Reversible Functionalization of Clickable Polyacrylamide Gels with Protein and Graft Copolymers.

Modular strategies to fabricate gels with tailorable chemical functionalities are relevant to applications spanning from biomedicine to analytical chemistry. Here, the properties of clickable poly(acrylamide-co-propargyl acrylate) (pAPA) hydrogels are modified via sequential in-gel copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. Under optimized conditions, each in-gel CuAAC reaction proceeds with rate constants of ~0.003 s-1, ensuring uniform modifications for gels < 200 µm thick. Using the modular functionalization approach and a cleavable disulfide linker, pAPA gels were modified with benzophenone and acrylate groups. Benzophenone groups allow gel functionalization with unmodified proteins using photoactivation. Acrylate groups enabled copolymer grafting onto the gels. To release the functionalized unit, pAPA gels were treated with disulfide reducing agents, which triggered ~50 % release of immobilized protein and grafted copolymers. The molecular mass of grafted copolymers (~6.2 kDa) was estimated by monitoring the release process, expanding the tools available to characterize copolymers grafted onto hydrogels. Investigation of the efficiency of in-gel CuAAC reactions revealed limitations of the sequential modification approach, as well as guidelines to convert a pAPA gel with a single functional group into a gel with three distinct functionalities. Taken together, we see this modular framework to engineer multifunctional hydrogels as benefiting applications of hydrogels in drug delivery, tissue engineering, and separation science.

Kinetic Analysis of Enzymes Immobilized in Porous Film Arrays.

Measuring the catalytic activity of immobilized enzymes underpins development of biosensing, bioprocessing, and analytical chemistry tools. To expand the range of approaches available for measuring enzymatic activity, we report on a technique to probe activity of enzymes immobilized in porous materials in the absence of confounding mass transport artifacts. We measured reaction kinetics of calf intestinal alkaline phosphatase (CIAP) immobilized in benzophenone-modified polyacrylamide (BPMA-PAAm) gel films housed in an array of fluidically isolated chambers. To ensure kinetics measurements are not confounded by mass transport limitations, we employed Weisz's modulus (Φ), which compares observed enzyme-catalyzed reaction rates to characteristic substrate diffusion times. We characterized activity of CIAP immobilized in BPMA-PAAm gels in a reaction-limited regime (Φ â‰ª 0.15 for all measurements), allowing us to isolate the effect of immobilization on enzymatic activity. Immobilization of CIAP in BPMA-PAAm gels produced a ∼2× loss in apparent enzyme-substrate affinity (Km) and ∼200× decrease in intrinsic catalytic activity (kcat) relative to in-solution measurements. As estimating Km and kcat requires multiple steps of data manipulation, we developed a computational approach (bootstrapping) to propagate uncertainty in calibration data through all data manipulation steps. Numerical simulation revealed that calibration error is only negligible when the normalized root-mean-squared error (NRMSE) in the calibration falls below 0.05%. Importantly, bootstrapping is independent of the mathematical model, and thus generalizable beyond enzyme kinetics studies. Furthermore, the measurement tool presented can be readily adapted to study other porous immobilization supports, facilitating rational design (immobilization method, geometry, enzyme loading) of immobilized-enzyme devices.

A lateral electrophoretic flow diagnostic assay.

Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.