Onsite fabrication of centrifugal microfluidic cartridges is one way to provide laboratory and diagnostics platforms for extreme point-of-care (EPOC) settings. This paper presents a rapid fabrication process of centrifugal microfluidic cartridges (discs) using only a cutter plotter that is as low-cost, portable and rapid as conventional printers. Moreover, we devised an active valving mechanism to enable the development of complex sequential fluidic processes. The valves are engraved during disc manufacture itself by the cutter plotter. These embedded valves prevent the need for additional fabrication equipment and materials for the inserts that are usually required for other active valves. The valves are actuated by an external mechanical force, and are called active mechanical valves (M-valve). The M-valve is robust over a wide range of spinning speeds (e.g. up to 7000 rpm or 2470 rcf) and can be actuated manually, or automatically by a robotic arm. To demonstrate our approach, we developed two fluidic systems for immunoassay and chromatography. The first microfluidic platform was developed to automate a fluidic protocol usually required for immunoassays from whole blood. In this microfluidic disc, non-biological liquids were used to demonstrate the application of M-valves for robust control over retention and release of reagents. The chromatography microfluidic cartridge is a miniaturized experimental system for testing the capability of a modified resin (Sepharose 6B-PEG5000) for the isolation of monoPEGylated ribonuclease (RNase). The fabrication of microfluidic discs and M-valves by a simple cutter plotter is the fastest and least expensive method for the onsite development and onsite manufacturing of diagnostic kits for research and actual use even in EPOC settings.

Onsite fabrication of centrifugal microfluidic cartridges is one way to provide laboratory and diagnostics platforms for extreme point-of-care (EPOC) settings. This paper presents a rapid fabrication process of centrifugal microfluidic cartridges (discs) using only a cutter plotter that is as low-cost, portable and rapid as conventional printers. Moreover, we devised an active valving mechanism to enable the development of complex sequential fluidic processes. The valves are engraved during disc manufacture itself by the cutter plotter. These embedded valves prevent the need for additional fabrication equipment and materials for the inserts that are usually required for other active valves. The valves are actuated by an external mechanical force, and are called active mechanical valves (M-valve). The M-valve is robust over a wide range of spinning speeds (e.g. up to 7000 rpm or 2470 rcf) and can be actuated manually, or automatically by a robotic arm. To demonstrate our approach, we developed two fluidic systems for immunoassay and chromatography. The first microfluidic platform was developed to automate a fluidic protocol usually required for immunoassays from whole blood. In this microfluidic disc, non-biological liquids were used to demonstrate the application of M-valves for robust control over retention and release of reagents. The chromatography microfluidic cartridge is a miniaturized experimental system for testing the capability of a modified resin (Sepharose 6B-PEG5000) for the isolation of monoPEGylated ribonuclease (RNase). The fabrication of microfluidic discs and M-valves by a simple cutter plotter is the fastest and least expensive method for the onsite development and onsite manufacturing of diagnostic kits for research and actual use even in EPOC settings.

In this study, the bioelectrocatalytic reduction of molecular oxygen by two highly thermostable laccase isoforms from a native strain of Pycnoporus sanguineus CS43 were evaluated and compared to commercially available laccase from Trametes versicolor (TvL). The laccase isoforms (LAC1 and LAC2) and TvL laccase were immobilized by orientation onto anthracene-modified multiwalled carbon nanotubes (AC-MWCNT), which were subsequently immobilized onto carbon nanofiber mat electrodes fabricated using a carbon MEMS (C-MEMS) process. The performances of the isoforms were evaluated at differing pHs, temperatures, and with various inhibitors under hydrodynamic and hydrostatic conditions. Both LAC1 and LAC2 had onset potentials of over +650 mV vs Ag/AgCl at pH 4.0, which are among the highest reported to date for any laccase bioelectrode. High current densities were also obtained, producing 825 ± 88 μA/cm2 and 1220 ± 106 μA/cm2 with LAC1 and LAC2, respectively. The bioelectrodes also demonstrated remarkable operational ranges in pH and temperature as well as increased resistance to common laccase inhibitors. In both cases, they maintained over 70% of their maximum current densities after 12 h of continuous operation at 20 °C and over 20% of their maximum current densities after 6 h of continuous operation at 45 °C. In comparison, the TvL cathodes maintained 50% of their maximum current densities after 12 h at 20 °C and lost all catalytic activity after 2 h at 45 °C. The high stability, onset potential, current densities, and increased inhibitor resilience demonstrated by the results of this study make these isoforms very attractive for applications such as biofuel cell cathodes.Keywords: bioelectrocatalysis; C-MEMS; enzymatic biofuel cells; inhibitor resistance; laccase; oxygen reduction reaction; Pycnoporus sanguineus; thermostability

