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.     

毛细管区带电泳技术快速分离检测爆炸残留物中的无机离子

利用间接紫外毛细管区带电泳方法完成了对爆炸残留物中7种无机离子(K+,NH+4,NO-2,NO-3,SO2-4,ClO-3,ClO-4)的分离检测。阳离子测定采用的缓冲体系为10 mmol/L吡啶（pH 4.5）-3 mmol/L冠醚,K+和NH+4在2.6 min内达到基线分离,检出限分别为0.25 mg/L和0.10 mg/L(S/N=3)。阴离子测定采用的缓冲体系为40 mmol/L硼酸-1.8 mmol/L重铬酸钾-2 mmol/L硼酸钠（pH 8.6）,氢氧化四甲铵为电渗流改性剂,5种阴离子在4.6 min内达到基线分离,检出限为0.10～1.85 mg/L。该方法已成功地应用于实际爆炸物样品种类的判定分析,取得了很好的结果。     

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.     

Fabrication of microfluidic devices using photopatternable hybrid sol-gel coatings

UV-patternable organic-inorganic hybrid sol-gel coating was used to develop microchannel on silicon and glass wafers by photolithography processing. The sol-gel coating was formed with 3-methacryloxypropyltrimethoxysilane (MPTS) as photosensitive component and zirconium propoxide as property modifier. In order to enhance the UV light efficiency during photolithography processing for glass substrates, a thin copper layer with thickness of 100–200 nm was deposited on one side of the glass wafer. The closed microchannels were formed by bonding of two developed surface channels of compensating patterns by wafer bonding technology. The bonding material is the same as the channel body ensuring a uniform surface property for the microchannels. A thin layer of about 2 μm was applied on the developed channels by spin coating. The depth of the surface channels on each wafer is about 10–12 μm, and the height of the closed channels is therefore in the range of 22–24 μm. Different microfluidic devices such as chaotic micromixers and microsplitters were fabricated.     

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.     

Polyimide-based microfluidic devices

This paper describes the development of polyimide-based microfluidic devices. A layer transfer and lamination technique is used to fabricate flexible microfluidic channels in various shapes and with a wide range of dimensions. High bond strengths can be achieved by cure cycle adaptation and surface treatment of the polyimide layers prior to bonding. The polyimide microchannels can be combined with metallization layers to fabricate electrodes inside and outside channels. The resulting devices can be used for flexible fluidic and electrical connectors, implantable fluid delivery devices, microelectrodes with embedded fluidic channels, chip-based flow cytometry and for a great variety of other applications in medical, chemical or biological research.     

Wirelessly powered microfluidic dielectrophoresis devices using printable RF circuits

We report the first microfluidic device integrated with a printed RF circuit so the device can be wirelessly powered by a commercially available RFID reader. For conventional dielectrophoresis devices, electrical wires are needed to connect the electric components on the microchip to external equipment such as power supplies, amplifiers, function generators, etc. Such a procedure is unfamiliar to most clinicians and pathologists who are used to working with a microscope for examination of samples on microscope slides. The wirelessly powered device reported here eliminates the entire need for wire attachments and external instruments so the operators can use the device in essentially the same manner as they do with microscope slides. The integrated circuit can be fabricated on a flexible plastic substrate at very low cost using a roll-to-roll printing method. Electrical power at 13.56 MHz transmitted by a radio-frequency identification (RFID) reader is inductively coupled to the printed RFIC and converted into 10 V DC (direct current) output, which provides sufficient power to drive a microfluidic device to manipulate biological particles such as beads and proteins via the DC dielectrophoresis (DC-DEP) effect. To our best knowledge, this is the first wirelessly powered microfluidic dielectrophoresis device. Although the work is preliminary, the device concept, the architecture, and the core technology are expected to stimulate many efforts in the future and transform the technology to a wide range of clinical and point-of-care applications.     

Three-dimensional paper microfluidic devices assembled using the principles of origami

We report a method, based on the principles of origami (paper folding), for fabricating three-dimensional (3-D) paper microfluidic devices. The entire 3-D device is fabricated on a single sheet of flat paper in a single photolithographic step. It is assembled by simply folding the paper by hand. Following analysis, the device can be unfolded to reveal each layer. The applicability of the device to chemical analysis is demonstrated by colorimetric and fluorescence assays using multilayer microfluidic networks.     

Lamination-based rapid prototyping of microfluidic devices using flexible thermoplastic substrates

Transposing highly sensitive DNA separation methods (such as DNA sequencing with high read length or the detection of point mutations) to microchip format without loss of resolution requires fabrication of relatively long (approx. 10 cm) microchannels along with sharp injection bands. Conventional soft lithography methods, such as mold casting or hot-embossing in a press, are not convenient for fabricating long channels. We have developed a lamination-based replication technique for rapid fabrication of sealed microfluidic devices with a 10 cm long, linear separation channel. These devices are fabricated in thin cyclo-olefin copolymer (COC) plastic substrates, thus making the device flexible and capable of assuming a range of 3-D configurations. Due to the good optical properties of COC, this new family of devices combines multiple advantages of planar microfluidics and fused-silica capillaries.     

