An infrared sensor analysing label‐free the secondary structure of the Abeta peptide in presence of complex fluids

The secondary structure change of the Abeta peptide to beta‐sheet was proposed as an early event in Alzheimer's disease. The transition may be used for diagnostics of this disease in an early state. We present an Attenuated Total Reflection (ATR) sensor modified with a specific antibody to extract minute amounts of Abeta peptide out of a complex fluid. Thereby, the Abeta peptide secondary structure was determined in its physiological aqueous environment by FTIR‐difference‐spectroscopy. The presented results open the door for label‐free Alzheimer diagnostics in cerebrospinal fluid or blood. It can be extended to further neurodegenerative diseases.

An immunologic ATR‐FTIR sensor for Abeta peptide secondary structure analysis in complex fluids is presented.     

Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin‐producing Escherichia coli strains by a time‐resolved fluorescence immunoassay

A rapid and sensitive two‐step time‐resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin‐producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu³⁺) chelate was then used as a detector, followed by fluorescence measurements using time‐resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1–1000 ng/ml). The intra‐ and inter‐assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2‐specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2‐specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation‐based platform and aid in the accurate and prompt diagnosis of STEC infections.     

Fourier‐transform infrared spectroscopy study of dehydrated lipases from Candida antarctica B and Pseudomonas cepacia

Fourier‐transform infrared (FT‐IR) spectroscopy was employed to investigate potential lyophilization‐induced changes in the secondary structure of lipases from Candida antarctica B and Pseudomonas cepacia. The secondary structure elements were determined by curve fitting of the amide III bands of the two lipases in the lyophilized state in KBr pellets and in solution. It was found that lyophilization decreased the α‐helix and increased the β‐sheet content. However, FT‐IR analysis of crosslinked enzyme crystals of Pseudomonas cepacia lipase also indicated an increase in the β‐sheet content, which appears despite the fact that the enzyme, being in the crystallized state, should possess native conformation. This result partially questions the suitability of FT‐IR for analysis of the structure of solid proteins, at least as far as the β‐sheet content is concerned, because it is possible that the method overestimates the β‐sheets by measuring other hydrogen‐bonded nonperiodic intermolecular structures. No significant modification was observed when lipase from Pseudomonas cepacia was lyophilized in the presence of methoxypoly(ethylene glycol). © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 545–551, 1999.     

Application of ATR‐FTIR spectroscopy to the study of thermally induced changes in secondary structure of protein molecules in solid state

FTIR spectroscopy in combination with ATR sampling technique is the most accessible analytical technique to study secondary structure of proteins both in solid and aqueous solution. Although several studies have demonstrated the applications of ATR‐FTIR to study conformational changes of solid dried proteins due to dehydration, there are no reports that demonstrate the application of ATR‐FTIR in the study of thermally induced changes of secondary structure of biomolecules directly on the solid state. In this study, four biomolecules of pharmaceutical interest, lysozyme, myoglobine, chymotripsin and human growth hormone (hGH), were studied on the solid state before and after different thermal treatments in order to relate changes of secondary structure to partial or total thermal denaturation processes. The results obtained provide experimental evidence that protein thermal denaturation in the solid state can be detected by displacement of carbonyl bands which correspond to conformational transformations between α–helix to β‐sheet or intermolecular β‐sheet; the molecules studied undergo this transformation when exposed to a temperature close to their denaturation temperature which may become irreversible depending on the extent of the heating treatment. These findings demonstrate that ATR‐FTIR is an effective and time efficient technique that allows the monitoring of the protein thermal denaturation process of solid samples without further reconstitution or prior sample preparation. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 574–584, 2015.     

Investigation of the source‐detector separation in near infrared spectroscopy for healthy and clinical applications

Understanding near infrared light propagation in tissue is vital for designing next generation optical brain imaging devices. Monte Carlo (MC) simulations provide a controlled mechanism to characterize and evaluate contributions of diverse near infrared spectroscopy (NIRS) sensor configurations and parameters. In this study, we developed a multilayer adult digital head model under both healthy and clinical settings and assessed light‐tissue interaction through MC simulations in terms of partial differential pathlength, mean total optical pathlength, diffuse reflectance, detector light intensity and spatial sensitivity profile of optical measurements. The model incorporated four layers: scalp, skull, cerebrospinal‐fluid and cerebral cortex with and without a customizable lesion for modeling hematoma of different sizes and depths. The effect of source‐detector separation (SDS) on optical measurements' sensitivity to brain tissue was investigated. Results from 1330 separate simulations [(4 lesion volumes × 4 lesion depths for clinical +3 healthy settings) × 7 SDS × 10 simulation = 1330)] each with 100 million photons indicated that selection of SDS is critical to acquire optimal measurements from the brain and recommended SDS to be 25 to 35 mm depending on the wavelengths to obtain optical monitoring of the adult brain function. The findings here can guide the design of future NIRS probes for functional neuroimaging and clinical diagnostic systems.