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Intracellular Fluorescent Temperature Probe Based on Triarylboron Substituted Poly N‑Isopropylacrylamide and Energy Transfer Jun Liu,† Xudong Guo,† Rui Hu,† Jian Xu,† Shuangqing Wang,† Shayu Li,*,† Yi Li,*,‡ and Guoqiang Yang*,† †

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡ Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *

ABSTRACT: A novel hydrophilic fluorescence temperature probe (PNDP) based on polarity-sensitive triarylboron compound (DPTB) and PNIPAM is designed and synthesized. In order to overcome the shortcomings of the single-intensitybased sensing mechanism and obtain more robust signals, ratiometric readout is achieved by designing an efficient FRET system (PNDP-NR) between DPTB and Nile Red (NR). PNDP-NR possesses some excellent features, including wide temperature range, good linear relationship, high temperature resolution, excellent reversibility, and stability. Within a sensing temperature range of 30−55 °C, the fluorescence color of PNDP-NR experiences significant change from red to greenblue. PNDP-NR is also introduced into NIH/3T3 cells to sense the temperature at the single-cell level. It gave excellent photostability and low cytotoxicity in vivo. Up until now, only a few fluorophores could meet the requirements and exhibit good properties for temperature sensing, which had high sensitivity, instant response, excellent reversibility, and stability for temperature variations.17−19 It is necessary to develop new fluorophores to create some hydrophilic fluorescent thermometer to meet the needs of intracellular temperature sensing and gain in depth understanding of PNIPAM chain conformations. In this work, following our interest in the design of a novel fluorescent sensor with high temperature resolution and a wide temperature range, we chose dipyren-1-yl(2, 4, 6triisopropylphenyl)borane (DPTB) as the fluorophore considering that DPTB possessed excellent fluorescence features, including high quantum yields and extremely sensitive to the change of its surroundings.20−22 DPTB was modified and covalently connected to PNIPAM nanogel to get a new fluorescent temperature probe (PNDP), which displays a wide temperature range, good linear relationship, better temperature resolution, excellent reversibility, and very good stability. However, we realized that PNDP had the same shortcomings with previous reported fluorescent thermometers, i.e., the signal of the single emission readout was affected by some other factors, such as the distribution of the probes, excitation power,

T

emperature is a basic physical parameter that affects chemical reactions and biological processes. In particular, many cell activities are involved with the change of intracellular temperature, e.g., enzymatic reactions, gene expression, cell division, and energy metabolism.1 Cancer and malignant cells are reported to have higher temperature than normal cells due to enhanced metabolic activity.2 Therefore, accurate temperature detection of living cells is very important to the explanation of many biological processes and treatment of some diseases. In the past decade, fluorescent thermometers have received considerable attention due to their high spatial resolution and high throughput compared with conventional thermocouple thermometers.3−7 However, it is still a great challenge to measure temperature on the nanometer scale or at the single-cell level.8,9 Recently, poly N-isopropylacrylamide (PNIPAM), as a thermo responsive polymer, has been investigated in nanotechnology and biotechnology applications.10−12 PNIPAM displays a lower critical solution temperature (LCST) around 32 °C in water, above which PNIPAM undergoes a coil-to-globule phase transition due to the breakage of intermolecular hydrogen bonding between water and PNIPAM chains.13 It has been considered as a promising candidate for a nanothermometer. Uchiyama et al. incorporated the polarity-sensitive oxdiazole fluorophores into PNIPAM.14−16 The emission of the fluorophores would change with the process of phase transition. In this case, the polymer phase transition had translated into a fluorescent signal. © XXXX American Chemical Society

Received: November 2, 2014 Accepted: March 10, 2015

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DOI: 10.1021/acs.analchem.5b00887 Anal. Chem. XXXX, XXX, XXX−XXX

