Dual mode fluorometric and colorimetric detection of matrix metalloproteinase MMP-9 as a cancer biomarker based on AuNPs@gelatin/AuNCs nanocomposite.

Matrix metalloproteinase (MMPs) are proteolytic enzymes which considered as important enzymes and their higher expression is associated with some malignant progression. In the present study, a novel and sensitive method was developed for detection of MMP-9 based on its gelatinase activity. Green emitting gold nanoclusters (AuNCs) has been synthesized by gelatin substrate and then AuNPs@gelatin/AuNCs nanocomposite structure were prepared through coating of gold nanoparticles (AuNPs) with gelatin/AuNCs. Characterization and morphology of synthesized nanocomposite showed successful formation of AuNPs@gelatin/AuNCs. Constructed nanocomposite exhibited internal FRET occurrence and fluorescence quenching of AuNCs due to proximity of AuNCs/AuNPs structures. Hydrolysis effect of MMPs enzyme on AuNPs@gelatin/AuNCs in saline buffer induced changes in surface plasmon resonance (SPR) of gold nanoparticles and also enhanced the emitted fluorescence by AuNCs due to inhibition of internal FRET process. The proposed platform served as the efficient approach for semi-quantitative detection of MMP-9 enzyme by naked eye and precise activity detection with LOD of 2 ng/mL calorimetrically and 0.25 ng/mL fluorometrically. The approach was also showed convenient recovery for detection of MMP-9 enzyme in human serum matrix. Our work provides an efficient, convenient and practical tool for simple identification and precise detection of MMP-9 enzyme.

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.