Microfluidic impedance cytometry device

Abstract

A microfluidic impedance cytometry device for determining the position and impedance measurement of particles per second in a fluid carrying the particles, comprising: a microfluidic impedance flow channel for enabling the flow of the fluid; an upstream section; a downstream section a sensing region for receiving the fluid for sensing one or more parameters of the fluid, the sensing region including one or a plurality of pairs of electrodes, each pair forming a current path from an operating side upper part to an operating side lower part, each of the pairs being formed by an operating side upper electrode and an operating side lower electrode, a sensing region, a potential being applied to the operating upper electrode, each electrode of a particular pair being aligned in parallel, being symmetric with respect to each other, the same positive potential being applied to each of the upper electrodes, each of the lower electrodes being substantially grounded with respect to the pair, and a configuration of an amplifier.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering. In particular, the present invention relates to the field of cytometry. Specifically, the present invention relates to a microfluidic impedance cytometry device.

Background Art

[0002] Impedance cytometry is the measurement of the dielectric properties of cells using single or multiple frequencies. Recent advances in impedance cytometry research have shown that different types of cells present in human blood can be distinguished using multiple frequencies. The advantage of impedance cytometry is that the magnitude of impedance is not very dependent on the position of the particles. In cytometry using an optical system, sheath flow is required to keep the cells in the center of the channel, resulting in a large amount of fluid waste, a complex and bulky system, and high costs. Cytometry is the enumeration of various types of cells and other characteristics, such as the size of cells, the presence or absence of proteins present in the cell membrane, and the difference in the shape of the nucleus present in white blood cells. However, impedance cytometry has the advantage that the signal is least dependent on the position of the particles compared to optical measurements. Therefore, in impedance cytometry, measurement is possible without sheath fluid. This technological innovation is advantageous when constructing a complete blood count (CBC) device at the point of care (PoC) because the size of the device is significantly reduced. Cells with membranes of different electrical conductivities can be distinguished using multiple frequencies. This technology discloses a technological innovation that can perform accurate impedance measurements of cells using a plurality of electrodes embedded in the upper and bottom walls of a microfluidic channel. According to the first prior art citation [US10267720], it is stated that two ground electrodes are used between each measurement electrode and the signal electrode. In this citation, the signal electrode is an operable upper electrode to which a potential is applied, and the measurement electrode is an operable lower electrode that is substantially grounded (at 0V). The flow of current from the signal electrode to the measurement electrode is measured. The difference between two measurement electrodes gives an antisymmetric peak signal with a secondary peak between the global maximum and global minimum of the peak. Here, a peak is defined as an electronic signal obtained by the passage of particles from the first electrode to the last electrode. The electronic signal varies depending on the position of the particle in the Z direction (where the defined axis is shown) if the particles are of the same size. 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. Since the current density is non-uniform in the Z direction, the electronic signal varies depending on the position in the Z direction. The current density is relatively higher near the ends of the electrodes and relatively lower in the central part of the channel. According to the first prior art citation [US10267720], the ratio of the primary peak to the secondary peak has been discussed, and this ratio can be used to correlate the vertical position of the particle and then correct the diameter of the particle. The drawback of the first prior art citation is that the size of the secondary peak is very small. For particles with a small size of 4 microns or less, in the case of a cross-section of 30μm x 30um and an electrode width of 30μm, the secondary peak is almost buried in the noise. Even when the separation from end to end of two consecutive electrodes is 10 microns or less, the signal-to-noise ratio (SNR) of