handled the data of accepting, provide the result of biological significance.
2, the detector that is adopted according to the described Nucteic acid amplification gene chip hybridization Intelligent and Parallel of claim 1 detection method is characterized in that: contain: frame (1), temperature cycler (3), hybridization instrument (4), three-dimensional transfer robots (5), the feeler mechanism (7) that has three-dimensional transfer robots, cleaning system (15), computer (8); Pcr chip carriage (21) in the described temperature cycler (3) and the pcr chip carriage (22) in the hybridization instrument (4) are coupling with frame (1) respectively: described cleaning system (15), temperature cycler (3) and hybridization instrument (4) are assigned on the frame (1); Described three-dimensional transfer robots (5) is assigned between temperature cycler (3) and the hybridization instrument (4): corresponding pcr chip position is provided with position transducer and photoelectric commutator in described feeler mechanism; Described position transducer and photoelectric commutator are electrically connected with computer (8) after the A/D conversion.
3, detector according to claim 2 is characterized in that: described temperature cycler (3) contains: pcr chip carriage (21), have pcr chip turnover window (30) working bin (31), be arranged on warm area dividing plate (23A), warm area dividing plate (24B) and warm area dividing plate (25C) in the working bin (31); Go up corresponding pcr chip position at described warm area dividing plate (23A), warm area dividing plate (24B), warm area dividing plate (25C) and be provided with chip channel (26); Be furnished with heater block, blower fan, temperature control sensor at sex change district (10), amplification region (11), renaturation area that described warm area dividing plate delimited respectively in (12); Described temperature control sensor is electrically connected with computer (8) after the A/D conversion; Described heater block, blower fan connect computer (8).
4, detector according to claim 2 is characterized in that: described hybridization instrument (4) contains: the hybridization working bin (33), the pcr chip carriage (22) that have pcr chip turnover window (32); In described hybridization working bin (33), be furnished with heater block, blower fan, temperature control sensor; Described temperature control sensor is electrically connected with computer (8) after the A/D conversion; Described heater block, blower fan connect computer (8).
5, according to claim 2 or 3 described detectors, it is characterized in that: described temperature cycler (3) is provided with the air channel regulating mechanism between heater block and pcr chip.
6, according to claim 2 or 3 described detectors, it is characterized in that: cleaning system contains: clean liquid storage bottle (16), pipeline (17), peristaltic pump (18), pcr chip interface (28); The inlet of described pipeline (17) communicates with scavenging solution liquid storage bottle (16) inner chamber, and its outlet is corresponding with the port of pcr chip behind peristaltic pump (18); Near described pipe outlet, is furnished with pressure transmitter (19); Described peristaltic pump (18), pressure transmitter (19) are electrically connected with computer (8).
7, according to claim 2 or 3 described detectors, it is characterized in that: described feeler mechanism (7) is contained: detection part, have the mechanical manipulator (29) of three-dimensional transmission capacity; Described detection part is installed on the mechanical manipulator (29).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01128122 CN1209466C (en) | 2001-09-03 | 2001-09-03 | Nucleic acid exparding gene chip hybridization intellectualization and inspecting instrument thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01128122 CN1209466C (en) | 2001-09-03 | 2001-09-03 | Nucleic acid exparding gene chip hybridization intellectualization and inspecting instrument thereof |
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| CN1407114A CN1407114A (en) | 2003-04-02 |
| CN1209466C true CN1209466C (en) | 2005-07-06 |
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| CN 01128122 Expired - Fee Related CN1209466C (en) | 2001-09-03 | 2001-09-03 | Nucleic acid exparding gene chip hybridization intellectualization and inspecting instrument thereof |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7575862B2 (en) * | 2003-12-09 | 2009-08-18 | Asiagen Corporation | Assay systems, kits and methods for detecting microorganisms |
| CN101405410B (en) * | 2006-03-21 | 2013-06-19 | 皇家飞利浦电子股份有限公司 | Microelectronic sensor device with sensor array |
| CN101063078B (en) * | 2007-04-20 | 2010-06-09 | 博奥生物有限公司 | Biological chip hybrid cleaning device |
| CN108642159B (en) * | 2018-06-27 | 2022-07-01 | 上海宏滩生物科技有限公司 | Biochip detection system |
| CN109022543B (en) * | 2018-06-27 | 2022-03-11 | 上海宏滩生物科技有限公司 | Detection method of biochip detection system |
| CN112538425B (en) * | 2020-10-23 | 2022-09-09 | 北京理工大学 | On-chip nucleic acid amplification detection system and method based on micro-fluidic chip |
| CN117050851B (en) * | 2023-09-08 | 2024-04-12 | 中拓生物有限公司 | Heating device of nucleic acid extraction instrument |
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