Dahai Ren*,
Jun Wang and
Zheng You
State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instrument, Tsinghua University, Beijing, 100084, China. E-mail: rendh@tsinghua.edu.cn
First published on 15th October 2014
Conventional probes used to detect proteases are susceptible to either photobleaching or low efficiency in cell penetration. Also it is difficult to achieve multiplexed detection. Based on QD, nanogold and EGF, a unique probe was studied in this paper. The sensing section of the probe was developed by linking the streptavidin-labeled QD and monomaleimide-functionalized nanogold via a substrate peptide. The QD fluorescence is partially quenched by the nanogold when they are connected. After the substrate peptide is cleaved by protease, the QD fluorescence can be partially recovered as the distance between two kinds of nanoparticles increases. Biotin-labeled EGF was used to carry the sensing section into the cells, making the transfection efficiency higher than former nanoprobes. After assays, we found the optimal ratios of nanogold to QD and to EGF to realize the high efficiency of both quenching and transfer. Also an algorithm was proposed to evaluate the relative activity of proteases. Finally, caspase-3 was used as the target protease to be detected. The activity of caspase-3 was successfully monitored during a longer time span compared to the reported probes.
Fluorescence resonance energy transfer (FRET) probes composed of dual proteins or dual dyes6–9 can be used to detect the activity of intracellular proteases like caspase-3. However, overlapping between the donor and accepter cannot be avoided and leads to low signal-to-noise ratios. Some cell penetrating single-color probes also occurred,10–13 but the weak resistance of photobleaching on these kinds of probes determines that all of them can only be observed for a limited time. Because of the excellent optical properties of quantum dots (QD), high quenching efficiency, and the outstanding bio-compatibility of nanogold, they have been employed respectively as donor14,15 and quencher16,17 to constitute FRET probes. This can avoid fluorescence overlapping and allow much longer detection time. However, unlike small molecules, the probes constituted by nanoparticles are unable to freely diffuse into the cells. As a result, the cell penetrating efficiency of these probes is low.18–21
Actually, the above mentioned FRET probes also cannot accurately reflect the activity of proteases as they may partially diffuse into the surrounding solution from the cytoplasm in a short time. This means that the measurement result derived from the fluorescence signal is not accurate. In 2008, Cen22 synthesized a new probe consisting of a fluorogenic DNA dye (NucView488) and a DEVD substrate moiety specific to caspase-3. The probe is both non-fluorescent and nonfunctional as a DNA dye, and can rapidly pass through cell membranes and enter the cytoplasm. This allows for the real-time detection of caspase-3 activities in live cells to some degree. However, it could not accurately reflect the activities of caspase-3, as not all the NucView488 are combined with the DNA. Also, like other dye-based probes, it is susceptible to severe photobleaching. To summarize, the reported probes are susceptible to either photobleaching or low efficiency in cell penetration, or both.23–26
In this paper, based on the advantages of the reported probes, a new kind of probe was developed to overcome the above mentioned deficiencies. As shown in Fig. 1, the probe is composed of streptavidin-labeled QD, monomaleimide-functionalized nanogold, substrate peptide and biotin-labeled EGF. The substrate peptide mainly contains four functional sections: biotinylated N-terminal, C-terminal cysteine, protease recognition sequence and flanks. N-terminal biotin is used for coupling to the streptavidin-labeled QD. Cysteine at the C-terminal is used for coordinating coupling to a monomaleimide-functionalized nanogold. Protease recognition confirms the sub-peptide to be specifically cleaved by the corresponding protease. The function of the flanks is to decrease the steric hindrance and increase the chance that the recognition sequence can be cleaved.
