4-1 Synthesis of AuNPs and AuNPs/MCH-gelatin
In the study, the 13 nm AuNPs was used to establishing an optical biosensing platform to assay proteinase activity. The 13 nm AuNP was produced by sodium citrate reduction method. The absorption spectra of 13 nm AuNPs and modified-AuNPs (AuNPs/gelatin and AuNPs/MCH-gelatin) were scanned by Ultrospec 3300 pro UV-Vis spectrophotometer (Amersham Biosciences). The extinction coefficients of the AuNPs are normally very high [Jena and Raj, 2008]. In the previous reports, the optical spectrum was used to estimate the particles size of AuNPs, and the maximum absorbance (λmax) of 13 nm AuNPs was located at 520nm [Elghanian et al., 1997].
Figure 4-1 shows the UV-Vis absorption spectra of 13 nm AuNPs, AuNPs/gelatin and AuNPs/MCH-gelatin. The red-colored AuNPs (13nm in diameter) had surface plasmon resonance band at 520 nm. When the 13 nm AuNPs was modified with gelatin, the particles size was increased, and the λmax of AuNPs/gelatin shifted from 520 to 525 nm. In addition, the absorption peak at 280 nm was correspondent to the absorption from functional group of amino acid. The UV-Vis spectrum of AuNPs/MCH-gelatin was similar to AuNPs/gelatin, and both of their λmax was located at 525 nm. Owing to the MCH as a small compound, when AuNPs was modified with MCH, the particles size of AuNPs had no significant change, and the λmax of AuNPs/MCH-gelatin did not shift. Nevertheless, compared
AuNPs/MCH-gelatin with AuNPs/gelatin, the surface plasmon (SP) band of
AuNPs/MCH-gelatin was broader than AuNPs/gelatin. Jena and Raj reported that the SP band depends on the shape, size and the surrounding medium of the particle [Jena and Raj, 2008]. The results revealed that the size of AuNPs/MCH-gelatin was similar with AuNPs/gelatin, but the physical property of AuNPs/MCH-gelatin was different from
AuNPs/gelatin.
From the data, after appropriate curve fitting and subtraction of the spectrum from the AuNPs absorption, the concentrations of AuNPs could be calculate from the absorbance by used the Beer–Lambert law:
Absorbance = εbc
where ε is molar extinction coefficient, b is the path length of the sample, and c is molar concentration. The ε of 13 nm AuNPs at λ520 is 2.7 × 108/M cm [Jin et al., 2003]. By using the Beer–Lambert law, the concentration of modified-AuNPs were calculated, and
modified-AuNPs was adjusted to 5 nM for the further assay of proteinase activity.
4-2 Identification of the size and morphology change of modified-AuNPs
The size change of modified-AuNPs was investigated by DLS (Figure 4-2) and the morphology change of modified-AuNPs was observed by SEM (Figure 4-3). In the process of the 13 nm AuNPs synthesized by sodium citrate reduction, citrate ions and free chloride ions were adsorbed on the surface to AuNPs, and provided negative charge to AuNPs surface [Saraiva and de Oliveira, 2002]. The non-modified AuNPs was dispersive on the chip (Figure 4-3) due to the negative charge of AuNPs would repel each other, and the average particles size of non-modified AuNPs was about 13.3 nm (Figure 4-2 A).
When the 13 nm AuNPs was modified with gelatin (whether MCH was modified on AuNPs later or not), the gelatin increased the steric repulsion of AuNPs which can prevented the AuNPs from aggregation (Figure 4-3), and the diameter of AuNPs/gelatin and
AuNPs/MCH-gelatin was estimated about 35.5 nm and 37.2 nm, respectively (Figure 4-2).
After proteinase digested the AuNPs/gelatin, AuNPs/gelatin showed slightly aggregated (Figure 4-3 C) due to the steric repulsion generated by gelatin was too difficult to overcome.
In contrast, as shown in Figure 4-3 E, while AuNPs/MCH-gelatin was digested by proteinase, MCH increased the attraction among the AuNPs and resulted in a dramatic aggregation.
Moreover, the DLS data shown in Figure 4-2 was similar with the results in SEM assay.
Nevertheless, the size of AuNPs samples measured by DLS would larger than that obtained from SEM study, which was mainly because DLS measures the hydrodynamic radius while SEM provided a more precise measurement of the hard AuNPs core.
