Immobilization Density Research Articles

Preparation and Characterization of Various Membrane Morphologies for Lateral Flow Immunoassay Development

Lateral flow in nitrocellulose membrane is widely used as the transport medium in immunological test strips. The immunoassay performance depends greatly on the binding mechanism on membrane surface. If the membrane structures could be controlled precisely, various kinds of immunological analysis could be performed effectively and accurately. This study is aimed at a quantitative understanding on (1) the sensitivity of protein quantification on membrane surface by using densitometer and (2) comparison of the protein binding ability on different membrane surface pore structures. Concentration of immobilised protein on membrane surface was measured as a function of colour intensity, which is initially stained with Ponceau S. The findings showed that membrane morphologies significantly affect the sensitivity of membrane protein binding ability. Depending on the membrane pore structures, different red spot sharpness and intensities were observed from the stained protein dot. Membrane with smaller pore creates higher protein immobilization density and thus, increases the sensitivity level of the assay. However, larger membrane pore structure is necessary to induce rapid migration rate for faster assay.

Effect of pre-existing immobile dislocations on the evolution of geometrically necessary dislocations during fatigue

The role of pre-existing mobile and immobile dislocation densities on the evolution of geometrical necessary dislocation densities (GNDs) during cyclic fatigue in shear is studied using a continuum dislocation-based model incorporated in a crystal plasticity finite element scheme. Clusters with different immobile dislocation densities are implemented in a homogeneous medium containing a certain mobile dislocation density. It is found that whether GND walls are formed around the initial immobile cluster (or not) strongly depends on the absolute values of initial mobile dislocation density and on the ratio between mobile and immobile densities. The results are discussed in terms of the apparent GND densities experimentally obtained using Laue micro-diffraction.

Influence of antibody immobilization strategies on the analytical performance of a magneto-elastic immunosensor for Staphylococcus aureus detection

Magneto-elastic (ME) sensors have a great advantage in microbiology due to their ability to be queried wirelessly. Staphylococcus aureus is one of the most common bacteria widespread in the environment and a major human pathogen related to numerous illnesses. Immunosensors are affinity-based assays where the analyte is highly selective. The immobilization of antibodies (Ab) is an important step in the development of such devices. This study compared the effects of two antibody immobilization strategies on the analytical performance of a magneto-elastic immunosensor: (1) random antibody covalent immobilization (CysAb) and (2) specific-oriented antibody covalent immobilization (PrGAb). Immunosensors were exposed to solutions containing S. aureus at different concentrations (104 to 108CFU/ml) and sensor resonant frequencies were measured. In order to confirm that the frequency shifts were mainly caused by the binding of S. aureus to the sensor's surface, scanning electron microscope (SEM) and indirect immunofluorescence (IIF) images were taken after bacteria exposure at 108CFU/ml. Sensor surfaces were further monitored by non-contact topographic atomic force microscopy (AFM) images. In the covalent-oriented strategy, PrG was first bound covalently to the surface, which in turn, then binds the anti-S. aureus antibody in an oriented manner. Topographic AFM images showed different surface patterns between the two antibody immobilization strategies. Specific-oriented antibody covalent immobilization (PrGAb) strategy gave the highest anti-S. aureus antibody immobilization density. Therefore, the covalent-oriented strategy presented the best performance for S. aureus capture, detecting 104CFU/ml.

