Multipoint monitor of beer fermentation.

This short communication is devoted to a fully-mechanized flow analysis system for the control of beer fermentation process. The developed system is based on microsolenoid flow controlling devices (valves and pumps) and a flow-through optoelectronic detector. All these components are powered and controlled by a Adruino-compatible microprocessor platform that creates an integrated, compact, and robust analytical tool. Multiplication of sample aspiration ports of the analytical system allows for simultaneous monitoring of several independently performed fermentation processes, as well as a single process at the different places of fermentation tank. To demonstrate its practical utility, the developed system has been applied for online and real-time monitoring of yeast propagation and distribution in beer worts in the course of various fermentation processes. Potentially, this flow analysis system can be easily expanded to the form of multianalyte monitor equipped with optoelectronic sensors and biosensors for the determination of other parameters and analytes.

Photometric flow system for the determination of serum lactate dehydrogenase activity.

The routine method for LDH (Lactate dehydrogenase) activity determination is to monitor the increase of NADH concentration at 340 nm. There are some inconvenience in taking measurements in the near-UV region, especially in the case of serum samples analysis. In this work, two modifications of the routine LDH activity assay based on the use of reducing properties of NADH have been compared. Both methods involved the reduction of compounds that can be easily determined by well-known methods, ferric ion (with ferrozine) and nitrotetrazolium blue (NBT). A fully-mechanized Multicommutated Flow Analysis-Paired Emitter Detector Diode (MCFA-PEDD) system based on solenoid devices was developed and applied for both methods. The linear ranges obtained for Fe-ferrozine and NBT methods are 6.0-200.0 U L-1 and 10.0-250.0 U L-1 with estimated detection limits at 0.2 U L-1 and 4.5 U L-1, respectively. The low LOQ values enabled 10-fold sample dilutions, which is advantageous for samples with limited available volume. The Fe-ferrozine method is more selective for LDH activity in the presence of glucose, ascorbic acid, albumin, bilirubin, copper and calcium ions than NBT method. To confirm the analytical usefulness of the proposed flow system, the analysis of real human serum samples was carried out. The statistic tests showed satisfactory correlation between the results obtained for both developed methods and those received using the reference method.

Sequential Injection Analysis for Rapid Determination of Mercury in Skincare Products Based on Fluorescence Quenching of Eco-Friendly Synthesized Carbon Dots.

This work reports the analysis of mercury using a spectrofluorometric method combined with a sequential injection analysis (SIA) system. This method is based on the measurement of fluorescence intensity of carbon dots (CDs), which is quenched proportionally after adding mercury ions. Herein, the CDs underwent environmentally friendly synthesis using a microwave-assisted approach that provides intensive and efficient energy and shortens reaction time. After irradiation at 750 W for 5 min in a microwave oven, a dark brown CD solution with a concentration of 2.7 mg mL-1 was obtained. The properties of the CDs were characterized by transmission electron microscopy, X-ray diffractometry, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, and UV-vis spectrometry. We presented for the first time the use of CDs as a specific reagent for the determination of mercury in skincare products with the SIA system to achieve rapid analysis and full automatic control. The as-prepared CD stock solution was diluted 10 times and used as a reagent in the SIA system. Excitation and emission wavelengths at 360 and 452 nm, respectively, were used to construct a calibration curve. Physical parameters affecting the SIA performance were optimized. In addition, the effect of pH and other ions was investigated. Under the optimum conditions, our method showed a linear range from 0.3 to 600 mg L-1 with an R 2 of 0.99. The limit of detection was 0.1 mg L-1. Relative standard deviation was 1.53% (n = 12) with a high sample throughput of 20 samples per hour. Finally, the accuracy of our method was validated by comparison using inductively coupled plasma mass spectrometry. Acceptable recoveries were also presented without a significant matrix effect. This method was also the first time that uses the untreated CDs for the determination of mercury(II) in skincare products. Therefore, this method could be an alternative for mercuric toxic control in other sample applications.

Multicommutation flow analysis system for non-enzymatic lactate determination based on light-driven photometric assay.

