Dibenzo[a,c]phenazine-derived near-infrared fluorescence biosensor for detection of lysophosphatidic acid based on aggregation-induced emission

Abstract

A new near-infrared sensitive and highly selective fluorescence turn-on detector for lysophosphatidic acid has been developed based on a dibenz[a,c]phenazine unit with aggregation-induced emission, in which the limit of detection is as low as 4.47 × 10⁻⁷ M.

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Lysophosphatidic acid (LPA), the simplest phospholipid, plays a key role in biochemistry, such as stimulating the proliferation of cancer cells and promoting the aggregation of platelets.^1,2 It is known that the physiological concentrations of LPA in plasma are approximately 0.1–6.3 μM. Recently, some studies have reported that the LPA level increases in malignant effusions in patients with cancer, especially for ovaries.³ Therefore, plasma LPA levels may represent a potential biomarker for the early detection of malignant cancer.^4,5 As a result of the close relationship between LPA and human health, the development of reliable detection methods for LPA is highly desired and urgent. To date, a number of methods for LPA detection have been invented, including chromatography, capillary electrophoresis, and immunochemical methods.^6–10 However, some of these detection methods are expensive instrumentation as well as time-consuming and labor-intensive procedures hampered their practical application. It is necessary to find new method for the detection of LPA, such as chemosensors. Fluorescent turn-on sensing probes, as noninvasive reporters, have attracted much attention because of their easy-operation and high sensitivity. Until now, only a few such chemosensors can detect LPA, and most methods use metal-complexes as fluorescent indicators or colorimetric fluorescent sensor for the detection of LPA.¹¹ Owing to the importance of early diagnosis of ovarian cancer, the development of a relatively easy and efficient method to detect LPA is desirable.

The aggregation-induced emission (AIE) effect is a unique photophysical phenomenon discovered by Tang and his colleagues,¹² where the AIE luminogens are nonemissive or weakly fluorescent in solution, but they become highly emissive upon aggregation owing to the restriction of intramolecular rotations. This is totally opposite to the conventional aggregation-caused quenching (ACQ) effects.¹³ By taking advantage of AIE effect, many of the chemo- and bioprobes have been synthesized for the detection of various species, including ions,¹⁴ DNA,¹⁵ proteins,¹⁶ enzyme substrates/inhibitors,¹⁷ and carbohydrates.¹⁸ However, the red/NIR emitting probes are few reported. Recently, dibenzo[a,c]phenazine (DBP) and its derivatives have been employed in the construction of organic solar cell,¹⁹ optical waveguide²⁰ and metal ion sensor²¹ due to its unique electron accepting properties. The DBP is a stronger acceptor, because the two phenyl rings are connected by a single bond between the ortho positions, and it will significantly increase the planarity of molecules and facilitate charge transport.²² Therefore, using DBP as an acceptor is conductive to lower bandgap and shift the spectral response reached to the NIR region, which may have potential advantages in the bio-applications. In addition, triarylamine (TPA) has good electron donating and special propeller starburst molecular structure, which has been used widely in the designment of AIE materials.²³

Thus, in this work, we introduced TPA as donor to the DBP acceptor and synthesized a new near-infrared fluorescence dye TDBP for the detection of LPA by taking advantage of AIE behaviour of the TDBP. The design rationale of TDBP toward LPA in the HEPES buffer solution (pH 7.4, DMSO/water = 1 : 1, v/v) is illustrated in Scheme 1 and is explained as follows: (1) two parts of long alkyl chain and pyridine salt make TDBP soluble in the DMSO/water (1/1, v/v) solution, and it is weakly emissive in the mixture solution; (2) the long alkyl and pyridine salt in TDBP can interact with LPA through hydrophobic and electrostatic interaction, respectively, and the multipoint interactions may render the TDBP-LPA show low solubility in the DMSO/water (1/1, v/v), thus aggregation can be formed with fluorescence turn on. Therefore, TDBP can be used as a fluorescence turn-on probe for the detection of LPA. Compared with previously reported works, this is the first time to use AIE mechanism to detect LPA, which is expected to offer advantages such as high selectivity and sensitivity, and enhance imaging contrast. Also, the probe emits fluorescent signal in the near-infrared region, which has low signal-noise interference in the biological applications.

Scheme 1 Chemical structures of compound TDBP and LPA and the design rationale for the fluorescence turn-on detection of LPA.

