crystals Article N-Doped Carbon Quantum Dots as Fluorescent Bioimaging Agents Shih-Fu Ou 1 , Ya-Yun Zheng 2 , Sin-Jen Lee 2 , Shyi-Tien Chen 3 , Chien-Hui Wu 2 , Chien-Te Hsieh 4 , Ruey-Shin Juang 5,6 , Pei-Zhen Peng 2 and Yi-Huang Hsueh 2, *   Citation: Ou,S.-F.; Zheng, Y.-Y.; Lee, S.-J.; Chen, S.-T.; Wu, C.-H.; Hsieh, C.-T.; Juang, R.-S.; Peng, P.-Z.; Hsueh, Y.-H. N-Doped Carbon Quantum Dots as Fluorescent Bioimaging Agents. Crystals 2021, 11, 789. https://doi.org/10.3390/cryst11070789 Academic Editor: F. Christopher Pigge Received: 23 May 2021 Accepted: 5 July 2021 Published: 6 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Mold and Die Engineering, National Kaohsiung University of Science and Technology, Kaohsiung 80778, Taiwan; m9203510@nkust.edu.tw 2 Department of SeaFood Science, National Kaohsiung University of Science, Kaohsiung 81157, Taiwan; 1061234212@nkust.edu.tw (Y.-Y.Z.); 1061234121@nkust.edu.tw (S.-J.L.); chwu@nkust.edu.tw (C.-H.W.); 1061237117@nkust.edu.tw (P.-Z.P.) 3 Department of Safety, Health, and Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung 81164, Taiwan; shyitien@nkust.edu.tw 4 Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan 32003, Taiwan; cthsieh@saturn.edu.tw 5 Department of Chemical and Materials Engineering, Chang Gung University, Guishan, Taoyuan 33302, Taiwan; rsjuang@mail.cgu.edu.tw 6 Division of Nephrology, Department of Internal Medicine, Chung Gung Memorial Hospital, Linkou, Taoyuan 333, Taiwan * Correspondence: yihhsueh@nkust.edu.tw Abstract: Graphene quantum dots, carbon nanomaterials with excellent fluorescence characteristics, are advantageous for use in biological systems owing to their small size, non-toxicity, and biocom- patibility. We used the hydrothermal method to prepare functional N-doped carbon quantum dots (N-CQDs) from 1,3,6-trinitropyrene and analyzed their ability to fluorescently stain various bacteria. Our results showed that N-CQDs stain the cell septa and membrane of the Gram-negative bacteria Es- cherichia coli, Salmonella enteritidis, and Vibrio parahaemolyticus and the Gram-positive bacteria Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus. The optimal concentration of N-CQDs was approximately 500 ppm for Gram-negative bacteria and 1000 ppm for Gram-positive bacteria, and the exposure times varied with bacteria. N-Doped carbon quantum dots have better light stability and higher photobleaching resistance than the commercially available FM4-64. When excited at two different wavelengths, N-CQDs can emit light of both red and green wavelengths, making them ideal for bioimaging. They can also specifically stain Gram-positive and Gram-negative bacterial cell membranes. We developed an inexpensive, relatively easy, and bio-friendly method to synthesize an N-CQD composite. Additionally, they can serve as a universal bacterial membrane-staining dye, with better photobleaching resistance than commercial dyes. Keywords: bioimaging; N-doped carbon quantum dots; hydrothermal synthesis; photoluminescence 1. Introduction Graphene quantum dots (GQDs) are a three-dimensional material made of graphene or graphite and other graphite derivatives and are synthesized using top–down methods [1–4]. The material usually has a layered structure, with a lateral size of up to 20 nm [4,5]. The physical and chemical characteristics of GQDs are the same as those of graphene. Graphene quantum dots formed from graphene sheets have a lateral size of <20 nm; however, as the sheets are prepared using the bottom–up method, the maximum size reached is less than 10 nm [6]. Carbon nanodots (CDs) of a diameter less than 10 nm are also called carbon quantum dots (CQDs) [7]. Carbon quantum dots have potential applications in biomedicine [3,8–12] owing to their attractive fluorescent properties such as photostability and excitation-related emis- sion. Recently, they have been increasingly used as nanometers in sensing, photocatalysis, Crystals 2021, 11, 789. https://doi.org/10.3390/cryst11070789 https://www.mdpi.com/journal/crystals