Raman spectroscopy assisted tear analysis: A label free, optical approach for noninvasive disease diagnostics.

In recent times, tear fluid analysis has garnered considerable attention in the field of biomarker-based diagnostics due to its noninvasive sample collection method. Tears encompass a reservoir of biomarkers that assist in diagnosing not only ocular disorders but also a diverse list of systemic diseases. This highlights the necessity for sensitive and dependable screening methods to employ tear fluid as a potential noninvasive diagnostic specimen in clinical environments. Considerable research has been conducted to investigate the potential of Raman spectroscopy-based investigations for tear analysis in various diagnostic applications. Raman Spectroscopy (RS) is a highly sensitive and label free spectroscopic technique which aids in investigating the molecular structure of samples by evaluating the vibrational frequencies of molecular bonds. Due to the distinct chemical compositions of different samples, it is possible to obtain a sample-specific spectral fingerprint. The distinctive spectral fingerprints obtained from Raman spectroscopy enable researchers to identify specific compounds or functional groups present in a sample, aiding in diverse biomedical applications. Its sensitivity to changes in molecular structure or environment provides invaluable insights into subtle alterations associated with various diseases. Thus, Raman Spectroscopy has the potential to assist in diagnosis and treatment as well as prognostic evaluation. Raman spectroscopy possesses several advantages, such as the non-destructive examination of samples, remarkable sensitivity to structural variations, minimal prerequisites for sample preparation, negligible interference from water, and the aptness for real-time investigation of tear samples. The purpose of this review is to highlight the potential of Raman spectroscopic technique in facilitating the clinical diagnosis of various ophthalmic and systemic disorders through non-invasive tear analysis. Additionally, the review delves into the advancements made in Raman spectroscopy with regards to paper-based sensing substrates and tear analysis methods integrated into contact lenses. Furthermore, the review also addresses the obstacles and future possibilities associated with implementing Raman spectroscopy as a routine diagnostic tool based on tear analysis in clinical settings.

Visualization and characterisation of microplastics in aquatic environment using a home-built micro-Raman spectroscopic set up.

Microplastics (MP) which are tiny plastic particles of sizes range from 1 µm (µm) to 5 mm (mm), have become a growing cause of concern due to their widespread presence in the environment and their potential impacts on ecosystems and human health. Marine organisms have the potential to consume microplastics, which could lead to physical injuries, blockages, or the transfer of harmful substances up the food chain. Humans may indirectly consume microplastics through contaminated seafood and water, although the complete scope of health risks is currently under investigation. An essential step in gaining a comprehensive understanding of microplastic pollution in waterbodies is the identification of microplastics, which is also crucial for further development of effective environmental regulations to address its adverse impacts. Majority of the researchers are accomplishing it globally using commercial platforms based on Raman spectroscopy. However, the development of indigenous Raman systems, which can enable microplastic identification, particularly in developing nations, is the need of the hour due to the outrageous cost of commercial platforms. In the current study, a custom-designed micro-Raman spectroscopy system was developed to detect and characterize microplastics from waterbodies. The developed system enabled visualization, size measurement and characterization of microplastics. Experimental parameters were fine-tuned, and a standardized Raman database was established for each type of plastic. This system exhibited high resolution which was capable of analysing microparticles of size up to 5 µm. Principal component analysis was carried out on the experimental Raman data, demonstrating good classification amongst different kinds of plastics. The performance of the developed system in analysing real samples was evaluated through experiments conducted on water samples obtained from the shore of Malpe Beach in Udupi district. The results revealed the presence of polyethylene and polyethylene terephthalate in the samples, along with the detection of pigments like copper phthalocyanine and indigo blue.

Laser induced fluorescence spectroscopy analysis of kidney tissues: A pilot study for the identification of renal cell carcinoma.

