Surface enhanced Raman spectroscopy based analysis of SARS-CoV-2 spike protein binding to ACE2 receptor

The SARS-CoV-2 virus is still a challenge because of its diversity and mutations. The binding interactions of the angiotensin converting enzyme 2 (ACE2) receptor and the spike protein are relevant for the SARS-CoV-2 virus to enter the cell. Consequently, it is important and helpful to analyze binding activities and the changes in the structure of the ACE2 receptor and the spike protein. Surface enhanced Raman spectroscopy is able to analyze small concentrations of the proteins without contact, non-invasively and label-free. In this work, we present a SERS based approach in the visible wavelength range to analyze and study the binding interactions of the ACE2 receptor and the spike protein. SERS measurements of the ACE2 receptor, the spike protein and the ACE2-spike complex were performed. Additionally, an inhibitor was used to prevent the spike protein from binding to ACE2 and to compare the results. The analysis of the measured SERS spectra reveals structural differences and changes due to binding activities. Thus, we show that the performed SERS based approach can help for rapid and non-invasive analysis of binding interactions of the ACE2-spike complex and also of protein binding in general. © 2023 SPIE.

SERS Signature of SARS-CoV-2 in Saliva and Nasopharyngeal Swabs: Towards Perspective COVID-19 Point-of-Care Diagnostics.

In this study, the intrinsic surface-enhanced Raman spectroscopy (SERS)-based approach coupled with chemometric analysis was adopted to establish the biochemical fingerprint of SARS-CoV-2 infected human fluids: saliva and nasopharyngeal swabs. The numerical methods, partial least squares discriminant analysis (PLS-DA) and support vector machine classification (SVMC), facilitated the spectroscopic identification of the viral-specific molecules, molecular changes, and distinct physiological signatures of pathetically altered fluids. Next, we developed the reliable classification model for fast identification and differentiation of negative CoV(-) and positive CoV(+) groups. The PLS-DA calibration model was described by a great statistical value-RMSEC and RMSECV below 0.3 and R2cal at the level of ~0.7 for both type of body fluids. The calculated diagnostic parameters for SVMC and PLS-DA at the stage of preparation of calibration model and classification of external samples simulating real diagnostic conditions evinced high accuracy, sensitivity, and specificity for saliva specimens. Here, we outlined the significant role of neopterin as the biomarker in the prediction of COVID-19 infection from nasopharyngeal swab. We also observed the increased content of nucleic acids of DNA/RNA and proteins such as ferritin as well as specific immunoglobulins. The developed SERS for SARS-CoV-2 approach allows: (i) fast, simple and non-invasive collection of analyzed specimens; (ii) fast response with the time of analysis below 15 min, and (iii) sensitive and reliable SERS-based screening of COVID-19 disease.

Exploring the authentication of COVID-19 vaccines using Surface-enhanced handheld Raman spectroscopy (SERS) equipped with orbital Raster scattering and machine learning

COVID-19 is a novel coronavirus first emerging in Wuhan, China in December 2019 and has since spread rapidly across the globe escalating into a worldwide pandemic causing millions of fatalities. Emergency response to the pandemic included social distancing and isolation measures as well as the escalation of vaccination programmes. The most popular COVID-19 vaccines are nucleic acid-based. The vast spread and struggles in containment of the virus has allowed a gap in the market to emerge for counterfeit vaccines. This study investigates the use of handheld Raman spectroscopy as a method for nucleic acid-based vaccine authentication and utilises machine learning analytics to assess the efficacy of the method. Conventional Raman spectroscopy requires a large workspace, is cumbersome and energy consuming, and handheld Raman systems show limitations with regards to sensitivity and sample detection. Surface Enhanced Raman spectroscopy (SERS) however, shows potential as an authentication technique for vaccines, allowing identification of characteristic nucleic acid bands in spectra. SERS showed strong identification potential through Correlation in Wavelength Space (CWS) with all vaccine samples obtaining an r value of approximately 1 when plotted against themselves. Variance was observed between some excipients and a selected number of DNA-based vaccines, possibly attributed to the stability of the SERS colloid where the colloid-vaccine complex had been measured over different time intervals. Further development of the technique would include optimisation of the SERS method, stability studies and more comprehensive analysis and interpretation of a greater sample size. © 2023 IEEE.

