Quantification of biomolecules responsible for biomarkers in the surface-enhanced Raman spectra of bacteria using liquid chromatography-mass spectrometry.

Recently, specific biomarkers in the surface-enhanced Raman scattering (SERS) spectra of bacteria have been successfully exploited for rapid bacterial antibiotic susceptibility testing (AST) - dubbed SERS-AST. The biomolecules responsible for these bacterial SERS biomarkers have been identified as several purine derivative metabolites involved in bacterial purine salvage pathways (W. R. Premasiri, J. C. Lee, A. Sauer-Budge, R. Theberge, C. E. Costello and L. D. Ziegler, Anal. Bioanal. Chem., 2016, 408, 4631). Here we quantified these metabolites in the SERS spectra of Staphylococcus aureus and Escherichia coli using ultra-performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS). The time dependences of the concentrations of these molecules were measured using 13C- or 12C-purine derivatives as internal and external standards respectively in UPLC/ESI-MS measurements. Surprisingly, a single S. aureus and an E. coli cell were found to release millions of adenine and hypoxanthine into a water environment in an hour respectively. Furthermore, simulated SERS spectra of bacterial supernatants based on the mixtures of purine derivatives with measured concentrations also show great similarity with those of the corresponding bacterial samples. Our results not only provide a quantitative foundation for the emerging SERS-AST method but also suggest the potential of exploiting SERS for in situ monitoring the changes in bacterial purine salvage processes in response to different physical and chemical challenges.

Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers.

Rapid bacterial antibiotic susceptibility test (AST) and minimum inhibitory concentration (MIC) measurement are important to help reduce the widespread misuse of antibiotics and alleviate the growing drug-resistance problem. We discovered that, when a susceptible strain of Staphylococcus aureus or Escherichia coli is exposed to an antibiotic, the intensity of specific biomarkers in its surface-enhanced Raman scattering (SERS) spectra drops evidently in two hours. The discovery has been exploited for rapid AST and MIC determination of methicillin-susceptible S. aureus and wild-type E. coli as well as clinical isolates. The results obtained by this SERS-AST method were consistent with that by the standard incubation-based method, indicating its high potential to supplement or replace existing time-consuming methods and help mitigate the challenge of drug resistance in clinical microbiology.

Rapid detection of copper chlorophyll in vegetable oils based on surface-enhanced Raman spectroscopy.

The addition of copper chlorophyll and its derivatives (Cu-Chl) to vegetable oils to disguise them as more expensive oils, such as virgin olive oils, would not only create public confusion, but also disturb the olive oil market. Given that existing detection methods of Ch-Chl in oils, such as LC-MS are costly and time consuming, it is imperative to develop economical and fast analytical techniques to provide information quickly. This paper demonstrates a rapid analytical method based on surface-enhanced Raman spectroscopy (SERS) to detect Cu-Chl in vegetable oils; the spectroscopic markers of Cu-Chl are presented and a detection limit of 5 mg kg(-1) is demonstrated. The analysis of a series of commercial vegetable oils is undertaken with this method and the results verified by a government agency. This study shows that a SERS-based assessment method holds high potential for quickly pinpointing the addition of minute amounts of Cu-Chl in vegetable oils.

Looking into meta-atoms of plasmonic nanowire metamaterial.

Nanowire-based plasmonic metamaterials exhibit many intriguing properties related to the hyperbolic dispersion, negative refraction, epsilon-near-zero behavior, strong Purcell effect, and nonlinearities. We have experimentally and numerically studied the electromagnetic modes of individual nanowires (meta-atoms) forming the metamaterial. High-resolution, scattering-type near-field optical microscopy has been used to visualize the intensity and phase of the modes. Numerical and analytical modeling of the mode structure is in agreement with the experimental observations and indicates the presence of the nonlocal response associated with cylindrical surface plasmons of nanowires.

Custom-designed arrays of anodic alumina nanochannels with individually tunable pore sizes.

We demonstrate a process to selectively tune the pore size of an individual nanochannel in an array of high-aspect-ratio anodic aluminum oxide (AAO) nanochannels in which the pore sizes were originally uniform. This novel process enables us to fabricate arrays of AAO nanochannels of variable sizes arranged in any custom-designed geometry. The process is based on our ability to selectively close an individual nanochannel in an array by using focused ion beam (FIB) sputtering, which leads to redeposition of the sputtered material and closure of the nanochannel with a capping layer of a thickness depending on the energy of the FIB. When such a partially capped array is etched in acid, the capping layers are dissolved after different time delays due to their different thicknesses, which results in differences in the time required for the following pore-widening etching processes and therefore creates an array of nanochannels with variable pore sizes. The ability to fabricate such AAO templates with high-aspect-ratio nanochannels of tunable sizes arranged in a custom-designed geometry paves the way for the creation of nanophotonic and nanoelectronic devices.

Enhancing bright-field image of microorganisms by local plasmon of Ag nanoparticle array.

Inspecting biological cells with bright-field light microscopy often engenders a challenge, owing to their optical transparency. We show that imaging contrast can be greatly enhanced as yeast cells are placed on a silver nanoparticle array. Its near- and far-field traits, revealed by electrodynamic simulations, illustrate that the enhancement is attributed to the sensitivity of its plasmonic characteristics to the attached cells. This study demonstrates that the silver nanoparticle array can serve as the agent for concurrently enhancing Raman scattering and imaging contrast of microorganisms for identification and examination.

Label-free and culture-free microbe detection by three dimensional hot-junctions of flexible Raman-enhancing nanohybrid platelets.

