When imaging protein solutions (1ug/ml,10ug/ml,100ug/ml,1000ug/ml ; diluted with PBS buffer 7.4) on a gold surface, what is the optimal pretreatment(s) to separate out the effect of the buffer (in my case PBS) which interferes with bands of interest for proteins?
10ul of each concentration was dropped on a clean gold slide and allowed to dry for 24hr under Nitrogen purge, followed by FTIR-reflectance imaging.
Since samples with 1000ug/ml are highly concentrated, their signal appears very clear (Amide I, Amide II, Amide III, Amide A, Amide B), however at concentrations below and = 100ug/ml, protein signature is dominated by PBS buffer bands.
What kind of univariate or multivariate methods would you apply to
(a) identify protein pixels (remove interference of buffer, slide background if any)
(b) quantify protein pixels (eg. make PLSR model on 0ug,1ug,10ug,100ug,1000ug) and predict concentration level of an unknown dried protein sample?
Dear Sir. Concerning your issue about the optimal pretreatments to quantify protein concentration ranges on gold using FTIR (reflectance mode). Surface-sensitive quantitative studies of competitive molecular adsorption on nanoparticles were conducted using a modified attenuated total reflectanceFourier transform infrared (ATR-FTIR) spectroscopy method. Adsorption isotherms for thiolated poly(ethylene glycol) (SH-PEG) on gold nanoparticles (AuNPs) as a function of molecular mass (1, 5, and 20 kDa) were characterized. We find that surface density of SH-PEG on AuNPs is inversely proportional to the molecular mass (Mm). Equilibrium binding constants for SH-PEG, obtained using the Langmuir adsorption model, show the binding affinity for SH-PEG is proportional to Mm. Simultaneous competitive adsorption between mercaptopropionic acid (MPA) and 5 kDa SH-PEG (SH-PEG5K) was investigated, and we find that MPA concentration is the dominant factor influencing the surface density of both SH-PEG5K and MPA, whereas the concentration of SH-PEG5K affects only SH-PEG5K surface density. Electrospray differential mobility analysis (ES-DMA) was employed as an orthogonal characterization technique. ES-DMA results are consistent with the results obtained by ATR-FTIR, confirming our conclusions about the adsorption process in this system. Ligand displacement competitive adsorption, where the displacing molecular species is added after completion of the ligand surface binding, was also interrogated by ATR-FTIR. Results indicate that for SH-PEG increasing Mm yields greater stability on AuNPs when measured against displacement by bovine serum albumin (BSA) as a model serum protein. In addition, the binding affinity of BSA to AuNPs is inhibited for SH-PEG conjugated AuNPs, an effect that is enhanced at higher SH-PEG Mm values I think the following below links may help you in your analysis:
https://ws680.nist.gov/publication/get_pdf.cfm?pub_id=907586
https://spiral.imperial.ac.uk/bitstream/10044/1/24835/1/Glassford-SE-2013-PhD-Thesis.pdf https://www.azom.com/article.aspx?ArticleID=10963
Thanks
After drying you most probably get a coffee ring effect, meaning that the thickness of you layers is strongly changing from point to point. Therefore imaging and measuring individual points might in any way not be the correct method for reasons explained in Article The electric field standing wave effect in infrared transfle...
The electric field standing wave effect can be corrected as explained in Article The electric field standing wave effect in infrared transfle...
My feeling is, however, that you should better use a different technique, maybe to coat an ATR crystal and see if you could obtain a calibration line, in particular also, because for near normal incidence and thin layers, you might see nothing at all on your metallic substrate due to the fact that a metallic surface suppresses electric fields parallel to its surface. Certainly, this still does not solve the problem of overlapping bands.
a) I don't think that any univariate approach would be helpful at all. I suggest a multivariate classification approach, like PCA. Of course, success would largely depend on intesity of spectral interference. The multivariate approach could be performed as outlined in:
Article Detecting semen stains on fabrics using near infrared hypers...
b) PLS is worth a try. Again, LOD possibly would depend on the intensity of spectral interference.
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