Self-Assembled Chromogenic Polymeric Nanoparticle-Laden Nanocarrier as a Signal Carrier for Derivative Binary Responsive Virus Detection.

In the current biosensor, the signal generation is limited to single virus detection in the reaction chamber. An adaptive strategy is required to enable the recognition of multiple viruses for diagnostics and surveillance. In this work, a nanocarrier is deployed to bring specific signal amplification into the biosensor, depending on the target viruses. The nanocarrier is designed using pH-sensitive polymeric nanoparticle-laden nanocarriers (PNLNs) prepared by sequential nanoprecipitation. The nanoprecipitation of two chromogens, phenolphthalein (PP) and thymolphthalein (TP), is investigated in three different solvent systems in which PNLNs demonstrate a high loading of the chromogen up to 59.75% in dimethylformamide (DMF)/dimethyl sulfoxide (DMSO)/ethanol attributing to the coprecipitation degree of the chromogens and the polymer. The PP-encapsulated PNLNs (PP@PNLNs) and TP-encapsulated PNLNs (TP@PNLNs) are conjugated to antibodies specific to target viruses, influenza virus A subtype H1N1 (IV/A/H1N1) and H3N2 (IV/A/H3N2), respectively. After the addition of anti-IV/A antibody-conjugated magnetic nanoparticles (MNPs) and magnetic separation, the enriched PNLNs/virus/MNPs sandwich structure is treated in an alkaline solution. It demonstrates a synergy reaction in which the degradation of the polymeric boundary and the pH-induced colorimetric development of the chromogen occurred. The derivative binary biosensor shows feasible detection on IV/A with excellent specificities of PP@PNLNs on IV/A/H1N1 and TP@PNLNs on IV/A/H3N2 with LODs of 27.56 and 28.38 fg mL-1, respectively. It intrigues the distinguished analytical signal in human serum with a variance coefficient of 25.8% and a recovery of 93.6-110.6% for one-step subtype influenza virus detection.

Metal-Polydopamine Framework: An Innovative Signal-Generation Tag for Colorimetric Immunoassay.

In this work, an innovative enzyme-free colorimetric immunoassay was proposed for the sensitive detection of alpha-fetoprotein (AFP) by introducing thymolphthalein-modified metal-polydopamine framework (MPDA@TP) for the signal generation and amplification. Using zeolitic imidazolate framework (ZIF-67) as the template, the hollow-structured metal-polydopamine framework (MPDA) with high surface recovery and abundant groups was synthesized and functionalized with thymolphthalein (TP) molecules via typical π-stacking reaction. In the presence of target AFP, an MPDA@TP-linked immunosorbent assay (MLISA) was implemented on the capture antibody-modified microplate by using detection antibody-labeled MPDA@TP as the secondary antibody. Upon alkaline solution introduction, the coated hydrophobic TP on the MPDA was deprotonated into hydrophilic TP2- ion and dissolved in the solution, thereby resulting in the color change of the solution from nearly colorless to deep blue, and the increasing absorbance of the solution at 595 nm. Importantly, the MPDA@TP-based immunoassay could exhibit high sensitivity for the quantitative detection of target AFP on the basis of the absorbance within a linear range of 10-1000 pg mL-1 at a low detection limit of 2.3 pg mL-1. Furthermore, this system was validated preliminarily to screen human serum specimens with well-matched results for the referenced AFP ELISA kit. Taking advantage of simplicity, enzyme-free, convenience, and sensitivity, MPDA@TP-linked immunosorbent assay has the potential for the application in scientific research and clinical diagnosis.

Enzyme-Free Immunosorbent Assay of Prostate Specific Antigen Amplified by Releasing pH Indicator Molecules Entrapped in Mesoporous Silica Nanoparticles.

An enzyme-free titer plate-based colorimetric assay utilizing functionalized mesoporous silica nanoparticles (MSNs) entrapping pH-indicator molecules has been developed. Pores in the silica nanoparticles were functionalized with phenyltrimethyloxysilane so that pH indicator molecules (thymolphthalein or TP in the present case) can be tightly entrapped through π-π conjugation. To detect prostate specific antigen (PSA), the TP-containing MSNs were coated with polyethylenimine (PEI), which favors the attachment of the negatively charged secondary anti-PSA antibody. The entrapped thymolphthalein molecules can be readily released from the pores with a simple addition of alkaline solution. The resultant bifunctional MSNs were used for signal-amplified detection of PSA captured by the primary antibody preimmobilized in the wells of a plate. Our method possesses a wide dynamic range (0.5 to 8000 pg/mL) wherein the adsorption of the bifunctional MSNs obeys a modified Langmuir isotherm. A detection limit (LOD) down to as low as 0.36 pg/mL can be attained. Owing to the size uniformity of the MSNs and the obviation of enzyme molecules employed in the enzyme-linked immunosorbent assay (ELISA), excellent reproducibility (RSD = 1.12%) was achieved. The selective detection of PSA in human serum samples demonstrates the amenability of our method to detect important biomarkers in complex biological samples, whereas the performance of the assay in a titer plate ensures high throughput and obviates the use of expensive instruments. Both of these features are prerequisites for clinical settings wherein a great number of samples need to be analyzed in a timely fashion.

