Modeling and optimization of a multi-enzyme electrokinetically driven multiplexed microchip for simultaneous detection of sugars

A model-based methodology was developed to optimize microfluidic chips for the simultaneous enzymatic quantification of sucrose, d-glucose and d-fructose in a single microfluidic channel with an integrated optical detection system. The assays were based on measuring the change in concentration of the reaction product NADH, which is stoichiometrically related to the concentration of those components via cascade of specific enzymatic reactions. A reduced order mathematical model that combines species transport, enzyme reaction, and electrokinetic bulk flow was developed to describe the operation of the microfluidic device. Using this model, the device was optimized to minimize sensor response time and maximize signal output by manipulating the process conditions such as sample and reagent volume and flow rate. According to this simulation study, all sugars were quantified within 2.5 min in the optimized microchip. A parallel implementation of the assays can further improve the throughput. In addition, the amount of consumed reagents was drastically reduced compared to microplate format assays. The methodology is generic and can easily be adapted to other enzymatic microfluidic chips.     

Design optimization of an enzymatic assay in an electrokinetically-driven microfluidic device

Microfluidic systems are increasingly popular for rapid and cheap determinations of enzyme assays and other biochemical analysis. In this study reduced order models (ROM) were developed for the optimization of enzymatic assays performed in a microchip. The model enzyme assay used was β-galactosidase (β-Gal) that catalyzes the conversion of Resorufin β-d-galactopyranoside (RBG) to a fluorescent product as previously reported by Hadd et al. (Anal Chem 69(17): 3407–3412, 1997). The assay was implemented in a microfluidic device as a continuous flow system controlled electrokinetically and with a fluorescence detection device. The results from ROM agreed well with both computational fluid dynamic (CFD) simulations and experimental values. While the CFD model allowed for assessment of local transport phenomena, the CPU time was significantly reduced by the ROM approach. The operational parameters of the assay were optimized using the validated ROM to significantly reduce the amount of reagents consumed and the total biochip assay time. After optimization the analysis time would be reduced from 20 to 5.25 min which would also resulted in 50% reduction in reagent consumption.     

Construction and characterisation of a modular microfluidic system: coupling magnetic capture and electrochemical detection

This work presents the fabrication and characterisation of a versatile lab-on-a-chip system that combines magnetic capture and electrochemical detection. The system comprises a silicon chip featuring a series of microband electrodes, a PDMS gasket that incorporates the microfluidic channels, and a polycarbonate base where permanent magnets are hosted; these parts are designed to fit so that wire bonding and encapsulation are avoided. This system can perform bioassays over the surface of magnetic beads and uses only 50 μL of bead suspension per assay. Following detection, captured beads are released simply by sliding a thin iron plate between the magnets and the chip. Particles are captured upstream from the detector and we demonstrate how to take further advantage of the system fluidics to determine enzyme activities or concentrations, as flow velocity can be adjusted to the rate of the reactions under study. We used magnetic particles containing β-galactosidase and monitored the enzyme activity amperometrically by the oxidation of 4-aminophenol, enzymatically produced from 4-aminophenyl-β-d-galactopyranoside. The system is able to detect the presence of enzyme down to approximately 50 ng mL⁻¹.     

Controlling droplet size variability of a digital lab-on-a-chip for improved bio-assay performance

Although several applications of electrowetting on dielectric digital lab-on-a-chips are reported in literature, there is still a lack of knowledge about the influence of operational and design parameters on the performance of an analytical assay. This paper investigates how droplet size variability, introduced by droplet dispensing and splitting, influences the assay performance with respect to repeatability and accuracy and presents a novel method to reduce this variability. Both a theoretical and experimental approach were followed. Monte Carlo simulations were applied to study the cumulative effect of the variability caused by different droplet manipulations on the final assay performance. It is shown that a highly controllable droplet generation and manipulation is achieved with respect to droplet size variability through an accurate control of actuation voltage, activation time, relaxation time, and electrode size. As a case study, it is illustrated that through optimization of these parameters a complete on-chip calibration curve is obtained for a d-glucose assay with an average CV-value of 2%. These new insights aim to bring the digital lab-on-a-chip technology closer to researchers in the field of diagnostics offering them a valuable and accessible alternative to standard analysis platforms.     

