Modification of proteins with cyclodextrins prevents aggregation and surface adsorption and increases thermal stability

This work describes a general approach for preventing protein aggregation and surface adsorption by modifying proteins with β-cyclodextrins (βCD) via an efficient water-driven ligation. As compared to native unmodified proteins, the cyclodextrin-modified proteins (lysozyme and RNase A) exhibit significant reduction in aggregation, surface adsorption and increase in thermal stability. These results reveal a new chemistry for preventing protein aggregation and surface adsorption that is likely of different mechanisms than that by modifying proteins with poly(ethylene glycol).     

Antibody immobilization to phospholipid polymer layer on gold substrate of quartz crystal microbalance immunosensor

To modify gold electrode for immunosensor to construct an artificial cell membrane structure, water-soluble amphiphilic phospholipid polymer, poly[2-methacryloyloxyehtyl phosphorylcholine-co-n-butyl methacrylate-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (PMBN)] was applied. The polymer had active ester groups for immobilization of biomolecules and it was converted partially to thiol groups for binding to gold substrates. The partially thiolated PMBN was adsorbed on a gold electrode of quartz crystal microbalance (QCM). Surface characterization of adsorbed PMBN layers was thoroughly investigated with reflectance anisotropy spectroscopy, ellipsometry spectroscopy, dynamic contact angle and X-ray photoelectron spectroscopy measurements. Among several PMBN, having different degree of thiolation, it was concluded that 21.5% thiolated PMBN layer had the most well-ordered phosphorylcholine groups in its outer surface. The proteins adsorption test revealed that the phosphorylcholine group on the outer side of PMBN layers, which was substituted their active ester groups by glycine, showed suppress the non-specific adsorption of proteins, such as bovine serum albumin and γ-globulin. Also, through antigen–antibody binding evaluation, the anti-C-reactive protein antibody immobilized on the PMBN surface worked well and it was confirmed that denaturation of the antibody on the PMBN layers was hardly occurred in spite of 60 days storage at 4 °C. The antibody conjugated phospholipid polymer layer with well-ordered phosphorylcholine group could be outstanding functional membrane for biomedical diagnostic devices without non-specific binding and reduction of immunologic activity of immobilized antibody.     

Directly observed Au-S bond breakage due to swelling of the anchored polyelectrolyte

A weak polyelectrolyte coating, carboxylated poly(oligo(ethylene glycol)methacrylate-co-2-hydroxyethylmethacrylate), was prepared via surface initiated polymerization (SIP) from initiators immobilized to gold surface through the Au-S bonds. When dry thickness increased up to 75 nm, this polyelectrolyte coating was pulled off the Au substrate by simply exposing to phosphate buffer saline (PBS, pH = 7.4, [Na(+)] = 150 mM). This covalent bond breaking (CBB) behavior was monitored in situ using a quartz crystal microbalance (QCM) and CBB was associated with the swelling of the anchored polyelectrolyte chains.     

Rapid microarray detection of DNA and proteins in microliter volumes with surface plasmon resonance imaging measurements

A four-chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The 12-element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm(2) area; 45 nm thickness), each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA, respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (ssDNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements are also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss is proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18?000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 s in the microliter volume format.     

Tunable micropatterned substrates based on poly(dopamine) deposition via microcontact printing

A simple technique was developed to fabricate tunable micropatterned substrates based on mussel-inspired surface modification. Polydopamine (PDA) was developed on polydimethylsiloxane (PDMS) stamps and was easily imprinted to several substrates such as glass, silicon, gold, polystyrene, and poly(ethylene glycol) via microcontact printing. The imprinted PDA retained its unique reactivity and could modulate the chemical properties of micropatterns via secondary reactions, which was illustrated in this study. PDA patterns imprinted onto a cytophobic and nonfouling substrates were used to form patterns of cells or proteins. PDA imprints reacted with nucleophilic amines or thiols to conjugate molecules such as poly(ethylene glycol) for creating nonfouling area. Gold nanoparticles were immobilized onto PDA-stamped area. The reductive ability of PDA transformed silver ions to elemental metals as an electroless process of metallization. This facile and economic technique provides a powerful tool for development of a functional patterned substrate for various applications.     

