Microwave-accelerated ultrafast nanoparticle aggregation assays using gold colloids

In this paper, the proof of principle of microwave-accelerated aggregation assay technology, which shortens the solution-based aggregation assays' run time to seconds (>100-fold increase in kinetics) with microwave heating, was demonstrated using a model aggregation assay based on the well-known interactions of biotin and avidin. Biotinylated gold colloids were aggregated in solution with the addition of streptavidin, which takes 20 min at room temperature to reach >90% completion and only 10 s with microwave heating. The initial velocity (after 1-s microwave heating) of the biotinylated gold colloids reaches up to 10.5 m/s, which gives rise to greater sampling of the total volume but not a large increase in bulk temperature. The room-temperature, steady-state velocity of the colloids was <0.5 microm/s. In control experiments, where streptavidin preincubated with d-biotin in solution is added to biotinylated gold colloids and microwave heated, gold colloids did not aggregate, demonstrating that nonspecific interactions between biotinylated gold colloids and streptavidin were negligible.     

Durable metal-enhanced fluorescence flexible platform by in-situ growth of micropatterned Ag nanospheres

Here, we demonstrate an in-situ growth of micropatterned silver nanospheres(Ag NS) array on transparent polyimide(PI), namely PI-Ag substrate, applied as a flexible platform for metal-enhanced fluorescence(MEF). The scheme proposed here can easily control the grid formation of Ag NSs and the particle size inside, thus achieving patterned fluorescence imaging and MEF efficiency optimization. Meanwhile, the magnitude of enhanced intensity, relying on the distance between Ag NSs and emissive molecules, was systematically investigated by exploring diverse polyethyleneimine(PEI) spacer thickness. Consequently,an optimal enhancement factor of 7.9 and a pattern of grid fluorescence imaging was obtained with an insertion of 10 nm PEI on the PI-Ag(25 nm) platform. Moreover, owing to robust adhesion between Ag NSs and PI film by in-situ growth, this flexible PI-Ag MEF platform maintained a stable MEF efficiency even after taking mechanical bending for 1000 cycles. This new surface-confined micropatterned Ag NSs PI film provides a promising candidate in design flexible MEF platforms for future analytical and clinical sensing applications.     

Confeito-like assembly of organosilicate-caged fluorophores: ultrabright suprananoparticles for fluorescence imaging

We report ultrabright, photostable, sub-25 nm nanoparticle agglomerates (suprananoparticles) assembled from a few hundred 3.3 ± 0.9 nm units, each hosting on average a single rhodamine 6G (Rh6G) dye molecule encased in a thin organosilicate cage. These individual Rh6G-doped nanoparticle (DOSNP) units consist of a hydrophobic core containing the dye and an ultrathin, conformal silicate shell modified by CO(2) plasma to confer a beneficial 'cage effect' as well as surface hydrophilicity. The isolation of the dye within individual DOSNP units in the final 22 ± 5 nm agglomerate avoids dimerization and related spontaneous molecular interactions that otherwise lead to self-quenching in closely co-localized fluorophores. The resulting suprananoparticles are over 200 times brighter than the free Rh6G molecules in the same volume. There is no observable dye leaching, and the labels are 20-fold more resistant to photobleaching than free Rh6G in solution. We demonstrate the attractive features of DOSNPs as labels in bioimaging applications.     

An integrated fluorescence array as a platform for lab-on-a-chip technology using multimode interference splitters

We present the design and fabrication of 1-to-N multimode interference (MMI) splitters, suitable for use in integrated optical fluorescence array sensing, with particular applications in lab-on-a-chip (micro-TAS) technologies. Electron beam irradiation of germanium-doped flame hydrolysis deposited silica was used to define the MMI waveguide regions. The splitters were integrated with microfluidic channels to form direct-excitation fluorescence sensor chips for use at visible wavelengths. Characterization of the waveguides shows that predictable splitting ratios can be achieved. Two devices are presented: a 1/spl times/2 splitter integrated with one analytical chamber and a 1/spl times/4 array device for multipoint excitation. A photomultiplier tube was used to assess the analytical performance of the chip, in response to standard aliquots of fluorophore (31 nM to 1.25 /spl mu/M).     

