Sample injection and electrophoretic separation on a simple laminated paper based analytical device.

We described a strategy to perform multistep operations on a simple laminated paper-based separation device by using electrokinetic flow to manipulate the fluids. A laminated crossed-channel paper-based separation device was fabricated by cutting a filter paper sheet followed by lamination. Multiple function units including sample loading, sample injection, and electrophoretic separation were integrated on a single paper based analytical device for the first time, by applying potential at different reservoirs for sample, sample waste, buffer, and buffer waste. As a proof-of-concept demonstration, mixed sample solution containing carmine and sunset yellow were loaded in the sampling channel, and then injected into separation channel followed by electrophoretic separation, by adjusting the potentials applied at the four terminals of sampling and separation channel. The effects of buffer pH, buffer concentration, channel width, and separation time on resolution of electrophoretic separation were studied. This strategy may be used to perform multistep operations such as reagent dilution, sample injection, mixing, reaction, and separation on a single microfluidic paper based analytical device, which is very attractive for building micro total analysis systems on microfluidic paper based analytical devices.

Fabrication of a microfluidic paper-based analytical device by silanization of filter cellulose using a paper mask for glucose assay.

We developed a novel, low-cost and simple method for the fabrication of microfluidic paper-based analytical devices (µPADs) by silanization of filter cellulose using a paper mask having a specific pattern. The paper mask was penetrated with trimethoxyoctadecylsilane (TMOS) by immersing into TMOS-heptane solution. By heating the filter paper sandwiched between the paper mask and glass slides, TMOS was immobilized onto the filter cellulose via the reaction between cellulose OH and TMOS, while the hydrophilic area was not silanized because it was not in contact with the paper mask penetrated with TMOS. The effects of some factors including TMOS concentration, heating temperature and time on the fabrication of µPADs were studied. This method is free of any expensive equipment and metal masks, and could be performed by untrained personnel. These features are very attractive for the fabrication and applications of µPADs in developing countries or resource-limited settings. A flower-shaped µPAD was fabricated and used to determine glucose in human serum samples. The contents determined by this method agreed well with those determined by a standard method.

Analysis of intracellular reducing levels in human hepatocytes on three-dimensional focusing microchip.

A novel three-dimensional hydrodynamic focusing microfluidic device integrated with high-throughput cell sampling and detection of intracellular contents is presented. It has a pivotal role in maintaining the reducing environment in cells. Intracellular reducing species such as vitamin C and glutathione in normal and tumor cells were labeled by a newly synthesized 2,2,6,6-tetramethyl-piperidine-1-oxyl-based fluorescent probe. Hepatocytes are adherent cells, which are prone to attaching to the channel surface. To avoid the attachment of cells on the channel surface, a single channel microchip with three sheath-flow channels located on both sides of and below the sampling channel was developed. Hydrostatic pressure generated by emptying the sample waste reservoir was used as driving force of fluid on the microchip. Owing to the difference between the liquid levels of the reservoirs, the labeled cells were three-dimensional hydrodynamically focused and transported from the sample reservoir to the sample waste reservoir. Hydrostatic pressure takes advantage of its ease of generation on a microfluidic chip without any external pressure pump, which drives three sheath-flow streams to constrain a sample flow stream into a narrow stream to avoid blockage of the sampling channel by adhered cells. The intracellular reducing levels of HepG2 cells and L02 cells were detected by home-built laser-induced fluorescence detector. The analysis throughput achieved in this microfluidic system was about 59-68 cells/min.

Sequential operation droplet array: an automated microfluidic platform for picoliter-scale liquid handling, analysis, and screening.

This contribution describes a sequential operation droplet array (SODA) system, a fully automated droplet-based microfluidic system capable of performing picoliter-scale liquid manipulation, analysis, and screening. The SODA system was built using a tapered capillary-syringe pump module and a two-dimensional (2D) oil-covered droplet array installed on an x-y-z translation stage. With the system, we developed a novel picoliter-scale droplet depositing technique for forming a 2D picoliter-droplet array. On this basis, an automated droplet manipulation method with picoliter precision was established using the programmable combination of the capillary-based liquid aspirating-depositing and the moving of the oil-covered droplet array, the so-called "aspirating-depositing-moving" (ADM) method. Differing from the previously reported droplet systems based on microchips, microcapillaries, or digital microfluidics, this method can achieve complete and flexible droplet manipulations, including droplet assembling, generation, indexing, transferring, splitting, and fusion in the picoliter range, endowing the present system with ultralow sample/reagent consumptions and substantial versatility in analysis and screening for multiple different samples. To demonstrate its feasibility and versatility, we applied the SODA system in multiple experiments required in drug screening, including the screening of inhibitors for capases-1 from a chemical library, the measurement of IC50 values for the identified inhibitors, and the screening of the synergistic effect of multiple inhibitors. In the experiments, the consumptions of samples and reagents are only 60-180 pL for each droplet microreactor, which are commonly 3-5 orders of magnitude lower than those of conventional multiwell plate systems, and 1-2 orders of magnitude lower than other droplet-based microfluidic systems for multiple sample screening. The ability of the SODA system in performing complicated and multistep droplet manipulations was further demonstrated in the serial dilution of nanoliter-scale inhibitor droplets with concentrations spanning 6 orders of magnitude for IC50 profiling, which includes droplet generation, indexing, splitting, transferring, and fusion with picoliter precision.

