Orientation Control of Photo‐Immobilized Antibodies on the Surface of Azobenzene‐Containing Polymers by the Introduction of Functional Groups

In our photo‐induced immobilization technique for an antibody (IgG) using azopolymers, the introduction of COOH and NMe₂ into the azopolymers, which can introduce surface charges, strongly affected the immobilization properties such as the efficiency of immobilization and the activity of the immobilized IgG (i.e., the orientation of the immobilized IgG). The introduction of COOH promoted a more active orientation of the immobilized IgG. The orientation was determined during the adsorption process onto the azopolymer surface in solution before photo‐immobilization, and was maintained during the photo‐immobilization. The surface charge of the azopolymer appears to be an important factor for IgG orientation, which involves electrostatic interactions between its Fab and the azopolymer surface.

     

RNA Aptamers That Reversibly Bind Photoresponsive Azobenzene‐Containing Peptides

Modulation of biological networks assembled by diverse interactions among biologically active molecules has provided a platform for innovative biotechnologies. Here, we report RNA aptamers that bind to a photoresponsive peptide (KRAzR; Lys‐Arg‐azobenzene‐Arg) containing azobenzene chromophore, which can change its structure by photoirradiation. Aptamers were identified after 10 cycles of an in vitro selection procedure starting with a DNA library containing a 70 nt random region. Surface plasmon resonance (SPR) analysis demonstrated that interactions between aptamers and KRAzR were fully controlled by appropriate photoirradiation to the SPR sensor chip. Upon irradiation of 360 nm on the KRAzR‐immobilized surface, the binding of each aptamer to the surface was significantly decreased. Subsequent photoirradiation of the same surface with 430 nm restored the aptamer binding to the surface. We also observed that direct photoirradiation of the aptamer–peptide complex on a gold surface actively promoted dissociation of the complex. Furthermore, a doped reselection method was applied to acquire structural and sequence information of aptamer 66. From a data analysis of the conserved region and the mutation frequency, we were able to select a plausible secondary structure among three candidates predicted by computational folding simulation.     

Amplified Immunoassay of Human IgG Using Real—time Biomolecular Interaction ANalysis （BIA） Technology

An automated biomolecular interaction analysis instrument(BIAcore)based on surface plasmon resonance(SPR)has been used to determine human immunoglobulin G(IgG) in real time.Polyclonal anti-human IgG antibody was covalently immobilized to a carboxymethyldextran-modified gold film surface.The samples of human IgG prepared in HBS buffer were poured over the immpbilized surface.The signal amplification antibody was applied to amplify the response signal.After each measurement,the surface was regenerated with 0.1mol/L H3PO4.The assay was rapid,requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding,antibody amplification and regeneration.The antibody immobilized surface had good response to human IgG in the range of 0.12-60 nmol/L with a detection limit of 60 pmol/L.The same antibody immobilized surface could be used for more than 110 cycles of binding,amplification and regeneration.The results demonstrate that the sensitivity,specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.     

Photosensitive Azopolymer Brushes via Atom Transfer Radical Polymerization for Protein Sensing

Novel photosensitive azopolymer brushes were synthesized via surface initiated atom transfer radical polymerization using initiator self‐assembled on Au surface. The chemical structures of azobenzene derivatives were confirmed by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). The surface morphology of azopolymers via atom transfer radical polymerization (ATRP) for different time was investigated by atomic force microscopy (AFM). Additionally, the photoisomerization of azopolymer was measured by ultraviolet‐visible spectroscopy (UV‐Vis). The results indicate that such azopolymers can undergo trans‐cis‐trans photoisomerization efficiently by photo‐irradiation with UV light. Furthermore, this photoisomerization property could also induce the reversible adsorption of bovine serum albumin (BSA) adsorption on azopolymer brush surfaces. This adsorption kinetics of the reversible process can be measured by surface plasmon resonance (SPR) spectroscopy in situ. It suggests that the protein biochips could be regenerated safely by UV irradiation rather than by being rinsed with chemical reagents.     

