Microfluidic impedance cytometer

Abstract

A microfluidic impedance cytometer for determining the location of cells in a fluid carrying cells and measuring cell impedance, comprising: a microfluidic impedance flow channel for allowing the fluid to flow; an upstream portion; a downstream portion; the sensing area is used for receiving the fluid in the channel and sensing one or more parameters of the fluid, the sensing area comprises one or more sets of electrode pairs, each pair of electrodes forms a current path from an operating top electrode to an operating bottom electrode, each pair of electrodes is composed of an operating top electrode and an operating bottom electrode, and the operating top electrode and the operating bottom electrode form a current path from the operating top electrode to the operating bottom electrode. A potential is applied to the operation top electrode, the electrodes in each pair of electrodes are aligned in parallel and are symmetrical to each other, the top electrode of each pair of electrodes is applied with the same positive voltage, and the bottom electrode of each pair of electrodes is virtually grounded; and an amplifier structure.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, in particular to the field of particle measurement, and in particular to a microfluidic impedance cytometer. Background Art

[0002] Impedance particle measurement is the measurement of the dielectric properties of particles using single or multiple frequencies. Recent advances in impedance cytometry studies have shown that different types of particles present in human blood can be distinguished using multiple frequencies. The advantage of impedance particle measurement over optical measurements is that the impedance size is less dependent on the position of the particle. When measuring particles using optical methods, sheath flow is required to keep the particles in the center of the channel, which results in a lot of liquid waste and makes the system complex, bulky and costly.

[0003] Particle measurement counts different types of particles and measures various other properties, such as the size of the particles, the presence of certain proteins on the particle membrane, and the different shapes of the particle cores within the white granules. However, the advantage of impedance particle measurement compared to optical measurements is that the signal is minimally dependent on the position of the particles. Therefore, impedance cytometer measurements can be performed without any sheath fluid. Due to the significant reduction in the size of the instrument, the existing technology has an advantage in building a complete blood cell (CBC) instrument for point-of-care (PoC). Using multiple frequencies, particles with different membrane conductivity can be distinguished.

[0004] The prior art discloses an innovation that allows accurate impedance measurement of particles by embedding multiple pairs of electrodes on the top and bottom walls within a microfluidic channel. According to the first prior art reference document [US10267720], the reference document discusses the use of dual ground electrodes between each measuring electrode and the signal electrode. In the reference document, the signal electrode is an operating top electrode to which a voltage is applied, and the measuring electrode is an operating bottom electrode to which a virtual ground (0V) is applied. The current from the signal electrode to the measuring electrode is measured. The difference between the two measuring electrodes produces an antisymmetric peak signal with a secondary peak, which is located between the global maximum and global minimum of the peak. Here, the electrical signal obtained due to the flow of particles from the first electrode to the last electrode is defined as the secondary peak.

[0005] When the particle size is the same, the electrical signal changes with the position of the particle in the Z direction (please indicate the definition of the axis). Therefore, in order to accurately measure the diameter of the particle, it is very important to measure the position of the particle in the Z direction. The electrical signal changes with the position in the Z direction because the current density is non-uniform in the Z direction. The current density is relatively high near the edge of the electrode, while the current density is relatively low in the center of the channel.

[0006] According to the first prior art reference document [US10267720], the reference document discusses the ratio of the main peak to the secondary peak, which can be used to check the position of the particles in the vertical direction, thereby correcting the particle diameter. The disadvantage of the first prior art reference document is that the amplitude of the secondary peak is very low. For smaller particles less than 4 microns, the secondary peak is almost submerged by noise at a cross-section of 30 microns × 30 microns and an electrode width of 30 microns. Even if the edge spacing between two adjacent electrodes is less than 10 microns, the signal to noise ratio (SNR) of the secondary peak is not high. In addition, the amplitude of the secondary peak can be enhanced by reducing the height of the channel. However, this increases the risk of clogging of the microfluidic channel because smaller particles may clog the sensing area.

[0007] The second prior art reference [US20220034781A1] discusses “improving the sensitivity of microfluidic instruments” for counting particles smaller than 2 microns. The main application of the second prior art is to count bacteria smaller than 1 micron in a wider channel.

[0008] The electrodes manufactured in the first prior art always have a certain degree of misalignment, and the electrode geometry also varies slightly, so the peaks are not completely antisymmetric and the offset current is not zero. The offset current is defined as the difference between the currents from the bottom of the two signal electrodes when there are no particles between the two signal electrodes. Since the offset current is not zero and the thermal noise from the two bottom signal electrodes is large after subtraction, the impedance measurement of submicron particles becomes a problem. The second prior art discloses the idea of ​​applying two equal-amplitude voltages with the same frequency but a phase difference of 180 degrees. Two negative voltages are applied to the two electrodes at the top, and two positive voltages are applied to the other two electrodes at the top. The two currents with a phase angle difference of 180 degrees are summed through a summing amplifier, and similarly, the currents of the other two pairs of electrodes are summed in the same way. The outputs of the two summing amplifiers are subtracted through differential amplifiers (DA). Since the current is subtracted three times, the value of the voltage offset (the final output of the differential amplifier when there are no particles) is always smaller than that of the first prior art. Ideally, when there is no misalignment and the geometry of both electrodes is identical, the voltage offset should be zero.

[0009] The second prior art comparative document does not disclose how to determine the position of the particles, and the finite element analysis (FEM) shows that the peak impedance signal changes with the position of the particles in the Z direction. The first prior art comparative document and the second prior art comparative document are completely different because the voltages in the first prior art comparative document are in phase, while voltages of opposite amplitudes are used in the second prior art comparative document. Therefore, the first prior art comparative document and the second prior art comparative document cannot be used in combination. In addition, in the second prior art, the value of the theoretical anti-phase summed current is not zero because the two electric fields are not exactly the same. If the electrode geometry and spacing of all four pairs of electrodes are the same, the first and last electrodes will have the same electric field distribution, and the two middle electrodes will also have the same electric field distribution.

[0010] An instrument and method are needed to measure the position of the particle and then correct for the diameter of the particle. The present invention discusses how to determine the position of the particle in the Y direction and the Z direction. The advantage of the present invention is that the amplitude of "M" defined below is much larger than the amplitude of the secondary peak disclosed in Patent 1.

[0011] In addition, it is necessary to improve the sensitivity of the microfluidic instrument to make it superior to the second prior art comparative document.

[0012] The present invention does not use any grounding electrode and reverse voltage source. The electronic measurement system in the present invention is much simpler than the system in the second prior art reference document, and only requires a single voltage source of the same phase and the same frequency. The present invention also discusses how to measure the position of particles in the "Y" direction and the "Z" direction. The first prior art reference document discloses a method for measuring the position of particles in the Z direction by associating simulation data after finite element analysis (FEM) with experimental raw data. Its main disadvantage is that it requires huge computing power. However, in the present invention, the values ​​of "M", "A1" and "A2" defined later can be easily used to determine the position of particles in the Z direction. The present invention is the most economical in calculation.

[0013] Using more than four electrodes, preferably seven electrodes, wherein the innermost four electrodes make the electric field lines more symmetrical than the second prior art. Compared with the second prior art, the reduction of voltage offset is significantly improved. The present invention is completely different from the first prior art and the second prior art.

