CN110479391B - Low-voltage high-performance electroosmosis micropump chip based on solid-state track etching nano hole - Google Patents

Low-voltage high-performance electroosmosis micropump chip based on solid-state track etching nano hole Download PDF

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CN110479391B
CN110479391B CN201910719460.2A CN201910719460A CN110479391B CN 110479391 B CN110479391 B CN 110479391B CN 201910719460 A CN201910719460 A CN 201910719460A CN 110479391 B CN110479391 B CN 110479391B
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channel
chip
electroosmosis
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pump chip
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陈琴华
任芳玲
王册明
罗丹
冉凤英
张斌强
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Hubei University of Medicine
Sinopharm Dongfeng General Hospital
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    • B01L3/50273Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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Abstract

The invention discloses a low-voltage high-performance electroosmosis pump micro-fluidic chip based on solid-state track etching nano holes. The chip consists of a micro channel, a micro electro-osmotic pump, an electrode and an electrode reaction column. The electroosmosis pump chip is designed by selecting ultrathin nanometer porous materials, improving the arrangement position of electrodes, optimizing reaction solution and filling agarose gel, so that the electroosmosis pump has lower working voltage (1-10V) and more stable flow rate. The chip type electroosmosis pump provided by the invention can accurately control the flow speed and direction of a solution by adjusting the applied voltage, the flow speed range is 0-100 mu l/min, and the electroosmosis pump is integrated into a chip, so that the chip type electroosmosis pump has the advantages of miniaturization, easy operation, no moving part, low cost and the like, can further integrate the integration of an external channel and the separation, mixing, analysis and the like of other unit samples of a microfluidic chip laboratory, and realizes the real microfluidic chip integrated laboratory without the support of an external fluid pump.

Description

Low-voltage high-performance electroosmosis micropump chip based on solid-state track etching nano hole
Technical Field
The invention belongs to the field of micro-electromechanical systems, and relates to an electroosmosis pump microfluidic chip applied to the microfluidic field in the fields of medicine and chemical analysis, and a fluid driving system using the electroosmosis pump chip.
Background
The microfluidic chip has the advantages of precise fluid control, small sample requirement, rapid reaction and large-scale integration, so that the microfluidic chip becomes a powerful tool for clinical diagnosis and disease screening. In recent years, microfluidic chips have been widely used in nucleic acid and protein analysis, cell culture, sorting, drug screening, etc., and can provide an accurate, high-throughput, easily-integrated application platform for biomedical research, and integrated and automated microfluidic chip systems have been developed into a new research field in the interdiscipline of biomedicine, electronics, materials, fluids, etc.
The manufacture and testing of various types of micropumps, of which electroosmotic pumps operating on the electroosmotic flow principle have received increasing attention in recent years, is performed for microfluidic applications. Electroosmotic pumps have several prominent features: it can produce constant and pulse-free flow in a compact structure; the size and the direction of the flow are convenient to control; there are no moving parts. The electroosmosis pump is integrated into the chip and improved, so that the problems of complex operation, high working voltage, low flow speed and the like in the fluid driving process are solved, and the better performance is obtained.
Disclosure of Invention
The invention aims to solve the problems of complex operation, high working voltage, low flow rate, difficulty in integration with a microfluidic chip and the like in the fluid driving process of an electroosmotic pump, and discloses a low-voltage high-performance electroosmotic micropump chip based on a solid-state track etching nanopore and a preparation method thereof. Compared with the prior art, the invention has the advantages that the solid-state track etching nanopore electroosmosis pump is miniaturized, the flow rate and the long-time stability of the electroosmosis pump are obviously improved, the flow rate of the electroosmosis pump chip can reach 7 mu L/min under the low voltage of 10V, and the flow rate is stable and has no attenuation.
In order to solve the problems, the invention adopts the following technical scheme:
step one, preparing solid nano-pores: the preparation of the solid-state nanometer hole selects a track etching method, which is a preparation method based on high-energy heavy ion irradiation and chemical etching. Firstly, irradiating a high-molecular film material by using high-energy heavy ions to form a nanoscale latent track consisting of a damaged area in the high-molecular film material; and then, selectively etching the latent tracks by using a chemical etching method to obtain the pore channel structure with the nanometer size. The diameter, hole type and density of the track etching nano hole can be prepared in a highly controllable manner by adjusting the irradiation condition and the etching condition.
