WO2014036914A1

WO2014036914A1 - Method and device for controlling, based on electrophoresis, charged particles in liquid

Info

Publication number: WO2014036914A1
Application number: PCT/CN2013/082765
Authority: WO
Inventors: 吴传勇
Original assignee: 上海衡芯生物科技有限公司
Priority date: 2012-09-04
Filing date: 2013-09-02
Publication date: 2014-03-13
Also published as: CN102879453A; CN102879453B

Abstract

A method and a device for controlling, based on electrophoresis, charged particles in liquid. The method comprises: applying a voltage to two electrodes of a microflow device respectively to form a first voltage difference, changing the voltages on the two electrodes to form a second voltage difference after a first time duration, and continuing a second time duration so as to differently move the charged particles in a liquid drop to be measured, thereby enabling the particles to be separated, at least part of the charged particles in the liquid drop to be measured being capable of moving by means of the amplitude of at least one of the first voltage difference and the second voltage difference. By means of effects such as electrowetting, electrophoresis and the like, the operation on a liquid drop can be implemented, and in addition, charged particles suspended in the liquid drop, especially different particles with the same charge can be controlled.

Description

Method and device for controlling charged particles in liquid based on electrophoresis

Field of the Invention This invention relates to the field of microfluidic devices, and more particularly to a method and apparatus for manipulating charged particles in a liquid based on electrophoresis. Background technique

In recent years, microfluidic devices, also known as Lab-on-a-Chip and Micro Total Analysis Systems, have low sample usage, fast detection speed, and low experimental cost. The advantages of easy automation, high detection repetition rate, and good data quality have attracted attention from various industries.

The traditional liquid handling requires a large amount of sample, many steps and cumbersome, and the digital microfluidic device based on electrowetting-on-dielectric on the medium can not only control the liquid as independent control unit. Operation, thereby greatly increasing the ability to perform parallel processing and parallel detection on multiple samples; Moreover, by controlling the electrodes contained in the device, it is also possible to automate extremely small amounts of liquid, such as droplet movement, merging , splitting, incubation (incubation ^ mixing, reaction, waste collection, etc. Since there are no (and not required) moving parts on the digital microfluidic device, the stability and reliability of the device and handling are greatly improved.

The inventors of the present application have proposed a microfluidic device having a multilayer control electrode structure in the document of the patent No. WO 2008/147568, which not only makes a general-purpose microfluidic device possible, but also produces a low-cost, high-quality microfluid. In terms of devices, it is also a leap; in addition, it greatly simplifies the microfluidic control process. However, this patent document (W0 2008/147568) mainly deals with the operation of droplets, but the control of the particles contained in the droplets is not mentioned, but the control of the particles in the liquid, especially the control of charged particles, Sample preparation and biochemical analysis are important.

Electrophoresis is an important means in biochemical analysis. It refers to the effect of charged particles in a liquid (or gel) moving under the action of a uniform electric field. Electrophoretic effects can be effectively used for the separation of substances including DNA, proteins, cells, etc. due to the different migration speeds of different constituents in the suspension in liquid (or colloid) under electric field. It can also be used for material molecules. Analysis of the structure. For example, in a normal pH solution, the cells are usually negatively charged and thus move toward the positive electrode; typically red blood cells move at a rate of about 1 micron per second with an electric field of 1 V/cm (1 volt per centimeter). ). Electrophoresis can be achieved naturally in the microfluidics of the pipeline. The document numbered W0 2007/032789 describes the manner in which immunoassays are performed by electrophoresis in a microfluidic conduit. Summary of the invention SUMMARY OF THE INVENTION A primary object of the present invention is to provide a method and microfluidic device that can operate and detect charged particles in a liquid.

To achieve the above and other objects, the present invention provides a method of manipulating charged particles in a liquid based on electrophoresis, the method being applied to a microfluidic device having at least two electrodes, the method comprising at least the steps of:

Applying a voltage across the two electrodes to form a first voltage difference, and after a first duration, changing the voltage across the two electrodes to form a second voltage difference for a second duration, The charged particles in the droplet to be tested are subjected to different displacements for separation of the particles; wherein, the amplitude of at least one of the first voltage difference and the second voltage difference enables at least a portion of the charged particles in the droplet to be tested to move.

Preferably, step a is repeated a plurality of times, and at least one of the charged particles continues to move in the direction of one electrode in the droplet, eventually remaining at a position adjacent to the electrode in the droplet.

Preferably, the first voltage difference is opposite to the polarity of the second voltage difference.

Preferably, one of the first duration and the second duration is longer than the other.

The microfluidic device for controlling charged particles in a liquid based on electrophoresis provided by the invention includes at least:

a first substrate and a second substrate;

a first electrode structure layer disposed on the first substrate, a second electrode structure layer disposed on the surface of the first electrode structure layer, a third electrode structure layer disposed on the second substrate, and on the first substrate The electrode structure layer is disposed opposite to the electrode structure layer on the second substrate so as to have a space for accommodating the liquid therebetween;

Wherein, in the second electrode structure layer, the width of the two electrophoretic electrodes ranges from 1 micrometer to 1 millimeter, the pitch ranges from 10 micrometers to 20 millimeters, and the width range and spacing of other electrodes are in the range of 100 micrometers. Between 20 mm.

The invention provides a method for manipulating charged particles in a liquid based on electrophoresis, which comprises at least the steps of:

Applying a voltage of opposite polarity and an amplitude such that the charged particles move on the two electrophoretic electrodes of the aforementioned microfluidic device, so that the charged particles in the droplet to be tested move toward the electrophoretic electrode whose polarity is opposite to the polarity of the self charge. .

