Prostheses that can restore limited vision in the profoundly blind have been under investigation for several decades. Studies using epicortical macroelectrodes and intracortical microelectrodes have validated that electrical stimulation of primary visual cortical can serve as the basis for a vision prosthesis. However, neither of these approaches has resulted in a clinically viable vision prosthesis. Epicortical macroelectrodes required high levels of electrical current to evoke visual percepts, while intracortical microelectrodes faced challenges with longevity and stability. We hypothesized that epicortical microelectrodes could evoke visual percepts at lower currents than macroelectrodes and provide improved longevity and stability compared with intracortical microelectrodes. To test this hypotheses we implanted epicortical microelectrode arrays over the primary visual cortex of a nonhuman primate. Electrical stimulation via this array was used to evaluate the ability of epicortical microstimulation to evoke differentiable visual percepts. Visual percepts were evoked using the epicortical microelectrode array, and at electrical currents notably lower than those required to evoke visual percepts on macroelectrode arrays. The electrical current thresholds for evoking visual percepts on the epicortical microelectrode array were consistent across multiple array implants and over several months. Normal vision of light perception was not impaired by multiple array implants or chronic electrical stimulation, demonstrating that no gross visual deficit resulted from the experiments. We specifically demonstrate that epicortical microelectrode interfaces can serve as the basis for a vision prosthesis and more generally may provide an approach to evoking perception in multiple sensory modalities. One Sentence Summary Electrical stimulation of the brain via microelectrodes resting on the surface of primary visual cortex can evoke multiple differentiable visual percepts.

Prostheses that can restore limited vision in the profoundly blind have been under investigation for several decades. Studies using epicortical macroelectrodes and intracortical microelectrodes have validated that electrical stimulation of primary visual cortical can serve as the basis for a vision prosthesis. However, neither of these approaches has resulted in a clinically viable vision prosthesis. Epicortical macroelectrodes required high levels of electrical current to evoke visual percepts, while intracortical microelectrodes faced challenges with longevity and stability. We hypothesized that epicortical microelectrodes could evoke visual percepts at lower currents than macroelectrodes and provide improved longevity and stability compared with intracortical microelectrodes. To test this hypotheses we implanted epicortical microelectrode arrays over the primary visual cortex of a nonhuman primate. Electrical stimulation via this array was used to evaluate the ability of epicortical microstimulation to evoke differentiable visual percepts. Visual percepts were evoked using the epicortical microelectrode array, and at electrical currents notably lower than those required to evoke visual percepts on macroelectrode arrays. The electrical current thresholds for evoking visual percepts on the epicortical microelectrode array were consistent across multiple array implants and over several months. Normal vision of light perception was not impaired by multiple array implants or chronic electrical stimulation, demonstrating that no gross visual deficit resulted from the experiments. We specifically demonstrate that epicortical microelectrode interfaces can serve as the basis for a vision prosthesis and more generally may provide an approach to evoking perception in multiple sensory modalities. One Sentence Summary Electrical stimulation of the brain via microelectrodes resting on the surface of primary visual cortex can evoke multiple differentiable visual percepts. ### Competing Interest Statement The authors have declared no competing interest.

Advances in neural prosthetic technologies demand ever increasing novelty in material composition to enhance the mechanical and electrochemical properties of existing microelectrode arrays. Conductive polymers present advantages such as mechanical flexibility, outstanding biocompatibility, remarkable electrical properties and, most of all, cellular agreement. However, for long-term chronic applications, they fall short in their electrochemical endurance and mechanical adhesion to their substrate materials. Multiple electrochemical approaches have been investigated to improve the adherence of Poly(3,4-ethylenedioxythiophene) (PEDOT) to underlying metallic thin films. In this work, an electrochemical treatment of diazonium salt on platinum microelectrodes is incorporated as an electrochemical adhesion promoter for PEDOT and it is further combined with using the highly microporous geometry of Platinum Grey (Pt-Grey); a technology developed by Second Sight Medical Products Inc (SSMP). The intertwined mechanical integration of Pt-Grey and PEDOT molecules together with the covalent binding agency of diazonium salt demostrate a composite coating technology with long-term stability of more than 452 days while providing >70× enhancement to the interfacial capacitive impedance.