the secondary peak is not good. Furthermore, by reducing the height of the channel, the size of the secondary peak can be improved. However, in this case, there is a possibility that small particles may block the detection region, increasing the likelihood of clogging of the microfluidic channel. The second prior art [US20220034781A1] discusses "improving the sensitivity of a microfluidic device" for counting particles of 2 microns or less. The main use of the second prior art is to count bacteria of 1 micron or less in a wider channel. The electrodes fabricated with the first prior art always have a certain degree of misalignment and slight variations in the electrode shape. Therefore, the peak is not exactly asymmetric, and the offset current is not zero. The offset current is defined as the difference in current coming from the lower parts of two signal electrodes when there are no particles between the electrodes. Since the offset current is not zero and becomes larger when subtracting the two thermal noises coming from the two lower signal electrodes, impedance measurement of submicron-sized particles becomes difficult. The second prior art discloses the idea of applying two equal voltages with a 180-degree phase difference at the same frequency. Two negative voltages are applied to any two upper electrodes, and two positive voltages are applied to the other two upper electrodes. Two currents with a 180-degree phase angle are summed using an adder amplifier, and similarly, the other two currents are summed from the other two pairs of electrodes in the same way. The outputs from the two adder amplifiers are subtracted using a differential amplifier (DA). Since the current is subtracted three times, the value of the offset voltage (the final output from the DA when there are no particles) is always smaller compared to the first prior art. Ideally, when there is no misalignment and the shapes of the two electrodes are the same, the offset voltage should be zero. Citation Document 2 does not disclose a method for determining the position of particles. FEM analysis shows that the impedance peak signal changes depending on the position of the particles in the Z direction. The first prior art citation document and the second prior art citation document are completely different because the voltage is in the same phase in the first prior art citation document, while the second prior art citation document uses voltages of opposite magnitudes. Therefore, the citation of the first prior art and the citation of the second prior art cannot be combined. Also, in the second prior art, since the two electric fields are not exactly the same, the value of the sum of the out-of-phase currents is not theoretically zero. If the shape and separation of the electrodes are the same for all four sets of electrodes, the first and last electrodes have the same electric field distribution, and the two middle electrodes have the same electric field distribution. There is a need for an apparatus and method that can measure the position of particles and then correct the diameter of the particles. In this technical field, methods for determining the position of particles in the Y and Z directions are discussed. The advantage of the present invention is the magnitude of 'M' defined in the following section, which is much larger than the magnitude of the secondary peak disclosed in Patent Document 1. Furthermore, it is required that the sensitivity of the microfluidic device be improved compared to the citation of the second prior art. The present invention does not use a ground electrode and a voltage source of opposite phase. The electronic measurement system of the present invention is not more complex than the second prior art, which only requires a single voltage source of the same phase and the same frequency. Also, the present invention discusses a method for measuring the position of particles in both 'Y' and 'Z'. The first prior art discloses a technique for measuring the position of particles in the Z direction by comparing simulation data and experimental data after FEM analysis. The main drawback is that it requires a huge computational power. However, with the current technology, the position of particles in the Z direction can be easily obtained using the values of "M", "A1", and "A2" defined in the following section. This technology has the lowest computational cost. By using four or more electrodes, preferably seven electrodes, four inner electrodes are used where the electric field lines are more symmetric compared to the second prior art. The reduction of the offset voltage is much improved compared to the second prior art. The present invention is completely different from the first prior art and the second prior art. Object of the invention

