CN103323383B - Particle counting system of micro-fluidic chip based on electric resistance technology - Google Patents
Particle counting system of micro-fluidic chip based on electric resistance technology Download PDFInfo
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- CN103323383B CN103323383B CN201310217688.4A CN201310217688A CN103323383B CN 103323383 B CN103323383 B CN 103323383B CN 201310217688 A CN201310217688 A CN 201310217688A CN 103323383 B CN103323383 B CN 103323383B
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Abstract
The invention discloses a particle counting system of a micro-fluidic chip based on an electric resistance technology. The system comprises a micro-fluidic chip and a signal detection circuit. The micro-fluidic chip comprises a glass substrate, a PDMS substrate and two pairs of metal needles used as electrodes. Normal conductive metal needles are utilized as the electrodes of the micro-fluidic chip to produce an electric field which is across a micro-channel, therefore the preparation process of the micro-fluidic chip is comparatively simple and the production cost is comparatively low. The signal detection circuit comprises two I/V conversion circuit, a differential circuit, an envelope detection circuit, a high-pass filter circuit, a low-pass filter circuit and an amplifying circuit. The resistance signals are detected in the signal detection circuit by utilizing signal differential detection method to ensure precision of signal detection.
Description
Technical field
Invention relates to a kind of particle counting system, is specifically related to a kind of micro-fluidic chip particle counting system based on electrical impedance technology, belongs to electrical detection technical field.
Background technology
Electrical impedance counting technology is the earliest for particulate/Cytometric automatic technology.Because particulate/cell can be used as insulating bodies to a certain extent, when particulate/cell passes through a certain fixed area electric field, the impedance in this stationary electric field region can be made to become large; When particulate/cell leaves electric field region, impedance recovers again normal; The size of impedance signal and the volume of particulate/cell are directly proportional.Therefore people utilize this characteristic count particulate/cell and classify.But present commercial electrical impedance counter is generally relatively large, cannot meet portable requirement; And based on the counting technology of microfluidic chip technology, provide the possibility of solution for this requirement.
Microfluidic chip technology refers to integrated for the basic operation unit such as sample preparation, reaction, separation, detection involved in the fields such as biological and chemical or be substantially integrated on the chip of a piece several square centimeters (even less), network is formed by microchannel, whole system is run through, in order to replace a kind of technology platform of the various functions of standard biologic or chemical laboratory with controlled fluid.Obviously, microfluidic chip technology has the series of features such as volume is little, portability is high, application is convenient, and make immediately to detect the scene of some pathological parameters to become possibility, application space is very extensive.Nearly ten years, microfluidic chip technology development is very rapid, especially in the research field for particulate/cell count and sorting.
But the micro-fluidic chip counting micro particles device of present stage all to need on micro-fluidic chip plating or splash-proofing sputtering metal as electrode, and by electrode for generation of the electric field across microchannel.This design makes chip manufacturing process and cost relative complex, and cost compare is high.This design simultaneously, electrode is spaced very near, easily causes passage internal induction electric field intensity uneven.Even if the particulate of same class size, because the position flowing through microchannel is different, signal intensity also can be caused to differ; Have a strong impact on the embody rule of counting assembly.In order to solve this kind of problem, researchist introduces various focus method, and such as dielectrophoresis focuses on, and the methods such as focusing ultrasonic wave focus on particulate/cell, although improve signal stabilization so to a certain extent, also improve complexity and the cost of package unit simultaneously.
Meanwhile, in the micro-fluidic chip counting micro particles device of present stage, because signal to noise ratio (S/N ratio) is lower, so detection system generally adopts the technology of more complicated, such as synchronous detection technology, or use very expensive instrument, such as lock-in amplifier etc. realize the detection to feeble signal.Make the cost burden of whole device very large, simultaneously special instrument, also cannot meet portability, the requirement of low cost.
Summary of the invention
In view of this, the invention provides a kind of micro-fluidic chip particle counting system based on electrical impedance technology, this system architecture is simple, easily manufactured, cost is low and be easy to carry.
This system comprises micro-fluidic chip and signal deteching circuit; Described micro-fluidic chip comprises: glass substrate, polydimethylsiloxane substrate and two are to the metal needle being used as electrode; Described signal deteching circuit comprises: two I/V change-over circuits, difference channel, envelope detection circuit, high-pass filtering circuit, low-pass filter circuit and amplifying circuits.
In described micro-fluidic chip, PDMS substrate is bonded in the upper surface of glass substrate, is processed with sample inflow entrance, fluid outlet, sprue, sheath liquid flow inlet and electrode mounting hole respectively at PDMS substrate upper surface; Be specially: in the center of PDMS substrate upper surface, process strip groove along its length as sprue.At the symmetria bilateralis processing two arrays of electrodes mounting hole of sprue, be embedded in a metal needle at each electrode mounting hole.Four through by rectangular recess between electrode mounting hole and sprue; At the sample inflow entrance that the processing of the end, one end of sprue is through with sprue, the end of the other end processes the fluid outlet through with sprue.Be processed with sheath liquid flow inlet in the symmetria bilateralis of described sample inflow entrance, two sheath liquid flow inlet and sprue through.The degree of depth of above-mentioned sample inflow entrance, fluid outlet, sheath liquid flow inlet and electrode mounting hole all with the consistency of thickness of PDMS substrate.Sprue on described micro-fluidic chip and the passage through with sprue are referred to as microchannel, and the degree of depth of described microchannel is all consistent; In two pairs of metal needles on described micro-fluidic chip, two metal needles being positioned at sprue side are connected with outside sine wave AC driving source respectively, and two metal needles of opposite side are respectively connected with an I/V change-over circuit.