As can be seen from the above, the present invention proposes a method for manipulating charged particles (especially different particles with the same charge) in a droplet based on an electrophoretic effect; and also proposes a use and patent proposed in WO 2008/147568 A multi-layered control electrode is similar in structure to a digital microfluidic device. On the basis of not being limited by theory, electrophoresis is mainly used to manipulate charged particles in a liquid medium, and redistribution or separation of charged particles in a liquid medium can be realized. In combination with the prior patents of the present inventors (TO 2008/147568, W0 2009/003184, and PCT/CN2012/070594), the digital microfluidic device of the present invention has a more complete function, and many liquid sample operations can be realized, for example. Droplet generation, movement, merging, mixing, separation, position and size measurement, incubation, heat treatment, etc., and for further convenience The treatment process, the charged particles in the liquid sample can also be redistributed or separated. The present invention enables the separation and identification of biomarkers (such as antibodies or other proteins, DNA or RNA, etc.), viruses, in complex liquid samples (eg, blood, serum, plasma, sweat, saliva, urine, etc.) using a digital microfluidic system. Bacteria and cells are possible. DRAWINGS

1 is a flow chart of a method for manipulating charged particles in a liquid based on electrophoresis according to the present invention;

2A is a schematic cross-sectional view of a digitized microfluidic device for controlling charged particles in a liquid based on electrophoresis according to the present invention; FIG. 2B is a three-dimensional view of the microfluidic device shown in FIG. 2A;

2C to 2G show a flow diagram of redistribution of two differently charged particles in a droplet under electrophoretic effect, and splitting the droplet into two using an electrowetting effect;

Fig. 3 is a flow chart showing the process of extracting DNA from a whole blood sample on a digital microfluidic device of the present invention and performing real-time PCR reaction on the device. All of the steps, including sample preparation, sample handling (such as heating, mixing, and moving), and signal measurements, are all implemented on the device. detailed description

The embodiments of the present invention are described below by way of specific specific examples, and those skilled in the art can readily understand other advantages and effects of the present invention from the disclosure of the present disclosure. The invention may be practiced or applied in various other specific embodiments, and the details of the invention may be variously modified or changed without departing from the spirit and scope of the invention.

Please refer to Figure 1 to Figure 3. It should be noted that the illustrations provided in this embodiment merely illustrate the basic concept of the present invention in a schematic manner, and only the components related to the present invention are shown in the drawings, instead of the number and shape of components in actual implementation. Dimensional drawing, the actual type of implementation of each component's type, number and proportion can be a random change, and its component layout can be more complicated.

Some terms are explained below:

In the present invention, the term "particle" is used to mean an entity of the order of micrometers or nanometers, which may be natural or artificial, such as cells, subcellular components, viruses, liposomes (l ipo) _SO me ), nanospheres, and microspheres, or smaller entities such as biomacromolecules, proteins, DNA, and RNA, which may also refer to droplets that are not fused to the suspension medium, and may also refer to liquids. Small bubbles, etc. The (linear) size of "particles" can range from a few nanometers to a few hundred microns.

The term "electrowetting" is used to mean that the contact angle of a liquid with a solid surface varies with the applied electric field. Effect. It should be noted that when the applied voltage or electric field is AC, the "electrowetting" effect and the "dielectrophoresis" effect exist simultaneously. When the frequency of the voltage or electric field increases, the relative specific gravity of the "dielectrophoresis" effect is correspondingly enhanced. . The "electrowetting" effect and the "dielectrophoresis" effect are not strictly distinguished in the present invention.

It is a primary object of the present invention to achieve methods and devices that can operate and detect charged particles in a liquid reagent. The term "manipulation" can include one or more combinations of the following steps:

1. Selection - Separation of one of the particles containing a plurality of particles.

2, reordering - rearrange the spatial position of the particles.

3. Union - Move two or more particles spatially to a similar or identical location (sometimes a particle can contain another particle).

4. Separation - Separation of particles that are originally in contact with each other, separated by a certain distance, or evenly distributed in the medium.

5. Trapping or focusing - Move particles to a specified location and control those particles at that location for a certain period of time.

As another specific implementation of the present invention, electrophoresis utilizes a uniform electric field to generate force on the particles, and the charged particles are

(One, several, or groups) Move to the lowest potential. In the design of the present invention, the positively charged particles move toward the negative potential electrode, and the negatively charged particles move toward the positive potential electrode.

For the purposes of this disclosure, the term "microfluidic" refers to at least one dimension

(dimension) A device or system that operates on liquids ranging from a few to a few hundred microns.

For the purposes of the present disclosure, the term "droplet" refers to a liquid, or other surface, or other solid (usually referred to as non-fused) liquid, or solid surface (eg, the inner surface of a digitized microfluidic device). ) A certain amount of liquid (one or several kinds of mixtures) separated. The volume of "droplets" is very large: generally from a few liters

( femtol iter, femtoliter) to a few hundred microliters (microl iters). The "droplet" may have any shape such as a sphere, a hemisphere, a flat circle, an irregular shape, or the like.

The present invention provides devices, methods, and systems for detecting analytes in a sample solution. Those skilled in the art are aware that examples of non-limiting sample solutions are body fluids (including, but not limited to, blood, serum, plasma, saliva, urine, etc.); purified samples (eg, purified) DNA, RNA, protein, etc.; environmental samples (including, but not limited to, water, air, agricultural related samples, etc.); biological warfare agent samples

( biological warfare agent sample ) and so on. The body fluid may be a body fluid of any organism, but the present invention is more interested in the body fluids of mammals, especially humans. For the purposes of the present disclosure, the term "analyte" refers to a substance or chemical component to be tested in an assay or test. The "analyte" can be an organic or inorganic substance. It can refer to biomolecules (such as proteins, lipids, cytokines, hormones, carbohydrates, etc.), viruses (such as herpesviruses, retroviruses, adenoviruses, lentiviruses), intact cells (including prokaryotic and eukaryotic cells), Environmental pollutants (including toxins, pesticides, etc.), drug molecules (such as antibiotics, therapeutic drugs and drug abuse, and drugs), nucleus, spores, and so on.

