Multicolor Phenylenediamine Carbon Dots for Metal-Ion Detection with Picomolar Sensitivity

Carbon dots keep attracting attention in multidisciplinary fields, motivating the development of new compounds. Phenylenediamine C6H4(NH2)2 dots are known to exhibit colorful emission, which depends on size, composition, and the functional surface groups, forming those structures. While quite a few fabrication protocols have been developed, the quantum yield of phenylenediamine dots still does not exceed 50% owing to undesired fragment formation during carbonization. Here, we demonstrate that an ethylene glycol-based environment allows obtaining multicolor high-quantum-yield phenylenediamine carbon dots. In particular, a kinetic realization of solvothermal synthesis in acidic environments enhances carbonization reaction yield for meta phenylenediamine compounds and leads to quantum yields, exciting 60%. Reaction yield after the product’s purification approaches 90%. Furthermore, proximity of metal ions (Nd3+, Co3+, La3+) can either enhance or quench the emission, depending on the concentration. Optical monitoring of the solution allows performing an accurate detection of ions at picomolar concentrations. An atomistic model of carbon dots was developed to confirm that the functional surface group positioning within the molecular structure has a major impact on dots’ physicochemical properties. The high performance of new carbon dots paves the way toward their integration in numerous applications, including imaging, sensing, and therapeutics.

solution was treated more for further extraction, carbon dots solution is dried at the chemical hood. Drying may be accelerated by mild heating to ensure that there is no significant thermal degradation. Then the remaining solution is transferred to vacuum chamber for 48h. Each reaction volume was monitored at each time point of collection. The missing volume was mainly evaporation at first 24hours.

Reaction medium impact on fluorescence properties of CDs
Photoluminescence (PL), photoluminescence excitation spectroscopy (PLE), and absorbance spectra were measured with a plate reader Synergy H1. The working spectral range of the H1 is 300-700nm for excitation and emission, while 230-900nm band, sampled with 1 nm step, is available for absorption measurements. Horiba Jobin Yvon FL3-11 spectrofluorometer was used for Fluorescence measurments. Lifetime measurements were done with a PicoQuant system, which uses Taiko picosecond diodes as a 375 nm excitation.
The collected light was split by 50:50 beam splitter to CMOS camera (Thorlabs DCC1240C) for imaging and fiber-coupled Andor Kymera 193i spectrometer equipped with iDus 401 TEC-cooled CCD) for Spectrum analysis, and PicoQuant PDM photon counter for lifetime measurements.
Reaction types are summarized in Table S1.   Fig.S1.

Fluorescence evolution in presence of high acid contents
CDs formation output was monitored by taking fluorescence spectra (420 nm excitation) of samples collected at different times after reaction was initiated.
Reaction was carried in 250mL three-necked flask with attached refluxed system. 0.5mg PD was dissolved in 50mL EG with 10 mL concentrated 12M hydrochloric acid.
Reaction temperature was set to 433K. Carbon do ts obtained under these conditions are referred as γm-CDs, γp-CDs, and γo-CDs respectively. Results are summarized in Fig. S5.

Acid and solvent impact
Reaction mixtures were prepared in 20mL scintillation vials by adding 10% (1.5mL) concentrated acids to total volume of 15ml (6.6mg/mL) mPD solution.
The best acid was chosen in terms of QY for testing different solvents. We chose hydrochloric acid as 10% of aqueous content with 90% different solvents. Various solvents interact differently with acids and PD molecules. The affinity between reaction components, global steric factors of Solvent-EG, and protonation dynamics are all a matter of solvent. Emission characteristics and intensities in the set ε1-ε4, for both for mPD and pPD, are recorded ( Fig S8B).
Generally, both ε4 m/p CDs attained the highest emission properties RY-QY. The ε1 environment of propylene glycol showed similar results (Fig S8B), as predicted, due a physicochemical similarity with EG. Spectral emission and intensity are almost identical for ε1, ε2, and different for ε3. Supporting the assumption on chemical environment similarity. To avoid decoherence in the work, and for consistency, we stick to the higher QY results of dots prepared in EG. Further investigation concerning the solvent nature is yet to be established.
Thus, this could be a question to be addressed in a future research.

Viscosity impact on CD carbonization
Viscosity changes were obtained by using identical chemical affinity solvents to the reacting molecules thus PEG 200(ζ1)-PEG 400(ζ2)-PEG 600(ζ3), EG (ζ4) gave the desired conditions. The reaction was held in 1.2M HCl concentration and a total volume of 15mL.
mPD reaction was done also in a refluxed system at 250mL at round threenecked flask with temperature adjusted to 150 0 C. 1g of mPD in 100mL reaction volume, 90mL EG and 10mL aqueous HCl to gain the initial concentration of [ 0.12M (η7) -0.6M (η8) -1.2M (η9).