In described signal deteching circuit, the output terminal of two I/V change-over circuits is connected with two input ends of difference channel respectively, the output terminal of difference channel is connected with envelope detection circuit, envelope detection circuit is connected with low-pass filter circuit by high-pass filtering circuit, the amplifying circuit of the output termination afterbody of low-pass filter circuit.Described I/V change-over circuit is used for the current signal collected to be converted to voltage signal, and two-way voltage signal produces into differential signal by difference channel; Described envelope detection circuit for extracting the impedance signal in differential signal, and carries out the amplification of the first order to the impedance signal extracted; After flip-flop in impedance signal received by high-pass filtering circuit filtering, impedance signal is carried out to the amplification of the second level; After carrier signal in the impedance signal that low-pass filter circuit filtering receives, by amplifying circuit, impedance signal is carried out to the amplification of afterbody, the number of the last impedance signal according to exporting realizes the counting to particulate.
The degree of depth of described sprue and width are 1.5 times of mean particle dia to be detected to 3 times; Between through electrode mounting hole and sprue, the width of rectangular recess is 10 μm to 50 μm; Described electrode mounting hole and and sprue between distance be 20 μm; Distance H between two groups of metal needles
1it is 100 μm to 200 μm.
In described signal deteching circuit, the version of two I/V change-over circuits is identical; I/V change-over circuit comprises operational amplifier A and sampling resistor; The wherein electrode input end ground connection of operational amplifier A, negative input is connected with the metal needle being used as electrode, is connected sampling resistor R0 between its output terminal with negative input; Described difference channel comprises differential amplifier U1, operational amplifier C, resistance R3 and resistance R4; In two I/V change-over circuits, the output terminal of operational amplifier is connected with two input ends of differential amplifier U1 respectively, the output terminal of differential amplifier U1 is connected with the electrode input end of operational amplifier C, the negative input of operational amplifier C passes through resistance R3 ground connection, simultaneously contact resistance R4 between its negative input and output terminal; The output terminal of operational amplifier C is connected with envelope detection circuit.
Described envelope detection circuit comprises diode D6, resistance R2, electric capacity C1 and operational amplifier D; The output terminal of operational amplifier C is connected with the negative electrode of diode D6, and the anode of diode D6 is connected with the electrode input end of operational amplifier D; Be connected between the anode of diode and ground after resistance R2 and electric capacity C1 parallel connection; The output terminal of described operational amplifier D is connected with high-pass filtering circuit.
Described high-pass filtering circuit comprises operational amplifier E, resistance R5, resistance R6, electric capacity C2 and electric capacity C3; The output terminal of operational amplifier D is connected with the electrode input end of operational amplifier E with after resistance R6 by resistance R5 successively, the electrode input end of operational amplifier E is by electric capacity C3 ground connection simultaneously, one end of electric capacity C2 is connected with the output terminal of operational amplifier E, and the other end is connected between resistance R5 and resistance R6; Contact resistance R7 between the negative input and ground of operational amplifier E, the indirect resistance R8 of its negative input and output terminal; The output terminal of operational amplifier E is connected with low-pass filter circuit.
Described low-pass filter circuit comprises resistance R9, resistance R12, electric capacity C4, electric capacity C5 and operational amplifier F; The output terminal of operational amplifier E is connected with the electrode input end of operational amplifier F with electric capacity C5 by electric capacity C4, and the electrode input end of operational amplifier F is by resistance R9 ground connection simultaneously; One end of resistance R12 is connected with the output terminal of operational amplifier F, and the other end is connected between electric capacity C4 and electric capacity C5; Contact resistance R10 between operational amplifier F negative input and ground, the indirect resistance R11 of operational amplifier F negative input and output terminal; The output terminal of operational amplifier F is connected with amplifying circuit.
Described amplifying circuit comprises operational amplifier H, resistance R13 and resistance R14; The output terminal of described operational amplifier F is connected with the electrode input end of operational amplifier H, and the negative input of operational amplifier H passes through resistance R13 ground connection, simultaneously contact resistance R14 between the output terminal and negative input of operational amplifier H.
Described sine wave AC driving source frequency is 500KHz ~ 1.2MHz, and peak value is 3V ~ 5V.
In described I/V change-over circuit, the resistance of sampling resistor is 30K Ω ~ 300K Ω, and the enlargement factor of described differential amplifier U1 is less than or equal to 10 times, and described low-pass filter circuit cutoff frequency is 1/10th of sine-wave excitation frequency input signal.
The described metal needle being used as electrode is acupuncture needle or platinum pin.
