Nitrogen-Doped Carbon Quantum Dots as a Fluorescent Sensor for Hydrolysed Gluten

I am very interested in quantum chemistry and biomedical engineering. I did some initial research on quantum dots and discovered their uses extend from solar cells to sensors. I learned that carbon quantum dots could be used to detect gluten and I extended this to look at how to detect hydrolysed gluten. This is an important area to explore because there is currently no way to detect hydrolysed gluten in fermented foods, so the FDA can only regulate it in gluten free labeling based on manufacturing records. In other words, manufacturers must prove that their fermented food is gluten free before processing. On top of that I have many relatives who are celiac so I was inspired to create a product that could help them. I thought that nitrogen-doped carbon quantum dots (N-CQDs) might be able to detect hydrolysed gluten so I gave it a try. A sensor using my research and research done by scientists could detect both gluten and hydrolysed gluten reliably. This could have both industry and commercial uses. In industry, it could be used to make sure companies are verifying that there is no gluten in their products to a higher degree of certainty. Additionally, people with a gluten intolerance or those with Celiac disease could use this to test their own food, specifically for foods that say "may contain gluten" or for foods that they are unsure about.

N-CQD SYNTHESIS AND RATIO TEST: Varying ratios of urea and citric acid were combined with water and microwaved to produce the N-CQDs. Each ratio was tested to determine which had the maximum fluorescence, and this ratio was used for subsequent tests.3

GLUTEN HYDROLYSIS: Vital wheat gluten was refluxed in 2.0 M HCl then neutralized with 2.0 M NaOH.4

GELATIN HYDROLYSIS: Gelatin was acidified to pH 3 with 0.1 M HCl. Pepsin was added before incubating. After incubation, it was neutralized with 0.1 M NaOH.

CHEMICAL ASSAYS: All tests in the table above used the following: 2.000 mL of 0.18 mg/mL N-CQDs and 0.300 mL of pH 7.1 phosphate buffer was added to a cuvette. 0.000 mL to 0.700 mL of the analyte being tested was then added. It was diluted to 3.000 mL with distilled water. The fluorescence (405 nm excitation) and absorbance (434.5 to 950.0 nm) spectra were collected.

pH: Five different pH’s (pH 3, 5, 7, 9, 11) were tested. To do this 0.1 M HCl and 0.1 M NaOH was used to adjust the pH in each different cuvette.

DATA PROCESSING: If there was no change in the fluorescence with changing concentrations of analyte, the integral of the fluorescence from 434.5 nm to 950.0 nm was found. If there was a change in fluorescence, I used absorbance measurements to correct for the inner-filter effect. The following was used:

Icorrected = Iobserved × 10(Aₑₓ + Aₑₘ)/2

Source: 5,6,7

I corrected fluorescence at each wavelength using absorbance at the emission wavelength, absorbance at the excitation wavelength, and fluorescence at the emission wavelength.5,6,7

My main findings from this project is that hydrolysed gluten results in an increased fluorescence (when corrected with the inner filter effect) with N-CQDs and based on my chemical interference tests, it is displaying specificity, although it is not yet conclusive. I have tested NaCl, KCl, CaCl2, FeCl2, MgCl2, albumin, casein, gelatin, hydrolysed gelatin, lactose, sucrose, and pH 7.1 phosphate buffer. None of these substances have any fluorescent effect with N-CQDs. pH has an effect of increasing fluorescence around neutral pH (5-7). I therefore used a pH 7.1 phosphate buffer to maximize fluorescence and to control for pH differences in the analytes.

The graph above shows two sections. The first section (0-2.4 mg/mL) shows the linearity of the data before saturation (when the ratio of N-CQDs to HG is too high which means that the added HG cannot find a place to bond with the N-CQDs). The limit of detection was calculated for the linear area of the graph which has a slope of 13.7 RFU nm per mg/mL. My LOD is 0.19 mg/mL (~190 ppm). This does not meet the standard for gluten free labeling (20 mg/kg, 20 ppm).2,8 My LOD is not optimized and my research merely shows that it is possible to reliably detect hydrolysed gluten and more research is needed to optimize it.

