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Micropatterned aptasensors for continuous monitoring of cytokine release from human leukocytes.

ABSTRACT: We report the development of a microdevice for detecting local interferon gamma (IFN-?) release from primary human leukocytes in real time. Our microdevice makes use of miniature aptamer-modified electrodes integrated with microfluidics to monitor cellular production of IFN-?. The aptamer species consists of a DNA hairpin molecule with thiol groups on the 3'-end for self-assembly onto Au electrodes. A redox reporter is covalently attached at the 5'-end for electrochemical sensing. This aptasensor has excellent sensitivity for IFN-? (<60 pM detection limit) and responds to the target analyte in real time without additional washing or labeling steps. Aptamer-functionalized electrode arrays are fabricated on glass slides containing poly(ethylene glycol) (PEG) hydrogel patterns designed to expose glass regions adjacent to electrodes while protecting the remainder of the surface from nonspecific adsorption. The micropatterned substrates are integrated with PDMS microfluidic channels and incubated with T-cell-specific antibodies (Ab) (anti-CD4). Upon injection of blood, leukocytes are bound to Ab-modified glass regions in proximity to aptasensors. Cytokine release from captured cells is triggered by mitogenic activation and detected at the aptamer-modified electrodes using square wave voltammetry (SWV). The IFN-? signal is monitored in real time with signal appearing as early as 15 min poststimulation from as few as 90 T cells. The observed IFN-? release profiles are used to calculate an initial IFN-? production rate of 0.0079 pg cell(-1) h(-1) upon activation. The work described here represents an important step toward development of aptasensors for immune cell analysis and blood-based diagnostics.

SUBMITTER: Liu Y

PROVIDER: S-EPMC3235337 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Micropatterned aptasensors for continuous monitoring of cytokine release from human leukocytes.

Liu Ying Y Yan Jun J Howland Michael C MC Kwa Timothy T Revzin Alexander A

Analytical chemistry 20111013 21

We report the development of a microdevice for detecting local interferon gamma (IFN-γ) release from primary human leukocytes in real time. Our microdevice makes use of miniature aptamer-modified electrodes integrated with microfluidics to monitor cellular production of IFN-γ. The aptamer species consists of a DNA hairpin molecule with thiol groups on the 3'-end for self-assembly onto Au electrodes. A redox reporter is covalently attached at the 5'-end for electrochemical sensing. This aptasenso ...[more]

PMID: 21942846

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Project description:Controlled molecule release from scaffolds can dramatically increase the scaffold ability of directing tissue regeneration in vitro and in vivo. Crucial to the regeneration is precise regulation over release direction and kinetics of multiple molecules (small genes, peptides, or larger proteins). To this end, we developed gradient micropatterns of electrospun nanofibers along the scaffold thickness through programming the deposition of heterogeneous nanofibers of poly(?-caprolactone) (PCL) and poly(D,L-lactide-co-glycolide) acid (PLGA). Confocal images of the scaffolds containing fluorophore-impregnated nanofibers demonstrated close matching of actual and designed gradient fiber patterns; thermal analyses further showed their matching in the composition. Using acid-terminated PLGA (PLGAac) and ester-terminated PLGA (PLGAes) to impregnate molecules in the PCL-PLGA scaffolds, we demonstrated for the first time their differences in nanofiber degeneration and molecular weight change during degradation. PLGAac nanofibers were more stable with gradual and steady increase in the fiber diameter during degradation, resulting in more spatially confined molecule delivery from PCL-PLGA scaffolds. Thus, patterns of PCL-PLGAac nanofibers were used to design versatile controlled delivery scaffolds. To test the hypothesis that molecule-impregnated PLGAac in the gradient-patterned PCL-PLGAac scaffolds can program various modalities of molecule release, model molecules, including small fluorophores and larger proteins, were respectively used for time-lapse release studies. Gradient-patterns were used as building blocks in the scaffolds to program simultaneous release of one or multiple proteins to one side or, respectively, to the opposite sides of scaffolds for up to 50 days. Results showed that the separation efficiency of molecule delivery from all the scaffolds with a thickness of 200 ?m achieved >88% for proteins and >82% for small molecules. In addition to versatile spatially controlled delivery, micropatterns were designed to program sequential release of proteins. The hierarchically structured materials presented here may enable development of novel multifunctional scaffolds with defined 3D dynamic microenvironments for tissue regeneration.

Project description:BackgroundWuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases.Principal findingsIn vitro, PMNs expel large amounts of NETs that capture but do not kill B. malayi Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill B. malayi Mf in vitro and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf in vitro, which may reflect the pathological heterogeneity associated with filarial parasitic infections.Conclusions/significanceHuman innate immune cells are able to recognize, attach to and kill B. malayi microfilariae in an in vitro system. This suggests that, in vivo, the parasites can evade this ability, or that only some human hosts support an infection with circulating Mf.

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Micropatterned aptasensors for continuous monitoring of cytokine release from human leukocytes.

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Micropatterned aptasensors for continuous monitoring of cytokine release from human leukocytes.

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