In this work, we want to apply the microgels-based platform to design innovative immunoassay biosensors for antibodies and antigens detection, afterwards miniaturized into lab-on-chip devices. The immunoassay is based on the non-competitive sandwich format for immunoglobulin IgG detection. The detection of the target occurs due to the interaction between two particles: microgels and magnetic particles. Microgels are used as the carrier of both anti-Fab antibodies and horseradish peroxide HRP enzyme. Magnetic particles functionalized with anti-Fc antibodies are involved acting as capture particles. The target is recognized by antibodies to produce the “sandwich structure”. After magnetic separation, the HRP enzyme on microgels produces an optical signal to show the target presence. The HRP enzymes confined to the small volume of the microgels results in signal amplification.
Microgels are multifunctional particles with chemical flexibility and a highly tunable nature. Recently, we had synthesized microgels with a core−shell molecular architecture with two different fluorescent dyes as an optical barcode. They were endowed with a fluorescent probe for miRNA detection chosen as cancer biomarkers. Oligonucleotide assay with microgels-based platform had high sensitivity due to the confinement of probes onto the surface of nanometric particles producing an enhancement of fluorescent signal. This represents a modular platform that can be generalized for any direct detection applied to a wide spectrum of biomedical applications.
For the immunoassay biosensors presented here, microgels composed of PEGDMA copolymerized with a fluorescent dye were synthesized by suspension polymerization. The polyacrylic acid was introduced during the synthesis to create pendant groups for post-modifications. Microgels were characterized to determine their physical, chemical, and optical properties. The functionalization of microgels with fluorescent test anti-IgG and HRP enzyme was performed. The capture magnetic particles were functionalized with test IgG. As a proof of concept, interactions between capture and carrier particles were carried out.
The synthesized microgels showed a sub-micrometric size and the optical barcode recovered by fluorescence emission. The bioconjugation reactions on microgels of fluorescent test anti-IgG antibodies and enzyme had shown a modulable functionalization degree by changing the number of particles involved in the reaction. Such a parameter is fundamental to improving the sensitivity and the limit of detection of the assay. The signal produced by biomolecules onto microgels was amplified due to their confinement on the particles small volume. Even magnetic particles had shown a good functionalization degree with test antibodies IgG. To prove the feasibility of interactions, carrier and capture particles were mixed. The binding was shown by recovering the microgel optical barcode at the confocal laser scanning microscope. After the immunoassay optimization, its miniaturization into lab-on-chip will give the possibility to improve its sensitivity to reach the best performance of the assay. The on-chip biosensor allows improving microgels abilities linked to the assay sensitivity and the limit of the detection. Moreover, the microfluidic biosensors offer faster analysis times and advantages in portability and ease of use.