Biomedical subjects

Shaoyi Jiang

Publications and source records attributed to Shaoyi Jiang.

At least 19 recordsLinked to original sources

PubMed2007

Controlling the orientation of bone osteopontin via its specific binding with collagen I to modulate osteoblast adhesion.

Osteopontin (OPN) is an important matricellular protein that modulates cell functions. It is potentially an excellent surface-coating component for engineered biomaterials. It is believed that in its preferred orientation and conformation on a surface, the functional domains of OPN such as the arginine-glycine-aspartic acid (RGD) motif will be presented to cells to the greatest extent. Previously, the authors demonstrated that OPN orientation could be modulated by surface charge. In this work, the authors attempt to control the orientation/conformation of bone OPN via its specific interactions with type I collagen. Surface plasmon resonance was used to confirm the specific binding between bone OPN and collagen I. A radiolabeled OPN adsorption assay was used to determine the amount of adsorbed OPN on tissue culture polystyrene (TCPS) surfaces with or without collagen I as an interlayer. An in vitro cell adhesion assay using osteoblast MC3T3-E1 was performed to compare the functionality of collagen-bound OPN and adsorbed OPN on TCPS. With the same amount of OPN on the surfaces, the number of cells adhered to collagen-bound OPN is significantly higher than to OPN alone on TCPS. A cell inhibition assay using soluble GRGDSP peptides showed that a higher GRGDSP concentration was needed to completely block osteoblast adhesion to collagen-bound OPN than to OPN directly on TCPS. Enhanced cell adhesion and higher blocking peptide concentration suggest that collagen-bound bone OPN has a preferable orientation/conformation for cell adhesion compared with OPN alone on TCPS. Thus, the specific binding of OPN to collagen I may naturally orient OPN, thus influencing osteoblast adhesion.

Animals↗

PubMed2007

Development of biocompatible interpenetrating polymer networks containing a sulfobetaine-based polymer and a segmented polyurethane for protein resistance.

Interpenetrating polymer networks (IPNs) were prepared by the modification of a segmented polyurethane (SPU) with a cross-linked sulfobetaine methacrylate (SBMA) polymer. The IPN films that were prepared can effectively resist nonspecific protein adsorption when the distribution of SBMA units within the SPU film is well controlled, and they retain high mechanical strengths inherent from the base SPU films. Furthermore, the zwitterionic and biomimetic nature of sulfobetaine and the ease of SBMA preparation make SBMA-based materials very attractive for a wide range of applications. It is challenging to control the diffusion of highly polar SBMA into the hydrophobic network of SPU. In this study, various parameters governing the formation of IPNs containing SBMA were studied. The chemical composition depth profile of the IPN films was determined by confocal Raman microscopy. The morphology and thickness of these IPN films were examined by atomic force microscopy and scanning electron microscopy. The amount of adsorbed proteins on the IPN films was determined by an enzyme-linked immunosorbent assay. Results show that the amount of adsorbed proteins on the IPN films depends on the incubation conditions, including solvent polarity, incubation time, SBMA monomer ratio, and incubation concentration. It appears that the IPN films prepared in a mixed solvent of higher polarity with long incubation time lead to very low protein adsorption. This study not only introduces a new IPN system containing SBMA, but also provides a fundamental understanding of various parameters governing the formation of IPNs.

Adsorption↗

PubMed2006

Understanding the nonfouling mechanism of surfaces through molecular simulations of sugar-based self-assembled monolayers.

This paper presents a molecular simulation study of the interactions of a protein (lysozyme) with self-assembled monolayers (SAMs) of mannitol and sorbitol terminated alkanethiols in the presence of explicit water molecules and ions. The all-atom simulations were performed to calculate the force generated on the protein as a function of its distance above the SAM surfaces. The structural and dynamic properties of water molecules both above the SAM surfaces and around the SAM head groups were analyzed to provide a better understanding of the nonfouling behavior of the sugar-based SAM surfaces. Results from this work suggest that both mannitol and sorbitol SAMs generate a tightly bound, structured water layer around the SAM chains. This hydration layer creates a repulsive force on the protein when it approaches the surface, resulting in a nonfouling surface despite the presence of hydrogen-bond donor groups. This work demonstrates the importance of strong surface-water interactions for surface resistance to nonspecific protein adsorption.

