in buffer of pH 4.0. Fig. 4-7D also indicates the spectra of negatively charged modification process of 11.0A/1466p-FITC probe; however, the spectra of pH 11.0 modification before removal process gained no smooth curve. The 11.0A/1466p-FITC probe seems stable in all pH of buffer but in pH 4.0.
4-1-6. Effect of peptide substrate charges on AuNPs probes
The effect of 1466p-FITC charges at function of pH (pH 4.0, 5.6, 7.4, 10.0 and 11.0) was shown in Fig. 4-8A. Except pH 4.0 of AuNPs modification caused serious aggregation (A625
/A525 0.8), the others conditions of A625 /A525 were obtained around 0.18 ~ 0.22. After the removal excess peptide substrates, the A/1466p-FITC probe was suspended in 0.1 % BSA solution. As shown in Fig. 4-8B, the best stable conditions of A/1466p-FITC are pH 7.4 and 10.0 of AuNPs modification (A625 /A525 0.21). Also, the effect of 1477p-FITC charges at function of pH (pH 4.0, 5.6, 7.4, 10.0 and 11.0) was shown in Fig. 4-9A. The positively charged of pH 4.0 and 5.6 showed serious aggregation, but the pH 7.4 of AuNPs predicted with slightly positive charges still remain stable. After removal- suspension process, the best stable conditions of A/1466p-FITC as shown in Fig. 4-9B are pH 10 and 11 of AuNPs modification (A625 /A525 around 0.24 ~ 0.26), and the pH 7.4 of AuNPs modification shows aggregation (A625 /A525 0.45).
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4-1-7. Conjugation of peptide substrates to AuNPs
The peptide substrate conjugations to AuNPs were discussed. During the modification, the peptides substrates were existed either on the AuNPs or in the discarded supernatant. In this study, dithiothreitol (DTT)-based displacement were used to discard the peptide substrates conjugated on AuNPs. Therefore, pure peptide substrates (1466p-FITC, 1477p-FITC and 1482p-FITC), the first discarded supernatant of each A/p-FITC (7.4A/1466p-FITC, 10.0A/1477p-FITC and 5.6A/1482p-FITC) and peptide substrates
acquired after processed with DTT displacement (1466p-FITC, 1477p-FITC and 1482p-FITC) were diluted to proper concentrations. Hence, the linear correlation between emission of FITC and peptide substrates in different states were recorded in Table 4-1. The ratios of peptide substrates at each part to total loading amount were compared in Table 4-2. The conjugation rate (%) was acquired from dividing DTT displacement caused fluorescence intensity by pure peptide substrates part of fluorescence intensity. The conjugation rate (%) times the amount of loading of pure peptide substrates (10 μg/mL) to gain total peptide substrates on total AuNPs used in modification. The number of peptides substrates per AuNPs was obtained by the ratio of the number of peptide-FITC molecules to the number of particles molecules of solution (Table 4-3).
4-1-8. Zeta potentials of AuNPs and AuNPs probes
Zeta potentials of citrate-capped AuNPs and AuNPs probes were measured. AuNPs probes were divided into two groups of different process products, modification and
purification. The modification group represents the peptide substrates conjugated on AuNPs without further treatment. The purification group is modified AuNPs undergoing
centrifugation and suspension treatments. As shown in Table 4-4, that citrate reduction methods produced citrate-capped AuNPs with zeta potential value of -34.7 mV. The 7.4A/1466p-FITC probe has the highest zeta potential (-52.2 mV) but after purification
57
reduces to -17.2 mV. The 10.0A/1477p-FITC probe also shows good stability before purification, but also reduced to -15 mV of the purification product. The 5.6A/1482p-FITC probe shows a mild improvement (-39.2 mV) compared with AuNPs, and remain the zeta potential (-34.3 mV) after conducted with purification process.
