Light-degradable nanocomposite hydrogels for antibacterial wound dressing applications.

Skin injuries infected by bacteria can cause life-threatening human diseases if not treated properly. In this work, we developed a light-degradable nanocomposite hydrogel to achieve both controlled antibiotic delivery and hydrogel degradation using light as the sole stimulus. Specifically, we incorporated triclosan-loaded, poly(N-isopropylacrylamide)-based nanogels (TCS-NGs) that exhibited potent antibacterial efficacy, into a light-degradable poly (ethylene glycol) (PEG)-based hydrogel matrix via simple physical entrapment method. Upon exposure to 365 nm light, the hydrogel matrix could rapidly degrade, which subsequently released the entrapped TCS-NGs into the surrounding environment. Our results demonstrated that TCS-NGs released from light-degradable nanocomposite hydrogels still possessed remarkable antibacterial efficacy by inhibiting the growth of Staphylococcus aureus both in solution (a fivefold reduction in optical density compared to the blank control) and on bacteria-infected porcine skins (a fivefold reduction in colony-forming units compared to the blank control). Finally, using an alamarBlue assay on human dermal fibroblasts, we determined that each component of the nanocomposite hydrogel exhibited excellent biocompatibility (>90% cell viability) and would not cause significant cytotoxicity. Overall, the fabricated light-degradable nanocomposite hydrogels could serve as novel material for antibacterial wound dressing applications.

Self-assembled poloxamer-legumin/vicilin nanoparticles for the nanoencapsulation and controlled release of folic acid.

Plant-based food proteins are a promising choice for the preparation of nanoparticles (NPs) due to their high digestibility, low cost, and ability to interact with various compounds and nutrients. Moreover, nanoencapsulation offers a potential solution for protecting nutrients during processing and enhancing their bioavailability. This study aimed to develop and evaluate nanoparticles (NPs) based on legumin/vicilin (LV) proteins extracted from fava beans, with the goal of encapsulating and delivering a model nutraceutical compound, folic acid (FA). Specifically, NPs were self-assembled from LV proteins extracted from commercially available frozen fava beans using a pH-coacervation method with poloxamer 188 (P188) and chemically cross-linked with glutaraldehyde. Microscopy and spectroscopy studies were carried out on the empty and FA-loaded NPs in order to evaluate the particle morphology, size, size distribution, composition, mechanism of formation, impact of FA loading and release behavior. In vitro studies with Caco-2 cells also confirmed that the empty and FA-loaded nanoparticles were non-toxic. Thus, the LV-NPs are good candidates as food additives for the delivery and stabilization of nutrients as well as in drug delivery for the controlled release of therapeutics.

Method for Glucose and Fructose Quantitation in Beverages Using an Off-the-Shelf Glucose Test Strip.

A method was developed for quantifying both glucose and fructose in solutions and grape juice using commercially available glucose test strips connected to a mini-potentiostat. The first step of this sensing approach involved exposing the sample solution to an Accu Chek Aviva glucose test strip, which allowed for the direct quantitation of glucose. To quantify fructose, the solution was exposed to glucose isomerase, which led to the conversion of glucose to fructose and vice versa until an equilibrium was reached. Once equilibrium was reached, the solution was exposed to another glucose test strip; the signal obtained was shown to be related to the total amounts of glucose and fructose in solution. Finally, fructose was quantified by subtracting the glucose concentration (from the initial measurement) from the total concentration of glucose and fructose (from the second measurement after the reaction with glucose isomerase). The method yielded a limit of detection of 0.047 g L-1 for glucose and 0.49 g L-1 for fructose. Importantly, this method was shown to work well for analyzing glucose and fructose concentration in grape juice, which contains >60 g L-1 glucose and fructose. Since the ratio of glucose and fructose concentration in ripe grapes is close to 1, this method can be used to aid in the determination of grape ripeness to guide harvesting times.

Microgel-based etalon immunoassay for IgG detection.

We developed an immunoassay for mouse immunoglobulin (IgG) quantitation using poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) microgel-based etalon devices. To achieve this, a biotinylated primary antibody specific to mouse IgG was immobilized on the top Au layer of an etalon device via its interaction with a streptavidin-modified etalon surface. Mouse IgG captured on the etalon surface from the solution was quantified using an HRP-conjugated secondary antibody. HRP catalyzed the oxidation of 4-chloro-1-naphthol (4CN) to form insoluble 4-chloro-1-naphthon (4CNP), resulting in a concentration change of 4CN in solution. The etalon was able to detect the 4CN concentration change by monitoring the extent of the etalon's reflectance peak shift, which allows the quantitation of mouse IgG. The etalon-based assay can detect mouse IgG down to 0.018 nM with a linear range of 0.02-5 nM.

