Sulfuric Acid Assisted Preparation of Red-Emitting Carbonized Polymer Dots and the Application of Bio-Imaging

Red-emitting carbonized polymer dots (CPDs) was prepared from p-phenylenediamine (p-PD) aqueous solution with the assistance of sulfuric acid (H2SO4), and the optical properties and bio-imaging application were studied in this paper. Compared with other strong acids-assisted systems, SA-CPDs (prepared from H2SO4-assisted system, average diameter is ~ 5 nm) is the brightest. The photoluminescence Quantum Yields (QYs) is 21.4% (in water), and the product yield is 16.5%. SA-CPDs aqueous solution emits at 600 nm when excited by the light from 300 to 580 nm. The emission wavelength is independent on the excitation wavelength. Formation energies of CPDs in two ways were calculated to show that longitudinal growth (forming polymers) is difficult, and the transverse growth (forming CPDs) is easy. In addition, the two-photon photoluminescence properties (emitting at 602 nm when excited by 850 nm femtosecond pulse laser) of SA-CPDs were also utilized in the experiments for HeLa cells staining and shown to have potential applications in bio-imaging. Electronic supplementary material The online version of this article (10.1186/s11671-018-2657-4) contains supplementary material, which is available to authorized users.

CDs (QYs = 10.8%, in water) as "carbonized polymer dots (CPDs)." Unlike the traditional carbon dots, the CPDs' emission wavelengths do not depend on the excitation wavelength, and thus the PD-based "CDs" should be named more accurately as CPDs.
Herein, we report a facile and high-efficient method of strong acid-assisted hydrothermal route to prepare red-emitting CPDs and the application of bio-imaging with two-photon photoluminescence properties. Mechanism for the formation of CPDs is proposed by using Gaussian 09 program package.

Synthesis of Red CPDs From Acid-Assisted p-PD Systems
Based on our previous work [25], we selected sulfuric acid (H 2 SO 4 ), hydrochloric acid (HCl), and perchloric acid (HClO 4 ) as the assistants for the preparation of red CPDs, the corresponding CPDs were labeled as SA-CPDs, HC-CPDs, and PA-CPDs, respectively. In order to optimize the experimental conditions of H 2 SO 4 -assisted system, we selected several parameters, such as c (acid) to c (p-PD) ratio, c (p-PD), temperature (T), and reaction time (t). CPDs products were washed by hexane to remove the unreacted p-PD and by ethyl alcohol to remove acids, centrifuged at 14000 rpm for 30 min to remove polymer precipitation, and filtered through a 0.22 μm filter membrane. If powder is desirable, the purified CPDs solution can be further evaporated by rotary evaporator to near dry state at 80°C and a low vacuum condition (the remaining will be of powder form).

Characterization and Measurement
High-resolution TEM (HR-TEM) images were recorded on JEM-2100 transmission microscope operating at 200 kV. Infrared spectra of CPDs solutions were collected using Prestige-21 FT-IR spectrometer by use of KRS-5 window slices (mixture of TlBr and TlI), typically, liquid phases were dropped on one slice and dried. The slice was covered by the other slice and fixed on the testing stand. Then the infrared spectra were recorded.
The fluorescence spectra of CPDs were measured on F-2500 fluorescence spectrophotometer. UV-Vis absorption spectra were recorded on Lambda 950 UV/VIS/NIR Spectrometer. The two-photon emission spectra of CPDs were recorded by a fiber spectrograph (QE65000, Ocean Optics) in the microscope system. SA-CPDs aqueous solution and the powders re-dissolved solution were spun on slides, and the two-photon photoluminescence properties were then measured.
Photoluminescence quantum yields (QYs) of the CPDs were measured with Rhodamine B (QYs = 56% in ethanol) as the reference dye at the emission range of 580-610 nm excited by 365 nm UV light [25,28], the procedure of QYs measurements were shown in the Additional file 1.

