Surface-enhanced Raman spectroscopy (SERS) is a transformative tool for infectious disease diagnostics, offering rapid and sensitive species identification. However, background spectra in biological samples complicate analyte peak detection, increase the limit of detection, and hinder data augmentation. To address these challenges, we developed a deep learning framework utilizing dual neural networks to extract true virus SERS spectra and estimate concentration coefficients in water for 12 different respiratory viruses. The extracted spectra showed a high similarity to those obtained at the highest viral concentration, validating their accuracy. Using these spectra and the derived concentration coefficients, we augmented spectral data sets across varying virus concentrations in water. XGBoost models trained on these augmented data sets achieved overall classification and concentration prediction accuracy of 92.3% with a coefficient of determination (R2) > 0.95. Additionally, the extracted spectra and coefficients were used to augment data sets in saliva backgrounds. When tested against real virus-in-saliva spectra, the augmented spectra-trained XGBoost models achieved 91.9% accuracy in classification and concentration prediction with R2 > 0.9, demonstrating the robustness of the approach. By delivering clean and uncontaminated spectra, this methodology can significantly improve species identification, differentiation, and quantification and advance SERS-based detection and diagnostics.

Raman spectroscopy in biological applications faces challenges due to complex spectra, characterized by peaks of varying widths and significant biological background noise. Convolutional neural networks (CNNs) are widely used for spectrum classification due to their ability to capture local peak features. In this study, we introduce a multiscale CNN designed to detect weak biomolecule signals and differentiate spectra with features that cannot be statistically distinguished. The approach is further enhanced by a new visualization technique tailored for multiscale spectral analysis, providing clear insights into classification results. Using the classification of cholera toxin B subunit (CTB)-treated versus untreated cell membrane samples, whose spectra cannot be statistically differentiated, the optimized multiscale CNN achieved superior performance compared to traditional machine learning methods and existing multiscale CNNs, with accuracy (99.22%), sensitivity (99.27%), specificity (99.16%), and precision (99.20%). Our new visualization method, based on gradients of activation maps with respect to class scores, generates saliency scores that capture sample variations, with decision-making relying on consistently identified peak features. By visualizing the effects of different kernel sizes, Grad-AM highlights features at varying scales, aligning closely with spectral features and enhancing CNN interpretability in complex biomolecular analysis. These advancements demonstrate the potential of our method to improve spectral analysis and reveal previously hidden peaks in complex biological environments.

The integration of Artificial Intelligence (AI) techniques with medical kits has revolutionized disease diagnosis, enabling rapid and accurate identification of various conditions. We developed a novel deep learning model, namely DeepATsers based on a combination of CNN and GAN to employ a one-pot SERS biosensor to rapidly detect COVID-19 infection. The model accurately identifies each SARS-CoV-2 protein (S protein, N protein, VLP protein, Streptavidin protein, and blank signal) from its experimental fingerprint-like spectral data introduced in this study. Several augmentation techniques such as EMSA, Gaussian-noise, GAN, and K-fold cross-validation, and their combinations were utilized for the SERS spectral dataset generalization and prevented model overfitting. The original experimental dataset of 126 spectra was augmented to 780 spectra that resembled the original set by using GAN with a low KL divergence value of 0.02. This significantly improves the average accuracy of protein classification from 0.6000 to 0.9750. The deep learning model deployed optimal hyperparameters and outperformed in most measurements comparing supervised machine learning methods such as RF, GBM, SVM, and KNN, both with and without augmented spectral datasets. For model training, a whole range of spectra wavenumbers ([Formula: see text] to [Formula: see text]) as well as wavenumbers ([Formula: see text] and [Formula: see text]) only for fingerprint peak spectra were employed. The former led to highly accurate 0.9750 predictions in comparison to 0.4318 for the latter one. Finally, independent experimental spectra of SARS-CoV-2 Omicron variant were used in the model verification. Thus, DeepATsers can be considered a robust, generalized, and generative deep learning framework for 1D SERS spectral datasets of SARS-CoV-2.

