The realm of non-invasive disease diagnosis is a critical area of research and development. It primarily focuses on monitoring distinct biochemical constituents within the body fluids. These constituents, often referred to as biomarkers, undergo alterations in concentration and structure that signify changes in physiological or pathological conditions. Monitoring biomarkers, therefore, offer invaluable insights into diseases or health statuses, paving the way for timely and targeted interventions. Monitoring these biomarkers in body fluids during disease progression and therapy, can also provide insight into the efficacy of a particular treatment. Human body fluids, including blood serum/plasma, tears, urine, saliva, and cerebrospinal fluid etc are considered as a valuable source of disease indicators due to their minimal invasiveness and ease of sample collection and processing. They offer an intriguing alternative to tissues and cells for identifying various health abnormalities. Body fluid analysis not only enables disease diagnosis but also allows for the assessment of disease progression using advanced and highly sensitive tools. In summary, biomarker analysis in body fluids has the potential to provide critical information about an individual's health status and offer essential clues pointing to the presence of a range of health disorders.

The eye is considered as one of the most sensitive organs of the visual system. The cornea together with the eye lens and retina respectively are responsible for the collection and detection of light that enters the eyes. A healthy lifestyle is required to maintain good eye health and thereby, minimizing risks to this sensitive organ. Tears play a crucial role in maintaining the health and function of the eyes. The tear film is a complex and dynamic structure consisting of three layers. They perform functions for avascular corneal surfaces similar to those of blood for the rest of the body, including delivering oxygen and nutrients, eliminating waste, protecting against infections and repairing ocular surface damage (Barmada and Shippy, 2020). Tear fluid analysis, in particular, is generating significant interest in the development of new biomarker-based diagnostics due to its noninvasive sample collection method. Tear sampling from the ocular surface can be easily and noninvasively performed in a matter of seconds, allowing point-of- care devices to quickly and continuously monitor patient health both within the clinic and outside the hospital setting (Hagan et al., 2016). Schirmer strips and capillaries are considered as conventional sampling tools (Ponzini et al., 2022). Nevertheless, other absorbent techniques, such as sponges and ocular flush methods have also been utilized in previous investigations. (Ma et al., 2021; Nättinen et al., 2022). In addition to the other benefits, the convenience and ease of accessibility of Schirmer strips are noteworthy. While the use of capillary tubes is time-consuming and requires expertise, Schirmer strips on the other hand can be readily integrated into clinical practice, necessitate minimal manipulation during sample collection, and furnish valuable insights into the dry-eye condition of the patient (Bachhuber et al., 2021).

However, capillary tubes offer the benefit of straightforward post-collection handling. A rapid centrifugation step allows the collection of the entire undiluted volume of tear fluid (Bachhuber et al., 2021). On the other hand, Schirmer strips necessitate the extraction of tear fluid. The strips possess the ability to retain proteins to different degrees, which is determined by their molecular properties (Choy et al., 2001). The diffusion-based processing of samples after collection impacts the absolute and relative concentrations of human tear proteins obtained using Schirmer strips (Denisin et al., 2012). The extraction protocols involve centrifugation, centrifugation combined with a washing step, or diffusion-based elution (Posa et al., 2013; Hümmert et al., 2019; Calais et al., 2010). The efficiency of extraction is influenced by factors such as the choice of solvent, time, temperature, and volume (Aass et al., 2015). In addition, the physical contact between Schirmer strips and the cornea and conjunctiva can cause mechanical irritation, leading to the release of vascular transudation or cell damage (Choy et al., 2001; Van Setten et al., 1990). This can result in the leakage of cellular or plasma proteins into the collected sample. On the other hand, the use of capillary tubes eliminates any contact with the surface of the eye. The tear collection using capillary tubes offers a non-invasive and efficient method for obtaining tear samples. Capillary tubes, typically ranging between 5 and 10 μm in diameter and made of glass, are utilized for this purpose. Tears are gathered by delicately placing the tube parallel to the lower meniscus. Once collected, tears are effortlessly transferred from the capillary tubes into glass vials, primed for subsequent analysis (Lam et al., 2014; Tham et al., 2023). This approach obviates the need for additional extraction procedures. However, the approach takes a few minutes to collect 4–5 μL of tear. Post-collection, tears undergo centrifugation for a few seconds. Nevertheless, centrifugation is not obligatory for obtaining tears from capillary tubes. In this case, the tears are collected and immediately stored at −80 °C until subsequent analysis (Lam et al., 2014; Tham et al., 2023; Ami et al., 2021). It is important to note that centrifugation is not performed to collect tears, but rather to remove air bubbles trapped at the bottom due to the minute volumes of tear fluid (Ami et al., 2021).

