Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions

Microbial contamination is not only a medical problem, but also plays a large role in pharmaceutical clean room production and food processing technology. Therefore many techniques were developed to achieve differentiation and identification of microorganisms. Among these methods vibrational spectroscopic techniques (IR, Raman and SERS) are useful tools because of their rapidity and sensitivity. Recently we have shown that micro-Raman spectroscopy in combination with a support vector machine is an extremely capable approach for a fast and reliable, non-destructive online identification of single bacteria belonging to different genera. In order to simulate different environmental conditions we analyzed in this contribution different Staphylococcus strains with varying cultivation conditions in order to evaluate our method with a reliable dataset. First, micro-Raman spectra of the bulk material and single bacterial cells that were grown under the same conditions were recorded and used separately for a distinct chemotaxonomic classification of the strains. Furthermore Raman spectra were recorded from single bacterial cells that were cultured under various conditions to study the influence of cultivation on the discrimination ability. This dataset was analyzed both with a hierarchical cluster analysis (HCA) and a support vector machine (SVM).     

Characterization of the surface enhanced raman scattering (SERS) of bacteria

The surface enhanced Raman scattering (SERS) of a number of species and strains of bacteria obtained on novel gold nanoparticle (approximately 80 nm) covered SiO(2) substrates excited at 785 nm is reported. Raman cross-section enhancements of >10(4) per bacterium are found for both Gram-positive and Gram-negative bacteria on these SERS active substrates. The SERS spectra of bacteria are spectrally less congested and exhibit greater species differentiation than their corresponding non-SERS (bulk) Raman spectra at this excitation wavelength. Fluorescence observed in the bulk Raman emission of Bacillus species is not apparent in the corresponding SERS spectra. Despite the field enhancement effects arising from the nanostructured metal surface, this fluorescence component appears "quenched" due to an energy transfer process which does not diminish the Raman emission. The surface enhancement effect allows the observation of Raman spectra of single bacterial cells excited at low incident powers and short data acquisition times. SERS spectra of B. anthracis Sterne illustrate this single cell level capability. Comparison with previous SERS studies reveals how the SERS vibrational signatures are strongly dependent on the morphology and nature of the SERS active substrates. The potential of SERS for detection and identification of bacterial pathogens with species and strain specificity on these gold particle covered glassy substrates is demonstrated by these results.     

Identification and classification of respiratory syncytial virus (RSV) strains by surface-enhanced Raman spectroscopy and multivariate statistical techniques

There is a critical need for a rapid and sensitive means of detecting viruses. Recent reports from our laboratory have shown that surface-enhanced Raman spectroscopy (SERS) can meet these needs. In this study, SERS was used to obtain the Raman spectra of respiratory syncytial virus (RSV) strains A/Long, B1, and A2. SERS-active substrates composed of silver nanorods were fabricated using an oblique angle vapor deposition method. The SERS spectra obtained for each virus were shown to posses a high degree of reproducibility. Based on their intrinsic SERS spectra, the four virus strains were readily detected and classified using the multivariate statistical methods principal component analysis (PCA) and hierarchical cluster analysis (HCA). The chemometric results show that PCA is able to separate the three virus strains unambiguously, whereas the HCA method was able to readily distinguish an A2 strain-related G gene mutant virus (ΔG) from the A2 strain. The results described here demonstrate that SERS, in combination with multivariate statistical methods, can be utilized as a highly sensitive and rapid viral identification and classification method.     

Towards a receptor-free immobilization and SERS detection of urinary tract infections causative pathogens

Based on molecular-specific surface-enhanced Raman scattering (SERS) spectroscopy we were able to discriminate between rough and smooth strains of Escherichia coli and Proteus mirabilis bacteria. For this purpose, bacteria have been immobilized through electrostatic forces by inducing a positive charge on the glass slide. This way, SERS spectra on bacterial biomass and also on single bacteria could be recorded in less than 2 h, by using concentrated silver nanoparticles as SERS-active substrate. Single-bacterium SERS spectral fingerprints showed to be sensitive to the presence of the O-antigen at strain level and to the microorganisms growth phase. By using principal component analysis (PCA) on the SERS spectra recorded from E. coli and P. mirabilis, these two uropathogens could be fairly discriminated.     