4-2. Optimize the fluorescence assays by AuNPs probes 4-2-1. Effect of stabilizers to proteinase K sensitivity to AuNPs probe
For improving stability of the AuNPs probe, the stabilizers such as PEG and BSA were used; however, do these stabilizers cause negative effect to the sensitivity of AuNPs probe to proteinase? The first experiment is to determine the effect of shorty immersed in PEG solution on AuNPs probe’s sensitivity. AuNPs probe washed with different concentration of PEG (0.5, 1, 2 and 5% (w/w)) and suspended with 0.1% BSA (w/w) were prepared. The 1.25 nM 7.4A/1466p-FITC was incubated with fixed concentration proteinase K (100 ng/mL) for 1 hr at 37°C. As showed in Fig. 4-10A, the concentration of PEG has no effect to proteinase K activity, except 5% PEG is significant lower compared with 0% PEG.
The second experiment is to determine the effect of BSA as stabilizer on AuNPs probe’s sensitivity. AuNPs probe washed with 2% PEG (w/w) and suspended with different
concentrations of BSA (0.1, 0.5, 1 and 7% (w/w)). The 1.25 nM 7.4A/1466p-FITC was incubated with fixed concentration proteinase K (100 ng/mL) for 1 hr at 37°C. It clearly shows that the proteinase K activities decrease with the increase of BSA concentrations (Fig.
4-10B).
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4-2-2. pH optimization of proteinase K sensitivity to AuNPs probe
The 7.4A/1466p-FITC (1.25 nM ) was incubated with fixed concentration proteinase K (100 ng/mL) for 1 hr at 37°C under various pH (pH 7.4, 8.0, 9.0 and 10.0). Fig. 4-11 displayed that proteinase K has optimal activity at pH 9 condition.
4-2-3. AuNPs probes concentration optimization
The concentration of AuNPs probe was concerned because of the quenching effect of AuNPs is distance dependence. Different concentrations of 7.4A/1466p-FITC (0.156, 0.313, 0.625, 1.25, 2.5 and 3.75 nM) were incubated with fixed concentration proteinase K (100 ng/mL) for 1 hr at 37°C. In Fig. 4-12A, the optimal concentrations are 0.29 to 1.25 nM 7.4A/1466p-FITC, while higher or lower concentration lead to lower proteinase K sensitivity responded on fluorescence intensity change. Furthermore, different concentrations of
10.0A/1477p-FITC (0.156, 0.313, 0.625, 1.25, 2.5 and 3.75 nM) were incubated with fixed concentration proteinase K (400 ng/mL) for 1 hr at 37°C. At the higher concentration of proteinase K case, the need of more AuNPs probe. As shown in Fig. 4-12B, the optimal concentration is 1.25 nM.
4-2-4. Proteinase K activity assay by AuNPs probe (7.4A/1466p-FITC)
The 7.4A/1466p-FITC (1.25 nM) was incubated with different concentrations of proteinase K (10, 25, 50, 100, 150, 200, 300, 350 and 400 ng/mL) for 1 hr at 37°C, pH 9.0.
Delta fluorescence intensity was correlated to proteinase K concentration, which is given in Fig. 4-13A. The result showed that delta fluorescence intensity increased with the increasing concentration of proteinase K in linear correlation. The linear correlation ranged from 10 to 400 ng/mL proteinase K was confident, of which y = 8.32 x + 433.82 and R² = 0.96. The time course of proteinase K activity also was given in Fig. 4-13B, which was obtained by
7.4A/1466p-FITC (1.25 nM ) incubated with fixed concentrations proteinase K (100 ng/mL)
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for different time (0.25, 0.5, 0.75, 1, 1.5, 2, 3 and 4 hr) at 37°C, pH 9.0.
4-2-5. Comparison of different AuNPs probes to proteinase K sensitivity
The two AuNPs probes sensitivity to proteinase K was also compared in short detection time. The 7.4A/1466p-FITC and 10.0A/1477p-FITC (1.25 nM ) were incubated with different concentrations proteinase K (10, 25, 50, 100, 150, 200, 300, 350 and 400 ng/mL) for 15 min at 37°C, pH 9.0. The results displayed in Fig. 4-14A and showed that both AuNPs probes had great linear correlation between fluorescence intensity change and proteinase K concentration.
The 7.4A/1466p-FITC probe had the linear correlation ranged from 25 to 400 ng/mL proteinase K, of which y = 3.14 x + 6.82 and R² = 0.99; while 10.0A/1477p-FITC had the range from 10 to 400 ng/mL proteinase K, of which y = 10.56 x + 396.76 and R² = 0.99.