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Fig. 1 Schematic principle of caspase-3 detection in living cells with the EGF–QD–peptide–nanogold probe. |
The sensing section of the probe is developed by linking the streptavidin-labeled QD and monomaleimide-functionalized nanogold via a substrate peptide. When the distance between the QD and nanogold is short enough (less than 20 nm), the QD fluorescence will be partially quenched by nanogold as a result of energy transfer or some other mechanisms. After the substrate peptide is cleaved by protease, the distance between the two kinds of nanoparticles would increase. As a result, the QD fluorescence can be partially recovered. Just like the substrate peptide, the biotin-labeled EGF also couples to the streptavidin-labeled QD. It can efficiently carry the sensing section into the cells by bonding with the EGFR,27,28 which is ubiquitously expressed in normal cells and preferentially over-expressed on the surface of many cancer cells.29,30
Assembling this probe is simple. It can be used to detect intracellular proteases during a prolonged time span and give accurate results, and is a most promising probe that can be used in real-time monitoring of protease activity in living cells.
70 μl nanogolds solution (2 nmol) was then pipetted and added into the 500 μl reduced sub-peptide solution. The ratio of sub-peptide to nanogolds is about 50. The excess amount of peptide compared to the nanogold was used to ensure sufficient binding of the peptides to the nanogolds.15 The resulting solution was incubated for 20 hours at 4 °C without stirring.
After the reaction, the resulting solution was spin-filtered using a centrifugal filter (microcentricon, YM-10) with an exclusion cutoff of 10 kDa to separate the unbound peptides from the nanogold conjugates (nanogold clusters typically have a molecular weight of 15 kDa according to product specifications). Then, double-distilled water was added, and the solution was spin-filtered. The concentrated conjugates were resuspended in 335 μl PBS and added into an appropriate quantity of BSA (the final concentration of BSA is 0.1%).33 The concentration of the conjugated nanogolds is 5 pmol μl−1, and is determined according to specifications in the manual. At this time, the nanogolds conjugated with the sub-peptides (sub-nanogold) were obtained and the resulting conjugates were stored at 4 °C until use. As a control group, the remaining free nanogolds were reserved without any treatment.
The SA-QD605 was mixed with certain amounts of sub-nanogolds and biotinylated EGF in a 384-well plate for 30 minutes at room temperature to allow specific association between SA and biotin.15 The ratios of EGF to QD and sub-nanogold to QD are about 3 and 12.5 respectively.
The final concentration of the probe in an aqueous solution was typically 5 nM (corresponding to 0.4 pmol in 80 μl reaction volume). The probe fluorescence intensity was measured at an excitation wavelength of 360 nm by using a microplate reader (Envision, PerkinElmer, USA).
HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Macgene, China) and supplemented with 15% (vol/vol) FBS (Gibco, USA), and 1% penicillin/streptomycin solution. The cells were cultured in a humidified atmosphere of 5% (vol/vol) CO2 and 95% air (vol/vol) at 37 °C.
To determine the ideal concentration ratio of EGF to QD, we conducted assays on EGF-QD entering into cells. Similarly, fixed amounts of QD (0.4 μl, 1 μM) were mixed with varying amounts of EGF (0, 0.05 μl, 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 0.6 μl, 0.8 μl, and 1 μl). The EGF concentration was 6 μM. The results showed that there was no clear difference in transfer efficiency when the amount of EGF was more than 0.2 μl. To make sure that the QDs can, to a large degree, combine with and be quenched by nanogolds, in later assays, we fixed the ratio of EGF to QD as 3. According to the manual for the QDs, one QD is covered by 3 to 8 streptavidins. One streptavidin is able to be combined with 4 biotins on average. Therefore, in theory one QD can combine 12 to 32 biotins, namely 1 pmol (1 μl) QDs which can fully react with 22 pmol biotins. Given steric hindrance, the actual number may be less than 22, and 0.4 μl QD (1 μM) can still fully react with 1.52 μl sub-nanogold (5 μM). Actually, 0.5 μl of nanogold is enough to quench 0.4 μl QD. The quenching efficiency of nanogold to QD did not change greatly when 0.2 μl EGF was added (Fig. 2b).
