Optimization of a portable ligand-free optical spectroscopy method for SARS-CoV-2 protein detection

We have developed a simple and potentially a low-cost method for the sensitive detection of target proteins via surface-enhanced Raman scattering (SERS). The immunosensor constructed by the conjugation of monoclonal antibodies to 20 nm diameter gold nanoparticles via the bifunctional Raman reporter molecule, 5, 5'dithiobis (succinimidyl-2-nitrobenzoate) (DSNB) is the basis of a membrane-bound detection system. Traditionally, a common laboratory technique called a dot blot, which is a colorimetric method where detection of proteins is accomplished through the application of assorted dyes followed by their measurement via a densitometer. Dot blotting is a convenient and time saving method that involves the spotting of a protein onto an immobilizing matrix, such as nitrocellulose (NC) or polyvinylidene fluoride (PVDF) membrane. We found that for detection via SERS spectroscopy NC is the matrix of choice because it offers low background, minimal preparation prior to protein application, and optimal position of Raman bands. Furthermore, SERS detection of protein on NC requires only minimal sample preparation and demonstrates increased sensitivity when compared to other dot blot detection methods. Depending on the dye used for visualization, dot blots analyzed by commonly used optical methods have limits of detection in the nanogram range, some as low as 20 pg/ml. Here we demonstrate the use of the dot blot method for detecting target proteins (e.g., protein A and prostate specific protein (PSA)) by SERS spectroscopy down to a concentration of 100 fg/ml.

The rapidly growing field of proteomics and related applied sectors in the life sciences demands convenient methodologies for detecting and measuring the levels of specific proteins as well as for screening and analyzing for interacting protein systems. Materials utilized for such protein detection and measurement platforms should meet particular specifications which include ease-of-mass manufacture, biological stability, chemical functionality, cost effectiveness, and portability. Polymers can satisfy many of these requirements and are often considered as choice materials in various biological detection platforms. Therefore, tremendous research efforts have been made for developing new polymers both in macroscopic and nanoscopic length scales as well as applying existing polymeric materials for protein measurements. In this review article, both conventional and alternative techniques for protein detection are overviewed while focusing on the use of various polymeric materials in different protein sensing technologies. Among many available detection mechanisms, most common approaches such as optical, electrochemical, electrical, mass-sensitive, and magnetic methods are comprehensively discussed in this article. Desired properties of polymers exploited for each type of protein detection approach are summarized. Current challenges associated with the application of polymeric materials are examined in each protein detection category. Difficulties facing both quantitative and qualitative protein measurements are also identified. The latest efforts on the development and evaluation of nanoscale polymeric systems for improved protein detection are also discussed from the standpoint of quantitative and qualitative measurements. Finally, future research directions towards further advancements in the field are considered.

The paper considers a problem of detection and identification of unmanned aerial vehicles (UAVs) against the animate and inanimate objects and identification of their load by optical and spectral optical methods. The state-of-the-art analysis has shown that, when using the radar methods to detect small UAVs, there is a dead zone for distances of 250-700 m, and in this case it is important to use optical methods for detecting UAVs.The application possibilities and improvements of the optical scheme for detecting UAVs at long distances of about 1-2 km are considered. Location is performed by intrinsic infrared (IR) radiation of an object using the IR cameras and thermal imagers, as well as using a laser rangefinder (LIDAR). The paper gives examples of successful dynamic detection and recognition of objects from video images by methods of graph theory and neural networks using the network FasterR-CNN, YOLO and SSD models, including one frame received.The possibility for using the available spectral optical methods to analyze the chemical composition of materials that can be employed for remote identification of UAV coating materials, as well as for detecting trace amounts of matter on its surface has been studied. The advantages and disadvantages of the luminescent spectroscopy with UV illumination, Raman spectroscopy, differential absorption spectroscopy based on a tunable UV laser, spectral imaging methods (hyper / multispectral images), diffuse reflectance laser spectroscopy using infrared tunable quantum cascade lasers (QCL) have been shown.To assess the potential limiting distances for detecting and identifying UAVs, as well as identifying the chemical composition of an object by optical and spectral optical methods, a described experimental setup (a hybrid lidar UAV identification complex) is expected to be useful. The experimental setup structure and its performances are described. Such studies are aimed at development of scientific basics for remote detection, identification, tracking, and determination of UAV parameters and UAV belonging to different groups by optical location and spectroscopy methods, as well as for automatic optical UAV recognition in various environments against the background of moving wildlife. The proposed problem solution is to combine the optical location and spectral analysis methods, methods of the theory of statistics, graphs, deep learning, neural networks and automatic control methods, which is an interdisciplinary fundamental scientific task.

