Enhancing conjugation rate of antibodies to carboxylates: Numerical modeling of conjugation kinetics in microfluidic channels and characterization of chemical over-exposure in conventional protocols by quartz crystal microbalance     

Sasan Asiaei  Brendan Smith  Patricia Nieva 《Biomicrofluidics》2015,9(6)

This research reports an improved conjugation process for immobilization of antibodies on carboxyl ended self-assembled monolayers (SAMs). The kinetics of antibody/SAM binding in microfluidic heterogeneous immunoassays has been studied through numerical simulation and experiments. Through numerical simulations, the mass transport of reacting species, namely, antibodies and crosslinking reagent, is related to the available surface concentration of carboxyl ended SAMs in a microchannel. In the bulk flow, the mass transport equation (diffusion and convection) is coupled to the surface reaction between the antibodies and SAM. The model developed is employed to study the effect of the flow rate, conjugating reagents concentration, and height of the microchannel. Dimensionless groups, such as the Damköhler number, are used to compare the reaction and fluidic phenomena present and justify the kinetic trends observed. Based on the model predictions, the conventional conjugation protocol is modified to increase the yield of conjugation reaction. A quartz crystal microbalance device is implemented to examine the resulting surface density of antibodies. As a result, an increase in surface density from 321 ng/cm², in the conventional protocol, to 617 ng/cm² in the modified protocol is observed, which is quite promising for (bio-) sensing applications.Microfluidics have been implemented in various bio-medical diagnostic applications, such as immunosensors and molecular diagnostic devices.¹ In the last decade, a vast number of biochemical species has been detected by microfluidic-based immunosensors. Immunosensors are sensitive transducers which translate the antibody-antigen reaction to physical signals. The detection in an immunosensor is performed through immobilization of an antibody that is specific to the analyte of interest.² The antibody is often bound to the transducing surface of the sensor covered by self-assembled monolayers (SAMs). SAMs are organic materials that form a thin, packed and robust interface on the surface of noble metals like that of gold, suitable for biosensing applications.³ Thiolic SAMs have a “head” group that shows a high affinity to being chemisorbed onto a substrate, typically gold. The SAMs'' carboxylic functional group of the “tail” end can be linked to an amine terminal of an antibody to form a SAM/antibody conjugation.^3,4 The conjugation process is usually accomplished in the presence of carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). A yield increasing additive, N-Hydroxysuccinimide (NHS), is often used to enhance the surface loading density of the antibody.^4,5A typical reaction for coupling the carboxylic acid groups of SAMs with the amine residue of antibodies in the presence of EDC/NHS is depicted in Figure ​Figure11.⁴ NHS promotes the generation of an active NHS ester (k₂ reaction path). The NHS ester is capable of efficient acylation of amines, including antibodies (k₃ reaction path). As a result, the amide bond formation reaction, which typically does not progress efficiently, can be enhanced using NHS as a catalyst.⁴Open in a separate windowFIG. 1.NHS catalyzed conjugation of antibodies to carboxylic-acid ended SAMs through EDC mediation (Adapted from G. T. Hermanson, Bioconjugate Techniques, 2nd. Edition. Copyright 2008 by Elsevier⁴). EDC reacts with the carboxylic acid and forms o-acylisourea, a highly reactive chemical that reacts with NHS and forms an NHS ester, which quickly reacts with an amine (i.e., antibody) to form an amide.A number of groups have studied EDC/NHS mediated conjugation reactions such as the ones depicted in Figure ​Figure1.1. The general stoichiometry of the reaction involves a carboxylic acid (SAM), an amine (antibody), and EDC to produce the final amide (antibody conjugated SAM) and urea. However, the recommended concentration ratio of the crosslinking reagents inside the buffer, i.e., the ratio of EDC and NHS with respect to adsorbates and each other, varies from one study to another.⁶ The kinetics of the reactions outlined in Figure ​Figure11 have also been investigated,^4,6–8 but only in the absence of NHS for EDC or carboxylic acids in aqueous solutions.⁸ A relatively recent experimental study verified the catalytic role of the yield-increasing reagent N-hydroxybenzotriazole (HOBt), which acts similarly to NHS.⁷ In this study, the amide formation rate (k₃ reaction path, Figure ​Figure1)1) was found to be dependent on the concentration of the carboxylic acid and EDC in the buffer solution, and independent of the amine and catalyst reagent concentration. The same group also showed that the amide bond formation kinetics is controlled by the reaction between the carboxylic acid and the EDC to give the O-acylisourea, which they marked as the rate-determining step (k₁ reaction path, Figure ​Figure11).The k₁ reaction path, or the conjugation reaction, is usually a lengthy process and takes between 1 and 3 h.