The surface chemistry of nanomaterials, particularly the density of functional groups, governs their behavior in applications such as bioanalysis, bioimaging, and environmental impact studies. Here, we report a precise method to quantify carboxyl groups per nanoparticle by combining anisotropically collapsing agarose gels for nanoparticle immobilization with fluorescence microscopy and acid-base titration. We applied this approach to photon-upconversion nanoparticles (UCNPs) coated with poly(acrylic acid) (PAA) and fluorescence-labeled polystyrene nanoparticles (PNs), which serve as models for bioimaging and environmental pollutants, respectively. UCNPs exhibited 152 ± 14 thousand carboxyl groups per particle (∼11 groups/nm2), while PNs were characterized with 38 ± 3.6 thousand groups (∼1.7 groups/nm2). The limit of detection was 6.4 and 1.9 thousand carboxyl groups per nanoparticle, and the limit of quantification was determined at 21 and 6.2 thousand carboxyl groups per nanoparticle for UCNP-PAAs and PNs, respectively. High intrinsic luminescence enabled direct imaging of UCNPs, while PNs required fluorescence staining with Nile Red to overcome low signal-to-noise ratios. The study also discussed the critical influence of nanoparticle concentration and titration conditions on the assay performance. This method advances the precise characterization of surface chemistry, offering insights into nanoparticle structure that extend beyond the resolution of electron microscopy. Our findings establish a robust platform for investigating the interplay of surface chemistry with nanoparticle function and fate in technological and environmental contexts, with broad applicability across nanomaterials.

Department of Chemistry Faculty of Science Masaryk University 625 00 Brno Czech Republic

Institute of Analytical Chemistry of the Czech Academy of Sciences Veveří 97 602 00 Brno Czech Republic

See more in PubMed

Zhang, P. ; Ye, G. ; Xie, G. ; Lv, J. ; Zeng, X. ; Jiang, W. . Research Progress of Nanomaterial Drug Delivery in Tumor Targeted Therapy. Front. Bioeng. Biotechnol. 2023, 11, 10.3389/fbioe.2023.1240529. PubMed DOI PMC

Algar W. R., Massey M., Rees K., Higgins R., Krause K. D., Darwish G. H., Peveler W. J., Xiao Z., Tsai H.-Y., Gupta R., Lix K., Tran M. V., Kim H.. Photoluminescent Nanoparticles for Chemical and Biological Analysis and Imaging. Chem. Rev. 2021;121(15):9243–9358. doi: 10.1021/acs.chemrev.0c01176. PubMed DOI

Yao J., Yang M., Duan Y.. Chemistry, Biology, and Medicine of Fluorescent Nanomaterials and Related Systems: New Insights into Biosensing, Bioimaging, Genomics, Diagnostics, and Therapy. Chem. Rev. 2014;114(12):6130–6178. doi: 10.1021/cr200359p. PubMed DOI

Astruc D.. Introduction: Nanoparticles in Catalysis. Chem. Rev. 2020;120(2):461–463. doi: 10.1021/acs.chemrev.8b00696. PubMed DOI

Lin W.. Introduction: Nanoparticles in Medicine. Chem. Rev. 2015;115(19):10407–10409. doi: 10.1021/acs.chemrev.5b00534. PubMed DOI

Beach M. A., Nayanathara U., Gao Y., Zhang C., Xiong Y., Wang Y., Such G. K.. Polymeric Nanoparticles for Drug Delivery. Chem. Rev. 2024;124(9):5505–5616. doi: 10.1021/acs.chemrev.3c00705. PubMed DOI PMC

Ma Z., Mohapatra J., Wei K., Liu J. P., Sun S.. Magnetic Nanoparticles: Synthesis, Anisotropy, and Applications. Chem. Rev. 2023;123(7):3904–3943. doi: 10.1021/acs.chemrev.1c00860. PubMed DOI

