Ultra sensitive affinity chromatography on avidin-functionalized PMMA microchip for low abundant post-translational modified protein enrichment

Xia, Hui; Murray, Kermit K.; Soper, Steven A.; Feng, June

doi:10.1007/s10544-011-9586-7

Cited by 15 publications

(14 citation statements)

References 66 publications

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“…43 Ligand-receptor multivalent interactions can be also studied in these systems by the nanopatterning strategy presented here, as the ligand coupling chemistry can be applied to any amine-bearing molecule. 31,[44][45][46] In conclusion, our results could proof that nanopatterned surfaces are much faster and more efficient at eliciting the formation of cell receptor clusters compared to related surface-bound ligand presentation strategies. While nanopatterns presented a 9-fold lower overall ligand surface density compared to related configurations, the presence of nanodomains with locally elevated ligand densities facilitated multivalent interactions that could tune the receptor aggregation response.…”

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confidence: 56%

Nanopatterns of Surface-Bound EphrinB1 Produce Multivalent Ligand–Receptor Interactions That Tune EphB2 Receptor Clustering

et al. 2017

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Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate-with molecular sensitivity-the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems.

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confidence: 56%

Nanopatterns of Surface-Bound EphrinB1 Produce Multivalent Ligand–Receptor Interactions That Tune EphB2 Receptor Clustering

et al. 2017

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“…This apparent difference is a result of the high proportion of carboxyl groups (16%) generated on PMMA surfaces during IPA sonication when compared to pristine PMMA (7%, Figure S4), as predicted by previous studies. 32,33 As the penetration depth of X-rays used in XPS is usually between 10−100 nm, 34 the relative percentages of the functional groups do not provide an absolute estimate of the functional groups present on the outermost surface of PMMA, that is accessible to the biomolecules. Hence, the reduction of carboxyl groups cannot be significantly proven by XPS data alone.…”

Section: Resultsmentioning

confidence: 99%

Air Plasma-Enhanced Covalent Functionalization of Poly(methyl methacrylate): High-Throughput Protein Immobilization for Miniaturized Bioassays

Sathish

Ishizu

Shen

2019

ACS Appl. Mater. Interfaces

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Miniaturized systems, such as integrated microarray and microfluidic devices, are constantly being developed to satisfy the growing demand for sensitive and highthroughput biochemical screening platforms. Owing to its recyclability, and robust mechanical and optical properties, poly(methyl methacrylate) (PMMA) has become the most sought after material for the large-scale fabrication of these platforms. However, the chemical inertness of PMMA entails the use of complex chemical surface treatments for covalent immobilization of proteins. In addition to being hazardous and incompatible for large-scale operations, conventional biofunctionalization strategies pose high risks of compromising the biomolecular conformations, as well as the stability of PMMA. By exploiting radio frequency (RF) air plasma and standard 1ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry in tandem, we demonstrate a simple yet scalable PMMA functionalization strategy for covalent immobilization (chemisorption) of proteins, such as green fluorescent protein (GFP), while preserving the structural integrities of the proteins and PMMA. The surface density of chemisorbed GFP is shown to be highly dependent on the air plasma energy, initial GFP concentration, and buffer pH, where a maximum GFP surface density of 4 × 10 −7 mol/m 2 is obtained, when chemisorbed on EDC−NHS-activated PMMA exposed to 27 kJ of air plasma, at pH 7.4. Furthermore, antibody-binding studies validate the preserved biofunctionality of the chemisorbed GFP molecules. Finally, the coupled air plasma and EDC−NHS PMMA biofunctionalization strategy is used to fabricate microfluidic antibody assay devices to detect clinically significant concentrations of Chlamydia trachomatis specific antibodies. By coupling our scalable and tailored air plasma-enhanced PMMA biofunctionalization strategy with microfluidics, we elucidate the potential of fabricating sensitive, reproducible, and sustainable high-throughput protein screening systems, without the need for harsh chemicals and complex instrumentation.

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“…Most importantly, it allows reuse of the matrix and offers the potential for the development of automated high-throughput protein purification systems. SAVSBPM18 can also be immobilized to different platforms such as protein arrays, ELISA plates, magnetic beads, biosensor chips, lab-on-a-chip micro-devices and bioreactors for different applications [47]. It is important to note that proteins (or protein libraries) tagged with other tags including His-tag [48], Halo-tag [49] and Snap-tag [50] can, in fact, be easily converted to the biotinylated format.…”

Section: Discussionmentioning

confidence: 99%

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

Wong

2013

PLoS ONE

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Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.

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Ultra sensitive affinity chromatography on avidin-functionalized PMMA microchip for low abundant post-translational modified protein enrichment

Cited by 15 publications

References 66 publications

Nanopatterns of Surface-Bound EphrinB1 Produce Multivalent Ligand–Receptor Interactions That Tune EphB2 Receptor Clustering

Nanopatterns of Surface-Bound EphrinB1 Produce Multivalent Ligand–Receptor Interactions That Tune EphB2 Receptor Clustering

Air Plasma-Enhanced Covalent Functionalization of Poly(methyl methacrylate): High-Throughput Protein Immobilization for Miniaturized Bioassays

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

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