The Experts below are selected from a list of 20310 Experts worldwide ranked by ideXlab platform
Amy E Herr - One of the best experts on this subject based on the ideXlab platform.
-
microchamber western blotting using poly l lysine conjugated polyacrylamide gel for blotting of sodium dodecyl sulfate coated Proteins
Analytical Chemistry, 2013Co-Authors: Minsub Chung, Amy E HerrAbstract:We report a novel strategy to immobilize sodium dodecyl sulfate (SDS)-coated Proteins for fully integrated microfluidic Western blotting. Polyacrylamide gel copolymerized with a cationic polymer, poly-l-lysine, effectively immobilizes all sized Proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and enables SDS-PAGE and subsequent immuno-probing in an automated microfluidic chip. Design of a poly-l-lysine conjugated polyacrylamide gel allows optimization of SDS-Protein Immobilization strength in the blotting gel region of the microchamber. The dependence of Protein capture behavior on both the concentration of copolymerized charges and poly-lysine length is studied and gives important insight into an electrostatic Immobilization mechanism. Based on analysis of Protein conformation, the immobilized Proteins bind with partner antibody after SDS dilution. We demonstrate each step of the microchamber Western blot, including injection, separation, transfer, Immobilization, bloc...
-
Protein Immobilization techniques for microfluidic assays
Biomicrofluidics, 2013Co-Authors: Dohyun Kim, Amy E HerrAbstract:Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled Immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, Protein Immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on Immobilization surface, Protein properties, and specific assay goals. In this review, we present trade-offs considerations for common Immobilization surface materials. We overview Immobilization methods and chemistries, and discuss studies exemplar of key approaches-here with a specific emphasis on immunoassays and enzymatic reactors. Recent "smart Immobilization" methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach Proteins on demand with precise spatial control are highlighted. Spatially encoded Protein Immobilization using DNA hybridization for multiplexed assays and reversible Protein Immobilization surfaces for repeatable assay are introduced as Immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to Protein Immobilization.
-
microfluidic western blotting
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Alex J Hughes, Amy E HerrAbstract:Rapid, quantitative Western blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western blotting assay conducted in a single glass microchannel under purely electronic control. The μWestern blot is comprised of multiple steps: sample enrichment, Protein sizing, Protein Immobilization (blotting), and in situ antibody probing. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance advances are achieved, including: short durations of 10–60 min, multiplexed analyte detection, mass sensitivity at the femtogram level, high-sensitivity 50-pM detection limits, and quantitation capability over a 3.6-log dynamic range. Performance gains are attributed to favorable transport and reaction conditions on the microscale. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. This hydrophilic polymer constitutes both a separation matrix for Protein sizing and, after brief UV exposure, a Protein Immobilization scaffold for subsequent antibody probing of immobilized Protein bands. We observe Protein capture efficiencies exceeding 75% under sizing conditions. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. Taken together, the μWestern blot establishes a foundation for rapid, targeted proteomics by merging exceptional specificity with the throughput advantages of multiplexing, as is relevant to a broad range of biological inquiry.
-
electrostatic Protein Immobilization using charged polyacrylamide gels and cationic detergent microfluidic western blotting
Analytical Chemistry, 2012Co-Authors: Dohyun Kim, Kelly Karns, Samuel Tia, Amy E HerrAbstract:We report a novel Protein Immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic Immobilization gel (EIG) enables Immobilization of all Proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The “pan-analyte” capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte Immobilization and detection. Sandwich approaches limit analyte Immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte Immobilization on the EIG. We note that Protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength ...
Shaoyi Jiang - One of the best experts on this subject based on the ideXlab platform.