Athermal and Soft Multi-Nanopatterning of Azopolymers: Phototunable Mechanical Properties

Imprinting nanopatterns on flexible substrates has diverse applications in advanced fabrication. However, the traditional thermal nanoimprint lithography (T-NIL) often causes shrinkage upon cooling. Here, a simple yet versatile method is introduced to fabricate multiple nanopatterns on a flexible substrate coated with an azopolymer by combining athermal nanoimprint lithography (AT-NIL) and photolithography. The azopolymer has various mechanical properties upon photoirradiation: 1) phototunable glass-transition temperatures (T_g) and concomitantly photoinduced switch from glassy plastic to viscoplastic polymer; 2) prominent modulation of viscoplasticity under light illumination at different wavelengths. Regionally selective multiple nanopatterns are conveniently fabricated, presenting angle-dependent structural color images on poly(ethylene terephthalate) (PET) substrates. The flexible, athermal and multiple nanopatterning method has the potential for on-demand fabrication of complex nanopatterns.     

Athermal and Soft Multi‐Nanopatterning of Azopolymers: Phototunable Mechanical Properties

Imprinting nanopatterns on flexible substrates has diverse applications in advanced fabrication. However, the traditional thermal nanoimprint lithography (T‐NIL) often causes shrinkage upon cooling. Here, a simple yet versatile method is introduced to fabricate multiple nanopatterns on a flexible substrate coated with an azopolymer by combining athermal nanoimprint lithography (AT‐NIL) and photolithography. The azopolymer has various mechanical properties upon photoirradiation: 1) phototunable glass‐transition temperatures (T_g) and concomitantly photoinduced switch from glassy plastic to viscoplastic polymer; 2) prominent modulation of viscoplasticity under light illumination at different wavelengths. Regionally selective multiple nanopatterns are conveniently fabricated, presenting angle‐dependent structural color images on poly(ethylene terephthalate) (PET) substrates. The flexible, athermal and multiple nanopatterning method has the potential for on‐demand fabrication of complex nanopatterns.     

Kinetics of adsorption and proteolytic cleavage of a multilayer ovalbumin film by subtilisin Carlsberg

Adsorption and proteolytic activity of the enzyme subtilisin Carlsberg have been studied on an immobilized, multilayer ovalbumin film. The cross-linked multilayer substrate permits protease adsorption to be examined unencumbered by the surface inhomogeneity typically observed in monolayer studies of protease surface kinetics. Decline of the protein film was measured over time using ellipsometry. Resulting kinetic data as a function of aqueous enzyme concentration and temperature were well fit by a Langmuir-Michaelis-Menten model for surface proteolysis. We observed that both the protein degradation kinetics and the in situ adsorption data were well described by the proposed model. The temperature dependence of the kinetic rate parameter yielded an activation energy of 12 kcal/mol. Further, the apparent Langmuir adsorption equilibrium constant of the enzyme at the protein/aqueous interface was 0.11 L/mg at 22 degrees C, 0.034 L/mg at 36 degrees C, and 0.011 L/mg at 50 degrees C. Although enzyme adsorption at a given aqueous enzyme concentration decreased at higher temperature, the enzyme cleaved the substrate more rapidly, leading to a net increase in the ovalbumin film degradation rate. We observed that the maximum enzyme coverage on the immobilized protein surface was approximately 40% of a close-packed monolayer at ambient temperature (22 degrees C).     

Functional regulation of an immobilized redox protein on an oriented metal coordinated peptide monolayer as an electron mediator

