This was the title of my PhD dissertation work. I successfully defended it on Tuesday. Now it feels really great!!! Just to give you some idea about my dissertation work, below is the general description of it. Details of the research work can be found in the dissertation which will be available online soon and in upcoming publications as journal articles.
Enzyme-linked immunosorbent assay (ELISA) is arguably one of the most practiced techniques used for reliable quantitation of biological analytes in complex sample matrices. The advantages of ELISA arise mainly from the specific interaction between an antigen and its corresponding antibody, and the signal amplification due to an enzyme reaction that produces multitude of detectable species per binding event of the target molecule to the assay surface. The conventional methods of performing ELISA are predominantly based on polystyrene microtiter plates that require relatively large amounts (~100 µL) of expensive and/or precious samples or reagents. This volume may not be large itself but it becomes an issue when one has to deal with expensive reagents and limited amount of sample. For example, analysis of multiple biomarkers from the content of a single cell. There is a need to miniaturize these ELISA methods. Moreover, the limited ability of this assay format to measure biomarker concentrations (e.g., antigens/antibodies) circulating in bodily fluids restricts its use in the detection of several fatal diseases (e.g., cancers) at an early stage. Therefore, there is a need to improve the minimum analyte concentration detectable (limit of detection) by these techniques. In addition to this insufficient assay sensitivity, the microtiter plate version of ELISA requires that the enzyme substrate undergoes a change in its spectral signature during the enzyme reaction necessitating that its fluorophore be acted upon by the enzyme-label. This constraint, in turn, limits the number of possible enzyme-substrate couples available for use in the ELISA method.
In my dissertation work, these major limitations of conventional ELISA methods have been addressed to broaden their utility in biomedical applications. The sample/reagent volume requirement of microtiter plate based ELISA has been reduced by simultaneously miniaturizing this assay and enhancing its multiplexing capabilities using the microfluidic platform. In an effort to improve the limit of detection, we have developed a pre-concentration ELISA method based on field amplified stacking of enzyme reaction product molecules in the ELISA micro-channel using two buffers of different ionic strengths.
In addition, we have demonstrated an ELISA method for the first time in which the enzyme substrate does not need to undergo a change in its spectral properties to allow its distinction from the enzyme reaction product in the assay chamber. We have accomplished this goal by synthesizing a rhodamine B based substrate molecule for the alkaline phosphatase enzyme-label, and then applying it to measuring the concentration of human TNF-α in a microfluidic device.
Lastly, a microfluidic method for simultaneous pre-concentration of cationic and anionic chemical species is described in my dissertation. This method is again based on the field amplified sample stacking process, and has been realized by injecting a relatively large plug of a sample prepared in a low-conductivity buffer into a microfluidic channel.