16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article www.small-journal.com Graphene Nanobeacons with High-Affinity Pockets for Combined, Selective, and Effective Decontamination and Reagentless Detection of Heavy Metals David Panáček, Lukáš Zdražil, Michal Langer, Veronika Šedajová, Zdeněk Baďura, Georgio Zoppellaro, Qiuyue Yang, Emily P. Nguyen, Ruslan Álvarez-Diduk, Vítězslav Hrubý, Jan Kolařík, Nikolaos Chalmpes, Athanasios B. Bourlinos, Radek Zbořil, Arben Merkoçi, Aristides Bakandritsos,* and Michal Otyepka* 1. Introduction Access to clean water for drinking, sanitation, and irrigation is a major sustainable development goal of the United Nations. Thus, technologies for The increasing impact on the worldwide eco-systems of anthropogenic activities cleaning water and quality-monitoring must become widely accessible and (transportation, industrial, agricultural, of low-cost, while being effective, selective, sustainable, and eco-friendly. To and mining activities[1,2]) contributes meet this challenge, hetero-bifunctional nanographene fluorescent beacons substantially to the accumulation of with high-affinity pockets for heavy metals are developed, offering top-rated toxic heavy metals in the earth’s upper and selective adsorption for cadmium and lead, reaching 870 and 450 mg g-1, crust,[3] making the availability of high quality water for drinking and sanitation respectively. The heterobifunctional and multidentate pockets also operate as (a major sustainable development goal of selective gates for fluorescence signal regulation with sub-nanomolar sensi- the United Nations) into a growing chal- tivity (0.1 and 0.2 nm for Pb2+ and Cd2+, respectively), due to binding affinities lenge.[4] Several billions of people have as low as those of antigen-antibody interactions. Importantly, the acid-proof limited access to clean water (with esti- nanographenes can be fully regenerated and reused. Their broad visible-light mates approaching half of the popula- absorption offers an additional mode for water-quality monitoring based on tion by 2025),[5,6] thus the development of advanced materials and technologies is ultra-low cost and user-friendly reagentless paper detection with the naked- pivotal for efficient remediation and user- eye at a limit of detection of 1 and 10 ppb for Pb2+ and Cd2+ ions, respectively. friendly/on-site water quality-monitoring, This work shows that photoactive nanomaterials, densely-functionalized in order to secure a sustainable, and tech- with strong, yet selective ligands for targeted contaminants, can success- nologically advanced civilization. fully combine features such as excellent adsorption, reusability, and sensing Undoubtedly, advances in the design of tailored inorganic and carbon-based capabilities, in a way to extend the material’s applicability, its life-cycle, and materials have provided access to new value-for-money. and efficient nanostructures for sorption D. Panáček, L. Zdražil, M. Langer, V. Šedajová, Z. Baďura, G. Zoppellaro, D. Panáček, L. Zdražil, M. Langer, V. Šedajová, V. Hrubý V. Hrubý, J. Kolařík, R. Zbořil, A. Bakandritsos, M. Otyepka Department of Physical Chemistry Regional Centre of Advanced Technologies and Materials Faculty of Science Czech Advanced Technology and Research Institute (CATRIN) Palacký University Olomouc Palacký University Olomouc 17. listopadu 1192/12, Olomouc 771 46, Czech Republic Šlechtitelů 241/27, Olomouc 783 71, Czech Republic D. Panáček, Q. Yang, E. P. Nguyen, R. Álvarez-Diduk, A. Merkoçi E-mail: a.bakandritsos@upol.cz; michal.otyepka@upol.cz Catalan Institute of Nanoscience and Nanotechnology Autonomous University of Barcelona CSIC and BIST, Campus UAB, Bellaterra, Barcelona 08193, Spain The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.202201003. Z. Baďura Department of Experimental Physics © 2022 The Authors. Small published by Wiley-VCH GmbH. This is an Faculty of Science open access article under the terms of the Creative Commons Attribu- Palacký University Olomouc tion License, which permits use, distribution and reproduction in any 17. listopadu 1192/12, Olomouc 771 46, Czech Republic medium, provided the original work is properly cited. N. Chalmpes Department of Materials Science & Engineering University of Ioannina DOI: 10.1002/smll.202201003 Ioannina 45110, Greece Small 2022, 18, 2201003 2201003 (1 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com technologies, the latter remaining among the most facile and target analytes is particularly challenging.[17–20] For example, a effective methods for water decontamination due to their sim- Ca2+-MOF exhibited both adsorption and voltametric detection plicity and efficiency.[7,8] However, the next generation of sorb- of Pb2+ and Cd2+, with one of the highest Pb2+ adsorption capac- ents has to integrate the advantages of large accessible surface ities (≈522 mg g−1; and 220 mg g‑1 for Cd2+) and LoD of 3.0 and with chemical functionalization, acting as potent and selec- 11.6 nm for Pb2+ and Cd2+, respectively.[17] However, the MOF tive ligands for targeted contaminants.[9–15] A prerequisite for was unstable in the highly acidic regeneration medium and the broad application of such sophisticated and costly sorbents thus not reusable. Furthermore, sensing in presence of com- is the integration of features of high added-value, such as sus- petitive cations from drinking water (such as Ca2+ and Mg2+) tainability (i.e., sorbent regeneration and reuse),[10,16] catalytic might also be an issue since the material exhibited similar sen- activity after metal adsorption,[1,12,16] or utilization of the same sitivities for a wide variety of metal ions (with both low and high material for water-quality monitoring.[17–20] The combination of toxicities). In another case, exploiting the fluorescence, porosity, water decontamination and user‑friendly water‑quality moni- and thioether-functionalization of covalent organic frameworks toring is particularly attractive, since the two applications are (COFs), the highly selective fluorometric detection and facile highly complementary to each other, both from mechanistic removal of Hg2+ was achieved.