Carbon Quantum Dots from Amino Acids Revisited: Survey of Renewable Precursors toward High Quantum-Yield Blue and Green Fluorescence
從氨基酸重新探討碳量子點：可再生前驅體的調查以實現高量子產率的藍色和綠色螢光

Anna Kolanowska,* Grzegorz Dzido, Maciej Krzywiecki, Mateusz M. Tomczyk, Dariusz Łukowiec,
安娜·科拉諾夫斯卡，格熱戈日·季多，馬切伊·克日維茨基，馬特烏什·M·湯奇克，達里烏什·盧科維茨Szymon Ruczka, and Sławomir Boncel*
西門·魯茨卡，斯瓦沃米爾·邦策爾*

Cite This: ACS Omega 2022, 7, 41165-41176
引用此文：ACS Omega 2022, 7, 41165-41176

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Abstract 摘要

Carbon quantum dots (CQDs) were synthesized via a green, one-step hydrothermal method. As CQD precursors, nine amino acids of different structural descriptors (negatively/ positively charged in water, polar, hydrophobic, sulfur-containing, and other/complex ones) were surveyed: Asp, Cys, Gly, His, Leu, Lys, Phe, Pro, and Ser. The reactions were performed in an autoclave in the presence of citric acid at $180^{\circ} C$ for 24 h and yielded core-shell CQDs. CQDs were comprehensively characterized by transmission electron microscopy, dynamic light scattering, Raman, UV/Vis, infrared, X-ray photoelectron spectroscopy, and fluorescence spectroscopy. At the excitation wavelength of $λ_{ex} = 350 nm$ , Cys-, Phe-, Leu-, and Lys-based CQDs displayed the highest quantum yield blue fluorescence- 90 $\pm 5, 90 \pm 4, 87 \pm 5$ , and $67 \pm 3 %$ , respectively-superior to the conventional fluorescent dyes. Strikingly, for Lys- and Phe-CQDs, dissimilar trends in the excitation-emission wavelength relationships were identified, that is, constantly strong red shifts versus excitation wavelength-independent emission. Cys- and Lys-CQDs were water-dispersible toward the narrow unimodal distribution of hydrodynamic diameters- 0.6 and 2.5 nm , respectively. Additionally, Lys- and Cys-CQDs, with high absolute zeta potential values, formed stable aqueous colloids in a broad range of $pH (2, 7$ , and 12). The results constitute important premises for water-based applications of CQDs, such as bioimaging or photocatalysis.
碳量子點（CQDs）是通過一種綠色的一步水熱法合成的。作為 CQD 前驅體，調查了九種具有不同結構描述符的氨基酸（在水中帶負/正電、極性、疏水性、含硫及其他/複雜的）：天冬氨酸、半胱氨酸、甘氨酸、組氨酸、亮氨酸、賴氨酸、苯丙氨酸、脯氨酸和絲氨酸。反應在自壓釜中進行，並在檸檬酸的存在下於 $180^{\circ} C$ 下進行 24 小時，產生了核殼 CQDs。CQDs 通過透射電子顯微鏡、動態光散射、拉曼光譜、紫外/可見光、紅外線、X 射線光電子能譜和螢光光譜進行了全面表徵。在激發波長為 $λ_{ex} = 350 nm$ 時，基於半胱氨酸、苯丙氨酸、亮氨酸和賴氨酸的 CQDs 顯示出最高的量子產率藍色螢光-90 $\pm 5, 90 \pm 4, 87 \pm 5$ 和 $67 \pm 3 %$ ，優於傳統螢光染料。值得注意的是，對於賴氨酸和苯丙氨酸 CQDs，發現了激發-發射波長關係的不同趨勢，即持續強烈的紅移與激發波長無關的發射。半胱氨酸和賴氨酸 CQDs 在水中可分散，具有狹窄的單峰水動力直徑分佈-0.6 和 2。5 奈米，分別。此外，具有高絕對電位值的賴氨酸和半胱氨酸量子點，在廣泛的 $pH (2, 7$ 和 12）範圍內形成了穩定的水性膠體。這些結果為量子點的水基應用，如生物成像或光催化，提供了重要的前提。

■ INTRODUCTION ■ 介紹

Carbon quantum dots (CQDs) are fluorescent nanomolecules or nanoparticles smaller than

10 nm .^{1, 2}

The most studied 0 D CQDs consist of a more graphitized,

{sp}^{2}

-carbon rich core surrounded with a 5-50 wt % amorphous shell of polar functional groups.

^{3 - 6}

The structure of CQDs results in their excellent solubility in water, negligible cytotoxicity, and biocompatibility,

^{7}

and while bearing carboxylic groups, CQDs are conveniently functionalizable.

^{8}

CQDs combine the unique optical properties of quantum dots (QDs) with the electronic properties of carbon (nano)materials. Importantly, CQDs display a quantum limitation effect, translating into tunable absorption and emission.

^{9}

It means that after excitation, the energy of the emitted photons depends on the CQD size and its molecular structure. Hence, small CQDs fluoresce in blue while the emission wavelengths increase, with the CQD diameter spanning the entire range of visible light up to infrared (IR).

^{10}

Theoretical calculations of the emission wavelength of pristine zigzag-edged CQDs showed that CQDs of diameters 0.5 and 2.31 nm fluoresced at 235.2 to 999.5 nm wavelengths, respectively.

^{11}

At the same time, altering the core-shell CQD composition by N -, S-, P-, or B-doping enhances the fluorescence quantum yield (QY). Such doping alters the energy between the lowest unoccupied (LUMO) and the highest occupied molecular orbitals (HOMO).

