Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging

Polyacrylamide-based, ratiometric, spherical, optical nanosensors, or polyacrylamide PEBBLEs (Probes Encapsulated By Biologically Localized Embedding), have been fabricated, aimed at real-time glucose imaging in intact biological systems, i.e. living cells. These nanosensors are prepared using a microemulsion polymerization process, and their average size is about 45 nm in diameter. The sensors incorporate glucose oxidase (GOx), an oxygen sensitive fluorescent indicator (Ru[dpp(SO3Na)2]3)Cl2, and an oxygen insensitive fluorescent dye, Oregon Green 488-dextran or Texas Red-dextran, as a reference for the purpose of ratiometric intensity measurements. The enzymatic oxidation of glucose to gluconic acid results in the local depletion of oxygen, which is measured by the oxygen sensitive ruthenium dye. The small size and inert matrix of these sensors allows them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. The PEBBLE matrix protects the enzyme and 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 larger variety of dyes can be used in optimal fashion. Furthermore, the PEBBLE matrix enables the synergistic approach in which there is a steady state of local oxygen consumption, and this cannot be achieved by separately introducing free enzyme and dyes into a cell. The work presented here describes the production and characterization of glucose sensitive PEBBLEs, and their potential for intracellular glucose measurements. The sensor response is determined in terms of the linear range, ratiometric operation, response time, sensor stability, reversibility and immunity to interferences.     

Luminescent nanobeads for optical sensing and imaging of dissolved oxygen

A variety of luminescent oxygen nanosensors were prepared by addressable staining of poly(styrene-block-vinylpyrrolidone) nanobeads with metal–ligand complexes whose luminescence is quenched by oxygen. They display optimal sensitivity in responding to dissolved oxygen in concentrations from 0 to 100% air saturation. The nanobeads based on cyclometallated iridium(III) complexes with coumarins are especially promising due to excellent brightnesses. The nanosensors respond virtually in real time to altering oxygen concentration and are capable of recording even very rapid changes in oxygen partial pressure. Signals are obtained by determination of luminescence lifetime in the frequency domain and in the time domain, and by ratiometric measurement of luminescence intensity. The nanosensors have been applied to sensing and imaging of dissolved oxygen, to monitor the consumption of oxygen during enzymatic oxidation of glucose, and to monitoring dissolved oxygen in a growing culture of E. coli.     

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.     

Nanometre-sized molecular oxygen sensors prepared from polymer stabilized phospholipid vesicles

Nanometre-sized, chemically-stabilized phospholipid vesicle sensors have been developed for detection of dissolved molecular oxygen. Sensors were prepared by forming 150 nm phospholipid vesicles from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or DOPC doped with small (<1%) mole percentages of 1,2-dioleoyl-sn-glycero-3-phosphoethanol amine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE). Sensors were stabilized via cross-linking polymerization of hydrophobic methacrylate monomers partitioned into the hydrophobic interior of the DOPC bilayer. The resultant unilamellar, nanometre-sized, polymer-lipid vesicles are spherical, biocompatible and protect sensing components that are loaded into the aqueous interior of the vesicle from interfering species in the exterior environment. For O(2) detection, the oxygen-sensitive fluorescent dye, tris(1,10-phenanthroline)ruthenium(II) chloride (Ru(phen)(3)) was encapsulated into the aqueous interior of the polymerized phospholipid vesicle. NBD-PE was introduced into the phospholipid bilayer of the sensor as a reference dye, allowing ratiometric sensors to be constructed. The resultant sensors show high sensitivity, excellent reversibility and excellent linearity over a physiological range of dissolved oxygen concentrations. These results suggest that polymerized phospholipid vesicle sensors can be used for monitoring intracellular O(2) dynamics.     

Intrinsic Artefacts in Optical Oxygen Sensors—How Reliable are our Measurements?

Optical oxygen sensing is of broad interest in many areas of research, such as medicine, food processing, and micro‐ and marine biology. The operation principle of optical oxygen sensors is well established and these sensors are routinely employed in lab and field experiments. Ultratrace oxygen sensors, which enable measurements in the sub‐nanomolar region (dissolved oxygen), are becoming increasingly important. Such sensors prominently exhibit phenomena that complicate calibration and measurements. However, these phenomena are not constrained to ultratrace sensors; rather, these effects are inherent to the way optical oxygen sensors work and may influence any optical oxygen measurement when certain conditions are met. This scenario is especially true for applications that deal with high‐excitation light intensities, such as microscopy and microfluidic applications. Herein, we present various effects that we could observe in our studies with ultratrace oxygen sensors and discuss the reasons for their appearance, the mechanism by which they influence measurements, and how to best reduce their impact. The phenomena discussed are oxygen photoconsumption in the sensor material; depletion of the dye ground state by high‐excitation photon‐flux values, which can compromise both intensity and ratiometric‐based measurements; triplet–triplet annihilation; and singlet‐oxygen accumulation, which affects measurements at very low oxygen concentrations.     

