Sulfuric‑Acid‑Assisted Synthesis of Bright Red Carbonized Polymer Dots for Two‑Photon Bio‑Imaging

Background

Carbon dots (CDs) have attracted widespread interest due to their excellent water solubility, optical stability, low toxicity, and cost‑effective synthesis. They are explored in electrochemical sensors, bio‑imaging, catalysis, light‑emitting devices, and optoelectronics. Bottom‑up routes from organic monomers, polymers, or natural products are preferred for large‑scale production. Functional groups such as –OH, –COOH, –C=O, and –NH₂ undergo dehydration and carbonization at elevated temperatures to yield fluorescent nanoparticles.

Red‑emitting CDs are particularly valuable for bio‑imaging because of their deeper tissue penetration and minimal background interference. However, most CDs display excitation‑dependent emission, making a single, stable red photon source rare. Phenylenediamine isomers (o‑, m‑, p‑) have been used to generate blue, green, and red CDs, respectively. Recent work identified the p‑PD‑based dots as carbonized polymer dots (CPDs) due to their excitation‑independent emission.

Here we present a facile, high‑yield, sulfuric‑acid‑assisted hydrothermal synthesis of red‑emitting CPDs and demonstrate their two‑photon imaging capability in HeLa cells.

Methods

Synthesis of Red CPDs From Acid‑Assisted p-PD Systems

We investigated H₂SO₄, HCl, and HClO₄ as proton donors, labeling the resulting CPDs as SA‑CPDs, HC‑CPDs, and PA‑CPDs. Reaction variables—acid‑to‑p-PD ratio, precursor concentration, temperature, and time—were systematically optimized. After hydrothermal treatment (200 °C, 2 h), the product was purified by hexane (removing unreacted p-PD) and ethanol (removing acid), followed by centrifugation at 14,000 rpm, filtration through a 0.22 µm membrane, and optional rotary evaporation to yield dry powders.

Characterization and Measurement

High‑resolution TEM (JEM‑2100, 200 kV) assessed morphology and lattice fringes. FT‑IR spectra were recorded with a Prestige‑21 spectrometer using KRS‑5 windows. Fluorescence and UV‑Vis spectra were obtained with an F‑2500 spectrophotometer and Lambda 950 spectrometer, respectively. Two‑photon emission was measured on a fiber spectrograph (QE65000, Ocean Optics) coupled to a femtosecond laser. Quantum yields were calculated using Rhodamine B (QY = 56 % in ethanol) as a reference, following the procedure in Supplementary File 1.

Calculation Methods

Density‑functional theory (DFT) calculations were performed with Gaussian 09 (B3LYP/6‑311++G(d)) in the PCM water model to evaluate the energetics of longitudinal and transverse growth pathways.

Cell Culture and Treatment

HeLa cells (4 × 10⁴ cells mL⁻¹) were seeded in DMEM (Gibco) and incubated at 37 °C with 5 % CO₂ for 24 h. SA‑CPDs (40 µg mL⁻¹) were added for 12 h, followed by PBS washes. Imaging was performed on a confocal microscope using an 850‑nm femtosecond laser (30 mW).

Results and Discussion

Optimizing Preparation for Red CPDs

All acid systems yielded red CPDs at temperatures above 180 °C (2 h). Higher temperatures (240 °C, 4–12 h) increased particle size and diminished fluorescence. The optimal conditions were 200 °C, 2 h, with an acid‑to‑p-PD ratio of 1 for H₂SO₄, and 3 for HCl and HClO₄. The concentration range of p-PD (0.02–0.20 mol L⁻¹) was tolerant. Under these parameters, SA‑CPDs achieved a QY of 21.4 % and a 16.5 % yield, outperforming HC‑CPDs and PA‑CPDs.

TEM Characterization and FT‑IR Analysis

HR‑TEM revealed monodispersed spherical CPDs (average diameter ≈5 nm) with a 0.21 nm lattice spacing corresponding to graphene (100) planes. FT‑IR spectra indicated higher surface oxidation in SA‑CPDs, with pronounced –OH and –COOH features and reduced –NH₂ signals, suggesting a more oxidized, hydrophilic surface.

Proposed Mechanism for the Formation of CDs

DFT calculations show that transverse growth (−1,406 kJ mol⁻¹) is significantly more favorable than longitudinal growth (−616 kJ mol⁻¹), leading to planar intermediates that self‑assemble into spherical CPDs.

Optical Properties

SA‑CPDs exhibit strong UV absorption at 290 nm (π→π*), with additional bands at 430 and 510 nm (phenazine–amine transitions). Excitation spectra peak near 580 nm, matching the 600 nm emission, and the fluorescence remains excitation‑independent across 220–580 nm, confirming the CPD nature.

Cellular Imaging

Two‑photon excitation at 850 nm produced a 602 nm emission before powdering and a 529 nm emission after, with increased intensity. Confocal images of HeLa cells incubated with SA‑CPDs showed cytoplasmic localization, with stronger green channel fluorescence due to the blue‑shifted emission.

Conclusions

We demonstrated a simple, scalable, sulfuric‑acid‑assisted hydrothermal method for producing bright red CPDs (≈5 nm, QY = 21.4 %, yield = 16.5 %). The CPDs emit at 600 nm under 300–580 nm excitation, and at 602 nm under 850 nm two‑photon excitation. Their excitation‑independent, high‑brightness fluorescence and efficient cellular uptake make them promising candidates for deep‑tissue imaging and other bio‑applications.

Abbreviations

CDs:

Carbon dots

CPDs:

Carbonized polymer dots

HC-CPDs:

CPDs synthesized from p-PD with HCl assistance

PA-CPDs:

CPDs synthesized from p-PD with HClO₄ assistance

SA-CPDs:

CPDs synthesized from p-PD with H₂SO₄ assistance

QYs:

Quantum yields

p-PD:

p‑Phenylenediamine