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NH 3 and CO 2 are often involved in the spoilage of foods. In this study, a colorimetric array indicator was evaluated for the detection of NH 3 and CO 2 using pH-sensitive dyes (dimethyl yellow, methyl red, chlorophenol red, methyl orange, phenol red, thymol blue, m -cresol purple) encapsulated within ethylcellulose matrices. The pH dyes were doped either with hydrochloric acid or tetrabutylammonium hydroxide to manipulate the colorimetric responses. The sensing dyes were applied onto a plastic substrate to produce array indicators consisting of 4 × 4 elements. Digital images of the indicator array, before and after the exposure to NH 3 , CO 2 or NH 3 /CO 2 mixtures, were taken to monitor the change in color for each of the array elements. Unique ΔRGB patterns for NH 3 , CO 2 , and their mixture were observed. At 4 °C, principal component analysis discriminated samples into two clusters that were higher and lower than 15 μg/mL NH 3 respectively. For CO 2 , at both 4 and 25 °C, the array indicator discriminated air samples with lower than 5% CO 2 from those higher than 20% CO 2 . For NH 3 /CO 2 mixtures, the array indicator differentiated air samples of high NH 3 /low CO 2 and low NH 3 /high CO 2 combinations. These results show that the array indicator may be useful for intelligent packaging application of food products.

The use of coliforms and Escherichia coli as indicator species for assessing the quality of water is well established and a large variety of methods based on β-galactosidase (B-GAL) activity, inherent to the microbes within this classification, have arisen to enable their detection and enumeration. Chlorophenol red (CPR) is widely used as a chromogenic label, but its capacity for translation to electroanalytical devices has yet to be fully explored. The CPR moiety is capable of undergoing oxidation at carbon substrates (+0.7 V) giving rise to a variety of phenolic intermediates. Electrochemical, XPS and enzymatic techniques were employed to characterise the underpinning chemistry and the intermediate identified as a 1,2-quinone derivative in which the chlorine substituent is retained. The latter was found to accumulate at the electrode and, in contrast to the parent CPR, was found to be detected at a significantly less positive potential (+0.3 V). Bacterial hydrolysis of a CPR labelled substrate was demonstrated with the 1,2-quinone oxidation product found to accumulate at the electrode and detected using square wave voltammetry. Proof of concept for the efficacy of the alternative electrode pathway was established through the detection of E.coli after an incubation time of 2.5 h with no interference from the labelled substrates.

Biosensors have been used as first-step monitoring tools to detect on-site samples in a simple and cost-effective manner. Numerous recombinant microbial biosensors have been exploited for monitoring on-site toxic chemicals and biological signals. Herein, a recombinant microbial biosensor was constructed for monitoring cadmium. The cadmium responding cadC regulatory gene and it’s promoter from Staphylococcus aureus was amplified through PCR, fused with the lacZ gene, and transformed into Escherichia coli BL21 (DE3) cells. In the presence of cadmium, the biosensor cells express β-galactosidase showing red color development with chlorophenol red β-galactopyranoside (CPRG) as the enzymatic substrate. The biosensor cells showed the best β-galactosidase activity after 3 hr induction with cadmium at pH 5 and a detection range from 0.01 μM to 10 mM cadmium with a linearity from 0.01 to 0.1 μM cadmium (y = 0.98 x + 0.142, R² = 0.98). Among the heavy metals, cadmium and lead showed good responses, tin and cobalt showed medium responses, and mercury and copper showed no responses. The biosensor cells showed good responses to several waste waters similar to buffer solution, all spiked with cadmium. The biosensor described herein could be applied for on-site cadmium monitoring in a simple and cost-effective manner without sample pretreatments.