Salivary Analysis for Evidence based Ayurvedic Diagnosis

Ayurveda is called Mother of all medical sciences. It's the oldest therapeutic and medicinal treatment invented in ancient India. Ayurveda or Ayurvedic treatment is bit different from modern medical science. It believes in Nadi Pariksha and many subjective parameters are included to start diagnosis of disease. Whereas modern medical science has different approach of disease diagnosis. It utilizes different tools and testing to diagnose a disease effectively. Saliva analysis is already accepted in modern medical as an important bio-substance, as we see in COVID-19, but not in ayurveda. This paper shows how salivary analysis can act as an evidential proof for diagnosing a disease, in the ayurvedic way. The salivary contents can be analyzed use various biosensors. One of these is Surface Enhanced Raman Spectroscopy (SERS) platform. It allows molecular detection in bio fluids like saliva, sweat, urine, etc. The saliva analysis using SERS technique will help to detect various trace level molecules which is likely to assist the Ayurvedic diagnosis more accurately and dependency on subjective parameters will reduce to evaluate patient's condition. © 2023 IEEE.

Raman spectroscopy for viral diagnostics.

Raman spectroscopy offers the potential for fingerprinting biological molecules at ultra-low concentration and therefore has potential for the detection of viruses. Here we review various Raman techniques employed for the investigation of viruses. Different Raman techniques are discussed including conventional Raman spectroscopy, surface-enhanced Raman spectroscopy, Raman tweezer, tip-enhanced Raman Spectroscopy, and coherent anti-Stokes Raman scattering. Surface-enhanced Raman scattering can play an essential role in viral detection by multiplexing nanotechnology, microfluidics, and machine learning for ensuring spectral reproducibility and efficient workflow in sample processing and detection. The application of these techniques to diagnose the SARS-CoV-2 virus is also reviewed. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-023-01059-4.

A Multiplexed SERS Microassay for Accurate Detection of SARS-CoV-2 and Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 variants play an important role in predicting patient outcome during postinfection, and with growing fears of COVID-19 reservoirs in domestic and wild animals, it is necessary to adapt detection systems for variant detection. However, variant-specific detection remains challenging. Surface-enhanced Raman scattering is a sensitive and multiplexing technique that allows the simultaneous detection of multiple targets for accurate identification. Here we propose the development of a multiplex SERS microassay to detect both the spike and nucleocapsid structural proteins of SARS-CoV-2. The designed SERS microassay integrates gold-silver hollow nanobox barcodes and electrohydrodynamically induced nanomixing which in combination enables highly specific and sensitive detection of SARS-CoV-2 and the S-protein epitopes to delineate between ancestral prevariant strains with the newer variants of concern, Delta and Omicron. The microassay allows detection from as low as 20 virus/µL and 50 pg/mL RBD protein and can clearly identify the virus among infected versus healthy nasopharyngeal swabs, with the potential to identify between variants. The detection of both S- and N-proteins of SARS-CoV-2 and the differentiation of variants on the SERS microassay can aid the early detection of COVID-19 to reduce transmission rates and lead into adequate treatments for those severely affected by the virus.

Clinical Evaluation of an Antigen Home Test Using Surface-Enhanced Raman Spectroscopy and Stacking Pad for SARS-CoV-2 Screening with Nasal and Salivary Swab Samples.

