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.     

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.     

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 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.     

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.     

Polyamine-functionalized carbon quantum dots as fluorescent probes for selective and sensitive detection of copper ions

A novel sensing system has been designed for Cu(2+) ion detection based on the quenched fluorescence (FL) signal of branched poly(ethylenimine) (BPEI)-functionalized carbon quantum dots (CQDs). Cu(2+) ions can be captured by the amino groups of the BPEI-CQDs to form an absorbent complex at the surface of CQDs, resulting in a strong quenching of the CQDs' FL via an inner filter effect. Herein, we have demonstrated that this facile methodology can offer a rapid, reliable, and selective detection of Cu(2+) with a detection limit as low as 6 nM and a dynamic range from 10 to 1100 nM. Furthermore, the detection results for Cu(2+) ions in a river water sample obtained by this sensing system agreed well with that by inductively couple plasma mass spectrometry, suggesting the potential application of this sensing system.     

Controlled release of Fe3O4 nanoparticles in encapsulated microbubbles to tumor cells via sonoporation and associated cellular bioeffects

Fe(3)O(4) nanoparticles embedded in the shells of encapsulated microbubbles could be used therapeutically as in situ drug-delivery vehicles. Bioeffects on liver tumor cells SMMC-7721 due to the excitation of Fe(3)O(4) nanoparticles attached to microbubbles generated by ultrasound (US) are studied in an in vitro setting. The corresponding release phenomenon of Fe(3)O(4) nanoparticles from the shells of the microbubbles into the cells via sonoporation and related phenomena, including nanoparticle delivery efficiency, cell trafficking, cell apoptosis, cell cycle, and disturbed flow of intracellular calcium ions during this process, are also studied. Experimental observations show that Fe(3)O(4) nanoparticles embedded in the shells of microbubbles can be delivered into the tumor cells; the delivery rate can be controlled by adjusting the acoustic intensity. The living status or behavior of Fe(3)O(4) -tagged tumor cells can then be noninvasively tracked by magnetic resonance imaging (MRI). It is further demonstrated that the concentration of intracellular Ca(2+) in situ increases as a result of sonoporation. The elevated Ca(2+) is found to respond to the disrupted site in the cell membrane generated by sonoporation for the purpose of cell self-resealing. However, the excessive Ca(2+) accumulation on the membrane results in disruption of cellular Ca(2+) cycling that may be one of the reasons for the death of the cells at the G1 phase. The results also show that the Fe(3)O(4) -nanoparticle-embedded microbubbles have a lower effect on cell bioeffects compared with the non-Fe(3)O(4) -nanoparticle-embedded microbubbles under the same US intensity, which is beneficial for the delivery of nanoparticles and simultaneously maintains the cellular viability.     

Silica-coated S(2-)-enriched manganese-doped ZnS quantum dots as a photoluminescence probe for imaging intracellular Zn2+ ions

Detection of intracellular Zn(2+) has gained great attention because of its biological significances. Here we show the fabrication of silica-coated S(2-)-enriched Mn-doped ZnS quantum dots (SiO(2)-S-Mn-ZnS QDs) by enriching S(2-) with a silica shell on the surface of Mn-doped ZnS QDs via a sol-gel process for imaging intracellular Zn(2+) ions. The developed probe gave a good linearity for the calibration plot (the recovered PL intensity of the SiO(2)-S-Mn-ZnS QDs against the concentration of Zn(2+) from 0.3 to 15.0 μM), excellent reproducibility (1.2% relative standard deviation for 11 replicate measurements of Zn(2+) at 3 μM), and low detection limit (3s; 80 nM Zn(2+)). The SiO(2)-S-Mn-ZnS QDs showed negligible cytotoxicity, good sensitivity, and selectivity for Zn(2+) in a photoluminescence turn-on mode, being a promising probe for photoluminescence imaging of intracellular Zn(2+).     