The article describes a simple and rapid method for colorimetric detection of bacteria. It is based on competitive binding of positively charged polyethyleneimine-coated gold nanoparticles (PEI-AuNPs) to negatively charged enzymes and bacteria. The PEI-AuNPs are electrostatically attracted by both the bacterial surface and the enzyme β-galactosidase (β-Gal). Binding to the latter results in the inhibition of enzyme activity. However, in the presence of a large number of bacteria, the PEI-AuNPs preferentially bind to bacteria. Hence, the enzyme will not be inhibited and its activity can be colorimetrically determined via hydrolysis of the chromogenic substrate chlorophenol red β-D-galactopyranoside (CPRG). The detection limit of this assay is as low as 10 cfu·mL−1, and the linear range extends from 106 to 108 cfu·mL−1. The assay is applicable to both Gram-negative (such as enterotoxigenic Escherichia coli; ETEC) and Gram-positive (Staphylococcus aureus; S. aureus) bacteria. Results are obtained within 10 min using an optical reader, and within 2–3 h by bare-eye detection. The method was applied to the identification of ETEC contamination at a level of 10 cfu·mL−1 in spiked drinking water. Given its low detection limit and rapidity (sample preconcentration is not required), this method holds great promise for on-site detection of total bacterial contamination.

Aromatic hydrocarbons are toxic environmental pollutants that are detrimental to the ecosystem and human health. Among them, chlorotoluene and nitrotoluene are toxic to hydrobios and irritate the skin, eyes, and respiratory organs of humans. We herein report the development of recombinant microbial biosensors for cheap and rapid monitoring of chlorotoluene and nitrotoluene compounds. Plasmids were constructed by inserting the xylR regulatory gene for BTEX (benzene, toluene, ethylbenzene, and xylene) degradation into upstream of Po’ (the DmpR activator promoter Po with the deletion of its own upstream activating sequences) or Pu (the cognate promoter of XylR)::lacZ (the β-galactosidase gene) and transformed into Escherichia coli DH5α. In the presence of inducers, the biosensor cells immobilized in agarose developed a red color in 1-2 h due to the hydrolysis of chlorophenol red β-D-galactopyranoside (CPRG), a substrate of β-galactosidase that was expressed by the inducers. Among BTEX, high responses were specifically observed with o-, m-, p-chlorotoluene (0.1 ㎛-100 mM) and o-, m-, p-nitrotoluene (0.1 mM-100 mM). Po’ demonstrated higher responses than those with Pu. The biosensors immobilized in agarose showed good stability after 21 days’ storage at 4℃, and responses in untreated wastewater spiked with chlorotoluene and nitrotoluene, suggesting they can be used to detect compounds in wastewater.

A simple, accurate, and rapid extraction spectrophotometric procedure has been developed for the determination of some phenothiazine drugs such as chlorpromazine hydrochloride (CPH) and trifluoperazine dihydrochloride (TFPH) in pure form and their pharmaceutical preparations. The procedures are based on the reaction between the examined drugs (CPH and TFPH) and Alizarin Red S (AR), Bromocresol Purple (BCP), Chlorophenol Red (CPR) and Cresol Red (CR) producing ion-pair complexes which can be measured at the optimum wavelengths. The optimizations of the reaction conditions were investigated. Beer’s law is obeyed in the concentration ranges 1.77–96 μg ml −1 . The molar absorptivity and Sandell sensitivity are also calculated. The correlation coefficient for both drugs was ⩾0.9995 ( n = 5) with a relative standard deviation (R.S.D.) ⩽0.38. The methods are successfully applied to determine CPH and TFPH in pharmaceutical formulations.

We herein report the development of an agarose-gel-immobilized recombinant bacterial biosensor simple system for the field monitoring of phenolic compounds. Escherichia coli cells harboring the pLZCapR plasmid, which was previously designed to express the -galactosidase reporter gene in the presence of phenolic compounds, were co-immobilized with a substrate [chlorophenol red -galactopyranoside (CPRG) in agarose gel, and dispensed to the wells of a 96-well plate. Field samples were added to the wells and color development was monitored. In the presence of 5 to 10 mM of phenol, the biosensor developed a red (representing hydrolysis of CPRG) color. Other phenolic compounds were also detected by this immobilized system, with the pattern resembling that previously reported for the corresponding non-immobilized biosensor. The immobilized cells showed optimum activity when the gel was simultaneously supplemented with 6% dimethyl formamide (DMF), 0.1% SDS and 10 mM . The immobilized biosensor described herein does not require the addition of a substrate or the use of unwieldy instruments or sample pretreatments that could complicate field studies.