Expanding upon recent applications of interfacing electricity with centrifugal microfluidic platforms, we introduce electrochemical velocimetry to monitor flow in real-time on rotating fluidic devices. Monitoring flow by electrochemical techniques requires a simple, compact setup of miniaturized electrodes that are embedded within a microfluidic channel and are connected to a peripherally-located potentiostat. On-disc flow rates, determined by electrochemical velocimetry, agreed well with theoretically expected values and with optical measurements. As an application of the presented techniques, the dynamic process of droplet formation and release was recorded, yielding critical information about droplet frequency and volume. Overall, the techniques presented in this work advance the field of centrifugal microfluidics by offering a powerful tool, previously unavailable, to monitor flow in real-time on rotating microfluidic systems.

Bacterial bioreporters are limited in their abilities to detect large polar molecules due to their membrane selectivity. In this study, the activity of serum complement was used to bypass this undesired selectivity. Initially, the serum complement activity was assessed using the responses of a bacterial bioreporter harboring a recA::luxCDABE transcriptional fusion when exposed to the chemotherapy drug, mitomycin C (MMC). Using 50 °C-treated serum, the limit of detection for this bacterial sensor was lowered by nearly 450-fold, from 31 μg/L to 0.07 μg/L MMC. Real-time quantitative PCR demonstrated that serum-treated cultures responded more strongly to 100 μg/L MMC, with 3.1-fold higher recA expression levels. Subsequent experiments with other bioreporter strains also found enhanced sensitivities and responses. Finally, combining each of the above findings, tests were performed to demonstrate the potential application of the recA::luxCDABE bioreporter within a lab-on-a-CD platform as a point-of-care diagnostic to measure chemotherapeutic drug concentrations within blood.Highlights► Bioreporter responses to genotoxins are improved by serum complement activity. ► Mitomycin C can be detected down to 70 parts-per-trillion (ppt). ► This bioreporter system is more sensitive than HPLC for mitomycin C. ► Enhanced effector permeability was verified by fluorescence microscopy/RT-qPCR. ► Demonstrated use within point-of-care diagnostic devices using a centrifugal system.

Single suspended carbon nanowires (CNWs) integrated on carbon-MEMS (CMEMS) structures are fabricated by electrospinning of SU-8 photoresist followed by pyrolysis. These monolithic CNW-CMEMS structures enable fabrication of very high aspect ratio CNWs of predefined length. The CNWs thus fabricated display core–shell structures having a graphitic shell with a glassy carbon core. The electrical conductivity of these CNWs is increased by about 100% compared to glassy carbon as a result of enhanced graphitization. We suggest some tunable fabrication and pyrolysis parameters that may improve graphitization in the resulting CNWs, making them a good replacement for several carbon nanostructure-based devices.Keywords: carbon nanowire; carbon-MEMS; electrospinning; graphitization; photolithography;