Besides, the comparison in time course of low concentration proteinase K activity between two AuNPs probes also was given in Fig. 4-14B. The 7.4A/1466p-FITC and
10.0A/1477p-FITC (1.25 nM ) incubated with low concentrations proteinase K (25 ng/mL) for different time (0.25, 0.5, 0.75, 1, 1.5, 2, 3 and 4 hr) at 37°C, pH 9.0.
4-2-6. Proteinase K activity assay by AuNPs probe (5.6A/1482p-FITC)
To lower the detection limits and in short detection time, the 5.6A/1482p-FITC was applied. The 5.6A/1482p-FITC (1.25 nM ) was incubated with various concentrations
proteinase K (0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 ng/mL) for 15 min at 37°C, pH 9.0. Delta fluorescence intensity was correlated to proteinase K
concentration, which is given in Fig. 4-15A. The result showed that delta fluorescence intensity increased with the increasing concentration of proteinase K in linear correlation and saturated in higher concentration. The linear correlation ranged from 0.1 to 12.5 ng/mL proteinase K was confident, of which y = 570.36 x + 209.17 and R² = 0.99. The time course of proteinase K activity also given in Fig. 4-15B, which was obtained by 5.6A/1482p-FITC
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(1.25 nM ) incubated with fixed concentrations proteinase K (1 and 5 ng/mL) for various time (5, 10, 15, 20, 25, 30, 45, 60, 75 and 90 min) at 37°C, pH 9.0.
4-3. Evaluation of characteristic change in AuNPs probes 4-3-1. Gel electrophoresis analysis of AuNPs
To confirm the morphology change of AuNPs after modification and proteinase digestion, the gel electrophoresis by which the change of visual gel and UV-light excited fluorescence band could be observed were applied. The visual gel shown in Fig. 4-16A indicated that the migration difference of citrate-capped AuNPs, 5.6A/1482p-FITC and the AuNPs probe activated by proteinase K and chymotrypsin. The quench ability of AuNPs observed in Fig. 4-17B, the UV-light excited fluorescence band of 5.6A/1482p-FITC without activated by proteinase was very weak and was considered the unbounded peptide substrates effect. The evidence of both free and bound peptide substrate cleaved by proteinase also was given in Fig. 4-17B.
4-4. Chymotrypsin assay by AuNPs probes
4-4-1. pH optimization of chymotrypsin sensitivity to AuNPs probe
The 10.0A/1477p-FITC (1.25 nM) was incubated with fixed concentration chymotrypsin (200 ng/mL) for 1hr at 37°C under different pH (pH 6.0, 7.0, 7.4, 8.0, 9.0 and 10.0). Fig. 4-18 displayed that the optimal activity condition of chymotrypsin was at pH 8.
4-4-2. Comparison of different AuNPs probes to chymotrypsin sensitivity
The sensitivity to chymotrypsin of the two AuNPs probes was compared. The
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7.4A/1466p-FITC and 10.0A/1477p-FITC (1.25 nM) were incubated with different
concentrations chymotrypsin (25, 50, 100, 150, 200, 300, 400 and 500 ng/mL) for 1 hr at 37°
C, pH 8.0. Fig. 4-19A showed that using 10.0A/1477p-FITC probe to detect chymotrypsin could acquire confident linear correlation while 7.4A/1466p-FITC probe showed very low sensitivity. The 10.0A/1477p-FITC probe could be used to detect the concentration of chymotrypsin ranged from 25 to 500 ng/mL, of which it y = 6.75 x + 87.34 and R² = 0.96.
The comparison in time course of the activity of fixed concentration chymotrypsin (200 ng/mL) between two AuNPs probes was also given in Fig. 4-19B, and the detection time differs from 0.25 to 4 hr at 37°C, pH 8.0. The results also corresponded to the concentration results; that the 10.0A/1477p-FITC probe showed time correlated to delta fluorescence intensity, while 7.4A/1466p-FITC probe showed very low increase instead.