Depolarization of surface-enhanced fluorescence: an approach to fluorescence polarization assays

Localized surface plasmons of metallic particles of subwavelength sizes strongly modify the spectral properties of nearby fluorophores. The enhanced radiative decay rate leads to high fluorescence efficiencies and decreased fluorescence lifetimes. In this report we show that metal-enhanced fluorescence generated by the presence of the silver islands on the glass substrate displays high depolarization. Intensities, lifetimes, and emission anisotropies of several fluorophore protein conjugates have been studied in the absence and presence of metallic nanostructures. Despite highly decreased lifetimes of about 10-fold and immobilization of conjugates on the solid substrate, the observed emission anisotropies for all fluorophores on the metal-enhanced substrate decreased 300-500% compared to that in solution. This observation implies a new generation of fluorescence polarization immunoassays with broad applications because of no restrictions to the lifetime of the probe and the size of labeled biomolecules. The changes in polarization are due to binding that occur on the bioactive surface localized near the metal particles.     

Magnetic levitation as a platform for competitive protein-ligand binding assays

This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater than approximately 65 kDa.     

Distance-dependent metal-enhanced quantum dots fluorescence analysis in solution by capillary electrophoresis and its application to DNA detection

Here the distance dependence of metal-enhanced quantum dots (QDs) fluorescence in solution is studied systematically by capillary electrophoresis (CE). Complementary DNA oligonucleotides-modified CdSe/ZnS QDs and gold nanoparticles (Au NPs) were connected together in solution by the hybridization of complementary oligonucleotides, and a model system (QD-Au) for the study of metal-enhanced QDs fluorescence was constructed, in which the distance between the QDs and Au NPs was controlled by adjusting the base number of the oligonucleotide. In our CE experiments, the metal-enhanced fluorescence of the QDs solution was only observed when the distance between the QDs and Au NPs ranged from 6.8 to 18.7 nm, and the maximum enhancement by a factor of 2.3 was achieved at 11.9 nm. Furthermore, a minimum of 19.6 pg of target DNA was identified in CE based on its specific competition with the QD-DNA in the QD-Au system. This work provides an important reference for future study of metal-enhanced QDs fluorescence in solution and exhibits potential capability in nucleic acid hybridization analysis and high-sensitivity DNA detection.     

RCADiA: simple automation platform for comparative multidimensional protein identification technology

Multidimensional liquid chromatography in combination with tandem mass spectrometry has been used to analyze a variety of biological structures including protein complexes. Incorporating this approach with autosampling devices presents a number of problems including decreased sensitivity due to exposure to extra surfaces, carryover from run to run, and increased dead volume. We developed a device, termed Radial Column Array for Distribution and Automation (RCADiA), to automate multiple MuDPIT experiments while eliminating many of these problems and maintaining a high resolution and sensitive analysis. The design, which places each sample downstream of any common fluid path, presents a low risk of carryover between successive analyses. Beyond the convenience of automation, the RCADiA platform also produces data of similar quality to the standard method of performing individual MuDPIT experiments. We demonstrate this device by performing a comparative analysis of mitochondria enriched from rat liver and spinal cord.     

Nanoparticle fluorescence based technology for biological applications

Fluorescence is widely used in biological detection and imaging. The emerging luminescent nanoparticles or quantum dots provide a new type of biological agents that can improve these applications. The advantages of luminescent nanoparticles for biological applications include their high quantum yield, color availability, good photo-stability, large surface-to-volume ratio, surface functionality, and small size. In this review article, we first introduce quantum size confinement, photoluminescence and upconversion luminescence of nanoparticles, then describe the preparation and conjugation of water soluble nanoparticles and introduce the applications of luminescence nanoparticles for in vitro and in vivo imaging, fluorescence resonance energy based detection, and the applications of luminescence nanoparticles for photodynamic activation.     

