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.     

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.     

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.     

Ratiometric optical PEBBLE nanosensors for real-time magnesium ion concentrations inside viable cells

This paper presents the development and characterization of a highly selective magnesium fluorescent optical nanosensor, made possible by PEBBLE (probe encapsulated by biologically localized embedding) technology. A ratiometric sensor has been developed by co-immobilizing a dye that is sensitive to and highly selective for magnesium, with a reference dye in a matrix. The sensors are prepared via a microemulsion polymerization process, which entraps the sensing components inside a polymer matrix. The resultant spherical sensors are approximately 40 nm in diameter. The Coumarin 343 (C343) dye, which by itself does not enter the cell, when immobilized in a PEBBLE is used as the magnesium-selective agent that provides the high and necessary selectivity over other intracellular ions, such as Ca2+, Na+, and K+. The dynamic range of these sensors was 1-30 mM, with a linear range from 1 to 10 mM, with a response time of <4 s. In contrast to free dye, these nano-optodes are not perturbed by proteins. They are fully reversible and exhibit minimal leaching and photobleaching over extended periods of time. In vitro intracellular changes in Mg2+ concentration were monitored in C6 glioma cells, which remained viable after PEBBLE delivery via gene gun injection. The selectivity for Mg2+ along with the biocompatibility of the matrix provides a new and reliable tool for intracellular magnesium measurements.     

Nanoparticle PEBBLE sensors for quantitative nanomolar imaging of intracellular free calcium ions

Ca(2+) is a universal second messenger and plays a major role in intracellular signaling, metabolism, and a wide range of cellular processes. To date, one of the most successful approaches for intracellular Ca(2+) measurement involves the introduction of optically sensitive Ca(2+) indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca(2+) indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most-sensitive indicators, cytotoxicity of the indicators, interference from nonspecific binding caused by cellular biomacromolecules, challenging calibration, and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca(2+) indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine-based Ca(2+) fluorescence indicators. The rhod-2-containing PEBBLEs presented here show a stable sensing range at near-neutral pH (pH 6-9). Because of the protection of the PEBBLE matrix, the interference of protein-nonspecific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (K(d) = 478 nM) and intracellular (K(d) = 293 nM) measurements. These nanosensors are useful quantitative tools for the measurement and imaging of the cytosolic nanomolar free Ca(2+) levels.     

Design of a low-cost optical instrument for pH fluorescence measurements

This paper describes the electronic design and the performance of a low-cost fiber-optic instrument for pH fluorescent measurements. The chemical sensing phase consists of an organic pH indicator (mercurochrome) immobilized in a sol-gel matrix placed at the end of a fiber optic by means of a steel grid. The active phase was excited by means of a high-intensity blue light-emitting diode. The light signal was modulated to avoid external interference. Fluorescence emission is detected by a low-cost photodiode. To avoid drifts in excitation light emission intensity, a ratiometric measurement was proposed. To perform such measurements, two fiber-optic measurement channels were used. One of them was employed to measure only the pH indicator fluorescent emission intensity. The second channel was employed to measure only the intensity of the excitation light reflected by the sensing phase. The ratio between both signals is only proportional to pH and proved to be independent of excitation light intensity. The sensor is useful over the pH range of 4-8, providing highly reliable results.     

Ratiometric and fluorescence-lifetime-based biosensors incorporating cytochrome c' and the detection of extra- and intracellular macrophage nitric oxide.

Ratiometric and lifetime-based sensors have been designed for cellular detection of nitric oxide. These sensors incorporate cytochrome c', a hemoprotein known to bind nitric oxide selectively. The cytochrome c' is labeled with a fluorescent reporter dye, and changes in this dye's intensity or fluorescence lifetime are observed as the protein binds nitric oxide. The ratiometric sensors are composed of dye-labeled cytochrome c' attached to the optical fiber via colloidal gold, along with fluorescent microspheres as intensity standards. These ratiometric sensors exhibit linear response, have fast response times (< or = 0.25 s), and are completely reversible. The sensors are selective over numerous common interferents such as nitrite, nitrate, and oxygen species, and the limit of detection is 8 microM nitric oxide. The lifetime-based measurements are made using free, dye-labeled cytochrome c' in solution and have a limit of detection of 30 microM nitric oxide. The use of these two techniques has allowed measurement of intra- and extracellular macrophage nitric oxide. Employing the ratiometric fiber sensors gave a multicell culture average extracellular nitric oxide concentration of 210 +/- 90 microM for activated macrophages, while an average intracellular concentration of 160 +/- 10 microM was determined from the lifetime-based measurements of dye-labeled cytochrome c' in the macrophage cytosol. Microscopic adaptation of the lifetime-based methods described here would allow direct correlation of intracellular nitric oxide levels with specific cellular activities, such as phagocytosis.     

