Detection of 100 aM fluorophores using a high-sensitivity on-chip CE system and transient isotachophoresis

We present a highly sensitive capillary electrophoresis (CE) assay that combines transient, single-interface on-chip isotachophoresis (ITP) and a laser-induced confocal fluorescence detection setup. We performed experimental parametric studies to show the effects of microscope objective specifications and intensity of excitation laser on optimization of a high-sensitivity on-chip CE detection system. Using the optimized detection system, single-molecule detection of Alexa Fluor 488 was demonstrated, and signal data were validated with autocorrelation analysis. We also demonstrated a separation and detection of 100 aM fluorophores (Alexa Fluor 488 and bodipy) in a fast assay using a high-sensitivity on-chip CE detection system and an ITP/CE protocol with no manual buffer exchange steps. This is, to the knowledge of the authors, the highest electrophoretic separation sensitivity ever reported.     

Genetic engineering of an allosterically based glucose indicator protein for continuous glucose monitoring by fluorescence resonance energy transfer

Real-time monitoring of blood glucose could vastly reduce a number of the long-term complications associated with diabetes. In this article, we present a novel approach that relies on a glucose-binding protein engineered such that a 20% reduction in fluorescence due to the fluorescence resonance energy transfer occurs as a result of glucose binding. This change in fluorescence provides a signal for the optical detection of glucose. The novel glucose indicator protein (GIP) was created by fusing two fluorescent reporter proteins (green fluorescent proteins) to each end of an Escherichia coli glucose-binding protein in such a manner that the spatial separation between the fluorescent moieties changes when glucose binds, thus generating a distinct optical signal that can be used for glucose detection. By placing the GIP within a dialysis hollow fiber sensor, a microsensor has been developed for continuous monitoring of glucose. The sensor had a response time to sudden glucose changes within 100 s and was reversible. The sensor was shown to have an optional range on the order of 10 microM of glucose.     

An Active Core Fiber-Optic Temperature Sensor Using an Eu(III)-Doped Sol-Gel Silica Fiber as a Temperature Indicator

An active core fiber-optic temperature sensor has been developed by using an Eu(III)-doped sol-gel silica fiber as a temperature indicator and has been tested for sensing temperature in the range from 80 degC to 500 degC. The fluorescence of Eu(III) doped inside the sol-gel silica fiber was excited with a simple LED with peak wavelength at 420 nm and the fluorescence intensity was recorded as a sensing signal. The fluorescence intensity decreases with the increase of temperature. In the temperature range from 140 degC to 300 degC, the attenuation of the fluorescence intensity of the fiber-optic sensor in decibel has a linear relationship with temperature     

Characterization of the chemical architecture of carbon-fiber microelectrodes. 1. Carboxylates

A new method to characterize the chemical architecture of a carbon-fiber microelectrode surface is described. Derivatization of carboxyl groups on the carbon surface with a poly(oxyalkalene)diamine (Jeffamine ED-600), followed by biotinylation of the free amine, allowed the attachment of a fluorescein isothiocyanate (FITC) conjugate of ExtrAvidin. The fluorescence observed after excitation at 488 nm was imaged with a fluorescence microscope equipped with a CCD camera, yielding a spatial map of the distribution of modified carboxyl groups on the surface of the carbon fiber with 0.5-micron resolution. Colloidal gold particles (15 nm diameter) coated with ExtrAvidin were used in place of the FITC-ExtrAvidin, and the carbon-fiber surface was imaged with scanning electron microscopy on a submicron scale. This selective information regarding surface-bound functional groups (i.e. carboxylates) has proven invaluable toward the rational design of novel sensors based on surface-modified ultramicroelectrodes.     

Selective separation of fluorescent-magnetic nanoparticles with different magnetite-doping levels

Fluorescent-labeled magnetic nanoparticles were explored as a biomedical agent for selective magnetic separation. By adjusting the loading volume of citrate-stabilized magnetites during a sol-gel reaction with silicon alkoxide, magnetites were simultaneously embedded into both the surface and inside the silica matrix, consequently leading to magnetic nanoparticles with different doping levels of magnetites. For endowing them with multifunctional tools in biomedical fields, magnetic nanoparticles were further encapsulated with silica thin layer labeled with fluorescent organic dyes (such as Alexa Fluor 488 and 594). Fluorescent-magnetic nanoparticles with different magnetism successfully displayed the differential separation of fluorescence spectra under an external magnetic field.     

