Detection of microRNA by Electrocatalytic Amplification: A General Approach for Single-Particle Biosensing

Abstract

Here we report a sensing scheme for detection of microRNA (miRNA) using electrocatalytic amplification (ECA). ECA is a method in which nanoparticles (NPs) that are catalytic for a specific electrochemical reaction collide with an inert electrode surface. Each collision results in a detectable current transient. In the present article, we show that this general approach can be extended to detection of miRNA. Specifically, PtNPs are modified with a single-strand DNA (ssDNA) shell that is complementary to the miRNA target. Next, the ssDNA:miRNA conjugate is formed, which passivates the PtNP surface. In the presence of an enzyme called duplex specific nuclease (DSN), however, a fraction of the surface-bound DNA is removed thereby exposing some of the PtNP surface. In other words, the electrocatalytic properties of the PtNPs are reactivated only if miRNA complementary to ssDNA is present. This methodology resolves a number of problems that have rendered ECA ineffective for biosensing applications. Moreover, the results suggest that the underlying chemistry is broadly applicable to nucleic acid sensing.

Electroanalytic Aspects of Single‐Entity Collision Methods for Bioanalytical and Environmental Applications

“…Crooks and collaborators used electrocatalytic amplification at ssDNA‐modified PtNPs to quantitatively detect microRNA. The PtNPs were functionalized with a ssDNA shell (26 nucleotides) that is complementary to the miRNA target . Formation of the ssDNA‐miRNA conjugate on the PtNP surface passivates the PtNP surface (e. g. the PtNPs ssDNA‐miRNA conjugate is electrocatalytically inactive).…”

“…Furthermore, duplex‐specific nuclease (DSN) enzyme was used to remove the DNA from the NP surface; thus, the NPs which regained their electrocatalytic activity enabling measurement of miRNA. The general methodology is presented schematically in Figure . Blocking and reactivation occurred only when the ssDNA was present.…”

“…In the presence of the complementary DNA strand labeled with PtNP catalyst, an electrocatalytic current was observed due to oxidation of hydrazine; the collision event was sensitive to the concentration of hydrazine. Single NP‐collision events of DNA hybridization was also investigated at the surface of individual Ptand Ag NPs, by measuring the electrocatalytic oxidation of hydrazine (for Pt), or by monitoring oxidation and disaggregation of AgNPs upon binding of the complementary DNA or the DNA target.…”

Nanomaterials for molecular signal amplification in electrochemical nucleic acid biosensing: recent advances and future prospects for point-of-care diagnostics

“…6). 86 Specifically, nanoparticles were initially functionalized with ssDNA complementary to the target microRNA. This was followed by incubation with the target probe and a nuclease enzyme.…”

Ultrasensitive Bioaffinity Electrochemical Sensors: Advances and New Perspectives

“…To avoid the challenges of enzymatic amplification of mRNA, an amplification-free detection assay was reported for the analysis of FAM134B mRNA in tissues samples from patients with oesophageal carcinomas [180]. A sensing scheme was proposed for detection of micro-RNA (miRNA) using electrocatalytic amplification (ECA) [187]. Then, the amount of surface-attached mRNA is measured by differential pulse voltammetry (DPV) in the presence of [Fe(CN) 6 ] 4À / 3À redox system.…”