Platform Technologies for In vitro Point-of- Care Diagnostics
In vitro diagnostic (IVD) tests are critical for making informed clinical decisions. These tests are performed outside the body using specimens such as blood, sputum, saliva, and urine. IVD tests are usually performed at centralized labs with complex and expensive instrumentation, and associated protocols. There are only a few commercially available point-of-care (POC) devices for performing IVD Tests. This thesis presents platform technologies for POC testing of three categories of IVDs: Clinical Biochemistry, Immunodiagnostics, and Molecular diagnostics. Additionally, a Biosafety Level 2+ (BSL- 2+) Mobile Laboratory was designed & developed for testing of infectious diseases at the point-of-need. Clinical biochemistry tests are quantitative tests that measure the concentration of biochemicals in body fluids. Most clinical biochemistry tests involve the testing of serum. A POC serum separation device from micro-volumes of blood using an air-displacement pipette and a chemical-preloaded disposable pipette-tip cartridge is introduced in Chapter- 2. To address the issue of reagent stabilization and room-temperature storage, trehalosebased enzyme reagent matrix (TERM) cartridge with dried reagents was developed. The cartridge was tested for triglycerides measurement in blood and found to offer good shelflife. In addition, the development of reagent kits for conducting sickle-cell disease confirmatory test at point-of-need was reported. Finally, as an alternative to conventional biochemical analyzers, a portable multi-spectral absorbance reader capable of detecting the results at POC of a wide range of clinical biochemical assays was developed and validated. Immunodiagnostics includes tests that can detect and quantify antigens and antibodies in a sample. Chapter-3 of the thesis proposes the development of platform technologies in this category based on introducing innovative (i) protocols and (ii) processes: (i) Counting target CD4+ T-Cells in blood was demonstrated using anti-CD4 antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPIONs) and a commercially available haematology analyser. (ii) A bare fibre Bragg grating (bFBG) sensor-based technique is developed for pathogen detection, and demonstrated its ability in detecting E. coli in water by coating anti-E. Coli antibodies onto the bFBG sensor. Molecular diagnostics comprises of techniques that can detect the presence or modification of nucleic acid (DNA/RNA). These tests usually require sophisticated and iv expensive instruments. To address this issue, Chapter-4 reports the development of Portable PCR System: i) Isothermal Amplification Device (LAMP assays), ii) Handheld Thermal Cycler (tested for HCV & Dengue), and iii) Fluorescence Reader (tested for CoViD-19). In Chapter-5, a mobile laboratory for infectious diseases testing and reporting (MITR Lab) was conceptualized, designed, fabricated, and found to offer 100% match with static lab results in testing CoViD-19. The MITR lab is the first ICMR approved Mobile BSL2+ lab for COVID-19 testing and aided in conducting few lakh tests during the past one year. All the technologies developed as part of the thesis are validated with clinical samples and showed sensitivity and specificity above 90%. These innovative, affordable platform technologies are expected to create huge social impact by providing timely diagnoses at the point-of-need.