The Characterisation and Quantification of Immobilised Concanavalin A on Quartz Surfaces Based on The Competitive Binding to Glucose and Fluorescent Labelled Dextran

Abstract

The competition between various carbohydrates in the binding to Concanavalin A (Con A) can be exploited in gravimetric microsensors that detect changes in mass or viscoelasticity as a function of glucose concentration. Such sensors are based on the immobilisation of Con A as the ligand specific element, and a successful application requires that the binding property of Con A is retained. This paper presents a simplified immobilisation procedure of Con A on a quartz surface, a common material for gravimetric microsensors. Structural assessment with atomic force microscopy confirmed that the surface was covered with a layer of macromolecules. This layer shows the presence of entities of various sizes, presumably monomers, dimers and tetramers among which dimers of the Con A are the most dominant structure. Functional assessment using fluorescent labelled dextran (FITC and Alexa 488) suggests a surface coverage ranging from 1.8 × 1011 to 2.1 × 1012 immobilised fluorescent molecules per cm2. The assay was responsive to glucose over a concentration range from 0–40 mM, but became gradually saturated above 20 mM. Hence, the immobilised Con A is able to bind dextran, which is displaced by glucose in a concentration dependent manner, thus triggering a mass change proportional to the MW of dextran.

Ultrasensitive and Label-Free Detection of Cell Surface Glycan Using Nanochannel-Ionchannel Hybrid Coupled with Electrochemical Detector

“…After introduction of Con A, the aldehyde absorption peaks disappear (SI Figure S2). Due to the active group of Con A is amine group, 45 the disappearance of aldehyde absorption peaks indicates amine groups in Con A interacted with aldehyde in GA, and Con A has been favorably immobilized on PAA. Meanwhile, confocal Raman microscopy also shows the typical vibrations of different groups in Con A attached to PAA surface, including phenylalanine ring breathing mode (1003 cm −1 ) and various backbone amide bands (1233 cm −1 , 1556 cm −1 , 1670 cm −1 ) (SI Figure S3).…”

“…Before doing this, the detection time was optimized. As seen in Figure 5A, when cell solution is incubated with PAA for different time (5,10,20,30,45, 60, 90 min) at 37 °C, the I−V properties change continuously and then reach stable at ∼45 min. The captured cells could efficiently cover the outer surface of PAA, and impede the ionic flow through PAA, which results in a decreased current response.…”

Plasma Treatment of PDMS for Microcontact Printing (μCP) of Lectins Decreases Silicone Transfer and Increases the Adhesion of Bladder Cancer Cells

“…In this case, there is less chance that random molecular orientation of the lectin molecule on the substrate will result in a reduced number of functional binding sites. In addition, Hoang et al demonstrated by binding capacity of dextran to Con A covalently attached to a quartz surface that the density of active immobilized Con A was about 1/3 of the theoretical maximum surface coverage estimate. Previous studies suggest that adsorption to hydrophobic surfaces does not drastically reduce lectin sugar binding capacity, and that 15 min lectin deposition time is sufficient to obtain homogeneously covered substrates. , Both approaches result in the formation of lectin layers of different organization.…”

Special Issue on Nano-Biointerface for Biosensing

“…In the article on The Characterisation and Quantification of Immobilised Concanavalin A on Quartz Surfaces Based on The Competitive Binding to Glucose and Fluorescent Labelled Dextran [13] authored by T. Bich Hoang and coworkers, a simple protocol for the immobilization of Concanavalin A (Con A) on a quartz substrate, a common material for gravimetric microsensors, was developed. The binding activity of the immobilized Con A was assessed by measuring the intensity from fluorescent labelled dextran (FITC and Alexa 488; with a surface coverage ranging from 1.8 × 10 11 to 2.1 × 10 12 immobilized fluorescent molecules per cm 2 ) over a glucose concentration range from 0 to 40 mM, whereas any structural changes to the surface topography were investigated using AFM.…”