Toward Rapid and Automated Immunoassays: Using a Localized Electrochemical pH Modulation Platform to Perform a Single-Step Immunoassay.

An electrochemical platform for generating and controlling a localized pH microenvironment on demand is proposed by employing a closed-loop control algorithm based on an iridium oxide pH sensor input. We use a combination of solution-borne quinones and galvanostatic excitation on a prepatterned indium tin oxide (ITO) working electrode to modulate pH within a very well confined, small volume of solution close to the electrode surface. We demonstrate that the rate of pH change can be controlled at up to 2 pH s-1 with an excellent repeatability (±0.004). The desired pH microenvironment can be stably maintained for longer than 2 h within ±0.0012 pH. As a high-impact application of the platform technology, we propose a single-step immunoassay and demonstrate its utility in measuring C-reactive protein (CRP), a critical inflammatory marker in various conditions such as myocardial infarction and even SARS-Cov-2. Utilizing pH modulation technology along with pH-sensitive fluorescence dye simplifies the immunoassay process into a single-step, where a mixture of all of the reagents is incubated only for 1 h without any washing steps or the need to change solution. This simplified immunoassay process minimizes the hands-on time of the end-user and thus decreases technician-driven errors. Moreover, the absence of complicated liquid-handling hardware makes it more suitable and attractive for an ultracompact platform to ultimately be used in a point-of-care diagnostic assay.

Synthesis and Spectral Characteristics of BODIPY Dyes with Two or Three Dipyrrin Domains.

Several boron-dipyrrin (BODIPY) based fluorophores with two and three dipyrrin cores were synthesized and investigated in solvents under the concentration variation. Comparative analysis of spectral and photophysical changes under increasing the number of the cores in the dye molecule was made. Mutual influence of dipyrrin cores was detected leading to the increasing of the compounds rigidity and, thus, the absence of fluorescent molecular rotor effects under the viscosity variation. Aggregation induced quenching, which was observed for many mono-domain BODIPY dyes is reduced in case of investigated poly-domain compounds.

Biocompatible polymeric nanoparticles degrade and release cargo in response to biologically relevant levels of hydrogen peroxide.

Oxidative stress is caused predominantly by accumulation of hydrogen peroxide and distinguishes inflamed tissue from healthy tissue. Hydrogen peroxide could potentially be useful as a stimulus for targeted drug delivery to diseased tissue. However, current polymeric systems are not sensitive to biologically relevant concentrations of H(2)O(2) (50-100 µM). Here we report a new biocompatible polymeric capsule capable of undergoing backbone degradation and thus release upon exposure to such concentrations of hydrogen peroxide. Two polymeric structures were developed differing with respect to the linkage between the boronic ester group and the polymeric backbone: either direct (1) or via an ether linkage (2). Both polymers are stable in aqueous solution at normal pH, and exposure to peroxide induces the removal of the boronic ester protecting groups at physiological pH and temperature, revealing phenols along the backbone, which undergo quinone methide rearrangement to lead to polymer degradation. Considerably faster backbone degradation was observed for polymer 2 over polymer 1 by NMR and GPC. Nanoparticles were formulated from these novel materials to analyze their oxidation triggered release properties. While nanoparticles formulated from polymer 1 only released 50% of the reporter dye after exposure to 1 mM H(2)O(2) for 26 h, nanoparticles formulated from polymer 2 did so within 10 h and were able to release their cargo selectively in biologically relevant concentrations of H(2)O(2). Nanoparticles formulated from polymer 2 showed a 2-fold enhancement of release upon incubation with activated neutrophils, while controls showed a nonspecific response to ROS producing cells. These polymers represent a novel, biologically relevant, and biocompatible approach to biodegradable H(2)O(2)-triggered release systems that can degrade into small molecules, release their cargo, and should be easily cleared by the body.

NMR-derived models of amidopyrine and its metabolites in complexes with rabbit cytochrome P450 2B4 reveal a structural mechanism of sequential N-dealkylation.

