Huai-Hsien Wang

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

Summary form only given. Detecting bacteria in clinical samples without the time-consuming culture process is most desired for rapid diagnosis. Such a culture-free detection needs to capture and analyze bacteria from a body fluid usually containing complicate constituents. Here we show that vancomycin (Van) coating of a special substrate with arrays of Ag-nanoparticles, which can provide label-free analysis of bacteria via surface enhanced Raman spectroscopy (SERS), leads to 1000 folds increase in its capability to capture bacteria without introducing significant spectral interference. Bacteria spiked in 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 test. Our results represent a critical step towards the creation of SERS-based multifunctional biochips for rapid culture/label-free detection and drug-resistant testing of microorganisms in clinical samples.

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.

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.

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.

Ideally ordered 10 nm channel arrays grown by anodization of focused-ion-beam patterned aluminum

Arrays of ideally ordered alumina nanochannels with unprecedented ∼10nm pore size, 40–50 nm interpore spacing, and improved channel uniformity have been fabricated by anodizing an aluminum substrate with a guiding pattern on its surface. The pattern is an array of hexagonally close-packed concaves fabricated by focused ion beam direct sputtering; and its lattice constant is carefully matched to the conditions of the subsequent anodization process in order to achieve effective guiding in the growth of the nanochannels and therefore the ordering of an array.

Long-range ordered nanoaperture array with uniform diameter and interpore spacing

In application of focused-ion-beam lithography and grazing Ar+ milling on the U-shape barrier layer of anodic alumina nanochannels, we fabricated a hexagonally symmetry aperture array with nominal diameter of 12±2nm and interspacing of 100±2nm. Besides long-range spatial ordering, the focused-ion-beam guided-grown process has also significantly improved uniformity of both the interpore spacing and the aperture size. This aperture array membrane can be applied to the fabrication of nanostructures, such as a lithographic contact mask for ordered quantum-dot array.

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.

AbstractThe 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 (R2 > 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, Ki. 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. FigureAutomated quantitative analysis for the label-free detection of lipid content in living cells

Fabrication of an ordered nanoparticle array with a nanoaperture membrane used as a contact-mask

In this paper, we provide a useful technology to fabricate a long-range ordered nanoparticle array with a feature size under 30 nm. By adjusting the incident angle of Ar + beam milling on a U-shaped barrier layer of anodic alumina oxide, we can create a long-range ordered nanoaperture array with samples prepared by a focused-ion-beam-guided process. Compared to the naturally self-organized alumina nanochannels, the FIB-guided process has increased long-range ordering and uniformity of aperture size, and the aperture size can be varied by changing the grazing angle. The nanoaperture membrane can be used as a contact-mask and its undercut structure has another advantage for nanolithography. This technique could be extensively applied to the manufacturing of advanced nanodevices in large areas and as a catalyst to fabricate one-dimensional nanosized materials. (Some figures in this article are in colour only in the electronic version)

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.