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.     

Ratiometric Nanoparticle Probe Based on FRET‐Amplified Phosphorescence for Oxygen Sensing with Minimal Phototoxicity

Luminescent oxygen probes enable direct imaging of hypoxic conditions in cells and tissues, which are associated with a variety of diseases, including cancer. Here, a nanoparticle probe that addresses key challenges in the field is developed, it: i) strongly amplifies room temperature phosphorescence of encapsulated oxygen‐sensitive dyes; ii) provides ratiometric response to oxygen; and iii) solves the fundamental problem of phototoxicity of phosphorescent sensors. The nanoprobe is based on 40 nm polymeric nanoparticles, encapsulating ≈2000 blue‐emitting cyanine dyes with fluorinated tetraphenylborate counterions, which are as bright as 70 quantum dots (QD525). It functions as a light‐harvesting nanoantenna that undergoes efficient Förster resonance energy transfer to ≈20 phosphorescent oxygen‐sensitive platinum octaethylporphyrin (PtOEP) acceptor dyes. The obtained nanoprobe emits stable blue fluorescence and oxygen‐sensitive red phosphorescence, providing ratiometric response to dissolved oxygen. The light harvesting leads to ≈60‐fold phosphorescence amplification and makes the single nanoprobe particle as bright as ≈1200 PtOEP dyes. This high brightness enables oxygen detection at a single‐particle level and in cells at ultra‐low nanoprobe concentration with no sign of phototoxicity, in contrast to PtOEP dye. The developed nanoprobe is successfully applied to the imaging of a microfluidics‐generated oxygen gradient in cancer cells. It constitutes a promising tool for bioimaging of hypoxia.     

Polyelectrolyte microshells as carriers for fluorescent sensors: loading and sensing properties of a ruthenium-based oxygen indicator

A strategy for the design and fabrication of microcapsule-based fluorescent biosensors containing indicators and internal references is described. The rationale for this work is the physical immobilization and chemical separation of assay chemistry for use in biological environments. Using the general approach of depositing oppositely charged species on colloidal micro/nanotemplates, a sensor system employing polyelectrolyte microshells for uptake of functional molecules is proposed, and experiments to demonstrate the feasibility of nanoengineering the sensor properties are described in the context of an oxygen sensor. Methods for immobilization and entrapment of fluorescent indicator and reference dyes are shown, along with the pH dependence of this process. Embedded dyes are shown to be stable and retain their function, as demonstrated with oxygen-sensitivity experiments of loaded microcapsules. Although oxygen sensitivity is presented as an example of a specific application, the overall strategy is likely more generally useful. The work suggests that polyelectrolyte microshells may be used as a platform to develop novel sensors by entrapment of functional materials.     

Molecular oxygen-sensitive fluorescent lipobeads for intracellular oxygen measurements in murine macrophages

Intracellular oxygen concentration is of primary importance in determining numerous physiological and pathological processes in biological systems. This paper describes the development and application of micrometer-sized oxygen-sensitive fluorescence lipobeads for intracellular measurements of molecular oxygen in J774 murine macrophages. A ruthenium diimine complex [Ru(bpy-pyr)(bpy)2]C12 (bpy = 2,2'-bipyridine, bpy-pyr = 4-(1"-pyrenyl)-2,2'-bipyridine) is used as the oxygen indicator. The indicator exhibits high chemical and photostability and high sensitivity to oxygen. The indicator molecules are immobilized in a phospholipid membrane that coats polystyrene microparticles. The fluorescence of the lipobeads is effectively quenched by molecular oxygen. The fluorescence intensity of the oxygen-sensitive lipobeads is 3 times higher in a nitrogenated solution than in an oxygenated solution. The lipobeads are internalized by murine macrophages through phagocytosis. They maintain their spectral properties for 24 h in living cells when the cells are stored in phosphate-buffered saline at pH 7.4. The photostability, reversibility, and effect of hypoxia, hyperoxia, and oxidative stress on the intracellular level of oxygen in J774 murine macrophages are described.     

Estimating weak ratiometric signals in imaging data. I. Dual-channel data

Ratiometric fluorescent indicators are becoming increasingly prevalent in many areas of biology. They are used for making quantitative measurements of intracellular free calcium both in vitro and in vivo, as well as measuring membrane potentials, pH, and other important physiological variables of interest to researchers in many subfields. Often, functional changes in the fluorescent yield of ratiometric indicators are small, and the signal-to-noise ratio (SNR) is of order unity or less. In particular, variability in the denominator of the ratio can lead to very poor ratio estimates. We present a statistical optimization method for objectively detecting and estimating ratiometric signals in dual-wavelength measurements of fluorescent, ratiometric indicators that improves on standard methods. With the use of an appropriate statistical model for ratiometric signals and by taking the pixel-pixel covariance of an imaging dataset into account, we are able to extract user-independent spatiotemporal information that retains high resolution in both space and time.     

