Prototype Microfluidic Device Research Articles

Integrated Plastic Microfluidic Device for Heavy Metal Ion Detection.

The presence of heavy metal ions in soil, air and water constitutes an important global environmental threat, as these ions accumulate throughout the food chain, contributing to the rise of chronic diseases, including, amongst others, cancer and kidney failure. To date, many efforts have been made for their detection, but there is still a need for the development of sensitive, low-cost, and portable devices able to conduct on-site detection of heavy metal ions. In this work, we combine microfluidic technology and electrochemical sensing in a plastic chip for the selective detection of heavy metal ions utilizing DNAzymes immobilized in between platinum nanoparticles (PtNPs), demonstrating a reliable portable solution for water pollution monitoring. For the realization of the microfluidic-based heavy metal ion detection device, a fast and easy-to-implement fabrication method based on the photolithography of dry photosensitive layers is proposed. As a proof of concept, we demonstrate the detection of Pb2+ ions using the prototype microfluidic device.

Rapid microfluidics prototyping through variotherm desktop injection molding for multiplex diagnostics.

In this work, we demonstrate an inexpensive method of prototyping microfluidics using a desktop injection molding machine. A centrifugal microfluidic device with a novel central filling mechanism was developed to demonstrate the technique. We overcame the limitations of desktop machines in replicating microfluidic features by variotherm heating and cooling the mold between 50 °C and 110 °C within two minutes. Variotherm heating enabled good replication of microfeatures, with a coefficient of variation averaging only 3.6% attained for the measured widths of 100 μm wide molded channels. Using this methodology, we produced functional polystyrene centrifugal microfluidic chips, capable of aliquoting fluids into 5.0 μL reaction chambers with 97.5% accuracy. We performed allele-specific loop-mediated isothermal amplification (AS-LAMP) reactions for genotyping CYP2C19 alleles on these chips. Readouts were generated using optical pH sensors integrated onto chips, by drop-casting sensor precursor solutions into reaction chambers before final chip assembly. Positive reactions could be discerned by decreases in pH sensor fluorescence, thresholded against negative control reactions lacking the primers for nucleic acid amplification and with time-to-results averaging 38 minutes. Variotherm desktop injection molding can enable researchers to prototype microfluidic devices more cost-effectively, in an iterative fashion, due to reduced costs of smaller, in-house molds. Designs prototyped this way can be directly translated to mass production, enhancing their commercialization potential and positive impacts.

Microporous affinity membranes and their incorporation into microfluidic devices for monitoring of therapeutic antibodies

Control of monoclonal antibody (mAb) concentrations in serum is important for maintaining the safety and efficacy of these lifesaving therapeutics. Point-of-care (POC) quantification of therapeutic mAbs could ensure that patients have effective mAb levels without compromising safety. This work uses mimotope-functionalized microporous alumina affinity membranes in vertical flow assays for detection and quantitation of therapeutic mAbs. Selective capture of bevacizumab from 1000:1 diluted serum or plasma and binding of a fluorescently labelled anti-human IgG secondary antibody enable fluorescence-based analysis of bevacizumab at its therapeutically relevant concentration range of ∼50–300 μg/mL. The assay results in a linear relationship between the fluorescence intensity of the antibody capture spot and the bevacizumab concentration. A simple prototype microfluidic device containing these membranes allows washing, reagent additions and visualization of signal within 15 min using a total of 5 mL of fluid. The prototype devices can monitor physiologically relevant bevacizumab levels in diluted serum, and future refinements might lead to a POC device for therapeutic drug monitoring.

Rapid detection of SARS-CoV-2 RNA in saliva via Cas13.