Rapid detection of infectious diseases like COVID-19, flu, and dengue is crucial for healthcare professionals preparing for contagious outbreaks. Given the constant mutations in viruses and the recurring emergence of threats like Nipah and Zika, there is an urgent demand for a technology capable of distinguishing between infections that share similar symptoms. In this paper, we utilize laser-based Raman scattered signals from a drop of dried blood plasma, combined with generative artificial intelligence, to provide a rapid and precise diagnosis. Our optimized model exhibits exceptional performance, yielding high predictive scores of 96%, 98%, and 100% for flu, COVID-19, and dengue, respectively. The proposed Raman spectroscopic analysis, with a rapid turnaround time, can ensure a near-accurate diagnosis and proper quarantining of highly infectious cases. Furthermore, the potential extension of our method to include other viral diseases offers an alternative to the challenge of developing different diagnostic kits for each disease.

Veterinary drug residues in poultry and livestock products present persistent challenges to food safety, necessitating precise and efficient detection methods. Surface-enhanced Raman scattering (SERS) has been identified as a powerful tool for veterinary drug residue analysis due to its high sensitivity and specificity. However, the development of reliable SERS substrates and the interpretation of complex spectral data remain significant obstacles. This review summarizes the development process of SERS substrates, categorizing them into metal-based, rigid, and flexible substrates, and highlighting the emerging trend of multifunctional substrates. The diverse application scenarios and detection requirements for these substrates are also discussed, with a focus on their use in veterinary drug detection. Furthermore, the integration of deep learning techniques into SERS-based detection is explored, including substrate structure design optimization, optical property prediction, spectral preprocessing, and both qualitative and quantitative spectral analyses. Finally, key limitations are briefly outlined, such as challenges in selecting reporter molecules, data imbalance, and computational demands. Future trends and directions for improving SERS-based veterinary drug detection are proposed.

Campylobacter jejuni is a leading foodborne pathogen that may enter a viable but nonculturable (VBNC) state to survive under environmental stresses, posing a significant health concern. VBNC cells can evade conventional culture-based detection methods, while viability-based assays are usually hindered by low sensitivity, insufficient specificity, or technical challenges. There are limited studies analyzing VBNC cells at the single-cell level for accurate detection and an understanding of their unique behavior. Here, we present a culture-independent approach to identify and characterize VBNC C. jejuni using single-cell Raman spectra collected by optical tweezers and machine learning. C. jejuni strains were induced into the VBNC state under osmotic pressure (7% w/v NaCl solution) and aerobic stress (atmospheric condition). Using single-cell Raman spectra and a convolutional neural network (CNN), VBNC C. jejuni cells were distinguished from their culturable counterparts with an accuracy of ∼92%. There were no significant spectral differences between the VBNC cells formed under different stressors or induction periods. Furthermore, we utilized gradient-weighted class activation mapping to highlight the spectral regions that contribute most to the CNN-based classification between culturable and VBNC cells. These regions align with previously identified changes in proteins, nucleic acids, lipids, and peptidoglycan in VBNC cells, providing insights into the molecular characterization of the VBNC state of C. jejuni.

A single Raman spectrum reflects limited molecular information. Effective fusion of the Raman spectra of serum and urine source domains helps to obtain richer feature information. However, most of the current studies on immunoglobulin A nephropathy (IgAN) based on Raman spectroscopy are based on small sample data and low signal-to-noise ratio. If a multi-source data fusion strategy is directly adopted, it may even reduce the accuracy of disease diagnosis. To this end, this paper proposes a data enhancement and spectral optimization method based on variational autoencoders to obtain reconstructed Raman spectra with doubled sample size and improved signal-to-noise ratio. In the diagnosis of IgAN in multi-source domain Raman spectra, this paper builds a global and local feature decoupled variational autoencoder (DMSGL-VAE) model based on multi-source data. First, the statistical features after spectral segmentation are extracted, and the latent variables obtained by the variational encoder are decoupled through the decoupling module. The global representation and local representation obtained represent the global shared information and local unique information of the serum and urine source domains, respectively. Then, the cross-source reconstruction loss and decoupling loss are used to constrain the decoupling, and the effectiveness of the decoupling is proved quantitatively and qualitatively. Finally, the features of different source domains were integrated to diagnose IgAN, and the results were analyzed for important features using the SHapley Additive exPlanations algorithm. The experimental results showed that the AUC value of the DMSGL-VAE model for diagnosing IgAN on the test set was as high as 0.9958. The SHAP algorithm was used to further prove that proteins, hydroxybutyrate, and guanine are likely to be common biological fingerprint substances for the diagnosis of IgAN by serum and urine Raman spectroscopy. In summary, the DMSGL-VAE model designed based on Raman spectroscopy in this paper can achieve rapid, non-invasive, and accurate screening of IgAN in terms of classification performance. And interpretable analysis may help doctors further understand IgAN and make more efficient diagnostic measures in the future.

