Breath-based pneumonia diagnostics using ultrasensitive detection of synthetic breath biomarkers using digital colloid-enhanced Raman spectroscopy

Introduction: Pneumonia is responsible for 14% of deaths among children under five years old worldwide. The lack of sputum makes timely diagnosis difficult, ultimately resulting in the overprescription of antibiotics and prolonged illness. Recently, inhalable synthetic breath biomarkers that are cleaved by disease-specific proteases to release volatile organic compounds (VOCs) have been developed as alternative noninvasive diagnostics. However, there is a need for a point-of-care detection scheme as mass spectroscopy used to detect these VOCs is bulky, expensive to operate, and not conducive for use in resource-limited environments where this technology would be most beneficial. Raman spectroscopy is an ideal solution as it enables molecular detection based on the inelastic scattering of photons, generating a unique spectral fingerprint. Raman can be deployed in handheld microscope or probe-based systems that are easy to transport, and the addition of plasmonic metal nanoparticles through surface enhanced Raman spectroscopy (SERS) enables limits of detection around 1-10 ppb required for VOC detection. Here, we demonstrate SERS-based detection of VOCs for breath diagnostics using a 10x confocal microscope. We fabricated quartz capillary tubes internally coated with Au nanorods to electrostatically contain isopropylamine gas over the course of analysis. For trace concentrations, we performed line scans down the length of the capillary with a 10μm step size and digitally searched each spectra for Raman signature peaks of isopropylamine. This design minimizes sample processing steps, requiring only a breath or gas sample to be injected into the fabricated SERS capillary and utilizes simple computational techniques for rapid, automated diagnosis.

Materials and

Methods: VOC capture capillaries were fabricated using quartz tubes (ID 3mm, OD 5mm) and fitted rubber septa caps. Prior to placing rubber caps, the inside of the tube was coated with a thin layer of synthesized Au nanorods (length 48nm, diameter 14nm, localized surface plasmon resonance peak wavelength 770nm) and dried overnight. These nanorods were synthesized to have a positive charge to induce electrostatic adsorption of VOCs with amine groups that act as a Lewis base. To demonstrate VOC detection, isopropylamine was used as it evaporates less quickly at room temperature, allowing for controlled handling and precise concentrations. Different concentrations of gas were made by injecting 100uL of the liquid state of the isopropylamine into a vacuum sealed glass tube, heating to 80℃ to induce gas formation, sampling the headspace, and performing serial dilutions. Final concentrations were injected into the capillary tubes through the rubber septa. The ability of our fabricated tubes to contain VOCs was confirmed after 4 days using gas chromatography mass spectroscopy (GC-MS). For Raman interrogation, we used a commercially available confocal Raman microscope (WITec Alpha300). The sealed capillary tubes were secured on the microscope stage under a 10x objective lens, and Raman spectra were obtained via a point-by-point line scan down the length of the capillary with a 785nm laser at 10mW power over a 10s exposure time. The resulting spectra were subjected to baseline correction and iteratively searched for Raman signal above a given intensity threshold in a window containing the signature Raman peaks of isopropylamine.

Results, Conclusions, and Discussions: As confirmed by mass spectroscopy, our fabricated SERS capillaries effectively contained isopropylamine gas for at least 4 days, which is far beyond the time required for measurement acquisition (Figure 1A). Note that capillary tubes containing Au nanorods resulted in more consistent measurement at lower concentrations with tubes lacking nanorods reaching an apparent limit of detection after 0.12M. This result suggests that the addition of nanorods aids in containing the gas molecules inside the capillary tube for longer through electrostatic adsorption, whereas tubes without nanorods may lose gas molecules more readily leading to lower concentrations at the time of measurement. Unlike other VOCs, isopropylamine gas is difficult to detect with mass spectroscopy at lower concentrations due to the molecule’s small size and overlapping molecular weights with other molecules commonly found in ambient air. Given our desired limit of detection of 1-10ppb, this result further supports the need for Raman-based detection of isopropylamine gas for breath diagnostics. With the current acquisition parameters, we are able to detect the Raman signal of the isopropylamine gas molecules at a concentration as low as 0.12M (Figure 1B) noted by sharp peaks around 380cm⁻¹, 810cm⁻¹, and 980cm⁻¹ compared to broad noise peak in the negative control around 750cm⁻¹ and 1450cm⁻¹. By increasing laser power and optimizing nanorod concentration for greater, uniform enhancement, we are currently working to achieve detection lower than 1uM. Our proposed detection scheme will enable rapid, portable analysis of breath samples and enable more accurate diagnosis of lung diseases.

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