A fully-mechanized bioanalytical system for the photometric light-driven lactate determination is presented. The system is based on highly integrated four diode flow-through cell in which two UV-LEDs are used for the photochemical reduction of Fe(III) in the presence of lactate whereas LED-photodiode system allows the photometric detection of Fe(II)-Ferrozine complex to be registered. The developed multicommutation flow analysis system consisting of bifunctional optoelectronic device (photodegradation of Fe(III)-lactate complex and detection), solenoid micropumps and microvalves, is fully controlled and powered by the Arduino-compatible electronics. In consequence, independent and precise control of crucial parameters of the assay, including irradiation time and current supplying UV-LEDs is possible. Under optimized condition the system is useful for the lactate determination in the sub-milimolar range of concentration (6.0-300.0 µmol/L) with satisfactory sensitivity (597.1 AU·L/µmol) and detection limit (3.4 µmol/L), significant reduction of sample and reagents consumption (1.25 µL or 0.5 µL for 40- and 100-fold dilution of human serum samples, respectively) and high sample flow throughput (24 detections per hour). As the system offers the monitoring of detection process, the kinetic discrimination of effects from potential interferents is possible, improving the assay selectivity. The practical utility of the designed system was confirmed by its used for analysis of human serum standards. The recovery values were in the range of 92.1-104.2%. As the limit of quantitation offered by the system is significantly lower than physiological lactate levels, the serum samples have to be diluted what results in further decrease of both sample volume required for analysis and nonselective effects from matrix.

Multicommutated Flow Analysis System for Determination of Horseradish Peroxidase and Its Inhibitors.

A fully mechanized multicommutated flow analysis (MCFA) system dedicated to determining horseradish peroxidase (HRP) activity was developed. Detection was conducted using a flow-through optoelectronic detector-constructed of paired LEDs operating according to the paired emitter-detector diode (PEDD) principle. The PEDD-MCFA system is dedicated to monitoring the enzyme-catalyzed oxidation of p-phenylenediamine (pPD) by a hydrogen peroxide. Under optimized conditions, the presented bioanalytical system was characterized by a linear response range (33.47-200 U/L) with a detection limit at 10.54 U/L HRP activity and 1.66 mV·L/U sensitivity, relatively high throughput (12 signals recordings per hour), and acceptable precision (RSD below 6%). Additionally, the utility of the developed PEDD-MCFA system for the determination of HRP inhibitors allowing the detection of selected thiols at micromolar levels, is demonstrated. The practical utility of the flow system was illustrated by the analysis of some dietary supplements containing L-cysteine, N-acetylcysteine, and L-glutathione.

The comparison between light-scattering detectors based on LED and photodiode for immunoprecipitation assays of transferrin and ferritin.

Undoubtedly, light-emitting diodes (LEDs) and photodiodes (PDs) are indispensable optoelectronic devices in modern analytical chemistry. LEDs can serve as either light emitters or detectors, thus being an alternative to the most popular detection systems consisted of PD. In this contribution, a comparison between LED-LED and LED-PD detectors, operating in turbidimetric and nephelometric modes, has been carried out for immunoprecipitation detection of transferrin and ferritin. The greatest emphasis was placed on the study of detectors responses under different measurement conditions including current powering an emitter, amplification gain in the case of PD as detector or the construction of detection cells designed for the Multicommutated Flow Analysis (MCFA). The assumption was to obtain the fully-mechanized system with simple but efficient detection system to enable the determination of iron-binding proteins occurring at different concentration ranges in human body. As a result, the optimized arrangements of LED-LED and LED-PD setups were characterized by similar analytical characteristics, enabling the determination of transferrin with the detection limit (LOD) of 0.2 mg/L and RSDs of 2.8-4.8% for LED-LED, and LOD of 0.1 mg/L and RSDs of 0.9-3.6% for LED-PD. In the case of ferritin detection, only the response of the LED-PD detector was statistically distinguishable in the range of 130-198 µg/L of protein with recorded analytical signal change of 20 mV value. The addition of polymer for signal enhancement provided the increase of response range to 107-253 µg/L, enabling the developed system for detection of pathological serum ferritin levels.

Electrochemical Properties of Lipid Membranes Self-Assembled from Bicelles.

Supported lipid membranes are widely used platforms which serve as simplified models of cell membranes. Among numerous methods used for preparation of planar lipid films, self-assembly of bicelles appears to be promising strategy. Therefore, in this paper we have examined the mechanism of formation and the electrochemical properties of lipid films deposited onto thioglucose-modified gold electrodes from bicellar mixtures. It was found that adsorption of the bicelles occurs by replacement of interfacial water and it leads to formation of a double bilayer structure on the electrode surface. The resulting lipid assembly contains numerous defects and pinholes which affect the permeability of the membrane for ions and water. Significant improvement in morphology and electrochemical characteristics is achieved upon freeze-thaw treatment of the deposited membrane. The lipid assembly is rearranged to single bilayer configuration with locally occurring patches of the second bilayer, and the number of pinholes is substantially decreased. Electrochemical characterization of the lipid membrane after freeze-thaw treatment demonstrated that its permeability for ions and water is significantly reduced, which was manifested by the relatively high value of the membrane resistance.

