Introduction

Intracellular pH is a key target parameter in cell growth and death, muscle contraction, metabolism of organs, ion transport and homeostasis [1]. Abnormal intracellular pH values may cause various cardiopulmonary and neurological diseases, such as ischemic/reperfusion injury and Alzheimer’s and Parkinson’s disease [2]. Therefore, accurate intracellular pH monitoring is critical for developing a better understanding of cellular activities and providing crucial support in early disease identification. To date, the glass pH electrode is the most common tool used for pH measurement. However, the disadvantages of electrical interference or mechanical damage to cells cannot be avoided, making it unsuitable for intracellular pH studies [3]. In contrast, fluorescence-based sensors are more applicable to detection of intracellular pH changes because they are noninvasive and exhibit excellent spatial and temporal resolution, good sensitivity and ease of sample preparation [4, 5]. However, the majority of fluorescent pH sensors are dependent on changes of a single emission intensity, which can be affected by a range of parameters, such as fluorescence probe concentration and dye bleaching, thereby reducing the reliability of the measured data [6]. Keeping this in mind, considerable effort has been devoted to developing ratiometric pH sensors that can self-calibrate to overcome interference by environmental factors and successfully exceed the limits of single emission intensity measurements.

Carbon dots (CDs) have recently attracted much interest due to their small particle sizes, nonblinking properties, low toxicity, excellent solubility and biocompatibility [7,8,9,10,11]. In recent years, several groups have created many ratiometric fluorescent pH sensors based on CDs and used them to monitor intracellular pH [12,13,14,15,16,17,18]. Ma et al., for example, combined pH-insensitive rhodamine B isothiocyanate, pH-sensitive fluorescein isothiocyanate, and amino-coated CDs into a single device to create a programmable ratiometric pH sensor [19]. Using CD-fluorescein isothiocyanate composites, Wang et al. fabricated a fluorescent ratiometric pH probe [20]. However, these ratiometric pH-sensing devices involved cross-attachment or embedding of dyes in the CDs, and extensive separation and purification operations were still required [21]. To circumvent the disadvantages highlighted above, numerous ratiometric fluorescence nanosensors based on label-free CDs have been developed [22, 23]. For example, Yang et al. synthesized novel ratiometric fluorescent CDs for enantioselective sensing of L-lysine and pH in vivo and in vitro [24]. A novel ratiometric fluorescence nanosensor incorporating label-free CDs was constructed by Feng et al. for intracellular water pH monitoring and lysosome imaging [25]. Nevertheless, the development of CD-based fluorescent probes appropriate for measuring pHs in living cells is at an initial level [26, 27]. As a result, it is critical to construct CDs with pH-sensitive ratiometric fluorescence emission to develop ratiometric fluorescence nanoprobes for pH assessments in living cells.

A ratiometric fluorescent pH nanoprobe comprising label-free CDs was constructed in this work and utilized for intracellular pH sensing, as shown in Scheme 1. The preparation of CDs (p-CDs) exhibiting dual emission at 525 and 623 nm was achieved with a one-pot solvothermal treatment of p-phenylenediamine (p-PD) and urea. The p-CDs showed inherent ratiometric fluorescence emission (I525/I623) as a function of pH change without the necessity for dye labeling, and exhibited a strong linear relationship with pH over the response range 4.0−8.8 in buffer solutions. The CDs, owing to their unique emission features, were subsequently used as a ratiometric fluorescent pH nanoprobe to monitor the pHs of HeLa cells.

Scheme 1
scheme 1

Schematic illustration of p-CD preparation and application for ratiometric determination of pH

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Experimental Section

Materials

p-PD, urea, glutathione (GSH), cysteine (Cys), homocysteine (Hcy), alanine (Ala), arginine (Arg), serine (Ser), leucine (Leu), and metal ions were procured from Aladdin Ltd. (http://www.aladdin-e.com/, Shanghai, China). The rest of the chemicals were of analytical grade, and no additional purification was carried out before utilization in experiments. Throughout the research, distilled water was used.