Evaluation of three-dimensional microchannel glass biochips for multiplexed nucleic acid fluorescence hybridization assays

Three-dimensional, flow-through microchannel glass substrates have a potential for enhanced performance, including increased sensitivity and dynamic range, over traditional planar substrates used in medium-density microarray platforms. This paper presents a methodology for the implementation of multiplexed nucleic acid hybridization fluorescence assays on microchannel glass substrates. Fluorescence detection was achieved, in a first instance, using conventional low-magnification microscope objective lenses, as imaging optics whose depth-of-field characteristics match the thickness of the microchannel glass chip. The optical properties of microchannel glass were shown, through experimental results and simulations, to be compatible with the quantitative detection of heterogeneous hybridization events taking place along the microchannel sidewalls, with detection limits for oligonucleotide targets in the low-attomole range.     

Small unilamellar vesicles: a platform technology for molecular imaging of brain tumors

Molecular imaging enables the non-invasive investigation of cellular and molecular processes. Although there are challenges to overcome, the development of targeted contrast agents to increase the sensitivity of molecular imaging techniques is essential for their clinical translation. In this study, spontaneously forming, small unilamellar vesicles (sULVs) (30 nm diameter) were used as a platform to build a bimodal (i.e., optical and magnetic resonance imaging (MRI)) targeted contrast agent for the molecular imaging of brain tumors. sULVs were loaded with a gadolinium (Gd) chelated lipid (Gd-DPTA-BOA), functionalized with targeting antibodies (anti-EGFR monoclonal and anti-IGFBP7 single domain), and incorporated a near infrared dye (Cy5.5). The resultant sULVs were characterized in vitro using small angle neutron scattering (SANS), phantom MRI and dynamic light scattering (DLS). Antibody targeted and nontargeted Gd loaded sULVs labeled with Cy5.5 were assessed in vivo in a brain tumor model in mice using time domain optical imaging and MRI. The results demonstrated that a spontaneously forming, nanosized ULVs loaded with a high payload of Gd can selectively target and image, using MR and optical imaging, brain tumor vessels when functionalized with anti-IGFBP7 single domain antibodies. The unique features of these targeted sULVs make them promising molecular MRI contrast agents.     

Oligonucleotide array-in-well platform for detection and genotyping human adenoviruses by utilizing upconverting phosphor label technology

We have developed a robust array-in-well test platform based on an oligonucleotide array, combining advantages of simple instrumentation and new upconverting phosphor reporter technology. Upconverting inorganic lanthanide phosphors have a unique property of photoluminescence emission at visible wavelengths under near-infrared excitation. No autofluorescence is produced from the sample or support material, enabling a highly sensitive assay. In this study, the assay is performed in standard 96-well microtiter plates, making the technique easily adaptable to high-throughput analysis. The oligonucleotide array-in-well assay is employed to detect a selection of ten common adenovirus genotypes causing human infections. The study provides a demonstration of the advantages and potential of the upconverting phosphor-based reporter technology in multianalyte assays and anti-Stokes photoluminescence detection with an anti-Stokes photoluminescence imaging device.     

Nanoarrays: a method for performing enzymatic assays

Conventional enzymatic assays for alcohol dehydrogenase, pyruvate kinase, and enolase performed in 96-well microtiter plates were compared with assays monitored in 25-well nanoarrays. All miniaturized reactions could be performed in maximum volumes of 6.3-8 nL and were read out with a conventional fluorescence microscope system equipped with a scientific grade CCD camera. Substrate and cofactor were already present inside the wells after having been presprayed, or they were applied in solution to the wells of the nanoarray shortly before the assays started. For all of the assays, commercially available enzymes and enzymes present in cell-free extracts were used. Assays carried out in premixed nanoarrays gave results comparable to those performed in presprayed nanoarrays. Enzyme activities determined in nanoarrays by using two different methods were in good agreement with assays performed in microtiter plates. Also, good correspondence was found between expected and observed enzyme levels. In short, enzymatic assays performed in premixed and in particular in presprayed nanoarrays are a promising low-volume and low-reagent- and sample-consuming alternative to current methodology and could find applications in many different areas of analytical chemistry.     

