Development of ratiometric fluorescent pH sensors based on chromenoquinoline derivatives with tunable pKa values for bioimaging

In this work, we first studied the pH-dependent characteristic of chromenoquinoline. Based on this, we then designed and synthesized two novel chromenoquinoline derivatives that can act as fluorescent pH sensors. The pK_a values of two novel chromenoquinoline derivatives can be modulated from 2.32 to 4.38 and 6.27 by introducing EDG on the backbone of chromenoquinoline. Furthermore, we demonstrate that the sensor 4 can be used as a ratiometric fluorescent pH sensor for fluorescence imaging in living cells.     

A method for studying swelling kinetics based on measurement of electrical conductivity

A novel method was applied to the study of swelling kinetics of pH-responsive hydrogels. This technique is based on the pH-dependent electrical conductivity of these materials, which is measured by coating planar interdigitated electrode arrays with thin hydrogel membranes. To demonstrate the utility of the method, the swelling kinetics of a well-characterized pH-responsive hydrogel were studied. Cross–linked copolymers of 2-hydroxyethyl methacrylate (HEMA) with up to 20 mol% dimethylaminoethyl methacrylate (DMA) were studied as a function of copolymer composition in phosphate or triethanolamine buffer at buffer concentrations from 1 to 100 mM. The experiments consisted of measuring the change in electrical resistance of a hydrogel-coated electrode array following a small pH change in the external buffer medium. The characteristic response time to reach a new equilibrium following a pH change was proportional to the concentration of DMA within the polymer and was inversely proportional to the buffer concentration. The characteristic response times for devices tested in phosphate buffer were a function of the magnitude of the pH step, increasing from 2.6 to 5.6 min as the step size increased from 0.2 to 0.57 pH units. However, the response times for devices tested in triethanolamine were independent of step size. The observed dependences upon the values of the dissociation constant (pK_a) of the buffering ion, the apparent pK_a of DMA, and the pH of the external bath agreed with buffer-mediated diffusion–reaction theory, and as such this conductimetric method represents a powerful tool for the study of swelling kinetics of responsive hydrogels.     

Radiation synthesis of poly[(dimethylaminoethyl methacrylate)-co-(diallyl dimethyl ammonium chloride)] hydrogels and its application as a carrier for notoginsenoside delivery

Novel pH/temperature sensitive hydrogel was synthesized by radiation induced copolymerization and cross-linking of dimethylaminoethyl methacrylate (DMAEMA) and diallyldimethyl ammonium chloride (DADMAC). Reactivity ratio of DADMAC (r₁) and DMAEMA (r₂) was determined as 1.02 and 0.98, which means that poly(DMAEMA-co-DADMAC) is an azeotropic copolymer. Content of DADMAC, i.e., charge density of the hydrogel was found to influence their properties significantly. Compared with polyDMAEMA hydrogel, poly(DMAEMA-co-DADMAC) showed enhanced equilibrium degree of swelling (EDS). Low critical solution temperature (LCST) of the hydrogel increased with the charged density. Content of DADMAC had no effect on the pH dependence of the final gel. Aiming at its application as a carrier for Chinese herb extract delivery system, the embedment and pH/temperature dependence of controlled release were investigated using notoginsenoside as a model drug. The maximum embedment amount of notoginsenoside was obtained in a gel containing 3 mol.% DADMAC. The temperature dependence and pH dependence of notoginsenoside release followed the same trend as that of EDS, for instance, higher ratio of notoginsenoside release occurred at 25 °C and pH 1.7, at which higher EDS was obtained. By these means, the release of notoginsenoside can be controlled by adjusting the pH, ionic strength, temperature of solution as well as the composition and structure of the gel.     

