CN102604827B - System and method for detecting polymerase chain reaction (PCR) process through non-contact conductivity method in rear time - Google Patents
System and method for detecting polymerase chain reaction (PCR) process through non-contact conductivity method in rear time Download PDFInfo
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Abstract
The invention relates to a system and a method for detecting a polymerase chain reaction (PCR) process through a non-contact conductivity method in rear time. The system is characterized by comprising an integrated PCR microchip based on micro-electromechanical system (MEMS) integration, an alternating-current excitation power supply, a current-to-voltage converting and amplifying circuit, a temperature sensing circuit, a heating circuit, a digital-analog/analog-digital conversion interface DAQ and an upper Labview control center, wherein the integrated PCR microchip integrates a micro reaction cavity, a temperature sensing electrode, a heating electrode and an electrochemical detection electrode; the micro reaction cavity is etched on the front surface of a silicon substrate; the heating electrode and the temperature sensing electrode are integrated on the back surface of the silicon substrate; the electrochemical detection electrode is an interdigital electrode, is bonded with the silicon substrate through an insulating layer and forms a sealed structure with the micro reaction cavity; and temperature circulation required by amplification is realized through the temperature sensing electrode and the heating electrode which are arranged on the back surface of the silicon substrate. The sensitivity and the resolution ratio of the system provided by the invention can reach fg/muL below, the reusability of the electrodes is high, the service life is long, and the reliability of the detecting result is high.
Description
Technical field
The present invention relates to real-time detecting system and method that a kind of non-contact electric conductivity method realizes polymerase chain reaction (PCR) process, comprise polymerase chain reaction (PCR) microchip, non-contact electric conductivity electrochemical detection electrode, high-sensitivity readout circuit and analytical procedure etc., belong to electrochemical sensing technical field.
Background technology
The rapid detection of pathogenic microorganism and analysis are the technology of the field active demands such as food safety, environmental monitoring, public health, the detection analysis that Real-Time Fluorescent Quantitative PCR Technique is microorganism provides effective instrument, the major advantage of this technology is, in the amplification procedure of nucleic acid, nucleic acid is carried out to quantitative analysis, thereby has shortened the conventional needed time of end point determination method.Yet, all real-time quantitative PCRs are all based on optical detecting method at present, need to carry out fluorescent mark needs complexity and expensive Systems for optical inspection to carry out fluoroscopic examination simultaneously, Instrument structure is complicated, be difficult to miniaturization, be only suitable for carrying out in centralab, therefore still lack at present means and the instrument that is suitable for on-the-spot microorganism Fast Measurement.
The principle of electrochemical detection method is that to take the amplified production of thymus nucleic acid (DNA) or Yeast Nucleic Acid (RNA) be sensitive objects, electrochemical electrode is signal converter, take that electromotive force, electric current or electricity lead etc. is feature detection signal, realizes and detects in real time PCR reaction process.Detect according to being 1) some components of DNA have (for example guanine) of electrochemical activity under certain electromotive force window, can directly at electrode surface, realize transfer transport; 2) by outside some redox media (as methylene blue, Hoechst 33258, daunomycin and Quinomycin A etc.), realize electronics transmission, by means of the indicator that has electrochemical activity of these and DNA selective binding, carry out hybridization check.With the nm gold particles thing that makes marks, not only have better biocompatibility, the deposition that its surface can catalysis silver particle, has increased signal to noise ratio.Electrochemical detection method in recent years has reached higher sensitivity, as sensitivity reaches ng/uL level or even can reach Single Molecule Detection level with the hybridization of ssDNA probe mark.The bias light interference that is compared to fluorescent method is many, and the ground unrest of electrochemical process is less, and detection efficiency is higher.As mentioned above, that Electrochemical Detection has is highly sensitive, cost is low, be easy to the advantages such as integrated, microminiaturized, if electrochemical detection method and advanced micromachining technology can be matched, the potentiality with the scale operation of realizing, therefore adopt electrochemical method to detect in real time PCR process, can not only obtain the detected result that can match in excellence or beauty with quantitative fluorescent PCR, and the required equipment of Electrochemical Detection is simple, easy to operate, can and not need professional to operate in the simple and crude applications of condition.