Novel nanohybrid arrays of silver (Ag)-on-silicate platelets with flexibility and three-dimensional (3D) hot-junctions (particularly in z-direction) were discovered for improving the stability of free nanoparticles and the mobility of rigid (glass or silicon-based) substrates in surface-enhanced Raman scattering (SERS) detection technology. Since the Ag nanoparticles are adsorbed on both sides of few nanometer-thick silicate platelets (single-layer exfoliated clay), the geometric arrangement of Ag on both sides of the nanoplatelets (Ag/NSP) may induce strong hot-junctions (z-direction) in reference to the pristine montmorillonite clay (multi-layers) at the thickness of ∼20 nm, measured by small molecules (adenine of DNA) and bacteria (S. aureus). Enormous red-shifts (16 nm wavelength difference) were observed between single layer and multi-layer silicate platelets, showing that huge surface plasmon enhancement comes from hot junctions in the z-direction (∼7 times higher than 2D hot-junctions of traditional SERS biochips). Further, the Ag/NSP SERS substrate displays a free floating mobility and optical transparency (less background interference), which inherently increase the contacted surface-area between the substrate and microorganisms, to enhance the SERS sensitivity. The surface modulation with a surfactant could be complimentary towards a variety of microorganisms including hydrophobic microbes, irregular-shaped microorganisms and larger biological cells due to their mutual specific surface interactions. It was anticipated to apply in the rapid detection for varied microbes with label-free and culture-free characterizations.

Automated quantitative analysis of lipid accumulation and hydrolysis in living macrophages with label-free imaging.

The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R(2) > 0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K(i). Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders.

Functionalized arrays of Raman-enhancing nanoparticles for capture and culture-free analysis of bacteria in human blood.

Detecting bacteria in clinical samples without using time-consuming culture processes would allow rapid diagnoses. Such a culture-free detection method requires the capture and analysis of bacteria from a body fluid, which are usually of complicated composition. Here we show that coating Ag-nanoparticle arrays with vancomycin (Van) can provide label-free analysis of bacteria via surface-enhanced Raman spectroscopy (SERS), leading to a ~1,000-fold increase in bacteria capture, without introducing significant spectral interference. Bacteria from human blood can be concentrated onto a microscopic Van-coated area while blood cells are excluded. Furthermore, a Van-coated substrate provides distinctly different SERS spectra of Van-susceptible and Van-resistant Enterococcus, indicating its potential use for drug-resistance tests. Our results represent a critical step towards the creation of SERS-based multifunctional biochips for rapid culture- and label-free detection and drug-resistant testing of microorganisms in clinical samples.

Transparent Raman-enhancing substrates for microbiological monitoring and in situ pollutant detection.

Opaque Raman-enhancing substrates made of Ag nanoparticles on incompletely oxidized aluminum templates have been rendered transparent by an ion-drift process to complete the oxidation. The result shows that the transparent substrates exhibit high/uniform surface-enhanced Raman scattering (SERS) capability and good optical transmissivity, allowing for concurrent SERS characterization and high contrast transmission-mode optical imaging of S. aureus bacteria. We also demonstrate that the transparent substrates can used in conjunction with optical fibers as SERS sensors for in situ detection of malachite green down to 10(-9) M.

Identical-length nanowire arrays in anodic alumina templates.

Arrays of nanowires with identical length are fabricated by using ultrasound to remove the length fluctuation among nanowires, which are deliberately grown in burette-shaped nanochannels on an anodic anumina film. The process allows the fabrication of 10 micron Ag-nanowire arrays with length fluctuation as small as 0.09%. By integrating the process with a focused-ion-beam-based lithographic method to grow nanowires into selective nanochannels in an array, we fabricate arrays of uniform-length nanowires that are arranged in a custom-designed lateral geometry. The ability to fabricate such artificial nanomaterials paves the way for the exploitation of their unusual optical, electrical, and thermal properties.

A high speed detection platform based on surface-enhanced Raman scattering for monitoring antibiotic-induced chemical changes in bacteria cell wall.

Rapid and accurate diagnosis for pathogens and their antibiotic susceptibility is critical for controlling bacterial infections. Conventional methods for determining bacterium's sensitivity to antibiotic depend mostly on measuring the change of microbial proliferation in response to the drug. Such "biological assay" inevitably takes time, ranging from days for fast-growing bacteria to weeks for slow-growers. Here, a novel tool has been developed to detect the "chemical features" of bacterial cell wall that enables rapid identification of drug resistant bacteria within hours. The surface-enhanced Raman scattering (SERS) technique based on our newly developed SERS-active substrate was applied to assess the fine structures of the bacterial cell wall. The SERS profiles recorded by such a platform are sensitive and stable, that could readily reflect different bacterial cell walls found in Gram-positive, Gram-negative, or mycobacteria groups. Moreover, characteristic changes in SERS profile were noticed in the drug-sensitive bacteria at the early period (i.e., approximately 1 hr) of antibiotic exposure, which could be used to differentiate them from the drug-resistant ones. The SERS-based diagnosis could be applied to a single bacterium. The high-speed SERS detection represents a novel approach for microbial diagnostics. The single-bacterium detection capability of SERS makes possible analyses directly on clinical specimen instead of pure cultured bacteria.

Light scattering from 2D arrays of monodispersed Ag-nanoparticles separated by tunable nano-gaps: spectral evolution and analytical analysis of plasmonic coupling.

Two dimensional arrays of monodispersed Ag-nanoparticles separated by different gaps with sub-10 nm precision are fabricated on anodic alumina substrates with self-organized pores. Light scattering spectra from the arrays evolve with the gaps, revealing plasmonic coupling among the nanoparticles, which can be satisfactorily interpreted by analytical formulae derived from generic dipolar approximation. The general formulism lays down a foundation for predicting the Q factor of an array of metallic nano-particles and its geometric characteristics.