For the purposes of the present disclosure, the term "reagent" refers to reaction with a sample material, dilution of a sample material, mediation of a sample material, suspension of sample material, emulsification of sample material, encapsulation of sample material, and sample material. Any material that interacts, or is added to the sample material.

For the purposes of the present disclosure, the term "biomarker" refers to a substance that can be used to mark a disease state, a physiological state of a living organism, and a response of the body to a certain therapy. Non-limiting, the biomarker can be, but is not limited to, a certain protein in the blood (whose concentration reflects whether the organism has a certain disease, and the severity of the disease), DNA sequence, introduced into the organism for examination A substance that can track the measurement of an organ function or certain health indicators of the organism.

For the purposes of this disclosure, "amplification" refers to the process by which the amount or concentration of an analyte to be tested can be increased. Non-limiting examples include polymerase chain reaction (PCR) and variants thereof (eg, quantitative competitive PCR, immuno-PCR, reverse transcription PCR, etc.), Strand Displacement Amplification (SDA), based on nucleic acid sequences Nucleic Acid Sequence Based amplification or NASBA, Loop-mediated isothermal amplification or LAMP, and Helic-case adependent characterization (HAD).

For the purposes of the present disclosure, the terms "layer" and "film" are used interchangeably to refer to the structure of the body, which typically, but not necessarily, is planar or substantially planar, and is typically deposited , formed, coated or otherwise placed on another structure.

For the purposes of this disclosure, "electron selector" refers to any electronic device capable of setting an output electrical signal or changing it to a different voltage (or current) level, with or without intermediate electronics. . As a non-limiting example, a microprocessor, along with some driver chips, can be used to set different electrodes at different potentials at different times.

For the purposes of this disclosure, the term "ground" (such as for "ground electrode" or "ground voltage") means that the voltage of the corresponding electrode is zero or close enough to zero. All other voltage values, although typically less than 300 volts in amplitude, should be high enough to allow for adequate observation of electrophoresis, dielectrophoresis, and electrowetting effects. It should be noted that when the covered dielectric layer is disposed, the space between adjacent electrodes in the same layer is typically filled with the dielectric material. These spaces can also be left empty or filled with gases such as air, nitrogen, helium and argon. All of the electrodes in the same layer and the electrodes at the different layers are preferably electrically isolated.

For the purposes of the present disclosure, the term "communicate" (eg, the first component is "connected" to the second component or the first component is "connected to" the second component) refers to two or more components or components. Structural, functional, mechanical, electrical, optical, or fluid relationship, or any combination thereof. As such, the fact that one component is referred to as being in communication with the second component is not intended to exclude the presence of additional components and/or additional components between the first or second component that are operatively associated or coupled to the first or second component. The possibility.

For the purposes of this disclosure, it will be understood that when any form (eg, a droplet or a continuum, which may be moving or stationary), the liquid is described as being "on", "on" or "on" an electrode, array, matrix or surface. Upon "above", the liquid may be in direct contact with the electrode/array/matrix/surface, or may be in contact with one or more layers or membranes interposed between the liquid and the electrode/array/matrix/surface.

For the purposes of the present disclosure, it will be understood that when a given component such as a layer, region or substrate is referred to as being "positioned" or "in" or "in" It may be located directly on the other component, or alternatively, an intermediate component (eg, one or more buffer layers, interlayers, electrodes, or contacts) may also be present. It is also understood that the terms "on" and "on" can be used interchangeably to describe how a given component can be positioned or positioned relative to another component. Thus, the terms "placed on" and "formed on" are not intended to introduce any limitation as to the particular method of material transport, deposition or manufacture.

For the purposes of this disclosure, the terms "detection" and "measurement" are used interchangeably to derive physical quantities (eg, position, charge, temperature, concentration, pH, brightness, fluorescence, etc.) process. Under normal circumstances, at least one sensor (or detector) is used to acquire physical quantities and convert them to signals or information that can be recognized by adults or instruments. There may be other components between the object to be tested and the sensor, such as lenses, mirrors, filters, etc. used in optical measurements, and resistors, capacitors, transistors, etc. in electrical measurements. Moreover, in order to make the measurement possible or easier, other auxiliary devices or devices are often used in the measurement. For example, a light source such as a laser or a laser diode is used to excite particles from an electronic ground state to an electronically excited state, and the excited state particles sometimes emit fluorescence when they return to the ground state, and the fluorescence intensity measured here can be used to measure a liquid sample. The concentration of a certain particle. The sensor has CCD, photodiode, photomultiplier tube, etc. on the optical side, and has an operational amplifier, an analog-to-digital converter, a thermocouple, a thermistor, etc. in terms of electrical power.

Measurements can be performed on multiple parameters of multiple samples simultaneously or in a certain order. For example, while measuring the fluorescence of a certain particle in a droplet with a photodiode, the position of the droplet can also be obtained simultaneously by capacitance measurement. Sensor or detector It is usually connected to a computer, which is usually equipped with software to analyze the measured signal and usually convert it into information that can be read by adults or other instruments. For example, the measurement and analysis of the fluorescence intensity of a particle in a liquid can be used to infer the concentration of the particle.

For the purposes of the present disclosure, the term "extended electrode" has a length at least three times its width; preferably, the length is at least 5 times its width; more preferably, the length is at least 10 times its width.

As a non-limiting example, optical measurements include laser induced fluorescence measurement, infrared spectroscopy, Raman spectroscopy, chemi luminescence measurement, surface plasmon resonance Measurement (surface plasmon resonance measurement), absorption spectroscopy, etc.; electrical measurements include amperometry, voltammetry, photoelectrochemistry, coulometry, Capacitance measurement, and AC impedance measurement.

The following is a detailed description of the manipulation method and microfluidic device of the present invention. For convenience of explanation, the corresponding drawings (Figs. 1 to 3) will be mentioned as needed. It should be noted that the purpose of these examples is to help explain, not to limit the will and spirit of the invention.

Please refer to FIG. 1, which is a flow chart of a method for controlling charged particles in a liquid based on electrophoresis according to the present invention. Among them, the method of the present invention can be applied to a microfluidic device comprising at least two electrodes.