Although the electrochemical techniques that the DNA cloning on sheet is analyzed are more ripe, the Electrochemical Detection PCR in real time process of bibliographical information all adopts contact electrode directly to measure at present, no matter be with label-free or labelling method, based on principle be all detect end product in electrode surface enrichment or cause electrode surface ionic concn to change and cause the variation of electric current or electric impedance.Therefore electrode surface has residue, and its secondary detection does not far reach the performance while detecting for the first time.Although can carry out clean to electrode before secondary detection, such process is just complicated, and effect can not be fairly obvious.If use disposable electrode, has increased the cost of detection and the waste of resource.The advantage that contact process detects is that resolving power is higher, but surface adsorption is easily saturated, and sensing range is limited, easily pollutes, and reusability is not high, and work-ing life is short.And after electrode pressurization, molecules in solution is had to ionization hydrolytic action, be unfavorable for responsive detection.In the process of applying marking method, marker and target dna hybridization, all having there is variation in its space structure, steric restriction effect occurs, thereby affect joint efficiency.
Summary of the invention
The object of the present invention is to provide a kind of non-contact electric conductivity method to realize real-time detecting system and the method for PCR reaction process, feature of the present invention is with unconventional non-contact electric conductivity detection method (capacitively-coupled, contactless conductivity detection, C
4d) replace traditional contact electrochemical method, realize the real-time detecting system of the PCR process on integrated chip.Non-contact method is not because electrode directly contacts with electrolytic solution, and large size has reduced ground unrest, can reach less detectability.Also without the electrode surface that directly contact causes, produce in addition the problem of bubble.Easy of integration, and electrode and solution is all difficult for contaminatedly, and the reusability of electrode is very high.The detectability that the contactless electrode system that current bibliographical information is applied to electrophoresis chip can reach is 5 * 10
-8with 1 * 10
-7between M.The present invention is incorporated into contactless concept in integrated pcr amplification and detection chip, has not only greatly simplified the system (without marker and probe) of pcr amplification, and the work-ing life of having improved again electrode, has reduced thus the cost of equipment.And the required equipment of system is all very simple, easy of integration, can use and portable use at the simple and crude scene of condition.
The object of the invention is to reach by following measures: based on MEMS (micro electro mechanical system) (MEMS) technical project, manufacture PCR microchip, and integrated non-contact electric conductivity detector, design and produce even nano level dielectric insulation layer of micron order, significantly improve the sensitivity of electrical conductivity detector; PCR microchip is integrated temperature sensing electrode and heating electrode simultaneously, based on differential signal amplification principle and temperature compensation principle, significantly improves resolving power and the immunity from interference of sensing circuit; What working electrode adopted is interdigital electrode structure, and every electrode is all as working electrode, so the overall signal of its induction will, much larger than single electrode, have higher detection sensitivity and resolving power; The present invention adopts actual sample to analyze and verify the non-contact electric conductivity detection method of set up PCR reaction process.
The invention provides a kind of electrochemical detection method of effective detection real-time nucleic acid amplification process, by the method, can detect the starting point concentration of determined nucleic acid, and can be on PC Real-Time Monitoring amplification process.The cover glass that with thickness is 100 μ m is done preliminary experiment as insulation layer, take in the experiment that the aqueous dna of purifying is detected object, only need the sample volume of 10 μ L, can reach the resolving power of detection lower limit and the 0.5pg/ μ L of 0.1pg/ μ L, and can distinguish strand or double-stranded DNA (the impedance ratio double-stranded DNA of single stranded DNA is large, and the voltage difference producing with concentration double-stranded DNA is greater than single stranded DNA).Along with the optimization (silicon nitride that nano level is thick or silicon-dioxide) with material that reduces of thickness of insulating layer, the sensitivity of this non-contact detection system and resolving power are estimated can be improved to below fg/ μ L level.Correspondingly, the PCR of the template starting point concentration of fg/ μ L level level reaction is estimated at temperature cycle 5min with the interior voltage change can qualitative detection causing to obvious amplified production increase, effective very fast.By the template of known different starting point concentrations, carry out a series of pcr amplification reaction, record the voltage difference producing in same loop number and change, formulate typical curve, can be used as the foundation of quantitative PCR.The system that realizes the method is easy to control, easy and simple to handle, easy of integration, and volume is little, and power consumption is little, and cost is low, detecting electrode long service life.Realize the integrated system of the method because volume is little, a plurality of reaction chambers and Controlling System can be made on same device, realize high-flux parallel and detect a plurality of samples, and easy portability, can apply at the scene, there is very wide range of application.
