Nanoscale probes encapsulated by biologically localized embedding (PEBBLEs) for ion sensing and imaging in live cells

This review discusses the development and recent advances of probes encapsulated by biologically localized embedding (PEBBLEs), and in particular the application of PEBBLEs as ion sensors. PEBBLEs allow for minimally intrusive sensing of ions in cellular environments due to their small size (20 to 600 nm in diameter) and protect the sensing elements (i.e. fluorescent dyes) by encapsulating them within an inert matrix. The selectivity and sensitivity of these nanosensors are comparable to those of macroscopic ion selective optodes, and electrodes, while the response time and absolute detection limit are significantly better. This paper discusses the principles guiding PEBBLE design including synthesis, characterization, diversification, the advantages and limitations of the sensors, cellular applications and future directions of PEBBLE research.     

Poly(decyl methacrylate)-based fluorescent PEBBLE swarm nanosensors for measuring dissolved oxygen in biosamples

150-250 nm Poly(decyl methacrylate)(PDMA) fluorescent ratiometric nanosensors for dissolved oxygen have been developed. Platinum octaethylporphine ketone (PtOEPK), the oxygen-sensitive dye, and octaethylporphyrin (OEP), the oxygen-insensitive dye, have been incorporated into PDMA nanoparticles to make the sensors ratiometric. Based on the corresponding Stern-Volmer plot, these nanosensors exhibit almost complete linearity over the whole range of dissolved molecular oxygen from 0 to 42.5 ppm (deoxygenated to pure oxygen-bubbled water). The overall quenching response is up to 97.5%, the best so far for all dissolved oxygen optical sensors. These PEBBLE nanosensors also show very good reversibility and stability to leaching and photobleaching, as well as very short response times and no perturbation by proteins. In human plasma they demonstrate a robust oxygen sensing capability, little affected by light scattering and autofluorescence. Potential applications include intracellular oxygen imaging and microresolved pressure profiles in biological and other heterogenous environments.     

A fluorescent PEBBLE nanosensor for intracellular free zinc

The development and characterisation of a fluorescent optical PEBBLE (Probe Encapsulated By Biologically Localised Embedding) nanosensor for the detection of zinc is detailed. A ratiometric sensor has been fabricated that incorporates two fluorescent dyes; one is sensitive to zinc and the other acts as a reference. The sensing components are entrapped within a polymer matrix by a microemulsion polymerisation process that produces spherical sensors that are in the size region of 20 to 200 nm. Cellular measurements are made possible by the small sensor size and the biocompatibility of the matrix. The effects of reversibility, photobleaching and leaching have been examined, as well as the selectivity towards zinc over other cellular ions such as Na+, Ca2+, K+, and Mg2+. The dynamic range of these sensors was found to be 4 to 50 microM Zn2+ with a linear range from 15 to 40 microM. The response time for the PEBBLE is less than 4 s and the sensor is reversible. In addition, the nanosensors are photostable and leaching from the matrix, determined using a novel method, is minimal. These sensors are capable of real-time inter- and intra-cellular imaging and are insensitive to interference from proteins.     

Two-photon nano-PEBBLE sensors: subcellular pH measurements

Intracellular pH mapping is of great importance as it plays a critical role in many cellular events. Also, in tissue, pH mapping can be an indicator for the onset of cancer. Here we describe a biocompatible, targeted, ratiometric, fluorescent, pH sensing nano-PEBBLE (Photonic Explorer for Biomedical use with Biologically Localized Embedding) that is based on two-photon excitation. Two-photon excitation minimizes the photobleaching and cell autofluorescence drastically, leading to an increase in the signal-to-noise ratio. PEBBLE nanosensors provide a novel approach for introducing membrane impermeant dyes, like HPTS, into cells. We use both non-targeted and F3 peptide targeted PEBBLE nanosensors for intracellular pH measurement of 9L cells. The intracellular measurements suggest that the non-targeted nanosensors are mostly trapped in endosomes, whereas the F3 peptide targeting enables them to escape/avoid these acidic compartments. Combining the advantages of pH sensitive PEBBLE nanoparticles, including their specific targeting, with the advantages of two-photon microscopy provides an attractive and promising prospect for non-invasive real-time monitoring of pH inside cancer cells and tissues.     