Bovine Serum Albumin-Protected Gold Nanoclusters for Sensing of SARS-CoV-2 Antibodies and Virus.

An approach to assess severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (and past infection) was developed. For virus detection, the SARS-CoV-2 virus nucleocapsid protein (NP) was targeted. To detect the NP, antibodies were immobilized on magnetic beads to capture the NPs, which were subsequently detected using rabbit anti-SARS-CoV-2 nucleocapsid antibodies and alkaline phosphatase (AP)-conjugated anti-rabbit antibodies. A similar approach was used to assess SARS-CoV-2-neutralizing antibody levels by capturing spike receptor-binding domain (RBD)-specific antibodies utilizing RBD protein-modified magnetic beads and detecting them using AP-conjugated anti-human IgG antibodies. The sensing mechanism for both assays is based on cysteamine etching-induced fluorescence quenching of bovine serum albumin-protected gold nanoclusters where cysteamine is generated in proportion to the amount of either SARS-CoV-2 virus or anti-SARS-CoV-2 receptor-binding domain-specific immunoglobulin antibodies (anti-RBD IgG antibodies). High sensitivity can be achieved in 5 h 15 min for the anti-RBD IgG antibody detection and 6 h 15 min for virus detection, although the assay can be run in "rapid" mode, which takes 1 h 45 min for the anti-RBD IgG antibody detection and 3 h 15 min for the virus. By spiking the anti-RBD IgG antibodies and virus in serum and saliva, we demonstrate that the assay can detect the anti-RBD IgG antibodies with a limit of detection (LOD) of 4.0 and 2.0 ng/mL in serum and saliva, respectively. For the virus, we can achieve an LOD of 8.5 × 105 RNA copies/mL and 8.8 × 105 RNA copies/mL in serum and saliva, respectively. Interestingly, this assay can be easily modified to detect myriad analytes of interest.

Utilization of a Glucometer Test Strip and Enzymatic Reactions to Quantify Anti-SARS-CoV-2 Spike RBD IgG Antibody and SARS-CoV-2 Virus in Saliva and Serum.

A sensor capable of quantifying both anti-SARS-CoV-2 spike receptor-binding domain (RBD) antibody levels and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in saliva and serum was developed. This was accomplished by exploiting the enzymatic reaction of maltose and orthophosphate (PO43-) in the presence of maltose phosphorylase to generate an equivalent amount of glucose that was detected using a commercial glucometer test strip and a potentiostat. Important for this approach is the ability to generate PO43- in an amount that is directly related to the concentration of the analytes. RBD-modified magnetic microparticles were used to capture anti-SARS-CoV-2 spike RBD antibodies, while particles modified with anti-SARS-CoV-2 nucleocapsid antibodies were used to capture SARS-CoV-2 nucleocapsid protein from inactivated virus samples. A magnet was used to isolate and purify the magnetic microparticles (with analyte attached), and alkaline phosphatase-conjugated secondary antibodies were bound to the analytes attached to the respective magnetic microparticles. Finally, through enzymatic reactions, specific amounts of PO43- (and subsequently glucose) were generated in proportion to the analyte concentration, which was then quantified using a commercial glucometer test strip. Utilizing glucose test strips makes the sensor relatively inexpensive, with a cost per test of ∼US $7 and ∼US $12 for quantifying anti-SARS-CoV-2 spike RBD antibody and SARS-CoV-2, respectively. Our sensor exhibited a limit of detection of 0.42 ng/mL for anti-SARS-CoV-2 spike RBD antibody, which is sensitive enough to quantify typical concentrations of antibodies in COVID-19-infected or vaccinated individuals (>1 µg/mL). The limit of detection for the SARS-CoV-2 virus is 300 pfu/mL (5.4 × 106 RNA copies/mL), which exceeds the performance recommended by the WHO (500 pfu/mL). In addition, the sensor exhibited good selectivity when challenged with competing analytes and could be used to quantify analytes in saliva and serum matrices with an accuracy of >94% compared to RT-qPCR.

Design of hydrogel-microgel composites with tailored small molecule release profiles.

Stimuli-responsive hydrogel-microgel composites (HMC) were prepared by embedding poly(N-isopropylacrylamide)-based microgels in a poly(N-isopropylacrylamide)-based hydrogel. When the microgels were pre-loaded with the small molecule model drug crystal violet (CV) via electrostatics, the HMC was able to release the CV in a pH-triggered fashion. We found that the CV release rate was dependent on the solution temperature and the dimension of the material. Also, by changing the chemical composition and/or pore size of the hydrogel matrix, the CV release kinetics can be tuned. Moreover, when multiple microgels loaded with different model drugs were embedded in a single HMC, the HMC can be used to control the release rate of each drug analog individually in a pH-dependent fashion. By understanding how properties of a hydrogel can alter the release of small molecules from embedded microgels, new materials capable of controlled and triggered release of multiple small molecule drugs can be designed with myriad uses in the biomedical field.

