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Due to deteriorating water quality and the world’s increasing demand for clean water, the need for cheap, easy and portable techniques to characterize and quantify pollutants in waters is urgent. Hence, surface-enhanced Raman spectroscopy (SERS) have gained considerable attention in this field. Atrazine and bentazon are two of the most occurring pesticides causing pollution in Sweden, and where therefore examined in this study, along with 4-mercaptopyridine (mpy) as a reference molecule. In this project, silver and gold nanoparticles where synthesised and used as SERS substrates for detection of contaminants in water by using a handheld Raman device provided by Serstech AB. Sodium chloride (NaCl) and magnesium sulfate (MgSO4) where used as aggregation agents allowing the nanoparticles to form hot spots. Mpy was detected to 0.5 nM and an enhancement factor of 108 using silver nanoparticles aggregated with NaCl was obtained. No Raman signal was obtained from atrazine nor bentazon using the handheld Raman device with silver nanoparticles aggregated with NaCl. Therefore the Raman cross-section of the probe molecules where investigated using the handheld Raman device and a conventional Raman device. Bentazon was not detectable using the handheld Raman device but detectable using a conventional Raman device. Atrazine was detectable at high concentrations i.e. atrazine powder using the handheld Raman device and detectable at 100 nM using a conventional Raman device. Since bentazon was not detectable with the handheld Raman device, more focus was put on getting a detectable signal from atrazine using the handheld Raman device. Investigation of the adsorption of atrazine and bentazon to the silver nanoparticle surface was performed. Due to the weaker adsorption to the nanoparticle surface, MgSO4 was used aggregation agent instead of NaCl with mpy, atrazine and bentazon. Mpy was detectable using MgSO4 as aggregation agent, atrazine and bentazon was not. Measurements of mpy, atrazine and bentazon without any salt was performed. For these measurements, no detectable signal from neither molecule was obtained, indicating that the formation of hot spots is necessary to obtained a detectable Raman signal. Measurements of mpy and atrazine with gold nanostars where performed. Enhancement factor using the gold nanostars was calculated to 107, and a detectrable signal from mpy was obtained, not from atrazine. Measurements of atrazine and mpy simultaneously was performed, where mpy peaks was observed but no atrazine peaks. The affinity of the probe molecule and the nanoparticle is crucial to obtain a detectable signal. This study inducates that both the chemical enhancement and electromagnetic enhancement are needed to obtain a detectable signal. For that, strongly binding species is necessary. Considering the simplicity of this method and the limited optimization efforts, there is plenty of room for improvements, including different probe molecules and different SERS substrates. With the right conditions, the evaluated technique reveals a promising and accessible method using a commercially available handheld Raman spectrometer for detection and quantification of contaminants in water.