Step two, preparation of an electroosmosis pump chip: (a) preparation of a microchannel: preparing an electroosmosis pump chip micro-channel by using a low-cost polymethyl methacrylate (PMMA) material through the existing 3D numerical control micro-drilling technology; (b) preparation of electroosmotic pump chip: as shown in fig. 2, the electroosmotic pump chip is composed of six layers, wherein the bottom and top layer materials are Polycarbonate (PC) sheets. The fourth layer is a PMMA sheet with microchannels. The second, third and fifth layers are double-sided adhesive tapes with transparent microstructures to bond the materials. And a 3 x 3mm nano-pore film is clamped between the second double-sided adhesive tape and the third double-sided adhesive tape. 2 electrode reaction columns are bonded on the top sheet and communicated with the chip micro-channel, the column 1 is positioned at the top of the nanopore film, and the column 2 is positioned at the side channel; (c) to the electrode reaction column 2 and side channel was added 1000. mu.l of 2% agarose gel.
Step three, circuit connectivity test: TAE buffer solution (0.5-1. about. TAE) is filled in a microchannel and an electrode reaction column of the prepared electroosmosis pump chip, a platinum electrode is added to be connected with a power supply, the column 1 is connected with the anode of an external electric field, the column 2 is connected with the cathode of the external electric field, voltage 10V is set and the power supply is connected, and the current condition is observed.
In the first step, an ion track polymer film which is generated by UNILAC accelerator of 17MeV/u and bombarded by Ar ions and is generated by the UniLAC accelerator of the national institute of physics of Lanzhou of the Central academy of sciences is used, the type of the ion track polymer film is PET1238 (namely the thickness is 12 mu M, and the density is 3 x 10^8/cm2), and the polymer film is irradiated by ultraviolet rays for 3 hours and then etched in 2M 60 ℃ NaOH solution for 8.5 minutes to obtain the biconical solid nano-pore film with the outer diameter of about 200nm and the inner diameter of about 30 nm.
In the first step, the model of the polymer film for preparing the solid-state nano-pores is PET1238, and the thickness of the ultrathin nano-porous material is only 12 microns, so that a strong electric field can be formed in the nano-pores due to the small thickness of the film. The design of the biconical hole type further focuses the electric field in the central narrow area of the biconical hole.
In the second step (a), the micro-channel layout of the electroosmotic pump chip is designed in an Adobelluster, and then the PMMA sheet is cut by using a Silhauette CAMEO3 cutting plotter to obtain the product. The channel consists of a straight channel a connecting the electroosmotic pump function region, a trapezoidal side channel b filled with agarose gel, and a channel c. The straight channel a is mainly used for placing the solid-state nanopore membrane and connecting the anode and the cathode, the trapezoidal side channel b is mainly used for filling agarose gel, and the channel c is mainly used for observing liquid flow. The side channel b is designed into a trapezoid shape, so that the resistance of the side channel b can be reduced, the voltage drop loss is reduced, meanwhile, the loading part of the solid-state nano-pore film is required to be close to the side channel b as much as possible, so that the invalid channel length is reduced, the resistance of the solid-state nano-pore film is reduced, the voltage is applied to the nano-pore film more, and the electroosmotic flow is enhanced.
In the second step (b), the 3 x 3mm nanopore thin film clamped between the second and the third layers of double-sided adhesive tapes is prepared by the track etching method in the first step.
In the step two (b), the electrode reaction column is used for placing the electrode, so that the electrode can be prevented from extending into the chip and being too close to the nanopore film, and the influence of bubbles and pH value change generated by electrolytic reaction at the electrode on the performance of the electroosmosis pump is avoided.
In the second step (c), 1000. mu.l of 2% agarose gel was filled into the electrode reaction column 2 and the side channel in order to isolate bubbles generated by the electrolysis reaction and pH change.