A cortical visual prosthetic system bypasses the components of the visual pathway, whichmay be damaged due to injury or disease, by directly stimulating the visual cortex; therefore, cortical visual prostheses promise the capability of restoring a form of vision to patients who cannot benefit from other types of visual neural stimulators. A high data rate, multielectrode, implantable device, such as that utilized for a cortical visual prosthesis, requires continuous power provided by an external telemetry unit, which is nonnegligible, given the number of stimulating electrodes and the stimulation rate necessary to avoid flickering visual percepts. This aspect motivates the need to developmodels and methods that aid the development of such devices by assessing their compliance with electromagnetic safety standards. In this paper, the electromagnetic safety assessment of a cortical visual prosthetic system is considered, and the solutions employed to numerically treat the computational complexities associated with it are discussed. The specifics of the implementation of an actual visual cortical implant are discussed, and the parameters of such an implant are used as a test case to determine whether IEEE and ICNIRP electromagnetic standards are met in what can be considered a typical embodiment of the prosthesis. Results show that, for the considered implant, such a system meets IEEE and ICNIRP safety standards, thus enabling further development of similar neurorehabilitative devices.

A modular system for the reporting of drug-resistant strains of Mycobacterium tuberculosis (Mtb) in less than 30 min with full process automation was developed (see picture; MDR-TB=multi-drug-resistant tuberculosis). The fluidic cartridge uses a polymer chain reaction (PCR)/ligase detection reaction (LDR)/universal array assay that can detect, with high reliability, resistant strains that are a minority (less than 1 %) from a mixed population. Infectious diseases are a major global health burden accounting for approximately 15 million deaths annually, many from drug-resistant pathogenic agents, with a significant number of cases occurring in developing countries.1–7 In particular, the resurgence of tuberculosis (TB) has been accompanied by the rapid spread of multi-drug resistance TB (MDR-TB) resulting from Mycobacterium tuberculosis (Mtb) strains that fail to respond to the first-line drugs, rifampin and isoniazid. Currently, less than 5 % of around 0.5 million MDR-TB cases estimated globally are appropriately diagnosed and treated because in part of the long assay turnaround time associated with conventional culture-based drug susceptibility testing.8 Quantitative polymerase chain reaction (qPCR), line probe assays, or home-brewed nucleic acid amplification tests (NAATs), have recently been used to identify MDR-TB. These tests provide shorter assay turnaround times compared to culture, but depend on sophisticated laboratory infrastructure and well-trained personnel to ensure accurate, reliable, and reproducible results. The world health organization (WHO) expert group recently recommended two NAAT line probe assays, INNO-LiPA Rif.TB from Innogenetics and MTBDRplus from Hain Lifescience, both of which employ multiplexed PCR reverse hybridization approaches. Line probe assays, however, are not designed to interrogate a mixed population of drug-resistant and susceptible bacterial populations,9–11 because sequence-specific array hybridization is unable to detect low abundance single-base variations because of cross-hybridization artifacts, especially in high guanine-cytosine (GC) content regions. Several groups have developed partial or fully integrated microfluidic devices for carrying out NAATs for infectious diseases.12–17 For example, Cepheid's GeneXpert MDR-TB system performs qPCR using Taqman probes for Mtb and five rifampin-resistance mutations, providing results in nearly 2 h.18 However, many of these devices are made from silicon/glass materials and thus require direct photolithographic processing to manufacture the desired structures, which increases the production cost of devices. Herein, we describe a modular design approach for an assay and hardware to detect/identify MDR-TB. The molecular assay was designed to interrogate single-base variations in codons 516, 526, and 531 (using the numbering system of E. coli rpoB) in the rifampin-resistance determining region (RRDR) of the rpoB gene, which was used as surrogate markers for MDR-TB.19, 20 The multi-step assay was carried out within a modular thermoplastic fluidic cartridge operated by the accompanying support peripherals packaged into a small instrument (1′×1′×1′). No operator intervention was required once the clinical sample (sputum) was loaded into the fluidic cartridge. This system offered advantages compared to existing NAAT systems. For example, to accommodate low-resource setting scenarios where operator expertise is limited, the hardware provided full process automation. The fluidic system consisted of a cartridge made from thermoplastics with the desired structures fabricated in a high-production format using molding to keep chip cost low for one-time use applications (in vitro diagnostics) appropriate for resource limited settings. Additional design concepts employed in the fluidic cartridge included: 1) A hybrid modular architecture, which combined several task-specific modules interconnected to a fluidic motherboard with the material selected to optimize performance. 