[0014] Purpose of the Invention

[0015] It is an object of the present invention to provide a system, apparatus and method for performing accurate impedance measurements of particles or cells using single or multiple frequencies in a microfluidic impedance cytometer.

[0016] Another object of the present invention is to provide a system, apparatus and method for determining the operational vertical position of a particle (the vertical position is the Z axis, such as Fig.14 As shown), the position can be accurately calculated, allowing the volume of the particle to be accurately measured.

[0017] Another object of the present invention is to provide a system, apparatus and method to improve the detection sensitivity of counting particles smaller than 1 micron. Summary of the invention

[0018] According to the present invention, a microfluidic impedance cytometer is provided.

[0019] In at least one embodiment of the apparatus, the microfluidic resistive flow channel includes at least one inlet and at least one outlet opening for allowing flow of a conductive liquid that may carry non-conductive particles or particles having a different conductivity and dielectric constant than the liquid.

[0020] In at least one embodiment of the instrument, the instrument comprises at least one operating top electrode mounted on the top wall of the instrument and at least one operating bottom electrode mounted on the bottom wall of the instrument; a voltage is applied to the top electrode. Preferably, the instrument comprises a plurality of the operating top electrodes on the top wall and a plurality of the operating bottom electrodes on the bottom wall; the electrodes are in contact with the conductive liquid carrying the non-conductive particles.

[0021] The corresponding top electrodes and bottom electrodes form one or more sets of electrode pairs, providing a current path from top to bottom. When the conductive liquid carrying the non-conductive particles passes between the formed electrode pairs, the electric field lines are disturbed, resulting in a change in the current from the top to the bottom. The change in the current is proportional to the volume of the particles. However, the current density between each pair of electrodes is not uniform. Therefore, the change in the current depends on the position of the particles in the vertical direction. The current density near the edge of the electrode is stronger, so when the particles are close to the edge of the electrode, the change in the current is greater.

[0022] The present invention discloses a mechanism and a method, wherein:

[0023] The vertical position of the particle can be accurately calculated, and

[0024] The volume of the particles can then be accurately measured.

[0025] Furthermore, the position of the particles in the width direction may be measured.

[0026] The present invention comprises at least four pairs of said electrodes for measuring the position and impedance of said particles.

[0027] In addition, the system and the instrument also improve the detection sensitivity of counting the particles smaller than 1 micron.

[0028] According to the present invention, there is provided a microfluidic impedance cytometer for determining the position of particles in a particle-carrying liquid and measuring impedance, comprising:

[0029] A microfluidic impedance flow channel for allowing the flow of the liquid;

[0030] an upstream portion for directing the liquid in a predetermined direction;

[0031] a downstream portion for directing the liquid in a predetermined direction;

[0032] A sensing region is used to receive the guided liquid between the upstream portion and the downstream portion, and to sense one or more parameters of the liquid, wherein the sensing region includes one or more sets of electrode pairs, each set of the electrode pairs forms a current path from an operating top to an operating bottom, each pair of the electrodes consists of an operating top electrode and an operating bottom electrode, a voltage is applied to the operating top electrode, and for a particular pair of the electrodes, each of the electrodes is aligned in parallel and symmetrical to each other, a voltage is applied to each of the top electrodes.

[0033] With the same positive voltage, each of the bottom electrodes is virtually grounded, and for the electrode pair:

[0034] The distance between the edges of adjacent electrodes is at least 0.5 times the width of the electrodes, but the maximum distance is 5 times the width of the electrodes;

[0035] Wherein, the width of the channel is at least 1.5 times its height;

[0036] Amplifier construction, where:

[0037] a first summing amplifier configured to sum the current values ​​flowing from the first bottom electrode and the second bottom electrode to obtain a first summed value;

[0038] a second summing amplifier configured to add the current values ​​flowing from the third bottom electrode and the fourth bottom electrode to obtain a second summed value;

[0039] a first differential amplifier configured to obtain a first difference value using the first summed value and the second summed value within a defined time period, wherein the difference value includes at least three local maximum values ​​and three local minimum values;

[0040] The change in current between the electrodes is a function of the position of the particle in the vertical direction (Z direction) and the volume of the particle in the liquid, and when the liquid containing the particle passes through at least one pair of the sensing electrodes, it will cause the change in current; and for a given applied pressure of the liquid, the velocity of the particle is a function of the position of the particle in the width direction (Y direction) and the vertical direction (Z direction).

[0041] In at least one embodiment,

[0042] Each of the bottom electrodes is connected to a fixed precision resistor and the bottom electrode is grounded to act as a voltage divider, i.e.,

[0043] Each pair of electrodes is connected in series with a fixed resistance value, and the electrode pair is grounded;

[0044] The second differential amplifier is configured to output a second difference in voltage between the first electrode pair and the third electrode pair;

[0045] The third differential amplifier is configured to output a third difference in voltage between the second electrode pair and the fourth electrode pair;

[0046] The fourth differential amplifier is configured to output a fourth difference between the second difference and the third difference.

[0047] In at least one embodiment, the sensing area includes at least one detection chamber, which is defined as a region volume between the top electrode and the corresponding bottom electrode, and the top electrode and the corresponding bottom electrode together constitute a pair of electrodes.

[0048] In at least one embodiment, when the electrode group is an odd number of pairs, the center electrode is grounded.

[0049] In at least one embodiment, the width of the sensing area is at least 1.5 times the height of the sensing area in order to focus the particles on the operating level (Z plane).

[0050] In at least one embodiment, the width of the channel is 4 times the height of the sensing area in order to align the particles in the operating horizontal plane (Z plane).

[0051] The apparatus of claim 1 comprises a viscoelastic fluid for focusing the particles in the Z plane.

[0052] In at least one embodiment, each of the operational top electrodes is disposed on an operational top wall of the instrument such that the electrode is in communication with the liquid, which is disposed to pass through the resistive flow channel.

[0053] In at least one embodiment, each of the operational bottom electrodes is disposed on an operational bottom wall of the instrument such that the electrodes are in communication with the liquid, and the liquid is configured to pass through the resistive flow channel.

[0054] In at least one embodiment, each of the electrodes is made by coating a photoresist (SU-8) on a platinum electrode deposited on a silicon wafer or a glass wafer, and the electrode material is platinum or gold, so that all electrodes are in contact with the flowing liquid.

[0055] In at least one embodiment, from the three local maxima and the three local minima,

[0056] The difference between the third and fourth extreme values ​​gives the first (main) peak value, which has the value "A1";

[0057] The difference between the first extreme value and the sixth extreme value yields the second peak value, which has a value of "A2";

[0058] The difference between the second extreme value and the fifth extreme value gives a third peak value, whose value is "M";

[0059] therefore,

[0060] The amplitude difference between the "A1" value and the "A2" value can determine whether the particle in the liquid is above or below the center value;

[0061] A positive difference between the "A1" value and the "A2" value can determine whether the particles in the liquid are above or below the center value; and a negative difference between the "A1" value and the "A2" value can determine whether the particles in the liquid are above or below the center value.

