The COVID-19 pandemic demonstrated the critical need for accurate and rapid testing for virus detection. This need has generated a high number of new testing methods aimed at replacing RT-PCR, which is the golden standard for testing. Most of the testing techniques are based on biochemistry methods and require chemicals that are often expensive and the supply might become scarce in a large crisis. In the present paper we suggest the use of methods based on physics that leverage novel nanomaterials. We demonstrate that using Surface Enhanced Raman Spectroscopy (SERS) of virion particles a very distinct spectroscopic signature of the SARS-CoV-2 virus can be obtained. We demonstrate that the spectra are mainly composed by signals from the spike (S) and nucleocapsid (N) proteins. It is believed that a clinical test using SERS can be developed. The test will be fast, inexpensive, and reliable. It is also clear that SERS can be used for analysis of structural changes on the S and N proteins. This will be an example of application of nanotechnology and properties of nanoparticles for health and social related matters.

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Detection of SARS-CoV-2 and its S and N proteins using surface enhanced Raman spectroscopy

Rapid and accurate classification and discrimination of bacteria is an important task and has been highlighted recently for rapid diagnostics using real-time results. Coupled with a recent report by Jim O'Neill [] that if left unaddressed antimicrobial resistance (AMR) in bacteria could kill 10 million people per year by 2050, which would surpass current cancer mortality, this further highlights the need for unequivocal identification of microorganisms. Whilst traditional microbiological testing has offered insights into the characterisation and identification of a wide range of bacteria, these approaches have proven to be laborious and time-consuming and are not really fit for purpose, considering the modern day speed and volume of international travel and the opportunities it creates for the spread of pathogens globally. To overcome these disadvantages, modern analytical methods, such as mass spectrometry (MS) and vibrational spectroscopy, that analyse the whole organism, have emerged as essential alternative approaches. Currently within clinical microbiology laboratories, matrix assisted laser desorption ionisation (MALDI)-MS is the method of choice for bacterial identification. This is largely down to its robust analysis as it largely measures the ribosomes which are always present irrespective of how the bacteria are cultured. However, MALDI-MS requires large amounts of biomass and infrared spectroscopy and Raman spectroscopy are attractive alternatives as these physicochemical bioanalytical techniques have the advantages of being rapid, reliable and cost-effective for analysing various types of bacterial samples, even at the single cell level. In this review, we discuss the fundamental applications, advantages and disadvantages of modern analytical techniques used for bacterial characterisation, classification and identification.

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Discrimination of bacteria using whole organism fingerprinting: the utility of modern physicochemical techniques for bacterial typing.

A great enhancement in Raman scattering (SERS) from heme-containing submembrane biomolecules inside intact erythrocytes and functional mitochondria is demonstrated for the first time using silver–silica beads prepared using a new method involving aerosol pyrolysis with aqueous diamminesilver(I) hydroxide as a unique source of plasmonic nanoparticles for SiO2 microspheres. The recorded SERS spectra reveal a set of characteristic peaks at 750, 1127, 1170, 1371, 1565, 1585 and 1638 cm−1, resulting from the normal group vibrations of the pyrrole rings, methine bridges and side radicals in the heme molecules. The SERS spectra of functional mitochondria are sensitive to the activity of the mitochondrial electron transport chain, thus making the method a novel label-free approach to monitor the redox state and conformation of cytochromes in their natural cell environment. The developed nanocomposites are highly suitable for the analysis of biological objects due to their robust synthesis and superior spatial and temporal signal reproducibility, which was preserved for a period of at least one year.

New nanocomposites for SERS studies of living cells and mitochondria

Membrane proteins (MPs) play vital roles in the function of cells and are also major drug targets. Structural information on proteins is vital for understanding their mechanism of function and is critical for the development of drugs. However, obtaining high-resolution structures of membrane proteins, in particular, under native conditions is still a great challenge. In such cases, the low-resolution methods small-angle X-ray and neutron scattering (SAXS and SANS) might provide valuable structural information. However, in some cases small-angle scattering (SAS) provides ambiguous ab initio structural information if complementary measurements are not performed and/or a priori information on the protein is not taken into account. Understanding the nature of the limitations may help to overcome these problems. One of the main problems of SAS data analysis of solubilized membrane proteins is the contribution of the detergent belt surrounding the MP. Here, a comprehensive analysis of how the detergent belt contributes to the SAS data of a membrane-protein complex of sensory rhodopsin II with its cognate transducer from Natronomonas pharaonis (NpSRII-NpHtrII) was performed. The influence of the polydispersity of NpSRII-NpHtrII oligomerization is the second problem that is addressed here. It is shown that inhomogeneity in the scattering length density of the detergent belt surrounding a membrane part of the complex and oligomerization polydispersity significantly impacts on SAXS and SANS profiles, and therefore on 3D ab initio structures. It is described how both problems can be taken into account to improve the quality of SAS data treatment. Since SAS data for MPs are usually obtained from solubilized proteins, and their detergent belt and, to a certain extent, oligomerization polydispersity are sufficiently common phenomena, the approaches proposed in this work might be used in SAS studies of different MPs.

