1 School of Electrical and Automation Engineering, Jiangsu Key Laboratory of 3D Printing Equipment and Manufacturing, Nanjing Normal University, Nanjing 210023, China
2 Jiangsu Key Laboratory for Design and Manufacture of Micro-Nano Biomedical Instruments, Southeast University, Nanjing 211189, China
3 Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School,Nanjing 210008, China
Wenlai Tang
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Received
Revised
Published Online
2020-06-05
2020-06-28
2020-11-30
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Abstract
Single-cell level detection can distinguish rare abnormal cells from large cell populations, which plays a vital role in biomedical fields such as early diagnosis and treatment evaluation of diseases. By integrating microfluidics, electrical impedance spectroscopy and flow cytometry, the microfluidic impedance cytometer has attracted extensive attention, as it can realize non-destructive impedance detection of single cells in a continuous way with precise flow control. Compared with traditional single-cell detection methods, microfluidic impedance cytometer has significant advantages such as label-free, multi-parameters, low pollution and high throughput, providing a powerful tool for cell population identification and status monitoring. Therefore, various microfluidic impedance cytometers with different structures and functions have been successfully developed in recent years. In this review, we first summarize the working principles and recent developments of DC impedance cytometer, AC impedance cytometer and deformability impedance cytometer, and then focus on the latest applications of microfluidic impedance cytometer in the detection of biological samples such as blood cells, cancer cells and microorganisms. Later, the application prospect of microfluidic impedance cytometer in clinical real-time detection from the aspects of integration and miniaturization are also presented. Finally, the shortcomings of the existing microfluidic impedance cytometers and the future development trend in this field are discussed.
In recent years, more and more attention has been paid to the analysis of single cells. Traditional cell detection is usually carried out in a group of cells, and the average characteristics of the whole cell population are used to summarize all cells, which conceals the individual differences between single cells[1,2]. The intercellular differences caused by cellular heterogeneity widely exist in the growth process of cells, organs, and even individuals, and have a significant impact on the unit process of life activities[3,4]. The high heterogeneity of cell samples is a great challenge for clinical research and biomedical engineering. In recent years, there has been a great deal of interest in the isolation, enrichment, and enumeration of various types of "rare cells" (i.e., a very small subset of a large population of cells) or bacteria in the blood, in areas such as oncology, stem cell research, or infectious diseases[5]. Single cell detection can effectively identify a small number of abnormal cells in the cell population, which can provide a scientific basis for early diagnosis of diseases and drug screening, so as to guide targeted therapy[6].
Compared with traditional macroscopic methods, microfluidic diagnostic tools have many advantages in terms of sample size, cost, portability and integration[7]. Among the many advantages of microfluidic technology, the convenience of cell manipulation brought by the micron-sized channel is particularly prominent, which enables the microfluidic device to accurately control the microenvironment of cells and facilitate the manipulation and characterization of single cells[8⇓⇓~11]. In addition, the high throughput and precise control of microfluidics can provide more and more comprehensive cell information, so microfluidics technology has become an important tool for single cell analysis[12⇓~14]. Due to the complex composition of biological samples, single cell analysis based on microfluidic technology usually involves two aspects: sample sorting and enrichment and detection and characterization[15]. In the past two decades, researchers have developed a number of microfluidic technologies to achieve the separation of different types of cells or particles, such as inertial sorting, hydrodynamic sorting and other passive methods relying on channel structure and fluid flow, as well as active technologies using external fields such as light, magnetism, electricity and sound[16][17⇓~19][20⇓~22][23⇓~25][26⇓~28][29⇓~31].
For the detection and characterization of cells after sorting pretreatment, the existing research mainly uses optical methods, such as the use of fluorescent labeling methods to achieve molecular-specific detection, so as to calibrate and classify cells. For example, Hung et al. Developed a microfluidic sorter based on the principle of immunofluorescence detection, using field programmable gate arrays for communication and sorting decisions among all electronic devices, which greatly improved the throughput of fluorescence lifetime measurement and sorting[32]. However, the immunofluorescence detection method requires a tedious labeling step, and the process is time-consuming and the detection cost is high[33,34]. Therefore, more and more research has been devoted to the development of unlabeled cell detection and identification methods[35⇓~37]. Among them, the most promising ones are the microfluidic detection methods based on the electrical properties of cells, including the methods of characterizing the electrical parameters such as cell membrane capacitance and cytoplasmic conductance by using electric rotation and micro-impedance spectroscopy[38][39,40]. Although these techniques can measure the electrical properties of cells, they all have some shortcomings, such as low throughput of cell detection. For example, the electric rotation technology is difficult to achieve the effective rotation of cells in the buffer solution, and the cells need to be manipulated and fixed, which leads to time-consuming detection. The electrical impedance spectroscopy technology is to capture cells near the electrode, which is very easy to cause electrode contamination and slow experimental process[41]. By integrating microfluidic technology, electrical impedance technology and flow cytometry, microfluidic impedance cytometry can characterize the dielectric parameters of cells in a short time after a single cell suspension passes through the detection area[3]. Compared with the above two technologies, the microfluidic impedance cytometer significantly improves the detection throughput of single cells, and can obtain a large number of statistical information of cells in a relatively short time to eliminate the chance and randomness of cell detection, showing great application advantages.
