Development of a system for the rapid detection of protein biomarkers in whole human blood

Medical diagnostics contributes a large portion of both time and resources in the timeline of medical treatment. Point-of-Care diagnostics offers the ability to diagnose illness or the risk of certain conditions rapidly and at low cost, affording the opportunity of implementing immediate medical intervention. In order to provide appropriate Point-of-Care diagnostics systems, techniques and physical systems must be developed to ensure system portability and the ability to detect protein biomarkers in human samples. The objective of this study was twofold: to develop a process for the quantification of a protein biomarker, C-reactive protein, in whole human blood and to detect C-reactive protein using a lensless charge coupled device. Whole blood is a complex sample matrix rich with information for diagnostics and charge coupled devices are small and offer a portable detection tool. A microfluidic, lab-on-chip system was developed and a series of tests were conducted to develop a system and protocol capable of detecting C-reactive protein in whole human blood. A poly(dimethylsiloxane) microfluidic chip was used in combination with a plasma separation membrane to separate plasma from whole blood. The plasma was then treated to deplete high abundance proteins from the sample. High abundance proteins can be present in concentrations several orders of magnitude higher than biomarker proteins and it therefore becomes important to remove them from the sample to avoid interference and non-specific binding. C-reactive protein was then quantified with a fluorescent sandwich immunoassay using a fluorescent spectrometer. A limit of detection of 4.54 µg/mL was achieved within 50 minutes. A system utilizing the same microfluidic system components, but using a charge coupled device as the detection instrument to detect a fluorescent signal from the fluorescent immunoassay. The charge coupled device was placed directly below the detection site of the microfluidic chip to directly read the emission signal. In this case quantum dots were used as the molecule for optical detection through biotin/streptavidin affinity. A limit of detection of 5.76 µg/mL was achieved. Furthermore, a microfluidic chip was designed for the rapid, one step detection and quantification of C-reactive protein concentration in whole blood samples. The footprint of the microchannels was designed to incorporate the developed procedure for protein detection in whole blood and offer a user rapid, one-step Point-of-Care diagnostics with the use of a previously developed system