^{12}

By the interplay of CQD chemistry and morphology, it is possible to obtain fluorescence in the full spectral range from ultraviolet (UV) to near-IR (NIR). With the above characteristics, CQDs emerge as promising biosensors,

^{13}

imaging agents,

^{14}

and drug delivery systems,

^{15}

along with multi-modality. Due to their high electron mobility, long hot-electron lifetimes, ultrafast electron extraction, tunable band gaps, excellent electron donor/acceptor properties, and strong stable fluorescence, CQDs are considered as photocatalysts

^{16, 17}

and working elements in optoelectronic devices.

^{18}

碳量子點（CQDs）是小於

10 nm .^{1, 2}

的螢光納米分子或納米顆粒。最常研究的 0D CQDs 由一個更石墨化的、

{sp}^{2}

富碳核心組成，周圍包裹著 5-50 wt%的無定形極性官能團外殼。

^{3 - 6}

CQDs 的結構使其在水中的優異溶解性、微不足道的細胞毒性和生物相容性得以實現，

^{7}

而且由於含有羧基，CQDs 也方便進行功能化。

^{8}

CQDs 結合了量子點（QDs）的獨特光學特性和碳（納米）材料的電子特性。重要的是，CQDs 顯示出量子限制效應，轉化為可調的吸收和發射。

^{9}

這意味著在激發後，發射光子的能量取決於 CQD 的大小及其分子結構。因此，小型 CQDs 在藍色範圍內螢光，而隨著 CQD 直徑的增加，發射波長也隨之增加，涵蓋了可見光的整個範圍直至紅外線（IR）。

^{10}

對原始鋸齒邊 CQDs 的發射波長的理論計算顯示，直徑為 0.5 和 2.31 納米的 CQDs 分別在 235.2 至 999.5 納米的波長範圍內螢光。

^{11}

同時，通過 N、S、P 或 B 掺雜改變核心-殼 CQD 的組成可以增強螢光量子產率 (QY)。這種掺雜改變了最低未佔有分子軌道 (LUMO) 和最高佔有分子軌道 (HOMO) 之間的能量。

^{12}

通過 CQD 化學和形態的相互作用，可以在從紫外線 (UV) 到近紅外線 (NIR) 的全光譜範圍內獲得螢光。憑藉上述特性，CQD 成為有前景的生物傳感器，

^{13}

成像劑，

^{14}

和藥物傳遞系統，

^{15}

以及多模態。由於其高電子遷移率、長熱電子壽命、超快電子提取、可調帶隙、優秀的電子供體/受體特性和強穩定的螢光，CQD 被視為光催化劑

^{16, 17}

和光電設備中的工作元件。

^{18}

CQDs can be synthesized via a bottom-up approach from renewable sources such as fruit and vegetable peels,

^{19 - 22}

nuts,

^{23 - 25}

wastes,

^{26, 27}

or larger carbon (nano) materials in the top-down methods.

^{28, 29}

CQDs containing mainly carbon and oxygen (and hydrogen) suffer from low QY, while N -doping is the most frequently applied strategy to improve fluorescence. This modification introduces structural defects and new energy states and increases the number of electrons in the conduction band. Therefore, well-defined, small-molecule amino acids (AAs) emerge as promising candidates for the synthetic precursors of CQDs. AAs are renewable, abundant (global volume of the AA market reached 10.3 MT in 2021), relatively inexpensive (

110 - 1300

USD kg

^{- 1}

), and non-toxic.

^{30}

Zwitterionic and polyfunctional AAs can be variously charged depending on pH and equipped with hydrophilic (hydroxy -OH or mercapto -SH groups) or hydrophobic (aliphatic and/or aromatic) moieties, which, in turn, provides tunability of the optical properties of CQDs.

^{31, 32}

CQDs 可以通過自下而上的方法從可再生資源合成，例如水果和蔬菜的皮、

^{19 - 22}

堅果、

^{23 - 25}

廢料、

^{26, 27}

或者在自上而下的方法中使用較大的碳（納米）材料。

^{28, 29}

主要由碳和氧（以及氫）組成的 CQDs 受到低量子產率（QY）的影響，而氮摻雜是改善螢光的最常用策略。這種改性引入了結構缺陷和新的能量狀態，並增加了導電帶中的電子數量。因此，明確定義的小分子氨基酸（AAs）成為 CQDs 合成前驅體的有前景候選者。AAs 是可再生的，豐富的（2021 年全球氨基酸市場達到 10.3 百萬噸），相對便宜（

110 - 1300

美元/公斤

^{- 1}

），且無毒。

^{30}

兩性離子和多功能的 AAs 可以根據 pH 值而帶有不同的電荷，並配備親水性（羥基 -OH 或巰基 -SH 基團）或疏水性（脂肪族和/或芳香族）基團，這反過來又提供了 CQDs 光學性質的可調性。

^{31, 32}

Here, we propose a facile and sustainable one-step hydrothermal synthesis of CQDs from various AAs (hydrophilic, hydrophobic, aromatic, and aliphatic) and citric acid (CA) as the precursors of the core and shell, respectively. Our method covers a fully controlled four-stage synthesis, that is, dehydration, polymerization, passivation, and carbonization. The as-synthesized CQDs exhibit blue to green fluorescenceexhibiting red-shifts depending on the synthetic precursorwith merits of narrow size distribution and excellent water solubility, while the excitation wavelength falls in the range of 300 to 480 nm . Importantly, using cystein, phenylalanine, and leucine-as the synthetic precursors under the optimized conditions-we show that it is possible to obtain CQDs with high QYs superior to the conventional fluorescent dyes. LysCQDs emerged as also forming stable aqueous dispersions in a broad range of pH . The overall characteristics allow us to address the key prerequisites for numerous applications.
在此，我們提出了一種簡便且可持續的一步水熱合成方法，利用各種氨基酸（親水性、疏水性、芳香性和脂肪族）和檸檬酸作為核心和外殼的前驅物。我們的方法涵蓋了完全控制的四階段合成，即脫水、聚合、鈍化和碳化。合成的碳量子點顯示出從藍色到綠色的螢光，根據合成前驅物的不同而呈現紅移，具有狹窄的尺寸分佈和優異的水溶性，激發波長範圍在 300 至 480 納米之間。重要的是，使用半胱氨酸、苯丙氨酸和亮氨酸作為在優化條件下的合成前驅物，我們展示了獲得高量子產率的碳量子點的可能性，優於傳統螢光染料。LysCQDs 也在廣泛的 pH 範圍內形成穩定的水性分散體。整體特性使我們能夠滿足多種應用的關鍵前提條件。

