Aptamer-functionalized two-dimensional photonic crystal (2DPC) hydrogels are reported for the detection of adenosine (AD). As a molecular recognition group, an AD-binding aptamer was covalently ...attached to 2DPC hydrogels. This aptamer selectively and sensitively binds AD, changing the conformation of the aptamer from a long single-stranded structure (AD-free conformation) to a short hairpin loop structure (AD-bound conformation). The AD-binding-induced changes of aptamer conformation reduced the volume of the 2DPC hydrogels and decreased the interparticle spacing of the 2DPC embedded in the hydrogel network. The particle spacing changes being dependent on AD concentration were determined by measuring 2DPC light diffraction using a simple laser pointer. The 2DPC hydrogel sensor showed a large particle spacing decrease of ~ 110 nm in response to 1 mM AD in phosphate-buffered saline (PBS). The linear range of determination of AD was 0.1 nM to 1 mM and the limit of detection was 0.09 nM. The hydrogel sensor response for real samples was then validated in diluted fetal bovine serum (FBS) and human urine. The average % difference in particle spacing changes measured between diluted FBS and pure PBS was only 3.99%. In diluted human urine, the recoveries for the detection of AD were 95–101% and the relative standard deviations were 4.9–7.8%. The results demonstrate the potential applicability of the hydrogel sensor for real samples. This sensing concept, using the aptamer-functionalized 2DPC hydrogels, allows for a simple, sensitive, selective, and reversible detection of AD. It may enable sensor development for a wide variety of analytes by simply changing the aptamer recognition group.
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In situ-forming hydrogels are an attractive option for corneal regeneration, and the delivery of growth factors from such constructs have the potential to improve re-epithelialization and stromal ...remodeling. However, challenges persist in controlling the release of therapeutic molecules from hydrogels. Here, an in situ-forming bio-orthogonally crosslinked hydrogel containing growth factors tethered via photocleavable linkages (PC-HACol hydrogel) was developed to accelerate corneal regeneration. Epidermal growth factor (EGF) was conjugated to the hydrogel backbone through photo-cleavable (PC) spacer arms and was released when exposed to mild intensity ultraviolet (UV) light (2–5 mW/cm2, 365 nm). The PC-HACol hydrogel rapidly gelled within a few minutes when applied to corneal defects, with excellent transparency and biocompatibility. After subsequent exposure to UV irradiation, the hydrogel promoted the proliferation and migration of corneal epithelial cells in vitro. The rate of re-epithelialization was positively correlated to the frequency of irradiation, verified through ex vivo rabbit cornea organ culture studies. In an in vivo rat corneal wound healing study, the PC-HACol hydrogel exposed to UV light significantly promoted re-epithelialization, the remodeling of stromal layers, and exhibited significant anti-scarring effects, with minimal α-SMA and robust ALDH3A1 expression. Normal differentiation of the regenerated epithelia after healing was evaluated by expression of the corneal epithelial biomarker, CK12. The remodeled cornea exhibited full recovery of corneal thickness and layer number without hyperplasia of the epithelium.
Enhancement of corneal regeneration through photo-activated release from bio-orthogonally crosslinked hydrogels. Display omitted
•Epidermal growth factors were conjugated to bio-orthogonally crosslinked hydrogel through the o-nitrobenzene derivative, a photo-cleavable linker.•The conjugated growth factors were released from the hydrogels only under mild UV irradiation conditions, exhibiting highly controlled release patterns.•The rate of re-epithelialization was positively correlated to the frequency of irradiation, verified through ex vivo rabbit cornea organ culture model.•In vivo rat study, the hydrogel with UV irradiation significantly promoted re-epithelialization and stromal remodeling, with significant anti-scarring effects.
There is a need to develop at-home phenylalanine (Phe) test kits, analogous to home glucose meters, for phenylketonuria patients who must measure their blood Phe levels frequently to adjust their ...diet. Unfortunately, such test kits are not available yet because of the lack of simple and inexpensive Phe-sensing elements. With the goal of developing a Phe-sensing element, we fabricated two-dimensional photonic crystal (2DPC) hydrogels that quantify human serum phenylpyruvate (PhPY), which is the product of the reaction between Phe and the enzyme phenylalanine dehydrogenase. The PhPY-sensing hydrogels have oxyamine recognition groups that link PhPY to the hydrogel polymer network via chemoselective oxime ligation. This structural modification induces the hydrogel to swell, which then increases interparticle spacings within the embedded 2DPC. The PhPY-induced particle spacing changes are measured from light diffraction and used to quantify the PhPY concentrations. The estimated limit of detection of PhPY in human serum for a detection time of 30 min is 19 μM, which is comparable to the minimum blood Phe concentrations of healthy people. Besides the potential application for developing Phe-sensing elements, this new hydrogel sensing approach via chemoselective oxime ligation is generalizable to the development of other chemical sensors working in complex biological environments.