Rapid nucleic acid testing is central to infectious disease surveillance. Here, we report an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device. The assay, which we named DISCoVER (for diagnostics with coronavirus enzymatic reporting), involves extraction-free sample lysis via shelf-stable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore. The device consists of a single-use gravity-driven microfluidic cartridge inserted into a compact instrument for automated running of the assay and readout of fluorescence within 60 min. DISCoVER can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva with a sensitivity of 40 copies μl-1, and was 94% sensitive and 100% specific when validated (against quantitative PCR) using total RNA extracted from 63 nasal-swab samples (33 SARS-CoV-2-positive, with cycle-threshold values of 13-35). The device correctly identified all tested clinical saliva samples (10 SARS-CoV-2-positive out of 13, with cycle-threshold values of 23-31). Rapid point-of-care nucleic acid testing may broaden the use of molecular diagnostics.

Rapid prototyping of fluoropolymer microchannels by xurography for improved solvent resistance.

Microchannels made of fluoropolymers show potential merits due to their excellent solvent resistance, but such channels have not been widely used because of the complexity to fabricate them. This communication describes a method to prototype microfluidic devices using fluoropolymer films. The fabrication requires only two steps; cutting fluoropolymer films with a desktop cutting plotter and applying heat and pressure to laminate them. The method is rapid, simple, and low-cost. The conditions for heat press were identified for two common fluoropolymers: polytetrafluoroethylene and fluorinated ethylene propylene. The laminated films were confirmed to remain sealed with an internal pressure of at least 300 kPa. The fabricated devices were tested for the resistance to a set of organic solvents that would not be compatible with typical devices fabricated in polydimethylsiloxane. To highlight the potential of the fluoropolymer devices fabricated in this method, generation of droplets in a continuous stream of organic solvent using a T-junction channel was demonstrated. Our method offers a simple avenue to prototype microfluidic devices to conduct experiments involving organic solvents such as organic chemistry and in-channel synthesis of microparticles.

Electrode-assisted trapping and release of droplets on hydrophilic patches in a hydrophobic microchannel

In two-phase flow microfluidics, there is an increasing interest in technologies which enable the encapsulation of biological cells into aqueous drops and the subsequent study of their molecular (excretion or lysis) products. One not yet available but very promising analysis method is the use of biospecific surface patches embedded in the wall of microfluidic channels. In this paper, we tackle some technological challenges encountered in the development of such applications. In the detection protocol, each drop must be enabled to wet the designated patch, be held in contact long enough for biomolecular detection and subsequently be released. This is engineered via a combination of well-defined chemical sites in the walls of the flow channel and insulated microelectrodes. The tunability of the local electric field allows to modify the competition between chemical (pinning) forces which tend to immobilize the drop and hydrodynamic forces which oppose this process. We developed a prototype microfluidic device which offers this functionality. A channel structure is sandwiched between an actuation surface with electrowetting (EW) electrodes on one side and a detector surface with a hydrophilic patch amidst a hydrophobic environment on the other. Two pairs of carefully aligned EW electrodes are used: one for drop adherence and another one for the subsequent release. We demonstrate these operations and discuss the required voltage signals in terms of the forces on the drop. Finally, we discuss possible steps for further improvement in the device.

Fabrication of a Microfluidic Device with Boron-doped Diamond Electrodes for Electrochemical Analysis

A prototype microfluidic device using boron-doped diamond (BDD) electrodes patterned on an alumina chip was designed and fabricated. Electrochemical microfluidic devices have advantages in that the amount of sample required is small, the measurement throughput is high, different functions can be integrated on a single device, and they are highly durable. In using the device for the flow injection analysis of oxalic acid, the application of a brief conditioning step ensured that the reproducibility of the current signal was excellent.Furthermore, the fabricated system also performed as a prototype of “elimination-detection flow system”, in which interfering species are eliminated using “elimination electrodes” prior to the species reaching the “detection electrode”. The fabricated device reduced the current due to interfering species by 78%. Designs of devices to improve this efficiency are also discussed.