Computer-Readable DNAzyme Assay on Disc for ppb-Level Lead Detection

A method for the convenient detection of lead at the parts-per-billion (ppb)-level has been developed; it uses a conventional compact disc (CD) as the platform for preparing DNAzyme assays and an unmodified optical drive of ordinary desktop/laptop computers as the readout device. In particular, by immobilization of Pb(2+)-specific DNAzyme sensing constructs on the "transparent side" of a conventional CD-R via mild surface reactions, the Pb(2+) concentration can be determined by a free diagnostic program that checks the error distribution on the CD (i.e., it extracts the number of errors in a prerecorded audio file). The reading errors increase monotonically over a wide range of Pb(2+) concentrations (from 10 nM to 1 mM), and the selectivity is confirmed by testing several other divalent cations (Zn(2+), Ba(2+), Mg(2+), Ca(2+), Cu(2+), and Hg(2+)).     

Förster resonance energy transfer-based biosensors for multiparameter ratiometric imaging of Ca2+ dynamics and caspase-3 activity in single cells

As one of the principal cytoplasmic second messengers, the calcium ion (Ca(2+)) is central to a variety of intracellular signal transduction pathways. Accordingly, there is a sustained interest in methods for spatially- and temporally resolved imaging of the concentration of Ca(2+) in live cells using noninvasive methods such as genetically encoded biosensors based on F?rster resonance energy transfer (FRET) between fluorescent proteins (FPs). In recent years, protein-engineering efforts have provided the research community with FRET-based Ca(2+) biosensors that are dramatically improved in terms of enhanced emission ratio change and optimized Ca(2+) affinity for various applications. We now report the development and systematic optimization of a pair of spectrally distinct FRET-based biosensors that enable the simultaneous imaging of Ca(2+) in two compartments of a single cell without substantial spectral crosstalk between emission channels. Furthermore, we demonstrate that these new biosensors can be used in conjunction with previously reported caspase-3 substrates based on the same set of FRET pairs.     

"Molecular beacon"-based fluorescent assay for selective detection of glutathione and cysteine

We report on the development of a fluorescence turn-on "molecular beacon" probe for the detection of glutathione (GSH) and cysteine (Cys). The method is based on a competitive ligation of Hg(2+) ions by GSH/Cys and thymine-thymine (T-T) mismatches in a DNA strand of the self-hybridizing beacon strand. The assay relies on the distance-dependent optical properties of the fluorophore/quencher pair attached to the ends of the molecular beacon DNA strand. In a very selective coordination of Hg(2+) to GSH/Cys, the fluorophore/quencher distance increases concomitantly with the dehybridization and dissociation of the beacon stem T-Hg(2+)-T due to the extraction of Hg(2+) ions. This process results in switching the molecular beacon to the "on" state. The concentration range of the probe is 4-200 nM with the limit of detection (LOD) of 4.1 nM for GSH and 4.2 nM Cys. The probe tested satisfactorily against interference for a range of amino acids including sulfur-containing methionine.     

Dynamics of conformational Ca2+-switches in signaling networks detected by a planar plasmonic device

Ca(2+)-sensor proteins regulate a variety of intracellular processes by adopting specific conformations in response to finely tuned changes in Ca(2+)-concentration. Here we present a surface plasmon resonance (SPR)-based approach, which allows for simultaneous detection of conformational dynamics of four Ca(2+)-sensor proteins (calmodulin, recoverin, GCAP1, and GCAP2) operating in the vertebrate phototransduction cascade, over variations in Ca(2+) concentration in the 0.1-0.6 μM range. By working at conditions that quantitatively mimic those found in the cell, we show that the method is able to detect subtle differences in the dynamics of each Ca(2+)-sensor, which appear to be influenced by the presence of free Mg(2+) at physiological concentration and by posttranslational modifications such as myristoylation. Comparison between the macroscopic Ca(2+)-binding constants, directly measured by competition with a chromophoric chelator, and the concerted binding-conformational switch detected by SPR at equilibrium reveals the relative contribution of the conformational change process to the SPR signal. This process appears to be influenced by the presence of other cations that perturb Ca(2+)-binding and the conformational transition by competing with Ca(2+), or by pure electrostatic screening. In conclusion, the approach described here allows a comparative analysis of protein conformational changes occurring under physiologically relevant molecular crowding conditions in ultrathin biosensor layers.     

