Microfluidic paper-based analytical device for simultaneous determination of calcium and magnesium ions in human serum.

BACKGROUND: Calcium and magnesium ions are highly abundant and important cations in human body. At the same time, both dyscalcemia and dysmagnesemia are frequently encountered in the clinical practice. As deficiency or excess of Ca(II) or Mg(II) can cause severe symptoms, determining these ions in serum is of great importance. Concentration of these ions in biological samples is typically assayed in clinical laboratories with the use of expensive and specialized equipment. Since those methods cannot be easily adapted for self-diagnosis purposes, there is a great need to develop a convenient tool for reliable determination of calcium and magnesium in serum at the point-of-care. RESULTS: The colorimetric methods employed for calcium and magnesium analysis were o-cresophtalein complexone assay and xylidyl blue assay, respectively. Analytical signal acquisition was accomplished using an ordinary flatbed scanner or smartphone and free software. For increased user-friendliness the device was optimized to perform simultaneous determination of calcium and magnesium ions in only 10 min. In the optimized conditions, the limit of detection for calcium ions was 0.09 mmol L-1, while for magnesium it was 0.04 mmol L-1. Determination of both ions requires only 4 µL of serum sample. The developed paper-based sensors were validated with control human serum samples and the obtained relative errors for majority of samples were below 20 %. SIGNIFICANCE: In this paper, a microfluidic paper-based analytical device for simultaneous determination of calcium and magnesium ions in human serum is reported for the first time. Additionally, this is also the first report on colorimetric determination in serum of any of these ions in paper-based format. Simultaneous detection of both ions allows for fast and user-friendly screening of disturbance in calcium and magnesium homeostasis.

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.

Smartphone-Assisted Protein to Creatinine Ratio Determination on a Single Paper-Based Analytical Device.

Proteinuria is a condition in which an excessive amount of protein is excreted in urine. It is, among others, an indicator of kidney disease or risk of cardiovascular disease. Rapid and reliable diagnosis and monitoring of proteinuria is of great importance for both patients and their physicians. For that reason, a paper-based sensor for proteinuria diagnosis was designed, optimized, and validated utilizing smartphone-assisted signal acquisition. In the first step, a few commonly employed protein assays were optimized and compared in terms of analytical performance on paper matrix. The tetrabromophenol blue method was selected as the one providing a sufficiently low limit of detection (39 mg·L-1) on the one hand and appropriate long-term stability (up to 3 months) on the other hand. The optimized assay was employed for protein-to-creatinine ratio (PCR) determination on a single paper-based sensor. For both analytes the linear ranges were within the clinically relevant range. The analytical usefulness of the developed sensors was demonstrated by a PCR recovery study in artificial urine. The obtained PCR recoveries were from ca. 80 to 150%.

Prussian Blue (bio)sensing device for distance-based measurements.

In this research, microfluidic paper distance-based systems for the quantification of redox species are proposed. For the preparation of the sensing zone a Prussian Blue (PB) (convertible to Prussian White (PW)) layer was deposited in the channel manufactured by wax-printing technique. According to the chemical properties of PB/PW system, it is possible to develop optical sensors sensitive to both oxidizing and reducing agents. The created systems were evaluated for the determination of ascorbic acid and hydrogen peroxide, which were chosen and used as model analytes. The final versions of the proposed systems exhibited a linear response from 0.25 mmol L-1 to 4.0 and 2.0 mmol L-1 for ascorbic acid and H2O2, respectively. The analytical utility of the paper systems was confirmed by measuring the levels of ascorbic acid in dietary supplements. Results correlation obtained for the described systems and the reference method was over 0.98 (Pearson's R-coefficient). All measurements were characterized by satisfactory reproducibility and acceptable uncertainty (RSD (%) < 6%). Finally, it was demonstrated that the modification of the PW-strip systems with oxidoreductase led to an enzymatic assay for glucose up to 10 mmol L-1 range. Practical utility of the developed bio-strips was confirmed by quantifying glucose in drinks and dietary supplement samples.

From the bottom of an old jar: A fluorometric method for the determination of creatinine in human serum.