[0062] In at least one embodiment, each of the electrodes is connected to an input voltage signal, which is generated by a phase-locked board, and the output signal from the differential amplifier is fed to the input of the phase-locked amplifier for demodulation required for impedance measurement at different frequencies. The carrier frequency modulates the impedance signal, and the carrier frequency is a voltage with a sinusoidal frequency applied to the electrode. The output of the differential amplifier is the impedance signal carrying the particles and the modulated signal of the carrier frequency. The signal is demodulated using the phase-locked amplifier to obtain the impedance value at each frequency applied to the top electrode.

[0063] In at least one embodiment, a positive voltage is applied to the first top electrode and the fourth top electrode, and a negative voltage is applied to the second top electrode and the third top electrode, so as to obtain the three local maximum values ​​and the three local minimum values.

[0064] In at least one embodiment, a positive voltage is applied to the first top electrode and the fourth top electrode, a negative voltage of the same magnitude is applied to the second top electrode and the third top electrode, and all the bottom electrodes are virtually grounded, wherein the first summing amplifier is used to sum the current flowing out of the first bottom electrode and the third bottom electrode, and another summing amplifier is used to sum the current flowing out of the second bottom electrode and the fourth bottom electrode, and then a differential amplifier is used to find the difference between the two summing amplifier outputs to generate an output value related to the following: minimum-maximum-minimum, maximum-minimum-maximum, and / or maximum-minimum-maximum-minimum-maximum-minimum, wherein the output value has a higher sensitivity to provide the location of the particles and perform impedance correction.

[0065] In at least one embodiment, a positive voltage is applied to the first top electrode and the second top electrode, a negative voltage is applied to the third top electrode and the fourth top electrode, and all the bottom electrodes are connected to a resistor or a virtual ground to measure the sum of the currents flowing into the first bottom electrode and the fourth bottom electrode, and to measure the sum of the currents flowing into the second bottom electrode and the third bottom electrode, and then the two summed currents are subtracted using the differential amplifier to generate an output value related to: maximum-minimum-maximum-minimum, minimum-maximum-minimum-maximum, wherein the output value improves the detection limit but cannot be used to find the position of the particle.

[0066] In at least one embodiment, the first top electrode and the second top electrode are short-circuited and a positive voltage is applied to the first top electrode, the second top electrode uses only a single contact pad, the third top electrode and the fourth top electrode are electrically connected using two different contact pads, and the third top electrode and the fourth top electrode use only a single contact pad to apply a negative voltage of the same amplitude.

[0067] In at least one embodiment, the third bottom electrode and the second bottom electrode are short-circuited, the first bottom electrode and the fourth bottom electrode are electrically connected using two different pads, a total of three pads are used for current measurement, and all of the bottom electrodes are either virtually grounded or connected to a resistor.

[0068] In at least one embodiment, at least one of the operating top electrodes is applied with a carrier signal having at least two different frequencies, wherein the frequency range of the carrier signal is from 500kHz to 30MHz, so as to determine the size of the particles and the electrical properties (such as capacitance) of the particle film, thereby distinguishing the particles having the same size but different membrane electrical properties.

[0069] In at least one embodiment, the instrument has eight pairs of electrodes, wherein the innermost four pairs of symmetrical electrodes are used for measurement.

[0070] In some embodiments, the top surface of the microfluidic flow channel is made of glass and the bottom surface is made of silicon. The sidewalls of the microfluidic instrument are made of photoresist.

[0071] In some embodiments, the sensing region may be 30 microns to 600 microns in length for counting particles less than 20 microns in size.

[0072] In some embodiments, the height of the sensing region may be 10 microns to 100 microns.

[0073] In some embodiments, the width of the electrode may be 10 microns to 30 microns for counting submicron sized particles.

[0074] In some embodiments, the width of the sensing region may be between 30 microns and 200 microns.

[0075] In some embodiments, the number of the electrode pairs is at least 4, but can be increased to 7. All the electrode pairs have the same size.

[0076] In at least one embodiment, the protrusion of the impedance signal (final output signal) is calculated as (1-M / A2). The value of the protrusion is well correlated with the position of the particle in the vertical Z direction. The protrusion is well correlated with the following equation: Therein, for known fixed geometries "c1", "c2" and "c3" are known.

[0077] In at least one embodiment, substituting the Z position into the equation to obtain the true diameter of the particle. Where "d" is the diameter of the particle, "Z" is the vertical position of the particle, and "G" is a gain factor that depends on the electronic circuit and electrode geometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The present invention will now be described with reference to the accompanying drawings, in which:

[0079] Figure 1 a shows four pairs of electrodes with a width of 10 microns and an edge-to-edge spacing of 20 microns. A DA (differential amplifier) ​​is used to measure the current difference between the first pair and the third pair of electrodes, and another DA is used to measure the current difference between the second pair and the fourth pair of electrodes. The outputs of the two DAs are subtracted using a third DA;

[0080] Figure 1b shows a top view of the electrode, which includes three different regions: upstream region, sensing region, and downstream region. The current from the bottom electrode is measured using a Wheatstone bridge and a differential amplifier, as shown in Figure 1 As shown in a;

[0081] Figure 2 The relationship between the amplitude change of the particle (the final output comes from the third differential amplifier) ​​and the X coordinate of the particle is shown. The impedance signal shown in the figure corresponds to a particle with a diameter of 6 microns and a Z coordinate of -6 microns in a microfluidic chip with a height of 30 microns and a width of 30 microns. The center of the channel is located at (0, 0, 0). The amplitudes of "A1", "A2" and "M" are represented by black double-headed straight arrows;

[0082] Figure 3 The impedance signal of a 6-micron particle at different Z positions is shown. The unit of the impedance signal is microampere, and the unit of the Z position is micrometer;

[0083] Figure 4 The X-axis is shown as the Z position of the particle, and the Y-axis is the protrusion defined by Equation 2 described later;

[0084] Figure 5 Table 1 is shown, listing the equation fitting coefficients for different particle sizes, and these equations are described in detail later;

[0085] Figure 6 a shows that the solid line represents the fitting equation 1, and “o” represents the true value of A2 at different vertical positions after finite element analysis; Figure 6 b The solid line represents the fitting equation 2, and “o” represents the true value of the protrusion at different vertical positions after finite element analysis, as described in equation 2a;

[0086] Figure 7 The difference between the first and third pairs measured using a differential amplifier is shown, and similarly, the difference between the fourth and second pairs is also measured using a differential amplifier;

[0087] Figure 8 Five pairs of electrodes are shown, demonstrating the electrode wiring and the principle of current measurement used to calculate the Z position of the particle;

[0088] Fig. 9 Table 2 shows the Figure 8 Fitting coefficients of Eq. 1 and Eq. 2a for different particle sizes under the electrode geometries shown;

[0089] Fig.10 Current density field lines are shown for a four-pair electrode instrument system. The current density at the fourth pair of electrodes shows a change when the particle is located between them;

[0090] Fig.11A seven-pair electrode system is shown, demonstrating the electronic setup for particle impedance measurement. The inner five pairs of electrodes are used for the measurement;

[0091] Fig.12 shows that the same voltage is applied to the top electrodes and the currents from the first and second electrode pairs are summed using a summing amplifier. Another summing amplifier is used to sum the currents from the third and fourth electrode pairs. A differential amplifier is used to subtract the outputs of the two summing amplifiers to give the same Figure 2 Same output as shown;

[0092] Fig.13 A five-pair electrode system is shown, wherein the middle electrode pair is grounded; and

[0093] Fig.14 A cross section of the sensing area is shown. The axes are labeled as shown. The peak amplitude varies with the Z coordinate but is independent of the Y coordinate. The flow direction of the fluid is the X direction. DETAILED DESCRIPTION

[0094] According to the present invention, a microfluidic impedance cytometer is provided.

