Fluorescent nanosensors for intracellular chemical analysis: decyl methacrylate liquid polymer matrix and ion-exchange-based potassium PEBBLE sensors with real-time application to viable rat C6 glioma cells

Fluorescent spherical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), in the 500 nm-1 microm size range have been developed using decyl methacrylate as a matrix. A general scheme for the polymerization and introduction of sensing components creates a matrix that allows for the utilization of the highly selective ionophores used in poly(vinyl chloride) and decyl methacrylate ion-selective electrodes. We have applied these optically silent ionophores to fluorescence-based sensing by using ion-exchange and highly selective pH chromoionophores. This allows the tailoring of selective submicrometer sensors for use in intracellular measurements of important analytes for which selective enough fluorescent probes do not exist. The protocol for sensor development has been worked out for potassium sensing. It is based on the BME-44 ionophore (2-dodecyl-2-methyl-1,3-propanediylbis[N-[5'nitro(benzo-15-crown-5)-4'-yl]carbamate]). The general scheme should work for any available ionophore used in PVC or decyl methacrylate ion-selective electrodes, with minor adjustments to account for differences in ionophore charge and analyte binding constant. The reversible and highly selective sensors developed have a subsecond response time and an adjustable dynamic range. Applications to live C6 glioma cells demonstrate their utility; the intracellular potassium activity is followed in real time upon extracellular administration of kainic acid.     

Optical nanosensors for chemical analysis inside single living cells. 1. Fabrication, characterization, and methods for intracellular delivery of PEBBLE sensors

Spherical optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced in sizes including 20 and 200 nm in diameter. These sensors are fabricated in a microemulsion and consist of fluorescent indicators entrapped in a polyacrylamide matrix. A generalized polymerization method has been developed that permits production of sensors containing any hydrophilic dye or combination of dyes in the matrix. The PEBBLE matrix protects the fluorescent dye from interference by proteins, allowing reliable in vivo calibrations of dyes. Sensor response times are less than 1 ms. Cell viability assays indicate that the PEBBLEs are biocompatible, with negligible biological effects compared to control conditions. Several sensor delivery methods have been studied, including liposomal delivery, gene gun bombardment, and picoinjection into single living cells.     

Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors

Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective fluorescent indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of fluorescent indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Specifically, a distinct difference is noted between cells of different types (astrocyte vs neuron-derived cells) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle differences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB. Specifically, measurement of intracellular calcium, the precursor to cell death, has been achieved.     

A real-time ratiometric method for the determination of molecular oxygen inside living cells using sol-gel-based spherical optical nanosensors with applications to rat C6 glioma

The first sol-gel-based, ratiometric, optical nanosensors, or sol-gel probes encapsulated by biologically localized embedding (PEBBLEs), are made and demonstrated here to enable reliable, real-time measurements of subcellular molecular oxygen. Sensors were made using a modified St?ber method, with poly(ethylene glycol) as a steric stabilizer. The radii of these spherical PEBBLE sensors range from about 50 to 300 nm. These sensors incorporate an oxygen-sensitive fluorescent indicator, Ru(II)-tris(4,7-diphenyl-1,10-phenanthroline) chloride ([Ru(dpp)3]2+), and an oxygen-insensitive fluorescent dye, Oregon Green 488-dextran, as a reference for the purpose of ratiometric intensity measurements. The PEBBLE sensors have excellent reversibility, dynamic range, and stability to leaching and photobleaching. The small size and inert matrix of these sensors allow them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. Applications of sol-gel PEBBLEs inserted in rat C6 glioma cells for real-time intracellular oxygen analysis are demonstrated. Compared to using free dyes for intracellular measurements, the PEBBLE matrix protects the fluorescent dyes from interference by proteins in cells, enabling reliable in vivo chemical analysis. Conversely, the matrix also significantly reduces the toxicity of the indicator and reference dyes to the cells, so that a wide variety of dyes can be used in optimal fashion.     