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II-transducer complex.

The first member and eponym of the rhodopsin family was identified in the 1930s as the visual pigment of the rod photoreceptor cell in the animal retina. It was found to be a membrane protein, owing its photosensitivity to the presence of a covalently bound chromophoric group. This group, derived from vitamin A, was appropriately dubbed retinal. In the 1970s a microbial counterpart of this species was discovered in an archaeon, being a membrane protein also harbouring retinal as a chromophore, and named bacteriorhodopsin. Since their discovery a photogenic panorama unfolded, where up to date new members and subspecies with a variety of light-driven functionality have been added to this family. The animal branch, meanwhile categorized as type-2 rhodopsins, turned out to form a large subclass in the superfamily of G protein-coupled receptors and are essential to multiple elements of light-dependent animal sensory physiology. The microbial branch, the type-1 rhodopsins, largely function as light-driven ion pumps or channels, but also contain sensory-active and enzyme-sustaining subspecies. In this review we will follow the development of this exciting membrane protein panorama in a representative number of highlights and will present a prospect of their extraordinary future potential.

Rhodopsins: An Excitingly Versatile Protein Species for Research, Development and Creative Engineering

This is a review of relevant Raman spectroscopy (RS) techniques and their use in structural biology, biophysics, cells, and tissues imaging towards development of various medical diagnostic tools, drug design, and other medical applications. Classical and contemporary structural studies of different water-soluble and membrane proteins, DNA, RNA, and their interactions and behavior in different systems were analyzed in terms of applicability of RS techniques and their complementarity to other corresponding methods. We show that RS is a powerful method that links the fundamental structural biology and its medical applications in cancer, cardiovascular, neurodegenerative, atherosclerotic, and other diseases. In particular, the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS), which would help to further the development of protein structural crystallography and would result in a number of novel high-resolution structures of membrane proteins—drug targets; and, structural studies of photoactive membrane proteins (rhodopsins, photoreceptors, etc.) for the development of new optogenetic tools. Physical background and biomedical applications of spontaneous, stimulated, resonant, and surface- and tip-enhanced RS are also discussed. All of these techniques have been extensively developed during recent several decades. A number of interesting applications of CARS, resonant, and surface-enhanced Raman spectroscopy methods are also discussed.

Raman Scattering: From Structural Biology to Medical Applications

ATP synthase FOF1 structure, function, and structure-based drug design

Ferritin self-assembly, structure, function, and biotechnological applications.

F-type ATP synthases play a key role in oxidative and photophosphorylation processes generating adenosine triphosphate (ATP) for most biochemical reactions in living organisms. In contrast to the mitochondrial FOF1-ATP synthases, those of chloroplasts are known to be mostly monomers with approx. 15% fraction of oligomers interacting presumably non-specifically in a thylakoid membrane. To shed light on the nature of this difference we studied interactions of the chloroplast ATP synthases using small-angle X-ray scattering (SAXS) method. Here, we report evidence of I-shaped dimerization of solubilized FOF1-ATP synthases from spinach chloroplasts at different ionic strengths. The structural data were obtained by SAXS and demonstrated dimerization in response to ionic strength. The best model describing SAXS data was two ATP-synthases connected through F1/F1′ parts, presumably via their δ-subunits, forming “I” shape dimers. Such I-shaped dimers might possibly connect the neighboring lamellae in thylakoid stacks assuming that the FOF1 monomers comprising such dimers are embedded in parallel opposing stacked thylakoid membrane areas. If this type of dimerization exists in nature, it might be one of the pathways of inhibition of chloroplast FOF1-ATP synthase for preventing ATP hydrolysis in the dark, when ionic strength in plant chloroplasts is rising. Together with a redox switch inserted into a γ-subunit of chloroplast FOF1 and lateral oligomerization, an I-shaped dimerization might comprise a subtle regulatory process of ATP synthesis and stabilize the structure of thylakoid stacks in chloroplasts.

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Stepan D. Osipov

Papers

Raman Scattering: From Structural Biology to Medical Applications

ATP synthase FOF1 structure, function, and structure-based drug design

Ferritin self-assembly, structure, function, and biotechnological applications.

I-Shaped Dimers of a Plant Chloroplast FOF1-ATP Synthase in Response to Changes in Ionic Strength