BIOCATALYTIC SIGNAL AMPLIFICATION METHOD
FOR HIGHLY SENSITIVE SURFACE PLASMON RESONANCE
IMMUNOASSAYS
Júlia SZŰCS
Department of Inorganic and Analytical Chemistry
Budapest University of Technology and Economics
1111-Budapest, Hungary
E-mail address: szucsj@mail.bme.hu
Supervisors: Klára Tóth
Róbert E. Gyurcsányi
Surface plasmon resonance (SPR) is an optical method for characterizing
(bio)macromolecular interactions. Since the biological functions of (macro)molecules
depend on their ability to interact with other molecules in the past ten years the SPR
biosensors have become indispensable tools in biochemical and biophysical research
centers. The main advantage of this method is the possibility to monitor binding events
of the biomolecules (antibodies, receptors, nucleic acids etc.) in real-time without the
need of labeled reagents. [1]
Although SPR is a highly sensitive method, many diagnostic bioassays, especially
of low molecular weight compounds, require detection limit inaccessible with the
classical SPR methodologies. To address this problem, several signal amplification
approaches have been developed. Substantial interest has been focused on utilizing
secondary high molecular weight reagents, such as antibody-metal nanoparticle
conjugates, the binding of which to the analyte enhances the sensitivity of the basic
methodology. [2]
Here we are introducing a new amplification approach based on selective
biocatalytic reagents for SPR assays with ultimate sensitivity. The alkaline phosphatase
(ALP) labeled secondary reagents using a suitable substrate are catalyzing the formation
of a precipitate deposit, which by depositing onto the sensor surface increases the
refractive index of the adjoining layer. This results in a dramatic amplification of the
signal due to the binding events.
We used a model system in which the ALP has been directly assayed by using an
anti-ALP antibody modified sensor chip. For simultaneous detection of different samples
surfaces patterned with microcontact printing and microfluidic channels were used. The
lower detection limit was found to be 10-8 U/ml ALP, which equals 15,8 zmol/ml. This
means that only several thousands of ALP molecules can be readily detected by this
method. The wide dynamic range of the method (ca. six orders of magnitude) shows
perspective for its use for protein profiling analysis.
The method was applied for the sandwich type immunoassay of prostate-specific
antigen (PSA) is serum samples and validated with a standard ELISA immunoassay. The
use of the biocatalytic SPR sensing resulted in at least four orders of magnitude
improvement in the detection limit of the PSA assay when compared with the ELISA
method that uses the same immunoreagents. The method shows great promise for the invitro diagnostic of protein markers and ultrasensitive DNA analysis.
References
1.
Karlsson, R., SPR for molecular interaction analysis: a review of emerging application areas. Journal
of Molecular Recognition, 2004. 17(3): p. 151-161.
2.
Besselink, G.A.J., et al., Signal amplification on planar and gel-type sensor surfaces in surface plasmon
resonance-based detection of prostate-specific antigen. Analytical Biochemistry, 2004. 333(1): p. 165173.