Vibrational spectroscopic techniques, such as Raman spectroscopy, as a non-destructive method combined with machine learning (ML), were successfully tested as a quick method of plasticizer identification in poly(vinyl chloride) - PVC objects in heritage collection. ML algorithms such as Convolutional Neural Network (CNN), Random Forest (RF), Support Vector Machines (SVM), and Linear Discriminant Analysis (LDA) were applied to the classification and identification of the most common plasticizers used in the case of PVC. The CNN model was able to successfully classify the five plasticizers under study from their Raman spectra with a high accuracy of (98%), whereas the highest accuracy (100%) was observed with the RF algorithm. The finding opens doors for the development of robust and economical tools for conservators and museum professionals for fast identification of materials in heritage collections.

Using Raman spectroscopy (RS) signals for skin cancer tissue classification has recently drawn significant attention, because of its non-invasive optical technique, which uses molecular structures and conformations within biological tissue for diagnosis. In reality, RS signals are noisy and unstable for training machine learning models. The scarcity of tissue samples also makes it challenging to learn reliable deep-learning networks for clinical usages. In this paper, we advocate a Transfer Contrasting Learning Paradigm (TCLP) to address the scarcity and noisy characteristics of the RS for skin cancer tissue classification. To overcome the challenge of limited samples, TCLP leverages transfer learning to pre-train deep learning models using RS data from similar domains (but collected from different RS equipments for other tasks). To tackle the noisy nature of the RS signals, TCLP uses contrastive learning to augment RS signals to learn reliable feature representation to represent RS signals for final classification. Experiments and comparisons, including statistical tests, demonstrate that TCLP outperforms existing deep learning baselines for RS signal-based skin cancer tissue classification.

Surface-enhanced Raman spectroscopy (SERS) is a powerful tool that provides valuable insight into the molecular contents of chemical and biological samples. However, interpreting Raman spectra from complex or dynamic datasets remains challenging, particularly for highly heterogeneous biological samples like extracellular vesicles (EVs). To overcome this, we developed a tunable and interpretable deep autoencoder for the analysis of several challenging Raman spectroscopy applications, including synthetic datasets, chemical mixtures, a chemical milling reaction, and mixtures of EVs. We compared the results with classical methods (PCA and UMAP) to demonstrate the superior performance of the proposed technique. Our method can handle small datasets, provide a high degree of generalization such that it can fill unknown gaps within spectral datasets, and even quantify relative ratios of cell line-derived EVs to fetal bovine serum-derived EVs within mixtures. This simple yet robust approach will greatly improve the analysis capabilities for many other Raman spectroscopy applications.

Early detection and postoperative assessment are crucial for improving overall survival among lung cancer patients. Here, we report a non-invasive technique that integrates Raman spectroscopy with machine learning for the detection of lung cancer. The study encompassed 88 postoperative lung cancer patients, 73 non-surgical lung cancer patients, and 68 healthy subjects. The primary aim was to explore variations in serum metabolism across these cohorts. Comparative analysis of average Raman spectra was conducted, while principal component analysis was employed for data visualization. Subsequently, the augmented dataset was used to train convolutional neural networks (CNN) and Resnet models, leading to the development of a diagnostic framework. The CNN model exhibited superior performance, as verified by the receiver operating characteristic curve. Notably, postoperative patients demonstrated an increased likelihood of recurrence, emphasizing the crucial need for continuous postoperative monitoring. In summary, the integration of Raman spectroscopy with CNN-based classification shows potential for early detection and postoperative assessment of lung cancer.

Raman Spectroscopy promises the ability to encode in spectral data the significant differences between biological samples belonging to patients affected by a disease and samples of healthy patients (controls). However, the decoding and interpretation of the Raman spectral fingerprint is still a difficult and time-consuming procedure even for domain experts. In this work, we test an end-to-end deep-learning diagnostic pipeline able to classify spectral data from saliva samples. The pipeline has been validated against the SARS-COV-2 Infection and for the screening of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. The proposed system can be used for the fast prototyping of promising non-invasive, cost and time-efficient diagnostic screening tests.