In this paper we have investigated and optimized a non-enzymatic fluorometric creatinine assay. The method was originally described by Blass in the 90s, but we found that besides the reagents mentioned in the paper, an addition of hydrogen peroxide is required to obtain a fluorescent compound. The excitation and emission maxima of the fluorophore are 405 nm and 475 nm, respectively. The optimal conditions for creatinine quantification are as follows: 25 mmol L-1 3,5-dinitrobenzoic acid dissolved in 1,4-butanediol with 58 mmol L-1 H2O2 and aqueous solution of 2 mol L-1 NaOH mixed in 1:1 ratio. A linear calibration curve (y = 18,7x + 446 for 300 s of incubation) was obtained in the range from 2.6 to 750 µmol L-1 of creatinine with LOD and LOQ equal to 0.7 and 2.6 µmol L-1, respectively. The method was found to be selective towards the analyte in the presence of compounds such as urea, uric acid, bilirubin, albumin and glucose. The developed protocol was applied for creatinine determination in 13 real human serum samples. A correlation (y = (0.94 ± 0.03) x + (3.66 ± 3.22) with Pearson's r 0.996) and statistical agreement (two-tailed Student's t-test at 95% confidence interval with 12 degrees of freedom) was reached between the obtained results and the reference enzymatic method.

Multi-Substrate Biofuel Cell Utilizing Glucose, Fructose and Sucrose as the Anode Fuels.

A significant problem still exists with the low power output and durability of the bioelectrochemical fuel cells. We constructed a fuel cell with an enzymatic cascade at the anode for efficient energy conversion. The construction involved fabrication of the flow-through cell by three-dimensional printing. Gold nanoparticles with covalently bound naphthoquinone moieties deposited on cellulose/polypyrrole (CPPy) paper allowed us to significantly improve the catalysis rate, both at the anode and cathode of the fuel cell. The enzymatic cascade on the anode consisted of invertase, mutarotase, Flavine Adenine Dinucleotide (FAD)-dependent glucose dehydrogenase and fructose dehydrogenase. The multi-substrate anode utilized glucose, fructose, sucrose, or a combination of them, as the anode fuel and molecular oxygen were the oxidant at the laccase-based cathode. Laccase was adsorbed on the same type of naphthoquinone modified gold nanoparticles. Interestingly, the naphthoquinone modified gold nanoparticles acted as the enzyme orienting units and not as mediators since the catalyzed oxygen reduction occurred at the potential where direct electron transfer takes place. Thanks to the good catalytic and capacitive properties of the modified electrodes, the power density of the sucrose/oxygen enzymatic fuel cells (EFC) reached 0.81 mW cm-2, which is beneficial for a cell composed of a single cathode and anode.

Optoelectronic detectors for flow analysis systems manufactured by means of rapid prototyping technology.

Universal, customizable design of 3D printed photometric, and fluorometric flow-through detectors have been presented. The developed designs were fabricated with the use of the most affordable 3D printing technique, namely Fused Filament Fabrication, and require neither hardware nor tools to assemble. Numerous variants of detector geometries have also been presented. The designed parameters varied both in aperture (i.e., the internal diameter of the flow channel in an optical path) and in thickness of an absorbing layer. As expected, the geometry of the channels resulted in changes in the internal volumes. Two concepts of fluorometric detectors have also been described. The utility of all developed flow-through detectors was proven with the use of mechanized calibrations of both photometric and fluorometric experiments. Analytical parameters were characterized with the use of two model dyes: bromothymol blue and fluorescein for photometric and fluorometric experiments, respectively. The repeatability of the 3D printed vessels was found at 3.5-8.0% of the mean relative standard deviation (RSD), depending on the construction of the vessel, which is comparable to rather expensive commercially available flow cells. The compatibility of used 3D printing materials was also examined. For both variants of detection light emitting diodes were applied as light emitters. As the light detectors, both CCD spectrophotometers and light-emitting diodes were used.

Analytical aspects of smart (phone) fluorometric measurements.

Facing the problem of a growing number of analyses, the need for using simple equipment appears. Smartphone-based optical detection is one of the most widely applied ideas nowadays. A personal device such as a smartphone equipped with a camera is affordable even in the source-limited places. After a simple modification, providing the light source of both defined properties and orientation, a smartphone may become an efficient analytical device. In this work we present a uniform methodology of such a modification, offering a complete hand-held device for fluorometric measurements. Inducing the fluorescence of the tested analytes was done by ordinary light-emitting diodes, and phone camera was used as a detector. Then the obtained images were analyzed using the RGB colour model to get proper calibration curves. The demonstration of the system performing with the use of fluorescein preceded the examples of determination of quinine, rhodamine B, riboflavin and calcein in real-life circumstances. Example determinations of the calcium ions in mineral water and riboflavin in alcoholic beverages are provided. The results obtained with the designed device are fully comparable to the ones obtained with the conventional fluorometric equipment. The presented systems allow determination of all the investigated analytes with satisfactory detection limits, in some cases down to ppb levels. Thanks to the use of LEDs, the system could be adapted for both measuring and inducing fluorescence in different analytes, characterized by various excitation wavelengths.