Microenvironmental pH measurement during sodium naproxenate dissolution in acidic medium by UV/vis imaging.

Variable dissolution from sodium salts of drugs containing a carboxylic acid group after passing the acidic environment of the stomach may affect oral bioavailability. The aim of the present proof of concept study was to investigate pH effects in relation to the dissolution of sodium naproxenate in 0.01M hydrochloric acid. For this purpose a UV/vis imaging-based approach capable of measuring microenvironmental pH in the vicinity of the solid drug compact as well as monitoring drug dissolution was developed. Using a pH indicating dye real-time spatially resolved measurement of pH was achieved. Sodium naproxenate, can significantly alter the local pH of the dissolution medium, is eventually neutralized and precipitates as the acidic species naproxen. The developed approach is considered useful for detailed studies of pH dependent dissolution phenomena in dissolution testing.

Spectroscopic investigation on sonodynamic and sonocatalytic damage of BSA molecules by Thymol Blue (TB) derivants under ultrasonic irradiation.

In this paper, the Thymol Blue derivants including Thymol Blue (thymolsulfonphthalein), Thymol Blue-DA (3,3'-Bis [N,N-bis (carboxymethyl) aminomethyl] thymolsulfonphthalein) and Thymol Blue-DA-Fe(III) (3,3'-Bis [N,N-bis (carboxymethyl) aminomethyl] thymolsulfonphthalein-Ferrous(III)) were adopted as sonosensitizers to study the sonodynamic and sonocatalytic activities under ultrasonic irradiation. At first, the interaction of Thymol Blue derivants with bovine serum albumin (BSA) was studied by fluorescence spectroscopy. On that basis, the sonodynamic and sonocatalytic damages of Thymol Blue derivants to BSA under ultrasonic irradiation were investigated by the combination of UV-vis, circular dichroism (CD) and fluorescence spectroscopy. Meanwhile, some influenced factors (ultrasonic irradiation time, Thymol Blue derivants concentration and ionic strength) on the damaging degree of BSA molecules were also reviewed. In addition, synchronous fluorescence spectra were used to estimate the binding and damage sites of Thymol Blue derivants to BSA. Finally, the generation of ROS during sonodynamic and sonocatalytic processes was confirmed by the method of Oxidation-Extraction Spectrometry (OEP). Perhaps, this paper may offer some important subjects for the study of Thymol Blue derivants in sonodynamic therapy (SDT) and sonocatalytic therapy (SCT) technologies for tumor treatment and the effect of the amino acid and central metal.

Ultra-thin porous glass membranes--an innovative material for the immobilization of active species for optical chemosensors.

In addition to polymers, porous glasses can be used for the immobilization of indicators, chromoionophores or enzymes. Advantages of these materials include, among others, the photochemical and thermal stability. Porous glass membranes (CPG) based on phase-separated alkali borosilicate glasses with thicknesses of 250-300 µm and dimensions of approximately 9-13 mm² were used in this work. The average pore diameter was found to be between 12 and 112 nm. Initially, the membrane permeability for water was determined. Furthermore, the absorption spectra for the water-soaked membranes were recorded optically. CPG membranes which are pH-sensitive were prepared based on the covalent immobilization of thymol blue and a derivative of styryl acridine. In each case, the absorption spectra of the immobilized indicators are shown. The t90-times vary between 4 and 20 min and were determined for the thermodynamic equilibrium. The influence of the ionic strength on the characteristic curve is discussed and detailed results are given. After the storage time of about 900 days a pH-sensitivity for a CPG membrane styryl acridine derivative sample was still detectable.

Evaluation of a hydrophilic ionic liquid as a salting-out phase separation agent to a water-tetrahydrofuran homogeneous system for aqueous biphasic extraction separation.

The use of a hydrophilic ionic liquid (IL), 1-butyl-3-methylimidazolium chloride (C(4)mimCl), as a salting-out phase separation agent to a water-tetrahydrofuran homogeneous system was studied for possible applications to novel aqueous biphasic extraction separation. The IL showed a salting-out phase-separation ability. Also, differences in the polarity between the formed two phases were smaller than that when using NaCl as a salting-out agent. This result suggested that C(4)mimCl remaining in water-rich phase acts not only as a salting-out agent, but also a component of a mixed-solvent. Possible uses of C(4)mimCl/NaCl mixed salting-out agent system were also discussed.

