CN103471981B - A kind of high-throughput grain count device and method based on micro-fluidic chip - Google Patents
A kind of high-throughput grain count device and method based on micro-fluidic chip Download PDFInfo
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- CN103471981B CN103471981B CN201310393016.9A CN201310393016A CN103471981B CN 103471981 B CN103471981 B CN 103471981B CN 201310393016 A CN201310393016 A CN 201310393016A CN 103471981 B CN103471981 B CN 103471981B
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Abstract
The present invention disclose a kind of particle is carried out automatically on micro-fluidic chip, the device of high-throughput counting and size discrimination and method. The present invention comprises PDMS micro-fluidic chip and glass negative, the microchannel of testing sample circulation is formed between PDMS micro-fluidic chip and glass negative, PDMS micro-fluidic chip center position is provided with central authorities' inlet opening, along central authorities' some sample fluid holes matching used with microchannel of inlet opening circumference uniform distribution; All inserting platinum electrode in central authorities inlet opening, each fluid hole, the platinum electrode the other end in central authorities' inlet opening is connected with the positive pole of direct supply, and the platinum electrode the other end in fluid hole is all connected with the negative pole of direct supply with reference to resistance by one; Two ends with reference to resistance are all connected by the input terminus of wire with corresponding signal amplification component, and the output terminal of signal amplification component is all connected to signal acquisition control system. Present configuration is simple, it may also be useful to convenient, it may be achieved high throughput testing.
Description
Technical field
The present invention relates to a kind of high-throughput particle monitoring technique based on micro-fluidic chip, relate to specifically a kind of to be carried out by particle automatically on micro-fluidic chip, the device of high-throughput counting and size discrimination and method.
Background technology
Along with the development of economic society, develop portable particle Fast Detection Technique and device, for determining target compound, such as number and the particle diameter spectrum of bacterium, virus and marine microorganism etc., all there is comparatively urgent demand in fields such as public health detection, biomedical research and marine environmental monitorings.
Micro flow control chip device can on the micro-fluidic chip of a piece several square centimeters, construct complicated microchannel network, and carry out fluid (sample or reagent) wherein handling accurately and control, complete different biological or chemical reaction process, can in a large amount of biomolecules of short period of time inner analysis, accurately obtaining a large amount of information in sample, quantity of information is hundreds and thousands of times of traditional detection means, is the core support technology of portability instrument.
Ku Erte resistance pulse counting method a kind of particle and cell is detected, counts and the effective ways of size discrimination, and in recent years, along with the fast development of micro-fluidic chip processing technology, existing many research reports utilizing Coulter principle micro-nano particle, hemocyte, bacterium and virus etc. to be counted on micro-fluidic chip at present.
Owing to the sample channel of micro-fluidic chip is micron order, and in order to improve accuracy of detection, just require that the size of detection zone and the size of thing to be detected are close. In addition, under microscale, the transport velocity of sample is also lower, and these all cause the problem of lower detection flux in actual applications.
The usual method improving micro-fluidic chip particle detection flux at present arranges multiple parallel main passage, detection microelectrode is laid at the two ends of each main passage examination area, and distinguish, by applying the exchange signal of different characteristics frequency in every paths, the signal detecting passage from difference, in order to avoid the interference between counting channel signal. Though the method has higher detection flux, but chip manufacture program is complicated, it is necessary to expensive electrode sputtering equipment, and the signal generator Lai Duimei road detection passage needing large scale commercial product applies characteristic frequency, it is very difficult to obtain practical application.
Particle differential resistor impulse method detects, and by arranging detection arm (liquid detecting electrode) respectively at two ends, detection zone, though avoiding the processing difficulties of traditional splash-proofing sputtering metal electrode, has higher accuracy of detection. If but still adopt and the way that multiple parallel main passage improves detection flux is set, it lays the quantity of detection arm is limited, and the raising namely detecting flux is limited.
Summary of the invention
In view of prior art is improving the deficiency existed in detection flux, it is an object of the invention to be provided device and the detection method thereof of a kind of novel particle high-throughput based on micro-fluidic chip counting and size discrimination.
