Biocatalysis of large-sized substrates finds wide applications. Immobilizing the involved enzymes on solid supports improves biocatalysis yet faces challenges such as enzyme structural perturbation, leaching, and low cost-efficiencies, depending on immobilization strategies/matrices. Carbon nanotubes (CNTs) are attractive matrices but challenged by enzyme leaching (physical adsorption) or perturbation (covalent linking). Zeolitic imidazolate frameworks (ZIFs) overcome these issues. However, our recent study [J. Am. Chem. Soc., 2018, 140, 16032−16036] showed reduced costefficiency as enzymes trapped below the ZIF surfaces cannot participate in biocatalysis; the enzyme−ZIF composites are also unstable under acidic conditions. In this work, we demonstrate the feasibility of using ZIFs to immobilize enzymes on CNT surfaces on two model enzymes, T4 lysozyme and amylase, both of which showed negligible leaching and retained catalytic activity under neutral and acidic conditions. To better understand the behavior of enzymes on CNTs and CNT−ZIF, we characterized enzyme orientation on both matrices using site-directed spin-labeling (SDSL)−electron paramagnetic resonance (EPR), which is immune to the complexities caused by CNT and ZIF background signals and enzyme−matrix interactions. Our structural investigations showed enhanced enzyme exposure to the solvent compared to enzymes in ZIFs alone; orientation of enzymes in matrices itself is directly related to substrate accessibility and, therefore, essential for understanding and improving catalytic efficiency. To the best of our knowledge, this is the first time ZIFs and onepot synthesis are employed to anchor large-substrate enzymes on CNT surfaces for biocatalysis. This is also the first report of enzyme orientation on the CNT surface and upon trapping in CNT−ZIF composites. Our results are essential for guiding the rational design of CNT−ZIF combinations to improve enzyme stabilization, loading capacity, and catalytic efficiency.
We
report a rapid and highly sensitive approach based on gold-nanoparticle-decorated
silica nanorods (GNP-SiNRs) label and lateral-flow strip biosensor
(LFSB) for visually detecting proteins. Owing to its biocompatibility
and convenient surface modification, SiNRs were used as carriers to
load numerous GNPs, and the GNP-SiNRs were used as labels for the
lateral-flow assay. The LFSB detection limit was lowered 50 times
compared to the traditional GNP-based lateral-flow assay. Rabbit IgG
was used as a model target to demonstrate the proof-of-concept. Sandwich-type
immunoreactions were performed on the immunochromatographic strips,
and the accumulation of GNP-SiNRs on the test zone produced the characteristic
colored bands, enabling visual detection of proteins without instrumentation.
The quantitative detection was performed by reading the intensities
of the colored bands with a portable strip reader. The response of
the optimized device was highly linear for the range of 0.05–2
ng mL–1, and the detection limit was estimated to
be 0.01 ng mL–1. The GNP-SiNR-based LFSB, thus,
offered an ultrasensitive method for rapidly detecting trace amounts
of proteins. This method has a potential application with point-of-care
screening for clinical diagnostics and biomedical research.
We report a fluorescent carbon nanoparticle (FCN)-based lateral flow biosensor for ultrasensitive detection of DNA. Fluorescent carbon nanoparticle with a diameter of around 15 nm was used as a tag to label a detection DNA probe, which was complementary with the part of target DNA. A capture DNA probe was immobilized on the test zone of the lateral flow biosensor. Sandwich-type hybridization reactions among the FCN-labeled DNA probe, target DNA and capture DNA probe were performed on the lateral flow biosensor. In the presence of target DNA, FCNs were captured on the test zone of the biosensor and the fluorescent intensity of the captured FCNs was measured with a portable fluorescent reader. After systematic optimizations of experimental parameters (the components of running buffers, the concentration of detection DNA probe used in the preparation of FCN-DNA conjugates, the amount of FCN-DNA dispensed on the conjugate pad and the dispensing cycles of the capture DNA probes on the test-zone), the biosensor could detect a minimum concentration of 0.4 fM DNA. This study provides a rapid and low-cost approach for DNA detection with high sensitivity, showing great promise for clinical application and biomedical diagnosis.
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