Hydrogel-based microreactors as a functional component of microfluidic systems

A simple two-step method for fabricating poly(ethylene glycol) (PEG) hydrogel-based microreactors and microsensors within microfluidic channels is described. The intrachannel micropatches contain either a dye, which can report the pH of a solution within a fluidic channel, or enzymes that are able to selectively catalyze specific reactions. Analytes present within the microfluidic channel are able to diffuse into the micropatches, encounter the enzymes, and undergo conversion to products, and then the products interact with the coencapsulated dye to signal the presence of the original substrate. The micropatches are prepared by photopolymerizing the PEG precursor within the channel of a microfluidic system consisting of a poly(dimethylsiloxane) mold and a glass plate. Exposure takes place through a slit mask oriented perpendicular to the channel, so the size of the resulting micropatch is defined by the channel dimensions and the width of the slit mask. Following polymerization, the mold is removed, leaving behind the micropatch(es) atop the glass substrate. The final microfluidic device is assembled by irreversibly binding the hydrogel-patterned glass slide to a second PDMS mold that contains a larger channel. Multiple micropatches containing the same or different enzymes can be fabricated within a single channel. The viability of this approach is demonstrated by sensing glucose using micropatches copolymerized with glucose oxidase, horseradish peroxidase, and a pH-sensitive dye.     

Addressable Microfluidic Polymer Chip for DNA‐Directed Immobilization of Oligonucleotide‐Tagged Compounds

A microfluidic polymer chip for the self‐assembly of DNA conjugates through DNA‐directed immobilization is developed. The chip is fabricated from two parts, one of which contains a microfluidic channel produced from poly(dimethylsiloxane) (PDMS) by replica‐casting technique using a mold prepared by photolithographic techniques. The microfluidic part is sealed by covalent bonding with a chemically activated glass slide containing a DNA oligonucleotide microarray. The dimension of the PDMS–glass microfluidic chip is equivalent to standard microscope slides (76 × 26 mm²). The DNA microarray surface inside the microfluidic channels is configured through conventional spotting, and the resulting DNA patches can be conveniently addressed with compounds containing complementary DNA tags. To demonstrate the utility of the addressable surface within the microfluidic channel, DNA‐directed immobilization (DDI) of DNA‐modified gold nanoparticles (AuNPs) and DNA‐conjugates of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) are carried out. DDI of AuNPs is used to demonstrate site selectivity and reversibility of the surface‐modification process. In the case of the DNA–enzyme conjugates, the patterned assembly of the two enzymes allows the establishment and investigation of the coupled reaction of GOx and HRP, with particular emphasis on surface coverage and lateral flow rates. The results demonstrate that this addressable chip is well suited for the generation of fluidically coupled multi‐enzyme microreactors.     

Patterning enzymes inside microfluidic channels via photoattachment chemistry

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.     

Enzyme reactions in nanoporous, picoliter volume containers

Advancements in nanoscale fabrication allow creation of small-volume reaction containers that can facilitate the screening and characterization of enzymes. A porous, ～19 pL volume vessel has been used in this work to carry out enzyme reactions under varying substrate concentrations. Assessment of small-molecule and green fluorescent protein diffusion from the vessels indicates that pore sizes on the order of 10 nm can be obtained, allowing capture of proteins and diffusive exchange of small molecules. Glucose oxidase and horseradish peroxidase can be contained in these structures and diffusively fed with a solution containing glucose and the fluorogenic substrate amplex red through the engineered nanoscale pore structure. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Kinetic characteristics of the enzyme (K(m) and V(max)) were evaluated and compared with results from conventional scale reactions. These picoliter, nanoporous containers can facilitate quick determination of enzyme kinetics in microfluidic systems without the requirement of surface tethering and can be used for applications in drug discovery, clinical diagnostics, and high-throughput screening.     

Design and characterization of immobilized enzymes in microfluidic systems.

Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.     