In order to realize above-mentioned purpose, the technical scheme of the present invention:
A kind of high-throughput grain count device based on micro-fluidic chip, comprise the PDMS micro-fluidic chip being provided with some microchannels, and described PDMS micro-fluidic chip is recessed is carved with fluid channel side and glass negative sealing-in is structure as a whole, the microchannel of testing sample circulation is formed between described PDMS micro-fluidic chip and glass negative, it is characterized in that: described PDMS micro-fluidic chip center position is provided with central authorities' inlet opening, along central authorities' some sample fluid holes matching used with microchannel of inlet opening circumference uniform distribution; Platinum electrode is all inserted in described central inlet opening, each fluid hole, the platinum electrode the other end in described central authorities inlet opening is connected with the positive pole of direct supply, the platinum electrode the other end in described fluid hole is all connected with the negative pole of direct supply with reference to resistance by one, flows through the electroosmotic flow driving process of microchannel to fluid hole in order to realize testing sample from central authorities' inlet opening; The described two ends with reference to resistance are all connected by the input terminus of wire with corresponding signal amplification component, the output terminal of described signal amplification component is all connected with signal acquisition control system, in order to realize the amplification acquisition testing process of testing sample particle (cell etc.) number signal in each detection passage.
Described signal amplification component adopts differential amplifier element.
Described signal acquiring system adopts arm processor.
The signal that described signal acquiring system exports can show screen display at the arm processor that be connected with signal acquiring system, it is also possible to is directly stored in storage card, or be connected with PC end and analyze.
Employing said apparatus carries out the method that particle to be measured (cell etc.) high-throughput detects fast, comprises the steps:
1) sample drop adds: first drip the phosphate buffered saline buffer adding same volume in central authorities' inlet opening and each fluid hole, is then added dropwise to by a certain amount of testing sample in central authorities' inlet opening;
2) sample transport: connect direct supply, is transported in each fluid hole by the sample electric osmose in central authorities' inlet opening;
3) signal amplifies collection analysis: the number setting out fluid apertures is n, namely the quantity detecting passage is n, when sample particle detects microchannel surveyed area through i-th, corresponding reference resistance Ri two ends can produce voltage pulse signal, this signal enters the Fi differential amplifier being connected with reference to resistance Ri and amplifies, signal acquiring system it is sent to after amplification, record and show detection data, each detection passage of described detection data comprise obtains the number of sample particle and the size of this particle, 1 described��i��n, and be natural number.
The number detected value of described particle equals in all detection microchannels the number sum of the pulse signal produced.
The detected value of described particle size: the size judging particle according to the amplitude of the voltage pulse signal detected with reference to resistance Ri two ends, particularly when the diameter of the polystyrene standard particle of correction is dMarkTime, utilize this polystyrene standard particle to test, the amplitude recording the standard electric pressure pulse signal that resistance two ends detect is designated as; During sample test, if the amplitude of voltage pulse signal that resistance two ends detect is, according to formulaConvert, calculate the diameter value d obtaining this sample particle2��
Compared with prior art, the useful effect of the present invention:
1) adopt outer meeting resistance Differential Detection mode, it is not necessary to detection passage is set at two ends, detection zone, every road, more without the need to laying complicated electrode on micro-fluidic chip, while improving detection flux, greatly simplify chip structure;
2) use ARM signal acquiring system as data processing terminal, whole device volume is little, and cost is low, can be portable be applied to field quick detection;
3) every road detection signal inputs in arm processor respectively, does not interfere with each other, simplify testing process between signal.
4) sample adopts driven by electroosmosis, it is not necessary to the equipment such as additional pump, decrease the volume of device.
Accompanying drawing explanation
Fig. 1 is the microfluidic chip structure schematic diagram of the counting mechanism of the present invention;
Fig. 2 is embodiment of the present invention structure iron;
Fig. 3 is that the embodiment of the present invention first detects Air conduct measurement result figure.
In figure: 1��10, detect microchannel, A, central authorities' inlet opening, B��K, fluid hole, M, PDMS micro-fluidic chip, L, glass negative.