[18] Nevertheless, the sorbent and practical aspects. A potent sorbent requires a material that was unstable in water, showing better sensing in organic sol- can attract the target analyte onto its surface with high affinity, vents, and despite the exceptional selectivity, the sensitivity of significantly increasing locally the analyte’s concentration. This 120 nm was suboptimal, since the maximum allowance limits may greatly impact the sensing if the sorbent simultaneously for the toxic metals in drinking water (e.g., Hg2+, Pb2+, Cd2+) are offers an analyte-dependent signal. A variety of nanomaterials all bellow 80 nm.[46] Furthermore, the hydrazone bonds of the such as Ag and Au NPs,[21–23] inorganic quantum dots,[24–26] COF, although covalent, are highly labile, precluding the utiliza- graphene/carbon dots,[19,26–31] WS2 sheets,[32] metal organic tion of aggressive regeneration media, required for the desorp- frameworks (MOFs),[17] and 2D organic frameworks[33,34] have tion of the adsorbed metal ions and recycling of the sorbent. been utilized to develop nanosensors for the detection of metal Amino‑functionalized carbon dots were also successfully ions. Among them, tailor‑coated Au-NP films have demon- applied for dual adsorption and sensing reaching 0.5 nm LoD strated selective, attomolar detection for methylmercury, and for Pb2+.[19] Nevertheless, the detection was based on monitoring picomolar detection for Cd2+ (less selective) via changes in the absorbance at extremely low wavelength (215 nm, where tap tunneling currents.[23] Although extremely sensitive, the use and river water composition blocks this spectral window, as of noble metals and required methodology suggest the use of shown in Figure S1, Supporting Information), restricting the the sensor in dedicated high‑end applications, where detec- application in real samples. The response was found similar tion far below the drinking water allowance limits is required. to other metals (Fe3+ and Mn2+) with much lower toxicity than Other sensing schemes rely on specialized (bio)molecules Pb2+, further limiting the applicability. The challenge grows (DNA/aptamers,[22,28,35–39] proteins,[40] organic molecules,[41] enormously when such dual function should be combined metal ion-imprinted polymers[25]) to achieve selectivity and high with sorbent recyclability, high adsorption capacity, detection binding affinity. Carbon/graphene dots may offer low limits of selectivity, and sensitivity in order to bring added values and detection (LoDs; Table S1, Supporting Information) via simple high potential for applicability. Key properties to achieve this fluorometric or electrochemical sensing, owing to their particu- embrace structural stability, high binding affinities[10] for the larly small size, high surface area, and chemically rich shell, target metals, and an interaction mechanism intrinsically regu- allowing for coordination with metal ions or further derivatiza- lating the transduction sensing signal.[40] tion.[19,26,28–30] Along with their sustainability, excellent biocom- In order to overcome these challenges, we engineered dual patibility, and eco-friendly character,[42–44] they overall shape a functionalized graphene fluorescent nanobeacons equipped very attractive ground for innovation. with carboxyl groups and nitrogen doping. A synthesis of the However, high selectivity for the toxic metals is often an nitrogen-doped graphene derivative (NG, Figure 1a) was fol- issue,[19,21,45] while the combined adsorption and sensing of lowed by oxidation and size confinement, leading to a fluores- cent and carboxyl-functionalized nitrogen‑doped graphene acid (NGA, Figure 1b). The NGA proved a very efficient and selec- A. B. Bourlinos tive trap for Pb2+ and Cd2+ ions (Figure 1b1), facilitating excep- Physics Department tional water decontamination in presence of competing ions University of Ioannina (Table S2, Supporting Information), both in simulated and real Ioannina 45110, Greece samples. After a hydrothermal treatment of NGA and forma- R. Zbořil, A. Bakandritsos tion of 2–3 nm NGA dots (NGA-D, Figure 1c), to further con- Nanotechnology Centre Centre of Energy and Environmental Technologies fine the lateral dimensions and boost the photoluminescence VŠB-Technical University of Ostrava (PL) properties, the fast, reagentless, and selective detection 17. listopadu 2172/15, Ostrava-Poruba 708 00, Czech Republic of Pb2+ and Cd2+ was achieved via PL quenching (Figure 1c1). A. Merkoçi The obtained LoDs (0.1 and 0.2 nm for Pb2+ and Cd2+, respec- ICREA, Institució Catalana de Recerca i Estudis Avançats tively) were unprecedented, surpassing the state‑of‑the‑art Pg. Lluís Companys, 23, Barcelona 08010, Spain schemes based on fluorometric and potentiometric methods, M. Otyepka IT4Innovations even many sensors using aptamers and DNA strands for sen- VŠB-Technical University of Ostrava sitivity boosting,[38,47,48] (Table S1, Supporting Information). The 17. listopadu 2172/15, Ostrava-Poruba 708 00, Czech Republic high sensitivity of the NGA-D was ascribed to the very strong Small 2022, 18, 2201003 2201003 (2 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 1. Synthesis and application of nitrogen-doped graphene acid. a) Synthesis of NG and b) oxidation to NGA with its respective applications; b1) adsorption and b2) paper detection of Pb2+ and Cd2+. c) Synthesis of NGA-D and c1) application of NGA-D for selective PL detection of Pb2+ and Cd2+. binding for Pb2+ and Cd2+ inside a multivalent coordination (Figure 2c,d; Tables S5 and S6, Supporting Information). The pocket formed by the nitrogen doped-vacancies and the car- nitrogen content of NGA-D dropped to 4.9 at% (Figure 2b; boxyl groups, with association constants in the order of antigen Tables S4 and S6, Supporting Information). antibody-interactions (10−6–10−9 L m−1),[49] and with a direct Fourier‑transformed infrared spectroscopy (FT-IR, Figure 2e) binding mechanism involving the formation of dark com- showed that the NG spectrum was dominated by two bands plexes with the ground-state of the NGA dots. Importantly, the at 1560 cm−1, and between 1000 and 1210 cm−1, both corre- acid‑proof structure of NGA enabled its full regeneration and sponding to skeletal vibrations of the sp2 aromatic carbon reuse as sorbent, while its broad visible‑light absorption offered network and to aromatic rings.