Subnanomolar Detection of Oligonucleotides through Templated Fluorogenic Reaction in Hydrogels: Controlling Diffusion to Improve Sensitivity

Abstract

Oligonucleotide‐templated reactions are valuable tools for nucleic acid sensing both in vitro and in vivo. They are typically carried out under conditions that make any reaction in the absence of template highly unfavorable (most commonly by using a low concentration of reactants), which has a negative impact on the detection sensitivity. Herein, we report a novel platform for fluorogenic oligonucleotide‐templated reactions between peptide nucleic acid probes embedded within permeable agarose and alginate hydrogels. We demonstrate that under conditions of restricted mobility (that is, limited diffusion), non‐specific interactions between probes are prevented, thus leading to lower background signals. When applied to nucleic acid sensing, this accounts for a significant increase in sensitivity (that is, lower limit of detection). Optical nucleic acid sensors based on fluorogenic peptide nucleic acid probes embedded in permeable, physically crosslinked, alginate beads were also engineered and proved capable of detecting DNA concentrations as low as 100 pm.

Chemically Modified Hydrogel-Filled Nanopores: A Tunable Platform for Single-Molecule Sensing

“…Herein, we demonstrate that such tuning can be achieved by designing custom nanopore sensors using engineered hydrogel-filled nanopores (HFN). Hydrogels are traditionally used for biomedical applications including drug delivery, 25,26 tissue engineering, [27][28][29] but also in biosensors, acting as matrices to encapsulate receptor probes (e.g., enzymes, [30][31][32][33][34] nucleic acids, 35 and even protein nanopores 36 ) and enhance detection sensitivity. [37][38][39] Compared to other synthetic materials, hydrogels are biocompatible, antifouling (resistive to protein adsorption), and stable under physiological conditions.…”

Emerging Applications of Fluorogenic and Non‐fluorogenic Bifunctional Linkers

“…SCF8 was coupled to amine‐terminated ODN1 (NH 2 ‐ODN1) to afford nonfluorescent SCF8‐ODN1, which was then utilized in an ODN‐templated fluorogenic Heck reaction with vinyl‐functionalized ODN2 (ODN2‐vinyl; Figure ) . Other types of hetero‐bifunctional SCF linkers (SCF9‐SCF12) were also developed for ODN detection through template‐directed fluorogenic ODN ligation reaction (Figure ) –…”

Platforms for Bioorthogonal Oligonucleotide-templated Reactions: Translating Concepts into Devices

“…A recent study from Ladame et al reported the first examples of fluorogenic OTRs carried out within physical hydrogels [60]. In this novel platform, fluorogenic PNA probes were engineered that can detect NA biomarkers via OTR when embedded within permeable agarose and alginate hydrogels.…”

“…This resulted in lower background signals and higher signal-to-noise ratio (SNR) when compared to similar reactions carried out in solution. Probes Encapsulated by biologically localized embedding (PEBBLE) biosensors were also developed that consisted of spherical alginate beads containing a stoichiometric mixture of PNA probes and that could detect DNA concentrations as low as 100 pM [60]. Permeable hydrogels such as agarose or alginate could therefore represent low-cost, easily modifiable matrices for the next generation of OTR-based optical NA sensors.…”