Label-Free Impedimetric Detection of Glycan−Lectin Interactions

Abstract

A compact biosensor for a label-free, rapid (<80 s) detection of glycan-lectin interactions using ac impedance measurements was developed for the first time. A galactose-binding peanut agglutinin (PNA) and sialic acid-binding Sambucus nigra agglutinin (SNA) were covalently surface-immobilized on the layered Cu/Ni/Au printed circuit board (PCB) electrodes. Samples of artificial and natural glycoconjugates consisting of (1) gold glyconanoparticles encapsulated with approximately 90-100 copies of TF-antigen disaccharide Galalpha1-3GalNAc (TF-AuNP), (2) asialofetuin (ASF) containing both LacNAc (Galbeta1-4GlcNAc) and TF-antigen, and (3) fetuin (FET), the sialylated glycoform of ASF. The samples were run separately on PNA- and SNA-immobilized PCB electrodes. Our results indicate that TF-AuNP could be rapidly and reliably detected up to 1 pg/mL (13 fM) concentration on PNA electrode but, as expected, yielded no response on the SNA electrode. ASF and FET glycocoproteins were unambiguously detectable up to 10 pg/mL (150 fM) on PNA and SNA electrodes, respectively. Moreover, the technique allowed us to observe glyco-microheterogeneity of FET as well as establish the presence of two isoforms of SNA lectin, SNA-I and SNA-II, in one of the vendor's formulations. Further elaboration of the described technology into novel electrochemically driven lectin arrays may find applications in diagnosis of cancer and other diseases with multiple glycobiomarkers or as a rapid low-cost bioanalytical tool for glycoproteome analyses.

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells

“…Human Embryonic Stem Cell Culture-The National Institutes of Health-approved hESC lines, H1 and HSF-6, were maintained under feeder-free conditions using a chemically defined medium, X-VIVO 10 (Cambrex, Walkersville, MD), supplemented with human recombinant growth factors fibroblast growth factor and transforming growth factor-␤1 (80 and 0.5 ng/ml, respectively; R & D Systems (10). The cells are propagated on hESC-qualified Matrigel-coated plates (BD Biosciences).…”

Electrochemical detection of glycan and protein epitopes of glycoproteins in serum

“…[25][26][27]30 In contrast, a linear increase from baseline impedance was observed when the SNA lectin biosensor was incubated with an increasing concentration of fetuin expressing a correct glycan epitope. [25][26][27]30 Apart from showing very high binding specicity towards the glycan epitope, these SNA lectin biosensors have demonstrated sensitivity for glycoprotein detection (e.g. fetuin) up to attomolar concentration.…”

“…29 In fact, glycan structure specic interactions of a glycoprotein with lectins are very well demonstrated in the recent past mainly using sialic acid binding lectin biosensors. [25][26][27]30 Incubation of the SNA lectin biosensor with asialofetuin, a non-sialic acid expressing glycoform variant of glycoprotein fetuin, showed very minimal change in the baseline impedance. [25][26][27]30 In contrast, a linear increase from baseline impedance was observed when the SNA lectin biosensor was incubated with an increasing concentration of fetuin expressing a correct glycan epitope.…”

“…[25][26][27]30 Incubation of the SNA lectin biosensor with asialofetuin, a non-sialic acid expressing glycoform variant of glycoprotein fetuin, showed very minimal change in the baseline impedance. [25][26][27]30 In contrast, a linear increase from baseline impedance was observed when the SNA lectin biosensor was incubated with an increasing concentration of fetuin expressing a correct glycan epitope. [25][26][27]30 Apart from showing very high binding specicity towards the glycan epitope, these SNA lectin biosensors have demonstrated sensitivity for glycoprotein detection (e.g.…”

Trends and Perspectives in Immunosensors

“…Additionally, electrode geometry has proven important with interdigitated electrode arrays providing improved sensitivity [39,48]. Impedance-based sensors have been used to monitor protein-carbohydrate [49] and proteinprotein [50,51] interactions. For example, Yu et al [52] reported multiplexed protein monitoring with an array of gold electrodes.…”