Attomolar sensitivity in bioassays based on surface plasmon fluorescence spectroscopy

Area of Science:

• Biophysics
• Analytical Chemistry
• Biochemistry

Background:

• Metal-induced fluorescence loss and distance dependence of fluorescence yield complicate ultratrace detection.
• Surface plasmon fluorescence spectroscopy (SPFS) requires optimized conditions for sensitive detection.
• Dextran matrices are utilized in biosensing for immobilization and signal enhancement.

Purpose of the Study:

• To reduce metal-induced fluorescence loss and distance dependence in SPFS using a dextran matrix.
• To optimize the limit of detection (LOD) for SPFS.
• To demonstrate ultratrace detection capabilities of a direct immunoassay.

Main Methods:

• Utilized a carboxymethyl dextran matrix on a CM5 sensor chip for covalent immobilization of mouse IgG.
• Employed Alexa-Fluor 647 as the fluorescent label for the secondary antibody.
• Performed time-resolved ultratrace detection using surface plasmon fluorescence spectroscopy.

Main Results:

• Significantly reduced metal-induced fluorescence loss and convoluted distance dependence of fluorescence yield.
• Achieved a limit of detection of 500 aM (10^-18 M) for rabbit anti-mouse antibody.
• Demonstrated a binding rate of approximately 10 molecules mm^-2 min^-1.

Conclusions:

• The dextran matrix effectively mitigates fluorescence quenching in SPFS.
• The developed model system enables highly sensitive ultratrace detection of antibodies.
• This approach enhances the LOD assessment for SPFS, applicable to direct immunoassay development.