CN1588088A - Micro flow control chip detecting system for flowing cell detection - Google Patents
Micro flow control chip detecting system for flowing cell detection Download PDFInfo
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- CN1588088A CN1588088A CN 200410077992 CN200410077992A CN1588088A CN 1588088 A CN1588088 A CN 1588088A CN 200410077992 CN200410077992 CN 200410077992 CN 200410077992 A CN200410077992 A CN 200410077992A CN 1588088 A CN1588088 A CN 1588088A
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Abstract
The invention relates to a microflow control chip system for flow type cell detection, it character lies in: it includes microflow control chip driven by gravity, a laser focusing induced fluorescence detecting system which can be connected to a computer, charge coupling detector and high voltage power; the primary channel and assistant channel of the microflow chip are arranged on the top surface of the microflow chip, and which are connected to the sample pool and buffer liquid pool on the microflow control chip, three cell collectors are on the end of the primary channels, the microflow control chip is fixed on a base vertically, the focusing laser induced fluorescence detecting system is at one side of the microflow control chip, and its detecting area is at the middle or lower part of the primary channel, the charge coupling detector is at another side of the microflow control chip, the output of the high voltage powder is positive and negative voltage which are conducted to the cell collectors are two sides of the microflow control chip. The structure is simple, and the sample and reagent quantity can be reduced.
Description
Technical field
The present invention relates to a kind of fluidic chip detecting system, particularly about a kind of fluidic chip detecting system that fluidic cell detects that is used for that adopts segregation drive.
Technical background
(the micro-fluidic chip technology among the μ-TAS) is subjected to extensive concern to micro-total analysis system in recent years, development at home is also quite rapid, has applied for many pieces of patents, at present, many companies are with the micro-fluidic chip technology commercialization, as the cooperation of HP and Caliper, Hitachi, Nanogen etc.
Fluidic cell detection technique (Flow CytoMetry, FCM) be a kind of on functional level to detection means unicellular or that the other biological particle carries out quantitative test and sorting, have advantages such as speed is fast, precision is high, accuracy is good, its principle of work is to make single cell suspension behind the cell dyeing to be measured.With certain pressure testing sample is pressed into flow chamber, not celliferous phosphate buffer forms a fluid stream of definite shape under high pressure from the ejection of sheath fluid pipe, and cell to be measured single file under the bag quilt of sheath fluid is arranged, and passes through surveyed area successively.Flow cytometer usually with laser as excitation source, vertical irradiation under the irradiation of laser beam, is produced scattered light and fluorescence excitation by the cell of fluorescent dye on sample flow, received by the photomultiplier of forward light electric diode and 90 ° of directions simultaneously.The sorting of cell contains single celled drop by separation and realizes in the fluidic cell detection technique.Be furnished with a ultrahigh frequency electric crystal on the spout of flow chamber, the charging after vibration makes the liquid stream of ejection be fractured into uniform drop, and cell to be determined just is dispersed among these drops.These drops are filled with positive and negative different electric charge, when stream of liquid droplets when having the deflecting plate of several kilovolts, the effect deflect at high-voltage electric field falls into collection container separately, the waste fluid container of the drop that will not charge in the middle of falling into, thereby realize the separation of cell.But there is shortcoming such as cost an arm and a leg, bulky, complex operation, sample and reagent dosage greatly, easily pollute not easy cleaning in traditional flow cytometer.
Because traditional flow cytometer has above-mentioned shortcoming, therefore micro-fluidic chip system being used for cell analysis becomes in recent years a focus, receives increasing concern.Micro-fluidic chip system has characteristics such as highly microminiaturized, integrated and flexible design, can realize the functions such as cultivation, apoptosis and detection of cell by ingenious design and Precision Machining, the micro-fluidic chip technology is combined the volume of lowering apparatus greatly with the fluidic cell detection technique, reduce the probability of delay and cross-infection, can also reduce cost simultaneously.Recently, this technology is subjected to paying close attention to widely just gradually.