This prospective study aimed to evaluate the performance of the InstaView COVID-19 (coronavirus diseases 2019) Antigen Home Test (InstaView AHT) which detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens. In this test kit, surface-enhanced Raman spectroscopy was used, a stacking pad was inserted, and nasal swab and salivary swab samples were used simultaneously to improve performance. The clinical performance of the InstaView AHT was compared to that of RT-PCR using nasopharyngeal samples. The participants without any prior training were recruited and performed the sample collection, testing, and interpretation of the results by themselves. Of the 91 PCR-positive patients, 85 had positive InstaView AHT results. The sensitivity and specificity of the InstaView AHT were 93.4% (95% confidence interval [CI]: 86.2-97.5) and 99.4% (95% CI: 98.2-99.9). The sensitivity of the InstaView AHT was above 90% for all samples obtained from patients with Ct ≤ 20, 20 < Ct ≤ 25, and 25 < Ct ≤ 30 (100%, 95.1%, and 92.0%, respectively). The InstaView AHT can be used as an alternative to RT-PCR testing because of its relatively high sensitivity and specificity, especially when SARS-CoV-2 prevalence is high, and the availability of RT-PCR testing is limited.

Detection of live SARS-CoV-2 virus and its variants by specially designed SERS-active substrates and spectroscopic analyses.

A method using label-free surface enhanced Raman spectroscopy (SERS) based on substrate design is provided for an early detection and differentiation of spike glycoprotein mutation sites in live SARS-CoV-2 variants. Two SERS-active substrates, Au nanocavities (Au NCs) and Au NPs on porous ZrO2 (Au NPs/pZrO2), were used to identify specific peaks of A.3, Alpha, and Delta variants at different concentrations and demonstrated the ability to provide their SERS spectra with detection limits of 0.1-1.0% (or 104-5 copies/mL). Variant identification can be achieved by cross-examining reference spectra and analyzing the substrate-analyte relationship between the suitability of the analyte upon the hotspot(s) formed at high concentrations and the effective detection distance at low concentrations. Mutation sites on the S1 chain of the spike glycoprotein for each variant may be related and distinguishable. This method does not require sample preprocessing and therefore allows for fast screening, which is of high value for more comprehensive and specific studies to distinguish upcoming variants.

Label-free detection and discrimination of respiratory pathogens based on electrochemical synthesis of biomaterials-mediated plasmonic composites and machine learning analysis.

Seasonal outbreaks of respiratory viral infections remain a global concern, with increasing morbidity and mortality rates recorded annually. Timely and false responses contribute to the widespread of respiratory pathogenic diseases owing to similar symptoms at an early stage and subclinical infection. The prevention of emerging novel viruses and variants is also a big challenge. Reliable point-of-care diagnostic assays for early infection diagnosis play a critical role in the response to threats of epidemics or pandemics. We developed a facile method for specifically identifying different viruses based on surface-enhanced Raman spectroscopy (SERS) with pathogen-mediated composite materials on Au nanodimple electrodes and machine learning (ML) analyses. Virus particles were trapped in three-dimensional plasmonic concave spaces of the electrode via electrokinetic preconcentration, and Au films were simultaneously electrodeposited, leading to the acquisition of intense and in-situ SERS signals from the Au-virus composites for ultrasensitive SERS detection. The method was useful for rapid detection analysis (<15 min), and the ML analysis for specific identification of eight virus species, including human influenza A viruses (i.e., H1N1 and H3N2 strains), human rhinovirus, and human coronavirus, was conducted. The highly accurate classification was achieved using the principal component analysis-support vector machine (98.9%) and convolutional neural network (93.5%) models. This ML-associated SERS technique demonstrated high feasibility for direct multiplex detection of different virus species for on-site applications.

Fabrication of MERS-nanovesicle biosensor composed of multi-functional DNA aptamer/graphene-MoS2 nanocomposite based on electrochemical and surface-enhanced Raman spectroscopy.