Characterization

TEM micrographs were obtained using a GZF2.0 FEI Electron Optics transmission electron microscope (Thermo Scientific, America https://www.thermofisher.com/). An ESCALAB 250Xi multitechnique surface analyzer was used to perform X-ray photoelectron spectroscopy (XPS) studies (Thermo Scientific, America https://www.thermofisher.com/). KBr pellets were used to acquire Fourier transform infrared (FT-IR) spectra on a Tensor 27 FT-IR spectrometer (Bruker, Germany https://www.bruker.com/). A Shimadzu UV-2550 spectrophotometer was used to record absorption values (Shimadzu, Japan https://www.shimadzu.com). An RF5301 fluorescence spectrometer was used to measure all fluorescence spectra (Shimadzu, Japan https://www.shimadzu.com). The fluorescent lifetimes and absolute quantum yields were determined with an Edinburgh Analytical FLS1000 instrument (Techcomp, China) (Techcomp, China http://www.techcomp.cn/). A Sartorius PB-10 pH meter was used to measure pH (Sartorius, Germany https://www.sartorius.com.cn/). A Xiangyi H1650-W centrifuge was used to separate mixtures (Xiangyi, China http://www.xiangyihn.com/). A Carl Zeiss LSM 880 confocal laser scanning microscope was used for fluorescence imaging (Zeiss, Germany https://www.zeiss.com/corporate/int/home.html).

Preparation of CDs

The p-CDs were prepared through a one-pot solvothermal method according to literature reports [28, 29]. Carbonization of p-PD and urea with the help of concentrated HNO3 was used to generate p-CDs via a solvothermal technique. First, 0.1 g of p-PD and 0.1 g of urea were dissolved in C2H5OH (10 mL). Subsequently, the solution was poured into a 25 mL poly(tetrafluoroethylene)-lined stainless steel autoclave. Afterward, 40 µL of pure HNO3 was added dropwise with vigorous stirring. A dark-red suspension was formed after heating for 12 h at 180 °C, and the solution was left to cool to room temperature. To eliminate the nonfluorescent precipitates, the solution was subjected to centrifugation for 15 min at 12,000 rpm (with a 10,000×g centrifugation force). The products were purified on a silica column using methanol and methylene dichloride as the eluent, and the p-CDs were separated for use as nanosensors. The CD sample was eventually obtained following evaporation of the solvent and drying in a vacuum, and the absolute quantum yield of p-CDs emission was 8%.

Fluorescence Assays of pH Values

To make a stock solution, the p-CDs were dissolved in ethanol. pH values were determined using a 20 mM buffer containing disodium hydrogen phosphate-citric acid buffer at pHs 2.2−8.0 and a glycine-sodium hydroxide buffer at pHs 8.5−10.6. A 100 µL CD dispersion (1.0 mg/mL) was added to 4.90 mL of buffer at various pH levels in a series of test tubes. The resultant solutions were mixed well before fluorescence spectra were taken. By altering the pH values between 5.0 and 8.0 with HCl and NaOH solutions, the reversibility of CD suspension was studied after repetition for four continual cycles, and the fluorescence spectra were obtained. The tests were carried out in triplicate.

Cytotoxicity Assay

A conventional 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) test was employed to examine the cytotoxicity of the p-CDs. Ninety-six-well plates were used to seed HeLa cells for 24 h prior to treatment, and they were subsequently cultured for 12 h with various doses of p-CDs (0−60 µg/mL). Next, 20 µL of MTT solution (5.0 mg/mL) was added to the individual wells and incubated for 4 h at 37 °C. Then, 100 µL of supernatant was withdrawn, and 150 µL of dimethyl sulfoxide (DMSO) was added to the individual wells for solubilization of formazan. The plate was agitated for 10 min before a microplate reader was employed for absorbance measurements of the solutions at 562 nm. All MTT tests were carried out four times in total.