- MATERIALS AND METHODS - 材料與方法

Materials. Synthesis of CQDs. CA, quinine sulfate (QS), 7diethylamino-4-methylcoumarin (Coumarin 1), and AAs were purchased from Sigma-Aldrich. CQDs were synthesized using a one-step hydrothermal method. CA ( 1.5 mmol ) and AA (aspartic acid, cysteine, glycine, histidine, leucine, lysine, phenylalanine, proline, and serine) ( 1.5 mmol ) were dissolved in distilled water

(10 mL)

. The amount of water was adjusted to dissolve CA and AA at room temperature. The solution was heated in a Teflon-coated autoclave at

180^{\circ} C

for 24 h in a laboratory dryer. The autoclave was allowed to cool down to room temperature, and the post-reaction mixture was centrifuged at 5500 rpm for 15 min to separate the larger particles. The resulting supernatant was filtered through a 0.22

μ

m-syringe filter (Minisart NY hydrophilic polyamide, 25 mm ). Following purification, the solution was frozen in liquid nitrogen and lyophilized until dried.
材料。CQDs 的合成。CA、奎寧硫酸鹽（QS）、7-二乙氨基-4-甲基香豆素（香豆素 1）和氨基酸（AAs）均購自 Sigma-Aldrich。CQDs 是使用一步水熱法合成的。CA（1.5 毫摩爾）和氨基酸（天冬氨酸、半胱氨酸、甘氨酸、組氨酸、亮氨酸、賴氨酸、苯丙氨酸、脯氨酸和絲氨酸）（1.5 毫摩爾）溶解在蒸餾水中

(10 mL)

。調整水的量以在室溫下溶解 CA 和氨基酸。將溶液在 Teflon 塗層的高壓鍋中加熱至

180^{\circ} C

，在實驗室乾燥器中保持 24 小時。高壓鍋冷卻至室溫後，將反應後的混合物以 5500 轉/分鐘離心 15 分鐘，以分離較大的顆粒。得到的上清液通過 0.22

μ

微米注射器過濾器（Minisart NY 親水性聚酰胺，25 毫米）過濾。經過純化後，將溶液在液氮中冷凍並凍乾至乾燥。

Instrumentation. The characterization of CQDs was performed by transmission electron microscopy (TEM) (S/ TEM Titan 80-300 operated at 300 kV , Field Electron and Ion Company), combustional elemental analysis (PerkinElmer 2400 Series II CHNS/O, PerkinElmer), thermogravimetric analysis (TGA) (TGA 8000, PerkinElmer), Raman (inVia Confocal Raman microscope, Renishaw), UV-Vis (HP 8452A UV-Vis Diode Array Spectrophotometer, Hewlett Packard), fluorescence spectroscopy (SpectraMax i3x, Molecular Devices and FluoroMax Plus, Horiba Scientific), Fourier-transform IR
儀器。CQDs 的特徵化是通過透射電子顯微鏡（TEM）（S/TEM Titan 80-300，操作電壓 300 kV，Field Electron and Ion Company）、燃燒元素分析（PerkinElmer 2400 系列 II CHNS/O，PerkinElmer）、熱重分析（TGA）（TGA 8000，PerkinElmer）、拉曼光譜（inVia 共焦拉曼顯微鏡，Renishaw）、紫外-可見光（HP 8452A 紫外-可見光二極管陣列分光光度計，Hewlett Packard）、螢光光譜（SpectraMax i3x，Molecular Devices 和 FluoroMax Plus，Horiba Scientific）、傅立葉變換紅外光譜。
(FT-IR) (Nicolet 6700 FT-IR, Thermo Fischer Scientific), and X-ray photoelectron spectroscopy (XPS) (PreVac EA15, PreVac). Additionally, by applying dynamic light scattering (DLS), nanoparticle size and zeta-potential were determined (Zetasizer Nano S90, Malvern Panalytical).
(FT-IR) (Nicolet 6700 FT-IR, Thermo Fischer Scientific) 和 X 射線光電子能譜 (XPS) (PreVac EA15, PreVac)。此外，通過應用動態光散射 (DLS)，確定了納米顆粒的大小和 ζ 電位 (Zetasizer Nano S90, Malvern Panalytical)。

Transmission Electron Microscopy. The nanomorphology of CQDs was determined based on TEM images collected using a transmission electron microscope S/TEM TITAN 80300. The samples were prepared by dispersion and ultrasonication of CQDs in ultrapure ethanol and then placed on a copper TEM grid with lacey carbon films ( 200 mesh).
透射電子顯微鏡。CQDs 的納米形態是根據使用透射電子顯微鏡 S/TEM TITAN 80300 收集的 TEM 影像來確定的。樣品是通過在超純乙醇中分散和超聲處理 CQDs 來製備的，然後放置在帶有蕾絲碳膜的銅 TEM 網格上（200 目）。

Combustional Elemental Analysis. A sample of CQDs (ca.