We report on the functionalization of a micropatterned carbon electrode fabricated using the carbon-MEMS process for its use as a miniature diffusion-free glucose oxidase anode. Carbon-MEMS based electrodes offer precise manufacturing control on both the micro- and nanoscale and possess higher electron conductivity than redox hydrogels. However, the process involves pyrolysis in a reducing environment that renders the electrode surface less reactive and introduction of a high density of functional groups becomes challenging. Our functionalization strategy involves the electrochemical oxidation of amine linkers onto the electrode. This strategy works well with both aliphatic and aryl linkers and uses stable compounds. The anode is designed to operate through mediated electron transfer between 2,5-dihydroxybenzaldehyde (DHB) based redox mediator and glucose oxidase enzyme. The electrode was first functionalized with ethylene diamine (EDA) to serve as a linker for the redox mediator. The redox mediator was then grafted through reductive amination, and attachment was confirmed through cyclic voltammetry. The enzyme immobilization was carried out through either adsorption or attachment, and their efficiency was compared. For enzyme attachment, the DHB attached electrode was functionalized again through electro-oxidation of aminobenzoic acid (ABA) linker. The ABA functionalization resulted in reduction of the DHB redox current, perhaps due to increased steric hindrance on the electrode surface, but the mediator function was preserved. Enzyme attachment was then carried out through a coupling reaction between the free carboxyl group on the ABA linker and the amine side chains on the enzyme. The enzyme incubation for both adsorption and attachment was done either through a dry spotting method or wet spotting method. The dry spotting method calls for the evaporation of enzyme droplet to form a thin film before sealing the electrode environment, to increase the effective concentration of the enzyme on the electrode surface during incubation. The electrodes were finally protected with a gelatin based hydrogel film. The anode half-cell was tested using cyclic voltammetry in deoxygenated phosphate buffer saline solution pH 7.4 to minimize oxygen interference and to simulate the pH environment of the body. The electrodes that yielded the highest anodic current were prepared by enzyme attachment method with dry spotting incubation. A polarization response was generated for this anodic half-cell and exhibits operation close to maximum efficiency that is limited by the mass transport of glucose to the electrode.

We report on a continuous method for controlled electrospinning of polymeric nanofibers on two-dimensional (2D) and three dimensional (3D) substrates using low voltage near-field electrospinning (LV NFES). The method overcomes some of the drawbacks in more conventional near-field electrospinning by using a superelastic polymer ink formulation. The viscoelastic nature of our polymer ink enables continuous electrospinning at a very low voltage of 200 V, almost an order of magnitude lower than conventional NFES, thereby reducing bending instabilities and increasing control of the resulting polymer jet. In one application, polymeric nanofibers are freely suspended between microstructures of 3D carbon on Si substrates to illustrate wiring together 3D components in any desired pattern.

A novel active valving technique, whereby paraffin wax plugs in microchannels on a centrifugal microfluidic platform are actuated using focused infrared (IR) radiation is demonstrated in this report. Microchannels were simultaneously or sequentially opened using a stationary IR source by forming wax plugs with similar or differing melting points. The presented wax plugs offer key advantages over current active valving techniques, including a less involved fabrication procedure, a simpler actuation process, and the ability to multiplex experiment with active valves. In addition, a new technique for automated liquid reagent storage and release on the microfluidic disc platform, based on the formation and removal of a wax layer, is demonstrated. Overall, the techniques presented in this report offer novel methods for liquid handling, separation, and storage on the centrifugal microfluidic disc platform.

The pumping of fluids in microfluidic discs by centrifugal forces has several advantages, however, centrifugal pumping only permits unidirectional fluid flow, restricting the number of processing steps that can be integrated before fluids reach the edge of the disc. As a solution to this critical limitation, we present a novel pumping technique for the centrifugal microfluidic disc platform, termed the thermo-pneumatic pump (TPP), that enables fluids to be transferred the center of a rotating disc by the thermal expansion of air. The TPP is easy to fabricate as it is a structural feature with no moving components and thermal energy is delivered to the pump via peripheral infrared (IR) equipment, enabling pumping while the disc is in rotation. In this report, an analytical model for the operation of the TPP is presented and experimentally validated. We demonstrate that the experimental behavior of the pump agrees well with theory and that flow rates can be controlled by changing how well the pump absorbs IR energy. Overall, the TPP enables for fluids to be stored near the edge of the disc and transferred to the center on demand, offering significant advantages to the microfluidic disc platform in terms of the handling and storage of liquids.

We introduce the integration of a novel dielectrophoresis (DEP)-assisted filter with a compact disk (CD)-based centrifugal platform. Carbon-electrode dielectrophoresis (carbon-DEP) refers to the use of carbon electrodes to induce DEP. In this work, 3D carbon electrodes are fabricated using the C-MEMS technique and are used to implement a DEP-enabled active filter to trap particles of interest. Compared to traditional planar metal electrodes, 3D carbon electrodes allow for superior filtering efficiency. The system includes mounting modular 3D carbon-DEP chips on an electrically interfaced rotating disk. This allows simple centrifugal pumping to replace the large footprint syringe pump approaches commonly used in DEP systems. The advantages of the CD setup include not only a reduced footprint, but also complexity and cost reduction by eliminating expensive precision pumps and fluidic interconnects. To demonstrate the viability of this system we quantified the filter efficiency in the DEP trapping of yeast cells from a mix of latex and yeast cells. Results demonstrate selective filtering at flow rates up to 35 µl min−1. The impact of electrode height, DEP chip misalignment and particle sedimentation on filter efficiency and the advantages this system represents are analyzed. The ultimate goal is to obtain an automated platform for bioparticle sorting with application in different fields such as point-of-care diagnostics and cell-based therapies.