Intracellularly grown gold nanoislands as SERS substrates for monitoring chromate,sulfate and nitrate localization sites in remediating bacteria biofilms by Raman chemical imaging

Understanding the chemical composition of biofilm matrices is vital in different fields of biology such as surgery, dental medicine, synthetic grafts and bioremediation. The knowledge of biofilm development, composition, active reduction sites and remediation efficacy will help in the development of effective solutions and evaluation of remediating approaches prior to implementation. Surface-enhanced Raman spectroscopy (SERS) based imaging is an invaluable tool to obtain an understanding of the remediating efficacy of microorganisms and its role in the formation of organic and inorganic compounds in biofilms. We demonstrate for the first time, the presence of chromate, sulfate, nitrate and reduced trivalent chromium in soil biofilms. In addition, we demonstrate that SERS imaging was able to validate two observations made by previous studies on chromate/sulfate and chromate/nitrate interactions in Shewanella oneidensis MR-1 biofilms. Additionally, we show a detailed Raman mapping based evidence of the existence of chromate–sulfate competition for cellular entry. Subsequently, we use Raman mapping to study the effect of nitrate on chromate reduction. The findings presented in this paper are among the first to report – detection of multiple metallic ions in bacterial biofilms using intracellular SERS substrates. Such a detailed characterization of biofilms using gold nanoislands based SERS mapping substrate can be extended to study cellular localization of other metallic ions and chemical species of biological and toxicological significance and their effect on reduction reactions in bacterial biofilms.     

Towards single-microorganism detection using surface-enhanced Raman spectroscopy

The identification and discrimination of microorganisms is important not only for clinical reasons but also for pharmaceutical clean room production and food-processing technology. Vibrational spectroscopy such as IR, Raman, and surface-enhanced Raman scattering (SERS) can provide a rapid ‘fingerprint’ on the chemical structure of molecules and is used to obtain a ‘fingerprint’ from microorganisms as well. Because of the requirement that a single bacterium cell and noble metal nanoparticles must be in close contact and the lack of a significant physical support to hold nanoparticles around the single bacterium cell, the acquisition of SERS spectra for a single bacterium using colloidal nanoparticles could be a challenging task. The feasibility of SERS for identification down to a single bacterium is investigated. A Gram-negative bacterium, Escherichia coli, is chosen as a model for the investigation. Because the adsorption of silver nanoparticles onto the bacterial cell is an exclusive way for locating nanoparticles close to the bacterium cell, the absorption characteristics of silver nanoparticles with different surface charges are investigated. It is demonstrated that the citrate-reduced colloidal silver solution generates more reproducible SERS spectra. It is found that E. coli cells aggregate upon mixing with silver colloidal solution, and this may provide an additional benefit in locating the bacterial cell under a light microscope. It is also found that a laser wavelength in the UV region could be a better choice for the study due to the shallow penetration depth. It is finally shown that it is possible to obtain SERS spectra from a single cell down to a few bacterial cells, depending on the aggregation properties of bacterial cells for identification and discrimination.     

Time fluctuations and imaging in the SERS spectra of fungal hypha grown on nanostructured substrates

Surface-enhanced Raman scattering (SERS) spectroscopy is an emerging technique in biomolecular analysis that can have a tremendous impact in the life sciences. We report on the SERS imaging of fungal hyphae grown on nanostructured SERS active substrates engineered using semiconductor technologies. Time fluctuations in the intensity and band position in the SERS spectra measured on the same sample position with 1 s integration time have been observed indicating that the SERS signal arises from a limited number of molecules and that possibly single components are being detected.     

FT-Raman, FTIR and surface-enhanced Raman spectroscopy of the antiviral and antiparkinsonian drug amantadine

FT-Raman, FTIR and surface-enhanced Raman spectroscopy (SERS) are applied to the vibrational characterization of the antiviral and antiparkinsonian drug amantadine. SERS spectroscopy is employed for the first time for characterizing the interfacial behavior of this molecule and to study its interaction with colloidal silver. The comparison of SERS spectrum with the Raman spectra of amantadine in solid state and in aqueous solution reveals remarkable changes attributed to the interaction of the drug with the metal through the unprotonated amino group and the formation of a self-assembled amantadine layer on the metal surface. A tentative assignment of the obtained vibrational spectra is carried out on the basis of the vibrational spectra of the structurally related molecules adamantane and tert-buthylamine and the ab initio calculations accomplished for amantadine.     