Based on the job operation of micro-fluidic chip in the micro-fluidic chip particle counting system of electrical impedance technology be:
On step one, known micro-fluidic chip microchannel each several part size after, draw microchannel plane design drawing on computers;
Step 2, be worked on mask by the microchannel plane design drawing designed, the mask after processing is only transparent at channel portion;
Step 3, by negative photoresist uniform application to silicon chip upper surface, the thickness of negative photoresist is consistent with the depth value of microchannel; Then silicon chip is heated 1 minute under the constant temperature of 100 DEG C, make the negative photo adhesive curing of its upper surface;
Step 4, using the mask in step 2 as light shield, through light shield, uv-exposure 10 second is carried out to silicon chip, now ultraviolet irradiates the negative photoresist on silicon chip through the transparent part on mask, receives the negative photoresist generation cross-linking reaction of ultraviolet irradiation and is polymerized;
The developing solution dissolution that step 5, utilization are mated with the negative photoresist in step 3 falls the negative photoresist that silicon chip does not occur cross-linking reaction; Then adopt pure water rinsing silicon chip, after cleaning, dry up the pure water on silicon chip with nitrogen; The negative photo plastic structure that now silicon chip upper surface is left is the formpiston part of micro-fluidic chip, and described formpiston part refers to the spatial structure of microchannel, and its thickness is consistent with the degree of depth of microchannel;
Step 6, alkanisation process is carried out to the silicon chip that step 5 obtains;
Step 7, PDMS and rigidizer mixed with the ratio of mass ratio 10:1 and stirs, removing its air entrapment, then by the formpiston part of mixed liquid pouring on silicon chip, and cover formpiston part completely; Being heated under the constant temperature of 100 DEG C by silicon chip makes it solidify in 4 hours, then takes the PDMS layer of silicon chip upper surface off, obtains the PDMS substrate with microchannel thus;
Step 8, utilize card punch PDMS substrate upper surface mark have the position of sample inflow entrance, fluid outlet, sheath liquid flow inlet and electrode mounting hole to stamp through hole;
Step 9, PDMS substrate and glass substrate are carried out oxygen plasma surface treatment, then will accept the intimate surface laminating of plasma treatment in two substrates, the chemical bond on both surface of contact is bonded together mutually; Finally four conducting metal pins are embedded in four electrode mounting holes respectively, form micro-fluidic chip thus.
In described step 4, after having exposed, the silicon chip after exposure is heated 1 minute under the constant temperature of 100 DEG C.
In described step 9, the PDMS substrate be bonded together and glass substrate are heated 4 hours under the constant temperature of 100 DEG C.
Beneficial effect:
Conducting metal pin is directly arranged on as electrode on micro-fluidic chip by this system, thus produces across the electric field of microchannel, such that chip manufacturing process is simple, cost is low; This kind of design simultaneously, electrode interval is to each other determined by the width of sprue, and two electrode symmetries are installed on sprue both sides, makes the induction field even intensity in induction zone, ensures the stability of impedance signal.
For ensureing the accuracy of input, at the symmetria bilateralis processing sheath liquid flow inlet of sample inflow entrance in this system, utilizing sheath fluid stream particulate one by one to be gathered relatively more fixing flow location, thus being convenient to the detection of signal.
Adopt differential signal detection circuit to improve the degree of accuracy of detection simultaneously within the system, the sensitivity of input can not only be improved, make the cost of whole system little simultaneously, can portability be met, the requirement of low cost.
Accompanying drawing explanation
Fig. 1 is the structural representation of the micro-fluidic chip for counting micro particles;
Fig. 2 is the vertical view of PDMS substrate;
Fig. 3 is the enlarged drawing in induction of signal district;
Fig. 4 is the integrated connection figure of this system;
Fig. 5 is the circuit theory diagrams of I/V conversion, differential amplification and envelope detection part;
Fig. 6 is the circuit theory diagrams of high-pass filtering and low-pass filtering part;
Fig. 7 be adopt systems axiol-ogy of the present invention to particulate pass through the differential signal that electro-induction district formed;
Fig. 8 is the testing result comparison diagram adopting the present invention and prior art.
Wherein, 1-glass substrate, 2-PDMS substrate, 3-sample inflow entrance, 4-fluid outlet, 5-sprue, 6-sheath liquid flow inlet, 7-electrode mounting hole, 8-respond to core space, 9-sense blind area, 10-metal needle, 11-induction of signal district
Embodiment
To develop simultaneously embodiment below in conjunction with accompanying drawing, describe the present invention.
The present embodiment provides a kind of micro-fluidic chip particle counting system based on electrical impedance technology, this system utilizes common conducting metal pin as the electrode of micro-fluidic chip, and the method utilizing signal differential to detect detects its impedance signal, there is simple, the with low cost and feature such as to be easy to carry of chip manufacturing process.
This system comprises micro-fluidic chip and signal deteching circuit.Wherein micro-fluidic chip comprises: glass substrate, PDMS(dimethyl silicone polymer) substrate and two is to being used as the metal needle of electrode.Signal deteching circuit comprises: two I/V change-over circuits, difference channel, envelope detection circuit, high-pass filtering circuit, low-pass filter circuit and amplifying circuits.The integrated connection of this system closes and is: in two pairs of metal needles on micro-fluidic chip, two metal needles being positioned at sprue side are connected with outside sine wave AC driving source respectively, and two metal needles of opposite side are respectively connected with an I/V change-over circuit.Described sine wave AC driving source frequency range is chosen as between 500KHz ~ 1.2MHz, and peak ranges is between 3V to 5V.