In this paper, we present the design and characterization of a novel platform for mechanical cell lysis of even the most difficult to lyse cell types on a micro or nanoscale (maximum 70 μL total volume). The system incorporates a machined plastic circular disk assembly, magnetic field actuated microfluidics, centrifugal cells and tissue homogenizer and centrifugation system. The mechanism of tissue disruption of this novel cell homogenization apparatus derives from the relative motion of ferromagnetic metal disks and grinding matrices in a liquid medium within individual chambers of the disk in the presence of an oscillating magnetic field. The oscillation of the ferromagnetic disks or blades produces mechanical impaction and shear forces capable of disrupting cells within the chamber both by direct action of the blade and by the motion of the surrounding lysis matrix, and by motion induced vortexing of buffer fluid. Glass beads or other grinding media are integrated into each lysis chamber within the disk to enhance the transfer of energy from the oscillating metal blade to the cells. The system also achieves the centrifugal elimination of solids from each liquid sample and allows the elution of clarified supernatants via siphoning into a collection chamber fabricated into the plastic disk assembly. This article describes system design, implementation and validation of proof of concept on two samples—Escherichia coli and Saccharomyces cerevisiae representing model systems for cells that are easy and difficult to lyse, respectively.

A low-cost, disposable card for rapid polymerase chain reaction (PCR) was developed in this work. Commercially available, adhesive-coated aluminum foils and polypropylene films were laminated with structured polycarbonate films to form microreactors in a card format. Ice valves [1] were employed to seal the reaction chambers during thermal cycling and a Peltier-based thermal cycler was configured for rapid thermal cycling and ice valve actuation. Numerical modeling was conducted to optimize the design of the PCR reactor and investigate the thermal gradient in the reaction chamber in the direction of sample thickness. The PCR reactor was experimentally characterized by using thin foil thermocouples and validated by a successful amplification of 10 copy of E. coli tuf gene in 27 min.

We have successfully developed a DNA biosensor based on double layer capacitance variations due to DNA hybridization. The study was performed to demonstrate the feasibility of electrical detection of DNA hybridization using electrochemical impedance spectroscopy (EIS). At 100 Hz, electrochemical impedance measurements on a label-free DNA sensor show a 25% increase for dsDNA on a Au electrode compared with the same gold electrode with immobilized ssDNA. Differential experiments were carried out to establish the selectivity of the biosensor; no impedance change was observed at electrodes with non-specific DNA sites. An enzyme amplification scheme employing an impedance amplifying label (IAL) was developed to increase the sensitivity of the DNA sensor. In this approach, after hybridization, the enzymatic precipitation of an insoluble compound on the sensing interface causes a significant impedance change. The total impedance, again measured at 100 Hz, is 160% higher than the impedance with the label-free dsDNA on the electrode.

A dynamic DNA hybridization microfluidic system was developed for a compact disc (CD) platform. The compact disc was used as the fluidic platform for sample and reagent manipulation using centrifugal force. Chambers for reagent storage and conduits for fluidic functions were replicated from polydimethylsiloxane (PDMS) using a SU-8 master mold fabricated by a 2-level lithography process which we developed specially for the microfluidic structures used in this work. For capture probes, we used self-assembled DNA oligonucleotide monolayers on gold pads patterned on glass slides. The PDMS flow cells were aligned with and sealed against the glass slides to form the DNA hybridization units. Hybridization was detected using an enzymatic-labeled fluorescence technique. An analytical model was introduced to quantitatively predict the accumulation of target DNA molecules. The flow-through hybridization units were tested using DNA samples (25-mers) of various concentrations down to 100 pM and passive assays (no flow) using samples of the same concentrations were performed for comparison. For the same concentration, with the same hybridization time (3 min), a fluorescence intensity increase up to threefold was observed in the flow-through hybridization unit compared to the passive hybridization assays. Furthermore, at the lowest sample concentration, the signal intensity from the passive assay is at the same level of the background while the signal from the flow-through assay is appreciably above the noise level. Besides the fast hybridization rate, the CD-based method has the potential for enabling highly automated, multiple and self-contained assays for DNA detection.

