带电悬浮粒子胶体晶体的固定化

采用光固化技术, 以丙烯酰胺单体与亚甲基双丙烯酰胺交联剂在紫外光的照射下发生光聚合反应, 嵌入聚苯乙烯胶体晶体, 实现了胶体晶体的固定化. 结合反射光谱和Kossel衍射技术研究对照了固定化前后胶体晶体的变化, 实验结果表明, 通过这种水凝胶固定化的胶体晶体保存了未固定前悬浮液中胶体晶体的结构. 但固定化后的胶体晶体的晶面间距和晶体的尺寸都略微减小. 通过对固定化后的水凝胶长时间的反射光谱观测, 发现固定化后胶体晶体在Milli-Q水中起初会发生溶胀, 经过2-5天溶胀-消溶胀过程达到平衡, 平衡后的水凝胶胶体晶体十分稳定, 可以长时间保持胶体晶体的结构. 因此, 胶体晶体固定化不但极大地提高了悬浮液中胶体晶体的抗剪切能力, 还克服了悬浮液中胶体晶体对离子、外界干扰的敏感性, 扩大了胶体晶体的实际应用价值.     

An Amperometric Glucose Sensor Based on Isoporous Crystalline Protein Membranes as Immobilization Matrix

Abstract

S-layer ultrafiltration membranes (SUMs) with an active filtration layer composed of coherent two-dimensional, isoporous protein crystals (S-layers) have been used as matrix for immobilizing monolayers of enzymes. Since S-layers are formed by periodic repetition of identical protein subunits, functional groups are present on the crystalline array in an identical position and orientation. As a consequence monolayers of enzymes can bind in a geometrically well defined way. For the covalent immobilization of enzymes carboxyl groups from the S-layer protein were activated with carbodiimide and allowed to react with amino groups of the enzyme. SUMs were employed as a new type of immobilization matrix for the developement of an amperometric glucose sensor using glucose oxidase (GOD) as the biologically active component. Glucose oxidase covalently bound to the surface of the S-layer protein retained approximately 40% of its activity. The enzyme loaded SUMs were covered with a layer of gold or platinum to function as working electrodes. These sensors yielded high signals (150nA/mm²/mmol glucose), fast response times (10–30s) and a linearity range up to 12 mM glucose. The stability under working conditions was more than 48 hours. There was no loss in activity after a storage period of 6 month.     

Real-time QCM-D immunoassay through oriented antibody immobilization using cross-linked hydrogel biointerfaces

This report presents the development of pre-cross-linked and in situ cross-linked polyethyleneimine-carboxymethylcellulose antibody immobilization platforms for real-time QCM-D immunoassay of sepsis-related biomarkers. These platforms differ significantly from recent trends in QCM-based assays, a rapidly expanding field given the affordability and sensitivity of the transduction system, by providing ultrafast biointerface deposition through cross-linking of polysaccharides. Using rhIL-1ra (17 kDa), a known sepsis biomarker, for development, various immunoassay modifications to increase sensitivity were investigated, including the use of Protein A, Protein G, and anti-IgG Fc specific antibody capture ligands for oriented antibody immobilization, higher-frequency QCM-D crystals, and amplification using secondary antibodies. The optimized assay employs Protein A oriented immobilization on pre-cross-linked polymer and secondary antibodies to achieve a detection limit of 25 ng/mL on 5 MHz crystals. Assay repeatability using the optimized chemistry is robust, with no loss in 100 ng/mL antigen detection over 20 cycles of the 10 min sandwich assay. Nonspecific adsorption of human serum albumin, as characterized by ToF-SIMS, is minimal and negligible for the pre-cross-linked and in situ cross-linked compositions, respectively.     

Effects of particle volume fraction on distortion of particle-arrayed structure during immobilization of colloidal crystals formed by poly(methyl methacrylate)-grafted silica in acetonitrile

Changes of particle array structure with particle volume fraction during immobilization of colloidal crystals, formed by poly(methyl methacrylate)-grafted silica in acetonitrile, were investigated. Immobilization of colloidal crystals formed in acetonitrile was carried out by two-step photo-radical copolymerization of methyl methacrylate and ethylene dimethacrylate to make organogel, followed by solidification after exchanging the solvent with methyl methacrylate. Crystallite size in colloidal crystals formed in acetonitrile was mostly unchanged with particle volume fraction in the range of 0.11–0.18, while the size and number of single crystals decreased during gelation. Disordering in particle array in immobilized colloidal crystals in gel and poly(methyl methacrylate) matrix was observed to decrease with increasing particle volume fraction less than 0.18 due to strong electrostatic repulsion between particles.     

