Multicommutated Flow Analysis Research Articles

Turbidimetric flow analysis system for the investigation of microbial growth

The monitoring of life of microbial populations is of the uttermost importance in environmental and food analysis, agriculture, as well as in medicine. The duration of bacteria adaptation to new environmental conditions, its lifetime and the divisions’ pace are the key information in many studies. It was found that the fully-mechanized flow analysis system based on solenoid valves and pumps, paired with a dedicated flow-through optoelectronic detector can be successfully applied for monitoring of bacteria growth. The applicability of the designed multicommutated flow analysis (MCFA) system was proved by analysis of solutions containing bacteria cells proceeded by tests of McFarland (McF) standards. The developed setup allowed modelling and simulation of microbial growth, as well as monitoring of the bacteria growth in real-time manner to be carried out. The monitor is useful for the quantitative estimation of the basic parameters of bacteria population like its size, the rate of bacteria multiplication, as well as the times of lag, log and stationary phases of microbial growth.

On-line ‘protein shaker’: A multicommutated flow analysis system for fluorometric creatinine determination in deproteinized serum

A fully mechanized multicommutated flow analysis (MCFA) system for fluorometric determination of creatinine in serum samples is introduced in this paper. The flow system was constructed with microsolenoid pumps and valves and with a 3D-printed flow cell. Fluorometric assay relied on creatinine reaction with 3,5-dinitrobenzoic acid and hydrogen peroxide in an alkaline environment. To overcome significant interference from protein, a flow reactor for serum deproteinization was designed and implemented in the flow system. The deproteinization was carried out by precipitation with trichloroacetic acid and the addition of sodium chloride facilitated the precipitate sedimentation. The supernatant representative sample was pumped out and subjected to fluorometric creatinine assay. The obtained linear range was from 1.6 to 500 μmol L−1 and the precision, expressed as RSD, was below 3%. The proposed MCFA system was used to determine creatinine concentration in control serum samples. The results obtained with flow deproteinization correlated well with results obtained with conventional deproteinization (y = (0.91 ± 0.09) x + (37 ± 28)) with Pearson's r 0.979.

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.

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.

Multicommutated Flow System for the Determination of Glyphosate Based on Its Quenching Effect on CdTe-Quantum Dots Fluorescence

Glyphosate is the most widely used herbicide at the moment. It presents a broad spectrum of action, hence its use for many different crops. Regulatory agencies have constantly mentioned the low hazard potential to mammals. However, the International Agency for Research on Cancer concluded in 2015 that glyphosate is “probably carcinogenic to humans.” For this reason, it is important to develop reliable analytical methods to study the fate and levels of glyphosate in environmental samples. In this work, we propose a multicommutated flow analysis method for the determination of glyphosate, based on the quenching effect produced by this herbicide on the fluorescence of CdTe quantum dots (λexc/λem 400/548 nm/nm). The proposed analytical method presents detection and quantitation limits of 0.5 and 1.7 μg mL−1, respectively. A sample throughput of 30 samples per hour was achieved using a 2.5 mL min−1 flow rate. Interference studies were carried out to assess the selectivity of the method, observing no interference from other common pesticides. Finally, we carried out recovery experiments in water and cereal samples (amaranth, barley, oat, and quinoa). We obtained recovery yields between 92 and 108% in all the analyzed samples. HPLC-MS/MS was used as reference method to assess the accuracy of the proposed methodology.

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.

A comparison of photometric methods for serum iron determination under flow analysis conditions

In this contribution, a multicommutated flow analysis (MCFA) system for serum iron determination is presented. The construction of the system has enabled sequential measurements with three different determination methods with 1,10-phenantroline, Ferrozine and Ferene S using separate analytical units (modules). For each method a dedicated optoelectronic detector has been developed. Under optimal flow conditions, investigated methods were analytically characterized by linear range and sensitivity: 1.5 − 100.0μmolL−1 and 4.9mVL μmol−1, 3.3 − 50.0μmolL−1 and 6.6mVL μmol−1, 4.0 − 40.0μmolL−1 and 13.3mVL μmol−1 for 1,10-phenantroline, ferrozine and ferene S methods, respectively. In the course of investigations also clinical usefulness of each method has been discussed. The analysis of serum samples proved that Ferrozine and Ferene S methods are sufficient for serum iron determination with relatively low consumption of samples (less than 50μL of serum).

A multicommutated tester of bioreactors for flow analysis

Enzymes are often used in the modern analytical procedures allowing selective recognition and conversion of target analytes into easily detected products. In flow analysis systems, enzymes are predominantly applied in the immobilized forms as flow-through bioreactors. In this research the multicommutated flow analysis (MCFA) system for evaluation and comparison of analytical parameters of bioreactors has been developed. The MCFA manifold allows simultaneous testing up to four bioreactors, but if necessary their number can be easily increased. The system allows comparison of several parameters of tested bioreactors including activity, repeatability, reproducibility, operational and storage stability. The performance of developed bioreactor tester is presented using urea-urease model system based on plastic open-tubular bioreactor with covalently immobilized enzyme. Product of enzymatic reaction is detected using two different chemical methods and by dedicated optoelectronic ammonium detectors. Moreover, the utility of developed MCFA manifold for evaluation of other enzyme bioreactors is demonstrated.