Paeonia lactiflora Pall. (PLP) is a plant with excellent ornamental and therapeutic value that can be utilized in traditional Chinese medicine as Paeoniae Radix Alba (PRA) and Paeoniae Radix Rubra (PRR). PRA must undergo the "peeling" process, which involves removing the cork and a portion of the phloem. PLP's biological function is strongly linked to its secondary metabolites, and the distribution of metabolites in different regions of the PLP rhizome causes changes in efficacy when PLP is processed into various therapeutic compounds.

The metabolites of the cork (cor), phloem (phl), and xylem (xyl) were examined in the roots of PLP using a metabolomics approach based on UPLC-Q-Exactive-Orbitrap-MS/MS (UPLC-MS/MS), and the differential metabolites were evaluated using multivariate analysis.

Significant changes were observed among the cor, phl, and xyl samples. In both positive and negative ion modes, a total of 15,429 peaks were detected and 7366 metabolites were identified. A total of 525 cor-phl differential metabolites, 452 cor-xyl differential metabolites, and 328 phl-xyl differential metabolites were evaluated. Flavonoids, monoterpene glycosides, fatty acids, sugar derivatives, and carbohydrates were among the top 50 dissimilar chemicals. The key divergent metabolic pathways include linoleic acid metabolism, galactose metabolism, ABC transporters, arginine biosynthesis, and flavonoid biosynthesis.

The cor, phl, and xyl of PLP roots exhibit significantly different metabolite types and metabolic pathways; therefore, "peeling" may impact the pharmaceutical effect of PLP. This study represents the first metabolomics analysis of the PLP rhizome, laying the groundwork for the isolation and identification of PLP pharmacological activity, as well as the quality evaluation and efficacy exploration of PLP.

We propose an approach utilizing gamma-distributed random variables, coupled with log-Gaussian modeling, to generate synthetic datasets suitable for training neural networks. This addresses the challenge of limited real observations in various applications. We apply this methodology to both Raman and coherent anti-Stokes Raman scattering (CARS) spectra, using experimental spectra to estimate gamma process parameters. Parameter estimation is performed using Markov chain Monte Carlo methods, yielding a full Bayesian posterior distribution for the model which can be sampled for synthetic data generation. Additionally, we model the additive and multiplicative background functions for Raman and CARS with Gaussian processes. We train two Bayesian neural networks to estimate parameters of the gamma process which can then be used to estimate the underlying Raman spectrum and simultaneously provide uncertainty through the estimation of parameters of a probability distribution. We apply the trained Bayesian neural networks to experimental Raman spectra of phthalocyanine blue, aniline black, naphthol red, and red 264 pigments and also to experimental CARS spectra of adenosine phosphate, fructose, glucose, and sucrose. The results agree with deterministic point estimates for the underlying Raman and CARS spectral signatures.

Surface-enhanced Raman spectroscopy (SERS) has wide diagnostic applications due to narrow spectral features that allow multiplex analysis. We have previously developed a multiplexed, SERS-based nanosensor for micro-RNA (miRNA) detection called the inverse molecular sentinel (iMS). Machine learning (ML) algorithms have been increasingly adopted for spectral analysis due to their ability to discover underlying patterns and relationships within large and complex data sets. However, the high dimensionality of SERS data poses a challenge for traditional ML techniques, which can be prone to overfitting and poor generalization. Non-negative matrix factorization (NMF) reduces the dimensionality of SERS data while preserving information content. In this paper, we compared the performance of ML methods including convolutional neural network (CNN), support vector regression, and extreme gradient boosting combined with and without NMF for spectral unmixing of four-way multiplexed SERS spectra from iMS assays used for miRNA detection. CNN achieved high accuracy in spectral unmixing. Incorporating NMF before CNN drastically decreased memory and training demands without sacrificing model performance on SERS spectral unmixing. Additionally, models were interpreted using gradient class activation maps and partial dependency plots to understand predictions. These models were used to analyze clinical SERS data from single-plexed iMS in RNA extracted from 17 endoscopic tissue biopsies. CNN and CNN-NMF, trained on multiplexed data, performed most accurately with RMSElabel = 0.101 and 9.68 × 10-2, respectively. We demonstrated that CNN-based ML shows great promise in spectral unmixing of multiplexed SERS spectra, and the effect of dimensionality reduction on performance and training speed.

