Thesis

Fluorescence lifetime activated cell screening (FLACS) platform for cancer cell study

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Awarding institution
  • University of Strathclyde
Date of award
  • 2024
Thesis identifier
  • T16826
Person Identifier (Local)
  • 201857344
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Qualification Name
Department, School or Faculty
Abstract
  • The lack of sensitive and affordable tools for rapid and reliable cancer diagnosis remains a major obstacle to reducing cancer mortality. Biopsy, the most commonly used diagnostic tool, involves lengthy multiple processes and costly reagents. Additionally, the outcome of a biopsy depends on the experience of assessors, and interpretation is often subjective. To address these issues, we developed a rapid and high throughput fluorescence lifetime-activated cell screening (FLACS) platform to distinguish between cancer and noncancer cells based on a flow cytometer (FCM) setup and straightforward time-domain measurements using the novel single photon avalanche diode (SPAD) camara cooperating with the fast fit-free lifetime calculation method including centre of mass (CMM) method and phasor plot. The FLACS prototype combines FCM with a wide-field microscope equipped with a 192x128 SPAD camera, which is characterised and configured as a Megapixel. The sample is injected by a syringe pump and hydrodynamically focused into a narrow stream inside a flow cell down to 45 μm using sheath fluid, such as distilled water or PBS, driven by a peristaltic pump. The flow cell is based on the microscope slide design with 210 x 70 μm microfluidic channel. A graphic user interface (GUI) software has been developed to control and configured the SPAD camera, receive time-resolved fluorescence data, and analyse using fast and fit-free methods. Moreover, multiple FCM parameters can be graphically displayed as distribution and scattering plots, and the software also allows users to selectively identify some areas of the plots to specifically analyse sub-population. This helps the users to understand the insight of the sample and to easily distinguish sample populations. The current development of the platform shows speedy performance with a maximum detection rate of 1147 frames per second (fps), while the optimal speed is 309 fps with the least possibility of miscounted particles due to the dead time from data transferring and processing. In order to distinguish between cancer and non-cancer cells, an RNA nanoprobe for cancer cell detection is synthesised and tested with complementary deoxyribonucleic acid (cDNA) prior to human embryonic kidney 293 (HEK293) and human prostate cancer (PC3) cell lines. The nanoprobe design is based on a gold nanorod core with hairpin oligonucleotides (hpDNA) labelled with Cyanine-5 (Cy5), which is initially in a quenched state through Förster resonance energy transfer (FRET) and becomes fluorescent after hybridisation with target ribonucleic acid (RNA). The 291% and 76% increase in fluorescence intensity and lifetime, respectively, were observed after incubating the nanoprobe with the target RNA. To ensure the accuracy and reliability of measuring before proceeding to the precious nanoprobe with cell lines, the platform undergoes testing using C- and YG-fluorescent microspheres (C- and YG-beads). These beads mock-up positive and negative cell lines by mimicking their long and short fluorescence lifetimes, as well as strong and weak intensity characteristics, respectively. By flowing a single type of bead, the platform can detect the fluorescence lifetime of 1.89 ±} 0.10 ns and 1.49 ±} 0.66 ns for C- and YG- beads, respectively. For mixed bead samples, the data is extracted using an intensity threshold of > 285 photons and using phasor position acquired from single-type bead measurement to neglect the events without beads, and to filter the events with C- and YG- beads showing the extracted lifetime of 1.87 ±} 0.13 ns and 1.64 ±} 0.43 ns, respectively. By combining the prototype with the RNA nanoprobe, this platform can successfully detect the flowing PC3 cell lines incubated with the RNA nanoprobe for 2 hours, which shows a lifetime of 1.34 ±} 0.34 ns, longer than 1.14 ±} 0.26 ns in the case of HEK293 cell lines. This trend follows the results acquired from the fluorescence lifetime imaging microscopy (FLIM) technique, which shows the lifetime values of 1.25 ± 0.43 ns and 0.93 ± 0.02 ns for PC3 and HEK293 cases, respectively, demonstrating the capability for differentiating cancer cells from non-cancer cells using the FLACS platform. Our FLACS platform has the potential to improve cancer diagnosis by providing a rapid, reliable, and alternative measurement to traditional biopsy methods. Future development will focus on improving the platform's sensitivity and specificity, validating its performance in clinical settings, and optimising the RNA nanoprobe for improved cancer detection.
Advisor / supervisor
  • Birch, David J. S.
  • Chen, Yu, 1973 December 28-
Resource Type
Note
  • This thesis was previously held under moratorium from 12th February 2024 until 12th February 2026.
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