Cell Fluorescence Imaging

HeLa cells were seeded on 6-well culture plates with sterile coverslips and left to adhere for 24 h before investigation. The media was withdrawn, and the cells were subjected to treatment for 5 h at 37 °C with p-CDs (40 µg/mL) in RPMI medium. Phosphate buffered saline (PBS) (pH 7.4) was then utilized to wash the cells three times. It was subsequently incubated for 30 min with 5 µg/mL nigericin in a high concentration K+ buffer at various pH values (5.0, 7.0, and 8.0). A Carl Zeiss LSM 880 confocal laser scanning microscope was used to image the cells immediately. The excitation wavelength was 458 nm, while the emission collection wavelength range was 475–575 nm.

Results and Discussion

Characterization of p-CDs

TEM was first employed to assess the morphological features of the p-CDs. The p-CDs were spherical and exhibited good dispersion in aqueous solutions, as shown in Fig. 1. Furthermore, their size distribution was determined to be 2.6−5.4 nm, with a mean size of 3.8 nm (based on measurements of more than 100 particles in the TEM photograph) (inset of Fig. 1).

Fig. 1
figure 1

TEM image and histogram showing the particle size distribution (inset) of the p-CDs.

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The surface functional groups and elemental compositions were then determined using XPS analysis and FT-IR spectroscopy. According to the survey spectrum, the main components of the targeted CDs were carbon, oxygen, and nitrogen: 78.48% carbon, 7.69% oxygen, and 13.83% nitrogen (Fig. 2 A). The high-resolution C 1s XPS spectrum (Fig. 2B) was deconvoluted into three peaks at 284.78, 287.13, and 290.43 eV that were ascribed to C-C/C = C, C-N/C = O, and O-C = O bonds, respectively [24, 30]. The high-resolution N 1s spectrum (Fig. 2 C) showed a pair of peaks at 399.48 and 402.08 eV, corresponding to pyridinic and amino N bonds, respectively [31, 32]. Oxygens on carbon atoms (C = O/C-OH) (Fig. 2D) were identified in the high resolution O 1s spectrum with a peak at 531.63 eV [33]. FT-IR characterization was also carried out for analysis of chemical bonding on the surfaces of the p-CDs. The FT-IR spectrum of the p-CDs is shown in Fig. 3 A, which demonstrates that the p-CDs contained –NH2 (3301 and 3203 cm− 1), –CH2 (3008 cm− 1), O = C − O− (1759 cm− 1), C = C (1630 cm− 1), –C = N (1517 cm− 1), C − N−C (1261 cm− 1), and C-O-C (1128 cm− 1) functional groups [34,35,36]. The results of the FT-IR and XPS analyses were similar, indicating that –NH2 and –COOH groups existed on the p-CD surface, rendering it highly water-soluble. The chemically reactive entities also allowed further functionalization and surface passivation.

Fig. 2
figure 2

Structural characterization of the p-CDs. (A) XPS survey spectrum; high-resolution XPS survey scans for (B) C 1s, (C) N 1s and (D) O 1s binding energies of the p-CDs.

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Fig. 3
figure 3

(A) FT-IR spectrum of p-CDs; (B) Absorption (Abs) and fluorescence emission spectra (Em) of p-CDs with various excitation wavelengths

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Optical Features of the p-CDs

The optical features of the p-CDs were examined by collecting their absorption and fluorescence spectra. The p-CDs in aqueous solutions exhibited numerous absorption bands at 241, 296, and 400–550 nm, as shown in Fig. 3B. The aromatic C = C bonds gave rise to n-π* transitions, which were responsible for the absorption bands at 241 and 296 nm [37], and the wide absorption between 400 and 550 nm was ascribed to n-π* transitions occurring with the surface states of p-CDs [38, 39]. Next, the fluorescence characteristics of the p-CDs were thoroughly investigated. The fluorescence intensities of the p-CDs at 525 and 623 nm increased with increasing excitation wavelength, as shown in Fig. 3B. However, the emission spectrum at 525 nm was not fully displayed when the wavelength excitation was 510 nm. Therefore, an excitation wavelength of 500 nm was selected as the excitation maximum in this study, which was facilitated by ratiometric detection of pH. Furthermore, when the excitation wavelength was varied from 325 to 405 nm, the emission wavelengths of the p-CDs remained nearly unchanged. This excitation independence may be attributed to the remarkable chemical composition and content uniformity of the p-CDs [40], which may enable expanded biological applications by avoiding autofluorescence [41].