In the third step, the prepared electroosmosis pump chip is subjected to a circuit connection test by using TAE buffer solution. TAE buffer has the effect of maintaining a suitable pH during this process. During electroosmotic flow, electrolytic reaction occurs between the positive electrode and the negative electrode, the positive electrode is subjected to oxidation reaction (4 OH-4 e- ═ 2H2O + O2), the negative electrode is subjected to reduction reaction (4H + +4e- ═ 2H2), and long-time electroosmotic flow causes the pH value of the positive electrode to be reduced and the pH value of the negative electrode to be increased. The TAE has stronger buffering capacity, and can keep the pH value of two poles of the solution basically unchanged. The TAE buffer solution has another function of making the solution have certain conductivity, so that an electric field is uniformly applied to the nanopore to generate electroosmotic flow.
In step three, the prepared electroosmotic pump chip was subjected to a circuit connection test using 1 × TAE buffer. If no current (generally less than 0.1mA), the circuit is not communicated, and the electroosmosis pump chip is abnormal; if current (generally 0.1 mA-0.2 mA) exists, the circuit is communicated, and the electroosmotic pump chip is normal.
And in the third step, performing a circuit connection test on the prepared electroosmosis pump chip by using a platinum electrode. Platinum is an inert noble metal and is not susceptible to chemical reactions with other substances.
In addition, the invention provides a preparation method of the low-voltage high-performance electroosmotic pump chip based on the solid-state nano holes, and the preparation steps are as described above.
Compared with the prior art, the invention has the following characteristics:
1. high efficiency and good stability: the electroosmosis pump chip has the advantages that the working voltage is lower, the flow speed is higher, and the flow speed is stable without obvious attenuation by using a novel ultrathin solid nano-pore material, improving the electrode arrangement position, replacing a reaction solution, filling agarose gel and the like.
2. The operation is simple, the flow rate is controllable: the electroosmosis pump chip has simple preparation method and compact functional structure, can drive the solution to flow at high speed by applying constant direct current voltage, and can regulate and control the flow rate and direction by voltage.
3. The microchip is miniaturized, and the practical applicability is strong: the electroosmotic pump chip can be integrated with an external channel and other units of a microfluidic chip to provide a real-time, rapid, accurate and high-flux application platform.
Drawings
FIG. 1 is a working principle diagram of the low-voltage high-performance electroosmosis micro-pump chip based on solid-state track etching nano-pores.
FIG. 2 is a channel structure diagram of the low-voltage high-performance electroosmosis micro-pump chip based on solid-state track etching nano-pores.
FIG. 3 is a schematic diagram of the design of the low-voltage high-performance electroosmotic micropump chip based on the solid-state track etching nanopore.
FIG. 4 shows the current conditions of the electroosmotic pump chip under different reaction solutions when the operating voltage is 50V.
FIG. 5 shows the flow rate of the electroosmotic pump chip under different reaction solutions and different voltages.
FIG. 6 shows the flow rate of the electroosmotic pump chip at different voltages when the reaction solution was 0.5 TAE.
Detailed Description
The invention is further illustrated by the following examples, which are intended to provide a better understanding of the invention, but which are not intended to limit the scope of the invention:
the electroosmotic pump microfluidic chip system was constructed as shown in the steps of FIG. 2.
Step one, preparing solid nano-pores: the preparation of the solid-state nanometer hole selects a track etching method, which is a preparation method based on high-energy heavy ion irradiation and chemical etching. Firstly, irradiating a high-molecular film material by using high-energy heavy ions to form a nanoscale latent track consisting of a damaged area in the high-molecular film material; and then, selectively etching the latent tracks by using a chemical etching method to obtain the pore channel structure with the nanometer size. The diameter, hole type and density of the track etching nano hole can be prepared in a highly controllable manner by adjusting the irradiation condition and the etching condition.
The ion-track polymer film after Ar ion bombardment of 17MeV/u produced by UNILAC accelerator of the national institute of Physics of Lanzhou, the academy of sciences, was used as described above, and its model number was PET1238 (i.e., thickness of 12 μm and density of 3 x 10^8/cm2), and the polymer film was cut into a number of 2 cm-diameter wafers and irradiated in a CL-1000 UV crosslinking apparatus (power: 2mW/cm2, duration: 3 hours for each of the positive and negative sides). And then, filling 30ml of 2M NaOH solution into a conical flask, putting the conical flask into a 62 ℃ water bath kettle, heating until the temperature of the NaOH solution is constant at 60 ℃, soaking the ultraviolet irradiated polymer film into NaOH for etching, taking out the polymer film after 8.5 minutes, and washing the polymer film with deionized water to obtain the biconical solid nano-pore film with the outer diameter of about 200nm and the inner diameter of about 30 nm.
