Water-Dispersible Carboxymethyl Dextran-Coated Melamine Nanoparticles for Biosensing Applications.

In this study, we developed a simple method for preparing highly dispersed, stable, and streptavidin (SA)-functionalized carboxymethyl dextran (CMD)-coated melamine nanoparticles (MNPs) in an aqueous buffer at neutral pH. Dynamic light scattering (DLS) revealed the agglomeration of MNPs in an aqueous buffer at neutral pH. When CMD, N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were simultaneously mixed with the MNPs, CMD was bound to the MNPs, promoting their dispersibility. Preparation of SA-CMD-MNPs was accomplished simply by adding SA solution to the CMD-MNPs. The amount of SA bound to the CMD-MNPs was quantified by the bicinchoninic assay, and the amount of SA molecules bound to each CMD-MNP was 417 ± 4. SA-CMD-MNPs exhibited high dispersity (polydispersity index = 0.058) in a neutral phosphate buffer and maintained it for 182 days with dispersion using a probe sonicator (5 s) before DLS characterization. The performance of the SA-CMD-MNPs in biosensing was evaluated by immunohistochemistry, which revealed that the nanoparticles could specifically stain MCF-7 cells derived from breast cancer cells with low HER2 expression. This study provides an effective method for synthesizing highly dispersible nanoparticles for biosensing.

Exploration of interactions between membrane proteins embedded in supported lipid bilayers and their antibodies by reflectometric interference spectroscopy-based sensing.

A microfluidic reflectometric interference spectroscopy (RIfS)-based sensor was fabricated to investigate the activity of multidrug resistance-associated protein 1 (MRP1), applied as a model membrane protein. Vesicles containing MRP1 were immobilized simply by injecting a vesicle solution (50 µg mL(-1)) onto a zirconium oxide (ZrO2) chip under constant flow conditions. Monitoring the shift of the minimum reflectance wavelength (Δλ) of the RIfS demonstrated that the vesicles were adsorbed onto the ZrO2 chip in a Langmuir-like fashion and suggested that the lipid bilayer structure was preserved on the ZrO2 chip. The theoretical maximum physical thickness of the layer was 4.97 nm, which was close to the values previously reported for supported lipid bilayers (4.2 to 5.2 nm). When a model protein, the anti-MRP1 antibody (1-50 µg mL(-1)), was injected onto the MRP1-immobilizing ZrO2 chip a concentration-dependent increase in Δλ was observed. In contrast, a ZrO2 chip on which the supported lipid bilayers did not contain MRP1 exhibited no response. Moreover, an anti-human IgG antibody generated no change in Δλ, confirming that anti-MRP1 antibodies were selectively bound to the MRP1 immobilized on the chip. These results show that the RIfS sensor can follow specific binding events of biologically active membrane proteins and represents a simple, label-free system capable of facilitating biomedical investigations.

Microfluidic reflectometric interference spectroscopy-based sensing for exploration of protein-protein interaction conditions.

A microfluidic reflectometric interference spectroscopy (RIfS) system was adopted for the investigation of protein-protein interaction (PPI). The influence of reaction conditions (pH and temperature) on the antigen-antibody reaction of alpha-fetoprotein (AFP) and its monoclonal antibody (anti-AFP) as a model of PPI was investigated in real time with a label-free fusion, where anti-AFP was covalently immobilized on the carboxylated silicon nitride sensor chips via amide bonds. Optimal pH and temperature were rapidly found by successive and alternate injections of AFP and the regeneration solution (glycine-HCl, pH 1.5) onto the anti-AFP immobilized sensor chip. The resultant optimized reaction conditions (30°C, pH 5.0) gave a 10 times higher detection limit, compared with the response under the commonly employed conditions (25°C, pH 7.4). The proposed system was revealed to provide rapid tracking response against the change in temperature and pH. Consequently, the proposed RIfS system has a potential for the effective tool towards PPI analyses.

Fabrication of carboxylated silicon nitride sensor chips for detection of antigen-antibody reaction using microfluidic reflectometric interference spectroscopy.

In this study, we report label-free detection of alpha-fetoprotein (AFP), which has been used as a biomarker for hepatocellular carcinoma, by a microfluidic reflectometric interference spectroscopy (RIfS) system adopting a simple halogen light source and an inexpensive silicon-based sensor chip. Introduction of carboxy groups on a silicon nitride sensor chip to immobilize anti-AFP monoclonal antibody (anti-AFP) was carried out simply by immersion in aqueous solution containing triethoxysilylpropylmaleamic acid bearing a carboxy group and a silanol group. The RIfS system with the anti-AFP-immobilized sensor chip was found to give a reversible response through 100 on/off cycles using a regeneration buffer with high reproducibility (coefficient of variation (CV) = 5.7%). The limit of detection (LOD) of AFP was 100 ng mL(-1), and the measurement range spanned 3 orders of magnitude. Furthermore, the sensor chip showed no cross-reactivity with human serum albumin, Immunoglobulin G, transferrin, or fibrinogen at 100 µg mL(-1) without the use of blocking reagents such as bovine serum albumin. Consequently, the proposed RIfS system is a potentially effective tool for biomarker detection and in vitro diagnostics.

Label-free detection of C-reactive protein using reflectometric interference spectroscopy-based sensing system.

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique, and suitable for detecting antibody-antigen interaction. This work describes a continuous flow biosensor for C-reactive protein (CRP), involving an effective immobilization method of a monoclonal antibody against CRP (anti-CRP) to achieve highly sensitive RIfS-based detection of CRP. The silicon nitride-coated silicon chip (SiN chip) for the RIfS sensing was first treated with trimethylsilylchloride (TMS), followed by UV-light irradiation to in situ generation of homogeneous silanols on the surface. Following amination by 3-aminopropyltriethoxysilane, carboxymethyldextran (CMD) was grafted, and subsequently, protein A was immobilized to create the oriented anti-CRP surface. The immobilization process of protein A and anti-CRP was monitored with the RIfS system by consecutive injections of an amine coupling reagent, protein A and anti-CRP, respectively, to confirm the progress of each step in real time. The sensitivity was enhanced when all of the processes were adopted, suggesting that the oriented immobilization of anti-CRP via protein A that was coupled with the grafted CMD on the aminated surface of TMS-treated SiN chip. The feasibility of the present sensing system was demonstrated on the detection of CRP, where the silicon-based inexpensive chips and the simple optical setup were employed. It can be applied to other target molecules in various fields of life science as a substitute of surface plasmon resonance-based expensive sensors.