Preferably, the two electrodes have a width ranging from 1 micrometer to 1 millimeter and a pitch ranging from 10 micrometers to 20 millimeters.

In step S101, voltages are respectively applied to the two electrodes to form a first voltage difference, and after the first duration, the voltages on the two electrodes are changed to form a second voltage difference, and the first For two durations, the charged particles in the droplet to be tested are displaced differently to separate the particles; wherein, the amplitude of at least one of the first voltage difference and the second voltage difference enables at least a portion of the charged particles in the droplet to be tested mobile.

Among them, the types of particles that the droplet to be tested may contain may include the following situations:

The first case: The droplet to be tested may contain two kinds of particles, one of which has a positive charge and the other of which has a negative charge;

The second case: The droplet to be tested may contain two or more kinds of particles, and each of the particles has the same type of charge, for example, both positively or negatively charged;

Third case: The droplet to be tested may contain three or more particles, and at least one of the particles is positively charged, one particle is negatively charged, and the remaining species are either positively or negatively charged. When the particles to be tested may contain particles in the first case, if the first voltage difference and the second voltage difference have the same polarity, for example, both positive values or negative values, the liquid droplets to be tested are The direction of displacement of the positively charged particles is the same as the direction of the electric field between the two electrodes, and the direction of the displacement of the negatively charged particles is opposite to the direction of the electric field. Therefore, the particles in the droplet to be measured are displaced in different directions; if the first voltage difference and the first If the two voltage differences are the same but the polarities are opposite, the direction of the electric field between the two electrodes corresponding to the elder in the first duration and the second duration determines the direction of displacement of the particles in the droplet to be tested, for example, If the duration is longer than the second duration, the electric field direction of the two electrodes corresponding to the first duration is the displacement direction of the positively charged particles, and the opposite direction of the electric field is the displacement direction of the negatively charged particles, so the droplet to be tested The particles in the middle also produce displacements in different directions.

When the particles to be tested may contain particles in the third case, the corresponding voltage may be applied to the two electrodes in the manner described in the first case above to move the positively charged particles and remain in the droplet to be tested. Near the position of one electrode, the negatively charged particles move and stay at the position of the liquid to be tested adjacent to the other electrode; then apply a corresponding voltage on the two electrodes to separate the droplet to be tested into two based on the electrowetting effect. If there are a plurality of particles in the sub-droplet, the types of charges of the plurality of particles are the same, that is, the second case.

The following is a detailed description of the second case where the particles to be tested may be included: When the droplet to be tested contains different particles with the same charge, the liquid to be tested after the first duration and the second duration The total displacement direction of the different particles of the same charge in the drop is determined based on the first duration, the second duration, the first voltage difference, and the second voltage difference.

For example, when the droplet D1 to be tested may contain the particles a1 and the particles bl1 which are both positively charged, a voltage is respectively applied to the electrodes El1 and E12 in the first duration ti1 to form a first voltage difference Ul1. Changing the voltages on the electrodes El1, E12 to form a second voltage difference U12 during the second duration t12, wherein the first voltage difference U11 and the second voltage difference U12 both move the charged particles, and the two are the same size On the contrary, the first duration ti l is less than the second duration t12, and after the first duration ti l and the second duration t12, the total displacement directions of the positively charged particles al l and bl l are all oriented. In the second duration t12, the polarity of the voltage is opposite to the polarity of the positively charged particles a1, bl1, and since the mass-to-charge ratios of the particles al l and bl l are different, the particles al l, bl l The displacement of the medium to large charge-to-charge ratio is greater than the displacement of the larger mass-to-charge ratio.

Therefore, based on the above description, those skilled in the art should understand that, after selecting the appropriate first duration length, second duration duration, first voltage difference, and second voltage difference, the liquid to be tested after the second duration The displacement of at least one of the droplets in the droplet should be greater than (preferably much greater than) the displacement of the other particles of the droplet to be tested that are of the same type as the charge of the species; more preferably, after the second duration, At least one of the particles in the droplet is measured to have a significant displacement, while the other species in the droplet to be tested remain substantially in place, or the displacement is negligible. Wherein the frequency of the voltage applied across the electrodes is typically less than 10,000 Hz, preferably less than 100 Hz; more preferably, the frequency is less than 1 Hz.

Based on the above description, if step S101 is repeated a plurality of times, at least one charged particle in the droplet to be tested moves toward one electrode direction after each step S101 is performed, and finally stays in the droplet to be tested adjacent to the electrode. At the position, the distribution of particles in the droplets can be changed, which facilitates subsequent processing or measurement of the particles.

For example, following the above-mentioned liquid droplet D1 to be tested, after each step S101 is performed, the mass-to-charge ratios of the particles al l and bl l have a larger orientation toward the electrode E11 and the mass-to-charge ratio of the particles al l and bl l . The larger one is basically kept in place or the displacement is negligible. After the step S101 is performed multiple times, the mass-to-charge ratio of the particles al l and bl l is moved and staying at the position of the adjacent electrode E11 in the liquid droplet D1 to be tested. Then, in the droplet D1 to be measured, the concentration of particles having a small mass-to-charge ratio at a position adjacent to the electrode E11 is remarkably increased. Subsequently, if a voltage is applied to the electrodes El l, E12 so that the droplet D1 to be measured is separated into two sub-droplets based on the electrowetting effect, particles having a small mass-to-charge ratio are concentrated in the sub-droplets adjacent to the electrode E11.

Preferably, if a positive voltage is applied to one electrode and a negative voltage is applied to the other electrode before the step S101 is performed, the charged particles in the droplet to be tested are moved and finally retained in the droplet. At the position of the electrode with the opposite polarity of charge; then, the operation of step S101 is started again, so that at least one of the droplets to be tested is away from the position of the droplet, and the position of the droplet adjacent to the other electrode Moved and eventually stayed at that location.