2 - 10 mg

) was accurately weighed into small tin capsules. At elevated temperatures, in the presence of excess oxygen, the organic materials combusted into

{CO}_{2}, H_{2} O, {SO}_{2}

, and

N_{x} O_{y}

compounds (next reduced by fine copper particles in the reduction tube to

N_{2}

). For quantitative analysis,

{CO}_{2}, H_{2} O

{SO}_{2}

, and

N_{2}

content represent carbon, hydrogen, sulfur, and nitrogen content, respectively. Oxygen content was calculated indirectly from the difference between the sample weight and the sum of the other element contents.
燃燒元素分析。將一樣 CQDs（約

2 - 10 mg

）準確稱量到小錫膠囊中。在高溫下，在過量氧氣的存在下，這些有機材料燃燒成

{CO}_{2}, H_{2} O, {SO}_{2}

和

N_{x} O_{y}

化合物（接著在還原管中被細銅顆粒還原為

N_{2}

）。對於定量分析，

{CO}_{2}, H_{2} O

、

{SO}_{2}

和

N_{2}

的含量分別代表碳、氫、硫和氮的含量。氧含量則是通過樣品重量與其他元素含量總和之間的差異間接計算得出的。

TGA Analysis. TGA curves were acquired under nitrogen (flow rate of

40 mL \min^{- 1}

). The samples (

1 - 5 mg

) were heated in alumina crucibles up to

800^{\circ} C

at a heating rate of 20

^{\circ} C min^{- 1}

.
TGA 分析。TGA 曲線是在氮氣下獲得的（流速為

40 mL \min^{- 1}

）。樣品（

1 - 5 mg

）在鋁土礦坩埚中加熱至

800^{\circ} C

，加熱速率為 20

^{\circ} C min^{- 1}

。

Raman Spectroscopy. Raman spectra were obtained at 514 nm (a green laser) with a laser power of

5 %

, a 2400 line per mm grating,

20 \times

magnification, and an exposure time of 15 s. For each material, three accumulations were collected in three locations within the sample. The spectra were averaged and normalized to the G-band.
拉曼光譜學。拉曼光譜是在 514 納米（綠色激光）下獲得的，激光功率為

5 %

，2400 條/mm 的光柵，

20 \times

倍放大，曝光時間為 15 秒。對於每種材料，在樣品的三個位置收集了三次累積數據。光譜被平均並標準化到 G 帶。

FT-IR Spectroscopy. Spectra were collected in the range of

400 - 4000 {cm}^{- 1}

, with 16 scans for each sample with a resolution of

4 {cm}^{- 1}

. Lyophilized CQDs were mixed with dry KBr in an agate mortar and then pressed in an evacuable slot to form a pellet under 40 MPa pressure for 2 min using a hydraulic press.
FT-IR 光譜學。光譜在

400 - 4000 {cm}^{- 1}

範圍內收集，每個樣本進行 16 次掃描，解析度為

4 {cm}^{- 1}

。冷凍乾燥的量子點與乾燥的 KBr 在瑪瑙研缽中混合，然後在可抽氣的槽中以 40 MPa 的壓力壓制 2 分鐘，形成顆粒。

X-ray Photoelectron Spectroscopy. XPS measurements were performed in a UHV multi-chamber experimental setup with a PreVac EA15 hemispherical electron energy analyzer fitted with a 2D multi-channel plate detector. The system base pressure was equal to

9 \times 10^{- 9} Pa

. An

Mg - K α

X-ray source (PreVac dual-anode XR-40B source, excitation energy of 1253.60 eV ) was used to excite the sample. Pass energy was set to 200 eV for the survey spectra collection (scanning step of 0.9 eV ) and to 100 eV for high-accuracy energy regions (scanning step of 0.06 eV ). All measurements were done with a normal take-off angle and the curved analyzer exit slit (

0.8 \times

25 mm ) choice for the highest energy resolution. The binding energy scale of the analyzer was calibrated to the

{Au}_{4} f_{7 / 2}

(84.0 eV ) region of the gold-covered sample placed at the same sample stage.

^{33}

The acquired spectra were fitted using CasaXPS software. The components were fitted with the sum of Gauss (30%) and Lorenz (70%) functions, while the Shirley function was applied for background subtraction.
X 射線光電子能譜。XPS 測量是在一個超高真空多腔體實驗設置中進行的，配備有 PreVac EA15 半球形電子能量分析儀和 2D 多通道板檢測器。系統的基準壓力為

9 \times 10^{- 9} Pa

。使用

Mg - K α

X 射線源（PreVac 雙陽極 XR-40B 源，激發能量為 1253.60 eV）來激發樣品。通過能量設置為 200 eV 以收集調查光譜（掃描步長為 0.9 eV），高精度能量區域設置為 100 eV（掃描步長為 0.06 eV）。所有測量均以正常的起飛角度進行，並選擇了曲面分析儀的出口狹縫（

0.8 \times

25 mm）以獲得最高的能量分辨率。分析儀的束縛能量刻度已校準至

{Au}_{4} f_{7 / 2}

（84.0 eV）區域，該區域的金覆蓋樣品放置在同一樣品台上。

^{33}

獲得的光譜使用 CasaXPS 軟件進行擬合。組件使用高斯（30%）和洛倫茲（70%）函數的總和進行擬合，同時應用 Shirley 函數進行背景扣除。

UV-Vis Spectroscopy. UV-Vis spectra were obtained in quartz cuvettes ( 2 mL ) with a 10 mm optical path at a scanning rate of 1.0 nm from 250 to 800 nm .
紫外-可見光光譜。紫外-可見光光譜是在石英比色皿（2 mL）中以 10 mm 的光路，在 250 至 800 nm 範圍內以 1.0 nm 的掃描速率獲得的。

Fluorescence Spectroscopy. The fluorescence spectra were measured under different excitation wavelengths (from 250 to 480 nm ) for

200 μ L

of the sample transferred to a clear bottom 96 -well plate (scan speed

20 nm min^{- 1}

).
螢光光譜學。螢光光譜在不同的激發波長（從 250 到 480 納米）下測量，樣本轉移到透明底部的 96 孔板中（掃描速度

20 nm min^{- 1}

）。

Figure 1. Skeletal molecular formulae of AAs with different structural descriptors as the CQD precursors, including the net charge in water (a) and the general synthetic pathway toward CQDs-here illustrated by the hydrothermal transformation of Asp via the four-stage decomposition (b).
圖 1. 具有不同結構描述符的氨基酸的骨架分子式，作為量子點前驅物，包括在水中的淨電荷(a)以及通往量子點的一般合成途徑-這裡通過天冬氨酸的四階段分解的水熱轉化來說明(b)。

The QY

(φ)

of CQDs was calculated using QS

(φ = 54 %)

0.1 M H_{2} {SO}_{4 (aq)}

and Coumarin

1 (φ = 59 %)

in ethanol as the references by comparing the integrated photoluminescence intensity and absorbance.