Based on the latest progress of microfluidic impedance cytometry in single cell detection, this paper mainly reviews the system construction of impedance cytometry and its clinical application. The working principle, system type and electrode structure of different detection methods are described in detail.According to the cell type, the clinical application of microfluidic impedance cytometry is divided into blood cells, cancer cells and bacteria, and the integration and miniaturization of microfluidic impedance cytometry are summarized. Finally, the application prospect of microfluidic impedance cytometry in single cell detection is discussed, which provides more possibilities for the related research of single cell analysis.
2 Microfluidic impedance flow cytometer
Flow cytometry is widely used in rapid and high-throughput single-cell statistical analysis, but traditional flow cytometry requires large instruments, complex excitation devices and special biomarkers, which can cause irreversible damage to cells[42,43]. In contrast, microfluidic Impedance Flow Cytometry (Electrical Impedance Flow Cytometry, IFC) combined with microfluidic technology and Electrical impedance detection technology is a label-free, non-invasive single cell detection technology, which has the advantages of high sample integrity and fast cell detection[6].
The IFC can perform real-time impedance detection on the single cell suspension passing through the detection area, and the obtained impedance signal and the characteristic cell property are related to the applied excitation electric signal. The electrical impedance is defined as the ratio of the excitation voltage to the response current of the suspended cell fluid, namely:[44]
Where the Z* is the electrical impedance, the V* is the excitation voltage, the I* is the current response, and the superscript * is a complex number.
According to Maxwell's mixing theory, the complex permittivity of a cell suspension can be determined by three parameters: the complex permittivity of the cell, the complex dielectric constant of the suspending medium, and the volume fraction of the ratio of cell volume to channel volume, namely:
Where, the is the complex permittivity, and the is the volume fraction, that is, the ratio of the cell volume to the medium volume in the detection channel. The subscripts "mix", "cell", and "med" denote the mix, cell, and medium, respectively. The impedance (Zmix) of the mixture containing cells can be calculated from equation (3):
Where G is a constant to correct for the effects of the inhomogeneous electric field and the fringing field, and l is the width of the channel. The correction constants for different electrode configurations can be calculated from known research literature.
To improve the accuracy of cell impedance detection, the researchers also used the deformability of the cell itself to seal the contraction channel to reduce the leakage of electrical signals. Therefore, this section will describe the basic principles and detection methods of different types of microfluidic impedance cytometry according to the classification of DC/low frequency, AC and deformation impedance flow cytometry.
2.1 DC/low frequency impedance flow cytometer
DC/low frequency impedance cytometers are used to measure and count the volume of cells suspended in a flowing fluid,and cell counting is a fundamental requirement for laboratory research and clinical applications.the principle of DC impedance cytometry was proposed by Coulter in 1956,and the early device was called Coulter counter[45]。 Its working principle is that when the cells suspended in the electrolytic cell pass through the detection area(a small hole in a typical Coulter counter),the cells will replace the electrolyte in the detection area,and by applying a constant voltage circuit to two electrodes near the detection area,the insulation of the cells under the action of a direct current signal or a low-frequency alternating current signal makes the resistance between the pores change instantaneously to form current pulses,and the number of the measured current pulses is proportional to the peak value,the number of single cells and the volume.in the late 1990s,This principle was applied to Microsystems.Larsen et al.First used a planar silicon structure to make a micro Coulter particle counter,and used a four-electrode structure on the microchannel of the silicon substrate to detect the resistance change in the pore.At the same time,the influence caused by electrode/liquid contact is reduced.this experiment proves the ability of micro-Coulter principle to count and classify living cells and particles in liquid suspension,and lays the foundation for the application of micro-Coulter counter[46]。
Later,Choi et al.Proposed a micro-Coulter counter based on DC impedance,which consists of an integrated microfluidic chip,an impedance detection circuit and a signal acquisition system.the microfluidic chip consists of a sample storage chamber,a sheath pinching unit and two polyelectrolyte gel electrodes.the system can detect the DC impedance changes of the two electrodes under low DC voltage,and effectively count the rare CTCs in blood by using the size difference between Circulating Tumor Cells(CTCs)and blood Cells[47]。 Although a single channel Coulter counter gives reliable results,the analysis time is too long to handle a large number of analysis samples in a very short time.Therefore,Zhe et al.developed a high-throughput and high-sensitivity Coulter counter with a multi-channel structure,and adopted a unique design of arranging the sensing electrode in the center of the microchannel,which effectively prevented the coupling problem between multi-channel detections,but the circuit design was relatively complex[48]。 in order to simplify the circuit of the multi-channel micro Coulter counter,Khodaparastasgarabad et al.Proposed a microfluidic Coulter counter consisting of a main channel(300μm wide)and two sensing channels(40μm wide),as shown In Figure 1(a)[49]。 the working principle is that when the fluid is forced from the main channel into Two narrower sensing channels,two pairs of coplanar metal electrodes(100μm wide)in the sensing channels are used to count.two sensing channels detect particles simultaneously,increasing throughput and eliminating the need for hydrodynamic focusing of the particles.the device was able to successfully count and distinguish between 10μm and 20μm polystyrene particles and cancer cells.Compared with the single-channel Coulter counter,the multi-channel structure design can analyze particles through multiple sensing channels at the same time,providing higher detection throughput。