The use of silver nanoparticles (Ag NPs) as substrates to obtain satisfactory Raman spectra of native proteins is a simple and valuable but challenging process. Herein, the Ag NPs modified with aluminum and iodide ions (Ag IANPs) were introduced for Raman detection of proteins, including acidic BSA (PI 4.7), catalase (PI 5.4), β-casein (PI 4.5), α-casein (PI 4.0), insulin (PI 5.35), basic myoglobin (PI 6.99), and lysozyme (PI 11.2). The Raman signals of all the detected proteins were significantly improved in comparison with the reported spectra obtained by using Ag NPs containing Na2SO4, I-, and Mg2+. Specifically, detection sensitivities of the acidic proteins were drastically increased. The limit of detection (LOD) of bovine serum albumin (BSA), α-casein, and β-casein was 0.03 ng/mL. The LOD of insulin and catalase were 0.3 and 3 ng/mL, respectively. As the bands corresponding to disulfide bonds, α-helices, residues of Phe, Trp, and Tyr, and carboxyl groups were also greatly enhanced, it was easy to monitor the folding of native protein and the denaturation of protein under acidic and heated conditions. Thus, Ag IANPs as substrates open a way for surface-enhanced Raman spectroscopy (SERS) detection of proteins. Hence, the method can provide more valuable information about protein and, therefore, has the potential for wide applications.

This work aims to evaluate the application of optical and X-ray spectroscopy methods to determine the effect of alpha-emitting radionuclides on the properties of solid-state nuclear track detectors (SSNTD) based on nitrocellulose during their detection. The proposed estimation methods are alternative methods to standard technologies, making it possible to determine the concentration of radon and its decay products without the chemical etching of film detectors and subsequent direct counting of the formed latent tracks from interacting particles. During the research, it was found that the use of optical spectroscopy and X-ray diffraction methods makes it possible to qualitatively determine the irradiation effect on changes in the properties of film detectors when α-particles with different energies pass through them. At the same time, a comparison of the data of optical spectroscopy, X-ray diffraction and the visualization of latent tracks after chemical etching made it possible to establish that a part of the registered α-particles in living quarters has an energy of less than 2.5 MeV, which is not enough to pass through the polymer film of the detector, as a result of which well-like tracks are formed. An increase in the intensity of the interference bands in the region above 700 nm and a decrease in the intensity of diffraction reflection characterized the changes in optical transmission. The penetration of the α-particles through the detecting film decreases the film’s transmission capacity, forming an anisotropic change in diffraction reflections associated with a change in the film’s structure and defective fractions distorting the molecular structure.

The requirement to measure the stable isotopic compositions of saline pore fluids by optical methods has prompted a re-evaluation of the isotopic salt effect for common salts. Hydrogen and oxygen isotopic salt effects were measured at room temperature (21°C) by optical methods. For hydrogen isotopes, our results agree well with those of previous studies and better define these effects at low temperatures. In contrast, measured oxygen isotope salt effects disagree within error for NaCl and CaCl2 solutions from those reported previously. Subtle differences between measurement methods may account for the discrepancy. In studies that involve highly saline fluids, the isotopic salt effect must be taken into account because modern methods that measure stable isotopic compositions as activities or concentrations may be not directly comparable to historical data sets.

Proteins are useful biomarkers for a wide range of applications such as cancer detection, discovery of vaccines, and determining exposure to viruses and pathogens. Here, we present a low-noise front-end analog circuit interface towards development of a portable readout system for the label-free sensing of proteins using Nanowell array impedance sensing with a form factor of approximately 35cm2. The electronic interface consists of a low-noise lock-in amplifier enabling reliable detection of changes in impedance as low as 0.1% and thus detection of proteins down to the picoMolar level. The sensitivity of our system is comparable to that of a commercial bench-top impedance spectroscope when using the same sensors. The aim of this work is to demonstrate the potential of using impedance sensing as a portable, low-cost, and reliable method of detecting proteins, thus inching us closer to a Point-of-Care (POC) personalized health monitoring system. We have demonstrated the utility of our system to detect antibodies at various concentrations and protein (45 pM IL-6) in PBS, however, our system has the capability to be used for assaying various biomarkers including proteins, cytokines, virus molecules and antibodies in a portable setting.