Melt-and-mold fabrication (MnM-Fab) of reconfigurable low-cost devices for use in resource-limited settings

Interest in low-cost analytical devices (especially for diagnostics) has recently increased; however, concomitant translation to the field has been slow, in part due to personnel and supply-chain challenges in resource-limited settings. Overcoming some of these challenges require the development of a method that takes advantage of locally available resources and/or skills. We report a Melt-and-mold fabrication (MnM Fab) approach to low-cost and simple devices that has the potential to be adapted locally since it requires a single material that is recyclable and simple skills to access multiple devices. We demonstrated this potential by fabricating entry level bio-analytical devices using an affordable low-melting metal alloy, Field's metal, with molds produced from known materials such as plastic (acrylonitrile–butadiene–styrene (ABS)), glass, and paper. We fabricated optical gratings then 4×4 well plates using the same recycled piece of metal. We then reconfigured the well plates into rapid prototype microfluidic devices with which we demonstrated laminar flow, droplet generation, and bubble formation from T-shaped channels. We conclude that this MnM-Fab method is capable of addressing some challenges typically encountered with device translation, such as technical know-how or material supply, and that it can be applied to other devices, as needed in the field, using a single moldable material.

Method of mould alignment for double‐sided hot embossing of microfluidic devices using kinematic constraints

Demand has increased for the double-sided moulding of microfluidic devices for a series of biochemical analyses. Accurate alignment of mould inserts is a critical aspect to transfer doubled-sided patterns on the top and bottom surfaces of a moulded part. A method of mould alignment was studied for double-sided hot embossing of polymer microfluidic devices. An alignment method was designed using kinematic constraints, and a set of three locating pins. To validate the alignment method, two brass mould inserts with fluidic reservoirs were used for the double-sided moulding of prototype microfluidic devices. The kinematic constraints were used to align the mould inserts with each other on the hot embossing machine. The misalignment of double-sided features on the moulded parts was measured using a measuring microscope. The X- and Y-magnitudes of the mismatches ranged from −28 to 118 µm along the X-axis of the moulded parts. The X- and Y-magnitudes of the mismatches along the Y-axis of the moulded parts varied from −38 to 141 µm. The results of the experiments showed that the mould alignment using kinematic constraints is applicable to the fabrication of double-sided patterns for microfluidic devices.

High reliability nanosandwiched Pt/Ti multilayer electrode actuators for on-chip biomedical applications

Microactuators provide the main driving force for fluid pumping in microfluidic devices and thus play an important role in on-chip biomedical applications. Interdigitated electrode based electrochemical actuators have provided a viable choice for effective actuation with advantages of flexible controllability, biocompatibility and ease of fabrication. However, the current feature size of a typical electrode structure is around 100 μm, which is relatively large for device miniaturization and integration. Further decrease in the feature size will lead to dramatic decrease in the reliability and lifetime of the actuators, caused by metal delamination. In this work, we propose a novel design of electrodes to fabricate a new type of microactuator with high reliability. To prevent the occurrence of delamination, a nanosandwiched multilayer structure of titanium/platinum is used to construct the conductive metal layer for the interdigitated electrodes. The feature size of the electrodes is greatly reduced to 20 μm where we have reduced the size by 80% of the similar structures reported previously. At the same time, the lifetime of the new electrodes has been dramatically increased to over 400% as compared to the conventional design. With these remarkable improvements in the electrode design, we have fabricated a prototype microfluidic device integrating the new microactuator for drug tests in cancer therapy, demonstrating its usefulness for on-chip biomedical applications.

A microfluidic optical beam steerer

This paper reports the fabrication and characterization of a prototype microfluidic device that can act as a periodic beam steerer. The prototype is formed by a simple T-junction followed by a serpentine channel that allows generation of a periodical segmented flow of air and water bubbles. If light hits the channel wall with a suitable angle, it can be either transmitted or reflected by the segmented flow, giving rise to an alternating beam steerer. The duty cycle, switching frequency, and overall stability and reproducibility of this prototype system are presented and discussed.