Surface-enhanced Raman spectroscopy (SERS), well acknowledged as a fingerprinting and sensitive analytical technique, has exerted high applicational value in a broad range of fields including biomedicine, environmental protection, food safety among the others. In the endless pursuit of ever-sensitive, robust, and comprehensive sensing and imaging, advancements keep emerging in the whole pipeline of SERS, from the design of SERS substrates and reporter molecules, synthetic route planning, instrument refinement, to data preprocessing and analysis methods. Artificial intelligence (AI), which is created to imitate and eventually exceed human behaviors, has exhibited its power in learning high-level representations and recognizing complicated patterns with exceptional automaticity. Therefore, facing up with the intertwining influential factors and explosive data size, AI has been increasingly leveraged in all the above-mentioned aspects in SERS, presenting elite efficiency in accelerating systematic optimization and deepening understanding about the fundamental physics and spectral data, which far transcends human labors and conventional computations. In this review, the recent progresses in SERS are summarized through the integration of AI, and new insights of the challenges and perspectives are provided in aim to better gear SERS toward the fast track.

The lack of specific and sensitive early diagnostic options for pancreatic cancer (PC) results in patients being largely diagnosed with late-stage disease, thus inoperable and burdened with high mortality. Molecular spectroscopic methodologies, such as Raman or infrared spectroscopies, show promise in becoming a leader in screening for early-stage cancer diseases, including PC. However, should such technology be introduced, the identification of differentiating spectral features between various cancer types is required. This would not be possible without the precise extraction of spectra without the contamination by necrosis, inflammation, desmoplasia, or extracellular fluids such as mucous that surround tumor cells. Moreover, an efficient methodology for their interpretation has not been well defined. In this study, we compared different methods of spectral analysis to find the best for investigating the biomolecular composition of PC cells cytoplasm and nuclei separately. Sixteen PC tissue samples of main PC subtypes (ductal adenocarcinoma, intraductal papillary mucinous carcinoma, and ampulla of Vater carcinoma) were collected with Raman hyperspectral mapping, resulting in 191,355 Raman spectra and analyzed with comparative methodologies, specifically, hierarchical cluster analysis, non-negative matrix factorization, T-distributed stochastic neighbor embedding, principal components analysis (PCA), and convolutional neural networks (CNN). As a result, we propose an innovative approach to spectra classification by CNN, combined with PCA for molecular characterization. The CNN-based spectra classification achieved over 98% successful validation rate. Subsequent analyses of spectral features revealed differences among PC subtypes and between the cytoplasm and nuclei of their cells. Our study establishes an optimal methodology for cancer tissue spectral data classification and interpretation that allows precise and cognitive studies of cancer cells and their subcellular components, without mixing the results with cancer-surrounding tissue. As a proof of concept, we describe findings that add to the spectroscopic understanding of PC.

Raman spectroscopy has been extensively utilized as a marker-free detection method in the complementary diagnosis of cancer. Multivariate statistical classification analysis is frequently employed for Raman spectral data classification. Nevertheless, traditional multivariate statistical classification analysis performs poorly when analyzing large samples and multicategory spectral data. In addition, with the advancement of computer vision, convolutional neural networks (CNNs) have demonstrated extraordinarily precise analysis of two-dimensional image processing.

Combining 2D Raman spectrograms with automatic weighted feature fusion network (AWFFN) for bladder cancer detection is presented in this paper. Initially, the s-transform (ST) is implemented for the first time to convert 1D Raman data into 2D spectrograms, achieving 99.2% detection accuracy. Second, four upscaling techniques, including short time fourier transform (STFT), recurrence map (RP), markov transform field (MTF), and grammy angle field (GAF), were used to transform the 1D Raman spectral data into a variety of 2D Raman spectrograms. In addition, a particle swarm optimization (PSO) algorithm is combined with VGG19, ResNet50, and ResNet101 to construct a weighted feature fusion network, and this parallel network is employed for evaluating multiple spectrograms. Class activation mapping (CAM) is additionally employed to illustrate and evaluate the process of feature extraction via the three parallel network branches. The results demonstrate that the combination of a 2D Raman spectrogram along with a CNN for the diagnosis of bladder cancer obtains a 99.2% accuracy rate，which indicates that it is an extremely promising auxiliary technology for cancer diagnosis.