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Cell Culture and Viability Assay. Mouse fibroblast cells (NIH/3T3) were cultured and plated on glass bottomed dishes at 37 °C under 5% CO2 atmosphere. The cells, prewashed twice, were incubated with 0.05 mg/mL PNDP (or PNDP-NR) in cultured medium. After 30 min, the cells were washed with PBS to remove unbounded probes six times before imaging by an Olympus FV1000-IX81 confocal laser scanning microscope using an oil objective, with excitation by a 405 nm laser, and 450−500 nm emission light was collected as the green channel and 600−650 nm emission light was collected as the red channel. Cell viability was measured by MTT assay. NIH/3T3 cells were cultured on a 96-well plate. After 24 h, the medium was replaced with fresh medium (200 μL) containing varied concentrations of PNDP-NR and the cells were cultured another 24 h. The medium was replaced with fresh medium containing MTT (0.5 mg/mL) and incubated for 4 h. The supernatant was removed, and 100 μL of DMSO was added to each well to dissolve the formed formazan and the absorbance of the solution was measured to assess the relative viability of the cells. The absorbance values (A) were read at a wavelength of 490 nm. Relative cell viability was expressed as A/A0 × 100%, where A is the absorbance of the experimental group and A0 is the absorbance of the control group.

and detectors.23,24 Meanwhile, the slightly changes in single intensity are rather difficult to be distinguished either by the naked eye or the aid of an instrument. To measure temperature more accurately and overcome difficulties encountered by conventional fluorescent thermometers, an emerging trend is to develop a dual-emission and wavelength-ratiometric sensing schemes.25,26 In contrast with single- intensity-based sensing, the ratiometric fluorescent probe can display a self-calibrating readout and significant color switching. Therefore, for a further improvement of the performance of the fluorescent temperature probe and obtaining more robust signals, we developed a fluorescent nanogel temperature probe with ratiometric readout. The dual fluorescent signal (green and red) was rendered by efficient fluorescence resonance energy transfer (FRET) between DPTB and freely distributed Nile Red (NR) in the hydrophobic domains of the nanogel. The ratiometric readout is measured directly with a colorimeter or even distinguished easily by the naked eye since the fluorescent color changes. This fluorescent temperature probe is reversible and demonstrates good stability. Compared with the previously reported ratiometric temperature probes,24 PNDP-NR possesses some advantages, such as long excitation wavelength, instant response to temperature, significant color changes, and a wide temperature range. Moreover, we applied the ratiometric fluorescent probe to measure the temperature at the single-cell level. The results of the cell experiments indicate that the probe has good photostability and low cytotoxicity.



RESULTS AND DISCUSSION In order to obtain the polymer of PNIPAM with DPTB, we synthesized DPTB-2OH according to the Scheme S1 in Supporting Information. The cross-linked nanogel of P (NIPAM-co-HA) was synthesized as the previous reported method.14 We prepared the PNDP (Figure 1) by the successful



EXPERIMENTAL SECTION General Information. N-Isopropylacrylamide (NIPAM), 2-hydroxyethyl acrylate (HA), and N,N′-methylenebis(acrylamide) (MBAM) were obtained from Alfa Aesar; NIPAM was purified by recrystallization from n-hexane prior to use. All other reagents were purchased from J&K (Beijing, China) and used without further purification. Dialysis bag, 1200−1400; absorption spectra were recorded on Hitachi UV3010. The fluorescence spectra were obtained on a Hitachi F4500. The transmission electron microscopy (TEM) image was obtained by a JEM-2010HT (200 keV) transmission electron microscope. Dynamic light scattering (DLS) was performed on ALV/DLS/SLS-5022F. Cells were analyzed using a confocal microscope (OLYMPUS FV1000-IX81). 1H NMR spectra were obtained on BrukerAvance III 400H (400 MHz) spectrometers. A detailed description of the synthesis of fluorophores is provided in the Supporting Information. P (NIPAM-co-HA) nanogel and DPTB was synthesized according to the reported procedure previously.14,20 Preparation of Polymeric Fluorescent Probe PNDP. P (NIPAM-co-HA) nanogel (115 mg) is dissolved in dry CH2Cl2 (2.5 mL), and the solution is added dropwise over 20 min to the solution of 7 (20 mg) in dry CH2Cl2 (2.5 mL) under a 0 °C N2 atmosphere. When the addition was completed, the mixture was allowed to warm to room temperature for 5 h. Then 5 μL of water is added. The nanogel is reprecipitated three times using Et2O to obtain PNDP as a yellow powder. PNDP can be redispersed into water by using a disperser and further purified by dialysis for 24 h. Preparation of Polymeric Fluorescent Probe PNDPNR. PNDP (10 mg) and 1 mL solution of NR (10−4 M) in THF were codissolved in 2 mL of THF with vigorous stirring for 1 h; the solution was vacuum-dried to remove the THF. PNDP-NR can be obtained as a dark green solid and redispersed into water by using a disperser.