2) Most of the active elements were poised off-chip to keep the chip costs low. 3) The embossing step was used to not only create the fluidic network, but other necessary device components as well, such as the DNA extraction bed, thermal isolation grooves, valve seats, and waveguides. The fluidic cartridge could easily be reconfigured without requiring re-engineering to accommodate alternative assays. For example, a universal array module could be replaced with a colorimetric readout module to simplify the readout hardware. Compared to monolithic12–16 or LEGO approaches,21, 22 the hybrid modular architecture balanced the efficiency and flexibility in fluidic cartridge design and material selection. Collectively, more than 95 % of resistance to rifampin is associated with missense, insertion, and deletion mutations in the RRDR of the rpoB gene. Single-base variations in codons 516, 526, or 531 are most frequently found in rifampin-resistant Mtb strains world-wide. We therefore developed a PCR/ligase detection reaction (LDR)/universal array assay to detect sequence variations harbored within these codons (see Figure 1).23 The molecular assay. Following a primary PCR to generate a 193 bp rpoB amplicon spanning the RRDR, three sets of ligase detection reaction (LDR) primers were used for interrogating missense mutations in codons 516, 526, and 531 of the rpoB gene. The discriminating primer contained a base complementary to either the wild-type or mutation sequence at its 3′-end. The common primer was phosphorylated at its 5′-end and contained a fluorescent dye, Cy5, at its 3′-end. A thermally stable ligase covalently links the two primers hybridized to the target if there was a perfect match at the locus being interrogated. The discriminating primer also carried a unique complementary "zipcode" sequence (cZip) at its 5′-end to direct the LDR product to a specific location on the "zipcode" universal array. The PCR/LDR/universal array approach employed a high fidelity Taq ligase and decoupled the mutation discrimination step from the amplification and hybridization steps.23–27 The assay demostrated the following important attributes: 1) The closely clustered drug resistance mutations of MDR-TB could be interrogated using a uniplex PCR. Therefore, careful design of primers with similar melting temperatures (Tms) and problems with uneven amplification produced by different targets was negated. 2) Only the specific drug-resistant sequence variations generate positive results. Silent mutations, which do not confer drug resistance, did not generate false positive results. 3) Hetero-resistant Mtb containing a low abundance of drug-resistant strains (around 1 %) in patients with emerging drug resistance could be identified because of the high discriminatory capability of the ligase enzyme. 4) Two amplification steps were used, an exponential amplification associated with the PCR and a linear amplification resulting from the LDR, which improved the limit of detection of the assay. The fluidic cartridge shown in Figure 2, S1, and S2 (see the Supporting Information) was designed to include five different processing steps; cell lysis, solid-phase DNA extraction (SPE), PCR, LDR, and universal array hybridization. The thermal steps (cell lysis, PCR, and LDR) were situated on a fluidic motherboard while the SPE and universal array were placed on two different modules interconnected to the motherboard. A detailed description of how each module and the motherboard were fabricated is included in the Supporting Information. The modules and motherboard were hot embossed from a particular thermoplastic, selected to optimize performance of each specific processing step. To minimize post-assembly steps, embossing was used not only to generate fluidic networks, but also specific components required for proper operation of the module or motherboard. For example, the SPE module required the use of polycarbonate, which was suitable for the selective capture of nucleic acids to its surface following photoactivation.28 Further, to increase the available surface area required to increase the DNA load, high-aspect ratio micropillars were manufactured into the SPE bed during embossing of the fluidic network, thus obviating the need for packing the SPE bed with externally added beads (see Figure S1 in the Supporting Information). Integrated, modular fluidic cartridge for TB analysis. A) 3D rendering of the cartridge and the array. 