Inkjet-printed microfluidic multianalyte chemical sensing paper

This paper presents an inkjet printing method for the fabrication of entire microfluidic multianalyte chemical sensing devices made from paper suitable for quantitative analysis, requiring only a single printing apparatus. An inkjet printing device is used for the fabrication of three-dimensional hydrophilic microfluidic patterns (550-mum-wide flow channels) and sensing areas (1.5 mm x 1.5 mm squares) on filter paper, by inkjet etching, and thereby locally dissolving a hydrophobic poly(styrene) layer obtained by soaking of the filter paper in a 1 wt % solution of poly(styrene) in toluene. In a second step, the same inkjet printing device is used to print "chemical sensing inks", comprising the necessary reagents for colorimetric analytical assays, into well-defined areas of the patterned microfluidic paper devices. The arrangement of the patterns, printed inks, and sensing areas was optimized to obtain homogeneous color responses. The results are "all-inkjet-printed" chemical sensing devices for the simultaneous determination of pH, total protein, and glucose in clinically relevant concentration ranges for urine analysis (0.46-46 muM for human serum albumin, 2.8-28.0 mM for glucose, and pH 5-9). Quantitative data are obtained by digital color analysis in the L*a*b* color space by means of a color scanner and a simple computer program.     

Capillary-assembled microchip for universal integration of various chemical functions onto a single microfluidic device

A novel concept for assembling various chemical functions onto a single microfluidic device is proposed. The concept, called a capillary-assembled microchip, involves embedding chemically functionalized capillaries into a lattice microchannel network fabricated on poly(dimethylsiloxane) (PDMS). The network has the same channel dimensions as the outer dimensions of the capillaries. In this paper, we focus on square capillaries to be embedded into a PDMS microchannel network having a square cross section. The combination of hard glass square capillary and soft square PDMS channel allows successful fabrication of a microfluidic device without any solution leakage, and which can use diffusion-based two-solution mixing. Two different types of chemically modified capillaries, an ion-sensing capillary and a pH-sensing capillary, are prepared by coating a hydrophobic plasticized poly(vinyl chloride) membrane and a hydrophilic poly(ethyleneglycol) membrane containing functional molecules onto the inner surface of capillaries. Then, they are cut into appropriate lengths and arranged on a single microchip to prepare a dual-analyte sensing system. The concept proposed here offers advantages inherent to using a planar microfluidic device and of chemical functionality of immobilized molecules. Therefore, we expect to fabricate various types of chemically functionalized microfluidic devices soon.     

Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants

Control of surface chemistry and protein adsorption is important for using microfluidic devices for biochemical analysis and high-throughput screening assays. This paper describes the control of protein adsorption at the liquid-liquid interface in a plug-based microfluidic system. The microfluidic system uses multiphase flows of immiscible fluorous and aqueous fluids to form plugs, which are aqueous droplets that are completely surrounded by fluorocarbon oil and do not come into direct contact with the hydrophobic surface of the microchannel. Protein adsorption at the aqueous-fluorous interface was controlled by using surfactants that were soluble in fluorocarbon oil but insoluble in aqueous solutions. Three perfluorinated alkane surfactants capped with different functional groups were used: a carboxylic acid, an alcohol, and a triethylene glycol group that was synthesized from commercially available materials. Using complementary methods of analysis, adsorption was characterized for several proteins (bovine serum albumin (BSA) and fibrinogen), including enzymes (ribonuclease A (RNase A) and alkaline phosphatase). These complementary methods involved characterizing adsorption in microliter-sized droplets by drop tensiometry and in nanoliter plugs by fluorescence microscopy and kinetic measurements of enzyme catalysis. The oligoethylene glycol-capped surfactant prevented protein adsorption in all cases. Adsorption of proteins to the carboxylic acid-capped surfactant in nanoliter plugs could be described by using the Langmuir model and tensiometry results for microliter drops. The microfluidic system was fabricated using rapid prototyping in poly(dimethylsiloxane) (PDMS). Black PDMS microfluidic devices, fabricated by curing a suspension of charcoal in PDMS, were used to measure the changes in fluorescence intensity more sensitively. This system will be useful for microfluidic bioassays, enzymatic kinetics, and protein crystallization, because it does not require surface modification during fabrication to control surface chemistry and protein adsorption.     