[0003] An object of the present invention is to provide a system, apparatus, and method for accurately measuring the impedance of particles or cells within a microfluidic impedance cytometry device using single or multiple frequencies. Another object of the present invention is to provide a system, apparatus, and method capable of accurately calculating the operable vertical position of a particle (as seen in FIG. 14, the vertical position is the Z-axis), and then measuring the exact volume of the particle. Yet another object of the present invention is to provide a system, apparatus, and method for improving the detection sensitivity for counting particles less than 1 micron. Summary of the Invention

[0004] According to the present invention, a microfluidic impedance cytometry device is provided. In at least one embodiment of the device, the microfluidic impedance flow channel is composed of at least an inlet and at least an outlet opening to allow the flow of a conductive liquid, and the liquid can carry non-conductive particles or particles having a conductivity and permittivity different from that of the liquid, or particles having a conductivity or permittivity different from that of the liquid. In at least one embodiment of the present device, the device is composed of at least one upper operating electrode provided on the upper wall of the device and at least one lower operating electrode provided on the lower wall of the device, and a potential is applied to these upper electrodes. Preferably, the device is composed of a plurality of upper electrodes provided on the upper wall and a plurality of lower electrodes provided on the bottom wall, which are in contact with a conductive liquid carrying non-conductive particles. The corresponding upper and lower electrodes form one or more pairs that provide a current path flowing from top to bottom. When a conductive liquid containing non-conductive particles passes between the formed electrode pairs, the electric field lines are disturbed, and as a result, the current flowing from top to bottom changes. The change in current is directly proportional to the volume of the particles. However, the current density between each pair is non-uniform. Therefore, the change in current depends on the vertical position of the particles. Since the current density is stronger closer to the ends of the electrodes, the closer the particles are to the ends of the electrodes, the greater the change in current. The present invention discloses the following mechanisms and methods: - The vertical position of the particles is accurately calculated; and - Then, the exact volume of the particles is measured. - Then, the position of the particles in the width direction is measured. The present invention consists of at least four pairs of electrodes for particle position and impedance measurement. Furthermore, this system and device also improve the detection sensitivity for counting particles smaller than 1 micron. According to the present invention, there is provided a microfluidic impedance cytometric device for particle positioning and impedance measurement in a fluid carrying particles, the device comprising: - A microfluidic impedance flow channel that enables the flow of the fluid; - An upstream section configured to flow the fluid in a predefined direction; - A downstream section configured to flow the fluid in a predefined direction; - A sensing region configured to receive the flowed fluid and sense one or more parameters of the fluid, between the upstream section and the downstream section, the sensing region including one or more sets of electrode pairs, each pair forming a current path from an operating upper part to an operating lower part, each of the pairs being formed by an operating upper electrode and an operating lower electrode, a potential being applied on the operating upper electrode, each electrode for a particular pair being aligned parallel to and symmetric with each other, the same positive potential being applied to each of the upper electrodes, and each of the lower electrodes being substantially grounded with respect to the pair; ○ Here, the end-to-end spacing between adjacent electrodes is at least 0.5 times the electrode width, but the maximum spacing is 5 times the electrode width; 〇 Here, the width of the channel is at least 1.5 times its height; - A configuration of an amplifier 〇 A first summing amplifier configured to sum the current values flowing from a lower first electrode and a lower second electrode to obtain a first summing value; A second summing amplifier configured to sum the current values flowing from a lower third electrode and a lower fourth electrode to obtain a second summing value; 〇 A first differential amplifier configured to obtain a first differential value consisting of at least three local maximum values and three local minimum values over a defined time using the first summing value and the second summing value; ■ A function of the position of particles in the vertical direction (Z direction) between electrodes and the volume of the particles in the fluid in the current, causing the change in the current when the fluid containing the particles passes through the at least one pair of electrodes of the detection electrodes; and The velocity of the particles is a function of the position of the particles in the width direction (Y direction) and the vertical direction (Z direction) with respect to a predetermined applied pressure of the fluid. In at least one embodiment, - Each lower electrode is connected to a fixed precision resistor grounded to function as a voltage divider, wherein each pair of electrodes is connected in series with a grounded fixed resistance value; - A second differential amplifier configured to output a second difference value between voltage values of the first electrode pair and the third electrode pair; - A third differential amplifier configured to output a third difference value between the voltage value of the second pair of electrodes and the voltage value of the fourth pair of electrodes; - A fourth differential amplifier configured to output a fourth difference value between the second difference value and the third difference value. In at least one embodiment, the detection region includes at least a detection chamber defined as the volume of the region between an upper electrode forming a pair of electrodes together with a corresponding lower electrode. In at least one embodiment, the set of pairs of electrodes is odd, and at this time, the central electrode is grounded. In at least one embodiment, the width of the sensing region is at least 1.5 times the height of the sensing region to focus the particles on the operating horizontal plane (Z-plane) at a higher flow rate. In at least one embodiment, the width of the channel is 4 times the height of the sensing region to align the particles on the operating horizontal plane (Z-plane). The device according to claim 1 includes a fluid having viscoelasticity for focusing particles in the Z-plane. In at least one embodiment, each of the operating upper electrodes is provided on the operating