Clickable Enzyme-Linked Immunosorbent Assay

Abstract

Click chemistry is explored as a potential cost-effective and selective immobilization method for the production of an enzyme-linked immunosorbent assay (ELISA). Coatings were formulated containing either a terminal alkyne or a bicyclo[6.1.0]non-4-yne (BCN) chemical handle, and a diagnostic peptide was subsequently immobilized onto these coatings by the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) or copper-free strain-promoted azide-alkyne 1,3-dipolar cycloaddition (SPAAC), respectively. The terminal alkyne-containing coating showed high background levels in subsequent ELISA's due to the copper catalyst used in the immobilization step. The BCN-containing coating, however, was successfully employed and presents a cost-effective alternative to existing (strept)avidin-biotin immobilization methods. This technology was illustrated with an ELISA used for the diagnosis of rheumatoid arthritis (RA) but could be easily applied to a wide range of diagnostic tests.

Exploring Strain‐Promoted 1,3‐Dipolar Cycloadditions of End Functionalized Polymers

“…Although DIBO is rather labile, we were encouraged by a recent report that indicated that cyclooctynes tolerate free radical polymerization conditions. [18] Furthermore, CTAs containing sensitive norbornene moieties which bear a strained double bond have also been used in free radical polymerizations. [19] …”

“…With the DIBO‐modified RAFT agent in hand, polymerization of styrene under standard conditions was attempted using 1,4‐dioxane as the solvent and azobisisobutyronitrile (AIBN) as the initiator at 70 °C (Table 1, condition A). Although DIBO is rather labile, we were encouraged by a recent report that indicated that cyclooctynes tolerate free radical polymerization conditions 18. Furthermore, CTAs containing sensitive norbornene moieties which bear a strained double bond have also been used in free radical polymerizations 19…”

Single‐molecule pull‐down (SiMPull) for new‐age biochemistry

“…There are many commercial ELISA kits, manufactured by several diagnostic companies, available for different analytes detection . And new technological integrations are being pursued . SiMPull has the ultimate detection limit of single‐molecules; its sensitivity is about a few picomolar in concentration for reliable scoring above background fluorescence, and has the added capability to perform biochemical measurements alongside detection of the analyte, which is currently lacking for ELISA.…”

Azide‐rich peptides via an on‐resin diazotransfer reaction

“…The bioorthogonal strain‐promoted azide‐alkyne cycloaddition (SPAAC) reaction and the Staudinger ligation also exploit the azide functional group to expand the range of non‐natural functional groups that can be incorporated into amino acid sequences . Several amino acids with azide groups in the side chain have been reported for use in SPPS (e.g., N α ‐Fmoc‐protected derivatives of azidonorleucine, azidonorvaline, azidohomoalanine, and azidoalanine), and these have been routinely used to site‐specifically introduce into peptides a single bioorthogonal reaction site. Peptides in which several azides have been site‐specifically incorporated have also been reported, and these serve as key intermediates in the syntheses of tools for chemical biology and of hybrid biomaterials .…”