Neuromorphic Processing for Optical Microbead Arrays: Dimensionality Reduction and Contrast Enhancement

This paper presents a neuromorphic approach for sensor-based machine olfaction that combines a portable chemical detection system based on microbead array technology with a biologically inspired model of signal processing in the olfactory bulb. The sensor array contains hundreds of microbeads coated with solvatochromic dyes adsorbed in, or covalently attached on, the matrix of various microspheres. When exposed to odors, each bead sensor responds with corresponding intensity changes, spectral shifts, and time-dependent variations associated with the fluorescent sensors. The bead array responses are subsequently processed using a model of olfactory circuits that capture the following two functions: chemotopic convergence of receptor neurons and center on-off surround lateral interactions. The first circuit performs dimensionality reduction, transforming the high-dimensional microbead array response into an organized spatial pattern (i.e., an odor image). The second circuit enhances the contrast of these spatial patterns, improving the separability of odors. The model is validated on an experimental dataset containing the responses of a large array of microbead sensors to five different analytes. Our results indicate that the model is able to significantly improve the separability between odor patterns, compared to that available from the raw sensor response     

DNA hybridization and discrimination of single-nucleotide mismatches using chip-based microbead arrays

The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of < or = 10(2) different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10000 and limit of detection values of approximately 10(-13) M are obtained readily with this microbead array system.     

Development of ultrabright semiconducting polymer dots for ratiometric pH sensing

Semiconducting polymer-based nanoparticles (Pdots) have recently emerged as a new class of ultrabright probes for biological detection and imaging. This paper describes the development of poly(2,5-di(3',7'-dimethyloctyl)phenylene-1,4-ethynylene) (PPE) Pdots as a platform for designing Fo?rster resonance energy transfer (FRET)-based ratiometric pH nanoprobes. We describe and compare three routes for coupling the pH-sensitive dye, fluorescein, to PPE Pdots, which is a pH-insensitive semiconducting polymer. This approach offers a rapid and robust sensor for pH determination using the ratiometric methodology where excitation at a single wavelength results in two emission peaks, one that is pH sensitive and the other one that is pH insensitive for use as an internal reference. The linear range for pH sensing of the fluorescein-coupled Pdots is between pH 5.0 and 8.0, which is suitable for most cellular studies. The pH-sensitive Pdots show excellent reversibility and stability in pH measurements. In this paper, we use them to measure the intracellular pH in HeLa cells following their uptake by endocytosis, thus demonstrating their utility for use in cellular and imaging experiments.     

Epiallele quantification using molecular inversion probes

The location and level of DNA methylation within a genome is emerging as an important biomarker for cancer diagnosis. Despite its potential, it is difficult to comprehensively analyze the epialleles that are often found in a biological sample. Therefore, an assay utilizing molecular inversion probes was designed and used to expose and quantify epialleles in heterogeneously methylated bisulphite treated genomic DNA. Different CpG dinucleotides were able to be rapidly quantified with high resolution, sensitivity and specificity over a large dynamic range using rapid flow cytometric readout of multiplexable microbead DNA biosensors.     

Quantitating Fluorescence Intensity from Fluorophore: The Definition of MESF Assignment

The quantitation of fluorescence radiance may at first suggest the need to obtain the number of fluorophore that are responsible for the measured fluorescence radiance. This goal is beset by many difficulties since the fluorescence radiance depends on three parameters 1) the probability of absorbing a photon (molar extinction), 2) the number of fluorophores, and 3) the probability of radiative decay of the excited state (quantum yield). If we use the same fluorophore in the reference solution and the analyte then, to a good approximation, the molar extinction drops out from the comparison of fluorescence radiance and we are left with the comparison of fluorescence yield which is defined as the product of fluorophore concentration and the molecular quantum yield. The equality of fluorescence yields from two solutions leads to the notion of equivalent number of fluorophores in the two solutions that is the basis for assignment of MESF (Molecules of Equivalent Soluble Fluorophore) values. We discuss how MESF values are assigned to labeled microbeads and by extension to labeled antibodies, and how these assignments can lead to the estimate of the number of bound antibodies in flow cytometer measurements.     

Enhanced emission of highly labeled DNA oligomers near silver metallic surfaces

Fluorescein is a widely used fluorescent probe in DNA analysis. One difficulty with fluorescein is its self-quenching due to resonance energy transfer between the residues, which results in decreased intensities with increasing labeling density. We examined the emission spectral properties of DNA oligomers labeled with one or five fluorescein residues. The emission intensity of the more highly labeled oligomer was decreased due to self-quenching. The self-quenching was mostly eliminated when this oligomer was held approximately 90 A from the surface of metallic silver particles. The intensities increased 7- and 19-fold for the oligomers with one or five fluoresceins, respectively. The increased intensity did not result in increased photobleaching. These results suggest the use of substrates coated with silver particles for increased sensitivity on DNA arrays or for DNA analysis.     