As shown in Figure 1, wherein metal needle requires to conduct electricity the concrete structure of the micro-fluidic chip for counting micro particles that the present embodiment provides, and has certain biocompatibility and stability, adopts acupuncture needle in the present embodiment.PDMS substrate is bonded in the upper surface of glass substrate, PDMS substrate is processed with sample inflow entrance, fluid outlet, sprue, sheath liquid flow inlet and electrode mounting hole respectively, as shown in Figure 2.Be specially: in the center of PDMS substrate upper surface, process strip groove along its length as sprue; Be processed with two groups of (often organizing two) electrode mounting holes (two along sprue symmetry is a group) in the symmetria bilateralis of sprue, be embedded in a metal needle at each electrode mounting hole.The degree of depth of described electrode mounting hole and the consistency of thickness of PDMS substrate; Four through by rectangular recess between electrode mounting hole and sprue.The circular hole through at the processing of the end, one end of sprue and sprue is used as sample inflow entrance, and the circular hole that the end processing of the other end is through with sprue is used as fluid outlet.Some particulate, such as cells etc. are easy to be sticked together, and affect the detection of signal; For ensureing the accuracy of input, sheath fluid stream is utilized particulate one by one to be gathered relatively more fixing flow location in the present embodiment, for this reason sample inflow entrance symmetria bilateralis processing circular hole be used as sheath liquid flow inlet, described two sheath liquid flow inlet by tilt bar-shaped trough and sprue through.The degree of depth of above-mentioned sample inflow entrance, fluid outlet and sheath liquid flow inlet all with the consistency of thickness of PDMS substrate.
Below the size Selection of influential system sensitivity direct on PDMS substrate is described in detail.
On PDMS substrate, along sprue symmetry two metal needles between region be induction of signal district, the enlarged drawing in induction of signal district is as shown in Figure 3.In impedance signal detects, the volume in induction of signal district and the volume ratio of particulate/cell to be detected are very important parameters.If this ratio is too large, input sensitivity can be caused low.Induction of signal district comprises induction core space and sense blind area, and wherein the region of sprue between symmetrical two metal needles is induction core space; Coupling part between sprue and electrode mounting hole is sense blind area.The width wherein responding to the degree of depth of volume by sprue of core space, width and the rectangular recess between electrode mounting hole and sprue determines, ideally, the volume of particulate/cell to be detected is more close better with the volume of induction core space; But in a practical situation, if too close, be easy to cause sprue to get clogged.Therefore, in the present embodiment, the degree of depth of sprue and width are all taken as 1.5 times of mean particle dia to be detected, sprue can not be blocked while guarantee detection sensitivity.Between electrode mounting hole and sprue, the width of rectangular recess then defines the coverage of electric field, if too little, is unfavorable for that processing realizes; If too large, then make the induction volume of core space and the volume ratio of particulate to be detected increase, and then reduce the sensitivity of system; In the present embodiment, the width of this rectangular recess is set to 20 μm.The volume of sense blind area by the degree of depth of the rectangular recess between electrode mounting hole and sprue, width and electrode mounting hole and and sprue between distance determine.In theory, the distance between electrode mounting hole and sprue is the smaller the better.Be confined to micro fabrication, if be worked into very little, yield rate can be caused too low, therefore guarantee compared with under the prerequisite of high finished product rate, in the present embodiment electrode mounting hole and and sprue between distance be chosen as 20 μm.。By the sprue on described micro-fluidic chip and and the through passage of sprue (comprising the rectangular recess between electrode mounting hole and sprue, bar shaped between two sheath liquid flow inlet and sprue) be referred to as microchannel, the degree of depth of microchannel is consistent.
For improving the degree of accuracy detected, this system carries out difference processing to two groups of impedance signals, and the distance H between two groups of metal needles directly affects the effect of input.Although the position of electric field limit by the size of sprue, electric field outwards itself needs an attenuation process at induction core space; If two pairs of electrode contacts are too near, the electric field produced between two pairs of electrodes easily interferes with each other, and is unfavorable for input; If but at a distance of too far away, then the detection rates of reduction particulate that can be serious.Distance H in the present embodiment between two groups of metal needles
1be 100 μm, under this distance, namely ensure that the detection rates of particulate, the electric field simultaneously produced between two pairs of electrodes also can not interfere with each other.
When the nonconductors such as particulate/cell pass through induction of signal district, the anti-increase of overall resistance in induction of signal district; Once particulate/cell leaves, the electrical impedance in induction of signal district recovers again normal; A projection on time impedance signal has reacted above-mentioned whole process.Owing to being an AC field at the electric field of induction zone, and particulate/cell flow rate is comparatively fast corresponding, so impedance signal is characteristically, and a closely amplitude-modulated signal.For this judgement, the present invention utilizes the principles of modulation and demodulation of envelope detection, designs respective impedance signal deteching circuit.Simultaneously in order to improve accuracy of detection and sensitivity, utilizing difference detecting method, utilizing two pairs of electrode needle combinations to realize differential signal.In signal deteching circuit, wherein the output terminal of two I/V change-over circuits is connected with two input ends of difference channel respectively, the output terminal of difference channel is connected with envelope detection circuit, the output signal of envelope detection circuit is after amplifying circuit amplifies, by high-pass filtering circuit and low-pass filter circuit, signal is processed again, the amplifying circuit of the output termination afterbody of low-pass filter circuit.