Choi S., Lee S., Kim M.-K., Yu E.-S., Ryu Y.-S.. Challenges and Recent Analytical Advances in Micro/Nanoplastic Detection. Anal. Chem. 2024;96(22):8846–8854. doi: 10.1021/acs.analchem.3c05948. PubMed DOI

Kelpsiene E., Ekvall M. T., Lundqvist M., Torstensson O., Hua J., Cedervall T.. Review of Ecotoxicological Studies of Widely Used Polystyrene Nanoparticles. Environ. Sci. Process. Impacts. 2022;24(1):8–16. doi: 10.1039/D1EM00375E. PubMed DOI

Nowack B., Bucheli T. D.. Occurrence, Behavior and Effects of Nanoparticles in the Environment. Environ. Pollut. 2007;150(1):5–22. doi: 10.1016/j.envpol.2007.06.006. PubMed DOI

Dale A. L., Casman E. A., Lowry G. V., Lead J. R., Viparelli E., Baalousha M.. Modeling Nanomaterial Environmental Fate in Aquatic Systems. Environ. Sci. Technol. 2015;49(5):2587–2593. doi: 10.1021/es505076w. PubMed DOI

Auffan M., Rose J., Bottero J.-Y., Lowry G. V., Jolivet J.-P., Wiesner M. R.. Towards a Definition of Inorganic Nanoparticles from an Environmental, Health and Safety Perspective. Nat. Nanotechnol. 2009;4(10):634–641. doi: 10.1038/nnano.2009.242. PubMed DOI

Hochella M. F., Lower S. K., Maurice P. A., Penn R. L., Sahai N., Sparks D. L., Twining B. S.. Nanominerals, Mineral Nanoparticles, and Earth Systems. Science. 2008;319(5870):1631–1635. doi: 10.1126/science.1141134. PubMed DOI

Geißler D., Nirmalananthan-Budau N., Scholtz L., Tavernaro I., Resch-Genger U.. Analyzing the Surface of Functional NanomaterialsHow to Quantify the Total and Derivatizable Number of Functional Groups and Ligands. Microchim. Acta. 2021;188(10):321. doi: 10.1007/s00604-021-04960-5. PubMed DOI PMC

Jayawardena H. S. N., Liyanage S. H., Rathnayake K., Patel U., Yan M.. Analytical Methods for Characterization of Nanomaterial Surfaces. Anal. Chem. 2021;93(4):1889–1911. doi: 10.1021/acs.analchem.0c05208. PubMed DOI PMC

Delille F., Pu Y., Lequeux N., Pons T.. Designing the Surface Chemistry of Inorganic Nanocrystals for Cancer Imaging and Therapy. Cancers. 2022;14(10):2456. doi: 10.3390/cancers14102456. PubMed DOI PMC

Zhang J., Mou L., Jiang X.. Surface Chemistry of Gold Nanoparticles for Health-Related Applications. Chem. Sci. 2020;11(4):923–936. doi: 10.1039/C9SC06497D. PubMed DOI PMC

Bhattacharjee K., Prasad B. L. V.. Surface Functionalization of Inorganic Nanoparticles with Ligands: A Necessary Step for Their Utility. Chem. Soc. Rev. 2023;52(8):2573–2595. doi: 10.1039/D1CS00876E. PubMed DOI

Shang J., Gao X.. Nanoparticle Counting: Towards Accurate Determination of the Molar Concentration. Chem. Soc. Rev. 2014;43(21):7267–7278. doi: 10.1039/C4CS00128A. PubMed DOI PMC

Holzmeister P., Acuna G. P., Grohmann D., Tinnefeld P.. Breaking the Concentration Limit of Optical Single-Molecule Detection. Chem. Soc. Rev. 2014;43(4):1014–1028. doi: 10.1039/C3CS60207A. PubMed DOI

Zhu S., Yang L., Long Y., Gao M., Huang T., Hang W., Yan X.. Size Differentiation and Absolute Quantification of Gold Nanoparticles via Single Particle Detection with a Laboratory-Built High-Sensitivity Flow Cytometer. J. Am. Chem. Soc. 2010;132(35):12176–12178. doi: 10.1021/ja104052c. PubMed DOI