^4,9 Compared to k₁, the k₂ and ?k₃ reactions are considerably faster. Microfluidics has the potential to enhance the kinetics of these reactions using the flow-through mode.^10,11 This improvement occurs because while conventional methods rely only on diffusion as the primary reagent transport mode, microfluidics adds convection to better replenish the reagents to the reaction surfaces. However, there are many fundamental fluidic and geometrical parameters that might affect the process time and reagents consumption in a microfluidics environment, such as concentration of antibodies and reagents, flow rate, channel height, and final surface density of antibodies. A model that studies the kinetics of conjugation reaction against all these parameters would therefore be helpful for the optimization of this enhanced kinetics.There are a number of reports on numerical examination of the kinetics of binding reactions in microfluidic immunoassays.^12–15 All these models developed so far couple the transport of reagents, by diffusion and convection, to the binding on the reaction surface. Myszka''s model assumes a spatially homogeneous constant concentration of reagents throughout the reaction chamber, thus fails to describe highly transport-limited conditions due to the presence of spatial heterogeneity and depletion of the bulk fluid from reagents.^16,17 In transport-limited conditions, the strength of reaction is superior to the rate of transport of reagents to the reaction surface.^18,19 More recently, the convection effects were included in a number of studies, describing the whole kinetic spectrum from reaction-limited conditions to transport-limited reactions.^20–22 Immunoreaction kinetics has also been examined with a variety of fluid driving forces, from capillary-driven flows,²⁰ to electrokinetic flows in micro-reaction patches,²¹ pressure-driven flows in a variety of geometric designs.²² Despite these comprehensive numerical investigations, the fundamental interrelations between the constitutive kinetic parameters, such as concentration, flow velocity, microchannel height, and antibody loading density, have not been studied in detail. In addition, the conjugation kinetics has not yet been exclusively examined.In this paper, a previous model for immunoreaction is modified to study the antibody/SAM conjugation reaction in a microfluidic system. Model findings are used to examine the process times recommended in the literature and possible modification scenarios are proposed. The new model connects the convective and diffusive transport of reagents in the bulk fluid to their surface reaction. The conjugation reaction is studied against fluidic and geometrical parameters such as flow rate, concentration, microchannel height and surface density of antibodies. Damköhler number is used to compare the reaction and fluidic phenomena present and justify the kinetic trends observed. Model predictions are discussed and the main finding on possible overexposure of carboxylates to crosslinking reagents, in conventional protocols, is verified by comparing the resultant antibody loading densities obtained using a quartz crystal microbalance (QCM) set up. The results demonstrate an improved receptor (antibody) loading density which is quite promising for a number of (bio-) sensing applications.^23,24 Major application areas include antibody-based sensors for on-site, rapid, and sensitive analysis of pathogens such as Bacillus anthracis,²³ Escherichia coli, and Listeria monocytogenes, and toxins such as fungal pathogens, viruses, mycotoxins, marine toxins, and parasites.²⁴  相似文献   

EFL teachers’ conceptions of speaking competence in English     

Sasan Baleghizadeh  Mohammad Naseh Nasrollahi Shahri 《Teachers and Teaching》2013,19(6):738-754

The present article lies at the intersection of research on teacher cognition and speaking competence in a second language. It is a qualitative analysis of teacher accounts of speaking in the context of English as a foreign language (EFL) in Iran. More specifically, the study is an exploration of three EFL teachers’ conceptions of learning and teaching the speaking skill in English. The teachers represented varying levels of experience and education and were classified as experts and novices. Through juxtaposing teachers’ views of how students should learn to speak and how the teachers themselves learned to speak English, the research study uncovers the interwoven nature of learning experience with teaching conceptions. It also unveils the images underpinning their conceptions of how to learn and teach speaking in an EFL context. The results also portray teachers as having unique ways of thinking of speaking while at the same time sharing certain patterns.  相似文献