For example, following the aforementioned droplet D1 to be tested, if a positive voltage is applied to the electrode E11 and a negative voltage is applied to the electrode E12 before the step S101 is performed, the positively charged particles a1 in the droplet D1 to be tested are Bl l is moved to the position of the droplet D1 adjacent to the electrode E12 and finally stays at the position; subsequently, the operation of the step S101 is performed, and the particle with a small mass-to-charge ratio in the particle a K bl l is moved and finally stays in the waiting The position of the droplet D1 adjacent to the electrode El l is measured, and the particle having a large mass-to-charge ratio is still retained at the position of the adjacent electrode E12 in the droplet D1 to be tested, thereby realizing the same kind of charged particles in the same droplet. Separation. Then, a voltage is applied to the electrodes El l and E12 so that the droplet D1 to be measured is separated into two sub-droplets based on the electrowetting effect, so as to detect the particles al l and bl l , respectively.

Further, moving the droplet to be tested to a desired position by applying a voltage on the electrode included in the microfluidic device (for example, at a liquid outlet, at a position overlapping an electrode performing the foregoing operation of step S101, etc.), The application of the in-phase voltage on the two electrodes to change the shape of the droplet to be tested based on the electrowetting effect, etc., is known to those skilled in the art and will not be described in detail herein.

2A is a schematic cross-sectional view of a digitized microfluidic device of the present invention for manipulating charged particles in a liquid based on electrophoresis. In this embodiment, the droplet D is sandwiched between the lower deck 202 and the upper deck 204. The terms "upper" and "lower" are used in this context only to distinguish between the lower deck 202 and the upper deck 204, and not as a limitation of the orientation of the lower deck 202 and the upper deck 204 relative to the ground plane. The lower layer plate 202 is provided with a first electrode structure layer and a second electrode structure layer, A third electrode structure layer is disposed on the layer plate 204. The first electrode structure layer disposed on the first substrate 201 includes an electrode E1 and a dielectric layer 203B. The second electrode structure layer disposed on the surface of the first electrode structure layer includes two elongated electrophoretic electrodes E2E-1. E2E-2, electrode E2, and dielectric layer 203B. The third electrode structure layer disposed on the second substrate 205 includes an electrode L and a dielectric layer 207.

Preferably, the spacing between the lower deck 202 and the upper deck 204 is less than 1 mm; more preferably, less than 0.3 mm.

Wherein, the electrophoretic electrodes E2E-1, E2E-2 have a width ranging from 1 micrometer to 1 millimeter, and the spacing between the two ranges from 10 micrometers to 20 millimeters, and the width range of each electrode E2 and between adjacent electrodes The pitch ranges from 100 micrometers to 20 millimeters; preferably, the electrophoretic electrodes E2E-1, E2E-2 have a width ranging from 5 micrometers to 500 micrometers, a pitch ranging from 100 micrometers to 5 millimeters, and each electrode E2 The width range and adjacent electrode spacing range from 200 microns to 2 mm.

Wherein, the width range of each electrode E1 in the first electrode structural layer and the spacing between adjacent electrodes range from 1 micrometer to 10 millimeters.

Preferably, each of the electrodes El, E2, E2E-1 and E2E-2 employs an elongated electrode.

Among them, the electrodes E2E-1 and E2E-2 can be used to generate an electric field (between them) and operate on the suspended charged particles in the droplet. Of course, the primary use of control electrodes E1 and E2 is to create an electrowetting effect to control charged particles in the droplets. It will be appreciated that in constructing devices that benefit from the present invention, the control electrodes El, E2, E2E-1, or E2E-2 are typically part of a large number of control electrodes that together form a two-dimensional electrode array or grid.

Preferably, at least a portion of the surfaces of the control electrodes E2E-1 and E2E-2 are not covered by the dielectric layer 203C, thereby being in direct contact with the droplets, as shown in Fig. 2A.

Fig. 2B is a three-dimensional view of the electrode L in the upper layer 204 of the microfluidic device shown in Fig. 2A and the control electrode embedded in the lower layer plate 202. Fig. 2C is a plan view showing the control electrode embedded in the lower layer plate 202.

The material used to make the first substrate or the second substrate is not critical except that the area in which the electrodes are placed is not electrically conductive. The material should have a certain hardness so that the basic shape of the substrate and the spacing between the two can remain substantially unchanged. The first substrate or the second substrate may be made of, but not limited to, quartz, glass, or a polymer such as a polycarbonate or a cycl ic olefin copolymer.

The number of control electrodes E1 and E2 is between 1 and 10,000, but is preferably from 2 to 1000, more preferably from 2 to 200. 1微米。 The number of the electrodes in the upper layer 204 is between 1 and 10000, preferably between 2 and 1000, more preferably between 2 and 200, the distance between adjacent electrodes L is in the range of 0.1 micron. Between 20 mm, preferably between 1 and 2 mm. The control electrodes El, E2, E2E-1, and E2E-2 can be connected to a DC or AC power source through conventional conductive leads. Each power supply can be controlled independently, or a power supply can be used to control multiple electrodes using a transfer switch. Typical voltage amplitudes are typically less than 300 volts. The frequency of the alternating voltage used to generate the electrowetting effect is typically less than 10,000 Hz. When it is desired to produce an electrophoretic effect, the electrodes E2E-1, and E2E-2 can be connected by a conventional conductive lead and a direct current or alternating current power source. The frequency of the alternating current is usually less than 10,000 Hz, but preferably less than 100 Hz, more preferably Less than 1 Hz.

The electrode can be made of any conductive material such as copper, chromium, indium antimony oxide (IT0), and the like. For drawing and display convenience, the electrode shapes in Figs. 2A to 2C are drawn in a rectangular shape, but they may be in any other shape. In fact, the shape, width, and spacing of the electrodes can vary based on different locations on the device, allowing for more efficient operation of particles of different sizes and shapes at different locations on the device.