Based on the analyte tests, it is likely that big proteins (albumin, gelatin, and casein), carbohydrates (lactose and sucrose), and smaller proteins (hydrolysed gelatin) do not cause a change in the fluorescence of N-CQDs. It is likely that the exclusivity of a fluorescent effect with N-CQDs is due to the functional groups of gluten.

My results suggest that it is possible to reliably detect HG in foods with N-CQDs by measuring the fluorescence. While more interference tests are needed, so far the effect looks to be specific. I tested other large and small proteins, various salts, and carbohydrates and none affected the fluorescence. Therefore, it is possible to create a reliable HG sensor for people with a gluten intolerance or Celiac disease. In a working sensor, I could detect HG by taking a baseline test of N-CQDs then adding the food sample in question. I would take fluorescence and absorbance again, adjusting for the inner filter effect as needed. If the sample gives higher fluorescence, then that means it would have hydrolysed gluten in it. Currently, because there is no reliable method to detect HG, and it is not fully regulated in gluten free foods even though it causes an immune response. The limit for gluten in foods with gluten free labeling is 0.02 mg/mL, and my LOD is 0.19 mg/mL. Once my LOD is improved enough to meet or exceed 0.02 mg/mL, hydrolysed gluten could finally be regulated in gluten free food.

The first thing I will do is test more mole ratios of citric acid and urea in the synthesis of N-CQDs to determine the maximum fluorescence. I also want to collect images of the N-CQDs to determine their diameter. I will also do many practical experiments such as putting food into a prototype, testing specific peptides of gluten, and other chemical interference tests to see issues that I can’t see with my current experiments. I would need to optimize my LOD to make a reliable test. This could be done by optimizing the concentration and excitation wavelength.

I would like to thank Glenlyon Norfolk School and Ms. Van Sprengel for providing resources and equipment. I would also like to thank Ms. Smook for helping me with organizing the personal project side of it. Lastly, I would like to thank my mom for helping me organize, plan and explain scientific concepts and words.

Citations

Daley, Sharon F., and Muhammad Haseeb. 2025. “Celiac Disease - StatPearls - NCBI Bookshelf.” NCBI. https://www.ncbi.nlm.nih.gov/books/NBK441900/.

U.S. Food and Drug Administration. 2020. “Food Labeling; Gluten-Free Labeling of Fermented or Hydrolyzed Foods.” Federal Register. https://www.federalregister.gov/documents/2020/08/13/2020-17088/food-labeling-gluten-free-labeling-of-fermented-or-hydrolyzed-foods.

Vallan, Lorenzo, and Hiroshi Imahori. 2022. “Citric Acid-Based Carbon Dots and Their Application in Energy Conversion.” ACS Applied Electronic Materials 4, no. 11 (11). https://doi.org/10.1021/acsaelm.2c01117.

Gabler, Angelika M., and Katharina A. Scherf. 2020. “Comparative Characterization of Gluten and Hydrolyzed Wheat Proteins.” Biomolecules, (8). https://doi.org/10.3390/biom10091227.

Lakowicz, Joseph R. 2006. Principles of fluorescence spectroscopy. Edited by Joseph R. Lakowicz. New York: Springer.

Kubista, Mikael, Robert Sjöback, Svante Eriksson, and Bo Albinsson. 1994. “Experimental Correction for the Inner-Filter Effect in Fluorescence Spectra.” Analyst 119, no. 3 (3): 3. https://doi.org/10.1039/AN9941900417.

Parker, C. A., and W. T. Rees. 1960. “Correction of Fluorescence Spectra and Measurement of Fluorescence Quantum Efficiency.” Analyst 85, no. 1013 (8): 14. DOI https://doi.org/10.1039/AN9608500587.

Codex Alimentarius Commission. 1979. “Standard for Foods for Special Dietary Use for Persons Intolerant to Gluten.” FAO / WHO Codex Alimentarius. https://www.fao.org/input/download/standards/291/CXS_118e_2015.pdf.