Adsorption of Apollo reactive dyes on poly(N,N dimethylamino ethylmethacrylate) hydrogels

Poly(N,N-dimethylamino ethylmethacrylate) [P(DMAEMA)] hydrogels were prepared by irradiating the ternary mixtures of dimethylamino ethylmethacrylate (DMAEMA)/ethyleneglycol dimethacrylate (EGDMA)/water (H₂O) by γ-rays at ambient temperature. The swelling of four types of DMAEMA hydrogels in distilled water is higher than the swelling of these hydrogels in dye solutions. The value of equilibrium swelling of P(DMAEMA)1 hydrogel was 338% at pH 7.0 in distilled water, while it was 325% and 326% at pH 7.0 in Apollofix Red (AR) and Apollofix Yellow (AY) solutions, respectively. The adsorption capacity of P(DMAEMA)1 hydrogel was found to increase from 85 to 131 mg for AR g⁻¹ dry gel and from 58 to 111 mg for AY g⁻¹ dry gel with decreasing pH of the dye solutions.     

Synthesis and Swelling Behavior of Acrylate‐Based Hydrogels

The purpose of this investigation was to report the synthesis of a novel pH‐sensitive acrylate‐based hydrogel by polymerizing the comonomers 2‐hydroxyethyl methacrylate, HEMA, acrylic acid, AA, and sodium acrylate, NaAc. The NaAc component was obtained by neutralization of AA with sodium hydroxide. Hydrogels were obtained by free radical copolymerization in aqueous solution in the presence of redox initiators, Na₂S₂O₈/Na₂S₂O₅, and ethylene glycol dimethacrylate, EGDMA, crosslinker. The copolymers were synthesized by varying neutralization percent of AA in the range of 10–100. The swelling behavior of the copolymeric gels were investigated as a function of pH, temperature, ionic strength, and AA neutralization percent. The polymer mesh size, ξ, molecular weight between crosslinks, M_c , and crosslinking density, q, were determined by using the Flory‐Rehner equation in the pH range of 2–8 as 8.78–48.8 Å, 209–2667 g/mol, and 0.046–0.59, respectively. The diffusional exponent value, n, of the synthesized hydrogel was found to be 0.59, indicating a non‐Fickian diffusion mechanism. It can be concluded that the hydrogel demonstrated a sharp change in its water absorbency, mesh size and molecular weight between crosslinks of the network with a change in pH of the swelling media. The latter properties suggest strong consideration of these hydrogels for use as oral drug delivery systems and ion‐exchangers for removal of metal ions from aqueous media, owing to the carboxylate groups within the polymeric network.     

A new sensor for thermometric titrations

A new thermometric sensor, which is a transistor (OC71), has been introduced to follow thermometric titrations successfully to clear end points. The sensor was suitable in both normal and differential modes of titration. It is possible to titrate down to 1.32 μmol of HCl and 26.4 μmol of H₃BO₃in a final 20 ml solution with accuracy and precision of 1%, 2.2% and 1.4%, 2.2%, respectively. The sensor, in association with a pH glass electrode, was used for the determination of pK values of some well established weak acids such as, acetic acid (4.77), phosphoric acid (pK₁ = 2.18, pK₂ = 7.20 and pK₃ = 12.32) as well as for a very weak acid of uncertain pK values H₃BO₃ (pK₁ = 9.20, pK₂ = 12.7 and pK₃ = 13.80). The sensor was also examined for kinetic catalytic determination of iron(III) in water, milk and pharmaceuticals.     

Monolayer Co3O4 Inverse Opals as Multifunctional Sensors for Volatile Organic Compounds

Monolayers of periodic porous Co₃O₄ inverse opal (IO) thin films for gas‐sensor applications were prepared by transferring cobalt‐solution‐dipped polystyrene (PS) monolayers onto sensor substrates and subsequent removal of the PS template by heat treatment. Monolayer Co₃O₄ IO thin films having periodic pores (d≈500 nm) showed a high response of 112.9 to 5 ppm C₂H₅OH at 200 °C with low cross‐responses to other interfering gases. Moreover, the selective detection of xylene and methyl benzenes (xylene+toluene) could be achieved simply by tuning the sensor temperature to 250 and 275 °C, respectively, so that multiple gases can be detected with a single chemiresistor. Unprecedentedly high ethanol response and temperature‐modulated control of selectivity with respect to ethanol, xylene, and methyl benzenes were attributed to the highly chemiresistive IO nanoarchitecture and to the tuned catalytic promotion of different gas‐sensing reactions, respectively. These well‐ordered porous nanostructures could have potential in the field of high‐performance gas sensors based on p‐type oxide semiconductors.     