Ratiometric singlet oxygen nano-optodes and their use for monitoring photodynamic therapy nanoplatforms

Ratiometric photonic explorers for bioanalysis with biologically localized embedding (PEBBLE) nanoprobes have been developed for singlet oxygen, using organically modified silicate (ORMOSIL) nanoparticles as the matrix. A crucial aspect of these ratiometric singlet-oxygen fluorescent probes is their minute size. The ORMOSIL nanoparticles are prepared via a sol-gel-based process and the average diameter of the resultant particles is about 160 nm. These sensors incorporate the singlet-oxygen-sensitive 9,10-dimethyl anthracene as an indicator dye and a singlet-oxygen-insensitive dye, octaethylporphine, as a reference dye for ratiometric fluorescence-based analysis. We have found experimentally that these nanoprobes have much better sensitivity than does the conventional singlet-oxygen-free dye probe, anthracene-9,10-dipropionic acid disodium salt. The much longer lifetime of singlet oxygen in the ORMOSIL matrix, compared to aqueous solutions, in addition to the relatively high singlet oxygen solubility because of the highly permeable structure and the hydrophobic nature of the outer shell of the ORMOSIL nanoparticles, results in an excellent overall response to singlet oxygen. These nanoprobes have been used to monitor the singlet oxygen produced by "dynamic nanoplatforms" that were developed for photodynamic therapy. The singlet oxygen nanoprobes could potentially be used to quantify the singlet oxygen produced by macrophages.     

Quantitative generalized ratiometric fluorescence spectroscopy for turbid media based on probe encapsulated by biologically localized embedding

PEBBLE (probe encapsulated by biologically localized embedding) nanosensor encapsulating an intensity-based fluorescence indicator and an inert reference fluorescence dye inside the pores of stable matrix can be used as a generalized wavelength-ratiometric probe. However, the lack of an efficient quantitative model render the choices of inert reference dyes and intensity-based fluorescence indicators used in PEBBLEs based generalized wavelength-ratiometric probes rather limited. In this contribution, an extended quantitative fluorescence model was derived specifically for generalized wavelength-ratiometric probes based on PEBBLE technique (QFM_GRP) with a view to simplify the design of PEBBLEs and hence further extend their application potentials. The effectiveness of QFM_GRP has been tested on the quantitative determination of free Ca²⁺ in both simulated and real turbid media using a Ca²⁺ sensitive PEBBLE nanosensor encapsulating Rhod-2 and eosin B inside the micropores of stable polyacrylamide matrix. Experimental results demonstrated that QFM_GRP could realize precise and accurate quantification of free Ca²⁺ in turbid samples, even though there is serious overlapping between the fluorescence excitation peaks of eosin B and Ca²⁺ bound Rhod-2. The average relative predictive error value of QFM_GRP for the test simulated turbid samples was 5.9%, about 2–4 times lower than the corresponding values of partial least squares calibration model and the empirical ratiometric model based on the ratio of fluorescence intensities at the excitation peaks of Ca²⁺ bound Rhod-2 and eosin B. The recovery rates of QFM_GRP for the real and spiked turbid samples varied from 93.1% to 101%, comparable to the corresponding results of atomic absorption spectrometry.     

A fluorescence ratiometric nano-pH sensor based on dual-fluorophore-doped silica nanoparticles

We have synthesized dual-fluorophore-doped core-shell silica nanoparticles used as ratiometric pH sensor. The nanoparticles were prepared with a reverse microemulsion technique by simultaneously encapsulating two different fluorophores, the pH-sensitive dye fluorescein as a pH indicator and the pH-insensitive dye phenosafranine as an internal reference for fluorescence ratiometric measurement, into silica shell. The nanoparticles prevent the fluorescence dyes leaching from the silica matrix when immersed inside water. The hydrophilic silica shells were made by hydrolysing and polymerizing tetraethoxysilane (TEOS) in water-in-oil microemulsion. The fluorescence intensity ratio of the two dyes varied linearly as a function of pH in the range from 4.0 to 8.0. The sensor was also applied to measure pH of real water samples. The results are in good agreements with that using the conventional glass electrode method. The as-prepared fluorescent nanoparticles showed rapid response, excellent stability and high reproducibility as pH sensors.     

Alexa Fluor 488 as an iron sensing molecule and its application in PEBBLE nanosensors

Molecular Probes' Alexa Fluor dyes are generally used for biological labeling because of their ideal fluorescent properties, but here we detail Alexa Fluor 488's nanomolar sensitivity to free iron. Furthermore, the dye has been encapsulated into a polymer nanosphere by a microemulsion method, producing <100 nm particles. These nanosensors, PEBBLEs (Probe Encapsulated By Biologically Localized Embedding) have micromolar sensitivity and are non-responsive to other metal ions of biological interest.     