A new model for dynamic recrystallization under hot working conditions based on critical dislocation gradients

Abstract Dynamic recrystallization (DRX) occurs during hot working of metals and alloys with a sufficiently low stacking-fault energy. It is characterized by the nucleation and growth of new grains, which takes place concurrently to plastic deformation. DRX processes are used in industrial hot forming processes to control the microstructure and properties of the workpiece. Various models have been developed that consider the coupled evolution of microstructure and flow stress during hot deformation processes. It is widely accepted that the onset of DRX is characterized by an inflection point of the strain hardening rate as a function of stress. This so-called second-derivative criterion was derived by Poliak and Jonas in the 1990s using results from the thermodynamics of irreversible processes. Recent results show that models which use classical Avrami kinetics to describe the evolution of the recrystallized volume fraction violate the criterion if the Avrami exponent assumes a value of 3 or less. This inconsistency has its root in the assumptions made for the nucleation and growth of DRX nuclei and was shown to affect the accuracy of models used to design and control metal forming processes. This paper presents a new, consistent model for the nucleation of DRX. Typical nucleation models assume a constant dislocation density throughout the parent grains to derive a critical dislocation density for nucleation. However, it is clear from crystal plasticity that the dislocation density is higher at the grain boundaries than inside the grains. In order to involve dislocation gradients, coupled evolution equations for mobile and immobile dislocation densities are derived and solved under hot working conditions in this work. A new nucleation criterion is developed based upon the strain-dependent dislocation density gradient in parent grains. Once the critical gradient is reached, nucleation starts. The results of the newly developed criterion are compared with conventional models and experimental data. The comparison shows that the new criterion predicts more realistic critical dislocation densities in contrast to conventional models which over-estimate the critical dislocation density. As a consequence, more accurate predictions in the design and control of metal forming processes may be achieved with the new model.

Comparison of antibody immobilization strategies in detection of Vibrio cholerae by surface plasmon resonance.

Surface plasmon resonance immunosensor for the detection of bacterial cells was first reported in 1998 with high detection limit as much as 107 cfu/ml. Since then, many efforts have been made aiming to lower the detection limit and improve the sensitivity of detection. The aim of this study was to compare the effect of four most frequently used immobilization strategies, including direct physical adsorption (physisorption), covalent immobilization via self-assembled monolayer (SAM) formation, bioaffinity immobilization using protein G-mediated immobilization and using mixed SAM of alkane thiols on signal strength of detection of Vibrio cholerae using these modified surfaces. The most widely used strategy, covalent binding of antibodies to sensor chip via SAM formation, gave the highest immobilization density and mixed SAM of 20/80 (v/v) of 11-mercaptoundecanoic acid (11-MUA)/9-mercapto-1-nonanol resulted in the least surface coverage in antibody immobilization step. To optimize surface density in covalent immobilization, four different concentrations (12.5, 25, 50, and 100 μg/ml) of anti-OmpW were immobilized on 11-MUA modified gold chips and maximum interaction response was achieved at 25 μg/ml. The interaction response signals for detection of V. cholerae using immobilized anti-OmpW were in this order: Oriented immobilization using protein G/antibody complex > mixed SAM of 11-MUA and 9-mercapto-1-nonanol > homogenous 11-MUA SAM > direct physical adsorption. In order to evaluate interaction studies in real sample condition, waste water samples that were artificially spiked with V. cholerae were tested and the authors concluded that for real samples, it is better to setup experiment with low surface coverage such as mixed SAM to overcome nonspecific adsorption.

Development of enzyme-loaded PVA microspheres by membrane emulsification

Enzyme applications have a considerable role at the heart of biotechnological processes. A large number of these biotechnological processes require successful enzyme immobilization in terms of resistance to leaking, retention of enzyme activity over long-term storage, operational stability under adverse environmental conditions, accessibility to substrates, fast catalysis, and, in general, proper enzyme immobilization density with adequate orientation. Among the different methods of immobilization, enzyme encapsulation in a network matrix of polymeric materials in the form of particles is of particular interest. Polyvinyl alcohol (PVA) microspheres produced by a membrane emulsification/cross-linking method have been used as enzyme carriers. The enzyme was immobilized during particle formation (by entrapment) or after particle production (by adsorption and cross-linking). The entrapment during particle solidification from a water in oil (W/O) emulsion, allowed the enzyme to be distributed at the interface in its active form. The best operating conditions to control particle size, particle size distribution and particle structure for PVA microspheres production as well as the most suitable method for lipase immobilization on/in PVA microspheres have been identified. The immobilization method that does not affect enzyme intrinsic biocatalytic activity, whilst allowing at the same time confinement with balance between molecular mobility and heterogenization has been identified.