Real-time measurements of dissolved oxygen inside live cells by organically modified silicate fluorescent nanosensors

Optical PEBBLE (probes encapsulated by biologically localized embedding) nanosensors have been developed for dissolved oxygen using organically modified silicate (ormosil) nanoparticles as a matrix. The ormosil nanoparticles are prepared via a sol-gel-based process, which includes the formation of core particles with phenyltrimethoxysilane as a precursor followed by the formation of a coating layer with methyltrimethoxysilane as a precursor. The average diameter of the resultant particles is 120 nm. These sensors incorporate the oxygen-sensitive platinum porphyrin dye as an indicator and an oxygen-insensitive dye as a reference for ratiometric intensity measurement. Two pairs of indicator dye and reference dye, respectively, platinum(II) octaethylporphine and 3,3'-dioctadecyloxacarbocyanine perchlorate, and platinum(II) octaethylporphine ketone and octaethylporphine, were used. The sensors have excellent sensitivity with an overall quenching response of 97%, as well as excellent linearity of the Stern-Volmer plot (r(2) = 0.999) over the whole range of dissolved oxygen concentrations (0-43 ppm). In vitro intracellular changes of dissolved oxygen due to cell respiration were monitored, with gene gun injected PEBBLEs, in rat C6 glioma cells. A significant change was observed with a fluorescence ratio increase of up to 500% after 1 h, for nine different sets of cells, which corresponds to a 90% reduction in terms of dissolved oxygen concentration. These results clearly show the validity of the delivery method for intracellular studies of PEBBLE sensors, as well as the high sensitivity, which is needed to achieve real-time measurements of intracellular dissolved oxygen concentration.     

One- and two-photon excited optical ph probing for cells using surface-enhanced Raman and hyper-Raman nanosensors

We demonstrate spatially resolved probing and imaging of pH in live cells by mobile and biocompatible nanosensors using surface-enhanced Raman scattering (SERS) of 4-mercaptobenzoic acid (pMBA) on gold nanoaggregates. Moreover, we also show that this concept of pH nanosensors can be extended to two-photon excitation by using surface-enhanced hyper-Raman scattering (SEHRS). In addition to the advantages of two-photon excitation, the SEHRS sensor enables measurements over a wide pH range without the use of multiple probes.     

Plasmonic nanosensors for imaging intracellular biomarkers in live cells

Here we present the first intracellular molecular imaging platform using multifunctional gold nanoparticles which incorporate both cytosolic delivery and targeting moieties on the same particle. The utility of these intracellular sensors was demonstrated by monitoring actin rearrangement in live fibroblasts. We observed a strong molecular specific optical signal associated with effective targeting of actin filaments. These multifunctional nanosensors can be adapted to target various intracellular processes especially where transfection or cytotoxic labels are not feasible.     

Encapsulation of enzymes within polymer spheres to create optical nanosensors for oxidative stress

We describe the fabrication and characterization of poly(ethylene glycol) (PEG) hydrogel spheres containing the enzyme horseradish peroxidase (HRP) for application as optical nanosensors for hydrogen peroxide. HRP was encapsulated in PEG hydrogel spheres by reverse emulsion photopolymerization, yielding spheres with a size range from 250 to 350 nm. Encapsulated HRP activity and sensitivity to hydrogen peroxide were investigated by the Amplex Red assay based on the fluorescence response as a function of H2O2. These HRP-loaded spheres were then introduced to murine macrophages with Amplex Red in the culture media. After phagocytosis, the biocompatibility of spheres was determined by live cell staining using calcein AM (5 microM). The HRP-loaded PEG hydrogel spheres were activated (i.e., fluorescent oxidized Amplex Red produced within the spheres) by oxidative stresses such as exogenous H2O2 (100 microM) and lipopolysaccharide (1 microg/mL), which induced the production of endogenous peroxide inside macrophages. The results presented here indicate that after polymerization, the enzyme activity of HRP was still maintained and that using these HRP-containing nanospheres, peroxide production could be sensed locally within cells.     