Fluorometric detection of ca(2+) based on an induced change in the conformation of sol-gel entrapped parvalbumin

We report the development of a fluorometric detection strategy for Ca(2+) based on induced changes in the conformation of cod III parvalbumin entrapped within a sol-gel processed glass. The detection scheme utilizes a fluorescent allosteric signal transduction (FAST) strategy wherein conformational changes induced by Ca(2+) binding result in alterations in the intrinsic fluorescence from the single tryptophan residue at position 102. Intrinsic fluorescence was also used to examine chemically induced changes in protein structure to ascertain the effects of entrapment on the conformational motions and stability of the protein. Fluorescence analysis indicated that the behavior of the protein depended on the entrapment protocols used. The entrapped protein retained conformational flexibility similar to that observed in solution and remained accessible to analytes such as Ca(2+). Entrapment also caused improvements in protein stability against chemical denaturants. However, entrapment caused the apparent affinity constant for binding of Ca(2+) to decrease substantially with aging time. Even so, in optimum cases, fluorometric detection of Ca(2+) could be done over a 600 μM range with a limit of detection of 3 μM and with no interference from divalent ions such as Mg(2+), Sr(2+), or Cd(2+), indicating the viability of using sol-gel entrapped FAST proteins for the detection of Ca(2+).     

Identification of Toxin Inhibitors Using a Magnetic Nanosensor‐Based Assay

A magnetic nanosensor‐based method is described to screen a library of drugs for potential binding to toxins. Screening is performed by measuring changes in the magnetic relaxation signal of the nanosensors (bMR nanosensors) in aqueous suspension upon addition of the toxin. The Anthrax lethal factor (ALF) is selected as a model toxin to test the ability of our bMR nanosensor‐based screening method to identify potential inhibitors of the toxin. Out of 30 molecules screened, sulindac, naproxen and fusaric acid are found to bind LF, with dissociation constants in the low micromolar range. Further biological analysis of the free molecules in solution indicate that sulindac and its metabolic products inhibited LF cytotoxicity to macrophages with IC₅₀ values in the micromolar range. Meanwhile, fusaric acid is found to be less effective at inhibiting LF cytotoxicity, while naproxen does not inhibit LF toxicity. Most importantly, when the sulindac and fusaric acid‐bMR nanosensors themselves are tested as LF inhibitors, as opposed to the corresponding free molecules, they are stronger inhibitors of LF with IC₅₀ values in the nanomolar range. Taken together, these studies show that a bMR nanosensors‐based assay can be used to screen known drugs and other small molecules for inhibitor of toxins. The method can be easily modified to screen for inhibitors of other molecular interactions and not only the selected free molecule can be study as potential inhibitors but also the bMR nanosensors themselves achieving greater inhibitory potential.     

High-basicity determination in mixed water-alcohol solutions by a dual optical sensor approach

The activity of NaOH is known to be significantly affected by the presence of an alcohol in aqueous solutions. A novel linear relationship between (deltaA/deltaC(alcohol)) and C(base) was found in the highly alkaline, mixed H2O-ROH solutions (R = Me, Et, i-Pr). The use of this linear relationship led to a dual-transducer approach to decompose the optical signals of optical base sensors and to give base and alcohol concentrations in concentrated NaOH-H2O-ROH solutions ([OH-] = 0.05-3.6 M). The scope of the new dual-sensor approach was evaluated, and errors in C(base) and C(alcohol) were analyzed. The optical base sensors consist of sol-gel SiO2-ZrO2-organic polymer composites doped with high-pKa indicators. The pKa(s) of the indicators encapsulated in the composite films were determined and found to be affected by the composition of the sol-gel composites. Optical sensors and their uses in multicomponent systems are of intense current interest.( 1-7) In the multicomponent systems, the activity of the analyte and sensor response are often affected by change in ionic strength. For optical sensors that are based on indicator equilibria involving the analyte as their transducing mechanism, such effect is particularly significant. The concentrations of both the analyte and other chemicals affect ionic strength, and the sensor response to concentration of the analyte is thus often indistinguishable from those of other chemicals. An accurate measurement of each component in these multicomponent systems is actively studied. Several approaches have been developed to correct ionic strength in optical sensing for the pH region and solutions of low-to-medium ionic strength. (1-9) We recently reported a dual-transducer approach to measure acid concentrations (2-9 M HCl) in salt-containing, concentrated strong acids such as MClx-HCl (M = Li, Ca, Al) solutions. (10) This approach was shown to reduce the error in C(acid) from, for example,     