in The multi-channel microfluidic chip,a special Coulter counter is assigned to each channel,or the electrical detection is completed by time-sharing driving the multiplexing of each microfluidic channel,which means that more electrodes need to be connected,and the structure of the chip is easy to be complicated.and the number of the interfaces connected with the electrodes increases linearly with the increase of the microfluidic channels,so that the expandability of the whole chip is limited.to solve these problems,Liu et al used Code Division Multiple Access(CDMA)spread spectrum communication technology to orthogonally detect particles In Multiple microfluidic channels.each multiplexed micro Coulter counter generates different digital codes when detecting particles,which are called microfluidic codes.the codes are orthogonal to each other and can distinguish overlapping codes by calculation[50]。 the chip uses only three electrodes,is simple and scalable,and is theoretically applicable to the orthogonal detection of particles in any number of microfluidic channels.using a device with four microfluidic channels,they demonstrated the detection of a single human ovarian cancer cell in phosphate buffered saline(PBS)per microfluidic channel.However,although the method of Using Gold sequences to maintain mutual orthogonality under asynchronous transmission can successfully identify signals disturbed by coincident particles,relying on specific code sequences will bring challenges to the system.For example,the orthogonality constraint not only limits the number of Coulter counters in the network,but also requires a complex design to scale,and the complex calculation of the iterative method hinders the implementation of real-time detection.Faced with these problems,Wang et al.proposed a network composed of 10 microfluidic Coulter counters,coded it to generate random non-orthogonal waveforms,and established a signal processing algorithm based on convolutional neural network deep learning structure to extract sensor signals.the working process of the whole system is shown in Figure 1(B)[51]。
in conventional microfluidic cell counting,the possibility of two or more particles entering the detection area at the same time is minimized by focusing the sample flow into a very narrow channel through a surrounding sheath flow.In order to avoid the introduction of sheath pinching and further simplify the chip structure,Tang et al combined inertial focusing and liquid electrode technology,used inertial prefocusing technology to arrange cells In a single row to reduce adhesion,and used Ag/AgCl liquid electrode to optimize the system to achieve a detection throughput of 5000 cells per second[52]。 They also proposed a novel 3D printing method to produce micro-channels with semi-elliptical,triangular,square and circular cross sections in a low-cost and efficient way.It is found that both semi-elliptical and triangular microchannels can achieve three-dimensional single-row focusing near the bottom of the channel in a large flow range,and this lower single-row particle focusing will be very valuable for microfluidic impedance cell counters with detection electrodes at the bottom of the channel[53]。
Recently,Kong et al.developed a micro-Coulter Counter(μCC)to enrich and count rare CTCs in order to overcome the lack of pretreatment function of Coulter Counter when analyzing cell samples[54]。 the device consists of a glass substrate containing electrodes,a PDMS microchamber,and a sensing well,as shown in Figure 1(C),and integrates affinity enrichment and unlabeled counting functions to separate and count CTCs from a mixture of healthy donor blood samples and CTCs.The method was proved to be able to detect biological cells with a diameter larger than 5.9μm,and The cells did not need to be labeled to maintain their original state for subsequent cell analysis and culture。
Direct current impedance flow cytometry has the advantages of simple structure,high throughput and high sensitivity,which is widely used in various types of cell counting and classification,and has become an indispensable tool in the field of biological and medical research.However,the DC/low-frequency signal of Coulter counter can only obtain the size information of cells,and can not analyze their internal structure and composition,so it is difficult to distinguish biological cells with similar size and volume。
2.2 AC impedance flow cytometer
In the AC impedance flow cytometer,the impedance detection circuit measures the cell suspension passing through the detection area In real time.under the AC electric signal,the obstruction of the cell plasma membrane is significantly reduced,so that the electric signal can penetrate part of the cell membrane and intracellular fluid.the measured impedance value reflects the cell size and internal information.In addition,the effect of the electric double layer on the surface of the metal electrode Under an alternating current signal is very weak,so that the design of the metal electrode of the microfluidic chip can be simplified and the detection sensitivity can be improved.Han et al.Proposed the first conceptual AC impedance analyzer,which used a circular hole at the bottom of the capture cavity to apply negative pressure to the cavity to complete cell capture,and a pair of microelectrodes on both sides of the hole could obtain AC impedance measurements,and successfully distinguished normal breast cells from abnormal breast cancer cells[55]。
Microelectrodes in impedance flow cytometer systems have always been the key to design,so Iqbal et al systematically studied the effects of microelectrode size,dielectric impedance,circuit resistance and microchannel size on the output of electrical impedance sensing[56]。 the cell impedance in the suspension medium was modeled using an equivalent circuit model,and the overall circuit of the cell suspension is shown in Figure 2(a).A voltage signal with a frequency ranging from 100 Hz to 100 MHz is applied as an excitation signal through the microelectrode to realize the broadband impedance measurement of the cell sample.the results show that the area of the microelectrode can have a significant effect on the electrical opacity of cells(the ratio of cell impedance at high and low frequencies),which provides valuable guidance for the sensor design and characterization of AC impedance flow cytometry.in most microfluidic impedance cytometers,the fabrication of devices relies on advanced microfabrication techniques in clean rooms,such as Metal sputtering/vapor deposition to fabricate Metal electrodes on substrates(glass or silicon).and the substrate is bonded with the flow channel to complete the assembly of the coplanar microelectrode integrated microchannel,which is a complex and expensive microelectrode manufacturing process and limits the application of the microfluidic impedance cytometry in clinical diagnosis.in order to bypass the traditional gold(platinum)electrode preparation method,Panwar et al.fabricated a coplanar"contact"Field metal microelectrode("in-contact"Field's metal",icFM)in a multilayer elastomer device using single-step lithography,as shown in Figure 2(B)[57]。 the microelectrode show excellent performance in detecting single red blood cell,has high sensitivity and long-term working stability,and can further reduce that detection volume by using non-conductive fluid focusing or sheath fluid pinch to realize higher sensitivity,thereby realizing more challenging applications such as cell classification,microorganism detection and The like。
