Quantification of Metabolic Products from Microbial Hosts in Complex Media Using Optically Diffracting Hydrogels.

We herein describe a highly versatile platform approach for the in situ and real-time screening of microbial biocatalysts for enhanced production of bioproducts using photonic crystal hydrogels. This approach was demonstrated by preparing optically diffracting films based on polymerized N-isopropylacrylamide that contracted in the presence of alcohols and organic acids. The hydrogel films were prepared in a microwell plate format, which allows for high-throughput screening, and characterized optically using a microwell plate reader. While demonstrating the ability to detect a broad range of relevant alcohols and organic acids, we showed that the response of the films correlated strongly with the octanol-water partition coefficient (log P) of the analyte. Differences in the secretion of ethanol and succinic acid from strains of Zymomonas mobilis and Actinobacillus succinogenes, respectively, were further detected via optical characterization of the films. These differences, which in some cases were as low as ∼3 g/L, were confirmed by high-performance liquid chromatography, thereby demonstrating the sensitivity of this approach. Our findings highlight the potential utility of this multiplexed approach for the detection of small organic analytes in complex biological media, which overcomes a major challenge in conventional optical sensing methods.

Integrated Methods to Manufacture Hydrogel Microparticles with High Protein Conjugation Capacity and Binding Kinetics via Viral Nanotemplate Display.

Genetically modified tobacco mosaic virus (TMV) can serve as a potent nanotemplate for high capacity protein conjugation through covalent coupling to its coat proteins with precise nanoscale spacing. TMV's own genomic RNA can also be exploited for orientationally controlled assembly onto various platforms with sequence and spatial selectivity via nucleic acid hybridization. Here we describe detailed methods for fabrication of hydrogel microparticles with capture DNA sequences, chemical activation and programming of TMV templates, TMV assembly with the microparticles and protein conjugation via bio-orthogonal click reactions.

High-throughput double emulsion-based microfluidic production of hydrogel microspheres with tunable chemical functionalities toward biomolecular conjugation.

Chemically functional hydrogel microspheres hold significant potential in a range of applications including biosensing, drug delivery, and tissue engineering due to their high degree of flexibility in imparting a range of functions. In this work, we present a simple, efficient, and high-throughput capillary microfluidic approach for controlled fabrication of monodisperse and chemically functional hydrogel microspheres via formation of double emulsion drops with an ultra-thin oil shell as a sacrificial template. This method utilizes spontaneous dewetting of the oil phase upon polymerization and transfer into aqueous solution, resulting in poly(ethylene glycol) (PEG)-based microspheres containing primary amines (chitosan, CS) or carboxylates (acrylic acid, AA) for chemical functionality. Simple fluorescent labelling of the as-prepared microspheres shows the presence of abundant, uniformly distributed and readily tunable functional groups throughout the microspheres. Furthermore, we show the utility of chitosan's primary amine as an efficient conjugation handle at physiological pH due to its low pKa by direct comparison with other primary amines. We also report the utility of these microspheres in biomolecular conjugation using model fluorescent proteins, R-phycoerythrin (R-PE) and green fluorescent protein (GFPuv), via tetrazine-trans-cyclooctene (Tz-TCO) ligation for CS-PEG microspheres and carbodiimide chemistry for AA-PEG microspheres, respectively. The results show rapid coupling of R-PE with the microspheres' functional groups with minimal non-specific adsorption. In-depth protein conjugation kinetics studies with our microspheres highlight the differences in reaction and diffusion of R-PE with CS-PEG and AA-PEG microspheres. Finally, we demonstrate orthogonal one-pot protein conjugation of R-PE and GFPuv with CS-PEG and AA-PEG microspheres via simple size-based encoding. Combined, these results represent a significant advancement in the rapid and reliable fabrication of monodisperse and chemically functional hydrogel microspheres with tunable properties.

Remotely Triggered Locomotion of Hydrogel Mag-bots in Confined Spaces.

In this study, soft hydrogel crawlers with remote magnetic-responsive motility in confined spaces have been developed. Inspired by the motion of maggots, the hydrogel crawlers can reversibly contract and elongate their body controlled by repeatedly switching on/off an alternating magnetic field. Based on the cyclic deformation, the hydrogel crawlers can move peristaltically in a confined space that is coated with asymmetric micro-patterns. The dependence of the hydrogel motility on the pattern structures and lubrication is characterized using experimental measurements. Such a hydrogel system pioneers the study of active motile systems in porous media and has the potential to impact the fields of targeted drug delivery and active actuators.

Enhanced Optical Sensitivity in Thermoresponsive Photonic Crystal Hydrogels by Operating Near the Phase Transition.