[52,53] The feature at 1395 cm−1 an additional sensing mode for the two toxic metals based on indicated heteroatom substitution in aromatic rings, for reagentless detection with naked-eye on paper, offering a solu- example, to pyridinic ring vibrations.[53,54] In agreement with tion for ultra‑low cost, and user-friendly water quality moni- XPS, the spectra of NGA and NGA-D revealed the characteristic toring (Figure 1b2). stretching band of the carbonyl group at 1720 cm−1 (Figure 2e) from the carboxyls. The broad band at 1230 cm−1 changed its pattern in comparison to NG, comprising now also the CO 2. Results and Discussion stretching modes from the carboxylic groups.[55] Ionization of the carboxylic groups to carboxylates gave rise to additional NG was obtained by the reaction of fluorographene with vibrations at 1600 cm−1 and at 1420/1350 cm−1, ascribed to sodium azide, as previously reported,[50] leading to extensive CO2− asymmetric and symmetric stretching, respectively.[56] defluorination, and to 11.6 at% nitrogen doping, according to The band in NGA at 1140 cm−1 reflects the presence of COH X-ray photoelectron spectroscopy results (XPS, Figure 2a,b). groups, which significantly decreased upon further oxidation The deconvoluted C 1s domain of the high-resolution XPS in NGA-D, corroborating the XPS results. The aromatic ring (HR‑XPS, Figure 2c) spectra showed that NG consisted mostly vibrations at 1560 cm−1 were present in both NGA and NGA-D, of sp2 carbons (66.3 at%), CNC carbons (25.9 at%), and in agreement to the sp2 carbon components observed in XPS. CO groups (5.2 at%, (Table S3, Supporting Information). The new band at 1650 cm−1 in NGA-D was attributed to a small The deconvolution of N 1s spectrum (Figure S2, Supporting amount of organic nitrate groups (R−O−NO2 Figure S3 and Information) showed a typical three‑component pattern char- Table S4, Supporting Information), which typically have a very acteristic for nitrogen doped graphene, reflecting pyridinic, strong asymmetric band in this region.[57] Further details sup- pyrrolic, and graphitic nitrogen configurations (Table S4, Sup- porting the FT‑IR analysis are provided in the Supporting Infor- porting Information).[51] Oxidation of NG with nitric acid mation (Figure S3, Supporting Information). afforded the NGA product, where the oxygen content increased The morphology and microstructure of NG, NGA, and NGA-D from 3.0 at% to 32.7 at% and nitrogen decreased to 5.2 at%; were analyzed by transmission electron microscopy (TEM), (Figure 2a,b, Table S5, Supporting Information). According showing that NG comprised few-layered graphene flakes with to the deconvolution of the C 1s and O 1s HR-XPS spectral size around 600 nm (Figure 3a). The NGA flakes were smaller, regions, 15.2 at% of the total carbons corresponded to carbox- ≈50 nm, due to the oxidative cutting from the action of concen- ylic carbons, 10.5 at% to CO carbons, and another ≈10 at% trated nitric acid, showing high proclivity to interact with each to CO carbons (Figure 2c,d; Tables S5 and S6, Supporting other (Figure 3b). Elemental mapping of NGA confirmed the Information). NGA sheets were downsized by hydrothermal homogeneous coverage of the flakes with nitrogen and oxygen treatment into graphene dots of 2.5 nm (NGA-D product), (Figure 3c–f), coinciding with the spatial distribution of carbon. accompanied by further oxidation, as evidenced by the increase After hydrothermal treatment at 90 °C, graphene dots of very in oxygen content (Figure 2a,b), ascribed to the oxidative cutting small size in the range of 2–3 nm were formed (NGA-D), with of the sheets. HR-XPS results showed that 25.2 at% of the car- a lattice spacing of 0.21 nm corresponding to that of graphene bons corresponded to carboxylic groups, and 11.7 at% to CO {1100} lattice fringes[28] (Figure 3h, Figure S4a–d, Supporting groups, while all COH groups were transformed to carboxyls Information). After measurement of lattice spacings from more Small 2022, 18, 2201003 2201003 (3 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 2. a) XPS survey spectra, b) Atomic contents of the nitrogen doped-graphene (NG), after its treatment with nitric acid (NGA) and after hydro- thermal treatment (NGA-D), c) HR-XPS C 1s, d) HR-XPS O 1s and e) FT-IR spectra of NG, NGA, and NGA-D. dots (Figure S4c,d, Supporting Information), only these fringes after oxidation NGA exhibited a PL maximum at 523 nm were observed, confirming the dominant presence of single or under 470 nm excitation (Figure 4a). The appearance of PL few-layered graphene dots sitting flat on the grid, rather than can be ascribed to quantum confinement effects due to the carbon dots, which are multilayered with interlayer spacing of development of small sp2 islands isolated by the sp3 carbons ≈3.4 nm. The abundance of single-layered graphene dots was bonded to the oxygen functionalities, overall forming the further confirmed from the selected area diffraction from many PL-active surface states. The thermal treatment of NGA resulted dots, showing the {1100} diffraction peak much brighter than to PL enhancement in the NGA-D product, keeping the same the {2110}[58,59] (Figure S4b, Supporting Information). Atomic photophysical characteristics (Figure 4a), which could be thus force microscopy (AFM, Figure 3i) showed an average thick- attributed to the significant reduction of the lateral dimensions ness of 3 nm, corresponding to single-layer sheets, considering of the NGA sheets in the form of discrete dots, increasing fur- that unfunctionalized single layer graphene shows AFM thick- ther the content in quantum-confined areas.[60–62] The increased ness up to 2.6 nm.