upper wall of the device so as to communicate with the fluid configured such that the electrode passes through the impedance flow channel. In at least one embodiment, each of the operating lower electrodes is provided on the operating lower wall of the device so as to communicate with the fluid configured such that the electrode passes through the impedance flow channel. In at least one embodiment, each of the electrodes is fabricated by coating a platinum electrode deposited on a silicon or glass wafer with a photoresist (such as SU-8), and the electrode material is platinum or gold such that they are all in contact with the fluid flowing therethrough. In at least one embodiment, from the three local maxima and three local minima, - The difference between the third and fourth extrema gives a first (main) peak value with an 'A1' value; - The difference between the first and sixth extrema gives a second peak value with an 'A2' value; - The difference between the second and fifth extrema gives a third peak value with an 'M' value; In this way, ■ The amplitude difference between the 'A1' value and the 'A2' value provides a determination as to whether the particles in the fluid are above or below the central value; ● A positive difference between the 'A1' value and the 'A2' value provides a determination that the particles in the fluid are above or below the central value; and ● A negative difference between the 'A1' value and the 'A2' value provides a determination that the particles in the fluid are above the central value. In at least one embodiment, each of the electrodes is connected to an input voltage signal, the input voltage signal is generated from a lock-in board, the output signal from the differential amplifier is supplied to the input of the lock-in amplifier for demodulation required for impedance measurement at different frequencies, the impedance signal is modulated at a carrier frequency, the carrier frequency is a sine wave voltage applied to the electrodes at a sine wave frequency on the electrodes, the output of the differential amplifier is a modulated signal carrying the impedance signal of the particles and the carrier frequency, and the demodulation of the signal is performed using the lock-in amplifier to obtain the impedance value at each applied frequency applied to the upper electrode. In at least one embodiment, a positive voltage is supplied to the first and fourth upper electrodes and a negative voltage is supplied to the second and third upper electrodes in order to obtain three local maxima and three local minima. In at least one embodiment, a positive voltage is supplied to the first upper electrode and the fourth upper electrode, a negative potential of the same magnitude is supplied to the second upper electrode and the third upper electrode, all the lower electrodes are substantially grounded, the currents flowing from the first lower electrode and the third lower electrode are added by a first summing amplifier, the currents flowing from the second lower electrode and the fourth lower electrode are added by another summing amplifier, the difference between the outputs from the two summing amplifiers is obtained by a differential amplifier, and a minimum-maximum-minimum or a maximum-maximum-maximum-maximum is generated. This configuration not only increases the sensitivity but can also be used to find the position of the particle, and then impedance correction can be performed. In at least one embodiment, a positive voltage is supplied to the first upper electrode and the second upper electrode, a negative voltage is supplied to the third upper electrode and the fourth upper electrode, and for measuring the sum of the currents flowing through the first lower electrode and the fourth lower electrode and the sum of the currents flowing through the second first lower electrode and the third lower electrode, all the lower electrodes are connected to resistors or substantially grounded, and then a differential amplifier is used to subtract the two summed currents. This configuration generates a maximum-minimum-maximum-minimum, or a minimum-maximum-minimum-maximum. This configuration increases the limit of detection (LOD) but cannot be utilized to find the position of the particle. In at least one embodiment, the first upper electrode and the second upper electrode are short-circuited with a positive voltage applied to the first upper electrode and the second upper electrode using only a single contact pad, the third upper electrode and the fourth upper electrode are provided with two different pads for electrical connection, and the third upper electrode and the fourth upper electrode are applied with a negative voltage of the same magnitude using only a single contact pad. In at least one embodiment, the third lower electrode and the second lower electrode are short-circuited, the first lower electrode and the fourth lower electrode have two different contact pads for current measurement and have a total of three contact pads, and in this case, all the lower electrodes are substantially grounded or connected to resistors. In at least one embodiment, at least one of the upper operating electrodes is applied with a carrier signal having at least two different frequencies in the range of 500 kHz to 30 MHz to find electrical cell membrane properties such as capacitance for distinguishing the size of the particles and particles having the same size but different membrane electrical properties. In at least one embodiment, the device has eight pairs of electrodes, and only the innermost symmetric four pairs of electrodes are used for measurement. In some embodiments, the microfluidic flow channel has a glass upper surface and a silicon lower surface. The side walls of the microfluidic device are made of photoresist. In some embodiments, the length of the detection region can be from 30 microns to 600 microns for counting micron-sized particles less than 20 microns. In some embodiments, the height of the sensing region can be from 10 microns to 100 microns. In some embodiments, the width of the electrode can be from 10 microns to 30 microns for counting sub-micron particle sizes. In some embodiments, the width of the sensing region can be from 30 microns to 200 microns. In some embodiments, the number of electrode pairs is at least four pairs, but can be increased to seven pairs. The dimensions of all pairs of electrodes are the same. In at least one embodiment, the prominence of the impedance signal (final output signal) is calculated as (1 - M / A2). The value of the prominence is well correlated with the position of the particles in the vertical Z direction. The prominence is well correlated with P = c1 + c2(2Z / H)^2 + c3(2Z / H)^4. c1', 'c2', 'c3' are known for a fixed known geometry. In at least one embodiment, the true diameter of the particle is obtained by substituting the Z position into the formula A2 = Gd^3(1 + a1(2Z / H) + a2(2Z / H)^2). 'd' is the diameter of the particle, 'Z' is the vertical position of the particle, and 'G' is the gain coefficient depending on the electronic circuit and the electrode shape.