Due to the inherent heterogeneity of biological samples,cell separation is an essential step in various biomedical applications.Usually,researchers use optical observation to evaluate the lateral distribution of particles or cells in the microchannel,but this method requires transparent materials and processing of a large number of image data,and can not achieve the quantitative characterization of cells in the cross section of the microchannel.Due to the convenience and spatial correlation of electrical signal measurement and analysis,cell location detection based on electrical impedance method effectively solves the above problems in optical detection system.For example,Brazey et al.Proposed a real-time sensor based on impedance measurement to characterize the longitudinal position of particles along the length of the channel[58]。 Solsona et al.Utilize gradients in the electric field to detect lateral particle positions along the width of the channel[15]。 Reale et al.Made electrical measurements of the cross-sectional position of a single particle with two different sets of electrodes,and the lateral position of the particle was determined by five pairs of electrodes and the resulting two differential currents[59]。 in order to overcome the defect that most of the existing studies detect the particle position along one direction,Reale et al.Made further research on the detection of the particle cross-section position(along the width and height of the channel),and used a non-traditional arrangement scheme to enhance the electrical signal to measure the transverse and longitudinal positions of the particles in the channel cross-section,as shown in Figure 2(C)[60]。
the microfluidic impedance cytometer achieves high-throughput detection of the cross-sectional position of a single flowing red blood cell,and is expected to become an effective tool for studying microscale particle motion,microfluidic sorting and separation applications,and particle focusing system design optimization.Recently,Yang et al.Proposed a novel microfluidic impedance flow cytometer using an N-type electrode design to measure the lateral position of individual cells and particles,and the differential current collected from the N-type electrode encodes the flow trajectory of individual particles[61]。 the transverse position of a single particle or cell in the N-shaped electrode can be calculated from the current signal and the geometric relationship between the positions of the flowing particle,the electrode,and the microchannel.the system measures the lateral position of particles and human red blood cells flowing in a 200μm wide channel at different flow rates,and the results show good correlation and consistency with the traditional microscopic imaging method.the system not only has the advantages of fast,accurate and high-throughput electrical signal processing,but also can be easily integrated with other microfluidic platforms to measure the lateral position and physical characteristics of single cells in real time。
in order to realize the detection of cells in high-speed flow process,it is necessary to develop high-throughput real-time signal processing methods.to this end,Honrado et al.Proposed to apply the neural network method to the AC microfluidic impedance analyzer to extract multiple performance parameters of cells from the measured impedance signal,as shown in fig.2(d)[62]。 A design of microfluidic impedance chip for multi-parameter data extraction is proposed.the measured signal waveform containing time series is input into the Neural network.the Recurrent Neural Network(RNN)suitable for processing data with time dimension is selected and trained to predict the size,velocity and cross section position(i.e.Horizontal and longitudinal position)of cells.Comprehensive experimental data show that the trained Network can accurately describe the size,velocity and cross-sectional position of particles,red blood cells and yeast,with a prediction time of 0.4 ms,which indicates that the combination of microfluidic impedance cell detection and machine learning can be used as an effective cornerstone for real-time single cell analysis and classification。
AC impedance cytometry is a technique that can characterize the internal electrical properties of cells,which has the advantages of high throughput and label-free,and is widely used in cell status analysis and stem cell differentiation detection.However,because the cells suspended in the fluid only occupy part of the detection area in the detection process,it is easy to cause the leakage of the detection signal,which makes the sensitivity of the whole detection system not high。
2.3 Deformation impedance cytometer
A deformation impedance flow cytometer is a device that measures the impedance of a single cell as it is squeezed through a constricted microchannel.A common configuration is to place two electrodes on either side of the constricted channel,apply an AC voltage to one electrode,and measure the current from the other electrode[63]。 the principle is that when a single cell flows through the constriction channel,the cell temporarily blocks the electric field of the constriction channel,resulting in an increase in the impedance measured on both electrodes.the time interval between the change in impedance as the cell enters and leaves the constricted channel can also be used as an indicator of the ability of the cell to deform.as measured by Yang et al.,cell deformability was measured by evaluating the transit time of each red blood cell as it was squeezed through a microscale constricted channel(cross section 5μm×5μm),and more than 30,000 red blood cells could be analyzed in 1 minute for quantitative analysis of plasmodiaemia at a rate of 500 cells/second[64]。
