Development of an aptasensor for electrochemical detection of exosomes.

Exosomes are small (50-100 nm in diameter) vesicles secreted from various mammalian cells. Exosomes have been correlated with tumor antigens and anti-tumor immune responses and may represent cancer biomarkers. Herein, we report on the development of an aptamer-based electrochemical biosensor for quantitative detection of exosomes. Aptamers specific to exosome transmembrane protein CD63 were immobilized onto gold electrode surfaces and incorporated into a microfluidic system. Probing strands pre-labeled with redox moieties were hybridized onto aptamer molecules anchored on the electrode surface. In the presence of exosomes these beacons released probing strands with redox reporters causing electrochemical signal to decrease. These biosensors could be used to detect as few as 1×10(6) particles/mL of exosomes, which represents 100-fold decrease in the limit of detection compared to commercial immunoassays relying on anti-CD63 antibodies. Given the importance of exosome-mediated signal transmission among cells, our study may represent an important step towards development of a simple biosensor that detects exosomes without washing or labeling steps in complex media.

Microfluidic compartments with sensing microbeads for dynamic monitoring of cytokine and exosome release from single cells.

Monitoring activity of single cells has high significance for basic science and diagnostic applications. Here we describe a reconfigurable microfluidic device for confining single cells along with antibody-modified sensing beads inside 20 picoliter (pL) microcompartments for monitoring cellular secretory activity. An array of ∼7000 microchambers fabricated in the roof of the reconfigurable microfluidic device could be raised or lowered by applying negative pressure. The floor of the device was micropatterned to contain cell attachment sites in registration with the microcompartments. Using this set-up, we demonstrated the detection of inflammatory cytokine IFN-γ and exosomes from single immune cells and cancer cells respectively. The detection scheme was similar in both cases: cells were first captured on the surface inside the microfluidic device, then sensing microbeads were introduced into the device so that, once the microcompartments were lowered, single cells and microbeads became confined together. The liquid bathing the beads and the cells inside the compartments also contained fluorescently-labeled secondary antibodies (Abs). The capture of cell-secreted molecules onto microbeads was followed by binding of secondary antibodies - this caused microbeads to become fluorescent. The fluorescence intensity of the microbeads changed over time, providing dynamics of single cell secretory activity. The microdevice described here may be particularly useful in the cases where panning upstream of sensing is required or to analyze secretory activity of anchorage-dependent cells.

Coating of electrospun poly(lactic-co-glycolic acid) nanofibers with willemite bioceramic: improvement of bone reconstruction in rat model.

We have investigated the combination effects of bioceramics and poly(lactide-co-glycolide) (PLGA) on bone reconstruction in calvarial critical size defects using a rat model. Willemite (Zn2SiO4) ceramics were prepared and coated on the surface of electrospun fabricated scaffolds. After scaffolds and nanoparticles characterization, osteoconductivity of the construct was analyzed using digital mammography, multislice spiral-computed tomography (MSCT) imaging, and histological analysis. Eight weeks after implantation, no sign of inflammation was observed at the site of the osseous defect. The results showed that the ceramics supported bone regeneration and highest bone reconstruction were observed in willemite-coated PLGA. This suggests that electrospun PLGA nanofibers coated with BG are potential candidate implants for bone tissue engineering applications.

Photodegradable hydrogels for capture, detection, and release of live cells.

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T-cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV-induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95% purity of CD4 and CD8 T-cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti-CD4 and anti-TNF-α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence "halo" after immunofluorescent staining and could be retrieved by site-specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.

Clinical Evaluation of the Torq Zero Delay Centrifuge System for Decentralized Blood Collection and Stabilization.

Blood sample collection and rapid separation-critical preanalytical steps in clinical chemistry-can be challenging in decentralized collection settings. To address this gap, the Torq™ zero delay centrifuge system includes a lightweight, hand-portable centrifuge (ZDrive™) and a disc-shaped blood collection device (ZDisc™) enabling immediate sample centrifugation at the point of collection. Here, we report results from clinical validation studies comparing performance of the Torq System with a conventional plasma separation tube (PST). Blood specimens from 134 subjects were collected and processed across three independent sites to compare ZDisc and PST performance in the assessment of 14 analytes (K, Na, Cl, Ca, BUN, creatinine, AST, ALT, ALP, total bilirubin, albumin, total protein, cholesterol, and triglycerides). A 31-subject precision study was performed to evaluate reproducibility of plasma test results from ZDiscs, and plasma quality was assessed by measuring hemolysis and blood cells from 10 subject specimens. The ZDisc successfully collected and processed samples from 134 subjects. ZDisc results agreed with reference PSTs for all 14 analytes with mean % biases well below clinically significant levels. Results were reproducible across different operators and ZDisc production lots, and plasma blood cell counts and hemolysis levels fell well below clinical acceptance thresholds. ZDiscs produce plasma samples equivalent to reference PSTs. Results support the suitability of the Torq System for remotely collecting and processing blood samples in decentralized settings.