Fluorescence detection of lead(II) ions through their induced catalytic activity of DNAzymes

We have developed a fluorescence approach for the highly selective and sensitive detection of Pb(2+) ions using AGRO100, a G-quadruplex DNAzyme. The sensing strategy is based on Pb(2+) ions inducing increased DNAzyme activity of AGRO100 in the presence of hemin, which acts as a cofactor to catalyze H(2)O(2)-mediated oxidation of Amplex UltraRed (AUR). A test of eight aptamers of various sequences for the detection of Pb(2+) ions revealed that AGRO100 performed the best in terms of sensitivity. The AGRO100-AUR probe exhibited high selectivity (>100-fold) toward Pb(2+) ions over other tested metal ions. The fluorescence intensity (excitation/emission maxima, ca. 561/592 nm) of the AUR product was proportional to the concentration of Pb(2+) ions over the range 0-1000 nM, with a linear correlation (R(2) = 0.98). For 5 mM Tris-acetate (pH 7.4) solutions in the presence and absence of 100 mM NaCl, the AGRO100-AUR probe provided limits of detection (signal-to-noise ratio = 3) for Pb(2+) ions of 1.0 and 0.4 nM, respectively. We validated the practicality of the use of the AGRO100-AUR probe for the determination of the concentrations of Pb(2+) ions in soil samples. This approach allows the determination of the concentrations of Pb(2+) ions with simplicity, selectivity, and sensitivity.     

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.     

Responsive polymers-based dual fluorescent chemosensors for Zn2+ ions and temperatures working in purely aqueous media

We report on the fabrication of responsive double hydrophilic block copolymers (DHBCs)-based dual fluorescent chemosensors for Zn(2+) ions and temperatures and investigate the effects of thermo-induced micellization and detection conditions on the probing sensitivity and binding reversibility of Zn(2+) ions. A novel quinoline-based polarity-sensitive and Zn(2+)-recognizing fluorescent monomer (ZQMA, 6) was synthesized at first. Well-defined DHBCs bearing quinoline-based Zn(2+)-recognizing moieties (ZQMA) in the thermoresponsive block, PEG-b-P(MEO(2)MA-co-OEGMA-co-ZQMA), were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-(2-methoxyethoxy)ethyl methacrylate (MEO(2)MA), oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA), and ZQMA in the presence of PEG-based macroRAFT agent. The OEGMA contents in the thermoresponsive block varied in the range of 0-12.0 mol % to tune their lower critical solution temperatures (LCSTs). At 20 °C, almost nonfluorescent PEG-b-P(MEO(2)MA-co-ZQMA) molecularly dissolved in water and can selectively bind with Zn(2+) ions over other common metal ions, leading to prominent fluorescence enhancement due to the coordination of ZQMA with Zn(2+). At a polymer concentration of 0.2 g/L, the Zn(2+) detection limit can be down to ~3.0 nM. PEG-b-P(MEO(2)MA-co-ZQMA) self-assembles into micelles possessing P(MEO(2)MA-co-ZQMA) cores and well-solvated PEG coronas upon heating to above the LCST, and the fluorescence intensity exhibit ~6.0-fold increase due to the fact that ZQMA moieties are now located in a more hydrophobic microenvironment. Compared to the unimer state at 20 °C, although PEG-b-P(MEO(2)MA-co-ZQMA) micelles possess a slightly decreased detection limit for Zn(2+) (~14 nM), reversible binding between ZQMA moieties and Zn(2+) ions at 37 °C can be achieved, as evidenced by the on/off switching of fluorescence emission via the sequential addition of Zn(2+) and EDTA. In vitro fluorescence imaging studies suggested that the micelles can effectively enter into living cells and sensitively respond to Zn(2+) ions. This work represents the first example of a purely aqueous-based polymeric Zn(2+) sensing system by integrating the well-developed small molecule Zn(2+)-sensing moieties with stimuli-responsive DHBCs.