Next-Generation NASA Airborne Oceanographic Lidar System

The complete design and flight test of the next-generation Airborne Oceanographic Lidar (AOL-3) is detailed. The application of new technology has allowed major reductions in weight, volume, and power requirements compared with the earlier AOL sensor. Subsystem designs for the new AOL sensor include new technology in fiber optics, spectrometer detector optical train, miniature photomultiplier modules, dual-laser wavelength excitation from a single small laser source, and new receiver optical configuration. The new design reduced telescope size and maintained the same principal fluorescence and water Raman bands but essentially retained a comparable measurement accuracy. A major advancement is the implementation of single-laser simultaneous excitation of two physically separate oceanic target areas: one stimulated by 532 nm and the other by 355 nm. Backscattered fluorescence and Raman signals from both targets are acquired simultaneously by use of the same telescope and spectrometer-detector system. Two digital oscilloscopes provide temporal- and depth-resolved data from each of seven spectral emission bands.     

Nanoscale control of Ag nanostructures for plasmonic fluorescence enhancement of near-infrared dyes

Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based detection techniques. Metal induced fluorescence enhancement offers the possibility of increasing the sensitivity of protein detection in clinical applications. We report the use of tunable plasmonic silver nanostructures for the fluorescence enhancement of a near-infrared (NIR) dye (Alexa Fluor 790). Extensive fluorescence enhancement of ～2 orders of magnitude is obtained by the nanoscale control of the Ag nanostructure dimensions and interparticle distance. These Ag nanostructures also enhanced fluorescence from a dye with very high quantum yield (7.8 fold for Alexa Fluor 488, quantum efficiency (Qy) = 0.92). A combination of greatly enhanced excitation and an increased radiative decay rate, leading to an associated enhancement of the quantum efficiency leads to the large enhancement. These results show the potential of Ag nanostructures as metal induced fluorescence enhancement (MIFE) substrates for dyes in the NIR “biological window” as well as the visible region. Ag nanostructured arrays fabricated by colloidal lithography thus show great potential for NIR dye-based biosensing applications.      

Characterization of gradient-index lens-fiber spacing toward applications in two-photon fluorescence endoscopy

We report on the experimental investigation into the characterization of two-photon fluorescence microscopy based on the separation distance of a single-mode optical fiber coupler and a gradient-index (GRIN) rod lens. The collected two-photon fluorescence signal exhibits a maximum intensity at a defined separation distance (gap length) where the increasing effective excitation numerical aperture is balanced by the decreasing confocal emission collection. A maximum signal is found at gap lengths of approximately 2, 1.25, and 1.75 mm for GRIN lenses with pitches of 0.23, 0.25, and 0.29 wavelength at 830 nm. The maximum two-photon fluorescence signal collected corresponds to a threefold reduction of axial resolution (38.5 microm at 1.25 mm), compared with the maximum resolution (11.6 microm at 5.5 mm), as shown by the three-dimensional imaging of 10 microm beads. These results demonstrate an intrinsic trade-off between signal collection and axial resolution.     

Wide-field depth-sectioning fluorescence microscopy using projector-generated patterned illumination

We present a simple and cost-effective wide-field, depth-sectioning, fluorescence microscope utilizing a commercial multimedia projector to generate excitation patterns on the sample. Highly resolved optical sections of fluorescent pollen grains at 1.9 microm axial resolution are constructed using the structured illumination technique. This requires grid excitation patterns to be scanned across the sample, which is straightforwardly implemented by creating slideshows of gratings at different phases, projecting them onto the sample, and synchronizing camera acquisition with slide transition. In addition to rapid dynamic pattern generation, the projector provides high illumination power and spectral excitation selectivity. We exploit these properties by imaging mouse neural cells in cultures multistained with Alexa 488 and Cy3. The spectral and structural neural information is effectively resolved in three dimensions. The flexibility and commercial availability of this light source is envisioned to open multidimensional imaging to a broader user base.     