Imaging the binding ability of proteins immobilized on surfaces with different orientations by using liquid crystals

We report an investigation of the binding ability of a protein immobilized on surfaces with different orientations but in identical interfacial microenvironments. The surfaces present mixed self-assembled monolayers (SAMs) of 11-[19-carboxymethylhexa(ethylene glycol)]undecyl-1-thiol, 1, and 11-tetra(ethylene glycol) undecyl-1-thiol, 2. Whereas 2 is used to define an interfacial microenvironment that prevents nonspecific adsorption of proteins, 1 was activated by two different schemes to immobilize ribonuclease A (RNase A) in either a preferred orientation or random orientations. The binding of the ribonuclease inhibitor protein (RI) to RNase A on these surfaces was characterized by using ellipsometry and the orientational behavior of liquid crystals. Ellipsometric measurements indicate identical extents of immobilization of RNase A via the two schemes. Following incubation of both surfaces with RI, however, ellipsometric measurements indicate a 4-fold higher binding ability of the RNase A immobilized with a preferred orientation over RNase A immobilized with a random orientation. The higher binding ability of the oriented RNase A over the randomly oriented RNase A was also apparent in the orientational behavior of nematic liquid crystals of 4-cyano-4'-pentylcyanobiphenyl (5CB) overlayed on these surfaces. These results demonstrate that the orientations of proteins covalently immobilized in controlled interfacial microenvironments can influence the binding activities of the immobilized proteins. Results reported in this article also demonstrate that the orientational states of proteins immobilized at surfaces can be distinguished by examining the optical appearances of liquid crystals.     

Direct electrochemistry and superficial characterization of DNA-cytochrome c-MUA films on chemically modified gold surface

Cytochrome c (Cyt. c) was immobilized on the 11-mercaptohendecanoic acid (MUA)-modified gold electrode. The electrode was stable and sensitive to Cyt. c. Later, DNA was also immobilized on the two-layer modified electrode. Cyclic voltammetry studies show that Cyt. c can interact with dsDNA and ssDNA. The binding site sizes were determined to be 15 base pairs per Cyt. c molecule with dsDNA and 30 nucleotides binding 1 Cyt. c molecule with ssDNA. The modified electrodes were characterized by quartz crystal microbalance (QCM), impedance spectroscopy and atomic force microscope (AFM). The modified electrode can be used for determining DNA.     

Towards the optimization of an optical DNA sensor: control of selectivity coefficients and relative surface affinities

Short single-stranded DNA (ssDNA) oligonucleotides can be grown on the surface of fused silica by automated nucleic acid synthesis. The immobilized ssDNA can be deposited at a desired average density. The density of ssDNA provides a controlled parameter that in combination with temperature, ionic strength and pH, can be used to define the selectivity of hybridization. Furthermore, the density of ssDNA can be used to control the affinity of complementary DNA so that it associates with the nucleic acids on the surface rather than areas that are not coated with ssDNA. The characteristic melt temperature observed for immobilized double-stranded DNA (dsDNA) 20mer shifts by up to 10 °C when a single base pair mismatch is present in the center of a target oligonucleotide. Optimization of quantitative analysis of such single base pair mismatches requires use of select experimental conditions to maximize the formation of the fully matched target duplex while minimizing the formation of the mismatched duplex. Results based on fiber optic biosensors that are used to study binding of fluorescein-labeled complementary DNA demonstrate that it is possible to achieve a selectivity coefficient of fully matched to single base pair mismatch of approximately 85-1, while maintaining >55% of the maximum possible signal that can be obtained from the fully matched target duplex.     