[0095] The present invention relates to accurate impedance measurement of particles at multiple frequencies. The main applications of the instrument include blood cell counting, E. coli counting and impedance spectroscopy analysis of cancer cells to obtain electrical parameters such as conductivity and dielectric constant of the particles, and to measure the electrical properties of cell membranes.

[0096] In at least one embodiment of the apparatus, the microfluidic resistive flow channel comprises at least one inlet and at least one outlet opening for allowing the flow of a conductive liquid carrying non-conductive particles therein.

[0097] In at least one embodiment of the apparatus, the apparatus comprises at least one operating top electrode mounted on the top wall of the apparatus and at least one operating bottom electrode mounted on the bottom wall of the apparatus; a voltage is applied to these electrodes. Preferably, the apparatus comprises a plurality of operating top electrodes on its top wall and a plurality of operating bottom electrodes on its bottom wall; these electrodes are in contact with the conductive liquid carrying the non-conductive particles.

[0098] The corresponding top electrodes and bottom electrodes form one or more sets of electrode pairs, providing a current path from top to bottom. When a conductive liquid carrying non-conductive particles passes through the formed electrode pairs, the electric field lines are disturbed, causing the current from top to bottom to change. The change in current is proportional to the volume of the particles. However, the current density between each pair of electrodes is not uniform. Therefore, the change in current depends on the position of the particles in the vertical direction. The current density near the edge of the electrode is stronger, so when the particle is close to the edge of the electrode, the change in current is greater.

[0099] In a preferred embodiment, the microfluidic device of the present invention is prepared by depositing platinum electrodes on a silicon wafer or a glass wafer and coating it with a photoresist (SU-8), such as Figure 1 The electrode material of the instrument can be platinum or gold.

[0100] In at least one embodiment, the microfluidic instrument of the present invention has three parts, such as Figure 1 Shown in b: upstream part, sensing area and downstream part.

[0101] In a preferred embodiment, the sensing region of the instrument may be between 10 microns and 600 microns in length, with at least four pairs of electrodes. The top and bottom electrodes are aligned to ensure that the electric field lines are parallel and symmetrical, thereby reducing any errors caused by electrode misalignment.

[0102] In a preferred embodiment, the width of the electrodes can vary from 5 microns to 100 microns depending on the size of the particles to be counted. The edge spacing between each pair of electrodes can be from 5 microns to 60 microns. The high current density near the top electrode and the bottom electrode will cause changes in the amplitude of the impedance signal. However, the position of the particle can be calculated from the impedance signal of the particle by using three factors, namely "A2", "M" and "A1", as shown in FIG. Figure 2 as shown, as described below; thereafter, the impedance value can be corrected. Figure 2 A plot of voltage recorded across an electrode versus position is shown; multiple values ​​can be obtained from it. For a given electrode configuration, the plot shows three maxima and three minima.

[0103] The sensitivity of the instrument is defined as the ratio of the minimum volume of countable particles to the volume of the detection chamber. Here, the detection chamber is defined as the volume of the area between the top electrode and its corresponding bottom electrode, which together form a pair of electrodes. The electrode size can be reduced to increase the sensitivity of the instrument. The signal sensitivity discussed in the first prior art is limited due to the following factors:

[0104] (a) The higher potential cannot exceed a certain value because the current-to-voltage conversion circuit will be saturated and the high voltage gradient in the height direction will damage the electrode material.

[0105] (b) The electrodes always have misalignment and geometric variations, which increase the voltage offset in the electronic circuit, resulting in high noise and saturation if the circuit gain is increased.

[0106] In at least one embodiment, the same potential is applied to all of the top electrodes, and all of the bottom electrodes are connected to a fixed resistor value and ground, such as Figure 1 When the particle is close to the wall or located between the top electrode and the bottom electrode, the current flowing from the top to the bottom will change. Figure 1 a shows four pairs of electrodes; but in general, more electrodes can be made. Figure 3 Shows Figure 1 The impedance signal of the particle at different heights in the microfluidic device shown in a clearly shows that the impedance value is higher when the particle is close to the edge of the electrode. The impedance size of the particle represented by "A2" is related to the position and diameter of the particle, as described in Equation 1.

[0107]

[0108] in,

[0109] A2 is the impedance amplitude of the signal,

[0110] "a1", "a2", "c1", and "c2" are the fitting parameters,

[0111] Z is the vertical position of the particle Fig.14 As shown in

[0112] "d" is the diameter of the particle,

[0113] "G" is the gain of the electronic circuit,

[0114] "H" is the height of the channel.

[0115] The coefficients “a1” and “a2” are obtained by least squares fitting, since A2 and the different Z positions are known after solving the Laplace equation in voltage and applying appropriate boundary conditions on the walls of the sensing area, given the known microfluidic instrument geometry and particle diameter.

[0116] Here, “P” is called prominence, which is defined as shown in Equation 3 below.

[0117]

[0118] The characteristics of the impedance signal, showing M and A2, in Figure 2 Marked out in.

[0119]

[0120] Among them, Y=y / L, Z=z / l, A=l / L, U=u / u m and

[0121] Fig.14 The coordinate axes and the definitions of l and L are shown.

[0122] Here, U is the scaled velocity defined as the ratio of the velocity at locations "X" and "Y" to the average flow velocity within the sensing area.

[0123] The operational vertical position of the particle can be above or below the center of the channel. The difference in amplitude between A1 and A2 determines whether the particle is above or below the center. A positive difference between A1 and A2 indicates that the particle is below the center, while a negative difference indicates that the particle is above the center of the channel. "P" is defined as another height factor (protrusion, "P") that can be used to calculate the position of the particle in the vertical direction.

[0124] Equation 2 relates "P" to the position of the particle in the vertical direction. For a known geometry, the impedance signal of the particle at different Z positions is obtained after finite element analysis (FEM) discussed below. For a known geometry, the value of "P" for different positions of the particle in the vertical direction is known, and it should be noted that the impedance value is independent in the Y direction (coordinate axis is in Figure 7 (defined in ).

[0125] Equation 2a is fitted by least squares to obtain the fitting coefficients c1, c2, and c3 for known electrode geometry. The electrode width and the edge-to-edge spacing between electrodes are selected to have a significant impact on the value of "M", thereby allowing better discrimination of the operational vertical position of the particle.

[0126] Another obtained value "P" is equal to 1-M / A2, which is related to the position of the particle in the "Z" direction inside the sensor. Here, the "Z" direction is defined as the direction of the height of the channel (the sensing area). The "Z" position of the particle can be calculated by fitting the equation:

[0127]

[0128] Where "H" is the height of the channel, and "c1", "c2", and "c3" are fitting parameters.