Orthophosphate Quantification in Water Utilizing an Enzymatic Reaction and a Commercial Glucometer Test Strip.

A sensor for the determination of orthophosphate (PO43-) concentration in water was developed that utilizes the enzymatic cleavage of maltose in the presence of PO43- to generate an equivalent amount of glucose. The glucose produced from this reaction was quantified using a commercial glucometer test strip coupled to a miniature potentiostat using amperometry. This sensing approach yielded a limit of detection of 1.45 µM (0.1 ppm) glucose/PO43-, which is lower than that of a commercial glucometer (≥600 µM glucose), and a dynamic range of 10 µM-3 mM. The selectivity of the approach to PO43- was evaluated by determining the device's response to known components in natural water samples. Finally, we showed that the approach can be used to determine PO43- in tap and river water samples.

Enhancing the Sensitivity of Surface Plasmon Resonance Measurements Utilizing Polymer Film/Au Assemblies.

Surface plasmon resonance (SPR) is used to infer information about a sample that is in contact with an Au-coated glass slide coupled to the SPR prism. Shifts in the angle of the "SPR minimum reflection" can be related to changes in the refractive index (and/or thickness) of the sample that is in contact with the Au film, which can then be used to determine the concentration of an analyte in that sample. Here, we show that by depositing a layer of poly(N-isopropylacrylamide-co-acrylic acid) [p(NIPAm-co-AAc)] microgel on the SPR's Au film, with a subsequent layer of Au deposited on top of the microgels, the sensitivity of SPR to changes in solution properties can be enhanced. We investigated the sensitivity of the SPR to changes in the temperature of water in contact with the SPR's Au film as a function of the microgel immobilization density and the thickness of the Au layer deposited on the microgel layer. The data revealed that the SPR's Au film densely coated with microgels, with 5 nm of Au deposited, exhibited the maximal enhancement. The plasmon coupling effect between the additional Au film on the microgels and the SPR's Au film was further confirmed by 3D finite difference time domain simulations.

Portable point-of-care diagnostic devices: an updated review.

The global pandemic caused by the SARS-CoV-2 (COVID) virus indiscriminately impacted people worldwide with unquantifiable and severe impacts on all aspects of our lives, regardless of socioeconomic status. The pandemic brought to light the very real possibility of pathogens changing and shaping the way we live, and our lack of preparedness to deal with viral/bacterial outbreaks. Importantly, the quick detection of pathogens can help prevent and control the spread of disease, making the importance of diagnostic techniques undeniable. Point-of-care diagnostics started as a supplement to standard lab-based diagnostics, and are gradually becoming mainstream. Because of this, and their importance in detecting pathogens (especially in the developing world), their development has accelerated at an unprecedented rate. In this review, we highlight some important and recent examples of point-of-care diagnostics for detecting nucleic acids, proteins, bacteria, and other biomarkers, with the intent of making apparent their positive impact on society and human health.

Controlled Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Dexamethasone-Loaded Light-Responsive Microgels.

Human mesenchymal stem cells (hMSCs), which have the ability to differentiate into osteoblasts, show promise for bone tissue engineering and bone defect treatment. While there are a number of approaches currently available to accomplish this, e.g., utilizing biodegradable materials loaded with the synthetic glucocorticoid osteogenic inducer dexamethasone (DEX), there are still many disadvantages with the current technologies. Here, we generated light-responsive microgels that we showed are capable of loading and releasing DEX in a light-triggered fashion, with the released DEX being able to induce hMSC differentiation into osteoblasts. Specifically, light-responsive poly(N-isopropylacrylamide-co-nitrobenzyl methacrylate) (pNIPAm-co-NBMA) microgels were synthesized via free radical precipitation polymerization and their size, morphology, and chemical composition were characterized. We then went on to show that the microgels could be loaded with DEX (via what we think are hydrophobic interactions) and released upon exposure to UV light. We went on to show that the DEX released from the microgels was still capable of inducing osteogenic differentiation of hMSCs using an alamarBlue assay and normalized alkaline phosphatase (ALP) activity assay. We also investigated how hMSC differentiation was impacted by intermittent DEX released from UV-exposed microgels. Finally, we confirmed that the microgels themselves were not cytotoxic to hMSCs. Taken together, the DEX-loaded light-responsive microgels reported here may find a use for niche clinical applications, e.g., bone tissue repair.

