Chemical introduction of disulfide groups on glycoproteins: a direct protein anchoring scenario.
The present work reports a direct glycoprotein immobilization protocol where the protein is chemically modified with disulfide groups which act as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The specificity of the chemical reaction, for disulfide introduction, toward carbohydrate moieties prevents any cross-reaction with other functional groups present in the protein structure. Horseradish peroxidase (HRP) was chosen as a model glycoprotein, and a biologically active densely packed SAM was obtained on gold, as demonstrated by spectrophotometry and surface plasmon resonance (SPR) spectroscopy. A hydrogen peroxide amperometric biosensor was designed using a freely diffusing mediator which exhibited high sensitivity (196 mA M-1 cm-2) and low apparent Michaelis-Menten constant (67 microM). By extension, a mixed bienzymatic monolayer, obtained by simultaneous cochemisorption of modified HRP and glucose oxidase (GOD), on a clean gold electrode displayed a high sensitivity toward glucose (13 mA M-1 cm-2). Far from competing with the versatility of the classic SAM scenario or the precision of genetic engineering, this work presents a rational and particularly rapid approach where the selectivity of chemical reactions takes advantage of the specific location of carbohydrates on glycosylated protein and antibody structures for creating highly active biological interfaces directly chemisorbed onto bare gold detection devices.
Suárez
Guillaume
G
Diagnostic and Therapeutic Technologies, School of Clinical and Laboratory Sciences, The Medical School, University of Newcastle-upon-Tyne, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, United Kingdom.
Jackson
Richard J
RJ
Spoors
Julia A
JA
McNeil
Calum J
CJ
D015374:Biosensing Techniques; D002241:Carbohydrates / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005947:Glucose / Q000032:analysis; D006023:Glycoproteins / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000032:analysis
D002241:Carbohydrates; D004220:Disulfides; D004800:Enzymes, Immobilized; D006023:Glycoproteins; D006046:Gold; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D005947:Glucose