Adsorption↗

PubMed2006

Superlow fouling sulfobetaine and carboxybetaine polymers on glass slides.

This work describes the superlow fouling properties of glass slides grafted with zwitterionic polymers to highly resist the adsorption of proteins and the adhesion of mammalian cells. Glass slides were first silanized using 2-bromo-2-methyl-N-3-[(triethoxysilyl)propyl]propanamide (BrTMOS). Two zwitterionic polymers, poly(sulfobetaine methacrylate) (polySBMA) and poly(carboxybetaine methacrylate) (polyCBMA), were then grafted from the silanized glass substrates using the atom-transfer radical polymerization (ATRP) method. X-ray photoelectron spectroscopy (XPS) was used to analyze the surfaces of the silanized glass substrates and the substrates grafted with the polymers. An enzyme-linked immonosobrbent assay (ELISA) using polyclonal antibodies was used to measure fibrinogen adsorption on these surfaces. The surfaces with polySBMA or polyCBMA layers were shown to reduce fibrinogen adsorption to a level comparable with that of adsorption on poly(ethylene glycol)-like films. Bovine aortic endothelial cells (BAECs) were seeded on these surfaces. The attachment and spreading of the cells were observed only on unpolymerized glass surfaces. This work further demonstrates that zwitterionic polymers highly resist nonspecific protein adsorption and cell adhesion and provides an effective method to modify glass slides or other oxide surfaces to achieve superlow fouling.

Adsorption↗

PubMed2006

Molecular simulation studies of the structure of phosphorylcholine self-assembled monolayers.

We report a study of the structure of phosphorylcholine self-assembled monolayers (PC-SAMs) on Au(111) surfaces using both molecular mechanics (MM) and molecular dynamics (MD) simulation techniques. The lattice structure (i.e., packing densities and patterns) of the PC chains was determined first, by examining the packing energies of different structures by MM simulations in an implicit solvent. The chain orientation (i.e., antiparallel and parallel arrangements of the PC head groups) was then evaluated. The initial azimuthal angles of the PC chains were also adjusted to ensure that the optimal lattice structure was found. Finally, the two most probable lattice structures were solvated with explicit water molecules and their energies were compared after 1.5 ns of MD simulations to verify the optimal structures obtained from MM. We found that the optimal lattice structure of the PC-SAM corresponds to a radical7 x radical7 R19degree lattice structure (i.e., surface coverage of 50.4 A(2)molecule) with a parallel arrangement of the head groups. The corresponding thickness of the optimal PC-SAM is 13.4 A which is in agreement with that from experiments. The head groups of the PC chains are aligned on the surface in such a way that their dipole components are minimized. The P-->N vector of the head groups forms an angle of 82 degrees with respect to the surface normal. The tilt direction of molecular chains was observed to be towards their next nearest neighbor.

Adsorption↗

PubMed2006

Method for generation of in vivo biotinylated recombinant antibodies by yeast mating.

We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and Western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of K(d)=4.8 x 10(-9) M and K(d)=5.1 x 10(-9) M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.

Antibodies↗

PubMed2006

Supramolecular assembly of cyclodextrin-based nanoparticles on solid surfaces for gene delivery.

