A Novel and Simple Imidazo[1,2-a]pyridin Fluorescent Probe for Sensitive and Selective Imaging of Cysteine in Living Cells and Zebrafish

Abstract

Cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play many crucial physiological roles in organisms. Their abnormal levels can cause and indicate various diseases. In the present study, a small-molecule fluorescent probe 2-(imidazo[1,2-a]pyridin-2-yl)phenyl acrylate (IPPA) was designed, synthesized and characterized by NMR, FT-IR and HRMS. IPPA can selectively detect Cys over other analytes because of an approximately 76 times enhancement in fluorescence intensity. The limit of detection of IPPA for Cys was 0.33 μM. The pseudo-first-order rate constant of the reaction between IPPA and Cys was approximately 10 times that of the reaction between IPPA and Hcy (K_Cys 3.18 × 10⁻³ S⁻¹ vs K_Hcy 4.92 × 10⁻⁴ S⁻¹), indicating that Cys can be distinguished from Hcy. In addition, IPPA exhibits strong anti-interference ability, small molecular weight, high efficiency, low toxicity and good cell permeability. It was successfully used in imaging HepG2 cells and zebrafish. The fluorescence response of IPPA for calf serum are powerful proofs for practical application. Therefore, IPPA has high potential for bioassay applications.

Graphical Abstract

1. Introduction

Cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are three biological thiols present in organisms [1–4]. Their metabolism and transport are closely related to the functional expression of many significant enzymes and proteins in the body [5]. Abnormal levels of biothiols in vivo can cause and indicate various diseases. The sulfhydryl group of Cys is an ideal nucleophile in reactions, undergoing a reversible redox reaction under physiological conditions, which is necessary for the formation of disulfide bonds that maintain the tertiary and quaternary structure of proteins [6–9]. Hcy is a key intermediate product derived from Cys by methionine and is closely related to the health of the cardiovascular system [10–12]. GSH participates many cellular activities, such as maintenance of intracellular redox homeostasis, heterogeneous metabolism, intracellular signaling, and gene regulation [9, 13, 14]. Therefore, quantitative detection of biothiols in organisms can help us understand the role of thiol-containing enzymes or proteins in physiological and pathological processes. It can also provide important information for the prevention and diagnosis of various diseases, theoretically laying the foundation for clinical applications of quantitative biothiol detection methods [15–18].

Traditional methods to detect biothiols include high-performance liquid chromatography [19], mass spectrometry [20], and capillary electrophoresis [19, 21]. However, these methods need high-cost equipment, complicated sample processing and long runtime, and are therefore not suitable for high-throughput routine clinical trials and scientific research applications [22–24]. Because of their low detection limit and high selectivity, fluorescent probes have been used for the quantitative detection of biothiols [25–28]. Cho’s group [29] reported a two-photon fluorescent probe for the detection of thiols based on a thiol/disulfide exchange. Two-photon microscopy, using two near-infrared photons as the excitation source, has the advantages of a high tissue-penetration depth, localized excitation, and prolonged observation time, thereby allowing tissue imaging. Pang et al. [30] reported a ratiometric biothiol fluorescent probe with an acryloyl group as a recognition site, which can selectively recognize Cys in cells and can be applied to bioluminescence imaging. Kim’s group [31, 32] developed a chromene molecule that can be used to detect thiol content in solid tumors. The probe exhibits high selectivity, high sensitivity and a ratiometric response in which its yellow fluorescence emission (550 nm) changes to blue fluorescence emission (496 nm), which can effectively eliminate interference from background autofluorescence [31, 32]. On the basis of tandem conjugate addition and intramolecular cyclization, Yang et al. [33] developed a fluorescent probe for detection of Cys and the probe shows selectivity for Cys over other biothiols. Up to now, some fluorescent probes can selectively monitor Cys [34–36]. However, most of these probes have complicated structures and large molecular weight, which may not enter the cells or organisms easily in the real applications. Therefore, it is meaningful to develop simple fluorescent probes which can recognize Cys over other analytes.