Materials for forming the dielectric layers 203B, 203C, and 207 include, but are not limited to, Teflon, Cytop, Parylene C, silicon nitride, silicon oxide, and the like. Dielectric layers 203B and 207 are preferably hydrophobic, which can be achieved by applying a layer of Teflon, Cytop, or other hydrophobic material to dielectric layers 203C and 207.

The control electrodes El, E2, E2E-1, and E2E-2 are embedded or formed on the first substrate 201. Dielectric layer 203A is coated on each electrode E1 to electrically isolate each electrode E1, and also to electrode E1 (belonging to the first electrode structure layer) and each electrode E2, E2E-1, and E2E-2 (belonging to Two-electrode structure layer) Electrically isolated. The other dielectric layer 203C covers at least a portion of the control electrode E2, and thereby electrically isolates the electrodes E2, E2E-1, and E2E-2. The upper layer 204 includes a control electrode L embedded in or formed on the second substrate 205. Preferably, a hydrophobic insulating layer 207 covers the electrodes L and thereby electrically isolates the electrodes L.

Standard IC or LCD production processes can be used to create digital microfluidic devices that are compatible with bioanalysis. For example, techniques for making thin layers include, but are not limited to, deposition, such as plasma enhanced chemical vapor deposition (PECVD), sputtering, or spinning coating, and the like; Techniques for removing thin layers are, but are not limited to, etching, such as wet etching, plasma etching, etc.; film patterning techniques are (but not limited) )) UV lithography, electron beam lithography, and the like.

The microfluidic device shown above is a digital microfluidic device that may also include other microfluidic components and/or microelectronic components. For example, the device may also include resistive heating regions, microchannels, micropumps, pressure sensors, optical waveguides, and/or metal oxide semiconductors. (Metal Oxide Semiconductor, or MOS) Circuit-connected biosensing or chemosensing components. As a preference, the microfluidic device of the present invention further includes an electrode selection unit. The electrode selection unit is respectively connected to the address electrodes in the first electrode structure layer, the second electrode structure layer and the third electrode structure layer of the second substrate of the first substrate for selecting from the address electrodes The electrode to which the voltage is applied is applied to apply the corresponding voltage.

As still another preferred, the device of the present invention may further include at least one temperature control element to control the temperature of the partial region of itself or the like. The temperature control component, such as a semiconductor cooler (Peltier), may be disposed outside the integrated chip to which the device belongs, which is in contact with at least one region of the chip to which the microfluidic device 100 belongs; or integrated on the integrated chip to which the device belongs, such as directly fabricated on The thin film electric resistance heater on the outer surface of the device; in addition, the device may include both temperature control elements disposed outside the integrated chip to which it belongs, and temperature control elements integrated on the integrated chip to which it belongs. The temperature control element can stably control the temperature of the area in which it contacts from 0 degrees Celsius to about 100 degrees Celsius.

Further, the microfluidic device of the present invention further includes a liquid inlet, a liquid outlet, and the like in communication with the space in which the liquid is housed. Figure 2D shows that positive electrode VI is applied to electrode E2E-1, negative electrode V2 is applied to electrode E2E-2, and charged particles in droplet D are redistributed, that is, positively charged particles are near electrode E2E-2, with negative charge The particles are near the electrode E2E-1. Among them, the amplitudes of the voltages VI, V2 should be large enough to move the charged particles.

Figure 2E and Figure 2F show that after the charged particles are redistributed and the appropriate voltages are applied to electrodes E2 - 1 and E2 - 2 (for example, V3 and V4, respectively), droplet D is divided based on the electrowetting effect. Two smaller sub-droplets.

Figure 2G shows that the two sub-droplets become naturally oblate after the voltages on electrodes E2 - 1 and E2 - 2 are removed. It can be seen that the two electrophoretic electrodes of the microfluidic device according to the present invention can manipulate the charged particles in the droplets, in particular, the positively charged particles can be separated from the negatively charged particles.

Further, by performing the operations of the foregoing step S101 based on the two electrophoretic electrodes of the above-described microfluidic device, separation of different particles having the same kind of charges in the droplets can also be realized.

Because of its high affinity and specificity, immunoassays are sensitive and commonly used methods for quantitative detection, such as viruses, peptides, polynucleotides, proteins (such as antibodies, Toxins, cytokines, etc.) and other small molecules. In clinical laboratories, immunoassays are still used to detect heart disease markers, tumor markers, hormones, drugs, infectious agents, and immune responses; and new tests are constantly being used. Add it in. Among the different immunoassay formats, heterogeneous immunoassay has higher sensitivity and is therefore the most commonly used. Heterogeneous immunoassays have three typical steps: first, capture - the reaction that produces the labeled antigen-antibody complex; second, separation - the process of separating the bound antigen-antibody complex from the free antigen; third, detection - The signal emitted from the bound antigen-antibody complex is measured.

In common heterogeneous immunoassays, antigen-antibody complexes are usually immobilized on the surface of solids (plates or micromagnetics) Beads), then the unbound molecules are washed away. With the present invention, participation in bound and unbound molecules can be achieved by electrophoresis on a digital microfluidic device, thus eliminating the need to use a solid surface to immobilize the analyte. This can reduce the complexity and experimental cost of the entire system.

The operation of the charged particles in the liquid can be realized by the digital microfluidic device of the present invention, and the required particle separation can be realized by controlling the electrodes in the device, so that it is not necessary to use wel l-plates or Microbeads and the like are used to immobilize the antigen-antibody complex. This brings great benefits: including reliable measurements, more economical inspections, easy-to-use systems, and more.

To date, samples analyzed on microfluidic devices require pretreatment before they are placed in the device, ie sample preparation^ For most analytical methods, sample preparation is an important part because the analysis may Insensitive to analytes in situ, it is also possible that the results of the analysis are susceptible to interference with other substances that coexist with the analyte. Traditionally, sample preparation usually involves concentration and solvent exchange of analytes prior to analysis. (the exchange of solvent), removal of interfering substances, etc. In biochemical analysis, sample preparation is usually a time-consuming process that requires many steps, such as from raw samples (eg whole blood, saliva, urine, sweat, cerebrospinal fluid) , feces, etc.) Collect the DNA, RNA, or protein required.