Described real-time detecting system comprises based on the integrated PCR microchip of MEMS, ac-excited power supply, electric current and turns voltage and amplifying circuit, temperature sensing circuit, heating circuit, digital-to-analogue/analog to digital conversion interface DAQ and upper Labview control center; Wherein, ac-excited power supply connects the electrochemical detection electrode in PCR microchip; Heating control circuit connects the heating electrode in PCR microchip; The electrochemical detection electrode the other end turns voltage with electric current and amplifying circuit is connected; Temperature sensing circuit connects the temperature sensing electrode in PCR microchip; Temperature sensing circuit, electric current turn voltage amplifier circuit and are connected with modulus/digital-to-analog conversion interface DAQ again, modulus/digital-to-analog conversion interface DAQ is connected with Labview control center and the heating control circuit of upper computer respectively, described integrated PCR microchip is integrated reaction microchamber, temperature sensing electrode and heating electrode and electrochemical detection electrode, the positive etching reaction microchamber of silicon base, back side integrated heating electrode and temperature sensing electrode; Electrochemical detection electrode is interdigital electrode, by insulation layer and silicon base bonding, forms closed structure with reaction microchamber, by controlling the temperature sensor electrode reconciliation thermode at the silicon base back side, realizes the required temperature cycle of amplified reaction.
Non-contact electric conductivity provided by the invention detects the method for real-time nucleic acid amplification process and traditional nucleic acid amplification electrochemical detection method has following characteristics:
1, the present invention adopts non-contact electric conductivity to detect, and has avoided the mutual pollution between detecting electrode and object to be measured, and without the electrode clean complex steps such as live again, easy and simple to handle, electrode reusability is high, long service life, and detected result reliability is high.
2, electrochemical detection electrode of the present invention adopts interdigital electrode as working electrode, and detection efficiency is far longer than common single working electrode.The serviceability of interdigital electrode is by interdigital length, interdigital spacing, interdigital number, effectively the many kinds of parameters such as working area determines, optimization in several ways all can improve detection resolution and the sensitivity of interdigital electrode more greatly, and upgrading is convenient.
3, using plasma enhancing chemical vapor deposition (PECVD) technology sputter silicon-dioxide of the present invention or silicon nitride film replace traditional polydimethylsiloxane (PDMS) material and common slide to make dielectric layer and the insulation layer of electrode, both can play buffer action, can make again solution and electrode very approaching, reduce the contribution that insulation layer itself is led electrochemistry electricity, to improve sensitivity and resolving power, material surface characteristic is more suitable for pcr amplification process simultaneously.
4, single reaction integrated system volume of the present invention is little, power consumption is little, and by a plurality of reaction chambers and control system integration, a plurality of DNA samples of augmentation detection, shorten and detect the time of analyzing simultaneously, have improved the information flux detecting.
5, realization of the present invention can adopt microminiaturized power supply and circuit assembly, and simple in structure, low in energy consumption, voltage request is low, is that a kind of portability detects Analytical equipment.
6, temperature controlling system of the present invention adopts Labview to complete, the resistance signal of thermometric electrode converts voltage signal to through amplifying, this voltage signal carries out analog to digital conversion by data collecting card and becomes digital voltage signal, and by Labivew, carry out data analysis and generate feedback signal, via rising or the decline of the D/A fuction output voltage control heating electrode temperature of data card.Feedback signal can adopt PID s operation control scheduling algorithm, can improve temperature controlled accuracy and intellectuality.
7, the present invention adopts the multi-functional data card simultaneously with digital-to-analog conversion and analog-digital conversion function, and the communication of conductance signal, temperature signal and temperature feedback signal and PC is integrated, and simplifies the structure of whole system.
8, the present invention is owing to having adopted MEMS (micro electro mechanical system) (MEMS) technology to manufacture micro-reaction chamber, heating electrode, temperature sensor and non-contact electric conductivity detector, its temperature control device structure and temperature control program are simple, amplified reaction is rapid, electricity is led detection resolution and highly sensitive, and can control software by PC and realize the sequencing of whole reaction process, intellectuality, graphical operation friendly interface is convenient to man-machine dialogue.
Accompanying drawing explanation
Fig. 1. system design structural representation of the present invention.
Fig. 2. the structure iron of micro-integrated chip.Wherein, (A) schematic cross-section of PCR microchip; (B) reaction cavity structure; (C) integrated temperature sensor heated by electrodes electrode structure; (D) CD
4with interdigital electrode structure.
