Search

This work presents a colorimetric dye-based array for naked-eye detection of chicken meat spoilage. The array is obtained by fixing five acid-base indicators, m-cresol purple (1), o-cresol red (2), bromothymol blue (3), thymol blue (4), and chlorophenol red (5), and a sensing molecule specific for thiols, 5,5'-dithiobis(2-nitrodibenzoic acid), called Ellman's reagent (6), on a cellulose-based support. The dyes, being permanently charged, are fixed on the support via ion-exchange. The entire degradation process of beast poultry meat, at ambient temperature and in a domestic fridge, is followed by the change of the color of the array, placed in the headspace over the meat samples. The device is set after selection of the most suitable starting form, which could be the acidic or the basic color of indicators, being the proper dye concentration and the dimension of the spots already established. Basing on sensors colors, we identified three levels of the degradation process of chicken meat, named SAFE, WARNING, and HAZARD. By instrumental analysis, we demonstrated that sensors response was correlated to volatile organic compounds (VOCs) composition in the headspace and, thus, to meat spoilage progress. We demonstrated that biogenic amines (BAs), commonly considered a critical spoilage marker, are indeed produced into the samples but never present in the headspace, even in traces, during the investigated time-lapse. The VOC evolution nevertheless allows one to assign the sample as WARNING and further HAZARD. Some indicators turned out to be more informative than others, and the best candidates for a future industrial application resulted in a bromothymol blue (3)-, chlorophenol red (5)-, and Ellman's reagent (6)-based array.

A colorimetric strip sensor array based on the naked-eye detection has been reported as a simple instrument-free device able to visually display a concentration level of gaseous analytes with basicity. The array has a vertically aligned configuration of five sensing elements and their detection sensitivities to basic analytes are controlled to decrease downwardly. The colorimetric sensors were fabricated by drop-casting a dye solution containing a pH indicator, an acid compound, and hydrophilic polyethylene glycol on a polyacrylonitrile nanofiber mat. The detection sensitivity was regulated by using different amounts of the acid compound in addition to variation in pH Indicator. When sensors containing a pH indicator of bromophenol blue (BPB), bromocresol green (BCG), or chlorophenol red (CPR) were exposed to ammonia gas, coloration happens considerably in the regions of 0–1, 0–5 and 10–100 ppm for BPB, BCG and CPR sensors, respectively. Furthermore, the detection sensitivity could be controlled to decrease by increasing an amount of citric acid, which reveals that the color-changing acid-base reactions between pH indicator and basic analytes are reduced by competitive interactions between the analytes and acid additive. As illustrations, two strip sensor arrays based on BPB and BCG dyes were fabricated and confirmed the successful naked-eye discrimination of a NH 3 concentration level in the ranges of 1–50 and 5–100 ppm, respectively. The concentration level can be determined by a number of coloration elements in the strip device while its resolution relies on a total number of sensing elements.

A simple on package indicator label for grapes (Vitis vinifera L.) ripeness has been developed based on chlorophenol red (CPR). The CPR was immobilized onto the filter paper via absorption method to make the CPR membrane. The membrane as a colorimetric label for grape ripeness works based on pH increase as the volatile organic acids decreased gradually in the package headspace due to the formation of sugars during berry ripening process. Subsequently, the color of the indicator label will change from white to beige then finally to yellow for over-ripe indication, which is easily detected by naked eye. The results show that the indicator label could be used to monitor the ripeness of grapes since the color changes of indicator label toward the ripeness of the grapes are in similar tendency and have a good linear correlation. Thus, the indicator label can be used as an effective tool for on-line ripeness monitoring of grapes packaging. Finally, the indicator label was successfully used as on-package indicator label for on-line ripeness monitoring of grapes in ambient and chilled conditions.