Core-shell hydrogel microcapsules enable formation of human pluripotent stem cell spheroids and their cultivation in a stirred bioreactor.

Cellular therapies based on human pluripotent stem cells (hPSCs) offer considerable promise for treating numerous diseases including diabetes and end stage liver failure. Stem cell spheroids may be cultured in stirred bioreactors to scale up cell production to cell numbers relevant for use in humans. Despite significant progress in bioreactor culture of stem cells, areas for improvement remain. In this study, we demonstrate that microfluidic encapsulation of hPSCs and formation of spheroids. A co-axial droplet microfluidic device was used to fabricate 400 µm diameter capsules with a poly(ethylene glycol) hydrogel shell and an aqueous core. Spheroid formation was demonstrated for three hPSC lines to highlight broad utility of this encapsulation technology. In-capsule differentiation of stem cell spheroids into pancreatic ß-cells in suspension culture was also demonstrated.

Microencapsulated Immunoassays for Detection of Cytokines in Human Blood.

Cytokines are produced by leukocytes in blood and may be used as indicators of malignancies or infections. The objective of this study was to develop a strategy for immunosensing cytokines in whole, unprocessed human blood. Microfluidic droplet generation was employed to fabricate ∼400 µm diameter microcapsules with a hydrogel shell and an aqueous core containing sensing microbeads. The hydrogel shell was composed of poly(ethylene glycol) forming a thin (∼10 µm) immunoisolation layer protecting antibody-modified microbeads inside the capsule from immune cells on the outside. The microbeads were functionalized with antibodies against cytokines of interest: interferon (IFN)-γ and tumor necrosis factor (TNF)-α. While nonfouling, a hydrogel shell was permeable to cytokine molecules; these molecules were captured on microbeads and were detected with fluorescently labeled secondary antibodies. Calibration of encapsulated immunoassays with known concentrations of cytokines revealed a limit of detection of 14.8 and 14.4 pM for IFN-γ and TNF-α, respectively. We also demonstrated the concept of multi-cytokine detection by fabricating distinct populations of capsules carrying either anti-IFN-γ or anti-TNF-α microbeads and dispensing these capsules into a solution containing both cytokine types. Importantly, when placed into whole blood for 16 h, microcapsules were free of leukocytes, effectively protecting sensing beads from the blood components. To further demonstrate utility of this strategy, encapsulated microbeads were used for detection of IFN-γ in blood of patients with latent tuberculosis infection (LTBI) and unexposed healthy controls. When compared to gold standard technology (interferon gamma release assay or IGRA), our encapsulated immunoassay accurately predicted LTBI diagnosis in 11 out of 14 patients. Overall, encapsulation of immunoassays represents a promising strategy for keeping sensing elements operational in a highly fouling complex environment such as blood.

Detecting cell-secreted growth factors in microfluidic devices using bead-based biosensors.

Microfluidic systems provide an interesting alternative to standard macroscale cell cultures due to the decrease in the number of cells and reagents as well as the improved physiology of cells confined to small volumes. However, the tools available for cell-secreted molecules inside microfluidic devices remain limited. In this paper, we describe an integrated microsystem composed of a microfluidic device and a fluorescent microbead-based assay for the detection of the hepatocyte growth factor (HGF) and the transforming growth factor (TGF)-ß1 secreted by primary hepatocytes. This microfluidic system is designed to separate a cell culture chamber from sensing chambers using a permeable hydrogel barrier. Cell-secreted HGF and TGF-ß1 diffuse through the hydrogel barrier into adjacent sensing channels and are detected using fluorescent microbead-based sensors. The specificity of sensing microbeads is defined by the choice of antibodies; therefore, our microfluidic culture system and sensing microbeads may be applied to a variety of cells and cell-secreted factors.

