Internal standard fluorogenic probe based on vibration-induced emission for visualizing PTP1B in living cells

Abstract

Herein, as a proof of concept, we developed the first enzymatic VIE fluorogenic probe for protein tyrosine phosphatase 1B (PTP1B). The detection and imaging of PTP1B using VIE in living cells were both realized. Particularly importantly, the designed probe herein provides a guideline and platform for the development of new VIE-based enzymatic probes.

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Vibration-induced emission (VIE), a specific fluorescence phenomenon that involves the intrinsic display of orange-red fluorescence for the free state but abnormal display of blue fluorescence in the constrained state, has drawn considerable attention because of its exploitation in flexible multi-color emission.^1,2 Biological imaging based on the VIE phenomenon generates great interest for us since a distinctive change in fluorescence (large Stokes shift up) can be caused by an intrinsic change in the solubility of the molecule in question or by a change in its planarity. Hence, developing a VIE-based probe for visualizing enzyme activity is extraordinarily fascinating.

Phosphatases constitute a large and structurally diverse class of signalling enzymes that catalyze the removal of the phosphate group from their substrates and play important roles in human diseases.³ Protein phosphatases (PPs) are the most common and important phosphatases. Protein tyrosine phosphatases (PTPs, a major class of PPs) in particular are associated with tumorigenesis in numerous types of cells and tissues.⁴ Acid and alkaline phosphatases (ACP, ALP) are other important examples, as their levels of expression have been routinely used for disease diagnosis.⁵ Thus the biological fluorescence imaging/detection of the activities of phosphatases have attracted a lot of attention.⁶ Herein, as a proof of concept and taking advantage of the simply designed strategy used for phosphatase probes, we developed the first enzymatic VIE fluorogenic probe for a phosphatase. Very interestingly, this new probe exhibited a novel type of VIE performance. In contrast to the aforementioned VIE phenomenon, the new probe showed VIE before and after enzymatic reaction with protein tyrosine phosphatase 1B (PTP1B, a common phosphatase), i.e., the intensity of the red-light emission at a wavelength of about 600 nm remained almost constant during the reaction and acted as an “internal standard” while the intensity of the blue-light emission at about 450 nm increased as the reaction proceeded. Such “internal standard” probes show several advantages, e.g., no need for an “external standard” and independence of the fluorescence ratio.⁷ Meanwhile, the currently developed probe exhibited good selectivity towards PTP1B over other phosphatases and fast response features. Imaging of PTP1B in living cells by using this VIE trait was also realized.

As shown in Fig. 1a, the phosphatase probe (Q1) was constructed by introducing a phosphate radical to the VIE matrix. The probe when showing good solubility in solvent exhibited the “free” vibration state, namely an emission of red light. And when the probe reacted with phosphatase, the fluorophore (3) showed a “fixed” vibration state that led to an emission of blue light. Importantly, the intensity of the emission of red light at about 600 nm remained almost constant during the course of the reaction while the intensity of the blue-light emission increased, which reflected the different solubilities to some extent. This hypothetical phenomenon was also in good accordance with the aforementioned VIE behavior and suggested that the probe can be used as a VIE-based imaging tool (Fig. 1b). Meanwhile, the structures of the intermediate product and Q1 were well characterized from ¹H/¹³C-NMR and high-resolution mass spectra (ESI,† Fig. S1–S7).

Fig. 1 (a) The synthetic route to probe Q1: i. 4-iodoanisole, K₂CO₃, (CF₃SO₃)₂Cu, 1,3,5-trichlorobenzene, 210 °C for 8 h; ii. BBr₃, DCM, −78 °C to room temperature overnight; iii. POCl₃, pyridine, DCM, 0 °C for 2 h. (b) Schematic illustration of the use of Q1, which showed the VIE phenomenon before and after reacting with phosphatase in living cells.

