Real-time Raman analysis of the hydrolysis of formaldehyde oligomers for enhanced collagen fixation.

Formaldehyde (FA) is widely applied as a fixative for proteins such as collagen. Current studies have confirmed that the reversible oligomer-to-monomer equilibrium of FA in aqueous solution and the proportion of FA monomer is a significant factor affecting tissue fixation. Since the hydrolysis of FA oligomers is a dynamic process affected jointly by different factors, its real time monitoring has proved to be challenging. In this work, by utilizing the well-established Raman technique as an analytical platform, we identified the factors affecting the hydrolysis of FA oligomers by rationally examining the νs (OCO) and νas (OCO) modes with varying conditions, such as time, pH, temperature, and FA concentration. The optimized conditions of the highest hydrolysis rate of oligomers into monomers for fixation on collagen and tissues have been found to be relatively low FA concentration (≤5%) in phosphate-buffered saline at pH 9.0 in room temperature. In order to compare the fixation quality of the optimized conditions to that of the conventional conditions used by current medical practices (4% FA concentration in tap water under room temperature), Raman spectroscopy and chemical derivatization methods with o-phthalaldehyde and fluorescent probe FAP-1 have been investigated, and our results revealed that the FA molecules under our optimized conditions have reacted with at least 15% more amino groups within collagen compared to those under the conventional conditions mentioned above. This study provides direct evidence of the FA equilibrium in solution by Raman spectroscopy, which could be applied for the optimal use of FA in medicine, even at an industrial scale.