Ionic liquid "buffers"-pH control in ionic liquid systems.

We describe hydrated ionic liquids which have an intrinsic proton buffering action and hence an ability to control proton activity in an ionic medium.

A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities.

The standard assay for iodoperoxidase activity is based on the spectrophotometric detection of triiodide formed during the enzymatic reaction. However, chemical instability of I3- has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometric assay for the determination of iodoperoxidase activities of vanadium haloperoxidases based on the halogenation of thymol blue. The relation between color and chemical entities produced by the vHPO/H(2)O(2)/I(-) catalytic system was characterized. The method was extended to bromine and, for the first time, allowed measurement of both iodo- and bromoperoxidase activities using the same assay. The kinetic parameters (K(m) and k(cat)) of bromide and iodide for vanadium bromoperoxidase from Ascophyllum nodosum were determined at pH 8.0 from steady-state kinetic analyses. The results are concordant with an ordered two-substrate mechanism. It is proposed that halide selectivity is guided by the chemical reactivity of peroxovanadium intermediate rather than substrate binding. This method is superior to the standard I3- assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxidases.

Experimental correlation between the pKa value of sulfonphthaleins with the nature of the substituents groups.

This work presents the results obtained from a spectrophotometry study performed on some indicators of the sulfonphtaleins like phenol red (PR), thymol blue (TB), bromothymol blue (BTB), xylenol orange (XO) and methylthymol blue (MTB). During the first stage the acidity constants of some of the indicators were determined using the data from spectrophotometry, potentiometry and with the use of the software SQUAD. These were as follows: for the equilibrium 2H+BTB<-->H(2)BTB, log beta(2)=15.069+/-0.046 and for H+BTB<-->HBTB, log beta(1)=8.311+/-0.044. For the XO and the MTB five values were calculated for each, namely, for MTB: log beta(5)=42.035, log beta(4)=38.567+/-0.058, log beta(3)=32.257+/-0.057, log beta(2)=23.785+/-0.057, and log beta(1)=12.974+/-0.045 while for XO: log beta(5)=40.120+/-0.102, log beta(4)=35.158+/-0.062, log beta(3)=29.102+/-0.053, log beta(2)=21.237+/-0.044, and log beta(1)=11.682+/-0.044. During the second stage, a study was conducted on the effect of the substituents present in the indicators to determine the effect of different functional groups on the pK(a) value corresponding to the last indicator's dissociation.

Water-soluble pH-responsive dendritic core-shell nanocarriers for polar dyes based on poly(ethylene imine).

A simple general synthetic concept to build dendritic core-shell architectures with pH-labile linkers based on hyperbranched PEI cores and biocompatible PEG shells is presented. Using these dendritic core-shell architectures as nanocarriers, the encapsulation and transport of polar dyes of different sizes is studied. The results show that the acid-labile nanocarriers exhibit much higher transport capacities for dyes than unfunctionalized hyperbranched PEI. The cleavage of imine bonds and controlled release of the polar dyes revealed that weak acidic condition (pH approximately 5.0) could cleave the imine bonds linker and release the dyes up to five times faster than neutral conditions (pH = 7.4).

Determination of the complexation constants of Pb(II) and Cd(II) with thymol blue using spectrophotometry, SQUAD and the HSAB principle.

This paper presents the results concerning the determination of the formation constants of the complexes between thymol blue, TB, and the metal ions Pb(II) or Cd(II). The experimental procedure was carried out in the presence of a nitrogen atmosphere at 25 degrees C. The spectrophotometry data obtained were processed through the software SQUAD to calculate the complexation constants of the metal-indicator and to establish an adequate base of the models which considered the structure of the indicator, and the actual metal species in the aqueous solutions. For the Pb(II)-TB-H2O system the logK value calculated for the PbTB complex was 5.591+/-0.057 while for the Cd(II)-TB-H2O system, the logK value of the CdTB complex was 5.099+/-0.008. Also, supporting theoretical chemistry results on the chemical hardness of TB molecule were performed to enable establishment of a relative prediction scale of the TB complexation constants ranking in the framework of the Principle of Hard and Soft Acids and Bases or HSAB Principle.

Simultaneous determination of copper, bismuth and lead by adsorptive stripping voltammetry in the presence of thymolphthalexone.