Fig. 3. signal conversion and modulate circuit in the present invention.(A) temperature signal conversion and amplifying circuit, R
sensorfor the resistance of temperature sensor, Vsensor be temperature signal transform and amplify after voltage signal, this end is directly connected with the analog to digital conversion input aperture of DAQ; (B) chip heating control circuit, intermediate rectangular frame is voltage regulator chip, V
heaterfor being directly carried in the heater voltage at heating electrode two ends, V
controlthe signal cut-offfing for controlling voltage regulator chip voltage, is connected with the digital-to-analog conversion interface of DAQ; (C) the output signal change-over circuit of interdigital electrode, I
ifor the outward current of interdigital electrode, V
ovoltage signal for current conversion and after amplifying, is connected with the analog to digital conversion interface of DAQ.
The operation interface of Fig. 4 .PC software control.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described.
What Fig. 1 showed is the constructional device schematic diagram of whole system of the present invention.This system comprises that integrated PCR microchip (dotted line frame in), ac-excited power supply, electric current turn the Labview control center of voltage and amplifying circuit, temperature-control circuit (sensor signal conversion and amplify and add thermal control), digital-to-analogue/analog to digital conversion interface (DAQ) and upper computer.
Wherein, integrated PCR microchip structure, the integrated modules (as shown in Figure 2) such as reaction microchamber (B), temperature sensor electrode and heating electrode (C), electrochemical detection electrode (D).In Fig. 2, (A) be structural representation, this size is not actual size, and actual integrated PCR microchip planar dimension is 15mm * 12mm.The practical structures of the cavity at detecting electrode in each layer, PCR reaction place and integrated temperature control electrode and size respectively corresponding (B), (C), (D).(B) in, reaction chamber center area is about 50mm
2, 2 circular injection port radiuses are 1mm, the wide 50 μ m of microfluid pipeline.(C) electrode materials in is metal platinum.(D) the interdigital electrode material in is metallic gold, interdigital wide 3mm, and 60mm is long, and adjacent interdigital spacing is 9mm, and circular injection port radius is 2mm, during bonding, aims at the injection port in (B).Figure (A) shows that total two layers of substrate forms.The method of making is that first in the silicon base one side of two polishings oxidations, AZ4620 is graphical by mask plate with photoresist, after developing, with HF solution, dissolves exposed SiO
2, with glue or blue film, protect the other one side SiO of not photoetching simultaneously
2layer; The Si substrate of cavity patterned surface is come out, then in 50 ℃ of constant temperature water baths, etch the cavity with certain depth with 50%KOH.With acetone, except glue clean silicon base again, one side is made Pt metal electrode (C) by lift-off technique in addition, as integrated heating electrode and temperature sensing electrode, in real time accurate temperature, controls.For example, on electrode supporting thing (common glass sheet) surface, by same lift-off technique, make gold electrode as electrochemical detection electrode (D) and punch as inlet and outlet, then with standard technology at this electrode surface very Si of book that grows
3n
4or SiO
2layer is as insulation layer.The reaction microchamber dignity of interdigital electrode face and silicon base is by ultraviolet glue bonding, and the circular injection port of two substrates is aimed at respectively, so just can form closed structure with micro-reaction chamber.(C) be connected with peripheral control circuit by the metal lead wire on weld with electrode in (D).In reality detects, two micro-integrated chips carry out pcr amplification reaction the real-time conductance signal that detects two-way simultaneously.
As shown in fig. 1, ac-excited source produces the sinusoidal voltage of 35kHz, 10V, and access interdigital electrode makes it produce induced current.This induced current can not directly be gathered by data card, therefore need to convert voltage signal to by simple calculations amplifier circuit (Fig. 3 C).Because the electric current that condenser coupling produces is less, the voltage signal converting to is also fainter, can not directly measure.By the resistance R resistance of adjusting Fig. 3 C, realize the amplification of suitable multiple, within the voltage after transforming so just can be controlled at the sample range and sampling precision of data collecting card.For avoiding interfering with each other between high frequency ac signal, the actuation signal line of interdigital electrode and signal sense wire are as far as possible away from also isolation mutually.By high-performance interface DAQ, (sample frequency is at least 500kHz to voltage signal after conditioning, amplitude-10V～10V) sampling is transferred to the Labview control center of upper computer, carry out the digital processings such as denoising, filtering, wave test, finally with atomic little time delay, show that the electricity of solution in current microchip is led and change the voltage change causing.Detect in sample, the carrying out of pcr amplification causes the release of various ions simultaneously, thereby causes the variation of solution conductivity, produces faradic difference.So the signal difference real time reaction that the Labview control center of upper computer shows pcr amplification situation.
