3-1 Reagents and solutions
All chemicals were of analytical grade and were used without further purification.
Sodium citrate (C6H5Na3O7·2H2O) was obtained from Merck (Darmstadt, Germany). Tris HCl was purchased from Chemicon (Invitrogen, San Diego, LA, USA). Sodium chloride (NaCl) was purchased from USB (Cleveland, OH, USA). Agarose and 10 X
Tris-Borate-EDTA buffer (TBE) were purchased from Amresco (Cleveland, OH, USA).
ONO-4817 (C22H28N2O6) was purchased from Tocris (Ellisville, MO, USA). Galardin (Ilomastat; GM6001; C20H28N4O4) was purchased from USBiological (Swampscott, MA).
40% acrylamide/Bis solution (37.5:1) was purchased from Bio-Rad (Hercules, CA, USA).
Calcium chloride (CaCl2), trypsin, 6-mercapto-1-hexanol (MCH), triton X-100, dimethyl sulfoxide (DMSO), p-Aminophenylmercuric Acetate (APMA), type A gelatin, glycine, hydrogen tetrachloroaurate(III) (HAuCl4 · 3H2O), coomassie brilliant blue R250, matrix metalloproteinase-2 human (expressed in mouse NSO cells) were obtained from
Sigma-Aldrich (St. Louis, MO, USA).
Nanopure water was obtained by passing twice-distilled water through a Milli-Q system (18 MΩ· cm; Millipore, Bedford, MA, USA).
3-2 Preparation of the AuNPs/MCH-gelatin
3-2-1 Synthesis of 13 nm AuNPs
The 13 nm AuNPs was prepared by citrate reduction of HAuCl4．3H2O according to the
literature procedure [Saraiva and de Oliveira, 2002]. A 50 mL aqueous solution consisting of 2.5 mM HAuCl4．3H2O was brought to a vigorous boil with stirring in a conical flask, and then 38.8 mM sodium citrate (5 mL) was added to the solution rapidly. This solution was boiled with vigorous stirring for another 15 min, resulted in the color change from the originally yellow solution to deep red. The solution was cooled to room temperature with continuous stirring for 15 min. The colloidal AuNPs with an average diameter of 13 nm was produced, and was stored at 4ºC.
Diameter of the prepared 13 nm AuNPs was measured by a scanning electron
microscope (SEM) and dynamic light scattering (DLS) particle size analyzer. Moreover, the absorption spectrum of AuNPs was measured by a spectrophotometer (SpectraMax 190;
Molecular Devices Corporation, Sunnydale, CA, USA).
3-2-2 Modification of AuNPs/MCH-gelatin
The process of modified gelatin and MCH on AuNPs were monitored by observing the spectral change after the addition of gelatin and MCH to colloidal AuNPs. The gelatin and MCH were attached to the AuNPs according to below procedures:
The 50 μL of aqueous gelatin solution (0.1%) was added to 950 μL of the aqueous 13 nm AuNPs solution. After careful mixing, the mixture was incubated and shaken at 37°C for 2 hr. The mixture was then centrifuged for 6 min at 14,000 × g to remove the excess gelatin.
After two centrifuge/wash cycles, the colloids was resuspended in 200 μL of NTTC buffer (50 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl2, and 0.05 % Triton X-100, pH 7.5), and this colloid solution was regarded as AuNPs/gelatin. The synthesis procedure of AuNPs/MCH-gelatin was similar with AuNPs/gelatin. After 950 μL AuNPs solution and gelatin (0.1%, 50 μL) incubated at 37°C for 2 hr, 10 μL of MCH (1 mM) was added into the solution. After
vortexing, the mixture was incubated and shaken at 37°C for another 2 hr. The mixture was then centrifuged for 6 min at 14,000 × g to remove the excess gelatin and MCH. After two centrifuge/wash cycles, the colloid solution was resuspended in 200 μL of NTTC buffer, and this colloid was regarded as AuNPs/MCH-gelatin.
3-3 Confirm size and morphology change of AuNPs/MCH-gelatin
3-3-1 Dynamic light scattering
Dynamic light scattering (DLS; also known as Photon Correlation Spectroscopy or Quasi-Elastic Light Scattering) is a technique which can be used to determine the size
distribution of small particles in solution. The method utilizes laser as light source which is monochromatic and coherent, and observes a time-dependent fluctuation in the scattering light intensity to determine the translational diffusion coefficient of small particles.