The 325 nm-excited autofluorescence spectra from cancerous and normal renal tissues were collected ex vivo biopsy tissue samples, through an optical fiber probe-based system. Noticeable changes in intensity/wavelength were observed in the fluorescence emissions from endogenous fluorophores such as collagen, Nicotinamide adenine dinucleotide (NADH), Vitamin A (retinol), and flavin adenine dinucleotide, in pathological conditions with respect to the normal state. The energy metabolism involved in clear cell renal cell carcinoma (ccRCC) and chromophobe renal cell carcinoma (chRCC) are reflected in the fluorescence emission band at 445 nm due to bound NADH attributed to enhanced oxidative phosphorylation in chRCC and emission at 465 nm contributed by free NADH showing higher glycolytic action in ccRCC. The principal component analysis and one-way ANOVA effectively discriminate ccRCC from chRCC. It is shown that laser induced fluorescence technique with 325 nm excitation can be a suitable technique for optical pathology and in vivo surgical boundary demarcation in renal cell carcinoma.

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.

Serum protein profile study of myocardial infarction using a LED induced fluorescence based HPLC system.

Cardiovascular diseases (CVDs) are the major health conditions for high mortality and morbidity in humans. Delay in the diagnosis of CVDs effect patients long and short-term health condition. In -house assembled UV-light emitting diode (LED) based fluorescence detector for high -performance liquid chromatography (HPLC) (HPLC-LED-IF) system is used to record serum chromatograms of three categories of samples namely, before medicated- myocardial infarction (B-MI), after medicated- MI (A-MI), and normal. The sensitivity and performance of HPLC-LED-IF system is estimated using commercial serum proteins. Statistical analysis tools like, descriptive statistics, principal component analysis (PCA), and Match/ No Match test were applied to visualize the variation in three groups of samples. Statistical analysis of the protein profile data showed fairly good discrimination among the three categories. The receiver operating characteristic (ROC) curve also supported the reliability of the method to diagnose MI.

Micro-Raman spectroscopy study of optically trapped erythrocytes in malaria, dengue and leptospirosis infections.

Malaria, dengue and leptospirosis are three tropical infectious diseases that present with severe hematological derangement causing significant morbidity and mortality, especially during the seasonal monsoons. During the course of these infectious diseases, circulating red blood cells are imperiled to the direct ill-effects of the infectious pathogen in the body as well as to the pro-inflammatory cytokines generated as a consequence of the infection. RBCs when exposed to such inflammatory and/or pathogenic milieu are susceptible to injuries such as RBC programmed eryptosis or RBC programmed necrosis. This research aimed to explore the Raman spectra of live red cells that were extracted from patients infected with malaria, dengue, and leptospirosis. Red cells were optically trapped and micro-Raman probed using a 785 nm Diode laser. RBCs from samples of all three diseases displayed Raman signatures that were significantly altered from the normal/healthy. Distinct spectral markers that were common across all the four groups were obtained from various standardized multivariate analytical methods. Following comprehensive examination of multiple studies, we propose these spectral wavenumbers as "Raman markers of RBC injury." Findings in our study display that anemia-triggering infections can inflict variations in the healthy status of red cells, easily identifiable by selectively analyzing specific Raman markers. Additionally, this study also highlights relevant statistical tools that can be utilized to study Raman spectral data from biological samples which could help identify the very significant Raman peaks from the spectral band. This approach of RBC analysis can foster a better understanding of red cell behavior and their alterations exhibited in health and disease.

In vivo spectroscopy: optical fiber probes for clinical applications.

INTRODUCTION: Fiber optic probe-based in vivo spectroscopy techniques are fast and highly objective methods for intraoperative diagnoses and minimally invasive surgical interventions for all procedures where endoscopic observations are carried out for cancers of different types. The Raman spectral features provide molecular fingerprint-type information and can reveal the subjects' pathological state in label-free manner, making endoscopy multiplexed fiber optic probe-based devices with the potential for translation from bench to bedside for routine applications. AREAS COVERED: This review provides a general overview of different fiber-optic probes for in vivo measurements with emphasis on Raman spectroscopy for biomedical application. Various aspects such as fiber-optic probe, radiation source, detector, and spectrometer for extracting optimum spectral features have also been discussed. EXPERT OPINION: Optical spectroscopy-based fiber probe systems with 'Chip-on-Tip' technology, combined with machine learning, can in the near future, become a complementary diagnostic tool to magnetic resonance imaging (MRI), computed tomography (CT) scan, ultrasound, etc. Hyperspectral imaging and fluorescence-based devices are in the advanced stage of technology readiness level (TRL), and with advances in lasers and miniature spectroscopy systems, probe-based Raman devices are also coming up.