Towards mechanized biparametric ceruloplasmin assay.

Two analytical strategies for determination of both biocatalytic activity and concentration of ceruloplasmin conditions have been proposed. For this purpose, two constructions of fully-mechanized Multicommutated Flow Analysis (MCFA) systems were designed. The versatility of solenoid pumps and valves arrangement enabled to construct both manifolds using similar flow units, taking into account the different requirements for each method. In the case of ceruloplasmin catalytic activity assay, the kinetic measurements with the use of p-phenylenediamine and hydrogen peroxide were performed. The optimization process was focused on the selection of substrate and oxidizer concentration, incubation time as well as solving the issue of substrate autoxidation. It led to the development of the flow bioanalytical system characterized by following analytical parameters: LOD - 0.07 U mL- 1, LOQ - 0.38 U mL-1, RSD ≤6% with 8 µL consumption of human serum. In turn, for examination of ceruloplasmin concentration, the light-scattering detector was used in MCFA system adapted for immunoprecipitation measurements. In this case, the use of potentiator (polyethylene glycol) turned out to be necessary to obtain satisfactory analytical signals. Such a method allowed to obtain 35 measurements per hour with LOD and LOQ of 0.9 mg L-1 and 3.2 mg L-1, respectively. The usefulness of both MCFA systems was successfully examined by performing analyses of real human serum samples.

A remote-controlled immunochemical system for nephelometric detection of human serum transferrin.

The fully-mechanized Multicommutated Flow Analysis (MCFA) system for determination of transferrin in human serum has been developed. The analytical methodology concerns immunoprecipitation measurements with the use of LED-based nephelometric flow-through detectors with polymeric light guides. To improve the mechanization degree of the MCFA system, the Do-It-Yourself electronic module for mobile phone control has been developed. The design and structure of an Android software (build using open source application) controlling the operation of presented flow system has been presented. To bypass the problem of nonlinearity of calibration curve caused by the nature of antigen-antibody interactions, the on-line dilution module has been integrated into the presented manifold. Under optimized conditions, the developed flow analysis system is characterized by relatively low limit of detection and quantitation (2.0 and 4.9 mg L-1, respectively), good precision (RSD < 4%), low reagent and sample consumption per one measurement (16 µL of undiluted reagent and 20 µL of undiluted sample without further on-line dilution, respectively) and relatively high throughput (approximately 35 signal recordings per hour). The developed MCFA system enabled to analyze 16 serum samples with the transferrin concentration from 90 to almost 350 mg dL-1 with statistical agreement to the reference method.

Microfluidic Analysis with Front-Face Fluorometric Detection for the Determination of Total Inorganic Iodine in Drinking Water.

A microfluidic method with front-face fluorometric detection was developed for the determination of total inorganic iodine in drinking water. A polydimethylsiloxane (PDMS) microfluidic device was employed in conjunction with the Sandell-Kolthoff reaction, in which iodide catalyzed the redox reaction between Ce(IV) and As(III). Direct alignment of an optical fiber attached to a spectrofluorometer was used as a convenient detector for remote front-face fluorometric detection. Trace inorganic iodine (IO3- and I-) present naturally in drinking water was measured by on-line conversion of iodate to iodide for determination of total inorganic iodine. On-line conversion efficiency of iodate to iodide using the microfluidic device was investigated. Excellent conversion efficiency of 93 - 103% (%RSD = 1.6 - 11%) was obtained. Inorganic iodine concentrations in drinking water samples were measured, and the results obtained were in good agreement with those obtained by an ICP-MS method. Spiked sample recoveries were in the range of 86%(±5) - 128%(±8) (n = 12). Interference of various anions and cations were investigated with tolerance limit concentrations ranging from 10-6 to 2.5 M depending on the type of ions. The developed method is simple and convenient, and it is a green method for iodine analysis, as it greatly reduces the amount of toxic reagent consumed with reagent volumes in the microfluidic scale.

Photometric flow analysis system for biomedical investigations of iron/transferrin speciation in human serum.