The AuNPs samples were diluted with NTTC buffer (filtered through 0.45 μm syringe filters) and filled into the light scattering cuvette. Light scattering experiments were
performed using the BI-200SM Goniometer (Brookhaven Instruments Corporation, Holtsville, NY, USA) at a temperature of 20°C. The laser wavelength was 532 nm, and measurements were conducted at an angle of 90°. The DLS data were analyzed by Brookhaven
Instruments-Dynamic Light Scattering software.
3-3-2 Scanning electron micrographs
High resolution scanning electron microscopic (SEM) images of modified-AuNPs were obtained with a field-emission SEM instrument JSM-6700F (JEOL, Tokyo, Japan) and operated at 15 kV. The samples were prepared by dropping 10 μl of AuNPs solution onto a
gold chip and incubated samples at 37°C for 30 min. Finally, the chips were rinsed thoroughly with distilled water and air-dried for scanning.
3-3-3 The electrophoresis analysis of modified-AuNPs
To observe the size change of modified-AuNPs which digested by proteinase, the gel electrophoresis was used to separate AuNPs according to the gelatin of attached.
Agarose gel 0.5 % (w/v) was prepared with and immersed in 0.5 X TBE buffer
(Tris-Borate-EDTA buffer, prepared by diluting 10 x stock solutions). The gel was run in a horizontal electrophoresis system (Mini-Sub Cell GT, Biorad, electrode spacing 15 cm). The gel image was recorded by a consumer digital camera and processed the images only with small linear contrast adjustments in order to give a true representation of the visual gel appearance.
Before loading the gel of the modified-AuNPs samples, the modified-AuNPs were coated with a layer of sodium dodecyl sulfate (SDS) which imparts a negative charge on the gelatin of AuNPs, and forced all of modified-AuNPs run to positive electrode. After trypsin digested at 37°C for 10 min, each of digested-AuNPs samples was loaded into one well of the gel, and the gel was ran for 30 min at 110 V in 0.5 X TBE buffer.
3-4 Investigation the stability of AuNPs/MCH-gelatin
To study the stability of AuNPs/MCH-gelatin in strict envionmrnt, 50 μL of different buffer solution was added into 200 μL of AuNPs/MCH-gelatin solution. The 1N HCl and the 1N NaOH were used to analyze the stability of AuNPs/MCH-gelatin under extreme pH
condition, and 10 X PBS buffer (1,370 mM NaCl, 27 mM KCl, 100 mM Na2HPO4, 20 mM KH2PO4, pH 7.4) was used to analyze the influence of high salt concentration buffer on AuNPs/MCH-gelatin. As a control, 50 μl of NTTC buffer solution was mixed with 200 μL of AuNPs/MCH-gelatin. All of samples were incubated at 37°C for 30 min, and the
absorbance wavelength was analyzed with UV-Vis absorption spectrophotometer.
3-5 Assay of proteinase activity
3-5-1 Activation of MMP-2
Preliminary experiments were undertaken to determine the concentrations MMP-2 required for maximal activation of each sample. The lyophilized powder MMP-2 was resuspended in 0.1 mL of TCNB buffer (composed of 50 mM Tris, 10 mM CaCl2, 150 mM NaCl2, and 0.05% triton-X100, pH 7.5), and activated by p-Aminophenylmercuric acetate (APMA).
APMA is an organomercurial agent used for the activation of latent MMPs in vitro. The procedure for activation was as follows: The stock solution of APMA was prepared by dissolve 3.5 mg APMA in 1 mL 0.1 M NaOH, this stock solution should be 10 mM. Then neutralize the high base by diluting 4 folds in Tris-Triton-Calcium buffer (50 mM Tris-HCl, 1 mM CaCl2, 0.05% triton X-100 pH 7.5). For activated MMP-2, the APMA solution was mix with MMP-2 sample to give a final concentration of 0.25 mM. Then activation times will vary depending upon the samples. MMP-2 generally requires short activation time. In this study, MMP-2 samples were activated at 37°C for 2 hr. This final activated MMP-2 solution can be used directly without dialyzing away the APMA [Sellers et al., 1977].
