Improvement of Ficin-Based Inhibitive Enzyme Assay for Toxic Metals Using Response Surface Methodology and Its Application for Near Real-Time Monitoring of Mercury in Marine Waters

Abstract

Potentially toxic metals pollution in the Straits of Malacca warrants the development of rapid, simple and sensitive assays. Enzyme-based assays are excellent preliminary screening tools with near real-time potential. The heavy-metal assay based on the protease ficin was optimized for mercury detection using response surface methodology. The inhibitive assay is based on ficin action on the substrate casein and residual casein is determined using the Coomassie dye-binding assay. Toxic metals strongly inhibit this hydrolysis. A central composite design (CCD) was utilized to optimize the detection of toxic metals. The results show a marked improvement for the concentration causing 50% inhibition (IC50) for mercury, silver and copper. Compared to one-factor-at-a-time (OFAT) optimization, RSM gave an improvement of IC50 (mg/L) from 0.060 (95% CI, 0.030–0.080) to 0.017 (95% CI, 0.016–0.019), from 0.098 (95% CI, 0.077–0.127) to 0.028 (95% CI, 0.022–0.037) and from 0.040 (95% CI, 0.035–0.045) to 0.023 (95% CI, 0.020–0.027), for mercury, silver and copper, respectively. A near-real time monitoring of mercury concentration in the Straits of Malacca at one location in Port Klang was carried out over a 4 h interval for a total of 24 h and validated by instrumental analysis, with the result revealing an absence of mercury pollution in the sampling site.

Microbial Sensing and Removal of Heavy Metals: Bioelectrochemical Detection and Removal of Chromium(VI) and Cadmium(II)

“…For detecting acute toxicity caused by heavy metals, pesticides and organic solvents, several biochemical assays were applied based on the function of microorganisms [ 13 ], bacteria [ 14 ], antibodies [ 15 ] and enzymes [ 16 ]. Indicator microbes provide a simpler method [ 17 ] because microorganisms have biosorption capabilities as well as they are easy to culture in a short generation time so their response to toxic substances is quite rapid [ 18 ].…”

Ficin–Cyclodextrin-Based Docking Nanoarchitectonics of Self-Propelled Nanomotors for Bacterial Biofilm Eradication

“…Contents of the capping unit and cargo were also completed for control nanodevices ( Figure S4 and Table S3 ). In addition, the protein degradation capacity of ficin was evaluated by running a protease activity assay against casein, 36 with a result of 1.02 × 10 –5 U mg –1 of NM VF .…”

“…NM VF protease activity was studied by a casein degradation assay . 1 g of casein was solved in 9.9 mL of 100 mM PBS pH 7.5 at 60 °C for 15 min to ensure complete dissolution.…”

“…NM VF protease activity was studied by a casein degradation assay. 36 1 g of casein was solved in 9.9 mL of 100 mM PBS pH 7.5 at 60 °C for 15 min to ensure complete dissolution. Then, 1 mL of 1% casein was mixed with 500 mL of NM VF (1 mg mL –1 ) and incubated at 40 °C.…”

Nanomotores Janus de platino y sílice mesoporosa como plataforma para la entrega de fármacos en aplicaciones biomédicas

“…La actividad proteolítica de las nanopartículas se determinó mediante un ensayo de degradación de caseína. 330 Para ello, se disolvió 1 g de caseína en 10 mL de PBS (100 mM, pH 7.5) a 60 °C durante 15 min. Después, se añadió 1 mL de esa disolución a 500 L de NM CHX-F (1 mg mL -1 , PBS 100 mM, pH 7.5) y la mezcla se incubó a 40 °C durante 20 min.…”