1–7: fluidic inlets and outlets: 1=sample inlet, 2=PCR cocktail inlet, 3=LDR cocktail inlet, 4=ethanol and air inlet, 5=array wash inlet, 6=vacuum connection, and 7=waste. V1-V6=on-chip membrane valves (note that V2 is positioned next to the SPE module on the cell lysis microchannel and is not visible in the current view). B) Close-up of the SPE bed showing the DNA capture bed filled with an array of high-aspect ratio pillars. C) Schematic operation of the on-chip membrane valve with mechanical actuation—electrically actuated solenoid presses on the polymer membrane closing the passage of fluid from the bottom layer through the valve and back to the bottom layer. D) Geometry of the continuous flow PCR reactor with dual-depth microchannels for extended residence time in the extension-zone; Den=denaturation, Ext=extension, and PA=primer annealing. E) Schematic representation of the detection mode. The laser excitation is coupled to a waveguide through an integrated prism. Light travelling through the waveguide excites the labeled LDR products hybridized to the zip code array spotted on the waveguide surface. Polycarbonate has a relatively high glass transition temperature, which makes it a suitable material for thermal reactions and thus, the ideal material for the fluidic motherboard, which performed thermal reactions requiring operation temperatures of 65 to 95 °C. Polycarbonate also has a relatively large elongation at break threshold such that it can be used as a microfluidic valve membrane. Using polycarbonate as the cover plate for the motherboard obviated the need for an additional post-molding assembly step as the valve membranes were attached to the fluidic network in the same lamination step used to enclose the fluidic network with the cover plate. However, polycarbonate is not compatible with ultrasensitive fluorescence detection because of its relatively high background. Poly(methyl methacrylate), PMMA, shows good optical clarity and minimal nonspecific adsorption artifacts, making it an ideal material for construction of the universal array module.29 The universal array was poised within a microchannel resident on this module to reduce the time for probe addressing by minimizing diffusional constraints.30–32 Waveguides were fabricated using double-sided embossing with simple plasma activation of the PMMA surface to allow for covalent attachment of the zipcode probes to the waveguide (Figure S3 in the Supporting Information).33 Finally, printing of the zipcode probes could be performed using conventional DNA spotting equipment prior to enclosure of the fluidic network because of the low temperature required for bonding the cover plate to the substrate containing the probes.34 Figure 2 B and Figure S1 B provide schematic drawings and SEM images of the SPE module. Design specifics on the SPE module can be found in the Supporting Information. Figure 2 C shows a schematic of the operation of the polycarbonate membrane valves with solenoid actuation. These microvalves could withstand head pressures up to 105 psi without leakage, which is a more than an order of magnitude higher pressure load relative to that of polydimethylsiloxane (PDMS) valves.35 The thermal reactors situated on the fluidic motherboard (Figure 2 A, and Figures S1 A and S1 C in the Supporting Information) incorporated a continuous flow format, which provided ultrafast PCR amplification because extension times are limited by the kinetics of the polymerase for properly designed thermal reactors.36 Several thermal management structures were also included to further improve amplification efficiency (Figure S4 in the Supporting information), such as backside thermal isolation grooves, a thin substrate, and copper plates to give a uniform temperature distribution throughout a particular thermal zone.37 Further, a dual-depth channel (200 and 100 μm) was employed to provide sufficient residence time within the polymerase extension zone to generate full-length PCR products during each cycle while operating at a fixed linear flow velocity (Figure 2 D and Figure S1 C in the Supporting information). For a detailed discussion on the operation of the fluidic cartridge, see the Supporting Information. We performed a series of assays using different positive and negative controls that were processed using the modular fluidic cartridge. The results concurred with those expected, as shown in Figure S5 in the Supporting Information. For example, supplying an input of E. coli cells did not result in postive fluorescence signals on the universal array. However, input of a wild-type Mtb strain generated fluorescence signatures at the appropriate spots of the array (Figure S5 in the Supporting Information). We were interested in evaluating the minimum number of Mtb cells required to provide positive results using our system and whether results could meet the needed level for clinical diagnosis. To determine the limit-of-detection, assays were conducted using a serial dilution of cultured H37Rv cells as the input. As few as 50 Mtb cells could be successfully detected, representing a 100-fold improvement in sensitivity compared to current clinical smear tests, which require 5000–10 000 bacilli in 1 mL of sputum. Hetero-resistant Mtb is defined as the coexistence of mixed populations of drug-susceptible and resistant Mtb strains in the same patient, which is considered as a preliminary stage to full resistance. These cases result either from super-infection or from selection pressure during antibiotic treatment. The occurrence of hetero-resistance can be as high as 20 %.10, 11 To evaluate the ability of our system to identify a minority population of drug-resistant Mtb strains, a