4-4-3. AuNPs probes (10.0A/1477p-FITC) to chymotrypsin sensitivity
Fig. 4-20 displayed different reaction time to different concentrations of chymotrypsin relation of delta fluorescence intensity. The 10.0A/1477p-FITC (1.25 nM) probe were incubated with different concentrations chymotrypsin (25, 50, 100, 150, 200, 300, 400 and 500 ng/mL) for 30 min and 1hr at 37°C, pH 8.0. The results showed that shorten the detection time to 30 min also could gain a perfect linear correlation range from 25 to 300 ng/mL, of which y = 5.07 x - 105.87, R² = 1.
4-4-4. AuNPs probes (10.0A/1477p-FITC) specificity to chymotrypsin
The specificity of 10.0A/1477p-FITC probes was investigated. Serine protease like trypsin was compared with chymotrypsin. The 10.0A/1477p-FITC (1.25 nM) probe were incubated with various concentrations trypsin (50, 100, 200, 300, 400, 500 and 600 ng/mL) for 1 hr at 37°C, pH 8.0. Although the results given in Fig. 4-21 showed that the linear
correlation ranged from 200 to 600 ng/mL chymotrypsin was confident, of which y = 0.62 x -
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76.48 and R² = 0.99; compared with the specificity of chymotrypsin is relatively low about 10 fold fluorescence intensity change.
4-4-5. BTEE assays of chymotrypsin
The chymotrypsin assay commonly is used by measuring an increase in absorbance at 256 nm resulting from the hydrolysis of BTEE. One unit is defined it hydrolyzes one μmole of BTEE per min at pH 7.8 and 25°C under the specified conditions. To compare with AuNPs probes detection limit, the BTEE assays detection limit was investigated. As shown in Fig. 4-22, various concentrations of chymotrypsin (100- 1000 ng/mL) react with BTEE and obtained a change in A256. The BTEE assays could acquire a linear correlation range from 200 to 600 ng/mL, of which y = 7E-05x + 0.0034 and R² = 0.995.
4-4-6. Chymotrypsin activity assay by AuNPs probe (5.6A/1482p-FITC)
To lower the detection limits and in short detection time for chymotrypsin activity assay, the 5.6A/1482p-FITC applied. Fig. 4-23A acquired different reaction time to various
concentrations of chymotrypsin relation of delta fluorescence intensity. The time course of chymotrypsin activity obtained by 5.6A/1482p-FITC (1.25 nM) incubated with fixed
concentrations (1 and 5 ng/mL) for various time (5, 10, 15, 20, 25, 30, 45, 60 and 75 min) at 37°C, pH 9.0. The concentration correlation also displayed in Fig. 4-23B, the
5.6A/1482p-FITC (1.25 nM) was incubated with various concentrations chymotrypsin (0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 ng/mL) for 15 and 30 min at 37°C, pH 8.0. The results showed that delta fluorescence intensity increased with the
increasing concentration of chymotrypsin. In 15 min detection time, the linear correlation ranged from 0.25 to 10 ng/mL chymotrypsin was confident, of which y = 506.69x - 90.82 and R² = 0.99.
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4-5. Animal experiments
4-5-1. Islet isolation from mouse pancreas
The pancreas should be carefully isolated since that is easy confused with fatty tissue.
Therefore, the dithizone (DTZ) staining was performed to confirm the isolation tissue is pancreas. Dithizone binds zinc ions present in the islet's beta cells, and therefore stains the islets red. Fig. 4-24A showed the image of islet and the size is about 250 μm in diameter;
while Fig. 4-24B showed the image of beta cells and the size is about 20 μm in diameter.
4-5-2. Effect of fasting/feeding treatments to intestinal chymotrypsin of mouse
The pancreas can produce proteinases which help in the digestion of food. It is believed that the amount of enzyme would be different in different situation; therefore, fast and feeding conditions were discussed. The mice were both treated with 12 hr fasting and 1 hr feeding for feeding group before being sacrificed. The intestine was divided into five parts: duodenum (about 4 cm), jejunum (part 1, J1; about 7.5 cm), jejunum (part 2, J2; about 7.5 cm), ileum (part 1, I1; about 5 cm) and ileum (part 2, I2; about 5 cm). Every part of intestinal fluid was collected by DPBS washing and conducted in chymotrypsin assay by 5.6A/1482p-FITC probe.