The fluorescence of Q1 in the absence/presence of PTP1B was studied first. (The absorption spectrum of Q1 is shown in Fig. S8, ESI.†) As shown in Fig. 2a, before the enzymatic reaction, Q1 itself showed fluorescence emissions both at 450 nm (quantum yield: 0.08) and 600 nm. Interestingly, after the enzymatic reaction with PTP1B (from lower concentration to higher concentration), the intensity of the emission at 450 nm increased evidently (quantum yield: 0.16), while that at 600 nm remained approximately constant, indicating that it can act as an “internal standard”. This curious phenomenon was similar to the previous VIE performance, but different from aggregation-induced emission (AIE). In the AIE case, the probe exhibits almost no fluorescence before the enzymatic reaction, and only after the reaction does the system show enhanced fluorescence.⁸ Meanwhile, in the current work, the color of solution changed from light red to blue (inset of Fig. 2a). The fluorescence intensity ratio I₄₅₀/I₆₀₀ was then used to judge the solubility of Q1 or 3 as well as to provide a quantitative determination of the concentration of PTP1B (i.e., the higher the I₄₅₀/I₆₀₀, the poorer the solubility of the compound or the higher the concentration of PTP1B in the reaction system). The I₄₅₀/I₆₀₀ values of both Q1 and 3 increased with decreasing content of DMSO, while the probe showed a lower I₄₅₀/I₆₀₀ value in the presence of the preferable co-solvent Triton-100 (Fig. S9, ESI†), i.e., Q1 displayed better solubility than did 3 here. This important phenomenon proved that the above fluorescence changes arose from the VIE phenomenon. Q1 also exhibited excellent response to PTP1B, with a fluorescence intensity showing a good linear relationship with PTP1B concentration in the concentration range of 0.01–0.1 μg μL⁻¹ (Fig. 2b). The detection limit was determined to be 5.7 ng μL⁻¹ of PTP1B. Notably, Q1 also showed a rapid response and good selectivity (Fig. 2c and d) under the optimized conditions (Fig. S10, ESI†) as well as good stability (Fig. S11, ESI†). Note that the enzymatic cleavage product was confirmed using mass spectral analysis (m/z = 449.4 [M]⁻; Fig. S12, ESI†). At the same time, the inhibitor and high-temperature experiments (i.e., where PTP1B was first treated with inhibitor or at high temperature, Fig. S13 and S14, ESI†) showed that the changes of I₄₅₀/I₆₀₀ values indeed originated from the enzymatic reaction. According to the Michaelis–Menten equation (Fig. S15, ESI†), the corresponding Michaelis constant (K_M), the maximum of the initial reaction rate (V_max), K_cat and K_cat/K_M for the present enzymatic reaction were determined to be 94.56 μM, 0.4 μM s⁻¹, 0.4 s⁻¹ and 4.23 × 10³ s⁻¹ M⁻¹, respectively. Moreover, Q1 and 3 showed good biocompatibility (Fig. S16, ESI†).

Fig. 2 (a) Fluorescence response of Q1 (at 2 μM) to PTP1B at various concentrations (from bottom to top: 0–0.4 μg μL⁻¹). The inset shows the fluorescence of the solution before (1) and after (2) the reaction. (b) The I₄₅₀/I₆₀₀ fluorescence intensity ratio of the reaction system as a function of PTP1B concentration. (The inset shows a plot of I₄₅₀/I₆₀₀versus the PTP1B concentration at low PTP1B concentrations.) (c) Effect of reaction time on the fluorescence of Q1 (2 μM) in the presence of PTP1B (150 ng μL⁻¹). (d) Values of I₄₅₀/I₆₀₀ of Q1 (2 μM) in the presence of various species: 1. Q1 (2 μM), 2. 150 mM KCl, 3. 2.5 mM CaCl₂, 4. 100 μM ZnCl₂, 5. 100 μM CuSO₄, 6. 5 mM GSH, 7. 0.3 μg μL⁻¹ alanine, 8. 0.3 μg μL⁻¹ glutamine, 9. 0.3 μg μL⁻¹ DPP4, 10. 0.3 μg μL⁻¹ PGP-1, 11. 0.3 μg μL⁻¹ lipase, 12. 0.3 μg μL⁻¹ protease, 13. 0.1 μg μL⁻¹ BSA, and 14. 300 ng μL⁻¹ PTP1B. Conditions: λ_ex = 360 nm, 10 mM phosphate-buffered saline (PBS; pH 7.4) containing 1% DMSO and 0.01% Triton-100 as co-solvent.

Surprisingly, we found that Q1 displayed a better response to PTP1B than to other common phosphatases. As shown in Fig. 3a and Fig. S17a (ESI†), the probe showed good selectivity for PTP1B over the other common phosphatases tested, namely Gleep, StepCD, N12, SSH2, PPM1A, PPM1B, PPMAK, ACP and ALP. (Except for ALP and ACP (purified), the other phosphatases were extracted and purified, Fig. S18, ESI†). On the other hand, Q1 showed no selectivity for TCPTP (data not shown). However, the commercial phosphatase probe (DiFMUP) exhibited poor selectivity for these phosphatases (Fig. 3b and Fig. S17b, ESI†).

Fig. 3 (a) I₄₅₀/I₆₀₀ values of Q1 (2 μM, λ_ex = 360 nm) and (b) intensity values of DiFMUP (2 μM) in the presence of various phosphatases (10 ng μL⁻¹): Gleep, PTP1B, StepCD, N12, SSH2, PPM1A, PPM1B, and PPM1K. λ_ex = 350 nm; λ_em = 450 nm. All samples were incubated at 37 °C in 10 mM PBS (pH 7.4) containing 1% DMSO and 0.01% Triton-100 as co-solvent for 40 min.