CD emission lifetimes
CDs emission lifetimes were measured using a home built TCSPC setup around SPAD detector (MPD photonics) coupled to spectrograph (Andor Kymera 193) with 375nm picosecond pulsed laser (PicoQuant) used for excitation. Reaction set η was used for CDs emission analysis. The η-mCDs have emission bands around 520nm. ηoCDs peak appears around 485nm with two bands at 450 and 530nm respectively (Fig S9a-c). ηpCDs contribute to a broad emission from 530nm to 600nm with apparently two overlapping peaks (Fig S9b).

Structural characterization
. In order to understand the changes addressed to phenylenediamine molecules during carbonization, we have done FTIR, mass spectra and XPS analyses. We believe, all of these will enable to introduce a hypothetical model for CDs formation.

Transmission electron microscopy (TEM)
Transmission electron microscopy (TEM) images were collected with a Jeol JEM 1011 (Jeol, Japan) electron microscope operating at an acceleration voltage of 100 kV and recorded with an 11 Mp fiber optical charge-coupled device (CCD) camera (GatanOrius SC-1000). For the sample preparation, 1 µL of the diluted sample was dropped onto a carbon-coated copper grid, and the solvent was removed by evaporation at room temperature.

Dynamic light scattering (DLS) and Zeta potential measurements
The size distribution of particles and their surface zeta potential was measured in DLS mode with Malvern Zetasizer Nano Series running DTS software.A He-Ne laser source operates at 633 nm with maximum otput power of 4 mW..
Analysis performed at an angle of 173 and a temperature of 25C. Since CD is a small molecule with a low scattering cross-section at the laser wavelength, the measurement setup detection limit is 0.1mg/mL all the samples were done in an aqueous solution at 1-2mg/mL concentration.

Liquid chromatography Mass Spectra
The mass-spectra of each reaction product was taken after different reaction times. Using the C18 column to charge and separate the phases. The peak at 107-109 a.m.u. corresponds to the initial unreacted product (phenylene diamine isomer). The peak intensity appears in percent of the fraction mass from other similar fractions and not from the total mass distribution Figure S13. Mass spectra dynamics in LCMS instruments. A,B,C,D are 30min, 2hours,   24hours, and 48hours respectively. The numbers 1,2,3

X-ray Photoelectron Spectroscopy
XPS samples were prepared from dried carbon dots placed over carefully cleaned silicon slides. Canning 5600 AES/XPS multi-technique system (PHI, USA) is a state-of-the-art analytical tool for chemical analysis of any solid material, ranging from Li to U. It can determine the chemical composition of surfaces not only by their atomic content but also by the chemical bonding of the surface atoms. In our case, the elemental analysis yielded.

H 1 NMR Hydrogen Nuclear Magnetic Resonance
Dried and purified mCDs powder are dissolved in D 2 O. Then inserted to NMR Bruckers Ascennd 500 High Resolution NMR machine. The field was at 500MHz 11.7Tesla. The spectra obtained from sample m-η8CDs.

Carbon dot synthesis kinetics
Our simple model is based on the first order consecutive reactions: According to the model, x refers to the initial isomer of Phenylenediamine (x = ortho, meta, para), corresponds to the photoactive reaction product of the respective 1 isomer, corresponds to the non-fluorescent product obtained from Same fitting properties obtained in both regular fit and with normalized data to the maximal peak value of the intensivist point ( Fig S15).

High-performance liquid chromatography (HPLC)
Samples of 10mg/mL of a standard solution containing mPD in EtOH. The sample was mixed 1/100 v/v with acetonitrile then centrifuged for 5min. Then the sample runs through the C18 column with KH2PO4.

minutes respectively (D) in terms of fraction from the mixture, RY1 corresponds to overall (blue line) corresponds to fragments that maintain the same absorbance spectra RY 2(purple) corresponds to brightest fractions only, RY 3 (green) is the abundance of the brightest fraction at
10.25minutes

Quantum yield measurements
In order to calculate the relative QY of the resulting product, different references were used and then an averaged value was introduced, the main overlapping spectra is that of fluorescein thus, used as the primary reference. Fluorescein QY in 0.1M NaOH was referenced to that of Rhodamine 6G in pure was used to cross corelate the Ethanol. The relation -is the is the QY under study is a reference QY, and references.
∅ , ∅ -are the absorbance of the , ratio of refractive indices of the solvents . measured sample and the reference sample respectively. Fluorescein QY Value appeared to be 91%.
The gradient method that averages over a range of concentration is also used to obtain statistical data to increase the accuracy of QY measurements.
The quantum yield was fixed for 550nm excitation to 100% then the QY value at 420-450nm was the intensity percentage of the 550nm excitation.