Calculation Methods
The Gaussian 09 package was used for the density function theory (DFT) calculations [29]. The equilibrium structures were optimized by B3LYP method in conjunction with the 6-311++G (d) basis set level [30]. To investigate the role of solvent effects, water was utilized in polarized continuum model (PCM). Frequency analyses were done with the same level for confirming that each optimized structure corresponded to a stationary point.
Cell Culture and Treatment

mL of HeLa cells in Dulbecco's Modified Eagle
Medium (DMEM; Gibco) at an initial density of 4 × 10 4 cell per milliliters were seeded in each dish and cultured at 37°C for 24 h under a humidified atmosphere containing 5% CO 2 . SA-CPDs powders were re-dissolved in water to prepare the reserve solution (400 μg mL − 1 ). 1350 μL cells were cultured with 150 μL SA-CPDs reserve solution (the final concentration is 40 μg mL − 1 ) for 12 h and then washed three times with PBS to remove the free SA-CPDs. Finally, the cellular imaging results were collected with a confocal microscope under 850-nm femtosecond laser excitation (30 mW).

Optimizing Preparation for Red CPDs
In basic experiments, different acid-assisted systems with various concentration ratios, reaction temperatures, and times were investigated (see Additional file 1: Figure S1). We found that red CPDs can be formed above 180°C (reacting for 2 h) for different acids systems, and the reactions are not affected by anions in the solutions. Long-time (4-12 h, 240°C for H 3 PO 4 and HF systems, see Additional file 1: Figure S1f ) reaction will increase the particle size, and the red fluorescence will be eventually faded, while the fluorescence change is not obvious for HCl system (2-6 h, 200°C, see Additional file 1: Figure S2). Considering the energy saving and upper temperature limit of Teflon liner, optimum temperature and reaction time are selected as 200°C and 2 h respectively. Based on the optimization strategy of p-PD + HCl system (see Additional file 1: Figure S2), we optimized the p-PD + H 2 SO 4 and p-PD + HClO 4 systems and obtained the optimization results shown as Table 1.
SA-CPDs, HC-CPDs, and PA-CPDs were prepared from p-PD solution with the assistance of H 2 SO 4 , HCl, and HClO 4 , respectively. The optimized c (acid) to c (p-PD) ratios of H 2 SO 4 -, HCl-, and HClO 4 -assisted systems are 1, 3, and 3, respectively (see Additional file 1: Figure S3a). The suitable c (p-PD) range for the preparation of red CPDs is wide (from 0.02 to 0.20 mol L − 1 ). The optimized temperature (T) and the reaction time (t) are 200°C and 2 h. SA-CPDs is the brightest red CPDs with a high QYs of 21.4% (Additional file 1: Figure S3b).
There are two reasons why H 2 SO 4 -assisted carbon dots have a better quality compared to HCl-, HClO 4 -, and HNO 3 -assisted ones (published in our previous work [25]). First, H 2 SO 4 is a non-volatile strong acid which maintains its acidity in a high temperature and high pressure reaction solution. Second, H 2 SO 4 -assisted system is the only one that can form ammonium salt precipitation in precursor, and the precipitates release the free reactants slowly, avoiding the formation of large particle polymer precipitation and further promoting the formation of high quality carbon dots. HA-CPDs and PA-CPDs are dark-red-brown thick solutions and emit dark red PL under 365 nm UV light irradiation, while the as-prepared SA-CPDs is bright-red transparent thin solution and emits bright red light (Additional file 1: Figure S3c). After being purified by washing, concentration, filtering, and evaporation, dark-red-brown powders of SA-CPDs (Additional file 1: Figure S3d) were obtained with the product yield of 16.5%. The powders can be re-dissolved in water and the solution emits bright and red fluorescence (Additional file 1: Figure S3e).
Surface states of CPDs may affect the optical properties. The surface chemical groups of sample OpPD and SA-CPDs were characterized by FT-IR spectra (Fig. 1d). Two samples have several similar groups, such as Ar-H (2700-3200 cm − 1 [33], belongs to aromatic C-H stretching vibration), C-C (~1433 cm − 1 , belongs to aromatic C-bone stretching vibration, reveals the presence of Table 1 The selected experimental parameters of p-PD + acids systems

