ABSTRACT
An ultrasensitive cardiac troponin I antibody conjugated with graphene quantum dots (GQD) and polyamidoamine (PAMAM) nanohybrid modified gold electrode based sensor was developed for the rapid detection of heart attack (myocardial infarction) in human. Screen printed gold (Au) electrode was decorated with 4-aminothiophenol for amine functionalization of the Au surface. These amino groups were further coupled with carboxyl functionalities of GQD with EDC-NHS reaction. In order to enhance the sensitivity of the sensor, PAMAM dendrimer was successively embedded on GQD through carbodiimide coupling to provide ultra-high surface area for antibody immobilization. The activated cardiac troponin I (cTnI) monoclonal antibody was immobilized on PAMAM to form nanoprobe for sensing specific heart attack marker cTnI. Various concentrations of cardiac marker, cTnI were electrochemically measured using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in human blood serum. The modifications on sensor surface were characterized by FTIR and AFM techniques. The sensor is highly specific to cTnI and showed negligible response to non-specific antigens. The sensitivity of the sensor was 109.23µAcm-2µg-1 and lower limit of detection of cTnI was found 20fgmL-1.
Subject(s)
Antibodies, Monoclonal/immunology , Biosensing Techniques/methods , Myocardial Infarction/diagnosis , Troponin I/immunology , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antibodies, Monoclonal/chemistry , Biosensing Techniques/instrumentation , Dendrimers/chemistry , Electrodes , Gold/chemistry , Graphite/chemistry , Humans , Immunoassay , Limit of Detection , Quantum Dots/chemistry , Time FactorsABSTRACT
Anti-VEGF drugs that are used in conjunction with laser ablation to treat patients with diabetic retinopathy suffer from short half-lives in the vitreous of the eye resulting in the need for frequent intravitreal injections. To improve the intravitreal half-life of anti-VEGF drugs, such as the VEGF decoy receptor sFlt-1, we developed multivalent bioconjugates of sFlt-1 grafted to linear hyaluronic acid (HyA) chains termed mvsFlt. Using size exclusion chromatography with multiangle light scattering (SEC-MALS), SDS-PAGE, and dynamic light scattering (DLS), we characterized the mvsFlt with a focus on the molecular weight contribution of protein and HyA components to the overall bioconjugate size. We found that mvsFlt activity was independent of HyA conjugation using a sandwich ELISA and in vitro angiogenesis assays including cell survival, migration and tube formation. Using an in vitro model of the vitreous with crosslinked HyA gels, we demonstrated that larger mvsFlt bioconjugates showed slowed release and mobility in these hydrogels compared to low molecular weight mvsFlt and unconjugated sFlt-1. Finally, we used an enzyme specific to sFlt-1 to show that conjugation to HyA shields sFlt-1 from protein degradation. Taken together, our findings suggest that mvsFlt bioconjugates retain VEGF binding affinity, shield sFlt-1 from enzymatic degradation, and their movement in hydrogel networks (in vitro model of the vitreous) is controlled by both bioconjugate size and hydrogel network mesh size. These results suggest that a strategy of multivalent conjugation could substantially improve drug residence time in the eye and potentially improve therapeutics for the treatment of diabetic retinopathy.
Subject(s)
Biocompatible Materials/chemistry , Hyaluronic Acid/chemistry , Vascular Endothelial Growth Factor Receptor-1/metabolism , Cell Movement , Chromatography, Gel , Dynamic Light Scattering , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Matrix Metalloproteinase 7/metabolismABSTRACT
Cardiac immunosensor for early detection of heart attack (myocardial infarction) was developed using amine functionalized graphene quantum dots (afGQDs) conjugated with antibody anti-cardiac Troponin I (anti-cTnI) to detect cardiac marker antigen Troponin I (cTnI) in blood based on fluorescence resonance energy transfer (FRET) between conjugate and graphene (quencher) only in 10 min. The anti-cTnI was covalently conjugated to afGQDs through carbodiimide coupling reaction. The conjugate was characterized by zeta potential UV-vis spectroscopy and field emission scanning electron microscopy (FESEM). The sensing performance of the sensor was studied with respect to changes in the photon count and photoluminescence of GQDs based on interaction of target cTnI with its specific anti-cTnI antibody. The sensor is highly specific and shows negligible response to non-specific antigens. The sensor displayed a linear response to cTnI from 0.001 to 1000 ng mL(-1) with a limit of detection of 0.192 pg mL(-1).
