Burcu Gumuscu

Photopatterning of hydrogel microarryas in closed microchips

To date, optical lithography has been extensively used for in situ patterning of hydrogel structures in a scale range from hundreds of microns to a few millimeters. The two main limitations which prevent smaller feature sizes of hydrogel structures are (1) the upper glass layer of a microchip maintains a large spacing (typically 525 μm) between the photomask and hydrogel precursor, leading to diffraction of UV light at the edges of mask patterns, (2) diffusion of free radicals and monomers results in irregular polymerization near the illumination interface. In this work, we present a simple approach to enable the use of optical lithography to fabricate hydrogel arrays with a minimum feature size of 4 μm inside closed microchips. To achieve this, we combined two different techniques. First, the upper glass layer of the microchip was thinned by mechanical polishing to reduce the spacing between the photomask and hydrogel precursor, and thereby the diffraction of UV light at the edges of mask patterns. The polishing process reduces the upper layer thickness from ∼525 to ∼100 μm, and the mean surface roughness from 20 to 3 nm. Second, we developed an intermittent illumination technique consisting of short illumination periods followed by relatively longer dark periods, which decrease the diffusion of monomers. Combination of these two methods allows for fabrication of 0.4 × 10(6) sub-10 μm sized hydrogel patterns over large areas (cm(2)) with high reproducibility (∼98.5% patterning success). The patterning method is tested with two different types of photopolymerizing hydrogels: polyacrylamide and polyethylene glycol diacrylate. This method enables in situ fabrication of well-defined hydrogel patterns and presents a simple approach to fabricate 3-D hydrogel matrices for biomolecule separation, biosensing, tissue engineering, and immobilized protein microarray applications.

Highly Sensitive Determination of 2,4,6-Trinitrotoluene and Related Byproducts Using a Diol Functionalized Column for High Performance Liquid Chromatography

In this work, a new detection method for complete separation of 2,4,6-trinitrotoluene (TNT); 2,4-dinitrotoluene (2,4-DNT); 2,6-dinitrotoluene (2,6-DNT); 2-aminodinitrotoluene (2-ADNT) and 4-aminodinitrotoluene (4-ADNT) molecules in high-performance liquid-chromatography (HPLC) with UV sensor has been developed using diol column. This approach improves on cost, time, and sensitivity over the existing methods, providing a simple and effective alternative. Total analysis time was less than 13 minutes including column re-equilibration between runs, in which water and acetonitrile were used as gradient elution solvents. Under optimized conditions, the minimum resolution between 2,4-DNT and 2,6-DNT peaks was 2.06. The recovery rates for spiked environmental samples were between 95–98%. The detection limits for diol column ranged from 0.78 to 1.17 µg/L for TNT and its byproducts. While the solvent consumption was 26.4 mL/min for two-phase EPA and 30 mL/min for EPA 8330 methods, it was only 8.8 mL/min for diol column. The resolution was improved up to 49% respect to two-phase EPA and EPA 8330 methods. When compared to C-18 and phenyl-3 columns, solvent usage was reduced up to 64% using diol column and resolution was enhanced approximately two-fold. The sensitivity of diol column was afforded by the hydroxyl groups on polyol layer, joining the formation of charge-transfer complexes with nitroaromatic compounds according to acceptor-donor interactions. Having compliance with current requirements, the proposed method demonstrates sensitive and robust separation.

Complete dissipation of 2,4,6-trinitrotoluene by in-vessel composting†

We demonstrate complete removal of 2,4,6-trinitrotoluene (TNT) in 15 days using an in-vessel composting system, which is amended with TNT-degrading bacteria strains. A mixture of TNT, food waste, manure, wood chips, soil and TNT-degrading bacteria consortium are co-composted for 15 days in an aerobic environment. Variations in the TNT degradation rates are assessed when composting reactors are operated at different carbon/nitrogen ratios (C/N), aeration rates, TNT concentrations and TNT-degrading bacteria inoculum loads. Changes in TNT concentrations are measured using high performance liquid chromatography, and C/N are determined using elemental analysis every 5 days. Temperature and moisture of the system are measured every 6 hours. Optimum TNT degradation performance is achieved by combining C/N of 20/1 and a 5 L min−1 aeration rate. Complete removal is achieved for TNT concentrations of 2, 10, and 100 g kg−1 in 15 days by the help of Citrobacter murliniae STE10, Achromobacter spanius STE11, Kluyvera cryocrescens STE12, and Enterobacter amnigenus STE13 bacteria strains. The final products of composting are used to cultivate four different plant seedlings for 10 weeks and showed no toxic effect, which is promising for the potential agricultural use of TNT-contaminated lands after remediation.

Lab-on-a-chip devices with patterned hydrogels

Hydrogels are considered to be in the class of smart materials that find application in diagnostic, therapeutic, and fundamental science tools for miniaturized total analysis systems. The use of patterned hydrogels in closed fluidic microchips for different research fields depends crucially on the ease and accessibility of their fabrication technology. In this work, two simple fabrication procedures are developed to pattern hydrogel microarrays. First, intermittent illumination is applied on mechanically polished microchips for the photopatterning of hydrogels. Second, capillary pressure barriers are used for controlling the position of the liquid-air meniscus in microchip channels, allowing the subsequent patterning of hydrogels by photopolymerization and thermo-gelation. As the first major application of hydrogels, we describe a novel method for concurrent continuous flow fractionation and purification of DNA fragments in a microfluidic device filled with agarose gel. We exploit the variation in the field-dependent mobility of DNA molecules with DNA length for the fractionation. Since this new mechanism can be applied using agarose gel, it provides a low-cost, robust, and versatile separation matrix. We propose and demonstrate an in vitro microfluidic cell culture platform that consists of periodic 3D hydrogel structures as the second major application of hydrogels. The unique architecture of the microchip enables culturing of human intestine cells, which spontaneously grow into 3D structures on the 3rd day of cell culturing. On the 8th day of culture, Caco-2 cells are co-cultured for 36 hours with intestinal bacteria E.coli, which adhered to the cells without affecting the cell viability. Different compartment geometries lead to a difference in the proliferation and cell spread profile of Caco-2 cells. Microelectrodialysis is explored as the last major application of hydrogels in this thesis. We firstly show that parallel streams of concentrated and ion-depleted water are formed in continuous flow when a potential difference is applied across the microchip containing alternating rows of patterned cation- and anion-selective hydrogels. The device could remove approximately 75% of the 1 mM sodium chloride salt introduced via the inlet streams. Secondly, the microchip enables ion transport visualization in the ion selective hydrogels and microchannels.