Different with traditional flow cytometer is, micro-fluidic chip system comprises negative pressure, electric osmose (electrophoresis) and handle and drive pattern based on various kinds of cell such as dielectrophoresis that except adopting positive pressure drives, also having adopted these methods respectively have relative merits.Wherein the electric field pair cell has lethal effect to a certain degree, can produce false positive results when the detection cell survives probability.Pressure-driven is the method that the conventional cell detecting instrument usually adopts, but when being used for the very little micro-fluidic chip system of channel size, the flow velocity that then requires forcing pump to provide wants enough low and stable to adapt to the requirement of chip cell analysis.Based on the cellular driven pattern of dielectrophoresis is a kind of method that is fit to very much cell operation on the chip, has the little characteristics such as can accurately control of damage, but will process microelectrode array on chip, and cost is higher comparatively speaking.Segregation drive once was used in the micro-fluidic chip, and such system has the equipment characteristic of simple, but was not used for the fluidic cell detection as yet with the micro-fluidic chip of segregation drive.
Summary of the invention
At the problems referred to above, but the object of the present invention is to provide a kind of volume of reduction equipment, reduce equipment cost, reduce the consumption of sample and reagent, adopt the fluidic chip detecting system that fluidic cell detects that is used for of segregation drive.
For achieving the above object, the present invention takes following technical scheme: a kind of micro-fluidic chip system that is used for the fluidic cell detection, it is characterized in that: it comprises the micro-fluidic chip with segregation drive, the confocal laser induced fluorescence detecting system that can be connected with computing machine, charge-coupled detector(CCD) and high-voltage power supply; The main channel of described micro-fluidic chip and accessory channel all are opened on the upper surface of described micro-fluidic chip, and connect the sample cell and the buffering solution pool that are positioned at described micro-fluidic chip upper surface respectively, three cell harvestors are communicated with the end of described main channel respectively, described micro-fluidic chip is vertically fixed on the pedestal, described confocal laser induced fluorescence detecting system is positioned at a side of described micro-fluidic chip, and its detection zone is positioned at the middle and lower part of described main channel, described charge-coupled detector(CCD) is located at the opposite side of described micro-fluidic chip, and described high-voltage power supply output just, negative voltage connects the cell harvestor that is positioned at both sides on the described micro-fluidic chip respectively by lead.
Described confocal laser induced fluorescence detecting system comprises LASER Light Source, exciting light optical filter, dichroic mirror, microcobjective, emission light optical filter, condenser lens, pin hole and the photomultiplier of installing according to the light path order.
Described micro-fluidic chip is made by glass.
Described micro-fluidic chip is made by the macromolecule polymeric material of saturating visible light.
Described macromolecule polymeric material is poly-dimethoxy silane.
Described macromolecule polymeric material is a polymethylmethacrylate.
The present invention is owing to adopt above technical scheme, it has the following advantages: 1, the present invention is different from other micro-fluidic chip fluidic cell detection system, owing to main channel and accessory channel all are opened on the upper surface of micro-fluidic chip, be connected with the buffer solution pond with the sample cell that is arranged on the upper surface equally, when therefore operating, the vertical placement of chip is fixed on the pedestal of detection platform, just can be that driving force is to the motion in chip channel with the cell self gravitation, carry out the quantitative test and the sorting of cell, save auxiliary devices such as impressed pressure pump, dwindled the volume of equipment.2, since the density of cell much larger than water, just can settle down very soon in aqueous solution, therefore when cell during, adopt gravity just can obtain analysis speed faster, simultaneously because cell is solid so the problem that also can not have sample diffusion as type of drive as analytic sample.3, the present invention is owing to be provided with the confocal laser induced fluorescence detecting system that can be connected with computing machine, charge-coupled detector(CCD) and high-voltage power supply, therefore the very little the present invention of volume can carry, in the place that computing machine is arranged, install software just can be operated after connecting each equipment, and can monitor in real time operating process by charge-coupled detector(CCD) and cooperating of computing machine.4, the present invention can apply certain electric field by computer control high-voltage power supply pair cell gatherer, by control to electric field level and direction, under the effect of gravity and electrophoresis combination, will there be fluorescence to separate with the cell that does not have fluorescence, not only make cellular damage little, and can accurately control.The present invention can be widely used in various unicellular or other biomones are carried out in the detection of quantitative test and sorting.