Described I/V change-over circuit is used for current signal to be converted to voltage signal, and two-way voltage signal produces into differential signal by difference channel, and envelope detection circuit is for extracting the rough envelope of differential signal, and namely particulate/cell passes through the impedance signal of electric field; Then by high-pass filtering circuit filtering flip-flop, signal amplifies by amplifying circuit subsequently, then sends to the further filtering carrier signal of low-pass filter circuit, finally utilizes amplifying circuit to amplify signal, so that identify; Then just by signals collecting or can show.The parameter of envelope detection circuit and filtering circuit depends on the speed of particulate/cell to be measured through electro-induction district.The selection of operational amplifier in circuit, must meet the lower bias current input requirements of higher gain bandwidth (GB) sum, noise inputs error is also little as much as possible simultaneously.
The concrete structure of signal deteching circuit as shown in Figure 5 and Figure 6.Described I/V change-over circuit is realized by operational amplifier and sampling resistor.The integrated operational amplifier U2 containing four operational amplifiers (being respectively operational amplifier A, B, C, D) has been selected in the present embodiment.Wherein operational amplifier A and operational amplifier B are respectively used to two I/V change-over circuits, and two I/V change-over circuits are sampled identical version.For operational amplifier A, the electrode input end ground connection of operational amplifier A, negative input is connected with the metal needle being used as electrode, sampling resistor R0 is connected between its output terminal with negative input, in order to reduce error, R0 selects high-accuracy resistance, and its resistance is directly proportional to the volume in induction of signal district, and scope is between 30K Ω to 300K Ω.Described difference channel comprises differential amplifier U1 and operational amplifier C; Operational amplifier A is connected with two input ends of differential amplifier U1 respectively with the output terminal of operational amplifier B, and the enlargement factor of differential amplifier U1 can not arrange too large, is good within 10 times.The output terminal of differential amplifier U1 is connected with the electrode input end of operational amplifier C, the negative input of operational amplifier C is by resistance R3 ground connection, contact resistance R4 between its negative input and output terminal, utilizes operational amplifier C and resistance R3, R4 to amplify 23 times further to the output signal of differential amplifier U1 in the present embodiment simultaneously.The output terminal of operational amplifier C is connected with envelope detection circuit, and described envelope detection circuit comprises diode D6, resistance R2, electric capacity C1 and operational amplifier D.The output terminal of operational amplifier C is connected with the negative electrode of diode D6, and the anode of diode D6 is connected with the electrode input end of operational amplifier D; Between the anode being connected on diode D6 after resistance R2 and electric capacity C1 parallel connection and ground.In the envelope detection circuit of the present embodiment, in the direction of diode D6 and conventional envelope detector, diode direction is contrary; Because the signal of I/V change-over circuit collection is a current signal, due to particulate/cell flow through induction of signal district time, the impedance of this region increases, and current value reduces, and the signal therefore collected is the downward signal of a peak value; Here utilize reverse diode to arrange and process is swung to signal, make the peak value of signal upwards.The value of resistance R2 and electric capacity C1 determines the detection scope of envelope detection, and therefore the selection of its value much smaller than the incoming frequency of sine-wave excitation, should also be greater than particulate cutting induction zone electric field frequency simultaneously.Operational amplifier D is used for follower, plays the effect of impedance matching.
The output terminal of operational amplifier D is connected with high-pass filtering circuit.As shown in Figure 6, this part has selected the integrated operational amplifier U3 containing four operational amplifiers to the detailed construction of described high-pass filtering circuit and low-pass filter circuit, has only used wherein three, be respectively operational amplifier E, F, H in the present embodiment.Wherein operational amplifier E and resistance R5, resistance R6, electric capacity C2, electric capacity C3 form Order RC high-pass filtering circuit, the output terminal of operational amplifier D is connected with the electrode input end of operational amplifier E with after resistance R6 by resistance R5 successively, the electrode input end of operational amplifier E is by electric capacity C3 ground connection simultaneously, one end of electric capacity C3 is connected with the output terminal of operational amplifier E, and the other end is connected between resistance R5 and resistance R6.In the present embodiment, the cutoff frequency of high-pass filtering circuit is at about 10Hz, for the DC component after filtering envelope detection.Then utilize the resistance R7 that is connected between the negative input of operational amplifier E and ground, and the resistance R8 between negative input and output terminal is put to its output signal and is twice.
