Colorimetric and label free detection of gelatinase positive bacteria and gelatinase activity based on aggregation and dissolution of gold nanoparticles

A simple and sensitive method was developed for the detection of bacteria gelatinase activity based on their enzymatic hydrolysis effect on the surface plasmon resonance (SPR) of gelatin functionalized gold nanoparticles (Au@gelatin NPs) in bacteria supernatant. Characterization of synthesized NPs showed a very thin gelatin layer on the surface of about 20 nm AuNPs which modified the intrinsic SPR property of AuNPs. The extracted supernatants of applied bacteria were incubated with Au@gelatin NPs. Gelatinase activity of bacteria resulted in gradual gelatin shell removal and subsequent dissolution of bare AuNPs. The presence of inducer agents such as NaCl as the common ingredient in the bacterial medium led to the aggregation process of AuNPs and further bacterial activity resulted in AuNPs dissolution. AuNPs colloid solution color was changed from red to purple after addition of bacteria supernatants with gelatinase activity to the reaction. Also, the spectroscopic studies showed that the gelatinase activity of bacteria resulted in the gradual decrease of absorbance at 529 nm and subsequently led to extinction of SPR characteristics. So, the observed absorbance decrease in UV-Vis spectra at 529 nm was indicated as the gelatinase activity of applied bacteria. Different strains of gelatinase positive Bacillus strains were used as the real sample and their gelatinase activity was determined in the present study. Also, sensitivity analysis of the applied method was determined through this method and the obtained results showed Bacillus subtilis gelatinase activity in the linear range of 0-120 U/mL and detection limit of 0.5 U/mL. This method introduced label free, facile and sensitive assay of the bacterial gelatinase activity without any complicated instrument, affording convenience and simplicity.