Poly(styrene-block-vinylpyrrolidone) beads as a versatile material for simple fabrication of optical nanosensors

A versatile platform for designing optical nanosensors is proposed. The "sensing chemistries" are entrapped into the poly(styrene-block-vinylpyrrolidone) nanobeads having the average size of 245 nm in aqueous media. Addressable staining into the core or the shell of the beads results in nanosensors for essential analytes such as dissolved oxygen, temperature, pH, chloride, and copper ions. Two immobilization procedures are developed: staining in the polystyrene core is performed from a tetrahydrofuran/water mixture (50:50 v/v) and staining in the poly(vinylpyrrolidone) shell is achieved by using the ethanol/water mixture (70:30 v/v). The oxygen and temperature indicators should be preferably immobilized into the core, whereas nanosensors for ions are manufactured by staining into the shell. In the case of the lipophilic pH indicators both procedures result in similar pKa values. The unique properties of the beads make them promising for sensing and imaging even in very complex media, multianalyte sensing, and monitoring of very fast processes.     

Nanogap detector inside nanofluidic channel for fast real-time label-free DNA analysis

We report fabrication and characterization of a novel real-time, label-free DNA detector, that uses a long nanofluidic channel to stretch a DNA strand and a nanogap detector (with a gap as small as 9 nm) inside the channel to measure the electrical conduction perpendicular to the DNA backbone as it moves through the gap. We have observed electrical signals caused by 1.1 kilobase-pair (kbp) double-stranded (ds)-DNA passing through the gap in the nanogap detectors with a gap equal to or less than 13 nm.     

非接触式光纤内螺纹测试装置

为克服接触式测量内螺纹方法的不足,研制了基于强度调制型光纤传感器的非接触测量装置.通过与特制的机械结构相结合.实现了相对效率高、速度快、且便于应用的内螺纹参数的测量.该装置主要由进给平台和光纤探头组成,同时采用半导体激光光源(LD)作光源,光电二极管为光电转换器件.测量的信号经光路转换及前置放大后,通过采样保持送入数据采集系统,经数据处理软件Matlab进行处理得出螺距误差.实验表明,经适当改进后,该方法可用于工业生产过程中内螺纹的非接触测量.     

A CNN-based passive optical range finder for real-time roboticapplications

The paper presents a new cellular neural network (CNN) for real-time stereo vision, useful as a passive optical range finder for autonomous robots and vehicles. The stereo matching as energy minimization is discussed, and former neural approaches to the problem are analyzed. Experimental results with the new CNN both with synthetic and real images are reported, demonstrating the performance of the system     

Plastic crystal-incorporated magnesium ion conducting gel polymer electrolyte for battery application

Studies on a novel composition of magnesium ion conducting gel polymer electrolyte (GPE), comprising a solution of Mg-salt, magnesium trifluoromethanesulfonate (Mg-triflate or \(\hbox {Mg(Tf)}_{2})\) in a plastic crystal succinonitrile (SN), entrapped in a host polymer poly(vinylidenefluoride–hexafluoropropylene) (PVdF–HFP) was reported. Small amount of an ionic liquid, 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (EMITf) was added to stabilize the GPE composition. The electrolyte possesses excellent dimensional integrity in the form of free-standing thick film, which offers the ionic conductivity of \(4 \times 10^{-3} \hbox { S } \hbox {cm}^{-1}\) at room temperature \({\sim }26{^{\circ }}\hbox {C}\). The electrochemical potential window of the electrolyte, observed from the linear sweep voltammetry, is determined to be \({\sim }4.1 \hbox { V}\). The magnesium ion conduction in the GPE film is confirmed from cyclic voltammetry, electrochemical impedance spectroscopy and dc polarization techniques. Different structural, thermal and electrochemical studies demonstrate the promising characteristics of the polymer film, suitable as electrolyte in rechargeable magnesium batteries. The potential of the GPE as electrolyte/separator was ascertained by fabricating a prototype magnesium battery of the configuration Mg:graphite composite \(\hbox {anode}/\hbox {GPE}/\hbox {MnO}_{2}\)-cathode. The specific discharge capacity of \(40 \hbox { mAh g}^{-1}\) (with respect to the \(\hbox {MnO}_{2}\) cathode material) was obtained at the first discharge. The cell shows charge–discharge performance for eight cycles with a substantial fading in capacity.     