Detecting bacteria is important in the fields of human health, environmental monitoring, and food safety. Foodborne pathogens alone are estimated to cause 420 »000 deaths annually, with low-income regions affected most. Despite improvements in bacterial detection, fast, disposable, low-cost, sensitive, and user-friendly methods are still needed. Traditional methods for detecting bacteria rely primarily on cell culturing or polymerase chain reaction (PCR), which require highly trained personnel and a central laboratory and take several hours or even days to deliver results. Low-cost methods like lateral flow immunoassays exist but frequently suffer from poor sensitivity and/or lack quantitative results. Here, a rapid method for detecting bacteria at very low concentrations is presented using two sequential preconcentration steps. In the first preconcentration step, the sample is mixed with antibody-modified magnetic particles and free antibodies conjugated to β-galactosidase (β-gal). The target bacteria are isolated and concentrated using immunomagnetic separation. The isolated bacteria are then incubated with chlorophenol red-β-d-galactopyranoside (CPRG), which reacts with β-gal to produce chlorophenol red (CPR) in a bacteria concentration-dependent manner. In the second step, CPR and CPRG are separated and focused using an isotachophoretic microfluidic paper-based analytical device, significantly improving the final detection limit relative to paper-based devices lacking the focusing mechanism. Moreover, CPR and CPRG form two visible color bands that act as test and control bands, respectively, improving assay robustness. The method was tested with E. coli DH5-α and successfully detected concentrations as low as 9.2 CFU/mL in laboratory samples and 920 CFU/mL in apple juice samples in ∼90 min. Fil: Schaumburg, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Carrell, Cody S.. State University of Colorado - Fort Collins; Estados Unidos Fil: Henry, Charles S.. State University of Colorado - Fort Collins; Estados Unidos

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.

Amine-based volatile compounds released bymicroorganisms offer an alternative diagnostic approach for the identification of foodborne pathogens. Our objective has been to solvent cast cellulose acetate membranes to immobilise dyes and to use the resultant membranes as a plastic device to discriminate between different types of amines (triethylamine, isobutylamine, isopentylamine). The plastic device consisted of an array of membranes with five pH indicators (namely alizarin, bromophenol blue, chlorophenol red, methyl red and thymol blue). To analyse the data using a portable instrument, we used an iPhone (R) to obtain images and to extract red, green and blue colours (RGB) using in-house software before and after contact with each individual amine. All the RGB values extracted for each analyte allowed us to generate a unique colour pattern, which was used as input for non-supervised pattern recognition methods. Based on this analysis, it was possible to clearly discriminate between the amines studied without any misclassification, demonstrating that the device is well-suited for large-scale applications such as non-destructive methods to discriminate amines and, in future, for smart packaging applications in order to give early warning of rotting food that may lead to food poisoning. Additionally, a semi-quantitative analysis was performed and we have demonstrated that it is possible to quantify concentrations of amines down to 1 ppm.

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.

In this study we report a method for the rapid and sensitive estimation of bacterial cell concentration in solution based on a colorimetric enzyme/gold nanoshells conjugate system. The CTAB capped gold nanoshells are electrostatically attracted by both the bacterial surface and the enzyme β-galactosidase. The preferential binding of cationic (CTAB)-functionalized gold nanoshells to the more negative bacterial surfaces leaves active β-galactosidase in solution, providing an enzyme-amplified colorimetric response of the binding event. A progressive increase in the enzyme activity is evidenced by the conversion of the yellow-orange CPRG substrate into the red chromophore chlorophenol red, which can be correlated with increasing bacterial cell numbers. Using this strategy, the quantification of bacteria at concentrations as low as 10 bacteria/mL of solution has been achieved. The present method of bacterial cell load assessment offers a distinct potential advantage over other conventional methods such as plate counting in terms of ease of operation, rapidity, high sensitivity and quantitative detection of bacterial cells.

The pH of polar ice is important for the stability and mobility of impurities in ice cores and can be strongly influenced by volcanic eruptions or anthropogenic emissions. We present a simple optical method for continuous determination of acidity in ice cores based on spectroscopically determined color changes of two common pH-indicator dyes, bromophenol blue, and chlorophenol red. The sealed-system method described here is not equilibrated with CO2, making it simpler than existing methods for pH determination in ice cores and offering a 10–90% peak response time of 45 s and a combined uncertainty of 9%. The method is applied to Holocene ice core sections from Greenland and Antarctica and compared to standard techniques such as electrical conductivity measurement (ECM) conducted on the solid ice, and electrolytic meltwater conductivity, EMWC. Acidity measured in the Greenland NGRIP ice core shows good agreement with acidity calculated from ion chromatography. Conductivity and dye-based acidity Hdye+ are f...

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.