[0129] For known sensor dimensions and electrode geometry, "c1", "c2" and "c3" are parameters derived from a least squares fit and can be calculated after solving the Laplace equation and applying appropriate boundary conditions.

[0130] The Z position can be used to calculate the diameter of the particle. Under the channel geometry and the width of the electrode, the amplitude of the particle varies as follows

[0131] Where "d" is the diameter of the particle.

[0132] The value of "A2" and "Z" can be used to calculate the diameter of the particle.

[0133] The velocity of the particle flowing in the geometry can be obtained by finding the time difference between two consecutive peaks, because the sampling rate of the electrical signal is much higher than the transit time of the particle in the sensing area. The center-to-center distance of two consecutive electrodes is known, so the velocity of the particle can be easily measured using the geometry defined herein. The flow in the channel is parabolic, which is a function of the Z and Y coordinates. Here, the "Y" coordinate refers to the direction of the channel width. The "Z" position obtained according to claims 12 and 13 can be used to calculate the "Y" coordinate using:

[0134]

[0135] Among them, Y=y / L, Z=z / l, A=l / L, U=u / u m and

[0136] Where "2L" is the width of the sensing area and "2l" is the height of the channel.

[0137] The coordinate center is located at the geometric center of the sensing area defined in the claims.

[0138] Here "u" is the velocity of the fluid at any location within the defined geometry.

[0139] The geometry defined by the apparatus of the present invention can apply a positive voltage to the first top electrode and the fourth top electrode, while applying a negative potential of the same magnitude to the second top electrode and the third top electrode to obtain the same characteristics (A1, A2, M). All the bottom electrodes are virtual grounds. The currents flowing out of the first and third bottom electrodes are added using a first summing amplifier, and the currents flowing out of the second and fourth bottom electrodes are added using another summing amplifier. The difference between the outputs of the two summing amplifiers is obtained by a differential amplifier to generate the same characteristics (A1, A2, M).

[0140] Figure 4 The relationship between the "P" value after FEM analysis of particles with diameters of 4 microns, 5 microns and 6 microns and the position of the particles in the vertical direction is shown; it is clearly shown that the value of "P" depends only on the vertical position of the particles and has little to do with the size of the particles. The X-axis in the figure is the operating vertical position, and the Y-axis is the prominence "P" described in equation 2. The center of the cross section is located at (0, 0), as shown in Fig.14 shown.

[0141] Equation 2 is used to calculate the vertical position of the particle inside the microfluidic instrument after obtaining the coefficients c1, c2 and c3 from finite element analysis and least squares fitting.

[0142] Equation 2 is used to calculate the "Z" position of the particle for a known "M" value from the acquired electronic signal, and then the "Z" value is substituted into Equation 1 to obtain the correct diameter "d" of the particle for a known "A2" value. Here "d" is the diameter when the Z position of the particle is equal to zero.

[0143] Figure 5 The fitting coefficients of equation 1 and equation 2 are shown for the particle sizes of 3 microns, 4 microns, and 5 microns. The R2 factor of each curve clearly shows that the fitting coefficients can calculate the position of the particle in the vertical direction and the diameter of the particle well. For particles of 4 microns, 5 microns, and 6 microns, the coefficients of the two equations do not change much. Figure 6 The fitted equations 1 and 2 and the true values ​​of A2 and P are shown.

[0144] The current difference between the first electrode pair and the third electrode pair of the microfluidic device is found using a differential amplifier (DA), and the current difference between the fourth electrode pair and the third electrode pair is found using another differential amplifier, such as Figure 7 shown. Figure 7 The 1k resistor shown in Figure 1 is connected to ground and the bottom electrode surface, acting as a voltage divider in combination with the electrode pair, but generally the value of the resistor can vary. However, the difference in the current flowing between the first electrode pair and the third electrode pair and between the fourth electrode pair and the second electrode pair can also be found using a transimpedance amplifier and a differential amplifier, such as Fig.12 Another advantage of using a current electrode configuration is that the voltage offset caused by poor alignment of the top and bottom electrodes is reduced. The current difference between the first pair and the fourth pair of electrodes is non-zero. However, the double difference between the two different electrode pairs reduces the offset value and improves the sensitivity of the microfluidic instrument. Figure 2The factor "M" shown in Figure 1 can be increased or decreased depending on the edge spacing between two consecutive pairs of electrodes. Increasing the edge spacing will reduce "M" and when the edge spacing is greater than 5 times the width of the electrode, each electrode will be isolated from each other. Isolation here means that there is no cross current flowing through alternate electrodes.

[0145] The reduced edge-to-edge spacing causes some of the current to flow from the top electrode pair to the alternating bottom electrodes. By reducing the edge spacing between two consecutive pairs of electrodes, the cross-flowing current can be increased. This concept is not limited to four pairs of electrodes, but can also be applied to more than four pairs of electrodes.

[0146] In another embodiment of the present invention, the microfluidic instrument uses five pairs of electrodes, such as Figure 8 The center electrode is connected to a 1k resistor connected to ground. The impedance signal of the particle is calculated using the wiring scheme shown in the figure. For a given geometry, the fitting coefficients of Equation 1 and Equation 2 are as follows Fig. 9 shown. Figure 2 The impedance signal shown in is for a single frequency; however, in general, a sum of frequencies can be applied to the electrode surface and the concept is not limited to a single frequency. This technique not only provides information about the position of the particle in the Z direction, but also improves the sensitivity of the entire system; thus, enabling the microfluidic instrument to measure particles smaller than 1 micron in relatively wide channels.

[0147] The electrode size and spacing of the current instrument are known, and the transit time between the two peaks can be calculated from the known signal sampling rate. For the current microfluidic instrument, these two information data items can be used to calculate the velocity of the particle. In the hard microfluidic channel, the velocity exhibits a parabolic velocity distribution. Equation 3 can be used with the current instrument to calculate the Y position of the particle.

[0148] Another application of the instrument of the present invention is to determine the position of the particle in the Y and Z directions, which can provide useful insights into scientific studies of particle dynamics and can therefore serve as an alternative to very expensive particle image velocimeters.

[0149] The relationship of Equation 2 with respect to P and the height of the particle will be different for different electrode geometries. However, for a given geometry, as long as the volume of the detection chamber is much larger than the volume of the particle, Equation 2 will always have minimal dependence on the size of the particle. The current density curve of the instrument of the present invention is shown in Fig.10As shown; the two outermost electrodes show symmetrical behavior, and the two middle electrodes also show symmetrical behavior. The current microfluidic instrument has the same current density lines for the two middle electrode pairs and the same current density for the two outermost electrode pairs. However, not all electrode pairs have the same current density. The four-pair electrode system may not show better sensitivity due to the asymmetric behavior of the field lines. However, its sensitivity is better than the prior art of the first comparative document.

[0150] In another embodiment of the present invention, an eight-pair electrode system is provided to improve sensitivity and impedance correction due to different positions of particles in the Z direction. Fig.11 The inner 4 electrodes are shown, increasing the number of electrodes will make the field lines more symmetrical. The advantage of the current system is that the voltage offset caused by poor electrode alignment and electrode geometry variation is reduced to near zero.