We herein report the development of a recombinant bacterial biosensor for the rapid and easy detection of phenolic compounds in the field. A plasmid was designed to encode a β-galactosidase reporter gene under the control of capR, an activator involved in phenolic compound degradation. The construct was transformed into Escherichia coli , and transformed cells were stored after being freeze-dried in the presence of sucrose. For detection of phenolic compounds, the cells were rehydrated, and used instantly, without any growth step. In the presence of 0.1 μM–10 mM phenol, we observed a red color from hydrolysis of chlorophenol red β- d -galactopyranoside (CPRG) or an indigo color from hydrolysis of X-galactopyranoside (X-gal). Other phenolic compounds could be detected by this system, including catechol, 2-methylphenol, 2-chlorophenol, 3-methylphenol, 2-nitrophenol, and 4-chlorophenol. These results suggest that this novel bacteria biosensor may be useful for easy, on-site detection of phenolic compounds without the need for unwieldy equipment or sample pretreatment. Indeed, biosensor systems involving β-galactosidase-expressing freeze-dried recombinant bacteria could prove useful for the in situ detection of many more compounds in the future.

We report on a new fluorimetric assay for β-galactosidase (β-gal) and faecal coliform bacteria that utilizes a long-wavelength dye, chlorophenol red-β-d-galactopyranoside (CPRG), that has been widely used for colorimetric assays. The novel feature of this new assay is the unexpected development of a large fluorescence response from liberated chorophenol red (CPR) upon complexation with poly-l-arginine (pR) in solution. The binding of CPR to pR occurs through the sulphonate group of CPR, causing formation of a charge-transfer complex and up to a 70-fold increase in emission intensity. A major advantage of the assay is the ability to utilize excitation and emission wavelengths in the red end of the spectrum, which avoids common interferences obtained when using UV-absorbing dyes such as 4-methylumbelliferyl-β-d-galactopyranoside. We provide data on the utility of CPRG as a fluorimetric reporter for both β-gal and Escherichia coli ATCC 25922 and demonstrate optimized reaction conditions for rapid and sensitive detection of E. coli at a level of 1 colony-forming unit (cfu)/10 mL after 12 h of culture followed by a 1-h assay, which is below the regulatory limit for testing of recreational water.

A simple Yoghurt Culture Test (YCT) for the routine detection of antibiotic and other residues in raw milk was developed. It involved acidifying a sample of the milk to pH 6.0, adding Chlorophenol Red as an indicator and inoculating the milk (4%, v/v) with a culture containing a balanced mixture of Streptococcus thermophilus and Lactobacillus delbrueckii sub-sp. bulgaricus . In the absence of inhibitory substances, incubation at 42 °C for 2.5 h gave rise to sufficient acid production for the milk to gel and the indicator to change colour (PASS), but low levels of the antibiotics tested increased the incubation time beyond 4 h (FAILURE). A survey of 618 samples of raw milk collected from three dairies in Jordan identified a failure rate of 15% with the YCT at 2.5 h whereas, due to differences in sensitivity, the Delvotest-P failed only 12.3% of the milks. It was concluded that the YCT was a reliable and inexpensive approach for testing ex-farm milks for inhibitory residues.

A comparison between the different proposed methods to perform the standard addition in an on-line way (flow injection analysis) is made. The different assemblies (reverse and conventional FIA) and the employed standards in every method are compared. Three analyses were carried out, two of them were analyses of samples of pH indicators (methylorange and chlorophenol red) prepared at an acid pH, while the standards were prepared at a basic pH. The third analysis was the analysis of chloride in different synthetic waters with a perfectly known composition for all the ions present in the samples. Besides a new method to perform the on-line standard addition is proposed, trying to reduce the consumption of reagents and sample. This new method makes use of a 6-port multiposition valve electrically actuated.