A novel voltammetric sensor for ascorbic acid based on molecularly imprinted poly(o-phenylenediamine-co-o-aminophenol)

A molecularly imprinted copolymer, poly(o-phenylenediamine-co-o-aminophenol) (PoPDoAP), was prepared as a new ascorbic acid (AA) sensor. The copolymer was synthesized by incorporation of AA as template molecules during the electrochemical copolymerization of o-phenylenediamine and o-aminophenol, and complementary sites were formed after the copolymer was electrochemically reduced in ammonium aqueous solution. The molecularly imprinted copolymer sensor exhibited a high sensitivity and selectivity toward AA. Differential pulse voltammograms (DPVs) showed a linear concentration range of AA from 0.1 to 10 mM, and the detection limit was calculated to be 36.4 μM. Compared to conventional polyaniline-based AA sensors, the analytical performance of the imprinted copolymer sensor was improved due to the broadened usable pH range of PoPDoAP (from pH 1.0 to pH 8.0). The sensor also exhibited a good reproducibility and stability. And it has been successfully applied in the determination of AA in real samples, including vitamin C tablet and orange juices, with satisfactory results.     

Stabilization of glucose oxidase fromPenicillium vitale entrapped in high-concentration gels

Glucose ixidase fromPenicillium vitale was immobilized in a 2-hydroxyethyl methacrylate (HEMA) gel containing 0.3 to 2% of methacrylic acid (MAA) or MAA neutralized by allylamine (AA). Depending on the MAA quantity of MAA in the gel, the thermal irreversible inactivation(k _in) constants of the immobilized enzymes sharply decrease at gel concentrations higher than 29 to 50% at pH 5.8. A 220- to 250-fold decrease ofk _in was observed in 60 to 80% gel. The inactivation rate of enzyme in HEMA gel also decreases considerably under the action of urea. Over the range of pH 5.0 to 9.0 thek _in of the native enzyme depends on pH by a degree of 2.1, and of the immobilized enzyme, 0.3 to 0.55. Over the pH range of 5.2 to 5.7,k _in of the native and immobilized enzymes are approximate, whereas at pH 8 and over the difference betweenk _in values exceeds four orders of magnitude. The activity dependence of the immobilized enzyme on pH is shifted two units to the alkaline region. This shift depends on the ionic strength of the solution. This dependence is best reflected in the high gel concentrations. A mechanism of enzymes stabilization in the concentrated HEMA gel is discussed.     

Factors driving the protonation of poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole) hydrogels immersed in aqueous acid solutions produce an increment in the pH of the bath because of proton uptake by basic imidazole moieties, leading to hydrogel protonation. Both kinetic and equilibrium measurements of the pH of the bath have been performed under a variety of conditions and with different hydrogel samples. The kinetics of the xerogel protonation process (which includes solvent and titrant diffusion, the true protonation reaction or ion–dipole association, and the polymer relaxation to a new conformation) are mostly driven by the size of the hydrogel sample, whereas other magnitudes, such as the initial pH, the effective polymer concentration, and the network structure, governed by the crosslinker ratio and total comonomer concentration in the feeding, have a minor influence. pK_a changes with the degree of protonation (α), delimitating two different regions: (1) a broad α range in which pK_a decreases with increasing α but less pronouncedly with increasing ionic strength and (2) an α range close to α = 1 in which pK_a decreases abruptly, more markedly with sulfate than with chloride counteranions and with larger ionic strengths. In the first region, pK_a is determined by repulsive electrostatic interactions and so is larger for titration with H₂SO₄ than with HCl and increases as the effective polymer concentration and ionic strength increase. Two steps (i.e., two protonation sites) can be observed in the titration curves, the second one corresponding to abrupt changes in the basicity of the second pK_a-versus-α region. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 2294–2307, 2004     

A high-precision ratiometric fluorosensor for pH: implementing time-dependent non-linear calibration protocols for drift compensation

