Application of a fiber optic biosensor (FOB) to the real-time investigation of the interaction kinetics between FITC-conjugated monoclonal sheep anti-human C-reactive protein (CRP) antibody and CRP isoforms on the surface of optical fiber is described. Recently, both the native pentameric CRP (pCRP), an acute phase protein belonging to pentraxin family, and an isoform of pCRP, modified CRP (mCRP), have been suggested to have proinflammation effects on vascular cells in acute myocardial infarction (AMI). In current studies, we generate mCRP from pCRP, and use several methods including fluorescence spectral properties, circular dichroism, analytical ultracentrifuge, and Western blotting to demonstrate their differences in physical and chemical properties as well as the purity of pCRP and mCRP. In addition, we design and implement an FOB to study the real-time qualitative and quantitative biomolecular recognition of CRP isoforms. Specifically, the association and dissociation rate constants of the reaction between FITC-conjugated monoclonal sheep anti-human CRP antibody and the pCRP and mCRP are determined. The feasibility of our current approach to measure the association and dissociation rate constants of the reaction between tested CRP isoforms was successfully demonstrated.
We proposed and developed a novel fiber-optic biosensor based on localized surface plasmon coupled emission (LSPCE)
which consists of sandwich format of <antibody/antigen/Cy5-antibody-GNP> immuno-complex. It is immobilized on the
surface of optical fiber where <Cy5-antibody-GNP> is a fluorescence probe produced by mixing Cy5 labeled antibody
and protein A conjugated gold nanoparticles (Au-PA). The fluorophores are excited by localized surface plasmon (LSP)
on gold nanoparticle (GNP) surface where the evanescent field is applied near the core surface of unclad optical fiber.
Meanwhile, the fluorescence signal is detected by a photomultiplier tube being set beside the unclad optical fiber with
high collection efficiency. In the experiment, this novel LSPCE biosensor demonstrates the minimum detectable
concentration of mouse immunoglobulin G (IgG) at 1pg/ml (7fM) in the biomolecular interaction with anti-mouse IgG.
From the experimental result, it verifies that LSPCE biosensor is a very high sensitive biosensor which is capable of
measuring biomolecular interaction at very low concentration.
Previous work with amplitude-sensitive paired surface plasma waves biosensor (PSPWB) demonstrated that the
detection sensitivity of PSPWB is able to experimentally detect 0.001% sucrose-water solution and biomolecular
interaction of 10pg/ml mouse IgG interacting with immobilized anti-mouse IgG successfully.
Further development of the detection sensitivity of PSPWB has been conducted by using 20nm in diameter colloid gold
nanoparticles conjugated with target molecules that can result in a higher mass coverage and a larger resonant angle
change of plasmon resonance, thereby its detection sensitivity is further enhanced significantly. Bare gold nanoparticles,
which is randomly suspended in solution, is adopted to differentiate biospecific binding induced further signal
enhancement. Experimentally, the sensitivity at 330fg/ml of Au-nanoparticle conjugated protein A (PA-Au) interacting
with mouse IgG which is immobilized on a CM5 sensor chip was detected successfully. By this arrangement, 6-fold
signal amplification is demonstrated compared with the same concentration of PA without conjugated gold nanoparticles.
The proposed optical fiber biosensor is used to study the qualitative and quantitative aspects of biomolecular recognition in real time. This approach is able to apply for measuring the association and dissociation rate constants of the reaction between biomolecules.
A novel optical heterodyne surface plasmon resonance (SPR) biosensor using Zeeman laser is proposed. There are two surface plasma waves (SPWs) being excited by two correlated P polarized waves in an SPR device of Kretschmann configuration. The two reflected P waves are optically heterodyned so that the amplitude of the heterodyned signal is proportional to the multiplication of two attenuated reflected P waves. The detection sensitivity and the dynamical range based on this amplitude sensitive method are enhanced. In the experiment, the kinetics between mouse IgG and anti-mouse IgG is obtained according to the sensograms of different concentrations of anti-mouse IgG. The detection sensitivity corresponding to 0.2 nM is achieved. In addition, a concentration of 5 ng/ml of protein G interacting with mouse IgG is measured successfully.