Embodiment
The technical scheme of the present invention is described further below in conjunction with accompanying drawing and specific embodiment:
As shown in Figure 1, the present invention comprises the PDMS micro-fluidic chip M being provided with some fluid channel, and described PDMS micro-fluidic chip M is recessed is carved with fluid channel side and glass negative L sealing-in is structure as a whole, between described PDMS micro-fluidic chip M and glass negative L, form some microchannels for testing sample circulation.
Specific as follows: described PDMS micro-fluidic chip M center position is provided with central authorities' inlet opening, centre, the Yanzhong some fluid holes of inlet opening circumference uniform distribution, each microchannel that each fluid hole corresponds supports the use, and the quantity of microchannel detects demand with reality and is as the criterion;
The present invention utilizes differential principle to be amplified by signal, each parallel micro-detection Tandem one, with reference to resistance, is drawn detection signal from reference to resistance two ends, thus is reached differential amplification object, eliminate common background noise impact, it is to increase signal to noise ratio and accuracy of detection.
Specific as follows: in described central authorities inlet opening, each storage fluid apertures, all to insert platinum electrode, the platinum electrode the other end in described central authorities inlet opening is connected with the positive pole of direct supply, the platinum electrode the other end in described fluid hole is all connected with the negative pole of direct supply with reference to resistance by one, flows through each micro-detection passage to each fluid hole in order to driven by electroosmosis testing sample from central authorities' inlet opening; The described two ends with reference to resistance are all connected by the input terminus of wire with corresponding differential amplifier, the output terminal of described differential amplifier is all connected to the arm processor of Signal sampling and processing, in order to realize each differential amplification detecting testing sample detection signal in passage, collection, display and storage. Owing to each chip detection scope is different, described different with reference to resistance, it is necessary to determine according to concrete situation, generally between 100 ��-2M ��.
The described signal exported with arm processor can in the display screen display being connected with arm processor, it is also possible to is directly stored in storage card, or is connected with PC end and analyzes. Inexpensive arm processor is utilized to complete the acquisition and processing of signal so that volume of the present invention is little, portability and low cost.
Detect for polystyrene sample particle below, as shown in Figure 2:
The device parameter of this example: size 10*30*25 ��m (wide �� long �� high) in the chip detection region that the implementation case is used, the quantity of fluid hole B��K is 10, distance between central authorities inlet opening A and each sample fluid hole B��K is 2.5cm, and each detection microchannel 1��10 is of a size of 200*25 ��m (wide �� high); Detected sample is 5 ��m of granules of polystyrene solution; Damping fluid is phosphoric acid salt (2 �� PBS) buffered soln; Applying the voltage in central authorities inlet opening A and each fluid hole B��K is 20V;
Concrete, this device comprises the central inlet opening A, fluid hole B, fluid hole C, fluid hole D, fluid hole E, fluid hole F, fluid hole G, fluid hole H, fluid hole I, fluid hole J, fluid hole K and the microchannel that matches with it that are positioned on described PDMS micro-fluidic chip and forms 1st��No. 10 detection microchannels. Central authorities inlet opening A detects microchannel with 1st��No. 10 and is all connected; Central authorities inlet opening A and fluid hole B��K all inserts platinum electrode; The other end of platinum electrode in central authorities inlet opening A is connected with the positive pole of direct supply; The other end of the platinum electrode in fluid hole B is connected to the negative pole of direct supply by resistance R1; The other end of the platinum electrode in fluid hole C is connected to the negative pole of direct supply by resistance R2; The other end of the platinum electrode in fluid hole D is connected to the negative pole of direct supply by resistance R3; The other end of the platinum electrode in fluid hole E is connected to the negative pole of direct supply by resistance R4; The other end of the platinum electrode in fluid hole F is connected to the negative pole of direct supply by resistance R5; The other end of the platinum electrode in fluid hole G is connected to the negative pole of direct supply by resistance R6; The other end of the platinum electrode in fluid hole H is connected to the negative pole of direct supply by resistance R7; The other end of the platinum electrode in fluid hole I is connected to the negative pole of direct supply by resistance R8; The other end of the platinum electrode in fluid hole J is connected to the negative pole of direct supply by resistance R9; The other end of the platinum electrode in fluid hole K is connected to the negative pole of direct supply by resistance R10; Corresponding resistance Ri (i=1��10) two ends are connected to two input terminuss of differential amplifier Fi (i=1��10) respectively by wire; The output terminal of differential amplifier Fi (i=1��10) is connected to the input terminus of arm processor; Arm processor outputs signal the display screen display that can directly connect, it is also possible to be stored in SD card, or can be connected directly in computer and analyze.
