Fabrication and Characterization of Planar Plasmonic Substrates with High Fluorescence Enhancement

The use of plasmonic nanostructures for fluorescence signal amplification is currently a very active research field. The detection of submonolayers of proteins labeled with organic dyes is a widely used technique in surface-based immunoassays and DNA hybridization. There is a strong interest in the development of new optical and chemical methods to increase the signal from ultralow concentrations of dyes on the surface of sensor substrates. Herein, we have explored the possibility of using vacuum-deposited silver nanostructures on dielectric layers and silver mirrors as potential plasmonic substrates that effectively amplify fluorescence over a broad spectral range. By optimizing deposition parameters for dielectric layers and silver nanostructures and applying thermal annealing processes, we observed large fluorescence amplifications from three different dye-strept(avidin) conjugates: about 7-fold for a UV/blue dye AF350-Av, 49-fold for a blue-green dye AF488-SA, and up to 208-fold for red-emitting AF647-SA dye. The observed amplification factors for the ensemble of fluorophores are very promising for development of surface-based bioassays. These substrates can be prepared using simple vacuum deposition in which we circumvent using the expensive nanofabrication methods. In addition, unlike most nanofabrication methods, the present approach is appropriate for large scale fabrication of substrates with microscope slide surface area suitable for sensing applications.     

Automatic white matter lesion segmentation using an adaptive outlier detection method

White matter (WM) lesions are diffuse WM abnormalities that appear as hyperintense (bright) regions in cranial magnetic resonance imaging (MRI). WM lesions are often observed in older populations and are important indicators of stroke, multiple sclerosis, dementia and other brain-related disorders. In this paper, a new automated method for WM lesions segmentation is presented. In the proposed method, the presence of WM lesions is detected as outliers in the intensity distribution of the fluid-attenuated inversion recovery (FLAIR) MR images using an adaptive outlier detection approach. Outliers are detected using a novel adaptive trimmed mean algorithm and box-whisker plot. In addition, pre- and postprocessing steps are implemented to reduce false positives attributed to MRI artifacts commonly observed in FLAIR sequences. The approach is validated using the cranial MRI sequences of 38 subjects. A significant correlation (R=0.9641, P value=3.12×10(-3)) is observed between the automated approach and manual segmentation by radiologist. The accuracy of the proposed approach was further validated by comparing the lesion volumes computed using the automated approach and lesions manually segmented by an expert radiologist. Finally, the proposed approach is compared against leading lesion segmentation algorithms using a benchmark dataset.     

Distance-dependent metal-enhanced fluorescence from Langmuir-Blodgett monolayers of alkyl-NBD derivatives on silver island films

We described the effect of fluorophore distance from the silver island films (SIFs) on the metal-enhanced fluorescence (MEF) from two newly developed long-chain nitrobenzoxadiazole derivatives (NBD-C16 and NBD-C18). The well-established Langmuir-Blodgett technique is used to deposit the fluorophores at defined distances from the SIFs surface, and an inert amphiphilic stearic acid is used to control the distance. NBD probes deposited directly on the SIFs surface show the highest metal-enhanced fluorescence of approximately 32-fold, and both of the probes that were studied show a consistent decrease in metal-enhanced fluorescence when increasing the distance from the fluorophore to the SIFs surface. The lowest fluorescence enhancement of approximately 4-fold is observed for the probes located 90 nm from the SIFs surface. Additionally, we also have noticed the shortest fluorescence lifetimes for the NBD probes deposited directly onto the SIFs surface, and the lifetimes are consistently increased when increasing the distances between the fluorophore and SIFs surfaces. These contrasting spectral changes, enhanced fluorescence, and decreased fluorescence lifetimes are in accordance with an increase in the rate of radiative decay for fluorophores near the silver particles. The present study provides significant information on the effect of fluorophore distance on the metal-enhanced fluorescence phenomenon.     

Langmuir-Blodgett monolayers of long-chain NBD derivatives on silver island films: Well-organized probe layer for the metal-enhanced fluorescence studies

The metal-enhanced fluorescence (MEF) from the well-organized monolayers of two newly prepared long-chain alkylamine derivatives of nitrobenzoxadiazole (NBD-C16 and NBD-C18) deposited on silver island films (SIFs) has been investigated. The NBD derivatives were conveniently prepared by using a single step procedure in quantitative yields. The monolayers of the probes on SIFs as well as on bare-glass slides were obtained by using the Langmuir-Blodgett (LB) technique. Orientation of the NBD probe molecule in LB monolayer film was measured with polarized absorption spectroscopy. The observed tilt angle of the probe transition dipole moment with respect to the surface normal of approximately 67-68 degrees was evaluated. We observed that the NBD monolayers deposited in close proximity to silver islands show about a 16-fold increase in fluorescence intensity and shortened fluorescence lifetime compared to those on bare-glass, which are due to metal enhanced fluorescence. On the other hand, the corresponding MEF from randomly oriented film obtained by using spin coating of the probes on SIFs was only 2.5-fold. Further, we deposited mixed monolayers of NBD-C16 or NBD-C18 with various molar ratios of stearic acid to understand the polarity effect on MEF. Interestingly, we found a consistent increase in MEF efficiency with increasing molar ratio of stearic acid. Along with MEF, we also found a continuous blue-shift in emission band maxima of the probes with an increasing molar ratio of stearic acid. The observed increase in MEF efficiency is justified based on cooperative effects of (1) the modulations in electronic density of the surface plasmon absorption band of SIFs and (2) defined probe orientation that might lead to preferential excitation.     