The synthetic route of TDBP is shown in Scheme S1.† The compound 3 was prepared by the Suzuki coupling reaction between compound 2 and (4-(bis(4-methoxyphenyl)amino)phenyl)-boronic acid. Subsequently, the important intermediate 4 was also obtained by the Suzuki coupling reaction between 3 and pyridin-4-ylboronic acid. Finally, the target compound TDBP was prepared by the nucleophilic substitution reaction between 4 and 1-bromohexade-cane in the anhydrous tetrahydrofuran (THF). All the intermediates and target compound TDBP are characterized with ¹H and ¹³C NMR spectroscopy as well as HRMS (see the ESI†).

Fig. 1 shows the normalized absorption spectra of TDBP in THF and THF/hexane mixtures at a concentration of 1 × 10⁻⁴ M. The maxima (λ_max) absorption of TDBP is located at 517 nm in the THF solution and bathochromically shifted by 14 nm in aggregate form in the THF/hexane mixtures (90% hexane in volume, λ_abs = 531 nm). The absorption tails extending well into the long wavelength region implied the luminogens aggregated into particles in the presence of water, as it is well known that the Mie effect of nanoparticles causes such level-off tails in the absorption spectra.²⁴ To investigate the AIE attribute of the TDBP, we chose the anhydrous THF as the good solvent and hexane as the poor solvent to prepare stable nano-aggregates of TDBP by precipitation method.²⁵ As shown in the Fig. 2A, TDBP almost had no fluorescence in pure THF, but fluorescence began to increase with the hexane added slowly, and when hexane content reached to 80%, the fluorescent intensity was boosted to the maximum and the peaks located at 750 nm. Also, the fluorescence intensity of TDBP emits the strong NIR luminescence with a 26-fold increase in the I/I₀ ratio at the f_h = 80%, see the Fig. 2B. In addition, the fluorescence spectra of TDBP in DMSO/H₂O mixtures was also investigated (shown in Fig. S1†).

Fig. 1 Normalized absorption spectra of TDBP in THF and THF/hexane mixtures (90% hexane in volume) at a concentration of 1 × 10⁻⁴ M.

Fig. 2 (A) Fluorescence emission spectra of TDBP in THF and THF/hexane mixtures with different hexane fractions (f_h). (B) Plots of relative emission intensity (I/I₀) versus the composition of the THF/hexane mixtures of TDBP; I₀ = emission intensity in pure THF solution. Inset: photographs of TDBP in pure THF (0%) and THF/hexane mixtures (80%) under 365 nm UV irradiation. Solution concentration: 1 × 10⁻⁴ M; λ_ex: 530 nm.

With primary AIE properties determined, we further probed the ability of TDBP for detection of LPA in the NIR region, as shown in the Fig. 3A. As shown in Fig. S1,† the solution of TDBP dissolved in DMSO/water (1/1, v/v) was almost non-emissive in the absence of LPA. However, fluorescence started to increase gradually after addition of LPA. For instance, fluorescence intensity at 730 nm increased by more than 23 times when the concentration of LPA reached 100 μM. As depicted in the Fig. 3B, the detection produced satisfactory linearity at a low concentration range (5–30 μM) of LPA and the limits of detection (LOD) for LPA, defined as three times the standard deviation of background, was determined to be 4.47 × 10⁻⁷ M. As depicted by the Scheme 1, such fluorescence turn on was attributed to hydrophobic interaction and electrostatic interaction between TDBP and LPA, which reduced the solubility of TDBP in DMSO/water (1/1, v/v) solution and induced aggregation of the TDBP-LPA. The hypothesis was further confirmed by the scanning electron microscopic (SEM) study. In the Fig. 4, we observed from the images that there was almost no nano-aggregates formed before the addition of the LPA (Fig. 4A). In sharp contrast, the size of aggregation of the dye in the presence of the LPA increased to approximately 200 nm in the buffer solution in the presence of the LPA (Fig. 4B). This observation indicates that the fluorescence turn on of TDBP for LPA was based on the AIE mechanism.²⁶

Fig. 3 (A) Fluorescence titration spectra of TDBP (100 μM) in the presence of increasing lysophosphatidic acid (LPA) (from bottom to the top curve, 0, 5, 10, 20, 40, 60, 80 and 100 μM); inset: photographs of TDBP in the absence and presence of LPA (100 μM) in the DMSO/water solution (1/1, v/v) under 365 nm UV irradiation. (B) Plotting the fluorescence intensity (value of the emission maxima at 730 nm) as a function of LPA concentration for determination of the limit of detection (LOD) of TDBP, λ_em = 730 nm. All fluorescence spectra were measured in the HEPES buffer solution (pH = 7.4, DMSO/water = 1/1, v/v), λ_ex = 530 nm.

