(210l) Collagen Type II Quantification with Pd@Pt Nanoparticle-Linked Immunosorbent Assay

In this presentation, we show a method for quantitatively detecting a larger-sized analyte, Collagen type II, using a Pd@Pt nanoparticle-linked immunosorbent assay.

Extracellular matrix (ECM), including cartilage tissue, is made up of proteoglycan and collagens, which act as the main structural proteins. Collagen types I, II, and III are essential fibril-forming collagens that comprise 80 – 90% of all collagens found in the human body. Collagen type II makes up approximately 50% of the protein and 85 – 90% of collagen in articular cartilage. Articular cartilage is the tissue that connects diarthrodial joints, providing a cushion between the bones.

The Centers for Disease Control and Prevention (CDC) estimates that over 32.5 million US adults suffer from arthritis. The most common type of arthritis, osteoarthritis, is a degenerative joint disease frequently found in the hands, hips, and knees. Osteoarthritis causes symptoms such as pain, swelling, and less flexibility because of the erosion and damage of articular cartilage. When osteoarthritis is present in a joint, components of the ECM, Collage type II and aggrecan, are broken down by the enzyme collagenase. Therefore, Collage type II quantification is a potential method for observing the degree of cartilage damage in osteoarthritis patients.

There presently is no treatment or cure for osteoarthritis, which is why we are studying articular cartilage regeneration through the use of scaffold-based techniques. Collagen type II is one component of the regenerative medical scaffolding used for cartilage and tissue engineering applications. Therefore, the ability to produce Collage type II and quantify the production is needed.

Nanozymes have enzyme-like catalytic activity like that of peroxidase and catalase and finds usage in various field because nanoparticles have the capacity to be modified, functionalized, and mass produced at low cost, and are inherently stable. In this study we used mesoporous palladium@platinum (Pd@Pt) nanoparticles a nanozyme with peroxidase-like catalytic activity that consists of a palladium core with a platinum shell. In addition, these nanoparticles are highly stable with respect to pH and temperature, allow simple conjugation with biological components such as antibodies, and allow simple separation methods through centrifugation. These characteristics allow us to use Pd@Pt nanoparticles as detection agents instead of the enzymes in immunosorbent assays.

Since Collage type II is large enough to have two epitopes, a sandwich immunoassay was used to capture and label this larger analyte with two different antibodies. Assay conditions were optimized for Collage type II quantification using a Pd@Pt nanoparticle-linked immunosorbent assay. An optimal concentration of coating Abs was found to be 2.5 µg/mL at a pH of 8.4 – 8.6, Ab conjugated to Pd@Pt NPs at a level of 7.5 µg Ab to 1 mg/mL Pd@Pt, and a concentration of Ab – nanoparticle conjugates of 2 mg/mL. We plan to apply our sandwich detection method to engineered tissue samples. Our use of this method broadens the scope of Pd@Pt nanoparticle-linked immunosorbent assays beyond our previous study for small analytes such as pesticides. We expect this assay to be adopted for use in the fields of bioengineering, tissue engineering, and bioanalysis.