We fabricated a vertically and unidirectionally oriented metal coordinated α-helical peptide monolayer, Leu(2)Ala(Pyri)(Co(II))Leu(6)Ala(4-Pyri)(Co(II))Leu(6), by stepwise polymerization on a mixed self-assembled monolayer consisting of amino-alkanethiol, dialkyl disulfide, and ferrocenyl alkanethiol acted as a photoresponsive electron donor. Redox-active protein, nitrate reductase (NR), was fixed on the surface of the peptide monolayer. By contrast, we fixed NR on the mixed self-assembled monolayer directly. Upon photoirradiation, electron flow occurred from the excited ferrocenyl group on the substrate to the electron acceptor, NR, on the surface of the molecular layers. The activated NR on the molecular layers reduced the nitrate to nitrite. The amount of the bioelectrocatalytic product, nitrite, generated by the immobilized NR on the peptide monolayer was larger than that produced by the immobilized NR on the mixed self-assembled monolayer directly. That is to say, the NR on the peptide monolayer has been more activated rather than that on the peptide absent monolayer by photoirradiation. The effective activation of the NR on the peptide monolayer can be explained in terms of enhancement of the vectorial electron flow along the macro-dipole moment of the α-helical peptide that arranged unidirectionally. It suggested that the ordered metal coordinated α-helical peptide monolayer acted as an efficient electron mediator to achieve a communication between the electron donor and the redox-active moiety. Such a hybrid molecular system looks promising for novel nanodevices, such as nano-photoreactors.     

偶氮苯聚合物的光控图案化

将自组装得到的聚苯乙烯胶体晶体,利用反应离子刻蚀和软刻蚀法复形到偶氮苯聚合物膜表面,获得六方非紧密排列的偶氮苯聚合物半球状阵列微图案膜。采用场发射扫描电子显微镜(FESEM)、原子力显微镜(AFM)和接触角测量仪等对膜的微阵列结构和表面润湿性能进行了表征。研究了光照对膜微图案结构和润湿性能的影响。结果表明:基于偶氮苯基团的光致取向特性,偶氮苯聚合物膜的微图案在偏振光照射下,可由初始的半球状阵列微结构形变成纺锤状和椭球状等结构,这种微结构的改变可以改变膜表面润湿性,实现偶氮苯聚合物膜表面不同微图案和润湿性能的光照调控。     

Electrochemical Immunosensing Strategies Based on Immobilization of Anti-IgC on Mixed Self-Assembly Monolayers Carrying Surface Amide or Carboxyl Groups

Abstract

In the development of electrochemical immunosensing strategies, stability or activity of the immobilized biocomponents and signal amplification of the immunoconjugates are two key factors. In this study, a comparative study of immunoglobulin G antibody (anti‐IgG) immobilization, as a model, was performed on cysteine (Cys), 2‐aminoethane thiol (AET), and 11‐mercaptoundecanoic acid (MUA) monolayers. The change of anti‐IgG layer formation on the three base layers as a function of the anti‐IgG concentration was investigated in parallel via electrochemical impedance spectroscopy, cyclic voltammetry, surface plasmon resonance, and quartz crystal microbalance. Through the parallel measurements, we demonstrate that the Cys‐modified layer is more suitable for the immobilization of the anti‐IgG molecules than the MUA or AET‐modified layer. Based on the CV and EIS analyses, it was determined that the current responses decreased with the increment of anti‐IgG concentration, while the resistance responses increased with the concentration of anti‐IgG increased. Moreover, the current and resistance shifts were more remarkable on the Cys layer than that of the other two layers. In the SPR and QCM measurements, the SPR and QCM response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. With regard to the fundamental problem of comparing different measurement principles, the mechanism of the IgG immobilized on the three layers was proposed. Consequently, the surface concentration of anti‐IgG immobilized on the electrode should be optimized to improve the sensitivity of the immunosensors.     

Studies on Improving the Immobilized Bead Reusability and Alkaline Protease Production by Isolated Immobilized <Emphasis Type="Italic">Bacillus circulans</Emphasis> (MTCC 6811) Using Overall Evaluation Criteria

