What are the characterization technique available to ensure binding/ adsorption of BSA protein and Trypsin enzyme on citrate capped gold nanoparticles in colloidal form. It means I want to do the characterization of BSA conjugated AuNPs and Trypsin conjugated AuNPs in colloidal form (of AuNPs) only. The binding of BSA protein with AuNPs and Trypsin enzyme with AuNPs have been done by direct mixing BSA solution (0.1-0.3 mg/ml). Similarly done for the Trypsin enzyme.
Any suggestions will be a great help.
Dear Sibasish Dutta, maybe you can try these two methods below:
1. Us the UV-Vis spectrometer to check the BSA concentration variation after the absorption, and use the DLS and Zeta potential equipments to check whether the colloidal size surface charge has been varied.
2. Use the neturon scattering technique to test the real colloidal size before and after AuNPs absorbs BSA. But I think it is very difficult to find the available equipment .
Dear Sibasish
You can go for
1. Simple optical measurement by UV-VIS spectrophotometry- there should be a slight rightward shift in the spectrum.
2. Dynamic light scattering measurements.
3. NTA
4. FTIR
You can go for others....however these should be enough with TEM and HRTEM.
There are standard techniques in biochemistry to find percentage of binding. For each biomolecule there are standard reagents. For BSA you may refer my paper on Direct binding of BSA on magnetic Particle
This and other papers are available on RG.
Dear Sibasish: To solve your problem, use the method surface-enhanced Raman scattering (SERS). See method, our article. Kinetics and Catalysis, 2014, Vol. 55, No. 6, pp. 683–687.
FTIR, UV-Vis, XPS and Raman spectroscopy would ensure you about the binding of protein and enzyme with Au NPs.
CD spectroscopy, DLS, UV-Vis spectroscopy, Fluorescence spectroscopy etc.
We can use UV Vis Absorption spectroscopy and Fluorescence spectroscopy to study the binding of GNP with BSA.The absorption spectra shows shift in Wavelength in SPR region.In Fluorescence spectra, quenching occurs in 280 excitation of protein. But the concentration of BSA and GNP should be in nano molar to obtain the spectrum.We can also calculate binding efficiency from the spectra
Dear, you can use UV-Vis spectrometer or FTIR to analyse protein bound to nanoparticles. As starting point, increase in size can be detected by zetasizer.
You can use FTIR and compare the bound AuNPs data with nonbounded NPs, BSA and trypsin data.
As far as trypsin is concerned you should find improved performance in terms of enzyme activity for coated nanoparticles. Hence bradford assay may give more concrete inference along with other characterizations including FTIR/Size change/UV absorbance measurement directly.
Dear Sibashis, If you are interested in calculating percent binding efficiency, BCA assay works well for BSA. Quantify it in the supernatant obtained in conjugation procedure. Alternatively, HPLC can be used.
X-RAY PHOTOELECTRON SPECTROSCOPY can say to what is there on surface of nanoparticle...
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