Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform

Abstract

The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin-HRP). Hydrogen peroxide (H2O2) is then reduced by avidin-HRP in the presence of TMB (3,3',5,5'-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h.

Aptasensor with Expanded Nucleotide Using DNA Nanotetrahedra for Electrochemical Detection of Cancerous Exosomes

“…Moreover, as a consequence of the flexibility of single-stranded DNA, immobilized aptamers on electrodes tend to undergo self-assembled monolayer aggregation or entanglement, which largely impede accessibility of the target exosomes. 26 It is also difficult to control the spatial orientation of single-stranded aptamers with precision.…”

Designed Microdevices for In Vitro Diagnostics

“…[25a] Girault and co‐workers designed a type of soft interface functionalized by roughly half a monolayer of mirror‐like nanofilms of gold nanoparticles, which could be used for studying of the thermodynamic and kinetic aspects of interfacial redox catalysis ( Figure A) . Fan and co‐workers designed a approach to modify the biomolecular‐recognition interface using well‐defined, differentially sized tetrahedral DNA nanostructures, and improved the sensitivity of several magnitude orders for the detection of molecular targets (Figure B), such as DNA, RNA, and proteins . Yang et al developed a voltammetry sensor combined with a microfluidic device to perform real‐time electrochemical melting‐curve measurements.…”

Biological Functions and Current Advances in Isolation and Detection Strategies for Exosome Nanovesicles

“…Electrochemical detection is highly suitable for biomolecular analysis due to its inherent advantages, such as high sensitivity and specificity, compatibility with miniaturization, simplicity, and relatively low‐cost detection . After the clinical success of electrochemical glucose sensors, electrochemical systems for biomolecular analysis have received significant attention and have become the focus of interest for many research groups.…”