To intensively study this surprising phenomenon, the molecular docking method was used. Unfortunately, we found that the probe was modelled to form interactions with these phosphatases similar to those modelled with PTP1B (Fig. S17c and d, ESI†). So, the preferable selectivity of the probe toward PTP1B can be attributed to steric hindrance of the probe to access the respective active sites of the phosphatases.^6d,9

The potential of using the VIE probe to image living cells was a very attractive idea for us at this stage, so we utilized the probe to monitor PTP1B in living cells. Interestingly, as shown in Fig. S19 (ESI†), the ratio of the fluorescence intensity of the green channel (430–530 nm) to the nearly constant fluorescence intensity of the red channel (550–650 nm) for HeLa cells increased with incubation time (and the fluorescence of the merged images changed from yellow green to green), which indicated that Q1 can respond to PTP1B in living cells. Note that this phenomenon was in good accordance with the aforementioned VIE performance. A similar result was also found when using HepG2 cells (Fig. S20, ESI†).

In order to further highlight the imaging properties of the probe, the endogenous PTP1B in LO2 and HeLa cells was monitored by Q1. As shown in Fig. 4a and b, the green fluorescence of HeLa cells was brighter than that of LO2 cells while the red fluorescence remained almost constant (and the fluorescence of the corresponding merged images was yellow green and green, respectively); at the same time, introduction of inhibitor (Na₃VO₄) also decreased the intensity of the green fluorescence (Fig. 4c), which led to the change of the ratio of the intensity of the green channel to that of the red channel (Fig. 4d). The above results indicated that the HeLa cells contained more PTP1B than did the other cells tested, which was confirmed by the cell lysate and Western blot analysis (Fig. 4e). The introduction of inhibitor also decreased this ratio in HepG2 cells (Fig. S21, ESI†). All of the above results demonstrated that the VIE fluorogenic probe can be used to detect and image PTP1B in living cells.

Fig. 4 (a–c) Fluorescence images of LO2 and HeLa cells. Displayed for panels 1 to 4, i.e., left to right, are the green channel, red channel, DIC and overlap images, respectively. λ_ex = 405 nm; green channel: 430–530 nm; red channel: 550–650 nm. Scale bar 20 μm. (a1–4) LO2 cells incubated with Q1 (2 μM, 1% DMSO as co-solvent) at 37 °C for 60 min. (b1–4) HeLa cells incubated with Q1 (2 μM, 1% DMSO as co-solvent) at 37 °C for 60 min. (c1–4) HeLa cells first treated with inhibitor (Na₃VO₄, 1 mM) for 30 min, and then incubated with Q1 (2 μM, 1% DMSO as co-solvent) at 37 °C for 60 min. (d) The ratio of green channel fluorescence intensity to red channel fluorescence intensity (I_Green/I_Red) for each of the above cell images. (e) The normalized I₄₅₀/I₆₀₀ values of the cell lysate (total protein content: 100 μg μL⁻¹). The lysate was incubated with Q1 (2 μM) for 40 min. λ_ex = 360 nm. The inset shows the Western blot analysis of the cell lysate (with the total protein content of 30 μg determined using the BCA standard method). MW (PTP1B) = 50 kDa, MW (GAPDH) = 37 kDa.

In summary, as a proof of concept, we have developed the first enzymatic VIE probe with internal standard properties for detecting PTP1B. This new probe exhibits a novel VIE performance before and after the enzymatic reaction with PTP1B, good selectivity over other phosphatases, and rapid response. The imaging of PTP1B by VIE in living cells was also realized. More importantly, the designed probe herein provides a guideline and platform for the development of new VIE-based enzymatic probes.

This work was financially supported by the National Natural Science Foundation of China (81672508), Jiangsu Provincial Foundation for Distinguished Young Scholars (BK20170041), Natural Science Foundation of Shaanxi Province (2019JM-016), China-Sweden Joint Mobility Project (51811530018), Fundamental Research Funds for the Central Universities, GSK-EDB Trust Fund (R-143-000-688-592) and Synthetic Biology Research & Development Programme (SBP) of the National Research Foundation (SBP-P4 and SBP-P8), Singapore. We also warmly thank Prof. He Tian from East China University of Science and Technology for insightful comments.

Live subject statement: HepG2, LO2 and HeLa cells were purchased from American Type Culture Collection (ATCC). The procedures used to prepare all of the biological samples are described in the ESI.†

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc07680h

‡ These authors contributed equally to this work.

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