(2jx) Charge based high throughput fractionation and biosensing of exRNA nanocarriers (Extracellular vesicles, Lipoproteins and Ribonucleic protein)

My overarching research interests is the development of novel microfluidics technologies to probe the heterogeneity of Extracellular RNAs (exRNAs) nanocarriers classes as well as their subclasses for disease diagnostics and therapeutics applications.

exRNAs are actively secreted in physiological fluids (e.g., blood, urine, lymph fluids) by host cells and encode complex cellular communication signatures, thus serving as promising biomarkers for various disease states. Typically, exRNA are encased and protected by three general classes of nanoscale molecular shuttles – extracellular vesicle (EVs), lipoprotein (LLP s), and ribonucleic protein (RNPs) – many with several subtypes (e.g., small and large EVs, or HDL, LDL, VLDL). Nanocarrier intercellular signaling is hence fundamental to the cellular basis of disease progression, biomarker discovery and therapeutics applications. These nanocarriers have a great potential to be next generation disease biomarkers for liquid biopsy. The vast heterogeneity even in the nanocarrier subclasses, not only between various biofluids and source cell types, but also between donors and experiments, has been a major impediment to advances in understanding exRNA synthesis and function. Hence, bias free, high-throughput, high yield, and pure isolation of these nanocarriers coupled with subsequent sensitive detection is extremely important. However, the conventional isolation technologies have extremely low yield, laborious and susceptible to cross contamination from non-targets as well as other nanocarriers thus manifesting in inaccurate downstream analysis. The inherent challenge of physically separating and isolating these nanocarriers from a biofluid stems from their overlapping size and mass distributions.

Hence, my research direction will be focused on solving the above-mentioned issues and can be divided into two broad categories:

1. Isolation of nanocarrier subclasses: There is growing evidence some proteins are only active when present on specific nanocarriers. One such case is mitochondria enriched EVs which are highly over expressed in the blood plasma of various cancer patients. Leveraging my Post-doctoral work, I will utilize bipolar membranes based continuous isoelectric focusing microfluidics device to generate scalable and tunable linear pH gradient to fractionate mitochondria positive EVs from normal EVs based on their charge instead of size and mass. I have already developed such device can isolate RNPs from EVs and lipoproteins at high throughput (~ml/hour), yield (>78%) and purity (>93%). Also, other nanocarriers binary mixtures such as HDL-LDL, HDL-RNP and LDL-EVs were separated with similar performance. I also developed a machine learning platform for further optimizing this microfluidics device along with a theoretical model to gain greater mechanistic understanding of the system. Pure isolation of mitochondria derived EVs will open up new avenues not only for cancer but also for Alzheimer’s and Parkinson's disease where key proteins such as aggregated alpha synuclein, tau and beta-amyloid has been shown to be associated with mitochondria derived EVs.
2. Probing nanocarrier subclass heterogeneity using ultrasensitive biosensors: Once fractionated, I will develop highly sensitive biosensors for accurate detection of key proteins present on these nanocarrier surfaces. In my PhD and Post doc, I have shown that microwells can be used to concentrated biologics inside microwells and can be further analyzed very accurately. During my PhD, I did drug-protein interaction studies on individual molecules isolated in these pico litre size wells alongside with theoretical mechanistic studies. Moreover, in my Post doc, I developed a highly sensitive duplex digital ELISA platform for protein colocalization assay. Further development of these tools will allow multiplex detection of key proteins on the same nanocarrier along with realtime monitoring of their interactions with various biologics and even cells.

Teaching Interests:

I have a genuine interest in teaching and have explored all the opportunities, I have got in my PhD and Post-Doct work at KIST Europe (Saarbruecken, Germany), CEERI Pilani (Rajasthan, India), IIT Bombay (Maharashtra, India) and at Notre Dame (Indiana, USA). During my PhD work in Germany, I took 3 months praktikum for microfluidics at Department of Mechatronics, University of Saarland, Germany. At IIT Bombay, I worked as an instructor for selected lectures for Microfluidics course and worked as a T.A. for undergraduate lab (Measurements Lab) consisting of 150 students. I have keen interest in teaching core courses such as fluid mechanics, transport phenomena and heat transfer and have published some theoretical articles in this domain as well. Moreover, based on my electronics undergraduate background and my research experience, I also want to propose new elective courses at the undergraduate or graduate level, covering electrokinetics, microfluidics and bioelectronics. In these courses students apart from gaining theoretical knowledge, the students will be exposed to modern microfluidics fabrication methods such as 3D printing, soft lithography and finite element based COMSOL simulations.