Figure 1. Chemical compositions of nanogel PNDP.

combination of DPTB-2OH and P (NIPAM-co-HA) (Scheme S1 in the Supporting Information) using oxalyl chloride as the reaction site. Since DPTB-2OH contains two hydroxyl groups in the molecule, two acid chloride groups were formed when reacting with excess oxalyl chloride. One of them reacted with hydroxyl of P (NIPAM-co-HA) and the other transformed to carboxyl when dispersed into water. The remaining carboxyl group is strongly hydrophilic, which guarantees the hydrophilic distribution of DPTB. The appearance and size distribution of PNDB have been examined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). A TEM image indicates that the nanogel exist in spherical particles in the dried state (Figure S1a in the Supporting Information). DLS measurement at various temperatures is shown in Figure S1b in the Supporting B

DOI: 10.1021/acs.analchem.5b00887 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry Information. The significant shrinkage occurring from 32 to 34 °C is the result of cooperative dehydration of PNIPAM chains.27 The slight shrinkage with further increased temperature is attributed to rearrangement of the fine structures in the polymer. Figure 2a shows the temperature-dependent fluorescence spectroscopy. The spectra display an obvious blue shift from

Figure 3. Emission spectra of PNDP in aqueous solution at 25 and 45 °C; absorption and emission spectra of NR in ethanol.

simply by dissolving PNDP with NR in organic solvent (THF), and the organic solvent was removed with a vacuum pump to form the NR loaded nanogel PNDP-NR. Since the nanogel contains both hydrophilic and hydrophobic domains, oilsoluble NR is distributed in the hydrophobic domains when the nanogel is redispersed into aqueous media. Such a distribution of NR warrants that it will not disturb cellular events in vivo sensing. As expected, the two fluorophores give a dual (green and red) fluorescence signal with efficient FRET and easily to be ratioed. With a photoexcitation at 405 nm, the green emission of PNDP is induced and part of the emission is transferred to NR by FRET, which has a peak at 625 nm. Upon heating, the green fluorescence at 470 nm experiences the same increase as described in PNDP (Figure 4a). Meanwhile, the fluorescence intensity of NR at 625 nm experiences a slightly decrease rather than increase. The slightly decrease of the fluorescence intensity of NR at 625 nm is due to the decreasing efficiency of FRET. The content of NR in PNDP-NR has been optimized based on the following considerations: (1) NR should not influence the LCST of PNIPAM and (2) PNDP-NR

Figure 2. (a) Fluorescence spectra of PNDP in an aqueous solution (0.5 mg/mL) at various temperatures; excitation wavelength, 405 nm; the color change under UV radiation. (b) A correlation between the temperatures and FL intensity (black line, left axis; fluorescence intensity was normalized to the value at 30 °C) at 470 nm and good temperature resolution (blue line, right axis).

500 to 470 nm around the LCST of PNIPAM (32 °C) owing to sudden decrease in polarity around DPTB, which is associated with the heat-induced phase transition due to the polymer’s structural change from the coiled to globular state. With further increase of temperature, more and more fluorophores shifted from higher polar microenvironment to lower polar microenvironment derived from rearrangement of the fine structures in the polymer. Notably, the fluorescence intensity at 470 nm displays a 14-fold enhancement from 32 to 57 °C and even increases further when continue heating. Moreover, as shown in Figure 2b, the fluorescence intensity of PNDP displays a linear relationship from 34 to 56 °C. The large leap and wide temperature range can be ascribed to the strong sensitivity of DPTB to the polarity change of the surroundings, which confirms that merely minor change in the fine structure of PNIPAM can trigger significant fluorescence change. Therefore, high temperature resolution (0.2 °C) and a relatively wide temperature range is also achieved. Figure S2 in the Supporting Information shows multiple-run reversibility experiments of the fluorescence responses of PNDP to temperature variation, which indicates that PNDP has excellent reusability. According to the results above, we studied the cellular application of PNDP in a live-cell imaging mode. We introduced PNDP into live NIH/3T3 cells by incubating the cells with PNDP for 30 min without other additional reagents. A bright green fluorescence was observed in the cytoplasm of NIH/3T3 cells under confocal fluorescence microscopy with excitation at 405 nm (Figure S3 in the Supporting Information). This indicates that PNDP is a potential fluorescent thermometer capable of sensing intracellular temperature. In order to achieve the ratiometric readout, we designed a fluorescence resonance energy transfer (FRET) system between PNDP and a red fluorescent dye Nile Red (NR). NR has an absorption peak at 525 nm, which matches well with the fluorescence spectrum of PNDP. On the other hand, NR possesses a large stoke shift with an emission peak at 625 nm (in ethanol), avoiding the mutual influence of the fluorescence spectrum with PNDP (Figure 3). The system was prepared