Probing the response of poly (N-isopropylacrylamide) microgels to solutions of various salts using etalons.

The Hofmeister series is a qualitative ordering of ions according to their ability to precipitate proteins in aqueous solution and is extremely important to consider when trying to understand materials and biomolecular structure and function. Herein, we utilized optical devices (etalons) composed of poly(N-isopropylacrylamide) (pNIPAm)-co-10% acrylic acid (AAc) or pNIPAm-based microgels to investigate how various salts in the Hofmeister series influenced the microgel hydration state. Etalons were exposed to a series of salts solutions at different concentrations and the position of the peaks in the reflectance spectra monitored using reflectance spectroscopy. As expected, pNIPAm-co-10%AAc microgel-based etalons responded to the presence of ions, although in this case the response to cations deviated from the Hofmeister series. However, when using etalons prepared with pNIPAm-based microgels, the responses followed the Hofmeister series for both cation and anions. Finally, we observed that the sensitivity of etalons prepared with pNIPAm microgels was significantly higher than the response obtained from etalons composed of pNIPAm-co-10%AAc microgels. This was explained by considering the charge on the pNIPAm-co-10%AAc microgels that influences how osmotic and Hofmeister effects impacts hydration state.

Bioinspired tissue-compliant hydrogels with multifunctions for synergistic surgery-photothermal therapy.

Operation therapy is a common treatment for many cancers, but malignant tumors likely recur and metastasize after surgery, resulting in treatment failure. In this study, we aimed at synthesizing a multifunctional hydrogel patch that features multifunctions for synergistic surgery-photothermal therapy. Our polydopamine nanoparticle (PDA NP)-crosslinked poly(acrylamide-co-N-(3-aminopropyl)methacrylamide) hydrogels undergo several dynamic interactions (e.g., hydrogen bonds, π-π interactions, and imine bonds), which confer high stretchability (∼3430%) and adhesive strength to porcine skin (∼75 kPa) that mimics soft wound tissues. Furthermore, PDA NP incorporation into the hydrogel matrix endows it with photothermal responsivity under 808 nm irradiation. As a proof of concept, our hydrogel system was used to ablate residual tumors in 4T1 tumor-bearing mice models after surgery via photothermal therapy. We find that synergistic operation-photothermal therapy effectively eradicates solid tumors and prevents cancer recurrence in mice. We envision that our work provides an effective synergistic strategy for cancer treatment and offers great potential for clinical applications.

Stimuli-responsive polymer/nanomaterial hybrids for sensing applications.

Chemical and biological/biochemical sensors are capable of generating readout signals that are proportional to the concentration of specific analytes of interest. Signal sensitivity and limit of detection/quantitation can be enhanced through the use of polymers, nanomaterials, and their hybrids. Of particular interest are stimuli-responsive polymers and nanomaterials due to their ability to change their physical and/or chemical characteristics in response to their environment, and/or in the presence of molecular/biomolecular species of interest. Their individual use for sensing applications have many benefits, although this review focuses on the utility of stimuli-responsive polymer and nanomaterial hybrids. We discuss three main topics: stimuli-responsive nanogels, stimuli-responsive network polymers doped with nanomaterials, and nanoparticles modified with stimuli-responsive polymers.

Stimuli-responsive polymer-based systems for diagnostic applications.

Stimuli-responsive polymers exhibit properties that make them ideal candidates for biosensing and molecular diagnostics. Through rational design of polymer composition combined with new polymer functionalization and synthetic strategies, polymers with myriad responsivities, e.g., responses to temperature, pH, biomolecules, CO2, light, and electricity can be achieved. When these polymers are specifically designed to respond to biomarkers, stimuli-responsive devices/probes, capable of recognizing and transducing analyte signals, can be used to diagnose and treat disease. In this review, we highlight recent state-of-the-art examples of stimuli-responsive polymer-based systems for biosensing and bioimaging.

Microgel-Based Stretchable Reservoir Devices for Elongation Enhanced Small Molecule Release Rate.

