Microfluidic devices for sample clean-up and screening of biological samples

Abstract

Analytical chemistry plays an important role in the separation and identification of analytes from raw samples (e.g. plant extracts, blood), but the whole analytical process is tedious, difficult to automate and time consuming. To overcome these drawbacks, the concept of μTAS (miniaturized total analysis systems) was proposed by researchers in the early 90’s. The research described in this thesis, aimed towards the development of a microfluidic device that can be used for sample clean-up and screening of biological samples. Both solid phase extraction and liquid-liquid phase extraction have been investigated. The aim in chapter 2 to chapter 5 is to develop a microfluidic device that can selectively adsorb products from a process stream (plant extracts and serum samples) through molecular interactions. To realize this, first a protocol for carbohydrate immobilization on glass surfaces was developed and later the developed protocol was employed to prepare carbohydrate modified capillary columns to study specific carbohydrate-lectin interactions. However, due to low column capacity, the carbohydrate capillary columns are not efficient to bind lectins. To improve the column capacity, monoliths, a new generation of stationary phases, were chosen for carbohydrate immobilization. Two protocols were developed (a three-step protocol and a single step protocol). The advantage of the single step protocol is the lower amount of time needed to prepare an affinity monolithic column. The carbohydrate monolithic columns efficiently captured lectins (α-mannose column captured Concanavalin A and Lens culinaris, β-galactose column captured Arachis hypogaea), and antibodies (GM1 and GM2 columns specifically captured IgM antibodies) from serum samples of patients suffering from Guillain-Barré syndrome (GBS). These columns can also be used to study the dissociation constants (Kd) of carbohydrate-lectin interactions. The carbohydrate monolith prepared in a microchip gave identical results as the carbohydrate monolith in a capillary. The initial attempt to prepare a carbohydrate monolithic array microfluidic chip was successful. However, the fluid flow in the two channels varied due to the difference in permeability of the two affinity columns. Further fine tuning of the permeability of the two columns is necessary to use this array microfluidic chip for screening of analytes. The aim in chapter 6 is develop a three-phase microfluidic chip (liquid-liquid extraction) for efficient sample clean-up and screening of acidic or basic molecules in aqueous solution. The extraction of strychnine was studied using a two-phase microchip, followed by “simultaneous extraction and back extraction” of strychnine using a three-phase microchip. Maximum extraction of strychnine was achieved at longer residence times i.e. lower flow rates. A good correlation between experimental results and model data was found for both two-phase and three-phase microchips. Sample clean-up of a Strychnos seed extract was successful with the three-phase microchip. The developed model can be used to predict the extraction outcome by changing various parameters e.g. chip design, partition coefficient, and viscosity. On-line screening of strychnine was demonstrated by interfacing the two-phase microchip with nanospray ESI-MS. The initial interfacing of a three-phase microchip with nanospray ESI-MS led to the disturbance of the phase separation in the chip due to pressure differences in the three channels. Fine tuning of the interface connections between microchip and MS could enhance the chance of using this three-phase microchip for on-line screening of plant extracts. <br/