The output terminal of operational amplifier E is connected with low-pass filter circuit.Described low-pass filter circuit is the second order RC lowpass filter be made up of the operational amplifier F in resistance R9, resistance R12, electric capacity C4, electric capacity C5 and operational amplifier U3.The output terminal of operational amplifier E is connected with the electrode input end of operational amplifier F with electric capacity C5 by electric capacity C4, and the electrode input end of operational amplifier F is by resistance R9 ground connection simultaneously.One end of resistance R12 is connected with the output terminal of operational amplifier F, and the other end is connected between electric capacity C4 and electric capacity C5.Low-pass filter circuit is used for the sine wave exciting signal component in further filtered signal, and its cutoff frequency is relevant with input sine wave excitation components, elects 1/10th of sine-wave excitation frequency input signal in the present embodiment as.Then utilize the resistance R10 between operational amplifier F negative input and ground, and the resistance R11 between operational amplifier F negative input and output terminal outputs signal to put and is twice to it.
The output terminal of operational amplifier F is connected with the electrode input end of operational amplifier H, the negative input of operational amplifier F is by resistance R13 ground connection, contact resistance R14 between the output terminal and negative input of operational amplifier H simultaneously, operational amplifier H and resistance R13 and resistance R14 forms the amplification of final step to signal, and final signal outputs on display device or collecting device by the output terminal of operational amplifier G.
The processing technology of described micro-fluidic chip is:
On step one, known micro-fluidic chip microchannel each several part size after, draw microchannel plane design drawing as shown in Figure 7 on computers;
Step 2, be worked on mask by the microchannel plane design drawing designed, the mask after processing is transparent at channel portion, and remainder is then black, light tight;
Step 3, by negative photoresist (SU-8) uniform application to silicon chip upper surface, the thickness of negative photoresist is consistent with the depth value of microchannel; Then silicon chip is heated 1 minute at 100 DEG C, make the negative photo adhesive curing of its upper surface;
Step 4, using the mask in step 2 as light shield, ultraviolet exposure machine through light shield to silicon wafer exposure 10 second, ultraviolet irradiates the negative photoresist on silicon chip through the transparent part on mask, receive the negative photoresist generation cross-linking reaction of ultraviolet irradiation and be polymerized, then heating instrument is utilized to heat 1 minute at 100 DEG C the silicon chip after exposure, to strengthen polymerization effect;
Step 5, SU-8 developing solution dissolution is utilized to fall the part that silicon chip does not occur cross-linking reaction, then pure water rinsing silicon chip is utilized, pure water is dried up with nitrogen after cleaning, the SU-8 structure that now silicon chip upper surface is left is the formpiston part (i.e. the spatial structure of microchannel, its thickness is consistent with the degree of depth of microchannel) of micro-fluidic chip;
Step 6, alkanisation process is carried out to the silicon chip that step 5 obtains, the PDMS layer being separated formpiston after being convenient to and toppling over thereon;
Step 7, by PDMS and rigidizer with the mixing of the ratio of mass ratio 10:1, stir, removing bubble, is then poured over the formpiston part on silicon chip, and covers formpiston part completely; Then be placed on 100 ° of oven for solidifying 4 as a child, and took out, take PDMS layer off, obtain the PDMS substrate with microchannel thus;
Step 8, utilize card punch PDMS substrate upper surface mark have the position of sample inflow entrance, fluid outlet, sheath liquid flow inlet and electrode mounting hole to stamp through hole.
Step 9, by PDMS substrate with amass bigger glass substrate than PDMS substrate surface and carry out oxygen plasma surface treatment, then the intimate surface accepting plasma treatment in two substrates is fit together, the chemical bond on both surface of contact is bonded together mutually; For strengthening bonding effect, the PDMS substrate be bonded together and glass substrate can be put into 100 ° of baking boxs roasting 4 hours.Finally four conducting metal pins are embedded in four electrode mounting holes respectively, form micro-fluidic chip thus.
The principle of work of this particle counting system is:
Before measuring process starts, first in microchannel, be full of conducting solution, two electrodes of sprue one end connect sine wave AC excitation (frequency range is chosen as between 500KHz ~ 1.2MHz, and peak ranges is between 3V to 5V), and two electrodes of the other end connect impedance signal testing circuit.Like this, just define an electric field between every two symmetrical electrodes, this electric field and microchannel intersection form induction of signal district.
When the electric field that particulate/stream of cells builds through pair of electrodes, impedance signal is now had to produce, and in the electric field that second pair of electrode subsequently builds, owing to not having particulate/cell to flow through, so not any signal occurs.Signal deteching circuit utilizes the difference of this two paths of signals to count the particulate/cell flowing through induction of signal district, effectively can increase system sensitivity.
Fig. 7 is when adopting native system, and 10 μm of particulates, by the testing result behind electric field region, this curve represents a particulate and pass through electro-induction district and the differential signal that formed.
Fig. 8 is by native system and the further Experimental comparison results of existing flow cytometer, adopt hybrid fine particles sample (5 μm, 10 μm and 15 μm), then sample is divided into two parts, a part utilizes flow cytometer to test, and a part utilizes system of the present invention to test in addition.Fig. 8 (a) is the Output rusults of flow cytometer, and horizontal ordinate is forward scattering light, and ordinate is side scattered light; Clearly demonstrate three sample collection districts in figure, it represents the particulate samples of 5 μm (A), 10 μm (B) and 15 μm (C) respectively, and its ratio is 54.3%(9 respectively), 28.5%(10) and 12.6%(11).Measurement result is turned to the histogram based on forward scattering light, as shown in Figure 8 (b) shows.Adopt the measurement result of system of the present invention as shown in Fig. 8 (c), have obviously three piecemeals, the ratio that every block accounts for whole quantity is respectively 49.7%, 32.4%, 14,1%, very good with (b) consistance with Fig. 8 (a).