Vaclavek T., Prikryl J., Foret F.. Resistive Pulse Sensing as Particle Counting and Sizing Method in Microfluidic Systems: Designs and Applications Review. J. Sep. Sci. 2019;42(1):445–457. doi: 10.1002/jssc.201800978. PubMed DOI

Meng Z., Zheng L., Fang H., Yang P., Wang B., Li L., Wang M., Feng W.. Single Particle Inductively Coupled Plasma Time-of-Flight Mass SpectrometryA Powerful Tool for the Analysis of Nanoparticles in the Environment. Processes. 2023;11(4):1237. doi: 10.3390/pr11041237. DOI

Elsaesser A., Barnes C. A., McKerr G., Salvati A., Lynch I., Dawson K. A., Howard C. V.. Quantification of Nanoparticle Uptake by Cells Using An Unbiased Sampling Method and Electron Microscopy. Nanomed. 2011;6(7):1189–1198. doi: 10.2217/nnm.11.70. PubMed DOI

Moerner W. E., Fromm D. P.. Methods of Single-Molecule Fluorescence Spectroscopy and Microscopy. Rev. Sci. Instrum. 2003;74(8):3597–3619. doi: 10.1063/1.1589587. DOI

Xin H. L., Zheng H.. In Situ Observation of Oscillatory Growth of Bismuth Nanoparticles. Nano Lett. 2012;12(3):1470–1474. doi: 10.1021/nl2041854. PubMed DOI

Hlaváček A., Křivánková J., Brožková H., Weisová J., Pizúrová N., Foret F.. Absolute Counting Method with Multiplexing Capability for Estimating the Number Concentration of Nanoparticles Using Anisotropically Collapsed Gels. Anal. Chem. 2022;94(41):14340–14348. doi: 10.1021/acs.analchem.2c02989. PubMed DOI

Al Harraq A., Brahana P. J., Arcemont O., Zhang D., Valsaraj K. T., Bharti B.. Effects of Weathering on Microplastic Dispersibility and Pollutant Uptake Capacity. ACS Environ. Au. 2022;2(6):549–555. doi: 10.1021/acsenvironau.2c00036. PubMed DOI PMC

Ustunol I. B., Gonzalez-Pech N. I., Grassian V. H.. pH-Dependent Adsorption of α-Amino Acids, Lysine, Glutamic Acid, Serine and Glycine, on TiO2 Nanoparticle Surfaces. J. Colloid Interface Sci. 2019;554:362–375. doi: 10.1016/j.jcis.2019.06.086. PubMed DOI

Villacorta A., Cazorla-Ares C., Fuentes-Cebrian V., Valido I. H., Vela L., Carrillo-Navarrete F., Morataya-Reyes M., Mejia-Carmona K., Pastor S., Velázquez A., Arribas Arranz J., Marcos R., López-Mesas M., Hernández A.. Fluorescent Labeling of Micro/Nanoplastics for Biological Applications with a Focus on “true-to-Life″ Tracking. J. Hazard. Mater. 2024;476:135134. doi: 10.1016/j.jhazmat.2024.135134. PubMed DOI

Chatterjee S., Krolis E., Molenaar R., Claessens M. M. A. E., Blum C.. Nile Red Staining for Nanoplastic Quantification: Overcoming the Challenge of False Positive Counts Due to Fluorescent Aggregates. Environ. Chall. 2023;13:100744. doi: 10.1016/j.envc.2023.100744. DOI

Srivastava P., Tavernaro I., Genger C., Welker P., Hübner O., Resch-Genger U.. Multicolor Polystyrene Nanosensors for the Monitoring of Acidic, Neutral, and Basic pH Values and Cellular Uptake Studies. Anal. Chem. 2022;94(27):9656–9664. doi: 10.1021/acs.analchem.2c00944. PubMed DOI