In this work, a new approach for surface-mediated gene delivery based on inclusion complex formation between the solid surface and delivery vehicles is presented. beta-Cyclodextrin (CD) molecules form high-affinity inclusion complexes with adamantane. This complexation ability was used to specifically immobilize beta-CD-modified poly(ethylenimine) (CD-PEI) nanoparticles on adamantane- (AD-) modified self-assembled monolayers. To investigate the nanoparticle/surface interaction, CD-PEI-based and PEI-based nanoparticles were passed through a surface plasmon resonance flow cell containing the monolayers. CD-PEI nanoparticles are specifically immobilized on the chip surface by cyclodextrin-adamantane inclusion complex formation. Minimal nanoparticle adsorption was detected with PEI-based nanoparticles or on control surfaces. Competition studies with free cyclodextrins reveal that the multivalent interactions between CD-PEI nanoparticles and the adamantane-modified surface results in significantly higher binding affinity than single cyclodextrin-adamantane complexes. Immobilized nanoparticles were characterized by atomic force microscopy and quantified by fluorescence assay. Thus, the ability of CD-PEI nanoparticles to form inclusion complexes can be exploited to attain specific, high-affinity loading of delivery vehicles onto solid surfaces.

Adamantane↗

PubMed2006

Unbinding of the streptavidin-biotin complex by atomic force microscopy: a hybrid simulation study.

A hybrid molecular simulation technique, which combines molecular dynamics and continuum mechanics, was used to study the single-molecule unbinding force of a streptavidin-biotin complex. The hybrid method enables atomistic simulations of unbinding events at the millisecond time scale of atomic force microscopy (AFM) experiments. The logarithmic relationship between the unbinding force of the streptavidin-biotin complex and the loading rate (the product of cantilever spring constant and pulling velocity) in AFM experiments was confirmed by hybrid simulations. The unbinding forces, cantilever and tip positions, locations of energy barriers, and unbinding pathway were analyzed. Hybrid simulation results from this work not only interpret unbinding AFM experiments but also provide detailed molecular information not available in AFM experiments.

Biotin↗

PubMed2006

Strong resistance of a thin crystalline layer of balanced charged groups to protein adsorption.

Resistance of mixed self-assembled monolayers (SAMs) with various counter-charged terminal groups of different valence and protonation/deprotonation states to nonspecific protein adsorption is investigated. It is demonstrated that excellent nonfouling surfaces can be readily constructed from mixed positively and negatively charged components of equal valence in a wide range of thiol solution compositions. Furthermore, the lattice structure of one of the mixed SAM systems studied is revealed by atomic force microscopy (AFM) to be (5.2 +/- 0.2 A x 5.2 +/- 0.2 A)60 degrees . Results indicate that the packing structure of mixed charged SAMs is determined by strong charge-charge interactions of the terminal groups rather than S-Au and chain-chain interactions. This work provides direct evidence that conformational flexibility is not required for protein resistance of a surface and even a single compact layer of charged groups of balanced charge with a crystalline structure can resist nonspecific protein adsorption, suggesting that tightly bound water molecules on the topmost part of the mixed SAMs play a dominant role in surface resistance to nonspecific protein adsorption.

Adsorption↗

PubMed2006

Modulating cell adhesion and spreading by control of FnIII7-10 orientation on charged self-assembled monolayers (SAMs) of alkanethiolates.

In this work, we demonstrate that surface charge can be used to modulate cell adhesion/spreading through the control of the orientation of adsorbed FnIII(7-10), which is a cell-adhesive protein containing RGD residues. Carboxylic acid (COOH) and amine (NH(2))-terminated self-assembled monolayers (SAMs) of alkanethiolates were used as model negatively and positively charged surfaces, respectively. The adsorbed amount of FnIII(7-10) is controlled to be equivalent on both SAMs as confirmed by the adsorption isotherms determined using I(125)-radiolabeled FnIII(7-10.) The binding of a monoclonal antibody specific for the cell-binding domain of FnIII(7-10) was measured by surface plasmon resonance (SPR) to evaluate FnIII(7-10) orientations on different SAMs. Results indicate that adsorbed FnIII(7-10) on NH(2)-SAM has an orientation with more cell-binding domains accessible than on COOH-SAM, confirming our predictions from Monte Carlo simulations. Both phase contrast images and Vybrant MTT cell proliferation assays show that the adhesion/spreading of bovine aortic endothelial cells (BAECs) on the NH(2)-SAM is significantly better than that on the COOH-SAM coated with an equivalent amount of FnIII(7-10). These results indicate that surface charge can be used to specifically orient cell adhesive proteins such as FnIII(7-10), thus providing a promising strategy to increase the activity of materials incorporating biological moieties.