An alternative, single-point method for creatinine determination in urine samples with optoelectronic detector. Critical comparison to Jaffé method.

A rapid, single-point method for colorimetric creatinine determination has been developed as an alternative for a well-established Jaffé assay, which lacks analytical specificity. 3,5-dinitrobenzoic acid was employed in an alkaline environment as a chromophore instead of picric acid. Apart from the spectrophotometer, a simple optoelectronic detector was applied as an alternative detection method. The conditions of the reaction were optimized to provide satisfactory analytical parameters. The linear ranges of the proposed assays were 40-4000 and 10-300 µmol L-1 of creatinine for conventional spectrophotometry and a PEDD detector, respectively. A broad spectrum of potential interferents was examined. The presence of compounds such as glucose, urea or uric acid was the source of a significantly smaller bias in the examined method than in the case of the Jaffé method variants. The presented method was validated with the use of 10 human urine samples with the creatinine level determined by the recommended method. Two-tail paired Student's t-tests with 9 degrees of freedom at the 95% confidence level showed the agreement between the proposed and the reference methods.

3D printed flow-through cuvette insert for UV-Vis spectrophotometric and fluorescence measurements.

Rapid Prototyping technologies expand the availability of fabrication of plastic objects to non-skilled users that need sophisticated equipment for their research. In this communication, for the very first time, the universal design of photometric-fluorometric, UV-Vis compatible, 3D-printed flow-through cuvette with two optical paths (2 and 10 mm) is introduced. The cuvette insert was made with the use of the most economically viable Fused Material Deposition technology which enables truly one-step manufacturing and easy replicating of the device. A utility of the cuvette was presented in the example of the basic flow injection analysis experiments on the model photometric (bromothymol blue) and fluorometric (fluorescein) dyes and proven by investigation of solubility constant of calcium hydrophosphate dihydrate by determination of phosphate using fluorescence quenching of molybdenum blue-Rhodamine B ion pair formation and calcium reaction with calcein in basic environment.

Reticulated vitreous carbon as a scaffold for enzymatic fuel cell designing.

Three - dimensional (3D) electrodes are successfully used to overcome the limitations of the low space - time yield and low normalized space velocity obtained in electrochemical processes with two - dimensional electrodes. In this study, we developed a three - dimensional reticulated vitreous carbon - gold (RVC-Au) sponge as a scaffold for enzymatic fuel cells (EFC). The structure of gold and the real electrode surface area can be controlled by the parameters of metal electrodeposition. In particular, a 3D RVC-Au sponge provides a large accessible surface area for immobilization of enzyme and electron mediators, moreover, effective mass diffusion can also take place through the uniform macro - porous scaffold. To efficiently bind the enzyme to the electrode and enhance electron transfer parameters the gold surface was modified with ultrasmall gold nanoparticles stabilized with glutathione. These quantum sized nanoparticles exhibit specific electronic properties and also expand the working surface of the electrode. Significantly, at the steady state of power generation, the EFC device with RVC-Au electrodes provided high volumetric power density of 1.18±0.14mWcm-3 (41.3±3.8µWcm-2) calculated based on the volume of electrode material with OCV 0.741±0.021V. These new 3D RVC-Au electrodes showed great promise for improving the power generation of EFC devices.

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.

Biomedical analytical monitor of artificial kidney operation: Monitoring of creatinine removal.

A general concept for the development of flow analysis system for non-invasive, bloodless monitoring of uremic toxins' removal in the course of clinical hemodialysis treatment is presented. The monitor operates in both (discrete and continuous) modes of measurements. In this study as a model uremic marker creatinine has been chosen. The monitor is based on solenoid operated microdevices (pumps and valves) and an optoelectronic flow-through detector made of paired light emitting diodes allowing photometric determination of this metabolite using Jaffé method. Additionally, a simple two microsolenoid pump-based module allowing the modeling of toxin removal by artificial kidney has been developed. The developed monitor has been validated with real samples of postdialysate fluid produced by artificial kidney in the course of clinical hemodialysis treatment. The results of hemodialysis monitoring are fully comparable with those obtained using reference off-line method.