A novel, sensitive and selective adsorptive stripping procedure for simultaneous determination of copper, bismuth and lead is presented. The method is based on the adsorptive accumulation of thymolphthalexone (TPN) complexes of these elements onto a hanging mercury drop electrode, followed by reduction of adsorbed species by voltammetric scan using differential pulse modulation. The influences of control variables on the sensitivity of the proposed method for the simultaneous determination of copper, lead and bismuth were studied using the Derringer desirability function. The optimum analytical conditions were found to be TPN concentration of 4.0 microM, pH of 9.0, and accumulation potential at -800 mV vs. Ag/AgCl with an accumulation time of 80 s. The peak currents are proportional to the concentration of copper, bismuth and lead over the 0.4-300, 1-200 and 1-100 ng mL(-1) ranges with detection limits of 0.4, 0.8 and 0.7 ng mL(-1), respectively. The procedure was applied to the simultaneous determination of copper, bismuth and lead in the tap water and some synthetic samples with satisfactory results.

Spectrophotometric study of the system Hg(II)-thymol blue-H2O and its evidence through electrochemical means.

The detailed analysis of the experimental spectrophotometric data obtained from solutions containing the acid-base indicator thymol blue (TB) and mercury(II) (Hg(II)) coupled with data processing by means of the SQUAD program, a chemical model was determined that includes the formation of complexes indicator-metal ion (HgTB and HgOTB), dimer species (H3TB2 and H4TB2) and monomer species (HTB and TB). The values of the overall formation constants (log beta) were calculated for the chemical equilibria involved: TB+Hg<-->HgTB log beta=16.047 +/- 0.043, TB+Hg+H2O<-->HgOHTB+H log beta=7.659 +/- 0.049, 2TB+4H<-->H4TB2 log beta=31.398 +/- 0.083, 2TB+3H<-->H3TB2 log beta=29.953 +/- 0.084 and H+TB<-->HTB-log beta=8.900. To compliment the present research, the values of the absorptivity coefficients are included for all the species involved, within a wide range of wavelengths (250-700 nm). The latter were used subsequently to carry simulations of the absorption spectra at various pH values, thus corroborating that the chemical model proposed is fully capable to describe the experimental information. Voltammetric study performed evidenced the formation of a complex with a 1:1 stoichiometry Hg(II):TB.

Spectrophotometric determination of fluoxetine hydrochloride in bulk and in pharmaceutical formulations.

Two new rapid, sensitive and economical spectrophotometric methods are described for the determination of fluoxetine hydrochloride in bulk and in pharmaceutical formulations. Both methods are based on the formation of a yellow ion-pair complex due to the action of methyl orange (MO) and thymol blue (TM) on fluoxetine in acidic (pH 4.0) and basic (pH 8.0) medium, respectively. Under optimised conditions they show an absorption maxima at 433 nm (MO) and 410 nm (TB), with molar absorptivities of 2.12 x 10(-4) and 4.207 x 10(-3) l mol(-1) cm(-1) and Sandell's Sensitivities of 1.64 x 10(-2) and 0.082 microg cm(-2) per 0.001 absorbance unit for MO and TB, respectively. The colour is stable for 5 min after extraction. In both cases Beer's Law is obeyed at 1-20 microg mol(-1) with MO and 4-24 microg mol(-1) with TB. The proposal method was successfully extended to pharmaceutical preparations capsules. The results obtained by both the agreement and E.P. (3rd edition) were in good agreement and statistical comparison by Student's t-test and variance ratio F-test showed no significant difference in the three methods.

[Microbiological determination of netilmicin using a thin-layer chromatography scanner].

We presented a new colorimetric bioassay of aminoglycoside antibiotics, which were represented by netilmicin (NTL) in this study, based on the discoloration of thymolphthalein (TP) in paper (indicator-disc) by carbon dioxide produced by Bacillus subtilis. To evaluate the amount of the carbon dioxide, the following experiment was carried out. One milliliter of B. subtilis suspension containing 4.5 x 10(7) colony forming units/ml, 1 ml of nutrient broth, 0.9 ml of 0.1 M phosphate buffer (pH 8.0) and 0.1 ml NTL sample solution were added to an incubation container, which was then placed in a water-bath (37 degrees C) for 3 hours. The oxygen concentration in the head space of flask was determined using gas-chromatograph. The dose-response curves showed good correlation between amounts of NTL and carbon dioxide produced by B. subtilis. The indicator-disc containing TP and sodium hydroxide was placed into the Reacti-flask and then incubated in the same manner as described above. After incubation, concentration of blue colored TP was determined using a TLC scanner. The discoloration of blue color to white showed the proportionality between NTL concentrations and the degrees of discoloration of TP. The method can accurately measure NTL levels down to 2.5 micrograms/ml in water using 0.1 ml samples, and should be adequate for rapid bioassay.