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

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/

Zooplankton visualization system: design and real-time lossless image compression

In this thesis, I present a design of a small, self-contained, underwater plankton imaging system. I base the imaging system’s design on an embedded PC architecture based on PC/104-Plus standards to meet the compact size and low power requirements. I developed a simple graphical user interface to run on a real-time operating system to control the imaging system. I also address how a real-time image compression scheme implemented on an FPGA chip speeds up image transfer speeds of the imaging system. Since lossless compression of the image is required in order to retain all image details, I began with an established compression scheme like SPIHT, and latter proposed a new compression scheme that suits the imaging system’s requirements. I provide an estimate of the total amount of resources required and propose suitable FPGA chips to implement the compression scheme. Finally, I present various parallel designs by which the FPGA chip can be integrated into the imaging system

Recent developments in the rapid analysis of plants and tracking their bioactive constituents

Natural products chemistry has witnessed many new developments in the last 5 years like extractions with subcritical water and ionic liquids, LC/HRMS and LC/SPE/cryo-NMR, UHPLC, TLC/MS, MS-based preparative HPLC, comprehensive chromatography (GC × GC, LC × LC), high-throughput screening, introduction of monolithic columns, miniaturisation, and automated structure identification. Nevertheless identifying bioactive constituents in complex plant extracts remains a tedious process. The classical approach of bioassay guided fractionation is time-consuming while off-line screening of extracts does not provide information on individual compounds and sometimes suffers from false positives or negatives. One way out of this is by coupling chromatography with chemical or biochemical assays, so called high resolution screening. An example is the development of HPLC on-line assays for antioxidants. By the post-column addition of a relatively stable coloured radical like DPPH¿ or ABTS¿+, radical scavengers are detected as negative peaks because in a reaction coil they reduce the model radical to its reduced, non-coloured form. When combined with LC/DAD/MS and LC/SPE/NMR, reliable identification of active constituents becomes possible without the necessity of ever isolating them in a classical sense. Also for finding leads for new drugs, combining HPLC with biochemical assays is interesting but technically more difficult. Most enzymes do not work at the organic modifier concentrations commonly encountered in RP-HPLC and the reaction time is often longer requiring dilution and lengthy coils respectively. Therefore, new techniques have to be implemented to gain the required sensitivity for on-line enzyme assays. For stable analytes, high temperature LC offers a solution to the organic modifier problem. When enzymes are highly expensive, like those used in the screening for Cytochrome P450 inhibitors, miniaturisation to chip format may offer a way out. Microreactors (chips) are not only useful for miniaturising larger assays but also offer completely new prospects in phytochemical analysis. One such application is in the sample clean-up of acids and bases like alkaloids. In a lay-out of three parallel channels of 100 ¿m width with the middle one containing organic phase and the two outer ones water of high pH (feed phase) and low pH (trapping phase) such a chip replaces two classical LLE steps but is much faster and requires less solvents and less manpower input

Onion based zinc oxide nanoparticles ability as electrochemical sensor

This work describes the preparation of zinc oxide nanoparticles (ZnO NP) from onion extract through a combination of microwave irradiation and muffle furnace in less than 2 h. Characterization with UV-VIS, FT-IR, and XRD has confirmed successful formation of nanoparticles. TEM analysis displayed the nanoparticles to be spherical shaped with an average size of 6.4 nm. The developed ZnO NP film displayed good sensing capability of dopamine and had an oxidation potential of + 0.3 V. Under optimized conditions (pH and scan rate), the ZnO NP-GCE is stable even at 500 RPM, showed good linearity in sensing dopamine within the tested range of 3.9 - 250 µM, has a limit of detection of 3.53 µM and stable electrocatalytic activity even after 30 cycles. The newly developed electrode can distinguish dopamine in a satisfactory way in presence of ascorbic acid and glucose and with 94.9 % of detection in human urine. This study also highlighted the potential of IO Rodeostat (an open source electrochemical workstation) as a feasible option in low resource laboratories to perform sensing studies