References

Anuar, Nurul K., Hooi L. Tan, Yit P. Lim, Mohd S. So'aib, and Nurfatehah W. Bakar. 2021. “A Review on Multifunctional Carbon-Dots Synthesized from Biomass Waste: Design/Fabrication, Characterization and Applications.” Bioenergy and Biofuels 9 (4). https://doi.org/10.3389/fenrg.2021.626549.

Chu, Hsin-Wei, Balasubramanian Unnikrishnan, Anisha Anand, Yu-Wen Lin, and Chih-Ching Huang. 2020. “Carbon quantum dots for the detection of antibiotics and pesticides.” Carbon Quantum Dots for the Detection of Antibiotics and Pesticides 28, no. 4 (12): 537-557. https://doi.org/10.38212/2224-6614.1269.

Gao, Wensu, Shurong Zhang, Guiqiao Wang, Jinzhi Cui, Yaxin Lu, Xing Rong, Yawen Luo, Lichao Zhang, Zhongfa Cheng, and Canzhu Gao. 2023. “Nitrogen and Sulfur Co-Doped Carbon Quantum Dots as 'On-Off-On' Fluorescence Probes to Detect Hg2+ and MnO4- and Improving the Photostability of Rhodamine B.” Analytica Chimica Acta 1277 (10). https://doi.org/10.1016/j.aca.2023.341683.

Hangsarumba, Surendra, Kishori Yadav, Santosh K. Das, Suresh P. Gupta, and Saddam H. Dhobi. 2026. “Synthesis of N-Doped Carbon Quantum Dot and Its Application on Sensing.” Discover Sensors 2, no. 11 (2): 1-14. https://doi.org/10.1007/s44397-026-00041-5.

Kamal, Atahar, Sanghyun Hong, and Hyunhyub Ju. 2025. “Carbon Quantum Dots: Synthesis, Characteristics, and Quenching as Biocompatible Fluorescent Probes.” Biosensors 15, no. 2 (2). https://doi.org/10.3390/bios15020099.

McMurry, John. 2016. Organic Chemistry. Boston: Cengage Learning.

Pykal, Martin, Jana Nociarová, David Řeha, Juraj Filo, Marek Šebela, Petr Zajíček, Markéta Paloncýová, et al. 2025. “Thermodynamics and Kinetics of Early Stages of Carbon Dot Formation: A Case of Citric Acid and Ethylenediamine Reaction.” Nanoscale, no. 13 (2). https://doi.org/10.1039/D4NR04420G.

Sengottuvelu, Dharmaraj, Adil K. Shaik, Shivan Mishra, Hasan Ahmad, Milad Abbaszadeh, Nathan I. Hammer, and Santanu Kundu. 2022. “Multicolor Nitrogen-Doped Carbon Quantum Dots for Environment-Dependent Emission Tuning.” ACS Omega 7, no. 31 (8). https://doi.org/10.1021/acsomega.2c03912.

Sun, Ya-Ping, Bing Zhou, Yi Lin, Wei Wang, K. A. S. Fernando, Pankaj Pathak, Mohammed J. Meziani, et al. 2006. “Quantum-Sized Carbon Dots for Bright and Colorful Photoluminescence.” Journal of the American Chemical Society 128, no. 24 (5). https://doi.org/10.1021/ja062677d.

U.S. Food and Drug Administration. 2022. “Questions and Answers on the Gluten-Free Food Labeling Final Rule.” FDA. https://www.fda.gov/food/nutrition-food-labeling-and-critical-foods/questions-and-answers-gluten-free-food-labeling-final-rule.

Venkatesh, Yeduru, Parimi V. Naidu, Podilapu A. Rao, and Durga B. Kundrapu. 2025. “Green Synthesis of Multifunctional Nitrogen-Doped Carbon Quantum Dots (N-CQDs) Derived from Citrus Lemon Juice.” Research on Chemical Intermediates 51 (4): 2621–2643. https://doi.org/10.1007/s11164-025-05567-8.

