Design and application of hollow silica microspheres for density-based bioseparations
Challenges to the detection of low-abundance biological analytes from complex mixtures, such as biological fluids and food extracts, still exist and are critical for the management of infectious diseases like influenza and gastroenteritis. Typically, analytes must be separated and concentrated from complex sample matrices in order to meet the sensitivity and purity requirements of the downstream detection system. Unfortunately, many of the existing separation methods are time consuming and/or costly, which limits their use in point-of-care settings, such as a rural clinic or doctor’s office in developing countries, where rapid diagnostic testing is vital to initiating treatment. The goal of this dissertation work was to develop a bioseparation approach that is inexpensive and easy to use, with absolutely no external instrumentation required. Our “molecular buoy” approach used low-density hollow silica microspheres, functionalized with target-specific antibodies to bind and separate target biomolecules from a complex sample matrix by floatation. We characterized the size and floatation properties of the hollow microspheres in aqueous solutions of increasing density and viscosity. Separation times were found to be inversely proportional to the microsphere size and directly proportional to the solution viscosity. Methods for surface functionalization with protein G were established with an estimated binding capacity of 31 µg/mg for size-fractionated microspheres 38 µm in diameter, and 50 µg/mg for size-fractionated microspheres 81 µm in diameter. We then applied the molecular buoy bioseparation method to the isolation of an infectious disease pathogen Cryptosporidium parvum, a protozoan parasite that is a common cause of acute/persistent diarrheal illness. When spiked into buffer or watery stool at known C. parvum oocyst concentrations, we obtained a relatively high capture efficiency (average recovery rate 95.4%) in less than 5 minutes. In addition, we integrated this novel buoyancy-assisted separation approach with a colorimetric paper-based microfluidic test to ultimately demonstrate a low-cost and instrumentation-free method that sequentially achieves complete sample-to-answer diagnostics. It is expected that this research will establish new materials and methodologies for rapid bioseparation from complex matrices that are applicable to diverse protein analytes, biomarkers, and pathogens for improved detection and bioanalysis of infectious diseases.
bioseparation, sample preparation, analyte concentration and isolation, buoyancy cell separation, microsphere, cryptosporidium isolation, paper-based diagnostic device
Xiang, L. (2016). Design and application of hollow silica microspheres for density-based bioseparations [Doctoral dissertation, Texas State University].
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