-
functionalizable surface platform with reduced nonspecific Protein adsorption from full blood plasma material selection and Protein Immobilization optimization
Biosensors and Bioelectronics, 2009Co-Authors: Hana Vaisocherová, Marek Piliarik, Allen D Taylor, Zheng Zhang, Jiří Homola, Wei Yang, Gang Cheng, Shaoyi JiangAbstract:In this work, zwitterionic polymers are investigated as ultra-low fouling and functionalizable coatings for biosensors, nanoparticle-based diagnostics, and microarrays to enable detections in real-world complex media. The effect of the spacer length between the two charged groups on the nonfouling properties of zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) was studied in blood plasma and serum. The polyCBAA polymer with an ethylene spacer was selected for Protein Immobilization studies. A polyCBAA-coated surface was functionalized with antibodies using a simple and fast amino coupling chemistry for direct Protein Immobilization in two simple steps: surface activation and Protein Immobilization/background deactivation. The effect of pH was found to be very important for both steps and it was optimized. The functionalized polyCBAA surface exhibited very low fouling properties even when exposed to undiluted blood plasma for more than 6 h with <7 ng/cm2 of adsorbed Proteins. The biological activity of the immobilized Proteins was demonstrated with the detection of a model Protein in undiluted blood plasma. A recently developed highly sensitive four-channel surface plasmon resonance (SPR) sensor was used for the evaluation of specific and nonspecific Protein adsorption to these surfaces.
-
zwitterionic polymers exhibiting high resistance to nonspecific Protein adsorption from human serum and plasma
Biomacromolecules, 2008Co-Authors: Jon Ladd, Zheng Zhang, Shengfu Chen, Jason C Hower, Shaoyi JiangAbstract:This study examined six different polymer and self-assembled monolayer (SAM) surface modifications for their interactions with human serum and plasma. It was demonstrated that zwitterionic polymer surfaces are viable alternatives to more traditional surfaces based on poly(ethylene glycol) (PEG) as nonfouling surfaces. All polymer surfaces were formed using atom transfer radical polymerization (ATRP) and they showed an increased resistance to nonspecific Protein adsorption compared to SAMs. This improvement is due to an increase in the surface packing density of nonfouling groups on the surface, as well as a steric repulsion from the flexible polymer brush surfaces. The zwitterionic polymer surface based on carboxybetaine methacrylate (CBMA) also incorporates functional groups for Protein Immobilization in the nonfouling background, making it a strong candidate for many applications such as in diagnostics and drug delivery.
-
dual functional biomimetic materials nonfouling poly carboxybetaine with active functional groups for Protein Immobilization
Biomacromolecules, 2006Co-Authors: Zheng Zhang, Shengfu Chen, Shaoyi JiangAbstract:We introduce a dual-functional biocompatible material based on zwitterionic poly(carboxybetaine methacrylate) (polyCBMA), which not only highly resists Protein adsorption/cell adhesion, but also has abundant functional groups convenient for the Immobilization of biological ligands, such as Proteins. The dual-functional properties are unique to carboxybetaine moieties and are not found in other nonfouling moieties such as ethylene glycol, phosphobetaine, and sulfobetaine. The unique properties are demonstrated in this work by grafting a polyCBMA polymer onto a surface or by preparing a polyCBMA-based hydrogel. PolyCBMA brushes with a thickness of 10-15 nm were grafted on a gold surface using the surface-initiated atom transfer radical polymerization method. Protein adsorption was analyzed using a surface plasmon resonance sensor. The surface grafted with polyCBMA very largely prevented the nonspecific adsorption of three test Proteins, that is, fibrinogen, lysozyme, and human chorionic gonadotropin (hCG). The Immobilization of anti-hCG on the surface resulted in the specific binding of hCG while maintaining a high resistance to nonspecific Protein adsorption. Transparent polyCBMA-based hydrogel disks were decorated with immobilized fibronectin. Aortic endothelial cells did not bind to the polyCBMA controls, but appeared to adhere well and spread on the fibronectin-modified surface. With their dual functionality and biomimetic nature, polyCBMA-based materials are very promising for their applications in medical diagnostics, biomaterials/tissue engineering, and drug delivery.
