Studies On The Electrochemical Discrimination And Capture Of Cells In Microfluidic Chips

Cell is the basic unit both for the structure and the fuction of organism, which covers all the secrets of life. Study on biological cells has been the basis for the developments of life sciences. Serving as a novel research plateform, microfluidic technology plays more and more significant roles in the investigations of cells, by which not only the single cells can be analysed with high-throughput flow cytometry, but also cells can be captured and screened for a long-term. Electrical method is one of the typical approaches that commonly be integrated with microfluidic chips, and it is of great availability for the manipulation and analysis of cells. Unfortunately, some problems such as fabrication of microfluidic chips and microelectrodes as well as their integration still exsist in a general laboratory due to the lack of the MEMS techniques, which has been an obstacle for the progress of research on biological cells especially on the single cell level with microfluidic techniques. In this dissertation, we are aiming to cell research and trying to construct novel microfluidic devices with more practical and compatible methods for on-chip electrodes fabrication and integration, by which we intend to carry out effective control and fast analysis of cells, even of single cells, with electrical manipulation and sensing methods. Moreover, we try to strike out dynamically on-chip capture for several cells and single cells, expecting to integrate electrical tools further for real-time monitoring of cells and extend new field for cell investigation.Based on the studies in our group for microfabrication and cell patterning, the following research works have been carried out:1. A simple method to fabricate gold film patterns and gold microelectrodes by air plasma assisting micro-contact deprinting and printing transfer approaches has been presented. Chemical gold plating is employed instead of conventional metal evaporation or sputtering to obtain perfect gold film both on flat and topographic PDMS chips, and complicated SAM pre-coating is replaced by simple air plasma treatment to activate both the surface of gold film and PDMS. In this way, large area patterns of conductive gold film could be easily obtained on the elastomeric PDMS substrate. Both the chemical plating gold film and transferred gold film were of good electrochemical properties and similar hydrophilicity with smooth and conductive surface, which made it potentially useful in microfluidic devices and electronics. The gold transfer mechanism is discussed in detail. For typical applications, a cell patterning chip based on the gold pattern was developed to imply the interfacial property, and dielectrophoresis control of live cells was carried out with the patterned-gold as interdigital electrodes to show the conductivity.2. A label-free electrical flow cytometry for cell counting and discrimination has been developed based on a microfluidic chip with novel design. We designed a double-layer microchannel constructed by aligning a wide and shallow channel over a narrow and deep channel, between which a pair of opposite microband gold film electrodes were fabricated by a transfer printing method. The electrodes were connected to a Keithley measurement system for the discrimination of size and status of flowing single cells. Our experimental results demonstrated that this approach can be accurately used for the discrimination of HL-60 and SMMC-7721 cells. Moreover, the status of normal, apoptotic and necrotic SMMC-7721 cells can be efficiently discriminated based on the changes of measured capacitance and resistance. An equivalent circuit described the principle of this method for the simultaneous measurement of capacitance and resistance is presented. Meanwhile, the response mechanism of the changes of these parameters related to the cell size and status was briefly discussed. Based on the capacity of label-free discrimination of cells, this electrical measurement in microfluidic chip may be a promising approach for cell sorting and the screening of the cell status.3. Three approaches for the cell-trapping in microfluidic chips have been presented based on hydrodynamic principles. Microfluidic chips for the capture of several cells and single cells are fabricated with ordinary materials and simple methods. We use the printed methods previously developed to fabricate shallow microchannels, with which crossing over another deep channel, trapping positions for cell capture are construced in a two-layer microfluidic chip. Moreover, channel gaps for capture of several and single cells are made with alignment along the edges of two microchannels fabricated by ordinary microfabrication approaches as well as a sequencing orientation integration of the two layers. Using this device, single cells can be successfully captured in control with non-elaborate micro-structures. Additionally, carbon fibers are fixed on the substrate by a thin film, which is fabricated by evaporation of PVA solution. The carbon fiber then acts as a positive master to form microchannels with several microns, by which single cells can be dynamically captured like a patch clamp. These approaches are fast fabrication and easily controlled for cell capture, which bring great advantages for microfluidic single cell analysis, meanwhile, they provide more executable means for cell research on microfluidic chips in commom laboratories.