Photonic crystal hydrogels composed of analyte-responsive hydrogels and crystalline colloidal arrays have immense potential as reagentless chemical and biological sensors. In this work, we investigated a general mechanism to rationally tune the sensitivity of photonic crystal hydrogels consisting of stimuli-responsive polymers to small molecule analytes. This mechanism was based on modulating the demixing temperature of such hydrogels relative to the characterization temperature to in effect maximize the extent of phase separation behavior; thus, the volume changes in response to the target analytes. Using ethanol as a model analyte, we demonstrated that this mechanism led to a dramatic increase in the sensitivity of optically diffracting poly(N-isopropylacrylamide) (pNIPAM) hydrogel films that exhibit a lower critical solution temperature (LCST) behavior. The demixing temperature of the pNIPAM films was modulated by copolymerization of the films with relatively hydrophobic and hydrophilic comonomers, as well as by varying the ionic strength of the characterization solution. Our results showed that copolymerization of the films with 2.5 mol % of N-tert-butylacrylamide, which is hydrophobic relative to pNIPAM, enabled the detection limit of the pNIPAM films to ethanol to be lowered ∼2-fold at 30 °C. Additionally, increasing the ionic strength of the characterization solution above 200 mM resulted in a dramatic increase in the extent of contraction of the films in the presence of ethanol. Ultimately, it was demonstrated that as little as 16 g/L or 2 vol % of ethanol in water could reliably be detected, and that the sensitivity of the films to ethanol was predictably greatest when operating near the phase transition, such that even small additions of the analyte induced the start of demixing and the collapse of the hydrogel. Such a mechanism may be extended to photonic crystal hydrogel sensors prepared from other stimuli-responsive polymers and more broadly exploited to enhance the utility of these sensors for a broad range of analytes.

Improved Protein Conjugation with Uniform, Macroporous Poly(acrylamide-co-acrylic acid) Hydrogel Microspheres via EDC/NHS Chemistry.

We demonstrate a robust and tunable micromolding method to fabricate chemically functional poly(acrylamide-co-acrylic acid) (p(AAm-co-AA)) hydrogel microspheres with uniform dimensions and controlled porous network structures for rapid biomacromolecular conjugation. Specifically, p(AAm-co-AA) microspheres with abundant carboxylate functional groups are fabricated via surface-tension-induced droplet formation in patterned poly(dimethylsiloxane) molds and photoinduced radical polymerization. To demonstrate the chemical functionality, we enlisted rapid EDC/NHS (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)) chemistry for fluorescent labeling of the microspheres with small-molecule dye fluorescein glycine amide. Epifluorescence imaging results illustrate the uniform incorporation of carboxylate groups within the microspheres and rapid conjugation kinetics. Furthermore, protein conjugation results using red fluorescent protein R-phycoerythrin demonstrate the highly porous nature of the microspheres as well as the utility of the microspheres and the EDC/NHS scheme for facile biomacromolecular conjugation. Combined, these results illustrate the significant potential for our fabrication-conjugation strategy in the development of biofunctionalized polymeric hydrogel microparticles toward rapid biosensing, bioprocess monitoring, and biodiagnostics.

Integrated fabrication-conjugation methods for polymeric and hybrid microparticles for programmable drug delivery and biosensing applications.

Functionalized polymeric microparticles possess significant potential for controlled drug delivery and biosensing applications, yet current fabrication techniques face challenges in simple and scalable fabrication and biofunctionalization. For programmable manufacture of biofunctional microparticles in a simple manner, we have developed robust micromolding methods combined with biopolymeric conjugation handles and bioorthogonal click reactions. In this focused minireview, we present detailed methods for our integrated approaches for fabrication of microparticles with controlled 2D and 3D shapes and dimensions toward controlled release, and for biomacromolecular conjugation via strain promoted alkyne-azide cycloaddition (SPAAC) and tetrazine-trans-cyclooctene (Tz-TCO) ligation reactions utilizing a potent aminopolysaccharide chitosan as an efficient conjugation handle. We believe that the fabrication-conjugation methods reported here from a range of our recent reports illustrate the simple, robust and readily reproducible nature of our approaches to creating multifaceted microparticles in a programmable, cost-efficient and scalable manner toward a wide range of medical and biotechnological application areas.

Porosity-Tuned Chitosan-Polyacrylamide Hydrogel Microspheres for Improved Protein Conjugation.

