SERS Microsensors for the Study of pH Regulation in Cystic Fibrosis Patient-Derived Airway Cultures.

Acidification of the airway surface liquid in the respiratory system could play a role in the pathology of Cystic Fibrosis, but its low volume and proximity to the airway epithelium make it a challenging biological environment in which to noninvasively collect pH measurements. To address this challenge, we explored surface enhanced Raman scattering microsensors (SERS-MS), with a 4-mercaptobenzoic acid (MBA) pH reporter molecule, as pH sensors for the airway surface liquid of patient-derived in vitro models of the human airway. Using air-liquid interface (ALI) cultures to model the respiratory epithelium, we show that SERS-MS facilitates the optical measurement of trans-epithelial pH gradients between the airway surface liquid and the basolateral culture medium. SERS-MS also enabled the successful quantification of pH changes in the airway surface liquid following stimulation of the Cystic Fibrosis transmembrane conductance regulator (CFTR, the apical ion channel that is dysfunctional in Cystic Fibrosis airways). Finally, the influence of CFTR mutations on baseline airway surface liquid pH was explored by using SERS-MS to measure the pH in ALIs grown from Cystic Fibrosis and non-Cystic Fibrosis donors.

10†kHz Shifted-Excitation Raman Difference Spectroscopy with Charge-Shifting Charge-Coupled Device Read-Out for Effective Mitigation of Dynamic Interfering Backgrounds.

In this work we demonstrate an advanced concept of a charge-shifting charge-coupled device (CCD) read-out combined with shifted excitation Raman difference spectroscopy (SERDS) capable of operating at up to 10 kHz acquisition rates for the effective mitigation of fast-evolving interfering backgrounds in Raman spectroscopy. This rate is 10-fold faster than that achievable with an instrument we described previously and is overall 1000-fold faster than possible with conventional spectroscopic CCDs capable of operating at up to ∼10 Hz rates. The speed enhancement was realized by incorporating a periodic mask at the internal slit of an imaging spectrometer permitting a smaller shift of the charge on the CCD (8 pixels) to be required during the cyclic shifting process compared with the earlier design which employed an 80-pixel shift. The higher acquisition speed enables the more accurate sampling of the two SERDS spectral channels, enabling it to effectively tackle highly challenging situations with rapidly evolving interfering fluorescence backgrounds. The performance of the instrument is evaluated for heterogeneous fluorescent samples which are moved rapidly in front of the detection system aiming at the differentiation of chemical species and their quantification. The performance of the system is compared with that of the earlier 1 kHz design and a conventional CCD operated at its maximum rate of 5.4 Hz as previously. In all situations tested, the newly developed 10 kHz system outperformed the earlier variants. The 10 kHz instrument can benefit a number of prospective applications including: disease diagnosis where high sensitivity mapping of complex biological matrices in the presence of natural fluorescence bleaching restricts achievable limits of detection; accurate data acquisition from moving heterogeneous samples (or moving a handheld instrument in front of the sample during data acquisition) or data acquisition under varying ambient light conditions (e.g., due to casting shadows, sample or instrument movement). Other beneficial scenarios include monitoring rapidly evolving Raman signals in the presence of largely static background signals such as in situations where a heterogeneous sample is moving rapidly in front of a detection system (e.g., a conveyor belt) in the presence of static ambient light.

SERS microsensors for pH measurements in the lumen and ECM of stem cell derived human airway organoids.

Patient derived organoids have the potential to improve the physiological relevance of in vitro disease models. However, the 3D architecture of these self-assembled cellular structures makes probing their biochemistry more complex than in traditional 2D culture. We explore the application of surface enhanced Raman scattering microsensors (SERS-MS) to probe local pH gradients within patient derived airway organoid cultures. SERS-MS consist of solid polymer cores decorated with surface immobilised gold nanoparticles which are functionalised with pH sensitive reporter molecule 4-mercaptobenzoic acid (MBA). We demonstrate that by mixing SERS-MS into the extracellular matrix (ECM) of airway organoid cultures the probes can be engulfed by expanding organoids and report on local pH in the organoid lumen and ECM.