In this investigation we report on the influence of volumetric flow rate, flow velocity, complementary DNA concentration, height of a microfluidic flow channel and time on DNA hybridization kinetics. A syringe pump was used to drive Cy3-labeled target DNA through a polydimethylsiloxane (PDMS) microfluidic flow channel to hybridize with immobilized DNA from the West Nile Virus. We demonstrate that a reduction of channel height, while keeping a fixed volumetric flow rate or a fixed flow velocity, enhances mass transport of target DNA to the capture probes. Compared to a passive hybridization, the DNA hybridization in the microfluidic flow channel generates higher fluorescence intensities for lower concentration of target DNA during the same fixed period of time. Within a fixed 2 min time period the fastest DNA hybridization at a 50 pM concentration of target DNA is achieved with a continuous flow of target DNA at the highest flow rate and the lowest channel height.

Cell lysis was demonstrated on a microfluidic CD (Compact Disc) platform. In this purely mechanical lysis method, spherical particles (beads) in a lysis chamber microfabricated in a CD, cause disruption of mammalian (CHO-K1), bacterial (Escherichia coli), and yeast (Saccharomyces cerevisiae) cells. Interactions between beads and cells are generated in the rimming flow established inside a partially filled annular chamber in the CD rotating around a horizontal axis. To maximize bead–cell interactions in the lysis chamber, the CD was spun forward and backwards around this axis, using high acceleration for 5 to 7 min. Investigation on inter-particle forces (friction and collision) identified the following parameters; bead density, angular velocity, acceleration rate, and solid volume fraction as having the most significant contribution to cell lysis. Cell disruption efficiency was verified either through direct microscopic viewing or measurement of the DNA concentration after cell lysing. Lysis efficiency relative to a conventional lysis protocol was approximately 65%. In the long term, this work is geared towards CD based sample-to-answer nucleic acid analysis which will include cell lysis, DNA purification, DNA amplification, and DNA hybridization detection.

We present Suspended Carbon Nanofibers featuring a central nanometric gap fabricated by integration of Electro-Mechanical Spinning, pyrolysis of ultraviolet-cured microstructures and a Joule heating process. Photopatterned walls were used to suspend polymeric electrospun fibers based on a solution of SU-8. After pyrolysis, the complete structure was converted into a monolithic carbon microstructure featuring stable ohmic contact between the suspended fibers and the supporting walls without the need of further processing. By applying an electrical bias through the electrodes, the wire can be gradually thinned by excessive Joule heating, eventually forming a nanogap. The maximum supported current density in the fibers was found to be of the order of 105 A/cm2. Furthermore, the electrical characteristics of carbon nanofibers and the dimensions of the nanogaps were demonstrated to be dependent on the length of the fiber and atmosphere conditions, i.e. air or vacuum. A finite element simulation was used to gain insight into the influence of length on nanogap size. The shorter fibers modeled portrayed a significantly steeper temperature gradient, which agrees with the experimental observation of smaller gaps. The presented method facilitates an inexpensive manufacturing technique of nanogaps as small as 12 nm, holding potential for the preparation molecular electronics devices.

Carbon transmission electron microscope (TEM) grids were fabricated for the first time using Carbon-MEMS (C-MEMS) process. The C-MEMS process and its application in the fabrication of carbon TEM grids are detailed. The benefits of making carbon TEM grids this way are also explained. Commercially available TEM grids are often hard to use for many types of samples. Moreover sample preparation such as nanowire placement on conventional TEM grids remains very challenging. Here we demonstrate a novel fabrication method of carbon TEM grids. These grids serve as a support for the nanowire deposition process itself; no further nanowire placement is required. The method for constructing in-situ carbon TEM grids is illustrated for the study of suspended carbon nanowires. The method is generic as long as the nano-devices that are being built can survive the pyrolysis step. In this paper, a test-bed for CNW TEM study is fabricated using one step photolithography to pattern SU8 TEM grids and then fibers are electrospun over the grid. Both, grids and fibers are then converted to carbon at the same time by pyrolysis in an inert (N2) environment. The grids are made on a Si/SiO2 substrate and are released from the substrate using Buffered Oxide Etch.