Patterned solvent delivery and etching for the fabrication of plastic microfluidic devices

A very simple method for micropatterning flat plastic substrates that can be used to build microfluidic devices is demonstrated. Patterned poly(dimethylsiloxane) elastomer is used as a template to control the flow path of an etching solvent through a channel design to be reproduced on the plastic substrate. The etching solvent was a acetone/ethanol mixture for poly(methyl methacrylate) substrates or a dimethylformamide/acetone mixture for polystyrene. The method is extremely fast in that duplicate plastic substrates can be patterned in just a few minutes each. We identified conditions that lead to smooth channel surfaces and characterized the rate of etching under these conditions. We determined that, for sufficiently short etching times (shallow channel depths), the etch rate is independent of the linear flow rate. This is very important since it means that the etch depth is approximately constant even in complex channel geometries where there will be a wide range of etchant flow rates at different positions in the pattern to be reproduced. We also demonstrate that the method can be used to produce channels with different depths on the same substrate as well as channels that intersect to form a continuous fluid junction. The method provides a nice alternative to existing methods to rapidly fabricate microfluidic devices in rigid plastics without the need for specialized equipment.     

An Immobilized Enzyme Reactor for Spatiotemporal Control over Reaction Products

This paper describes a microfluidic chip wherein the position and order of two immobilized enzymes affects the type and quantity of reaction products in the flowing fluid. Assembly of the chip is based on a self‐assembled monolayer presenting two orthogonal covalent capture ligands that immobilize their respective fusion enzyme. A thiol‐tagged substrate is flowed over a region presenting the first enzyme—which generates a product that is efficiently transferred to the second enzyme—and the second enzyme's product binds to an adjacent thiol capture site on the chip. The amount of the three possible reaction products is quantified directly on the chip using self‐assembled monolayers for matrix‐assisted laser desorption/ionization mass spectrometry, revealing that the same microsystem can be spatiotemporally arranged to produce different products depending on the device design. This work allows for optimizing multistep biochemical transformations in favor of a desired product using a facile reaction and analytical format.     

Catalytic three-dimensional protein architectures

We demonstrate a strategy for microfabricating catalytically active, three-dimensional matrixes composed of cross-linked protein in cellular and microfluidic environments. In this approach, a pulsed femtosecond laser is used to excite photosensitizers via multiphoton absorption within three-dimensionally defined volumes, a process that promotes cross-linking of protein residue side chains in the vicinity of the laser focal point. In this manner, it is possible to fabricate protein microparticles with dimensions on the order of the multiphoton focal volume (less than 1 microm(3)) or, by scanning the position of a laser focal point relative to a specimen, to generate surface-adherent matrixes or cables that extend through solution for hundreds of micrometers. We show that protein matrixes can be functionalized either through direct cross-linking of enzymes, by decoration of avidin matrixes with biotinylated enzymes, or by cross-linking biotinylated proteins that then are linked to biotinylated enzymes via an avidin couple. Several formats are explored, including microparticles that can be translocated to desired sites of action (including cytosolic positions), protein pads that generate product gradients within cell cultures, and on-column nanoreactors for microfluidic systems. These biomaterial fabrication technologies offer opportunities for studying a variety of cell functions, ranging from single-cell biochemistry and development to perturbation and analysis of small populations of cultured cells.     

Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp protocols

Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABAA receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABAA receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABAA receptors.     

Characterization of functionalized nanoporous supports for protein confinement

Here we characterize a highly efficient approach for protein confinement and enzyme immobilization in NH(2)-?or HOOC-?functionalized mesoporous silica (FMS) with pore sizes as large as tens of nanometres. We observed a dramatic increase of enzyme loading in both enzyme activity and protein amount when using appropriate FMS in comparison with unfunctionalized mesoporous silica and normal porous silica. With different protein loading density in NH(2)-FMS, the negatively charged glucose oxidase (GOX) displayed an immobilization efficiency (I(e), the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in stock solution) in a range from 30% to 160%, while the same charged glucose isomerase (GI) showed an I(e) of 100% to 120%, and the positively charged organophosphorus hydrolase (OPH) exhibited I(e) of more than 200% in HOOC-FMS. The enzyme-FMS composite was stained with the charged gold nanoparticles and imaged by transmission electron microscopy (TEM). Fourier transform infrared (FTIR) spectroscopy showed no major secondary structural change for the enzymes entrapped in FMS. Thanks to the large, rigid, open pore structure of FMS, the reaction rate and K(m) of the entrapped enzymes in FMS were comparable to those of the free enzymes in solution. In principle, the general approach described here should be applicable to many enzymes, proteins, and protein complexes since both pore sizes and functional groups of FMS are controllable.     