Simultaneous determination of human Enterovirus 71 and Coxsackievirus B3 by dual-color quantum dots and homogeneous immunoassay

Human Enterovirus 71 (EV71) and Coxsackievirus B3 (CVB3) have high risks for morbidity and mortality. A virus quantitation immunoassay has been proposed by employing two colored quantum dots (QDs), antibodies of the virus, and graphene oxide (GO). The QDs are streptavidin-conjugated quantum dots (SA-QDs), and the antibodies are biotinylated antibodies. Biotinylated EV71 antibody (Ab1) was associated with 525 nm green colored SA-QDs via biotin-streptavidin interaction forming QDs-Ab1, whereas biotinylated CVB3 antibody (Ab2) was associated with 605 nm red colored SA-QDs via biotin-streptavidin interaction forming QDs-Ab2. GO was an excellent quencher to the fluorescence of both QDs-Ab1 and QDs-Ab2. The targets of EV71 and CVB3 can break up the complex of QDs-Ab and GO, recovering the fluorescence of QDs-Ab1 and QDs-Ab2, respectively. Using these two different colored QDs-Ab fluorescence recovery intensities upon the addition of targets EV71 and CVB3, the two enteroviruses can be simultaneously quantitatively determined with a single excitation light. The detection limits of EV71 and CVB3 are 0.42 and 0.39 ng mL(-1) based on 3 times signal-to-noise ratio, respectively. More importantly, this strategy can be further used as a universal method for any protein or virus determination by changing the conjugated antibodies in disease early diagnosis, which can provide a fast and promising clinical approach for virus differentiation and determination. In a word, a simple, fast, sensitive, and highly selective assay for EV71 and CVB3 has been developed. It could be applied in clinical sample analysis with a satisfactory result. It was notable that the sensor could not only achieve rapid and precise quantitative determination of protein/virus by fluorescent intensity but also could be applied in semiquantitative protein/virus determination by digital visualization.     

Fluorescence characteristics of Cu-ZSM-5 zeolites in reactive gas mixtures: mechanisms for a fiber-optic-based gas sensor

In situ measurements of the fluorescence spectra, intensity, and response time were made at elevated temperatures (~500 degrees C) for samples of Cu-ZSM-5 exposed to dilute mixtures of O(2) in N(2) and to various O(2)-reductant combinations in N(2). The results of these experiments are interpreted with a physical model of the oxidation/reduction kinetics of the copper ions. The fluorescence signal depends strongly on the gas composition, providing a mechanism for a gas composition sensor. A prototype sensor configuration is described that uses a visible (488-nm) excitation source and a fiber-optic geometry to generate and detect the fluorescence. The results indicate that for a weakly reducing gas the fluorescence signal correlates well with the oxygen concentration, whereas for strongly reducing gases the signal correlates more closely with the reductant-to-oxidant ratio.     

Characterization of time-resolved fluorescence response measurements for distributed optical-fiber sensing

A distributed optical-fiber sensing system based on pulsed excitation and time-gated photon counting has been used to locate a fluorescent region along the fiber. The complex Alq3 and the infrared dye IR-125 were examined with 405 and 780 nm excitation, respectively. A model to characterize the response of the distributed fluorescence sensor to a Gaussian input pulse was developed and tested. Analysis of the Alq3 fluorescent response confirmed the validity of the model and enabled the fluorescence lifetime to be determined. The intrinsic lifetime obtained (18.2±0.9 ns) is in good agreement with published data. The decay rate was found to be proportional to concentration, which is indicative of collisional deactivation. The model allows the spatial resolution of a distributed sensing system to be improved for fluorophores with lifetimes that are longer than the resolution of the sensing system.     

Enhancement of Raman signals with silver-coated tips

Silver coated SiN and SiO(2) tips have been fabricated for use with a bottom-illumination tip-enhanced Raman spectroscopy (TERS) setup with a 488 nm laser excitation. SiN tips with 50-60 nm of deposited Ag give the best TERS enhancements for brilliant cresyl blue test analyte spin-coated on a glass slide. Ag nanoparticles on SiN or SiO(2) rather than Si tips are better for TERS because of the proximity of the wavelengths of their surface plasmon resonance to 488 nm. Adjustments of tilt angle of the metallized tip with respect to the surface plane is shown to considerably raise the intensities of the TERS signals, even from tips that initially appear to be rather non-enhancing. This work helps to enable the more frequent use of the 488 nm laser for nanoscale chemical analysis with both TERS and fluorescence imaging in the same setup.     

