Quantitative biomarker assay with microfluidic paper-based analytical devices

This article describes the use of microfluidic paper-based analytical devices (μPADs) to perform quantitative chemical assays with internal standards. MicroPADs are well-suited for colorimetric biochemical assays; however, errors can be introduced from the background color of the paper due to batch difference and age, and from color measurement devices. To reduce errors from these sources, a series of standard analyte solutions and the sample solution are assayed on a single device with multiple detection zones simultaneously; an analyte concentration calibration curve can thus be established from the standards. Since the μPAD design allows the colorimetric measurements of the standards and the sample to be conducted simultaneously and under the same condition, errors from the above sources can be minimized. The analytical approach reported in this work shows that μPADs can perform quantitative chemical analysis at very low cost.      

USB powered microfluidic paper-based analytical devices

Microfluidic paper-based analytical devices (μPADs) allow user-friendly and portable chemical determinations, although they provide limited applicability due to insufficient sensitivity. Several approaches have been proposed to address poor sensitivity in μPADs, but they frequently require bulky equipment for power and/or read-outs. Universal serial buses (USB) are an attractive alternative to less portable power sources and are currently available in many common electronic devices. Here, USB-powered μPADs (USB μPADs) are proposed as a fusion of both technologies to improve performance without adding instrumental complexity. Two ITP USB μPADs were developed, both powered by a 5 V potential provided through standard USB ports. The first device was fabricated using the origami approach. Its operation was analyzed experimentally and numerically, yielding a two-order-of-magnitude sample focusing in 15 min. The second ITP USB μPAD is a novel design, which was numerically prototyped with the aim of handling larger sample volumes. The reservoirs were moved away from the ITP channel and capillary action was used to drive the sample and electrolytes to the separation zone, predicting 25-fold sample focusing in 10 min. USB μPADs are expected to be adopted by minimally-trained personnel in sensitive areas like resource-limited settings, the point-of-care and in emergencies.     

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.     

Simultaneous electrochemical detection in paper-based analytical devices

The growing need for reliable analytical tools to perform measurements at the point-of-need has prompted the development of novel sensors that are low cost, portable, sensitive, easy to use, and capable of multiplexed analysis. Miniaturization of the sensors into microfluidic platforms has become a promising approach to achieve these self-contained sensors. However, traditional microfluidics often require relatively expensive and complicated pumping mechanisms that increase the cost and limit the portability of the sensors. From a material perspective, paper is an attractive substrate for constructing point-of-need sensors because of its affordability, vast availability, and self-pumping ability, particularly when combined with electrochemical detection. In this mini-review, we discuss various strategies to achieve multiplexing or simultaneous detection of multiple analytes in electrochemical paper-based devices and provide a brief guide on selecting the detection strategy based on the electrochemical property of the analytes.     

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.     

Determination of nitrite in saliva using microfluidic paper-based analytical devices

Point-of-care platforms can provide fast responses, decrease the overall cost of the treatment, allow for in-home determinations with or without a trained specialist, and improve the success of the treatment. This is especially true for microfluidic paper-based analytical devices (μPAD), which can enable the development of highly efficient and versatile analytical tools with applications in a variety of biomedical fields. The objective of this work was the development of μPADs to identify and quantify levels of nitrite in saliva, which has been proposed as a potential marker of periodontitis. The devices were fabricated by wax printing and allowed the detection of nitrite by a colorimetric reaction based on a modified version of the Griess reaction. The presented modifications, along with the implementation of a paper-based platform, address many of the common drawbacks (color development, stability, etc.) associated with the Griess reaction and are supported by results related to the design, characterization, and application of the proposed devices. Under the optimized conditions, the proposed devices enable the determination of nitrite in the 10–1000 μmol L⁻¹ range with a limit of detection of 10 μmol L⁻¹ and a sensitivity of 47.5 AU [log (μmol L⁻¹)]⁻¹. In order to demonstrate the potential impact of this technology in the healthcare industry, the devices were applied to the analysis of a series of real samples, covering the relevant clinical range.     

High throughput method for prototyping three-dimensional, paper-based microfluidic devices

This paper describes an efficient and high throughput method for fabricating three-dimensional (3D) paper-based microfluidic devices. The method avoids tedious alignment and assembly steps and eliminates a major bottleneck that has hindered the development of these types of devices. A single researcher now can prepare hundreds of devices within 1 h.     

