Novel Micro fluidic Techniques for Point-of-Care Diagnostics
Point-of-care Diagnostics (POCD) is one of the rapidly growing areas of health-care sector that avails to the needs of the patient at the point-of-care. An ideal POCD device is required to be compact, portable, offer quick results, require no or minimal sample preparation, and inexpensive with a low cost per test. Micro fluidics has a potential to cater to these needs, thereby leading to a growing research interest to develop micro uidic POCD (POCD) devices. POCD devices can be broadly categorized into cellular diagnostics and non-cellular diagnostics. Micro fluidic flow cytometers (imaging and laser based) are the emerging diagnostics tools for biological cell identi cation, categorization, and counting. Despite the advances in this area these flow cytometers have not yet been turned into POCD devices. This thesis focuses on finding solutions for the major problems associated with these micro fluidic flow cytometers towards becoming POCD devices. However, the developed techniques are quite general and can be equally well applied for non-cellular diagnostics as well. More specfi cally this thesis presents techniques for focusing of cells in flow, in- flow decantation, and pumping along with the experimental demonstration of these techniques in the context of deformability estimation of cells in ow, blood cell counting, and quantitative microscopic urinalysis. Focusing of the cells while in flow is at the heart of the operation of flow cytometers. The developed technique reduces the complexity of fabrication and offers its applicability for a wide range of flow rates, thereby decreasing the cost per device and simultaneously offering the flexibility of its use in both imaging and laser-based flow cytometers. The in- flow decantation technique adds an extra dimension to the possibility of realizing sheath-free ow focusing, by separating the particle-free fluid from the sample itself. The simplicity of the design and the applicability of the technique for wide varieties of ow rates, particle concentrations, and sizes while having the ability to offer 100 % purity at high yields, offers its potential applications into the realization of POCD that can operate on plasma separated from whole blood for several biochemical assays, rare cell enrichment in cancer diagnostics, immunodiagnostics etc. Pumping is an unavoidable task to cause ow of either sample or sheath fluids into the micro fluidic device. The developed pump uses the mechanical energy from the fingers and stores inside an elastic block to cause pumping action when released subsequently. The pumping mechanism is very general and is independent of the type of elastomeric block involved. However, the low rate quantity and stability depend upon the nature of stress-strain curve and the stiffness of the elastomeric block used inside the pump. This pump is inexpensive (< 4 USD), compact, portable, and reusable (> 500 times) making it as an ideal choice for the POCDs. Using the developed ow focusing device, deformability and the associated elastic moduli of RBCs (that are obtained from healthy and diabetic subjects) and cancer cells (with and without Emodine Anticancer drug treatment) were obtained and the results were found to be in agreement with literature. To further demonstrate the applicability of the ow focusing device for blood cell imaging and counting, blood cells (Red Blood Cells - RBCs and White Blood Cells - WBCs) in ow were imaged and the counts were compared with standard hematological analyzer counts. Similar experiments were performed on urine samples to demonstrate the technique's applicability for quantitative microscopic urinalysis. Wide variety of cells that can be found in Urine such as RBCs, WBCs, Epithelial cells, and Casts were imaged, and a quantitative analysis was performed to infer the diagnosis and the observations were compared with clinical results. All these results indicate the robustness of the developed techniques and their excellent applicability for a wide range of POCDs.