Proposed Mechanism for the Formation of CDs
Formation energies were calculated using the Gaussian 09 program package. After being protonated by acid-assisting, bi-polymers may be polymerized in two ways that called longitudinal and transverse growth. The calculated formation energy of transverse growth (− 1406.07 kJ mol − 1 ) is significantly higher than that of longitudinal growth (− 616.25 kJ mol − 1 ). It shows that the fully protonated bi-polymers (pH 3, after excess H + was added) tend to be polymerized in transverse way to form planar structure (Fig. 2). These planar structures were then self-assembled to form spherical CPDs.

Optical properties
Although being prepared by the assistance of different acids, all CPDs have the similar optical properties [25].
For the UV-Vis spectra of SA-CPDs aqueous solution (Fig. 3a), the absorbance peak located at~290 nm is associated to the transitions in benzene ring, and the peaks located at 430 nm and 510 nm could be assigned to π-π* transition of substituted phenazine conjugated to the lone electron pairs on the adjacent amine group and the electron transition from the benzenoid ring to the quinoid rings, respectively [32]. The excitation curve describes a wide and a gradual upward trend at visible region, and the maximum excitation peak (~580 nm) is close to the emission peak (~600 nm). The CPDs emit at red light zone (600-700 nm) when they are excited from 220 nm to 310 nm, while emit at orange light (~600 nm) when excited from 310 nm to 580 nm (Fig. 3b). It is worth noting that the fluorescence of this kind of red-emitting CPDs is excitation-wavelength-independent [22,35].

Cellular Imaging
Two-photon photoluminescence properties of SA-CPDs before and after the powdering process are shown in Fig. 4a. There is a blue shift from 602 nm (before the Fig. 2 Formation energies of longitudinal and transverse growths a b Fig. 3 a UV-Vis absorption, excitation (peak at 600 nm), and b emission (excited 220-580 nm) spectra of SA-CPDs powdering process) to 529 nm (after the powdering process) at the same excitation wavelength of 850 nm by femtosecond pulse laser (30 mW). The PL intensity was increased after the powdering process. SA-CPDs powders were re-dissolved in PBS (1X) and was applied in HeLa cells imaging using confocal fluorescence microscopy and 850 nm femtosecond pulse laser (30 mW) (see Fig. 4b-e). After cultured with HeLa cells for 12 h, SA-CPDs were swallowed by HeLa cells, and CPDs entered cytoplasm. The FL intensity of red channel (645-675 nm) is weak while the green channel (526-574 nm) is bright, it is consistent with the blue shift in the powdering process.

Conclusions
A facile method of acid-assisted hydrothermal route to prepare carbon dots and the application of bio-imaging were reported. Within H 2 SO 4 -, HCl-and HClO 4assisted systems, SA-CPDs prepared from H 2 SO 4assisted system is the brightest CPDs with the average size of~5 nm, the QYs of 21.4%, and the product yield of 16.5%. SA-CPDs aqueous solution emits at 600 nm when excited by light from 300 to 580 nm. The emission wavelength is excitation-wavelength-independent. In addition, SA-CPDs have two-photon photoluminescence properties emitting at 602 nm when excited by 850 nm femtosecond pulse laser (30 mW). The method has also been utilized in imaging for HeLa cells and has the potential in, e.g., bio-imaging applications.

Additional file
Additional file 1: Figure S1. Photos of CPDs samples under UV light (365 nm). Figure S2. The optimization process for HA-CPDs preparation with HCl-assisted p-PD system. Figure S3. Abbreviations CDs: Carbon dots; CPDs: Carbonized polymer dots; HC-CPDs: Carbon Dots were prepared from p-PD with HCl-assisted system; PA-CPDs: Carbon Dots were prepared from p-PD with HClO 4 -assisted system; p-PD: Pphenylenediamine; QYs: Quantum Yields; SA-CPDs: Carbon Dots were prepared from p-PD with H 2 SO 4 -assisted system