Onion based zinc oxide nanoparticles ability as electrochemical sensor

1100-1107This work describes the preparation of zinc oxide nanoparticles (ZnO NP) from onion extract through a combination of microwave irradiation and muffle furnace in less than 2 h. Characterization with UV-visible, FTIR, and XRD has confirmed successful formation of nanoparticles. TEM analysis displays the nanoparticles to be spherical shaped with an average size of 6.4 nm. The developed ZnO NP film displays good sensing capability of dopamine and had an oxidation potential of + 0.3 V. Under optimized conditions (pH and scan rate), the ZnO NP-GCE is stable even at 500 rpm, showed good linearity in sensing dopamine within the tested range of 3.9-250 µM, has a limit of detection of 3.53 µM and stable electrocatalytic activity even after 30 cycles. The newly developed electrode can distinguish dopamine in a satisfactory way in presence of ascorbic acid and glucose and with 94.9 % of detection in human urine. This study also highlighted the potential of IO Rodeostat (an open source electrochemical workstation) as a feasible option in low resource laboratories to perform sensing studies

Palladium-catalysed synthesis of amidines and imidates

A novel palladium-catalysed three-component synthesis of alkenyl amidines and imidates from alkenyl bromides, isonitriles and amines or alcohols was developed. The amidine synthesis utilised CS2CO3 as the base and was limited to tert-buiyl isonitrile. In the imidate synthesis NaO'Bu was the base and different isonitriles were successfully utilised. The stereochemistry of the group in alkenyl bromide was retained in the product. The stereochemistry of the imine double bond in two of the amidines was shown to be the thermodynamically more stable (^-isomer by X- ray crystallography.</p

Mixed matrix membranes for efficient adsorption of copper ions from aqueous solutions

This work presents adsorption of copper (Cu2+) ions from aqueous solutions using mixed matrix membrane (MMM) and its elution afterwards. The developed flat sheet MMM, comprises of chitosan beads incorporated in Ethylene vinyl alcohol (EVAL) polymer porous matrix and exhibits static Cu2+ ion adsorption capacity of 410.5 mg Cu2+ ions per g chitosan bead, which is higher than earlier studies reported on chitosan as adsorbent (beads, membranes and hydrogel). Dynamic adsorption of Cu2+ ions from aqueous solution with MMM is similar to static adsorption but it is achieved much faster (15 min versus 60 min). Attempts to desorb Cu2+ ions from chitosan beads in packed bed column resulted in bead aggregation, restriction of Cu2+ ion desorption and reusability of the column. Under similar experimental conditions, Cu2+ ions desorbed successfully from the MMM without loss of beads from the membrane structure and the MMM was reusable. Overall, the MMM developed in this study shows superiority over packed bed columns in terms of low pressure drop, high adsorption capacity, successful desorption of Cu2+ ions and reusability for few cycles

Bioaffinity chromatography on monolithic supports

Affinity chromatography on monolithic supports is a powerful analytical chemical platform because it allows for fast analyses, small sample volumes, strong enrichment of trace biomarkers and applications in microchips. In this review, the recent research using monolithic materials in the field of bioaffinity chromatography (including immunochromatography) is summarized and discussed. After giving an introduction into affinity chromatography, information on different biomolecules (antibodies, enzymes, lectins, aptamers) that can act as ligands in bioaffinity chromatography is presented. Subsequently, the history of monoliths, their advantages, preparation and formats (disks, capillaries and microchips) as well as ligand immobilization techniques are mentioned. Finally, analytical and preparative applications of bioaffinity chromatography on monoliths are presented. During the last four years 37 papers appeared. Protein A and G are still most often used as ligands for the enrichment of immunoglobulins. Antibodies and lectins remain popular for the analysis of mainly smaller molecules and saccharides, respectively. The highly porous cryogels modified with ligands are applied for the sorting of different cells or bacteria. New is the application of aptamers and phages as ligands on monoliths. Convective interaction media (epoxy CIM disks) are currently the most used format in monolithic bioaffinity chromatograph