A rapid and highly sensitive immunoassay format for human lipocalin-2 using multiwalled carbon nanotubes

A sensitive and rapid sandwich immunoassay (IA) was developed for human lipocalin-2 (LCN2) by functionalizing a KOH-treated polystyrene microtiter plate with multiwalled carbon nanotubes (MWCNTs) dispersed in 3-aminoproyltriethoxysilane (APTES). The significantly increased surface area due to the presence of MWCNTs led to a high immobilization density of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) activated anti-LCN2 capture antibodies (Ab). The anti-LCN2 Ab-bound MTPs were stable for 6 weeks when stored in 0.1M PBS, pH 7.4 at 4°C. The IA detects LCN2 from 0.6 to 5120pgmL−1 with a limit of detection (LOD) and limit of quantification (LOQ) of 0.9pgmL−1 and 6pgmL−1, respectively. The assay offered a ~50-fold lower LOD and ~3-fold faster IA, compared to a commercial sandwich enzyme-linked immunosorbent assay kit.

Multi-biofunction of antimicrobial peptide-immobilized silk fibroin nanofiber membrane: Implications for wound healing

An antimicrobial peptide motif (Cys-KR12) originating from human cathelicidin peptide (LL37) was immobilized onto electrospun SF nanofiber membranes using EDC/NHS and thiol-maleimide click chemistry to confer the various bioactivities of LL37 onto the membrane for wound care purposes. Surface characterizations revealed that the immobilization density of Cys-KR12 on SF nanofibers could be precisely controlled with a high reaction yield. The Cys-KR12-immobilized SF nanofiber membrane exhibited antimicrobial activity against four pathogenic bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa) without biofilm formation on the membrane surface. It also facilitated the proliferation of keratinocytes and fibroblasts and promoted the differentiation of keratinocytes with enhanced cell-cell attachment. In addition, immobilized Cys-KR12 significantly suppressed the LPS-induced TNF-α expression of monocytes (Raw264.7) cultured on the membrane. These results suggest that a Cys-KR12-immobilized SF nanofiber membrane, which has multiple biological activities, would be a promising candidate as a wound dressing material. Statement of SignificanceThis research article reports various bioactivities of an antimicrobial peptide on electrospun silk fibroin nanofiber membrane. Recently, human cathelicidin peptide LL37 has been extensively explored as an alternative antibiotic material. It has not only a great antimicrobial activity but also a wide variety of bioactivities which can facilitate wound healing process. Especially, many studies on immobilization of LL37 or its analogues have shown the immobilization technique could improve performance of wound dressing materials or tissue culture matrices. Nevertheless, so far studies have only focused on the bactericidal effect of immobilized peptide on material surface. On the other hand, we tried to evaluate multi-biofunction of immobilized antimicrobial peptide Cys-KR12, which is the shortest peptide motif as an analogue of LL37. We fabricated silk fibroin nanofiber membrane as a model wound dressing by electrospinning and immobilized the antimicrobial peptide. As a result, we confirmed that the immobilized peptide can play multi-role in wound healing process, such as antimicrobial activity, facilitation of cell proliferation and keratinocyte differentiation, and inhibition of inflammatory cytokine expression. These findings have not been reported and can give an inspiration in wound-care application.

Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions.

Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end.