Description of drawings
Fig. 1 is the microfluidic chip structure synoptic diagram
Fig. 2 is traditional fluidic cell pick-up unit partial structurtes synoptic diagram
Fig. 3 adopts structural representation of the present invention
Fig. 4 is that the present invention is applied to the half-quantitative detection example of UV-irradiation to the HeLa cellular damage
Embodiment
As shown in Figure 1, micro-fluidic chip 1 of the present invention adopts glass or poly-dimethoxy silane (PDMS), polymethylmethacrylate visible light permeable macromolecule polymeric materials such as (PMMA) or plastic material to process.The microchannel that directly links with sample cell 2 is main channel 3, and 3 both sides, main channel are provided with accessory channel 4, two accessory channels 4 and are connected with buffer solution pond 5 respectively, and cell harvestor C1, C2, C3 are arranged on the end of main channel 3.Laser-Induced Fluorescence Detection zone 6 is positioned at the middle and lower part of main channel 3.Because main channel 3 and accessory channel 4 all are opened on chip 1 upper surface, during operation chip 1 is vertically placed, cell and buffering solution in sample cell 2 and the buffering solution pool 5 just can enter quantitative fluorescence analysis and the sorting that cell is carried out in main channel 3 under the effect of self gravitation.
Shown in Figure 2, traditional flow cytometer, buffer solution in cell in the sample cell 21 and the buffering solution pool 22 enters flow cytometer under the promotion of pressure system 23, by controlled pressure and flow velocity, cell is arranged in single file from the mouth of pipe 24 ejections, carry out the detection of fluorescence signal, carry out sorting by applying positive or negative high voltage electric field pair cell.The present invention is integrated into above-mentioned several parts 21,22,24 of traditional flow cytometer on the chip of several centimeter square, has dwindled the volume of instrument greatly, has reduced cost.Simultaneously since the present invention by the motion of segregation drive cell in chip microchannel, thereby saved pressure system 23, further simplified device.
As shown in Figure 3, detection system of the present invention comprises: micro-fluidic chip 1, confocal laser induced fluorescence detecting system, high-voltage power supply 15, charge-coupled detector(CCD) (CCD) 16.Micro-fluidic chip 1 vertically is fixed in (not shown) on the pedestal of detection platform, confocal laser induced fluorescence detecting system comprises microcobjective 10, bidirectional color mirror 9, emission light optical filter 11, condenser lens 12, pin hole 13 and the photomultiplier 14 that is successively set on micro-fluidic chip 1 one sides, and the light source 7, the exciting light optical filter 8 that are arranged on dichroic mirror 9 belows.16 of charge-coupled detector(CCD)s are arranged on the corresponding position of micro-fluidic chip 1 opposite side, the output terminal of high-voltage power supply 15 connects by lead and is fixed on cell harvestor C1, the C3, and be cell harvestor C1, C3 input 600V and 0V voltage, simultaneously, photomultiplier 14, high-voltage power supply 15 and charge-coupled detector(CCD) 16 also are connected a computing machine 17 by circuit respectively and connect, can not comprise computing machine 17 in the present device, only have various connecting lines, in the place that computing machine is arranged, load onto Control Software and just can operate.
During operation, laser instrument 7 sends beam of laser, filters parasitic light by exciting light optical filter 8, through dichroic mirror 9 reflections, focuses on the laser-Induced Fluorescence Detection district 6 of chip main channel 3 again by microcobjective 10.When the cell of crossing through fluorochrome label through out-of-date, sent the fluorescence of respective wavelength by laser excitation, fluorescence signal is collected by microcobjective 10, through dichroic mirror 9 transmissions, the interference of removing exciting light again through emission light optical filter 11, line focus lens 12, pin hole 13 are collected amplification and are converted electric signal to by photomultiplier 14, get off by computing machine 17 software records at last, and carrying out signal Processing, charge-coupled detector(CCD) 16 is used to the motion conditions of pair cell in the micro-fluidic chip passage and monitors in real time.