Cardiovascular biomarkers in body fluids: progress and prospects in optical sensors.

Cardiovascular diseases (CVD) are the major causative factors for high mortality and morbidity in developing and developed nations. The biomarker detection plays a crucial role in the early diagnosis of several non-infectious and life-threatening diseases like CVD and many cancers, which in turn will help in more successful therapy, reducing the mortality rate. Biomarkers have diagnostic, prognostic and therapeutic significances. The search for novel biomarkers using proteomics, bio-sensing, micro-fluidics, and spectroscopic techniques with good sensitivity and specificity for CVD is progressing rapidly at present, in addition to the use of gold standard biomarkers like troponin. This review is dealing with the current progress and prospects in biomarker research for the diagnosis of cardiovascular diseases. Expert opinion. Fast diagnosis of cardiovascular diseases (CVDs) can help to provide rapid medical intervention, which can affect the patient's short and long-term health. Identification and detection of proper biomarkers for early diagnosis are crucial for successful therapy and prognosis of CVDs. The present review discusses the analysis of clinical samples such as whole blood, blood serum, and other body fluids using techniques like high-performance liquid chromatography-LASER/LED-induced fluorescence, Raman spectroscopy, mainly, optical methods, combined with nanotechnology and micro-fluidic technologies, to probe patterns of multiple markers (marker signatures) as compared to conventional techniques.

Micro-Raman spectroscopy study of blood samples from myocardial infarction patients.

Acute myocardial infarction (MI) is found to be a major causative factor for global mortality and morbidity. This situation demands necessity of developing efficient and rapid diagnostic tools to detect acute MI. Raman spectroscopy is a non-destructive optical diagnostic technique, which has high potential in probing biochemical changes in clinical samples during initiation and progress of diseases. In this work, blood was taken as the sample to examine inflammation in acute MI patients using Raman spectroscopy. Ratio of Raman peak intensities that corresponds to phenylalanine (1000 cm-1) and tyrosine (825 cm-1) can facilitate indirect information about tetrahydrobiopterin (BH4) availability, which can indicate inflammatory status in patients. This ratio obtained was higher for MI patients in comparison with control subjects. The decrease in phenylalanine and tyrosine ratio (Phe-Tyr ratio) is attributed to the prognosis of standard of care (medications like antiplatelets including aspirin, statin and revascularisation) leading to inflammation reduction. Phe-Tyr ratio estimated from the Raman spectra of blood can be exploited as a reliable method to probe inflammation due to MI. The method is highly objective, require only microliters of sample and minimal sample preparation, signifying its clinical utility.

A micro-Raman spectroscopy study of inflammatory condition of human cervix: Probing of tissues and blood plasma samples.

The present study explores the application of the micro-Raman spectroscopy technique to discriminate normal and cervicitis condition from cervical malignancy by analyzing the Raman signatures of tissues and plasma samples of the same subjects. The Raman peaks from tissue samples at 1026 cm-1,1298 cm-1 and 1243 cm-1 are attributed to glycogen, fatty acids and collagen and are found to be reliable signatures capable of identifying cervicitis and normal condition from cervical cancer. The Raman signatures from plasma samples belonging to carbohydrates (578 cm-1), lipids (1059 cm-1) and nucleic acids (1077 cm-1,1341 cm-1 and 1357 cm-1) are quite useful to classify various pathological conditions of cervix at par with tissue based diagnosis. The PCA-SVM based classification of the spectral data indicates the potential of Raman spectroscopy based liquid biopsy to rule out false diagnosis of cervicitis as cervical malignancy.

