A microfluidic paper-based analytical device for rapid quantification of particulate chromium

Occupational exposure to Cr is concerning because of its myriad of health effects. Assessing chromium exposure is also cost and resource intensive because the analysis typically uses sophisticated instrumental techniques like inductively coupled plasma-mass spectrometry (ICP-MS). Here, we report a novel, simple, inexpensive microfluidic paper-based analytical device (μPAD) for measuring total Cr in airborne particulate matter. In the μPAD, tetravalent cerium (Ce(IV)) was used in a pretreatment zone to oxidize all soluble Cr to Cr(VI). After elution to the detection zone, Cr(VI) reacts with 1,5-diphenylcarbazide (1,5-DPC) forming 1,5-diphenylcarbazone (DPCO) and Cr(III). The resulting Cr(III) forms a distinct purple colored complex with the DPCO. As proof-of-principle, particulate matter (PM) collected on a sample filter was analyzed with the μPAD to quantify the mass of total Cr. A log-linear working range (0.23–3.75 μg; r² = 0.998) between Cr and color intensity was obtained with a detection limit of 0.12 μg. For validation, a certified reference containing multiple competing metals was analyzed. Quantitative agreement was obtained between known Cr levels in the sample and the Cr measured using the μPAD.     

Selective determination of homocysteine levels in human plasma using a silver nanoparticle-based colorimetric assay

The first use of silver nanoparticles (AgNPs) for the rapid, simple, and selective determination of homocysteine (Hcy) levels in human plasma was studied. Hcy and five other amino acids, including cysteine (Cys), could be distinguished by their different aggregation kinetics, which caused a change in the visible color and a shift in the UV-vis absorption spectra. The difference in the cross-linking (aggregation) rate between Hcy and Cys was used as the basis for developing a selective probe for Hcy and allowed the detection of Hcy in the linear range of 2-12 μM (R² = 0.9936). The limits of detection and quantification were found to be 0.5 μM and 1.7 μM, respectively. To investigate its selectivity and potential applicability, this AgNP-based method was successfully applied for the determination of Hcy levels in actual biological (human plasma) samples, where the determined levels of Hcy were within the error range of the measured level using the traditional chemiluminescence microparticle immunoassay (CMIA). Thus, the use of AgNPs is a feasible and potentially reliable method for the determination of Hcy levels in biological samples.     

Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen

A micro-magnetic chemiluminescence (CL) enzyme immunoassay with high sensitivity, selectivity, and reproducibility was developed for the determination of the tumor marker, carcinoembryonic antigen (CEA) in human serum. A sandwich scheme assay has been utilized with fluorescein isothiocyanate antibody (FITC)-labeled anti-CEA antibody and alkaline phosphate (ALP)-labeled anti-CEA antibody being used in the CL detection. The CL signal produced by the emission of photons from 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD) was directly proportional to the amount of analyte present in a sample solution. The influences of the reaction time of antigen with antibody, the reaction time of substrate with label, the dilution ratio of ALP-labeled anti-CEA antibody, the concentration of FITC-labeled anti-CEA antibody, and other relevant variables upon the CL signal were examined and optimized. The CL responses depended linearly on the CEA concentration over the range from 2 to 162 ng mL⁻¹ in a logarithmic plot. Assay sensitivity as low as 0.69 ng mL⁻¹ was achieved. A coefficient of variance of less than 13% was obtained for intra- and inter-assay precision. This method has been successfully applied to the analysis of CEA in human serum. According to the procedure based on spiked standards, the recoveries obtained were 80–110%. Comparison experiments were carried out with the commercially available CEA chemiluminescence immunoassay. Satisfactory results were obtained according to a paired t-test method (t value < t _critical at the 95% confidence level).     

Blood separation on microfluidic paper-based analytical devices

A microfluidic paper-based analytical device (μPAD) for the separation of blood plasma from whole blood is described. The device can separate plasma from whole blood and quantify plasma proteins in a single step. The μPAD was fabricated using the wax dipping method, and the final device was composed of a blood separation membrane combined with patterned Whatman No.1 paper. Blood separation membranes, LF1, MF1, VF1 and VF2 were tested for blood separation on the μPAD. The LF1 membrane was found to be the most suitable for blood separations when fabricating the μPAD by wax dipping. For blood separation, the blood cells (both red and white) were trapped on blood separation membrane allowing pure plasma to flow to the detection zone by capillary force. The LF1-μPAD was shown to be functional with human whole blood of 24-55% hematocrit without dilution, and effectively separated blood cells from plasma within 2 min when blood volumes of between 15-22 μL were added to the device. Microscopy was used to confirm that the device isolated plasma with high purity with no blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was 2.62% (n = 10) and 5.84% (n = 30) for within-day and between day precision, respectively. Our proposed blood separation on μPAD has the potential for reducing turnaround time, sample volume, sample preparation and detection processes for clinical diagnosis and point-of care testing.     

