A multiplex platform for digital measurement of circular DNA reaction products

Abstract

Digital PCR provides high sensitivity and unprecedented accuracy in DNA quantification, but current approaches require dedicated instrumentation and have limited opportunities for multiplexing. Here, we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy. Circular DNA strands, which may result from a wide range of molecular detection reactions, are captured on streptavidin-coated surfaces via hybridized biotinylated primers, followed by rolling circle amplification (RCA). The addition of 15% polyethylene glycol 4000 during RCA resulted in uniform, easily recorded reaction products. Immobilized DNA circles were visualized as RCA products with 100% efficiency, as determined by droplet digital PCR. We confirmed previous reports about the influence on RCA by sequence composition and size of RCA templates, and we developed an efficient one-step restaining procedure for sequential multiplexing using toehold-triggered DNA strand displacement. Finally, we exemplify applications of this digital readout platform by demonstrating more than three orders of magnitude improved sensitivity by digital measurement of prostate specific antigen (PSA) (detection threshold ∼100 pg/l), compared to a commercial enzyme-linked immunosorbent assay (ELISA) with analogue readout (detection threshold ∼500 ng/l), using the same antibody pair.

Functional nucleic acid biosensors utilizing rolling circle amplification

“…Incorporation of PEG 4000 at concentrations from 10-20% (w/w) can also improve RP generation and uniformity, particularly when RCA is performed on a solid surface. 177 Our groups observed that generating RCA on a nitrocellulose surface also led to improved RCA efficiency. 110 In each case, the authors attributed the improvement in RCA rates to a molecular crowding effect, which increased local concentrations of RCA reagents.…”

Rolling Circle Amplification Tailored for Plasmonic Biosensors: From Ensemble to Single-Molecule Detection

“…32 Research on manipulating the long RCA-generated DNA chains by introducing short DNA staples to form a compact globular conformation improved resolving the sensor signal of individually captured target molecules. 33 An RCA step performed on solid surfaces can be utilized for sensitive detection of chemical and biological species by using various transducing principles including electrochemical, gravimetric, or optical methods (overview with typical analytical performance characteristics presented in Supporting Information, Section S1). 34−38 Among them, using the metallic surface allows for efficient optical probing with the evanescent field of surface plasmons (SPs).…”

A multiplexed circulating tumor DNA detection platform engineered from 3D-coded interlocked DNA rings

“…And the topological constraint between reporter ring and recognition ring becomes weak so that the function of the released reporter ring can be regained [ 42 ]. ctDNA-specific amplifications were subsequently realized via the rolling circle amplification (RCA), an isothermal nucleic acid amplification technique with high fidelity and extreme processivity [ 43 , 44 ]. Please note that a sequence specific RE is extremely specific down to recognition sites.…”