Figure 4. (a) Fluorescence spectra of PNDP-NR in an aqueous solution (0.5 mg/mL) at various temperatures; (b) temperaturedependent ratio (normalized to the value at 30 °C) of fluorescence intensity (peak maximum) at 470 and 625 nm and good temperature resolution (blue line, right axis); (c) reversibility of the fluorescence responses of PNDP-NR to temperature variation (30 °C, CV% = 4.0%; 55 °C, CV% = 1.1%) (normalized to the value of 0 cycle); (d) fluorescence spectra of suspensions of fresh nanogel, 1 week and 2 week old nanogel (the samples are protected from light but not protected from air). Excitation wavelength: 405 nm. C

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Analytical Chemistry

it convenient for measuring directly with a colorimeter or even distinguishing easily by bare eyes. To explore the temperature sensing mechanism, we further measured the fluorescence spectra by photoexcitation at 525 nm (the maximum UV absorption of NR). The fluorescence intensity at 625 nm is much weaker than the emission of NR in PNDP-NR, which further demonstrates that the strong luminescence of NR in PNDP-NR results from FRET. With elevating temperature, instead of slightly decreasing by photoexcitation at 405 nm, the fluorescence intensity of NR increases about double from 32 to 50 °C resulting from the slightly decreasing polarity around NR (Figure S6 in the Supporting Information). This proves that the efficiency of FRET reduces with the temperature rising which is attributed to the slight decreasing overlap between the emission of the energy donor and the absorption of the energy acceptor (Figure 3). To demonstrate the application of PNDP-NR in vivo, it was introduced into NIH/3T3 cells by the same way with PNDP. The intracellular temperature sensing was also conducted with confocal fluorescence microscopy, and a heating stage was used to control the temperature of the cell solution. The green channel exhibits brighter fluorescence at 37 °C (Figure 6e)

should have to experience the widest range of color over the temperature range of 30−50 °C. The ratio of green fluorescence intensity at 470 nm and red fluorescence intensity at 625 nm also shows a 13-fold leap from 32 to 56 °C and a good linear relationship with temperatures from 34 to 56 °C (Figure 4b). The same high temperature resolution (0.2 °C) with PNDP is also achieved. For further evaluation of the reversibility of PNDP-NR, we conducted multiple-runs to examine the fluorescence responses of PNDP-NR to temperature change. The result indicates that PNDP-NR has excellent reversibility (Figure 4c).The improvement of the reproducibility compared with the RSD without ratiometry may be attributed to the signal of the single emission readout affected by some other factors, such as the distribution of the probes, excitation power, and detectors. When the temperature rises to 70−80 °C, the solubility of PNIPAM becomes very small. Therefore, the reversibility cannot be maintained. Meanwhile, the fluorescence spectra of the fresh nanogel, 1 week and 2 week old nanogels also indicate minor differences, which show PNDP-NR has good stability (Figure 4d). The absorption and normalized emission spectra of PNDP-NR with different concentrations are recorded as Figure S4 in the Supporting Information. The highly consistent shape of the fluorescence spectra with different concentrations confirm that the process of FRET only occurs at the interior of single nanoparticle, which is further confirmed by another feature as shown in Figure S5 in the Supporting Information, that is, efficient FRET between PNDP and NR does not occur in the solution in which PNDP and NR are dissolved separately and they are too far from each other.28 Since the ratio changes greatly with raising temperature, the color may experience a significant change from red, yellow, white to green-blue over 30 to 55 °C as shown in Figure 5. The

Figure 6. Confocal fluorescence images of NIH/3T3 cells labeled with PNDP-NR at λexc = 405 nm at 25 °C (a−c) and 37 °C (e−g). The green channels shown in parts a and e were acquired by collecting the green fluorescence from 450 to 500 nm, while the red channels shown in parts b and f were from 600 to 650 nm. Parts c and g are the overlay of parts a and b, parts e and f, respectively. Parts d and h are their corresponding bright-field images.