-
dna directed Protein Immobilization for simultaneous detection of multiple analytes by surface plasmon resonance biosensor
Analytical Chemistry, 2006Co-Authors: Christina Boozer, Jon Ladd, Shengfu Chen, Shaoyi JiangAbstract:A versatile multichannel biosensor surface is prepared by site-directed Immobilization of single-stranded DNA-Protein conjugates onto a patterned self-assembled monolayer composed of ssDNA thiols and oligo(ethylene glycol)-terminated thiols. The conjugates each consist of an antibody chemically linked to a unique ssDNA target with a sequence complementary to the surface-bound ssDNA probes and are immobilized on the surface via sequence-specific hybridization. The exceptional specificity of DNA hybridization combined with the diversity of available sequences makes this platform perfect for multichannel sensors. Once the surface is patterned with the appropriate probe sequences, sequence-specific hybridization sorts out the target conjugates and directs them to the appropriate spots on the surface. Previously (Boozer, C. L.; Ladd, J.; Chen, S.; Yu, Q.; Homola, J.; Jiang, S. Anal. Chem. 2004, 76, 6967-6972), we performed proof-of-concept experiments demonstrating the feasibililty of using DNA-directed Protein Immobilization to produce a single channel biosensor. In this work, we extend this technique and employ DNA-directed Protein Immobilization to functionalize a multichannel biosensor, which was used for the simultaneous detection of a set of three fertility hormones: human chorionic gonadotropin, human luteinizing hormone, and follicle stimulating hormone by surface plasmon resonance sensor.
Giovanni Venturoli - One of the best experts on this subject based on the ideXlab platform.
-
Protein Immobilization capabilities of sucrose and trehalose glasses the effect of Protein sugar concentration unraveled by high field epr
Journal of Physical Chemistry Letters, 2016Co-Authors: Marco Malferrari, Anton Savitsky, Wolfgang Lubitz, K Mobius, Giovanni VenturoliAbstract:Disaccharide glasses are increasingly used to immobilize Proteins at room temperature for structural/functional studies and long-term preservation. To unravel the molecular basis of Protein Immobilization, we studied the effect of sugar/Protein concentration ratios in trehalose or sucrose matrixes, in which the bacterial photosynthetic reaction center (RC) was embedded as a model Protein. The structural, dynamical, and H-bonding characteristics of the sugar–Protein systems were probed by high-field W-band EPR of a matrix-dissolved nitroxide radical. We discovered that RC Immobilization and thermal stabilization, being independent of the Protein concentration in trehalose, occur in sucrose only at sufficiently low sugar/Protein ratios. EPR reveals that only under such conditions does sucrose form a microscopically homogeneous matrix that immobilizes, via H-bonds, the nitroxide probe. We conclude that the Protein Immobilization capability depends critically on the propensity of the glass-forming sugar to cr...
-
Protein Immobilization capabilities of sucrose and trehalose glasses the effect of Protein sugar concentration unraveled by high field epr
Journal of Physical Chemistry Letters, 2016Co-Authors: Marco Malferrari, Anton Savitsky, Wolfgang Lubitz, K Mobius, Giovanni VenturoliAbstract:Disaccharide glasses are increasingly used to immobilize Proteins at room temperature for structural/functional studies and long-term preservation. To unravel the molecular basis of Protein Immobilization, we studied the effect of sugar/Protein concentration ratios in trehalose or sucrose matrixes, in which the bacterial photosynthetic reaction center (RC) was embedded as a model Protein. The structural, dynamical, and H-bonding characteristics of the sugar-Protein systems were probed by high-field W-band EPR of a matrix-dissolved nitroxide radical. We discovered that RC Immobilization and thermal stabilization, being independent of the Protein concentration in trehalose, occur in sucrose only at sufficiently low sugar/Protein ratios. EPR reveals that only under such conditions does sucrose form a microscopically homogeneous matrix that immobilizes, via H-bonds, the nitroxide probe. We conclude that the Protein Immobilization capability depends critically on the propensity of the glass-forming sugar to create intermolecular H-bond networks, thus establishing long-range, homogeneous connectivity within the matrix.