The acryloyl group was first established as a recognition group in 2011 by Strongin’s group [37, 38]. It is a classic Michael acceptor with a polarized α-β unsaturated moiety, which can react with the mercapto group of biothiols through a Michael addition reaction [39, 40]. Due to the different nucleophilicities of the three biothiols, selective identification of biothiols can be achieved via their different addition reaction rates and cyclization cleavage processes [41–43]. To date, it is still a challenge to distinguish three biothiols completely with fluorescent probes [44–46]. Therefore, a new strategy for the design and development of fluorescent probes for selectively detecting Cys is highly desirable. The acrylate group has a strong electron-withdrawing ability and reactivity with biothiols, undergoing the cyclization and cleavage reaction. Thus, the fluorescence would change with the leaving of the electron-withdrawing groups.

Based on the above strategies, a small-molecule fluorescent probe, 2-(imidazo[1,2-a]pyridin-2-yl)phenyl acrylate (IPPA), was designed and synthesized, using 2-(imidazo[1,2-a]pyridin-2-yl)phenol (HPIP) as the fluorophore and the acryloyl group as the recognition group. IPPA can selectively detect Cys over Hcy, GSH and other analytes due to their different nucleophilicities, addition reaction rates and cyclization cleavage processes. In addition, IPPA exhibits low cytotoxicity and good cell permeability and was successfully applied to imaging studies of HepG2 cells and zebrafish.

2. Experimental

2.1. Chemicals and instruments

Cys, Hcy and GSH were purchased from Sigma-Aldrich (St. Louis, MO, USA). Proline (Pro), tryptophan (Try), tyrosine (Tyr), arginine (Arg), histidine (His), lysine (Lys), aspartic acid (Asp), threonine (Thr), glutamic acid (Glu), glycine (Gly), KNO₃, Ca(NO₃)₂·4H₂O, NaNO₃, Cu(NO₃)₂·3H₂O, Mg(NO₃)₂·6H₂O, Zn(NO₃)₂·6H₂O, and Fe(NO₃)₃·9H₂O were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and J&K (Beijing, China). All reagents were used without further purification, unless otherwise specified. Aqueous solutions of metal ions were prepared from the corresponding purchased nitrate salt. The deionized water was obtained from a Milli-Q Plus ultrapure water system (Billerica, MA, USA). HepG2 cells were provided by the Provincial Hospital of Anhui in China.

During the experiment, all fluorescence spectra were recorded in a 1-cm quartz cuvette with a volume of 3.0 mL on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a xenon lamp. The slit width was at 10 nm. UV-Vis absorption spectra were measured with a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). Mass spectra (MS) and HRMS were collected on a UPLC/XevoTQ MS/MS (Waters, Milford, MA, USA) and an Agilent Accurate-Mass-Q-TOF MS 6520 system equipped with an electrospray ionization (ESI) source. ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) spectra were collected on a spectrometer (Agilent Technologies, Santa Clara, CA, USA) using tetramethylsilane (TMS) as an internal standard. The pH values were measured by a model pH-25 digital display pH meter (Shanghai REX Instrument Factory, Shanghai, China). Fluorescence imaging of HepG2 cells and zebrafish was performed on an EVOS FL Auto inverted fluorescence microscope (Massachusetts, USA) and a Nikon inverted Ti-S epifluorescence microscope (Tokyo, Japan), respectively.