Blood is considered one of the most important types of forensic evidence found at a crime scene. The use of surface-enhanced Raman spectroscopy (SERS) provides a potentially non-destructive and highly sensitive technique for the confirmation of blood and this method can be applied using a portable Raman device with quick sample preparation and processing. Crime scenes are inherently complex and the impact of SERS analysis provides easy use and practical application for in-field sample analysis. SERS is one of the few confirmatory techniques employed for the identification of blood at a crime scene or in the forensic laboratory. This method is able to distinguish between blood and other body fluids by collecting a SERS spectrum from a sample placed on a surface that has been embedded with gold nanoparticles (AuNPs). The AuNPs create an electric field surface enhancement that produces an intense molecular vibrational signal, leading to a SERS enhancement. The SERS enhancement allowed for sensitive blood detection at dilutions greater than 1:10,000. A stain transfer method to the SERS substrate was optimized by extracting dried bloodstains with water, saline, and various acid solutions. Fifty percent aqueous acetic acid solutions was found to be the most efficient in retaining the blood components and releasing the hemoglobin component of blood for detection. The SERS spectrum of blood is a robust signature of hemoglobin that does not significantly change between donors nor over time. Characteristic peaks for the identification of blood are 754, 1513, and 1543 wavenumbers (cm-1), attributed to a pyrrole ring breathing mode (15) and two Cβ-Cβ stretches (11, 38), respectively. These key SERS peaks, high sensitivity, and signal enhancement are favorable when compared to normal Raman spectroscopy. A quick and easy-to-use procedure for on-site sample analysis for the detection of blood on different substrates was developed and applied on a portable Raman device. Various nonporous and porous substrates including glass, ceramic tile, cotton, denim, fleece, nylon, acetate, wool, polyester, wood, and coated wood yielded strong results for identification of bloodstains. In addition, different commercial and in-house SERS substrates were tested to determine effectiveness for the detection and identification of blood. SERS identification of blood for forensic work is a potentially non-destructive and portable tool that can be applied for quick and easy examination of evidence at a crime scene. The high sensitivity and selectivity of SERS provides a robust spectroscopic signature that aids in the confirmation of blood, even when it is not visible to the naked eye. It is a more favorable method when compared to current presumptive and confirmatory tests for blood and can be applied to stains on different SERS substrates and a variety sample surfaces for universal testing.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21113号 / 工博第4477号 / 新制||工||1696(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 田中 勝久, 教授 三浦 清貴, 教授 作花 哲夫 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Self-assembly of organothiols (OTs) and thiolated biomolecules onto gold nanoparticle (AuNP) surfaces remains one of the most intense areas of nanoscience research and understanding molecular interfacial phenomena is crucial. Investigation of OT adsorption onto AuNPs, including OT structure and orientation on nanoparticle surfaces, is of fundamental importance in understanding the structure and function relationship of functionalized nanoparticles. Despite the great importance of the interfacial interaction of AuNPs, the exact mechanism of OT interactions with AuNPs has remained unclear and quantitative investigation of OT adsorption has been very limited. The research reported here focused on developing a fundamental and quantitative understanding of OT interactions with AuNPs in water. In studies of OT interactions with AuNPs in water, we found that the OTs form an adsorbed monolayer on AuNPs by releasing the sulfur-bound hydrogen as a proton and acidifying the ligand binding solution. The pH measurements suggest that there is a substantial fraction (up to 45%) of the protons derived from the surface adsorbed OTs retained close to the gold surface, presumably as the counter-ion to the negatively- charged, thiolate-covered AuNPs. Charge-transfer between the surfacesorbed thiolate and the AuNPs is demonstrated by the quenching of the OT UV-vis absorption when the OTs are adsorbed onto the AuNPs. Using a combination of surface enhanced Raman spectroscopy (SERS), density function calculations, and normal Raman spectroscopy, the pH dependence of mercaptobenzimadazole (MBI) adsorption onto AuNPs was systematically studied. By using the ratiometric SERS ligand quantification technique, MBI adsorption isotherms were constructed at three different pHs (1.4, 7.9, and 12.5). The Langmuir isotherms indicate that MBI thione has a higher saturation packing density (~­631 pmol/cm2) than MBI thiolate (~­568 pmol/cm2), but its binding constant (2.14 x 106 M-1) is about five times smaller than the latter (10.12 x 106 M-1). The work described in this dissertation provides a series of new insights into AuNP-OT interaction, and structure and properties of OTs on AuNPs.

Malondialdehyde (MDA) is the commonly accepted biomarker of lipid peroxidation. We reported the surface-enhanced Raman (SERS) detection of MDA using Thiobarbituric acid (TBA) as a molecular probe. The lowest concentration of HPLC purified TBA-MDA adduct that can be determined with reasonable signal to noise ratio is 0.45 nM. The specificity of the SERS technique has been demonstrated by comparing the SERS spectrum of TBA-MDA adduct with other TBA-aldehyde adducts. As a small organosulfur compound, TBA exhibits tremendous structural complexity. Discussed in this thesis is the drastic pH and concentration dependence of TBA SERS spectral features. To understand the origins of the TBA SERS spectral variations, UV-Vis spectra of TBA were also acquired under same experimental conditions of SERS. Density function calculations (DFT) were performed for different TBA tautomers with different charge states to facilitate the SERS spectral interpretation, allowing us to speculate the type of tautomers dominating the nanoparticle surfaces.