deformability characterization devices based on electrical impedance usually use a single constriction channel to characterize the Deformability and electrical impedance characteristics of a single cell after it passes through.For example,Zhou et al.Developed a single-constriction microfluidic device that can simultaneously characterize the mechanical and electrical characteristics of a single biological cell in a high-throughput manner[65]。 the experiment proves that the combination of mechanical properties and electrical impedance information can significantly improve the ability and efficiency of distinguishing different cell populations.However,the strain impedance cytometer,which uses a single constriction channel to measure the biophysical properties of cells,usually has a low detection throughput.Increasing the number of constriction channels can effectively improve the speed and accuracy of single cell characterization.For example,Ren et al.Proposed a new microfluidic device containing four parallel sensing constriction channels,each of which includes two constriction structures separated by cell recovery zones[66]。 All channels share a pair of electrodes to record electrical impedance to obtain the magnitude and phase of single cell impedance at different frequencies。
as shown in fig.3(a).the device was used To distinguish breast cancer cell lines from normal epithelial cell lines,and the detection accuracy of single cell level reached 97%.It is proved that the multi-channel microfluidic device can study the deformation and recovery behavior of cells,and improve the efficiency of distinguishing cancer cells from normal cells compared with the single-channel device.However,existing multi-pinch-channel microfluidic devices capable of evaluating cell deformation properties rely on high-speed microscopic imaging techniques.to solve this problem,Yang et al.developed a differential multi-pinch channel microfluidic device with self-aligned three-dimensional electrodes using a low-melting alloy,As shown in Figure 3(B)[67]。 Electrical impedance method is used to study the deformation and recovery process of cells instead of image analysis method.the deformation index of single cell is studied by comparing the information of cell passage time during continuous contraction.A back propagation neural network is also developed and applied to the quantification of cell type classification.the results showed that the classification accuracy was significantly improved with high sensitivity and specificity,which provided great potential for single cell detection and classification。
Although the above-mentioned methods can effectively record the impedance signal and complete the characterization of single cells,they can only collect the original physical markers(such as the passage time of cells through the constriction channel and the change of impedance signal),but can not characterize the intrinsic biophysical properties of cells(such as cytoplasmic conductance and membrane capacitance).To this end,Zhao et al.designed a crossed constriction channel including a side channel,and when cells are continuously squeezed through the constriction channel,electrodes arranged in the side channel monitor the electrical impedance changes of the detection area in real time.Combined with the established equivalent circuit model,the performance parameters such as cell membrane capacitance and cytoplasmic conductance can be obtained without microscopic imaging[68]。 However,when the cells pass through the constriction channel,they are prone to deform towards the inside of the side channel,which destroys the corresponding equivalent electrical model and leads to inaccurate electrical characteristics of the cells.in order to solve the above problems,Zhang et al.Improved the device by introducing sheath flow in the side channel,so that the position of cells in the process of their movement was properly controlled[63]。 more specifically,the sheath flow is introduced into the side channel in advance,and when cells enter the cross constriction channel along the main constriction channel,the cells cannot enter the side channel due to the pressure effect of the sheath flow on both sides,thereby ensuring the reliability of cell impedance measurement.the experimental results show that the specific membrane capacitance and cytoplasmic conductivity of 100,000 A549 tumor cells can be obtained by the developed microfluidic system,which is More accurate than the previous experimental results.in addition,the device can distinguish between low-metastatic and high-metastatic cancer cell types,which confirms the potential application value of cell electrical characteristics in cell type classification。
Recently,Liu et al.Have further improved the deformation impedance cytometer with cross constriction channel,that is,when cells enter the cross constriction channel along the main constriction channel,Negative pressure was applied to the side channel to suck the cells into the side channel.Combined with the established electrical model,the measured impedance change signals were converted into inherent characteristics such as cell viscosity,cytoplasmic conductance and cell membrane capacitance,as shown in Figure 3(C)[69]。 the system enabled the quantification of hundreds of HL-60 cells and the successful classification of HL-60 from HL-60+CD cells and HL-60-ConA.Compared with previous methods,this method can obtain multiple biomechanical and bioelectrical properties of a single cell at the same time,and can significantly improve the success rate of cell type classification。
Deformation impedance flow cytometry can continuously characterize the deformability and impedance characteristics of single cells.This technology has the advantages of high precision and simple data processing,and can be used for rapid,real-time and accurate analysis of single cells,which has a very broad application prospect in cell classification and single cell detection。
3 Application of Impedance Cytometry in Single Cell Detection
In recent years, the research and development of microfluidic chip systems for biomedical research and disease diagnosis have attracted much attention. These Microsystems can detect, analyze and characterize the biochemical and biophysical properties of single cells or particles with high sensitivity while reducing the cost and size. Among them, the microfluidic impedance cytometer can characterize multiple dielectric parameters of cells in a short time after the single cell suspension passes through the detection area, providing researchers with a non-invasive, high-throughput single cell analysis method. Therefore, this paper will introduce the application of microfluidic impedance cytometry in the detection of blood cells (white blood cells, red blood cells), cancer cells and bacteria.
