br To investigate the e ect of pH on the
To investigate the eﬀect of pH on the luminescence of the nanop-robe in aqueous media, the emission intensity of [email protected] in 50 mM PBS solutions having diﬀerent pH values was determined. As shown in Fig. S4, both steady-state luminescence (λem = 584 nm) and TGL (λem = 612 nm) intensities of [email protected] were unchanged with pH changes ranged from 3.0 to 11, indicating the pH-independent emissions of the nanoprobe. Furthermore, steady-state luminescence and TGL intensities of the nanoprobe remained a constant value upon incubation for 7 days in PBS at pH 7.4 (Fig. S5), revealing the high stability of the nanoprobe in buﬀer without dye-leakage from nanoparticles. The photo-stability of the nanoprobe was examined by continuously irradiating the nanoprobe in PBS for a long time. As shown in Fig. S6, both CTMR and BHHBCB-Eu3+ in the nanoprobe exhibited high resistances to the photobleaching, suggesting the use-fulness of the nanoprobe for long-term “double-check” luminescence imaging of live cells.
Prior to use for cell imaging, cytotoxicity of the nanoprobe to HeLa and L-02 SB 203580 was evaluated by the MTT assay method. As shown in the Fig. S7, after incubating HeLa and L-02 cells with increased con-centrations of [email protected] (0, 15, 30, 60 100 μg/mL) for 24 h, the cell viability was kept to be greater than 86% (HeLa) and 83% (L-02). These results indicate that the nanoprobe [email protected] is low cytotoxic, allowing it to be used for staining live cells. Steady-state luminescence and TGL imaging experiments were conducted to investigate the performance of the nanoprobe for cancer cell-recogni-tion and imaging detection.
As shown in Fig. 4A, after HeLa cells were incubated with [email protected]
imaging was then performed to confirm (“double-check”) that the lu-minescence signals were produced by the cellular internalization of the nanoprobe. As shown in right column of Fig. 4A, after introducing a delay time of 33 μs, TGL images of HeLa cells showed still strongly red luminescence signals, which indicates that the nanoprobe [email protected] BHHBCB-Eu-FA is suitable to be used for the TGL imaging of FR-overexpressed cancer cells. Specific recognition of FR-overexpressed HeLa cells over normal L-02 cells was then confirmed by confocal mi-croscopy imaging, where only HeLa cells showed intense cellular lu-minescence (Fig. S8), demonstrating the feasibility of the nanoprobe for imaging of cancer cells.
In a control experiment, L-02 cells (FR-) that were incubated with [email protected] for 1 h showed no luminescence signals from all three channels. This can be attributed to the insuﬃcient cell binding (FA-FR binding) and internalization of the nanoprobe due to the lack of FR on the membrane of L-02 cells. To confirm this, other two control experiments were further performed, and in which, (i) HeLa cells were pre-treated with FA to block FR before incubation with the nanoprobe;
(ii) HeLa cells were incubated with the FA-free nanoparticles [email protected] BHHCBT-Eu. In these two cases, HeLa cells showed almost no lumi-nescence both under steady-state and TGL imaging modes (Fig. 4C and D). The above results demonstrate that the nanoprobe [email protected] BHHBCB-Eu-FA can specifically recognize FR-overexpressed HeLa cells through a FA-FR mediated binding process, which enables the probe to be potentially used for the cancer cell-targeted luminescence imaging.
In summary, a FR-targeted nanoprobe, [email protected], has been prepared for the recognition and detection of cancer cells through “double-check” luminescence imaging. The nanoprobe was fabricated by covalently doping CTMR and BHHBCB-Eu3+ in core and shell of silica nanoparticles, followed by conjugating FA molecules on the surface of nanoparticles. This nanoprobe features several distinct advantages including: (i) well monodisperse in buﬀer with high pH and photo-stability; (ii) unique steady-state and time-gated emissions at diﬀerent wavelengths; (iii) low cytotoxicity; and (iv) specific binding ability to FR-overexpressed HeLa cells through FA-FR interactions. Using the nanoprobe, the “double-check” imaging of HeLa cells was successfully performed, which could be expected to contribute to future clinical and pre-clinical cancer cell detections in biological samples.