Stretchable poly(N-isopropylacrylamide)-co-acrylic acid (pNIPAm-co-10% AAc) microgel-based reservoir devices were fabricated and used to control the release rate of the small molecule model drug tris(4-(dimethylamino)phenyl)methylium chloride (crystal violet, CV) to solution by varying the Au layer thickness coating the microgels and device elongation. Specifically, we showed that CV could be loaded into the microgel layer of the devices via electrostatic interactions at pH 6.5, and the release could be triggered upon exposure to a pH 3.0 solution, which breaks the microgel-CV electrostatic interactions. We demonstrated that the rate of release could be increased by decreasing the Au layer thickness coating microgels and by stretching, that is, thin Au and high elongation promoted the relatively fast release of CV from the device. We found that the Au overlayer thickness (and porosity) dominated the observed release rate profiles when the device was not stretched (or at low elongation), while elongation-induced cracks dominated the release rate at high elongation. We also showed that the CV release kinetics could transition from low ("off") to high ("on"), which enhanced when the devices are stretched. This behavior could be exploited in the future for autonomous release systems that release small molecules when stretched by natural processes, for example, movement of joints and muscles.

Stimuli-responsive microgels for controlled deposition of gold nanoparticles on surfaces.

A variety of gold nanoparticle (AuNP) core/poly(N-isopropylacrylamide) (pNIPAm) shell microgels (Au@pNIPAm) were generated using seed-mediated polymerization. The shell thickness and AuNP core diameter were easily tunable at the time of synthesis. The resultant Au@pNIPAm microgels were characterized via photon-correlation spectroscopy, transmission electron microscopy and ultraviolet-visible spectroscopy. AuNP arrays were generated by "painting" the microgels on a surface, using the shell thickness to define the distance between the AuNPs, followed by shell removal via plasma etching. We found that when the pNIPAm shell thickness decreased (via its tuning at the time of synthesis or deposition at elevated temperature at which the shell is collapsed) the AuNPs were closer to one another. We also showed that via sequential deposition Au@pNIPAm microgels with different AuNP core sizes could be deposited on a single surface. The presented "painting protocol" offers a facile way to coat large area surfaces quickly which is not easily achievable using other approaches. We envision that this approach is extremely versatile, allowing a number of different nanomaterials embedded in pNIPAm shells to be deposited/patterned on surfaces. With the control over the deposition on the surface that we show here, we hope that the Au@pNIPAm microgels will find use in lithography/surface patterning applications.

Alkanethiol Molecular Barriers for Controlling Small Molecule Release Kinetics from a Microgel-Based Reservoir Device.

Poly(N-isopropylacrylamide)-co-acrylic acid microgel-based reservoir devices were constructed by "sandwiching" a single layer of microgels between two thin Au layers (all on a glass support). The microgels were loaded with the model drug crystal violet (CV) utilizing the electrostatic interactions between deprotonated acrylic acid (AAc) and the positively charged CV; release can be triggered from the microgels by neutralizing the deprotonated AAc groups at acidic conditions. Alkanethiols of different alkyl chain lengths and polarities were immobilized on the upper Au layer of the device, and the release rate of the model drug CV from the microgel layer, after acid neutralization, was assessed. We found that the CV release rate was the highest when the alkyl chain length was short and contained a hydrophilic moiety. Conversely, the release rate was hindered by the presence of thiols with long alkyl chain lengths and with no hydrophilic moiety. We explain this phenomenon by quantifying the thiol's ability to hinder acid penetration into the microgel layer, and the ability of free CV to pass through the upper Au layer and into the solution. Utilizing various thiols and mixed thiol layers, we are able to tune release profiles from these reservoir devices to potentially achieve array devices with precisely tuned small molecule release profiles.

Poly(N-isopropylacrylamide) microgel-based etalons for the label-free quantitation of estradiol-17ß in aqueous solutions and milk samples.

A novel estradiol-17ß (E2) biosensor was constructed from poly(N-isopropylacrylamide) (pNIPAm) microgel-based etalons by modification of their outermost Au layer with an E2 binding 75-mer DNA aptamer. When E2 is not present in the solution, the aptamer forms a loose/linear structure that allows ions to pass through and into the microgel layer. The ions can change the solvation state of the microgels, which changes the optical properties of the etalon. When E2 is present in the solution, the aptamer binds the E2 and undergoes a conformational change to a form that can block the diffusion of salt ions into the microgel layer. This blocking decreases the response of the device to salt exposure, which can be related to the concentration of E2 in solution. Using this approach, E2 sensor showed a dynamic range of 0.9-200 pg/mL with a calculated detection limit of 0.9 pg/mL (3.2 pM) E2, and the lowest measured concentration of E2 is 5.0 pg/mL. This sensor also showed low cross reactivity with progesterone, a similar steroid hormone. Moreover, this sensor could be regenerated five times without losing its sensitivity. Finally, we demonstrated that the sensor could also be used to quantify E2 in commercial skim and 2% milk, as well as farm milk directly without any pre-treatment. The successful quantitation of E2 in unprocessed milk demonstrates its potential use as a "cow-side" testing device for the dairy industry. Graphical abstract ᅟ.