Femtomolar Detection of Thrombin in Serum and Cerebrospinal Fluid via Direct Electrocatalysis of Oxygen Reduction by the Covalent G4-Hemin-Aptamer Complex

Abstract

Thrombin, a serine protease playing a central role in the coagulation cascade reactions and a potent neurotoxin produced by injured brain endothelial cells, is a recognized cardiac biomarker and a critical biomarker for Alzheimer’s disease development. Both in vivo and in vitro, its low physiological concentrations and nonspecific binding of other components of physiological fluids complicate electroanalysis of thrombin. Here, femtomolar levels of thrombin in serum and an artificial cerebrospinal fluid (CSF) were detected by the indicator-free electrochemical methodology exploiting the O2 reduction reaction directly, with no electron transfer mediators, electrocatalyzed by the covalent G4-hemin DNAzyme complex naturally self-assembling upon thrombin binding to the hemin-modified 29-mer DNA aptamer sequence tethered to gold via an alkanethiol linker. Coadsorbed PEG inhibited nonspecific protein binding and allowed the sought signal resolution. The proposed assay exploiting the “oxidase” activity of G4-hemin DNAzyme does not require any coreactants necessary for the traditional peroxidase activity-based assays with this DNAzyme, such as H2O2 and redox mediators, or solution deaeration and allows fast, overall 30 min analysis of thrombin in aerated buffer, CSF, and 1% human serum solutions. This pioneer approach exploiting the oxidase activity G4-hemin DNAzyme is simple, sensitive, and selective and represents a new tool for ultrasensitive electrocatalytic assays based on simple and efficient O2-dependent DNAzyme labels.

De novo designed peptides form a highly catalytic ordered nanoarchitecture on a graphite surface

“…Notably, the K s value for the assemblies formed by 1 is comparable to the highest value reported to date in all the systems where heme is not in direct contact with a conducting surface. [36][37][38][39][40] In addition, peptide 1 had a stable reaction over multiple cycles under cyclic voltammetry for both single and multi-substrate reactions with H 2 O 2 and TMB (Fig. S15 †).…”

Critical Design Factors for Electrochemical Aptasensors Based on Target-Induced Conformational Changes: The Case of Small-Molecule Targets

“…In the target-recognition process via various types of interactions, the aptamer molecules can adopt a large variety of structural patterns such as stem-loops, internal loops, bulges, pseudoknots, and G-quadruplexes (schematically depicted in Figure 2 ), which can be exploited for transduction [ 16 , 90 , 91 , 92 , 93 , 94 , 95 ]. These patterns arise frequently during in vitro selection of the aptamers, and are usually indicators of the potential binding sequences in the selected aptamers [ 96 , 97 ].…”

Recent Progress on Highly Selective and Sensitive Electrochemical Aptamer-based Sensors

“…The detection limit of long non-coding modified aptamers [78][79][80] . Amide bond has the advantages of strong stability, diverse generation strategies, flexible design and wide application system [101][102][103] . It can be applied to the modification of carbon electrodes, emerging carbon materials and metal nanoparticles [104][105][106] .…”