Brief Description of the Drawings

[0005] Next, the present invention will be described in relation to the accompanying drawings: Figure 1(a) shows four pairs of electrodes with a width of 10 microns and a distance of 20 microns between the ends. The current difference between the first and third pairs of electrodes is obtained using a differential amplifier DA, and the current difference between the second and fourth pairs of electrodes is obtained using another DA. The outputs from the two DAs are subtracted using a third DA; Figure 1(b) is a top view of the electrodes, and there are three different regions: an upstream region, a detection region, and a downstream region. The current from the lower electrode is measured using a Wheatstone bridge and a differential amplifier as shown in Figure 1(a); Figure 2 shows the amplitude change of the particle (the final output from the third DA) with respect to the x coordinate of the particle. The impedance signal shown in the figure is for the case where the particle diameter is 6 microns and Z = -6 microns in a microfluidic chip with a height of 30 μm and a width of 30 μm. The center of the channel is located at (0,0,0). The amplitudes of 'A1', 'A2', and M are indicated by black double-headed straight arrows; Figure 3 shows the impedance signals of 6-micron particles at various Z positions. The unit of the impedance signal is microampere, and the unit of the Z position is micron; Figure 4 shows that the X-axis is the Z position of the particle and the Y-axis is the prominence defined by Equation 2 described later; Figure 5 shows the fitting coefficients according to the difference in particle diameter for the equation defined in the later section in Table 1; Figure 6(a) shows that the solid line represents the fitting formula 1, and 'o' represents the true value of A2 after FEM analysis for different vertical positions (b). The solid line represents the fitting formula 2, and 'o' is the discrete point representing the true value of the prominence described by Equation 2a after FEM analysis for different vertical positions; FIG. 7 shows the difference between the first pair and the third pair obtained using a differential amplifier, and similarly the difference between the fourth pair and the second pair; FIG. 8 shows five pairs of electrodes demonstrating the concept of electrode wiring and the principle of current measurement for calculating the Z position of particles; FIG. 9 shows Table 2 indicating the fitting coefficients of both Equation 1 and Equation 2a for different particle sizes with respect to the electrode shape shown in FIG. 8; FIG. 10 shows the current density magnetic field lines of a four - pair electrode device system. The current density of the fourth electrode pair shows a change when the particle is between the fourth electrode pair; FIG. 11 shows a seven - pair electrode system demonstrating the electronic setup - impedance measurement of particles. The inner five pairs of electrodes are used for the measurement; FIG. 12 shows the sum of the currents flowing from the first electrode pair and the second electrode pair using an adder amplifier with the same voltage applied to the upper electrode. The currents flowing from the third electrode pair and the fourth electrode pair are summed using another adder amplifier. The outputs from the two adder amplifiers are subtracted using a differential amplifier to obtain the same output as shown in FIG. 2; FIG. 13 shows a five - pair electrode system in which the central electrode pair is grounded; FIG. 14 is a cross - sectional view of the sensing region. The axes are labeled as shown in the figure. The peak amplitude varies with the Z - coordinate but is independent of the Y - coordinate. The fluid flow is in the X - direction. Detailed description of the accompanying drawings

