Mouse and human islets survive and function after coating by biosilicification

Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to medium containing saturating silicic acid levels for 9-15 min. The resulting tissue constructs were then cultured for up to 4 wk under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. A cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function were assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice

Tunable, spatially addressable functionalization strategies for micro/nano scale, multi-analyte biosensors

Physiological sensing is inherently a multi-scale problem involving measurements of multiple analytes, spanning a broad range of spatial and temporal scales from either single cells or complex cell networks. Addressing multiple scales requires novel high throughput sensing paradigms capable of multiplexed, multi-modal sensing with high spatial and temporal resolution. In recent years, novel nanomaterials and fabrication techniques have enabled a new generation of electrical and optical sensing elements that have significantly enhanced sensor sensitivity and temporal resolution. However, biofunctionalization of these sensing elements still rely on relatively simple drop-coating or dip-coating methods to achieve coupling with analyte-specific transducers. Use of such passive functionalization methods limits the spatial resolution, precludes multiplexing and restricts use in high throughput schemes. We have developed novel electrochemical and optical sensor functionalization strategies that utilize the sensing element as an active component in the functionalization process. In both modalities the sensing element is used to actively direct polymerization of selected monomers to produce polymer matrices that entrap the analyte-specific transducer within the active zone of the sensor element. Electrochemically and optically activated monomers are used to achieve tunable doping of transducer molecules such as enzymes, organic dyes and fluorescent sensor proteins at resolutions limited only by the size and spacing of the fabricated sensing element. These methods represent a significant advancement towards multiplexed, multimodal sensing of a wide range of analytes at high spatial and temporal resolution

Robust Functionalization of Large Microelectrode Arrays by Using Pulsed Potentiostatic Deposition

Surface modification of microelectrodes is a central step in the development of microsensors and microsensor arrays. Here, we present an electrodeposition scheme based on voltage pulses. Key features of this method are uniformity in the deposited electrode coatings, flexibility in the overall deposition area, i.e., the sizes and number of the electrodes to be coated, and precise control of the surface texture. Deposition and characterization of four different materials are demonstrated, including layers of high-surface-area platinum, gold, conducting polymer poly(ethylenedioxythiophene), also known as PEDOT, and the non-conducting polymer poly(phenylenediamine), also known as PPD. The depositions were conducted using a fully integrated complementary metal-oxide-semiconductor (CMOS) chip with an array of 1024 microelectrodes. The pulsed potentiostatic deposition scheme is particularly suitable for functionalization of individual electrodes or electrode subsets of large integrated microelectrode arrays: the required deposition waveforms are readily available in an integrated system, the same deposition parameters can be used to functionalize the surface of either single electrodes or large arrays of thousands of electrodes, and the deposition method proved to be robust and reproducible for all materials tested.ISSN:1424-822

Light-directed functionalization methods for high-resolution optical fiber based biosensors

Recent advances in miniaturization and analyte-sensitive fluorescent indicators make optical fiber biosensors promising alternatives to microelectrodes. Optical sensing offers several advantages over electrochemical methods including increased stability and better spatial control to monitor physiological processes at cellular resolutions. The distal end of an optical fiber can be functionalized with different fluorophore/polymer combinations through mechanical, dip-coating or photopolymerization techniques. Unlike mechanical and dip-coating schemes, photopolymerization can spatially confine the sensing layer in the vicinity of light in a more reproducible and controllable manner. The objective of this study was to fabricate microscale fluorescence lifetime based optrodes using UV-induced photopolymerization. Six commercially available acrylate based monomers were investigated for stable entrapment of the oxygen sensitive porphyrin dye (PtTFPP) dye via photopolymerization at the end of optical fibers. Of these, the acrylate-functionalized alkoxysilane monomer, 3-methacryloxypropyl-trimethoxysilane (tradename Dynasylan MEMO) showed maximal response to changes in oxygen concentration. Dye-doped polymer microtips were grown at the ends 50 mu m optical fibers and sensitivity and response time were optimized by varying both the concentration of doped dye and the excitation power used for polymerization. The resulting sensors showed linear response within the physiologically relevant range of oxygen concentrations and fast response times. While applied here to oxygen sensing, the photopolymer formulation and process parameters described are compatible with a wide range of available organic dyes and can be used to pattern arrays of spots, needles or more complex shapes at high spatial resolution