than that at 25 °C (Figure 6a). Meanwhile, the fluorescence intensity in the red channel basically keeps with rising temperature, which is consistent with the results in vitro (Figure 4a). The two fluorophores inside the nanogel particles are revealed by the overlay of the two images (Figure 6c,g) and their bright field images are shown in parts d and h of Figure 6, respectively. The results demonstrate that PNDP-NR could sense the intracellular temperature change in living cells with ratiometric readout. Photobleaching and cytotoxicity are other problems that limit the application of many organic dyes in cells. Good photostability can effectively reduce the impact of photobleaching. Figure 7 and Figure S7 in the Supporting Information show that the fluorescence intensity in the green and red channels is almost unchanged even after 30 scans with a laser (405 nm) power of 100 μW. Fluctuations of light sources during the process of scanning also can be well eliminated by the ratio of green and red fluoresce intensities (blue line in Figure S7 in the Supporting Information).The cytotoxicity of PNDP-NR on NIH/3T3 cells was examined by standard 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium

Figure 5. CIE chromaticity diagram showing the temperature dependence of the (x, y) color coordinates of PNDB-NR; photographs of PNDB-NR in aqueous solution at different temperatures.

temperature-dependent spectra are transformed to the Commission Internationale de L’Eclairage (CIE) 1931 coordinates. Figure 5 also shows the color changes of the luminescence in the CIE (x, y) chromaticity diagram at different temperatures. Such an obvious color switching makes D

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ACKNOWLEDGMENTS We are grateful for funding from the National Basic Research Program (Grants 2013CB834703, 2013CB834505, 2011CBA00905, and 2009CB930802) and the National Natural Science Foundation of China (Grant Nos. 21233011, 91123033, 21273252, 21205122, 21261160488, and 21072196).



Figure 7. Fluorescent images of living NIH/3T3 cells stained with PNDP-NR with increasing number of scans (1, 10, 20, and 30 times). Excitation wavelength, 405 nm (laser power at the sample plane, 100 μW); emission wavelength, 450−500 nm (green channel) and 600− 650 nm (red channel); irradiation time, 15 s/scan.

bromide (MTT) assay. PNDP-NR has no obvious effects on the cell viability even at a high concentration of 0.4 mg/mL (Figure S8 in the Supporting Information), which indicated that PNDP-NR has good biocompatibility. A thermal mapping shows the temperature distribution in the cytoplasm of NIH/ 3T3 cells is basically consistent (Figure S9 in the Supporting Information).



CONCLUSIONS A novel luminescent temperature probe (PNDP) has been developed by covalently labeling PNIPAM with a triarylboron compound (DPTB). By loading the PNDP nanogel with NR, the ratiometric readout was achieved by efficient FRET between DPTB and NR. PNDP-NR gives some advantages. (1) The dual emission (green and red) provide a self-calibrating readout and more robust signal, which could overcome some difficulties encountered by conventional fluorescent thermometers. (2) Since a 13-fold enhancement of the ratio from 32 to 56 °C, PNDP-NR has both high temperature resolution and relatively wide temperature range matching well with the physiologically relevant temperatures. (3) PNDP-NR displays a good linear relationship between temperature and the emission from 34 to 56 °C, good reversibility, and stability, which make it adaptable for use in different research areas, such as biological research, marine research, and underground geochemistry, etc. A significant color change from red, yellow, white to green makes it possible to be observed directly with the bare eye. Finally, such ratiometric readout can also be applied into sensing temperature at the single-cell level. It has good photostability and biocompatibility, making it suitable for potential applications in biology and medicine.



ASSOCIATED CONTENT

S Supporting Information *

Additional information a snoted in text. This material is available free of charge via the Internet at http://pubs.acs.org.



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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] Fax: (+86) 10-8261-7315. *E-mail: [email protected] Fax: (+86) 10-8261-7315. *E-mail: [email protected] Fax: (+86) 10-8254-3518. Notes

The authors declare no competing financial interest. E

DOI: 10.1021/acs.analchem.5b00887 Anal. Chem. XXXX, XXX, XXX−XXX

Intracellular fluorescent temperature probe based on triarylboron substituted poly N-isopropylacrylamide and energy transfer.

A novel hydrophilic fluorescence temperature probe (PNDP) based on polarity-sensitive triarylboron compound (DPTB) and PNIPAM is designed and synthesi...
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