Feeding group owns significant higher amount of chymotrypsin in jejunum (J2) and ileum (I2) than in fast group as shown in Fig. 4-25.
4-5-3. Effect of fasting/feeding treatments to fecal chymotrypsin of mouse
The mice were treated with 3 hr fasting and 5 hr feeding and the fecese were collected during the process. The feces were conducted with lysis and the supernatant was applied in 5.6A/1482p-FITC probe activated for 15 min at 37°C. Fig. 4-26 represents the distribution of chymotrypsin amount in fecal of each period. As the results showed that the 5th hr of fecal had the lowest chymotrypsin activity.
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4-5-4. Cerulein-induced acute pancreatitis mouse model - analysis by plasma amylase and lipase
AP was induced by 4 doses of intraperitoneal injections of cerulein (200 μg/kg/2 hr). All three groups contain four mice. The control subjects were equally treated with 0.9% NaCl (10μL/mg/2 hr for 4 injections). The mice were sacrificed at 8, 10, or 24 hr after the first administration of saline or cerulein. Mice were fasted for 6 hr before being sacrifice. Then the plasma, duodenal fluid, and pancreas were collected.
The collected plasma was analyzed by Fujifilm clinical chemistry analyzer, which of the amylase and lipase levels were determined. The plasma amylase of cerulein-induced group was significantly higher than saline group over 6 and 12 folds in the 8th and 10th hr sacrificed mice, respectively (Fig. 4-27A). Besides, the plasma lipase also showed same phenomenon, which cerulein-induced group was significantly higher than saline group over 4 and 6 folds in the 8th and 10th hr mice, respectively (Fig. 4-27B). The saline group in each sacrificed time had coordinated the amount of plasma amylase (about 4000 U/L) and lipase (about 500 U/L).
It was noted that both plasma amylase and lipase decrease to normal level as saline group in the 24th hr cerulein-induced group.
4-5-5. Cerulein-induced acute pancreatitis mouse model - analysis by chymotrypsin in duodenum and pancreas
The intestinal fluid of duodenum were collected and washed by 1 mL DPBS. The
collected solutions were conducted with 5.6A/1482p-FITC probe to analyze the concentration of chymotrypsin in 15 min at 37°C. The amount of chymotrypsin in cerulein-induced mice showed significantly decrease in duodenum fluid for all three periods compared with saline subjects as given in Fig. 4-28A. The normal level of chymotrypsin is about 45 μg in
duodenum, and decrease to about 11 μg in cerulein-induced mice.
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The pancreas was isolated and lysed to obtain protein in 1 mL of protein extraction solution. The total protein was determined by Bio-Rad protein assay. The chymotrypsin activity in pancreas was analyzed by 5.6A/1482p-FITC probe activated for 15 min at 37°C.
The results shown in Fig. 4-28B displayed that chymotrypsin activity significantly increased in pancreas of cerulein-induced mice than in saline subjects. It was noted that both in the 10th hr cerulein-induced mice that chymotrypsin in duodenum fluid and pancreas had the most significantly change.
4-5-6. Cerulein-induced acute pancreatitis mouse model - analysis by fecal chymotrypsin
The feces were collected at each hour for 24 hr time course after the first administration of saline or cerulein. The supernatant of feces protein extraction was applied in
5.6A/1482p-FITC probe (1.25 nM) activated for 15 min at 37°C. Every 5 hr periods were classified into one group, the groups were: 0 ~ 4th hr, 5 ~ 9th hr, 10 ~ 14th hr, 15 ~ 19th hr and 20~24th hr (Fig. 4-29). The saline subject shows cycle change of chymotrypsin activity due to activity of diet. The groups of 0 ~ 4th hr, 5 ~ 9th hr and 20 ~ 24th hr from cerulein-induced mice had significant lower chymotrypsin activity compared with those of fecal chymotrypsin in saline subjects.
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Figure 4-1. Fluorescence emission spectra of peptide-FITC and adsorption spectra of AuNPs
There is a significant overlap between the emission of FITC (as donor) and the absorbance of 15 nm AuNPs (2.5 nM, as quencher) implies that energy transfer between the two is a
significant quenching mechanism.