Step two, preparation of an electroosmosis pump chip: (a) preparation of a microchannel: preparing an electroosmosis pump chip micro-channel by using a low-cost polymethyl methacrylate (PMMA) material through the existing 3D numerical control micro-drilling technology; (b) preparation of electroosmotic pump chip: as shown in fig. 2, the electroosmotic pump chip is composed of six layers, wherein the bottom and top layer materials are Polycarbonate (PC) sheets. The fourth layer is a PMMA sheet with microchannels. The second, third and fifth layers are double-sided adhesive tapes with transparent microstructures to bond the materials. And a 3 x 3mm nano-pore film is clamped between the second double-sided adhesive tape and the third double-sided adhesive tape. 2 electrode reaction columns are bonded on the top sheet and communicated with the chip micro-channel, the column 1 is positioned at the top of the nanopore film, and the column 2 is positioned at the side channel; (c) to the electrode reaction column 2 and side channel was added 1000. mu.l of 2% agarose gel.
The electroosmotic pump chip microchannel layout is designed in Adobe Illustrator, and then cut into PMMA sheet by using Silhouette CAMEO3 cutting plotter. The channel consists of a straight channel a connecting the electroosmotic pump function region, a trapezoidal side channel b filled with agarose gel, and a channel c.
And a 3 x 3mm biconical solid nano-pore film with the outer diameter of about 200nm and the inner diameter of about 30nm, which is prepared by using a track etching method, is clamped between the second double-sided adhesive tape and the third double-sided adhesive tape.
The 2% agarose gel should be placed into the oven after preparation, then quickly take 1000 u l to fill in the side channel and the electrode reaction column 2, check the filling is tight and bubble-free, then put the chip into the refrigerator to make the gel solidify, 5min later take out, in the column 2 filled with TAE to moisten the gel, prevent shrinking. The 2% agarose gel should be ready for use.
Step three, circuit connectivity test: the prepared electroosmotic pump chip was subjected to a connectivity test using 1 × TAE buffer. If no current (generally less than 0.1mA), the circuit is not communicated, and the electroosmosis pump chip is abnormal; if current (generally 0.1 mA-0.2 mA) exists, the circuit is communicated, and the electroosmotic pump chip is normal.
Example 1 at a working voltage of 50V, the current test of the electroosmotic pump chip under different reaction solutions was carried out as follows:
(1) adding 0.1 × TAE (total volume of about 2 ml) into the electrode reaction columns 1 and 2 and the chip straight channel (a) by using a Pasteur pipette, sealing the sample inlet by using a sealing anti-collision paste, and avoiding the generation of bubbles in the operation process as much as possible; platinum electrodes are inserted into the electrode reaction columns 1 and 2, a power supply and an ammeter are connected, the voltage is set to be 50V, and timing is started after the circuit is connected. The current was recorded every two minutes for one hour. During the experiment, the condition of liquid leakage of the electroosmosis pump chip is noticed.
(2) After the recording is completed, the voltage is turned off, the electrodes are taken out, the solution in the chip is emptied, and the electroosmotic pump chip is cleaned by using the TAE before the next TAE test is carried out.
Repeating the steps (1) and (2), and testing the current stability of the three TAE concentrations at 50V under the conditions of 0.1 TAE, 0.3 TAE, 0.5 TAE, 0.8 TAE and 1.0 TAE.
As shown in fig. 4, at 50V, when the reaction solution was 0.1 × TAE and 0.3 × TAE, the generated current was small, about 0.43mA and 0.48mA, respectively, and the current tended to decrease with time, and the current decreased more significantly with the lower concentration of TAE; when the reaction solution was 0.8 × TAE and 1.0 × TAE, the generated currents were large, about 0.87mA and 1.16mA, respectively, and increased with time; when the reaction solution was 0.5 × TAE, the current generated was centered at about 0.57mA, and was stable with no significant change over time. Therefore, it is assumed that the current condition of the electroosmotic pump chip under the 0.5 × TAE solution is stable, and the 0.5 × TAE solution can be selected as the optimal reaction solution of the electroosmotic pump chip system.