Post-COVID syndrome screening through breath analysis using electronic nose technology.

There is an urgent need to have reliable technologies to diagnose post-coronavirus disease syndrome (PCS), as the number of people affected by COVID-19 and related complications is increasing worldwide. Considering the amount of risks associated with the two chronic lung diseases, asthma and chronic obstructive pulmonary disease (COPD), there is an immediate requirement for a screening method for PCS, which also produce symptoms similar to these conditions, especially since very often, many COVID-19 cases remain undetected because a good share of such patients is asymptomatic. Breath analysis techniques are getting attention since they are highly non-invasive methods for disease diagnosis, can be implemented easily for point-of-care applications even in primary health care centres. Electronic (E-) nose technology is coming up with better reliability, ease of operation, and affordability to all, and it can generate signatures of volatile organic compounds (VOCs) in exhaled breath as markers of diseases. The present report is an outcome of a pilot study using an E-nose device on breath samples of cohorts of PCS, asthma, and normal (control) subjects. Match/no-match and k-NN analysis tests have been carried out to confirm the diagnosis of PCS. The prediction model has given 100% sensitivity and specificity. Receiver operating characteristics (ROC) has been plotted for the prediction model, and the area under the curve (AUC) is obtained as 1. The E-nose technique is found to be working well for PCS diagnosis. Our study suggests that the breath analysis using E-nose can be used as a point-of-care diagnosis of PCS.Trial registrationBreath samples were collected from the Kasturba Hospital, Manipal. Ethical clearance was obtained from the Institutional Ethics Committee, Kasturba Medical College, Manipal (IEC 60/2021, 13/01/2021) and Indian Council of Medical Research (ICMR) (CTRI/2021/02/031357, 06/02/2021) Government of India; trials were prospectively registered.

Development of a spectroscopic technique that enables the saliva based detection of COVID-19 at safe distances.

Research activities are in full swing globally to translate the use of saliva as a non-invasive and highly potential specimen for clinical diagnostics, particularly for COVID-19 detection. Being comprised of a pool of biomarkers also enriched with ACE-2 receptors, saliva can provide vital information regarding the state of the human body. Advancements in biophotonics tools for saliva investigation may offer promise for developing rapid, highly objective, optical modalities for COVID- 19 detection. This article presents concept/design study, which propose the use of Raman/laser induced fluorescence spectroscopic device that have the potential for viral detection via saliva from a safer distance. Noticeable changes of biomarkers present in saliva in response to viral infection can reflect the pathological state, thus can altogether affect the Raman spectral pattern. Monitoring these spectral patterns of saliva, which are further enhanced by using cost effective and reproducible Surface Enhanced Raman Spectroscopy substrates can be a viable option for sensitive and non-invasive viral detection. The spectral information acquired from the optical device can be processed using various multivariate statistical analytical tools, which ultimately facilitate effective viral detection in few minutes. This method doesn't demand the necessity of qualified professionals and sample processing with reagents unlike in RT-PCR test. The proposed optical device can be further modified into a portable form, which can be easily transported for field applications. The stand-off observation, contactless and highly non-invasive technique can be of paramount importance in the current context, where the safer screening of a large population for viral infection by maintaining social distances is a necessity. The proposed stand-off spectroscopic technique can also address the major concern of nosocomial viral transmission amongst healthcare workers during sample collection in a pandemic scenario.

Photonics of human saliva: potential optical methods for the screening of abnormal health conditions and infections.

Human saliva can be treated as a pool of biological markers able to reflect on the state of personal health. Recent years have witnessed an increase in the use of optical devices for the analysis of body fluids. Several groups have carried out studies investigating the potential of saliva as a non-invasive and reliable clinical specimen for use in medical diagnostics. This brief review aims to highlight the optical technologies, mainly surface plasmon resonance (SPR), Raman, and Fourier transform infrared (FTIR) spectroscopy, which are being used for the probing of saliva for diverse biomedical applications. Advances in bio photonics offer the promise of unambiguous, objective and fast detection of abnormal health conditions and viral infections (such as COVID-19) from the analysis of saliva.