Investigation of pH-Response Properties

Using 20 mM buffer with various pH values in the range 4.0 to 8.8, standard fluorescence pH titrations of the p-CDs were carried out. As shown by the 3D emission-pH spectra (Fig. 4 A), the fluorescence emission intensity at 525 nm underwent a progressive increase with increasing pH values, whereas the emission intensity at 623 nm for the p-CDs remained more or less constant. Fluorescence emission spectra also confirmed this result (Fig. S1). The intensity ratios of the two emission channels (I525/I623) manifested a linear relationship with pH over the range 4.0 to 8.8 and were fit with the linear regression equation I525/I623 = 0.1279pH + 0.3057, with a linear coefficient of 0.9968 (Fig. 4B). The linear range was wide enough to suit the needs of intracellular pH monitoring and in vivo imaging. The aforementioned findings showed that p-CDs might be used as a novel nanoprobes for quantitatively determining pH levels.

Fig. 4
figure 4

(A) 3D emission-pH spectra of p-CDs in 20 mmol/L buffer at different pH values; (B) Linear relationship of the fluorescence intensity ratio (I525 nm/I620 nm) versus pH. λex = 500 nm

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We initially looked at the UV–vis spectra of p-CDs as the pH was increased from 4.0 to 8.8 to see if we could determine the cause of the response to pH. There was no discernible change, as indicated in Fig. 5 A. As a result, we concluded that the increased fluorescence of the p-CDs at high pH levels could not be due to aggregation [42]. Then, fluorescence lifetime measurements were measured (Fig. 5B). The fluorescence decays of the p-CD solutions remained biexponential as the pH was increased from 4.0 to 8.0, and the mean lifetimes rose from 1.70 to 4.31 ns, showing that the rate of nonradiative transitions was reduced. As a result, we hypothesized that the increased fluorescence intensity was due to the carboxylate-based negatively charged protective shell present on the surfaces of the p-CDs, which, in turn, restricted the rate of nonradiative decay from the photoexcited state of the p-CDs and resulted in a significant improvement in fluorescence [43].

Fig. 5
figure 5

(A) UV‒vis absorption spectra of the as-prepared p-CDs in different pH buffers; (B) Normalized transient intensity of p-CDs on the nanosecond timescale

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Photostability and Reversibility of p-CDs

Since excellent photostability and reversibility are essential for long-term and real-time cellular imaging, we further investigated the photostability and reversibility of the p-CDs. Timed scans for measurements of fluorescence intensity in 20 mM PBS buffer with varying pH values (5.8, 7.0, and 8.0) were conducted for 12 h to examine the photostability. The ratiometric fluorescence intensities (I525/I623) of the p-CDs remained uniform during the scanning process (Fig. 6 A), which performed that they were stable to air and light at appropriate pH values. The fluorescence reversibility as a function pH was then tested by repeatedly altering the pH between 5.0 and 8.0. The ratiometric fluorescence intensity (I525/I623) of the carbon dots showed high consistency for the alkaline (pH 8.0) and acidic (pH 5.0) solutions, as shown in Fig. 6B. Thus, the pH-sensitive CDs demonstrated satisfactory fluorescence reversibility and photostability in solutions with a range of pH values, indicating that they might be utilized as intracellular pH nanoprobes.

Fig. 6
figure 6

(A) Time-dependent fluorescence intensity changes of p-CDs in buffer solutions with different pH values in daylight for 12 h; (B) Fluorescence reversibility versus pH for repeated changes of pH between 5.0 and 8.0

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Influence of Potentially Interfering Substances in Solutions with Varying pH Values

The possible interference resulting from high quantities of amino acids (Ala, Arg, Ser, and Leu), biothiols (GSH, Cys, and Hcy), and metallic ions (Ca2+, Mg2+, Al3+, Zn2+, Cu2+, Ni2+, Mn2+, Pb2+, Hg2+, Fe2+, Fe3+, Cd2+, and Ag+) was explored. As demonstrated in Fig. 7, there were no substantial variations in the fluorescence intensities of the p-CDs in the presence of these interfering chemicals, indicating remarkable selectivity for the nanoprobe.