A Trachea‐Inspired Bifurcated Microfilter Capturing Viable Circulating Tumor Cells via Altered Biophysical Properties as Measured by Atomic Force Microscopy

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina‐inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid–structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead‐conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 μL min^?1, an 8 μm filter gap optimizes the recovery rate and purity. MCF‐7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re‐collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.     

Equilibrium and kinetic measurements of muscarinic receptor antagonism on living cells using bead injection spectroscopy

Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay. Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands. A flow injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber. The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BIS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response. Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.     

Effects of microbead surface chemistry on DNA loading and hybridization efficiency

Polymer microbeads are witnessing renewed interest for performing biomolecule recognition assays with distinct advantages over planar microarray technology. In this study, DNA hybridization assays are performed on the surfaces of 1-microm-diameter, synthetically modified polystyrene microbeads. The microbead surfaces contain varying amounts of poly(acrylic acid) as a source of carboxylate groups to which a DNA capture strand may bind. Through a series of controlled experiments in which the microbead carboxylate density and DNA:surface area ratios are systematically altered, we find that the density of carboxylate groups on the microbead surface may be the most important parameter affecting not only the total number of DNA strands that may bind to the microbead surface but, surprisingly, also the efficiency of DNA hybridization with complementary strands. These studies are aimed directly at understanding the physical interactions between DNA strands and an anionic microbead surface.     

Bead Capture on Magnetic Sensors in a Microfluidic System

The accumulation of magnetic beads by gravitational sedimentation and magnetic capture on a planar Hall-effect sensor integrated in a microfluidic channel is studied systematically as a function of the bead concentration, the fluid flow rate, and the sensor bias current. It is demonstrated that the sedimentation flux is proportional to the bead concentration and has a power law relation to the fluid flow rate. The mechanisms for the bead accumulation are investigated and it is found that gravitational sedimentation dominates the bead accumulation, whereas the stability of the sedimented beads against the fluid flow is defined by the localized magnetic fields from the sensor.      

Flow cytometric analysis to detect pathogens in bacterial cell mixtures using semiconductor quantum dots

Compared to a common green organic dye, semiconductor quantum dots (QDs) composed of CdSe/ZnS core/shell bioconjugates display brighter fluorescence intensities, lower detection thresholds, and better accuracy in analyzing bacterial cell mixtures composed of pathogenic E. coli O157:H7 and harmless E. coli DH5alpha using flow cytometry. For the same given bacterial mixture, QDs display fluorescence intensity levels that are approximately 1 order of magnitude brighter compared to the analogous experiments that utilize the standard dye fluorescein isothiocyanate. Detection limits are lowest when QDs are used as the fluorophore label for the pathogenic E. coli O157:H7 serotype: limits of 1% O157:H7 in 99% DH5alpha result, corresponding to 106 cells/mL, which is comparable to other developing fluorescence-based techniques for pathogen detection. Finally, utilizing QDs to label E. coli O157:H7 in cell mixtures results in greater accuracy and more closely approaches the ideal fluorophore for pathogen detection using flow cytometry. With their broader absorption spectra and narrower emission spectra than organic dyes, QDs can make vast improvements in the field of flow cytometry, where single-source excitation and simultaneous detection of multicolor species without complicating experimental setups or data analysis is quite advantageous for analyzing heterogeneous cell mixtures, both for prokaryotic pathogen detection and for studies on eukaryotic cell characteristics.     

Spectral bar coding of polystyrene microbeads using multicolored quantum dots

This paper focuses on encoding polystyrene microbeads, 10-100 microm in diameter, with a luminescent spectral bar code composed of mixtures of quantum dots (QDs) emitting at different wavelengths (colors). The QDs are encapsulated in the bead interior during the bead synthesis using a suspension polymerization, and the bar code is constructed by varying both the number of colors included in the bead and, for each color, the number of QDs of that color. Confocal laser scanning microscopy images of the beads demonstrate that the multicolored QDs are pushed together into inclusions within the bead interior. The encoded bead emission spectrum indicates that the peak position of the included colors does not shift relative to the corresponding peaks of the spectra recorded for the nonaggregated QDs at identical loading concentrations. Due to the spatial proximity of the QDs in the inclusions, electronic energy transfer from the lower wavelength emitting QDs to the higher emitting QDs changes the relative intensities of the colors compared to the values in the nonaggregated spectra. We show that this energy transfer does not obscure the spectral uniqueness of the different codes. Ratiometric encoding, in which the bar code is read as relative color intensity, is shown to remove the dependence of the code on the bead size.     