series of control hetero-resistance samples containing increasing percentages of a rifampin-resistant strain (S531L)—0, 1, 2, 5, 10, and 100 %—in a drug-susceptible strain of H37Rv were prepared and analyzed (Figure 3). The fluorescence intensity from the 531 mutant spot increased with an increased percentage of the rifampin-resistant strain present in the sample while the spot intensity from the 531Wt decreased (Figure 3 C). The fluorescence from the 531 mutant spots run with samples containing as little as 1 % of rifampin-resistant strain was calculated to be around 10-fold higher than the fluorescence from those run with samples containing 0 % rifampin-resistant strains (Figure 3 C). Therefore, less than 1 % of the drug-resistant strain could still be discriminated based on a three-fold standard-deviation higher signal than a nonspecific signal. Very slight levels of fluorescence were detected from the 531Wt spots run with 100 % of the drug-resistant strain, which could be attributed to misligation and/or nonspecific hybridization.38 Identification of a mixed-population of drug-susceptible (H37Rv) and drug-resistant (S531L) Mtb strains. A) Layout of the universal array. Spots labeled C were printed with 5′-Cy5 oligonucleotides, and used as quality controls (C) of the printing process. Spots labeled as Hyb were hybridization controls. Spots labeled as Pos were positive controls (spiked plasmids). Spots labeled Mtb were Mtb-specific probes targeting an IS6110 insertion fragment. B–G) Universal array images for different amounts of the drug-resistant and drug-susceptible strains. The percentage of the sample containing the drug resistant strain was 0 (B), 1 (C), 2 (D), 5 (E), 10 (F), and 100 % (G). The modular system was challenged with six clinical samples containing either rifampin-resistant or susceptible Mtb strains. Figure 4 represents results that were consistent with the phenotype and the genotype of those clinical samples. The analysis of samples YE69 (A516V), 3976-83 (A516V), YE68 (H526Y), 3908-83 (S531L), and 94-2219 (Wt), Figure 4 B–G, identified the correct mutation for each drug-resistant strain as determined by sequence analysis of the rpoB gene of these clinical isolates. Universal array hybridization results from clinical samples (sputum). A) Layout of the universal array with the same designations as that given in Figure 3 A. B) Mtb(+) clinical isolate (YE69) harbors a mutation in codon 516 of rpoB. C) Mtb(+) sputum sediment (3976-83) harbors a mutation in codon 516 of rpoB. D) Mtb clinical isolate (YE68) possesses a mutation in codon 526 of rpoB. E) Mtb(+) sputum sediment (3908-83) harbors a mutation in codon 531 of rpoB. F) Mtb(+) sputum sediment (94-2219) contained no mutations in rpoB RRDR (Wt). G) Mtb(−) sputum sediment. A colorimetric zipcode array module was developed as detailed in the Supporting Information to simplify the readout by eliminating the need for laser-induced fluorescence (Figure S6 in the Supporting Information). PMMA wafers containing immobilized zipcode probes were prepared using a PDMS stencil (Figure S6 B in the Supporting Information). Also, 1.4 nm nanogold-labeled common primers were used instead of fluorescent dye-labeled primers as shown in Figure S6 A in the Supporting Information. After LDR and hybridization, the nanogold labels acted as a catalytic site for silver deposition, which was visible to the naked eye and could be recorded by a digital camera. A drug-susceptible Mtb stain (H37Rv) and a rifampin-resistant strain (S531L) were tested and the results are shown in Figure S6 C in the Supporting Information. Using this colorimetric test for detection, the Mtb cell limit-of-detection was found to be 50 cells, similar to that observed using laser-induced fluorescence detection. We have reported the development of a modular design approach for both the software (molecular assay) and hardware (fluidic cartridge and support peripherals) to determine sequence variations in reporter sequences to detect and identify MDR-TB directly from sputum samples. The system provided full-process automation minimizing operator expertise requirements and also, generated results in less than 30 min, which will be a key operating metric by providing rapid results to allow proper treatment of patients, potentially minimizing the generation of additional drug-resistant strains because of the implementation of a full treatment regimen. Recently, extremely drug-resistant TB (XDR-TB) has been reported. XDR-TB strains contain mutations responsible for MDR-TB as well as sequence variations resulting in resistance to second-line drugs. By incorporating a multiplexed PCR and additional LDR primers, the system presented here can easily be configured to detect sequence variations responsible for XDR-TB without hardware redesign. The system, both software and hardware, holds the potential to be a universal platform for identifying genetic sequence signatures in a variety of applications, including cancer diagnostics,23, 26, 27 forensic testing, and biothreat pathogen detection, and identification in both developing and developed countries24 because of the modular design. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