[59] PL intensity could be also ascribed to the improved quality of PL measurements at room temperature of NG, NGA, and the dispersion in the NGA-D product in comparison to the NGA-D dispersed in water did not show any PL for NG, but NGA system, since interparticle interactions can cause PL Small 2022, 18, 2201003 2201003 (4 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 3. TEM images of a) NG and b) NGA. EDS chemical mapping of c) carbon, d) nitrogen, and e) oxygen and combined chemical mapping of f) carbon plus nitrogen and g) carbon plus oxygen of NGA. h) HR-TEM image of NGA-D, with insets presenting resolved crystalline lattice (top right) and size distribution of NGA-D (bottom left). i) AFM image of NGA-D with height profiles along the indicated white line. quenching.[63] The excitation-emission map of NGA-D showed for Cd2+ and Pb2+.[10,12,65] Indeed, NGA showed a substantially excitation-independent emission (Figure 4b), suggesting that enhanced adsorption capacity, reaching 872 mg g–1 for Pb2+ (at PL originates from dots with very similar band-gap states, due 100 ppm Pb2+ feed, pH = 3.5), while NG reached 170 mg g–1 to their monodispersity.[61,63] Along with the small Stokes-shift (Table S2 Supporting Information), confirming the key role of ≈50 nm (Figure 4c), these observations suggest the absence of carboxyl groups in NGA. As a result, NGA outperformed of interlayer interactions, which usually lead to formation of state-of-the-art sorbents (Figure 5a). The same trend was excimers and energy-transfer (responsible for the large Stokes- observed for the case of Cd2+, with NG reaching a capacity shift and excitation-dependent PL, respectively) in carbon of 97 mg g−1 which was only a fraction of NGA’s capacity of dots.[64] The absorption spectra of the products (Figure 4c, 448 mg g−1 (at 100 ppm Cd2+ feed, pH = 3.5), surpassing the at 0.5 mg mL−1) at the long wavelengths (i.e., above 700 nm, capacity of most of the recently reported sorbents (Figure 5b). where light scattering phenomena mainly dominate) showed The sorption isotherms showed higher affinity for Pb2+ than decreasing absorbance going from NG to NGA and to NGA-D, for Cd2+, as indicated from the Langmuir and Freundlich con- verifying that the flake size also decreases with the same order stants, and from magnitudes of mean free sorption energy from NG to NGA and to NGA-D. The absorption spectrum derived from data fitting analysis (Figure S6, Supporting Infor- of NGA-D showed a perturbation between 400 and 500 nm, mation). The higher affinity for Pb2+ was also evident from the with a maximum at 460 nm, as verified by the derivative curve adsorption rate that was higher for Pb2+ than for Cd2+ based (Figure 4c), coinciding well with the excitation maximum of on the kinetic monitoring of the process analyzed by fitting the 467 ± 2 nm. The band-gap value of 2.8 eV (i.e., 440 nm) for the data to three different kinetic models (Figure S7, Supporting NGA-D, as calculated from the Tauc plot (Figure S5, Supporting information). A highly detailed information about the analysis Information), was also found in agreement with the spectral of the adsorption processes is in the Supporting information. features. The PL emission peak of NGA-D under the excitation The adsorption capacities at higher pH values were further of 467 nm was centered at 527 nm (FWHM of 119 nm) with a improved, but the results are affected by metal-ion hydrolysis PL lifetime of 1.1 ns, as obtained from fitting the PL decay by a (in more detail in the Supporting information). The adsorption stretched-exponential function (Figure 4d). properties of NGA were further tested in presence of competing The carboxyl groups are very strong metal-coordinating ions that are found in mineral water (Ca2+, Mg2+, Na+, K+; the ligands, and exhibit particularly high adsorption capacities concentrations are available in the Supporting Information) and Small 2022, 18, 2201003 2201003 (5 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 4. a) Comparison of PL spectra of NG, NGA, and NGA-D (λex = 470 nm, 0.5 mg mL−1). b) Excitation-emission color map of NGA-D. c) UV–vis absorption spectra of NG, NGA, and NGA-D (with its derivative), along with the excitation (λex = 467 nm) and PL (λem = 527 nm) spectra of NGA-D. The inset in the PL panel shows the aqueous NGA-D dispersion under ambient light and under 365 nm irradiation. d) Time-resolved PL decay (λem = 527 nm) with corresponding stretched-exponential fit (blue line) for derivation of the PL lifetime. in river water (Figure 5c). The results highlighted a very low its structure and thus its adsorption properties. Therefore, decrease of the adsorption capabilities (15% and 20% decrease the NGA, collected after the adsorption experiments, was for Pb2+ and Cd2+, respectively, in river water), suggesting the regenerated by acidic washing to desorb the metals, treated high selectivity of NGA toward the heavy metal ions. To fully with 1% w/v NaOH to establish the carboxylate form, washed, validate the witnessed high selectivity, adsorption experiments dried, and then reused for the next adsorption experiments. in presence of Pb2+ and Ca2+ (both at 100 ppm feed, pH = 3.5) Indeed, NGA retained its adsorption capacities for both Pb2+ were carried out, which showed only 10% decrease in the Pb2+ and Cd2+ for at least six regeneration cycles at levels higher adsorption (from 872 to 780 mg g−1, Figure 5c). According to the than 90% (Figure S8, Supporting Information), eliminating the principle of hard and soft acids and bases, the selectivity can relatively increased costs of the sorbent. be explained as H2O molecules are considered as “hard base” Motivated by these adsorption results, which suggested that ligands, therefore coordinating and keeping in solution the very NGA can selectively concentrate the Pb2+ and Cd2+ cations on its “hard acid” metal ions of Mg2+ and Ca2+ (which is also reflected surface, we identified that its PL signal was quenched upon inter- by the high hydration energy of these cations), rather than the action with Pb2+ and Cd2+, suggesting its applicability as a heavy- intermediate “soft acid” cations of Cd2+ and Pb2+, which coor- metal sensor. NGA clearly showed quenching in presence of dinate better with the carboxyl groups.