Lensfree sensing on a microfluidic chip using plasmonic nanoapertures

We demonstrate lensfree on-chip sensing within a microfluidic channel using plasmonic nanoapertures that are illuminated by a partially coherent quasimonochromatic source. In this approach, lensfree diffraction patterns of metallic nanoapertures located at the bottom of a microfluidic channel are recorded using an optoelectronic sensor-array. These lensfree diffraction patterns can then be rapidly processed, using phase recovery techniques, to back propagate the optical fields to an arbitrary depth, creating digitally focused complex transmission patterns. Cross correlation of these patterns enables lensfree on-chip sensing of the local refractive index surrounding the near-field of the plasmonic nanoapertures. Based on this principle, we experimentally demonstrate lensfree sensing of refractive index changes as small as ～2×10(-3). This on-chip sensing approach could be quite useful for development of label-free microarray technologies by multiplexing thousands of plasmonic structures on the same microfluidic chip, which can significantly increase the throughput of sensing.     

Microfluidic device for the discrimination of single-nucleotide polymorphisms in DNA oligomers using electrochemically actuated alkaline dehybridization

This work describes an integrated microfluidic (mu-fl) device that can be used to effect separations that discriminate single-nucleotide polymorphisms (SNP) based on kinetic differences in the lability of perfectly matched (PM) and mismatched (MM) DNA duplexes during alkaline dehybridization. For this purpose a 21-base single-stranded DNA (ssDNA) probe sequence was immobilized on agarose-coated magnetic beads, that in turn can be localized within the channels of a poly(dimethylsiloxane) microfluidic device using an embedded magnetic separator. The PM and MM ssDNA targets were hybridized with the probe to form a mixture of PM and MM DNA duplexes using standard protocols, and the hydroxide ions necessary for mediating the dehybridization were generated electrochemically in situ by performing the oxygen reduction reaction (ORR) using O2 that passively permeates the device at a Pt working electrode (Pt-WE) embedded within the microfluidic channel system. The alkaline DNA dehybridization process was followed using fluorescence microscopy. The results of this study show that the two duplexes exhibit different kinetics of dehybridization, rate profiles that can be manipulated as a function of both the amount of the hydroxide ions generated and the mass-transfer characteristics of their transport within the device. This system is shown to function as a durable platform for effecting hybridization/dehybridization cycles using a nonthermal, electrochemical actuation mechanism, one that may enable new designs for lab-on-a-chip devices used in DNA analysis.     

Microfluidics: Accelerated Biofluid Filling in Complex Microfluidic Networks by Vacuum‐Pressure Accelerated Movement (V‐PAM) (Small 33/2016)

Rapid fluid transport and exchange are critical operations involved in many microfluidic applications. However, conventional mechanisms used for driving fluid transport in microfluidics, such as micropumping and high pressure, can be inaccurate and difficult for implementation for integrated microfluidics containing control components and closed compartments. Here, a technology has been developed termed Vacuum–Pressure Accelerated Movement (V‐PAM) capable of significantly enhancing biofluid transport in complex microfluidic environments containing dead‐end channels and closed chambers. Operation of the V‐PAM entails a pressurized fluid loading into microfluidic channels where gas confined inside can rapidly be dissipated through permeation through a thin, gas‐permeable membrane sandwiched between microfluidic channels and a network of vacuum channels. Effects of different structural and operational parameters of the V‐PAM for promoting fluid filling in microfluidic environments have been studied systematically. This work further demonstrates the applicability of V‐PAM for rapid filling of temperature‐sensitive hydrogels and unprocessed whole blood into complex irregular microfluidic networks such as microfluidic leaf venation patterns and blood circulatory systems. Together, the V‐PAM technology provides a promising generic microfluidic tool for advanced fluid control and transport in integrated microfluidics for different microfluidic diagnosis, organs‐on‐chips, and biomimetic studies.     