X-ray absorption spectroscopy (XAS) is an extremely valuable tool for the characterization of the electronic and geometric properties of nanoparticles. However, there are drawbacks when it comes, for example, to time-resolved in situ measurements of wet-chemical synthetic reactions or when X-ray absorption near edge structure (XANES) spectra are used for characterizing atoms that occupy different sites in a nanoparticle. In this paper, we report results of test experiments using resonant inelastic X-ray scattering (RIXS) for obtaining high-resolution spectra that allow in principle site- and/or valency-specific XANES measurements. For a detailed analysis of wet-chemical reactions, a microreactor system is used in which time resolution is obtained by spatial resolution, i.e., by measuring spectra at various points along the microreactor. This system provides significantly better resolution (in the order of milliseconds) compared to conventional techniques.

Mixed-scale nano- and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 µm in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared via HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective T(g) was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: ∼6% for PMMA and ∼9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 ± 0.7 × 10(-4) cm(2) V(-1) s(-1) and 7.6 ± 0.6 × 10(-4) cm(2) V(-1) s(-1) for PMMA and COC, respectively, at a field strength of 25 V cm(-1). The extension factors for λ-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2-6% standard deviation).

A high throughput, multi-well (96) polymerase chain reaction (PCR) platform, based on a continuous flow (CF) mode of operation, was developed. Each CFPCR device was confined to a footprint of 8 x 8 mm(2), matching the footprint of a well on a standard micro-titer plate. While several CFPCR devices have been demonstrated, this is the first example of a high-throughput multi-well continuous flow thermal reactor configuration. Verification of the feasibility of the multi-well CFPCR device was carried out at each stage of development from manufacturing to demonstrating sample amplification. The multi-well CFPCR devices were fabricated by micro-replication in polymers, polycarbonate to accommodate the peak temperatures during thermal cycling in this case, using double-sided hot embossing. One side of the substrate contained the thermal reactors and the opposite side was patterned with structures to enhance thermal isolation of the closely packed constant temperature zones. A 99 bp target from a lambda-DNA template was successfully amplified in a prototype multi-well CFPCR device with a total reaction time as low as similar to 5 min at a flow velocity of 3 mm s(-1) (15.3 s cycle(-1)) and a relatively low amplification efficiency compared to a bench-top thermal cycler for a 20-cycle device; reducing the flow velocity to 1 mm s(-1) (46.2 s cycle(-1)) gave a seven-fold improvement in amplification efficiency. Amplification efficiencies increased at all flow velocities for 25-cycle devices with the same configuration.

