High Quantum Yield Amino Acid Carbon Quantum Dots with Unparalleled Refractive Index

Carbon quantum dots (CQDs) are one of the most promising types of fluorescent nanomaterials due to their exceptional water solubility, excellent optical properties, biocompatibility, chemical inertness, excellent refractive index, and photostability. Nitrogen-containing CQDs, which include amino acid based CQDs, are especially attractive due to their high quantum yield, thermal stability, and potential biomedical applications. Recent studies have attempted to improve the preparation of amino acid based CQDs. However, the highest quantum yield obtained for these dots was only 44%. Furthermore, the refractive indices of amino acid derived CQDs were not determined. Here, we systematically explored the performance of CQDs prepared from all 20 coded amino acids using modified hydrothermal techniques allowing more passivation layers on the surface of the dots to optimize their performance. Intriguingly, we obtained the highest refractive indices ever reported for any CQDs. The values differed among the amino acids, with the highest refractive indices found for positively charged amino acids including arginine-CQDs (∼2.1), histidine-CQDs (∼2.0), and lysine-CQDs (∼1.8). Furthermore, the arginine-CQDs reported here showed a nearly 2-fold increase in the quantum yield (∼86%) and a longer decay time (∼8.0 ns) compared to previous reports. In addition, we also demonstrated that all amino acid based CQD materials displayed excitation-dependent emission profiles (from UV to visible) and were photostable, water-soluble, noncytotoxic, and excellent for high contrast live cell imaging or bioimaging. These results indicate that amino acid based CQD materials are high-refractive-index materials applicable for optoelectronic devices, bioimaging, biosensing, and studying cellular organelles in vivo. This extraordinary RI may be highly useful for exploring cellular elements with different densities.

PL emission spectra were recorded at 360 nm in order to determine the integrated fluorescence intensity, which is defined as the area under the PL curve over the wavelength range of 370 to 700 nm.As a next step, the integrated fluorescence intensity was plotted against the absorbance value.In order to calculate QY values, the following equation was used: (1) where QY is the fluorescence quantum yield, A is the absorbance, PL is the fluorescence emission peak, and n is the RI, where the RI of the quinine sulphate is 1.33.To reduce the effects of re-absorption, we kept the absorbance below 0.1 OD (optical density) in a 10 mm quartz cuvette.

Fluorescence decay time:
AA-CQD suspensions were placed in quartz cuvettes and the fluorescence/PL decay time was measured using a FluoroMax-4 Spectrofluorometer (Horiba Jobin Yvon, Kyoto, Japan).This spectrometer is equipped with double monochromators, a 450 W Xe lamp, an AGILE source, time-correlated single photon counting capability, a standard cuvette holder, and a standard PMT-900 detector.Fluoracle's standard option for acquiring decay times based on excitation provides automatic acquisition of decay times based on excitation.A standard streak camera (Hamamatsu) was used in conjunction with a femtosecond laser excitation source able to produce several wavelengths of excitation to measure the excitonic decay lifetime of all twenty AA-CQDs.In this study, we measured time-resolved PL at wavelengths of 400 and 440 nm.

Refractive index measurements:
The RI of the AA-CQD suspensions were determined by refractometer (ATAGO, PAL-RI), for a single wavelength of 598 nm, and by interferometric phase microscopy (IPM) for 8 different wavelengths across the visible spectrum.
For the IPM technique, the solutions were inserted into a 100 µm tall microfluidic channel (Ibidi, µ-Slide VI 0.1) and fluorescence microscopy imaging was performed to verify that the fluorescent AA-CQD suspension (fully transparent and with no precipitation) had filled the channel.Next, off-axis holograms of the channel filled with the suspension were acquired using our shearing IPM system 37 and a supercontinuum laser source (NKT SuperK EXTREME) coupled to an acousto-optical filter (NKT SuperK SELECT), thereby enabling acquisition of holograms with 8 different illumination wavelengths (490, 500, 515, 530, 620, 641, 650, and   680 nm). 37Additionally, background holograms of the channel containing only water were also acquired for each wavelength.Following this, optical path delay (OPD) maps of the channel filled with each sample suspension and illuminated with each wavelength were reconstructed from their respective holograms while using the corresponding background holograms to negate the OPD contribution of the water in the suspension.The OPD of a sample at a given point (x,y) and for an illumination wavelength λ is defined by: (2) where n s is RI of the sample, n m is the RI of the surrounding medium (i.e. the medium of the background hologram), and h is the sample height.By rearranging the equation, we determined the RI of the suspension, n s , using the average OPD value of the channel in each hologram, as follows: (3) where h is the known channel height, 100 µm, and n m is the known RI of water for each wavelength, taken from literature. 38Finally, the RI of the suspended AA was determined using the Lorentz-Lorenz mixture rule 39 (4) where n 1 and Φ 1 are the RI and the volume fraction of the suspended AA, respectively, and n 2 and Φ 2 are the RI and the volume fraction of the suspension medium, respectively.The volume fractions were calculated using the known sample concentrations of the AA-CQDs density, and the values of n 2 are identical to n m from earlier.