Raman and Surface-enhanced Raman Scattering of Chlorophenols

Raman spectrum is a powerful analytical tool for determining the chemical information of compounds. In this study, we obtained analytical results of chlorophenols(CPs) molecules including 4-chlorophenol(4-CP), 2,6-dich- lorophenol(2,6-DCP) and 2,4,6-trichlorophenol(2,4,6-TCP) on the surface of Ag dendrites by surface-enhanced Raman scattering(SERS) spectra. SEM images indicate that the SERS substrate of Ag dendrites is composed of a large number of polygonal nanocrystallites, which self-assembled into a 3D hierarchical structure. It was found that there were distinct differences for those three molecules from Raman and SERS spectra. This indicates that SERS could be a new tool of detection technique regarding trace amounts of CPs.     

一步法制备银溶胶用于临床嗜麦芽窄食单胞菌的表面增强拉曼光谱快速检测

该文利用一步法快速制备银溶胶,实现了基底的现用现制。对嗜麦芽窄食单胞菌进行表面增强拉曼散射(SERS)检测,得到18组拉曼特征峰的生物指纹谱图。方法能够检测到的最低菌悬液浓度为5×106菌落形成单位(CFU)/mL,重现性的相对标准偏差为6.8%~16%,满足生物医学领域定性分析的基本要求。采用该方法获得的阳性病人血液中的病原细菌细胞的拉曼信号与纯培养菌株接近,进一步证明这种快速(10 min)、简单、便携式和低成本的方法在临床诊断中具有广阔的应用前景。     

青蒿素的Raman光谱研究

本文对固态青蒿素的FT-Raman光谱和普通Raman光谱以及低浓度(1×10^-^5mol/L)青蒿素水溶液的表面增强Raman散射(SERS)光谱进行了检测, 着重观察了1756cm^-^1六元内酯环振动谱带, 发现其在FT-Raman和普通Raman光谱中表现为强振动, 而在SERS光谱中此振动谱带消失, 说明分子内酯环发生了破裂。根据1756cm^-^1谱带的变化以及724cm^-^1过氧基团振动频率的位移, 对青蒿素在银表面上的吸附取向进行了研究。还研究了青蒿素与氯高铁血红素的作用情况, 发现两者作用后, 明显改变了青蒿素在银表面上的吸附。     

Excitation wavelength dependent surface-enhanced Raman spectra of a dipping film of azobenzene-containing long-chain fatty acid on a silver mirror

Surface-enhanced Raman scattering (SERS) of dipping films of azobenzene-containing long-chain fatty acids, nAmH (n=8, 12, m=3, 5), on silver mirrors measured with a wide range of excitation wavelengths in the 457.9-1064 nm region is reported. The obtained Raman spectra show great SERS effect even with the 1064 nm excitation, and the excitation with 457.9, 476.5, and 488.0 nm gives surface-enhanced resonance Raman scattering (SERRS) due to the resonance effect of the symmetry-forbidden n-pi* transition of the azo group. Of particular note in the present study is that the SERS spectra with the excitation in the 532-1064 nm region yield Raman bands whose frequencies are almost identical to those bands in Raman spectra of nAmH in solid state while the SERRS spectra with the excitation in the 457.9-514.5 nm region show not only a set of bands which correspond to those of nAmH in the solid state but also a set of bands whose frequencies show a significant shift from those of the bands of nAmH in the solid state. These observations lead us to conclude that there are two kinds of molecular aggregates in the dipping films of azobenzene-containing long-chain fatty acid in which azobenzene moieties are condensed to form small bundles.     

Raman and SERS investigation of isolated sp carbon chains

Carbon chains with sp hybridization and a known distribution of lengths have been studied by Raman and Surface Enhanced Raman Scattering (SERS). With the support of density functional theory simulations the observed Raman features have been assigned to active modes of individual chains. SERS spectra show remarkable differences, attributed to the interaction between sp chains and Ag nanoparticles used as a SERS active medium. The results obtained for this model system permit in principle to explain a large number of Raman data from molecular and solid sp/sp² carbon systems.     

银纳米粒子簇的设计、 制备和表面增强拉曼光谱

通过匹配激光光斑直径与胶体微球的尺寸, 设计制备了银纳米粒子的表面增强拉曼散射(SERS)基底, 并将其用于研究单个银纳米粒子簇的表面增强拉曼光谱. 在制备纳米粒子的过程中, 考察了等离子体刻蚀时间与银沉积厚度对“单”银纳米粒子结构与形貌的影响. 将吡啶、 巯基苯和罗丹明R6G作为SERS探针分子, 研究了其SERS效应, 通过荧光共振能量转移(FRET)机理, 实现了染料分子在单银纳米粒子簇上的SERS效应. SERS光谱测试与相关计算结果表明, 单个银纳米粒子簇的拉曼增强因子能够达到约10⁶.     