2.2. Synthesis and characterization of IPPA

2-(Imidazo[1,2-a]pyridin-2-yl)phenol (HPIP) [47]. 1-(2-Hydroxyphenyl)ethanone (200 mg, 1.47 mmol), pyridin-2-amine (318 mg, 3.38 mmol) and I₂ (448 mg, 1.78 mmol) were mixed in a round-bottom flask. The mixture was stirred at 110 °C for 4 h and then stirred overnight at 70 °C. After the reaction was completed, an excess amount of NaOH (aq, 45%) was added and the reaction mixture was stirred at 100 °C for 1 h. Then the reaction mixture was cooled to room temperature and diluted with 25 mL of CH₂Cl₂. The pH of the reaction solution was made neutral using HCl (aq, 10%). The mixture was extracted with CH₂Cl₂ and washed with deionized water (3 × 10 mL). The organic phase was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The product was isolated using a chromatography column (SiO₂, CH₂Cl₂/n-hexane = 3:1), and recrystallized from an ethanol-water system to give an orange-yellow solid (235 mg, 60%). ¹H NMR (600 MHz, DMSO-d₆): δ 12.03 (s, 1H), 8.61–8.57 (m, 1H), 8.46 (s, 1H), 7.86–7.85 (dd, J = 7.7, 1.7 Hz, 1H), 7.65–7.63 (dd, J = 9.0, 1.1 Hz, 1H), 7.33–7.30 (m, 1H), 7.18–7.16 (m, 1H), 6.98–6.95 (dd, J = 6.7, 1.2 Hz, 1H), 6.92–6.86 (m, 2H). HRMS (ESI, m/z) calculated for [C₁₃H₁₀N₂O + H]⁺: 211.0866, found: 211.0866. IR (KBr, cm⁻¹): 3445.69, 3132.31, 3042.06, 1587.13, 1403.44, 1355.23, 1253.99, 1146.47, 1063.47, 1023.53, 915.54, 840.33, 749.21, 660.50.

2-(Imidazo[1,2-a]pyridin-2-yl)phenyl acrylate (IPPA)

2-(Imidazo[1,2-a]pyridin-2-yl)phenol (500 mg, 2.4 mmol) was dissolved in anhydrous CH₂Cl₂ (30 mL) and cooled to 0 °C. Then two equivalents of triethylamine were added to the mixture and acryloyl chloride (270 mg, 3.0 mmol) was slowly added dropwise to the solution. Finally, the temperature of the solution was slowly raised to room temperature and stirred overnight. The progress of the reaction was monitored by a thin-layer chromatography silica gel plate (TLC). The solvent was evaporated to dryness when the reaction was finished. The desired product was obtained by puritication of the crude material on a silica gel column (V_Hexane : V_{Ethyl acetate} = 6:1), white solid, 420 mg, yield: 66.2%, solubility: 46.25 μg/ml in water (25 °C). ¹H NMR (600 MHz, DMSO-d₆) δ 8.54 (dt, J = 6.8, 1.2 Hz, 1H), 8.26–8.19 (m, 1H), 8.15 (s, 1H), 7.57–7.52 (m, 1H), 7.42–7.34 (m, 2H), 7.23 (m, 2H), 6.87 (td, J = 6.7, 1.1 Hz, 1H), 6.59 (d, J = 1.7 Hz, 2H), 6.20 (dd, J = 6.6, 5.0 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 164.59, 147.71, 144.52, 139.96, 134.26, 129.38, 128.91, 128.28, 127.48, 127.03, 126.76, 125.74, 123.78, 117.08, 112.73, 111.75. HRMS (ESI, m/z) calculated for [C₁₆H₁₂N₂O₂ + H]⁺: 265.0972, found: 265.0971. IR (KBr, cm⁻¹): 3442.83, 3152.68, 3074.06, 1740.18, 1633.80, 1499.35, 1405.49, 1368.19, 1276.49, 1244.21, 1152.40, 1072.95, 1007.07, 902.81, 836.05, 755.37, 741.84, 674.33.

2.3. Fluorescence quantum yield

The fluorescence quantum yield of IPPA was calculated by comparison with a quinine sulfate standard solution containing 0.1 M H₂SO₄. The formula for calculating the fluorescence quantum yield is as follows [48–50]:

$${\varphi }_X={\varphi }_S\left(F_X/F_S\right)\left(A_S/A_X\right)\left({\eta }_X^2/{\eta }_S^2\right)$$ (1)

where Φ represents the fluorescence quantum yield, and F and A indicate the area of the fluorescent peak after correction and the absorbance at the maximum excitation wavelength, respectively. η is the refractive index of the solvent used, and “s” and “x” represent data for the quinine sulfate standards and calculated samples, respectively.