One step fabrication of hydrogel microcapsules with hollow core for assembly and cultivation of hepatocyte spheroids.

3D hepatic microtissues can serve as valuable liver analogues for cell-based therapies and for hepatotoxicity screening during preclinical drug development. However, hepatocytes rapidly dedifferentiate in vitro, and typically require 3D culture systems or co-cultures for phenotype rescue. In this work we present a novel microencapsulation strategy, utilizing coaxial flow-focusing droplet microfluidics to fabricate microcapsules with liquid core and poly(ethylene glycol) (PEG) gel shell. When entrapped inside these capsules, primary hepatocytes rapidly formed cell-cell contacts and assembled into compact spheroids. High levels of hepatic function were maintained inside the capsules for over ten days. The microencapsulation approach described here is compatible with difficult-to-culture primary epithelial cells, allows for tuning gel mechanical properties and diffusivity, and may be used in the future for high density suspension cell cultures. STATEMENT OF SIGNIFICANCE: Our paper combines an interesting new way for making capsules with cultivation of difficult-to-maintain primary epithelial cells (hepatocytes). The microcapsules described here will enable high density suspension culture of hepatocytes or other cells and may be used as building blocks for engineering tissues.

Nanowire Aptasensors for Electrochemical Detection of Cell-Secreted Cytokines.

Cytokines are small proteins secreted by immune cells in response to pathogens/infections; therefore, these proteins can be used in diagnosing infectious diseases. For example, release of a cytokine interferon (IFN)-γ from T-cells is used for blood-based diagnosis of tuberculosis (TB). Our lab has previously developed an atpamer-based electrochemical biosensor for rapid and sensitive detection of IFN-γ. In this study, we explored the use of silicon nanowires (NWs) as a way to create nanostructured electrodes with enhanced sensitivity for IFN-γ. Si NWs were covered with gold and were further functionalized with thiolated aptamers specific for IFN-γ. Aptamer molecules were designed to form a hairpin and in addition to terminal thiol groups contained redox reporter molecules methylene blue. Binding of analyte to aptamer-modified NWs (termed here nanowire aptasensors) inhibited electron transfer from redox reporters to the electrode and caused electrochemical redox signal to decrease. In a series of experiments we demonstrate that NW aptasensors responded 3× faster and were 2× more sensitive to IFN-γ compared to standard flat electrodes. Most significantly, NW aptasensors allowed detection of IFN-γ from as few as 150 T-cells/mL while ELISA did not pick up signal from the same number of cells. One of the challenges faced by ELISA-based TB diagnostics is poor performance in patients whose T-cell numbers are low, typically HIV patients. Therefore, NW aptasensors developed here may be used in the future for more sensitive monitoring of IFN-γ responses in patients coinfected with HIV/TB.

Comparative evaluation between two methods of induced hypotension with infusion of Remifentanil and Labetalol during sinus endoscopy.

OBJECTIVE: This study aimed to compare two methods of controlled hypotension using labetalol and remifentanil in terms of capability to create controlled hypotension and to investigate the obtained complications, and satisfaction rate of surgeon and patient during functional endoscopic sinus surgery. METHODS: In this prospective clinical trial, 62 patients underwent endoscopic sinus surgery in Al-Zahra and Ayatollah Kashani Hospitals of Isfahan were divided into two groups: in the first group, 20 mg bolus dose of labetalol and then infusion of it, at a rate of 0.5-2.0 mg/min and in the second group, remifentanil with dose of 0.5-1 µg/kg started and then 0.25-0.5 µg/kg/min were prescribed. Hemodynamic parameters during anesthesia and recovery time, surgeon and patient satisfaction, and recovery time were measured and recorded. FINDINGS: Hemodynamics variable were comparable between two groups at different times of the study. The mean of bleeding and the frequency of side effects were higher in labetalol group (P = 0.033 and P < 0.0001, respectively). The median of surgeon satisfaction score in remifentanil group was statistically higher in labetalol group (P < 0.0001). Recovery time, fluid requirement, and pain score in labetalol group reported significantly more than remifentanil group. Richmond Agitation-Sedation Scale status at time points in the postanesthetic care unit showed differences between groups. CONCLUSION: With infusion of labetalol and remifentanil after a bolus dose we can induce effective controlled hypotension under general anesthesia. Remifentanil is a short-acting narcotic drug; then, patient satisfaction was better and recovery time was shorter. From the economic aspect, labetalol prefers to remifentanil.

