Traditional methods for analyzing the activity of MMPs include the zymography and fluorescein-labeled synthetic peptides [Leber and Balkwill, 1997; Netzel-Arnett et al., 1991].
The zymography is time consuming and complicated for MMPs activity and inhibition studies.
Moreover, fluorescein-labeled synthetic peptides increase the cost of diagnosis and need expensive instruments to operate and analyze. Therefore, the SPR property of AuNPs is used to establish an AuNPs-based optical biosensing platform for measuring proteinase activity and screening the inhibitors of proteinase.
Most of AuNPs-based diagnoses for the detection of enzyme activity mainly depend on the enzyme properties to induce the change in AuNPs aggregation [Wang et al., 2006; Xu et al., 2007]. However, using AuNPs to establish a platform for detection of enzyme activity may encounter the following problems. First, ion concentration effects enzyme activity and AuNPs aggregation. When enzymes carry out their function, changing in the niche (such as pH, temperature, metal ion, or salt concentration) would affect the activity of enzymes;
therefore the diagnostic system should provide an adequate environment for enzyme working.
In terms of AuNPs, AuNPs would easily aggregate while surface charge is neutralized by counterions. In previous AuNPs-based sensing systems, AuNPs aggregation relies on electrostatic interaction, while the surface charges of receptor-modified AuNPs become neutral upon the addition of target analyte [Sato et al., 2003; Zhao et al., 2007; Chen et al., 2008]. Combining above problems, when the AuNPs-based sening system is applied to assay enzyme activity, not only the analyte would promote monodispersed AuNPs to
aggregate, but also the cation which are supplied by the enzyme buffer may induce AuNPs to aggregate, it would result in false-positive results and interfere the analysis of enzyme
activity.
Moreover, according to the DLVO theory, the stability of colloidal system is determined by the balance between two opposing forces - electrostatic repulsion and van der Waals attraction [Craig et al., 1998; Malvern Instruments]. When the bio-recognition element was modified onto AuNPs as the substrate, the bioelement molecule provides the steric repulsion and prevents the AuNPs coming into close contact [Glomm, 2005; Persoons and Verbiest, 2006]. In a detection of protein-protein interaction like proteinase digestion, the steric hindrance between the proteins would lead AuNPs dispersion and the slow enzyme kinetics would prolong the detection time. In addition, AuNPs have high affinity for biomolecules [Lu et al., 2007], which can conjugate with amino acids that have thiol, amino, carboxylic, or hydroxyl groups in their side chains. Therefore, the protein which was digested by
proteinase would adsorb on AuNPs again and interfere with the aggregation of AuNPs.
To overcome these arduous problems, in this study, the colloidal AuNPs was modified with gelatin as proteinase substrate, and then modified with 6-mercapto-1-hexanol (MCH) not only as inducer but also for blocking of this system. Figure 2-1 illustrates the schema of this platform and Figure 2-2 shows the experimental flowchart of this study.
When the AuNPs was modified with gelatin, the gelatin adsorbed onto the AuNPs surface not only as proteinase substrate but also increase the steric repulsion of AuNPs which can prevent the particle surfaces coming into close contact. After proteinase digested the AuNPs/gelatin, the AuNPs/gelatin still can suspend stably in the solution and retain red-wine due to the steric repulsion generated by gelatin was too difficult to overcome. Hence, the MCH) was used in this system for solve the problem.
The chemical compound, MCH, played an important role as an inducer. The MCH has two functional groups, one side is thiol group (-SH), and another is hydroxyl group (-OH).
Both functional group of MCH can conjugate with AuNPs by covalent bond and the -SH group has stronger attraction with AuNPs than -OH group. Therefore, in the modified
process, the MCH predominantly functionalized with gelatin AuNPs by SH-Au covalent bond and the -OH group was been exposed on AuNPs surface [Sato et al., 2003; Chen et al., 2008].
In addition, the MCH can remove nonspecifically adsorbed of gelatin from the AuNPs surface, which helps to improve subsequent biomolecular recognition efficiency [Zhao et al., 2008].
When the AuNPs was modified with gelatin and MCH, the AuNPs/MCH-gelatin could suspend stably. After proteinase digested gelatin, the AuNPs/MCH-gelatin lost the shelter, the repulsion of steric hindrance generation by gelatin was decreased and MCH attracted other AuNPs by OH-Au covalent bond [Zhu et al., 2008]. MCH induced the AuNPs to
irreversibly aggregated and resulted in shift in the surface plasmon spectrum and a consequent color change of the AuNPs from red to purple. Besides, MCH modified on the AuNPs also plays the role as a blocker [Huang et al., 2007]. The MCH possessed the ability to bind on the surface of AuNPs where not conjugated with gelatin through the covalent bond, and prevent the gelatin which digested by proteinase to conjugate with AuNPs again.
In the system, the color change of AuNPs can be observed with the naked eye, and the maximum wavelength (λmax) can be measured by UV/Vis spectroscopy. In addition, the method could serve as an alternative platform for efficient screening of the proteinase
inhibitors. When the proteinase was inhibited by candidate drugs, the drugs block activity of proteinase, the AuNPs/MCH-gelatin are intact and stably in the solution without the color change. Therefore, the novel AuNPs-based optical biosensing platform can not only detect the activity of proteinase rapidly, but also can screen a great deal of effective inhibitor for proteinase.