2.4. Fluorescence and UV-Vis spectra

The measuring conditions of all fluorescence spectra during the experiments were as follows: E_x= 330 nm, slit width of 10 nm, and emission wavelength range of 300–500 nm. The measurement conditions of the UV-Vis spectrum were as follows: the scanning wavelength range is 200–500 nm, and the scanning step is 1.0 nm. IPPA stock solution (100 μM) was prepared by dissolving IPPA in DMSO solution, and amino acids, metal ions, H₂O_2, NaHS and glucose solutions were prepared using deionized water. The solutions required in the subsequent experiments were all formulated using the above stock solutions, and probe solution was prepared using 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) as the buffer solution.

2.5. Cytotoxicity

First, 96-well plates were seeded with HepG2 cells at a density of 4 × 10⁴ cells per well. Then the plates were incubated with IPPA at a series of concentrations (10, 20, 30, 40 μM) and cells in wells to which no IPPA was added were used as a control group. After 24 h of incubation, the cells were rinsed with PBS three times. Then, a CCK-8 assay was used to analyze cell viability. The absorbance was measured at 450 nm by using a microtiter plate reader (ELx808, BioTek, USA).

2.6. Cell imaging of IPPA

HepG2 cells (liver hepatocellular cells) were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin, and 100 μg/mL streptomycin and incubated at 37 °C under a humidified atmosphere containing 5% CO₂. A stock solution of the probe IPPA having a concentration of 10 mM in DMSO was formulated. The above stock solution of the probe was diluted with cell culture solution to 20 μM for cell culture (DMSO content < 0.2%). Next, the cells were incubated with the probe, N-ethylmaleimide (NEM, is mainly used as a block agent for biothiols) or biothiols in the incubator at 37°C and then rinsed three times with PBS. Fluorescent images were obtained under a fluorescence microscope after three washes with PBS to remove the probe residue.

2.7. Study of zebrafish in vivo imaging

Zebrafish eggs were incubated in E3 embryo medium (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO₄, 1 mM CaCl₂, 0.15 mM, KH₂PO₄, 0.05 mM Na₂HPO₄, 0.7 mM NaHCO₃, and 10⁻⁵% methylene blue at pH 7.5). The incubation rate was accelerated by a 12-hour light/12-hour dark cycle. After 3 days of incubation, the normal-growing zebrafish were selected for the experiment. Zebrafish were incubated with and without 200 μM NEM in E3 medium for 15 min, washed three times with E3 medium to remove the remaining NEM, and further incubated with 20 μM of IPPA (DMSO content < 0.2%) in E3 medium for 30 min (28 °C). After washing three times with E3 medium, the zebrafish were imaged under a fluorescence microscope, and untreated zebrafish were used as blank controls.

3. Results and discussion

3.1. Synthesis of IPPA

This work mainly utilizes the strong nucleophilicity of sulfhydryl groups in small-molecule biothiols [51–53]. Considering the Michael addition mechanism [54, 55], 2-(imidazo[1,2-a]pyridin-2-yl)phenol is used as the fluorophore, and the acrylate group is selected as the recognition site. The acrylate group is a classic Michael acceptor with a polarized α-β unsaturated moiety, which can react with the mercapto group of the biothiols through the Michael addition reaction. Thus, the biothiol fluorescent probe IPPA was designed and synthesized. Due to the different nucleophilicities of the three biothiols, selective identification of biothiols can be achieved via their different addition reaction rates and cyclization cleavage processes [56, 57]. IPPA may enter cells or living tissues easily because of the low molecular weight. The synthetic pathway of IPPA is shown in Scheme 1, and IPPA was characterized by ¹HNMR, ¹³CNMR, HRMS and IR (Figures S1–6).

Scheme 1.

Synthetic scheme of 2-(imidazo[1,2-a]pyridin-2-yl)phenyl acrylate (IPPA).

3.2. Optimization of determination condition

In the present work, the response time of the probe IPPA to biothiols was studied. Cys, Hcy and GSH were separately added to DMSO-HEPES buffer solutions (pH 7.4, 8:2, v/v) containing IPPA. The response time was measured based on the fluorescence emission spectrum (Figure 1A). The results showed that the probe IPPA can react with Cys rapidly and that the fluorescence intensity was stable in approximately 12 min. However, the reaction time of Hcy and GSH with IPPA were significantly (14-fold) lower than that of Cys, and their reaction time was approximately 50 min.

