ABSTRACT
Gold nanoparticles (AuNPs) can be applied in biosensors using fluorescence resonance energy transfer (FRET) technique. Based on this technique, we have established a sensitive and efficient biosensing method by modifying a peptide-probe onto AuNPs to detect proteinase enzyme activity in this study. This biosensing method was designed for chymase activity detection and applied in kidney disease diagnosis. In this study, 16 nm-AuNPs were used to construct the AuNPs-based fluorescence peptide probe (named AuNPs-peptide probe) for chymase activity determination. The peptide sequence is FITC-Acp-DRVYIHPFHLDDDDDC, which comprises a fluorophore at the N-terminal end, an enzyme (chymase) substrate (DRVYIHPFHL), a spacer (DDDDD) and cysteine (C) to conjugate to AuNPs surface. When the enzyme catalyzes the substrate sequence, the fluorophore drifts away from AuNPs and the fluorescence emitting signal can be excited at 495 nm and detected at 515 nm. The results indicate that the time required for the AuNPs-peptide probe for activity detection of chymase was only 15 min, and a linear correlation from 10 to 100 ng mL-1 of chymase was acquired. The chymase reaction would be significantly inhibited by addition of specific chymase inhibitor chymostatin. The AuNPs-peptide probe was tested for the detection of high concentrations of trypsin and chymotrypsin, but only minor emitted fluorescence intensity was detected. According to these results, sensitivity and specificity of the AuNPs-peptide probe for chymase detection have been confirmed. AuNPs-peptide probe was successfully used for the detection of renal chymase activity; and the results indicate the pathogenically increased chymase activity in kidney tissue of nephropathic mice from aristolochic acid I treatment.
ABSTRACT
Thrombin plays the role in cardiovascular diseases and regulates many processes in inflammation and could be a feature of many pathological conditions, including the thromboembolic disease, cancer and neurodegenerative diseases. An ultrasensitive and amplified electrochemical sandwich assay using screen-printed carbon electrode (SPCE) strips for thrombin detection was established in this study. The conductivity and sensing performance of the carbon electrodes were enhanced by using gold nanoparticles (AuNPs). The aptamer addressed on the strips was used as a primary probe to capture thrombin in the detected samples. An amplifier was invented for recognizing thrombin captured on the SPCE, which is the multiple molecules of anti-thrombin antibody (Ab) and horseradish peroxidase (HRP) co-modified AuNPs (AuNPs/Ab-HRP). Hydrogen peroxide was used as the substrate for HRP and then the response current (RC) could be detected. The optimization of these AuNPs conjugates-amplified aptamer immunosensing SPCE strips was conducted for thrombin detection. The detection sensitivity showed a linear relation between RC and thrombin concentration in the range of 10 pM-100 nM, and limit of detection (LOD) was 1.5 pM. The fabricated AuNPs/Ab-HRP-amplified aptamer immunosensing SPCE strips were further used to detect thrombin in human serum with a linear range of 100 pM-100 nM. This study provided the promising SPCE strips with highly sensitive and rapid detection for thrombin by the electrochemical aptasensor combined with AuNPs conjugates for amplifying the detection signal.