Electrochemical detection of glucose from whole blood using paper-based microfluidic devices

Electrochemical paper-based analytical devices (ePADs) with integrated plasma isolation for determination of glucose from whole blood samples have been developed. A dumbbell shaped ePAD containing two blood separation zones (VF2 membranes) with a middle detection zone was fabricated using the wax dipping method. The dumbbell shaped device was designed to separate plasma while generating homogeneous flow to the middle detection zone of the ePAD. The proposed ePADs work with whole blood samples with 24–60% hematocrit without dilution, and the plasma was completely separated within 4 min. Glucose in isolated plasma separated was detected using glucose oxidase immobilized on the middle of the paper device. The hydrogen peroxide generated from the reaction between glucose and the enzyme pass through to a Prussian blue modified screen printed electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal detection potential for H₂O₂ (−0.1 V versus Ag/AgCl reference electrode) were proportional to glucose concentrations in the whole blood. The linear range for glucose assay was in the range 0–33.1 mM (r² = 0.987). The coefficients of variation (CVs) of currents were 6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration at 3.4, 6.3, and 15.6 mM, respectively. Because each sample displayed intra-individual variation of electrochemical signal, glucose assay in whole blood samples were measured using the standard addition method. Results demonstrate that the ePAD glucose assay was not significantly different from the spectrophotometric method (p = 0.376, paired sample t-test, n = 10).     

Disposable electrochemical paper-based devices fully fabricated by screen-printing technique

This work presents a novel method for the full fabrication of electrochemical paper-based cells by screen-printing technology. Firstly, a mixture of ultraviolet curable inks was used for patterning hydrophobic barriers into chromatography paper. Afterwards three-electrode systems were coupled for electrochemical detection by printing successively carbon and silver/silver chloride inks over the hydrophilic areas. The resulting electrochemical cells were characterized by cyclic voltammetry in different redox systems and used for amperometric detection of ferricyanide.     

Chelate titrations of Ca and Mg using microfluidic paper-based analytical devices

We developed microfluidic paper-based analytical devices (μPADs) for the chelate titrations of Ca²⁺ and Mg²⁺ in natural water. The μPAD consisted of ten reaction zones and ten detection zones connected through narrow channels to a sample zone located at the center. Buffer solutions with a pH of 10 or 13 were applied to all surfaces of the channels and zones. Different amounts of ethylenediaminetetraacetic acid (EDTA) were added to the reaction zones and a consistent amount of a metal indicator (Eriochrome Black T or Calcon) was added to the detection zones. The total concentrations of Ca²⁺ and Mg²⁺ (total hardness) in the water were measured using a μPAD containing a buffer solution with a pH of 10, whereas only Ca²⁺ was titrated using a μPAD prepared with a potassium hydroxide solution with a pH of 13. The μPADs permitted the determination of Ca²⁺ and Mg²⁺ in mineral water, river water, and seawater samples within only a few minutes using only the naked eye—no need of instruments.     

The electrochemical detection of droplets in microfluidic devices

This paper presents a new electrochemical method for the detection and characterisation of aqueous droplets in an organic carrier fluid (1,2-dichloroethane) formed in flow-focusing microfluidic devices. The devices consist of a conventional flow-focusing channel 250 microm wide and 250 microm deep cast out of poly(dimethylsiloxane) (PDMS) which is sealed onto a glass substrate containing a set of microelectrodes 100 microm long. Chronoamperometric analysis of a suitable electrolyte contained in the organic phase is presented for characterising the droplet frequency and size. This chronoamperometric method is then extended to a dual working electrode approach in order to determine the velocity of the droplet. Good agreement between experimental measurements and theory was observed.     

Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.     

"Fluidic batteries" as low-cost sources of power in paper-based microfluidic devices

This communication describes the first paper-based microfluidic device that is capable of generating its own power when a sample is added to the device. The microfluidic device contains galvanic cells (that we term "fluidic batteries") integrated directly into the microfluidic channels, which provides a direct link between a power source and an analytical function within the device. This capability is demonstrated using an example device that simultaneously powers a surface-mount UV LED and conducts an on-chip fluorescence assay.     

Separation and electrochemical detection of paracetamol and 4-aminophenol in a paper-based microfluidic device

The present work describes the construction and application of a simple, low cost and sensitive microfluidic paper-based device with electrochemical detection for the detection of paracetamol and 4-aminophenol. The separation channels of a width of 2.0 mm were created on paper using a wax printing process to define the regions of the device. A baseline separation level of the analytes can be obtained in 0.1 mol L⁻¹ acetate buffer solution at pH 4.5 and by injecting 500 nL of the standard solutions at 12 mm from the working electrode. The electrochemical detection system was created at the end of the channels through a process known as sputtering. The previously separated analytes were detected at the end of the hydrophilic separation channel by applying a potential of 400 mV vs. pseudo Au on the working electrode. Experimental variables such as type of paper (cation exchanger and n1), pH, sample volume, applied potential and distance of sample injection were evaluated and, under the conditions of higher response, it was possible to obtain detection limits of 25.0 and 10.0 μmol L⁻¹ for paracetamol and 4-aminophenol, respectively.     