Figure 1.

Time-dependent fluorescence response and pseudo-first-order kinetic plots of IPPA (10 μM) at 375 nm with and without biothiols (200 μM) in DMSO-HEPES buffer solution (8:2, v/v, pH 7.4, λ_ex = 330 nm, slit: 10 nm). Data are mean ± SE (bars) (n = 3).

The pH value is another important factor that can influence the reaction. We investigated the fluorescence intensity of the reaction between IPPA and three biothiols in the pH range of 2–12. The fluorescence intensity of the IPPA + Cys solution changes continuously with increasing pH value (Figure S7). Obviously, in the pH range from 2.0–4.0, the fluorescence intensity of IPPA + Cys is rapidly enhanced and slowly increases with the increase of pH value (4–7.4). While the pH value is over 7.4, the fluorescence intensity decreases significantly. In addition, the similar fluorescence change can be seen in the presence of Hcy/GSH. Therefore, the physiological pH of 7.4 were used in all experiments.

The effect of DMSO concentration on the reaction of IPPA and biothiols was also investigated. The fluorescence intensity of IPPA in the absence and presence of biothiols in solutions with different ratios of DMSO and HEPES is listed in Figure S8. Ultimately, we chose 8/2 DMSO/HEPES buffer (v/v, pH 7.4) and used it for all experiments.

3.3. Selectivity of IPPA

With the probe in hand, we investigated the response of IPPA to various analytes, including thiol-containing amino acids (Cys, Hcy and GSH), other amino acids (Pro, Try, Tyr, Arg, His, Lys, Asp, Thr, Glu, Gly), various ions (K⁺, Ca²⁺, Na⁺, Cu²⁺, Mg²⁺, Zn²⁺, Fe³⁺ and HS^-), H₂O₂ and glucose. As shown in Figure 2B, the UV-Vis spectrum of IPPA changes significantly with the addition of Cys/Hcy, with an absorption peak appearing at 330 nm. The absorption spectra of the solution recorded upon titration indicated that the absorption of the peak at 330 nm with the addition of Cys and Hcy (Figure S10). In Figure 1A, the probe IPPA has almost no fluorescence, but its fluorescence increases in the presence of biothiols. With the addition of Cys and Hcy to the solution of the probe IPPA (10 μM), the fluorescence intensity exhibited a 76- and 51-fold enhancement, respectively. However, compared to that of the solution without GSH, the fluorescence intensity of the solution with GSH increased only 14-fold. The addition of other amino acids and various analytes hardly caused any changes in the fluorescent signal (Figure 2A). In Figure 2A (inset), under excitation with 365 nm UV light, the color of the IPPA solution changed from weak blue to stronger blue-green after adding Cys/Hcy. By contrast, there was no significant change after adding GSH to the probe solution. The results indicate that the probe can specifically recognize Cys/Hcy over GSH. In addition, we calculated the pseudo first-order rate constants of reactions between the probe IPPA (10 μM) and the three biothiols. The pseudo-first-order rate constants from Cys, Hcy and GSH are obtained by the linear relationships in Figure 1 as K = 3.18 × 10⁻³, 4.33 × 10⁻⁴ and 4.92 × 10⁻⁴ S⁻¹, respectively. K_Cys is approximately 15 times K_Hcy and K_GSH. Comparison of the pseudo-first-order rate constants reveals that IPPA can selectively recognize Cys over Hcy and GSH.

Figure 2.

Fluorescence (A) and UV-Vis spectra (B) of the probe IPPA (10 μM) when multiple different analytes (200 μM) were added. The inset is a photograph of IPPA solutions with and without biothiols. Data are mean ± SE (bars) (n = 3).

We also investigated the effect of various interfering substances on the recognition of biothiols by IPPA, including thiol-containing amino acids (Cys, Hcy and GSH), other amino acids (Pro, Try, Tyr, Arg, His, Lys, Asp, Thr, Glu, Gly), various ions (K⁺, Ca²⁺, Na⁺, Cu²⁺, Mg²⁺, Zn²⁺, Fe³⁺ and HS^-), H₂O₂ and glucose. Figure 3 shows that except for the biothiols, most of analytes do not cause fluorescence enhancement. These results revealed that the probe IPPA can selectively detect Cys/Hcy over other analytes.