A facile strategy to integrate robust porous aluminum foil into microfluidic chip for sorting particles

High efficiency integration of functional microdevices into microchips is crucial for broad microfluidic applications. Here, a device-insertion and pressure sealing method was proposed to integrate robust porous aluminum foil into a microchannel for microchip functionalization which demonstrate the advantage of high efficient foil microfabrication and facile integration into the microfluidic chip. The porous aluminum foil with large area (10 × 10 mm²) was realized by one-step femtosecond laser perforating technique within few minutes and its pores size could be precisely controlled from 3 μm to millimeter scale by adjusting the laser pulse energy and pulse number. To verify the versatility and flexibility of this method, two kinds of different microchips were designed and fabricated. The vertical-sieve 3D microfluidic chip can separate silicon dioxide (SiO₂) microspheres of two different sizes (20 and 5 μm), whereas the complex stacking multilayered structures (sandwich-like) microfluidic chip can be used to sort three different kinds of SiO₂ particles (20, 10 and 5 μm) with ultrahigh separation efficiency of more than 92%. Furthermore, these robust filters can be reused via cleaning by backflow (mild clogging) or disassembling (heavy clogging).     

Development of high throughput microfluidic cell culture chip for perfusion 3-dimensional cell culture-based chemosensitivity assay

This study reports a microfluidic cell culture chip encompassing 36 microbioreactors for high throughput perfusion 3-dimensional (3D) cell culture-based chemosensitivity assays. Its advantages include the capability for multiplexed medium delivery, and the function for both efficient and high throughput micro-scale 3D culture construct preparation and loading. The results showed that the proposed medium pumping mechanism was able to provide a uniform pumping rates ranging from 1.2 to 3.9 μl h⁻¹. In addition, the simple cell/hydrogel loading scheme has been proven to be able to carry out 3D cell culture construct preparation and loading precisely and efficiently. Furthermore, a chemosensitivity assay was successfully demonstrated using the proposed cell culture chip. The results obtained were also compared with the same evaluation based on a conventional 2D monolayer cell culture. It can be concluded that the choice of cell culture format can result in different chemosensitivity evaluation results. Overall, because of the nature of miniaturized perfusion 3D cell culture, the cell culture chip not only can provide stable, well-defined and more biologically relevant culture environments, but it also features low consumption of research resources. All these traits are found particularly useful for high-precision and high-throughput 3D cell culture-based assays.     

Development of a capillary flow microfluidic Escherichia coli biosensor with on-chip reagent delivery using water-soluble nanofibers

Although the potential role of microfluidics in point of care diagnostics is widely acknowledged, the practical limitations to their use still limit deployment. Here, we developed a capillary flow microfluidic with on-chip reagent delivery which combines a lateral flow assay with microfluidic technology. The horseradish peroxidase tagged antibody was electrospun in a water-soluble polyvinylpyrrolidone nanofibers and stored in a microfluidic poly(methyl methacrylate) chip. During the assay, the sample containing Escherichia coli on immunomagnetic beads came in contact with the nanofibers causing them to dissolve and release the reagents for binding. Following hybridization, the solution moved by capillary flow toward a detection zone where the analyte was quantified using chemiluminescence. The limit of detection was found to be approximately 10⁶ CFU/mL of E. coli O157. More importantly, the ability to store sensitive reagents within a microfluidic as nanofibers was demonstrated. The fibers showed almost instant hydration and dissemination within the sample solution.     

A microfluidic device for array patterning by perpendicular electrokinetic focusing

This paper describes a microfluidic chip in which two perpendicular laminar-flow streams can be operated to sequentially address the surface of a flow-chamber with semi-parallel sample streams. The sample streams can be controlled in position and width by the method of electrokinetic focusing. For this purpose, each of the two streams is sandwiched by two parallel sheath flow streams containing just a buffer solution. The streams are being electroosmotically pumped, allowing a simple chip design and a setup with no moving parts. Positioning of the streams was adjusted in real-time by controlling the applied voltages according to an analytical model. The perpendicular focusing gives rise to overlapping regions, which, by combinatorial (bio) chemistry, might be used for fabrication of spot arrays of immobilized proteins and other biomolecules. Since the patterning procedure is done in a closed, liquid filled flow-structure, array spots will never be exposed to air and are prevented from drying. With this device configuration, it was possible to visualize an array of 49 spots on a surface area of 1 mm². This article describes the principle, fabrication, experimental results, analytical modeling and numerical simulations of the microfluidic chip.     