[66] NGA appears to have 1 ppb (≈5 nm) of Pb2+ (Figure S9a, Supporting Information). This similar adsorption capacity for Pb2+ and Cd2+ in terms of moles property was boosted significantly in NGA-D, which has very (4.2 and 4 mmol g−1, respectively). Nevertheless, competing similar composition, but it is more finely dispersed, has stronger adsorption experiments with simultaneous presence of Pb2+ PL, and smaller size, predisposing it as a better candidate for the and Cd2+ at 100 ppm feed for both meal ions, showed that Pb2+ application, showing quenching even at 0.05 ppb (≈0.25 nm) of adsorption dropped only by 7% and by 40% for Cd2+ in compar- Pb2+ (Figure S9b, Supporting Information). The quenching was ison to the single metal ion experiments, suggesting the higher also highly selective (Figure 5d), even in natural mineral water affinity for Pb2+ than for Cd2+, as confirmed from the Langmuir and tap water samples, which contained much higher con- constants after fitting the sorption isotherms (Figure S6, Sup- centrations of other divalent metals (Figure 5d). The LoDs (at porting Information). a signal-to-noise ratio of 3) were as low as 0.1 nm for Pb2+ and To render NGA applicable and competitive against the low- 0.2 nm for Cd2+, as determined from PL intensity versus metal cost active carbons, complementary properties are required, concentration curves, (Figure S10, Supporting Information), such as reusability. Owing to its covalently bound, non-labile outperforming previously reported systems (Figure 6e,f and metal-coordination functionalities, NGA could be treated Table S1, Supporting Information), including electroluminescent under harsh acidic conditions (2% v/v HCl), without affecting graphene dots (LoD(Cd2+) = 13 nm[67]) and DNA based-sensors Small 2022, 18, 2201003 2201003 (6 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 5. a,b) Comparative graph showing the adsorption capacities (qmax) for Pb2+ and Cd2+ for NGA (star symbols) and for previously reported sorbents at various metal feeds and pH values: (α) MOF-808(Zr) modified with EDTA,[10] (β) GO,[68] (γ) GO/polymer hydrogel,[69] (δ) carbon nanotube sheets,[70] (ε) GO, amino-modified and decorated with Fe3O4 nanoparticles,[71] (ζ) copolymer modified Fe3O4 nanoparticles,[72] (η) silica gel modified with nitrilotriacetic acid,[73] (θ) GO decorated by iron-based nanoparticles,[74] (ι) GO,[75] (κ) GO,[76] (λ) 2D Ca2+-MOF,[17] (μ) FJI-H9 MOF modified with amino groups,[77] (π) α-FeOOH,[78] (ρ) LDH modified with MoS42-,[79] (ψ) graphene acid.[12] c) Adsorption capacities at pH = 3.5 for Pb2+ and Cd2+ in distilled, mineral, and river water, as well in combined Pb2+ and Cd2+ solutions in distilled water. In all experiments, the initial metal feed was 100 ppm. d) Comparison of fluorescence quenching of NGA-D induced by Pb2+, Cd2+, and other metal ions at 5 µm concentration in ultra-pure water (blue), tap water (green), and mineral water (purple). e) Comparative graph showing the LoD values for Pb2+ for NGA-D and from the state-of-the-art material sensors. (α) GQDs modified with AuNPs,[21] (β) CDs,[19] (ζ) CDs,[29] (ε) ZnSe QDs,[25] (η) CDs,[45] (θ) GO dots modified with Fe3O4,[80] (ρ) GO combined with CDs,[81] (ψ) GO combined with QDs,[38] (π) CDs,[30] (λ) Rhodamine–Quinoline Conjugates.[82] f) Comparative graph showing the limit of detection for Cd2+ for NGA-D and for top-rated results from literature. (α) ZnSe/CDs,[25] (ρ) CdTe QDs,[26] (γ) DPDB ligand,[83] (η) CdTe QDs,[84] (β) ZnSe/CdS,[24] (ζ) CdTe QDs,[85] (θ) GO combined with CDs,[86] (ε) CDs modified with gold nanoclusters[87] and (ψ) GOQD.[38] Small 2022, 18, 2201003 2201003 (7 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com Figure 6. Interaction of three divalent ions (Ca2+, Cd2+, and Pb2+) with molecular models of NGA-D, displaying two energetically favorable binding positions comprising a) carboxyl groups only and b) carboxyl groups plus nitrogen atoms, along with their corresponding binding energies (Figures S13 and S14, Supporting Information for further details). X-band CW-EPR and CW-LEPR spectra of NGA-D dispersed in H2O and measured in frozen matrix (T = 80 K) of c) pure NGA-D recorded under dark conditions (red line) and under UV irradiation (orange line) and NGA-D with adsorbed Ca2+ cations recorded under dark conditions (black line) and under UV irradiation (purple dashed line). d) NGA-D with adsorbed Cd2+ cations recorded under dark conditions (blue line) and under UV irradiation (orange line). e) NGA-D with adsorbed Pb2+ cations recorded under dark conditions (blue line) and under UV irradiation (orange line). (LoD(Cd2+) = 10 nm;[47] LoD(Pb2+) = 0.3[48] or 4.4 nm[38]). In some detection.[25] Despite this, the LoD values were significantly infe- cases, low LoD values have been reported (e.g., 0.2 and 2.2 nm rior to the NGA-D sensor. Finally, such materials are not suit- for Pb2+ and Cd2+, respectively[21,25]) via the PL turn-on of gold able for heavy metal-sorption and water decontamination, unlike nanoparticles released from the graphene surface upon inter- NGA-D, whose precursor (NGA) exhibits top-rated sorption for action with Pb2+. Apart from the use of noble metals, the Pb2+ the two heavy metal ions. Due to the high selectivity, NGA-D sensor showed similar selectivity for the much less toxic Mn2+ sensing ability was further challenged in mineral water and ions, limiting its applicability.[21] The sensor with 2.2 nm LoD tap water, showing the same LoD values as in ultrapure water for Cd2+ relied on the coating of the ZnSe dots with metal cat- (Figure S11, Supporting Information), which were found well ion-imprinted polymers, and, apart from the PL spectrometer, below the drinking water European concentration limits of 72 nm a sophisticated microfluidic chip was required for analysis and (15 ppb) and 44 nm (5 ppb) for Pb2+ and Cd2+, respectively,[46] Small 2022, 18, 2201003 2201003 (8 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com rendering NGA-D a very effective material for practical To shed more light on the nature of the interactions, we sensing of toxic heavy metals, not affected by other metals probed the PL decay curves with and without metals and ana- which are less toxic and with higher drinking water limits. lyzed the PL quenching results with the Stern–Volmer plots of The high selectivity and PL sensitivity of the NGA-D in F0/F versus metal concentration using the functions based on presence of Pb2+ and Cd2+ ions should be related to the highly bimolecular theory. The derived Stern–Volmer-constants (Ksv) specific electronic interactions rather than to the non-specific were 0.046 nm−1 (4.6 × 107 m−1) and 0.022 nm−1 (2.2 × 107 m−1) aggregation-induced quenching due to electrostatic interac- for Pb2+ and Cd2+, respectively (Figure S16, Supporting Infor- tions, as implied by the absence of PL emission peak shift.