Abstract 5265: Development of a microfluidic device for tagless analysis of cell surface proteins directing lung metastasis

Abstract Diagnostic analysis of the few tumor cells obtained by needle aspirates is currently limited to the cells’ morphologic characteristics. We are investigating tagless analysis of cell surface protein composition, when limited cell numbers are available, using a flow retardation microfluidic device. Expression of biologically significant cell surface proteins by individual cells is scored positive by achievement of flow retardation due to transient binding of cell surface proteins to cognizant antibodies immobilized on tandem patches of the channel. This permits repetitive queries of the same cell for several proteins that predict specific malignant behavior. We are adapting the model to LM2 cells, derived from lung metastases of MDA-MB-231 breast cancer cells, which selectively metastasize to the lung*. The original study identified 95 unique genes either overexpressed or underexpressed in LM2 cells compared to parental cells. Of these we identified three cell surface proteins, VCAM1, IL13RA2, and EMP1, as heterogeneously upregulated in a subset of LM2 cells and CXCR4, as downregulated in a subset of LM2 cells when compared with MDA-MB-231 cells. VCAM1 and IL13RA2 promote lung metastasis*. We sterile sorted LM2 cells into fractions expressing none, medium and high levels of these four proteins. We built a prototype microfluidic device able to retard the flow of streptavidin-coated silica oxide beads by immobilized biotin and used optical imaging and a tracking program for simultaneous analysis of multiple particles. We developed efficient and stable protein coating of microchannels and are generating patches with cognizant antibodies to these 4 antigens and to endothelial, macrophage, lymphocyte, fibroblast and endothelial cell markers as controls. We will correlate antigen expression levels with flow retardation efficiency. We will optimize shear rates affecting bond formation and shear stress affecting bond dissociation by optimizing velocity, viscosity, detergent and divalent cation concentrations to maximize cell separation. We will adapt the channel to select LM2 cells from excess controls in vitro and from tumor xenograft aspirates. *Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL and Massague J (2005) Genes that mediate breast cancer metastasis to lung. Nature 436:518-524. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5265. doi:10.1158/1538-7445.AM2011-5265

Time correlated fluorescence characterization of an asymmetrically focused flow in a microfluidic device

In this study, we explore and model the behavior of a prototype microfluidic device which employs two non-mixing fluids (sheath and inlet fluids) displaying an asymmetric focused flow, in the presence of a fluorescent dye. Fluorescence correlation spectroscopy is employed, allowing the precise measure of flow speeds across the channels and of the concentration profile of the central focused flux along the flow direction. The system is modeled via a standard Navier–Stokes finite-element approach, coupled to convection–diffusion equations for the solute. Simulations reproduce accurately the shape, the position, and the width of the velocity and concentration profiles along the central channel and across the transversal and vertical sections of the microfluidic device. The observed asymmetric flow with respect to the center of the channel is reproduced numerically with an error in the position determination smaller than 1%.

High-throughput droplet PCR

The polymerase chain reaction has facilitated the ready analysis of nucleic acids. A next challenge requires the development of means to unravel the complexity of heterogeneous tissues. This has presented the task of producing massively parallelized quantitative nucleic acid data from the cellular constituents of tissues. The production of aqueous droplets in a two phase flow is shown to be readily and routinely facilitated by miniaturized fluidic devices. Droplets serve as ideal means to package a future generation of PCR, offering an enhanced handling potential by virtue of reactant containment, to concurrently eliminate both contamination and sample loss. This containment also enables the measurement of nucleic acids from populations of cells, or molecules by means of high throughput, single cell analysis. Details are provided for the production of a prototype micro-fluidic device which shows the production and stable flow of droplets which we suggest will be suitable for droplet-based continuous flow micro-fluidic PCR. Suggestions are also made as to the optimal fabrication techniques and the importance of device calibration.

Continuous dielectrophoretic cell separation microfluidic device

We present a prototype microfluidic device developed for the continuous dielectrophoretic (DEP) fractionation and purification of sample suspensions of biological cells. The device integrates three fully functional and distinct units consisting of an injector, a fractionation region, and two outlets. In the sheath and sample injection ports, the cell sample are hydrodynamically focused into a stream of controlled width; in the DEP fractionation region, a specially shaped nonuniform (isomotive) electric field is synthesized and employed to facilitate the separation, and the sorted cells are then delivered to two sample collection ports. The microfluidic behavior of the injector region was simulated and then experimentally verified. The operation and performance of the device was evaluated using yeast cells as model biological particles. Issues relating to the fabrication and operation of the device are discussed in detail. Such a device takes a significant step towards an integrated lab-on-a-chip device, which could interface/integrate to a number of other on-chip components for the device to undertake the whole laboratory procedure.

