Two-layer reflectometric interference spectroscopy-based immunosensing for C-reactive protein

Abstract

We report on dual interference layers for use in reflectometric interference spectroscopy (RIfS)-based immunosensing. The layers consist of (a) an antibodyembedded organic/inorganic hybrid titanium dioxide (TiO 2 ) layer (~60 nm thick) as a sensitive layer, and (b) a TiO 2 layer (~110 nm thick) as an interference base layer placed on a silicone substrate by liquid phase deposition. A monoclonal antibody against C-reactive protein (anti-CRP) was coembedded with poly(L-lysine) as an organic binder during deposition of the TiO 2 layer. The optical properties of layer with the antibody were optimized to achieve high sensitivity. Atomic force microscopy images of the sensor surface showed it to consist of a mesoporous structure with nano-scale unevenness. The binding of CRP to the sensor chips was monitored by RIfS, and CRP was repeatedly detected by this biosensor with a detection limit of~60 ng mL −1 . We presume that this two-layer method has a wide scope in that various kinds of biochips can be constructed for use in label-free optical biosensing.

Synthesis of grafted phosphorylcholine polymer layers as specific recognition ligands for C-reactive protein focused on grafting density and thickness to achieve highly sensitive detection

“…The sensitivity of this system is therefore ten times larger than our previous report of 50 ng mL À1 . [14][15][16] These results suggested that the thickness difference affected the CRP binding behavior via SPR measurement, and in this study, PMPC layer with 4.6 nm of thickness showed the sensitive and accurate detection for CRP.…”

“…8,9 However, these methods are limited to a threshold of 0.3 mg mL À1 . [14][15][16] Although using antibody recognition elements enables highly sensitive and selective detection of target proteins, the production and purification of the antibodies are complex and expensive. [10][11][12][13] In addition, we have reported highly sensitive CRP detection with an anti-CRP immobilized substrate utilizing surface plasmon resonance (SPR) and reflectometric interference spectroscopy, with a limit of detection (LOD) of 50 ng mL À1 having been achieved.…”

“…[10][11][12][13] In addition, we have reported highly sensitive CRP detection with an anti-CRP immobilized substrate utilizing surface plasmon resonance (SPR) and reflectometric interference spectroscopy, with a limit of detection (LOD) of 50 ng mL À1 having been achieved. [14][15][16] Although using antibody recognition elements enables highly sensitive and selective detection of target proteins, the production and purification of the antibodies are complex and expensive. In addition, antibodies readily denature due to inherent instability.…”

Critical assessment of relevant methods in the field of biosensors with direct optical detection based on fibers and waveguides using plasmonic, resonance, and interference effects

“…Further applications are the quantification of sarcosine [360], the determination of myoglobin at 0.1 mg L-1 by immobilizing the antibody via histidine-tagged recombinant protein A [361], or the analysis of biological toxins and E. coli ( [362]. By optimizing the layer system C-reactive protein was measured [363].…”

A plasmonic chip-based bio/chemical hybrid sensing system for the highly sensitive detection of C-reactive protein

“…560 pM, respectively. 15,16 We recently demonstrated the highly sensitive detection of CRP based on non-immunological interactions. 17,18 The phosphorylcholine group is a specific ligand for CRP in the presence of Ca 2+ ions [19][20][21] and thus our methodology utilizes protein-ligand interactions between a polymeric ligand, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), and CRP.…”