Middle East respiratory syndrome coronavirus (MERS-CoV) is one of the most harmful viruses for humans in nowadays. To prevent the spread of MERS-CoV, a valid detection method is highly needed. For the first time, a MERS-nanovesicle (NV) biosensor composed of multi-functional DNA aptamer and graphene oxide encapsulated molybdenum disulfide (GO-MoS2) hybrid nanocomposite was fabricated based on electrochemical (EC) and surface-enhanced Raman spectroscopy (SERS) techniques. The MERS-NV aptamer was designed for specifically binding to the spike protein on MERS-NVs and it is prepared using the systematic evolution of ligands by exponential enrichment (SELEX) technique. For constructing a multi-functional MERS aptamer (MF-aptamer), the prepared aptamer was connected to the DNA 3-way junction (3WJ) structure. DNA 3WJ has the three arms that can connect the three individual functional groups including MERS aptamer (bioprobe), methylene blue (signal reporter) and thiol group (linker) Then, GO-MoS2 hybrid nanocomposite was prepared for the substrate of EC/SERS-based MERS-NV biosensor construction. Then, the assembled multifunctional (MF) DNA aptamer was immobilized on GO-MoS2. The proposed biosensor can detect MERS-NVs not only in a phosphate-buffered saline (PBS) solution (SERS LOD: 0.176 pg/ml, EIS LOD: 0.405 pg/ml) but also in diluted 10% saliva (SERS LOD: 0.525 pg/ml, EIS LOD: 0.645 pg/ml).

Research Progress of Raman Spectroscopy and Raman Imaging in Pharmaceutical Analysis.

The analytical investigation of the pharmaceutical process monitors the critical process parameters of the drug, beginning from its development until marketing and post-marketing, and appropriate corrective action can be taken to change the pharmaceutical design at any stage of the process. Advanced analytical methods, such as Raman spectroscopy, are particularly suitable for use in the field of drug analysis, especially for qualitative and quantitative work, due to the advantages of simple sample preparation, fast, non-destructive analysis speed and effective avoidance of moisture interference. Advanced Raman imaging techniques have gradually become a powerful alternative method for monitoring changes in polymorph distribution and active pharmaceutical ingredient distribution in drug processing and pharmacokinetics. Surface-enhanced Raman spectroscopy (SERS) has also solved the inherent insensitivity and fluorescence problems of Raman, which has made good progress in the field of illegal drug analysis. This review summarizes the application of Raman spectroscopy and imaging technology, which are used in the qualitative and quantitative analysis of solid tablets, quality control of the production process, drug crystal analysis, illegal drug analysis, and monitoring of drug dissolution and release in the field of drug analysis in recent years.

In-situ fabrication of 3D interior hotspots templated with a protein@Au core-shell structure for label-free and on-site SERS detection of viral diseases

Nanoscale plasmonic hotspots play a critical role in the enhancement of molecular Raman signals, enabling the sensitive and reliable trace analysis of biomedical molecules via surface-enhanced Raman spectroscopy (SERS). However, effective and label-free SERS diagnoses in practical fields remain challenging because of clinical samples' random adsorption and size mismatch with the nanoscale hotspots. Herein, we suggest a novel SERS strategy for interior hotspots templated with protein@Au core-shell nanostructures prepared via electrochemical one-pot Au deposition. The cytochrome c and lysates of SARS-CoV-2 (SLs) embedded in the interior hotspots were successfully functionalized to confine the electric fields and generate their optical fingerprint signals, respectively. Highly linear quantitative sensitivity was observed with the limit-of-detection value of 10-1 PFU/ mL. The feasibility of detecting the targets in a bodily fluidic environment was also confirmed using the proposed templates with SLs in human saliva and nasopharyngeal swabs. These interior hotspots templated with the target analytes are highly desirable for early and on-site SERS diagnoses of infectious diseases without any labeling processes.

Surface Enhanced Raman Spectroscopy activity of hepcidin hormone over Au and Ag nanostructurated substrates: contribution for hyperinflammation screening

The hormone hepcidin present in saliva is a hyperinflammation markers for COVID-19 and other pathological states. Here we present DFT-based vibrational calculations that enabled assign the experimental vibrational spectra of hepcidin and predict its SERS-activiy. © Optica Publishing Group 2022 The Authors.