Analysis of Electrically Induced Swirling Flow of Isotonic Saline in a Mixing Microchannel

We have designed a prototype microfluidic device to mix suspended particles with isotonic saline by use of electrically induced swirling flow in the microchannel. However, the principles underlying microfluidic rotation induced by AC electrodes are not well understood, and the characteristics of the rotation velocity are unpredictable. Furthermore, these properties have not been studied using a highly conductive liquid like isotonic saline, which is an important fluid in the medical and biological fields. The lack of such studies causes uncertainty in the design required for high-performance microfluidic devices. We have examined the electrical rotational properties of the microfluid at an isotonic concentration of saline using computer simulation, and here we show that buoyant flow, which has previously been largely ignored, has a significant effect in channels of 100-μm depth or deeper, and that AC electroosmotic flow is not induced at isotonic saline concentrations.

Microfluidic electroporation of robust 10-μm vesicles for manipulation of picoliter volumes

We present a new way to transport and handle picoliter volumes of analytes in a microfluidic context through electrically monitored electroporation of 10–25 μm vesicles. In this method, giant vesicles are used to isolate analytes in a microfluidic environment. Once encapsulated inside a vesicle, contents will not diffuse and become diluted when exposed to pressure-driven flow. Two vesicle compositions have been developed that are robust enough to withstand electrical and mechanical manipulation in a microfluidic context. These vesicles can be guided and trapped, with controllable transfer of material into or out of their confined environment. Through electroporation, vesicles can serve as containers that can be opened when mixing and diffusion are desired, and closed during transport and analysis. Both vesicle compositions contain lecithin, an ethoxylated phospholipid, and a polyelectrolyte. Their performance is compared using a prototype microfluidic device and a simple circuit model. It was observed that the energy density threshold required to induce breakdown was statistically equivalent between compositions, 10.2 ± 5.0 mJ/m 2 for the first composition and 10.5 ± 1.8 mJ/m 2 for the second. This work demonstrates the feasibility of using giant, robust vesicles with microfluidic electroporation technology to manipulate picoliter volumes on-chip.

Xurography: rapid prototyping of microstructures using a cutting plotter

This paper introduces xurography, or razor writing, as a novel rapid prototyping technique for creating microstructures in various films. This technique uses a cutting plotter traditionally used in the sign industry for cutting graphics in adhesive vinyl films. A cutting plotter with an addressable resolution of 10 /spl mu/m was used to cut microstructures in various films with thicknesses ranging from 25 to 1000 /spl mu/m. Positive features down to 35 /spl mu/m and negative features down to 18 /spl mu/m were cut in a 25 /spl mu/m thick material. Higher aspect ratios of 5.2 for positive features and 8 for negative features were possible in a 360 /spl mu/m thick material. A simple model correlating material properties to minimum feature size is introduced. Multilayered microstructures cut from pressure sensitive and thermal activated adhesive films were laminated in less than 30 min without photolithographic processes or chemicals. Potential applications of these microstructures are explored including: shadow masking, electroplating, micromolds for PDMS, and multilayered three-dimensional (3-D) channels. This inexpensive method can rapidly prototype microfluidic devices or tertiary fluid connections for higher resolution devices. [1488].

The potential of autofluorescence for the detection of single living cells for label‐free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.

Direct Drawing of Suspended Filamentary Micro- and Nanostructures from Liquid Polymers

Using various nanomanipulating instruments, solvated polymers are simultaneously formed into fibers, adhered to solid supports, and interconnected in real-time to create suspended fiber bridges and networks of specified geometries. Fibers from 50 nm to 20 μm diameter have been drawn individually and in parallel using single tips and tip arrays. The speed and ease of producing suspended three-dimensional structures recommends the method for application to custom fabrication of prototype microfluidic and microoptical devices.