Benzoyl peroxide (BPO) is a common bleaching agent used in wheat flour. Due to its ability to damage existing nutrients in food and potential adverse effect to health, BPO have been strictly banned as a food additive in several countries and regions, such as China and Europe. However, the United States specifies that BPO is generally recognized as safe (GRAS). So, the WHO/FAO created a Codex Alimentarius Commission (CAC) to regulate the international BPO usage standard. According to the CAC, it is restricted at 75 mg/kg or parts per million (ppm). BPO is very unstable and easily converts to benzoic acid (BA), which places the analytical challenge for accurate BPO quantification. The objective of this study is to develop a reliable method for BPO quantification in flour. Raman spectroscopy was first explored to detect BPO and BA on an aluminum foil slide. The result showed BPO and BA produced distinct Raman peaks that can be discriminated against. However, the sensitivity was not satisfactory to reach the regulation limit. To improve sensitivity, surface-enhanced Raman spectroscopy (SERS) was applied using silver nanoparticles as the substrate. Although the signals did enhance significantly using SERS, the characteristic peaks of BPO disappeared as BPO converted to BA during the sample preparation. We then went back to Raman spectroscopy but focused on optimizing the sample preparation to enhance the signal intensity. Using a hydrophobic surface (i.e., parafilm) which can hold the droplet and minimize the spread, the Raman signal was enhanced significantly after repeating multiple droplets on the same surface. A standard curve was created for BPO from 25 ppm to 250 ppm and for BA from 250 ppm to 1000 ppm, respectively. To detect BPO in wheat flour, we applied a more advanced Raman imaging instrument and focused on the analysis of Raman maps instead of spectra for the analysis of effect flour matrix to BPO extraction and detection. We firstly tried an in situ method, which scanned the pellet of flour spiked with different amounts of BPO without extraction. However, we could not detect BPO at 0.1% or lower in flour samples. We then tried an extraction method using acetonitrile as the solvent, which showed a lower detection limit compared to the in situ method. However, this extraction method yielded inconsistent results for BPO that is under 0.05% in flour. The extraction method developed was further improved with an evaporating step and a C18 solid phase extraction (SPE) spin column. This improved the extraction efficacy and provided a roughly 60% recovery percentage for detecting BPO in wheat flour without decomposing into BA. In conclusion, we developed a simple sample preparation protocol coupled with Raman spectroscopy to quantify BPO in flour without converting to BA, which would meet the regulation requirement. This method also shortened the experiment time including both sample preparation and detection time compared to current methods.

Pathogenic microbial contamination of the environment poses a significant threat to human health. Accordingly, microbial surveillance is needed to ensure safe drinking water and air quality. Current analytical methods for microbes are generally either culture-based, gene amplification-based, or sequencing-based. However, these approaches require centralized facilities, well-trained personnel, and specialized instruments that result in high costs and long turnaround times. Surface-enhanced Raman spectroscopy (SERS)-based techniques have been proposed to overcome these limitations. In this dissertation, we discuss work conducted to develop novel SERS-based methods to enable both sensitive microbial quantification and analysis of the interactions of pathogens, their hosts, and the surrounding environment. We first developed a labeled SERS-based lateral flow test for virus quantification. Optimization of the lateral flow design and digital signal analysis enabled high sensitivity towards SARS-CoV-2. To elicit a comprehensive understanding of pathogen infection, label-free living-cell SERS sensors were engineered by incubating host cells with nanoparticles. SERS spectral changes in host cellular components and metabolites during infection were used for viral quantification and offered inherent insights into the temporal and spatial molecular-level mechanisms of infection. These biosensors were validated using bacteriophage Phi6 and then developed for infectious H1N1 influenza. To understand microbial survival in the environment, living-cell SERS methods were applied under various conditions. Results showed cell inactivation and antibiotic treatment induced significant cellular and metabolic responses in the living whole-cell sensors, implying their potential applicability to various environmental conditions. Our research achieves rapid and on-site pathogen quantification and infection mechanism identification. / Doctor of Philosophy / Pathogenic microbes, such as the SARS-CoV-2 virus, can spread through air and water and are potentially harmful to human health. Monitoring the concentrations of these microbes in the environment is crucial to track their presence and provide an early warning of their spread. Unfortunately, current microbial detection methods are often expensive and take a long time since they typically require professional facilities and expert elicitation. Our research relies on a technique called surface-enhanced Raman spectroscopy (SERS) to address these challenges. SERS enables identification and quantification of microbes by analyzing specific features (i.e., peak position, peak intensity) in the spectra. We first applied this technique by modifying a commercial SARS-CoV-2 antigen test kit with a label molecule that provides SERS signals. We achieve accurate and sensitive quantification, even in the presence of high levels of environmental interference. To better understand how these harmful microbes interact with our bodies, we developed sensors that can measure SERS signal changes in host cells before and after infection. These sensors were tested using the bacteriophage virus Phi6 that infects bacteria and infectious H1N1 influenza virus. Furthermore, we applied these sensors to study how bacteria respond to different environmental conditions, providing valuable insights into their survival and behavior under various conditions. In summary, our research introduces methods that are more accessible to identify and quantify harmful microbes that can be potentially used by the general public. The methods provide us with molecular level understanding of pathogen interactions with humans and the environment.

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