^{34, 35}

Samples of aqueous CQD suspensions of different concentrations were prepared by keeping the absorbance values less than 0.1 at their excitation wavelengths (similar to different CQD concentrations). Next, the integrated photoluminescence intensities for all samples were measured. The integrated photoluminescence intensity was plotted against absorbance, and the slope values of the obtained linear plots were measured. The QY was calculated using the below equation
QY

(φ)

的 CQDs 是通過比較整合的光致發光強度和吸收度，使用 QS

(φ = 54 %)

在

0.1 M H_{2} {SO}_{4 (aq)}

和乙醇中的香豆素

1 (φ = 59 %)

作為參考來計算的。

^{34, 35}

以保持其激發波長下的吸收值低於 0.1（類似於不同的 CQD 濃度），準備了不同濃度的水相 CQD 懸浮液樣品。接下來，測量了所有樣品的整合光致發光強度。將整合的光致發光強度與吸收度繪製成圖，並測量所獲得的線性圖的斜率值。QY 是使用以下方程計算的。

φ_{x} = φ_{QS} \cdot \frac{S_{x}}{S_{QS}} \cdot \frac{I_{QS}}{I_{x}} \cdot \frac{η_{x}^{2}}{η_{QS}^{2}}

where:

φ

-QY; S-integrated fluorescence intensity (area under spectrum);

I

-fluorescence intensity;

η

-refractive index; and

x

-CQD sample.
在哪裡：

φ

-QY；S-綜合螢光強度（光譜下的面積）；

I

-螢光強度；

η

-折射率；以及

x

-CQD 樣本。
DLS Measurement. The hydrodynamic diameter and zeta potential of CQDs were measured by DLS using a monochromatic coherent

He - Ne

laser with a fixed wavelength of 633 nm . The measurements were performed in triplicate for 2 mL of sample (

1 mg {mL}^{- 1}

) in distilled water. The zeta potential for each sample was measured for three pH values: 2.0, 7.0, and 12.0. The pH of the suspension was adjusted by adding

{HCl}_{(aq)}

{NaOH}_{(aq)}

.
DLS 測量。CQDs 的水動力直徑和 ζ 電位是使用波長為 633 nm 的單色相干

He - Ne

激光進行 DLS 測量的。對於 2 mL 的樣本 (

1 mg {mL}^{- 1}

) 在蒸餾水中進行了三次重複測量。每個樣本的 ζ 電位在三個 pH 值下測量：2.0、7.0 和 12.0。通過添加

{HCl}_{(aq)}

或

{NaOH}_{(aq)}

調整懸浮液的 pH 值。

RESULTS AND DISCUSSION 結果與討論

The molecular structure of the AA substrates and the conditions represent the most important variables in the properties-by-design synthesis of CQDs. As optimized, white to yellowish mat CQD powders were synthesized via the hydrothermal method, lasting 24 h at

180^{\circ} C

-employing as substrates nine different AAs and CAs (as the main carbon core precursor) (Figure 1). Our synthetic protocol was inspired by numerous earlier studies. For instance, Chahal et al. proved that the application of both CA and AAs is necessary for higher yields in the CQD synthesis, displaying high QYs.

^{36}

Indeed, in the absence of CA, the synthesis of CQDs proceeds at low yields. The authors claimed that CA played two roles in the CQD preparation. First, CA emerged as a multifunctional compound bearing three carboxyl groups and one hydroxyl group, indicating several sites to react with AAs and also with other CA molecules. Second, CA served as a Brønsted acidic catalyst in the addition-elimination reactions.
AA 基質的分子結構和條件是 CQDs 設計合成中最重要的變數。經過優化，白色至淡黃色的 CQD 粉末是通過水熱法合成的，持續 24 小時，使用九種不同的 AA 和 CA 作為基質（作為主要碳核心前驅物）（圖 1）。我們的合成方案受到許多早期研究的啟發。例如，Chahal 等人證明了同時應用 CA 和 AA 對於提高 CQD 合成的產量是必要的，顯示出高的量子產率（QY）。事實上，在缺乏 CA 的情況下，CQDs 的合成產量較低。作者聲稱 CA 在 CQD 製備中扮演了兩個角色。首先，CA 作為一種多功能化合物，具有三個羧基和一個羥基，顯示出多個與 AA 和其他 CA 分子反應的位點。其次，CA 在加成-消除反應中作為布朗斯特酸催化劑。

Here, the rationale behind the selection of AAs was to cover their most important structural descriptors (Figure 1a). The CQD products of the synthesis from the particular AA (in the form of three-letter international codes) are denoted as AACQDs such as, for example, Phe-CQD, representing Lphenylalanine-derived CQDs. The unique colors of molecular formulae of AAs are consequently applied in the analyses and spectra throughout the entire work for the sake of clarity and unambiguity.
在這裡，選擇氨基酸的理由是涵蓋它們最重要的結構描述符（圖 1a）。來自特定氨基酸的合成產物（以三個字母的國際代碼形式）被稱為 AACQDs，例如 Phe-CQD，代表 L-苯丙氨酸衍生的 CQDs。因此，氨基酸的分子式獨特顏色在整個工作中被應用於分析和光譜，以便於清晰和明確。