We present a versatile time-dependent non-linear calibration protocol for optical sensors, implemented on the pH sensitive ratiometric fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) immobilized in ethyl-cellulose. The calibration protocol individually compensated for the progressive drift of calibration parameters, whereby sensor precision and accuracy, as well as applicable lifetime were improved. A severely reduced photoacidity was observed for the immobilized fluorophore, for which excited state dynamics was characterized and benefited from during measurements. Due to the significantly reduced photoacidity of HPTS immobilized in the ethyl-cellulose sensing membrane, a dual excitation/dual emission (F₁, ex/em: 405/440 nm and F₂, ex/em: 465/510 nm) ratiometric (RF₁,F₂ = F₁/F₂) sensing scheme could be used to amplify sensor response. The signal to noise (S/N) ratio was enhanced by ∼400% utilizing the dual excitation/dual emission ratiometric sensing scheme, rather than the more commonly used protocol of dual excitation/single emission for HPTS fluorescence. Apparent pK_a of the fluorophore ranged from 6.74 to 8.50, mainly determined by the immobilization procedure. The repeatability (IUPAC, pooled standard deviation) over three pH values (6.986, 7.702 and 7.828) was 0.0044 pH units for the optical sensor, compared to 0.0046 for the electrode used for standardization. Sensor analytical characteristics were thereby in principle limited by the performance of the standardization procedure.     

BODIPY-derived ratiometric fluorescent sensors: pH-regulated aggregation-induced emission and imaging application in cellular acidification triggered by crystalline silica exposure

Modification of classic fluorophore to possess the emission transitions between aggregation-induced emission (AIE) and intrinsic emission offers reliable approach to the design of ratiometric fluorescent sensors. In this study, a proton acceptor benzimidazole was integrated with BODIPY to form three compounds, BBI-1/2/3, which demonstrated the AIE (~595 nm, I_agg) in neutral aqueous medium and intrinsic BODIPY emission (~510 nm, I_int) in acidic medium. All the three showed the ratiometric pH sensing behavior in a dual excitation/dual emission mode, yet BBI-3 displayed still the dual emission ratiometric pH sensing ability. The pH-dependent emission ratio I_int/I_agg of the three were fully reversible, and no interference was observed from normal abundant chemical species in live cells. Their different pK_a (BBI-1, pK_a 4.4; BBI-2, pK_a 2.7; BBI-3, pKa 3.6) suggested that the substituents on benzimidazole moiety were essential to govern their functioning pH range. The ratiometric imaging of BBI-1 in A549 cells provided an effective intracellular pH (pHi) calibration formula corresponding to emission ratio of I_int/I_agg. Ratiometric pH_i imaging in A549 cells upon small particle exposure confirmed the particle-induced cellular acidification with this formula. Both particle size and the chemical nature of the particle contribute to the observed acidification effect. The synchronization of lysosome disruption to cellular acidification in A549 cells upon crystalline silica exposure was directly observed for the first time with BBI-1, showing the potential application of BBI-1 in the study of silicosis and other related diseases. This study demonstrated that endowing fluorophore with AIE/intrinsic emission transition could be a promising strategy for ratiometric sensor design.     

Effect of Acetylene on the γ-Ray Crosslinking Polymerization of Various Kinds of Acrylic Monomers

The γ-radiation-induced crosslinking polymerization of methyl methacrylate (MMA), methacrylic acid (MAA), 2-hydroxyethyl methacrylate (HEMA), methyl acrylate (MA), and acrylic acid (AA) was carried out in bulk. The polymerization rates of AA and HEMA were much larger than those of other monomers. Acetylene had no influence on the polymerization rate in the initial stage, and the rate was dependent only on the kinds of monomers. In the absence of acetylene, gelation took place in PMA, PAA, and PHEMA obtained at complete conversion, but not in PMMA and PMAA. By the addition of acetylene, complete gelation of PMMA was observed, but no gel formation was observed for PMAA. Volume and weight swelling ratios were measured for PMA, PAA, PHEMA, and PMMA with complete gelation. It was found that by the addition of acetylene the molar concentration of crosslinks increased as much as about twice for PMA and PHEMA, and over about one hundred times for PMMA AND PAA. The relation between volume S_v and weight swelling ratio S_w was studied theoretically. S_v was expressed by the the equation, S_v = (p_p /p+)S_w+ (1 -p_p/p_s), as a function of S_w and the densities of pure polymer (p_p) and solvent (p_s) in swollen polymer.     