Temperature control modules in Fig. 1 is realized signal conversion and signal condition function, bridge circuit and the signal amplification circuit of Fig. 3 A, consists of.Along with the variation of chip temperature, the Pt temperature sensor resistance on chip also produces linear change, and this resistance change changes into voltage signal by bridge circuit.But this voltage signal is fainter, can not directly measure, so within voltage signal need to be amplified to the sample range and precision of DAQ equally; Ground unrest is further removed by low-pass filter circuit to the temperature signal gathering through DAQ by the Labview control center of upper computer, has improved the signal to noise ratio of voltage signal; The digital voltage of processing the most at last is reduced into by signal the temperature signal that temperature sensor detects, thereby realizes the mensuration to chip temperature.Then this temperature signal be take as input feedback signal in the Labview control center of upper computer, according to the requirement of the temperature cycle of demarcating, calculate, by pulsed modulation algorithm (PWM), obtain the control signal of heated by electrodes, through DAQ, form simulating signal service voltage setter (Fig. 3 B); Voltage regulator controlled loading is at the voltage switch off time at heating electrode two ends, thereby regulating power realizes the gradient of temperature of different rates.PWM algorithm can guarantee being rapidly heated of temperature, prevents temperature overshot equilibrium temperature.Direct supply is directly powered by integrated DC/DC module.The artificial circuit part of system (Fig. 3) is all integrated on the pcb board that about 15cm * 10cm is large, and is isolated from the outside by shielding box, effectively resists external interference.
What Fig. 4 showed is the Labview control center operation interface of upper computer in the present invention.The signal flow of the interdigital electrode ，Bing You Labview control center that is connected with upper computer by same multichannel DAQ with temperature adjusting signal realizes time-sharing multiplex function to data collecting card, guarantees can realize compared with high sampling rate at single passage again under the prerequisite of hyperchannel work.The processing such as the filtering of numerary signal, calculating are all carried out at Labview program rear end platform, at operation interface, do not show.Control interface for operations such as temperature controlled startup, the demonstration of actual temperature and the real-time demonstration of PCR process voltage change and storages, very directly perceived and operation is very easy.
System operation step:
1, reaction microchamber body first uses the BSA solution of 0.5% (quality) to fill with semiclosed 1 hour, can reduce being adsorbed of PCR reactant, improves the reaction efficiency of chip PCR.
2, BSA is extracted totally, by gas driven injection mode, reaction mixture is injected to reaction microchamber, for importing and exporting, paraffin oil or PDMS sealing, prevent the volatilization of sample in temperature cycle process.A microchip adds the PCR reaction mixture with template.Another one microchip adds the PCR reaction mixture of not being with template, as negative control sample.Paraffin oil or mineral oil sealing for sample introduction product.
3, operation Labview program, after seeing that stable temperature and voltage waveform show, selects the file address of storage, and then start-up temperature is controlled function, can carry out pcr amplification reaction, and the conductance signal detecting is in real time changed and shown and storage.Pcr amplification reaction is without marker and probe.
The time that 4, can finish according to the autonomous selective reaction of reaction result.Stop, after temperature control, reaction mixture being extracted from microcavity body, more repeatedly cleaning microcavity with damping fluid and deionized water.Because electrode does not directly contact with detecting sample, so the performance of electrode remains unchanged always, the chip after cavity cleaning can be used for detection next time.
Claims (9)
1. non-contact electric conductivity method realizes a real-time detecting system for PCR process, it is characterized in that comprising the Labview control center that turns voltage and amplifying circuit, temperature sensing circuit, heating control circuit, digital-to-analogue/analog to digital conversion interface DAQ and upper computer based on the integrated PCR microchip of MEMS, ac-excited power supply, electric current;
Wherein, ac-excited power supply connects the electrochemical detection electrode in PCR microchip; Heating control circuit connects the heating electrode in PCR microchip; The electrochemical detection electrode the other end turns voltage with electric current and amplifying circuit is connected; Temperature sensing circuit connects the temperature sensing electrode in PCR microchip; Temperature sensing circuit, electric current turn voltage and amplifying circuit is connected with modulus/digital-to-analog conversion interface DAQ, modulus/digital-to-analog conversion interface DAQ is connected with heating control circuit with the Labview control center of upper computer respectively, described integrated PCR microchip is integrated reaction microchamber, temperature sensing electrode and heating electrode and electrochemical detection electrode, the positive etching reaction microchamber of silicon base, back side integrated heating electrode and temperature sensing electrode; Electrochemical detection electrode is interdigital electrode, and it is make gold electrode as electrochemical detection electrode and punch as inlet and outlet by Lift-off technique on electrode supporting thing surface, then uses standard technology at the Si of this electrode surface growing nano grade
3n
4or SiO
2layer is as insulation layer, the reaction microchamber body of interdigital electrode face and silicon substrate is by ultraviolet glue bonding, during bonding, the injection port of interdigital electrode face is aimed at reaction cavity injection port, form closed structure with reaction microchamber, by controlling temperature sensing electrode and the heating electrode at the silicon base back side, realize the required temperature cycle of amplified reaction.
