Optical Biosensing Technology Based On Metal Nanomaterials For Detection Of Enzyme Activity

In recent years, with the development of nanotechnology, various kinds ofnanomaterials including gold nanoparticles, quantum dots, magnetic nanoparticals andmetal nanoclusters have been studied. Because of their unique physical, chemical,mechanical, magnetic and optical properties, there has been great progress in thenanometerial based biosensors. Nanomaterial based biosensor research is amultidiscipline area integrating material science, molecular engineering, chemistry andbiotechnology. The nanomaterial based biosensors can markedly improve thesensitivity and specificity of biomolecule detection, and have great potential in manyresearch fields such as biomolecular recognition, pathogenic diagnosis andenvironment monitoring. Here, we developed a series of optical biosensors based onnovel nanomaterials such as Au nanopartical, silver nanocluster and coppernanocluster, to sensitively detect bacillus licheniformis protease, cholesterol andalkaline phosphatase （ALP）. The detailed methods are as follows:（1） A convenient and fast colorimetry method was developed to detect bacilluslicheniformis protease based on gold nanoparticals aggregation. A peptide with C-andN-terminal cysteine （Cys） and contained two D-leucines was designed as substrate forbacillus licheniformis protease assay. Cys strongly interacted with gold nanoparticleby Au-S bond, causing the aggregation of gold nanoparticles and the color of itssolution was changed from red to blue. Whereas, in the presence of target protease, theD-leucine residue selectively recognized the target protease and the peptide was cut off.The cleaved peptides were unable to induce gold nanoparticle aggregation; and as aresult there was no change in the color of the solution. A linear correlation between theratio of the absorbance at520and600nm （A520nm/A600nm） and the logarithm ofprotease concentration ranging from0.1to5μg/mL, with a detection limit of0.09μg/mL was obtained. Comparing to the traditional detection method, the presentanalytical strategy was simple, fast and demonstrated desirable selectivity.（2） A novel fluorescence sliver nanocluster synthesized by ssDNA template withtwo guanines at the3’. G-DNA-Ag NCs was developed to detect enzyme-basedsubstrate, cholesterol. In this assay, cholesterol was oxidized by dissolved oxygen （O2）in the presence of cholesterol oxidase to produce H₂O₂. Since H₂O₂could quench thefluorescence signal of the prepared G-DNA-Ag NCs, cholesterol was determined indirectly by the amount of generated H₂O₂according to the fluorescence quenchingsignal. The fluorescence decrease was found to be directly related to the cholesterolconcentration, which showed a linear detection range from0.2to200M with adetection limit of0.15M. The present method was convenient and demonstrateddesirable selectivity. Moreover, this method provided an alternative platform fordetecting other substrates through the oxidation by its O2dependent oxidases whichcould generate H₂O₂. The platform also held a potential application for the detection ofO2dependent oxidases assay in the biological complex system.（3） On the basis of double strand DNA templated synthesized fluorescent coppernanoclusters （Cu NCs）. A novel label-free fluorescent strategy for alkalinephosphatase （ALP） assay was developed. This method relied on the strong interactionbetween pyrophosphoric acid （PPi） substrate and Cu²⁺, which would hamper theeffective formation of fluorescent Cu NCs, leading to a low fluorescence signal. In thepresence of ALP, ALP catalyzed the hydrolysis of PPi and hence prohibited thecomplexation between PPi and Cu²⁺, facilitating the fromation of fluorescent Cu NCsthrough the reduction by ascorbate in the presence of dsDNA templates. Thus thefluorescence intensity was recovered, and the fluorescence enhancement wascorrelated to the concentration of ALP. The detection limit of ALP was estimated to be0.096nM and detection range was from0.1to2.5nM. The present method waslabel-free, simple, fast and high selective and sensitive for ALP assay. Additionally,this method worked well in complex biological samples. The proposed assay using PPisubstrate may hold potential applications in the diagnosis of ALP-related diseasesunder physiological conditions.