Abstract

Cancer is a group of diseases involving abnormal growth of cells with a potential to spread to the other parts of the body. It is predicted that one in two will be diagnosed with Cancer by 2030 and one in four would die. The continuous growth and metastasis of cancer cells depend upon cell-to-cell communication. This communication largely involves the secretion of soluble factors by cancer cells within the tumor microenvironment, although these cell types have also shown to export membrane-encapsulated particles containing regulatory molecules that contribute to cell-to-cell communication. These particles are known as extracellular vesicles (EVs) and include exosomes and microvesicles. EVs are a group of extracellular communication organelles enclosed by a phospholipid bilayer secreted by all types of cells throughout the animal kingdom. These vesicles are in the range of 30 to 1000 nm containing a myriad of substances like RNA, DNA, proteins, and lipids from their origin cells, offering a good source of biomarkers. Although their characteristics provide EVs great potential as biomarkers, efficient isolation and detection techniques are still challenging. Currently, the standard method for their isolation and quantification from body fluids is by ultracentrifugation, which is not a practical method to be implemented in a clinical setting. Thus, a versatile and cutting-edge platform is required to detect and isolate exosomes selectively for further analysis at a clinical level.
This dissertation is focused on developing lab-on-chip devices for the capture, detection, and isolation of cancerous extracellular vesicles from MCF7 (breast cancer cell lines) culture media. Here, we have explored three platforms based on gold nanoparticles, namely, in-situ synthesized nanocomposites, gold nano-islands on a substrate, and colloidal platform with suspended gold nanoparticles (AuNPs).
The first step was to identify the suitable platform for the capture and isolation of EVs, based on the sensitivity of the platform. Initially, gold – poly (dimethylsiloxane) (PDMS) nanocomposites, were created by the in-situ synthesis of nanoparticles on the surface of PDMS substrate by reducing gold precursor solution with curing agent of PDMS. The Au-PDMS nanocomposites are a special category of composites, with gold nanoparticles segregated in a sub-surface layer of the polymer. The second platform is the gold nano-island platform, where AuNPs are synthesized using Turkevich’s method and then deposited on a glass substrate by thermal convection. Then, the deposited nanoparticles are heat-treated at 560°C for an hour to form gold nano-islands. The refractive index sensitivity of nanocomposite and gold nano-islands is found to be around 50 and 111nm/RIU, respectively. The lower sensitivity in the nanocomposites as the nanoparticles are under the surface covered by a thin PDMS layer, whereas the in case of gold nanoislands, they are above the surface and available to interact directly with the analyte. Considering the sensitivities, the gold nano-island platform was utilized further for the capture and detection of EVs. The gold nano-islands on a substrate platform, where only half of the islands are available for sensing and capture, exhibited a better sensitivity meaning that utilizing the whole surface of nanoparticle would result in even better sensitivity as in the case of the third platform where gold nanoparticles suspended in a colloidal solution.
This dissertation then focuses on the biosensing protocol developed based on localized surface plasmon resonance (LSPR) of gold nano-islands, for the detection of EVs. The protocol involves the adsorption or binding of multiple bio-entities to the gold nano-island and absorption spectrum measurement after each step. The molecular interactions shift the gold plasmon band in the visible spectrum toward longer wavelengths and the shift is proportional to the concentration of the entity. The sensing protocol utilizes the strong affinity between EVs, and polypeptide called Venceremin or Vn96, which is specifically designed to capture EVs by binding the heat shock proteins (HSPs) present on their surface. Physical modeling, based on the characteristics of the gold nano-islands and the bio-entities involved in the sensing, is developed to determine the detection capability of the platform, which is optimized experimentally at each stage of the sensing protocol. The results and modeling present a relationship between the plasmonic shift and the concentration of exosomes. Further, a simple microfluidic device is designed for detecting EVs consists of a glass substrate with gold nano-islands, sealed by a PDMS film that contains a microchannel with a collection chamber in between. The same biosensing protocol has been transferred to a microfluidic environment by infusing biosensing entities into the device and measurement of the spectrum after each step. The capture and detection ability of the device is validated by Atomic Force Microscopy (AFM) and the measurement of gene copy numbers using droplet digital PCR (ddPCR). The results indicate that the developed device can capture and isolate the EVs from a very low sample volume, in less than 30 minutes, without affecting their size and shape, a major advantage compared to existing methods. Later, to increase the sensitivity, a two-level microfluidic device is fabricated utilizing a double-sided gold nano-island substrate, where AuNPs are deposited on both sides. This technique can be extended to multi-level microfluidics by introducing an intermediate channel in between the nanoisland substrates.
Parallelly, the gold nanoparticles suspended in a colloidal solution were utilized for the capture, detection, and isolation of EVs using the same protocol. Initially, this technique was studied at the cuvette stage by mixing the desired volume and concentrations of bio-entities manually and measuring the absorption spectrum at each stage for LSPR shift. As the nanoparticles are freely suspended in the solution, the binding of EVs on the entire surface area contributes the nanoparticles to sediment at the bottom of the cuvette. Thus, the precipitation method was used to isolate EVs. Then, a liquid biopsy chip is designed and fabricated using soft-lithography consisting of a 3D mixer, a collection chamber, and gravity assisted sedimentation unit. The streptavidin-coated magnetic particles are utilized in the liquid biopsy chip for the isolation of EVs. The device consists of a 3D mixer to ensure capture efficiency and it also consists of a sedimentation unit, which allows EV-captured magnetic particles to settle in it. The sedimented EV-captured magnetic particles are then isolated from the chip for elution of EVs and their validation using Nanoparticle Tracking Analysis (NTA), AFM, and gene amplification. The results clearly show that the proposed liquid biopsy chip can isolate the EVs without affecting their morphology. Thus, this chip can be considered as a potential point-of-care device for diagnostics in a clinical setting and for the isolation of EVs for future therapeutics.