Highly efficient treatment of industrial wastewater by solution plasma with low environmental load.

Advanced oxidation techniques are efficient processes to dispose of organic contaminants in industrial wastewater with low secondary pollution. The solution plasma technique was featured as an advanced oxidation technique with low secondary pollution and high efficiency. However, the solution plasma technique reported previously could only treat wastewater of less than 200 mL owing to the limited plasma generated by only one pair of electrodes. In this work, multiple pairs of electrodes were installed at the bottom of the reaction vessel to generate plasma for decomposing methylene blue trihydrate (MB) and methyl orange (MO) solutions with a batch amount of 18 L/batch. The solution plasma technique was compared with direct ozonation in decomposition of MB and MO wastewater. A surprising phenomenon is that MO was more readily decomposed than MB by using direct ozonation, whereas the removal of MO was too low, and MB was more readily decomposed than MO by using the solution plasma technique.

Droplet-based microfluidic flow injection system with large-scale concentration gradient by a single nanoliter-scale injection for enzyme inhibition assay.

We described a microfluidic chip-based system capable of generating droplet array with a large scale concentration gradient by coupling flow injection gradient technique with droplet-based microfluidics. Multiple modules including sample injection, sample dispersion, gradient generation, droplet formation, mixing of sample and reagents, and online reaction within the droplets were integrated into the microchip. In the system, nanoliter-scale sample solution was automatically injected into the chip under valveless flow injection analysis mode. The sample zone was first dispersed in the microchannel to form a concentration gradient along the axial direction of the microchannel and then segmented into a linear array of droplets by immiscible oil phase. With the segmentation and protection of the oil phase, the concentration gradient profile of the sample was preserved in the droplet array with high fidelity. With a single injection of 16 nL of sample solution, an array of droplets with concentration gradient spanning 3-4 orders of magnitude could be generated. The present system was applied in the enzyme inhibition assay of ß-galactosidase to preliminarily demonstrate its potential in high throughput drug screening. With a single injection of 16 nL of inhibitor solution, more than 240 in-droplet enzyme inhibition reactions with different inhibitor concentrations could be performed with an analysis time of 2.5 min. Compared with multiwell plate-based screening systems, the inhibitor consumption was reduced 1000-fold.

Modification of Microfluidic Paper-Based Devices with an Oxidant Layer for Distance Readout of Reducing Substances.

We developed a novel strategy for modification of paper cellulose with water-insoluble oxidants for distance readout of reducing substances on microfluidic paper-based analytical devices (µPADs). Water-insoluble oxidants were formed and modified onto paper cellulose through the redox reaction that occurred between paper cellulose and potassium permanganate deposited on the paper channel, developing a yellowish-brown color on the channel. As aqueous solutions containing reducing substances flowed along the channel, reducing substances were consumed owing to the redox reaction that occurred between oxidants and reducing substances until the reducing substances were depleted, forming a discolored zone on the yellowish-brown channel. The redox reaction between insoluble oxidants and reducing substances on the paper cellulose could be used for distance-based detection of a wide variety of reducing substances, which is similar to the classical potassium permanganate titration that employs the redox reaction that occurred between potassium permanganate and reducing substances. We believe that this method will broaden the analytical applications of distance-based detection on µPADs. This method was applied to ascorbic acid assay and captopril assay in real samples with analytical results comparing well with the labeled values, demonstrating its great potential in real sample analysis.

Indicator-Free Argentometric Titration for Distance-Based Detection of Chloride Using Microfluidic Paper-Based Analytical Devices.

