An electrochemical aptasensor for detection of prostate-specific antigen-based on carbon quantum dots-gold nanoparticles.

In this work, an electrochemical aptasensor was described for the determination of prostate-specific antigen (PSA). Aptamer chains were decorated on the surface of a glassy carbon electrode (GCE) via carbon quantum dots/Au nanoparticles (Au/CQD). Structural analysis that was used to characterize the prepared materials shows that Au/CQD nanoparticles synthesized in a spherical shape with an average size of 70 nm. Furthermore, the combination of Au nanoparticles with CQD resulted in formation of crystalline the structure of the Au/CQD composite. To study the electrochemical performance of the prepared aptasensor, cyclic voltammetry, square wave voltammetry, and electrochemical impedance spectroscopy were used. The results show that the aptasensor has a good selectivity to PSA over other biomaterials with the time optimized about 30 min. K4 [Fe(CN)6 ] was used as an electrochemical probe with the limit of detection about 2 fg⋅mL-1 . To avoid the hazardous nature of K4 [Fe(CN)6 ], a label-based aptasensor was prepared using methylene blue as an electrochemical signal producer. They provide the capability of electrochemical detection in buffer phosphate solution with high sensitivity.

Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris.

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD). Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains' interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

An electrochemical aptasensor for detection of prostate-specific antigen using reduced graphene gold nanocomposite and Cu/carbon quantum dots.

We report a label-free electrochemical aptamer-based biosensor for the detection of human prostate-specific antigen (PSA). The thiolate DNA aptamer against PSA was conjugated to the reduced graphene oxide/Au (RGO-Au) nanocomposite through the self-assembly of Au-S groups. Owing to the large volume to surface ratio, the RGO-Au nanocomposite provides a large surface for aptamer loading. The RGO-Au/aptamer was combined with a Nafion polymer and immobilized on a glassy carbon electrode. The interaction of aptamer with PSA was studied by cyclic voltammetry, square wave voltammetry, and electrochemical impedance spectroscopy. The detection of limit for prepared electrode was obtained about 50 pg/mL at the potential of 0.4 V in potassium hexacyanoferrate [K4 Fe(CN)6 ] medium. To decrease the limit of detection (LOD) and applied potential of the prepared nanoprobe Cu/carbon quantum dots (CuCQD) is introduced as a new redox. The results show that this new electrochemical medium provides better conditions for the detection of PSA. LOD of a nanoprobe in CuCQD media was obtained as 40 pg/mL at the potential of -0.2 V. Under optimal conditions, the aptasensor exhibits a linear response to PSA with a LOD as small as 3 pg/mL. The present aptasensor is highly selective and sensitive and shows satisfactory stability and repeatability.

A highly-sensitive vascular endothelial growth factor-A(165) immunosensor, as a tool for early detection of cancer.

Biomarkers can be ideal indicators for assessing the risk of the presence of a disease. In this study, a label-free electrochemical biosensor was designed to quantify the vascular endothelial growth factor A (165) (VEGF-A(165)) antigen, using reduced graphene oxide-gold nanoparticle for early detection of breast cancer. The conductivity of gold nanoparticle along with its biocompatibility provide an enhanced surface, suitable for anti-VEGF antibody immobilization. 11-mercaptoundecanoic acid was used to facilitate a single-step and convenient bonding of the antibodies to the surface, compared to previous studies. The dynamic range of the biosensor was between 20 to 120 pg/ml and its limit of detection of the biomarker VEGF-A(165) was obtained to be about 0.007 pg/ml, using different electric signal transduction modes. Hence, the biosensor is a beneficial immunosensor with high sensitivity and ideal dynamic range for early-stage diagnosis of breast cancer and other cancers diseases associated with expression of VEGF-A(165). The as-prepared immunosensor could be efficiently employed for designing a point-of-care diagnostic platform.

Electrospun poly-caprolactone/graphene oxide/quercetin nanofibrous scaffold for wound dressing: Evaluation of biological and structural properties.

AIMS: In this study, for the first time, the effect of quercetin (Q) on the characteristic properties, antimicrobial activity, and cell viability of polycaprolactone (PCL)/graphene oxide (GO) electrospun scaffold was investigated. MAIN METHODS: Quercetin loaded graphene oxide nanoparticles have been incorporated into the poly-caprolactone solution, and their mixture has been electrospun to be applied as a nanofibrous scaffold for wound dressing and tissue engineering applications. The properties of scaffolds, like their morphology, tensile strength, hydrophilicity, and in vitro biological performance, are investigated. KEY FINDINGS: The SEM micrographs reveal the uniform bead-free nanofibers with smooth structures have been successfully fabricated via the electrospinning procedure. The overall average of cell viability of NIH/3 T3 fibroblast cells on scaffolds is 95% that means the scaffolds have no toxicity, and FESEM shows cells attach and proliferate on scaffolds. Moreover, among all the fabricated scaffolds, the maximum release of quercetin belongs to PCL/GO/Q 0.5 with about 70% after 15 days, and this scaffold reduces bacterial growth by about 50% after 12 h shows the excellent effect of GO/Q on the antibacterial activity of PCL nanofibers. SIGNIFICANCE: The results confirm that more than 1% of GO has some cytotoxicity, which limits its concentration; therefore, a second antibacterial agent is essential to improve the antibacterial activity of PCL/GO scaffold, and quercetin shows that it is an excellent candidate for this purpose.