The proposed two-dimensional Raman spectroscopy method has an improved precision than one-dimensional spectroscopic data, which presents a potential methodology for assisted cancer detection and providing crucial technical support for assisted diagnosis.

Raman spectroscopy is widely used for its exceptional identification capabilities in various fields. Traditional methods for target identification using Raman spectroscopy rely on signal correlation with moving windows, requiring data preprocessing that can significantly impact identification performance. In recent years, deep-learning approaches have been proposed to leverage data augmentation techniques, such as baseline and additive noise addition, in order to overcome data scarcity. However, these deep-learning methods are limited to the spectra encountered during training and struggle to handle unseen spectra. To address these limitations, we propose a multi-input hybrid deep-learning model trained with simulated spectral data. By employing simulated spectra, our method tackles the challenges of data scarcity and the handling of unseen spectra encountered in traditional and deep-learning methods. Experimental results demonstrate that our proposed method achieves outstanding identification performance and effectively handles spectra obtained from different Raman spectroscopy systems.

Electron energy loss spectroscopy (EELS) is a well established technique in electron microscopy that yields information on the elemental content of a sample in a very direct manner. One of the persisting limitations of EELS is the requirement for manual identification of core-loss edges and their corresponding elements. This can be especially bothersome in spectrum imaging, where a large amount of spectra are recorded when spatially scanning over a sample area. This paper introduces a synthetic dataset with 736,000 labeled EELS spectra, computed from available generalized oscillator strength tables, that represents 107 K, L, M or N core-loss edges and 80 chemical elements. Generic lifetime broadened peaks are used to mimic the fine structure due to band structure effects present in experimental core-loss edges. The proposed dataset is used to train and evaluate a series of neural network architectures, being a multilayer perceptron, a convolutional neural network, a U-Net, a residual neural network, a vision transformer and a compact convolutional transformer. An ensemble of neural networks is used to further increase performance. The ensemble network is used to demonstrate fully automated elemental mapping in a spectrum image, both by directly mapping the predicted elemental content and by using the predicted content as input for a physical model-based mapping.

The development of wearable sensors for remote patient monitoring and personalized medicine has led to a revolution in biomedical technology. Plasmonic metasurfaces that enhance Raman scattering signals have recently gained attention as wearable sensors. However, finding a flexible, sensitive, and easy-to-fabricate metasurface has been a challenge for decades. In this paper, a novel wearable device, the flexible, stretchable, and single-molecule-sensetive SERS-active sensor, is proposed. This device offers an unprecedented SERS enhancement factor in the order of 1011, along with other long-desired characteristics for SERS applications such as a high scattering to absorption ratio (∼2.5) and a large hotspot volume (40 nm × 40 nm × 5 nm). To achieve flexibility, we use polydimethylsiloxane (PDMS) as the substrate, which is stable, transparent, and biologically compatible. Our numerical calculations show that the proposed sensor offers reliable SERS performance even under bending (up to 100° angles) or stretching (up to 50% stretch). The easy-to-fabricate and flexible nature of our sensor offers a promising avenue for developing highly sensitive wearable sensors for a range of applications, particularly in the field of personalized medicine and remote patient monitoring.

