We describe a technique to physically isolate single/individual cells from their surrounding environment by fabricating three-dimensional microchambers around selected cells under biocompatible ...conditions. Isolation of targeted cells is achieved via rapid fabrication of protein hydrogels from a biocompatible precursor solution using multiphoton lithography, an intrinsically 3D laser direct write microfabrication technique. Cells remain chemically accessible to environmental cues enabling their propagation into well-defined, high density populations. We demonstrate this methodology on gram negative (E. coli), gram positive (S. aureus), and eukaryotic (S. cerevisiae) cells. The opportunities to confine viable, single/individual-cells and small populations within user-defined microenvironments afforded by this approach should facilitate the study of cell behaviors across multiple generations.
A versatile and simple method is introduced for formation of maleimide-functionalized surfaces using maleimide-activated aryl diazonium salts. We show for the first time electrodeposition of ...N-(4-diazophenyl)maleimide tetrafluoroborate on gold and carbon electrodes which was characterized via voltammetry, grazing angle FTIR, and ellipsometry. Electrodeposition conditions were used to control film thickness and yielded submonolayer-to-multilayer grafting. The resulting phenylmaleimide surfaces served as effective coupling agents for electrode functionalization with ferrocene and the redox-active protein cytochrome c. The utility of phenylmaleimide diazonium toward formation of a diazonium-activated conjugate, followed by direct electrodeposition of the diazonium-modified DNA onto the electrode surface, was also demonstrated. Effective electron transfer was obtained between immobilized molecules and the electrodes. This novel application of N-phenylmaleimide diazonium may facilitate the development of bioelectronic devices including biofuel cells, biosensors, and DNA and protein microarrays.
Holding cells: Individual gold electrodes can be activated by phenylboronic acid diazonium salts for the facile and reversible immobilization of eukaryotic cells (see scheme). This platform provides ...a simple method for on‐demand release of captured cells (yeast and macrophage) and can be used in single‐cell or array‐based studies.
A proof of concept procedure for the electroaddressable covalent immobilization of DNA and protein on arrayed electrodes along with simultaneous detection of multiple bioagents in the same sample ...solution is described. Carboxyphenyldiazonium was selectively deposited onto five of nine individually addressable electrodes in an array via bias assisted assembly. Amine functionalized DNA probes were covalently coupled to the carboxyl surface via carbodiimide chemistry. This was followed by the covalent immobilization of diazonium-antibody conjugates into the remaining four electrodes via cyclic voltammetry. Simultaneous electrochemical detection of a DNA sequence related to the breast cancer BRCA1 gene and the human cytokine protein interleukin-12, which is a substantial component in the immune system response and attack of tumor cells, is reported. These results demonstrate the possibility of selective patterning of diverse biomolecules on a single device and may have significant implications for future development of microarrays and biosensors.
Experimental dog model of acute spinal cord injury.
To compare the relative value of methylprednisolone, surgical decompression, or both for the treatment of traumatic spinal cord injury.
Acute ...spinal cord injury results from both primary damage to the spinal cord at the time of the initial injury as well as a deleterious secondary cascade of events, which leads to further damage. Surgical decompression is known to improve clinical outcomes, but the timing of surgical decompression remains controversial.
A nylon tie was used to constrict the spinal cord in 18 adult male beagle dogs. The animals were then prospectively randomized to 3 groups: 1) surgical decompression at 6 hours and intravenous methylprednisolone; 2) surgical decompression at 6 hours and intravenous saline; and 3) intravenous methylprednisolone without surgical decompression. Each animal was evaluated by somatosensory-evoked potentials, daily neurologic assessment, and histologic examination at 2 weeks following injury.
Immediately following spinal cord constriction, all animals were paraplegic, incontinent, and the somatosensory-evoked potentials were abolished. Surgical decompression 6 hours after injury, with or without methylprednisolone, led to significantly better neurologic function at 2 weeks than methylprednisolone alone.
In the setting of acute and persistent spinal cord compression in beagle dogs, surgical decompression 6 hours after injury, with or without methylprednisolone, is more effective for improving neurologic recovery than methylprednisolone alone.
Rare hematopoietic stem cell populations are responsible for the transplantation engraftment process. Umbilical cord blood (UCB) is usually processed to the total nucleated cell (TNC), but not to the ...mononuclear cell (MNC) fraction. TNC counts are used to determine UCB unit storage, release for transplantation and correlation with time to engraftment. However, the TNC fraction contains varying concentrations of red blood cells, granulocytes, platelets and other cells that dilute and mask the stem cells from being detected. This does not allow the quality and potency of the stem cells to be reliably measured.