Alkanesulfonates↗

PubMed2006

Surface grafted sulfobetaine polymers via atom transfer radical polymerization as superlow fouling coatings.

One of the sulfobetaine methacrylate (SBMA) monomers, N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine, was polymerized onto initiator-covered gold surfaces using atom transfer radical polymerization (ATRP) to form uniform polymer brushes. Self-assembled monolayers (SAMs) with ATRP initiators were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The thickness of grafted poly(SBMA) films was measured by ellipsometry. Fibrinogen adsorption on poly(SBMA) grafted surfaces was measured with a surface plasmon resonance (SPR) sensor. Two approaches were compared to graft ATRP initiators onto gold surfaces for surface polymerization and subsequent protein adsorption on these polymer grafted surfaces. The first was to prepare a SAM from omega-mercaptoundecyl bromoisobutyrate onto a gold surface. Superlow fouling surfaces with well-controlled poly(SBMA) brushes were achieved using this approach (e.g., fibrinogen adsorption <0.3 ng/cm2). The second approach was to react bromoisobutyryl bromide with a hydroxyl-terminated SAM on a gold surface. Although protein adsorption decreased as the density of surface initiators increased, the surface prepared using the second approach was not able to achieve as low protein adsorption as the first approach. Key parameters to achieve superlow fouling surfaces were studied and discussed.

Adsorption↗

PubMed2006

Controlling DNA orientation on mixed ssDNA/OEG SAMs.

We present and characterize a mixed self-assembled monolayer (SAM) consisting of single-stranded oligonucleotide (ssDNA)- and oligo(ethylene glycol) (OEG)-terminated thiols. The ssDNA/OEG SAMs are prepared by simultaneous coadsorption from a common thiol solution over a broad range of compositions. Electron spectroscopy for chemical analysis (ESCA) is used to measure the surface coverage of ssDNA, whereas surface plasmon resonance (SPR) sensor is used to measure the hybridization of complementary ssDNA and protein resistance. Through the complementary use of these techniques, we find that the composition of OEG in the assembly solution controls a key parameter: the surface coverage of ssDNA on the surface. There is evidence that it influences the orientation of the immobilized ssDNA probes. Lower OEG concentrations yield a surface with higher ssDNA coverage and less favorable orientation, whereas higher OEG concentrations produce a surface with lower DNA coverage and more favorable orientation. Competition between these two effects controls the hybridization efficiency of the ssDNA surface. Compared to ssDNA surfaces prepared with other diluent thiols, the use of OEG improves the protein resistance of the surface, making it more broadly applicable.

Adsorption↗

PubMed2006

Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor.

We report the quantitative and simultaneous detection of four species of bacteria, Escherichia coli O157:H7, Salmonella choleraesuis serotype typhimurium, Listeria monocytogenes, and Campylobacter jejuni, using an eight-channel surface plasmon resonance (SPR) sensor based on wavelength division multiplexing. Detection curves showing SPR response versus analyte concentration were established for each species of bacteria in buffer at pH 7.4, apple juice at native pH 3.7, and apple juice at an adjusted pH of 7.4, as well as for a mixture containing all four species of bacteria in buffer. Control experiments were performed to show the non-fouling characteristics of the sensor surface as well as the specificity of the amplification antibodies used in this study. The limit of detection (LOD) for each of the four species of bacteria in the tested matrices ranges from 3.4 x 10(3) to 1.2 x 10(5) cfu/ml. Detection curves in buffer of an individual species of bacteria in a mixture of all four species of bacteria correlated well with detection curves of the individual species of bacteria alone. SPR responses were higher for bacteria in apple juice at pH 7.4 than in apple juice at pH 3.7. This difference in sensor response could be partly attributed to the pH dependence of antibody-antigen binding.

