Femtomolar Detection of Thrombin in Serum and Cerebrospinal Fluid via Direct Electrocatalysis of Oxygen Reduction by the Covalent G4-Hemin-Aptamer Complex.
Kamila MaleckaElena E FerapontovaPublished in: ACS applied materials & interfaces (2021)
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.
Keyphrases
- label free
- cerebrospinal fluid
- electron transfer
- living cells
- gold nanoparticles
- endothelial cells
- high throughput
- single molecule
- binding protein
- heart failure
- cognitive decline
- small molecule
- left ventricular
- white matter
- hydrogen peroxide
- cell free
- resting state
- blood brain barrier
- sensitive detection
- protein protein
- high glucose
- circulating tumor
- atrial fibrillation
- cerebral ischemia
- anti inflammatory
- dna binding
- subarachnoid hemorrhage
- quantum dots