AAs bear amino and carboxylic acid groups, enabling the formation of a variety of nitrogen and oxygen functionalities
氨基酸具有氨基和羧酸基團，使得能夠形成多種氮和氧的官能團

Figure 2. Structural features of the as-synthesized CQDs. TEM images of Phe-CQDs; top inset shows the particle size distribution estimated from 100 individual CQDs, and the bottom inset presents a higher magnification image showing isolated Phe-CQDs (a); combustion elemental analysis of CQDs (b); TGA curves of CQDs ©; analysis of the corresponding step-wise thermal degradation of CQDs (d); Raman spectra of CQDs at the critical regions: D-, G-, and 2D-bands (e); summary of the key Raman spectra parameters for all CQDs (f). FWHM-full width at half maximumfor D-, G-, and 2D-peaks.
圖 2. 合成的 CQDs 的結構特徵。Phe-CQDs 的透射電子顯微鏡（TEM）圖像；上方插圖顯示從 100 個單獨 CQDs 估算的粒徑分佈，下方插圖呈現更高放大倍率的圖像，顯示孤立的 Phe-CQDs（a）；CQDs 的燃燒元素分析（b）；CQDs 的熱重分析（TGA）曲線（c）；CQDs 的相應逐步熱降解分析（d）；CQDs 在關鍵區域的拉曼光譜：D、G 和 2D 帶（e）；所有 CQDs 的關鍵拉曼光譜參數摘要（f）。FWHM-全寬半最大值，針對 D、G 和 2D 峰。
within the CQD shell, while Cys also provides sulfur moieties. Upon hydrothermal synthesis, the AA molecules first assemble as a result of hydrogen bonding. Next, upon heating and subsequent dehydration, polymerization occurs, leading to a short single burst of nucleation. The resulting nuclei grow by the diffusion of solutes toward CQD surfaces.

^{37}

Such a mechanism describing the synthetic route for the “bottom-up” methods has been proposed by many researchers. Accordingly, synthesis of CQDs includes polymerization (polycondensation via dehydration), nucleation, carbonization, and growth. In our attempt, the polymer carbon skeleton is proposed as a crosslinking agent after dehydration, whereas upon carbonization, a
在 CQD 殼內，半胱氨酸也提供硫基團。在水熱合成過程中，AA 分子首先因氫鍵作用而組裝。接著，在加熱和隨後的脫水過程中，發生聚合，導致短暫的成核爆發。生成的核通過溶質向 CQD 表面的擴散而增長。

^{37}

許多研究人員提出了這種描述“自下而上”方法合成路徑的機制。因此，CQD 的合成包括聚合（通過脫水的聚縮），成核，碳化和生長。在我們的嘗試中，聚合碳骨架被提議作為脫水後的交聯劑，而在碳化過程中，

Figure 3. XPS spectra of CQDs. Signals and their deconvolution for Asp-CQDs in the C 1s (a), O 1s (b), and N 1s BE regions ©. Signals and their deconvolution for Leu-CQDs in the

C 1 s (d), O 1 s (e)

, and N 1 s BE regions (f). Signals and their deconvolution for Cys-CQDs in the C 1 s (g), O 1s (h), N 1s (i), and S 2p BE regions (inset in i).
圖 3. CQDs 的 XPS 光譜。Asp-CQDs 在 C 1s (a)、O 1s (b)和 N 1s BE 區域(c)的信號及其解卷積。Leu-CQDs 在

C 1 s (d), O 1 s (e)

和 N 1s BE 區域(f)的信號及其解卷積。Cys-CQDs 在 C 1s (g)、O 1s (h)、N 1s (i)和 S 2p BE 區域(i 的插圖)的信號及其解卷積。
fraction of the precursors is consumed to further modify the carbon core.

^{38}

前驅物的部分被消耗以進一步改變碳核心。

^{38}

The general morphological, compositional, and structural features of the as-synthesized CQDs were analyzed (Figure 2). The morphology and size distribution of the as-synthesized CQDs were analyzed by TEM (Figure S1). The imaging showed that CQDs were composed predominantly of quasispherical and amorphous shells (revealing a “halo-effect” at the CQD edges that are less graphitized and hence rich in

{sp}^{3}

carbon atoms). The size distribution of Phe-CQDs, further selected as one of the most perspective ones in terms of high QY, was narrow, that is, in the range of

1 - 5 nm

with the abundance peak at 3 nm (as determined from the population of 100 CQDs) (Figure 2a and the insets). TEM images of other CQDs frequently showed nanoparticle agglomerates larger than 10 nm , presumably formed upon lyofilization. The selected area electron diffraction (SAED) patterns of CQDs were primarily composed of diffused rings (Figure S2). This behavior stayed in good agreement with the literature dataCQDs prepared using hydrothermal methods were generally found to be amorphous.