The work reported in this dissertation discusses the recent advancements in the development of 2DPC hydrogel sensors for biomolecular detection. The 2DPC hydrogel sensors are fabricated by ...incorporating 2DPC into responsive hydrogels. Each hydrogel sensor is functionalized with specific recognition groups that interact with the desired target, causing the hydrogel to undergo volume phase transitions (VPTs). We measure the target-induced hydrogel VPTs by monitoring the 2DPC light diffraction and report the analyte concentrations. This sensing platform is simple, does not require sophisticated instrumentation, and could be used in resource-limited environments and point-of-care testing.We demonstrated the use of oxyamine recognition groups for the detection of phenylpyruvate, an enzymatic reaction product between phenylalanine and phenylalanine dehydrogenase. The chemoselective oxime reaction between the hydrogel oxyamines and phenylpyruvate covalently modifies the hydrogel structure and induces the hydrogel VPTs. This 2DPC hydrogel sensor could be further used to develop a phenylalanine sensor for patients with phenylketonuria and to develop a lactate sensor to evaluate the development of sepsis.We also developed two other mechanisms for small molecule detection utilizing DNA aptamers as molecular recognition groups. Upon binding targets, the DNA aptamers that are attached to the hydrogel network undergo conformational changes, triggering the hydrogel VPTs. We hypothesize that this sensing motif is generalizable and that other sensors can be easily fabricated by simply exchanging the aptamer recognition group. We plan to continue to develop this sensing platform for the detection of other small molecules and proteins of interest.
Cerebral aneurysm embolization is a therapeutic approach to prevent rupture and resultant clinical sequelae. Current, non-biodegradable metallic coils (platinum or tungsten) are the first-line choice ...to secure cerebral aneurysms. However, clinical studies report that up to 17% of aneurysms recur within 1 year after coiling, leading to retreatment and additional surgery. It would be ideal for the aneurysm coiling material to induce acute thrombotic occlusion, contribute to a tissue development process to fortify the degenerated vessel wall, and ultimately resorb to avoid leaving a permanent foreign body. With these properties in mind, a new fatty amide-based polyurethane urea (PHEUU) elastomer was synthesized and coated on biodegradable metallic (Mg alloy) coils to prepare a bioabsorbable cerebral saccular aneurysm embolization device. The chemical structure of PHEUU was confirmed using two-dimensional nuclear magnetic resonance spectroscopy. PHEUU showed comparable physical properties to elastomeric biodegradable polyurethanes lacking fatty amide immobilization, modest enzymatic degradation profiles in the first 8 wks, inherent antioxidant activity (>70% at 48 h), no cytotoxicity, and better protection for the underlying Mg alloy than poly(lactic-co-glycolic acid) (PLGA) against surface corrosion and cracking. Rat aortic smooth muscle cell attachment and platelet deposition were higher with the PHEUUs compared to bare or PLGA coated Mg alloy in vitro. PHEUU-coated Mg alloy coils showed the potential to design a fully bioabsorbable embolization coil amenable to clinical placement conditions based on computational mechanics modeling and blood-contacting test using an in vitro aneurysm model. In vivo studies using a mouse aneurysm model elicited comparable inflammatory cytokine expression to a commercially available platinum coil.
There is a need to develop versatile sensing motifs that can be used to detect a variety of chemical targets in resource-limited settings, for example, at the point of care. While numerous sensing ...technologies have been developed toward this effort, these technologies can be overly complex and require a skilled technician, extensive sample preparation, or sophisticated instrumentation to use, limiting their generalizability and application in resource-limited settings. Here, we report a novel sensing motif that utilizes DNA-crosslinked two-dimensional photonic crystal (2DPC) hydrogels. These hydrogel sensors contain a DNA aptamer recognition group that binds a target analyte. As proof of concept, we fabricated 2DPC hydrogels using a well-studied adenosine-binding aptamer. This adenosine aptamer is duplexed with a partially complementary strand and forms responsive crosslinks in the hydrogel polymer network. When adenosine is introduced, aptamer–adenosine binding occurs, breaking the DNA crosslinks and causing the hydrogel to swell. This in turn increases the particle spacing of an embedded 2DPC array, shifting the 2DPC Bragg diffraction. Thus, adenosine concentration can be monitored through 2DPC Bragg diffraction measurements. A linear range of 20 μM to 2 mM was observed. The detection limits were calculated to be 13.9 μM in adenosine-binding buffer and 26.7 μM in fetal bovine serum. This reported sensing motif has a readout that is simple and rapid and requires minimal equipment. We hypothesize that this sensing motif is generalizable and that other sensors can be easily fabricated by simply exchanging the aptamer that serves as a molecular recognition group.
