We have previously investigated the application of cellulose dialysis tubing (dialysis membrane) as a biosensor scaffold medium for the forthcoming detection with the UV spectrophotometer. This was achieved by using small volumes of biological materials, through drying the samples on the dialysis membrane surface. The outcomes proved the feasibility of this approach as a novel quantitative detection scheme of the bovine serum albumin (BSA) protein. However, standard deviations seemed to be high, which revealed the necessity of optimization. Here it is shown that the standard deviations diminish significantly, when dialysis membrane tubing is eliminated and sample is dried directly on the quartz cuvette of the spectrophotometer, in addition to performing the analysis by baseline correction of the data. These tests were performed also with the BSA protein, for comparability with the previous work that utilized dialysis membrane as the sample application surface. The detection limit of the current measurements was found to be 5 micromolar, corresponding to approximately 165 nanogram of protein in 0.5 microliter of the sample. The presented approach is appropriate for the detection of the presence of protein in minute amounts of samples. It is advantageous also due to the availability of the instrument in almost all laboratories and the presence of portable designs of the instrument as well. In the future, applicability of the technique is aimed to be tested for DNA detection, to see the possibility of obtaining results with trace amounts of DNA, without using any sample enrichment procedures such as polymerase chain reaction.
Thanks in advance.
First, thanks a lot for these nice questions and for finding this work interesting.
You initially asked whether the drying conditions (temperature, heating rates, or even the type of drying e.g. lyophilization) would significantly affect the BSA structure. Before coming to that point, it is better first to mention that the protein structure attains a specific form together with its environment, namely the environment’s being water (including the extent and type of ions), air, other proteins, and other materials like the surfaces of different types. So, all these can have an influence on the protein structure. This point is important and relevant to your question since drying changes the environment of the proteins and therefore must result in protein structure changes. However, significance of the effects is depending on what you call as significant and how sensitive your measurement for protein structure determination is. If there is a drying process by heating, it should be kept in mind that heating by itself can denature the protein. Therefore, if the effect of drying rates will be studied by heating, I guess one must ensure that small sample volumes are used because otherwise the sample will be heated up to temperatures that would denature the protein before the sample is dried. I did not come across with such a study. Protein-drying rates are generally studied through lyophilization. Lyophilization is different than drying by heating. You can find published work on the characterization and effects of lyophilization and lyophilization rates. They generally report that there is no observable difference in the secondary structure of BSA, but those results are generally according to the comparison of the relevant regions of the protein FTIR spectrum or according to the secondary structure analysis by curve-fitting of those regions. FTIR technique is very nice. However, curve-fitting gives the secondary structure percentages. FTIR signal is normally blind to the position of the protein residues. Namely, by just looking at their FTIR spectrum, you will typically not be able to tell the identity of the residues that contribute to the peak that characterizes certain secondary structure parts of the proteins. It means that one must be careful during interpretation. For instance, if some residues of the proteins with helix structure change and attain beta-sheet structure, you will probably not detect that if the same amount of other protein residues with the beta-sheet structure change and attain helix structure at the same time. This is simply because the secondary structure percent of the protein with the altered-structure will be the same of that protein with the unaltered structure. I think the rate of drying can influence the protein structure if the drying-rate can really be varied dramatically, without allowing enough time to the residues in the protein for reorientation. Besides, maybe water can also be confined because of accelerated drying rates and that might have an influence on the proteins’ structure. Such studies may already be present. Additionally, the work by Rombouts and co-workers (Rombouts et al. 2015. Scientific Reports 5, article number 12210) can be interesting for you. It is not involving lyophilization but there is heating...
You also asked if the structural changes of the protein would affect the UV signal (that is correlated with the BSA concentration). I did not yet analyze my results accordingly but that is possible since UV-vis spectroscopy of tyrosine side-groups can be utilized in the protein-structure studies (Antosiewicz and Shugar. 2016. Biophys. Rev. 8:151; Antosiewicz and Shugar. 2016. Biophys. Rev. 8:163).
I hope these explanations answer your questions.
Kind regards & thanks again,
Yekbun