The goal of this project is to build a miniaturized, user-friendly cytometry setup (Datta et al. in Microfluidic platform for education and research. COMS, Baton Rouge, 2008; Frische et al. in Development of an miniaturized flow cytometry setup for visual cell inspection and sorting. Baton Rouge, Project Report, 2008) by combining a customized, microfluidic device with visual microscope inspection to detect and extract specific cells from a continuous sample flow. We developed a cytological tool, based on the Coulter particle counter principle, using a microelectrode array patterned on a borosilicate glass chip as electrical detection set-up which is fully embedded into a polymeric multi-layer microfluidic stack. The detection takes place between pairs of coplanar Cr/Au microelectrodes by sensing an impedance change caused by particles continuously carried within a microfluidic channel across the detection area under laminar flow conditions. A wide frequency range available for counting provides information on cell size, membrane capacitance, cytoplasm conductivity and is potentially of interest for in-depth cell diagnostic e.g. to detect damaged or cancerous cells and select them for extraction and further in-depth analysis.

In order for Magnetoresistive Biosensor technology to become a mainstream product for clinical and consumer use, several outstanding technical issues must be solved. This paper will focus on one of those issues, which is the need to adapt standard semiconductor packaging processes to fall within some biosensor fabrication process constraints. A set of materials and interconnection methods that meet these biosensor requirements are presented. The resulting architecture is compatible with laboratory assembly, but can be scaled up to small and medium manufacturing quantities by using larger 2-dimensional areas per production batch.

The fabrication and characterization of a novel cyclic olefin copolymer (COC) waveguide embedded in a poly(methyl methacrylate), PMMA, fluidic chip configured in a multi-channel format with an integrated monolithic prism for evanescent fluorescence excitation are reported. The fabrication approach allowed the embedded waveguide to be situated orthogonal to a series of fluidic channels within the PMMA wafer to sample fluorescent solutions in these channels using the evanescence properties of the waveguide. Construction of the device was achieved using several fabrication techniques including high precision micromilling, hot embossing and stenciling of a polymer melt to form the waveguide and coupling prism. A waveguide channel was fabricated in the fluidic chip's cover plate, also made from PMMA, and was loaded with a COC solution using a pre-cast poly(dimethylsiloxane), PDMS, stencil containing a prism-shaped recess. The PMMA substrate contained multiple channels (100 microm wide x 30 microm deep with a pitch of 100 microm) that were situated orthogonal to the waveguide to allow penetration of the evanescent field into the sampling solution. The optical properties of the waveguide in terms of its transmission properties and penetration depth of the evanescent field in the adjacent solution were evaluated. Finally, the device was used for laser-induced fluorescence evanescent excitation of a dye solution hydrodynamically flowing through multiple microfluidic channels in the chip and processed using a microscope equipped with a charge-coupled device (CCD) for parallel readout. The device and optical system were able to image 11 channels simultaneously with a limit-of-detection of 7.1 x 10(-20) mol at a signal-to-noise ratio of 2. The waveguide was simple to manufacture and could be scaled to illuminate much higher channel numbers making it appropriate for high-throughput measurements using evanescent excitation.

Integration of electronic wiring with microfluidic chips is an important process as it allows electrical interactions with the fluidic media, for example required for resistive and capacitive sensing. It is also necessary in order to implement various actuation and control mechanisms such as pumping, electrophoresis and temperature control. Typically electrical wire traces are added to microfabricated fluidic chips using metal deposition processes that are carried out after the fluidic chip has been fabricated. The process for adding the wiring is complicated and is limited to select metals that can be deposited by evaporation or sputtering. We present a single step method for integrating electrical wires into polymer microfluidic chips that are fabricated by a hot embossing process. This process can flexibly embed any kind of commercially available metal wire with a microfluidic chip and the wiring may be integrated to come into surface contact with the fluid or may be embedded in close proximity to (but insulated from)the fluid paths for example for local heating purposes. This method significantly reduces total processing time and is thus a valuable method for wire integration into polymer chips. We demonstrate two applications—a microelectrolysis chip and a heater chip that were fabricated using this methodology. The design, fabrication process and the initial test results are presented.