Controlled association and delivery of nanoparticles from jet-sprayed hybrid microfibrillar matrices

To develop bioactive scaffolds of targeted properties for tissue repair or biomedical applications, hybrid microfiber-nanoparticle (MF-NP) matrices capable of controlled nanoparticle (NP) delivery were prepared through two novel approaches. In a first strategy, the suppleness of the jet-spraying method to produce polymer microfibers (MF) was used to deposit poly(d,l-lactide) (PLA) NP on poly(lactic-co-glycolic acid) (PLGA) MF by direct co-projection. The second approach relied on the post-incubation of PLA NP aqueous dispersion with MF preliminarily prepared by jet-spraying. NP coverage density onto MF and NP release was assessed by scanning electron microscopy and fluorescence measurements using coumarin-6 loaded NP. The first process was shown to allow high coverage density of NP onto MF (300μg/mg MF) and strong association, with no NP release observed over time. In the second approach, direct incubation of PLA NP with PLA MF led to lower NP coverage density (40μg/mg MF) with very fast release of NP from MF. The pre-coating of MF with poly-l-lysine (PLL) or the one of NP with lysozyme as a model protein drug afforded a higher coverage density and stronger association, coupled with a more sustained release of NP from MF over time. These results show the possibility to control the immobilization density and release of NP through appropriate preparation process and surface modification, and are of prime interest for the development of complex scaffolds with orchestrated bioactivity.

Analytic model of the remobilization of pinned glide dislocations from quasi-static to high strain rates

In this paper, we construct an analytic model describing mobile-immobile dislocation intersections using a mean-first-passage-time (MFPT) framework. By applying MFPT theory to dislocation intersection, the deformation mechanics at high strain rates is more reliably described than in traditional models based on Van't Hoff-Arrhenius thermal activation. The plastic strain rate is expressed as a function of the applied stress, mobile and immobile dislocation densities, material density, and temperature. This kinetic equation is applicable at strain rates from quasi-static to rates in excess of 1012 s−1, pressures from ambient to about 1000 GPa, and temperatures ranging from zero to the melt temperature.

A Sensitive DNA Biosensor Using Graphene Oxide as the Multi-Site Platform for Probe DNA Immobilization

An ultrasensitive DNA biosensor was constructed in this work by using graphene oxide nanosheets (GONs) as the multi-site platform for probe DNA immobilization. First, L-cysteine (L-cys) was self-assembled on a gold electrode surface based on the Au-S chemistry. Then the GONs and amino modified probe DNA was covalently grafted to L-cys/AuE surface in turn, with the aid of 1-(3-Dimethylanminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxy succinimide (NHS). The layer upon layer assembly process was characterized with atomic force microscopy and electrochemical methods. Due to the nano-size effect and multiple site characteristic of GONs, the immobilization density of the probe DNA was determined to be as high as 5.72 × 10−10 mol cm−2 using methylene blue as the redox probe. The hybridization experiments showed that the biosensor can quantitatively detect target DNA in a wide range from 1.0 × 10−15 M to 1.0 × 10−9 M with a detection limit of 5.0 × 10−16 M. The selectivity experiments showed that the sensing system could accurately discriminate the complementary sequence from the base mismatched and non-complementary sequences.

SPRi determination of inter-peptide interaction by using 3D supramolecular co-assembly polyrotaxane film

Accurate measurement of inter-peptide interactions is beneficial for in-depth understanding disease-related protein folding and peptide aggregation, and further for designing and selecting potential peptide drugs to the target antigen. Herein, we demonstrate a 3D polyrotaxane (PRX) surface for detecting peptides interactions by surface plasmon resonance imaging (SPRi). This surface is supramolecular self-assembly monolayer (SAM) structure fabricated by threading α-cyclodextrans (α-CD) through a linear polyethylene glycol (PEG) chain fixed on gold chip surface to form pseudopolyrotaxane, and further capping the pseudopolyrotaxane with bulky terminated group to form PRX film. The hydroxyl groups of α-CD can provide more active sites to increase molecules immobilization density, and PEG chain has unique protein non-fouling feature. We chose Alzheimer's disease marker β-amyloid 40 (Aβ40) as model peptide, and detected the interaction between it and its inhibitors KLVFFK6 by SPRi. As a striking result, the specific adsorption of KLVFFK6 solution at the concentration of 352μM on Aβ40-PRX was 700RU, whereas PEG SAM surface gave no significant binding. Interaction between other lower molecular weight peptides was detected via PRX surface, and the relatively weak interactions (KD=1.73×10−4M) between LPFFD (Mw=0.6kDa) and amylin20-29 (Mw=1.0kDa) are successfully detected.