The Multicommutated Flow Analysis (MCFA) system for the estimation of clinical iron parameters: Serum Iron (SI), Unsaturated Iron Binding Capacity (UIBC) and Total Iron Binding Capacity (TIBC) has been proposed. The developed MCFA system based on simple photometric detection of iron with chromogenic agent (ferrozine) enables a speciation of transferrin (determination of free and Fe-bound protein) in human serum. The construction of manifold was adapted to the requirements of measurements under changing conditions. In the course of studies, a different effect of proteins on SI and UIBC determination has been proven. That was in turn the reason to perform two kinds of calibration methods. For measurements in acidic medium for SI/holotransferrin determination, the calibration curve method was applied, characterized by limit of determination and limit of quantitation on the level of 3.4 µmol L-1 and 9.1 µmol L-1, respectively. The determination method for UIBC parameter (related to apotransferrin level) in physiological medium of pH 7.4 forced the use of standard addition method due to the strong influence of proteins on obtaining analytical signals. These two different methodologies, performed in the presented system, enabled the estimation of all three clinical iron/transferrin parameters in human serum samples. TIBC corresponding to total transferrin level was calculated as a sum of SI and UIBC.

Optoelectronic iron detectors for pharmaceutical flow analysis.

Compact flow-through optoelectronic detectors fabricated by pairing of light emitting diodes have been applied for development of economic flow analysis systems dedicated for iron ions determination. Three analytical methods with different chromogens selectively recognizing iron ions have been compared. Ferrozine and ferene S based methods offer higher sensitivity and slightly lower detection limits than method with 1,10-phenantroline, but narrower ranges of linear response. Each system allows detection of iron in micromolar range of concentration with comparable sample throughput (20 injections per hour). The developed flow analysis systems have been successfully applied for determination of iron in diet supplements. The utility of developed analytical systems for iron release studies from drug formulations has also been demonstrated.

Bianalyte multicommutated flow analysis system for microproteinuria diagnostics.

In this work a bianalyte multicommutated flow analysis (MCFA) system for determination of microproteinuria is presented. The developed MCFA system is based on two dedicated optoelectronic flow-through detectors which allow estimation of urinary protein creatinine ratio. For total protein determination, turbidimetric Exton's method was used, whereas creatinine was determined by the photometric Jaffe reaction. The developed analytical system is fully-mechanized, easy to operate, economic in reagent consumption and characterized by satisfactory analytical parameters. It allows protein determination in the range 36-300 mg L(-1) with 33 mg L(-1) detection limit and simultaneous determination of creatinine in the range 0.045-2.50 mmol L(-1) with 0.025 mmol L(-1) detection limit. The measurement procedure for the presented MCFA system offers performing 30 peaks per hour for both analytes. To prove the analytical usefulness of the system, real human urine samples have been analyzed. The correlation and agreement between results offered by the developed system and clinical analyzers are fully acceptable.

An immunoprecipitation assay in the multicommutated flow analysis format.

In this contribution, a multicommutated flow analysis (MCFA) system for immunoprecipitation assays is presented. The developed bioanalytical system constructed from microsolenoid devices coupled with an optoelectronic flow-through nephelometric detector is sufficient to monitor the kinetics of antigen-antibody interactions, the effect of which is turbidity. Because of the uncommon shape of the immunoprecipitation response curve, the developed MCFA system contains a special module for on-line dilution of the analyzed samples and for single-standard calibration. As a model analyte and an immunoreceptor for the presented investigations, human albumin and antibody for this albumin were used, respectively. The proposed MCFA system is characterized by robust construction, low reagent consumption (maximum 42 µL of undiluted reagent for the analysis of one sample) and relatively high throughput under optimal conditions (approximately 18 measurements per hour). The analytical usefulness of the developed MCFA system for clinical analysis has been experimentally confirmed. Samples of human urine were analyzed for the needs of microalbuminuria diagnostics and satisfactory analytical results have been obtained.

Multicommutated flow analysis system for determination of total protein in cerebrospinal fluid.

A fully mechanized, computer-controlled, multicommutated flow analysis (MCFA) system dedicated for total protein determination in cerebrospinal fluid samples has been developed. For the protein determination the Exton method has been applied. Dedicated turbidimetric and nephelometric flow-through detectors operating according to paired-emitter detector diode principle have been fabricated by integration of two or three respective light emitting diodes. The developed MCFA system is characterized by robust, compact design and low consumption of the sample (72 µ L). The limits of detection for turbidimetric and nephelometric detection mode are 65 mg L(-1) and 9 mg L(-1), respectively. For turbidimetric measurements the range of linear response offered by the MCFA system is 72-900 mg L(-1), whereas in the case of nephelometric detection 18-500 mg L(-1) linear range is obtained. The throughput of the MCFA system is over 30 injection per hour. The analytical system was optimized with bovine serum albumin standards and successfully validated with real samples of human cerebrospinal fluid.