A voltammetric sensor prepared by the immobilization of metallophthalocyanine complexes onto a glassy carbon electrode has been developed for the detection of β- d -galactosidase (B-GAL) of faecal origin in water. Electrooxidation of chlorophenol red, a breakdown product of the chromogenic substrate chlorophenol red β- d -galactopyranoside, was used as a measure of β- d -galactosidase activity. At metallophthalocyanine modified electrodes, in particular copper(II) phthalocyanine, a decrease in electrode fouling was observed. The sensor was sensitive to fluctuations in pH, not significantly affected by temperature variations and could detect one colony forming unit/100 mL in 15 min. Loss of 40% sensitivity was observed over a period of 30 days. A strong correlation between sensor sensitivity and colony forming units was observed. The sensor is capable of detecting viable but nonculturable bacteria, overcoming this drawback of the use of culture media for detection of coliforms.

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 solid-state, diffuse reflectance-based fiberoptic sensor is described for quantifying ammonia. This sensor is constructed by immobilizing chlorophenol red, a weak acid chromophoric indicator dye, in a microporous polypropylene membrane. A flow-injection analysis system is used to carry a 10-μL aliquot of the sample across the treated membrane. Ammonia in the sample diffuses through the air-filled pores within the membrane structure before reacting with the indicator dye. A reversible acid-base reaction between ammonia and chlorophenol red results in a measurable change in the reflectance at 560 nm. Response characteristics include a peak response within 20 s, a limit of detection of 0.2 ± 0.1 mM ammonia, and a dynamic range of up to 60 mM ammonia. The analytical utility of this sensor is demonstrated by measuring ammonia levels in 48 individual samples collected during the growth of PC3 human prostate cancer cells in a typical serum-containing growth medium. Accuracy of the proposed sensor is verified by a comparison of results from this sensor to those from a conventional enzyme assay.

The preparation and characterization of three different plastic thin-film colorimetric sensors for gaseous ammonia is described. In the film sensors, the neutral form of a pH-sensitive dye (Bromophenol Blue, Bromocresol Green or Chlorophenol Red) was encapsulated in a plastic medium, either poly(vinyl butyral) or ethylcellulose plasticized with tributyl phosphate. Each of these film optodes gave a reproducible and reversible response towards gaseous ammonia. The sensitivity of the film sensors towards ammonia was found to be strongly dependent upon the pK a of the encapsulated dye. Thus, the film with Chlorophenol Red (pK a = 6.25), proved to be very insensitive (operating range: 0.29% < %NH3 < 100%), whereas the film with Bromophenol Blue (pK a = 4.1), was much more sensitive (operating range: 0.0003% < %NH3 < 0.11%). The sensitivity of a plastic film sensor decreased markedly with increasing operating temperature and the 90% response (15–38 s) and recovery (820-127 s) times were slow and activation-controlled.

In this study, recombinant bacterial biosensors were immobilized in an agarose matrix and used for the simple and disposable field monitoring of phenolic compounds. In brief, Escherichia coli cells harboring the pLZCapR plasmid, which was previously designed to express the β-galactosidase reporter gene in the presence of phenolic compounds, were immobilized in agarose gel with or without a substrate [chlorophenol red β-galactopyranoside (CPRG)] and dispensed to the wells of a 96-well plate. Analytes were added to the wells, and color development was monitored either directly from wells containing intact cells co-immobilized with CPRG (SYS I), or using cells that were lysed prior to the addition of CPRG (SYS L). SYS L showed relatively higher intensities and faster color development than SYS I. However, both systems developed a red color (representing hydrolysis of CPRG) in the presence of 10 μM to 10~100 mM phenol, with maximum responses seen at 1~5 and 50 mM phenol for SYS I and SYS L, respectively. Other phenolic compounds (2-chlorophenol, 2-methylphenol, 3-methylphenol, 4-chlorophenol, 2-nitrophenol, resorcinol, catechol, and 2,5-dimethylphenol) were also detected by the systems, with varied detection ranges and responses. The agarose-immobilized biosensors were stable for 28 days, retaining 39~69% of their activities when stored at 4°C without nutrients or additives. The immobilized biosensors described herein do not require the on-site addition of a substrate (in the case of SYS I), the pretreatment of samples, or the use of unwieldy instruments for the on-site monitoring of phenolic compounds from environmental samples.

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

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.