Combined in situ and ex situ optical data analysis of magnesium fluoride coatings deposited by plasma ion assisted deposition

The combination of in situ spectrophotometry during film deposition and ex situ spectrophotometry allows insight into the depth distribution of optical losses in plasma ion assisted deposition coatings. An adapted optical characterization strategy for absorbing coatings using only in situ transmittance data has been developed and is exemplified in application to magnesium fluoride coatings. Measurements and simulation results strongly indicate an increased absorption caused by local understoichiometry of the fluoride material close to the fused silica substrate.     

Digital error-signal extraction technique for real-time automatic control of optical interferometers

We describe an efficient and robust method for the extraction of the longitudinal error signal for the automatic control of optical interferometers, which can also be applied when the uncontrolled optical system spans hundreds of fringes. The method is based on classic modulation techniques (phase modulation, mechanical modulation, etc.), but extends their performances by the use of the information available only at the output photodiode. We digitally implemented such a method by following modular hardware and software architectures. We then tested the whole procedure in the automatic control of a suspended Michelson interferometer, showing its feasibility and the good performances.     

Phase-sensitive optical low-coherence reflectometry for the detection of analyte concentrations

Optical techniques may potentially be used for noninvasive glucose sensing. We investigated the application of phase-sensitive optical low-coherence reflectometry (PS-OLCR) to the measurement of analyte concentrations. The dependence of the PS-OLCR signal on the concentration of various analytes, including aqueous solutions of glucose, calcium chloride, magnesium chloride, sodium chloride, potassium chloride, potassium bicarbonate, urea, bovine serum albumin, and bovine globulin, were determined in clear and turbid media. Obtained results demonstrated (1) a high degree of sensitivity and accuracy of the phase measurements of analyte concentrations with PS-OLCR; (2) a concentration-dependent change in the phase-shift for glucose that is significantly greater than that of other analytes sampled over the same physiological range; and (3) a high submillimolar sensitivity of PS-OLCR for the measurement of glucose concentration. Further exploration of the application of PS-OLCR to the noninvasive, sensitive, and specific monitoring of glucose concentration seems warranted.     

Multicolor quantum dot encoding for polymeric particle-based optical ion sensors

Multicolor quantum dot-encoded polymeric microspheres are prepared with controllable and uniform doping levels that function as chemical sensors on the basis of bulk optode theory. TOP/TOPO-capped CdSe quantum dots and CdTe quantum dots capped with CdS (lambdaem = 610 and 700 nm, lambdaex = 510 nm) are blended with a THF solution of poly(methyl methacrylate-co-decyl methacrylate), poly(n-butylacrylate), or poly(vinyl chloride) plasticized with bis(2-ethylhexyl) sebacate without a need for ligand exchange. Polymeric microspheres are generated under mild, nonreactive conditions with a particle caster that breaks down a polymer stream containing the quantum dots into fine droplets by the vibration of a piezocrystal. The resulting microspheres exhibit uniform size and fluorescence emission intensities. Fluorescent bar codes are obtained by subsequent doping of two quantum dots with different colors and mass ratios into the microspheres. A linear relationship is found between the readout fluorescence ratio of the two types of nanocrystals and the mixing ratio. Quantum dot-encoded ion sensing optode microspheres are prepared by simultaneous doping of sodium ionophore X, chromoionophore II, a lipophilic tetraphenylborate cation exchanger, and TOPO-capped CdSe/CdS quantum dot as the fluorescent label. A net positive charge of the quantum dots is found to induce an anion-exchange effect on the sensor function, and therefore, an increased concentration of the lipophilic cation exchanger is required to achieve proper ion sensing properties. The modified quantum dot-labeled sodium sensing microspheres show satisfactory sodium response between 10(-4) and 0.1 M at pH 4.8, with excellent selectivity toward common interferences. The amount of the carried positive charges of the CdSe quantum dots is estimated as 2.8 mumol/g of quantum dots used in this study.