3-5-2 Proteinase activity assay by AuNPs-based optical biosensing platform
For the assay of proteinase activity, the measurement were performed in NTTC buffer as a control experiment and observed result. NTTC buffer would not influence the absorbance of AuNPs, and NTTC buffer would not promote the AuNPs/MCH-gelatin to aggregate. The concentration of gelatin modified-AuNPs was adjusted to 5 nM for the further assay of proteinase activity. In the proteinase activity assay, an amount of 50 μL trypsin or MMP-2 samples solution with different concentrations was added into 200 μL of modified-AuNPs and then the mixture was incubated at 37°C. The activity of MMP-2 were much lower than the ones of trypsin, in order to amplify the detection signal of MMP-2, the incubation time was adjusted to 30 min. After proteinase digested, 200 μL of mixture solutions were transferred into 96-well plate. All of samples were analyzed with UV-Vis absorption spectrophotometer and recorded their wavelength and A625 nm/A525 nm. The tests were performed in triplates.
3-5-3 Proteinase activity assay by
zymography
The activated MMP-2 solution was mixed with zymography buffer (composed of 0.5 M Tris-HCl, pH 6.8, glycerol, 10% (w/v) SDS, and 0.1% bromophenol blue) and stood at room temperature for 10 min. Then loaded on 8% SDS-polyacrylamide gel containing 0.1 mg/mL gelatin, and ran the gel with Tris-Glycine SDS running buffer at 80 V for 4 hr.
Following electrophoresis, the gel was washed twice in renaturing buffer (2.5% trixton X-100) with gentle agitation at room temperature for 30 min in order to exchange SDS to Triton-X100. And decant the renaturing buffer, then replaced with developing buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 5 mM CaCl2) at room temperature for 30 min, then replaced with fresh developing buffer and incubated at 37°C for 18 hr.
The gel was stained with coomassie staining solution (0.125% Coomassie Brilliant Blue
R250, 50% (v/v) methanol, 10% (v/v) acetic acid) for 30 min, then was destained with destain buffer (25% (v/v) methanol, 7.5% (v/v) acetic acid in ddH2O).
Gelatinolytic activities were identified as clear bands against a dark blue background where the protease has digested the substrate. Gelatinase activities in the gels were
quantified using Scion imageTM which quantifies both the surface and the intensity of lysis bands after scanning the gel [Stawowy et al., 2004].
3-6 Assay the efficiency of MMPs inhibitors
In order to apply for drug screening, two broad-spectrum MMP inhibitors were used in this study. MMPIs are synthetic molecules and behave as MMPs competitive inhibitors [Augé et al., 2004]. Galardin is a potent hydroxamate-type MMP inhibitor with a broad inhibitory profile. ONO-4817 is a novel synthetic hydroxamic acid-based nonpeptide compound designed to be administered orally. ONO-4817 binds reversibly to the
zinc-binding region of MMPs and has a selective inhibitory spectrum [Yamamoto et al., 2003].
Both of them have been used to inhibit the MMP-2 activity through binding to its active site and develope the treatment of MMP-related diseases [Galardy et al., 1994; Yamada et al., 2000; Yamamoto et al., 2003; Bai et al., 2005].
3-6-1 Assay the efficiency of MMPs inhibitors by AuNPs-based optical biosensing platform
For drug screening assay of MMP-2, the procedure was similar with proteinase activity assay. In this inhibitors screening assay, 50 μL of MMP-2 (250 ng/mL) with different concentrations of inhibitor was added into 200 μL of AuNPs/MCH-gelatin (5nM) and the
mixtures were incubated at 37°C for 30 min. Finally, the mixture solution was transferred into 96-well plate, then the color change and UV-Vis absorption spectra were collected by SpectraMax 190 UV-Vis spectrophotometer. A625 nm/A525 nm of AuNPs/MCH-gelatin was used to quantitatively estimate the inhibitors activity.
3-6-2 Assay the efficiency of MMPs inhibitors by zymograph
The condition of zymography electrophoresis for inhibitors screening was the same as previously narrated except the composition of developing buffer. After washed twice in renaturing buffer and replaced with developing buffer for 30 min, the gel was replaced with fresh developing buffer which was added with different concentrations of inhibitor and incubated at 37°C for 48 hr. Finally, the gel was stained with coomassie staining solution, and then was destained with the destain buffer.