Biparametric multicommutated flow analysis system for determination of human serum phosphoesterase activity.

A multicommutated flow analysis (MCFA) system constructed of microsolenoid valves and pumps offering simultaneous determination of activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) in human serum samples has been developed. The MCFA system is based on optoelectronic flow-through detector made of two light emitting diodes and operating according to paired emitter detector diode (PEDD) principle. This photometric PEDD device has been dedicated for detection of p-nitrophenol (NP) generated in the course of enzymatic hydrolysis of p-nitrophenyl phosphate and optimized for the determination of NP in human serum samples. The developed PEDD-based MCFA system allows independent optimization of conditions for reaction and detection steps of photometric ACP and ALP bioassays. Moreover, it allows elimination of photometric interferences from serum matrix components according to two-points kinetic mode of measurement. The single measurement cycle takes 12 min, consists of four measurements (two for each phosphoesterase) and enables determination of serum ACP and ALP activities at physiological and pathological levels. The real analytical utility of the developed MCFA system has been confirmed by analysis of control sera as well as real human serum samples from healthy persons and oncological patients.

Multicommutated flow analysis system for determination of creatinine in physiological fluids by Jaffe method.

For the needs of photometric determination of creatinine according to Jaffe protocol a dedicated paired emitter detector diode (PEDD) detector has been developed. This PEDD device has been constructed in the compact form of flow-through cell (30 µL total volume and 7 mm optical pathlength) integrated with 505 nm LED-based emitter and 525 nm LED-based detector compatible with multicommutated flow analysis (MCFA) system. This fully mechanized MCFA system configured of microsolenoid valves and pumps is operating under microprocessor control. The developed analytical system offers determination of creatinine in the submillimolar range of concentrations with detection limit at ppm level. The throughput offered by the system operating according to multi-point fixed-time procedure for kinetic measurements is 15-40 samples per hour depending on the mode of measurements. The developed PEDD-based MCFA system has been successfully applied for the determination of creatinine in real samples of human urine as well as serum. The developed sampling unit used the system is free from effects caused by differences in sample viscosity.

Nephelometry and turbidimetry with paired emitter detector diodes and their application for determination of total urinary protein.

Two miniature and compact optoelectronic devices fabricated by means of integration of light emitting diodes have been developed for turbidimetric and nephelometric measurements. These devices are operating according to paired-emitter-detector-diode (PEDD) principle. The detectors have been characterized using bovine serum albumin and Exton protein assay as a model analyte and a model analytical method, respectively. The developed detectors have been adapted for measurements under conditions of flow injection analysis (FIA). Under optimized conditions the turbidimetric flow system offers the range of linear response up to 400 mg L(-1) with the detection limit at 20 mg L(-1). The linear range and detection limit found for optimized nephelometric FIA system are 15-500 mg L(-1) and 8 mg L(-1), respectively. The PEDD-based FIA systems with the detector operating according to both modes of measurements have been successfully applied for urinalysis offering total protein determination at physiological and pathological levels with high throughput (over 60 injections per hour).

Serum alkaline phosphatase assay with paired emitter detector diode.

A simple multicommutated flow system based on optoelectronic detector, three valves and peristaltic pump only has been developed for photometric determination of alkaline phosphatase activity in human serum. A miniaturized, compact flow-through detector dedicated to selective photometric detection of product formed in the course of the enzyme assay has been constructed using two paired light emitting diodes. The proposed analytical procedure based on kinetic methodology of enzyme activity detection and stopped-flow methodology of two-point measurements eliminates interferences caused by intense color of real samples and impurities present in commercial reagents. After optimization the system allows reproducible, mechanized analysis of human serum in relatively short time (8-9 samples per hour). Volume of serum required for single determination is 0.05mL only. The system validated with real clinical samples is useful for determination of enzyme activity in human serum at physiological and pathological levels.

Fluorimetric detector and sensor for flow analysis made of light emitting diodes.

Two compact optoelectronic fluorimetric devices operating according to the paired-emitter-detector-diode concept have been developed. The fluorimetric detector, fabricated of three light emitting diodes only, has been applied for the development of fluorimetric optosensor by further integration with sensing solid phase. In these investigations as a model analyte and as a model sensing layer useful for solid phase spectrometry, riboflavin and C18-silica have been chosen, respectively. Both developed analytical devices have been applied for non-stationary fluorimetric measurements performed under conditions of flow injection analysis. The presented flow-through detector and sensor operating under given flow conditions offer riboflavin determination in mg L(-1) and µg L(-1) ranges of concentration, respectively.