Micro-fluidic chip counting micro particles device of the present invention, simplicity of design, easily manufactured, with low cost, the needs that people detect immediately to scene can be met, in environment measuring, biological study and medical diagnosis etc., have wide using value.
In sum, these are only preferred embodiment of the present invention, be not intended to limit protection scope of the present invention.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. based on a micro-fluidic chip particle counting system for electrical impedance technology, it is characterized in that, comprise micro-fluidic chip and signal deteching circuit; Described micro-fluidic chip comprises: glass substrate, polydimethylsiloxane substrate and two are to the metal needle being used as electrode; Described signal deteching circuit comprises: two I/V change-over circuits, difference channel, envelope detection circuit, high-pass filtering circuit, low-pass filter circuit and amplifying circuits;
In described micro-fluidic chip, PDMS substrate is bonded in the upper surface of glass substrate, is processed with sample inflow entrance, fluid outlet, sprue, sheath liquid flow inlet and electrode mounting hole respectively at PDMS substrate upper surface; Be specially: in the center of PDMS substrate upper surface, process strip groove along its length as sprue; At the symmetria bilateralis processing two arrays of electrodes mounting hole of sprue, be embedded in a metal needle at each electrode mounting hole; Four through by rectangular recess between electrode mounting hole and sprue; At the sample inflow entrance that the processing of the end, one end of sprue is through with sprue, the end of the other end processes the fluid outlet through with sprue; Be processed with sheath liquid flow inlet in the symmetria bilateralis of described sample inflow entrance, two sheath liquid flow inlet and sprue through; The degree of depth of above-mentioned sample inflow entrance, fluid outlet, sheath liquid flow inlet and electrode mounting hole all with the consistency of thickness of PDMS substrate; Sprue on described micro-fluidic chip and the passage through with sprue are referred to as microchannel, and the degree of depth of described microchannel is all consistent; In two pairs of metal needles on described micro-fluidic chip, two metal needles being positioned at sprue side are connected with outside sine wave AC driving source respectively, and two metal needles of opposite side are respectively connected with an I/V change-over circuit;
In described signal deteching circuit, the output terminal of two I/V change-over circuits is connected with two input ends of difference channel respectively, the output terminal of difference channel is connected with envelope detection circuit, envelope detection circuit is connected with low-pass filter circuit by high-pass filtering circuit, the amplifying circuit of the output termination afterbody of low-pass filter circuit; Described I/V change-over circuit is used for the current signal collected to be converted to voltage signal, and two-way voltage signal produces into differential signal by difference channel; Described envelope detection circuit for extracting the impedance signal in differential signal, and carries out the amplification of the first order to the impedance signal extracted; After flip-flop in impedance signal received by high-pass filtering circuit filtering, impedance signal is carried out to the amplification of the second level; After carrier signal in the impedance signal that low-pass filter circuit filtering receives, by amplifying circuit, impedance signal is carried out to the amplification of afterbody, the number of the last impedance signal according to exporting realizes the counting to particulate.
2. a kind of micro-fluidic chip particle counting system based on electrical impedance technology according to claim 1, is characterized in that: the degree of depth of described sprue and width are 1.5 times of mean particle dia to be detected to 3 times; Between through electrode mounting hole and sprue, the width of rectangular recess is 10 μm to 50 μm; Described electrode mounting hole and and sprue between distance be 20 μm; Distance H between two groups of metal needles
1it is 100 μm to 200 μm.
3. a kind of micro-fluidic chip particle counting system based on electrical impedance technology according to claim 1, it is characterized in that: in described signal deteching circuit, the version of two I/V change-over circuits is identical; I/V change-over circuit comprises operational amplifier A and sampling resistor; The wherein electrode input end ground connection of operational amplifier A, negative input is connected with the metal needle being used as electrode, is connected sampling resistor R0 between its output terminal with negative input; Described difference channel comprises differential amplifier U1, operational amplifier C, resistance R3 and resistance R4; In two I/V change-over circuits, the output terminal of operational amplifier is connected with two input ends of differential amplifier U1 respectively, the output terminal of differential amplifier U1 is connected with the electrode input end of operational amplifier C, the negative input of operational amplifier C passes through resistance R3 ground connection, simultaneously contact resistance R4 between its negative input and output terminal; The output terminal of operational amplifier C is connected with envelope detection circuit;
Described envelope detection circuit comprises diode D6, resistance R2, electric capacity C1 and operational amplifier D; The output terminal of operational amplifier C is connected with the negative electrode of diode D6, and the anode of diode D6 is connected with the electrode input end of operational amplifier D; Be connected between the anode of diode and ground after resistance R2 and electric capacity C1 parallel connection; The output terminal of described operational amplifier D is connected with high-pass filtering circuit;
Described high-pass filtering circuit comprises operational amplifier E, resistance R5, resistance R6, electric capacity C2 and electric capacity C3; The output terminal of operational amplifier D is connected with the electrode input end of operational amplifier E with after resistance R6 by resistance R5 successively, the electrode input end of operational amplifier E is by electric capacity C3 ground connection simultaneously, one end of electric capacity C2 is connected with the output terminal of operational amplifier E, and the other end is connected between resistance R5 and resistance R6; Contact resistance R7 between the negative input and ground of operational amplifier E, the indirect resistance R8 of its negative input and output terminal; The output terminal of operational amplifier E is connected with low-pass filter circuit;
Described low-pass filter circuit comprises resistance R9, resistance R12, electric capacity C4, electric capacity C5 and operational amplifier F; The output terminal of operational amplifier E is connected with the electrode input end of operational amplifier F with electric capacity C5 by electric capacity C4, and the electrode input end of operational amplifier F is by resistance R9 ground connection simultaneously; One end of resistance R12 is connected with the output terminal of operational amplifier F, and the other end is connected between electric capacity C4 and electric capacity C5; Contact resistance R10 between operational amplifier F negative input and ground, the indirect resistance R11 of operational amplifier F negative input and output terminal; The output terminal of operational amplifier F is connected with amplifying circuit;
Described amplifying circuit comprises operational amplifier H, resistance R13 and resistance R14; The output terminal of described operational amplifier F is connected with the electrode input end of operational amplifier H, and the negative input of operational amplifier H passes through resistance R13 ground connection, simultaneously contact resistance R14 between the output terminal and negative input of operational amplifier H.