DNA-coated microcrystals

Coprecipitation leads to self-assembly of bioactive DNA on the surface of salt, sugar or amino-acid crystals and provides a rapid inexpensive immobilization method suitable for preparing dry-powder formulations of nucleic acids, useful for storage, imaging and drug delivery.     

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.     

Chemical silicon surface modification and bioreceptor attachment to develop competitive integrated photonic biosensors

Methodology for the functionalization of silicon-based materials employed for the development of photonic label-free nanobiosensors is reported. The studied functionalization based on organosilane chemistry allowed the direct attachment of biomolecules in a single step, maintaining their bioavailability. Using this immobilization approach in probe microarrays, successful specific detection of bacterial DNA is achieved, reaching hybridization sensitivities of 10?pM. The utility of the immobilization approach for the functionalization of label-free nanobiosensors based on photonic crystals and ring resonators was demonstrated using bovine serum albumin (BSA)/anti-BSA as a model system.     

Comparative Assessment of Oriented Antibody Immobilization on Surface Plasmon Resonance Biosensing

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody‐protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.     

Supramolecular Protein Immobilization on Lipid Bilayers

Protein immobilization on surfaces, and on lipid bilayers specifically, has great potential in biomolecular and biotechnological research. Of current special interest is the immobilization of proteins using supramolecular noncovalent interactions. This allows for a reversible immobilization and obviates the use of harsh ligation conditions that could denature fragile proteins. In the work presented here, reversible supramolecular immobilization of proteins on lipid bilayer surfaces was achieved by using the host–guest interaction of the macrocyclic molecule cucurbit[8]uril. A fluorescent protein was successfully immobilized on the lipid bilayer by making use of the property of cucurbit[8]uril to host together a methylviologen and the indole of a tryptophan positioned on the N‐terminal of the protein. The supramolecular complex was anchored to the bilayer through a cholesterol moiety that was attached to the methylviologen tethered with a small polyethylene glycol spacer. Protein immobilization studies using a quartz crystal microbalance (QCM) showed the assembly of the supramolecular complexes on the bilayer. Specific immobilization through the protein N‐terminus is more efficient than through protein side‐chain events. Reversible surface release of the proteins could be achieved by washing with cucurbit[8]uril or buffer alone. The described system shows the potential of supramolecular assembly of proteins and provides a method for site‐specific protein immobilization under mild conditions in a reversible manner.     

Immobilization of proteins on agarose beads, monitored in real time by bead injection spectroscopy

A novel approach to real-time monitoring of protein immobilization resulted in the surprising finding that current immobilization protocols are far from optimized.     

Immobilization of proteins on carboxylic acid functionalized nanopatterns

The immobilization of proteins on nanopatterned surfaces was investigated using in situ atomic force microscopy (AFM) and ex situ infrared reflectance–absorption spectroscopy (IRAS). The AFM-based lithography technique of nanografting provided control of the size, geometry, and spatial placement of nanopatterns within self-assembled monolayers (SAMs). Square nanopatterns of carboxylate-terminated SAMs were inscribed within methyl-terminated octadecanethiolate SAMs and activated using carbodiimide/succinimide coupling chemistry. Staphylococcal protein A was immobilized on the activated nanopatterns before exposure to rabbit immunoglobulin G. In situ AFM was used to monitor changes in the topography and friction of the nanopatterns in solution upon protein immobilization. Complementary studies with ex situ IRAS confirmed the surface chemistry that occurred during the steps of SAM activation and subsequent protein immobilization on unpatterned samples. Since carbodiimide/succinimide coupling chemistry can be used for surface attachment of different biomolecules, this protocol shows promise for development of other aqueous-based studies for nanopatterned protein immobilization.     