Biomedical monitoring of phosphate removal by hemodialysis.

A compact flow analysis system for non-invasive, dialysate-side monitoring of phosphate removal in the course of clinical hemodialysis treatment is presented. The monitor is based on solenoid operated micro-pumps and extremely cheap optoelectronic flow-through detector allowing photometric determination of phosphate in spent dialysate using a molybdenum blue method. The monitor can operate in both, discrete and continuous modes of measurement. The analytical utility of monitor has been tested with samples of spent dialysate produced by artificial kidney in the course of real hemodialysis sessions. The results of monitoring are comparable with those obtained using reference off-line method recommended for clinical analysis. Additionally, the possibility of two-side (dialysate and blood) monitoring of hemodialysis treatments with optoelectronic flow-through detectors has been announced.

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.

Towards optoelectronic urea biosensors.

Integration of immobilized enzymes with light-emitting diodes (LEDs) leads to the development of optoelectronic enzyme-based biosensors. In this work, urease, used as a model enzyme, immobilized in the form of an open-tubular microbioreactor or biosensing membrane that has been integrated with two red LEDs. It forms complete, fiberless, miniaturized, and extremely economic biooptoelectronic devices useful for nonstationary measurements under flow analysis conditions. Both enzyme-based biodevices, operating according to the paired emitter detector diode (PEDD) principle, allow relatively fast, highly sensitive, and well-reproducible urea detection in the millimolar range of concentrations. Potential analytical applications of the developed urea bioPEDDs have been announced. Both presented constructions will be easily adapted for the development of other optoelectronic biosensors exploring various enzyme-based schemes of biodetection.

Hybrid flow system integrating a miniaturized optoelectronic detector for on-line dynamic fractionation and fluorometric determination of bioaccessible orthophosphate in soils.

An integrated Sequential Injection (SI)/Flow Injection (FI) system furnished with a miniaturized LED-based fluorometric detector is presented in this work for expedient bioaccessibility tests of orthophosphate in soils. Equipped with a microcolumn of conical shape containing 50 mg of soil, the hybrid flow system was resorted to on-line dynamic leaching and real-time quantification of pools of mobilizable orthophosphate using a bi-directional syringe pump and multiposition valve. The flexibility of the flow manifold was harnessed to explore both bi-directional and uni-directional flow extraction modes with the added degree of freedom of on-line dilution of extracts whenever needed. Bioaccessible orthophosphate was split in three fractions, the so-called NH4Cl fraction containing labile exchangeable phosphates, the alkaline fraction with Fe and Al-bound phosphates and the acidic fraction containing Ca-bound phosphates. The prevailing molybdenum blue photometric detection method is replaced by spectrofluorometric detection based on the ion pair formation between the phosphomolybdate heteropolyacid and rhodamine B with the subsequent quenching of the dye fluorescence. The dedicated optoelectronic detector was integrated in a secondary FI manifold and operated according to the fluorometric paired emitter-detector diode (FPEDD) principle involving two light emitting diodes as fluorescence inductors and one as detector of LED-induced fluorescence. Demonstrated with the analysis of a standard reference material (SRM 2711) and a real agricultural soil, the developed FI/SI fractionation system with FPEDD detection is proven reliable against the standard molybdenum blue method (p>0.05), and useful for investigation of the leaching kinetics of orthophosphate in bioaccessibility tests through in-line recording of the extraction profiles.

A bimodal optoelectronic flow-through detector for phosphate determination.

A miniature flow-through detector useful for bimodal, photometric and fluorimetric, determination of phosphates has been developed. This optoelectronic device made of four light emitting diodes (LEDs) integrated in the form of 85 µL optical cell is easily applied in flow analysis manifolds. These LEDs play the roles of light source for photometric measurements, fluorescence inductors and detector of absorbance and fluorescence. For photometric mode of determinations a phosphomolybdenum blue method has been applied. The fluorimetric method of phosphate determination is based on quenching of rhodamine fluorescence by the heteropolyacid. The developed detector used in a simple three-channel flow injection analysis (FIA) system allows photometric or fluorimetric determination of phosphate in the wide range of concentration. The detection limits found for photometric and fluorimetric modes of FIA measurements are 5.5 mg L(-1) and 10.4 µg L(-1), respectively. The potential utility of the flow-through detector for the needs of food and clinical analysis has been demonstrated.

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.