Xu, Xiaoyou, Robert Ray, Yunlong Gu, Harry J. Ploehn, Latha Gearheart, Kyle Raker, and Walter A. Scrivens. 2004. “Electrophoretic Analysis and Purification of Fluorescent Single-Walled Carbon Nanotube Fragments.” Journal of the American Chemical Society 126, no. 40 (9). https://doi.org/10.1021/ja040082h.

Zhou, Jigang, Christina Booker, Ruying Li, Xingtai Zhou, Tsun-Kong Sham, Xueliang Sun, and Zhifeng Ding. 2007. “An Electrochemical Avenue to Blue Luminescent Nanocrystals from Multiwalled Carbon Nanotubes (MWCNTs).” Journal of the American Chemical Society 129, no. 4 (1). https://doi.org/10.1021/ja0669070.

Kong, Jichuan, Yihui Wei, Feng Zhou, Liting Shi, Shuangjie Zhao, Mengyun Wan, and Xiangfeng Zhang. 2024. “Carbon Quantum Dots: Properties, Preparation, and Applications.” Molecules 29, no. 9 (4). https://doi.org/10.3390/molecules29092002.

Elugoke, Saheed E., Gloria E. Uwaya, Taiwo W. Quadri, and Eno E. Ebenso. 2024. “Carbon Quantum Dots: Basics, Properties, and Fundamentals.” Carbon Dots: Recent Developments and Future Perspectives 1465 (4): 3-42. https://doi.org/10.1021/bk-2024-1465.ch001.

Yadav, Pradeep K., Subhash Chandra, Vivek Kumar, Keepak Kumar, and Syed H. Hasan. 2023. “Carbon Quantum Dots: Synthesis, Structure, Properties, and Catalytic Applications for Organic Synthesis.” Catalysts 13, no. 2 (2). https://doi.org/10.3390/catal13020422.

Dsouza, Slavia D., Marius Buerkle, Paul Brunet, Chiranjeevi Maddi, Dilli B. Padmanaban, Alessio Morelli, Amir F. Payam, Paul Maguire, Davide Mariotti, and Vladimir Svrcek. 2021. “The Importance of Surface States in N-Doped Carbon Quantum Dots.” Carbon 183 (10): 1-11. https://doi.org/10.1016/j.carbon.2021.06.088.

Nguyen, Kiem G., Ioan-Alexandru Baragau, Radka Gromicova, Adela Nicolaev, Stuart A. Thomson, Alistair Rennie, Nicholas P. Power, Muhammed T. Sajjad, and Suela Kellici. 2022. “Investigating the Effect of N-Doping on Carbon Quantum Dots Structure, Optical Properties and Metal Ion Screening.” 12 (8): 1-12. https://doi.org/10.1038/s41598-022-16893-x.

Shabbir, Hasan, Edit Csapó, and Marek Wojnicki. 2023. “Carbon Quantum Dots: The Role of Surface Functional Groups and Proposed Mechanisms for Metal Ion Sensing.” Inorganics 11, no. 6 (June). https://doi.org/10.3390/inorganics11060262.

Nguyen, Kiem G., Matej Huš, Ioan-Alexandru Baragau, James Brown, Tobias Heil, Adela Nicolaev, Laura E. Abramiuc, Andrei Sapelkin, Muhammad T. Sajjad, and Suela Kellici. 2024. “Engineering Nitrogen-Doped Carbon Quantum Dots: Tailoring Optical and Chemical Properties through Selection of Nitrogen Precursors.” Nano Micro Small 20, no. 24 (3). https://doi.org/10.1002/smll.202310587.

Karamdoust, Sanaz, Mohammad-Reza Milani-Hosseini, and Farnoush Faridbod. 2023. “Simple Detection of Gluten in Wheat-Containing Food Samples of Celiac Diets with a Novel Fluorescent Nanosensor Made of Folic Acid-Based Carbon Dots through Molecularly Imprinted Technique.” Food Chemistry 410 (1): 1-9. https://doi.org/10.1016/j.foodchem.2022.135383.