Development of two analyticalapproaches based on solution and paper based system for chlorpyrifos detection using graphene quantum dot capped gold nanoparticles for colorimetric assay

This thesis presents the development of a highly sensitive and selective colorimetric assay for chlorpyrifos pesticide in vegetable samples. Colorimetric assay was developed based on the reaction using graphene quantum dot capped with gold nanoparticles (GQDs-AuNPs) as reporter. AuNPs were synthesized using GQDs as a reducing agent and stabilizing agent. The principle of assay was based on acetylcholinesterase (AChE) enzyme catalyzed hydrolysis of an acetylthiocholine (ATCh) substrate to produce thiol-bearing thiocholine. Thiocholine causes the aggregation of GQDs-AuNPs, to generate a purple-blue colored product. The hydrolysis step was inhibited in the presence of chlorpyrifos, resulting in anti-aggregation of red colored GQDs-AuNPs. Development of colorimetric assay for chlorpyrifos determination was carried out based on two approaches; (i) UV-Visible spectrophotometry for chlorpyrifos detection in solution, and (ii) three-dimensional microfluidic paper-based analytical device (3D-microPAD) detected by image captured by digital camera compiled to ImageJ program.
UV-Visible spectrophotometry was used to monitor changes in absorbance during the reaction. Chlorpyfifos inhibited AChE by binding to the active site of an enzyme. This suppresses ATCh hydrolysis, thereby blocking the generation of thiocholine. The distinctive color change of GQDs-AuNPs, from red to blue, and the appearance of a maximum absorption wavelength at 520 nm. Optimal conditions were under, using ATCh and AChE concentrations of 50 microM and 200 mU mL-1, PBS solution (50 mM, pH 7.0), and 30 minutes of incubation time. The developed colorimetric in solution assay exhibits linear calibration over the range of 0.1-50 micro gram mL-1, with a linear correlation coefficient (r2) of 0.996. The limit of detection (LOD) calculated based on [3 S.D.]/slope is 0.046 micro gram mL-1. The simple colorimetric method provides good precision (%RSD=0.03) for chlorpyrifos detection with ten replicates.
Three-dimensional microfluidic paper-based analytical device (3D-microPAD) is an alternative technology for development of affordable, portable, disposable and low-cost diagnostic tools. The 3D-microPAD was fabricated by one-step polymer-screen-printing, using rubber latex (RL) wasted as a hydrophobic reagent for low-cost and simple manufacture. 3D-microPAD for colorimetric chlorpyrifos assay was designed by having two parts on paper. (i) the testing sheet, consisting of two circles; one circle forms the detection zone, for placing the GQDs-AuNPs and AChE-enzyme mixture, and the other circle is a buffer loading area, (ii) the sampling sheet in the shape of the dumbbell design, the bottom circle area of the dumbbell-shape was used to apply the mixing solution of sample/standard (chlorpyrifos) and ATCh (substrate). Chlorpyrifos was determined in the 3D-microPAD using image captured by digital camera coupled by ImageJ software. The assay provided a linear range between 0.001 to 1.0 micro gram mL-1, with a detection limit of 0.0007 micro gram mL-1, without sophisticated instrumentation. The developed 3D-microPAD was applied to detect chlorpyrifos in vegetable samples. Recovery study gave percent recoveries ranging from 93.0% to 104.6%. Our developed device provides good precision (%RSD ranges from 0.3 to 1.6). The calculated relative error comparison with HPLC ranges from 1.0% to 5.2%, indicating a high degree of accuracy. The 3D-microPAD exhibits good sensitivity and selectivity for a low-cost and rapid-screening test for the presence of insecticides, and might be useful on-site applications.