Predicting future left anterior descending artery events from non-culprit lesions: insights from the Lipid-Rich Plaque study.

AIMS: The left anterior descending (LAD) artery is the most frequently affected site by coronary artery disease. The prospective Lipid Rich Plaque (LRP) study, which enrolled patients undergoing imaging of non-culprits followed over 2 years, reported the successful identification of coronary segments at risk of future events based on near-infrared spectroscopy-intravascular ultrasound (NIRS-IVUS) lipid signals. We aimed to characterize the plaque events involving the LAD vs. non-LAD segments. METHODS AND RESULTS: LRP enrolled 1563 patients from 2014 to 2016. All adjudicated plaque events defined by the composite of cardiac death, cardiac arrest, non-fatal myocardial infarction, acute coronary syndrome, revascularization by coronary bypass or percutaneous coronary intervention, and rehospitalization for angina with >20% stenosis progression and reported as non-culprit lesion-related major adverse cardiac events (NC-MACE) were classified by NIRS-IVUS maxLCBI4 mm (maximum 4-mm Lipid Core Burden Index) ≤400 or >400 and association with high-risk-plaque characteristics, plaque burden ≥70%, and minimum lumen area (MLA) ≤4 mm2. Fifty-seven events were recorded with more lipid-rich plaques in the LAD vs. left circumflex and right coronary artery; 12.5% vs. 10.4% vs. 11.3%, P = 0.097. Unequivocally, a maxLCBI4 mm >400 in the LAD was more predictive of NC-MACE [hazard ratio (HR) 4.32, 95% confidence interval (CI) (1.93-9.69); P = 0.0004] vs. [HR 2.56, 95% CI (1.06-6.17); P = 0.0354] in non-LAD segments. MLA ≤4 mm2 within the maxLCBI4 mm was significantly higher in the LAD (34.1% vs. 25.9% vs. 13.7%, P < 0.001). CONCLUSION: Non-culprit lipid-rich segments in the LAD were more frequently associated with plaque-level events. LAD NIRS-IVUS screening may help identify patients requiring intensive surveillance and medical treatment.

A SERS-Active Electrospun Polymer Mesh for Spatially Localized pH Measurements of the Cellular Microenvironment.

Extracellular pH (pHe) is an important chemical factor in many cellular processes and disease pathologies. The routine sampling of pHe in vitro could lead to innovative advances in therapeutics. To this end, we have fabricated a novel gold-coated polymer mesh, which facilitates the real-time measurement of pHe via surface-enhanced Raman scattering (SERS). In this proof of concept study, we apply our SERS sensor to measure metabolically induced changes in the pHe of carcinoma-derived cell line HepG2/C3A. We demonstrate that gold-coated polyurethane electrospun nanofibers (AuNF) have strong and reproducible SERS spectra of surface-adsorbed analytes. By functionalizing AuNF with pH-responsive reporter 4-mercaptobenzoic acid (MBA), we have developed an accurate pH SERS sensor for the extracellular microenvironment. We cultured HepG2/C3A on the surface of MBA-AuNF and measured an acidic shift in pHe at the cell-fiber interface. Following exposure to staurosporine, an apoptosis-inducing drug, we observed changes in the HepG2/C3A cellular morphology indicative of controlled cell death, and detected an increase in the pHe of HepG2/C3A. These results demonstrate how subtle changes in pHe, induced by the metabolic activity of cells, can be measured with our novel SERS sensor MBA-AuNF. The excellent pH measurement performance of MBA-AuNF provides a unique platform to study extracellular pH on the microscale and will help to deepen our understanding of pHe in disease pathology.

Fabrication of a Wearable Flexible Sweat pH Sensor Based on SERS-Active Au/TPU Electrospun Nanofibers.