3.1 Blood cell
There is considerable evidence that the biophysical properties of blood cells are altered during the development of diseases such as sickle cell anemia,sepsis,malaria,inherited blood disorders,cancer,and diabetes[70][71][72][73,74][65][75]。 Therefore,understanding the biophysical and electrical characteristics of blood cells is a necessary means for early diagnosis of related diseases and evaluation of treatment effectiveness.This section summarizes studies using microfluidic impedance flow cytometry to study the dielectric properties of various blood cells,mainly white blood cells and red blood cells。
3.1.1 White blood cell
white blood cells are present in the immune system and are involved in protecting the body from infection and foreign invasion.Traditional white blood cell analysis methods,such as fluorescence and magnetic sorting methods for white blood cell separation,provide valuable and accurate information for medical experts,but these methods require complex and expensive antibody labeling[76]。 Petchakup et al.Developed a high-throughput integrated platform for continuous leukocyte sorting and single cell analysis in a label-free manner.the platform can directly separate neutrophils and monocytes from diluted or lysed blood samples in a few minutes,and then concentrate the inertia of white blood cells in a serpentine channel for impedance detection,providing a basis for the development of a rapid and label-free white blood cell analysis platform[77]。
In order to solve the problem of white blood cell counting,Subramaniam et al.used the microfluidic impedance cell counter to measure the impedance of each cell through the microelectrode on the chip,and counted the white blood cell subsets after recording the data,which could also distinguish three different white blood cell groups.the scatter diagram of the original data is shown in Figure 4(a)[78]。 Cell impedance measurement at different frequency facilitates accurate identification of that three leukocyte without the need for any biomarkers,and the reduced cost of the reagent enable its widespread use in resource-poor environments。
Macrophages are white blood cells differentiated from monocytes and located in various tissues.These cells play an important role in body development,homeostasis,tissue repair and pathogen immune response.Zhang et al.proposed a dielectrophoresis based method for rapid isolation and purification of macrophages using liquid biopsies and in situ assays using a microfluidic device that can be easily manipulated mechanically for direct isolation[79]。 the dielectrophoretic force of macrophages was measured under different electric field conditions,and macrophages were successfully separated from the mixture of macrophages and breast cancer cells by microfluidic chip,with a purity of more than 99%.Further study on the effects of flow rate and voltage on the separation efficiency of macrophages will provide a technical basis for monitoring and real-time rapid diagnosis of macrophage diseases。
3.1.2 Erythrocyte
In the detection of red blood cells,Gawad et al.First found a difference in the impedance signal at 15 MHz between normal and abnormal red blood cells(whose cytoplasm was replaced by a hypotonic solution),and learned that the difference between the two types of red blood cells was due to the conductivity of the cytoplasm[80]。 Later,Cheung et al.studied the effect of impedance signals at different frequencies on red blood cells mixed with glutaraldehyde,and used a parallel electrode design to distinguish red blood cells at three different concentrations:healthy red blood cells,abnormal red blood cell,and red blood cell mixed with glutaraldehyde[81]。 the results showed that the impedance signal of mixed cells was higher than that of normal red blood cells,which proved that the cytoplasmic conductivity of mixed red blood cells decreased,that is,the opacity increased.Bilican et al.Developed an unfocused microfluidic detection system,which was geometrically optimized by a combination of analytical,numerical and experimental methods[82]。 Erythrocytes and leukemic cells were experimentally detected and differentiated using the developed system,and the results are shown in Figure 4(B),which is a scatter plot of transit time and peak amplitude for cell types,with erythrocyte peak accumulation around 0.65±0.3 mV.the results show that reliable analysis of cell impedance can be achieved without labeling and focusing。
Compared with healthy people,the whole blood viscosity of patients with cardiovascular disease is increased,and hematocrit and erythrocyte deformability are the main determinants of whole blood viscosity.Therefore,Kim et al used impedance flow cytometry to reveal the electrical characteristics of erythrocytes,such as cytoplasmic resistance,plasma resistance and cell membrane capacitance,so as to analyze the whole blood indicators[83]。 Parasite invasion of red blood cells can change the dielectric properties of malaria red blood cells.Honrado et al.Developed a microfluidic flow cytometer with a highly sensitive graphene transistor.Antibody-functionalized transistors can sense malaria-infected red blood cells to selectively capture malaria-infected cells,and measuring the conductance before releasing the cells can effectively distinguish between two different stages of malaria infection[84]。
3.2 Cancer cell
Single cell analysis can study cancer stem cells by measuring the physical characteristics of the cells to understand the progression of the disease and detect it early so that more effective chemotherapy can be carried out[85,86]。 Circulating tumor cells In the blood can provide valuable information for the detection,diagnosis and monitoring of cancer,and the microfluidic device developed by Zhou et al can simultaneously characterize the mechanical and electrical properties of single cells in a high-throughput manner.in this study,the deformability of MCF-7 breast cancer cells was determined based on the transit time required for a single cell to pass through a constricted channel smaller than the cell size[65]。 Fig.4(C)shows the difference between untreated MCF-7 cells and MCF-7 cells treated with different doses of PMA,showing that cells treated with higher doses of PMA showed a significant increase in impedance compared to other populations.the microfluidic device can measure the deformability and electrical impedance of cancer cells,thereby obtaining the mechanical properties and electrical properties of the cancer cells。