To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T(1)) relaxation NMR. Results of STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme's active site. The model-dependent relaxation rates (R(M)) determined from T(1) relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine the orientation(s) of AP and its metabolites within the P450 2B4 active site, we used distances calculated from the relaxation rates to constrain the metabolites to the X-ray crystal structure of P450 2B4. Simulated annealing of the complex revealed that the metabolites do indeed occupy similar hydrophobic pockets within the active site, while the N-linked methyls are free to rotate between two binding modes. From these bound structures, a model of N-demethylation in which the N-linked methyl functional groups rotate between catalytic and noncatalytic positions was developed. This study is the first to provide a structural model of a drug and its metabolites complexed to a cytochrome P450 based on NMR and to provide a structural mechanism for how a drug can undergo sequential oxidations without unbinding. The rotation of the amide functional group might represent a common structural mechanism for N-dealkylation reactions for other drugs such as the local anesthetic lidocaine.

UV and near-IR triggered release from polymeric nanoparticles.

A new light-sensitive polymer containing multiple light-sensitive triggering groups along the backbone and incorporating a quinone-methide self-immolative moiety was developed and formulated into nanoparticles encapsulating a model pharmaceutical Nile Red. Triggered burst release of the payload upon irradiation and subsequent degradation of the nanoparticles were observed. This system is designed to be versatile where the triggering group can be sensitive to a number of wavelengths.

Electron-Transport Characteristics through Aluminum Oxide (100) and (012) in a Metal-Insulator-Metal Junction System: Density Functional Theory-Nonequilibrium Green Function Approach.

Al2O3 is commonly used in modern electronic devices because of its good mechanical properties and excellent electrical insulating property. Although fundamental understanding of the electron transport in Al2O3 is essential for its use in electronic device applications, a thorough investigation for the electron-transport mechanism has not been conducted on the structures of Al2O3, especially in nanometer-scale electronic device settings. In this work, electron transport via Al2O3 for two crystallographic facets, (100) and (012), in a metal-insulator-metal junction configuration is investigated using a density functional theory-based nonequilibrium Green function method. First, it is confirmed that the transmission function, T(E), decreases as a function of energy in (E - E F) < 0 regime, which is an intuitively expected trend. On the other hand, in the (E - E F) > 0 regime, Al2O3(100) and Al2O3(012) show their own characteristic behaviors of T(E), presenting that major peaks are shifted toward lower energy levels under a finite bias voltage. Second, the overall conductance decay rates under zero bias are similar regardless of the crystallographic orientation, so that the contact interface seemingly has only a minor contribution to the overall conductance. A noteworthy feature at the finite bias condition is that the electrical current drastically increases as a function of bias potential (>0.7 V) in Al2O3(012)-based junction compared with the Al2O3(100) counterpart. It is elucidated that such a difference is due to the well-developed eigenchannels for electron transport in the Al2O3(012)-based junction. Therefore, it is evidently demonstrated that at finite bias condition, the contact interface plays a key role in determining insulating properties of Al2O3-Pt junctions.

DFT based Monte Carlo simulations of poly(9,9-dialkylfluorene-2,7-diyl) polymers in solution.

The dilute solution properties of poly(9,9-dihexylfluorene-2,7-diyl) (PDHF) were studied by coupled SEC/light scattering and MALDI-TOF over a large molecular weight (MW) span ranging from PDHF oligomers (1-8-mer) to high MW polymer. The results were compared with Monte Carlo simulations based on realistic PDHF models obtained from X-ray data and density functional theory (DFT) calculations and with a DFT based Kratky-Porod-Benoit-Doty (KPBD) worm-like chain. The simulations called "selective random walk" (SRW) and the corresponding "selective self-avoiding random walk" (SSAW) explicitly take into account the rotationally labile bonds between the fluorene units in that four distinct torsion angles (+/-37.5 and +/-143 degrees) between the units are chosen randomly. The simulations better account than the KPBD model for the experimental data obtained by us and others for various poly(9,9-dialkylfluorene-2,7-diyl) polymers but still give somewhat larger values for the radii of gyration and hydrodynamic volumes. The torsion angle selectivity of the SRW and SSAW simulations predict long chain sections punctuated by sudden sharp loops.

Light-responsive nanoparticle depot to control release of a small molecule angiogenesis inhibitor in the posterior segment of the eye.

