# Single-Molecule Processing: Detection and Identification of Single DNAs, RNAs, and Proteins using Immobilized Nanoscale Enzymatic Reactors (INERs) and Nanoscale Electrophoresis

> **NIH NIH P41** · UNIVERSITY OF KANSAS LAWRENCE · 2021 · $268,552

## Abstract

TITLE: Biotechnology Resource Center of BioModular Multi-scale Systems (CBM2) for Precision Medicine
TR&D 1: Single-Molecule Processing: Detection and Identification of Single DNAs, RNAs, and Proteins using
Immobilized Nanoscale Enzymatic Reactors (INERs) and Nanoscale Electrophoresis
Abstract/Summary
The ability to process single molecules has already demonstrated its utility in a number of basic and translational
endeavors in biology and medicine. There are tangible examples of its success including digital PCR (dPCR)
and Next Generation Sequencing (NGS). In the case of dPCR, samples are parsed into nanoliter volumes such
that each reactor volume contains statistically a single molecule, which is subsequently amplified via PCR. This
technique shows exquisite analytical sensitivity by discerning subtle target copy number variations. For NGS,
bridge PCR is used to create clonal clusters of amplified targets for sequencing-by-synthesis. Unfortunately, both
do require a PCR step, which can be problematic. For example, amplification can mask epigenetic modifications
in DNA and/or RNA that can carry important diagnostic and/or prognostic information for disease management
(i.e., Precision Medicine). While amplification-free strategies are preferred, this can be problematic when
analyzing clinical markers that are sometimes low in abundance. This is the case when attempting to analyze
blood-borne markers, such as the liquid biopsy markers. For example, a single circulating tumor cell (CTC)
carries 6 pg of genomic DNA and thus, may not be detected by NGS without significant rounds of amplification.
In this P41 competitive renewal application of CBM2, the Center will develop a suite of tools that can process
single molecules (DNAs, RNAs, and proteins) harvested from liquid biopsy markers, such as CTCs, and
extracellular vesicles (EVs), using amplification-free strategies. The unique attributes of our tools is that they will
not only detect, but also identify unamplified single molecules with high efficiency. In TR&D 1, immobilized
nanoscale enzymatic reactors (INERs) will be realized that can enzymatically digest DNAs (using Exo I
processive exonuclease), RNAs (uses XRN1, a processive exoribonuclease), and proteins (trypsin, which is a
proteolytic enzyme). A fluidic network fabricated in a plastic via nanoimprint lithography (NIL) will be generated
that contains a sub-micron pillar to which the enzyme is surface immobilized. The INERs can be connected to
nanoscale electrophoresis that can monitor in real time the reaction products with high identification accuracy
via their electrophoretic mobility (i.e., Time-of-Flight, TOF) using fluorescence single-molecule tracking during
their electrokinetic transport through a plastic-based nano-column. Unique phenomena occurring in the
nanometer electrophoresis columns will produce molecular-dependent mobilities that are not observed using
microscale columns. Coupled with outputs from TR&D 2 (in-plane nanopore se...

## Key facts

- **NIH application ID:** 10172701
- **Project number:** 2P41EB020594-07A1
- **Recipient organization:** UNIVERSITY OF KANSAS LAWRENCE
- **Principal Investigator:** Steven Allan Soper
- **Activity code:** P41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $268,552
- **Award type:** 2
- **Project period:** 2015-09-16 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10172701

## Citation

> US National Institutes of Health, RePORTER application 10172701, Single-Molecule Processing: Detection and Identification of Single DNAs, RNAs, and Proteins using Immobilized Nanoscale Enzymatic Reactors (INERs) and Nanoscale Electrophoresis (2P41EB020594-07A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10172701. Licensed CC0.

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