Exosomes are nanosized (50-150nm) extracellular vesicles released by all types of cells in the body. They transport various biological molecules, such as DNAs, RNAs, proteins, and lipids from parent cells to recipient cells for intercellular communication. Exosomes, especially those from tumor cells, are actively involved in caner development, metastasis, and drug resistance. Recently, many studies have shown that exosomal proteins are promising biomarkers for cancer screening, early detection and prognosis. Among many detection techniques, surface plasmon resonance (SPR) is a highly sensitive, label-free, and real-time optical detection method. Commercial prism-based wavelength/angular-modulated SPR sensors afford high sensitivity and resolution, but their large footprint and high cost limit their adaptability for clinical settings. We have developed an intensity-modulated, compact SPR biosensor (25cm×10cm×25cm) for the detection of exosomal proteins. We have demonstrated the potential application of the compact SPR biosensor in lung cancer diagnosis using exosomal epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) as biomarkers. The compact SPR biosensor offers sensitive, simple, fast, user-friendly, and cost-effective detection of exosomal proteins, which may serve as an in vitro diagnostic test for cancer.

In the surgical treatment of malignant tumors, it is crucial to characterize the tumor as precisely as possible. The determination of the exact tumor location as well as the analysis of its properties is very important in order to obtain an accurate diagnosis as early as possible. In neurosurgical applications, the optical, non-invasive and in situ techniques allow for the label-free analysis of tissue, which is helpful in neuropathology. In the past decades, optical spectroscopic methods have been investigated drastically in the management of cancer. In the optical spectroscopic techniques, tissue interrogate with sources of light which are ranged from the ultraviolet to the infrared wavelength in the spectrum. The information accumulation of light can be in a reflection which is named reflectance spectroscopy; or interactions with tissue at different wavelengths which are called fluorescence and Raman spectroscopy. This review paper introduces the optical spectroscopic methods which are used to characterize brain tumors (neuro-oncology). Based on biochemical information obtained from these spectroscopic methods, it is possible to identify tumor from normal brain tissues, to indicate tumor margins, the borders towards normal brain tissue and infiltrating gliomas, to distinguish radiation damage of tissues, to detect particular central nervous system (CNS) structures to identify cell types using particular neurotransmitters, to detect cells or drugs which are optically labeled within therapeutic intermediations and to estimate the viability of tissue and the prediction of apoptosis beginning in vitro and in vivo. The label-free, optical biochemical spectroscopic methods can provide clinically relevant information and need to be further exploited to develop a safe and easy-to-use technology for in situ diagnosis of malignant tumors.

The biophysical characterization of globular proteins will almost always include some type of study of the unfolding of protein to obtain thermodynamic parameters. The basic idea is that a transition between a native and unfolded state, induced by temperature, pH, or denaturant concentration, can serve as a standard reaction for obtaining a thermodynamic measure of the stability of the native state. For example, the free energy change for the unfolding reaction can be used to compare the stability of a set of mutant forms of a protein (1-4). This type of analysis is based both on assumptions of the thermodynamic model for the unfolding process and on assumptions in the way the data are analysed; some of these assumptions and their limitations will be discussed below. There are a variety of methods that can be used to monitor an unfolding process. A common method is differential scanning calorimetry, DSC, which measures the variation in the specific heat of a protein-containing solution as a protein is thermally unfolded (5-7). DSC is a popular method for this purpose, but optical methods can also provide suitable information for tracking the unfolding of a protein The spectroscopic signals for the native and unfolded states of a protein can give some insight regarding the structure of the states, and often can provide advantages of economy, ease of measurement and amenability to a wide range of sample concentration. The optical spectroscopic methods that have been used most often for this purpose are absorption spectroscopy, circular dichroism and fluorescence, which will be discussed in this chapter. A key to each of these methods and their use in protein unfolding studies is that the signal is a mole fraction weighted average of the signals of each thermodynamic state. That is, the observed signal, S, can be expressed as . . . S = ∑XiSi . . . . . . 1 . . . where Xi is the mole fraction of species i and si is the intrinsic signal of species i. In order for a particular spectroscopic signal to be useful for tracking a N ↔ U transition of a protein, the signal must be sufficiently different for the N and U states.