[33] mation), being in the order of antigen antibody-interactions Since aggregation is a slow process, the previous statement was (0.058 nm−1[88] or 106–109 m−1[40,49]). Indeed, such KSV con- also clearly demonstrated by the instant quenching of PL after stants are two or three orders of magnitude larger than other addition of 1 or 10 µm of Pb2+ or Cd2+ (Figure S12a, Supporting CDs sensors,[45,82,86] but also than other potent sensors based Information). Only after 1 h, and after addition of 10 µm of on fluorescent MOFs,[33,89,90] all ranging between 3 × 104 and Pb2+ or Cd2+ there is evidence of aggregation (Figure S12b, 4 × 105 m−1. The rate constants from the Stern–Volmer plots Supporting Information). Interestingly, the addition of Ca2+ (3.5 × 1016 m−1 s−1 and 1.7 × 1016 m−1 s−1 for Pb2+ and Cd2+, did not cause PL quenching (Figure S12a, Supporting Infor- respectively) exceeded by far the kinetics of a purely diffu- mation) or aggregation (Figure S12b, Supporting Information, sion-controlled process (≈1010 m−1 s−1),[90] and the PL lifetimes despite it is also a divalent cation), even at 100 µm, verifying remained unchanged (Figure S16, Supporting Information) the previous results regarding the PL sensitivity and selectivity with or without the addition of metals, clearly indicating a of NGA-D. Density functional theory (DFT) calculations further static quenching mechanism. The static quenching mecha- corroborated the results from the adsorption experiments and nism suggests the strong and direct interaction of the metals from the above observations. Models were constructed to cor- with the ground state of NGA-D,[91] forming dark complexes respond to the XPS composition of NGA and NGA-D and the after bonding between the valence band of the dots with the spatial proximity of the carboxyl groups stemmed also from the empty orbitals of Pb2+ and Cd2+ ions, verifying experimen- actual experimental XPS data (Table S5, Supporting Informa- tally the theoretical results. The higher binding constants tion), showing a ratio of sp2/COOH of 3 and 1.6 for NGA and obtained from the PL quenching results (Figure S17, Sup- NGA-D, respectively, suggesting the presence of one carboxyl porting Information, 4.5 × 108 m−1 and 3.4 × 107 m−1 for Pb2+ group in almost every C6 ring. Two binding modes of metal cat- and Cd2+, respectively) than from the sorption isotherms ions at two different binding sites were analyzed in detail; one (Figure S6, Supporting Information), reflected very well the with carboxylic groups only and the other one with both carbox- theoretically obtained twofold more negative binding ener- ylic groups and nitrogen-doped vacancies (Figure 6a,b), which gies between the carboxyl-only and nitrogen-carboxyl com- were energetically most favorable. In both cases, Ca2+ had sig- plexes (Figure 6a,b), since the ratio of their logarithms was nificantly lower binding affinities, as indicated by the calculated also equal to two (logKb(Pb2+)/logKL(Pb2+) = 8.7/4.4 = 1.98, and binding energies which showed 2- or 3-fold stronger binding logKb(Cd2+)/logKL(Cd2+) = 7.5/3.4 = 2.20), highlighting the key for Cd2+, and Pb2+ showed ≈1.5-fold stronger binding even than role of the heterobifunctional and multivalent nitrogen-carboxyl Cd2+. The 1.5-fold stronger binding was in good agreement with binding sites at low metal concentrations. the experimentally determined higher association constant for The photophysics of NGA-D with Pb2+ or Cd2+, was fur- Pb2+ than for Cd2+ (logKL(Pb2+)/logKL(Cd2+) = 4.4/3.4 = 1.3), as ther studied by probing the spin-active electronic transitions obtained from the Langmuir isotherms (Figure S6, Supporting with continuous wave electron paramagnetic resonance spec- Information). The same trends were observed even when all troscopy (CW-EPR) with and without in situ light irradiation. four carboxylic groups were deprotonated (Figure S13, Sup- NGA-D was found EPR inactive under dark conditions, thus porting Information). The metal ion binding was dominated by not containing free radical sites (Figure 6c). Upon irradiation, electrostatic interaction and contribution from O and N moie- photoexcited spin-active species were clearly formed, as wit- ties electron donation to vacant metal ions orbitals; the N-doped nessed in the appearance of the strong EPR resonance line vacancies contributed more significantly, as evidenced by the at g = 1.999 (Figure 6c, NGA-D (light)). Therefore, during the natural bond orbital analysis (Figures S14,S15 and Table S7, photo­excitation process, part of the excited electrons undergo Supporting Information). The key role of the N-doped vacan- intersystem crossing, forming either triplet excimers or spa- cies in the coordination of the metal ions was also suggested tially separated polarons. Due to the witnessed shape of the by the analysis of the second-order perturbation estimates of photogenerated paramagnetic resonance signal, we suggest that donor-acceptor interactions, showing stronger donation to the formation upon photoexcitation of polaron pairs is preferred the vacant orbitals of the metal ions than the carboxylic oxy- compared to the possible formation of excimer triplet states. gens (Figures S14,S15 and Table S7, Supporting Information). Furthermore, excimer formation and relaxation (a common Overall, NGA and NGA-D exhibit two types of binding sites, PL relaxation mechanism in carbon dots) is usually accompa- with the minority, but stronger ones, represented by carbox- nied by a large Stokes-shift, which is not observed in NGA-D.