The present invention can add certain density dispersion medium in cell sample solution and buffering solution, make cell in solution, be scatter well, reduce reunion and settlement action, so cell admission passage one by one, do not had the phenomenon of blocking channel inlet.The cell sorting process is to realize by the size and Orientation of computing machine 17 On-line Control high-voltage power supplies 15 output voltages, cell just enters the branch constituency after by detecting device, high-voltage power supply 15 is exported 600V and 0V respectively in C1 and C3 (as shown in Figure 1), cell can be according to the positive and negative character of surface charge and is entered respectively among C1 and the C3, when detecting device obtains surpassing the fluorescence response signal of certain intensity, just can close the high voltage control program, so cell only enters among the C2, realize the separation of cell under action of gravity.
Example: the HeLa cell grows into the logarithmic growth after date and removes the double dish lid, inhales and removes nutrient solution, and after shining 10,20,40 minutes respectively under the ultraviolet light (254nm), the digestion collecting cell is also used TO-PRO-3 mark 1 hour.The single cell suspension 40 μ l of mark of learning from else's experience during detection add and to contain the phosphate buffered solution 200 μ l of 0.4%HPMC as sample solution, in the buffer solution pond 5 of micro-fluidic chip 1, add phosphate buffered solution 20 μ l respectively, in sample cell 2, add the good cell sample of mark of equivalent, carry out fluoroscopic examination.The result is respectively the testing result through ultraviolet ray irradiation 10min, 20min and 40min among the figure as shown in Figure 4 from bottom to up.As can be seen from the figure along with radiated time increases, apoptotic cell number and apoptosis degree all increase thereupon, and the cell number and the fluorescence intensity that detect also increase.
Claims (6)
1, a kind of micro-fluidic chip system that is used for the fluidic cell detection, it is characterized in that: it comprises the micro-fluidic chip with segregation drive, the confocal laser induced fluorescence detecting system that can be connected with computing machine, charge-coupled detector(CCD) and high-voltage power supply; The main channel of described micro-fluidic chip and accessory channel all are opened on the upper surface of described micro-fluidic chip, and connect the sample cell and the buffering solution pool that are positioned at described micro-fluidic chip upper surface respectively, three cell harvestors are communicated with the end of described main channel respectively, described micro-fluidic chip is vertically fixed on the pedestal, described confocal laser induced fluorescence detecting system is positioned at a side of described micro-fluidic chip, and its detection zone is positioned at the middle and lower part of described main channel, described charge-coupled detector(CCD) is located at the opposite side of described micro-fluidic chip, and described high-voltage power supply output just, negative voltage connects the cell harvestor that is positioned at both sides on the described micro-fluidic chip respectively by lead.
2, a kind of fluidic chip detecting system that fluidic cell detects that is used for as claimed in claim 1, it is characterized in that: described confocal laser induced fluorescence detecting system comprises LASER Light Source, exciting light optical filter, dichroic mirror, microcobjective, emission light optical filter, condenser lens, pin hole and the photomultiplier of installing according to the light path order.
3, a kind of fluidic chip detecting system that fluidic cell detects that is used for as claimed in claim 1 or 2, it is characterized in that: described micro-fluidic chip is made by glass.
4, a kind of fluidic chip detecting system that fluidic cell detects that is used for as claimed in claim 1 or 2, it is characterized in that: described micro-fluidic chip is made by the macromolecule polymeric material of saturating visible light.
5, a kind of fluidic chip detecting system that fluidic cell detects that is used for as claimed in claim 4, it is characterized in that: described macromolecule polymeric material is poly-dimethoxy silane.
6, a kind of fluidic chip detecting system that fluidic cell detects that is used for as claimed in claim 4, it is characterized in that: described macromolecule polymeric material is a polymethylmethacrylate.
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| CNB2004100779924A CN1304846C (en) | 2004-09-23 | 2004-09-23 | Micro flow control chip detecting system for flowing cell detection |
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| CNB2004100779924A CN1304846C (en) | 2004-09-23 | 2004-09-23 | Micro flow control chip detecting system for flowing cell detection |
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