[0006] According to the present invention, a micro - fluidic impedance cytometry device is provided. The present invention relates to accurate impedance measurement of particles at multiple frequencies. Current applications of such devices are mainly for counting blood cells, counting bacteria such as Escherichia coli, impedance spectroscopy of cancer cells for deriving electrical parameters such as the conductivity or permittivity of particles, and finding the electrical properties of cell membranes. In at least one embodiment of the device, the micro - fluidic impedance flow channel consists of at least an inlet and at least an outlet opening to allow the flow of a conductive liquid, which contains non - conductive particles. In at least one embodiment of the present device, the present device is composed of at least one upper operating electrode provided on the upper wall of the present device and at least one lower operating electrode provided on the bottom wall of the present device, and a potential is applied through these electrodes. Preferably, the present device is composed of a plurality of upper electrodes provided on the upper wall and a plurality of lower electrodes provided on the bottom wall, and these electrodes are in contact with a conductive liquid carrying non-conductive particles. The corresponding upper and lower electrodes form one or more pairs that provide a current path flowing from the upper part to the lower part. When a conductive liquid containing non-conductive particles passes between the formed electrode pairs, the electric field lines are disturbed, and as a result, the current flowing from top to bottom changes. The change in current is directly proportional to the volume of the particles. However, the current density between each pair is non-uniform. Therefore, the change in current depends on the vertical position of the particles. Since the current density becomes stronger closer to the ends of the electrodes, the closer the particles are to the ends of the electrodes, the greater the change in current. In a preferred embodiment, the microfluidic device of the present invention is fabricated by coating a platinum electrode deposited on a silicon or glass wafer shown in FIG. 1a with a photoresist (SU-8). The electrode material of the device is platinum or gold. In at least one embodiment, the microfluidic device of the present invention has three sections: an upstream section, a sensing section, and a downstream section, as shown in FIG. 1b. In a preferred embodiment, the length of the detection part of the device can vary from 10 microns to 600 microns with at least four pairs of electrodes. The upper and lower electrodes are aligned so that the electric field lines are parallel and symmetric, reducing the error caused by the misalignment of the electrodes. In a preferred embodiment, the width of the electrodes can be from 5 microns to 100 microns, depending on the size of the particles required for counting. The gap from end to end between each pair can be from 5 microns to 60 microns. When the current density is high near the upper and lower electrodes, the magnitude of the impedance signal varies. However, the position of the particles can be calculated from the impedance signal of the particles using three factors, namely 'A2', 'M', and 'A1', as shown in Figure 2 and as described below, and then the impedance value can be corrected. Figure 2 shows a graph of the voltage recorded between the electrodes versus position. This graph shows three maxima and three minima for a particular electrode arrangement. The sensitivity of the device is defined as the ratio of the minimum volume of the particles measurable with respect to the volume of the detection chamber. Here, the detection chamber is defined as the volume of the region between the upper electrode, which together with a corresponding lower electrode forms a pair of electrodes. The dimensions of the electrodes can be made smaller to increase the sensitivity of the device. The sensitivity of the signal discussed in the first prior art is limited due to the following factors: (a) The current-voltage conversion circuit saturates and a high voltage gradient in the height direction deteriorates the electrode material, so a potential above a certain level cannot be applied. (b) Misalignment of the electrodes and variations in the electrode shape always exist, resulting in an increase in the offset voltage of the electronic circuit, an increase in noise, and saturation when the gain of the circuit becomes high. In at least one embodiment, as shown in FIG. 1a, the same potential is applied to all upper electrodes, and all lower electrodes are connected to a fixed resistance value that is grounded. When a particle approaches the wall or enters between the upper and lower electrodes, the current flowing from the upper to the lower electrode changes. Although FIG. 1a shows four pairs of electrodes, generally a plurality of electrodes can be fabricated. For the microfluidic device shown in FIG. 1a, the impedance signals of particles at various heights are shown in FIG. 3, and it is clear that the impedance value is higher as the particle is closer to the end of the electrode. The magnitude of the impedance of the particle denoted as 'A2' is related to the position and diameter of the particle described by Equation 1. A2 = Gd^3(1 + a1(2Z / H) + a2(2Z / H)^2) (1) P = c1 + c2(2Z / H)^2 + c3(2Z / H)^4 (2a) Here, A2 is the magnitude of the impedance of the signal, 'a1', 'a2', 'c1' and 'c2' are fitting parameters, Z is the vertical position of the particle shown in FIG. 14, 'd' is the diameter of the particle, 'G' is the gain of the electronic circuit, 'H' is the height of the channel. The coefficients 'a1' and 'a2' are obtained after least squares fitting because, after solving the Laplace equation in voltage and applying appropriate boundary conditions to the wall of the sensing region, for the known shape of the microfluidic device and the diameter of the particle, A2 and Z are known for various Z positions. Here 'P' is called the prominence defined by Equation 3. P = 1 - M / A2 (2b) The characteristics of the impedance signals showing M and A2 are shown in FIG. 2.