[0151] In another embodiment, the Fig.12 The electronic measurement scheme shown uses a summing amplifier to measure the impedance of the particle. Fig.12 As shown, a sine wave voltage is applied to the top electrode. The currents flowing out of the first two bottom electrodes are added using a first summing amplifier. The currents flowing out of the fourth bottom electrode and the third bottom electrode are added using a second summing amplifier. The outputs of the two summing amplifiers (first and second) are subtracted using a differential amplifier, as shown in FIG. Fig.12 The same measurement scheme can also be applied to the instrument with five pairs of electrodes.

[0152] In another embodiment of the present electronic measurement system, a combination of frequencies is applied. The applied voltage may be: Vsin(ω1t)+Vsin(ω2t)+Vsin(ω3t)+…Vsin(ω n t). where ω1, ω2, ω3 and…ω n is the angular sinusoidal frequency applied to the top electrode.

[0153] In at least one embodiment, an electronic instrument with a plurality of said electrodes is connected to an input voltage signal, said input voltage signal being generated by a phase lock board, and the output signal from said differential amplifier is fed into the input of the phase lock amplifier for demodulation to perform impedance measurements at different frequencies. The impedance signal is modulated by a carrier frequency, said carrier frequency being a sinusoidal frequency voltage applied to said electrodes. The output from said differential amplifier is a modulated signal of said impedance signal carrying said particles and said carrier frequency.

[0154] The signal is demodulated using the lock-in amplifier to obtain the impedance value for each frequency applied to the top electrode. The sinusoidal voltage applied to the top electrode is generated by the lock-in amplifier board. The difference in the current between the two pairs of electrodes is:

[0155]

[0156] Where ΔZ is the impedance change due to the presence of the particle between the two electrodes, and Z is the impedance of the conductive solution. The change in ΔZ depends on the volume, conductivity, and dielectric constant of the particle. The sum of the sinusoidal voltage signals will be applied to the top surface of all electrodes, not just a single electrode surface. It is important to note that the low frequency applied to the electrodes shows information about the cell size, and the high frequency applied to the electrode surface shows information about the dielectric constant of the cell membrane. Figure 7 , 8 The output signal shown in and 12 can be expressed by the following equation 4:

[0157]

[0158] Here "ω" is the carrier angular frequency and ΔZ(t) is the impedance signal due to the particle. The ΔZ(t) signal can be obtained by multiplying the above output signal with the carrier frequency of the same phase and then passing it through a low pass filter to obtain the real part of the ΔZ(t) signal. The imaginary part of the ΔZ(t) signal can be obtained in the same way, but using a 90 degree phase change of the generated carrier frequency when multiplying.

[0159] In at least one embodiment, the geometry defined by the apparatus of the present invention can apply a positive voltage to the first top electrode and the fourth top electrode, while applying a negative potential of the same magnitude to the second top electrode and the third top electrode to obtain the same characteristics (A1, A2, M). All bottom electrodes are virtual grounds. The currents flowing out of the first and third bottom electrodes are added using the first summing amplifier, and the currents flowing out of the second and fourth bottom electrodes are added using another summing amplifier. The difference between the two summing amplifier outputs is found by the differential amplifier to generate the same characteristics (A1, A2, M).

[0160] In at least one embodiment, the geometry defined by the apparatus of the present invention allows for a positive voltage to be applied to the top of the first and second electrodes and a negative voltage to be applied to the third and fourth electrodes. All of the bottom electrodes are virtual grounds. The currents flowing from the first and fourth bottom electrodes are summed using the first summing amplifier, and the currents flowing from the second and third bottom electrodes are summed using another summing amplifier. The difference between the two summing amplifier outputs is found by the differential amplifier. However, the output resulting from the particle flow will appear first as maximum-minimum-maximum-minimum or first as minimum-maximum-minimum, which is different from other embodiments.

[0161] The obtained impedance curve can be fitted with two antisymmetric Gaussian functions.

[0162]

[0163] in,

[0164] "sig" is defined here as the width of the peak,

[0165] "u1", "u2", "u3" and "u4" are the locations of the extreme values ​​of the impedance curve.

[0166] "A" is the amplitude of the fitted curve.

[0167] The correlation between the two antisymmetric fit curves and the impedance curve can be obtained. Here, the correlation factor can be used to correlate the signal buried in the noise to improve the counting of smaller particles slightly above or below the noise threshold level.

[0168] In at least one embodiment, the defined geometry of the apparatus of the present invention, the first top electrode and the second top electrode are shorted, and the third electrode and the fourth electrode are shorted to provide two different pads for electrical connection. A positive voltage is applied to the first top electrode and the second top electrode using only a single contact pad, and a negative voltage of the same magnitude is applied to the third top electrode and the fourth top electrode using only a single contact pad.

[0169] In at least one embodiment, the defined geometry of the apparatus of the present invention, the third bottom electrode and the second bottom electrode are shorted, while the first bottom electrode and the fourth bottom electrode will have two different pads. The arrangement has two pads for supplying positive and negative voltages, and three pads for electrical current measurement.

[0170] In at least one embodiment, the defined geometry of the apparatus of the present invention applies a carrier signal to the top electrode, which can be two different frequencies ranging from 500kHz to 30MHz, for determining the size of the particle and the electrical properties of the cell membrane (such as capacitance) to distinguish between particles of the same size but different membrane electrical properties. One such application is to distinguish between small red blood cells and platelets of the same size but different membrane electrical properties.

[0171] In at least one embodiment, the microfluidic instrument with multiple electrode systems of the present invention has side openings for liquid inlet and outlet.

[0172] In at least one embodiment, Figure 1 As shown, the microfluidic instrument with a multi-electrode system of the present invention can also be opened from the top of the system. However, drilling holes from the top requires more preparation time and a precise laser cutting or mechanical drilling system.

[0173] In at least one embodiment, the length of the sensing region of the apparatus of the present invention may be from 30 microns to 600 microns to accommodate multiple pairs of electrodes, and the height of the sensing region may be from 10 microns to 100 microns.

[0174] In at least one embodiment, the width of the sensing area of ​​the instrument of the present invention along the Y direction can be from 40 microns to 100 microns.

[0175] In at least one embodiment, the width of the electrodes within the sensing area may vary from 10 microns to 100 microns.

[0176] In at least one embodiment, the edge spacing between two consecutive pairs of the electrodes can vary from 5 microns to 60 microns.

[0177] In at least one embodiment, the edge spacing between two consecutive electrodes can be varied from 0.25 times to 2 times the width of the electrode to increase Figure 2 The value of "M" is shown.

[0178] In at least one embodiment, the edge spacing between two pairs of consecutive electrodes may be different.

[0179] In at least one embodiment, the length of the microfluidic impedance sensor is less than 6 mm to reduce pressure drop.

[0180] As mentioned above, the microfluidic instrument of the present invention can be used to determine whether the particle is located above or below the center. Figure 2 A difference of A1-A2 shown in >0 indicates that the particle is below the center. A difference of A1-A2 <0 indicates that the particle is above the center.