Construction of nanogold hollow balls with dendritic surface as immobilized affinity support for protein adsorption

This contribution introduced the construction of nanosize gold hollow balls (NGB) with dendritic surface as the immobilized affinity support for aflatoxin B(1) antibody (anti-AFB(1)) adsorption, as a model protein, in biorecognition interface. The interaction between nanogold hollow balls and anti-AFB(1) was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and quartz crystal microbalance (QCM) technique. The deposited mass of anti-AFB(1) on the nanogold hollow ball-modified electrodes was more than that on the nanogold particle-modified electrodes via QCM analysis. The cyclic voltammogram tends to be more irreversible with anti-AFB(1) concentration increased. The antigen-antibody interaction was examined by using the nanogold hollow ball-modified QCM probes. Experimental results show that the developed protein assay system is sensitive to the concentration of AFB(1) as low as 0.05 ng mL(-1). Thus, the nanogold hollow ball is a useful matrix, and can be used as the immobilized affinity support for the adsorption of other proteins.     

Fabrication of novel molecular recognition membranes by physical adsorption and self-assembly for surface plasmon resonance detection of TNT

Recent concern on international terrorism and weapons of mass destruction demands the development of novel analytical methods for identification and quantification of explosive molecules. In this article, we describe the development of high-performance immunosensors for detection of 2,4,6-trinitrotoluene (TNT), a prime component of the landmines and bombs used by terrorist and military forces. The immunosensors were constructed by physical adsorption and self-assembly methods, and their binding interactions with a monoclonal anti-TNT antibody were evaluated for TNT detection using the surface plasmon resonance technique. A home-made 2,4,6-trinitrophenyl-keyhole limpet hemocyanine conjugate was used for physical adsorption. A poly(ethylene glycol) hydrazine hydrochloride thiolate was used in the construction of self-assembled monolayer surface and was immobilized with trinitrophenyl-β-alanine by the amide coupling method. The immunosensors were highly selective, regenerable, rapid, and exhibited remarkable sensitivity down to the parts-per-trillion level for TNT by the indirect competitive inhibition principle.     

硅表面分子刷图案化及生长DNA纳米管

结合"自上而下"和"自下而上"技术构建微纳米器件是目前纳米科学和技术领域追逐的目标之一。本文首先采用硅氢化反应在硅表面共价偶联引发聚合的活性基团,接着实施表面原子转移自由基聚合(ATRP)反应形成高分子刷poly(PEGMA),采用"自上而下"的光刻技术在硅表面制备功能化的图案,最后利用"自下而上"的DNA自组装技术在图案部分原位生长DNA纳米管。上述组装过程通过多次透射反射红外光谱、凝胶电泳、透射电镜和扫描电镜进行了检测,证实了硅芯片表面定位生长DNA纳米管的可行性。     

Immobilization of sunflower trypsin inhibitor (SFTI-1) peptide onto a gold surface and analysis of its interaction with trypsin

Sunflower trypsin inhibitor (SFTI-1) derived peptide having one disulfide bond could be immobilized via a thiol-disulfide exchange reaction onto a gold surface on a quartz crystal microbalance (QCM) chip. This permitted quantitative analysis of the specific interaction with trypsin.     

硅表面分子刷图案化及生长DNA纳米管

结合“自上而下”和“自下而上”技术构建微纳米器件是目前纳米科学和技术领域追逐的目标之一。本文首先采用硅氢化反应在硅表面共价偶联引发聚合的活性基团,接着实施表面原子转移自由基聚合（ATRP）反应形成高分子刷poly（PEGMA）,采用“自上而下”的光刻技术在硅表面制备功能化的图案,最后利用“自下而上”的DNA自组装技术在图案部分原位生长DNA纳米管。上述组装过程通过多次透射反射红外光谱、凝胶电泳、透射电镜和扫描电镜进行了检测,证实了硅芯片表面定位生长DNA纳米管的可行性。     

Plasma protein adsorption and thrombus formation on surface functionalized polypyrrole with and without electrical stimulation