3D interior hotspots embedded with viral lysates for rapid and label-free identification of infectious diseases

In recent decades, biomedical sensors based on surface-enhanced Raman spectroscopy (SERS), which reveals unique spectral features corresponding to individual molecular vibrational states, have attracted intensive attention. However, the lack of a system for precisely guiding biomolecules to active hotspot regions has impeded the broad application of SERS techniques. Herein, we demonstrate the irreversible active engineering of three-dimensional (3D) interior organo-hotspots via electrochemical (EC) deposition onto metal nanodimple (ECOMD) platforms with viral lysates. This approach enables organic seed-programmable Au growth and the spontaneous bottom-up formation of 3D interior organo-hotspots simultaneously. Because of the net charge effect on the participation rate of viral lysates, the number of interior organo-hotspots in the ECOMDs increases with increasingly positive polarity. The viral lysates embedded in the ECOMDs function as both a dielectric medium for field confinement and an analyte, enabling the highly specific and sensitive detection of SARS-CoV-2 lysates (SLs) at concentrations as low as 10-2 plaque forming unit/mL. The ECOMD platform was used to trace and detect the SLs in human saliva and diagnose of the delta-type SARS-CoV-2 in clinical environments;the results indicate that the proposed platform can provide point-of-care diagnoses of infectious diseases.

Surface Enhanced Raman Spectroscopy activity of hepcidin hormone over Au and Ag nanostructurated substrates: contribution for hyperinflammation screening

The hormone hepcidin present in saliva is a hyperinflammation markers for COVID-19 and other pathological states. Here we present DFT-based vibrational calculations that enabled assign the experimental vibrational spectra of hepcidin and predict its SERS-activiy. © Optica Publishing Group 2022 The Authors.

Highly Adsorptive Au-TiO2 Nanocomposites for the SERS Face Mask Allow the Machine-Learning-Based Quantitative Assay of SARS-CoV-2 in Artificial Breath Aerosols.

Human respiratory aerosols contain diverse potential biomarkers for early disease diagnosis. Here, we report the direct and label-free detection of SARS-CoV-2 in respiratory aerosols using a highly adsorptive Au-TiO2 nanocomposite SERS face mask and an ablation-assisted autoencoder. The Au-TiO2 SERS face mask continuously preconcentrates and efficiently captures the oronasal aerosols, which substantially enhances the SERS signal intensities by 47% compared to simple Au nanoislands. The ultrasensitive Au-TiO2 nanocomposites also demonstrate the successful detection of SARS-CoV-2 spike proteins in artificial respiratory aerosols at a 100 pM concentration level. The deep learning-based autoencoder, followed by the partial ablation of nondiscriminant SERS features of spike proteins, allows a quantitative assay of the 101-104 pfu/mL SARS-CoV-2 lysates (comparable to 19-29 PCR cyclic threshold from COVID-19 patients) in aerosols with an accuracy of over 98%. The Au-TiO2 SERS face mask provides a platform for breath biopsy for the detection of various biomarkers in respiratory aerosols.

In-situ fabrication of 3D interior hotspots templated with a protein@Au core–shell structure for label-free and on-site SERS detection of viral diseases

Nanoscale plasmonic hotspots play a critical role in the enhancement of molecular Raman signals, enabling the sensitive and reliable trace analysis of biomedical molecules via surface-enhanced Raman spectroscopy (SERS). However, effective and label-free SERS diagnoses in practical fields remain challenging because of clinical samples' random adsorption and size mismatch with the nanoscale hotspots. Herein, we suggest a novel SERS strategy for interior hotspots templated with protein@Au core–shell nanostructures prepared via electrochemical one-pot Au deposition. The cytochrome c and lysates of SARS-CoV-2 (SLs) embedded in the interior hotspots were successfully functionalized to confine the electric fields and generate their optical fingerprint signals, respectively. Highly linear quantitative sensitivity was observed with the limit-of-detection value of 10−1 PFU/mL. The feasibility of detecting the targets in a bodily fluidic environment was also confirmed using the proposed templates with SLs in human saliva and nasopharyngeal swabs. These interior hotspots templated with the target analytes are highly desirable for early and on-site SERS diagnoses of infectious diseases without any labeling processes.