Comprehensively considering the quenching efficiency, the transfection efficiency and the reagent saving, we constructed the probes by mixing 0.4 μl QD (1 μM) with 0.2 μl EGF (12 μM) and 1 μl nanogold-sub (5 μM). Specifically, the ratios of EGF to QD and sub-nanogold to QD are about 3 and 12.5 respectively. The probe solution was stored at 4 °C until use.
To confirm that the free nanogold will not quench the QD fluorescence, we mixed an appropriate amount of QD with varying amounts of free nanogold (Fig. 2c). There was negligible change in the fluorescence intensity of the QD solution after 1 μl, 6 μM pure nanogold was added. Therefore, the effect of free nanogold on QD fluorescence can be ignored.
We also further confirmed that the quenching of nanogold to QD required participation of biotin at the N-terminal of a substrate peptide. Before adding the nanogold–substrate peptide conjugate into the QD solution, first we added superfluous free biotin into the streptavidin–QD solution, and then found that the QD's FI would not be quenched by the sub-nanogold again, fitting the reported result.37
The nanoparticles and probe were also characterized by TEM (Fig. 3). As shown in Fig. 3b, QDs were successfully conjugated to nanogolds.
As shown in Fig. 4, without caspase-3, the probe's FI was stable in the buffer solution, showing negligible fluorescence signal change within 90 minutes. The FI of the pure QD was the same. After adding caspase-3, an approximately 3-fold increase in the fluorescence intensity of the probe was observed. Since the fluorescence recovery was efficiently blocked in the control group by the adding caspase-3 inhibitor, we concluded that the increase in the fluorescence intensity of probes was caspase-3-specific.
The results showed that the probe fluorescence could be recovered by caspase-3. According to the control group, the probe fluorescence did not increase obviously, indicating that the activity of caspase-3 was effectively inhibited by the AC-DEVD-CHO and the recovery of fluorescence should be credited to caspase-3. However, recovery efficiency was not 100%, even though the probe was incubated with superfluous caspase-3. We presumed that this resulted from steric hindrance, namely that not all of the cleavable sites of the substrate peptides was accessible to caspase-3. Then, we conducted assays to confirm. First, we mixed the caspase-3 with the sub-nanogold, and the resulting solution was added with QD. The fluorescence intensity (FI) of this mixture was the same as that of the pure QD, even stronger (Fig. 5). The stronger FI may also result from the biotin at the substrate peptide. In any case, the result confirmed that the large QD size would hinder the cleavage of caspase-3 to the substrate peptide.
We could observe obvious intracellular fluorescence in group C (Fig. 6c) and all the cells were stained with red fluorescence. However, no red fluorescence could be observed in group A (Fig. 6a), and only weak fluorescence in several cells could be observed in group B (Fig. 6b).
Cell nucleus dyeing was employed to test whether EGF can bring quantum dots into the nucleus of cells. QD positions were observed with a confocal fluorescence microscope. Fig. 6(d)–(f) show the fluorescence locations at the nucleus which was stained with hochst33342, indicating that EGF carried QDs into the cell nucleus. As a result, it is difficult for QDs to diffuse into the surrounding solution from the cytoplasm in a short time. As mentioned in “Introduction”, diffusion into the surrounding solution from the cytoplasm is a disadvantage of reported probes. These results show that EGF is an ideal candidate for carrying nanoparticles into the cells.
Furthermore, we employed the anti-EGFR to occupy the EGFR before adding a probe, and we found that the efficiency of transfection became lower compared to not adding anti-EGFR. It was confirmed that the probe was carried into cells by EGF.32
According to our assays, most of the probes entered into the nucleus after one and a half hour (Fig. 6), if TNF-α and AcD was not added. However, if TNF-α and AcD were added into the culture medium about ten minutes after the probes was added, most of the probes would not enter into the nucleus (can be seen in Fig. 12). This can be explained by the fact that apoptosis of cell would decrease the rate of material transport.