A surface modification technique was developed in which heparin was covalently immobilized onto electrically conductive polypyrrole (PPY) film through poly(ethylene glycol) methacrylate (PEGMA) graft copolymerization and subsequent cyanuric chloride activation. In vitro plasma protein adsorption and thrombus formation experiments were carried out on the various films. The PEGMA-graft-copolymerized PPY surfaces with immobilized heparin have good bioactivity indicated by low level of protein adsorption, high ratio of albumin to fibrinogen adsorption, and low thrombus formation, making them potentially good candidates for biomedical applications. Since the PPY film retained significant electrical conductivity after surface modification, the effect of electrical stimulation on protein adsorption and thrombus formation was also evaluated. The covalently immobilized heparin on the PPY film was able to retain its bioactivity after 4 days of immersion in PBS. The film after long-term immersion in PBS also retained sufficient electrical conductivity for electrical stimulation still to be effective for reducing protein adsorption.     

DNA recognition interfaces: the influence of interfacial design on the efficiency and kinetics of hybridization

The effect of the surface chemistry of DNA recognition interfaces on DNA hybridization at a gold surface was investigated using both electrochemistry and the quartz crystal microbalance (QCM) technique. Different DNA recognition interfaces were prepared using a two-component self-assembled monolayer consisting of thiolated 20-mer probe single-stranded DNA (ss-DNA) containing either a 3'-mercaptopropyl or a 3'-mercaptohexyl linker group and an alcohol-terminated diluent layer with 2-, 6-, or 11-carbon length. The influence of the interfacial design on the hybridization efficiency, the affinity constant (Ka) describing hybridization, and the kinetics of hybridization was assessed. It was found that the further the DNA was above the surface defined by the diluent layer the higher the hybridization efficiency and Ka. The kinetics of DNA hybridization was assessed using both a QCM and an electrochemical approach to ascertain the influence of the interface on both the initial binding of target DNA to the surface and the formation of a complete duplex. These measurements showed that the length of the diluent layer has a large impact on the time taken to form a perfect duplex but no impact on the initial recognition of the target DNA by the immobilized probe DNA.     

Poly(l‐glutamic acid)‐based micellar hydrogel with improved mechanical performance and proteins loading

Soft tissues, such as fat and skin, present high flexibility and are capable of withstanding large deformation in various functions. Hydrogels that can resemble the mechanical performance of soft tissue are unique and widely demanded. In this study, micellar hydrogels based on biocompatible poly(l ‐glutamic acid) (PLGA) were designed with the enhanced capacity to bear large deformation. Amphipathic triblock copolymer poly(ethylene glycol) acrylate‐co‐poly(ε‐caprolactone)‐co‐poly (ethylene glycol) acrylate (APEG‐PCL‐APEG) with two terminal double bonds was synthesized and self‐assembled into micelles. At the same time, graft copolymers, poly(l ‐glutamic acid)‐g‐hydroxyethyl methacrylate (PLGA‐g‐HEMA) with double bonds were synthesized. APEG‐PCL‐APEG micelles and PLGA‐g‐HEMA were mixed to construct micellar hydrogel via radical polymerization. The crystalline structure and hydrophobic aggregation of copolymers (APEG‐PCL‐APEG) were found to associate with PCL molecular weight. Due to the hydrophobic stress dissipation and crystalline structure of the micelles, the softness and toughness of hydrogels were promoted, exhibiting a 25% increase in ultimate strain. Moreover, the micellar hydrogels were able to load proteins with long‐term retention. In addition, under dynamic mechanical stimulation, the release of proteins could be accelerated. Besides, the micellar hydrogels also supported rabbit adipose‐derived stem cells (rASCs) growth, thus exhibiting the potential toward soft tissue engineering. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019 , 57, 1115–1125     

Fabrication of a novel immunosensor using functionalized self-assembled monolayer for trace level detection of TNT by surface plasmon resonance