Figure 3.

Fluorescence intensity (I_375nm) of IPPA (10 μM) without and with biothiols (200 μM) in the presence of various analytes (200 μM) in a DMSO-HEPES buffer solution (8:2, v/v, pH 7.4, λ_ex = 330 nm, slit: 10.0 nm). Data are mean ± SE (bars) (n = 3).

3.4. Quantification of biothiols

To further study the quantitative response of IPPA to the presence of different concentrations of biothiols, fluorescence and UV-Vis titration experiments were performed. In Figure S9, the maximum absorption wavelength of IPPA is approximately 330 nm. The addition of Cys and Hcy results in an increase in the intensity of the absorption peak of IPPA. Figure S9A shows that the addition of Cys causes a slight redshift, while the addition of Hcy has no effect on the maximum absorption wavelength of IPPA (Figure S9B). The fluorescence quantum yield of IPPA was calculated as 0.011 and became 0.52/0.21 with the addition of Cys/Hcy, respectively; this strong fluorescence indicates that IPPA was converted into another product. The result was consistent with that of the titration experiment. As shown in Figure 4, IPPA exhibits no fluorescence at 375 nm. However, the fluorescence intensity increased as the concentration of biothiols increased, and has a good linear relationship with the biothiol concentration. In the presence of 30 equiv. Cys/Hcy/GSH, the fluorescence intensity of IPPA undergoes 76, 51 and 14-fold enhancement, respectively. The limits of detection (LOD) of Cys and Hcy were calculated using the detection limit formula (CDL = 3 Sb/m) to be 0.33 and 0.76 μM, respectively (Figure S10). Compared to the recently reported probes for the detection of biothiols (Table S2), IPPA exhibits low detection limits, low molecular weight and short detection time.

Figure 4.

Fluorescence titration spectra of IPPA (10 μM) vs Cys, GSH and Hcy (0~30 equiv.) in a DMSO-HEPES buffer solution (8:2, v/v, pH 7.4, λ_ex = 330 nm). Plot of fluorescence intensity at 375 nm as a function of biothiol equivalents. Data are the mean ± SE (bars) (n = 3).

3.5. Mechanism of detection

The mechanism of the reaction between IPPA and biothiols was investigated with NMR analysis of the DMSO-d₆ solution of IPPA and one of biothiols (mol ratio, IPPA: biothiols = 1:1) at ambient temperature. In Figure 5, the hydrogen atoms on the acrylate group were located at 6.59 ppm (2H) and 6.20 ppm (1H). After adding the biothiols, the hydrogen atoms on the acrylate group disappear, which is accompanied by the appearance of new peaks. The results indicated that biothiols react with IPPA at the acrylate group, which causes the acrylate group to fall off IPPA.

Figure 5.

¹H NMR spectrum of IPPA in d₆-DMSO and the resulting spectra after the addition of Cys and Hcy.

The reaction mechanism was also investigated via analysis of the products of the reaction between IPPA and biothiols by LC-HRMS (Table S1, Figures S12–13). The results indicate that the biothiol is conjugated with the olefinic double-bond in the probe to form a thioether (calcd [H⁺]: IPPA-Cys/IPPA-Hcy, 386.1169/400.1326; found m/z: Cys/Hcy, 386.1172/400.1322). IPPA-Cys and IPPA-Hcy can be further cyclized to release the former fluorophore HPIP. However, the thioether produced by GSH is stable and the subsequent cyclization efficiency is low. Therefore, in the IPPA-GSH system, minimal fluorescence was generated. The cyclization process of Cys generates a seven-membered ring (calcd [H⁺]: 176.0376; found m/z: 176.0390), while that of Hcy generates an eight-membered ring (calcd [H⁺]: 190.0532; found m/z: 190.0542). Generally, the strain in tension of the eight-membered ring is much higher than that in the seven-membered ring, and the activation entropy of the formation reaction of an eight-membered ring needs more energy (Scheme 2). Thus, the formation rate of the seven-membered ring is much faster than that of the eight-membered ring. As a result, the selectivity of IPPA for Cys can be achieved because of the difference in reaction rate.