4. a kind of micro-fluidic chip particle counting system based on electrical impedance technology according to claim 1, is characterized in that: described sine wave AC driving source frequency is 500KHz ~ 1.2MHz, and peak value is 3V ~ 5V.
5. a kind of micro-fluidic chip particle counting system based on electrical impedance technology according to claim 3, it is characterized in that: in described I/V change-over circuit, the resistance of sampling resistor is 30K Ω ~ 300K Ω, the enlargement factor of described differential amplifier U1 is less than or equal to 10 times, and described low-pass filter circuit cutoff frequency is 1/10th of sine-wave excitation frequency input signal.
6. a kind of micro-fluidic chip particle counting system based on electrical impedance technology according to claim 1, is characterized in that: the described metal needle being used as electrode is acupuncture needle or platinum pin.
7., based on the job operation of micro-fluidic chip in the micro-fluidic chip particle counting system of electrical impedance technology, it is characterized in that,
On step one, known micro-fluidic chip microchannel each several part size after, draw microchannel plane design drawing on computers;
Step 2, be worked on mask by the microchannel plane design drawing designed, the mask after processing is only transparent at channel portion;
Step 3, by negative photoresist uniform application to silicon chip upper surface, the thickness of negative photoresist is consistent with the depth value of microchannel; Then silicon chip is heated 1 minute under the constant temperature of 100 DEG C, make the negative photo adhesive curing of its upper surface;
Step 4, using the mask in step 2 as light shield, through light shield, uv-exposure 10 second is carried out to silicon chip, now ultraviolet irradiates the negative photoresist on silicon chip through the transparent part on mask, receives the negative photoresist generation cross-linking reaction of ultraviolet irradiation and is polymerized;
The developing solution dissolution that step 5, utilization are mated with the negative photoresist in step 3 falls the negative photoresist that silicon chip does not occur cross-linking reaction; Then adopt pure water rinsing silicon chip, after cleaning, dry up the pure water on silicon chip with nitrogen; The negative photo plastic structure that now silicon chip upper surface is left is the formpiston part of micro-fluidic chip, and described formpiston part refers to the spatial structure of microchannel, and its thickness is consistent with the degree of depth of microchannel;
Step 6, alkanisation process is carried out to the silicon chip that step 5 obtains;
Step 7, PDMS and rigidizer mixed with the ratio of mass ratio 10:1 and stirs, removing its air entrapment, then by the formpiston part of mixed liquid pouring on silicon chip, and cover formpiston part completely; Being heated under the constant temperature of 100 DEG C by silicon chip makes it solidify in 4 hours, then takes the PDMS layer of silicon chip upper surface off, obtains the PDMS substrate with microchannel thus;
Step 8, utilize card punch PDMS substrate upper surface mark have the position of sample inflow entrance, fluid outlet, sheath liquid flow inlet and electrode mounting hole to stamp through hole;
Step 9, PDMS substrate and glass substrate are carried out oxygen plasma surface treatment, then will accept the intimate surface laminating of plasma treatment in two substrates, the chemical bond on both surface of contact is bonded together mutually; Finally four conducting metal pins are embedded in four electrode mounting holes respectively, form micro-fluidic chip thus.
8. as claimed in claim 7 based on the job operation of micro-fluidic chip in the micro-fluidic chip particle counting system of electrical impedance technology, it is characterized in that, in described step 4, after having exposed, the silicon chip after exposure is heated 1 minute under the constant temperature of 100 DEG C.
9. as claimed in claim 7 based on the job operation of micro-fluidic chip in the micro-fluidic chip particle counting system of electrical impedance technology, it is characterized in that, in described step 9, the PDMS substrate be bonded together and glass substrate are heated 4 hours under the constant temperature of 100 DEG C.
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