[0181] Another application of the instrument of the present invention is to distinguish different types of cells of the same size but with different membrane dielectric constants. The current center can also be used to count E. coli and microplastics in wastewater using multiple frequencies.

[0182] The concepts described previously can be extended to more advanced systems such as Fig.13 As shown, due to the presence of the ground electrode, the edge current density is reduced. Fig.13 The presence of the ground electrode shown in reduces the impedance variation, but does not reduce the variation to zero. The current microfluidic instrument can have multiple ground electrodes, but in Fig.13 Only one is shown in Figure 2. The purpose of using the ground electrode is to minimize the variation of A1. The position can be inferred from the values ​​of M and A2. For smaller sized particles, minimizing the variation of A1 is necessary because the poor signal-to-noise ratio (SNR) may not give a high "P" value. (The "P" value may be suppressed below the noise level).

[0183] In at least one embodiment, the flow rate of the sample containing suspended particles in the microfluidic instrument can be constant. Since the flow rate in the channel is parabolic and the flow rate is known, the Y coordinate of the particle can be calculated based on the velocity, which is a function of the Y and Z positions given in Equation 3. Since the electrode size and geometry are known, the velocity of the particle is known. When the signal is captured at a fixed known sampling rate, the time required to pass through the continuous electrode can be found by the position of the two peaks. The Y position calculated according to Equation 3 will give the absolute value of the Y coordinate, not the true value. This means that the particle may be at the +Y or -Y position.

[0184] According to another aspect of the present invention, A2 20MHz / A2 1MHz The ratio of is another important parameter that can be used to distinguish many different types of cells. The multidimensional frequency impedance measurement method can give the conductivity and dielectric constant of the particles by fitting the Maxwell-Wagner equation using the least squares method. The conductivity and dielectric constant values ​​obtained show information about the dielectric constant of the cell membrane wall and the conductivity of the cytoplasm.

[0185] In at least one embodiment, the microfluidic instrument of the present invention can be a disposable instrument integrated in a disposable box for automatic sample preparation required for complete blood cell count (CBC). The electrode material currently used in the instrument can be platinum, gold or silver plated on a glass wafer or a silicon wafer. First, an electrode pattern is made on the wafer, and then a photoresist is spin-coated on the silicon wafer to form an open microfluidic channel. Afterwards, the glass wafer with the electrode pattern is aligned and bonded to the silicon wafer to close the open channel.

[0186] In at least one embodiment, the microfluidic instrument of the present invention can be used to distinguish E. coli from microplastics in wastewater. The frequency combination applied to the top electrode system can distinguish cells from hard particles based on the difference in dielectric constant and conductivity between beads and E. coli. The high frequency applied to the top electrode can penetrate the cell membrane of bacteria, thereby better distinguishing beads from bacteria. The microfluidic instrument of the present invention can be used for continuous online water quality measurement in water filtration processes.

[0187] In at least one embodiment, the present microfluidic instrument can use a combination of two frequencies or apply more than two frequencies to distinguish live cells from dead cells. The present microfluidic instrument can be used in the wine industry to distinguish live yeast from dead yeast in wine during fermentation. Continuous online measurement of live yeast and dead yeast can monitor the progress of fermentation throughout the wine or beer production process.

[0188] In at least one embodiment of the present invention, the microfluidic instrument can also be used to count somatic cells in milk. Somatic cell count is an indicator of milk quality. A cell count greater than 250,000 cells / ml in milk indicates the presence of pathogens in the milk.

[0189] In at least one embodiment, the width of the channel can be at least four times its height. The high aspect ratio of the channel focuses the particles at the Z=0 plane when they are suspended in a viscoelastic liquid. The fluid can be made viscoelastic by adding polyacrylic acid (PAA) particles with a molecular weight greater than 5000 kDa. Viscoelastic focusing reduces the variation in the amplitudes of A1 and A2, which improves the accuracy of the entire system in measuring the particle diameter.

[0190] The apparatus and method of the present invention can measure the position of the particle, which can then be corrected for the diameter of the particle. The advantage of the present invention is that the amplitude of "M" is much higher than the amplitude of the secondary peak disclosed in Patent 1. Therefore, the signal-to-noise ratio (SNR) of the signal "M" is much higher.

[0191] In addition, compared with the second prior art comparative document, the present invention improves the sensitivity of the microfluidic instrument.

[0192] The electronic measurement system in the present invention is much simpler than the second prior art, and only requires a voltage source with the same phase and the same frequency. The present invention also discusses how to measure the position of the particle in the "Y" direction and the "Z" direction. The first prior art discloses a method of measuring the position of the particle in the Z direction by correlating the simulation data after finite element analysis (FEM) with the experimental original data. Its main disadvantage is that it requires huge computing power. However, in the present invention, the values ​​of "M", "A1" and "A2" can be easily used to determine the position of the particle in the Z direction. The present invention is computationally economical.

[0193] Using three differential amplifiers can reduce the voltage offset caused by electrode misalignment.

[0194] Using three differential amplifiers improves the signal-to-noise ratio (SNR) of the signal by rejecting common-mode noise in the same electrode.

[0195] More than four electrodes, preferably eight electrodes, are used, wherein the innermost four electrodes make the electric field lines more symmetrical than the second prior art. The reduction of voltage offset is significantly improved compared to the second prior art. The present invention is completely different from the first prior art and the second prior art. The potential will be applied to all eight pairs of electrodes, which will produce more than six peaks (extreme values), but only the innermost six peaks (maxima and minima) will be used to determine the peak positions.

[0196] The technical advancement of the present invention is to provide an apparatus and method, wherein:

[0197] The vertical position of the particle can be accurately calculated; and

[0198] After this, the exact volume of the particles can be measured.

[0199] In addition, the system and the instrument also improve the detection sensitivity of particle counting less than 1 micron.

[0200] Although this detailed description has disclosed certain specific embodiments for illustrative purposes, those skilled in the art will appreciate that various modifications will not depart from the spirit and scope of the present invention, as defined in the following claims. It should be clearly understood that the foregoing descriptive content is only intended to illustrate the present invention and should not be regarded as limiting it.

Claims

1. A microfluidic impedance cytometer for determining the position of particles in a particle-carrying liquid and measuring impedance, characterized in that: include: A microfluidic impedance flow channel for allowing the flow of the liquid; an upstream portion for directing the liquid in a predetermined direction; a downstream portion for directing the liquid in a predetermined direction; A sensing region, for receiving the guided liquid between the upstream portion and the downstream portion, for sensing one or more parameters of the liquid, the sensing region comprising one or more electrode pairs, each electrode pair forming a current path from an operating top to an operating bottom, each electrode pair consisting of an operating top electrode and an operating bottom electrode, a voltage being applied to the operating top electrode, the electrodes in the electrode pair being aligned in parallel and symmetrical to each other, the same positive voltage being applied to each of the top electrodes, each bottom electrode being virtually grounded, and for the electrode pairs: The distance between the edges of adjacent electrodes is at least 0.5 times the width of the electrodes, but the maximum distance is 5 times the width of the electrodes; wherein the width of the channel is at least 1.5 times the height of the channel; Amplifier structure, where: a first summing amplifier, configured to add current values ​​flowing from the first bottom electrode and the second bottom electrode to obtain a first summing value; a second summing amplifier for adding the current values ​​flowing from the third bottom electrode and the fourth bottom electrode to obtain a second summed value; a first differential amplifier, configured to obtain a first difference value using the first sum value and the second sum value within a defined time period, wherein the difference value includes at least three local maximum values ​​and three local minimum values; The change in current between the electrodes is a function of the position of the particle in the vertical direction and the volume of the particle in the liquid, wherein the vertical direction is the Z direction, and when the liquid containing the particle passes through at least one pair of the electrodes in the sensing electrodes, the change in current will be caused; and For a given applied pressure of the liquid, the velocity of the particle is a function of the position of the particle in the width direction, which is the Y direction, and in the vertical direction, which is the Z direction.

