DEVELOPMENT OF CURCUMIN CARBON DOTS 200°C CONJUGATED WITH IGY POLYCLONAL ANTIBODIES FOR SARS-COV-2 DIAGNOSTIC KITS BASED ON FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) AGAINST GRAPHENE OXIDE

• Main Author: Kuncoroadi Y W, Ayodya
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/84610
• Institution: Institut Teknologi Bandung

Infectious diseases such as COVID-19 caused by the SARS-CoV-2 virus can emerge and spread rapidly, leading to outbreaks that can affect the global economy. The occurrence of an outbreak necessitates the need for fast and sensitive diagnostic strategies to prevent and/or control the outbreak. In the development of diagnostic kits, Carbon Dots (CDs) can be used as fluorescent labels to detect the presence of antigens from pathogens. Carbon Dots are carbon nanoparticles with a diameter of less than 10 nm, characterized by tunable optical properties, resistance to photobleaching, low toxicity, and relatively lower preparation costs compared to metal nanoparticles, making them an alternative to fluorescent dyes. Fluorescence resonance energy transfer (FRET) is a photoexcitation-based technique involving energy transfer from a donor fluorophore to an acceptor molecule in close proximity. CDs conjugated with bioreceptors can serve as excellent probes in biosensing systems when combined with carbon-based nanomaterials like graphene and graphene oxide, which act as efficient quenchers for the fluorescence of CDs. This research focuses on developing a diagnostic kit using polyclonal IgY antibodies from chicken eggs conjugated with curcumin carbon dots produced at 200?C (cur-CDs 200?C) to replace nanoparticle-conjugated antibodies for antigen detection. The conjugation between polyclonal antibodies and cur-CDs 200?C was performed using the EDC/NHS coupling method. The polyclonal antibodies conjugated with cur-CDs 200?C (pAb-cur-CDs 200?C) were then characterized by their fluorescent emission spectrum. Subsequently, pAb-cur-CDs 200?C were used as donor fluorophores and graphene oxide (GO) was used as the acceptor for FRET treatment. FRET was optimized by varying GO concentrations (0, 25, 50, 75, and 100 ?g) with incubation durations of 5 minutes each for 60 minutes, with 0 GO concentration as a positive control compared for fluorescence on a Glomax with 365 nm excitation at 415-445 nm emission. Afterward, the sensitivity of pAb-cur-CDs 200?C with added GO was tested to detect SARS-CoV-2 multiepitope antigens with varying antigen concentrations (100, 200, 500, 1000, 1500, 2000, 3000, 4000, and 5000 ng/mL). The research results showed successful conjugation of antibodies with cur-CDs 200?C by observing the shift in peak emission wavelength at 300-500 nm excitation from pAb-cur-CDs 200?C compared to cur-CDs 200?C as a control. FRET optimization indicated that 25 ?g GO concentration with 5-minute incubation time showed a significant difference compared to the control. Sensitivity tests revealed significant fluorescence differences between several treatment concentrations and the control. Based on the results, it can be concluded that polyclonal antibodies were successfully conjugated with cur-CDs 200?C and have potential for development as a diagnostic tool for detecting SARS-CoV-2.