The direct preventative detection of flow-induced atherosclerosis remains a significant challenge, impeding the development of early treatments and prevention measures. This study proposes a method for diagnosing atherosclerosis in the carotid artery using nanometer biomarker measurements through surface-enhanced Raman spectroscopy (SERS) from single-drop blood samples. Atherosclerotic acceleration is induced in apolipoprotein E knockout mice which underwent a partial carotid ligation and were fed a high-fat diet to rapidly induce disturbed flow-induced atherosclerosis in the left common carotid artery while using the unligated, contralateral right carotid artery as control. The progressive atherosclerosis development of the left carotid artery was verified by micro-magnetic resonance imaging (micro-MRI) and histology in comparison to the right carotid artery. Single-drop blood samples are deposited on chips of gold-coated ZnO nanorods grown on silicon wafers that filter the nanometer markers and provide strong SERS signals. A diagnostic classifier was established based on principal component analysis (PCA), which separates the resultant spectra into the atherosclerotic and control groups. Scoring based on the principal components enabled the classification of samples into control, mild, and severe atherosclerotic disease. The PCA-based analysis was validated against an independent test sample and compared against the PCA-PLS-DA machine learning algorithm which is known for applicability to Raman diagnosis. The accuracy of the PCA modification-based diagnostic criteria was 94.5%, and that of the machine learning algorithm 97.5%. Using a mouse model, this study demonstrates that diagnosing and classifying the severity of atherosclerosis is possible using a single blood drop, SERS technology, and machine learning algorithm, indicating the detectability of biomarkers and vascular factors in the blood which correlate with the early stages of atherosclerosis development.

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.

Extracellular vesicles (EVs) have emerged as promising diagnostic and therapeutic candidates in many biomedical applications. However, EV research continues to rely heavily on in vitro cell cultures for EV production, where the exogenous EVs present in fetal bovine (FBS) or other required serum supplementation can be difficult to remove entirely. Despite this and other potential applications involving EV mixtures, there are currently no rapid, robust, inexpensive, and label-free methods for determining the relative concentrations of different EV subpopulations within a sample. In this study, we demonstrate that surface-enhanced Raman spectroscopy (SERS) can biochemically fingerprint fetal bovine serum-derived and bioreactor-produced EVs, and after applying a novel manifold learning technique to the acquired spectra, enables the quantitative detection of the relative amounts of different EV populations within an unknown sample. We first developed this method using known ratios of Rhodamine B to Rhodamine 6G, then using known ratios of FBS EVs to breast cancer EVs from a bioreactor culture. In addition to quantifying EV mixtures, the proposed deep learning architecture provides some knowledge discovery capabilities which we demonstrate by applying it to dynamic Raman spectra of a chemical milling process. This label-free characterization and analytical approach should translate well to other EV SERS applications, such as monitoring the integrity of semipermeable membranes within EV bioreactors, ensuring the quality or potency of diagnostic or therapeutic EVs, determining relative amounts of EVs produced in complex co-culture systems, as well as many Raman spectroscopy applications.

Surface-enhanced Raman spectroscopy/scattering (SERS) has evolved into a popular tool for applications in biology and medicine owing to its ease-of-use, non-destructive, and label-free approach. Advances in plasmonics and instrumentation have enabled the realization of SERS's full potential for the trace detection of biomolecules, disease diagnostics, and monitoring. We provide a brief review on the recent developments in the SERS technique for biosensing applications, with a particular focus on machine learning techniques used for the same. Initially, the article discusses the need for plasmonic sensors in biology and the advantage of SERS over existing techniques. In the later sections, the applications are organized as SERS-based biosensing for disease diagnosis focusing on cancer identification and respiratory diseases, including the recent SARS-CoV-2 detection. We then discuss progress in sensing microorganisms, such as bacteria, with a particular focus on plasmonic sensors for detecting biohazardous materials in view of homeland security. At the end of the article, we focus on machine learning techniques for the (a) identification, (b) classification, and (c) quantification in SERS for biology applications. The review covers the work from 2010 onwards, and the language is simplified to suit the needs of the interdisciplinary audience.

Knowledge about pancreatic cancer (PC) biology has been growing rapidly in recent decades. Nevertheless, the survival of PC patients has not greatly improved. The development of a novel methodology suitable for deep investigation of the nature of PC tumors is of great importance. Molecular imaging techniques, such as Fourier transform infrared (FTIR) spectroscopy and Raman hyperspectral mapping (RHM) combined with advanced multivariate data analysis, were useful in studying the biochemical composition of PC tissue.

Here, we evaluated the potential of molecular imaging in differentiating three groups of PC tumors, which originate from different precursor lesions. Specifically, we comprehensively investigated adenocarcinomas (ACs): conventional ductal AC, intraductal papillary mucinous carcinoma, and ampulla of Vater AC. FTIR microspectroscopy and RHM maps of 24 PC tissue slides were obtained, and comprehensive advanced statistical analyses, such as hierarchical clustering and nonnegative matrix factorization, were performed on a total of 211,355 Raman spectra. Additionally, we employed deep learning technology for the same task of PC subtyping to enable automation. The so-called convolutional neural network (CNN) was trained to recognize spectra specific to each PC group and then employed to generate CNN-prediction-based tissue maps. To identify the DNA methylation spectral markers, we used differently methylated, isolated DNA and compared the observed spectral differences with the results obtained from cellular nuclei regions of PC tissues.