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Claims

1. A microfluidic impedance cytometry device for determining the position and measuring the impedance of particles in a fluid carrying the particles, the device comprising: - A microfluidic impedance flow channel that enables the flow of the fluid; - An upstream section configured to flow the fluid in a predefined direction; - A downstream section configured to flow the fluid in a predefined direction; - A sensing region configured to receive the flowed fluid between the upstream section and the downstream section and to sense one or more parameters of the fluid, the sensing region including one or more sets of electrode pairs, each pair forming a current path from an operating upper part to an operating lower part, each of the pairs being formed by an operating upper electrode and an operating lower electrode, a potential being applied on the operating upper electrode, each electrode for a particular pair being aligned parallel to each other, symmetric, the same positive potential being applied to each of the upper electrodes, and each of the lower electrodes being substantially grounded with respect to the upper electrodes; 〇 Here, the end-to-end spacing of adjacent electrodes is at least 0.5 times the electrode width, but the maximum spacing is 5 times the electrode width; 〇 Here, the width of the channel is at least 1.5 times its height; - A configuration of amplifiers 〇 A first summing amplifier configured to sum the current values flowing from a lower first electrode and a lower second electrode to obtain a first summed value; 〇 A second summing amplifier configured to sum the current values flowing from a lower third electrode and a lower fourth electrode to obtain a second summed value; 〇 A first differential amplifier configured to use the first summed value and the second summed value to obtain a first differential value consisting of at least three local maximum values and three local minimum values over a defined time; ■ Which is a function of the position of the particle in the vertical direction (Z direction) between the electrodes and the volume of the particle in the fluid in the current, and causes the change in the current when the fluid containing the particle passes through the at least one pair of electrodes of the detection electrodes; and 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) with respect to a predetermined applied pressure of the fluid.

2. The device according to claim 1, Each lower electrode is connected to a fixed precision resistor grounded so as to function as a voltage divider, wherein each pair of electrodes is connected in series with a grounded fixed resistance value; A second differential amplifier is configured to output a second difference value between the voltage values of the first electrode pair and the third electrode pair; A third differential amplifier is configured to output a third difference value between the voltage value of the second pair of electrodes and the voltage value of the fourth pair of electrodes; A fourth differential amplifier is configured to output a fourth difference value between the second difference value and the third difference value.

3. The apparatus according to claim 1, wherein the detection region includes at least a detection chamber defined as the volume of the region between an upper electrode forming a pair of electrodes and a corresponding lower electrode together.

4. The apparatus according to claim 1, wherein the number of pairs of electrodes is odd, and in that case, the central electrode is grounded.

5. The apparatus according to claim 1, wherein the width of the sensing region is at least 1.5 times the height of the sensing region in order to focus the particles on the operating horizontal plane (Z-plane) at a higher flow rate.

6. The apparatus according to claim 1, wherein the width of the channel is 4 times the height of the sensing region in order to align the particles on the operating horizontal plane (Z-plane).

7. The apparatus according to claim 1, comprising a fluid having viscoelasticity for focusing particles in the Z-plane.

8. The apparatus according to claim 1, wherein each of the operating upper electrodes is provided on the operating upper wall of the apparatus so as to communicate with the fluid configured such that the electrodes pass through the impedance flow channel.

9. The apparatus according to claim 1, wherein each of the operating lower electrodes is provided on the operating lower wall of the apparatus so as to communicate with the fluid configured such that the electrodes pass through the impedance flow channel.