^{39}

Nevertheless, SAED analysis also revealed diffraction spots assignable to the polycrystalline graphitic areas (Figure S2). Furthermore, we have performed combustion elemental analysis of CQDs (Figure 2b), which were found to be composed mainly of carbon (from

39 wt %

for Cys-CQDs to 59 wt % for the more graphitized PheCQDs). For all CQDs, the shell surface was rich in oxygen and nitrogen functional groups. The elemental oxygen content varied from

30 wt %

for Phe-CQDs to

44 wt %

for Asp-CQDs. In turn, the highest nitrogen content was observed for HisCQDs (

15 wt %

) with the lowest one for Pro-CQDs ( 4.5 wt

%

), which corresponds to the less pronounced gasification of the aromatic moieties for His-CQDs upon the synthesis. As predicted, in the case of Cys-CQDs, apart from carbon, oxygen, and nitrogen atoms, CQDs contained sulfur, although at as high as

10 wt %

content.
合成的量子點（CQDs）的一般形態、組成和結構特徵進行了分析（圖 2）。合成的 CQDs 的形態和尺寸分佈通過透射電子顯微鏡（TEM）進行了分析（圖 S1）。成像顯示，CQDs 主要由準球形和非晶殼組成（顯示出 CQD 邊緣的“光暈效應”，這些邊緣的石墨化程度較低，因此富含

{sp}^{3}

碳原子）。Phe-CQDs 的尺寸分佈進一步被選為在高量子產率（QY）方面最具潛力的之一，其分佈較窄，即在

1 - 5 nm

範圍內，豐度峰值在 3 納米（根據 100 個 CQDs 的數量確定）（圖 2a 及插圖）。其他 CQDs 的 TEM 圖像經常顯示出大於 10 納米的納米顆粒聚集體，這可能是在冷凍乾燥過程中形成的。CQDs 的選區電子衍射（SAED）圖樣主要由擴散環組成（圖 S2）。這一行為與文獻數據保持良好一致，使用水熱法製備的 CQDs 通常被發現是非晶的。

^{39}

然而，SAED 分析也顯示出可歸因於多晶石墨區域的衍射點（圖 S2）。此外，我們對 CQDs 進行了燃燒元素分析（圖 2b），發現其主要成分為碳（從

39 wt %

的 Cys-CQDs 到 59 wt %的更石墨化的 PheCQDs）。所有 CQDs 的外殼表面富含氧和氮功能團。元素氧含量從 Phe-CQDs 的

30 wt %

到 Asp-CQDs 的

44 wt %

不等。反過來，HisCQDs 的氮含量最高（

15 wt %

），而 Pro-CQDs 的氮含量最低（4.5 wt

%

），這與 His-CQDs 在合成過程中芳香基團的氣化程度較低相對應。如預測的那樣，在 Cys-CQDs 的情況下，除了碳、氧和氮原子外，CQDs 還含有硫，儘管其含量高達

10 wt %

。

TGA was applied to indirectly trace the chemical nature of CQDs via thermal degradation under pyrolytic conditions (Figure 2c). Depending on the precursor, CQDs are decomposed in two or three steps (Figure 2d). The weight loss below

200^{\circ} C

corresponds to the moisture evaporation, dehydration (including constitutional water), and the evolution of pyrogases

({CO}_{2}, CO

, etc.) from the CQD surface. The losses in the range of

200 - 350^{\circ} C

match the evolution of gasification products from different functional groups (hydroxyl, carboxyl, carbonyl, amide, and amine groups) from the exteriors (cores) of CQDs.

^{40}

The decomposition of the carbonaceous material occurred in the
TGA 被應用於通過熱降解在熱解條件下間接追蹤 CQDs 的化學性質（圖 2c）。根據前驅物的不同，CQDs 的分解分為兩步或三步（圖 2d）。低於

200^{\circ} C

的重量損失對應於水分蒸發、脫水（包括結構水）以及從 CQD 表面釋放的熱氣體

({CO}_{2}, CO

等。範圍在

200 - 350^{\circ} C

的損失與來自 CQDs 外部（核心）不同官能團（羥基、羧基、碳基、酰胺和胺基）釋放的氣化產物相匹配。

^{40}

碳質材料的分解發生在

Figure 4. FT-IR spectra of Ser- (left) and Cys-CQDs (right).
圖 4. Ser-（左）和 Cys-CQDs（右）的 FT-IR 光譜。
range of

300 - 450^{\circ} C

, while scission of the aromatic nitrogen functionalities began with a plateau-like run above

550^{\circ} C

;

^{41}

indeed, no further degradation was observed onward. The brownish to black residue content corresponded to the highly carbonized, polyaromatic core with the highest weight percentages for His- (

\sim 40

wt %), Lys-, Ser-, and Gly-derived CQDs (all

\sim 20 wt %

300 - 450^{\circ} C

的範圍，而芳香氮功能的分裂在

550^{\circ} C

以上開始呈現平臺狀的運行；

^{41}

確實，隨後未觀察到進一步的降解。棕色至黑色的殘留物含量對應於高度碳化的多芳香核心，His-（

\sim 40

wt %）、Lys-、Ser-和 Gly 衍生的 CQDs 的重量百分比最高（均為

\sim 20 wt %

）。
To gain further insights into the chemistry of CQDs, Raman spectra were acquired and divided into two distinctly different regions (Figure 2e,f). The spectra showed typical graphitic features: (a) D-mode (disorder) (1350-1364

{cm}^{- 1}

) activated by symmetry breaking at defects and edges, (b) G-band (graphitic)

(1580 - 1594 {cm}^{- 1})

arising from the in-plane

C - C

2 D, D + G

, and 2 G combination modes.

^{42}

In all cases, the intensity of the G-band was higher than that of the D-band, indicating the dominating abundance of

{sp}^{2}

-, with an addition of

{sp}^{3}

-carbon atoms. CQDs can thus be considered as composed of

{sp}^{2}

-graphitic and

{sp}^{2} C = O

carbon atoms

(COOH, COO, CONH

, etc.), with the admixture of

{sp}^{3}

carbon defects, including

{sp}^{3}

-carbon-based functionalities

(> CHOH, > CHO -, > CHNH -

, etc.). The

I_{D} / I_{G}

ratio identifies the nature of carbon atoms in CQDs and is typically used to determine the average size of the

{sp}^{2}

-graphitic domains in carbon (nano)materials. The highest

I_{D} / I_{G}

ratio, and as a consequence, the highest functionalization degrees/structural disorders and high N - and S -doping levels were observed for Cys- (0.97), Leu- (0.95), and His-CQDs (0.91). In turn, the lowest