Tears are a translucent, three-layered fluid that covers the eye's surface. They perform functions for avascular corneal surfaces similar to those of blood for the rest of the body, including delivering oxygen and nutrients, eliminating waste, protecting against viruses, and repairing ocular surface damage (Barmada and Shippy, 2020). In 2006, a mass spectrometry based study identified as many as 491 proteins in tears (de Souza et al., 2006). Later, in 2016, a proteomics study identified 1526 proteins in tears (Barmada and Shippy, 2020; Aass et al., 2015). This suggests that tears contain a wealth of clinical data that is relevant to seemingly unrelated body regions beyond the ocular surface and eye. Additionally, tears carry a pool of biomarkers for a range of systemic diseases, including cancer, multiple sclerosis, diabetes mellitus, Parkinson's disease, cystic fibrosis, Alzheimer's disease, and others. Ascendant Dx, a diagnostic enterprise, has been engaged in the development of a breast cancer screening test utilizing tear protein, which claims an accuracy rate of 90%. This represents a more auspicious alternative to conventional mammography (Morton et al., 2018). Despite these benefits, various analytical challenges remain in making tear analysis a routine screening approach, as the concentrations of many biomarkers are extremely low compared to blood. The protein (biomarker) concentration in tear fluid can be affected by several factors, such as reflex tearing, collection procedure, and the processing of samples after collection (Bachhuber et al., 2021). Reflex tearing, which occurs due to mechanical, sensory, or emotional stimulation, has been found to significantly decrease the concentration of many proteins in tear fluid (Fullard and Snyder, 1990). Using Schirmer strips for tear collection can influence the composition of samples as a result of physical contact with the cornea and conjunctiva (Choy et al., 2001). Additionally, when tears are retrieved from the collection tool, proteins may be lost to different extents (Denisin et al., 2012). It is highly crucial to optimize the processing and analytical methods to fully utilize the sample for investigating various biomarkers (Tamhane et al., 2019). It is also advisable to prevent the reflex tearing activation while collecting samples, as it can change the sample's composition and make the results challenging to interpret. Furthermore, tear composition can also be influenced by various factors including the utilization of artificial tears, contact lenses, etc. (Tamhane et al., 2019).

Sensitive and reliable screening techniques are necessary to utilize tear fluid as a potential noninvasive diagnostic sample in clinical applications. Considerable research has been undertaken to explore the potential of Raman spectroscopy-based investigations for tear analysis in diverse diagnostic applications. This has recently garnered significant clinical interest, particularly when utilized in conjunction with artificial Intelligence and machine learning algorithms. Raman spectroscopy is widely recognized as a highly sensitive and label-free spectroscopic technique that can provide valuable biochemical information about biological samples, including tissue, cells, and body fluids. Raman Spectroscopy offers valuable insights into the molecular vibrations of a given sample of interest without any exogenous dyes or labelling agents, and can be performed directly in aqueous solutions. This spectroscopic method entails gathering the inelastic scattering (Raman scattering) signal of the sample, which arises upon exposure to a monochromatic excitation wavelength falling within the UV, visible, or NIR spectrum. By examining the spectra obtained from the inelastic scattered light, one can gain insight into the molecular vibrations of the sample. Raman spectroscopy provides significant advantages over conventional techniques. These include the ability to examine samples without causing damage, exceptional sensitivity to structural variations, minimal sample preparation requirements, minimal water interference, and suitability for real-time investigation of tear samples. The change in chemical composition of tear can also be analyzed non-invasively using Infrared spectroscopy technique. Infrared spectroscopy utilizes infrared radiation to measure the absorption, emission, or reflection by molecules within a sample. The change in spectrum due to absorption can qualitatively provide information regarding the chemical bonds present in a sample. Tears, comprising primarily water, serve as the main constituent and are known for their strong absorption of infrared rays. This characteristic presents a significant challenge, as it can interfere with the analysis, potentially compromising our ability to fully realize the diagnostic potential of tear analysis using infrared spectroscopy (Nagase et al., 2005). Techniques such as attenuated total reflectance (ATR) spectroscopy can mitigate water absorption to some extent but may still result in reduced sensitivity (Travo et al., 2014; Aparna et al., 2022). Infrared spectroscopy often requires extensive sample preparation, including drying or desiccation of tear samples to reduce water content and improve spectral quality. On the other hand, Raman spectroscopy relies on the inelastic scattering of photons by molecular vibrations within a sample. Water molecules primarily exhibit symmetric and asymmetric stretching vibrations of the O–H bonds in the Raman spectrum. These vibrations typically occur at higher wavenumbers compared to those of biomolecules. This minimal interference between spectral features of biomolecules and water molecules makes Raman spectroscopy an attractive option for tear biomarker analysis. However, reports on tear investigations using Raman spectroscopy, which are accessible to clinicians, biochemists, and spectroscopists, are often scattered. This can pose a challenge to the precise Raman spectroscopic assessment of biochemical alterations reflected in tear that may arise during various abnormalities, whether for early diagnosis or monitoring the prognosis of treatment. This underscores the necessity of consolidating a comprehensive summary of the research findings pertaining to tear analysis utilizing Raman spectroscopy in a single location, thereby rendering it practicable for the clinical community especially ophthalmologists and spectroscopic communities to comprehend and stay aware of the potential of this spectroscopic modality for tear investigations.