We report a robust method to manufacture polyacrylamide-based functional hydrogel microspheres with readily tunable macroporous structures by utilizing a simple micromolding-based technique. Specifically, surface tension-induced droplet formation of aqueous solutions of chitosan and acrylamide in 2D-shaped micromolds followed by photoinduced polymerization leads to monodisperse microspheres. Pore sizes of the microspheres can be readily tuned by simple addition of inert long-chain poly(ethylene glycol) porogen at low content in the prepolymer solution. The as-prepared chitosan-polyacrylamide microspheres exhibit chemical functionality through chitosan's primary amines, rapid protein conjugation with selective tetrazine-trans-cyclooctene reaction, and nonfouling property. Combined with the potential to create anisotropic network structures, we envision that our simple fabrication-conjugation method would offer a potent route to manufacture a variety of biofunctionalized hydrogel microentities.

Shape-Encoded Chitosan-Polyacrylamide Hybrid Hydrogel Microparticles with Controlled Macroporous Structures via Replica Molding for Programmable Biomacromolecular Conjugation.

Polymeric hydrogel microparticle-based suspension arrays with shape-based encoding offer powerful alternatives to planar and bead-based arrays toward high throughput biosensing and medical diagnostics. We report a simple and robust micromolding technique for polyacrylamide- (PAAm-) based biopolymeric-synthetic hybrid microparticles with controlled 2D shapes containing a potent aminopolysaccharide chitosan as an efficient conjugation handle uniformly incorporated in PAAm matrix. A postfabrication conjugation approach utilizing amine-reactive chemistries on the chitosan shows stable incorporation and retained chemical reactivity of chitosan, readily tunable macroporous structures via simple addition of low content long-chain PEG porogens for improved conjugation capacity and kinetics, and one-pot biomacromolecular assembly via bioorthogonal click reactions with minimal nonspecific binding. We believe that the integrated fabrication-conjugation approach reported here could offer promising routes to programmable manufacture of hydrogel microparticle-based biomacromolecular conjugation and biofunctionalization platforms for a large range of applications.

Gintonin stimulates gliotransmitter release in cortical primary astrocytes.

Lysophosphatidic acid (LPA) is a simple and minor phospholipid, but serves as a lipid-derived neurotransmitter via activation of G protein-coupled LPA receptors. Astrocytes abundantly express LPA receptors and contain gliotransmitters that modulate astrocyte-neuron interactions. Gintonin is a novel ginseng-derived G protein-coupled LPA receptor ligand. Gintonin induces [Ca(2+)]i transients in neuronal and non-neuronal cells via activation of LPA receptors, which regulate calcium-dependent ion channels and receptors. A line of evidence shows that neurotransmitter-mediated [Ca(2+)]i elevations in astrocytes are coupled with gliotransmitter release. However, little is known about whether gintonin-mediated [Ca(2+)]i transients are coupled to gliotransmitter release in astrocytes. In the present study, we examined the effects of gintonin on adenosine triphosphate (ATP) and glutamate release in mouse cortical primary astrocytes. Application of gintonin to astrocytes induced [Ca(2+)]i transients in a concentration-dependent and reversible manner. However, ginsenosides, other active ingredients in ginseng, had no effect on [Ca(2+)]i transients. The induction of gintonin-mediated [Ca(2+)]i transients was attenuated/blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5-triphosphate receptor antagonist, and an intracellular Ca(2+) chelator. Gintonin treatment on astrocytes increased ATP and glutamate release in a concentration- and time-dependent manner. BAPTA and Ki16425 attenuated gintonin-mediated ATP and glutamate release in astrocytes. The present study shows that gintonin-mediated [Ca(2+)]i transients are coupled to gliotransmitter release via LPA receptor activation. Finally, gintonin-mediated [Ca(2+)]i transients and gliotransmitter release from astrocytes via LPA receptor activation might explain one mechanism of gintonin-mediated neuromodulation in the central nervous system.

An integrated approach for enhanced protein conjugation and capture with viral nanotemplates and hydrogel microparticle platforms via rapid bioorthogonal reactions.

We demonstrate significantly enhanced protein conjugation and target protein capture capacity by exploiting tobacco mosaic virus (TMV) templates assembled with hydrogel microparticles. Protein conjugation results with a red fluorescent protein R-Phycoerythrin (R-PE) show significantly enhanced protein conjugation capacity of TMV-assembled particles (TMV-particles) compared to planar substrates or hydrogel microparticles. In-depth examination of protein conjugation kinetics via tetrazine (Tz)-trans-cyclooctene (TCO) cycloaddition and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction demonstrates that TMV-particles provide a less hindered environment for protein conjugation. Target protein capture results using an anti-R-PE antibody (R-Ab)-R-PE pair also show substantially improved capture capacity of R-Ab conjugated TMV-particles over R-Ab conjugated hydrogel microparticles. We further demonstrate readily controlled protein and antibody conjugation capacity by simply varying TMV concentrations, which show negligible negative impact of densely assembled TMVs on protein conjugation and capture capacity. Combined, these results illustrate a facile postfabrication protein conjugation approach with TMV templates assembled onto hydrogel microparticles for improved and controlled protein conjugation and sensing platforms. We anticipate that our approach can be readily applied to various protein sensing applications.