Sodium dodecyl sulfate-modified electrochemical paper-based analytical device for determination of dopamine levels in biological samples

We report the development of an electrochemical paper-based analytical device (ePAD) for the selective determination of dopamine (DA) in model serum sample. The ePAD device consists of three layers. In the top layer, SU-8 photoresist defines a hydrophilic sample application spot on the filter paper. The middle layer was made from transparency film and contained two holes, one for sample preconcentration and the other for the surfactant to allow transfer to the third layer. A screen-printed carbon electrode formed the bottom layer and was used for electrochemical measurements. In the absence of the anionic surfactant, sodium dodecyl sulfate (SDS), the oxidation peaks of DA, ascorbic acid (AA) and uric acid (UA) overlapped. With the addition of SDS, the DA oxidation peak shifted to more negative values and was clearly distinguishable from AA and UA. The oxidation potential shift was presumably due to preferential electrostatic interactions between the cationic DA and the anionic SDS. Indeed, whilst the SDS-modified paper improved the DA current five-fold, the non-ionic Tween-20 and cationic tetradecyltrimethylammonium bromide surfactants had no effect or reduced the current, respectively. Furthermore, only the SDS-modified paper showed the selective shift in oxidation potential for DA. DA determination was carried out using square-wave voltammetry between −0.2 and 0.8 V vs. Ag/AgCl, and this ePAD was able to detect DA over a linear range of 1–100 μM with a detection limit (S/N = 3) of 0.37 μM. The ePAD seems suitable as a low cost, easy-to-use, portable device for the selective quantitation of DA in human serum samples.     

Highly sensitive determination of cadmium and lead using a low-cost electrochemical flow-through cell based on a carbon paste electrode

A low-cost thin-layer electrochemical flow-through cell based on a carbon paste electrode (CPE), was constructed for the highly sensitive determination of cadmium(II) (Cd(2+)) and lead(II) (Pb(2+)) ions. The sensitivity of the proposed cell for Cd(2+) and Pb(2+) ion detection was improved by using the smallest channel height without the need for any complicated electrode modification. Under the optimum conditions, the detection limits of Cd(2+) and Pb(2+) ions (0.08 and 0.07 μg dm(-3), respectively) were 13.8- and 11.4-fold lower than that of a commercial flow cell (1.1 and 0.8 μg dm(-3), respectively). Moreover, the percentage recoveries of Cd(2+) and Pb(2+) for the in-house designed thin-layer flow cell were higher than those for the commercially available cell in all tested water samples, and within the acceptable range. The proposed flow cell is promising as an inexpensive and alternative one for the highly sensitive monitoring of heavy metal ions.     

Novel, simple and low-cost alternative method for fabrication of paper-based microfluidics by wax dipping

Paper-based microfluidic devices are an alternative technology for fabricating simple, low-cost, portable and disposable platforms for clinical diagnosis. Hereby, a novel wax dipping method for fabricating paper-based microfluidic devices (μPADs) is reported. The iron mould for wax dipping was created by a laser cutting technique. The designed pattern was transferred onto paper by dipping an assembly mould into melted wax. The optimal melting temperature and dipping time were investigated. The optimal melting temperature was in the range of 120-130 °C, and the optimal dipping time was 1 s. The whole fabrication process could be finished within 1 min without the use of complicated instruments or organic solvents. The smallest hydrophilic channel that could be created by the wax dipping method was 639 ± 7 μm in size. The reproducibility of the μPAD fabrication for hydrophilic channel width of the test zone and sample zone was 1.48% and 6.30%, respectively. To verify the performance of the μPAD, multiple colorimetric assays for simultaneous detection of glucose and protein in real samples were performed. An enzymatic assay and the bromocresol green (BCG) method were conducted on the paper device to determine the presence of glucose and protein in a test solution. The results of the assays were not significantly different from those of the conventional methods (p > 0.05, pair t-test and one-way ANOVA method). The wax dipping provides a new alternative method for fabricating lab-on-paper devices for multiple clinical diagnostics and will be very beneficial for developing countries.     