Fig. 4 Scanning electronic microscope (SEM) imaging of TDBP (A) in the absence of LPA and (B) in the presence of LPA.

The pH effects of the TDBP on the sensing for LPA process were also investigated. As shown in the Fig. 5, the fluorescence intensity of TDBP and TDBP + LPA are all relatively stable for pH values ranging from 3 to 9, which indicted that TDBP can be used to detect LPA in a wide range of pH values. For TDBP + LPA, at pH >9, the fluorescence began to decrease slightly with increasing pH, which may be attributed to the coordination interaction of OH⁻ in the solution to the pyridine salt cations of the TDBP, reducing the electrostatic interactions between TDBP and LPA.

Fig. 5 Effect of pH on the fluorescence intensity at 730 nm of free TDBP (100 μM) and TDBP + LPA (100 μM) mixture at room temperature.

To evaluate the selectivity of the probe, the fluorescent response of TDBP to a variety of biological species and anions and cations was studied. As shown in Fig. 6, comparing the changes in the fluorescence intensity ratio (F/F_LPA) of TDBP caused by the LPA and other biologically important species, it can be observed that LPA has higher fluorescence response than other interfere species, thus proving the excellent selectivity of the TDBP for the detection of LPA. Also, competition experiment was conducted to further confirm selectivity of TDBP for the detection of LPA in the presence of various interfere substrates, and it is evident from the Fig. S3† that those interfere substrates have negligibly interference in the binding of TDBP and LPA. Such good selectivity was mainly ascribed to the synergistic effect of electrostatic and hydrophobic interaction. The zeta-potential of TDBP was +20.4 mV resulting from the mass of positive charges on its surface. Upon the addition of 50 μM LPA, the zeta-potential of the solution was reduced to +8.7 mV, indicating that the negatively charged LPA interacted with TDBP through the electrostatic interaction; however, other single anion, such as HPO₄²⁻, has relatively low fluorescence response although the existence of electrostatic interaction, which may be caused by the lack of the long alkyl chain so that it could not present hydrophobic interaction and significantly reduces the interaction with LPA. Thus, in order to obtain the high selectivity of probe TDBP for the LPA, it is essential for the co-existence of electrostatic and hydrophobic interaction.

Fig. 6 Selectivity of TDBP (100 μM) to LPA (100 μM) in the presence of a variety of proteins and miscellaneous interference cations and anions (100 μM). F/F_LPA represents the fluorescence intensity ratio of other interfere substrates (F) and LPA (F_LPA). For the original FL spectra, see Fig. S2.†

To test the real biological applicability of the TDBP, we further used TDBP for the quantification of LPA in fetal bovine serum. Firstly, we spiked different concentrations of LPA to the TDBP solution at the concentration of 100 μM. Then, according to the low-concentration plotting shown in Fig. 7A, the recovered LPA concentrations were also determined. As shown in Fig. 7B, the high recovery rate of LPA in FBS can be observed and the average deviation is 1.06%, indicating that TDBP can be used as the probe for the LPA in the serum.²⁷

Fig. 7 (A) Plotting the fluorescence intensity (value of the emission maxima at 730 nm) as a function of low LPA concentration (3–36 μM) for TDBP. (B) The LPA stock solution in the spiked and recovered measurement were dissolved in the HEPES buffer solution and fetal bovine serum solution, respectively. (Black bar: the spiked concentrations of LPA, from left to right: 6, 12 and 18 μM; red bar: the recovered concentrations of LPA, from left to right: 6.05, 12.13 and 18.23 μM). The average deviation is 1.06%.

Conclusions

In summary, we have developed a new near-infrared fluorescence turn on chemosensor TDBP for the detection of LPA. This fluorescence detection of LPA possesses the following features: (1) different from previously reported works, this is the first time to detect LPA based on AIE mechanism; (2) the good sensitivity and selectivity is probably due to the hydrophobic and electrostatic interaction between the TDBP and LPA; (3) the limit of detection for LPA is as low as 4.47 × 10⁻⁷ M. Also, the quantification of LPA in the fetal bovine serum of TDBP indicated that it has potential biological applicability.

Acknowledgements

For financial support of this research, we thank the National Basic Research 973 Program (2013CB733700), and NSFC/China (21421004, 21172073, 21372082, 21572062 and 91233207).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Detailed experimental procedures, supporting figures. See DOI: 10.1039/c5ra21408d

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