Development Of High-Precision Microfluidic Impedance Cytometer Based On Low Melting Point Metal Electrodes

Coulter counter,which can sense theelectrical impedance changes when cells translocates through the detection area,is one of the earliest automated devices for high throughput cell counting.Moreover,the microfluidic impedance cytometer(MIC)that integrates the Coulter principle with current microfluidic technology has obtained increasing attentions,since it possesses good potentials in constructing miniaturized and/or portable devices,and thus is more suitable for point-of-care testing(POCT).At present,there are mainly two forms of electrode arrangement in MIC,i.e.,coplanar electrodes and parallel electrodes.But both of them have the disadvantage of heterogeneous electric field in vertical direction.In addition,they also need the sophisticated steps such as electroplating nobel metals,lithography and subsequent etching are always required for every single chip,posing limitations on batch manufacturing and,at the same time,greatly increasing the cost.To address these issuess,we firstly carried out detailed studies on theoretical analysis and simulation,and then developed a novel microfluidic impedance cytometer based on low melting point metal electrodes.This greatly simplifies the fabrication of microfluidic chip,and more importantly,it can generate fairly even electric field in vertical direction.As a result,a higher detection accuracy and sensitivity can be achieved with the help of simple planar focusingtechnique.The main contents include(1)The Comsol multiphysics simulation is performed to make clear the electric field distribution in detection area,as well as the factors that may affect the detection accuracy and sensitivity.The results show that,by placing a pair of electrodes of the same height with themain channel on lateral sides,the distribution of eletric field along channel depth becomes more uniform when compared with paralled electrode format.Moreover,the spindle-shaped symmetric distribution of electric field along the width of the main channel suggests that focusing cells near the side walls can bring higher sensitivity,since the closer the distance to elctrode is,the stronger the electric field becomes.The simulation results also show that higher the detection accuracy and sensitivity can be achieved with narrower the main channel and/or the smaller the electrode width.,This is well consistent with the theoretical analysis of electrical impedance detection of cells,namely,the smaller the detection area enbles the more notable impedance change upon cells flowing through the detection area.The simulation work provides a useful guidance for the development of microfluidic impedance cytometer in our study.(2)A high-precision microfluidic impedance cytomeric chip based on low melting point metal electrodes was developed.After being melted under mild heating conditions,the low-melting-point metal can be simply infused i into the electrode channels,and naturally solidifies at ambient temperature.This process can generate a stable solid metal electrode,which is of the same height with the main channel,thus catering a homogeneous vertical distribution of the electric field,which greatly decreases the susceptibility of the impedance signal to the particle's depth.The microfluidic chip just involves a single-layer channel structure fabricated via standard lithography,meanwhile,the preparation of electrodes does not require precise control or any fine alignment.Therefore,the processing process is very simple and cost effectively.On this basis,a microfluidic impedance cytomeric system was further constructed,with a set of algorithms such as baseline correction and convolution filtering being proposed to calibrate background noise and measurement errors.Results show that the developed microfluidic impedance cytomeric system can efficietly identify polystyrene microspheres with a diameter difference of only 2 microns,and have been successfully used for the counting of red blood cells(?8 ?m in average),white blood cells(?10 ?m in average)and human breast cancer cells(greater than 10 ?m).(3)A methodology for further reducing the detection area of microfluidic impedance cytomeric chip based on in-situ photocurring was proposed.As known from the above simulation and theoretical analysis,the smaller the detection volume is,the higher sensitivity of the system can afford.However,the current soft lithography can hardly process microstructures with both smaller size and larger depth-to-width ratio.In this regard,this paper proposed a two-step stretagy for processing ultrafine microstructures.First,the standard soft lithography was used to fabricate the microfluidic chip required,and then in-situ photocuring was introduced for futher micropatterning photosensitive reagent inside the microfluidic channel.Using this method,microelectrode structures as small as 10 ?m(depth-to-width ratio of 3:1)can be readily achieved,which largely improves the detection sensitivity,with results showing that polystyrene microspheres of just 4 ?m(the average diameter of platelet)can be effectively identified,implicating a promising potential for the sensitive analysis of all kinds of blood cells.