I_{D} / I_{G}

values were observed for Pro- (0.19) and PheCQDs ( 0.51 ) as the most ordered, that is, the least defective, hence resulting in a more aromatic/conjugated structure. Raman spectra were dependent on the measurement nanoscale position on the sample, indicating that the samples were a mixture of CQDs with different sizes and functionalization degrees. Indeed, for all CQDs, both D- and G-band frequencies have shown size-dependent trends, each one redshifted with the CQD size. The lowest G-frequency was observed for Pro-CQDs; however, it was not connected with the lowest D-frequency. This behavior may again correspond to the inhomogeneous distribution of CQD sizes. The lowest D-frequencies were observed for Lys- and His-CQDs. The differences between D - and G -frequencies were found to be higher than for Pro-CQD, which could prove that the homogeneity of size distribution for the latter one is lower. The highest D- and G-frequencies were observed for LeuCQDs, which suggests the presence of the smallest CQDs. 2DBand, which is the second-order D-band, is broader and blueshifted with the CQD size. This feature is more sensitive to the carbon core size, while the shift in D- and G-frequencies is the effect of not only the carbon core size but is also connected with higher functionalizations at the CQD core.

^{43}

Therefore, we can speculate that the Leu-CQD samplefeaturing high D -, G -, and 2 D frequencies - is composed of the medium size core and low molecular-weight dangling functional groups. In turn, the Phe-CQD sample with low D-, G-, and 2D frequencies contains CQDs with a small core and functional groups of higher molecular weight.

^{43, 44}

為了進一步了解量子點的化學特性，獲取了拉曼光譜並將其分為兩個明顯不同的區域（圖 2e,f）。光譜顯示出典型的石墨特徵：（a）D 模式（無序）（1350-1364

{cm}^{- 1}

），由於缺陷和邊緣的對稱破壞而激活，（b）G 帶（石墨）

(1580 - 1594 {cm}^{- 1})

，源自平面

C - C

變形，以及（c）對應於

2 D, D + G

的二次特徵和 2G 組合模式。

^{42}

在所有情況下，G 帶的強度高於 D 帶，表明

{sp}^{2}

-的佔主導地位，並附加了

{sp}^{3}

-碳原子。因此，量子點可以被視為由

{sp}^{2}

-石墨和

{sp}^{2} C = O

碳原子

(COOH, COO, CONH

等組成，並混合了

{sp}^{3}

碳缺陷，包括

{sp}^{3}

-基於碳的功能性

(> CHOH, > CHO -, > CHNH -

等。

I_{D} / I_{G}

比率識別了量子點中碳原子的性質，通常用於確定碳（納米）材料中

{sp}^{2}

-石墨域的平均大小。觀察到 Cys-（0.97）、Leu-（0.95）和 His-CQDs（0）的最高

I_{D} / I_{G}

比率，因此，功能化程度/結構無序度和高 N-及 S-摻雜水平。91). 反過來，Pro-（0.19）和 PheCQDs（0.51）的最低

I_{D} / I_{G}

值被觀察到，這是最有序的，即缺陷最少，因此導致了更具芳香性/共軛結構。拉曼光譜依賴於樣品的測量納米尺度位置，表明樣品是不同大小和功能化程度的 CQDs 的混合物。事實上，對於所有 CQDs，D 帶和 G 帶頻率均顯示出與大小相關的趨勢，每個頻率隨著 CQD 大小而紅移。Pro-CQDs 的 G 頻率最低；然而，這與最低的 D 頻率並無關聯。這種行為可能再次對應於 CQD 大小的不均勻分佈。Lys-和 His-CQDs 的 D 頻率最低。D 頻率和 G 頻率之間的差異被發現高於 Pro-CQD，這可能證明後者的大小分佈均勻性較低。LeuCQDs 的 D 頻率和 G 頻率最高，這表明存在最小的 CQDs。2DBand，即二階 D 帶，隨著 CQD 大小變寬並藍移。此特徵對碳核心大小更為敏感，而 D 頻率和 G 頻率的變化不僅受到碳核心大小的影響，還與 CQD 核心的高功能化有關。因此，我們可以推測，具有高 D、G 和 2D 頻率的 Leu-CQD 樣品是由中等大小的核心和低分子量的懸掛功能團組成。反之，D、G 和 2D 頻率較低的 Phe-CQD 樣品則包含具有小核心和高分子量功能團的 CQD。

^{43, 44}

Figure 3 shows the decomposed XPS spectra of CQDs, most potentially from the applicability point-of-view. Figure 3a-c display XPS spectra of Asp-CQDs. Figure 3a shows the peak of photoemission for C 1 s with the main peak for the carbon atoms located at a bonding energy (BE) of ca. 285 eV . Due to the presence of

{sp}^{2 + ε}

-carbon atoms, the peak is broad with a long asymmetric tail toward higher BE values.

^{45}

With the effect of functionalization, the concentration of

{sp}^{3}

-carbon atoms increased, which resulted in the symmetric peak at 285.5 eV . The peaks corresponding to

C - N / C - C = O / {CONH}_{2} (286.5

eV), C = O (287.5 eV)

, and

COOH (288.5 eV)

Carbon Quantum Dots from Amino Acids Revisited: Survey of Renewable Precursors toward High Quantum-Yield Blue and Green Fluorescence 從氨基酸重新探討碳量子點：可再生前驅體的調查以實現高量子產率的藍色和綠色螢光

Abstract 摘要

■ INTRODUCTION ■ 介紹

- MATERIALS AND METHODS - 材料與方法

RESULTS AND DISCUSSION 結果與討論

Carbon Quantum Dots from Amino Acids Revisited: Survey of Renewable Precursors toward High Quantum-Yield Blue and Green Fluorescence
從氨基酸重新探討碳量子點：可再生前驅體的調查以實現高量子產率的藍色和綠色螢光