We have developed a new immunosensor based on self-assembly chemistry for highly sensitive and label-free detection of 2,4,6-trinitrotoluene (TNT) using surface plasmon resonance (SPR). A monolayer of amine terminated poly(ethylene glycol) hydrazinehydrochloride (PEG-NH₂) thiolate was constructed on an activated gold surface and immobilized with trinitrophenyl-β-alanine (TNPh-β-alanine) by amide coupling method. The binding interaction of a monoclonal anti-TNT Ab (M-TNT Ab) with TNPh-β-alanine immobilized thiolate monolayer surface was monitored and evaluated for detection of TNT based on the principle of indirect competitive immunoreaction. Here, the competition between the self-assembled TNT derivative and the TNT in solution for binding with antibody yields in the response signal that is inversely proportional to the concentration of TNT in the linear detection range. With the present immunoassay format, TNT could be detected in the concentration range from 0.008 ng/ml (8 ppt) to 30 ng/ml (30 ppb). The response time for an immunoreaction was 2 min and one immunocycle could be done with in 4 min including surface regeneration. Bound antibodies could be easily eluted from the self-assembled immunosurface at high recoveries (more than 100 cycles) using pepsin solution without any damage to the TNT derivatives immobilized on the surface. The compact self-assembled monolayer was highly stable and prevented the non-specific adsorption of proteins on the surface favoring error free measurement.     

金电极表面植物原生质体的固定化

近年来 ,基于聚电解质同携带相反电荷的基团之间的静电吸引作用组装超薄复合膜的研究受到广泛的关注 [1～ 7] .但是 ,利用聚电解质来固定生物活细胞的研究尚不多见 .Wen等 [8]曾在水溶液中利用聚电解质复合物构成的微囊来固定人体肿瘤活细胞 .本文将该技术应用于生物活细胞在电极表面的固定化 .利用吸附在金电极上的荷正电的聚二烯丙基二甲基氯化铵 [Poly( diallyldimethylammoniumchloride) ,PDADMAC]将携带负电荷的大麦细胞固定在金电极表面 ,运用扫描隧道显微镜 ( STM)、石英晶体微天平 ( QCM)及共焦激光扫描荧光显微镜对该过程进…     

Single probe nucleic acid immobilization on chemically modified single protein by controlling ionic strength and pH

In an effort toward determining the feasibility of single molecule analysis, we describe a case whereby the binding of one biotinylated DNA to one streptavidin molecule via electrostatic interactions was controlled by altering in pH 4.0-9.0 and 0.16 of the ion strength. The quantitative analysis of immobilized probe ssDNA was realized in real-time via a quartz crystal microbalance (QCM) and electrochemical (EC) measurement in the range 100 pM to 50 μM of probe oligonucleotide concentration. The variation amount of biotinylated ssDNA immobilized on the streptavidin-modified surface at pH 7.5 was about 0.16 pmol, giving a ratio of streptavidin to biotinylated ssDNA of about 1:1.1. On the other hand, at pH 4.9, it was immobilized about 0.29 pmol. From the shape of the Langmuir plot and QCM, the immobilization efficiency of biotinylated DNA via streptavidin at pH 4.9 was approximately twofold that at pH 7.5. In view points of the reaction velocity, it was increased with decreasing buffer solution pH, indicating a strong interaction of negatively charged probe DNA with the positively charged streptavidin. And also the EC response value of ΔI/I_streptavidin for the immobilized biotinylated ssDNA in pH 4.9 was about 49%, while the corresponding value for the pH 7.5 was approximately 34%. As DNA molecules possess negative charges, electrostatic repulsion occurred between streptavidin and biotinylated ssDNA at pH 7.5. At pH 4.9, the attraction between the biotinylated ssDNA and streptavidin resulted in increased adsorption which has an isoelectric point of about 5.9. It was deduced that the binding of biotinylated ssDNA to one or two of the four binding sites of streptavidin can be controlled by adjusting the pH-controlled electrostatic interaction.