Chiroptical spectroscopy and metabolomics for blood-based sensing of pancreatic cancer.

To enable the early diagnosis of pancreatic cancer, the search for and definition of reliable biomarkers remain a subject of great interest, with the specificity and sensitivity of the currently used biomarkers being below the required values. We tested a novel diagnostic approach for pancreatic cancer based on the specific molecular signature of blood plasma components. To acquire more detailed structural information, structure-sensitive chiroptical methods (electronic circular dichroism and Raman optical activity) were supplemented by conventional Raman and infrared spectroscopies. The obtained spectra were subsequently processed by linear discriminant analysis yielding high values of specificity and sensitivity. In addition, to monitor not only large biomolecules as potential biomarkers but also those of low molecular weight, we conducted an analysis of blood plasma samples by using metabolomics. The achieved results suggest a panel of promising biomarkers for a reliable detection of pancreatic cancer.

Cocaine Hydrochloride Structure in Solution Revealed by Three Chiroptical Methods.

Structure and flexibility of natural compounds determine their biological activity. In the present study, electronic circular dichroism (ECD), vibrational circular dichroism (VCD), and Raman optical activity (ROA) spectra of cocaine hydrochloride in aqueous solutions were measured and related to the structure with the aid of density functional theory (DFT) computations. Additional measurements in deuterated environment made assignment of vibrational bands easier. The results suggest that the prevalent cocaine conformation in solution differs from that adopted in hydrochloride crystal. The spectroscopic results and computational analysis are consistent with X-ray structures of known cocaine-receptor complexes, in which the compound adopts a variety of conformations. All three kinds of chiroptical spectra exhibited significantly greater conformational sensitivity than unpolarized absorption or Raman scattering. The ROA technique provided the largest number of well-resolved bands, bearing rich structural information.

The potential of chiroptical and vibrational spectroscopy of blood plasma for the discrimination between colon cancer patients and the control group.

Colorectal cancer is one of the most abundant causes of cancer deaths in the world. At an early stage, the established clinical procedures have low reliability and sensitivity. Therefore, we tested a novel approach based on chiroptical methods such as electronic circular dichroism (ECD) and Raman optical activity (ROA). These methods are suitable for detecting slight changes in the 3D structure of chiral biomolecules, some of which may be caused by pathological processes occurring during cancer growth. Fifty-five blood plasma samples were analyzed using the combination of ECD and ROA supplemented by conventional Raman and FT-IR spectroscopy. All obtained spectra were evaluated together by linear discriminant analysis. The accuracy of sample discrimination reached 100% and the subsequent leave-one-out cross-validation resulted in 93% sensitivity and 81% specificity. The achieved results indicate that chiroptical methods supplemented by Raman and FT-IR spectroscopy might be new supporting and minimally invasive tools in the clinical diagnosis of colon cancer.

Identification of spectral biomarkers for type 1 diabetes mellitus using the combination of chiroptical and vibrational spectroscopy.

The current diagnostic tools are insufficient for the early detection of many diseases, including type 1 diabetes mellitus. The disease is accompanied not only by a permanently elevated level of blood glucose and altered levels of other biomarkers, but also by changes in the conformation of blood plasma proteins and other biomolecules associated with the pathogenesis of diabetes. However, the observation of these structural changes by conventional Raman and infrared spectroscopy is limited. Therefore, we used chiroptical spectroscopy which is inherently sensitive to the 3D structure of chiral molecules and able to detect any possible structural changes. We investigated the blood plasma samples of diabetic patients and healthy controls by Raman optical activity and electronic circular dichroism. The measurements were combined with conventional methods of molecular spectroscopy, i.e. Raman and infrared spectroscopy. The obtained data sets were statistically evaluated using linear discriminant analysis focusing on the spectral ranges that correspond to the structure and conformation of proteins and other plasmatic biomolecules. Our results suggest that chiroptical spectroscopy gives more detailed information about the 3D structure of biomolecules; and therefore, might be a promising complement to conventional diagnostic methods.

The minimizing of fluorescence background in Raman optical activity and Raman spectra of human blood plasma.

Raman optical activity (ROA) is inherently sensitive to the secondary structure of biomolecules, which makes it a method of interest for finding new approaches to clinical applications based on blood plasma analysis, for instance the diagnostics of several protein-misfolding diseases. Unfortunately, real blood plasma exhibits strong background fluorescence when excited at 532 nm; hence, measuring the ROA spectra appears to be impossible. Therefore, we established a suitable method using a combination of kinetic quenchers, filtering, photobleaching, and a mathematical correction of residual fluorescence. Our method reduced the background fluorescence approximately by 90%, which allowed speedup for each measurement by an average of 50%. In addition, the signal-to-noise ratio was significantly increased, while the baseline distortion remained low. We assume that our method is suitable for the investigation of human blood plasma by ROA and may lead to the development of a new tool for clinical diagnostics.

Analysis of human blood plasma and hen egg white by chiroptical spectroscopic methods (ECD, VCD, ROA).

Chiroptical methods are widely used in structural and conformational analyses of biopolymers. The application of these methods to investigations of biofluids would provide new avenues for the molecular diagnosis of protein-misfolding diseases. In this work, samples of human blood plasma and hen egg white were analyzed using a combination of conventional and chiroptical methods: ultraviolet absorption/electronic circular dichroism (UV/ECD), Fourier transform infrared absorption/vibrational circular dichroism (FTIR/VCD), and Raman scattering/Raman optical activity (Raman/ROA). For comparison, the main components of these substances--human serum albumin (HSA) and ovalbumin (Ova)--were also analyzed by these methods. The ultraviolet region of the ECD spectrum was analyzed using the CDNN CD software package to evaluate the secondary structures of the proteins. The UV/ECD, FTIR/VCD, and Raman/ROA spectra of the substances were quite similar to those of the corresponding major proteins, while some differences were also detected and explained. The conclusions drawn from the FTIR/VCD and Raman/ROA data were in good agreement with the secondary structures calculated from ECD. The results obtained in this work demonstrate that the chiroptical methods used here can be applied to analyze not only pure protein solutions but also more complex systems, such as biological fluids.

Can chiroptical spectroscopy be used for the analysis of blood plasma?

As an improvement on currently used methods of molecular spectroscopy, we used chiroptical techniques (electronic circular dichroism, fluorescence detected circular dichroism, and Raman optical activity [ROA]) to investigate the human blood plasma. To avoid the degradation of plasma samples, we measured them directly without any further preparation. We also tested cutoff weight filters (Amicon Ultra 100, 30, 10, and 3 kDa by Merck Millipore) to reduce undesirable fluorescence in the ROA and Raman spectra and also to remove the most abundant protein in the plasma-human serum albumin. The obtained spectra show that the ultrafiltration has a positive effect on undesirable fluorescence in ROA and Raman and also could reduce the amount of albumin in the plasma. Our results suggest that blood plasma can be successfully measured by the aforementioned methods. Therefore, these methods can potentially be useful for following research in the development of new, noninvasive, and reliable screening methods of clinical diagnostics.