Constitutive Relations for Modeling Single Crystal GaN at Elevated Temperatures

Thermal–mechanical constitutive relations for bulk, single-crystal, wurtzite gallium nitride (GaN) at elevated temperatures, suitable for modeling crystal growth processes, are presented. A crystal plasticity model that considers slip and the evolution of mobile and immobile dislocation densities on the prismatic and basal slip systems is developed. The experimental stress–strain data from Yonenaga and Motoki (2001, “Yield Strength and Dislocation Mobility in Plastically Deformed Bulk Single-Crystal GaN,” J. Appl. Phys., 90(12), pp. 6539–6541) for GaN is analyzed in detail and used to define model parameters for prismatic slip. The sensitivity to the model parameters is discussed and ranges for parameters are given. Estimates for basal slip are also provided.

Dislocation dynamical approach to force fluctuations in nanoindentation experiments

We develop an approach that combines the power of nonlinear dynamics with the evolution equations for the mobile and immobile dislocation densities and force to explain force fluctuations in nanoindentation experiments. The model includes nucleation, multiplication, and propagation thresholds for mobile dislocations, and other well known dislocation transformation mechanisms. The model predicts all the generic features of nanoindentation such as the Hertzian elastic branch followed by several force drops of decreasing magnitudes, and residual plasticity after unloading. The stress corresponding to the elastic force maximum is close to the yield stress of an ideal solid. The predicted values for all the quantities are close to those reported by experiments. Our model allows us to address the indentation-size effect including the ambiguity in defining the hardness in the force drop dominated regime. At large indentation depths, the hardness remains nearly constant with a marginal decreasing trend.

Immobilization of heparin onto the novel cellulose:chitosan vascular grafts: A preliminary study

Purpose: The aim of this study was to prepare a compliant cellulose:chitosan hollow tube (with 6 mm in internal diameter), to be used as a coronary bypass graft, and to investigate its ability to covalently immobilize heparin onto its surface. Method: Cellulose and chitosan solutions were mixed in equal proportions, spread in an appropriate mold and allowed to solidify for 24 hours, where a hollow tube was obtained. Heparin was covalently immobilized onto these hollow tubes, where the results were compared with that of a plain cellulose one. In addition, a correlation between the heparin concentration and the immobilization efficiency was investigated. Results: The presence of chitosan was essential for the heparin immobilization onto the cellulose:chitosan hollow tubes as no heparin was immobilized onto the plain cellulose ones. The immobilization density showed to be linearly dependent upon the concentration of the activated heparin, where a maximum concentration of 4 mg/mL was reached. Higher concentrations of activated heparin did not provide a greater immobilization density, which indicates that the amine groups of chitosan have been saturated. Conclusion: The cellulose:chitosan hollow tubes were successfully prepared, where the presence of chitosan was required for an efficient immobilization of heparin. The obtained heparin-immobilized cellulose:chitosan hollow tube, with its mechanically strong, reasonably compliant and antithrombogenic properties seems to be a good candidate to be used as a vascular bypass graft.

Solid-phase covalent immobilization of upconverting nanoparticles for biosensing by luminescence resonance energy transfer.