Facile strategy for protein conjugation with chitosan-poly(ethylene glycol) hybrid microparticle platforms via strain-promoted alkyne-azide cycloaddition (SPAAC) reaction.

We demonstrate a facile fabrication-conjugation scheme for protein-conjugated biosensing platforms. Specifically, we utilize a chitosan-poly(ethylene glycol) hybrid system to fabricate highly uniform and chemically reactive microparticle platforms via simple replica molding. Strain-promoted alkyne-azide cycloaddition (SPAAC) reaction between azide-modified proteins and microparticles activated with strain-promoted cyclooctynes allows tunable protein conjugation under mild reaction conditions. Upon conjugation of a model red fluorescent protein, fluorescence and confocal micrographs show selective protein conjugation near the particle surfaces as well as long-term stability of the conjugation scheme. Fluorescence and AFM results upon conjugation with varying protein concentrations indicate controllable protein conjugation. Examination of protein-particle conjugation kinetics shows multiple reaction regimes; rapid initial, intermediate, and steady final stage. Lastly, we demonstrate antibody conjugation with the particles and selective and rapid target protein capture with antibody-conjugated particles. Combined, these results illustrate a facile fabrication-conjugation scheme for robust protein-conjugated platforms that can be readily enlisted in various protein sensing applications.

Current stimulator IC with adaptive supply regulator for visual prostheses.

The current stimulation method is preferred over the voltage stimulation method in the visual prostheses based on functional electrical stimulation (FES) due to its accurate charge control property. Previous current stimulators are generally implemented using a static high supply voltage, because current stimulations require high output voltage compliance. This high static supply voltage, however, may harm the tissues or damage the electrodes. This paper proposes a novel integrated circuit (IC) current stimulator with adaptive supply regulator (ASR). In the proposed circuit, the internal power supply voltage is not static, but adaptively regulated to the minimum required voltage for stimulation. The current feedback loop in the ASR adaptively increases the internal supply voltage when the monitored current is smaller than the desired current, and reduces the internal supply voltage when the monitored current is higher than the desired current. With this method, the internal supply voltage of the stimulator is minimized, and potential damages of the tissues due to high voltage (HV) stimulation can be reduced. Also the current feedback loop in ASR enhances the accuracy of the output current and the robustness to the load impedance. The stimulator IC is fabricated using 0.35 micro m bipolar-CMOS-DMOS (BCDMOS) process, and the size of the chip is 2000 micro m by 1500 micro m.

Multi-channel stimulator IC using a channel sharing method for retinal prostheses.

A retinal stimulator is an implantable device restoring vision by supplying a controlled, stimulating electrical signal to people blinded by retinal diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). The resolution requirements of artificial retina systems become increasingly significant in their design as well as their usefulness. At least 32 x 32 pixels are required to provide a minimal visual function. However, a retinal stimulator with a high resolution imposes severe constraints on interface electronics. In this paper, a new stimulator IC (integrated chip) using a channel sharing technique is developed to minimize the circuit size, power consumption, as well as overheating of retina tissues. The proposed current-mode stimulator is fabricated by a 0.35 microm 2-poly/4-metal BCDMOS technology. Attention is given to minimizing the silicon area so that higher channel numbers can be implemented. The stimulator for each channel can provide output current in the range of 0-350 muA. The effective chip area excluding the pads is 1.2 mm x 1.2 mm.

Fabrication of chitosan-poly(ethylene glycol) hybrid hydrogel microparticles via replica molding and its application toward facile conjugation of biomolecules.

We demonstrate a facile scheme to fabricate nonspherical chitosan-poly(ethylene glycol) (PEG) microparticle platforms for conjugation of biomolecules with high surface density. Specifically, we show that PEG microparticles containing short chitosan oligomers are readily fabricated via replica molding (RM). Fluorescence and FTIR microscopy results illustrate that these chitosan moieties are incorporated with PEG networks in a stable manner while retaining chemical reactivity toward amine-reactive chemistries. The chitosan-PEG particles are then conjugated with single-stranded (ss) DNAs via Cu-free click chemistry. Fluorescence and confocal microscopy results show facile conjugation of biomolecules with the chitosan-PEG particles under mild conditions with high selectivity. These ssDNA-conjugated chitosan-PEG particles are then enlisted to assemble tobacco mosaic virus (TMV) via nucleic acid hybridization as an example of orientationally controlled conjugation of supramolecular targets. Results clearly show controllable TMV assembly with high surface density, indicating high surface DNA density on the particles. Combined, these results demonstrate a facile fabrication-conjugation scheme for robust biomolecular conjugation or assembly platforms. We expect that our approach can be enlisted in a wide array of biomolecular targets and applications.