Its operation steps:
(1) first central authorities inlet opening and each fluid hole in drip add same volume PBS, then a certain amount of testing sample is added dropwise to central authorities inlet opening in;
(2) sample transport: connect direct supply, is transported to the sample electric osmose in central authorities inlet opening A in each fluid hole B��K. When sample particle detects microchannel surveyed area through i-th, corresponding reference resistance Ri resistance value changes, cause the voltage change at the resistance Ri two ends connected with this road further, produce detection signal, detection signal inputs to ARM after AD620 differential amplifier difference signal amplifies, and signal is gathered, analyzes and stores by ARM.
(3) control inerface Selection parameter, adds up number and the dimension information of particle in sample.
Its detected result: by selecting sequence of control, detected result can directly obtain on ARM, and Fig. 3 is the detected result figure of No. 1 micro-detection passage. Wherein, each downward pulse represents the detection signal of a granules of polystyrene. In sample, the number of particle equals the number sum of 1 to No. 10 detection passage pulse signal; The amplitude of the voltage pulse signal that the size size of particle then detects according to resistance two ends judges. When the diameter of the polystyrene standard particle of correction is dMarkTime, utilize this polystyrene standard particle to carry out testing the amplitude recording the standard electric pressure pulse signal that resistance two ends detect and it is designated as. During to particle (comprising cell etc.) sample test in sample, if the amplitude of voltage pulse signal that resistance two ends detect is �� Voutput2, according to formulaConvert, the diameter value d obtaining sample particle can be calculated2��
The above; it is only the present invention's preferably embodiment; but protection scope of the present invention is not limited thereto; any it is familiar with those skilled in the art in the technical scope that the present invention discloses; technical scheme and invention design thereof according to the present invention are equal to replacement or are changed, and all should be encompassed within protection scope of the present invention.
Claims (6)
1. the high-throughput grain count device based on micro-fluidic chip, comprise the PDMS micro-fluidic chip being provided with some fluid channel, and described PDMS micro-fluidic chip is recessed is carved with fluid channel side and glass negative sealing-in is structure as a whole, the microchannel of testing sample circulation is formed between described PDMS micro-fluidic chip and glass negative, it is characterized in that: described PDMS micro-fluidic chip center position is provided with central authorities' inlet opening, along central authorities' some sample fluid holes matching used with microchannel of inlet opening circumference uniform distribution; Platinum electrode is all inserted in described central inlet opening, each fluid hole, the platinum electrode the other end in described central authorities inlet opening is connected with the positive pole of direct supply, the platinum electrode the other end in described fluid hole is all connected with the negative pole of direct supply with reference to resistance by one, flows through the electroosmotic flow driving process of microchannel to fluid hole in order to realize testing sample from central authorities' inlet opening; The described two ends with reference to resistance are all connected by the input terminus of wire with corresponding signal amplification component, the output terminal of described signal amplification component is all connected with signal acquisition control system, in order to realize the amplification acquisition testing process of testing sample particle number signal in each detection microchannel; Described signal amplification component adopts differential amplifier element.
2. counting mechanism according to claim 1, it is characterised in that: described signal acquiring system adopts arm processor.
3. counting mechanism according to claim 1 and 2, it is characterized in that: the arm processor display screen display that the signal that described signal acquiring system exports can be connected with signal acquiring system, directly can also be stored in storage card, or be connected with PC end and analyze.