4-3 The electrophoresis mobility of modified-AuNPs
The technique of gel electrophoresis was successfully used to separate modified-AuNPs according to particles size. The modified-AuNPs were digested by trypsin for 10 min, and ran the sample in 0.5% agarose gel for 30 min. The electrophoresis mobility of
modified-AuNPs was showed in Figure 4-4. In Figure 4-4, the non-modified AuNPs was move fastest in the agarose gel due to the particles size of bare AuNPs was smallest compared with other modified-AuNPs.
After trypsin digested, the particles size of modified-AuNPs decreased, and the
electrophoresis mobility of those digested-AuNPs increased. However, the particles size of digested-AuNPs was still larger than bare ones, which indicated that the trypsin did not cleavage the gelatin on the surface of AuNPs completely. In contrast to AuNPs/gelatin, after trypsin digested, the AuNPs/MCH-gelatin aggregated and was difficult to move in agarose gel.
For this result, some aggregated-AuNPs remain stayed upon gel and resulted in one black
band. Consequently, a few no aggregation AuNPs/MCH-gelatin could move toward anode.
Therefore, the mobility of AuNPs in the gel was strongly depending on the particles size of AuNPs.
4-4 Effects of MCH on AuNPs-based optical biosensing platform
The Figure 4-5 was revealed the effect of MCH on AuNPs-based optical biosensing platform. After trypsin was added into AuNPs/gelatin, the λmax at 525 nm was slightly decreased, the spectrum of AuNPs/gelatin without significant red shift, and no defectable color change (Figure 4-5A). However, while trypsin was added into AuNPs/MCH-gelatin, the λmax around 525 nm decreased conspicuously and emerged an absorbance band among 600 nm to 650 nm. The color of the solution changed from wine-red to violet-purple, and could be visuzlized by naked eye (Figure 4-5B).
The difference between AuNPs/gelatin and AuNPs/MCH-gelatin was the function groups of MCH could enhance the attraction between AuNPs and overcome the steric stabilization induced by gelatin, resulted in the aggregation and the color of AuNPs/MCH-gelatin changed from wine-red to violet-purple.
Time-dependent changed of absorbance spectrum of AuNPs-based optical biosensing platform was shown in Figure 4-6. After trypsin digested the substrate of
AuNPs/MCH-gelatin, the absorption band around 525 nm decreased gradually, and
concomitantly, a new broad absorption above 625 nm emerged and its intensity increased by prolonging the reaction time. The new broad absorption above 625 nm emerged was due to the AuNPs/MCH-gelatin began aggregation and resulted in a red shift of absorption spectrum.
Figure 4-7 showed the optimum MCH concentration on AuNPs-based optical biosensing
platform. Increasing amounts of MCH caused increased red shifting of the absorbance spectrum of AuNPs/MCH-gelatin, due to the MCH could enhance the attraction between AuNPs and cause the AuNPs/MCH-gelatin to aggregate. MCH played an important role as an inducer when the MCH concentration above 6 μM (Figure 4-7B). As the concentration of MCH increased, the response time for AuNPs/MCH-gelatin to aggregate decreased, and the concentration above 15 μM was caused a hasty aggregation. Therefore, the 10 μM MCH was used to modify on the surface of AuNPs as inducer and blocker to establish an
AuNPs-based optical biosensing platform for assay proteinase activity, and the absorption ratio (A625 nm/A525 nm) of AuNPs/MCH-gelatin was used to quantity estimate the proteinase activity.
4-5 The stability of AuNPs/MCH-gelatin
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. In addition, AuNPs would begain aggregate when the pH or the buffer salt concentration changed. To explore the stability of AuNPs/MCH-gelatin, the AuNPs/MCH-gelatin was applied into different buffer solutions. When AuNPs modified with gelatin and MCH, the gelatin increased the steric repulsion of AuNPs, and prevented the AuNPs surfaces coming into close contact. For this reason, the AuNPs/MCH-gelatin exhibited a dramatic stability in strict environment (pH 1, pH 13 or high salt concentration). The λmax and waveform of AuNPs/MCH-gelatin were no change in different buffer condition (Figure 4-8). Consequently, the system could detect enzyme activity in niche environment which for maximum activity of the enzyme.
4-6 Assay of proteinase activity
4-6-1 Assay of trypsin activity by AuNPs-based optical biosensing platform
Trypsin is a serine proteinase found in the digestive system of many vertebrates; it predominantly cleaved peptide chains at the carboxyl side of the amino acids lysine and arginine, except when either is followed by proline. Trypsin has been applied in many biotechnological processes, such as used in cell culture to resuspend cells or in biological research during proteomics experiments to digest proteins.
