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

Abstract

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 Damk€ ohler 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/cm 2 , in the conventional protocol, to 617 ng/cm 2 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 selfassembled 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. 4,5 a) S. Asiaei, B. Smith, and P. Nieva contributed equally to this work. b)

Signal amplification of a quartz crystal microbalance immunosensor by gold nanoparticles-polyethyleneimine for hepatitis B biomarker detection

“…also utilized the EDC/NHS-activated self-assembled monolayer (SAM) (11-mercaptoundecanoic acid and 1-octane thiol) as a linkage layer for site-directed immobilization of antibodies to develop a Salmon HSP70 biosensor. At the optimal conditions, one-hour time was needed for the activation of the carboxyl groups by 5 mM EDC/NHS solution on the SAM 46 .…”

NaYF4:Yb/Ho upconversion nanoprobe incorporated gold nanoparticle (AuNP) based FRET immunosensor for the “turn-on” detection of cardiac troponin I

“…Simultaneously, the NHS acts as an additive to EDC and is employed in the crosslinking reaction on the antibody surface loading on the probe. 56 The emission spectra of the APTES@SiO 2 -NaYF 4 :Yb/Ho NPs and Ab-cTnI conjugated APTES@SiO 2 -NaYF 4 :Yb/Ho was obtained and is shown in Fig. S7 (ESI †).…”

Role of Defects of Carbon Nanomaterials in the Detection of Ovarian Cancer Cells in Label-Free Electrochemical Immunosensors

“…This step gives an 11-MUA self-assembly monolayer film with AuNPs on the GCE surface. Next, the electrode was immersed into 0.1 M EDC for 5 h at 25 • C and then 0.4 M NHS for another 5 h at 25 • C. In this reaction, the 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., Ab) to create an amide [41]. The GCE electrode were incubated with antibody (X306) overnight at 4 • C to form a covalent linkage.…”