Development of wearable sensing platforms is essential for the advancement of continuous health monitoring and point-of-care testing. Eccrine sweat pH is an analyte that can be noninvasively measured and used to diagnose and aid in monitoring a wide range of physiological conditions. Surface-enhanced Raman scattering (SERS) offers a rapid, optical technique for fingerprinting of biomarkers present in sweat. In this paper, a mechanically flexible, nanofibrous, SERS-active substrate was fabricated by a combination of electrospinning of thermoplastic polyurethane (TPU) and Au sputter coating. This substrate was then investigated for suitability toward wearable sweat pH sensing after functionalization with two commonly used pH-responsive molecules, 4-mercaptobenzoic acid (4-MBA), and 4-mercaptopyridine (4-MPy). The developed SERS pH sensor was found to have good resolution (0.14 pH units for 4-MBA; 0.51 pH units for 4-MPy), with only 1 µL of sweat required for a measurement, and displayed no statistically significant difference in performance after 35 days (p = 0.361). Additionally, the Au/TPU nanofibrous SERS pH sensors showed fast sweat-absorbing ability as well as good repeatability and reversibility. The proposed methodology offers a facile route for the fabrication of SERS substrates which could also be used to measure a wide range of health biomarkers beyond sweat pH.

Identification of patients and plaques vulnerable to future coronary events with near-infrared spectroscopy intravascular ultrasound imaging: a prospective, cohort study.

BACKGROUND: Near-infrared spectroscopy (NIRS) intravascular ultrasound imaging can detect lipid-rich plaques (LRPs). LRPs are associated with acute coronary syndromes or myocardial infarction, which can result in revascularisation or cardiac death. In this study, we aimed to establish the relationship between LRPs detected by NIRS-intravascular ultrasound imaging at unstented sites and subsequent coronary events from new culprit lesions. METHODS: In this prospective, cohort study (LRP), patients from 44 medical centres were enrolled in Italy, Latvia, Netherlands, Slovakia, UK, and the USA. Patients with suspected coronary artery disease who underwent cardiac catheterisation with possible ad hoc percutaneous coronary intervention were eligible to be enrolled. Enrolled patients underwent scanning of non-culprit segments using NIRS-intravascular ultrasound imaging. The study had two hierarchal primary hypotheses, patient and plaque, each testing the association between maximum 4 mm Lipid Core Burden Index (maxLCBI4mm) and non-culprit major adverse cardiovascular events (NC-MACE). Enrolled patients with large LRPs (≥250 maxLCBI4mm) and a randomly selected half of patients with small LRPs (<250 maxLCBI4mm) were followed up for 24 months. This study is registered with ClinicalTrials.gov, NCT02033694. FINDINGS: Between Feb 21, 2014, and March 30, 2016, 1563 patients were enrolled. NIRS-intravascular ultrasound device-related events were seen in six (0·4%) patients. 1271 patients (mean age 64 years, SD 10, 883 [69%] men, 388 [31%]women) with analysable maxLCBI4mm were allocated to follow-up. The 2-year cumulative incidence of NC-MACE was 9% (n=103). Both hierarchical primary hypotheses were met. On a patient level, the unadjusted hazard ratio (HR) for NC-MACE was 1·21 (95% CI 1·09-1·35; p=0·0004) for each 100-unit increase maxLCBI4mm) and adjusted HR 1·18 (1·05-1·32; p=0·0043). In patients with a maxLCBI4mm more than 400, the unadjusted HR for NC-MACE was 2·18 (1·48-3·22; p<0·0001) and adjusted HR was 1·89 (1·26-2·83; p=0·0021). At the plaque level, the unadjusted HR was 1·45 (1·30-1·60; p<0·0001) for each 100-unit increase in maxLCBI4mm. For segments with a maxLCBI4mm more than 400, the unadjusted HR for NC-MACE was 4·22 (2·39-7·45; p<0·0001) and adjusted HR was 3·39 (1·85-6·20; p<0·0001). INTERPRETATION: NIRS imaging of non-obstructive territories in patients undergoing cardiac catheterisation and possible percutaneous coronary intervention was safe and can aid in identifying patients and segments at higher risk for subsequent NC-MACE. NIRS-intravascular ultrasound imaging adds to the armamentarium as the first diagnostic tool able to detect vulnerable patients and plaques in clinical practice. FUNDING: Infraredx.