Bioactive paper dipstick sensors for acetylcholinesterase inhibitors based on sol–gel/ enzyme / gold nanoparticle composites

A bioactive paper-based colorimetric "dipstick" bioassay is reported that is based on acetylcholinesterase (AChE) catalyzed enlargement of gold nanoparticles that are co-entrapped with the enzyme in a sol-gel based silica material that is coated on a functionalized paper substrate. Test solutions containing acetylthiocholine (ATCh) and a Au(III) salt are spotted over the sensing area of the bioactive test strips containing small (3 nm diameter) primary gold nanoparticles (AuNP). Biocatalyzed hydrolysis of ATCh via AChE leads to formation of thiocholine, which in turn reduces the Au(III) onto the entrapped nanoparticles, producing particle growth and a concomitant increase in color intensity that can be correlated to the amount of substrate or inhibitor present in test solutions. The entrapped AuNP cannot leach from the silica material, leading to a bioactive paper assay that can utilize visual detection of a color change as a simple readout. Our results show that the dipstick based bioassay is sufficiently sensitive to allow for detection of Paraoxon over the concentration range of 500 nM to approximately 1 mM. Detection can be made by eye or using a digital camera and image analysis, making the assay suitable for remote analysis.