Raman and surface Raman spectroscopy with ultraviolet excitation

We record the accurate and reliable Raman spectra of benzoic acid (BA), p-nitrobenzoic acid (PNBA) and o-nitrobenzoic (ONBA) in aqueous solution with ultraviolet excitation. And we find that the ultraviolet (UV) Raman spectrum of aqueous BA solution has one-to-one correspondence to that of BA solid whereas the others are less resemble to the solid counterparts. We also report surface Raman spectroscopy of them in silver colloid without any enhancement in UV region and call it surface-unenhanced Raman spectroscopy (SUERS) while the surface-enhanced Raman scattering (SERS) effects are perfect in near infrared or visible regions. It demonstrates the SERS effects are strongly dependent on the excitation wavelength. On the basis of the experiments, we discuss the mechanism of SERS excited in different regions.     

Studies of sample preparation for surface-enhanced raman spectrometry on silver hydrosols

Surface-enhanced Raman spectra (SERS) on silver hydrosols of p-nitrobenzoic acid, p-aminobenzoic acid and aniline are reported. Several theoretical and practical aspects of the hydrosol preparation protocols and sample preparation procedures, and their effects on the sensitivity and reproducibility of the Raman signals are discussed. The effect of acidity on SERS signal intensity is shown to depend on the time of observation of the Raman spectra, illustrating the relevance of time to quantitative SERS data. the identification power of SERS at trace levels for closely related compounds (p-nitrobenzoic acid, p-aminobenzoic acid and aniline) is illustrated.     

Raman spectroscopic studies of terthiophenes for molecular electronics

The effect of thiol and selenol functionalization on the vibrational spectra and photochemical stability of terthiophene based molecular wires was investigated using surface-enhanced Raman scattering (SERS). The molecules were found to exhibit markedly different properties at the silver surface of the SERS substrate, despite having almost identical Raman spectra in solution and in the solid state. In contrast to terthiophene (3T), the bisthiolterthiophene (T3) and biselenol-terthiophene (Se3) molecules were stable against photoinduced structural changes when adsorbed to the metal surface at low concentrations. This indicates that the strong bonds to the silver surface, via S or Se terminal atoms, leads to a rapid decay of photoexcited states. Comparison with ab initio calculations shows that both T3 and Se3 bind with only one of the functional groups to the Ag surface.     

Controllable growth of silver nanostructures by a simple replacement reaction and their SERS studies

Hierarchical silver nanostructures, consisting of dendritic (symmetric branched) and fractal patterns (randomly ramified), were synthesized very easily by dropping a droplet of AgNO₃-HF solution on silicon wafers. Scanning electron microscopy (SEM), X-ray diffraction (XRD) and open circuit potential-time (Ocp-t) measurement demonstrated that the two nanostructures converted with the reaction composition. The structural evolution was tentatively explained with the theory that oriented growth was determined by the anisotropy of the solid–liquid interfacial energy and the oriented attachment-based aggregation mechanism. Results on surface-enhanced Raman scattering (SERS) signals of the silver films with hierarchical nanostructures demonstrate that SERS is sensitive to silver nanostructures.     

水溶液中碳纳米管的表面增强拉曼光谱研究

合成了碳纳米管和金纳米颗粒的复合物, 测量了水溶液相中复合物的表面增强拉曼光谱, 结果表明, 碳纳米管的巯基化修饰可以提高碳纳米管与金纳米颗粒复合的效率, 随着金纳米颗粒负载量的增加, 碳纳米管的拉曼信号逐渐增强. 加入己二胺分子可以减小金纳米颗粒之间的距离使表面增强效应更显著, 碳纳米管的拉曼光谱得到进一步的增强. 还可进一步在复合体系中加入对巯基苯胺和罗丹明B等小分子拉曼探针, 利用金纳米颗粒的表面增强效应, 这种多元复合体系有望作为多通道拉曼成像探针材料.     

Inherent complexities of trace detection by surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) are powerful optical scattering techniques used in such frontier areas of research as ultrasensitive chemical analysis, the characterization of nanostructures, and the detection of single molecules. However, measuring and, most importantly, interpreting SERS/SERRS spectra can be incredibly challenging. This is the result of modifications to the measured spectra that are due to of a variety of instabilities and contributions. These interferences and modifications arise from the nature of the enhancement itself, as well as the conditions used to attain SERS spectra. The present report is an attempt to collect in one place the analytical interferences that are most commonly found during the collection of SERS/SERRS spectra.