Hlaváček A., Farka Z., Mickert M. J., Kostiv U., Brandmeier J. C., Horák D., Skládal P., Foret F., Gorris H. H.. Bioconjugates of Photon-Upconversion Nanoparticles for Cancer Biomarker Detection and Imaging. Nat. Protoc. 2022;17(4):1028–1072. doi: 10.1038/s41596-021-00670-7. PubMed DOI

Dong A., Ye X., Chen J., Kang Y., Gordon T., Kikkawa J. M., Murray C. B.. A Generalized Ligand-Exchange Strategy Enabling Sequential Surface Functionalization of Colloidal Nanocrystals. J. Am. Chem. Soc. 2011;133(4):998–1006. doi: 10.1021/ja108948z. PubMed DOI

Behnke T., Würth C., Laux E.-M., Hoffmann K., Resch-Genger U.. Simple Strategies towards Bright Polymer Particles via One-Step Staining Procedures. Dyes Pigments. 2012;94(2):247–257. doi: 10.1016/j.dyepig.2012.01.021. DOI

Hlaváček A., Uhrová K., Weisová J., Křivánková J.. Artificial Intelligence-Aided Massively Parallel Spectroscopy of Freely Diffusing Nanoscale Entities. Anal. Chem. 2023;95(33):12256–12263. doi: 10.1021/acs.analchem.3c01043. PubMed DOI PMC

Speiser A., Müller L.-R., Hoess P., Matti U., Obara C. J., Legant W. R., Kreshuk A., Macke J. H., Ries J., Turaga S. C.. Deep Learning Enables Fast and Dense Single-Molecule Localization with High Accuracy. Nat. Methods. 2021;18(9):1082–1090. doi: 10.1038/s41592-021-01236-x. PubMed DOI PMC

Martiskainen I., Talha S. M., Vuorenpää K., Salminen T., Juntunen E., Chattopadhyay S., Kumar D., Vuorinen T., Pettersson K., Khanna N., Batra G.. Upconverting Nanoparticle Reporter-Based Highly Sensitive Rapid Lateral Flow Immunoassay for Hepatitis B Virus Surface Antigen. Anal. Bioanal. Chem. 2021;413(4):967–978. doi: 10.1007/s00216-020-03055-z. PubMed DOI PMC

Ansari A. A., Parchur A. K., Thorat N. D., Chen G.. New Advances in Pre-Clinical Diagnostic Imaging Perspectives of Functionalized Upconversion Nanoparticle-Based Nanomedicine. Coord. Chem. Rev. 2021;440:213971. doi: 10.1016/j.ccr.2021.213971. DOI

Velev O. D., Kaler E. W.. In Situ Assembly of Colloidal Particles into Miniaturized Biosensors. Langmuir. 1999;15(11):3693–3698. doi: 10.1021/la981729c. DOI

Mikhnev L. V., Bondarenko E. A., Chapura O. M., Skomorokhov A. A., Kravtsov A. A.. Influence of Annealing Temperature on Optical Properties of the Photonic-Crystal Structures Obtained by Self-Organization of Colloidal Microspheres of Polystyrene and Silica. Opt. Mater. 2018;75:453–458. doi: 10.1016/j.optmat.2017.10.052. DOI

Al Harraq A., Bharti B.. Microplastics through the Lens of Colloid Science. ACS Environ. Au. 2022;2(1):3–10. doi: 10.1021/acsenvironau.1c00016. PubMed DOI PMC

Trozzolo A. M., Winslow F. H.. A Mechanism for the Oxidative Photodegradation of Polyethylene. Macromolecules. 1968;1(1):98–100. doi: 10.1021/ma60001a019. DOI

Wiśniewska M., Urban T., Grza̧dka E., Zarko V. I., Gun’ko V. M.. Comparison of Adsorption Affinity of Polyacrylic Acid for Surfaces of Mixed Silica-Alumina. Colloid Polym. Sci. 2014;292(3):699–705. doi: 10.1007/s00396-013-3103-x. PubMed DOI PMC