EXAMPLE 2 flow Rate testing of electroosmotic Pump chips at different reaction solutions and different voltages
The specific experimental procedures are as follows:
(1) adding 0.1 TAE (total volume of acetic acid) of about 2ml into the electrode reaction columns 1 and 2 and the front section of the straight channel (a) by using a Pasteur pipette, sealing a sample inlet by using a sealing anti-collision paste, and avoiding bubbles in the operation process as much as possible; using a pipette to suck about 300 mu L of diluted red ink to be added into the rear section of the straight channel (a), and using a sealing anti-collision paste to seal the sample inlet, wherein the operation process avoids generating bubbles as much as possible; platinum electrodes were inserted into the electrode reaction columns 1 and 2, a power supply and an ammeter were connected, a voltage was set to 20V, and the timing was started after the circuit was connected. Recording the advancing distance of the red ink in the channel (c) every two minutes; setting the voltage to 50V for recording after 10 minutes; after 10 minutes, the voltage was set to 100V for recording. During the experiment, the condition of liquid leakage of the electroosmosis pump chip is noticed.
(2) After the recording is completed, the voltage is turned off, the electrodes are taken out, the solution in the chip is emptied, and the electroosmotic pump chip is cleaned by using the TAE before the next TAE test is carried out.
Repeating the steps (1) and (2), and testing the flow rate of five different TAEs of 0.1 TAE, 0.3 TAE, 0.5 TAE, 0.8 TAE and 1.0 TAE at different voltages.
As shown in fig. 5, at different concentrations of TAE, the flow rate increases with increasing voltage, and the flow rate and the voltage have a linear relationship; compared with other concentrations, the electroosmotic pump chip generates the maximum flow rate in a 0.5 TAE solution system under three different voltage sets, namely 120.6 muL/min, 48.6 muL/min and 16.9 muL/min, and the flow rate is increased obviously along with the increase of the voltage. Therefore, it can be seen that the electroosmotic pump chip can generate higher flow rate at lower voltage, and the flow rate at 0.5 × TAE solution is the largest, and 0.5 × TAE solution can be selected as the optimal reaction solution of the electroosmotic pump chip system.
Example 30.5 TAE flow rate stability test at different voltages
The specific experimental procedures are as follows:
(1) adding 0.5 TAE (total volume of acetic acid) of about 2ml into the electrode reaction columns 1 and 2 and the front section of the straight channel (a) by using a Pasteur pipette, sealing a sample inlet by using a sealing anti-collision paste, and avoiding bubbles in the operation process as much as possible; using a pipette to suck about 300 mu L of diluted red ink to be added into the rear section of the straight channel (a), and using a sealing anti-collision paste to seal the sample inlet, wherein the operation process avoids generating bubbles as much as possible; platinum electrodes are inserted into the electrode reaction columns 1 and 2, a power supply and an ammeter are connected, the voltage is set to 10V, and timing is started after the circuit is connected. The distance of travel of the red ink in channel (c) was recorded every two minutes until the entire channel was filled. During the experiment, the condition of liquid leakage of the electroosmosis pump chip is noticed.
(2) After the recording was completed, the voltage was turned off, the electrodes were removed, the chip was emptied of solution, and the electroosmotic pump chip was cleaned with 0.5 TAE before the next voltage test was performed.
And (3) repeating the steps (1) and (2), and testing the flow rate stability at 10V, 20V, 30V and 50V respectively.
As shown in FIG. 6, when the reaction solution was 0.5 × TAE, the electroosmotic pump chip produced flow rates of about 37.5 μ L/min, 19.3 μ L/min, 13.0 μ L/min, and 7.1 μ L/min at 10V, 20V, 30V, and 50V, respectively, and the flow rates were stable without significant attenuation at different voltages. Therefore, the electroosmotic pump chip can generate higher flow rate under lower voltage and has stable performance.