Submicrometer spatial resolution of metal-enhanced fluorescence

Enhanced fluorescence emission intensity from fluorescein was observed on glass slides covered with thin films of silver nanoparticles using a confocal laser-scanning microscope. The silver nanoparticle film increased the emission intensity of fluorescein by an average of at least three-fold in the area studied. Statistics are given on the enhancement of individual areas of the silver particle film with a resolution of approximately 210 nm. A histogram of intensity values indicates that the enhancement appears to occur without distinct subpopulations, with the exception that very high intensity subpopulations may occur but could not be resolved. Spatial features with dimensions near or smaller than the resolution limit of the confocal microscope, on the silver nanoparticle slide that enhanced the emission of fluorescein, were found using autocorrelation functions. These spatial features are of the same size as those found from the emission of slides containing silver nanoparticles only. These spatial features do not appear in control slides containing fluorescein without any silver nanoparticles. No long-range spatial ordering of the fluorescence enhancement on chemcially deposited silver nanoparticle slides was detected.     

Fade and quench-resistant emission in calcium phosphate nanoreactors

The fluorescence emission and photodegradation properties of fluorescein dye inside fluid-filled spherical nanoreactors ～ 150 nm in diameter and surrounded by a few nanometres thick layer of calcium phosphate are considered in detail. Steady state, stopped flow, and laser pulsed fluorescence spectroscopies, absorption spectroscopy, dynamic light scattering and electron microscopy were used to characterize the materials as a function of encapsulated dye concentration, particle concentration, illumination time, and pH. Fluorescein tends to form stable J-aggregates inside the nanoreactors. The molecular collision rate constants between the dye aggregates and between the dyes and soluble quenchers are greatly reduced inside the nanoreactors and are responsible for the observed resistance to photodegradation and reduced emission quenching. A model for dye behaviour in nanoreactors is suggested. Nanoreactors can be concentrated to a high suspension concentration, yielding exceptionally strong luminescence affected only by inner filter effects absent particle-particle crosstalk. These and similar nanoreactors can be utilized as building blocks for three-dimensional photo-optical devices, and as versatile and resilient supramolecular chromophores or tracers in complex fluids, cells and microfluidic systems where high resolution visualization is needed.     

Stability of alginate microbead properties in vitro

Alginate microbeads have been investigated clinically for a number of therapeutic interventions, including drug delivery for treatment of ischemic tissues, cell delivery for tissue regeneration, and islet encapsulation as a therapy for type I diabetes. The physical properties of the microbeads play an important role in regulating cell behavior, protein release, and biological response following implantation. In this research alginate microbeads were synthesized, varying composition (mannuronic acid to guluronic acid ratio), concentration of alginate and needle gauge size. Following synthesis, the size, volume fraction, and morphometry of the beads were quantified. In addition, these properties were monitored over time in vitro in the presence of varying calcium levels in the microenvironment. The initial volume available for solute diffusion increased with alginate concentration and mannuronic (M) acid content, and bead diameter decreased with M content but increased with needle diameter. Interestingly, microbeads eroded completely in saline in less than 3 weeks regardless of synthesis conditions much faster than what has been observed in vivo. However, microbead stability was increased by the addition of calcium in the culture medium. Beads synthesized with low alginate concentration and high G content exhibited a more rapid change in physical properties even in the presence of calcium. These data suggest that temporal variations in the physical characteristics of alginate microbeads can occur in vitro depending on synthesis conditions and microbead environment. The results presented here will assist in optimizing the design of the materials for clinical application in drug delivery and cell therapy.     