Design of a compact disk-like microfluidic platform for enzyme-linked immunosorbent assay

This paper presents an integrated microfluidic device on a compact disk (CD) that performs an enzyme-linked immunosorbent assay (ELISA) for rat IgG from a hybridoma cell culture. Centrifugal and capillary forces were used to control the flow sequence of different solutions involved in the ELISA process. The microfluidic device was fabricated on a plastic CD. Each step of the ELISA process was carried out automatically by controlling the rotation speed of the CD. The work on analysis of rat IgG from hybridoma culture showed that the microchip-based ELISA has the same detection range as the conventional method on the 96-well microtiter plate but has advantages such as less reagent consumption and shorter assay time over the conventional method.     

Quantitative mass spectrometric determination of methylphenidate concentration in urine using an electrospray ionization source integrated with a polymer microchip

We have demonstrated the use of a simple microfabricated electrospray ionization source for coupling microfluidic chips to mass spectrometry (MS). A polymer-based microchip, coupled to a triple quadrupole mass spectrometer, has been employed for direct infusion quantitative bioanalysis of methylphenidate (Ritalin) extracted from human urine samples. The approach used a microfabricated polymer electrospray emitter to couple a microfluidic channel to a stable electrospray ionization source. The microchip was fabricated from cycloolefin plastic plate by hot embossing and thermal bonding. This microfluidic chip contained two independent microfluidic channels, integrated with two corresponding electrospray emitters and an internal gold electrode. Liquid-liquid extraction was used to prepare urine samples, spiked with methylphenidate. A trideuterated analogue of methylphenidate (methylphenidate-d(3)) was used as the internal standard for the analysis. The system showed good electrospray stability and reproducibility with different spray tips. Four different electrospray tips were used to analyze the same sample, and the results showed very small variation with a relative standard deviation of 1.4%. A standard curve prepared for methylphenidate in urine (R(2) = 0.999) was linear over the range of 0.4-800 ng/mL. The precision of the quality control samples for three different concentrations ranged from 19.1% at 20 ng/mL, 3.2% at 200 ng/mL, to 3.5% at 667 ng/mL while the accuracy was 96.3% at 20 ng/mL, 101.2% at 200 ng/mL, and 101.6% at 667 ng/mL. No system carryover was detected even when the same device was used for sequential analysis. These results suggest the potential of this microdevice for MS-based quantitative analysis in drug discovery and development.     

Automated control of local solution environments in open-volume microfluidics

We present an open-volume microfluidic system capable of on-line modification of a patterned laminar flow by using programmable inlet valves. Each separate solution environment in the flow pattern can be independently exchanged between different preloaded input solutions where each exchange requires 20 s. The number of flow patterns that can be generated by one device is N(n), where N represents the number of valve inlets and n the number of microchannels in the microfluidic system. Furthermore, the system can be operated as a combinatorial mixer, in which mixture of the different input solutions can be obtained independently in each channel. Since the flow patterns are generated in an open volume, they are accessible to many different detection methods and types of probes, e.g., microelectrodes, cells, or cell fragments. This technology offers the possibility to adjust the flow pattern composition in response to an output from a probe. This is the first step toward creating an automated feedback device controlled by, for example, biological cells.     

A microfluidic paper-based electrochemical biosensor array for multiplexed detection of metabolic biomarkers

Abstract

Paper-based microfluidic devices have emerged as simple yet powerful platforms for performing low-cost analytical tests. This paper reports a microfluidic paper-based electrochemical biosensor array for multiplexed detection of physiologically relevant metabolic biomarkers. Different from existing paper-based electrochemical devices, our device includes an array of eight electrochemical sensors and utilizes a handheld custom-made electrochemical reader (potentiostat) for signal readout. The biosensor array can detect several analytes in a sample solution and produce multiple measurements for each analyte from a single run. Using the device, we demonstrate simultaneous detection of glucose, lactate and uric acid in urine, with analytical performance comparable to that of the existing commercial and paper-based platforms. The paper-based biosensor array and its electrochemical reader will enable the acquisition of high-density, statistically meaningful diagnostic information at the point of care in a rapid and cost-efficient way.