A Biotinylated Undecylthiophene Copolymer Bioconjugate for Surface Immobilization:  Creating an Alkaline Phosphatase Chemiluminescence-Based Biosensor

Abstract

Methodology is described for the creation of a molecular assembly consisting of the enzyme alkaline phosphatase immobilized onto a glass surface using a biotinylated conjugated copolymer, poly(3-undecylthiophene-co-3-thiophenecarboxaldehyde) 6-biotinamidohexanohydrazone. The biotinylated polymer is attached to the inside walls of a silanized glass capillary via hydrophobic interactions, and a streptavidin-conjugated alkaline phosphatase is interfaced with the polymer through the classical biotin-streptavidin interaction. Utilizing a simple optical setup, we can detect the activity of as little as approximately 0.1 fmol of alkaline phosphatase with this molecular assembly. The assembly is mechanically robust and retains the majority of bound enzyme activity for up to 30 days. We have utilized this molecular assembly for the detection of organophosphorus-based pesticides. Both paraoxon and methyl parathion inhibit the enzyme-mediated generation of chemiluminescence signal. We are able to detect paraoxon and methyl parathion concentrations down to 500-700 ppb.

Conjugated Polymers as Optical Probes for Protein Interactions and Protein Conformations

“…Pande et al showed the first CP modified with biotin in a supramolecular assembly together with a phosphatase. [15] Faid and Leclerc took the biotin modification one step further and developed a polythiophene for colorimetric measurements based on a chromic shift, from violet to yellow, upon binding of streptavidin ( Figure 2). [16] The authors claim that the chromic shift can be attributed to a planar-to-non-planar twisting of the polymer chains, which increases the energy of the p-p transition.…”

“…The binding between biotin and avidin or streptavidin is one of the strongest biological non‐covalent interactions known. Pande et al showed the first CP modified with biotin in a supramolecular assembly together with a phosphatase 15. Faid and Leclerc took the biotin modification one step further and developed a polythiophene for colorimetric measurements based on a chromic shift, from violet to yellow, upon binding of streptavidin (Figure 2).…”

Self-assembly of synthetic peptides control conformation and optical properties of a zwitterionic polythiophene derivative

“…Polythiophene derivatives that display biotin (1,14) and different carbohydrates (3) have been synthesized and shown to undergo colorimetric transitions in response to binding of streptavidin and different types of bacteria and viruses, respectively. The chromic transitions, from deep violet (absorption maximum Ϸ550 nm) to bright yellow (absorption maximum near 400 nm), are related to a planar-to-nonplanar (from highly conjugated to less conjugated) conformational transition of the backbone.…”

Biosensors from conjugated polyelectrolyte complexes

“…As a result of this sensitivity, conjugated polymers are promising as sensory materials (4,5); sensing may be accomplished by transducing and͞or amplifying physical or chemical changes into electrical, optical, or electrochemical signals. Conjugated polymers have been used to detect chemical species (chemosensors) (6), such as ions (7)(8)(9)(10)(11), gases (for example, trinitrotoluene) (6,(12)(13)(14), and other chemicals (15), or biomolecules such as proteins, antibodies (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27), and DNA (28-31), using electrical (13,15), chromic (7,8,(16)(17)(18)(19), electrochemical (7-9, 20-25, 28-31), photoluminescent (11,26), chemoluminescent (27), or gravimetric (14) responses.…”