Christian Siltanen

Biosensor technology: recent advances in threat agent detection and medicine

Biosensors are of great significance because of their capability to resolve a potentially large number of analytical problems and challenges in very diverse areas such as defense, homeland security, agriculture and food safety, environmental monitoring, medicine, pharmacology, industry, etc. The expanding role of biosensing in society and a real-world environment has led to an exponential growth of the R&D efforts around the world. The world market for biosensor devices, according to Global Industry Analysts, Inc., is expected to reach

Microfluidic fabrication of bioactive microgels for rapid formation and enhanced differentiation of stem cell spheroids

12 billion by 2015. Such expedient growth is driven by several factors including medical and health problems, such as a growing population with a high risk of diabetes and obesity, and the rising incidence of chronic diseases such as heart disease, stroke, cancer, chronic respiratory diseases, tuberculosis, etc.; significant problems with environmental monitoring; and of course serious challenges in security and military applications and agriculture/food safety. A review paper in the biosensor technology area may be structured based on (i) the principles of detection, such as the type of transducer platform, bioanalytical principles (affinity or kinetic), and biorecognition elements origin/properties (i.e. antibodies, enzymes, cells, aptamers, etc.), and (ii) the application area. This review follows the latter strategy and focuses on the applications. This allows discussion on how different sensing strategies are brought to bear on the same problem and highlights advantages/disadvantages of these sensing strategies. Given the broad range of biosensor related applications, several particularly relevant areas of application were selected for review: biological threat agents, chemical threat agents, and medicine.

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

UNLABELLED A major challenge in tissue engineering is to develop robust protocols for differentiating ES and iPS cells to functional adult tissues at a clinically relevant scale. The goal of this study is to develop a high throughput platform for generating bioactive, stem cell-laden microgels to direct differentiation in a well-defined microenvironment. We describe a droplet microfluidics system for fabricating microgels composed of polyethylene glycol and heparin, with tunable geometric, mechanical, and chemical properties, at kHz rates. Heparin-containing hydrogel particles sequestered growth factors Nodal and FGF-2, which are implicated in specifying pluripotent cells to definitive endoderm. Mouse ESCs were encapsulated into heparin microgels with a single dose of Nodal and FGF-2, and expressed high levels of endoderm markers Sox17 and FoxA2 after 5 days. These results highlight the use of microencapsulation for tailoring the stem cell microenvironment to promote directed differentiation, and may provide a straightforward path to large scale bioprocessing in the future. STATEMENT OF SIGNIFICANCE Multicellular spheroids and microtissues are valuable for tissue engineering, but fabrication approaches typically sacrifice either precision or throughput. Microfluidic encapsulation in polymeric biomaterials is a promising technique for rapidly generating cell aggregates with excellent control of microenvironmental parameters. Here we describe the microfluidic fabrication of bioactive, heparin-based microgels, and demonstrate the adsorption of heparin-binding growth factors for enhancing directed differentiation of embryonic stem cells toward endoderm. This approach also facilitated a ∼90-fold decrease in consumption of exogenous growth factors compared to conventional differentiation protocols.

Micropatterned Photodegradable Hydrogels for the Sorting of Microbeads and 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.

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

Microarrays of biomolecules and cells have played important roles in genomics, diagnostics, and drug screening.[1] Live-cell capture on micropatterned surfaces is particularly valuable for diagnostic or biosensing applications,[2] as cell arrays are amenable to rapid characterization and sorting. For example, antibody-coated micropost arrays have been used to detect and isolate rare circulating tumor cells from peripheral blood with high fidelity.[3] Microwell arrays have been used for the capture and analysis of single immune cells.[4] While it is often important to release or sort subsets of cells from these arrays for more thorough downstream analysis (e.g. by PCR or Western blot), methods for the retrieval of specific cells from micropatterned surfaces remain limited. Electrochemical stimulation can be exploited to release cells from particular regions within micropatterns,[5] but these approaches require that cell arrays be registered with electrodes, and they can become somewhat complicated for larger arrays. Arguably, light-activated cell-release strategies provide more flexibility, particularly for retrieval from highly dense arrays. Laser capture microdissection or laser catapulting can be used to remove specific cells from micropatterned surfaces,[4a, 6] but the former approach works with fixed cells while the latter involves harsh conditions, often resulting in cell injury. Another example of light-activated cell sorting is the micropallet array developed by Albritton et al., in which cells are cultured on polymer microstructures (e.g. photoresist patterns) that can later be dislodged from the surface by laser pulses.[7]

Microfluidic generation of alginate microgels for the controlled delivery of lentivectors

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.

Sensing Conductive Hydrogels for Rapid Detection of Cytokines in Blood

Lentivectors are widely used for gene delivery and have been increasingly tested in clinical trials. However, achieving safe, localized, and sufficient gene expression remain key challenges for effective lentivectoral therapy. Localized and efficient gene expression can be promoted by developing material systems to deliver lentivectors. Here, we address the utility of microgel encapsulation as a strategy for the controlled release of lentivectors. Three distinct routes for ionotropic gelation of alginate were incorporated into microfluidic templating to create lentivector-loaded microgels. Comparisons of the three microgels revealed marked differences in mechanical properties, crosslinking environment, and ultimately lentivector release and functional gene expression in vitro. Gelation with chelated calcium demonstrated low utility for gene delivery due to a loss of lentivector function with acidic gelation conditions. Both calcium carbonate gelation, and calcium chloride gelation, preserved lentivector function with a more sustained transduction and gene expression over 4 days observed with calcium chloride gelated microgels. The validation of these two strategies for lentivector microencapsulation may provide a platform for controlled gene delivery.

Photodegradable Hydrogels for Capture, Detection, and Release of Live Cells

Conducting polymer hydrogel is fabricated atop gold or ITO electrodes and is functionalized with monoclonal antibodies. Binding of interferon-γ molecules causes redox properties of conductive hydrogel to change in a concentration-dependent fashion without the need for washing or sample handling steps. This conductive hydrogel remains functional in a fouling media such as whole blood.