Thesis Title :  "From Molecular Detection to Single-Cell Sequencing: Engineering Biological Information for Nanopore Readout"

Name of the Student : Mr. Mayank Mitram

Degree Registered : Ph.D. Engineering 

Advisor : Prof. Manoj Varma, CeNSE

Date : 4th September 2026 (Friday)

Time : 4:00 PM

Venue : CeNSE Seminar Hall (Hybrid)
Abstract:

Biological systems contain information across multiple scales, from individual biomolecules to 
heterogeneous populations of cells. However, conventional analytical approaches often require 
different measurement strategies for different molecular classes and may obscure cell-to-cell 
heterogeneity. This thesis has focused on developing sequencing-enabled strategies to convert 
diverse forms of biological information into interpretable, nucleic-acid-based readouts, with an 
emphasis on Oxford Nanopore sequencing.

The first part of this work establishes a PCR-free platform for Multiplexed detection of proteins, 
microRNAs, and small biomolecules by integrating molecular recognition, Hybridization Chain Reaction 
(HCR), and DNA barcoding with nanopore sequencing. In this approach, target recognition initiates the 
formation of sequence-defined DNA products containing target-specific barcodes, thereby translating the 
presence of chemically diverse analytes into a common sequencing-readable format. The platform enables
multiplexed detection with minimal cross-reactivity and was further investigated in increasingly complex 
biological backgrounds, demonstrating the potential of nanopore sequencing as a unified readout for 
heterogeneous molecular targets.

While molecular encoding provides information about what is present, biological heterogeneity also requires 
knowledge of where that information originates. This motivates the second part of the thesis, in which a 
Microfabricated PDMS hydrophobic-barrier platform for bacterial single-cell capture is developed. The device 
incorporates patterned hydrophobic barriers and hydrophilic capture regions to generate spatially isolated 
microenvironments, enabling parallel isolation of individual bacterial cells while preserving their spatial 
identity. Microfabrication and surface characterization demonstrated the formation of well-defined structures 
suitable for high-density single-cell capture.

Building upon this platform, the third part of the thesis explores Bacterial single-cell sequencing using 
Oxford Nanopore technology. A workflow was developed to process individually captured bacterial cells for 
molecular recovery and sequencing, addressing challenges associated with bacterial RNA, including the absence 
of conventional poly(A) tails and the high abundance of ribosomal RNA. The resulting approach provides a route 
toward cell-resolved molecular characterization using nanopore sequencing.

Together, these studies represent a progression from molecular detection to spatially resolved single-cell analysis, 
unified by a common principle: Engineering biological information into formats that can be read by sequencing. 
This work establishes a foundation for future platforms capable of integrating multiplexed molecular detection 
with single-cell resolution to interrogate biological systems across multiple scales.