63 UCB segments and 10 UCB units plus segments were analyzed for the response of both primitive lympho-hematopoietic and primitive hematopoietic stem cells in both the TNC and MNC fractions. The samples were analyzed using a highly sensitive, standardized and validated adenosine triphosphate (ATP) bioluminescence stem cell proliferation assay verified against the colony-forming unit (CFU) assay. Dye exclusion and metabolic viability were also determined.
Regardless of whether the cells were derived from a segment or unit, the TNC fraction always produced a significantly lower and more variable stem cell response than that derived from the MNC fraction. Routine dye exclusion cell viability did not correspond with metabolic viability and stem cell response. Paired UCB segments produced highly variable results, and the UCB segment did not produce similar results to the unit.
The TNC fraction underestimates the ability and capacity of the stem cells in both the UCB segment and unit and therefore provides an erroneous interpretation of the of the results. Dye exclusion viability can result in false positive values, when in fact the stem cells may be dead or incapable of proliferation. The difference in response between the segment and unit calls into question the ability to use the segment as a representative sample of the UCB unit. It is apparent that present UCB processing and testing methods are inadequate to properly determine the quality and potency of the unit for release and use in a patient.
A simple one-step procedure is introduced for the preparation of diazonium−enzyme adducts. The direct electrically addressable deposition of diazonium-modified enzymes is examined for electrochemical ...sensor applications. The deposition of diazonium−horseradish peroxidase leads to the direct electron transfer between the enzyme and electrode exhibiting a heterogeneous rate constant, k s, of 10.3 ± 0.7 s-1 and a ΔE p of 8 mV (v = 150 mV/s). The large k s and low ΔE p are attributed to the intimate contact between enzyme and electrode attached by one to three phenyl molecules. Such an electrode shows high nonmediated catalytic activity toward H2O2 reduction. Future generations of arrayed electrochemical sensors and studies of direct electron transfer of enzymes can benefit from protein electrodes prepared by this method.
Since the introduction of micro total analytical systems (μTASs), significant advances have been made toward development of lab-on-a-chip platforms capable of performing complex biological assays ...that can revolutionize public health, among other applications. However, use of these platforms in low-resource environments (e.g. developing countries) has yet to be realized as the majority of technologies used to control microfluidic flow rely on off-device hardware with non-negligible size, cost, power requirements and skill/training to operate. In this paper we describe a magnetic-adhesive based valve that is simple to construct and operate, and can be used to control fluid flow and store reagents within a microfluidic device. The design consists of a port connecting two chambers on different planes in the device that is closed by a neodymium disk magnet seated on a thin ring of adhesive. Bringing an external magnet into contact with the outer surface of the device unseats and displaces the valve magnet from the adhesive ring, exposing the port. Using this configuration, we demonstrate on-device reagent storage and on-demand transport and reaction of contents between chambers. This design requires no power or external instrumentation to operate, is extremely low cost ($0.20 materials cost per valve), can be used by individuals with no technical training, and requires only a hand-held magnet to actuate. Additionally, valve actuation does not compromise the integrity of the completely sealed microfluidic device, increasing safety for the operator when toxic or harmful substances are contained within. This valve concept has the potential to simplify design of μTASs, facilitating development of lab-on-a-chip systems that may be practical for use in point-of-care and low-resource settings.
The direct electrically addressable deposition of diazonium-modified antibodies is examined for electrochemical immunosensing applications. The immobilized antibodies can be detected by the use of ...electroactive enzyme tags and nanoparticle-gold labeling. Control over antibody functionalization density and minimal spontaneous grafting of diazonium–antibody adducts is shown. The utility of the technique for a sandwich immunoassay as well as the ability to individually and selectively address closely spaced microelectrodes for multi-target protein detection in an array format is demonstrated.
In this article we review recent work in our laboratory towards the realization of a multianalyte microelectrode detection platform capable of discriminating chemicals and different biomolecules ...simultaneously. The functionalization of electrodes with aryl diazonium salts provides an electrically addressable deposition procedure capable of immobilizing a wide range of molecules. We demonstrate control over surface density and electron transfer kinetics as well as the activation of individual electrodes in an array. The direct electrically‐addressable immobilization of diazonium‐modified proteins is shown to be suitable for the construction of multianalyte immunosensors and the immobilization of horseradish peroxidase leading to the direct electron transfer between the redox enzyme and the electrode. The use of catalytic nanoparticles leads to the construction of a reagent‐less immunosensor and the simultaneous detection of DNA and proteins on the same electrode array is demonstrated.
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