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   

#MeToo,sexual harassment: an article,a forum,and a dream for the future     

Robin Patric Clair  Nadia E. Brown  Debbie S. Dougherty  Hannah K. Delemeester  Patricia Geist-Martin  William I. Gorden 《Journal of Applied Communication Research》2019,47(2):111-129

#MeToo has breathed new life into the women’s movement and especially into understanding and rectifying sexual harassment, abuse and assault. It has galvanized activists around the globe. And it has placed thousands of stories of the harassed in full view of the public. Sexual harassment, abuse and assault may occur within the organizational context or beyond; but sexual harassment, in particular has been legally labeled an organizational phenomenon. With this in mind, Robin Clair frames the early part of this article around the most recent organizational communication theories (see the appendix for an overview of these theories). Following the essay is a forum, in which invited scholars address questions related to the #MeToo movement.  相似文献   

What Does Learning Prepare for Today? Co-creating Knowledge for Action     

Patricia Wastiau 《European Journal of Education》2015,50(4):394-399

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To perceive and to be perceived: challenges of ethnography in elite schools     

Patricia Mercader  Jean-Pierre Durif-Varembont 《International journal of qualitative studies in education》2015,28(9):1097-1111

Drawing on a larger study on “Gender practices and violence between peers: the stakes of mixed-sex education”, this paper examines the methodological challenges specific to carrying out ethnography in an elite high school. The researcher’s subjective experience in the field reveals the power dynamics at play in the elite setting. We also examine how interdisciplinary collective analysis of the observation journals unveils less-examined challenges to conceptualization in the research process. We call for the heuristic value when contradiction and subjectivity are acknowledged and implicated both in the field and data analysis, which leads us to articulate different social identities in the elite school environment.  相似文献   

Why has there been a decrease in the job satisfaction of faculty at Spanish universities?     

Olaskoaga-Larrauri  Jon  González-Laskibar  Xabier  Díaz-De-Basurto-Uraga  Pablo  Ignacio-Gómez  Patricia 《Tertiary Education and Management》2015,21(4):293-305

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The impact of the first COVID-19 lockdown in the UK for doctoral and early career researchers     

Jackman  Patricia C.  Sanderson  Rebecca  Haughey  Tandy J.  Brett  Caroline E.  White  Naomi  Zile  Amy  Tyrrell  Katie  Byrom  Nicola C. 《Higher Education》2022,84(4):705-722

Higher Education - Doctoral researchers and early career researchers (ECRs) are crucial to producing scientific advancements and represent the future of academic leadership. Their research...  相似文献   

Environmental Education and Complexity     

Pereira  Brenda Braga  Silva  Luciano Fernandes  dos Santos  Janaina Roberta 《Science & Education》2022,31(1):1-20

Science & Education - The purpose of this study is to identify and analyze meanings that the term complexity assumes in Brazilian theses and dissertations in the field of environmental...  相似文献   

Simulating peers: Can puppets simulate peer interactions in studies on children's socio-cognitive development?     

Roman Stengelin  Daniel B. M. Haun  Patricia Kanngiesser 《Child development》2023,94(5):1117-1135

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Children's hypothetical reasoning about complex and dynamic systems     

Angela Nyhout  Hilary Sweatman  Patricia A. Ganea 《Child development》2023,94(5):1340-1355

Children's hypothetical reasoning about a complex and dynamic causal system was investigated. Predominantly White, middle-class 5- to 7-year-old children from the Greater Toronto Area learned about novel food chains and were asked to consider the effects of removing one species on the others. In Study 1 (N = 72; 36 females, 36 males; 2018), 7-year-olds answered questions about both direct and indirect effects with a high degree of accuracy, whereas 5-year-olds performed at chance. Six-year-olds showed intermediate performance. Using food chains with clearer constraints, Study 2 (N = 72; 35 females, 37 males; 2020–2021) replicated these findings. These results indicate that the ability to think about hypothetical changes to dynamic causal systems develops between 5 and 7 years. Implications for science education are discussed.  相似文献   

Instructional practices in secondary science: How teachers achieve local and standards-based success     

Beth A. Covitt  Elizabeth Xeng de los Santos  Qinyun Lin  Christie Morrison Thomas  Charles W. Anderson 《科学教学研究杂志》2024,61(1):170-202

This article reports on analyses of the instructional practices of six middle- and high-school science teachers in the United States who participated in a research-practice partnership that aims to support reform science education goals at scale. All six teachers were well qualified, experienced, and locally successful—respected by students, parents, colleagues, and administrators—but they differed in their success in supporting students' three-dimensional learning. Our goal is to understand how the teachers' instructional practices contributed to their similarities in achieving local success and to differences in enabling students' learning, and to consider the implications of these findings for research-practice partnerships. Data sources included classroom videos supplemented by interviews with teachers and focus students and examples of student work. We also compared students' learning gains by teacher using pre–post assessments that elicited three-dimensional performances. Analyses of classroom videos showed how all six teachers achieved local success—they led effectively managed classrooms, covered the curriculum by teaching almost all unit activities, and assessed students' work in fair and efficient ways. There were important differences, however, in how teachers engaged students in science practices. Teachers in classrooms where students achieved lower learning gains followed a pattern of practice we describe as activity-based teaching, in which students completed investigations and hands-on activities with few opportunities for sensemaking discussions or three-dimensional science performances. Teachers whose students achieved higher learning gains combined the social stability characteristic of local classroom success with more demanding instructional practices associated with scientific sensemaking and cognitive apprenticeship. We conclude with a discussion of implications for research-practice partnerships, highlighting how partnerships need to support all teachers in achieving both local and standards-based success.  相似文献