Poly(dimethylsiloxane) cross-linked carbon paste electrodes for microfluidic electrochemical sensing

Recently, the development of electrochemical biosensors as part of microfluidic devices has garnered a great deal of attention because of the small instrument size and portability afforded by the integration of electrochemistry in microfluidic systems. Electrode fabrication, however, has proven to be a major obstacle in the field. Here, an alternative method to create integrated, low cost, robust, patternable carbon paste electrodes (CPEs) for microfluidic devices is presented. The new CPEs are composed of graphite powder and a binder consisting of a mixture of poly(dimethylsiloxane) (PDMS) and mineral oil. The electrodes are made by filling channels molded in previously cross-linked PDMS using a method analogous to screen printing. The optimal binder composition was investigated to obtain electrodes that were physically robust and performed well electrochemically. After studying the basic electrochemistry, the PDMS-oil CPEs were modified with multi-walled carbon nanotubes (MWCNT) and cobalt phthalocyanine (CoPC) for the detection of catecholamines and thiols, respectively, to demonstrate the ease of electrode chemical modification. Significant improvement of analyte signal detection was observed from both types of modified CPEs. A nearly 2-fold improvement in the electrochemical signal for 100 μM dithiothreitol (DTT) was observed when using a CoPC modified electrode (4.0 ± 0.2 nA (n = 3) versus 2.5 ± 0.2 nA (n = 3)). The improvement in signal was even more pronounced when looking at catecholamines, namely dopamine, using MWCNT modified CPEs. In this case, an order of magnitude improvement in limit of detection was observed for dopamine when using the MWCNT modified CPEs (50 nM versus 500 nM). CoPC modified CPEs were successfully used to detect thiols in red blood cell lysate while MWCNT modified CPEs were used to monitor temporal changes in catecholamine release from PC12 cells following stimulation with potassium.     

Nanoparticle-based electrochemical detection in conventional and miniaturized systems and their bioanalytical applications: a review

With recent advances in nanotechnology making more easily available the novel chemical and physical properties of metal nanoparticles (NPs), these have become extremely suitable for creating new sensing assays. Many kinds of NPs, including metal, metal-oxide, semiconductor and even composite-metal NPs, have been used for constructing electrochemical sensors. This article reviews the progress of NP-based electrochemical detection in recent applications, especially in bioanalysis, and summarizes the main functions of NPs in conventional and miniaturized systems. All references cited here generally show one or more of the following characteristics: a low detection limit, good signal amplification and simultaneous-detection capabilities.     

Use of multiple colorimetric indicators for paper-based microfluidic devices

We report here the use of multiple indicators for a single analyte for paper-based microfluidic devices (μPAD) in an effort to improve the ability to visually discriminate between analyte concentrations. In existing μPADs, a single dye system is used for the measurement of a single analyte. In our approach, devices are designed to simultaneously quantify analytes using multiple indicators for each analyte improving the accuracy of the assay. The use of multiple indicators for a single analyte allows for different indicator colors to be generated at different analyte concentration ranges as well as increasing the ability to better visually discriminate colors. The principle of our devices is based on the oxidation of indicators by hydrogen peroxide produced by oxidase enzymes specific for each analyte. Each indicator reacts at different peroxide concentrations and therefore analyte concentrations, giving an extended range of operation. To demonstrate the utility of our approach, the mixture of 4-aminoantipyrine and 3,5-dichloro-2-hydroxy-benzenesulfonic acid, o-dianisidine dihydrochloride, potassium iodide, acid black, and acid yellow were chosen as the indicators for simultaneous semi-quantitative measurement of glucose, lactate, and uric acid on a μPAD. Our approach was successfully applied to quantify glucose (0.5-20 mM), lactate (1-25 mM), and uric acid (0.1-7 mM) in clinically relevant ranges. The determination of glucose, lactate, and uric acid in control serum and urine samples was also performed to demonstrate the applicability of this device for biological sample analysis. Finally results for the multi-indicator and single indicator system were compared using untrained readers to demonstrate the improvements in accuracy achieved with the new system.