Oxygen optrode for use in a fiber-optic glucose biosensor

An optical fiber oxygen sensor, based on the dynamic quenching of the luminescence of tris(1,10-phenanthroline)-ruthenium(II) cation by molecular oxygen, is presented. The complex is adsorbed onto silica gel, incorporated in a silicone matrix possessing a high oxygen permeability, and placed at the tip of the optical fiber. Oxygen has been monitored continuously in the 0-750 Torr range, with the detection limit being as low as 0.7 Torr. The device has been applied to the development of a fast responding and highly sensitive fiber-optic glucose biosensor based on this highly sensitive oxygen transducer. The sensor relates oxygen consumption (as a result of enzymatic oxidation) to glucose concentration. The enzyme is immobilized on the surface of the oxygen optrode; carbon black is used as an optical isolation in order to prevent ambient light and sample fluorescence to interfere. Measurements have been performed in a flow-through cell in air-equilibrated glucose standard solutions of pH 7.0. The effects of enzyme immobilization procedures (including enzyme immobilization on carbon black) as to response times (around 6 min), analytical ranges (0.06-1 mM glucose), reproducibility in sensor construction, and long-term stability have been studied as well.     

Tandem dye acceptor used to enhance upconversion fluorescence resonance energy transfer in homogeneous assays

In fluorescence resonance energy transfer (FRET)-based assays, spectral separation of acceptor emission from donor emission is a common problem affecting the assay sensitivity. The challenge derives from small Stokes shifts characteristic to conventional fluorescent dyes resulting in leakage of donor emission to the measurement window intended only to collect the acceptor emission. We have studied a FRET-based homogeneous bioaffinity assay utilizing a tandem dye acceptor with a large pseudo-Stokes shift (139 nm). The tandem dye was constructed using B-phycoerythrin as an absorber and multiple Alexa Fluor 680 dyes as emitters. As a donor, we employed upconverting phosphor particles, which uniquely emit at visible wavelengths under low-energy infrared excitation enabling the fluorescence measurements free from autofluorescence even without time-resolved detection. With the tandem dye, it was possible to achieve four times higher signal from a single binding event compared to the conventional Alexa Fluor 680 dye alone. Tandem dyes are widely used in cytometry and other multiplex purposes, but their applications can be expanded to fluorescence-based homogeneous assays. Both the optimal excitation and emission wavelengths of tandem dye can be tuned to a desired region by choosing appropriate fluorophores enabling specifically designed acceptor dyes with large Stokes shift.     

氟化物玻璃Yb~(3+)敏化稀土离子的上转换发光

研究了氟化物玻璃中 Ｙｂ３ ＋ 敏化 Ｐｒ３ ＋ 、 Ｔｍ ３ ＋ 、 Ｅｒ３ ＋ 或 Ｈｏ３ ＋ 产生的上转换发光． 在波长为８８０ｎｍ光的激发下, Ｙｂ３ ＋ 敏化 Ｐｒ３ ＋ 产生波长为４８２ ,５２０ ,５２９ ,６０５ 和６３５ｎｍ 的荧光． 用波长为９７０ｎｍ 的光激发,在 Ｙｂ３ ＋ 敏化作用下, Ｔｍ３ ＋ 产生波长为４７８ , ６４９ 和７９３ｎｍ 的上转换发光; Ｅｒ３ ＋ 产生波长为５４７ 和５２５ｎｍ 的绿光; Ｈｏ３ ＋ 产生５４３ｎｍ 的绿光     

Competitive binding assay for glucose based on glycodendrimer-fluorophore conjugates

A new fluorescent glucose assay has been created using Alexa Fluor 647-labeled concanavalin A (Con A) and a fourth-generation PAMAM Alexa Fluor 594-labeled glycodendrimer. This assay has been shown to have a large response to glucose within the biological range and to be capable of functioning within a polymer hydrogel. In this paper, the glucose response is shown to be a single fluorophore-based quenching reaction. Data showing that the sensor is fully reversible and specific through competitive binding between the dendrimer and glucose with Con A are presented. Overall, the assay is shown to have potential over the traditional dextran-based assay because it has a larger dynamic response to physiological glucose concentrations, incorporates longer wavelength dyes that improve signal penetration through dermal tissue, and provides an internal reference in the form of a nonreactive fluorescent label.     