Multicommutated flow analysis system based on fluorescence microdetectors for simultaneous determination of phosphate and calcium ions in human serum

A bianalyte multicommutated flow analysis (MCFA) system allowing simultaneous determination of calcium and phosphate ions is developed. The detection of ions is based on measurements of fluorescence of calcein and rhodamine B, respectively. For such measurements performed under flow analysis conditions two dedicated detectors made of three integrated light emitting diodes each and operating according to fluorometric paired emitter detector diode (FPEDD) principle, have been used. The developed 2FPEDD-MCFA system has been applied for serum analysis. The system provides low detection limits (16µM and 3.6µM for calcium and phosphate ions, respectively) and therefore small consumption of sample (8µL) for analysis. The throughput of the system allows about 20 bianalyte determinations per hour. The presented system was validated using human serum samples. The analysis results show good correlation with those from the clinical laboratory obtained using reference photometric methods. The results of real samples analysis show that it is necessary to determine both calcium and phosphate ions simultaneously because their levels is serum are rather weakly correlated.

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 36–300mgL−1 with 33mgL−1 detection limit and simultaneous determination of creatinine in the range 0.045–2.50mmolL−1 with 0.025mmolL−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 (72μL). The limits of detection for turbidimetric and nephelometric detection mode are 65mgL−1 and 9mgL−1, respectively. For turbidimetric measurements the range of linear response offered by the MCFA system is 72–900mgL−1, whereas in the case of nephelometric detection 18–500mgL−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 12min, 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 (30μL total volume and 7mm optical pathlength) integrated with 505nm LED-based emitter and 525nm 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.

Determination of Total Selenium in Infant Formulas: Comparison of the Performance of FIA and MCFA Flow Systems.

Two flow methods, based, respectively, on flow-injection analysis (FIA) and on multicommutated flow analysis (MCFA), were compared with regard to their use for the determination of total selenium in infant formulas by hydride-generation atomic absorption spectrometry. The method based on multicommutation provided lower detection and quantification limits (0.08 and 0.27 μg L−1 compared to 0.59 and 1.95 μ L−1, resp.), higher sampling frequency (160 versus. 70 samples per hour), and reduced reagent consumption. Linearity, precision, and accuracy were similar for the two methods compared. It was concluded that, while both methods proved to be appropriate for the purpose, the MCFA-based method exhibited a better performance.

Automatic Multipumping Flow System for Handling Viscous Solutions: Application to the Spectrophotometric Determination of Trimipramine

The present work describes the implementation, by using MultiPumping Flow Analysis System (MPFS), of a spectrophotometric method for the determination of trimipramine in commercially available pharmaceutical formulations, based on the reaction with ammonium monovanadate in acidic medium yielding a colored compound with a maximum of absorbance at 620 nm. The improved flow mixing conditions during sample and reagents insertion and transport, as a result of the chaotic movement of the solutions originated by the MPFS pulsed flow, assured a fast reaction zone homogenization in a reduced residence time, which was particularly advantageous for carrying out analytical determinations that involved highly viscous solutions, as is the case of the sulfuric acid solution used in the determination of trimipramine, without impairing the sampling rate. A linear working range for trimipramine concentrations of up to 50 mg L−1 (r = 0.9998; n = 6) was obtained, and the determined detection limit was about 1.15 mg L−1. The sampling rate was approximately 50 determinations per hour. The obtained results were in agreement with those furnished by the reference procedure, with relative deviations lower than 4.7%. With the developed MPFS, the consumption of the reagents ammonium monovanadate and sulfuric acid was reduced by approximately 41.02% and 54.29%, respectively, compared with a previously proposed multicommutated flow analysis system.

Multicommutated flow system for the determination of glucose in honey with immobilized glucose oxidase reactor and spectrophotometric detection

A new automated method for the determination of glucose in honey is proposed. The method is based on multicommutated flow analysis (MCFA) and employs an immobilized glucose oxidase reactor and spectrophotometric detection at 505 nm of the red quinoneimine formed (Trinder's method). The calibration curve obeyed a second order equation in the range 0–0.14 g L −1 ( h = −2.2199 C 2 + 1.3741 C + 0.0077, r 2 = 0.9991, where h is the peak height (absorbance) and C the concentration in g L −1). The method was validated analyzing eight commercial samples, both by the AOAC 954.11 and 977.20 official methods. According to Student's t-test of mean values, at the confidence level of 95% the results obtained with the proposed method were in agreement with those obtained by the official methods. Precision ( s r(%), n = 10) was 3% and the sampling frequency of the system was 20 samples h −1.

Desenvolvimento de um micro-aquecedor para sistemas de análise química em fluxo: determinação espectrofotométrica de manganês em plantas

In this work a micro-heater device to be used as an integral part of the flow analysis manifold is described. The usefulness of the device was demonstrated using it in the development of a multicommutated flow analysis procedure for the spectrophotometric determination of manganese in plant digest. The method was based on the manganese oxidation by periodate in phosphoric acid medium to form the permanganate anion. The reaction development is dependent on the temperature and it was observed that at 25 °C a time interval of ca. 15 min was necessary for the reaction to attain equilibrium. Setting the temperature to 70 oC, this time interval could be decreased to ca. 30 s. This condition was easily attained employing the proposed micro-heater device coupled to the manifold. The procedure was applied to manganese determination in soybean digests and results compared with those obtained by inductively coupled argon plasma optical emission spectrometry (ICP-OES). No significant difference at 90% confidence level was observed. A linear response for sample concentrations ranging from 5.0 to 30.00 mg L-1 Mn2+; a relative standard deviation of 1.3% (n = 6) for a typical sample containing 6.3 mg L-1 Mn2+; a sampling rate of 22 determinations per hour; a low reagent consumption, of 12.0 mg NaIO4 per determination; and a detection limit of 1.2 mg L-1 were achieved.