We have developed a new pharmacological screening assay for epimastigotes of Trypanosoma cruzi (clone CL-B5) that express the Escherichia coli LacZ gene. The assay is based on determining the activity of the cytoplasmic beta-galactosidase released into the culture on membrane lysis in the presence of the substrate chlorophenol red beta-D-galactopyranoside (CPRG). The experimental conditions were adjusted to find those in which the relationship between epimastigote number and CPRG absorbance was linear over the widest possible range. Absorbance was significantly correlated with the number epimastigote from 5x10(3) to 1.2x10(6) parasites/ml (r=0.98, P

In recent years, the use of chlorine dioxide as an alternative disinfectant for drinking water has become increasingly attractive. As a result, an accurate method for the determination of mg l(-1) concentrations of chlorine dioxide is needed. Improvements to chlorophenol red (CPR) spectrophotometry result in a selective method for ClO(2) with few interferences. CPR selectively reacts with 0.1-1.9 mg l(-1) ClO(2) at pH 7, yielding a linear response (0.9994) with a limit of detection of 0.12 mg l(-1) ClO(2). Several species, ClO(2)(-), ClO(3)(-), NH(2)Cl, and free available chlorine (FAC), were studied as potential interferents using this method. There was found to be less than 2% interference due to 1.38 mg l(-1) ClO(2)(-), 9.87 mg l(-1) ClO(3)(-1), and 5.31 mg l(-1) NH(2)Cl. The interference from up to 1.19 mg l(-1) FAC was 3.7% and could be further reduced by the addition of oxalic acid, sodium cyclamate or thioacetamide.

For the detection of indicator bacteria such as total coliforms or Escherichia coli (E. coli), microbial enzyme profiles are a preferred option compared to classical methods, because the reactions are more sensitive and rapid to perform and bacteria can be detected and enumerated through specific enzyme activities (Romprẻ et al., 2002). Chromogenic or fluorogenic enzyme substrates are used to detect the enzymes b-D-galactosidase (β-GAL) and b-D-glucuronidase (β-GUD). The chromogenic enzyme substrates are phenol-based, for example the enzyme substrates for β-GUD are p-nitrophenyl-b-D-glucuronide (PNPG) and 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (XGLU), where PNPG produces ρ-nitrophenol (a yellow colour) and XGLU produces a blue indoxyl product. The fluorogenic substrate used to detect β-GUD is 4-methylumbelliferyl-b- D-glucuronide (MUG) (Manafi, 1996; 2000). β-GAL is detected by using the chromogenic substrates chlorophenol red- β-galactopyranoside (CPRG) and o-nitrophenyl-b-D-galactopyranoside (ONPG) (Edberg et al., 1988; Cheng et al., 2002). As phenolic compounds have extensive industrial applications, they are often found to be present in the aquatic environment (Llompart et al., 2002; Asan and Isildak, 2003). Severalmajor sources are responsible for the presence of these phenolic compounds in the environment, e.g. pesticides, bactericides, wood preservatives and dyes. Phenolic compounds are also present in pulp processing, petroleum refining, leather tannery, textiles and plastics (Lee et al., 1996; Angelino and Gennaro, 1997; Pẽnalver et al., 2002; Asan and Isildak, 2003; Lupetti et al., 2004). Furthermore, phenols may also be found in fertilizers and explosives (Aktas et al., 2006).Keywords: coliforms, E. coli, faecal, b-D-galactosidase, b-D-glucuronidase, inhibition, wastewater

The procedures for the determination of phosphate are based on the reduction of molybdophosphoric heteropoly acids using ascorbic acid. Measurements of the complex products formed with rhodamine B and chlorophenol red dyes and the reduced molybdophosphoric acid are made at 490 and 510 nm with molar absorptivities of 28571 and 20949 l/mol. cm with each dye, respectively. Phosphate could be determined in the concentration ranges of 0.44 to 1.21 and 0.44 to 1.32 μg/l PO4-P with rhodamine B and chlorophenol red dyes, respectively. The results obtained are compared with those of molybdophosphoric acid with rhodamine B and chlorophenol red using stannous chloride as reducing agent. These procedures are used for the determination of phosphate in the Kingdom of Saudi Arabia.