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.     

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.     

Research of the relationship of intracellular acidification and apoptosis in Hela cells based on pH nanosensors

In this paper,the relationship of intracellular acidification and apoptosis in Hela cells induced by vin-cristine sulfate has been studied by use of the ratiometric pH nanosensors that have been developed by our group,employing fluorescein isothiocyanate(FITC) doped as the pH-sensitive dye and Tris(2,2'-bipyidyl) dichlororuthenium(II) hexahydrate(RuBpy) doped as reference dye. The pH change of the Hela cells induced by vincristine sulfate has been monitored in vivo,in situ and real time by use of the ratiometric pH nanosensors. The experimental results show that the pH of the apoptotic Hela cells induced by vincristine sulfate has been acidified from 7.11 to 6.51,and the percentage of intra-cellular acidification is correlated with the induced concentration and incubation time of the vincristine sulfate. The further study of the percentage of intracellular acidification and the percentage of apop-tosis of Hela cells at the same time reveals that apoptosis of Hela cells induced by vincristine sulfate is preceded by intracellular acidification. These results would provide theoretical foundation for the therapy of cancer through interfering the pH of cells by use of vincristine sulfate or other anti-cancer drugs.     

Thin-film oxygen sensors using a luminescent polynuclear gold(I) complex

Robust thin-film oxygen sensors were fabricated by encapsulating a lipophilic, polynuclear gold(I) complex, bis{m-(bis(diphenylphosphino)octadecylamine-P,P′)}dichlorodigold(I), in oxygen permeable polystyrene and ormosil matrices. Strong phosphorescence, which was quenched by gaseous and dissolved oxygen, was observed from both matrices. The polystyrene encapsulated dye exhibited downward-turning Stern–Volmer plots which were well fitted by a two-site model. The ormosil trapped complex showed linear Stern–Volmer plots for dissolved oxygen quenching but was downward turning for gaseous oxygen. No leaching was observed when the ormosil based sensors were immersed in flowing water over an 8 h period. Both films exhibited fully reversible response and recovery to changing oxygen concentration with rapid response times.     

Ratiometric pH sensor based on mesoporous silica nanoparticles and Förster resonance energy transfer

Incorporation of a dual-FRET dye pair into mesoporous silica nanoparticles yields sensitive and sensing-range tunable nanosensors with good reversibility that can be used for ratiometric pH measurements under a single-wavelength excitation.     

Ratiometric fluorescent detection of acidic pH in lysosome with carbon nanodots

It is significant for cell physiology to keep the homeostasis of p H, and it is highly demanded to develop ratiometric fluorescent sensors toward p H. In this work, under mild condition, through the electrostatic interaction between carbon nanodots(CDs) and organic molecules, two novel ratiometric fluorescence hybrid nanosensors were fabricated for sensing acidic p H. These nanohybrid systems possess dual emission peaks at 455 and 527 nm under a single excitation wavelength of 380 nm in acidic p H condition.With the increasing of p H, the fluorescence of the 1,8-naphthalimide derivative completely quenches,while the blue fluorescence of CDs keeps constant. Furthermore, the CDsàorganic molecular nanohybrids exhibit excellent anti-disturbance ability, reversible p H sensing ability, and a linear response range in wide p H range respectively. Besides the ability to target lysosome, with one of the nanosensor, stimulated p H change has been successfully tracked in a ratiometric manner via fluorescence imaging.     

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.     

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.     

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.     

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.

比率式荧光纳米氧传感器

比率式荧光传感通过在同一传感基质中掺杂两种或两种以上对分析物具有不同响应的发光体,根据不同的光发射信号比值构建起来的光化学传感体系,能更好满足实际检测的需要。近年来,比率式荧光纳米传感器的构建已引起了人们的广泛关注。该文介绍了比率式荧光纳米氧传感器的制备方法、传感特性和应用情况,并对其传感检测的发展和应用前景进行了分析和展望。     

Patterning, integration and characterisation of polymer optical oxygen sensors for microfluidic devices