Genotyping with microfluidic devices

In the past few years, electrophoresis microchips have been increasingly utilized to interrogate genetic variations in the human and other genomes. Microfluidic devices can be readily applied to speed up existing genotyping protocols, in particular the ones that require electric field-mediated separations in conjunction with restriction fragment analysis, DNA sequencing, hybridization-based techniques, allele-specific amplification, heteroduplex analysis, just to list the most important ones. As a result of recent developments, microfabricated electrophoresis devices offer several advantages over conventional slab-gel electrophoresis, such as small sample volume requirement, low reagent consumption, the option of system integration and easy multiplexing. The analysis speed of microchip electrophoresis is significantly higher than that of any other electric field-mediated separation techniques. State-of-the-art microfluidic bioanalytical devices already claim their place in most molecular biology laboratories. This review summarizes the recent developments in microchip electrophoresis methods of nucleic acids, particularly for rapid genotyping, that will most likely play a significant role in the future of clinical diagnostics.     

Fabrication of paper-based microfluidic sensors by printing

A novel method for the fabrication of paper-based microfluidic diagnostic devices is reported; it consists of selectively hydrophobizing paper using cellulose reactive hydrophobization agents. The hydrophilic–hydrophobic contrast of patterns so created has excellent ability to control capillary penetration of aqueous liquids in paper channels. Incorporating this idea with digital ink jet printing techniques, a new fabrication method of paper-based microfluidic devices is established. Ink jet printing can deliver biomolecules and indicator reagents with precision into the microfluidic patterns to form bio-chemical sensing zones within the device. This method thus allows the complete sensor, i.e. channel patterns and the detecting chemistries, to be fabricated only by two printing steps. This fabrication method can be scaled up and adapted to use high speed, high volume and low cost commercial printing technology. Sensors can be fabricated for specific tests, or they can be made as general devices to perform on-demand quantitative analytical tasks by incorporating the required detection chemistries for the required tasks.     

Polymer microfluidic devices

Since the introduction of lab-on-a-chip devices in the early 1990s, glass has been the dominant substrate material for their fabrication (J. Chromatogr. 593 (1992) 253; Science 261 (1993) 895). This is primarily driven by the fact that fabrication methods were well established by the semiconductor industry, and surface properties and derivatization methods were well characterized and developed by the chromatography industry among others. Several material properties of glass make it a very attractive material for use in microfluidic systems; however, the cost of producing systems in glass is driving commercial producers to seek other materials. Commercial manufacturers of microfluidic devices see many benefits in employing plastics that include reduced cost and simplified manufacturing procedures, particularly when compared to glass and silicon. An additional benefit that is extremely attractive is the wide range of available plastic materials which allows the manufacturer to choose materials' properties suitable for their specific application. In this article, we present a review of polymer-based microfluidic systems including their material properties, fabrication methods, device applications, and finally an analysis of the market that drives their development.     

Integrated microfluidic devices

“With the fundamentals of microscale flow and species transport well developed, the recent trend in microfluidics has been to work towards the development of integrated devices which incorporate multiple fluidic, electronic and mechanical components or chemical processes onto a single chip sized substrate. Along with this has been a major push towards portability and therefore a decreased reliance on external infrastructure (such as detection sensors, heaters or voltage sources).” In this review we provide an in-depth look at the “state-of-the-art” in integrated microfludic devices for a broad range of application areas from on-chip DNA analysis, immunoassays and cytometry to advances in integrated detection technologies for and miniaturized fuel processing devices. In each area a few representative devices are examined with the intent of introducing the operating procedure, construction materials and manufacturing technique, as well as any unique and interesting features.     

Integration of nanocapillary arrays into microfluidic devices for use as analyte concentrators

A nanocapillary array was integrated into a microfluidic device and its ability to concentrate analytes was characterized. Through the application of an electric field across the channel, large molecules were concentrated in front of the nanocapillary array, and a concentrated analyte band was ejected from the channel by reversing the polarity of the electric field. The effects of nanocapillary diameter, analyte charge, analyte concentration, analyte plug length, and analyte relative mobility were investigated. Concentration factors up to 300-fold were measured for fluorescein. By concentrating anionic FITC-labeled peptides, it was demonstrated that the magnitude of the electrophoretic mobility did not have a measurable effect on the concentration factor. Therefore, multiple analytes can be concentrated in front of the same nanocapillary array without adjusting the conditions, provided the analytes have the same net charge. In the presence of an electric field, a charge trapping effect was observed; small anionic molecules can be concentrated in front of nanocapillary array with channel diameters which are orders of magnitude above the molecular weight cut-offs for hydrodynamically driven systems. The concentrating process was found to be very efficient for fluorescein, as no leakage through the nanocapillary array or sorption of fluorescein to the nanocapillary array was observed. Due to their flexibility and efficiency, it is anticipated that nanocapillary arrays will find increased utility in electrokinetically driven microfluidic systems.