Fig. 7
figure 7

Fluorescence intensity of p-CDs in the presence of interfering substances in 20 mM PBS buffer with different pH values. 1, Blank; 2, Ca2+; 3, Mg2+; 4, Al3+; 5, Zn2+; 6, Cu2+; 7, Ni2+; 8, Mn2+; 9, Pb2+; 10, Hg2+; 11, Fe2+; 12, Fe3+; 13, Cd2+; 14, Ag+; 15, Ala; 16, Arg; 17, Ser; 18, Leu; each concentration was 1 mM. 19, 5 mM GSH; 20, 0.5 mM Hcy; 21, 0.5 mM Cys. (A) pH at 5.0, (B) pH at 8.0

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pH Imaging in Living Cells

The use of p-CDs for determining the pH values of HeLa cells was attempted after the abovementioned results were obtained. To assess the toxicity of p-CDs to live cells, a conventional MTT test was first used [44]. As demonstrated in Fig. S2, there was no substantial reduction in cell viability, demonstrating that p-CDs had limited cytotoxicity toward HeLa cells at concentrations below 60 µg/mL. HeLa cells were then treated with p-CDs (40 µg/mL) for 5 h at 37 °C to look for subtle alterations in the intracellular pH. The cells were then treated for 30 min with buffers establishing pH values of 5.0, 7.0, and 8.0, which allowed for H+/K+ exchange and maintained an equal pH in the buffer media and inside the cells [45, 46]. As depicted in Fig. 8, the emission intensity of the treated cells in channel 1 (green pseudocolor) increased progressively as the pH was raised from 5.0 to 8.0, but channel 2 (red pseudocolor) remained nearly unaltered. The merged graphs of channel 1 and channel 2 exhibited a noticeable hue shift with pH changes. In addition, Picture-Pro Plus software was used to create a fluorescence ratio image (Igreen/Ired) (Fig. S3). The intensity ratio (I525/I623) was linearly related to pH over range 5.0−8.0, as predicted, demonstrating that the CD-based nanoprobe is capable of detecting subtle changes in intracellular pH.

Fig. 8
figure 8

Confocal microscope fluorescence images of 40 µg/mL p-CDs incubated with HeLa cells in high concentration K+ buffer at different pH values. Excitation wavelength for p-CDs: 458 nm; emission collection: 475–575 nm. Scale bar: 20 μm

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Finally, comparisons between this ratiometric nanosensor and other published CD-based ratiometric sensors used for pH detection in materials, including their diameters, excitation and emission wavelengths, and measurement ranges, are summarized in Table 1 [16, 23, 25, 47,48,49,50,51,52,53]. This nanosensor presented longer excitation and emission wavelengths (λex = 500 nm; λem = 526/623 nm), a wider linear range (4.0−8.8) and simpler synthetic procedures (one-pot solvothermal method) than the other CD-based pH sensors, making this nanosensor comparable to previous CD-based ratiometric sensors. The relatively small Stokes shift is the major limitation of the p-CDs, and this can be modified by constructing CDs with larger Stokes shifts to extend the scope of applications involving sensitive measurements of intracellular pH.

Table 1  A comparison of CD-based methods for detection of pH value
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Conclusion

A simple one-pot solvothermal carbonization process was used to generate novel nanoprobe p-CDs. Detailed spectroscopic characterization revealed that this novel fluorescent nanoprobe may be utilized to monitor pH in ratiometrically by using emission intensities at the two wavelengths 525 and 623 nm. The fluorescence intensity ratios (I525/I623) for p-CDs increased with increasing pH from 4.0 to 8.8 and showed a linear relationship. This sensor could be used effectively to ratiometrically monitor pH changes in living cells due to its excellent selectivity, strong photostability, and fluorescence reversibility, suggesting practical applications for detection of pH-related diseases and disorders.