Cross-linkage urease nanoparticles: a high-efficiency signal-generation tag for portable pH meter-based electrochemical immunoassay of lipocalin-2 protein diagnostics.

An innovative signal-transduction tag based on cross-linked urease nanoparticles (CLENP) was designed for the development of a pH meter-based immunoassay of lipocalin-2 (LCN2). The CLENP was synthesized with a typical desolvation method using ethanol as desolvation agent, followed by functionalization with polyaspartic acid. The carboxylated CLENP were used as the signal-generation tags for the labelling of secondary antibodies via the carbodiimide coupling. Upon target LCN2 introduction, a sandwich-type immune reaction was performed between capture antibody-coated plate and the labeled secondary antibody on the CLENP. The conjugated CLENP in the microplate hydrolyzed urea into ammonia (NH4+) and carbonate (CO32-), resulting in the pH change of solution, which was determined with a handheld pH meter. The pH variation was proportional to target concentration in the sample. By monitoring the pH variation of the urea solution, the level of LCN2 at a concentration as low as 5.2 pg mL-1 was evaluated. The pH meter-based electrochemical immunoassay can be utilized for mass production of miniaturized lab-on-a-chip devices with handheld pH meter, thereby opening new opportunities for protein diagnostics and biosecurity. Graphical abstract An innovative signal-transduction tag based on cross-linked urease nanoparticles was designed for high-efficiency immunoassay of lipocalin-2 with pH meter readout.

Silver-functionalized g-C3N4 nanohybrids as signal-transduction tags for electrochemical immunoassay of human carbohydrate antigen 19-9.

A simple and feasible electrochemical immunosensing platform was developed for highly efficient screening of a disease-related protein (human carbohydrate antigen 19-9, CA 19-9 used in this case) using silver-functionalized g-C3N4 nanosheets (Ag/g-C3N4) as signal-transduction tags. Initially, Ag/g-C3N4 nanohybrids were synthesized by combining thermal polymerization of the melamine precursor with the photo-assisted reduction method. Thereafter, the as-synthesized Ag/g-C3N4 nanohybrids were utilized for the labeling of the anti-CA 19-9 detection antibody by using a typical carbodiimide coupling method. The assay was carried out on a capture antibody-modified glassy carbon electrode in a sandwich-type detection mode. The detectable signal mainly derived from the voltammetric characteristics of the immobilized nanosilver particles on the g-C3N4 nanosheets within the applied potentials. Under the optimal conditions, the voltammetric peak currents increased with the increasing amount of target CA 19-9, and exhibited a wide linear range from 5.0 mU mL(-1) to 50 U mL(-1) with a detection limit of 1.2 mU mL(-1). Our strategy also displayed good reproducibility, precision and specificity. The results of the analysis of clinical serum specimens were in good accordance with the results obtained by an enzyme-linked immunosorbent assay (ELISA) method. The newly developed immunosensing system is promising for enzyme-free and cost-effective analysis of low-abundance proteins.

Label-free electrochemical immunosensor for the determination of fetoprotein based on core-shell-shell nanocomposite particles.

A new approach toward the development of advanced immunosensors based on chemically functionalized core-shell-shell magnetic nanocomposite particles, and the preparation, characteristics, and measurement of relevant properties of the immunosensor useful for the detection of alpha-1-fetoprotein (AFP) in clinical immunoassays. The core-shell NiFe2O4/3-aminopropyltriethoxysilance (APTES) (NiFe2O4@APTES) was initially prepared by covalent conjugation, then gold nanoparticles were adsorbed onto the surface of NiFe2O4@APTES, and then anti-AFP molecules were conjugated on the gold nanoparticles. The core-shell-shell nanocomposite particles not only had the properties of magnetic nanoparticles, but also provided a good biocompatibility for the immobilization of biomolecules. The core-shell-shell nanostructure present good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. The analytical performance of the immunosensor was investigated by using an electrochemical method. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of AFP, and exhibits wide linear range from 0.9 to 110 ng/mL AFP with a detection limit of 0.5 ng/mL. Moreover, the proposed immunosensors were used to analyze AFP in human serum specimens. Analytical results, obtained for the clinical serum specimen by the developed immunosensor, were in accordance with those assayed by the standard ELISA. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.