2. The microfluidic impedance cytometer according to claim 1, characterized in that: Each of the bottom electrodes is connected to a fixed precision resistor and the bottom electrode is grounded to act as a voltage divider, i.e., Each pair of electrodes is connected in series with a fixed resistance value, and the electrode pair is grounded; The second differential amplifier is configured to output a second difference in voltage between the first electrode pair and the third electrode pair; The third differential amplifier is configured to output a third difference in voltage between the second electrode pair and the fourth electrode pair; The fourth differential amplifier is configured to output a fourth difference between the second difference and the third difference.

3. The microfluidic impedance cytometer according to claim 1, characterized in that: The sensing area includes at least one detection chamber, which is defined as a region volume between the top electrode and the corresponding bottom electrode, and the top electrode and the corresponding bottom electrode together constitute a pair of electrodes.

4. The microfluidic impedance cytometer according to claim 1, characterized in that: When the electrode pairs are odd-numbered pairs, the central electrode is grounded.

5. The microfluidic impedance cytometer according to claim 1, characterized in that: The width of the sensing area is at least 1.5 times the height of the sensing area in order to focus the particles on an operating level, which is the Z plane.

6. The microfluidic impedance cytometer according to claim 1, characterized in that: The width of the channel is 4 times the height of the sensing area in order to align the particles in the operating level, which is the Z plane.

7. The microfluidic impedance cytometer according to claim 1, characterized in that: A viscoelastic fluid is included to focus the particles in the Z plane.

8. The microfluidic impedance cytometer according to claim 1, characterized in that: Each of the operating top electrodes is disposed on the operating top wall of the microfluidic impedance cytometer, so that the electrode is in communication with the liquid, and the liquid is used to pass through the impedance flow channel.

9. The microfluidic impedance cytometer according to claim 1, characterized in that: Each of the operating bottom electrodes is disposed on the operating bottom wall of the microfluidic impedance cytometer, so that the electrodes are connected to the liquid, and the liquid is used to pass through the impedance flow channel.

10. The microfluidic impedance cytometer according to claim 1, characterized in that: Each of the electrodes is made by coating a photoresist on a platinum electrode deposited on a silicon wafer or a glass wafer, wherein the photoresist is SU-8 photoresist and the material of the electrode is platinum or gold, so that all electrodes are in contact with the flowing liquid.

11. The microfluidic impedance cytometer according to claim 1, characterized in that: From the three local maxima and the three local minima, The difference between the third extreme value and the fourth extreme value results in a first peak value, which has the value "A1"; The difference between the first extreme value and the sixth extreme value results in a second peak value, which has the value "A2"; The difference between the second extreme value and the fifth extreme value gives a third peak value, which has a value of "M"; therefore, The amplitude difference between the "A1" value and the "A2" value can determine whether the particle in the liquid is above or below the center value; A positive difference between the "A1" value and the "A2" value can determine whether the particle in the liquid is above or below the center value; and A negative difference between the "A1" value and the "A2" value can determine whether the particles in the liquid are above or below the center value.

12. The microfluidic impedance cytometer according to claim 1, characterized in that: Each of the electrodes is connected to an input voltage signal, which is generated by a phase-locked board. The output signal from the differential amplifier is fed to the input of the phase-locked amplifier for demodulation required for impedance measurement at different frequencies. The carrier frequency modulates the impedance signal, which is a voltage with a sinusoidal frequency applied to the electrode. The output of the differential amplifier is the impedance signal carrying the particles and the modulated signal of the carrier frequency. The signal is demodulated using the phase-locked amplifier to obtain the impedance value at each frequency applied to the top electrode.

13. The microfluidic impedance cytometer according to claim 1, characterized in that: Positive voltages are applied to the first top electrode and the fourth top electrode, and negative voltages are applied to the second top electrode and the third top electrode, so as to obtain the three local maximum values ​​and the three local minimum values.

14. The microfluidic impedance cytometer according to claim 1, characterized in that: A positive voltage is applied to the first top electrode and the fourth top electrode, a negative voltage of the same magnitude is applied to the second top electrode and the third top electrode, and all the bottom electrodes are virtually grounded, wherein the first summing amplifier is used to sum the currents flowing out of the first bottom electrode and the third bottom electrode, another summing amplifier is used to sum the currents flowing out of the second bottom electrode and the fourth bottom electrode, and then a differential amplifier is used to find the difference between the two summing amplifier outputs to generate an output value related to the following: Min-Max-Min, Max-Min-Max, and / or Maximum-minimum-maximum-minimum-maximum-minimum, The output value has a higher sensitivity to provide the position of the particle and perform impedance correction.

15. The microfluidic impedance cytometer according to claim 1, characterized in that: A positive voltage is applied to the first and second top electrodes, a negative voltage is applied to the third and fourth top electrodes, and all bottom electrodes are connected to a resistor or a virtual ground to measure the sum of the currents flowing into the first and fourth bottom electrodes, and the sum of the currents flowing into the second and third bottom electrodes, and then the two summed currents are subtracted using the differential amplifier to generate an output value related to: Max-min-max-min, min-max-min-max, Wherein, the output value improves the detection limit.

16. The microfluidic impedance cytometer according to claim 1, characterized in that: The first top electrode and the second top electrode are short-circuited and a positive voltage is applied to the first top electrode, the second top electrode uses only a single contact pad, the third top electrode and the fourth top electrode are electrically connected using two different contact pads, and the third top electrode and the fourth top electrode use only a single contact pad to apply a negative voltage of the same amplitude.

17. The microfluidic impedance cytometer according to claim 1, characterized in that: The third bottom electrode and the second bottom electrode are short-circuited, the first bottom electrode and the fourth bottom electrode are electrically connected using two different pads, a total of three pads are used for current measurement, and all the bottom electrodes are virtually grounded or connected to a resistor.

18. The microfluidic impedance cytometer according to claim 1, characterized in that: At least one of the operating top electrodes is applied with a carrier signal having at least two different frequencies, wherein the frequency of the carrier signal ranges from 500kHz to 30MHz to determine the size of the particles and the electrical properties of the particle film, including capacitance, thereby distinguishing particles of the same size but different film electrical properties.

19. The microfluidic impedance cytometer according to claim 1, characterized in that: The microfluidic impedance cytometer has eight pairs of electrodes, among which the innermost four pairs of symmetrical electrodes are used for measurement.

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