K Mobius - One of the best experts on this subject based on the ideXlab platform.
-
Protein Immobilization capabilities of sucrose and trehalose glasses the effect of Protein sugar concentration unraveled by high field epr
Journal of Physical Chemistry Letters, 2016Co-Authors: Marco Malferrari, Anton Savitsky, Wolfgang Lubitz, K Mobius, Giovanni VenturoliAbstract:Disaccharide glasses are increasingly used to immobilize Proteins at room temperature for structural/functional studies and long-term preservation. To unravel the molecular basis of Protein Immobilization, we studied the effect of sugar/Protein concentration ratios in trehalose or sucrose matrixes, in which the bacterial photosynthetic reaction center (RC) was embedded as a model Protein. The structural, dynamical, and H-bonding characteristics of the sugar–Protein systems were probed by high-field W-band EPR of a matrix-dissolved nitroxide radical. We discovered that RC Immobilization and thermal stabilization, being independent of the Protein concentration in trehalose, occur in sucrose only at sufficiently low sugar/Protein ratios. EPR reveals that only under such conditions does sucrose form a microscopically homogeneous matrix that immobilizes, via H-bonds, the nitroxide probe. We conclude that the Protein Immobilization capability depends critically on the propensity of the glass-forming sugar to cr...
-
Protein Immobilization capabilities of sucrose and trehalose glasses the effect of Protein sugar concentration unraveled by high field epr
Journal of Physical Chemistry Letters, 2016Co-Authors: Marco Malferrari, Anton Savitsky, Wolfgang Lubitz, K Mobius, Giovanni VenturoliAbstract:Disaccharide glasses are increasingly used to immobilize Proteins at room temperature for structural/functional studies and long-term preservation. To unravel the molecular basis of Protein Immobilization, we studied the effect of sugar/Protein concentration ratios in trehalose or sucrose matrixes, in which the bacterial photosynthetic reaction center (RC) was embedded as a model Protein. The structural, dynamical, and H-bonding characteristics of the sugar-Protein systems were probed by high-field W-band EPR of a matrix-dissolved nitroxide radical. We discovered that RC Immobilization and thermal stabilization, being independent of the Protein concentration in trehalose, occur in sucrose only at sufficiently low sugar/Protein ratios. EPR reveals that only under such conditions does sucrose form a microscopically homogeneous matrix that immobilizes, via H-bonds, the nitroxide probe. We conclude that the Protein Immobilization capability depends critically on the propensity of the glass-forming sugar to create intermolecular H-bond networks, thus establishing long-range, homogeneous connectivity within the matrix.
Katsuaki Shimazu - One of the best experts on this subject based on the ideXlab platform.
-
Succinimidyl Ester Surface Chemistry: Implications of the Competition between Aminolysis and Hydrolysis on Covalent Protein Immobilization
2016Co-Authors: Marc D Porter, Makoto Takahashi, Katsuaki ShimazuAbstract:ABSTRACT: N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., Proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent Protein immobiliza-tion in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of th
-
succinimidyl ester surface chemistry implications of the competition between aminolysis and hydrolysis on covalent Protein Immobilization
Langmuir, 2014Co-Authors: Nicholas A Owens, Ronald D Wampler, Yixin Ying, Jennifer H Granger, Marc D Porter, Makoto Takahashi, Katsuaki ShimazuAbstract:N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., Proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent Protein Immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simp...
-
succinimidyl ester surface chemistry implications of the competition between aminolysis and hydrolysis on covalent Protein Immobilization
Langmuir, 2014Co-Authors: China Y Lim, Nicholas A Owens, Ronald D Wampler, Yixin Ying, Jennifer H Granger, Marc D Porter, Makoto Takahashi, Katsuaki ShimazuAbstract:N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., Proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent Protein Immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to Protein Immobilization suggests that under coupling conditions in which low Protein concentrations and buffers of near physiological pH are used, Proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for Protein Immobilization due to effects that may arise from noncovalently linked Proteins.