Fast and reliable protein microarray production by a new drop-in-drop technique

In contrast to DNA microarrays, production of protein microarrays is an immense technological challenge due to high complexity and diversity of proteins. In this paper we investigate three essential aspects of protein microarray fabrication based on the highly parallel and non-contact TopSpot technology: evaporation of probes during long lasting production times, optimization of protein immobilization and improvement of protein microarray reproducibility. Evaporation out of the printhead reservoirs was reduced to a minimum by sealing the reservoirs with gas permeable foils or PDMS frames. This led to dramatically lowered setup times through the possibility of long-term, ready-to-print storage of filled printheads. To optimize immobilization efficiency 128 printing buffers were tested by printing two different proteins onto seven different microarray slide types. This way we were able to reduce the CV of spot diameter on the microarray slide below 1.14%. To remarkably increase protein immobilization efficiency on microarray slides the commonly used EDC-NHS system (a laboratory method for immobilization of proteins) was miniaturized by using a new drop-in-drop printing technique. Additionally the very fast UV cross-linking was used to immobilize antibodies. The optimized system was used to produce antibody microarrays and with it microarray ELISA experiments were performed successfully.     

Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support

Bacterial cytochrome P450s (P450s), which catalyze regio‐ and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost‐effective industrial manufacturing. However, immobilization of P450s is difficult because electron‐transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein‐mediated co‐immobilization of a bacterial P450, and its electron‐transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co‐immobilized enzymes catalyzed monooxygenation because the electron‐transfer protein fused to PCNA via a single peptide linker retained its electron‐transport function.     

Immobilization of histidine-tagged proteins on electrodes

The development of new enzyme immobilization techniques that do not affect catalytic activity or conformation of a protein is an important research task in biotechnology including biosensor applications and heterogeneous reaction systems. One of the most promising approaches for controlled protein immobilization is based on the immobilized metal ion affinity chromatography (IMAC) principle originally developed for protein purification. Here we describe the current status and future perspectives of immobilization of His-tagged proteins on electrode surfaces. Recombinant proteins comprising histidine-tags or histidine rich native proteins have a strong affinity to transition metal ions. For metal ion immobilization at the electrode surface different matrices can be used such as self-assembled monolayers or conductive polymers. This specific technique allows a reversible immobilization of histidine-tagged proteins at electrodes in a defined orientation which is an important prerequisite for efficient electron transfer between the electrode and the biomolecule. Any application requiring immobilized biocatalysts on electrodes can make use of this immobilization approach, making future biosensors and biocatalytic technologies more sensitive, simpler, reusable and less expensive while only requiring mild enzyme modifications.     

Direct Comparison of Lysine versus Site-Specific Protein Surface Immobilization in Single-Molecule Mechanical Assays**

Single-molecule force spectroscopy (SMFS) is powerful for studying folding states and mechanical properties of proteins, however, it requires protein immobilization onto force-transducing probes such as cantilevers or microbeads. A common immobilization method relies on coupling lysine residues to carboxylated surfaces using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS). Because proteins typically contain many lysine groups, this strategy results in a heterogeneous distribution of tether positions. Genetically encoded peptide tags (e.g., ybbR) provide alternative chemistries for achieving site-specific immobilization, but thus far a direct comparison of site-specific vs. lysine-based immobilization strategies to assess effects on the observed mechanical properties was lacking. Here, we compared lysine- vs. ybbR-based protein immobilization in SMFS assays using several model polyprotein systems. Our results show that lysine-based immobilization results in significant signal deterioration for monomeric streptavidin-biotin interactions, and loss of the ability to correctly classify unfolding pathways in a multipathway Cohesin-Dockerin system. We developed a mixed immobilization approach where a site-specifically tethered ligand was used to probe surface-bound proteins immobilized through lysine groups, and found partial recovery of specific signals. The mixed immobilization approach represents a viable alternative for mechanical assays on in vivo-derived samples or other proteins of interest where genetically encoded tags are not feasible.     

Affinity capturing for targeting proteins into micro and nanostructures

Protein immobilization into micro and nanoscaled patterns opens exciting possibilities in fundamental and applied research. Developing efficient capturing techniques while preserving the structural and functional integrity of the proteins on surfaces is a key challenge for surface scientists. In this paper, current techniques for site-specific protein immobilization into engineered surface architectures are reviewed. Fundamental principles for functional protein immobilization on solid supports are discussed and popular affinity-based recognition pairs and their application for capturing proteins into nano and microstructures are presented.     

