In-line monitoring of protein concentration with MIR spectroscopy during UFDF.

Rapid increase of product titers in upstream processes has presented challenges for downstream processing, where purification costs increase linearly with the increase of the product yield. Hence, innovative solutions are becoming increasingly popular. Process Analytical Technology (PAT) tools, such as spectroscopic techniques, are on the rise due to their capacity to provide real-time, precise analytics. This ensures consistent product quality and increased process understanding, as well as process control. Mid-infrared spectroscopy (MIR) has emerged as a highly promising technique within recent years, owing to its ability to monitor several critical process parameters at the same time and unchallenging spectral analysis and data interpretation. For in-line monitoring, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a method of choice, as it enables reliable measurements in a liquid environment, even though water absorption bands are present in the region of interest. Here, we present MIR spectroscopy as a monitoring tool of critical process parameters in ultrafiltration/diafiltration (UFDF). MIR spectrometer was integrated in the UFDF process in an in-line fashion through a single-use flow cell containing a single bounce silicon ATR crystal. The results indicate that the one-point calibration algorithm applied to the MIR spectra, predicts highly accurate protein concentrations, as compared with validated offline analytical methods.

Molecular Structure and Interactions of Lipids in the Outer Membrane of Living Cells Based on Surface-Enhanced Raman Scattering and Liposome Models.

The distribution and interaction of lipids determine the structure and function of the cellular membrane. Surface-enhanced Raman scattering (SERS) is used for selective molecular probing of the cell membrane of living fibroblast cells grown adherently on gold nanoisland substrates across their whole contact areas with the substrate, enabling mapping of the membrane's composition and interaction. From the SERS data, the localization and distribution of different lipids and their interactions, together with proteins in the outer cell membrane, are inferred. Interpretation of the spectra is mainly supported by comparison with the spectra of model liposomes composed of phosphatidylcholine, sphingomyelin, and cholesterol obtained on the same gold substrate. The interaction of the liposomes with the substrate differs from that with gold nanoparticles. The SERS maps indicate colocalization of ordered lipid domains with cholesterol in the living cells. They support the observation of ordered membrane regions of micrometer dimensions in the outer leaflet of the cell membrane that are rich in sphingomyelin. Moreover, the spectra of the living cells contain bands from the groups of the lipid heads, phosphate, choline, and ethanolamine, combined with those from membrane proteins, as indicated by signals assigned to prenyl attachment. Elucidating the composition and structure of lipid membranes in living cells can find application in many fields of research.

SERS Imaging of Mesenchymal Stromal Cell Differentiation.

Understanding the process of mesenchymal stromal cell (MSC) osteogenic differentiation is essential for a wide range of medical applications. However, these primary cells vary significantly from donor to donor, making it difficult to fully exploit their therapeutic potential. Although osteogenic differentiation has been studied extensively, there is still a shortage of standardized methods for the evaluation of the degree of differentiation. Here, we employ noninvasive surface-enhanced Raman scattering (SERS) for studying such cells, offering a better understanding of cellular processes in situ. We present the long-term differentiation of MSCs on biocompatible gold nanoisland SERS substrates, combining imaging of cells with spectroscopic detection of molecular species and chemical events occurring on the cellular membrane adjacent to the surface of the SERS substrate. We detect multiple signs of bone tissue formation, from an early stage to mature osteoblasts, without labeling. We show that the results correlate very well with classical differentiation-detecting assays, indicating that the SERS imaging technique alone is sufficient to study the progress of osteogenic differentiation of such cells, paving a way toward continuous label-free screening of live cells.

Gold nanoisland substrates for SERS characterization of cultured cells.

We demonstrate a simple approach for fabricating cell-compatible SERS substrates, using repeated gold deposition and thermal annealing. The substrates exhibit SERS enhancement up to six orders of magnitude and high uniformity. We have carried out Raman imaging of fixed mesenchymal stromal cells cultured directly on the substrates. Results of viability assays confirm that the substrates are highly biocompatible and Raman imaging confirms that cell attachment to the substrates is sufficient to realize significant SERS enhancement of cellular components. Using the SERS substrates as an in vitro sensing platform allowed us to identify multiple characteristic molecular fingerprints of the cells, providing a promising avenue towards non-invasive chemical characterization of biological samples.