Background In drug monitoring assays the most common interferences are due to hematocrit, other drugs or their metabolites, while the interference by heterophilic antibodies has been reported only when measuring endogenous molecules. In the present paper a heterophilic antibody interference in the tacrolimus measurement is described. Methods Samples from a patient treated with tacrolimus were analyzed on RxL Dimension analyzer. Ranging drug concentrations from 49 to 12.5 μg/L, even after the interruption of the treatment, confirmation analysis were performed using heterophilic blocking tubes before tacrolimus measurement on the same analyzer, then testing the samples on V-Twin System, finally incubating the samples with chlorophenol red β- d -galactopyranoside, β-galactosidase, polyclonal mouse IgG, protein A and Protein G resin. Results The elevated tacrolimus concentrations were due to the presence of an interference attributable to heterophilic antibodies, as confirmed by treating the samples with heterophilic blocking tubes and protein G resin. Conclusions a) The interference caused by heterophilic antibodies can be found not only in immunoassays measuring endogenous molecules, but also in those for exogenous molecules; b) the pre-treatment sample procedure, which represent the main difference between the methods affected and unaffected by the interference, is a fundamental step in removing the antibodies responsible of the interference.

An optical fiber biosensor for the determination of the pesticides propoxur (Baygon®) and carbaryl, two of the most commonly used carbamate insecticides in vegetable crops, is described. A pH indicator, chlorophenol red, is used as optical transducer of the inhibition of the enzyme acetylcholinesterase by the analytes. The biorecognition element is covalently immobilized onto controlled pore glass beads (CPG) and packed in a thermostatized bioreactor connected to a flow-through cell that contains CPG- immobilized chlorophenol red placed at the common end of a bifurcated fiber optic bundle. In the presence of a constant acetylcholine concentration, the colour of the pH sensitive layer changes and the measured reflectance signal can be related to the carbamate concentration in the sample solution. The performance of the biosensor has been optimized using a flow injection system. The linear dynamic range for the determination of carbaryl and propoxur spans from 0.8 to 3.0 mg l-1 and from 0.03 to 0.50 mg l-1, respectively. The detection limit (3 s) of the biosensor for propoxur (0.4 ng) is lower than that measured for carbaryl (25 ng). Reproducibility, stability and interference studies of the optical device are reported. The biosensor has been applied to the determination of propoxur in spiked vegetables (onion and lettuce) using ultrasound extraction, achieving recovery values between 93 and 95% for onion samples at the different concentration levels assayed. (C) 2000 Elsevier Science S.A. An optical fiber biosensor for the determination of the pesticides propoxur (Baygon) and carbaryl, two of the most commonly used carbamate insecticides in vegetable crops, is described. A pH indicator, chlorophenol red, is used as optical transducer of the inhibition of the enzyme acetylcholinesterase by the analytes. The biorecognition element is covalently immobilized onto controlled pore glass beads (CPG) and packed in a thermostatized bioreactor connected to a flow-through cell that contains CPG-immobilized chlorophenol red placed at the common end of a bifurcated fiber optic bundle. In the presence of a constant acetylcholine concentration, the colour of the pH sensitive layer changes and the measured reflectance signal can be related to the carbamate concentration in the sample solution. The performance of the biosensor has been optimized using a flow injection system. The linear dynamic range for the determination of carbaryl and propoxur spans from 0.8 to 3.0 mg l-1 and from 0.03 to 0.50 mg l-1, respectively. The detection limit (3 s) of the biosensor for propoxur (0.4 ng) is lower than that measured for carbaryl (25 ng). Reproducibility, stability and interference studies of the optical device are reported. The biosensor has been applied to the determination of propoxur in spiked vegetables (onion and lettuce) using ultrasound extraction, achieving recovery values between 93 and 95% for onion samples at the different concentration levels assayed.

The nude mouse xenograft model is commonly used to examine the growth and development of human cancer cells in vivo. Tumor cells transfected with the Lac-Z reporter gene for beta-galactosidase (beta-gal) enzyme activity can be used to quantify tumor and metastatic development in this model. The present study was designed to develop methodology to accurately measure beta-gal in tumor and tissue samples from a nude mouse model. In this study, we developed tissue extraction procedures and compared the sensitivity and accuracy of o-nitrophenyl-beta-D-galactopyranoside (ONPG) and chlorophenol red beta-D-galactopyranoside (CPRG); two beta-gal substrates. Our results demonstrated that the CPRG substrate is more sensitive and accurate in the measurement of beta-gal activity than the ONPG substrate. In addition, matrices and blood in tissue samples are less likely to interfere with the CPRG assay. We concluded that the CPRG substrate-based assay represents a reliable technique for the determination of beta-gal activity in transfected cancer cells present in tumor and tissue specimens from the nude mouse xenograft model.