Measurements of serum C-reactive protein levels in patients with gastric cancer and quantification using silicon nanowire arrays.

We examined the levels of serum alpha-fetoprotein, carcinoembryonic antigen, and carbohydrate antigen in 83 of 400 patients who had undergone surgery for gastric cancer and correlated these markers with stages of the disease. In addition, we measured C-reactive protein (CRP) concentrations in the sera of gastric cancer patients with silicon nanowire field-effect transistors (SiNW FETs) to determine whether SiNW FETs could be used to accurately sense CRP, a marker of inflammation and possible indicator of future progression of the cancer. We designed and fabricated SiNWs to be responsive to CRP. Of the 83 patients examined, six who showed marked elevation of CRP (>3 to 10 mg/dL, according to hospital laboratory measurements) were selected and subjected to measurement with the SiNW FETs. Our findings showed that SiNW-based sensors could be highly sensitive and specific in measuring CRP in the sera of postoperative patients and thus could represent a simple and quick method of prognostic evaluation in patients. FROM THE CLINICAL EDITOR: In this study, silicon nanowire field effect transistors were fabricated to be responsive to C-reactive protein. The new technology resulted in highly sensitive and specific CRP sensors, which may greatly simplify this serum test for a variety of conditions where rapid, accurate and easily repeatable CRP measurements are needed.

Quantitative measurements of C-reactive protein using silicon nanowire arrays.

A silicon nanowire-based sensor for biological application showed highly desirable electrical responses to either pH changes or receptor-ligand interactions such as protein disease markers, viruses, and DNA hybridization. Furthermore, because the silicon nanowire can display results in real-time, it may possess superior characteristics for biosensing than those demonstrated in previously studied methods. However, despite its promising potential and advantages, certain process-related limitations of the device, due to its size and material characteristics, need to be addressed. In this article, we suggest possible solutions. We fabricated silicon nanowire using a top-down and low cost micromachining method, and evaluate the sensing of molecules after transfer and surface modifications. Our newly designed method can be used to attach highly ordered nanowires to various substrates, to form a nanowire array device, which needs to follow a series of repetitive steps in conventional fabrication technology based on a vapor-liquid-solid (VLS) method. For evaluation, we demonstrated that our newly fabricated silicon nanowire arrays could detect pH changes as well as streptavidin-biotin binding events. As well as the initial proof-of-principle studies, C-reactive protein binding was measured: electrical signals were changed in a linear fashion with the concentration (1 fM to 1 nM) in PBS containing 1.37 mM of salts. Finally, to address the effects of Debye length, silicon nanowires coupled with antigen proteins underwent electrical signal changes as the salt concentration changed.

Fabrication of suspended silicon nanowire arrays.

A method to fabricate suspended silicon nanowires that are applicable to electronic and electromechanical nanowire devices is reported. The method allows for the wafer-level production of suspended silicon nanowires using anisotropic etching and thermal oxidation of single-crystal silicon. The deviation in width of the silicon nanowire bridges produced using the proposed method is evaluated. The NW field-effect transistor (FET) properties of the device obtained by transferring suspended nanowires are shown to be practical for useful functions.

Hard X-ray microscopy with Zernike phase contrast.

Zernike phase contrast has been added to a full-field X-ray microscope with Fresnel zone plates that was in operation at 6.95 keV. The spatial resolution has also been improved by increasing the magnification of the microscope objective looking at the CsI(Tl) scintillation crystal. Cu no. 2000 meshes and a zone plate have been imaged to see the contrast as well as the spatial resolution. A Halo effect coming from the Zernike phase contrast was clearly visible on the images of meshes.

Observations of a human hair shaft with an x-ray microscope.

We observed the internal structures of a human hair shaft using x-ray microscopes with a spatial resolution in the range from a few microns to less than 100 nm. The energy of the x-ray used is 6.95 keV. The Zernike phase contrast together with a spatial resolution better than 100 nm enabled us to see the cuticles of scales, the cortex of macrofibrils and the medulla. All these internal features and more can easily be observed with no sample preparation including staining.