Detecting multiple cell-secreted cytokines from the same aptamer-functionalized electrode.

Inflammatory cytokines are secreted by immune cells in response to infection or injury. Quantification of multiple cytokines in parallel may help with disease diagnosis by illuminating inflammatory pathways related to disease onset and progression. This paper describes development of an electrochemical aptasensor for simultaneous detection of two important inflammatory cytokines, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). To enable multiplexing, IFN-γ and TNF-α aptamers were labeled with anthraquinone (AQ) and methylene blue (MB) redox reporters respectively. Random immobilization of two aptamer on gold exhibited redox peaks at -0.37 V (AQ) and -0.15 V (MB) vs. Ag/AgCl reference. When challenged with either IFN-γ or TNF-α, redox signal of the appropriate reporter changed in concentration dependent manner. To demonstrate one possible application of this sensing approach, electrodes were integrated into microfluidic devices and used to dynamically monitor cytokine release from immune cells. Two cell types, primary human CD4 T-cells and U937 monocytic cells, were used to compare differences in cytokine secretions upon stimulation. These cells were infused into the microfluidic devices and stimulated to commence cytokine production. Release of IFN-γ and TNF-α was monitored concurrently from the same small group of cells over the course of 2h. The strategy of encoding specific aptamer types with unique redox reporters allows sensitive and specific detection of multiple protein biomarkers from the same electrode.

Reconfigurable microfluidic device with integrated antibody arrays for capture, multiplexed stimulation, and cytokine profiling of human monocytes.

Monocytes represent a class of immune cells that play a key role in the innate and adaptive immune response against infections. One mechanism employed by monocytes for sensing foreign antigens is via toll-like receptors (TLRs)-transmembrane proteins that distinguish classes of foreign pathogens, for example, bacteria (TLR4, 5, and 9) vs. fungi (TLR2) vs. viruses (TLR3, 7, and 8). Binding of antigens activates a signaling cascade through TLR receptors that culminate in secretion of inflammatory cytokines. Detection of these cytokines can provide valuable clinical data for drug developers and disease investigations, but this usually requires a large sample volume and can be technically inefficient with traditional techniques such as flow cytometry, enzyme-linked immunosorbent assay, or luminex. This paper describes an approach whereby antibody arrays for capturing cells and secreted cytokines are encapsulated within a microfluidic device that can be reconfigured to operate in serial or parallel mode. In serial mode, the device represents one long channel that may be perfused with a small volume of minimally processed blood. Once monocytes are captured onto antibody spots imprinted into the floor of the device, the straight channel is reconfigured to form nine individually perfusable chambers. To prove this concept, the microfluidic platform was used to capture monocytes from minimally processed human blood in serial mode and then to stimulate monocytes with different TLR agonists in parallel mode. Three cytokines, tumor necrosis factor-α, interleukin (IL)-6, and IL-10, were detected using anti-cytokine antibody arrays integrated into each of the six chambers. We foresee further use of this device in applications such as pediatric immunology or drug/vaccine testing where it is important to balance small sample volume with the need for high information content.

On-chip regeneration of aptasensors for monitoring cell secretion.

We report on the use of reconfigurable microfluidics for on-chip regeneration of aptasensors used for continuous monitoring of cell-secreted products.

Reconfigurable microfluidics with integrated aptasensors for monitoring intercellular communication.

We report the development of a microsystem integrating anti-TNF-α aptasensors with vacuum-actuatable microfluidic devices that may be used to monitor intercellular communications. Actuatable chambers were used to expose to mitogen a group of ~600 cells while not stimulating another group of monocytes only 600 µm away. Co-localizing groups of cells with miniature 300 µm diameter aptamer-modified electrodes enabled monitoring of TNF-α release from each group independently. The microsystem allowed observation of the sequence of events that included 1) mitogenic activation of the first group of monocytes to produce TNF-α, 2) diffusion of TNF-α to the location of the second group of cells and 3) activation of the second group of cells resulting in the production of TNF-α by these cells. Thus, we were able to experimentally verify reciprocal paracrine crosstalk between the two groups of cells secreting the same signalling molecule. Given the prevalence of such cellular communications during injury, cancer or immune response and the dearth of available monitoring techniques, the microsystem described here is envisioned to have significant impact on cell biology.