0 0.5 1 1.5
0 1000 2000 3000 4000 5000 6000 7000 8000
500 550 600 650 700
Abs (OD)
FL (A.U)
Wavelength (nm)
FITC AuNP
Aceptor (Abs) Donor (Em)
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Figure 4-2. Characteristics of citrate-capped AuNPs analyzed by adsorption spectrum and DLS
(A), the maximum adsorption peak of 15 nm AuNPs is around 518 ~ 520 nm. The spectra of synthesized AuNPs are the line-1X, and dilute to half of its concentration is line-0.5X. (B), the DLS analysis gains the average diameter of citrate-capped AuNPs which is about 21.9 nm.
0 0.5 1 1.5 2 2.5
400 450 500 550 600 650 700
A bs orbanc e (O D )
Wavelength (nm)
1X 0.5X
0 20 40 60 80 100
5.0 21.9 50.0
In tens ity (% )
Diameter ( nm )
(A)
(B)
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Figure 4-3. Size determination of citrate-capped AuNPs by TEM
(A), TEM image of citrate-capped AuNPs. The scale bar of left is 20 nm and right is 10 nm.
(B), histogram of AuNP diameters were determined by analysis of approximately 120 NPs located at different regions of the grid. The average diameter is 14.6 nm ± 2.4 nm.
0 5 10 15 20 25 30 35
5 11 13 19 25
N um ber o f A uN Ps
AuNPs diameter (nm)
(A)
(B)
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Figure 4-4. Adsorption spectra and aggregation levels of citrate-capped AuNPs under different salt stress
(A), the 15 nm AuNPs were treated with various concentrations of NaCl (0 ~ 150 mM) and incubated for 30 min at room temperature. Then the adsorption spectra from 400 ~ 700 nm wavelengths were recorded. (B), the aggregation level was estimated with the ratio of A625 to A525. Over 10 mM NaCl incubated with citrate-capped AuNPs causes significant increment in A625 / A525. Error bars (SD) represent data from three independent detections. **, statistically significant compared with 0 mM NaCl at p-value < 0.01.
0
400 450 500 550 600 650 700
A bs orbanc e (O D )
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Figure 4-5. Aggregation levels of AuNPs with different stabilizers under different salt stress
The citrate-capped AuNPs were mixed with various concentrations of stabilizer and further treated with various concentrations of NaCl (0 ~ 150 mM) and incubated for 30 min at room temperature. The aggregation level was estimated with the ratio of A625 to A525. (A),
polyethylene glycol (PEG) as stabilizer and the final concentrations were (0.1 to 5% (w/w)).
(B), bovine serum albumin (BSA) as stabilizer and the final concentrations of BSA were (0.05 to 7% (w/w)). Error bars (SD) represent data from three independent detections.
0
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Figure 4-6. Adsorption spectra and aggregation levels of citrate-capped AuNPs under different pH stress
The 15 nm AuNPs were adjusted with various concentrations of pH (pH 4.0 ~ 11.0) and incubated for 30 min at room temperature. (A), the adsorption spectra of from 400 to 700 nm wavelengths of AuNPs under different pH stress were recorded. (B), the aggregation level was estimated with the ratio of A625 to A525. Citrate-capped AuNPs with different pH shows no effect in A625 / A525. Error bars (SD) represent data from three independent detections.
0
400 450 500 550 600 650 700
A bs orbanc e (O D )
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Figure 4-7. UV-Vis spectra study on AuNPs probe of different charges peptide substrate After modification of AuNPs probes, the excess peptides substrates were removed and suspended in various pH of buffer (pH 4.0, 5.6, 7.4 and 11.0). (A), the positively charged modification process of 4.0A/1466p-FITC probe and suspended in buffer. (B), the neutral charged modification process of 5.6A/1466p-FITC probe and suspended in buffer. (C), the negatively charged modification process of 7.4A/1466p-FITC probe and suspended in buffer.