Portable bead-based fluorescence detection system for multiplex nucleic acid testing: a case study with Bacillus anthracis

This paper describes the design, functioning and use of a portable detection platform for multiplex nucleic acid testing. The system features a bead-supported DNA hybridization assay performed inside a microfluidic cartridge. Polystyrene particles modified with DNA capture probes are confined in the detection area and exposed to a solution of fluorescently labeled target DNA strands. The cartridge, fabricated from inexpensive thermoplastic polymers, allows for conducting up to eight assays in parallel. The detection instrument is equipped with a pneumatic module and a manifold lid serving as an interface to mediate fluid displacement on the cartridge. The fluorescence signal deriving from each assay is recorded by a semi-confocal fluorescence reader embedded in the detection platform. The compact design of the instrument and its level of integration make it possible to obtain an analytical result in less than 15 min, while only few manual steps need to be performed in between. A proof-of-concept demonstration involving Cy3-labeled, PCR-amplified genomic DNA confirms the ability to detect Bacillus anthracis in a multiplexed single-assay format using lef and capC genes. Limits of quantification are on the order of 1 × 10⁹ copies/μL for lef targets.     

Selective Ion Extraction for High Throughput Screening with a Microfluidic Enzyme Assay

This proof-of-concept paper describes the application of selective ion extraction to an assay of protein kinase A on a microfluidic chip platform. Selective ion extraction is a flux balance technique, where a combination of independent pressure control and voltage are used to selectively extract one ion from a mixture. The assay product is completely separated and diverted into a separate channel from the waste stream containing the unconverted substrate and enzyme. By detecting only product, background noise generated by the substrate is removed which increases the signal to noise ratio and assay sensitivity. This technique is intended for adapting kinase or protease assays with low conversion rates to an on-chip reaction format for HTS screening.     

Integrated liquid and droplet dielectrophoresis for biochemical assays

Surface microfluidic systems have emerged as an attractive alternative to conventional closed-channel microfluidic devices. In many such systems, electric fields are leveraged for the manipulation and transport of discrete nanoliter droplets on open planar surfaces. The present research work discusses dielectrophoretic liquid and droplet actuations, which provide an attractive methodology for dispensing and manipulating nanoliter and picoliter droplets on planar surfaces. We demonstrate the integration of two independent sample actuation schemes, namely liquid dielectrophoresis (L-DEP) and droplet dielectrophoresis, and furthermore validate its applicability through model biochemical assays (DNA-PicoGreen^® assay and DNA FRET assay). We also describe and present ‘tapering L-DEP’ actuation scheme, whereby we demonstrate how to simultaneously create multiple droplets of different sizes and volumes in the range of nanoliter and picoliters, from a given larger parent sample droplet.     

On the Weighted Mean of a Pair of Strings

String matching and string edit distance are fundamental concepts in structural pattern recognition. In this paper, the weighted mean of a pair of strings is introduced. Given two strings, x and y, where d(x, y) is the edit distance of x and y, the weighted mean of x and y is a string z that has edit distances d(x, z) and d(z, y)to x and y, respectively, such that d(x, z) _ d(z, y) = d(x, y). We’ll show formal properties of the weighted mean, describe a procedure for its computation, and give practical examples. Received: 26 October 2000, Received in revised form: 27 April 2001, Accepted: 20 July 2001     

Privacy-preserving boosting

We describe two algorithms, BiBoost (Bipartite Boosting) and MultBoost (Multiparty Boosting), that allow two or more participants to construct a boosting classifier without explicitly sharing their data sets. We analyze both the computational and the security aspects of the algorithms. The algorithms inherit the excellent generalization performance of AdaBoost. Experiments indicate that the algorithms are better than AdaBoost executed separately by the participants, and that, independently of the number of participants, they perform close to AdaBoost executed using the entire data set. Responsible Editor: Charu Aggarwal.     

