Exploring Enzymatic Conformational Dynamics at Surfaces through µ-FTIR Spectromicroscopy.

Immobilization of proteins onto solid supports has critical industrial, technological, and medical applications, and is a daily task in chemical research. Significant conformational rearrangements often occur due to enzyme-surface interactions, and it is of broad interest to develop methods to probe and better understand these molecular-level changes that contribute to the enzyme's catalytic activity and stability. While circular dichroism is a common method for solution-phase conformational study, the application to surface-supported proteins is not trivial and spatial mapping is not viable. On the other hand, a nonlinear laser spectroscopy technique used to analyze surfaces and interfaces is not often found in most laboratories, therefore requiring an alternative and reliable method. Here, we employed high-dimensional data spectromicroscopy analysis in the infrared region (µ-FTIR) to investigate the enzyme's conformational change when adsorbed onto solid matrices, across a ca. 20 mm2 area. Alcohol dehydrogenase (ADH) enzyme was adopted as a model enzyme to interact with CaF2, Au, and Au-thiol model substrates, strategically chosen for mapping the enzyme dynamics on solid surfaces with different polarity/hydrophobicity properties and extendable to other materials. Two-dimensional chemical maps indicate that the enzyme adsorbs with different patterns in which secondary structures dynamically adjust to optimize interprotein and enzyme-surface interactions. The results suggest an experimental approach to identify and map enzyme conformational dynamics onto different solid surfaces across space and provide insights into immobilized protein structure investigations for areas such as biosensing and bioenergy.

Monitoring cellulose oxidation for protein immobilization in paper-based low-cost biosensors.

The oxidation of paper by periodate was investigated and systematically characterized by Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy, X-ray diffraction, goniometry, and dynamic mechanical analysis. For the first time, in situ FTIR microscopy analysis was performed, yielding chemical images of carbonyl groups on the cellulose fibers. The enhancement of protein immobilization on oxidized paper was quantified by a colorimetric assay with Ponceau dye, demonstrating that 0.5-h oxidation suffices to functionalize the paper-based devices. The oxidized paper was applied as a sensor for protein quantification in urine, a test able to detect levels of proteinuria and even microalbuminuria. The quantification was based on the capture of proteins through covalent bonds formed with the carbonyl groups on the oxidized paper followed by the staining of the region with Ponceau dye. There is a linear dependency between human serum albumin (HSA) concentration and the length of the stained blot from 0.1 to 3 mg mL-1. This method correlated linearly with a reference method showing a higher sensitivity (0.866 cm mL mg-1) than the latter. The limit of quantification was 0.1 mg mL-1, three times lower than that of the commercial strip. Graphical abstract Paper oxidation with periodate and extensive characterization, including microspectroscopy. The conversion of cellulose hydroxyl groups to aldehyde enhances covalent immobilization of protein on paper for application as analytical device. The oxidized paper determined protein in urine, suitable for proteinuria diagnosis.

Recent advances in nano-based electrochemical biosensors: application in diagnosis and monitoring of diseases.

Based on biological molecules combined with nanostructured components, the news generations of electrochemical biosensors can employ different transducers (potentiometric, amperometric and impedimetric) converting the chemical information into a measurable amperometric signal. Following this contemporary theme, our main focus in this review is to discuss different methodologies for application in biosensing, whose signal transduction is based on electrochemical principles. We apply a discussion on recent trends involving different nanostructured materials, but without daring to contemplate all nanomaterials incessantly cited in literature, which leads us to believe that this moment is an unprecedented revolution in the preparation of electrochemical biodevices. Besides, some structures of bio-nano interface and different electrochemical biosensors involved in diagnosis systems are also discussed. We outline in several parts of the report how nanoscience technologies are emerging in diagnostic medicine, as well as convergence of electrochemistry and bio-nanoscience. Our hopes for this review are that it can help different categories of researchers to understand the broad application area of electrochemistry and bioelectrochemistry, in order to detecting several types of diseases and biological phenomena.