Analytical performance of an ultrasonic particle focusing flow cytometer

Creation of inexpensive small-flow cytometers is important for applications ranging from disease diagnosis in resource-poor areas to use in distributed sensor networks. In conventional-flow cytometers, hydrodynamics focus particles to the center of a flow stream for analysis, which requires sheath fluid that increases consumable use and waste while dramatically reducing instrument portability. Here we have evaluated, using quantitative measurements of fluorescent microspheres and cells, the performance of a flow cytometer that uses acoustic energy to focus particles to the center of a flow stream. This evaluation demonstrated measurement precision for fluorescence and side scatter CVs for alignment microspheres of 2.54% and 7.7%, respectively. Particles bearing 7 x 10(3) fluorophores were well resolved in a background of 50 nM free fluorophore. The lower limit of detection was determined to be about 650 fluorescein molecules. Analysis of Chinese hamster cells on the system demonstrated that acoustic focusing had no effect on cellular viability. These results indicate that the ultrasonic flow cytometer has the necessary performance for most flow cytometry applications. Furthermore, through robust engineering approaches and the combination of acoustic focusing with low-cost light sources, detectors, and data acquisition systems, it will be possible to achieve a low-cost, truly portable flow cytometer.     

Effect of sintering temperature on the emission-spectrum-shape-evolution of Sr<Subscript>2</Subscript>SiO<Subscript>4</Subscript>:Eu<Superscript>2+</Superscript> phosphor

Color rendering index and color temperature are the key factors for the LEDs application. The two points are closely related to the emission spectrum shape of phosphors. As the key factors for the LEDs application, both the above aspects are closely dependent on the emission spectrum shape of phosphors. In this study, the emission spectrum shape has been adjusted via a home designed route. A combination of structural, morphological, and optical characterization techniques has been used to study the shape evolution mechanism. The structural results show that the Sr₂SiO₄ phase has not been changed with the sintering temperature increasing, but the emission spectrum shape has changed dramatically, meanwhile, the colorimetric coordinate moves from blue-green to green region. Gaussian fitting method has been used to treat the emission spectrum, and the as-obtained results indicate the emission spectrum contains two single bands, which come from the 4f⁷(⁷S_7/2)–4f⁶(⁷F_J)5d¹ transition of Eu²⁺ on the different Sr sites in the Sr₂SiO₄ crystal. The intensity of the two single bands is driven by sintering temperature, because of the difference between the energy barrier of the Eu²⁺ occupying the different Sr sites in the matrix crystal. Moreover, the mechanism of the above phenomenon has also been studied by means of first principles method, and the obtained results agree well with the former deduction.     

Engineering of Lanthanide‐Doped Upconversion Nanoparticles for Optical Encoding

Lanthanide‐doped upconversion nanoparticles (UCNPs) are an emerging class of luminescent materials that emit UV or visible light under near infra‐red (NIR) excitations, thereby possessing a large anti‐Stokes shift property. Due to their sharp excitation and emission bands, excellent photo‐ and chemical stability, low autofluorescence, and high tissue penetration depth of the NIR light used for excitation, UCNPs have surpassed conventional fluorophores in many bioapplications. A better understanding of the mechanism of upconversion, as well as the development of better approaches to preparing UCNPs, have provided more opportunities to explore their use for optical encoding, which has the potential for applications in multiplex detection and imaging. With the current ability to precisely control the microstructure and properties of UCNPs to produce particles of tunable emission, excitation, luminescence lifetime, and size, various strategies for optical encoding based on UCNPs can now be developed. These optical properties of UCNPs (such as emission and excitation wavelengths, ratiometric intensity, luminescence lifetime, and multicolor patterns), and the strategies employed to engineer these properties for optical encoding of UCNPs through homogeneous ion doping, heterogeneous structure fabrication and microbead encapsulation are reviewed. The challenges and potential solutions faced by UCNP optical encoding are also discussed.     

A novel fluorescent label based on organic dye-doped silica nanoparticles for HepG liver cancer cell recognition

In this paper, we report a method for the recognition of HepG liver cancer cells with the use of a novel fluorescent label based on organic dye-doped fluorescent silica nanoparticles. The novel organic dye-doped silica nanoparticles are prepared with a water-in-oil microemulsion technique. The silica network is produced by the controlled synchronous hydrolysis of tetraethoxysilane and 3-amino-propyltriethoxysilane (APTES). The organic dye fluorescein isothiocyanate is doped inside as a luminescent signaling element, through covalent bonding to the amino group of APTES. The organic dye-doped core-shell nanoparticles are highly luminescent and exhibit minimal dye leaching and excellent photostability. A novel fluorescent label method based on biological fluorescent nanoparticles has been developed. The dye-doped fluorescent silica nanoparticles are covalently immobilized with anti-human liver cancer monoclonal antibody HAb18. We have used antibody-labeled fluorescent nanoparticles to recognize HepG liver cancer cells. It has been observed that the bioassay based on the organic dye-doped nanoparticles can identify the target cells selectively and efficiently. The fluorescent nanoparticle label also exhibits high photostability.