(D), the negatively charged modification process of 11.0A/1466p-FITC probe and suspended in buffer; however, the spectra of pH 11.0 modification before removal process gained not smooth curve. Besides, the UV-vis spectra show that the AuNPs probe is stable in neutral and negatively charged environments and the spectra show little red shift in positively charged environment. Buffer pH 5.6-4.0A Buffer pH 11-4.0A
0 Buffer pH 5.6-5.6A Buffer pH 11-5.6A
0 Buffer pH 5.6-7.4A Buffer pH 11-7.4A
0 Buffer pH 5.6-11.0A Buffer pH 11-11.0A
(A) (B)
(C) (D)
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Figure 4-8. The aggregation levels of 1466p-FITC in modification and suspension state of differently functionalized pH
(A), modification state: the 15 nm AuNPs were adjusted with various concentrations of pH (pH 4.0 ~ 11.0) and modified with 1466p-FITC to form A/1466p-FITC. (B), suspension state:
after the removal excess peptide substrates, the A/1466p-FITC probe was suspended in 0.1 % BSA solution. The adsorption spectra of 400 ~ 700 nm wavelengths were recorded at both states. The aggregation level was estimated by the ratio of A625 to A525. The positively charged of pH 4.0 showed serious aggregation. Error bars (SD) represent data from three independent detections.
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Figure 4-9. The aggregation levels of 1477p-FITC in modification and suspension state of differently functionalized pH
(A), modification state: the 15 nm AuNPs were adjusted with various concentrations of pH (pH 4.0 ~ 11.0) and modified with 1477p-FITC to form A/1477p-FITC. (B), suspension state:
after the removal excess peptide substrates, the A/1477p-FITC probe was suspended in 0.1 % BSA solution. The adsorption spectra of 400 ~ 700 nm wavelengths were recorded at both states. The aggregation level was estimated with the ratio of A625 to A525. The positively charged of pH 4.0 and 5.6 show serious aggregation, but the pH 7.4 remains stable. After removal- suspension process, the pH 7.4 of AuNPs modification shows aggregation. Error bars (SD) represent data from three independent detections.
0
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Table 4-1. The function of fluorescence intensity from peptide substrates (pH 8.0)
peptide-FITC R2 Supernatant R2 DTT R2
7.4A/1466p-F y = 64.01x+87.11 0.98 y = 133743x+279.96 0.98 y = 94705x-0.03 0.99 10.0A/1477p-F y = 65.97x+358.8 0.97 y = 200352x+53.67 1.00 y = 98333x+266 0.99 5.6A/1482p-F y = 68.91x-64.59 0.99 y = 326424x-320.17 0.99 y = 86764x+164.11 1.00
Table 4-2. The ratio of diffferent parts of peptide substrates to total loading peptide substrates (pH 8)
Table 4-3. Conjugation ratio of various peptide substrates per 15 nm AuNPs Mw of
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Table 4-4. Zeta potentials of citrate-capped AuNPs and AuNPs probes
Modification (ZP, mV) Purification (ZP, mV) citrate-capped AuNPs -34.7 (±13.8)
7.4A/1466p-FITC -52.2 (±8.25) -17.2 (±6.73)
10.0A/1477p-FITC -43.4 (±5.73) -15 (±5.03)
5.6A/1482p-FITC -39.2 (±15.9) -34.3 (±9.02)
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Figure 4-10. Effect of different stabilizers to proteinase K sensitivity by fluorescence assays of 7.4A/1466p-FITC
(A), the shorty immersed in PEG solution affection. AuNPs probe washed with different concentrations of PEG (0.5 to 5% (w/w)) and suspended with 0.1% BSA (w/w) were prepared.
The 7.4A/1466p-FITC was incubated with fixed concentration proteinase K (100 ng/mL) for 1 hr at 37°C. The concentration of PEG has no effect to proteinase K activity, except 5% PEG is significant lower compared with 0% PEG. (B), the BSA as stabilizer affection. AuNPs probe washed with 2% PEG (w/w) and suspended with different concentration of BSA (0.1 to
The 7.4A/1466p-FITC was incubated with fixed concentration proteinase K (100 ng/mL) for 1 hr at 37°C. The concentration of PEG has no effect to proteinase K activity, except 5% PEG is significant lower compared with 0% PEG. (B), the BSA as stabilizer affection. AuNPs probe washed with 2% PEG (w/w) and suspended with different concentration of BSA (0.1 to