As the probes are modified with EGF, they will enter into the endosome. It is hard to confirm that all the probes would be released from endosome. However, a portion of the probes would certainly be released, as the endosome would finally be melted by the lysosome. Particularly, the function of lysosome will increase when apoptosis happens. Therefore, (when cells are killed) the probes are able to characterize the activity of caspase-3 not only in endosome.
Another characteristic of the probes should be noted is that the probes will activate EGF signaling pathways and activate the receptor tyrosine kinase pathway. However, it will not negatively affect the working of the probes. First of all, the activity of tyrosine kinase pathway is not necessarily related to the activity of caspase-3. Besides, to some degree, the toxicity of nanoparticles or dyes could be decreased by EGF which will improve the activity of cells. We culture the cells with the probes for days, and cells could grow normally before induction. However, the growth of cells would be affected if cells were cultured with nanoparticles without modification by EGF or cultured with dyes. For example, their growth rate became slower and their shapes became abnormal. Because normal cell growth is preserved, the using of EGF could not be regarded as disadvantage, but, to some degree, advantage.
To further confirm that the probes were indeed intracellular, they were also observed using a transmission electron microscopy. The cells of the test group were incubated with the probe. The control group was pure cells without a probe. Details of sample preparation have already been described.38 We can clearly see that the nanoparticles were intracellular in Fig. 7a. However, most of the nanogolds were separated from the QDs. It probably resulted from the effect of sectioning. There are no nanoparticles in control group cells (Fig. 7b).
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Fig. 7 TEM images of a cell section. Intracellular nanoparticles conjugates observed by TEM. (a) Significant accumulation of nanoparticles (QDs). (b) HeLa cells without incubation with a probe. |
After induction for 12 hours, we detected the activity of caspase-3 in cells using the reagent kit purchased from Biovision and Biotium. According to the protocol, we separated cells into groups A (induced and then added substrate), B (induced and added inhibitor, then added substrate) and C (without induction but added substrate). Group A was induced without adding inhibitor. In group B, before adding the TNF-α and AcD, AC-DEVD-CHO and Z-VAD-FMK were added as a caspase inhibitor. Group C contained cells without induction.
Next, we added caspase-3 probes into the three groups and incubated them in a humidified atmosphere of 5% (vol/vol) CO2 and 95% air (vol/vol) at 37 °C for 45 min. Afterwards, the three groups were observed under the fluorescence microscope (Fig. 8). Group A (group induced without inhibitor) is shown in Fig. 8a, and 50% of the cells had shrunk. The picture shows the shrunken cells which are stained with the green fluorescence. It conforms to the fact that the caspase-3 would be activated when apoptosis occurs. In group B (Fig. 8b), the ratio of the stained cell is about 10%, lower than group A (50% ratio). It can be explained by the inhibition of caspase-3 activity, and could hinder cell apoptosis. Fig. 8c shows that the ratio of stained cells in group C was about 1%, and almost all non-stained cells were in good shape.
The conclusion from the assays was that after cells were induced by TNF-α and AcD, the caspase-3 was activated, and the shape of the corresponding cells shrank. A caspase-3 inhibitor could reduce their occurrence probability.
Twelve hours later, the three groups were observed under the fluorescence microscope. The results are shown in Fig. 9 are similar to Fig. 8. The induction time in Fig. 8 was the same as that in Fig. 9, which was 12 hours. In Fig. 9a (group A), nearly half of the cells shrank. The shrunken cells are stained with the red fluorescence. The red fluorescence was gathered at the cell nucleus. It confirms to the fact that the EGF could carry the QD into the nucleus. In group B, the ratio of stained cell is about 10%. It is lower than that of group A (40%). Therefore, inhibiting caspase-3 activity could hinder the apoptosis of the cells. From Fig. 9c, we can deduce that the ratio of stained cells in group C was about 4%, and almost all the cells without clear fluorescence were in good shape.