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.     

Bioanalysis in microfluidic devices

Microfabricated bioanalytical devices (also referred to as laboratory-on-a-chip or micro-TAS) offer highly efficient platforms for simultaneous analysis of a large number of biologically important molecules, possessing great potential for genome, proteome and metabolome studies. Development and implementation of microfluidic-based bioanalytical tools involves both established and evolving technologies, including microlithography, micromachining, micro-electromechanical systems technology and nanotechnology. This article provides an overview of the latest developments in the key device subject areas and the basic interdisciplinary technologies. Important aspects of DNA and protein analysis, interfacing issues and system integration are all thoroughly discussed, along with applications for this novel "synergized" technology in high-throughput separations of biologically important molecules. This review also gives a better understanding of how to utilize these technologies as well as to provide appropriate technical solutions to problems perceived as being more fundamental.     

Dynamic temperature measurement in microfluidic devices using thermochromic liquid crystals

Thermochromic liquid crystals (TLCs) are used to explore the temperature transients during thermal cycling for microchip-based polymerase chain reaction (PCR). By analyzing the reflected spectra of the TLCs over time, temperature vs. time trajectories were extracted and overshoots/undershoots were estimated. To our knowledge, this is the first report of TLC-based dynamic temperature measurements in a microfluidic device for all PCR temperature stages.     

Nonlithographic fabrication of microfluidic devices

A facile nonlithographic method for expedient fabrication of microfluidic devices of poly(dimethylsiloxane) is described. Positive-relief masters for the molds are directly printed on smooth substrates. For the formation of connecting channels and chambers inside the polymer components of the microfluidic devices, cavity-forming elements are adhered to the surfaces of the masters. Using this nonlithographic approach, we fabricated microfluidic devices for detection of bacterial spores on the basis of enhancement of the emission of terbium (III) ions.     

Determination of inorganic ions in drinking water by ion chromatography

Ion chromatography (IC) is now a well established methodology for the analysis of ionic species. The technique is applicable to the determination of a wide range of solutes in many sample types, although the determination of inorganic ions in drinking water continues to be the most widely used application of ion chromatography. Many regulatory and standard organizations, such as ASTM, AOAC, ISO, and US EPA, have approved methods of analysis based upon IC, most of which have been published within the last 10 years. Recent developments in the field of IC, such as the use of higher capacity columns, larger loop injections, more complex sample preparation and detection schemes, have been incorporated into new approved methods to allow the determination of inorganic contaminants, such as bromate, perchlorate, and chromate, at low μg/l levels in drinking waters. IC appears certain to remain an important technique for drinking water analysis and new methods based on IC will continue to be developed as more inorganic contaminants become regulated at lower limits in the future.     

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).     

Generation of dynamic temporal and spatial concentration gradients using microfluidic devices

This paper describes a microfluidic approach to generate dynamic temporal and spatial concentration gradients using a single microfluidic device. Compared to a previously described method that produced a single fixed gradient shape for each device, this approach combines a simple "mixer module" with gradient generating network to control and manipulate a number of different gradient shapes. The gradient profile is determined by the configuration of fluidic inputs as well as the design of microchannel network. By controlling the relative flow rates of the fluidic inputs using separate syringe pumps, the resulting composition of the inlets that feed the gradient generator can be dynamically controlled to generate temporal and spatial gradients. To demonstrate the concept and illustrate this approach, examples of devices that generate (1) temporal gradients of homogeneous concentrations, (2) linear gradients with dynamically controlled slope, baseline, and direction, and (3) nonlinear gradients with controlled nonlinearity are shown and their limitations are described.     

Millisecond treatment of cells using microfluidic devices via two-step carrier-medium exchange

We present herein a simple but versatile microfluidic system for the treatment of cells with millisecond chemical stimulus, by rapidly exchanging the carrier-medium of cells twice in a microchannel. A technique we refer to as 'hydrodynamic filtration' has been employed for the exchange of medium, in which the virtual width of flow in the microchannel determines the size of filtered cells/particles. The treatment time of cells could be rigidly adjusted by controlling the inlet flow rates and by changing the volume of the stimulating area in the microchannel. In the experiment, two types of microdevices were designed and fabricated, and at first, the ability for carrier-medium exchange was confirmed. As an application of the presented system, we examined the influence of the treatment time of HeLa cells with Triton X-100, a non-ionic surfactant used to solubilize the cellular membrane, on cell viability, varying the average treatment time from 17 to 210 ms. Both quantitative and qualitative analyses were performed to estimate the damage on cell membrane, demonstrating that the cell viability dramatically decreased when the treatment time was longer than approximately 40 ms. The obtained results demonstrated the ability of the presented system to conduct the rapid stimulation of cells, which would be useful for the analysis of biochemical reactions at the cell surface.     

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.