Cell Viability Measurement:
A methyl thiazolyl tetrazolium (MTT) assay was performed using human cervical carcinoma cells (HeLa cells) to assess the cytotoxicity of AA-CQD nanomaterials.The cells were seeded into 96-well microtiter plates and allowed to adhere overnight at 37 °C under 5% CO 2 .Following 24 hours of HeLa cell growth, all cell culture media was removed, and the cells were treated with AA-CQD nanomaterials without FBS (fetal bovine serum) medium.After this, the cells were washed with 7.4 pH phosphate buffer solution (PBS) and incubated with MTT solution for 3.5 hours.Following that, the MTT reagent was discarded, and 100 μL of DMSO was added to each well in order to dissolve the formazan crystals.We measured the absorbance at 570 and 680 nm using a microplate reader.Control experiments were conducted without AA-CQD nanomaterials.The experiments were conducted in quadruplicate.

Live cell imaging:
The live cell imaging of Hela cells was conducted using confocal microscopy after the cells were grown in glass bottom petri dishes.HeLa cells were cultured in high glucose media for two days, then 100 µg/mL of AA-CQDs were added into the cell culture media and incubation was continued for different lengths of time (2h, 6h and 24h).The imaging was performed using an SP8 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany).The excitation and emission ranges were as follows: λ ex = 480 nm, 543 nm, 630 nm, and λ em = 500-580 nm, 560-630 nm, and 650-740 nm, respectively.

Figure
Figure S1.Visual analysis of pristine AAs and AA-CQDs under white and UV light.(a) AA-CQDs under ambient white light.(b) AA-CQDs from (a) under UV light.Associated AA and AA-CQDs label letters are in white.(c) Schematic representation of a possible mechanism for the formation of AA-CQDs from AAs through dehydration, polymerization, carbonization and passivation.

Figure S2 :
Figure S2: The scheamatic reperesentation of chemical formula of all the amino acids, which were used for the synthesis of CQDs from all the amino acids.

Figure S3 .
Figure S3.Visual analysis of pristine AAs and AA-CQDs under white and UV lights.(a) Aqueous solutions of pristine AAs under ambient white light.(b) Solutions from (a) under UV light.Associated AA label letters are in green for AAs and in red for AA-CQDs.

Figure S25 .
Figure S25.A comparison of the UV-vis spectra of each of the 20 different amino acid-derived CQDs with different amino acid functionalizations.

Figure S26 :
Figure S26: A comparison of fluorescence analysis graphs for all the AAs derived CQDs and pristine solutions of all amino acids: (a) Arg-CQDs and pristine Arg, (b) His-CQDs and pristine histidine, (c) Lys-CQDs and pristine aqueous solutions of lysine, (d) Glu-CQDs and a pristine aqueous solution of glutamic acid, (e) Asp-CQDs and a pristine aqueous solution of aspartic acid, (f) Thr-CQDs and a pristine aqueous solution of threonine, (g) Asn-CQDs and a pristine aqueous solution of asparagine, (h) Gln-CQDs and a pristine aqueous solution of glutamine, (i) Ser-CQDs and a pristine aqueous solution of serine, (j) Pro-CQDs and pristine aqueous solution of proline, (k) Cys-CQDs and a pristine aqueous solution of cysteine, (l) Gly-CQDs and a pristine aqueous solution of glycine, (m) Ala-CQDs and a pristine aqueous solution of alanine, (n) Ile-CQDs and pristine aqueous solution of isoleucine, (o) Trp-CQDs and a pristine aqueous solution of tryptophan, (p) Phe-CQDs and a pristine aqueous solution of phenylalanine, (q) Val-CQDs and pristine aqueous solution of valine, Figure S24r for Leu-CQDs and a pristine aqueous solution of leucine, (s) Met-CQDs and a pristine aqueous solution of methionine, and (t) Tyr-CQDs and a pristine aqueous solution of tyrosine.

Figure S27 :
Figure S27: The quantum yeild of all 20 amino acid dervied CQDs at 360 nm and campared the QS.

Figure :
Figure: S29.A Graphical representation of RI of all amino acid by measuring the RI by refractometer at 589 nm.Red: positively charged, cyan: negatively charged, green: uncharged side chain, orange: special cases, blue: hydrophobic side chain.

Figure: S33 .
Figure: S33.Live cell imaging of Hela cell without any treatment with CQDs (as a control) using four fluorescence channels: bright field images, Hoechst (dye staining live cell nucleus), excitation at 480 nm.Scale bar: 20 µm.