Claims (3)

1. A low-voltage high-performance electroosmosis pump chip based on solid-state nano-pores is characterized in that the preparation method comprises the following steps:
step one, preparing solid nano-pores: the preparation of the solid-state nanopore uses a track etching method, high-energy heavy ions are used for irradiating a polymer film material, and a nanoscale latent track consisting of damaged areas is formed in the polymer film material; then, selectively etching the latent track by using a chemical etching method to obtain a pore channel structure with a nano size; the diameter, hole type and density of the track etching nanometer hole can be controllably prepared by adjusting the irradiation condition and the etching condition;
step two, preparation of an electroosmosis pump chip: (a) preparation of a microchannel: preparing an electroosmosis pump chip micro-channel by using a polymethyl methacrylate (PMMA) material through a 3D numerical control micro-drilling technology; (b) preparation of electroosmotic pump chip: the electroosmotic pump chip consists of six layers, wherein the bottom layer and the top layer are made of Polycarbonate (PC) sheets; the fourth layer is a PMMA sheet with a micro channel; the second, third and fifth layers are double-sided adhesive tapes with transparent microstructures and are made of adhesive materials; a 3 mm-3 mm nano-pore film is clamped between the second layer of double-sided adhesive tape and the third layer of double-sided adhesive tape; 2 electrode reaction columns are bonded on the top sheet and communicated with the chip micro-channel, the column 1 is positioned at the top of the nanopore film, and the column 2 is positioned at the side channel; (c) adding 1000 μ L of 2% agarose gel solution into the electrode reaction column 2 and the side channel;
step three, circuit connectivity test: filling 0.5-1 TAE buffer solution in a microchannel and an electrode reaction column of the prepared electroosmosis pump chip, adding a platinum electrode to be connected with a power supply, connecting a column 1 with the anode of an external electric field, connecting a column 2 with the cathode of the external electric field, setting a voltage of 10V and connecting the power supply, and observing the current condition;
in the first step, 17MeV/u of Ar ion bombardment ion track polymer film produced by UNILAC accelerator from the national institute of physics, Lanzhou, the Chinese academy of sciences, model No. PET1238, thickness 12 μm, density 3 x 108/cm2After the polymer film is irradiated by ultraviolet rays for 3 hours, the polymer film is etched in 2M 60 ℃ NaOH solution for 8.5 minutes to obtain a biconical solid nano-pore film with the outer diameter of 200nm and the inner diameter of 30nm, the thickness of the film is small, and a strong electric field can be formed in the nano-pores; the design of the biconical hole type focuses an electric field in a central narrow area of the biconical hole;
in the step two (b), the electrode reaction column is used for placing the electrode, so that the electrode can be prevented from extending into the chip and being too close to the nanopore film, and the influence of bubbles and pH value change generated by electrolytic reaction at the electrode on the performance of the electroosmosis pump is avoided.
2. The solid-state nanopore based low voltage high performance electroosmotic pump chip of claim 1, wherein in step two (a), the electroosmotic pump chip microchannel layout is designed in Adobe Illustrator, and then the PMMA sheet is cut using a silouette CAMEO3 cutting plotter; the channel consists of a straight channel (a) connected with the electroosmosis pump functional area, a trapezoidal side channel (b) filled with agarose gel and a channel (c); the straight channel (a) is used for placing the solid nanopore membrane and connecting the anode and the cathode, the trapezoidal side channel (b) is used for filling agarose gel, and the channel (c) is used for observing liquid flow; the trapezoidal side channel (b) is designed into a trapezoid, so that the resistance of the channel can be reduced, and the voltage drop loss is reduced; meanwhile, the loading part of the solid-state nano-pore film is close to the trapezoidal side channel (b) to reduce the length of an invalid channel, so that the resistance of the channel is reduced, voltage is applied to the nano-pore film, and electroosmotic flow is enhanced.
3. The solid-state nanopore based low voltage high performance electroosmotic pump chip of claim 1, wherein in step three, a circuit continuity test is performed on the prepared electroosmotic pump chip using 1 × TAE buffer; if no current exists, generally less than 0.1mA, the circuit is not communicated, and the electroosmosis pump chip is abnormal; if the current is 0.1 mA-0.2 mA, the circuit is connected and the electroosmosis pump chip is normal.
CN201910719460.2A 2019-08-05 2019-08-05 Low-voltage high-performance electroosmosis micropump chip based on solid-state track etching nano hole Expired - Fee Related CN110479391B (en)

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