A microfluidic chip for heterogeneous immunoassay using electrokinetical control

This article presents the development of a novel, automated, electrokinetically controlled heterogeneous immunoassay on a poly(dimethylsiloxane) (PDMS) microfluidic chip. A numerical method has been developed to simulate the electrokinetically driven, time-dependent delivery processes of reagents and washing solutions within the complex microchannel network. Based on the parameters determined from the numerical simulations, fully automated on-chip experiments to detect Helicobacter pylori were accomplished by sequentially changing the applied electric fields. Shortened assay time and much less reagent consumptions are achieved by using this microchannel chip while the detection limit is comparable to the conventional assay. There is a good agreement between the experimental result and numerical prediction, demonstrating the effectiveness of using CFD to assist the experimental studies of microfluidic immunoassay.     

基于电渗流的微流控混和芯片系统级建模技术研究

微流控混合芯片是微流控芯片系统中的重要组成部分,其混合效率直接影响后续反应产物的分布和反应体系的容量.文中对基于电渗流驱动的微流控混合芯片的系统级建模技术进行了研究,本研究首先结合基于电渗流驱动的微流控混合芯片的控制方程,对系统各组件的参数化行为模型进行了提取,在此基础上编程实现了系统各组件的多端口组件模型,构建了基于电渗流的微流控混和芯片系统级模型,模型仿真结果与有限元方法相比,相对误差为2.5%,而仿真速度却远远高于有限元方法.表明该方法在不显著损失系统精度的前提下,可以更加有效的对系统性能做出评价.     

Refolding of β-galactosidase: microfluidic device for reagent metering and mixing and quantification of refolding yield

We have developed a device that uses microfluidic valves and pumps to meter reagents for subsequent mixing with application to refolding of the protein β-galactosidase. The microfluidic approach offers the potential advantages of automation, cost-effectiveness, compatibility with optical detection, and reduction in sample volumes as opposed to conventional techniques of hand-pipetting or using robotic systems. The device is a multi-layered poly(dimethylsiloxane) on glass device with automated controls for reagent aliquoting and mixing. Refolding experiments have been performed off-chip using existing protocols on the protein β-galactosidase and the refolding yield has been quantified on-chip using fluorescein di-β-d-galactopyranoside, a caged-fluorescent molecule. This work provides the potential to reduce the cost of drug discovery and realization of protein pharmaceuticals.     

A simple and cost-effective method for fabrication of integrated electronic-microfluidic devices using a laser-patterned PDMS layer

We report a simple and cost-effective method for fabricating integrated electronic-microfluidic devices with multilayer configurations. A CO₂ laser plotter was employed to directly write patterns on a transferred polydimethylsiloxane (PDMS) layer, which served as both a bonding and a working layer. The integration of electronics in microfluidic devices was achieved by an alignment bonding of top and bottom electrode-patterned substrates fabricated with conventional lithography, sputtering and lift-off techniques. Processes of the developed fabrication method were illustrated. Major issues associated with this method as PDMS surface treatment and characterization, thickness-control of the transferred PDMS layer, and laser parameters optimization were discussed, along with the examination and testing of bonding with two representative materials (glass and silicon). The capability of this method was further demonstrated by fabricating a microfluidic chip with sputter-coated electrodes on the top and bottom substrates. The device functioning as a microparticle focusing and trapping chip was experimentally verified. It is confirmed that the proposed method has many advantages, including simple and fast fabrication process, low cost, easy integration of electronics, strong bonding strength, chemical and biological compatibility, etc.     

Development and characterization of a capillary-flow microfluidic device for nucleic acid detection

We have developed a capillary flow-driven microfluidic biosensor to meet the needs of diagnostics for resource-limited areas. The device combined elements of lateral flow assays and microfluidic technology resulting in a hybrid with benefits of both formats. The biosensor was achieved by bonding two pieces of polymethyl methacrylate with channels ablated by a CO₂ laser, and enclosing an absorbent pad. The channels were UV/ozone treated to increase hydrophilicity which enabled capillary flow. The absorbent pad allowed for continuous flow in the channels once filled. The application of biosensor was demonstrated by detection of DNA with a sandwich assay. The target DNA was hybridized with nucleic acid modified magnetic beads as well as Ru(bpy) ₃ ²⁺ doped silica nanoparticles. Fluorescent signals were quantified in a holder fabricated to fit in a fluorescent microtiter plate reader. The capillary flow microfluidic was capable to detect 1?pmol target. The assay format which features rapid analysis and does not require the use of pumps could allow for inexpensive point of care diagnostics in the future.