ArticleAnalytical chemistry2014
Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs.
Article in Analytical chemistry, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed.
- Nano-injection molding with resin mold inserts for prototyping of nanofluidic devices for single molecular detection.Lab on a chip · 2023Article
- Modifying surface charge density of thermoplastic nanofluidic biosensors by multivalent cations within the slip plane of the electric double layer.Colloids and surfaces. A, Physicochemical and engineering aspects · 2022Article
- Label-Free Identification of Single Mononucleotides by Nanoscale Electrophoresis.Small (Weinheim an der Bergstrasse, Germany) · 2021Article
- Tailoring Thermoplastic In-Plane Nanopore Size by Thermal Fusion Bonding for the Analysis of Single Molecules.ACS sensors · 2021Article
- Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing.Nucleic acids research · 2021Article
- Half a century of bacteriophage lambda recombinase: In vitro studies of lambda exonuclease and Red-beta annealase.IUBMB life · 2020Review
- Electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) through thermoplastic nanochannels.Analytica chimica acta · 2018Article
- Thermoplastic nanofluidic devices for biomedical applications.Lab on a chip · 2017Review
- Characterization of activated cyclic olefin copolymer: effects of ethylene/norbornene content on the physiochemical properties.The Analyst · 2016Article
- Interrogating Surface Functional Group Heterogeneity of Activated Thermoplastics Using Super-Resolution Fluorescence Microscopy.Analytical chemistry · 2016Article
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5 authors.
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Abstract
The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. We report the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA. Enzyme immobilization was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our results suggest that the efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. We obtained a clipping rate of 1.0 × 10(3) nucleotides s(-1) for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. The kinetic behavior of the solid-phase reactor could be described by a fractal Michaelis-Menten model with a catalytic efficiency nearly 17% better than the homogeneous solution-phase reaction. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.
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