2. by system claimed in claim 1, it is characterized in that interdigital electrode turns voltage by the metal lead wire on weld with peripheral electric current and amplifying circuit is connected.
3. by system claimed in claim 1, it is characterized in that:
1. the sinusoidal voltage of ac-excited power generation 35KHz, 10V, accesses interdigital electrode and produces induced current; Sensor current signal turns voltage circuit by electric current and changes and zoom into the voltage signal that can be detected by data collecting card;
2. described interface DAQ sample frequency is at least 500KHz, and amplitude is-10V～10V.
4. by system claimed in claim 1, it is characterized in that:
(a) described integrated PCR microchip is of a size of 15mm * 12mm;
(b) area of described reaction microchamber is 50mm
2, the wide 50 μ m of microfluid pipeline of connection injection port and reaction microchamber body.
5. by the system described in claim 1 or 2, it is characterized in that described interdigital electrode material is for gold, interdigital wide be 3mm, long is 60mm, adjacent interdigital spacing is 9mm, circular injection port radius is 2mm.
6. by system claimed in claim 3, it is characterized in that the voltage signal detecting is admitted to the Labview control center of upper computer, carry out denoising, filtering, wave test and process, finally with atomic little time delay, show that the electricity of solution in current microchip is led and change the voltage change causing; In detection, the carrying out of pcr amplification causes the release of various ions simultaneously, thereby causes the variation of solution conductivity, produces faradic difference, the signal difference real time reaction that the Labview control center of upper computer shows pcr amplification situation.
7. by the system described in claim 1 or 6, it is characterized in that the Labview control center of upper computer collects after temperature signal, the requirement according to the temperature cycle of demarcating, by pwm pulse modulation algorithm, provides the voltage control of heating electrode; PWM algorithm guarantees being rapidly heated of temperature, prevents temperature overshot equilibrium temperature.
8. use by the method for the system described in any one in claim 1-6, it is characterized in that operation steps is:
(1) reaction microchamber body is first filled with semiclosed 1 hour with 0.5% BSA, reduces being adsorbed of PCR reactant, improves the reaction efficiency of chip PCR;
(2) BSA is extracted totally, by gas driven injection mode, reaction mixture is injected to reaction microchamber, for importing and exporting, paraffin oil or PDMS sealing, prevent the volatilization of sample in temperature cycle process; A microchip adds the PCR reaction mixture with template; Another one microchip adds the PCR reaction mixture of not being with template, as negative control sample; Paraffin oil or mineral oil sealing for injection port;
(3) the Labview control center of operation upper computer, after showing stable temperature and voltage waveform, select the file address of storage, then start-up temperature is controlled function, can carry out pcr amplification reaction, and the conductance signal detecting is in real time changed and shown and storage;
(4) time finishing according to the autonomous selective reaction of reaction result; Stop, after temperature control, reaction mixture being extracted from microcavity body, more repeatedly cleaning microcavity with damping fluid and deionized water; Because electrode does not directly contact with detecting sample, so the performance of electrode remains unchanged always, the chip after cavity cleaning can be used for detection next time.
9. by method claimed in claim 8, it is characterized in that in step (3) that pcr amplification reaction is without marker and probe.
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| CN105296351A (en) * | 2015-12-02 | 2016-02-03 | 北京大学 | Chip for polymerase chain reaction (PCR), and real-time detection device and system |
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| CN109957492A (en) * | 2017-12-26 | 2019-07-02 | 安诺优达基因科技(北京)有限公司 | A kind of automatic fluid processing workstation for two generations sequencing DNA library building |
| CN112881493B (en) * | 2020-11-05 | 2023-02-17 | 北京大学 | Field effect transistor type biosensing device and biomolecule detection method |
| CN114632559B (en) * | 2022-01-26 | 2023-03-31 | 浙江大学 | On-chip micro-groove array digital PCR chip based on electrical impedance detection and manufacturing method thereof |
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