We described an indicator-free argentometric titration strategy using a microfluidic paper-based analytical device. This strategy was based on the formation of insoluble silver salts by reactions occurring between analytes and titrant (Ag+) on a paper channel. After the insoluble silver salts were formed and precipitated on the channel, the paper substrate modified with the surplus titrant on the channel turned reddish-brown by exposure of the devices to a simple and cheap UV light source for 5 min, generating a colored band on the channel. Distance-based detection of chloride was achieved by measuring the length of the colored band with a detection limit of 1.7 mg L-1 Cl-. This method was used to detect chlorides in tap water, with an analytical result (10.1 ± 1.2 mg L-1) agreeing well with that obtained by a classical conventional precipitation titration (9.8 mg L-1), which was based on the measurement of the consumed volume of titrant. This paper-based precipitation titration method is free of skilled personnel and has advantages of low reagent/sample consumption, disposability, portability, and simple operation over the conventional precipitation titration. More importantly, being free of any indicator, this method may be used to detect more species than the conventional precipitation titrations, which are limited by the indicator, for example, CO3 2- and SO4 2-, which could form insoluble silver salts in aqueous liquids. Additionally, comparing with most of those paper-based titrimetry reported previously, this presented precipitation titration is free of any indicator or ion selective electrode to detect the end point of titration.

Instrument-free detection of polyphenols with a thread-based analytical device.

We described an instrument-free method for quantitative analysis of the total content of tea polyphenols by measurement of the length of a coloured band. Polyphenols react with ferrous ions to form a colourless ferrous-polyphenols complex on cotton threads, which could be adsorbed on the threads. The complex was then oxidized to form a blue-black ferric-polyphenols complex, generating a blue-black band on the cotton thread. The length of this blue-black band was then measured to detect the total content of polyphenols. The advantages of this method include low cost, rapid analysis, low consumption, easy fabrication and operation. Furthermore, the digital instrument (scanner or camera) as well as the image processing software are not required. This proposed method was used to detect polyphenols in tea leaf extracts with an analytical result agreeing well with that obtained by a standard method, which demonstrates its potential in monitoring of tea leaf quality, especially in resource-limited regions and settings.

Simultaneous determination of trace iron and aluminum by catalytic spectrophotometry based on a novel oxidation reaction of xylene cyanol FF.

A new, simple, sensitive and selective method for the simultaneous determination of trace iron and aluminum by catalytic spectrophotometry was presented, based on the catalytic effects of iron and aluminum on the discoloring reaction of xylene cyanol FF proceeded by hydrogen peroxide and potassium periodate in weak nitric acid medium. No catalytic effect was obtained in the presence of hydrogen peroxide or potassium periodate only. With the conditional rate constants determined in reaction systems catalyzed by Al or Fe only, the concentrations of Fe and Al in the samples can be calculated. The method was applied to the simultaneous determination of trace Fe and Al in tap water, lake water, river water and tea leaves without separation and preconcentration.

Defining microchannels and valves on a hydrophobic paper by low-cost inkjet printing of aqueous or weak organic solutions.

We describe a simple and cost-effective strategy for rapid fabrication of microfluidic paper-based analytical devices and valves by inkjet printing. NaOH aqueous solution was printed onto a hydrophobic filter paper, which was previously obtained by soaking in a trimethoxyoctadecylsilane-heptane solution, allowing selective wet etching of hydrophobic cellulose to create hydrophilic-hydrophobic contrast with a relatively good resolution. Hexadecyltrimethylammonium bromide (CTMAB)-ethanol solution was printed onto hydrophobic paper to fabricate temperature-controlled valves. At low temperature, CTMAB deposited on the paper is insoluble in aqueous fluid, thus the paper remains hydrophobic. At high temperature, CTMAB becomes soluble so the CTMAB-deposited channel becomes hydrophilic, allowing the wicking of aqueous solution through the valve. We believe that this strategy will be very attractive for the development of simple micro analytical devices for point-of-care applications, including diagnostic testing, food safety control, and environmental monitoring.

A simple paper-based sensor fabricated by selective wet etching of silanized filter paper using a paper mask.

We developed a novel strategy for fabrication of microfluidic paper-based analytical devices (µPADs) by selective wet etching of hydrophobic filter paper using a paper mask having a specific design. The fabrication process consists of two steps. First, the hydrophilic filter paper was patterned hydrophobic by using trimethoxyoctadecylsilane (TMOS) solution as the patterning agent. Next, a paper mask penetrated with NaOH solution (containing 30% glycerol) was aligned onto the hydrophobic filter paper, allowing the etching of the silanized filter paper by the etching reagent. The masked region turned highly hydrophilic whereas the unmasked region remains highly hydrophobic. Thus, hydrophilic channels, reservoirs, and detection zones were generated and delimited by the hydrophobic barriers. The effects of some factors including TMOS concentration, etching temperature, etching time, and NaOH concentration on fabrication of µPAD were studied. Being free of any expensive equipment, metal mask and expensive reagents, this rapid, simple, and cost-effective method could be used to fabricate µPAD by untrained personnel with minimum cost. A flower-shaped µPAD fabricated by this presented method was applied to the glucose assay in artificial urine samples with good performance, indicating its feasibility as a quantitative analysis device. We believe that this method would be very attractive to the development of simple microfluidic devices for point-of-care applications in clinical diagnostics, food safety, and environmental protection.