Self‐Assembly of a Monochromophore‐Based Polymer Enables Unprecedented Ratiometric Tracing of Hypoxia

The accuracy of traditional bischromophore‐based ratiometric probes is always compromised by undesirable energy/charge transferring interactions between the internal reference moiety and the sensing chromophore. In this regard, ratiometric sensing with a monochromophore system is highly desirable. Herein, an unprecedented monochromophore‐based ratiometric probe, which consists of a hydrophilic backbone poly(N‐vinylpyrrolidone) (PVP) and single chromophore of platinum(II) tetraphenylporphyrin (Pt‐TPP) is reported. Combination of the specific assembled clustering‐triggered fluorescent emission (oxygen‐insensitive) with the original Pt‐TPP phosphorescence (oxygen‐sensitive) enables successful construction of a monochromophore‐based ratiometric nanosensor for directly tracing hypoxia in vivo, along with the preferable facilitation of enhanced permeation and retention effect and long excitation wavelength. The unique ratiometric signals enable the direct observation from normoxic to hypoxic environment in both living A549 cells and a tumor‐bearing mice model, providing a significant paradigm of a monochromophore‐based dual‐emissive system with the specific assembled cluster emission. The work satisfactorily demonstrates a valuable strategy for designing monochromophore‐based dual‐emissive materials, and validates its utility for in vivo ratiometric biological sensing without the common energy/charge interference in bischromophore‐based system.     

Core-referenced ratiometric fluorescent potassium ion sensors using self-assembled ultrathin films on europium nanoparticles

Nanoengineered fluorescent sensor coatings on colloidal carriers have been developed for use intracellularly. These nanosensors are fabricated via the electrostatic layer-by-layer self-assembly technique to form ultrathin polyelectrolyte films containing indicators on fluorescent nanoparticles. The fluorescent nanoparticle templates and the fluorescent indicator are chosen such that their optical properties are complementary, enabling the inert nanoparticle templates to serve as internal intensity references for the fluorescent probe. In this work, the potassium ion indicator, potassium-binding benzofuran isophthalate potassium-binding benzofuran isophthalate was immobilized within poly(styrene sulfonate)/poly(allylamine hydrochloride) films assembled on the surface of fluorescent europium nanoparticles. The indicator retains its sensitivity to potassium ions after immobilization within the films and exhibits sensitivity to increases in potassium concentration over a broad range. In addition, the sensors demonstrate excellent leaching stability, with less than 1% of loaded indicator leached after 14 days of wet storage. The core-referenced nanosensor scheme described here is a simple and elegant way to co-immobilize fluorescent indicator and intensity reference within a single nanoscale package, which may be deployed intracellularly; furthermore, the separation of fluorescent indicator from the cellular environment is attractive, as it may prevent complications due to use of liquid-phase fluorescent sensors intracellularly, such as cytotoxicity and probe compartmentalization.     

Ratiometric fluorescence detection of mercury ions in water by conjugated polymer nanoparticles

We present dye-doped polymer nanoparticles that are able to detect mercury in aqueous solution at parts per billion levels via fluorescence resonance energy transfer (FRET). The nanoparticles are prepared by reprecipitation of highly fluorescent conjugated polymers in water and are stable in aqueous suspension. They are doped with rhodamine spirolactam dyes that are nonfluorescent until they encounter mercury ions, which promote an irreversible reaction that converts the dyes to fluorescent rhodamines. The rhodamine dyes act as FRET acceptors for the fluorescent nanoparticles, and the ratio of nanoparticle-to-rhodamine fluorescence intensities functions as a ratiometric fluorescence chemodosimeter for mercury. The light harvesting capability of the conjugated polymer nanoparticles enhances the fluorescence intensity of the rhodamine dyes by a factor of 10, enabling sensitive detection of mercury ions in water at levels as low as 0.7 parts per billion.     