Smart thermometer style sensor with volume readout and visualization for pH detection

A novel visual volume-type hydrogel sensor like thermometer for pH response was developed. The multifunctional stimuli-responsive fluorescent hydrogel was fabricated by employing 5, 6-dicarboxylic fluorescein crosslinked partially ammoniated polyacrylamide (PAM). The polymer hydrogel was characterized with fluorescent inverted microscope (FIM) and scanning electron microscope (SEM), as well as N₂ adsorption–desorption analysis. The polymer hydrogel emitted fluorescence and exhibited volume phase transition (VPT) in response to pH in aqueous solution. The intelligent response hydrogel was put into an elaborately altered shower-like pipette with uniform holes in which the water can pass through without the swelling hydrogel. A visual thermometer style hydrogel sensor combination of chemical reaction, separation and detection was designed. It was accurately measure the volume of hydrogel instead of the pH value when response to different pH solution by reading the graduation with naked eye. Therefore, the challenge for direct measurement of hydrogel volume was overcome. Meanwhile, a scale bar was also designed to indicate pH according to the volume. We can directly read the pH from the bar, similar to a thermometer in daily life. The volume-type sensor paves the way for VPT hydrogel sensors with convenience, visualization, low-cost, portable, smartness and ease of operation.     

A new Vilsmeier-type reaction for one-pot synthesis of pH sensitive fluorescent cyanine dyes

A new Vilsmeier-type reaction is suggested for the synthesis of novel indocarbocyanine pH sensors, which are fluorescent when protonated but nonfluorescent upon proton abstraction. These sensors show significant ratiometric UV-visible as well as fluorescence spectral changes upon subtle variation of pHs with pK_a values near neutral.     

SAMPL6: calculation of macroscopic p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> values from ab initio quantum mechanical free energies

Macroscopic pK_a values were calculated for all compounds in the SAMPL6 blind prediction challenge, based on quantum chemical calculations with a continuum solvation model and a linear correction derived from a small training set. Microscopic pK_a values were derived from the gas-phase free energy difference between protonated and deprotonated forms together with the Conductor-like Polarizable Continuum Solvation Model and the experimental solvation free energy of the proton. pH-dependent microstate free energies were obtained from the microscopic pK_as with a maximum likelihood estimator and appropriately summed to yield macroscopic pK_a values or microstate populations as function of pH. We assessed the accuracy of three approaches to calculate the microscopic pK_as: direct use of the quantum mechanical free energy differences and correction of the direct values for short-comings in the QM solvation model with two different linear models that we independently derived from a small training set of 38 compounds with known pK_a. The predictions that were corrected with the linear models had much better accuracy [root-mean-square error (RMSE) 2.04 and 1.95 pK_a units] than the direct calculation (RMSE 3.74). Statistical measures indicate that some systematic errors remain, likely due to differences in the SAMPL6 data set and the small training set with respect to their interactions with water. Overall, the current approach provides a viable physics-based route to estimate macroscopic pK_a values for novel compounds with reasonable accuracy.     

One-pot synthesis of a new rhodamine-based dually-responsive pH sensor and its application to bioimaging

A new colorimetric and fluorescent sensor for the highly acidic pH was developed from rhodamine B, 1-(2-aminoethyl)piperazine, and 4-chloro-7-nitro-2,1,3-benzoxadiazole. The sensor could be synthesized in one pot with an 82.5% yield, and for the first time N,N-diisopropylethylamine was found to be crucial for the rhodamine spirolactam formation. The sensor responded to pH rapidly, visibly, reversibly, highly selectively, and sensitively. From pH 7.77 to 2.03, the absorption and fluorescence intensity of the sensor increased by 285 and 50.3 folds, respectively. The pK_a value based on the fluorescence titration was 2.87. Fluorescent imaging of living cells treated with the sensor in different pH media indicated that the sensor could provide intracellular pH information.     