Raillon et al.Proposed a label-free high-throughput microfluidic platform to enrich cancer cells through the eddy current chip and then measure the electric field fluctuations generated by the cells passing through the electrodes in the impedance chip[87]。 the experimental results show that the eddy current chip can effectively enrich cancer cells,the impedance chip can be used to count and identify cancer cells,and the combined device can fully integrate the separation and counting of unlabeled circulating tumor cells from cancer patient samples.Invasive metastasis of cancer cells is the main cause of death in cancer patients.Ruzycka et al.Tried to use microfluidic analysis system as an innovative tool for the study of lung cancer metastasis.Single cancer cell analysis,endothelial cell migration and neovascularization were studied,demonstrating the application of microfluidic systems in the study of the mechanism of intercellular interaction between lung cancer cells and the surrounding tumor microenvironment[88]。
3.3 Bacteria
Since the Coulter principle was proposed,it has been widely used in clinical testing and many other industries,such as Parker's attempt to use the Coulter counter to measure the size distribution of bacterial spores during germination and growth[89]。 After that,for bacterial detection,Haandback et al.investigated a novel resonance-enhanced impedance cytometry for the detection of submicron beads and bacteria[90]。 However,the proposed technique cannot be used to distinguish different types of bacteria because the dielectric properties of bacteria and 2μm beads are similar at low frequencies.Later,they also proposed a device capable of dielectric characterization of single cells at frequencies up to 500 MHz,which could distinguish wild type yeast from mutants,and found that the size of mutants and the distribution of vacuoles at frequencies around 250 MHz could be used to distinguish differences in dielectric properties[91]。
Claudel et al.Proposed a flow cytometer based on bioimpedance cell detection technology,which was tested under static and dynamic conditions of yeast.By measuring all the electrical properties of,for example,yeast cells in a low conductivity medium,the experimental results provide the possibility of fully characterizing the cells,enabling better detection of individual cells and measurement of their size as well as the electrical properties of the membrane and cytoplasm[92]。 Mernier et al.Proposed a liquid electrode structure to realize dielectrophoresis focusing and impedance detection of cells,and successfully analyzed the activity of yeast[93]。
4 Application of Impedance Cytometry in Clinical Immediate Detection
The occurrence of diseases is often accompanied by abnormal changes in the state of some cells in the human body. Rapid and effective detection of disease-related cell States is a powerful guarantee for disease prevention, disease treatment and disease control. Although traditional medical devices can detect medical samples very accurately, these devices often require complex operating procedures and long detection time, and in the course of disease treatment, it takes more time to adjust the treatment plan in time by multiple tests[94]. With the continuous development of microfluidic impedance flow cytometry, its advantages of small size and low cost are becoming more and more obvious, which makes it possible for lab-on-a-chip and clinical real-time detection applications[95⇓~97].
4.1 Integration of Impedance Detection Chip
single cell analysis has become an emerging field in biological and biomedical research,and flow cytometry is a high-throughput technique widely used for Single cell analysis,but its application is limited by the lack of limited environmental control capabilities[42]。 Based on this,Petchakup et al.Developed a new integrated platform combining spiral inertia microfluidics and impedance flow cytometry,as shown in Figure 5,in which different functional units are highlighted by different colored boxes.cell sorting(red)and impedance detection(blue).Since both Cell sorting and impedance measurement use non-labeling methods,cells after impedance measurement can be continuously collected off-chip for downstream detection[77]。 the integrated platform can not only be used for leukocyte classification,identification and counting,but also distinguish the significant differences between different diseases,which greatly improves the work efficiency。
Because CMOS technology can effectively reduce the size of electrical devices,it is possible to integrate the system into a single chip,which provides a basis for the integration of chips in impedance analysis and detection and the wide application of single cell analysis[98]。 In many biological and medical applications,the enrichment and detection of rare cells are essential.for example,Wang et al.Prepared an integrated microsystem device using a staggered microelectrode chip For the enrichment and detection of Escherichia coli[99]。 the microfluidic chip prepared by the micromachining method enables the microelectrode array to have enough contact area with the bacterial suspension so as to improve the enrichment efficiency and the detection sensitivity.the method was successfully applied to the detection of Escherichia coli,and the experimental results showed that the detection time was only 6 min,which showed that the detection speed of Escherichia coli was significantly improved after optimization.Nguyen et al.Detected circulating tumor cells in a microfluidic device using a combination of dielectrophoretic manipulation and circular microelectrode impedance measurement[100]。 the chip of the device is fabricated by standard soft lithography.Lung cancer cells(A549)are guided to the center of the working area by the proposed microchip,and then captured on the required sensing electrodes.Lung cancer cells are identified by measuring the impedance.These integrated,low-cost approaches open new avenues for the development of cellular diagnostic systems for future applications。
4.2 Miniaturization of Impedance Detection System
