Gergana G. Nestorova

Thermoelectric method for sequencing DNA

This study describes a novel, thermoelectric method for DNA sequencing in a microfluidic device. The method measures the heat released when DNA polymerase inserts a deoxyribonucleoside triphosphate into a primed DNA template. The study describes the principle of operation of a laminar flow microfluidic chip with a reaction zone that contains DNA template/primer complex immobilized to the inner surface of the devices lower channel wall. A thin-film thermopile attached to the external surface of the lower channel wall measures the dynamic change in temperature that results when Klenow polymerase inserts a deoxyribonucleoside triphosphate into the DNA template. The intrinsic rejection of common-mode thermal signals by the thermopile in combination with hydrodynamic focused flow allows for the measurement of temperature changes on the order of 10(-4) K without control of ambient temperature. To demonstrate the method, we report the sequencing of a model oligonucleotide containing 12 bases. Results demonstrate that it is feasible to sequence DNA by measuring the heat released during nucleotide incorporation. This thermoelectric method for sequencing DNA may offer a novel new method of DNA sequencing for personalized medicine applications.

Detection and quantification of 8-hydroxy-2′-deoxyguanosine in Alzheimer's transgenic mouse urine using capillary electrophoresis

8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) is one of the major forms of oxidative DNA damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this study was to develop a sensitive method to accurately and rapidly quantify the 8‐OHdG by using CE‐LIF detection. The method involved the use of specific antibody to detect the DNA lesion (8‐OHdG) and consecutive fluorescence labeling. Next, urinary 8‐OHdG fluorescently labeled along with other constituents were resolved by capillary electrophoretic system and the lesion of interest was detected using a fluorescence detector. The limit of detection was 0.18 fmol, which proved sufficient sensitivity for detection and quantification of 8‐OHdG in untreated urine samples. The relative standard deviation was found to be 11.32% for migration time and 5.52% for peak area. To demonstrate the utility of this method, the urinary concentration of 8‐OHdG in an Alzheimers transgenic mouse model was determined. Collectively, our results indicate that this methodology offers great advantages, such as high separation efficiency, good selectivity, low limit of detection, simplicity and low cost of analysis.

Thermoelectric lab-on-a-chip ELISA

We report a new, thermoelectric method for performing enzyme-linked immunosorbent assay (ELISA) in a microfluidic device. The concentration of the analyte is determined by measuring the heat of an enzymatic reaction between glucose and glucose oxidase using thin-film antimony/bismuth thermopile. The feasibility of lab-on-a-chip thermoelectric ELISA is demonstrated by measuring the concentration of 8-hydroxy-2-deoxyguanosine (8OHdG) in urine samples from amyloid precursor protein transgenic mice. The detection method is based on formation of a complex between 8OHdG, anti-8OHdG capture antibody and glucose oxidase linked IgG antibody. The complex is immobilized at the lower channel wall of the microfluidic device, over the measuring junctions of the thermopile. The amount of heat detected by the thermoelectric sensor is inversely proportional to the concentration of 8OHdG. Standard calibration curve was created using synthetic 8OHdG. The regression line equation of the standard calibration curve was used to estimate the concentration of 8OHdG in mouse urine.