4. a counting mechanism according to claim 1 carries out particle high-flux detection method to be measured, it is characterised in that: comprise the steps:
1) sample drop adds: first drip the phosphate buffered saline buffer adding same volume in central authorities' inlet opening and each fluid hole, is then added dropwise to by a certain amount of testing sample in central authorities' inlet opening;
2) sample transport: connect direct supply, is transported in each fluid hole by the sample electric osmose in central authorities' inlet opening;
3) signal amplifies collection analysis: the number setting out fluid apertures is n, the quantity of the microchannel namely detected is n, when sample particle is through the surveyed area of i-th detection microchannel, corresponding reference resistance Ri both end voltage can change, this voltage change signal enters the Fi differential amplifier being connected with reference to resistance Ri and amplifies, signal acquiring system it is sent to after amplification, recording and show detection data, each detection microchannel of described detection data comprise obtains the number of sample particle and the size of this particle.
5. detection method according to claim 4, it is characterised in that: the number detected value of described particle equals in all detection microchannels the number sum of the pulse signal produced.
6. detection method according to claim 4, it is characterized in that: the detected value of described particle size: the size judging particle according to the amplitude of the voltage pulse signal detected with reference to resistance Ri two ends, particularly when the diameter of the polystyrene standard particle of correction is dMarkTime, utilize this polystyrene standard particle to test, the amplitude recording the standard electric pressure pulse signal that resistance two ends detect is designated asDuring sample test, if the amplitude of voltage pulse signal that resistance two ends detect isAccording to formulaConvert, calculate the diameter value d obtaining this sample particle2��
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| CN103940707A (en) * | 2014-05-06 | 2014-07-23 | 江苏苏净集团有限公司 | Method and device for counting insoluble particles |
| CN105784570B (en) * | 2016-04-21 | 2018-07-10 | 大连海事大学 | Particle on-line measuring device and its detection method based on micro-fluidic chip |
| CN107153122A (en) * | 2017-05-27 | 2017-09-12 | 哈尔滨工业大学（威海） | Digital microcurrent-controlled biochip in situ rest structure and method based on serial communication |
| CN107213930B (en) * | 2017-07-27 | 2022-12-20 | 深圳中科芯海智能科技有限公司 | Microfluidic chip for particle analysis and particle analysis method |
| CN108279202B (en) * | 2018-03-28 | 2023-05-30 | 大连海事大学 | Particle counting device and method with adjustable detection precision |
| CN108680503A (en) * | 2018-05-18 | 2018-10-19 | 王闯 | Liquid controls chip and system |
| CN108982335A (en) * | 2018-06-29 | 2018-12-11 | 浙江大学 | A kind of suspension cell counting chip based on electrical impedance and preparation method thereof and method of counting |
| CN109569753B (en) * | 2018-12-29 | 2021-02-26 | 北京工业大学 | Micro-fluidic chip channel device for PM2.5 particle collection and experimental observation |
| CN111235021B (en) * | 2020-03-06 | 2022-09-27 | 大连海事大学 | Double-liquid-phase separation and detection device and method for circulating tumor cells in peripheral blood |
| CN117629996B (en) * | 2023-11-30 | 2024-06-25 | 深圳市睿迈生物科技有限公司 | Separation parameter detection method and device for microfluidic chip and computer equipment |
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| CN102989533A (en) * | 2012-12-20 | 2013-03-27 | 中国科学院上海微系统与信息技术研究所 | Micro-fluidic chip for high-flux automatically rationing and mixing and use method and application of chip |
| CN103105352A (en) * | 2013-01-28 | 2013-05-15 | 大连海事大学 | Device and method for rapidly detecting surviving unicellular organisms in ship ballast water |
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| CN102989533A (en) * | 2012-12-20 | 2013-03-27 | 中国科学院上海微系统与信息技术研究所 | Micro-fluidic chip for high-flux automatically rationing and mixing and use method and application of chip |
| CN103105352A (en) * | 2013-01-28 | 2013-05-15 | 大连海事大学 | Device and method for rapidly detecting surviving unicellular organisms in ship ballast water |
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