In this study, the AuNPs/MCH-gelatin was used to assay the trypsin activity. The 5 nM of AuNPs/MCH-gelatin solutions were incubated with different concentrations of trypsin (from 5 × 10-2 U to 5 × 103 U) at 37°C for 10 min. As the concentrations of trypsin raising, the aggregation degree of AuNPs/MCH-gelatin increased, and resuted in a remarkable color change from wine-red to purple (Figure 4-9). In addition, large absorption spectra variation would be observed (Figure 4-10 A). To estimate the activity of trypsin, the ratios of the absorbance at 625 and 525 nm (A625 nm/A525 nm) at 10 min after the addition of trypsin were plotted as a function of trypsin concentration (Figure 4-10 B). These two absorbances were chosen to represent the relative amount of aggregating and suspending AuNPs, respectively.
When plotting A625 nm/A525 nm against the trypsin concentration, it is found that the correlation coefficients (R2) was 0.9733 for the determination of trypsin in the concentration ranges from 5 × 10-1 U to 5 × 102 U, and the detection limit of this method could reach to as low as 5 × 10-1 U for trypsin.
4-6-2 Assay of MMP-2 activity by AuNPs-based optical biosensing platform
This AuNPs-based optical biosensing platform also can assay MMP-2 activity, and the
procedure was the same as trypsin activity assay. When extend the detection time from 10 min to 30 min, the detection limit of this system can reach 50 ng/mL for MMP-2, and has a linear relation between 50 to 600 ng/mL (y = 0.0009x + 0.2341, R2 = 0.9874) (Figure 4-11).
4-6-3 Assay of MMP-2 activity by zymography
Gelatin zymography was also used to detection of the MMP-2 activity (Figure 4-12), and has a linear relationship between 10 ng/mL to 700 ng/mL (y = 0.0497x + 1.2533, R2 = 0.9912).
4-7 Assay the efficiency of MMPs inhibitors
This convenient AuNPs-based optical biosensing platform was further applied for MMP-2 inhibitor screening. Galardin and ONO-4817, two well-known inhibitors for MMP-2 were selected as examples to demonstrate the application in MMP-2 inhibitor screening. The AuNPs/MCH-gelatin solutions containing MMP-2 (250 ng/mL) and presence of different concentrations of MMP-2 inhibitors were measured after the solutions were incubated at 37 for 1 hr.
In the presence of an efficient inhibitor in the AuNPs/MCH-gelatin solution, no
detectable color change occurs, and the solutions are indefinitely stable without showing signs of aggregation. These results can be accountable, since galardin and ONO-4817 can inhibit the activity of MMP-2, the aggreation of AuNPs/MCH-gelatin will become slow, and resulted in less absorption variation (and less color change). Figure 4-13 displays the A625 nm/A525 nm
which obtained during the MMPs inhibitors to block the MMP-2 activity. The half maximal inhibitory concentration (IC50; Concentration of inhibitor that reduces enzyme activity to 50%
of the activity of the native enzyme) were estimated by calculate the A625 nm/A525 nm of AuNPs/MCH-gelatin. Comparison ONO-4817 with galardin, galartin had better efficiency than ONO-4817. The galardin and ONO-4817 inhibition with MMPs activity for IC50 values were 1.87 nM and 17.76 nM, respectively.
The efficiency of MMP-2 inhibitors was also analyzed by zymography (Figure 4-14) and the IC50 values were estimated to be 3.48 and 14.33 nM for galardin and ONO-4817, respectively. The data of AuNPs-based optical biosensing platform was consistent with zymography. Nevertheless, comparing this platform with zymograpgy, high
surface-to-volume area of AuNPs provided more space that can not only enhance the immobilization density of gelatin, but also raised the chance for MMP-2 to digest gelatin.
The detection time for zymography to screening the inhibitor of MMP-2 needs 24 hr at least;
however, the platform can shorten detection time to 1 hr. On the other hand, when
zymography was used to analyze the activity of MMP-2, the SDS would active MMP-2 and original activity of MMP-2 would change, but the activity of MMP-2 would not be changed when AuNPs-based optical biosensing platform was used. Therefore, the AuNPs-based optical biosensing platform not only possible to perform a colorimetric assay for proteinase activity, but also has potential for further applications in anti-MMPs drugs screening.