This study uses an overall evaluation criterion for improving the immobilized bead reusability and extracellular enzyme production by immobilized cells by assigning relative weightage to bead reusability, enzyme production, and cell leakage. Initially, alkaline protease production by alginate-immobilized Bacillus circulans (MTCC 6811) was analyzed using L18 orthogonal array (OA). The resultant optimized parameters were further fine-tuned with L9 OA experimentation. At L18-OA analysis, inoculum level and CaCl(2) had least influence at individual level. At the interactive level, incubation time revealed maximum and minimum interaction with sodium alginate and glucose concentration, respectively. L9 experimentation indicated that glucose concentration contributed the major influence on protease production followed by matrix material and incubation time at the individual level, and at the interactive level, matrix concentration played a vital role by interacting with incubation time, inoculum, and CaCl(2) concentration. All selected input parameters showed significance either at individual level or interactive in both OAs. Scanning electron microscopy analysis showed bacterial morphology variation with variation of matrix concentration. Overall, glucose concentration depicted a major influence at the individual level for the enzyme production. Significant improvement, approximately 147%, in enzyme yield was observed. Economic enzyme production by immobilized B. circulans is regulated by interactive influence of fermentation parameters, which influence the immobilized bead stability, reusability, and enzyme yield.     

Reusable chromium-coated quartz crystal microbalance for immunosensing

The application of oxidized chromium as a reusable platform for the development of immunosensors is presented. Chromium films were deposited on quartz crystal microbalances to study the affinity interaction between rabbit immunoglobulin G (IgG) and goat anti-rabbit IgG. A covalent approach, based on the silane chemistry, was followed for the grafting of either the rabbit IgG or the anti-rabbit IgG on the silane-modified chromium surface. Next the differences between the deposition of rabbit IgG on immobilized anti-rabbit IgG and the deposition of anti-rabbit IgG on immobilized rabbit IgG were investigated. The chromium layer could be reused between experiments, after the proper removal of the organic layers with piranha etch, obtaining a high repeatability in the steps of the functionalization protocol.     

Atomic force microscopy of human serum albumin (HSA) on poly(styrene/acrolein) microspheres

Atomic Force Microscopy (AFM) in the tapping mode was used for the observation of bare poly (styrene/acrolein) P(SA) microspheres and microspheres with attached HSA. Prior to the AFM observations the P(SA) microspheres were immobilized covalently on the surface of quartz slides modified with -aminopropyltriethoxysilane. Atomic Force Microscopy pictures were registered for the dry samples. The partial coalescence of the P(SA) microspheres connected to the quartz surface with amino groups has been observed. The AFM pictures of the single P(SA) microspheres revealed that the surface of these particles is smooth and that any irregularities, if present, do not exceed 1 nm. The surface of microspheres with attached HSA has very clearly different morphology with regular pattern of HSA macromolecules. Cracks on the surfaces of some microspheres with HSA revealed that protein macromolecules are attached to these particles in several layers. In the case of some other microspheres the defects in protein attachment allowed the observation of the border between the bare surface of the P(SA) microspheres and the surface covered with protein macromolecules. Comparison of the thickness of the HSA layers on the P(SA) microspheres with the dimensions of HSA macromolecules, determined earlier from the x-ray studies, suggests that the first layer, 3.0±0.2 nm thick, is formed of the HSA macromolecules arranged flatly on the surface whereas protein macromolecules in the subsequent layers, each 8.6±1 nm thick, are adsorbed protruding from the surface.     

Theoretical model for photochemical or thermally activated immobilization of macromolecules

The covalent immobilization of macromolecules on surfaces and within 3-dimensional networks is quantitatively described using a model based on Poisson statistics. This model determines the immobilized density or layer thickness as a function of molecular weight of the macromolecule or radiant exposure prior to and following the surface deposition of the macromolecule. Measurements of immobilized layer thickness provide first-order rate constants for decomposition of the surface-bound linker molecules and an estimate of the surface-bound linker density. The model predicts the relative density of immunocomplexed antibodies as a function of the irradiation time used to immobilize antigens. By providing the average number of bonds to the immobilized molecule, the model enables studies of the effect of multiple bonds on the activity of biomolecules. Experimental data by the authors and from the literature validate the model.     