[64] ylic groups plus N-doped vacancies (since nitrogen amounts to These e−/h+ species are thought to remain mostly localized on 5 at%), operating at low metal concentrations (i.e., at sub- and defect sites close to the carboxyl groups with small contribution few-nm in the case of sensing), and carboxylic groups oper- from nitrogen sites (Figures 6a,b), because they do not exhibit, ating after the saturation of the former, that is, at metal cation besides signal broadening, well defined hyperfine splitting in concentrations of several µm, which is the case of the initial the resonance line that arises from strong coupling of the elec- part of the sorption isotherms. tronic spin moment with nitrogen nuclear spins (14N, I = 1). Small 2022, 18, 2201003 2201003 (9 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com The entrapment of Cd2+ and Pb2+cations (1 µm) in NGA-D the h+ electron spin moment and the vector spin anisotropy.[92] leads to a significant drop in the light-induced EPR signal Overall, the finding that the metal ions have an impact only intensity (Figure 6d,e). Therefore, coordination of both metals on the generated holes provides another proof about their onto NGA-D leads to significant electronic perturbations of interaction with the valence band of the NGA-D. Importantly, the e−/h+ photoexcited states compared to the neat material. the addition of Ca2+ at the same concentration (1 µm) did not In particular, through computer simulation of the experi- induce any changes, confirming the absence of interactions mental EPR resonance line, using second order perturbation (Figure 6c), in accordance with the previous theoretical and theory, we found that during the photoexcitation in presence experimental results. of Cd2+, two sets of hole species are formed, which express To meet the demands for on-site, user-friendly sensing, axial to weakly rhombic anisotropy ((i)gx = 2.040, gy,z = 1.960 without the need of any electronic device, allowing for very low and (ii) gx = 2.003, gy = 1.990, gz = 1.980), while the reso- cost and point-of-interest sensors, we developed a paper-based nance signal associated to the e− sites remains fully isotropic lateral flow sensor by leveraging the black color of NGA, along (gx,y,z = 1.990) (Figure S18, Supporting Information). Adsorp- with its selective interactions to Pb2+ and Cd2+ interactions. tion of Pb2+ cations onto NGA-D also impacted the electronic The selected material was common chromatography nitrocellu- fingerprint of the photogenerated e−/h+ species (Figure 6e), but lose paper, whereby the lateral flow of NGA was guided along- to less extend than that expressed by coordination of Cd2+. In side the hydrophilic channels formed by wax-printing. The addition to the decreased intensity of the EPR resonance signal resulting chromatogram was affected by the concentration of during irradiation, the anisotropic nature of the electron spin Pb2+ and Cd2+ ions (Figure 7), due to the strong interactions moment tensor associated to the hole species (at lower extent between NGA’s functional groups and the heavy metal ions. than for Cd2+) resulted in an asymmetric broadening of the This simple and fast method (requiring just 30 min for chro- EPR signal centered at g = 1.990, clearly seen in the high field matogram evolution, Supporting Information, Experimental region (Figure 6e, NGA-D+Pb2+(light)). Such different behavior part) provides selective and quantitative information with LoDs of the photogenerated polaron pairs in Cd2+ and Pb2+ can be of 0.5 and 9 µm for Pb2+ and Cd2+, respectively (Figure 7a,b). interpreted in terms of their different ability to significantly This method of detection is even feasible for different ambient modulate, upon coordination, the spatial distribution of the temperatures, ranging from 5 to 35 °C, allowing use in loca- photoexcited spin charges on NGA-D, by perturbation of the tions where temperatures are low or too high (Figure S19, Sup- system electric field gradient (EFG).[92] The EFG gradient is porting Information). Considerably lower detection limits of linked to −∇Φ(r), where Φ(r) represents the electrostatic poten- 5 nm for Pb2+ and 88 nm for Cd2+ could be achieved after simple tial present in an atom or molecule at point r caused by the evaporation of the testing sample (Figure 7e,f), which are below distribution of charges (electrons, nuclei); hence, significant or very close to the allowed concentrations in drinking water perturbation of Φ(r) on NGA-D upon metal coordination arises, of 72 nm (15 ppb) and 44 nm (5 ppb) for Pb2+ and Cd2+, respec- as a result, of their different degrees of charge transfer, leaving tively.[46] Importantly, considering the influence from real water a more positively charged coordinated Cd2+ cation (1.85 natural matrix is an open issue for heavy metal ions detection, river charge) compared to Pb2+ (1.70 Natural charge, see natural elec- water samples were also tested and successfully measured tron configurations in Figures S13 and S14, Supporting Infor- with the same sensitivity, because all the humic acids/organic mation). This effect, in turn, impacts the observed variance in matter in the river water were separated/trapped at the bottom Figure 7. Dose-dependent effect of Pb2+ and Cd2+ concentration on NGA’s flow on the paper after 30 min of analysis. a,b) Pb2+ and Cd2+ in the pres- ence of different concentrations detected by paper sensor in mineral water. c,d) Pb2+ and Cd2+ in the presence of different concentrations detected by paper sensor in river water. e,f) Pb2+ and Cd2+ in the presence of different concentrations detected by paper sensor in mineral water after evaporation. g) The paper sensor in a protective 3D printed case. Small 2022, 18, 2201003 2201003 (10 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.small-journal.com of the paper-sensor (Figure S20, Supporting Information) and heterobifunctional and multidentate coordination sites as gates thus not affecting the detection (Figure 7c,d). Therefore, the for sensitive and selective signal regulation. NGA paper sensor can deliver on-site rigorous and practical water quality control, requiring only the facile