A Glucose Sensing Contact Lens: A Non-Invasive Technique for Continuous Physiological Glucose Monitoring

We have developed a range of glucose sensing contact lenses, using a daily, disposable contact lens embedded with newly developed boronic acid containing fluorophores. Our findings show that our approach may be suitable for the continuous monitoring of tear glucose levels in the range 50–1000 M, which typically track blood glucose levels, which are 5–10 fold higher. Our non-invasive approach may well offer an alternative solution to current invasive glucose monitoring techniques for diabetes, such as finger pricking.     

Wavelength–Ratiometric Probes for the Selective Detection of Fluoride Based on the 6-Aminoquinolinium Nucleus and Boronic Acid Moiety

Herein we report a set of new water-soluble fluorescent probes (N-boronobenzyl-6-aminoquinolinium bromides, BAQBAs) sensitive to aqueous fluoride. These probes shows spectral shifts and intensity changes in the presence of fluoride, in a wavelength ratiometric and colorimetric manner, enabling the detection of fluoride concentrations at visible wavelengths, in the concentration range approximately 1-300 mM. Although the sensing mechanism is different for fluoride as compared to the other halides, we have tested the utility of these probes towards the other halides, and the results reveal that the BAQBAs are in fact potential candidates towards the sensing of the all the halides, but in different concentration ranges. As the probes are based on the boronic acid moiety, which is a well-known fluoride and sugar chelator group, we have investigated the response of sugars (such as glucose and fructose, which are present in biological fluids and foodstuffs) as interferences in fluoride detection using these probes. Interestingly, the BAQBAs show a suppressed sugar response potentially allowing for the predominant fluoride sensitivity. In addition to physiological sugars, we also have assessed the response of aqueous halides as potential interferents, or indeed analytes to be sensed, and show that the new boronic acid containing probes respond well to aqueous fluoride in the presence of a high background of other species, such as in a biological cocktail of 50 mM Glucose, 50 mM aqueous Chloride and 5 mM Fructose.     

Fluorescence sensors for monosaccharides based on the 6-methylquinolinium nucleus and boronic acid moiety: potential application to ophthalmic diagnostics

Continuous monitoring of glucose levels in human physiology is important for the long-term management of diabetes. New signaling methods/probes may provide an improved technology to monitor glucose and other physiologically important analytes. The glucose sensing probes, BMQBAs, fabricated using the 6-methylquinolinium moiety as a fluorescent indicator, and boronic acid as a chelating group, may have versatile applications in glucose sensing because of their unique properties. In this paper we discuss the design logic, synthesis, characterization and spectral properties of three new isomeric glucose sensors (BMQBAs), and a control compound (BMQ) in the presence and absence of sugars. The sensing ability of the new probes is based on a charge neutralization and stabilization mechanism upon sugar binding. The new probes have attractive fluorescence quantum yields, are highly water-soluble, and have spectral characteristics compatible with cheap and portable LEDs and LDs. One of the probes, o-BMQBA, has a sugar bound pK_a of 6.1, and a dissociation constant K_D of 100 mM glucose. These probes have been designed specifically to respond to tear glucose in a contact lens polymer for ophthalmic glucose monitoring, where the reduced sugar bound pK_a affords for sensing, in a lens environment that we have previously shown to be mildly acidic.     

Enhanced fluorescence cyanide detection at physiologically lethal levels: reduced ICT-based signal transduction

Three water-soluble fluorescent probes have been specifically designed to determine free cyanide concentrations up to physiologically lethal levels, >20 microM. The probes have been designed in such a way as to afford many notable sensing features, which render them unique with regard to signal transduction, photophysical characteristics, and their application to physiological cyanide determination and safeguard. The probes are readily able to reversibly bind free aqueous cyanide with dissociation constants around 4 microM3. Subsequent cyanide binding modulates the intramolecular charge transfer within the probes, a change in the electronic properties within the probes, resulting in enhanced fluorescence optical signals as a function of increased solution cyanide concentration. The ground-state chelation with cyanide produces wavelength shifts, which also enable the probes to sense cyanide in both an excitation and emission ratiometric manner, in addition to enhanced fluorescence signaling. This has enabled a generic cyanide sensing platform to be realized that is not dependent on fluorescent probe concentration, probe photodegradation, or fluctuations in the intensity of any employed excitation sources, ideal for remote cyanide sensing applications. Further, the >600 nm fluorescence emission of the probes potentially allows for enhanced fluorescence ratiometric cyanide sensing in the optical window of tissues and blood, facilitating their use for the transdermal monitoring of cyanide for mammalian safeguard or postmortem in fire victims, both areas of active research.