Liquid polymer nano-PEBBLEs for Cl- analysis and biological applications

The first nanometer scale anion sensing fluorescent spherical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding) have been developed for the intracellular monitoring of chloride. The 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 poly(decyl methacrylate) ion-selective electrodes. We have demonstrated that our previously developed scheme for cation sensors can be utilized to tailoring selective submicron sensors for use in intracellular measurements of biologically relevant anions for which selective enough fluorescent probes do not exist. Three schemes were attempted for the development of chloride sensitive PEBBLEs. The first two used the Chloride ionophore indium(III) octaethylporphyrin chloride (In(OEP)Cl) (1) as an ionophore working in tandem with a chromoionophore and (2) as a chromoionophore with a peak shift generated by chloride mediated breaking of hydroxide ion-bridged porphyrin dimer. The third method used the optically silent Chloride ionophore III (ETH 9033) working in tandem with chromoionophore III (ETH 5350) to indirectly monitor Cl- activity by reporting the H+ coextracted into the matrix. Method 3 gave the most promising results, at a pH of 7.2 these PEBBLEs have a limit of detection of 0.2 mM Cl- with a linear dynamic range of 0.4 mM-190 mM Cl-. These PEBBLEs were delivered into C6 glioma cells, utilizing a gene gun, and intracellular chloride levels were monitored during ion-channel stimulation by kainic acid.     

Optochemical Nanosensors and Subcellular Applications in Living Cells

What may be the smallest anthropogenic devices to date, spherical sensors (wireless and fiberless) with radii as small as 10?nm have been produced. This class of optochemical PEBBLE (Probe Encapsulated By Biologically Localized Embedding) sensors covers a wide range of analytes (pH, calcium, oxygen and potassium included here) with excellent spatial, temporal and chemical resolution. Examples of such sensors for the monitoring of intracellular analytes are given. Methods, such as pico-injection, liposomal delivery and gene gun bombardment, are used to inject PEBBLE sensors into single cells. These PEBBLEs have caused minimal perturbation when delivered and operated inside single mammalian cells, such as human neuroblastoma, mouse oocytes or rat alveolar macrophage.     

On the Design of Fluorescent Ratiometric Nanosensors

Advances in nanoparticle technology have recently offered new tools to the bioanalytical field of research. In particular, new nanoparticle‐based sensors have appeared able to give quantitative information about different species (ions, metabolites, biomolecules) in biosamples through ratiometric measurements. This article describes the methodologies developed so far in the design of such nanosensors. In particular, the different approaches to immobilize fluorescent chemosensor dyes to nanoparticles are presented. Concept designs of ratiometric nanosensors in terms of composition and architecture are also described and illustrated with examples taken from the literature.     

Synthesis of ratiometric fluorescent nanoparticles for sensing oxygen

A facile reprecipitation-encapsulation method is used for the preparation of ratiometric fluorescent nanoparticles (NPs) for sensing intracellular oxygen. The surface of the NPs is modified in-situ with poly-L-lysine, which renders good biocompatibility and enables easy internalization into living cells. The sensor NPs contain a red fluorescent probe whose fluorescence is sensitive to oxygen with a quenching response of 77 % on going from nitrogen saturation to oxygen saturation, and a reference dye giving a green signal that acts as an oxygen-independent reference. The ratio of the two emissions serves as the analytical information and is sensitive to dissolved oxygen in the 0–43?ppm concentration range. When incorporated into cells, the ratio of the signals increases by 400?% on going from oxygen-saturated to oxygen-free environment.

Microparticle ratiometric oxygen sensors utilizing near-infrared emitting quantum dots

Luminescent sensors incorporating two luminophores, an indicator and a reference, offer many advantages over intensity measurements from sensors made with one indicator dye. Quantum dots have yet to be widely employed as insensitive reference luminophores in such systems. This work describes the use of near-infrared emitting quantum dots in conjunction with a long-lifetime platinum(II) porphyrin phosphor in a microsphere-based, ratiometric oxygen sensor. The process for self-assembly of the nanocomposite system was developed, and the response and photostability of the prototypes were investigated. Results indicate the sensors possess excellent sensitivity (K(SV) = 0.00826 μM(-1)) at oxygen concentrations below 300 μM and were resistant to photobleaching. The sensor luminophores displayed minimal spectral overlap and little interference from excitation light, preventing the need for optical filters. A reversible photoenhancement of the quantum dot signal was also observed when exposed for extended periods of time. This work demonstrates the advantages of incorporating long-wavelength quantum dots into ratiometric intensity sensing schemes and highlights some key limitations that must be considered in their use.     