Therapies for macular degeneration and diabetic retinopathy require intravitreal injections every 4-8 weeks. Injections are uncomfortable, time-consuming, and carry risks of infection and retinal damage. However, drug delivery via noninvasive methods to the posterior segment of the eye has been a major challenge due to the eye's unique anatomy and physiology. Here we present a novel nanoparticle depot platform for on-demand drug delivery using a far ultraviolet (UV) light-degradable polymer, which allows noninvasively triggered drug release using brief, low-power light exposure. Nanoparticles stably retain encapsulated molecules in the vitreous, and can release cargo in response to UV exposure up to 30 weeks post-injection. Light-triggered release of nintedanib (BIBF 1120), a small molecule angiogenesis inhibitor, 10 weeks post-injection suppresses choroidal neovascularization (CNV) in rats. Light-sensitive nanoparticles are biocompatible and cause no adverse effects on the eye as assessed by electroretinograms (ERG), corneal and retinal tomography, and histology.

Low power upconverted near-IR light for efficient polymeric nanoparticle degradation and cargo release.

By encapsulating NaYF4 :Tm.Yb upconverting nanocrystals in UV-degradable polymer capsules, it is possible to access efficient polymer photodegradation and remotely controlled release using near-IR laser light at an unprecedentedly low power.

Photochemical mechanisms of light-triggered release from nanocarriers.

Over the last three decades, a handful of photochemical mechanisms have been applied to a large number of nanoscale assemblies that encapsulate a payload to afford spatio-temporal and remote control over activity of the encapsulated payload. Many of these systems are designed with an eye towards biomedical applications, as spatio-temporal and remote control of bioactivity would advance research and clinical practice. This review covers five underlying photochemical mechanisms that govern the activity of the majority of photoresponsive nanocarriers: 1. photo driven isomerization and oxidation, 2. surface plasmon absorption and photothermal effects, 3. photo driven hydrophobicity changes, 4. photo driven polymer backbone fragmentation and 5. photo driven de-crosslinking. The ways in which these mechanisms have been incorporated into nanocarriers and how they affect release are detailed, as well as the advantages and disadvantages of each system.

Increasing materials' response to two-photon NIR light via self-immolative dendritic scaffolds.

Photoactivation using two photons of NIR allows non-invasive biological manipulation. We applied the principle of dendritic amplification to improve the materials' sensitivity to NIR light. Light induced uncaging or release of L-glutamic acid was 2.8 fold higher when incorporating 4-bromo-7-hydroxycoumarin (Bhc) with self-immolative dendrimers compared with Bhc directly conjugated to L-glutamic acid.

A Single UV or Near IR Triggering Event Leads to Polymer Degradation into Small Molecules.

We report two polymers with UV- and NIR-removable end caps that respond to a single light activated event by complete cleavage of the polymer backbone via a self-immolative mechanism. Two photocleavable protecting groups were used to cap the polymers; o-nitrobenzyl alcohol (ONB) and bromo-coumarin (Bhc). GPC and (1)H NMR confirmed complete degradation of the ONB-containing polymer in response to UV. The polymers were formulated into nanoparticles; fluorescence measurements of encapsulated Nile red confirmed release upon photolysis of the endcaps. Contrary to previous work using a similar backbone structure that degrades upon hydrolysis, here, the disassembly process and burst release of the payload are only activated on demand, illustrating the powerful capacity of light to trigger release from polymeric nanoparticles. Our design allows the signal to be amplified in a domino effect to fully degrade the polymer into small molecules. Thus, polymers and nanoparticles can reach maximal degradation without having to use intense and/or long periods of irradiation.

Low power, biologically benign NIR light triggers polymer disassembly.

Near infrared (NIR) irradiation can penetrate up to 10 cm deep into tissues and be remotely applied with high spatial and temporal precision. Despite its potential for various medical and biological applications, there is a dearth of biomaterials that are responsive at this wavelength region. Herein we report a polymeric material that is able to disassemble in response to biologically benign levels of NIR irradiation upon two-photon absorption. The design relies on the photolysis of the multiple pendant 4-bromo7-hydroxycoumarin protecting groups to trigger a cascade of cyclization and rearrangement reactions leading to the degradation of the polymer backbone. The new material undergoes a 50% Mw loss after 25 sec of ultraviolet (UV) irradiation by single photon absorption and 21 min of NIR irradiation via two-photon absorption. Most importantly, even NIR irradiation at biologically benign laser power is sufficient to cause significant polymer disassembly. Furthermore, this material is well tolerated by cells both before and after degradation. These results demonstrate for the first time a NIR sensitive material with potential to be used for in vivo applications.