Monodisperse water-soluble upconverting nanoparticles (UCNPs) were immobilized onto modified glass substrates for development of biosensing surfaces that operated using luminescence resonance energy transfer (LRET). Amine modified UCNPs were prepared from oleic acid capped UCNPs by ligand exchange using o-phosphorylethanolamine (PEA). PEA-UCNPs were covalently immobilized on aldehyde functionalized coverslips. Environmental scanning electron microscopy (ESEM) images indicated a homogeneous distribution of UCNPs on surfaces with a high immobilization density of approximately 1.3 × 10(11) UCNP cm(-2). This is the first account of covalent immobilization of UCNPs for bioassay and biosensor development where the density is on par with the high immobilization densities reported for other types of nanoparticles. The functionality and stability of the immobilized NPs were demonstrated by examining an LRET-based bioassay. The well-known sandwich assay for the detection of thrombin was selected as a model in which UCNPs were used as donors and quantum dots (QDs) as acceptors. The closely packed UCNPs on the glass surface showed a 2.5-fold enhancement in assay sensitivity compared to less-densely packed surfaces. In addition, a 1.5-fold enhancement in energy transfer efficiency was shown for solid-phase compared to solution-phase LRET.

Direct Covalent Attachment of DNA Microarrays by Rapid Thiol–Ene “Click” Chemistry

A rapid strategy for the covalent immobilization of DNA onto silicon-based materials using the UV-initiated radical thiol-ene reaction is presented in this study. Following this approach, thiol- and alkene-modified oligonucleotide probes were covalently attached in microarray format, reaching immobilization densities around 6 pmol·cm(-2). The developed methodology presents the advantages of spatially controlled probe anchoring (using a photomask), direct attachment without using cross-linkers (one-pot fashion), and short irradiation times (20 min). Using the described strategy, hybridization efficiencies up to 65% with full complementary strands were reached. The approach was evaluated by scoring single-base pair mismatches with discrimination ratios around 15. Moreover, the efficacy of the proposed DNA detection scheme is further demonstrated through the assay on a genomic target of bacterial Escherichia coli.

An equipment-free polydimethylsiloxane microfluidic spotter for fabrication of microarrays.

This paper presents a low-cost, power-free, and easy-to-use spotter system for fabrication of microarrays. The spotter system uses embedded dispensing microchannels combined with a polydimethylsiloxane (PDMS) membrane containing regular arrays of well-defined thru-holes to produce precise, uniform DNA or protein microarrays for disease diagnosis or drug screening. Powered by pre-evacuation of its PDMS substrate, the spotter system does not require any additional components or external equipment for its operation, which can potentially allow low-cost, high-quality microarray fabrication by minimally trained individuals. Polyvinylpyrrolidone was used to modify the PDMS surface to prevent protein adsorption by the microchannels. Experimental results indicate that the PDMS spotter shows excellent printing performance for immobilizing proteins. The measured coefficient of variation (CV) of the diameter of 48 spots was 2.63% and that of the intensity within one array was 2.87%. Concentration gradient experiments revealed the superiority of the immobilization density of the PDMS spotter over the conventional pin-printing method. Overall, this low-cost, power-free, and easy-to-use spotting system provides an attractive new method to fabricate microarrays.

Liquid Crystal-Based Proton Sensitive Glucose Biosensor

A transmission electron microscopy (TEM) grid filled with 4-cyno-4-pentylbiphenyl (5CB) on the octadecyltrichloro silane-coated glass in an aqueous medium was developed to construct a glucose biosensor by coating poly(acrylicacid-b-4-cynobiphenyl-4-oxyundecylacrylate) (PAA-b-LCP) at the aqueous/5CB interface and immobilizing glucose oxidase (GOx) covalently to the PAA chains. The glucose was detected from a homeotropic to planar orientational transition of 5CB by polarized optical microscopy under crossed polarizers. The maximum immobilization density of the GOx, 1.3 molecules/nm(2) obtained in this TEM grid cell enabled the detection of glucose at concentrations as low as 0.02 mM with a response time of 10 s. This liquid crystal-based glucose sensor provided a linear response of birefringence of the 5CB to glucose concentrations ranging from 0.05 to 2 mM with a Michaelis-Menten constant (Km) of 0.32 mM. This new and sensitive glucose biosensor has the merits of low production cost and easy detection through the naked eye and might be useful for prescreening the glucose level in the human body.