A highly parallel, polymerase chain reaction (PCR) multireactor platform is in high demand to satisfy the high throughput requirements for exploiting the accumulated genetic information from the Human Genome Project. By incorporating continuous flow PCR (CFPCR) devices in a polymer 96-well titer plate format, DNA amplification can be performed with steady-state temperature control and faster reaction speed at lower cost. Prior to the realization of a PCR multi-reactor platform, consisting of a sample delivery chip, a PCR multireactor chip, and a thermal cycler, optimization of the geometry for CFPCR devices in a titer plate-based PCR multi-reactor chip based on manufacturing feasibility is necessary. A prototype PCR multi-reactor chip was designed in a 96-well titer plate format with twelve different CFPCR configurations. High quality metallic, large area mold inserts (LAMIs) were fabricated using an SU-8 based UV-LIGA technique by overplating nickel in SU-8 electroplating templates. Micro molding of polycarbonate (PC) was done using hot embossing, resulting in good replication fidelity over the large surface area. Thermal fusion bonding of the molded PC chips using a custom-made bonding jig yielded acceptable sealing results. The manufacturability investigation throughout the design and the process sequence suggested that the microchannel walls require a minimum width of at least 20 μm and an aspect ratio of 2 for structural rigidity. An optimal CFPCR device for use in a PCR multi-reactor chip can be selected with a series of amplification experiments with the development of a thermal cycler.

A method of collecting and delivering single or precise numbers of cells to assess the feasibility of capturing very rare circulating tumor cells for human breast cancer diagnosis and monitoring was developed. A PMMA device was assembled with minimal assembly variation using passive alignment. Thermoplastic fusion bonding was optimized to yield minimal deformation of the microfluidic channel. UV modification and an anti-epithelial cell adhesion molecule (anti-EpCAM) functionalization process were used to generate capture surfaces and maximized by control experiment. Single or precise numbers of target cells were collected using a cell collecting capillary tube and a hemacytometer and delivered into the microchannel without any loss. Cells from one of the human breast cancer cell lines, the MCF-7 cell line (ATCC, Manassas, VA) which strongly overexpresses EpCAM, were successfully captured on the anti-EpCAM coated microchannel surfaces. Successful capture of early stage breast cancer cells in whole blood may be feasible with further optimization of the microchannel geometry and flow velocity through the microfluidic device.

Incorporation of spiral continuous flow (CF) polymerase chain reaction (PCR) devices into a 96-well titer plate format will enable highly parallel analyses of many nucleic acids under steady-state temperature control for the realization of a high throughput CFPCR multi-reactor platform.Prior to realization of the full platform, verifying the feasibility of small footprint CFPCR devices, each device confined to a footprint of 8x8 mm 2 , throughout the manufacturing and amplification processes was essential.Small footprint, CFPCR devices in a CFPCR multi-reactor module were designed and double-side hot embossed on one side of a substrate and structures for thermal isolation on the other to improve thermal management.Successful amplification of 99 bp target DNA fragments from λ-DNA template was demonstrated in the small footprint, 20-and 25-turn CFPCR devices.

We describe the development of a fully integrated, modular microfluidic system for molecular analyses. The processing pipeline was carried out in a 3-D microfluidic system and included cell lysis, solid-phase extraction (SPE) of DNA, continuous-flow polymerase chain reaction (CFPCR), continuous-flow ligase detection reaction (CFLDR), and zip-code array detection. Chip operation was provided by electronic and hydraulic controls located off-chip. Optical detection of array events was achieved with a fluorescence reader consisting of a VCSEL and CCD. As an example, the detection of E. coli O157:H7 and Salmonella is presented.