Scheme 2.

Proposed mechanism of 2-(imidazo[1,2-a]pyridin-2-yl)phenyl acrylate (IPPA) for biothiol sensing.

3.6. The density functional theory (DFT) calculation

To better understand the fluorescence performance of IPPA, density functional theory (DFT) calculation was performed using the B3LYP method on the Gaussian 09 program [58–60], and the spatial distribution and orbital energy of HPIP and IPPA were optimized (Figures S13–S14). The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of HPIP and IPPA are shown in Figure 6. The LUMO is localized almost all over the IPPA molecule, while the HOMO is mainly distributed on the imidazo-pyridine moiety. Upon fluorescence excitation, the recognition group blocks the fluorescence emission of IPPA, resulting in fluorescence quenching. Notably, when IPPA reacts with one biothiol, the acrylate group is attacked due to the Michael addition cyclization reaction, and the product HPIP is formed. Moreover, the HOMO of HPIP is distributed on the fluorophore, indicating that IPPA will show strong fluorescence when reacted with an amino mercaptan. The above theoretical findings are consistent with the experimental results, and confirm the previously proposed mechanism.

Figure 6.

HOMO and LUMO of IPPA and HPIP.

3.7. Real application of IPPA

To study the application of IPPA in actual conditions, the fluorescence response of IPPA toward calf serum in an aqueous HEPES buffer (pH 7.4, DMSO content < 0.2%) at ambient temperature was investigated. We performed an experiment with NEM as the block agent in calf serum to verify that the increase in fluorescence is related to the biothiols content in calf serum. As shown in the Figure S15, there is no obvious fluorescent change in the presence of NEM in calf serum. However, observation of the increasing fluorescence intensity in the absence of NEM indicated this increase can be definitely confirmed to biothiols in calf serum. The fluorescence intensity of IPPA increased with the increase of calf serum content (Figure 7A). It is exciting that the fluorescence intensity has a good linear relationship with calf serum content (R², 0.958). Therefore, IPPA can determine the content of calf serum with the detection of the biothiols in calf serum.

Figure 7.

(A) Relative fluorescence intensity of calf serum with IPPA (10 μM) in an aqueous HEPES buffer (pH 7.4, DMSO content < 0.2%, λ_ex =330 nm); (B) the correlation curve between relative fluorescence intensity and calf serum content. Data are mean ± SE (bars) (n = 3).

In addition, a test strip experiment was carried out. First, the test paper was cut into small pieces and immersed in IPPA solution. Once the test trip was dry, various analytes (Pro, Asp, Try, Arg, Tyr, His, Glu, Lys, Thr, glucose, K⁺, Ca²⁺, Na⁺, Mg²⁺, Zn²⁺, Fe³⁺, Cu²⁺, H₂O₂, NaHS, Cys, Hcy and GSH) were separately dropped on the test strips. In Figure S16, the IPPA-Cys/IPPA-Hcy strips changed from colorless to an intense blue-green fluorescence under excitation with a 365 nm UV lamp, while the change in the color of the IPPA-GSH strip was not obvious. The other analyte changes were also negligible. Therefore, IPPA test strips can be effectively applied in the detection of Cys/Hcy.

3.8. Study of cytotoxicity and cell imaging

To investigate the effect of IPPA on cells, the cytotoxicity was studied by a CCK-8 assay. HepG2 cells were incubated in probe solutions (0, 10, 20, 30 and 40 μM) for 24 h to observe cell survival. As shown in Figure 8, even when the probe IPPA concentration was increased to 40 μM, the survival rate of HepG2 cells was still greater than 85%. The experimental results show that IPPA has low toxicity to cells and can be further used for cell imaging research.

Figure 8.

Cell viability values (%) estimated by a CCK-8 assay versus incubation concentrations of IPPA. HepG2 cells were cultured in the presence of 0–40 μM IPPA. Data are mean ± SE (bars) (n = 3).