The results showed significant differences among cancer tissues of the studied PC groups. The main findings are the varying content of β-sheet-rich proteins within the PC cells and alterations in the relative DNA methylation level. Our CNN model efficiently differentiated PC groups with 94% accuracy. The usage of CNN in the classification task did not require Raman spectral data preprocessing and eliminated the need for extensive knowledge of statistical methodologies.

Molecular spectroscopy combined with CNN technology is a powerful tool for PC detection and subtyping. The molecular fingerprint of DNA methylation and β-sheet cytoplasmic proteins established by our results is different for the main PC groups and allowed the subtyping of pancreatic tumors, which can improve patient management and increase their survival. Our observations are of key importance in understanding the variability of PC and allow translation of the methodology into clinical practice by utilizing liquid biopsy testing.

Brain disorders such as brain tumors and neurodegenerative diseases (NDs) are accompanied by chemical alterations in the tissues. Early diagnosis of these diseases will provide key benefits for patients and opportunities for preventive treatments. To detect these sophisticated diseases, various imaging modalities have been developed such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET). However, they provide inadequate molecule-specific information. In comparison, Raman spectroscopy (RS) is an analytical tool that provides rich information about molecular fingerprints. It is also inexpensive and rapid compared to CT, MRI, and PET. While intrinsic RS suffers from low yield, in recent years, through the adoption of Raman enhancement technologies and advanced data analysis approaches, RS has undergone significant advancements in its ability to probe biological tissues, including the brain. This review discusses recent clinical and biomedical applications of RS and related techniques applicable to brain tumors and NDs.

Machine learning has had a significant impact on the value of spectroscopic characterization tools, particularly in biomedical applications, due to its ability to detect latent patterns within complex spectral data. However, it often requires extensive data preprocessing, including baseline correction and denoising, which can lead to an unintentional bias during classification. To address this, we developed two deep learning methods capable of fully preprocessing raw Raman spectroscopy data without any human input. First, cascaded deep convolutional neural networks (CNN) based on either ResNet or U-Net architectures were trained on randomly generated spectra with augmented defects. Then, they were tested using simulated Raman spectra, surface-enhanced Raman spectroscopy (SERS) imaging of chemical species, low resolution Raman spectra of human bladder cancer tissue, and finally, classification of SERS spectra from human placental extracellular vesicles (EVs). Both approaches resulted in faster training and complete spectral preprocessing in a single step, with more speed, defect tolerance, and classification accuracy compared to conventional methods. These findings indicate that cascaded CNN preprocessing is ideal for biomedical Raman spectroscopy applications in which large numbers of heterogeneous spectra with diverse defects need to be automatically, rapidly, and reproducibly preprocessed.

Placental extracellular vesicles (EVs) play an essential role in pregnancy by protecting and transporting diverse biomolecules that aid in fetomaternal communication. However, in preeclampsia, they have also been implicated in contributing to disease progression. Despite their potential clinical value, current technologies cannot provide a rapid and effective means of differentiating between healthy and diseased placental EVs. To address this, a fabrication process called laser-induced nanostructuring of SERS-active thin films (LINST) was developed to produce scalable nanoplasmonic substrates that provide exceptional Raman signal enhancement and allow the biochemical fingerprinting of EVs. After validating the performance of LINST substrates with chemical standards, placental EVs from tissue explant cultures were characterized, demonstrating that preeclamptic and normotensive placental EVs have classifiably distinct Raman spectra following the application of advanced machine learning algorithms. Given the abundance of placental EVs in maternal circulation, these findings encourage immediate exploration of surface-enhanced Raman spectroscopy (SERS) of EVs as a promising method for preeclampsia liquid biopsies, while this novel fabrication process will provide a versatile and scalable substrate for many other SERS applications.

Raman spectroscopy combined with augmented reality and mixed reality to reconstruct molecular information of tissue surface.