The polarized absorption and emission spectra of fluorescent stilbene dye dissolved in two side chain liquid crystalline polysiloxanes have been recorded as a function of temperature in the mesomorphic and glassy phases. From these spectra the order parameters <P2> and <P4> as well as the orientational distribution function of the side mesogenic groups have been estimated. The results have been compared with the data obtained for low molecular weight liquid crystal by using the same optical methods.

Semiconducting carbon nanotubes (sc-SWCNT) sit amongst the most promising nanomaterials for a new generation of electronic devices. Progress towards commercial applications is combined with challenges related to materials development and characterization, fabrication methods as well as device modeling. This talk will focus primarily on the enrichment process and the related ink formulation for printed electronics and integrated circuits. [1] The quality of carbon nanotube ink solutions is closely tied with the rapid feedback provided by optical spectroscopy methods: UV-vis, Raman and fluorescence. [2] I will describe our efforts on improving the material’s assessment when purity exceeds well beyond 99%. [3] [1] Ding, J.; Li, Z.; Lefebvre, J.; Cheng, F.; Dubey, G.; Zou, S.; Finnie, P.; Hrdina, A.; Scoles, L.; Lopinski, G. P.; Kingston, C. T.; Simard, B.; Malenfant, P. R. L. Enrichment of Large-Diameter Semiconducting SWCNTs by Polyfluorene Extraction for High Network Density Thin Film Transistors. Nanoscale 2014, 6, 2328-2339.[2] J. Lefebvre, J.; Finnie, P.; Fagan, J.; Zheng, M.; Hight Walker, A. R. Metrological Assessment of Single-Wall Carbon Nanotube Materials by Optical Methods. Handbook of Carbon Nanomaterials 2019, 9, R. B. Weisman and J. Kono editors (World Scientific) ISBN 978-981-3235-45-8.[3] Lefebvre, J.; Ding, J.; Li, Z.; Finnie, P.; Lopinski, G.; Malenfant, P. R. L. High-Purity Semiconducting Single-Walled Carbon Nanotubes: A Key Enabling Material in Emerging Electronics. Acc. Chem. Res. 2017, 50 (10), 2479-2486. Figure 1

Supported silver clusters as nanoplasmonic transducers for protein sensing

Information on the penetration depth, pathways, metabolization, storage of vehicles, active pharmaceutical ingredients (APIs), and functional cosmetic ingredients (FCIs) of topically applied formulations or contaminants (substances) in skin is of great importance for understanding their interaction with skin targets, treatment efficacy, and risk assessment—a challenging task in dermatology, cosmetology, and pharmacy. Non-invasive methods for the qualitative and quantitative visualization of substances in skin in vivo are favored and limited to optical imaging and spectroscopic methods such as fluorescence/reflectance confocal laser scanning microscopy (CLSM); two-photon tomography (2PT) combined with autofluorescence (2PT-AF), fluorescence lifetime imaging (2PT-FLIM), second-harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and reflectance confocal microscopy (2PT-RCM); three-photon tomography (3PT); confocal Raman micro-spectroscopy (CRM); surface-enhanced Raman scattering (SERS) micro-spectroscopy; stimulated Raman scattering (SRS) microscopy; and optical coherence tomography (OCT). This review summarizes the state of the art in the use of the CLSM, 2PT, 3PT, CRM, SERS, SRS, and OCT optical methods to study skin penetration in vivo non-invasively (302 references). The advantages, limitations, possibilities, and prospects of the reviewed optical methods are comprehensively discussed. The ex vivo studies discussed are potentially translatable into in vivo measurements. The requirements for the optical properties of substances to determine their penetration into skin by certain methods are highlighted.

Protein Conformational Dynamics Probed Correlation Spectroscopy of Multiply Scattered Light