the miniaturization of the impedance detection system can make it easy to carry and detect,which provides favorable conditions for the commercial development of the product.Furniturewalla et al.proposed a wearable microfluidic impedance cytometer,which combines a flexible circuit wristband with online smartphone readings to obtain portable biological counting analysis[101]。 the wearable detection system is shown in Figure 6(a).the wristband is composed of a self-made lock-in amplifier circuit,an analog-to-digital converter(ADC)and a wireless Bluetooth module.The measured data on Android smartphones can be exported to researchers and medical professionals for in-depth analysis and remote health monitoring.Experiments have proved The ability of The system to count blood cells,which can be applied to real-time clinical testing。
Nguyen et al.proposed a microchip-integrated handheld electronic integrated circuit module to characterize cancer cells using dielectrophoretic impedance measurements[102]。 The structure of the chip is shown in Figure 6(B).the chip is composed of circular microelectrodes,which are used to detect the concentration and impedance of cells,while the handheld electronic module can provide sinusoidal electrical signals over a wide frequency range to measure the impedance of cancer cells in the microchip.At the same time,the linear relationship between the admittance changes of human lung cancer cells and human lung epithelial cells and the number of cells was observed.the difference of the slope of these characteristic lines can be used to distinguish the two States of lung cells.the device is simple to operate,high sensitivity,low price and easy to carry。
At present,diagnosis based on blood tests is still mainly concentrated in laboratories,relying on huge and expensive cytometers or microscopes,and requiring professional laboratory technicians,while effective basic medical security is not available in remote areas.Faced with this problem,Abbasi et al.Developed a portable complete blood cell counter to detect white blood cells,red blood cells,platelets and hemoglobin in blood for rapid diagnosis in resource-poor environments,which consists of a small disposable chip.the proposed design benefits from the development of microfluidics and lab-on-a-chip manufacturing technology,which makes the traditional cytometer miniaturized and greatly reduces the cost[103]。 in addition,Talukder et al.Also proposed a portable personalized blood cell counting system,which consists of a microfluidic impedance cytometer and portable analog readout electronics.Input to the analog-to-digital converter and transfer via Bluetooth to a mobile application accessible to the user.the data transfer is shown In Figure 6(C).All communication is controlled via the mobile interface on the handset[104]。 Although the sensing platform uses low-end hardware devices,it can detect impedance changes as small as 0.032%,and can detect particles with a diameter of 3μm in a 300μm wide channel。
Applications such as personalized health monitoring require stable operation and strong anti-blocking ability of equipment,and it is better to avoid using channels equivalent to the detected particles to maintain high reliability.the sensitivity of the system should be comparable to that of a high-end bench-top impedance spectrometer.the above studies can prove the potential of microfluidic impedance cytometry in low-cost health monitoring,and demonstrate the application of microfluidic impedance flow cytometry technology in simple and fast single cell detection and cell counting。
5 Conclusion and prospect
Advances in lab-on-a-chip technology have facilitated the widespread use of microfluidic impedance flow cytometry in academic research and commercial instrumentation, making it one of the most powerful techniques for rapid analysis of single cells and microparticles. Compared with the traditional technology, the microfluidic impedance cytometer does not need labeling, and can greatly reduce the time consumption and cost. When analyzing single cells, the rapid analysis and high throughput of microfluidic devices allow users to do more detection in less time. Another advantage of microfluidic impedance cytometry is that the processing of pure electrical signals is conducive to the integration and miniaturization of the system. Researchers have developed many different microfluidic platforms to analyze cells by taking advantage of this good compatibility, so that single cell impedance analysis can be used to assist clinical decision-making. For example, due to the high heterogeneity of tumor cells and the lack of biomarker information, it is difficult to identify some cancers by using traditional immunofluorescence methods to detect protein expression. When combined with single cell impedance detection technology, it can more accurately complete the clinical diagnosis task.
At present, most of the impedance measurements are based on low-frequency AC signals to analyze the size and membrane capacitance characteristics of cells, and the characterization of the impedance characteristics of vacuoles or nuclei in cells is still in its infancy. In future studies, the importance of single cell high frequency impedance measurement will become increasingly prominent, because high frequency impedance signals can provide internal information about the nucleus and organelles, which is helpful for developmental biology or genomics research. In order to improve the practical application of microfluidic impedance flow cytometry in clinical real-time diagnosis, researchers are trying to achieve high-throughput detection of single cells through multi-channel structure design.And the use of printed circuit boards or microneedle electrodes to reduce manufacturing costs by eliminating the reliance of point-of-care clinical testing systems on microelectrodes. In addition, in order to facilitate the operation of users in medical applications, a large number of future research work will focus on the functional integration of microfluidic impedance cytometry, such as focusing pretreatment of cell samples and automatic sorting of different cell types after detection. In general, microfluidic impedance flow cytometry has great potential in the application of single cell detection, and with the integration of chips and miniaturization of systems, it can be widely used in biomedicine and clinical real-time diagnosis.
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