10. The apparatus according to claim 1, wherein each of the electrodes is fabricated by coating a platinum electrode deposited on a silicon or glass wafer with a photoresist (such as SU-8), and the electrode material is platinum or gold such that they are all in contact with the flowing fluid.

11. The apparatus according to claim 1, from the three local maximum values and three local minimum values, - the difference between the third extreme value and the fourth extreme value gives a first (main) peak value having an 'A1' value; - The difference between the first extreme value and the sixth extreme value gives a second peak value with an ’A2’ value; - The difference between the second extreme value and the fifth extreme value gives a third peak value with an ’M’ value; In this way, ■ The amplitude difference between the ’A1’ value and the ’A2’ value provides a determination as to whether the particles in the fluid are above or below the central value; ● A positive difference between the ’A1’ value and the ’A2’ value provides a determination that the particles in the fluid are above or below the central value; and ● A negative difference between the ’A1’ value and the ’A2’ value provides a determination that the particles in the fluid are above the central value.

12. The apparatus according to claim 1, each of the electrodes is connected to an input voltage signal, the input voltage signal is generated from a lock-in board, the output signal from the differential amplifier is supplied to the input of the lock-in amplifier for demodulation required for impedance measurement at different frequencies, the impedance signal is modulated at a carrier frequency, the carrier frequency is a sine wave voltage applied to the electrodes at a sine wave frequency on the electrodes, the output of the differential amplifier is a modulated signal carrying the impedance signal of the particles and the carrier frequency, and the demodulation of the signal is performed using the lock-in amplifier to obtain the impedance value at each applied frequency applied to the upper electrode.

13. The apparatus according to claim 1, a positive voltage is supplied to the first upper electrode and the fourth upper electrode, and a negative voltage is supplied to the second upper electrode and the third upper electrode in order to obtain three local maximum values and three local minimum values.

14. The apparatus according to claim 1, a positive voltage is supplied to the first upper electrode and the fourth upper electrode, a negative potential of the same magnitude is supplied to the second upper electrode and the third upper electrode, all the lower electrodes are substantially grounded, the currents flowing from the first lower electrode and the third lower electrode are added by a first summing amplifier, the currents flowing from the second lower electrode and the fourth lower electrode are added by another summing amplifier, the difference between the outputs from the two summing amplifiers is obtained by a differential amplifier, generating a minimum value - maximum value - minimum value, maximum value - minimum value - maximum value and / or maximum value - minimum value - maximum value - minimum value - maximum value - minimum value, the output value provides the position of the particles, and the sensitivity is increased for impedance correction.

15. The apparatus according to claim 1, A positive voltage is supplied to the first upper electrode and the second upper electrode, a negative voltage is supplied to the third upper electrode and the fourth upper electrode, and all the lower electrodes are connected to resistors or substantially grounded for measuring the sum of the currents flowing through the first lower electrode and the fourth lower electrode and for measuring the sum of the currents flowing through the second lower electrode and the third lower electrode. Then, two added currents are subtracted using the differential amplifier, and a maximum-minimum-maximum-minimum or minimum-maximum-minimum-maximum output value is generated, and the output value increases the detection limit.

16. The apparatus according to claim 1, wherein the first upper electrode and the second upper electrode are short-circuited with a positive voltage applied thereto using only a single contact pad, the third upper electrode and the fourth upper electrode are provided with two different pads for electrical connection, and the third upper electrode and the fourth upper electrode are applied with negative voltages of the same magnitude using only a single contact pad.

17. The apparatus according to claim 1, wherein the third lower electrode and the second lower electrode are short-circuited, the first lower electrode and the fourth lower electrode have two different contact pads for current measurement, have a total of three contact pads, and all the lower electrodes are substantially grounded or connected to resistors.

18. The apparatus according to claim 1, wherein a carrier signal having at least two different frequencies in the range of 500 kHz to 30 MHz is applied to at least one of the operating upper electrodes to find electrical cell membrane characteristics such as capacitance for distinguishing the size of particles and particles having the same size but different film electrical characteristics.

19. The apparatus according to claim 1, wherein the apparatus has eight pairs of electrodes, and the most inner symmetric four pairs of electrodes are used for measurement.

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