Developing SERS-based point-of-care inflammatory monitoring for risk dentification of worsening progression of age-related disease

Abstract

With an ageing population1 we are seeing a rise in many age-related diseases which are having long-term health implications for patients and cost implications for our health services2. Age-related hearing loss is the most common sensory deficit among older adults and can lead to decline in well-being and quality of life. A chronic state of raised inflammation in older people known as inflammaging3,4 has been linked to many age-related diseases3. Evidence from research suggests a link between inflammatory status and hearing thresholds in older people5-7. Neopterin is a small inflammatory metabolite that can be monitored in either the blood or urine8-10. Neopterin levels reflect immune activation status as neopterin is released from monocyte-derived macrophages and dendritic cells following stimulation with the pro-inflammatory cytokine interferon-γ. Neopterin has been explored as a marker for chronic inflammation in several conditions11,12. Most patients will happily give regular urine samples as they are non-invasive to collect. Urinary neopterin levels can be measured in a laboratory setting using gold-standard HPLC13 which can provide highly accurate quantitative results12. A 3-year longitudinal study measuring changes in hearing level and inflammatory profile of 45 adults (aged 65-75) has been carried out. Urine was collected monthly over 12 months for analysis of neopterin concentration. Individuals were stratified into groups depending on how often their biomarker was raised during the collection period. Pure Tone Audiometry was performed at baseline, after one year, and at the study end. Results demonstrate a correlation between raised inflammatory state and greater change in hearing levels (r= 0.334, p = 0.027) and when stratified into groups, those with consistently raised (more than 50% of the time) inflammatory state, showed a greater deterioration in high-frequency hearing loss (W (3.000, 19.60) = 9.164, p = 0.0005). We also demonstrate that these individuals are more than twice as likely to have other co-morbidities. However, for long-term regular monitoring of a patient’s inflammatory state, HPLC is not cost or time effective. Therefore, there is a real need for a quick and cost-effective method of measuring urinary neopterin levels in patient urine samples long-term. Ideally, the method would enable point-of-care measurement that would give a real-time result within the patient consultation or at the patient’s home. The technique wouldn’t be required to compete with HPLC in terms of quantitative accuracy, but it would need to be sensitive enough to be used as a screening-like tool to identify those with consistently raised urinary neopterin levels compared to normative values. The aim is to develop a point-of-care inflammatory monitoring device which would allow population screening, leading to identification of those ‘at risk’ of worsening age-related disease (including hearing loss). With this knowledge, an individual may seek earlier intervention or may wish to make lifestyle changes which may reduce their risk. Such a technique has been explored utilising the technique of Surface-Enhanced Raman Spectroscopy. Raman spectroscopy utilises inelastic light scattering from a sample to identify the molecules present within it. By combing the sample with metallic nanostructures extremely large enhancement effects are seen which enable detection of molecular information as low as fM concentration14. One of the difficulties with performing SERS on biofluids is the matrix effect. This research demonstrates the simple application of paper chromatography to urine samples, in order to achieve separation of the matrix prior to SERS acquisition to further eliminate the matrix effect. This research also demonstrates the use of a novel SERS substrate in urine. Colloidal SERS-sensors are formed using a layer-by-layer process where the metal nanoparticles are structurally spaced, giving control over the SERS enhancement. Results demonstrate improved reproducibility of these SERS sensors in comparison to colloidal metal nanoparticles. Urine samples were analysed with both SERS and HPLC to validate, and the measured concentrations were generally in good agreement (R2 of 0.936 and 0.990 for creatinine and neopterin) at physiologically relevant concentrations. By having access to a technique that can provide fast but reliable urinary neopterin readings we can regularly monitor a cohort of older adults and identify those who are at increased risk of worsening age-related hearing loss so that they can be targeted for intervention.

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Identifiers

ORCID for Tracey Newman: orcid.org/0000-0002-3727-9258

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