This paper describes a process for the layer-by-layer fabrication and integration of luminescent dye-based optical oxygen sensors into microfluidic devices. Application of oxygen-sensitive platinum(ii) octaethylporphyrin ketone fluorescent dye dissolved in polystyrene onto glass substrates by spin-coating was studied. Soft lithography with polydimethylsiloxane (PDMS) stamps and reactive ion etching in oxygen plasma were used to produce sensor patterns with a minimum feature size of 25 microm. Sensors patterns were integrated into a PDMS microfluidic device by plasma bonding. No degradation of the sensor response as a result of the lithography and pattern-transfer processes was detected. Gaseous and dissolved oxygen (DO) detection was characterised using fluorescence microscopy. The intensity signal ratio of the sensor films was found to increase almost two-fold from 3.6 to 6.8 by reducing film thickness from 1.3 microm to 0.6 microm. Calibration of DO measurement showed linear Stern-Volmer behaviour that was constant for flow rates from 0.5 to 2 mL min(-1). The calibrated sensors were subsequently used to demonstrate laterally resolved detection of oxygen inside a microfluidic channel. The fabrication process provides a novel, easy to use method for the repeatable integration of optical oxygen sensors into cell-culture and lab-on-a-chip devices.     

Optimization of Ormosil Films for Optical Sensor Applications

Recent work has indicated that Ormosil films, fabricated from organically modified precursors, produce better sensor performance for some specific applications, compared to films fabricated from conventional sol-gel precursors such as TEOS or TMOS. This paper aims to compare film properties and sensor behavior for films fabricated from tetraethoxysilane (TEOS) and tetramethoxysilane (TMOS) silica precursors and both methyltrimethoxysilane (MTMS) and methyltriethoxysilane (MTES) organically modified precursors. Microstructural differences, for example, porosity changes due to the different precursor backbone structures, are interrogated by monitoring oxygen gas and aqueous-phase sensor response. Oxygen sensing using these films is enabled by incorporating in the films an oxygen-sensitive ruthenium dye whose fluorescence is quenched in the presence of oxygen. Film properties such as thickness, thickness stabilization time, as well as sensor response, are discussed in terms of relative hydrolysis and condensation behavior for the different precursors. Film hydrophobicity, an issue which has been identified as being of crucial importance for optimum dissolved oxygen sensor response, is discussed and contact angle measurements are used to investigate the degree of hydrophobicity for different film types. The main motivation for this work is film optimization for optical gas-phase and dissolved oxygen sensors.     

A Novel Ratiometric Oxygen Sensor Based On a Sextuple Hydrogen‐Bonding Self‐Assembly Molecular Heterodimer

A novel sextuple hydrogen‐bonding (HB) self‐assembly molecular heterodimer bearing an iridium complex as the indicator dye and two carbazoles as the reference dye, namely 6HB‐Irbt‐Cz , was synthesized, and its molecular structure was confirmed by ¹H NMR, ¹³CNMR, TOF‐MS and 2D NMR. Because of the inefficient energy transfer process between the carbazole and iridium complex units, 6HB‐Irbt‐Cz exhibits distinct ?uorescence/phosphorescence dual emission in neat film state. More importantly, the neat film sample of 6HB‐Irbt‐Cz could display linear ratiometric optical response toward oxygen in the full oxygen concentration range from 0 to 100 vol%, together with good stability, reversibility and rapid response‐recovery times. Note that this represents the first discovery of neat‐film‐based oxygen sensor capable of showing strictly linear ratiometric Stern‐Volmer behavior in the oxygen concentration of 0–100 vol%.     

Poly(2,5-dimethoxyaniline) film coating for corrosion protection of iron

A poly(2,5-dimethoxyaniline) (PDMA) film was coated on the iron surface by the electropolymerization of 2,5-dimethoxyaniline in neutral buffer solution (pH?6.86). The PDMA film strongly adhered to the surface because of the polar methoxy groups of the PDMA molecules. The fact that no electrochemical response of the PDMA film-coated iron electrode to dissolved Fe²⁺ exhibited that the PDMA film was less permeable to dissolved species, acting as a diffusion barrier against agents causing corrosion such as H₂O and O₂. The PDMA film coating greatly lowered the anodic current peak ascribed to the anodic dissolution of iron and the corrosion current in strongly acidic medium, 0.5?M H₂SO₄ aqueous solution (1?M?????mol?dm^??) as well as neutral medium (pH?6.86). The high anti-corrosion ability was due to a hybrid effect of the PDMA film not only as the diffusion barrier, but also as an in situ oxidant in spite of the slight redox activity of PDMA. In addition, the PDMA film is much more durable and adhesive than polyaniline film against over-oxidation.