Human tear fluid analysis for clinical applications: progress and prospects.

Introduction: Human blood and saliva are increasingly under investigation for the detection of biomarkers for early diagnosis of non-communicable (e.g.cancers) and communicable diseases like COVID-19. Exploring the potential application of human tears, an easily accessible body fluid, for the diagnosis of various diseases is the need of the hour.Areas covered: This review deals with a comprehensive account of applications of tear analysis using different techniques, their comparison and overall progress achieved till now. The techniques used for tear fluid analysis are HPLC/UPLC/SDS-PAGE, CE, etc., together with ELISA, Mass Spectrometry, etc. But, with advances in instrumentation and data processing methods, it has become easy to couple the various separation methods with highly sensitive optical techniques for the analysis of body fluids.Expert opinion: Tear analysis can provide valuable information about the health condition of the eyes since it contains several molecular constituents, and their relative concentrations may alter under abnormal conditions. Tear analysis has the advantage that it is totally non-invasive. This study recommends tear fluid as a reliable clinical sample to be probed by highly sensitive optical techniques to diagnose different health conditions, with special emphasis on eye diseases.

Single cell spectroscopy of red blood cells in intravenous crystalloid fluids.

Crystalloid fluids, a subset of intravenous (IV) fluid solutions are commonly used in clinical settings. The influence of these fluids on the functions of blood components are least explored. Raman spectroscopy combined with optical trapping has been widely used to evaluate the impact of external stress agents on red blood cells. The present study investigates the impact of commonly used crystalloid fluids on red blood cells in comparison with that of blood plasma using Raman Tweezers spectroscopy. The red blood cells suspended in crystalloid fluids undergo deoxygenation readily than that in blood plasma. In addition, cells in blood plasma were able to withstand laser induced deoxygenation comparatively better than that in crystalloid fluids at higher laser powers. Principle component analysis of the Raman spectral data has clearly demonstrated the discrimination of cells in plasma with that of crystalloid fluids demonstrating the effect of external induced stress on RBCs.

A review on human body fluids for the diagnosis of viral infections: scope for rapid detection of COVID-19.

Introduction: The unprecedented outbreaks of corona virus disease of 2019 (COVID-19) have highlighted the necessity of readily available, reliable, precise, and faster techniques for its detection. Nasopharyngeal swab has been the gold standard for the diagnosis of COVID-19. However, it is not an ideal screening procedure for massive screening as it implicates the patient's stay in the hospital or at home until diagnosis, thus causing crowding of the specimen at the diagnostic centers. Present study deal with the exploration of potential application of different body fluids using certain highly objective techniques (Optical and e-Nose) for faster detection of molecular markers thereby diagnosing viral infections.Areas covered: This report presents an evaluation of different body fluids, and their advantages for the rapid detection of COVID-19, coupled with highly sensitive optical techniques for the detection of molecular biomarkers.Expert opinion: Tears, saliva, and breath samples can provide valuable information about viral infections. Our brief review strongly recommends the application of saliva/tears and exhaled breath as clinical samples using technics such as high-performance liquid chromatography-laser-induced fluorescence, photoacoustic spectroscopy, and e-Nose, respectively, for the fast diagnosis of viral infections.

Optical technologies for the detection of viruses like COVID-19: Progress and prospects.

The outbreak of life-threatening pandemic like COVID-19 necessitated the development of novel, rapid and cost-effective techniques that facilitate detection of viruses like SARS-CoV-2. The presently popular approach of a collection of samples using the nasopharyngeal swab method and subsequent detection of RNA using the real-time polymerase chain reaction suffers from false-positive results and a longer diagnostic time scale. Alternatively, various optical techniques namely optical sensing, spectroscopy, and imaging shows a great promise in virus detection. Herein, a comprehensive review of the various photonics technologies employed for virus detection, particularly the SARS-CoV family, is discussed. The state-of-art research activities in utilizing the photonics tools such as near-infrared spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, fluorescence-based techniques, super-resolution microscopy, surface plasmon resonance-based detection, for virus detection accounted extensively with an emphasis on coronavirus detection. Further, an account of emerging photonics technologies of SARS-CoV-2 detection and future possibilities is also explained. The progress in the field of optical techniques for virus detection unambiguously show a great promise in the development of rapid photonics-based devices for COVID-19 detection.