Crab urinary bladder appears to possess several morphological and functional similarities to vertebrate renal proximal tubule. Sections of intermolt rock crab bladder accumulated PAH by a process that was concentrative (60 min tissue-to-medium ratio (T/M) for 10 microM PAH averaged 24), Na dependent, powered by glycolytic metabolism, and inhibitable by other organic anions. Initial section uptakes exhibited saturation kinetics and a double-reciprocal plot of uptake vs. concentration yielded a single line with a Km of 70 microM and a Vmax of 5 nmol . mg tissue-1 . h-1. Chlorophenol red and bromocresol green (BCG) competitively inhibited PAH uptake. When bladder sheets were mounted in a flux chamber, they exhibited a large, net lumen-to-serosa (L leads to S) flux of 10 microM PAH that was abolished by 1 mM BCG. The small unidirectional S leads to L flux was not BCG-inhibitable. Bladder sheets exhibited PAH T/M greater than 1 after luminal, but not serosal, exposure. BCG only reduced bladder sheet T/M after luminal exposure. The data are consistent with uphill, Na-dependent, and carrier-mediated entry of PAH at the luminal membrane and nonmediated exit at the serosal membrane.

An indicator dye attached to an electrostatically charged macromolecular surface generally has a pK value (pKb') different from that of uncombined dye (pKf'). The question if changes in (pKb' - pKf'), designated as increment incrementpK, records changes in the electrostatic potential at the binding site has been examined in spectrophotometric and binding experiments, using the interaction of Chlorophenol Red and Phenol Red with human serum albumin and cationic micelles as examples. (1) In serum albumin solutions increment incrementpK is decreased by a reduction of pH. The decrease is correlated with the increase in positive charges on the protein molecule, and the response is attenuated by high ionic strength in accordance with electrostatic theory. (2) Opposite changes in binding affinity to serum albumin and increment incrementpK as a function of pH are observed; the binding of basic (bivalent anion) dye is more susceptible to a change in pH than in the acidic (univalent anion) form. (3) Preferential uptake of the basic as compared to the acidic form of dye is observed by binding to cetyltrimethylammonium chloride and cetylpyridinium chloride micelles (mu equals 0.033, [Cl-] equals 0.033 M). An increase in the ionic strength is accompanied by a positive value of increment incrementpK. The results are consonant with the view that the observed increment incrementpK values reflect changes in the electrostatic potential at the binding site with consequently little, if any, effect on the intrinsic pK. The extension of the method to measure changes in the electrostatic potential at binding sites on cell membranes is briefly discussed.

Apparent pKa values for two hydrophilic indicators (2-hydroxy- 5-bromo-trimethylanilinium chloride, HBTCL; and 2-hydroxy-5-nitro sodium benzenesulfonate, HNBNa) and two hydrophobic ones (bromocresol green, chlorophenol red) in the presence of reversed micelles and water-in-oil microemulsions of the nonionic surfactant Arkopal N-060 (polyoxyethylene (6.2) nonylphenyl ether) in cyclohexane were determined spectrophotometrically. The pKa values calculated by using the starting pH of the aqueous buffer solutions (i.e., those measured before their solubilization in the micellar solution) were found to be dependent on the value of W/D (= [water]/ [detergent]) and on the buffer used (imidazole and phosphate). The properties of reversed micelle-solubilized water are different from those of bulk water, this difference should have an effect on the pKa of the solubilized buffers. Therefore, the initially obtained indicator pKa values were corrected for the effect of this transfer (from bulk water to “micellar” water) on the starting pHs of the buffer solutions, using aqueous ethanol as a model for the micellar water “pool”, WP.

SummaryThe observation that phenol red is brominated by the pregnant dogfish uterus in vivo was further studied and chlorophenol red, metacresol purple, and fluorescein were also found to be brominated. Some evidence was obtained that p-hydroxybenzoic acid and phenol were similarly brominated. The bromine was derived from an extraneous source. The site of bromination seemed to reside in the epithelium of the uterus. Bromination was also demonstrated by in vitro incubation, the pH optimum was found to be 7.4.