evaporation of the water sample, a cup as sample holder, and a 3D printed Supporting Information case (Figure 7g), enabling its straightforward application by untrained users. Such a sensor based on simple operation prin- Supporting Information is available from the Wiley Online Library or ciples, involving direct use of the nanographene (without any from the author. surface modification) and naked-eye detection with few ppb’s sensitivity and high selectivity is of particular interest, since such features have not been reported before. Some related sen- Acknowledgements sors rely on chemically functionalized paper with the heavy The work was supported by the ERDF/ESF project “Nano4Future” (No. metal-sensitive β-galactosidase (B-GAL) for the colorimetric vis- CZ.02.1.01/0.0/0.0/16_019/0000754). R.Z. and A.B. acknowledge the ualization of its enzymatic activity inhibition, delivering LoDs funding from the Czech Science Foundation, GA CR–EXPRO, 19-27454X. of 680 and 180 nm for Pb2+ and Cd2+, respectively.[93] In addi- D.P., L.Z., V.Š., M.L., and V.H. thank the Internal Student Grant Agency of tion, the sensor was similarly sensitive to the much less toxic the Palacký University in Olomouc, Czech Republic (IGA_PrF_2022_019). Cu2+ cations (allowance limit of 1.3 ppm). Colorimetric lateral M.O. acknowledges the ERC grant 2D-CHEM, No 683024 from H2020. Operation of XPS and TEM facilities was partly funded by the Research flow sensors, with the use of proteins, antibodies, or metal Infrastructure NanoEnviCz, supported by the Ministry of Education, specific DNA, offered detection of Cd2+ at 10 ppb (90 nm)[94] or Youth and Sports of the Czech Republic under Project No. LM2018124. for Pb2+ at 0.5 µm,[95] as the first lower concentrations discern- N.C. gratefully acknowledges the IKY foundation for the financial support. ible by the naked eye. It should be also noted that the naked- N.C. and A.B.B. also thank the European Social Fund-ESF (co-financed by eye detection via color intensity differences on the test-lines of Greece and the European Union) through the Operational Programme the strips (especially considering the inaccuracy from different “Human Resources Development, Education and Lifelong Learning” in the context of the project “Strengthening Human Resources Research light conditions during testing) is much more challenging than Potential via Doctorate Research” (MIS-5000432), implemented by detecting the differences in the distance traveled by the dark the State Scholarships Foundation (IKY). Ms. Jana Dzíbelová, Ms. colored NGA. We have not identified reports based on this prin- Kateřina Roháčová, Mr. Ondřej Tomanec, and Ms. Jana Stráská are ciple, which, along with the previous observations with respect acknowledged for TGA analysis, Raman spectroscopy, HR‑TEM, and to the current state of the art, underline the effectiveness of the TEM characterization of samples, respectively. Dr. Martin Pykal is presently developed NGA paper sensor. acknowledged for the development of the 3D printed cover case and its subsequent manufacturing. Computational resources were supplied by the project “e-Infrastruktura CZ” (e-INFRA CZ LM2018140) supported by the Ministry of Education, Youth, and Sports of the Czech Republic. This 3. Conclusion work was supported by the Ministry of Education, Youth, and Sports of the Czech Republic through the e-INFRA CZ (ID:90140). ICN2 is funded The present work introduces a design principle leading to by CERCA programme, Generalit de Catalunya. Grant SEV-2017-0706 by recyclable materials for potent and selective heavy metal MCIN/AEI/10.13039/501100011033. The authors also acknowledge the decontamination, effectively combined with high-performance project MAT2017-87202-P funded by MCIN/AEI/10.13039/501100011033 and FEDER Una manera de hacer Europa. R.A.-D. acknowledges funding water-quality monitoring technologies (for mineral, tap, or from the European Union Horizon 2020 Programme under Grant No. even complex-matrix samples like river water), using low and 881603 (Graphene Flagship Core 3). ultra-low-cost signal transduction methods. These properties were imprinted by engineering dense and covalent dual func- tionalization of the graphene backbone with carboxylic groups and nitrogen atoms as multidentate metal traps, accompanied Conflict of Interest with size confinement that contributed to the material’s photo­ A patent application related to this work is pending. luminescence properties. Carboxylic groups are highly effec- tive coordination ligands for Cd2+ and Pb2+ cations but are less effective toward coordination of common minerals in drinking water, thus making the reported material a highly selective Data Availability Statement and efficient sorbent. The carboxyl-nitrogen heterobifunctional The data that support the findings will be available in Mendeley at and multidentate sites offered antibody-level affinity pockets, https://www.10.17632/gjr838mv9s.1 following an embargo from the date forming dark complexes with Cd2+ and Pb2+ functioning as of publication to allow for commercialization of research findings. PL-quenching sensors with LoD of 0.1 and 0.2 nm for Pb2+ and Cd2+, respectively, outperforming not only state-of-the-art sen- sors of this type, but also sensors based on electrochemical sig- Keywords nals or operating via exploitation of metal-selective DNA and aptamer strands. The reported design principle could be further carboxylation, heavy metals, nitrogen-doped graphene acid, paper-based sensors, sensing, sorption expanded for sensing additional analytes, either by modifying the present material (thanks to its abundant carboxyl groups) Received: February 16, 2022 with other molecules with high affinity for other types of ana- Revised: March 22, 2022 lytes, or by synthesis of new materials bearing such potent, Published online: July 1, 2022 Small 2022, 18, 2201003 2201003 (11 of 13) © 2022 The Authors. Small published by Wiley-VCH GmbH 16136829, 2022, 33, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202201003 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [18/10/2022]. 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