Next, we performed live HepG2 cell imaging experiments with IPPA under a fluorescence microscope. In Figure 9, HepG2 cells that were not treated with NEM showed significant fluorescence after incubation with IPPA for 20 min, while no fluorescence changes were observed in HepG2 cells treated with NEM. To further investigate the ability of IPPA to detect the three biothiols in HepG2 cells, we designed three groups of controlled trials as follows: HepG2 cells were pretreated with NEM (1.0 mM) for 1 h and then incubated with GSH, Hcy or Cys (20 μM) for 20 min. The combined images indicate that IPPA has satisfactory cell membrane permeability, which allows it to sensitively detect Cys and Hcy at endogenous levels in HepG2 cells. The results of cell imaging reveal that IPPA has potential for bioapplication in detecting intracellular Cys/Hcy.

Figure 9.

Fluorescence imaging of Cys/Hcy in living HepG2 cells. From left to right: HepG2 cells incubated with IPPA (20 μM) for 20 min (A); HepG2 cells pretreated with NEM (1 mM) for 1 h and then incubated with IPPA (20 μM) for 20 min (B); the last three groups of HepG2 cells pretreated with NEM (1.0 mM) for 1 h, then incubated with GSH (C), Cys (D), or Hcy (E) (20 μM) for 20 min, and finally incubated with IPPA (20 μM) for 20 min. From top to bottom: Dark field (UV light), Bright field, Merge. Scale bar: 20 μm.

3.9. Zebrafish imaging

To further confirm the bioapplication potential of IPPA, zebrafish imaging was performed using the probe IPPA. As shown in Figure 10, when the zebrafish were incubated with the probe (20 μM) for 30 min, blue fluorescence was observed in the UV channel. However, when the zebrafish were incubated with NEM (200 μM) for 15 min and then incubated with IPPA (20 μM) for 30 min, no fluorescence was observed. NEM also blocks the interaction of IPPA and biothiols in the zebrafish. Clearly, the probe IPPA can be used to visualize Cys/Hcy in the zebrafish.

Figure 10.

Fluorescence imaging of zebrafish. From left to right: zebrafish incubated without IPPA (20 μM) (A); zebrafish pretreated with IPPA (20 μM) for 20 min (B); zebrafish pretreated with NEM (200 μM) for 15 min and then incubated with IPPA (20 μM) for 20 min (C). From top to bottom: Bright field, Dark field (UV light), Merge. Scale bar: 500 μm.

4. Conclusions

We designed and synthesized a small-molecule fluorescent probe using an acryloyl group as a recognition group. The probe IPPA can selectively and sensitively distinguish Cys from other interferents. In addition, the reaction mechanism was studied by DFT calculation, HRMS and NMR. IPPA can also be used to detect biothoils in the test strip and calf serum. Moreover, IPPA was successfully applied for imaging in live HepG2 cells and zebrafish. The simple, inexpensive and efficient preparation of IPPA showed potential advantages in the detection of Cys in vivo. This study can provide useful information for possibility of further structural optimization for improving the sensing behavior. In addition, red or near-infrared dual-light water-soluble fluorescent probes can be designed to improve in vivo diagnostic applications in rodents or mammalians in our future studies.

Highlights.

• 2-(Imidazo[1,2-a]pyridin-2-yl)phenyl acrylate (IPPA) is a novel fluorescent probe.

• IPPA can detect Cys over Hcy, GSH and other analytes.

• IPPA can selectively detect as low as 0.33 μM of Cys in DMSO/HEPES buffer.

• IPPA has been successfully utilized to image living cells and zebrafish.

Abbreviations

Cys

cysteine

Hcy

homocysteine

GSH

glutathione

Pro

proline

Try

tryptophan

Tyr

tyrosine

Arg

arginine

His

histidine

Lys

lysine

Asp

aspartic acid

Thr

threonine

Glu

glutamic acid

Gly

glycine

DFT

density functional theory

DMEM

dulbecco’s modified eagle medium

HPIP

2-(imidazo[1,2-a]pyridin-2-yl)phenol

IPPA

2-(imidazo[1,2-a]pyridin-2-yl)phenyl acrylate