Use of antibody-hapten complexes attached to optical sensor surfaces as a substrate for proteases: real-time biosensing of protease activity.
Fluorescent antibody protein (IgG) was attached to the surface of an integrated optical glass waveguide chip via specific binding to a covalently attached hapten and used as a substrate for the measurement of protease activities. Exposure of the optical chip to proteases resulted in digestion of the bound fluorescent antibody molecules and proportional decrease in the detectable fluorescence resulting from loss of fluorescence from the evanescent field. The bound fluorescent antibody protein was used as a unique universal protease substrate in which the combined biological activity and fluorescence signal were the basis of measurement. The action of proteases was monitored in real-time mode where the gradual decrease in evanescent fluorescence was recorded. The chip was regenerated by complete digestion of the antibody substrate by excess pepsin and recharged by incubation with a fresh sample of the labelled antibody. The biosensor was used to detect activity of several proteases including a bacterial protease preparation, Pronase E. The linear range of measurable Pronase E activity was from 0.03 to 2 units/mL. A measurement cycle took 40 min for samples with high protease concentration (>or=0.5 units/mL), when the concentration of the protease was less measurement times up to 100 min were required. The method demonstrates the principle of a new mode of real-time biosensing of proteases. The modular integrated optical glass waveguide biosensor system used in this study is compact and controlled by a laptop computer and could easily be miniaturised and utilized as a true probe device for detecting proteases with potential applications in a wide range of areas including research, clinical diagnostics, biotechnology processing and food and detergent manufacturing industries.
Wildeboer
Dirk
D
King's College London, Pharmaceutical Science Division, Analytical Sciences Research Group, School of Biomedical & Health Sciences, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.
Jiang
Pisu
P
Price
Robert G
RG
Yu
Siyuan
S
Jeganathan
Fiona
F
Abuknesha
Ramadan A
RA
D000906:Antibodies / Q000276:immunology*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D006241:Haptens / Q000276:immunology*; D055620:Optical Phenomena; D010447:Peptide Hydrolases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013305:Streptomyces griseus / Q000201:enzymology; D013499:Surface Properties; D013997:Time Factors
C519608:6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein; D000906:Antibodies; D005452:Fluoresceins; D006241:Haptens; D010447:Peptide Hydrolases