Robust Functionalization of Large Microelectrode Arrays by Using Pulsed Potentiostatic Deposition

Surface modification of microelectrodes is a central step in the development of microsensors and microsensor arrays. Here, we present an electrodeposition scheme based on voltage pulses. Key features of this method are uniformity in the deposited electrode coatings, flexibility in the overall deposition area, i.e., the sizes and number of the electrodes to be coated, and precise control of the surface texture. Deposition and characterization of four different materials are demonstrated, including layers of high-surface-area platinum, gold, conducting polymer poly(ethylenedioxythiophene), also known as PEDOT, and the non-conducting polymer poly(phenylenediamine), also known as PPD. The depositions were conducted using a fully integrated complementary metal-oxide-semiconductor (CMOS) chip with an array of 1024 microelectrodes. The pulsed potentiostatic deposition scheme is particularly suitable for functionalization of individual electrodes or electrode subsets of large integrated microelectrode arrays: the required deposition waveforms are readily available in an integrated system, the same deposition parameters can be used to functionalize the surface of either single electrodes or large arrays of thousands of electrodes, and the deposition method proved to be robust and reproducible for all materials tested

Studies on Binary Diffusion of the Gas Pairs O2-A, O2-Xe and O2-He

Genetically encoded molecular-protein sensors (GEMS) are engineered to sense and quantify a wide range of biological substances and events in cells, in vitro and even in vivo with high spatial and temporal resolution. Here, we aim to stably incorporate these proteins into a photopatternable matrix, while preserving their functionality, to extend the application of these proteins as spatially addressable optical biosensors. For this reason, we examined the fabrication of 3D hydrogel microtips doped with a genetically encoded fluorescent biosensor, GCaMP3, at the end of an optical fiber. Stable incorporation parameters of GCaMP3 into a photo-cross-linkable monomer matrix were investigated through a series of characterization and optimization experiments. Different precursor-solution formulations and irradiation parameters of in situ photopolymerization were tested to determine the factors affecting protein stability and sensor reproducibility during photoencapsulation. The microstructure and performance of hydrogel microtips were controlled by varying UV irradiation intensity as well as the molecular weight and concentration of the photocurable monomer, PEGDA (polyethylene glycol diacrylate), in precursor solution. Protein-doped hydrogel micro-optrodes (microtip sensors) were fabricated successfully and reproducibly at the distal end of optical fiber. Under optimized conditions, the bioactivity of GCaMP3 within a hydrogel matrix of micro-optrodes remained similar to that of the protein-free matrix in buffer. The limit of detection of protein optrodes for free calcium was also determined to be 4.3 nM. The hydrogel formulation and fabrication process demonstrated here using microtip optrodes can be easily adapted to other conformation-dependent protein biosensors and can be used in sensing applications

Separate vmPFC Ensembles Control Cocaine Self-Administration Versus Extinction in Rats

Recent studies suggest that the ventral medial prefrontal cortex (vmPFC) encodes both operant drug self-administration and extinction memories. Here, we examined whether these opposing memories are encoded by distinct neuronal ensembles within the vmPFC with different outputs to the nucleus accumbens (NAc) in male and female rats. Using cocaine self-administration (3 h/d for 14 d) and extinction procedures, we demonstrated that vmPFC was similarly activated (indexed by Fos) during cocaine-seeking tests after 0 (no-extinction) or 7 extinction sessions. Selective Daun02 lesioning of the self-administration ensemble (no-extinction) decreased cocaine seeking, whereas Daun02 lesioning of the extinction ensemble increased cocaine seeking. Retrograde tracing with fluorescent cholera toxin subunit B injected into NAc combined with Fos colabeling in vmPFC indicated that vmPFC self-administration ensembles project to NAc core while extinction ensembles project to NAc shell. Functional disconnection experiments (Daun02 lesioning of vmPFC and acute dopamine D1-receptor blockade with SCH39166 in NAc core or shell) confirm that vmPFC ensembles interact with NAc core versus shell to play dissociable roles in cocaine self-administration versus extinction, respectively. Our results demonstrate that neuronal ensembles mediating cocaine self-administration and extinction comingle in vmPFC but have distinct outputs to the NAc core and shell that promote or inhibit cocaine seeking

Improved methods for marking active neuron populations

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.status: publishe