Antibody colocalization microarray: a scalable technology for multiplex protein analysis in complex samples.

DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (1) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (2) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same two-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, conventional multiplex sandwich assay (MSA). Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable.

Autonomous microfluidic capillaric circuits replicated from 3D-printed molds.

We recently developed capillaric circuits (CCs) - advanced capillary microfluidic devices assembled from capillary fluidic elements in a modular manner similar to the design of electric circuits (Safavieh & Juncker, Lab Chip, 2013, 13, 4180-4189). CCs choreograph liquid delivery operations according to pre-programmed capillary pressure differences with minimal user intervention. CCs were thought to require high-precision micron-scale features manufactured by conventional photolithography, which is slow and expensive. Here we present CCs manufactured rapidly and inexpensively using 3D-printed molds. Molds for CCs were fabricated with a benchtop 3D-printer, poly(dimethylsiloxane) replicas were made, and fluidic functionality was verified with aqueous solutions. We established design rules for CCs by a combination of modelling and experimentation. The functionality and reliability of trigger valves - an essential fluidic element that stops one liquid until flow is triggered by a second liquid - was tested for different geometries and different solutions. Trigger valves with geometries up to 80-fold larger than cleanroom-fabricated ones were found to function reliably. We designed retention burst valves that encode sequential liquid delivery using capillary pressure differences encoded by systematically varied heights and widths. Using an electrical circuit analogue of the CC, we established design rules to ensure strictly sequential liquid delivery. CCs autonomously delivered eight liquids in a pre-determined sequence in <7 min. Taken together, our results demonstrate that 3D-printing lowers the bar for other researchers to access capillary microfluidic valves and CCs for autonomous liquid delivery with applications in diagnostics, research and education.

Integrated microfluidic probe station.

The microfluidic probe (MFP) consists of a flat, blunt tip with two apertures for the injection and reaspiration of a microjet into a solution--thus hydrodynamically confining the microjet--and is operated atop an inverted microscope that enables live imaging. By scanning across a surface, the microjet can be used for surface processing with the capability of both depositing and removing material; as it operates under immersed conditions, sensitive biological materials and living cells can be processed. During scanning, the MFP is kept immobile and centered over the objective of the inverted microscope, a few micrometers above a substrate that is displaced by moving the microscope stage and that is flushed continuously with the microjet. For consistent and reproducible surface processing, the gap between the MFP and the substrate, the MFP's alignment, the scanning speed, the injection and aspiration flow rates, and the image capture need all to be controlled and synchronized. Here, we present an automated MFP station that integrates all of these functionalities and automates the key operational parameters. A custom software program is used to control an independent motorized Z stage for adjusting the gap, a motorized microscope stage for scanning the substrate, up to 16 syringe pumps for injecting and aspirating fluids, and an inverted fluorescence microscope equipped with a charge-coupled device camera. The parallelism between the MFP and the substrate is adjusted using manual goniometer at the beginning of the experiment. The alignment of the injection and aspiration apertures along the scanning axis is performed using a newly designed MFP screw holder. We illustrate the integrated MFP station by the programmed, automated patterning of fluorescently labeled biotin on a streptavidin-coated surface.

Optimizing pacing lead design: redesign of the tined lead.

Technical data derived from experimental and clinical experience in the field of lead/catheter implants formed the basis for developing a set of technical design criteria for pacing leads. Four related levels of technical data were identified: Basic lead components, mode of installation, application site, and mode of use. When the technical data were evaluated, a joint goal between the medical and engineering community evolved--namely, to reduce the total transvenous lead-related reoperation rate (both acute and chronic) from 20-25 percent to below 5 percent. As a result, a new family of leads designed specifically to meet the less than or equal to 5 percent reoperation rate is now available; the first leads introduced are a radical redesign of an earlier tined lead. The development process and results, both experimental and clinical (N greater than 1,005), demonstrate the potential of careful technical definition and communication of biomedical problems between engineering and medical personnel.

Potassium influx in the frog atrium during the cardiac cycle.

A method for measuring inwardly directed transmembrane tracer flow during the cardiac cycle was developed and applied to a study of 42K influx in frog atrial trabeculae. A fine frog atrial fiber was suspended in a stream of nonisotopic perfusate into which a smaller tracer-containing bolus could be injected, subjecting the fiber to a brief, controlled exposure to the tracer at any desired point in the cardiac cycle. In an experiment, the tissue was exposed to a fixed number of radioactive pulses at a selected point in the cardiac cycle; a brief flush with nonradioactive perfusate removed ambient and extracellular label and an extended wash and removed the remaining intracellular tracer for radioassay. The same procedure was repeated at different points in the cycle, and the resulting tracer uptake at each point measured the relative influx of the particular ion. In this way, a characteristic and reproducible 42K influx profile was demonstrated which exhibited a marked drop below diastolic values during the first 500 msec or so of the action potential followed by a rise and an overshoot above resting values. The time of return to the resting level was somewhat uncertain but was tentatively placed in the vicinity of rapid repolarization. We suggest that the rise and the overshoot reflect the activity of the membrane Na+, K+-adenosinetriphosphatase.