Microfluidics technology and single cell analysis

Microfluidics is a relatively new and advancing technology that has emerged with the potential to overcome the limitations associated with CE and flow cytometry for high- throughput single cell analysis.9 The technology has been around since the 1980s68 but has come of interest for biomedical applications since the 1990s.13

Microfluidic devices, also referred to as microchips or lab-on-chip devices, are being developed for a wide variety of applications. In general, they are used to manipulate

micrometer regime.69 In a typical device, channels range from 1 m to 1 mm in dimension. More recent devices have features in the nanometer range as well.70 The channels can be fabricated from a variety of materials to form the channel networks. Glass and polymers are the most common materials used for microdevice fabrication, although alternative materials such as plastics are also used.71-72

Microfluidic techniques have several advantages over traditional macroscale systems. Some of the initial motivations for microchip development include the low sample volume requirements for analysis and the high separation efficiencies that can be achieved.24,48 The small channel dimensions result in low total volumes contained within the channels;

therefore, smaller amounts of reagents and samples are required for analysis compared to macroscale techniques. This is highly advantageous for applications where only a small amount of sample is available, such as with DNA samples, or in instances where it is difficult to acquire large sample volumes.

Electrophoresis is the most common mode of separation performed on microfluidic devices. Because of the small channel dimensions and short channel lengths, extremely high electric fields can be applied using relatively safe voltage levels.10 As shown in Equation 1, the efficiency of an electrophoretic separation varies linearly with electric field strength; therefore, highly efficient separations can be achieved on-chip.

Equation 1

N = µep E Ld 2Dm

Where: N = theoretical plates E = Electric field strength ep = electrophoretic mobility

Ld = length of capillary to detector Dm = solute diffusion coefficient

Because of the increased efficiency, separations within microchannels can be

performed on a faster timescale compared to conventional CE systems.9 Other advantages of microfluidic devices that have been cited include the ease of automation, portability, low power requirements, inexpensive fabrication and ability to manipulate precise fluid volumes.7,9,24,56,68,73-74 The small channel volumes allow for highly sensitive analyte detection as well. Common detection methods, including electrochemical, mass

spectrometry and fluorescence, have been interfaced for analyte detection on microchips. These combined separation and detection set-ups make it possible to develop total analysis systems that can control everything from sample preparation through detection and data collection.24

The application of microfluidic devices for high-throughput single cell analysis is feasible for many reasons. First, the small sample requirements give microchip analysis a distinct advantage over techniques that require much more sample for successful operation.59 Often, the limited quantities of diseased cells that can be obtained from a patient are

impossible to analyze using conventional techniques, such as flow cytometry, yet

microfluidic devices can handle analysis of these smaller sample volumes. Next, because microfluidic channel dimensions are compatible with typical mammalian cell sizes75, cells can be easily moved through the channel networks and multiple process steps can be incorporated onto a single device.10,14,24 Retention and isolation of an individual cell and subsequent analysis of that cell’s cellular content have also been demonstrated on

microchips.76-77 Because cells generally need to be moved through the device for analysis, microfluidics is the optimal technology for high-throughput analysis of non-adherent (i.e., circulatory) cells. Manipulation of adherent cells within a microfluidic channel network

would unfortunately cause too much stress on the cells and the resulting data from such an experiment would most likely be highly inaccurate. Because of this, conventional CE methods with modified lysate injection schemes still remain the best option for adherent cell analysis.56

Another advantage of microfluidic devices over conventional CE for non-adherent cell analysis is that fast buffer exchange can occur around the cell just prior to lysis.1,78-79 It is known that buffers that are “cell-friendly”, meaning buffers that maintain cell viability and place little stress on cells, are not usually optimal for performing electrophoretic

separations.80-81 Cell-friendly buffers generally contain high salt content to provide isotonic conditions for the cell. Even at low electric fields, this high salt content can lead to

significant Joule heating and can result in bubble formation within the channels. Because fast buffer exchange can be achieved on a microfluidic device, cells can be maintained in a high salt content, cell-friendly buffer up until the time of lysis.

Besides the above mentioned reasons there are several other distinct advantages to using microfluidic devices for cell analysis. The small footprints of microfluidic channel networks make them amenable to parallelization that allows many cells to be observed simultaneously.13,24,61,79 Highly sensitive detection techniques allow for detection of even the low copy number analytes that are often found within a single cell.10 Injection of cell lysates can be achieved without significant dilution of the cellular contents.10 Finally, the

microenvironment around the cells can be carefully controlled within a microchannel, which reduces unnecessary stress on the cells.7,13,74 Stress is known to invoke signaling

Although in theory microfluidic technology should be easily adapted for high- throughput single cell analysis applications, it has not yet been demonstrated in a practical manner.6-7 However, many research groups have demonstrated novel ways to analyze single cells on microchips.