Respected expert, as I have done antioxidant assay for zinc oxide nanoparticle using medicinal plant Serriphidium kurramense. In this assay I have used Ascorbic acid as standard and DPPH as free radical. In the assay for control 2ml methanol and 1ml DPPH is used, and different concentrations of nanoparticles and plant extract are used as test samples against DPPH. The question is, my test sample that is nanoparticle always give higher absorption value then control due which I get wrong result and my sample showing no antioxidant activity while the plant used in the synthesis show antioxidant activity. Is it possible that the plant showed antioxidant activity but the nanoparticles synthesized from that plant show no antioxidant activity. The change from pinkish color of DPPH to yellow or colorless is completely done in case of Plant Extract and standard Ascorbic acid but show antioxidant activity not more than 80% when calculated although color change is 100% in both plant extract and standard and in case of NPs no color change occurred.
File is attached for reference. Please help what should I do in this case?
I have repeated the assay many time but got no positive result.
Your precious time and comments will be highly appreciated.
Yes, it is very likely that your crude plant extract contains proteins and lipids (etc.) that compete with binding of your target antioxidant to your NP, especially if you are loading the particles via simple adsorption. You may be able to salt out these competitors (e.g., ammonium sulfate), but they may complex with the anti-oxidant and co-precipitate.
Be sure to use blank NP for your negative control.
Dear Abrar Hussain,
Thank you for reaching out, and I appreciate your detailed explanation of the antioxidant assay results for zinc oxide nanoparticles using Serriphidium kurramense. I have reviewed the attached file for reference.
It's not uncommon to encounter challenges in experimental results, and I understand your concern regarding the discrepancy between the plant extract and the nanoparticles in terms of antioxidant activity. The higher absorption values for the nanoparticles compared to the control are indeed intriguing.
It is advisable to conduct another assay. Consider assessing total antioxidant capacity using the method described by Prieto et al. (1999) or alternative assays like the ABT Assay and the Ferrous Reducing Assay.
Note: "[reference: Prieto, P., Pineda, M., & Aguilar, M. (1999). Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Analytical biochemistry, 269(2), 337–341. [https://doi.org/10.1006/abio.1999.4019]"
Considering your experimental design and the consistent lack of positive results, several factors might contribute to the observed discrepancy:
Ø Possible Interference from Nanoparticles:
Nanoparticles might introduce interference, affecting the accuracy of the assay.
Ø Purity of Nanoparticles:
Assess the purity of zinc oxide nanoparticles to ensure the absence of contaminants.
Ø Concentration and Dilution Effects:
Evaluate the concentration of nanoparticles, as high concentrations may impact the assay.
Ø DPPH Sensitivity to Nanoparticles:
Investigate the interaction between DPPH and nanoparticles, as nanoparticles may not produce the expected color change.
Ø Particle Size and Surface Area:
Consider the influence of nanoparticle size and surface area on reactivity in the assay.
Ø Use of Multiple Assays:
Employ additional assays to gain a comprehensive understanding of antioxidant properties.
It is essential to recharacterize the synthesized nanoparticles using techniques such as SEM, FTIR, XRD, DLS, Zeta potential, and UV spectra. These analyses provide insights into morphology, functional groups, purity, size distribution, and nanoparticle charges. Preliminary tests are crucial to understand if any physiochemical properties of the nanoparticles have changed, potentially leading to incorrect biological assay results.
WQhen one uses living or complex organisms to measure antioxidant acrivities , do not loose your precious time . So many things can happen
I would like to express my gratitude for your detailed answer and valuable suggestions, sir. I have conducted various characterization techniques, including SEM, EDX, FTIR, and XRD, on the ZnO NPs. If you wouldn't mind, may I please have your email address? I would appreciate the opportunity to share the data from these characterizations with you. I am not very experienced in analyzing these techniques, so your expertise would be invaluable in identifying any potential problems in the nanoparticle synthesis. Mohanprasanth Aruchamy
Dear Abrar Hussain,
Thank you for your kind words, and I'm delighted to hear that you found my suggestions valuable. I'm pleased to learn that you have conducted various characterization techniques, including SEM, EDX, FTIR, and XRD, on the ZnO NPs. I'm more than willing to assist you in analyzing the data and identifying any potential issues in the nanoparticle synthesis.
While I appreciate your request for my email address, I prefer to maintain communication through this platform for professional reasons. However, you can share the relevant data and details here, and I'll do my best to provide insights and guidance. Feel free to upload any specific findings or questions you have, and I'll be happy to assist you further.
Looking forward to collaborating with you on your nanoparticle research.
Personal E.Mail Id: [email protected] Abrar Hussain
I would like to express my sincere gratitude for the time and attention you have generously given me. Your expertise and guidance have been truly invaluable. I have shared the detail via email, if you kindly check it Mohanprasanth Aruchamy
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