In general, sample preparation can be divided into two major steps: First, cell or tissue lysis - lysing cells without deforming or degrading sensitive macromolecules such as DNA or Protein; second, extraction or separation - extracting the analyte from the lysed cells. In microfluidic systems, cell decomposition methods have the following broad categories:

a. Mechanical Method - Crush the cells with mechanical force that is in direct contact with the cells.

b. Heating method - use high temperature to destroy the cell membrane.

c. Chemical Method - Use a chemical buffer or enzyme to open the cells.

d. Electrical Method - Use a low-intensity electric field to create a porous membrane, or use a stronger electric field to decompose cells.

Without being bound by theory, with the present invention, cell lysis can be readily accomplished on digital microfluidic devices using heating, chemical, electrical, and the like. Electrophoresis, extraction or separation can also be achieved on the device. That is, the present invention makes the digital microfluidic device a truly integrated device - for sample preparation, detection, and analysis.

Figure 3 is an example of the use of the digitized microfluidic device of the present invention to extract and analyze DNA samples from whole blood. In step S301, a blood sample of the patient and reagents (such as DNA primers, DNA polymerase, dNTP, etc.) for performing real-time PCR measurement on the specific DNA are placed on the digitized microfluidic device. In step S302, a voltage is applied to the respective electrodes of the device to separate one or more sample droplets based on electrowetting of the blood sample, and pass through the phase A voltage is applied across the electrodes to move the sample droplets to a position on the device where they can be heated. In step S303, the temperature of the sample droplet is raised to 100 degrees Celsius by the temperature control element on the device and maintained at this temperature for a short period of time (e.g., 30 seconds) to effect thermal decomposition of the cells in the sample. In step S304, the sample droplet is moved to a position corresponding to the electrophoresis electrode E2-1 or E2-2 by applying a voltage on the corresponding electrode, and a voltage pair is applied by the electrophoresis electrode E2-1 or E2-2. The sample droplets are subjected to an electrophoresis operation to separate the DNA to be tested. In step S305, the sample droplet is divided into two by generating an electrowetting effect by applying a voltage on the corresponding electrode, so that the DNA to be tested is mainly in one of the droplets (DNA droplet). In step S306, a voltage is applied to the respective electrodes of the device to cause the reagent to generate one or more reagent droplets based on electrowetting, and the reagent droplets are moved by applying a voltage across the respective electrodes to cause the reagent droplets to merge with the DNA droplets. In step S307, real-time PCR measurement is performed on the combined mixed droplets on the device. In step S308, the measured droplets are moved to a waste collection point in the device.

Figure 3 shows only one of many examples of detection analysis that can be performed by placing untreated samples and corresponding reagents on the digitized microfluidic device of the present invention. The digital microfluidic devices here have a variety of functions, such as extracting a test substance from an untreated sample, measuring an analyte, and experimental analysis. Non-limiting examples include blood chemistry of whole blood, such as blood gases, electrolytes, electrolytes, urea, etc.; Trichomonas in the urine Measurement of vaginal is) to diagnose bladder cancer; measure of sweat electrolytes in cysts to diagnose cystic fibrosis, corresponding interleukin IL-1B and IL-8 in saliva Oral measurement to determine oral squamous cel l carcinoma and the like.

It can be seen that the present invention proposes very useful methods in many fields such as biochemical analysis and point-of-care testing, including automation of sample preparation (e.g., cell separation, cell lysis, molecular extraction). And purification, concentration, mixing with reagents, or amplification, etc.), measurement and analysis. Some of the advantages can be seen from some of the examples above.

In addition to inheriting some of the advantages of digital microflow, the present invention introduces additional advantages, such as:

a. There are electrophoresis electrodes mainly used for electrophoresis and electrodes for electrowetting operation with electrophoresis electrodes, which are convenient for the user to use.

b. The device is more fully functional, especially for sample preparation, which can be done on the device without the need to perform other methods before placing the device. Therefore, the raw materials can be directly measured.

c Since the charged particles suspended in the liquid can be redistributed or concentrated in the droplets, the sensitivity of the measurement can be correspondingly increased accordingly. d. Since the charged particles in the droplets can be separated by controlling the electrodes on the device, magnetic beads and external magnet devices usually used for particle separation are not required. This simplifies the use of the device and reduces the cost of the device.

e. The ability to redistribute or separate charged particles in the droplets on the device allows for improved flexibility and multiplicity of detection.

f. Many steps in biochemical analysis can be integrated and automated on the device, such as sampling, sample preparation, liquid transfer, mixing, dilution, concentration, separation, incubation, reaction, measurement, waste collection, and more.

g. Multiple analytes can be detected simultaneously.

h. Different types of analysis and testing can be performed simultaneously.

i. Using the electrophoresis process on the device, the mixing process between the sample and the reagent can be accelerated.

j. Experimental calibration and detection analysis can be performed simultaneously. The droplets used for calibration and the droplets detected can be generated and operated simultaneously, and the process of experimental calibration does not require the experiment to be stopped first.

It should be noted here that in order to reduce the side effects of Joule heating, temperature control of the digital microfluidic device (whole or partial) of the present invention can be performed.

Although not described in detail herein, it should be noted that the amplitude of the applied voltage on the electrodes can also be adjusted when using the electrophoretic effect, which is also often used for more efficient particle manipulation.

As can be seen from the above, the present invention provides a true point-of-care testing method and device, in particular for efficiently separating different particles of the same charge in a droplet. Based on the present invention, cell lysis and analyte extraction/separation are part of the device function. The digital microfluidic device of the present invention has quite a complete function including sample preparation, measurement, analysis, and diagnosis. By combining with the Internet and cloud computing, the present invention can provide a good basis for a health care system, including condition diagnosis, online medical knowledge support, and remote doctor patient interaction.