Multicenter Metal–Organic Framework-Based Ratiometric Fluorescent Sensors

Metal–organic frameworks (MOFs) with multiple emission centers are newly emerging as ratiometric sensors owing to their high sensitivity and high selectivity toward a wide range of targeted functional species. Energy transfer between the light-absorbing group and emission centers and between different emission centers is the key to rationally design and synthesize MOF-based ratiometric sensors. A good match between the energy levels of the light-absorbing groups and emission centers is the prerequisite for MOF-based sensors to exhibit multiple emissions, and a good match of the MOF-based sensors and those of the targeted species can increase the sensitivity and selectivity, but this match is highly challenging to obtain via synthesis. MOFs with multiple emission centers can be produced by functionalizing MOFs with multiple lanthanide centers, organic luminophores, dyes, carbon dots, and other such emissive groups. In this progress report, recent advances in the strategies for synthesizing MOFs with multiple emission centers and their applications for ratiometric sensing of solution conditions, including the pH value, and ion, organic molecule, and biomolecule concentrations, are summarized, as are the related sensing mechanisms.     

Doped Thin-Film Sensors via a Sol-Gel Process for High-Acidity Determination

An optical sensor has been developed for high-acidity ([H(+)] = 1-11 M) measurements. The sensor is made of thin films of silica sol-gels doped with an acid indicator. Acid- and base-catalyzed methods to make the sol-gel films have been studied, and the properties of the sol-gel sensors prepared by these methods are discussed. The acid-catalyzed method was found to give more robust films and has been optimized to prepare thin films which are mechanically and chemically stable for a period of at least 3 months. The performance of the sensors resulted in a relative standard deviation of less than 2.5%. The response time is short (1 s), and a small hysteresis was observed during reproducibility measurements with 2-10 M HCl solutions.     

In situ fiber-optic oxygen consumption measurements from a working mouse heart.

Luminescence-based imaging-fiber oxygen sensors (IFOSs) were utilized for the in situ measurement of oxygen consumption from intact perfused mouse hearts. IFOSs were fabricated using a technically expedient, photoinitiated polymerization reaction whereby an oxygen-sensitive polymer matrix was immobilized in a precise location on an imaging fiber's distal face. The oxygen-sensing layer used in this work comprised a transition metal complex, Ru(Ph2phen)3(2+), entrapped in a gaspermeable photopolymerizable siloxane membrane (PS802). The transduction mechanism was based upon the oxygen collisional quenching of the ruthenium complex luminescence; detection was performed utilizing an epi-fluorescence microscope/charge coupled device imaging system. IFOS measurements from working mouse hearts were validated through concurrent, blind, ex situ blood gas analyzer (BGA) measurements. The BGA and IFOS methodologies were utilized successfully to measure oxygen concentrations in aortic and pulmonary artery perfusates from the working mouse heart before and after isoproterenol administration. Coupled with coronary-flow measurements, these data were used to calculate myocardial oxygen consumption. Regression analysis of measurements of myocardial oxygen consumption showed that there was a strong correlation between the values generated by the BGA sampling and those obtained via in situ IFOS methods. To our knowledge, this research represents the first report of in situ fiber-optic sensor monitoring of oxygen content from the intact, beating mouse heart.     

Analysis of intracellular oxygen and metabolic responses of mammalian cells by time-resolved fluorometry

A simple, minimally invasive methodology for the analysis of intracellular oxygen in populations of live mammalian cells is described. Loading of the cells with the phosphorescent O(2)-sensing probe, MitoXpress, is achieved by passive liposomal transfer or facilitated endocytosis, followed by monitoring in standard microwell plates on a time-resolved fluorescent reader. Phosphorescence lifetime measurements provide accurate, real-time, quantitative assessment of average oxygen levels in resting cells and their alterations in response to stimulation. Analytical performance of the method is examined, optimized, and then demonstrated with different suspension and adherent cell lines including Jurkat, PC12, A549, HeLa, SH-SY5Y, and C2C12, by monitoring responses to mitochondrial uncouplers, inhibitors, plasma membrane depolarization, and Ca(2+) effectors. The assay provides relevant, information-rich data on cellular function and metabolism. It allows monitoring of small, rapid, and transient changes in cell respiration and screening of new chemical entities.     

Determining oxygen diffusion coefficients in polymer films by lifetimes of luminescent complexes measured in the frequency domain

Polymer films doped with luminescent ruthenium complexes are proving to be important oxygen sensors. We describe a technique using lifetime measurements in the frequency domain for determining the diffusion coefficient of oxygen through various polymer supports. These fundamental measurements will allow for more rational design of improved sensors. Three types of polymers were doped with [Ru(4,7-diphenyl-1,10-phenanthroline)3]Cl2. We monitored the luminescence versus time after applying a step increase in the oxygen pressure at the surface of the film. We modeled the decrease in apparent lifetime as a function of time using the diffusion coefficient of oxygen in the polymer as the only adjustable parameter. The model accurately predicted the lifetime versus time curves, and diffusion coefficients agreed well with those obtained from intensity measurements. The advantages and disadvantages of the lifetime technique to those used earlier are discussed.