Ascorbic acid assays of individual neurons and neuronal tissues using capillary electrophoresis with laser-induced fluorescence detection

Ascorbic acid is an important cellular metabolite involved in many biochemical pathways. A method to quantitate ascorbic acid and dehydroascorbic acid in individual neurons and neuronal tissues is described with detection limits of 320 pM (430 zmol). The method uses microvial sampling, derivatization with 4,5-dimethyl-1,2-phenylenediamine, capillary electrophoresis separation, and laser-induced fluorescence detection and quantifies the ascorbic acid and dehydroascorbic acid levels with less than a 15-min total analysis time including sample preparation and derivatization. Ascorbic acid and dehydroascorbic acid levels are measured using functionally characterized and identified neurons of Aplysia californica, Pleurobranchaea californica, and Lymnaea stagnalis -three well-recognized models in cellular and system neuroscience. Multiple assays of a particular identified neuron (e.g., metacerebral cells from Aplysia) show a high level of reproducibility, while endogenous intracellular concentrations of ascorbate are neuron-specific. Ascorbic acid concentrations in the neurons studied range from 0.19 to 6.2 mM for Aplysia and 0.12 to 0.22 mM for Lymnaea. In contrast, concentrations of ascorbic acid observed in heterogeneous tissues such as ganglia (with connective tissues, glia, blood vessels, neuropile, and areas with intercellular spaces), 4-190 microM, are significantly lower than the single-cell values.     

Cucurbiturils: molecular nanocapsules for time-resolved fluorescence-based assays

A new fluorescent host-guest system based on the inclusion of the fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) into the cavity of the molecular container compound cucurbit[7]uril (CB7) has been designed which possesses an exceedingly long-lived emission (690 ns in aerated water). The large binding constant of (4/spl plusmn/1)/spl times/10/sup 5/ M/sup -1/ along with the resistance of the CB7/spl middot/DBO complex toward external fluorescence quenchers allow the use of CB7 as an enhancer in time-resolved fluorescence-based assays, e.g., to screen enzyme activity or inhibition by using DBO-labeled peptides as substrates. The response of CB7/spl middot/DBO to different environmental conditions and possible quenchers are described.     

FPGA-based reconfigurable processor for ultrafast interlaced ultrasound and photoacoustic imaging

In this paper, we report, to the best of our knowledge, a unique field-programmable gate array (FPGA)-based reconfigurable processor for real-time interlaced co-registered ultrasound and photoacoustic imaging and its application in imaging tumor dynamic response. The FPGA is used to control, acquire, store, delay-and-sum, and transfer the data for real-time co-registered imaging. The FPGA controls the ultrasound transmission and ultrasound and photoacoustic data acquisition process of a customized 16-channel module that contains all of the necessary analog and digital circuits. The 16-channel module is one of multiple modules plugged into a motherboard; their beamformed outputs are made available for a digital signal processor (DSP) to access using an external memory interface (EMIF). The FPGA performs a key role through ultrafast reconfiguration and adaptation of its structure to allow real-time switching between the two imaging modes, including transmission control, laser synchronization, internal memory structure, beamforming, and EMIF structure and memory size. It performs another role by parallel accessing of internal memories and multi-thread processing to reduce the transfer of data and the processing load on the DSP. Furthermore, because the laser will be pulsing even during ultrasound pulse-echo acquisition, the FPGA ensures that the laser pulses are far enough from the pulse-echo acquisitions by appropriate time-division multiplexing (TDM). A co-registered ultrasound and photoacoustic imaging system consisting of four FPGA modules (64-channels) is constructed, and its performance is demonstrated using phantom targets and in vivo mouse tumor models.