Time-resolved microfluidic flow cytometry

Flow cytometry is extensively applied in life sciences and clinical practice for high-throughput identification and characterization of cells, particles or microorganisms suspended in fluid samples. However, conventional flow cytometry based on spectral detection suffers from two main disadvantages: interference from biological autofluorescence, and spectral crosstalk of fluorochromes. Time-resolved detection employing long-lived luminescent probes such as lanthanide complexes provides an effective strategy to overcome these issues. It effectively removes short-lived autofluorescence background arising from non-target specimens in the sample, resulting in high detection sensitivity and contrast. Multiplexing can be realised based on the luminescence lifetimes of the probes, avoiding spectral crosstalk between the detection channels. This thesis reports the design, development and demonstration of a time-resolved microfluidic flow cytometer (TRMFC) as a practical and low-cost technique aiming to achieve lifetime-based multiplexed biosensing. A microfluidic chip coupled with a piezoelectric transducer is used for two-dimensional acoustic focusing of the events at the centre of the flow channel. The sample flow rates are set so that sufficient signal from the long-decay luminescence can be acquired as targets transit the detection aperture, to ensure accurate lifetime measurement. The excitation source (i.e. UV LED) and detector (i.e. gatable PMT) are pulsed with a time-shift delay between them to discriminate the autofluorescent before luminescence signal detection. The current TRMFC system is able to achieve high counting efficiencies (>90%) of the luminescent targets passing through the microfluidic channel, and resolve lifetimes of individual targets at high accuracy (coefficient of variation ~2.4%). The acoustic focusing efficiency under varied flow velocity and the influence on time-resolved detection have been analysed in detail. The results indicate that a flow rate as high as 20 μl/min can be applied in the current system without affecting accurate counting of the events and measurement of their individual lifetime and intensity. Poisson statistics has been applied to calculate the overlap probability of two or more events presenting simultaneously in the detection aperture. A peak detection algorithm has been developed to resolve the overlap events containing two targets; whereas events with three and more targets overlapped with each other are discarded due to significantly increased computational complexity and reduced robustness for nonlinear fitting. The probability of three and more targets overlap is limited to <1% by proper configuration of key experimental parameters including the detection aperture size, the flow velocity and the sample concentration, so that optimum performance is achieved at highest sample throughput. Finally, a pilot multiplexing bioassay has been demonstrated using two cell lines stained with different europium complexes. Factors that influence the luminescence lifetimes and intensities of the stained cells, including different cell lines, cell growth phases, fixation of cells, chelate structures, ratio of chelators and Eu3+, and reaction solvents for the chelates, have been investigated experimentally. Samples containing the two distinct cell types have been successfully analysed using the TRMFC. This new technique, upon further optimisation, is expected to benefit on-site screening of fluidic samples containing multiple target populations, such as blood, urine and environmental water, opening a range of opportunities using lanthanide complex as biosensors for ultrasensitive biomedical diagnostics.Key