A novel capacitive immunosensor based on gold colloid monolayers associated with a sol-gel matrix

A capacitive sensing method based on self-assembling gold nanoparticles to the surface of the sol-gel modified electrode has been developed for the direct detection of the human IgG in human serum. The capacitance of the immunosensor corresponding to the concentration of human IgG is investigated by alternating current impedance. The formed mercaptopropyltriethoxysilane (MPTS) film is ultrathin; the immobilization density of antibodies is high because of high surface-volume area of the assembled gold nanoparticles and the biological macromolecules when immobilized on gold nanoparticles can retain their bioactivity. This capacitive immunosensor prepared with present method can provide high sensitivity. The linear calibration curve was obtained in the range 8.3-2128 ng/ml, with a detection limit of 3.3 ng/ml when plotted versus the logarithm of the antigen concentration. After each immunoassay, the regeneration of the electrode could be performed through washing in basic solution without obvious decrease in response. No cross-reactivity was observed with other protein species. The dependence of sol-gel modified electrode stability on the pH value and ion strength was studied. The insulating properties of the different layers of the immunosensor were also investigated.     

Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification.     

金表面自组装化学发光免疫传感器

用Ｎ－乙酰半胱氨酸金表面自组装技术及ＥＤＣ、ＮＨＳ偶联剂将兔ＩｇＧ固定于金表面制成免疫传感器探头，用碱性磷酸酯酶标记山羊抗兔ＩｇＧ加入待测的含有山羊抗兔ＩｇＧ的样品中，采用竞争头测定山羊抗兔ＩｇＧ含量，标记的碱性磷酸酯酶可催化底物ＡＭＰＰＤ产生化学发光，其发光强度与样品浓度成反比。测得山羊抗兔ＩｇＧ的最低检出限为０．８ｍｇ／Ｌ，线性测定范围为０．８－４０ｍｇ／Ｌ。     

Coupling Proteins to Amine-terminated Magnetic Particles Activated with 1,4-Phenylene Diisothiocyanate

The homobifunctional crosslinker 1,4‐phenylene diisothiocyanate (PDC) was coupled to amine‐terminated magnetic particles, and human IgG, streptavidin, protein G and protein A were immobilized on the activated magnetic particles. The coupling of PDC to the amine‐terminated magnetic particles was completed in 10 min, and 1 mg of activated magnetic particles was able to immobilize 95 (g of protein G, 120 µg of protein A, 160 µg of streptavidin and 280 µg of IgG. Ultraviolet‐visible spectroscopy, FTIR spectroscopy and electron micrography were used to characterize the functional particles. The results indicated that PDC was successfully coupled to the surface of the amine‐terminated magnetic particles, and the proteins were effectively immobilized on their surface. The activity of protein G immobilized on the activated magnetic particles was confirmed by its ability to purify IgG from plasma.     

Leakage of immobilized IgG from therapeutic immunoadsorbents

In developing therapeutic immunoadsorbents (IAs), antibodies (IgG molecules) covalently immobilized on porous carriers, a leak of IgG was determined both in the storage test with buffers at 25 and 4 degrees C and in contact with plasma at room temperature (RT). The amount of antibody released from therapeutic IAs must be minimized to avoid side effects during treatment. The amount of IgG released was a. Dependent on the amount of IgG immobilized b. Much greater with CNBr-activated Sepharose 4B, or Formyl-Cellulofine as a support material than with aminopropyl CPG (controlled pore glass) c. Found to yield again during another storage in buffers after the IAs were washed and their buffers replaced with fresh ones and d. Decreased after the IAs were treated with glutaraldehyde (GA) solutions. Whereas treating the IAs with GA solutions significantly reduced the amount of IgG released, it caused some deterioration of the adsorption characteristics of the IAs. An irradiation dose of 2.5 Mrad as a crosslinking procedure also reduced the amount of IgG released; its effect was comparable to that of 0.025% GA, the lowest concentration used.     

Enhancement of the sensitivity of surface plasmon resonance biosensor with colloidal gold labeling technique

In our experiment colloidal gold was used to enhance the signal obtained from the surface-plasmonresonance biosensor. Fab′ fragments of human IgG molecules were immobilized on the gold surface and reacted with the sheep-anti-human IgG(SAH-IgG) molecules in solution. It was found that when the colloidal gold labeling technique was introduced the sensitivity of surface plasmon resonance instruments increased by a factor of about 300.