Optical calcium sensors: development of a generic method for their introduction to the cell using conjugated cell penetrating peptides

Probes Encapsulated By Biologically Localised Embedding (PEBBLEs) are optical sensors with nanometer dimensions fabricated by microemulsion polymerisation. The most beneficial characteristic of these sensors is the protection offered by the sensor matrix which decreases interaction between the fluorophore and the cell. These sensors have been introduced to the cell by a number of methods; however this paper discusses the development of a generic method to facilitate inclusion of this type of sensor in the cell by a simple incubation step. This was achieved by covalent linkage of a synthetic Cell Penetrating Peptide (CPP) based on the Human Immuno-deficiency Virus (HIV) -1 Tat, to the external sensor matrix. Calcium sensors were used to demonstrate this approach to incorporate the sensors within the cell. Characterisation revealed the calcium sensors were approximately 30 +/- 7 nm in diameter with a slightly negative zeta potential. The sensors demonstrated a linear range of 0-50 microM with negligible interference from a range of cellular ions and protein. Leaching of entrapped dyes from the calcium sensors was determined as 3% in a 24 h period, while photobleaching of the entrapped dye was minimal over a 40 min period. The sensors ability to cross the cell membrane using the covalently attached synthetic Tat peptide is demonstrated. Cellular inclusion of the sensors occurred within a 30 min incubation period.     

Optical Ratiometric Sensor for Alcohol Measurements

Abstract

The need for low‐cost, robust alcohol sensors has increased with the renewed interest in alternative fuels as well as high‐throughput screening of biological processes involving the production of ethanol. The goal of this research was to develop a miniaturized optical ratiometric ethanol sensor to be used for in situ measurements. The sensor is based on the fluorescent dye Nile Blue Chloride. When in solution, the dye exhibits a single fluorescence peak. However, a dual emission peak is observed upon physical immobilization of the dye in the hydrogel poly(ethylene glycol) dimethacrylate. The dual emission allows for ratiometric measurements, thus circumventing drawbacks associated with fluorescence intensity measurements such as signal variations due to dye bleaching, source intensity fluctuations, etc. In developing this sensor we investigated ethanol sensitivity; alcohol selectivity; response time; and cross‐sensitivity with pH, polarity, and ionic strength. We found that the sensor is sensitive to a broad range of ethanol concentrations, namely 5% to 90% v/v. Due to the hydrogel's restrictive pore size, the sensor is sensitive to short‐chain alcohols such as methanol, ethanol, and propanol, but lacks sensitivity to larger alcohols such as butanol and hexanol. We also found the sensor maintains full functionality after autoclaving. Sensor sensitivity to alcohol in solutions of varying ionic strength is negligible, whereas the solvent's polarity must be controlled to maintain meaningful results. The sensor is most sensitive in acidic and neutral environments, indicating promising use for yeast‐ based alcohol fermentations.     

Delivery and subcellular targeting of dendrimer-based fluorescent pH sensors in living cells

Synthesis and targeted delivery of dendrimer-based fluorescent biosensors in living HeLa cells are reported. Following electroporation dendrimers are shown to display specific subcellular localization depending on their size and surface charge and this property is preserved when they are functionalized with sensing moieties. We analyze the case of double dendrimer conjugation with pH-sensitive and pH-insensitive molecules leading to the realization of ratiometric pH sensors that are calibrated in vitro and in living cells. By tuning the physicochemical properties of the dendrimer scaffold sensors can be targeted to specific cellular compartments allowing selective pH measurements in different organelles in living cells. In order to demonstrate the modularity of this approach we present three different pH sensors with tuned H(+) affinity by appropriately choosing the pH-sensitive dye. We argue that the present methodology represents a general approach toward the realization of targetable ratiometric sensors suitable to monitor biologically relevant ions or molecules in living cells.     