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Fig. 9 Characterization of the activity of caspase-3 in living HeLa cells by the probes. (a) Induced. (b) Induced with added inhibitor. (c) No induction. |
We characterized the activity of caspase-3 by the product of the mean relative red fluorescence intensity (MRRFI) in the cells and the ratio of cells with clear fluorescence to the total. First, we extracted the red channel of the image. Second, the background fluorescence of the image was erased and we targeted areas where the value of the color was higher than the threshold we set. It is shown on the right of Fig. 9. Third, we calculated the average of the color value in all areas, namely the mean relative red fluorescence intensity (Table 1). Then, we counted the ratio of cells with clear fluorescence to the total. Table 1 shows both the MRRFI and the ratio. Finally, the relative activity of caspase-3 was reflected by the relative fluorescence value, which is the product of the MRRFI and the ratio, namely relative fluorescence value = MRRFI × ratio. The result is shown in Fig. 10.
Group no. | A | B | C |
---|---|---|---|
MRRFI | 0.4736 | 0.4773 | 0.4373 |
Ratio (%) | 40 | 10 | 4 |
The cells were separated into groups A and B, and then the cells of group A were labeled with QD605, and group B with Streptavidin Alexa Fluor® 488 (SA-Alex488). Then we randomly selected a region and excited it continuously. At the same time, photos were taken at certain time point (Fig. 11). From (a), we can see that the STR-488 faded quickly when exposed to excitation by laser. Just 0.5 min later, the fluorescence intensity of STR-488 reduced obviously, and the fluorescence of STR-488 almost disappeared after being excited for 1 minute.
QD605 was quite different, which can be seen from the series of pictures (b). After excitation for 2 minutes, the red fluorescence of QD605 became clearer rather than faded, because of the decrease in background noise. Moreover, 6 minutes later, the probes' fluorescence intensity within the cells was almost unchanged. Actually, QDs are able to weather the excitation of a laser source for hours or even days.30,39 The conclusion is that the probe based on QD is able to meet the requirements of prolonged protease activity monitoring in living cells, which could not be achieved by conventional dye or fluorescent proteins.
As stated above, the long-term monitoring almost totally depends on the photobleaching resistance of fluorescence component contained in the probe. Therefore, conventional dye is not applicable to long-term continuously monitor the alteration of caspase-3 activities. Further experiments were taken to support the statement that one major advantage of this QD-based probe is long-term monitoring of caspase-3 activity.
We synthesized another kind of probe by replacing the SA-QD605 with SA-Alex488, and then we monitored the activity of caspase-3 using two kinds of probes. When the fluorescence of the cells were detected one time per hour and in each time the excitation lasted 3 seconds to complete the image acquisition, the results of caspase-3 characteristics were similar. As shown in (a and b) of Fig. 12, the fluorescence intensity of both groups increased. The difference between the results could be attributed to the difference in steric hindrance and photobleaching resistance.
However, continuous monitoring the activity of capase-3 over several minutes is impossible with conventional dyes as they were bleached quickly. As shown in (c and d) of Fig. 12, after excitation for 10 minutes, the red fluorescence of the probes based on QD605 was almost unchanged. However, the green fluorescence of the probes based on Alexa Fluor® 488 faded quickly.
Because the emission spectrum of QD is much narrower than conventional dye or fluorescent protein, the probe based on QD is also the best candidate for the parallel detection of proteases in living cells. This can be achieved by replacing the specific substrate peptide and QD. One kind of protease corresponds to one kind of specific substrate peptide and QD with a specific different color. The probe we developed has the potential to illustrate the mechanism of proteases by exerting their function, and may serve as a model for strategies aimed at monitoring the activity of other intracellular proteases. Moreover, this kind of probe is not limited to detecting the activity of proteases. It can also be used to detect other types of enzymes like DNA, and even certain kinds of molecules.
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