Probing Nanoparticle-Cell Interaction Using Micro-Raman Spectroscopy: Silver and Gold Nanoparticle-Induced Stress Effects on Optically Trapped Live Red Blood Cells.

Advancements in the field of nanotechnology have resulted in the emergence of a large variety of engineered nanomaterials for innumerable applications. Despite the ubiquitous use of nanomaterials in daily life, concerns regarding the potential toxicity and safety of these materials have also been raised. There is a high demand for assessing the unwanted effects of both gold and silver nanoparticles, which is increasingly being used in biomedical applications. This paper deals with the study of stress due to silver and gold nanoparticles of varying size on red blood cells (RBCs) using Raman tweezers spectroscopy. RBCs were incubated with nanoparticles of size in the 10-100 nm range with the same concentrations, and micro-Raman spectra were recorded by optically trapping the nanoparticle-treated live RBCs. Spectral modifications implicating hemoglobin deoxygenation were observed in all nanoparticle-treated RBCs. One of the probable reason for the deoxygenation trend can be the adhesion of nanoparticles onto the cell surface causing imbalance in cell functioning. Moreover, the higher spectral variations observed for silver nanoparticles indicate that oxidative stress is involved in cell damage. These mechanisms lead to the modification in the hemoglobin structure because of changes in the pH of cytoplasm, which can be detected using Raman spectroscopy.

Human red blood cell behaviour in hydroxyethyl starch: probed by single cell spectroscopy.

Hydroxyethyl starch (HES) is a commonly used intravenous fluid in hospital settings. The merits and demerits of its application is still a debatable topic. Investigating the interaction of external agents like intravenous fluids with blood cells is of great significance in clinical environments. Micro-Raman spectroscopy combined with an optical tweezers technique has been utilized for conducting systematic investigations of single live red blood cells (RBCs) under the influence of external stress agents. The present work deals with a detailed biophysical study on the response of human live red blood cells in hydroxyethyl starch using optical techniques. Morphological changes in red blood cells were monitored using quantitate phase imaging techniques. Micro-Raman studies suggest that there is a significant reduction in the oxy-haemoglobin level in red blood cells suspended in HES. The spectra recorded by using different probe laser powers has shown that the cells are more vulnerable in HES under the influence of externally induced stress than in blood plasma. In addition, the spectral results support the possibility of heme aggregation and membrane damage for red blood cells in HES under externally induced stress. Principle component analysis performed on the Raman spectra were able to effectively discriminate between red blood cells in HES and in blood plasma. The use of Raman tweezers can be highly beneficial in elucidating biochemical alterations happening in live, human red blood cell.

Red blood cells under varying extracellular tonicity conditions: an optical tweezers combined with micro-Raman study.

Extracellular tonicity has a significant influence on human red blood cell deformation capability. Advancements in the area of laser physics and optical trapping have opened up a plethora of applications for understanding cell structure and dynamics. Here, Raman Tweezers technique was employed to investigate the impact of extracellular tonicity by exposing human red blood cells to both hypertonic and hypotonic intravenous fluids. Heme aggregation was observed in hypertonic saline solution, accompanied with damage in membrane protein. Loss of intracellular hemoglobin in hypotonic solution was evident from the decrease in porphyrin breathing mode present at 752 cm-1. Oxygen binding to the central iron in the red blood cell heme was also affected under both hyper/hypo tonicity conditions. Morphological deviation of discocytes to echinocytes/spherocytes were also evident from quantitative phase imaging. Principal component analysis have showed clear differentiation of samples in order to classify the control erythrocytes and the tonicity stressed erythrocytes. Present study has also demonstrated the application of Raman Tweezers spectroscopy as a potential tool for probing red blood cell under different stress conditions.