Synthesis and evaluation of self-calibrating ratiometric viscosity sensors

We describe the design, synthesis and fluorescent profile of a family of self-calibrating dyes that provide ratiometric measurements of fluid viscosity. The design is based on covalently linking a primary fluorophore (reference) that displays a viscosity-independent fluorescence emission with a secondary fluorophore (sensor) that exhibits a viscosity-sensitive fluorescence emission. Characterization of fluorescent properties was made with separate excitation of the units and through Resonance Energy Transfer from the reference to the sensor dye. The chemical structures of both fluorophores and the linker length have been evaluated in order to optimize the overall brightness and sensitivity of the viscosity measurements. We also present an application of such ratiometric dyes for the detection of membrane viscosity changes in a liposome model.     

Simultaneous use of multiple fluorescent reporter dyes for glucose sensing in aqueous solution

The simultaneous use of several fluorescent reporter dyes in a multicomponent boronic acid-based glucose sensing system is reported. In one application, two dyes with widely different emission wavelengths are used to report changes in glucose concentration. A third glucose-insensitive dye was then added to act as a reference dye and provide for a ratiometric correction to the two reporter dye signals. The inclusion of such a reference dye reduces errors arising from sources such as fluctuations in lamp intensity and sample dilution. The simultaneous use of multiple fluorescent reporter dyes     

A unique approach to development of near-infrared fluorescent sensors for in vivo imaging

Near-infrared (NIR) fluorescent sensors have emerged as promising molecular tools for imaging biomolecules in living systems. However, NIR fluorescent sensors are very challenging to be developed. Herein, we describe the discovery of a new class of NIR fluorescent dyes represented by 1a/1c/1e, which are superior to the traditional 7-hydroxycoumarin and fluorescein with both absorption and emission in the NIR region while retaining an optically tunable hydroxyl group. Quantum chemical calculations with the B3LYP exchange functional employing 6-31G(d) basis sets provide insights into the optical property distinctions between 1a/1c/1e and their alkoxy derivatives. The unique optical properties of the new type of fluorescent dyes can be exploited as a useful strategy for development of NIR fluorescent sensors. Employing this strategy, two different types of NIR fluorescent sensors, NIR-H(2)O(2) and NIR-thiol, for H(2)O(2) and thiols, respectively, were constructed. These novel sensors respond to H(2)O(2) or thiols with a large turn-on NIR fluorescence signal upon excitation in the NIR region. Furthermore, NIR-H(2)O(2) and NIR-thiol are capable of imaging endogenously produced H(2)O(2) and thiols, respectively, not only in living cells but also in living mice, demonstrating the value of the new NIR fluorescent sensor design strategy. The new type of NIR dyes presented herein may open up new opportunities for the development of NIR fluorescent sensors based on the hydroxyl functionalized reactive sites for biological imaging applications in living animals.     

Ratiometric fluorescence-based dissolved carbon dioxide sensor for use in environmental monitoring applications

The focus of this work is on the development and characterisation of a fluorescence-based ratiometric sol–gel-derived dissolved carbon dioxide (dCO₂) sensor for use in environmental monitoring applications. Fluorescence-based dCO₂ sensors are attractive as they facilitate the development of portable and low-cost systems that can be easily deployed outside the laboratory environment. The sensor developed for this work exploits a pH fluorescent dye 1-hydroxypyrene-3,6,8-trisulfonic acid, ion-paired with cetyltrimethylammonium bromide (HPTS-IP), which has been entrapped in a hybrid sol–gel-based matrix derived from n-propyltriethoxysilane along with the liphophilic organic base. The sensor spot deposited on a cover slip has been interrogated with a robust, ratiometric optical probe that combines effective fluorescence excitation and detection and thus facilitates the production of a highly sensitive sensor system using low-cost optoelectronic components. The probe design involves the use of dual-LED excitation in order to facilitate ratiometric operation and uses a silicon PIN photodiode. HPTS-IP exhibits two pH-dependent changes in excitation bands, which allows for dual excitation ratiometric detection as an indirect measure of the dCO₂. Such measurements are insensitive to changes in dye concentration, leaching and photobleaching of the fluorophore and instrument fluctuations unlike unreferenced fluorescence intensity measurements. The performance of the sensor system is characterised by a high degree of repeatability, reversibility and stability. Calculated limit of detection for the sensor was 35 ppb. The sensor probe was used to monitor dCO₂ levels in a laboratory-based aquatic habitat, and the expected diurnal pattern was clearly visible. The influence of temperature, biofouling and photobleaching on sensor performance has been also investigated.