Magnetic Enzyme-linked Immunosorbent Assay Research Articles

Controllable synthesis of sandwich-type magnetic beads for procalcitonin detection on a magnetic enzyme-linked immunosorbent assay

Although magnetic beads have been widely utilized as a homogeneous carrier for immunoassays, the effectiveness of the immuno-response is often hindered by the physical and chemical characteristics of these beads, such as the challenge of achieving an optimal equilibrium among magnetic response, suspension performance and colloidal dispersibility. Herein, we present the fabrication of monodisperse polystyrene magnetic beads in a sandwich-like configuration and further explore their immunoassay capabilities as capturing substrates in the detection of procalcitonin (PCT) using the enzyme-linked immunosorbent assay (ELISA). The developed two-step magnetic particles assembly process effectively regulates the magnetic content, while the organic/inorganic double silica layer coating imparts desirable stability and dispersion to the assembly structure. Notably, the prepared magnetic beads exhibit high aqueous dispersity (PDI ∼ 0.2), rapid magnetic separation (25 s) and gradual half-sedimentation time (5 h). Leveraging the magnetic bead-based ELISA, we accomplish not only qualitative screening of PCT at an exceptionally low concentration by naked eye (cut-off value: 0.1 ng/mL) but also highly sensitive quantification of PCT using a microplate reader (limit of detection: 0.031 ng/mL). This investigation offers a solution for striking a harmonious balance among the diverse properties of magnetic beads and demonstrates the perspective of the magnetic capturing substrates in the field of immunoassays.

Sol-gel polymerization of silylated amino acids around a protein template yields selective biomimetic imprints

The development of artificial receptors able to selectively recognize a target protein is of particular interest in separation, diagnostics, and therapeutics fields. Herein, we disclose a method to prepare biomimetic and functionalized protein imprints in biocompatible conditions avoiding any protein denaturation. For that purpose, a set of different hybrid silylated amino acid derivatives were synthesized and used without tetraethyl orthosilicate to prepare our molecularly imprinted polymers, allowing to reduce to a minimum of the silicon amount, in order to obtain imprints made almost entirely of amino acids to mimic paratope surfaces of antibodies. Such functional building blocks were polymerized on the surface of magnetic silica nanoparticles at pH 8.5 in ultrapure water in the presence of two globular proteins: cytochrome C or lysozyme. The resulting imprinted hybrid materials were evaluated for their adsorption capacity, specificity, and selectivity by quartz-crystal microbalance with dissipation and magnetic enzyme-linked immunosorbent assay (ELISA) assays. High imprinting factors of 8.7 were measured for these biomimetic hybrid materials (corresponding to approximately 4000 and 450 ng of protein per cm2 immobilized on molecularly imprinted polymers and non-imprinted polymer nanoparticles, respectively), representing a significant breakthrough in sol-gel-based molecular imprinting materials. Moreover, competition experiments performed by magnetic ELISA (mELISA) show very good specificity of our imprints at the usual concentrations of ELISA measurements.

Poly-L-lysine brushes on magnetic nanoparticles for ultrasensitive detection of Escherichia coli O157: H7

We have developed a magnetic enzyme-linked immunosorbent assay (MELISA) based poly-L-lysine (PLL) mediated brushes on magnetic nanoparticles (MNPs) for detection of Escherichia coli O157:H7 in river water samples. In the MELISA, we couple PLL on the surface of magnetic nanoparticles to prepare the PLL brushes (MPLLBs). PLL with plentiful amine groups provides multi-binding sites to allow the binding of both horse radish peroxidase (HRP) and antibody (Ab) on the surface of the MPLLBs with high capacity. Compared with the conventional particles (the binding capacity of MNPs for HRP and Ab are 19μg/mg and 16μg/mg, respectively), MPLLBs have achieved an improvement of 43-fold and 24-fold in binding capacity for HRP (816μg/mg, protein μg per mg of MPLLBs) and Ab (387μg/mg), respectively. This multifunctional Ab-MPLLBs-HRP conjugate serves as not only an immune-carrier for magnetic enrichment but also an enzyme assembly for signal amplification system. Compared with the conventional ELISA (the detection of limit is 400 cfu/mL), MELISA shows enhanced sensitivity (8cfu/mL) and shortened the analysis time (within 2h) for the detection of Escherichia coli O157:H7 in river water sample, which provides an attractive candidate platform for the rapid and sensitive detection of pathogen in complex samples.