3D interior hotspots embedded with viral lysates for rapid and label-free identification of infectious diseases

In recent decades, biomedical sensors based on surface-enhanced Raman spectroscopy (SERS), which reveals unique spectral features corresponding to individual molecular vibrational states, have attracted intensive attention. However, the lack of a system for precisely guiding biomolecules to active hotspot regions has impeded the broad application of SERS techniques. Herein, we demonstrate the irreversible active engineering of three-dimensional (3D) interior organo-hotspots via electrochemical (EC) deposition onto metal nanodimple (ECOMD) platforms with viral lysates. This approach enables organic seed-programmable Au growth and the spontaneous bottom-up formation of 3D interior organo-hotspots simultaneously. Because of the net charge effect on the participation rate of viral lysates, the number of interior organo-hotspots in the ECOMDs increases with increasingly positive polarity. The viral lysates embedded in the ECOMDs function as both a dielectric medium for field confinement and an analyte, enabling the highly specific and sensitive detection of SARS-CoV-2 lysates (SLs) at concentrations as low as 10–2 plaque forming unit/mL. The ECOMD platform was used to trace and detect the SLs in human saliva and diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2);the results indicate that the proposed platform can provide point-of-care diagnoses of infectious diseases.

Using the SERS method and machine learning technology to detect the influenza A virus

The worldwide pandemic caused by Covid-19 demonstrates the need to develop new methods for the respiratory viral diseases' diagnosis, which should be fast and accurate. Surface-enhanced Raman spectroscopy (SERS) can be one of such methods. Our work demonstrated the possibility of using SERS technology and machine learning for fast (about 30 seconds) and accurate detection of the influenza A virus in a biological sample. © 2022 IEEE.

A non-invasive ultrasensitive diagnostic approach for COVID-19 infection using salivary label-free SERS fingerprinting and artificial intelligence.

Clinical diagnostics for SARS-CoV-2 infection usually comprises the sampling of throat or nasopharyngeal swabs that are invasive and create patient discomfort. Hence, saliva is attempted as a sample of choice for the management of COVID-19 outbreaks that cripples the global healthcare system. Although limited by the risk of eliciting false-negative and positive results, tedious test procedures, requirement of specialized laboratories, and expensive reagents, nucleic acid-based tests remain the gold standard for COVID-19 diagnostics. However, genetic diversity of the virus due to rapid mutations limits the efficiency of nucleic acid-based tests. Herein, we have demonstrated the simplest screening modality based on label-free surface enhanced Raman scattering (LF-SERS) for scrutinizing the SARS-CoV-2-mediated molecular-level changes of the saliva samples among healthy, COVID-19 infected and COVID-19 recovered subjects. Moreover, our LF-SERS technique enabled to differentiate the three classes of corona virus spike protein derived from SARS-CoV-2, SARS-CoV and MERS-CoV. Raman spectral data was further decoded, segregated and effectively managed with the aid of machine learning algorithms. The classification models built upon biochemical signature-based discrimination method of the COVID-19 condition from the patient saliva ensured high accuracy, specificity, and sensitivity. The trained support vector machine (SVM) classifier achieved a prediction accuracy of 95% and F1-score of 94.73%, and 95.28% for healthy and COVID-19 infected patients respectively. The current approach not only differentiate SARS-CoV-2 infection with healthy controls but also predicted a distinct fingerprint for different stages of patient recovery. Employing portable hand-held Raman spectrophotometer as the instrument and saliva as the sample of choice will guarantee a rapid and non-invasive diagnostic strategy to warrant or assure patient comfort and large-scale population screening for SARS-CoV-2 infection and monitoring the recovery process.