An ultrasensitive method of real time pH monitoring with complementary metal oxide semiconductor image sensor

CMOS sensors are becoming a powerful tool in the biological and chemical field. In this work, we introduce a new approach on quantifying various pH solutions with a CMOS image sensor. The CMOS image sensor based pH measurement produces high-accuracy analysis, making it a truly portable and user friendly system. pH indicator blended hydrogel matrix was fabricated as a thin film to the accurate color development. A distinct color change of red, green and blue (RGB) develops in the hydrogel film by applying various pH solutions (pH 1–14). The semi-quantitative pH evolution was acquired by visual read out. Further, CMOS image sensor absorbs the RGB color intensity of the film and hue value converted into digital numbers with the aid of an analog-to-digital converter (ADC) to determine the pH ranges of solutions. Chromaticity diagram and Euclidean distance represent the RGB color space and differentiation of pH ranges, respectively. This technique is applicable to sense the various toxic chemicals and chemical vapors by situ sensing. Ultimately, the entire approach can be integrated into smartphone and operable with the user friendly manner.     

Divalent metal ion-sensitive holographic sensors

A holographic sensor for real-time detection of divalent metal ions (Ca²⁺, Mg²⁺, Ni²⁺, Co²⁺ and Zn²⁺) has been fabricated by incorporating a chelating monomer into a hydrogel matrix. A methacrylated analogue of iminodiacetic acid (IDA) was prepared and co-polymerised with hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EDMA) as a cross-linker to form polymer films. A silver-based reflection hologram was incorporated into the hydrogel by diffusion followed by holographic recording using a frequency-doubled Nd/YAG laser. Changes in the replay wavelength of the hologram were used to characterise the swelling behaviour of the matrix as a function of its chemical composition and concentration of analyte in the media. The effects of active monomer, cross-linker, pH and ionic strength on the swelling of the matrix and on metal detection sensitivity have been studied. Polymers containing >10 mol% of chelating monomer and 6 mol% of cross-linker showed significant responses (46.3 nm) within 30 s at an ion concentration of 0-40 nm. The selectivity of the holograms towards the different ions tested was Ni²⁺>Zn²⁺>Co²⁺>Ca²⁺>Mg²⁺. The sensor showed fully reversible responses, permitting real-time monitoring of calcium ion efflux during the germination of Bacillus megaterium spores.     

Hexavalent chromium quantification in solution: Comparing direct UV–visible spectrometry with 1,5-diphenylcarbazide colorimetry

Quantification of Cr(VI) in an aqueous solution is conducted by direct UV–visible spectrophotometry based on the yellow coloring of the chromate ion. Measurements show that absorption follows the Beer–Lambert law over a wide range of concentrations. At pH below the pK_a of 6.4 (HCrO₄^?/CrO₄^?2), the absorption maximum lies at 350 nm wavelength and the linear range spans from 0.5 to 100 mg Cr(VI)/L; above the pK_a (pH 6.4), the absorption maximum is 373 nm and linearity occurs in the range of 0.5–25 mg/L. The wide range of validity of the Beer–Lambert law is advantageous for the measurement of concentrated samples. The standard method of analysis of aqueous Cr(VI) is by colorimetry with the 1,5-diphenylcarbazide (DPC)–Cr(VI) complex. This method, although very sensitive, bears a narrow range of linearity from 0 to 0.8 mg Cr(VI)/L. It is shown that when analyzing Cr(VI) solutions with concentrations in the range of 30–500 mg/L, the DPC method gives inaccurate results and relative standard deviations of 20–50%. This is due to high dilution factors. On the contrary, the direct method performs with high accuracy. Relative standard deviation is only 0.5% at 500 mg Cr(VI)/L. The direct method is fast, reliable, and nondestructive for the sample. The direct method is recommended for the quantification of Cr(VI) at concentrations greater than 1 mg/L.