Fiber-based UV laser-diode fluorescence sensor for commercial gasolines

We report on an optical fiber probe, coupled to a 404-nm laser diode, as a fluorescence sensor for monitoring of commercial gasolines. The principle of operation of the sensor is based on quantifying the intensity of the Stokes-shifted fluorescence from some of the heavier polycyclic aromatic hydrocarbons C/sub x/H/sub y/,(x,y) /spl ges/ (14,10) present in gasolines as minor constituents. The normalized efficiency of the optical fiber probe, as a function of its geometry, is calculated in the cases of single-fiber and parallel dual-fiber designs. The spatial and temporal resolutions achievable by the sensor are discussed as a function of design parameters The performance of the sensor is investigated experimentally for commercial gasolines in the liquid and gas phase. The optimal excitation wavelength for such sensors is investigated in the range of 350-400 nm. The linear sensitivity to vapor concentrations of retail gasoline fuel is demonstrated in the range of 4%-125% of combustion stoichiometry at 10 bar and 180/spl deg/C. Statistical processing of the data from the sensor allows distinction to be made between different forecourt gasoline suppliers, as well as fuel varieties (unleaded, low sulfur, etc.).     

Fabrication of non-enzymatic biosensor based on metallic catalyst-TiO2 hollow sphere nanocomposite for determining biomolecules

A PtRu@TiO2-hollow nanocomposite for the detection of biomolecules was synthesized by chemical reduction. First, poly(styrene-co-vinylphenylboronic acid), PSB, was prepared as a template (approximately 250 nm) by surfactant-free emulsion polymerization. Second, PSB/TiO2 core-shell spheres were prepared by sol-gel reaction. Finally, TiO2 hollow spheres (TiO2-H) were then formed after removing the PSB template by calcination at 450 degrees C under air atmosphere. To prepare the electrocatalyst, PtRu nanoparticles (NPs) were deposited onto the TiO2-H surface by chemical reduction. The prepared PtRu@TiO2-H nanocomposite was characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), and elemental analysis. A non-enzymatic sensor was fabricated by depositing the as-prepared PtRu@TiO2-H nanocomposite on the surface of a glassy carbon electrode (GCE), which was prepared by a hand casting method with Nafion solution as a binder. The sensor was tested as a biomolecule sensor, especially for the detection of glucose and dopamine. The cyclic voltammograms (CV) obtained during the oxidation studies revealed that the PtRu@TiO2-H nanocomposite showed better catalytic function toward the oxidation of dopamine. The sensing range of the non-enzymatic sensor for glucose was 5.0-100 mM in a phosphate buffer. The results demonstrated the potential usefulness of this bimetallic@TiO2-H bifunctional catalyst for biosensor applications.     

Miniature fiber-optic pressure sensor with a polymer diaphragm

The fabrication and experimental investigation of a miniature optical fiber pressure sensor for biomedical and industrial applications are described. The sensor measures only 125 microm in diameter. The essential element is a thin polymer diaphragm that is positioned inside the hollow end of an optical fiber. The cavity at the fiber end is made by a simple and effective micromachining process based on wet etching in diluted HF acid. Thus a Fabry-Perot interferometer is formed between the inner fiber-cavity interface and the diaphragm. The fabrication technique is described in detail. Different sensor prototypes were fabricated upon 125 microm-diameter optical fiber that demonstrated pressure ranges from 0 to 40 and from 0 to 1200 kPa. A resolution of less than 10 Pa was demonstrated in practice. The fabrication technique presented facilitates production of simple and low-cost disposable pressure sensors by use of materials with that ensure the required biocompatibility.