Bacteria↗

PubMed2006

Strong resistance of oligo(phosphorylcholine) self-assembled monolayers to protein adsorption.

In this work, we demonstrate the strong resistance of oligo(phosphorylcholine) (OPC) self-assembled monolayers (SAMs) to protein adsorption and cell adhesion. OPC SAMs were characterized using X-ray photoelectron spectroscopy (XPS), and protein adsorption was measured using a surface plasmon resonance (SPR) sensor. Results are compared with those of phosphorylcholine (PC) SAMs. Despite the existence of negative charge on OPC SAMs and the simple synthesis procedure of OPC thiols, OPC SAMs resist protein adsorption as effectively as or better than PC SAMs formed from highly purified PC thiols. The ease of their preparation and the effectiveness of their function make OPC SAMs an attractive alternative for creating nonfouling surfaces.

Adsorption↗

PubMed2006

DNA-directed protein immobilization for simultaneous detection of multiple analytes by surface plasmon resonance biosensor.

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.

Antibodies↗

PubMed2006

Highly protein-resistant coatings from well-defined diblock copolymers containing sulfobetaines.

Three well-defined diblock copolymers of poly(sulfobetaine methacrylate) [poly(SBMA)] and poly(propylene oxide) (PPO) were synthesized by the sequential addition of SBMA monomer to fixed amounts of PPO using an atom transfer radical polymerization method and varying poly(SBMA) lengths. These copolymers were characterized by 1H NMR and aqueous gel permeation chromatography. These copolymers were physically adsorbed onto a surface plasmon resonance (SPR) sensor surface covered by methyl-terminated self-assembled monolayers, followed by the in situ evaluation of protein adsorption on the adsorbed copolymers. It is found that the behavior of the protein adsorption depends on the molecular weight of the copolymers. Results show that the diblock copolymers containing poly(SBMA) can be highly protein resistant when surface SBMA densities are well controlled. Thus, copolymers containing zwitterionic groups are ideal for resisting protein adsorption when the surface density of zwitterionic groups is controlled.

Adsorption↗

PubMed2006

Studies of alphaB crystallin subunit dynamics by surface plasmon resonance.

The molecular chaperone activity of alphaB crystallin, an important stress protein in humans, is regulated by physiological factors, including temperature, pH, Ca2+, and ATP. In this study, the role of these factors in regulating the subunit dynamics of human alphaB crystallin was investigated using surface plasmon resonance (SPR). SPR experiments indicate that at temperatures above 37 degrees C, where alphaB crystallin has been reported to have higher chaperone activity, the subunit dynamics of alphaB crystallin were increased with faster association and dissociation rates. SPR experiments also indicate that interactions between alphaB crystallin subunits were enhanced with much faster association and slower dissociation rates at pH values below 7.0, where alphaB crystallin has been reported to have lower chaperone activity. The results suggest that the dynamic and rapid subunit exchange rate may regulate the chaperone activity of alphaB crystallin. The effect of Ca2+ and ATP on the subunit dynamics of alphaB crystallin was minimal, suggesting that Ca2+ and ATP modulate the chaperone activity of alphaB crystallin without altering the subunit dynamics. Based on the SPR results and previously reported biochemical data for the chaperone activity of alphaB crystallin under different conditions of temperature and pH, a model for the relationship between the subunit dynamics and chaperone activity of alphaB crystallin is established. The model is consistent with previous biochemical data for the chaperone activity and subunit dynamics of small heat shock proteins (sHSPs) and establishes a working hypothesis for the relationship between complex assembly and chaperone activity for sHSPs.

Adenosine Triphosphate↗

PubMed2006

Dual-functional biomimetic materials: nonfouling poly(carboxybetaine) with active functional groups for protein immobilization.

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.

Adsorption↗