Calix[4]crown-5-ether as a biolinker for immobilization of protein and DNA in fluorescence glass slide chip

Binding affinity between calix[4]crown-5-ether and amino acids have been compared by studying the complexation association constant, and the best value has been obtained from complex of calix[4]crown-5 ether, and HrSOD tagged with Arg and Lys were tested to investigate the effects of specific residues in protein immobilization on calix[4]crown-5-ether. The protein tagged with 9Args has been shown to have much better immobilization potential. Taking advantage of the similar structure of a moiety of guanine base to that of Arg side chain, different homo-oligonucleotides have been immobilized, and it was found that calix[4]crown-5-ether is an appropriate agent in the immobilization of dGTP homo-oligonucleotides. The results demonstrate that calix[4]crown-5-ether on glass slide chip could be applied as an excellently oriented immobilization agent for protein or for DNA microarray designing. It has the ability of single base differentiation in SNP sequence detection.     

Use of reversible denaturation for adsorptive immobilization of urease

Urease was chosen as a model multimeric protein to investigate the utility of reversible denaturation for immobilization to a hydrophobic support. Of the various procedures investigated, acidic denaturation provided the highest degree of immobilization and enzymatic activity with lowering of K _m (apparent). Exposure of hydrophobic clusters in the protein molecule induced by the acidic pH environment was confirmed by fluorescence studies using 8-anilino-1-naphtalene-sulfonate as a hydrophobic-reporter probe. The catalytic potential of the enzyme at low pH values was dramatically improved with significant heat and pH stability enhancement on immobilization. Furthermore, the immobilized preparation was used successfully in continuous catalytic transformations. Based on the results presented in this article and a recent report involving a relatively more simple monomeric protein, it is suggested that reversible denaturation may be of general utility for immobilization of proteins, which are not normally adsorbed on hydrophobic supports.     

Combination of cysteine- and oligomerization domain-mediated protein immobilization on a surface plasmon resonance (SPR) gold chip surface

Here we report an effective method for protein immobilization on a surface plasmon resonance (SPR) gold chip, describing the combination of cysteine- and oligomerization domain-mediated immobilization of enhanced green fluorescent protein (EGFP) as a model protein for the purpose of orientation-controlled surface density packing. In order to facilitate the oligomerization of EGFP, the dimeric and trimeric constructs derived from GCN4- leucine zipper domain were chosen for multimeric EGFP assembly. For orientation-controlled immobilization of the protein, EGFP modified with cysteine residues showing excellent orientation on a gold chip was used as a starting protein, as previously reported in our earlier study (Anal. Chem., 2007, 79, 2680-2687). Constructs of EGFP with oligomerization domains were genetically engineered, and corresponding fusion proteins were purified, applied to a gold chip, and then analyzed under SPR. The immobilized EGFP density on a gold chip increased according to the states of protein oligomerization, as dimeric and trimeric EGFPs displayed better adsorption capability than monomeric and dimeric forms, respectively. Fluorescence measurement corroborated the SPR results. Taken together, our findings indicated that the combination of cysteine- and oligomerization domain-mediated immobilization of protein could be used in SPR biosensor applications, allowing for an excellent orientation and high surface density simultaneously.     

Catalytically Active Hollow Fiber Membranes with Enzyme‐Embedded Metal–Organic Framework Coating

Metal–organic frameworks (MOFs) are suitable enzyme immobilization matrices. Reported here is the in situ biomineralization of glucose oxidase (GOD) into MOF crystals (ZIF‐8) by interfacial crystallization. This method is effective for the selective coating of porous polyethersulfone microfiltration hollow fibers on the shell side in a straightforward one‐step process. MOF layers with a thickness of 8 μm were synthesized, and fluorescence microscopy and a colorimetric protein assay revealed the successful inclusion of GOD into the ZIF‐8 layer with an enzyme concentration of 29±3 μg cm^?2. Enzymatic activity tests revealed that 50 % of the enzyme activity is preserved. Continuous enzymatic reactions, by the permeation of β‐d ‐glucose through the GOD@ZIF‐8 membranes, showed a 50 % increased activity compared to batch experiments, emphasizing the importance of the convective transport of educts and products to and from the enzymatic active centers.