It should be noted here that the above examples and the advantages mentioned above are not exhaustive. The flexibility of the present invention can be used in many applications and does have many advantages over other technologies such as single layer electrode based digital microflow or pipe based microfluidics.

All written patents and publications mentioned in this application are hereby incorporated by reference in their entirety.

While a preferred embodiment of the invention has been shown and described, it is understood that many modifications may be made to the present invention without departing from the spirit and scope of the invention.

The above-described embodiments are merely illustrative of the principles of the invention and its advantages, and are not intended to limit the invention. Modifications or variations of the above-described embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, all that is accomplished by those of ordinary skill in the art without departing from the spirit and scope of the invention disclosed herein Covered by the claims.

Claims

Claim

CLAIMS 1. A method for manipulating charged particles in a liquid based on electrophoresis, for a microfluidic device having at least two electrodes, characterized in that the method comprises at least the steps of:

Applying a voltage across the two electrodes to form a first voltage difference, and after a first duration, changing the voltage across the two electrodes to form a second voltage difference for a second duration, Causing the charged particles in the droplet to be tested to produce different displacements for separation of the particles;

Wherein the amplitude of at least one of the first voltage difference and the second voltage difference causes at least a portion of the charged particles in the droplet to be tested to move.

2. The method according to claim 1, wherein the step a is repeated a plurality of times to cause the partially charged particles to continuously move toward the one electrode in the liquid droplet, and finally stay at a position adjacent to the electrode in the liquid droplet.

3. Method according to claim 1 or 2, characterized in that the first voltage difference is opposite to the polarity of the second voltage difference.

4. Method according to claim 1 or 2, characterized in that one of the first duration and the second duration is longer than the other.

The method according to claim 1, wherein: before step a, the method further comprises the steps of:

Applying opposite voltages on the two electrodes respectively, so that the charged particles in the droplet to be tested are moved to the electrode whose polarity is opposite in polarity to the charge of the charge, and finally stay in the droplet to be tested. The position of the electrodes.

6. The method according to claim 5, further comprising the steps of: applying a direct current or low frequency alternating voltage on the respective electrodes included in the microfluidic device, so that the droplets to be tested are separated into at least two based on the electrowetting effect. Sub-droplets.

7. The method according to claim 1, further comprising the steps of: applying a direct current or a low frequency voltage to a corresponding electrode included in the microfluidic device to cause the liquid droplet to be tested to be driven to the ground The desired electrode corresponds to the location.

8. The method according to claim 1, wherein: the frequency of the voltage applied to the two electrodes in step a is less than 10,000 Hz.

9. Method according to claim 8, characterized in that the frequency is less than 100 Hz.

10. Method according to claim 9, characterized in that the frequency is less than 1 Hz.

11. A microfluidic device for controlling charged particles in a liquid based on electrophoresis, characterized by at least: a first substrate and a second substrate; a first electrode structure layer disposed on the first substrate, a second electrode structure layer disposed on the surface of the first electrode structure layer, a third electrode structure layer disposed on the second substrate, and on the first substrate The electrode structure layer is disposed opposite to the electrode structure layer on the second substrate so as to have a space for accommodating the liquid therebetween;

12. The microfluidic device according to claim 11, wherein: the electrophoretic electrode has a width ranging from 5 micrometers to 500 micrometers and a pitch ranging from 100 micrometers to 5 millimeters, wherein the second electrode structure layer The other electrodes have a width range and a pitch ranging from 200 microns to 2 mm.

13. The microfluidic device according to claim 11, wherein: at least part of the surface of each of the electrophoretic electrodes is in a bare state to be in contact with the liquid droplets.

14. The microfluidic device according to claim 11, wherein: each of the electrodes in the first electrode structure layer has a width range and a pitch ranging from 1 micrometer to 10 millimeters.

15. The microfluidic device according to claim 11, wherein: the electrode included in the first electrode structure layer and the second electrode structure layer comprises an extension electrode.

The microfluidic device according to claim 11, further comprising: an electrode selection unit respectively connected to the address electrodes in the electrode structure layers of the first substrate and the second substrate, The electrode to be applied is selected from the addressable electrodes to apply a corresponding voltage.

17. The microfluidic device of claim 11 further comprising: a liquid inlet in communication with the space in which the liquid is contained.

18. The microfluidic device of claim 11 further comprising: a liquid outlet in communication with the space in which the liquid is contained.

19. The microfluidic device of claim 11 further comprising: at least one temperature control element to control the temperature of at least a portion of the device.

20. The microfluidic device according to claim 11, wherein at least a portion of the dielectric layer included in the electrode structure layer on the surface is hydrophobic on the first substrate and the second substrate.

The microfluidic device according to claim 11, wherein a distance between a surface of the electrode structural layer on the surface of the first substrate and a surface of the electrode structural layer on the surface of the second substrate is less than 1 mm.

The distance between the surface of the electrode structure layer on the surface of the first substrate and the surface of the electrode structure layer on the surface of the second substrate is less than 0.3. Millimeter.

23. A method of manipulating charged particles in a liquid based on electrophoresis, characterized by at least comprising the steps of: a. applying a polarity to each of the two electrophoretic electrodes of the microfluidic device of any one of claims 11 to 22 Conversely, the magnitude of the voltage that causes the charged particles to move causes the charged particles in the droplet to be measured to move toward the direction of the electrophoretic electrode whose polarity is opposite in polarity to its own charge.

24. The method according to claim 23, wherein when the charged particles have been retained in the droplet at a position adjacent to the electrophoretic electrode having a polarity opposite to that of the self-charged one, the method further comprises the steps of:

A direct current or low frequency alternating voltage is applied to the two electrophoretic electrodes to separate the droplets to be measured into two sub-drops based on the electrowetting effect.

25. The method according to claim 23, wherein: the droplet to be tested comprises particles having only one charge or both positively charged particles and negatively charged particles.