The presence of coliforms in polluted water was determined enzymatically (in situ) by directly monitoring the activity of beta-d-galactosidase (B-GAL) through the hydrolysis of the yellow chromogenic subtrate, chlorophenol red beta-d-galactopyranoside (CPRG), which produced a red chlorophenol red (CPR) product. The objectives of this study were to monitor the effect of compounds commonly found in the environment and used in water treatment on a B-GAL CPRG assay and to investigate the differences between the environmental B-GAL enzyme and the pure commercial enzyme. Environmental B-GAL was optimally active at pH 7.8. Two temperature optima were observed at 35 and 55 degrees C, respectively. B-GAL activity was strongly inhibited by silver and copper ions. While calcium and ferrous ions at lower concentrations (50-100mgl(-1)) increased the enzyme activity, a reduction was observed at higher concentrations (200mgl(-1)). Sodium hypochlorite, normally used in rural areas to disinfect water gradually decreased B-GAL activity at concentrations between 0 and 5600ppm for both the commercial and environmental enzymes. B-GAL from the environment behaved differently from its commercially available counterpart.

"Onion-type" multilamellar micro-vesicles of phospholipids (spherulites) were doped with different amounts of a cationic cosurfactant ((-)N-dodecyl-N-methylephedrinium bromide) for the purpose of controlling the sustained release of anionic drugs. Three weak acid probes (methyl red, chlorophenol red, and ibuprofen) were encapsulated in the vesicles as drug models. The kinetics and rate of release were studied by absorption spectroscopy and HPLC. The effect of probe charge (pH above and below pKa of the probes), of cosurfactant concentration and of added salt was investigated. It was found that, above pKa (i.e., when the probes are anionic), the release can be almost totally inhibited by doping the vesicles with 2.4 wt% of cationic cosurfactant. The release properties can even be finely tuned by controlling the amounts of the cosurfactant. Salt and pH effects demonstrate the role of electrostatic interactions in sustaining the release.

Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter. The redesigned test comprises two steps. First, a large a-mount of yeast with estrogenic compounds is incubated for 24 hr. Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta -D-galactopyranoside (CPRG) is added. The cycloheximide stops protein synthesis and allows for an end-point measurement of P-galactosidase activity generated during the first 24 hr. CPRG is converted to chlorophenol red and reflects beta -galactosidase activity, which is indicative of the estrogenic activity. The modifications shorten the duration of the assay at least I day and avoid interference of the estrogenic CPRG or chlorophenol red. The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p ' -DDT, and isoflavones (genistein, daidzein, and glycitein). Bisphenol A, methoxychlor, and genistein triggered higher levels of beta -galactosidase activity in the redesigned protocol. Estrogenic activity of p,p ' -DDT could only be demonstrated with the redesigned protocol. Glycitein and daidzein failed to give a response with both protocols. We also studied deconjugation of beta -glycosidic isoflavones present in soygerm powder. Treatment of the soygerm powder with beta -glycosidase released isoflavones. The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present. The release of isoflavones under conditions prevailing in the intestines was studied. Bacterial beta -glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could lie demonstrated.

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.

Available methods of examination do not provide absolute certainty that two samples of paper have the same origin. A new technique for testing paper, known as inverse paper chromatography (IPC), is based on using a strip of paper as the chromatographic sorbent medium on which a mixture of known probes is separated under standardized conditions. The resulting chromatogram will uniquely designate the paper grade and composition. This principle has been validated with 36 types of paper in a standard test, using cresol red, chlorophenol red, bromothymol blue, fuchsine, and methylene blue as the probe mixture and n-butanol as the eluant. The machine direction in the paper is distinguishable by this method. The experimental technique is simple, inexpensive, and essentially nondestructive. Exposure of paper sheets to physical influences such as printing, wetting, humidity, and filtered sunlight does not alter their characteristic chromatographic behavior. However, baking (artificial aging) and exposure ...

The rate of entry of aliphatic acids into human red blood cells was studied using a new stopped-flow technique. Solutions of aliphatic acids and erythrocyte suspensions were rapidly mixed in a stopped-flow reaction apparatus. The change in extracellular pH resulting from entry of acid into the cell was monitored using chlorophenol red as an extracellular indicator. Extracellular pH of the mixture was linearly related to differential light transmission at 480 and 560 nm. Values of membrane permeability were fitted to data using a model of passive exchange with flux proportional to the transmembrane concentration gradient. At all temperatures the permeabilities were in the order: valeric acid greater than butyric acid greater than propionic acid. Permeabilities were highly temperature dependent and the activation energy (10.5 kcal/mol) remained constant between 7 and 37 degrees C. Despite rapid rates of exchange, permeability measurements did not exhibit evidence of artifact secondary to an unstirred solvent layer on the cell surface. The data are compatible with the hypothesis that aliphatic acid transport across the erythrocyte membrane is passive in nature and proportional to the transmembrane concentration gradient.

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.