Planar carbon electrodes for real-time quantification of hydrogen sulfide release from cells.

A planar electrode system was developed to permit the real-time, selective detection of hydrogen sulfide (H2S) from stimulated cells. Planar carbon electrodes were produced via stencil printing carbon ink through a laser cut vinyl mask. Electrodes were preconditioned using a constant potential amperometry methodology to prevent sensor drift resulting from elemental sulfur adsorption. Modification with a bilaminar coating (electropolymerized ortho-phenylenediamine and a fluorinated xerogel) facilitated high selectivity to H2S. To demonstrate the biological application of this planar sensor system, H2S released from 17ß-estradiol-stimulated human umbilical vein endothelial cells (HUVECs) was quantified in situ in real-time. Stimulated HUVECs released sustained H2S levels for hours before returning to baseline. Cellular viability assays demonstrated negligible cell cytotoxicity at the electrochemical potentials required for analysis.

Mechanisms of Foreign Body Response Mitigation by Nitric Oxide Release.

Implantable glucose biosensors provide real-time information about blood glucose fluctuations, but their utility and accuracy are time-limited due to the foreign body response (FBR) following their insertion beneath the skin. The slow release of nitric oxide (NO), a gasotransmitter with inflammation regulatory properties, from a sensor surface has been shown to dramatically improve sensors' analytical biocompatibility by reducing the overall FBR response. Indeed, work in a porcine model suggests that as long as the implants (sensors) continue to release NO, even at low levels, the inflammatory cell infiltration and resulting collagen density are lessened. While these studies strongly support the benefits of NO release in mitigating the FBR, the mechanisms through which exogenous NO acts on the surrounding tissue, especially under the condition of hyperglycemia, remain vague. Such knowledge would inform strategies to refine appropriate NO dosage and release kinetics for optimal therapeutic activity. In this study, we evaluated mediator, immune cell, and mRNA expression profiles in the local tissue microenvironment surrounding implanted sensors as a function of NO release, diabetes, and implantation duration. A custom porcine wound healing-centric multiplex gene array was developed for nanoString barcoding analysis. Tissues adjacent to sensors with sustained NO release abrogated the implant-induced acute and chronic FBR through modulation of the tissue-specific immune chemokine and cytokine microenvironment, resulting in decreased cellular recruitment, proliferation, and activation at both the acute (7-d) and chronic (14-d) phases of the FBR. Further, we found that sustained NO release abrogated the implant-induced acute and chronic foreign body response through modulation of mRNA encoding for key immunological signaling molecules and pathways, including STAT1 and multiple STAT1 targets including MAPK14, IRAK4, MMP2, and CXCL10. The condition of diabetes promoted a more robust FBR to the implants, which was also controlled by sustained NO release.

Nitric oxide diffusion through cystic fibrosis-relevant media and lung tissue.

A simplified diffusion cell methodology was employed to measure the diffusion coefficient of nitric oxide (NO) through phosphate buffered saline (PBS) and artificial sputum medium (ASM)-an in vitro analog for airway mucus. Diffusion through the proteinaceous ASM yielded a significantly lower diffusion coefficient compared to PBS, which is attributed to both the physical obstruction by the mucin mesh and reactive nature of NO radicals towards the biological compounds in ASM. To further confirm that ASM was restricting NO from diffusing freely, a macromolecular propylamine-modified cyclodextrin donor (CD-PA) was employed to release the NO more slowly. The NO diffusion characteristics in ASM via the NO donor were also slower relative to PBS. As NO is likely to interact with lung cells after passing through the mucus barrier, the diffusion of both NO and the CD-PA macromolecular NO donor through differentiated lung tissue was investigated with and without an ASM layer. Comparison of NO diffusion through the three diffusion barriers indicated that the lung tissue significantly impeded NO penetration over the course of the experiment compared to PBS and ASM. In fact, the diffusion of CD-PA through the lung tissue was hindered until after the release of its NO payload, potentially due to the increased net charge of the NO donor structure. Of importance, the viability of the tissue was not influenced by the NO-releasing CD-PA at bactericidal concentrations.

Microfluidics for the detection of minimal residual disease in acute myeloid leukemia patients using circulating leukemic cells selected from blood.

We report a highly sensitive microfluidic assay to detect minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) that samples peripheral blood to search for circulating leukemic cells (CLCs). Antibodies immobilized within three separate microfluidic devices affinity-selected CLC subpopulations directly from peripheral blood without requiring pre-processing. The microfluidic devices targeted CD33, CD34, and CD117 cell surface antigens commonly expressed by AML leukemic cells so that each subpopulation's CLC numbers could be tracked to determine the onset of relapse. Staining against aberrant markers (e.g. CD7, CD56) identified low levels (11-2684 mL(-1)) of CLCs. The commonly used platforms for the detection of MRD for AML patients are multi-parameter flow cytometry (MFC), typically from highly invasive bone marrow biopsies, or PCR from blood samples, which is limited to <50% of AML patients. In contrast, the microfluidic assay is a highly sensitive blood test that permits frequent sampling for >90% of all AML patients using the markers selected for this study (selection markers CD33, CD34, CD117 and aberrant markers such as CD7 and CD56). We present data from AML patients after stem cell transplant (SCT) therapy using our assay. We observed high agreement of the microfluidic assay with therapeutic treatment and overall outcome. We could detect MRD at an earlier stage compared to both MFC and PCR directly from peripheral blood, obviating the need for a painful bone marrow biopsy. Using the microfluidic assay, we detected MRD 28 days following one patient's SCT and the onset of relapse at day 57, while PCR from a bone marrow biopsy did not detect MRD until day 85 for the same patient. Earlier detection of MRD in AML post-SCT enabled by peripheral blood sampling using the microfluidic assay we report herein can influence curative clinical decisions for AML patients.