Perovskite Nanocrystal Fluorescence-Linked Immunosorbent Assay Methodology for Sensitive Point-of-Care Biological Test

Abstract

•Water dispersity and stability of perovskite NCs was effectively improved•The water dispersity is 3.4 mg/mL, and the powder stability lasts more than 1 year•Narrow emission of perovskite-antibody probes (FWHM ∼18 nm) was achieved•Immunoassay methodology using perovskite NCs as labels was successfully established In recent years perovskites have attracted tremendous attention for their appealing application in optoelectronic devices. On the other hand, their outstanding optical property with high color purity and high quantum yield has not been used in immunoassays for point-of-care (POC) testing. Challenges have arisen regarding how to solve their hydrophobic characteristics and attach them to antibodies to obtain probes. Here, we demonstrate water-dispersed perovskite nanocrystals (Pe-NCs) with high quantum yield and narrow emission. In addition, a quantitative immunoassay using Pe-NCs as labels is successfully constructed in both food safety and clinical fields. The fluorescent probe maintain the excellent optical properties of Pe-NCs and is expected to be a promising candidate for future multi-target detection. Quantitative fluorescence immunoassay is a vital and convenient method in point-of-care (POC) testing for food safety and clinical healthcare. However, current marking materials, from organic luciferin to quantum dots, still lack the requirement of increasing need for multiple targets of interest because of their too broad emission. Here, a new label material, perovskite nanocrystals (Pe-NCs), with a narrower photoluminescence spectrum is reported, and a sensitive and quantitative immunoassay method named the perovskite nanocrystal fluorescence-linked immunosorbent assay is preliminarily demonstrated. The key point of realizing water-dispersed and stable Pe-NCs was addressed by functionalizing with hydroxyl groups. The fluorescent probes (perovskite antibody) were constructed via electrostatic adsorption with narrow full-width at half-maximum (∼18 nm), laying the foundation for future study of multi-target detection. Both indirect competitive immunity and sandwich immunoassay were successfully demonstrated. This work provides a possible label material for POC tests and may open up a promising avenue of next-generation immunoassay. Quantitative fluorescence immunoassay is a vital and convenient method in point-of-care (POC) testing for food safety and clinical healthcare. However, current marking materials, from organic luciferin to quantum dots, still lack the requirement of increasing need for multiple targets of interest because of their too broad emission. Here, a new label material, perovskite nanocrystals (Pe-NCs), with a narrower photoluminescence spectrum is reported, and a sensitive and quantitative immunoassay method named the perovskite nanocrystal fluorescence-linked immunosorbent assay is preliminarily demonstrated. The key point of realizing water-dispersed and stable Pe-NCs was addressed by functionalizing with hydroxyl groups. The fluorescent probes (perovskite antibody) were constructed via electrostatic adsorption with narrow full-width at half-maximum (∼18 nm), laying the foundation for future study of multi-target detection. Both indirect competitive immunity and sandwich immunoassay were successfully demonstrated. This work provides a possible label material for POC tests and may open up a promising avenue of next-generation immunoassay. Point-of-care (POC) tests, through enlightening bedside or on-site detection, have become indispensable in biological disciplines, including medical diagnostics, food safety, and environmental monitoring.1Quesada-González D. Merkoçi A. Nanomaterial-based devices for point-of-care diagnostic applications.Chem. Soc. Rev. 2018; 47: 4697-4709Crossref PubMed Google Scholar, 2Zhan L. Guo S.Z. Song F. Gong Y. Xu F. Boulware D.R. et al.The role of nanoparticle design in determining analytical performance of lateral flow immunoassays.Nano Lett. 2017; 17: 7207-7212Crossref PubMed Scopus (102) Google Scholar, 3Speranskaya E.S. Beloglazova N.V. Abe S. Aubert T. Smet P.F. Poelman D. Goryacheva I.Y. De Saeger S. Hens Z. Hydrophilic, bright CuInS2 quantum dots as Cd-free fluorescent labels in quantitative immunoassay.Langmuir. 2014; 30: 7567-7575Crossref PubMed Scopus (70) Google Scholar Fluorescent POC tests are probably the most favorable, with simple measurements.4Bruns O.T. Bischof T.S. 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Although the first-generation fluorescent label materials such as fluorochrome, fluorescein isothiocyanate, and rhodamine 6G show high photoluminescence quantum yield (PLQY), the full-width at half-maximum (FWHM) exceeds 50 nm.7Wu Y. Wei P. Pengpumkiat S. Schumacher E.A. Remcho V.T. A novel ratiometric fluorescent immunoassay for human α-fetoprotein based on carbon nanodot-doped silica nanoparticles and FITC.Anal. Methods. 2016; 8: 5398-5406Crossref Google Scholar,8Verma V.K. Tapadia K. Maharana T. Sharma A. Convenient and ultra-sensitive fluorescence detection of bovine serum albumin by using Rhodamine-6G modified gold nanoparticles in biological samples.Luminescence. 2018; 33: 1408-1414Crossref PubMed Scopus (11) Google Scholar The second-generation labels, quantum dots (QDs), known for narrower emission and higher PLQY, are good enough for normal detection, especially the Cd-based QDs,9Tayebi M. Tavakkoli Yaraki M. Ahmadieh M. Tahriri M. Vashaee D. Tayebi L. 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Fortunately, perovskite nanocrystals (Pe-NCs), the new fluorescent nanomaterial now popular in the field of optoelectronics,12Chen Y. Lei Y. Li Y. Yu Y. Cai J. Chiu M.H. Rao R. Gu Y. Wang C. Choi W. et al.Strain engineering and epitaxial stabilization of halide perovskites.Nature. 2020; 577: 209-215Crossref PubMed Scopus (217) Google Scholar, 13Xie Y. Yin J. Zheng J. Fan Y. Wu J. Zhang X. Facile RbBr interface modification improves perovskite solar cell efficiency.Mater. Today Chem. 2019; 14: 100179Crossref Scopus (16) Google Scholar, 14Zhang X. Strain control for halide perovskites.Matter. 2020; 2: 294-296Abstract Full Text Full Text PDF Scopus (14) Google Scholar are promising for shortening the distance due to their high color purity (FWHM <20 nm), high PLQY, and facile synthesis.15Song J. Li J. Li X. Xu L. Dong Y. Zeng H. Quantum dot light-emitting diodes based on inorganic perovskite cesium lead halides (CsPbX3).Adv. 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Enhancing the stability of CH3NH3PbBr3 quantum dots by embedding in silica spheres derived from tetramethyl orthosilicate in "waterless" toluene.J. Am. Chem. Soc. 2016; 138: 5749-5752Crossref PubMed Scopus (413) Google Scholar and TiO2,23Li Z.-J. Hofman E. Li J. Davis A.H. Tung C.-H. Wu L.-Z. Zheng W. Photoelectrochemically active and environmentally stable CsPbBr3/TiO2 core/shell nanocrystals.Adv. Funct. Mater. 2018; 28: 1705380Google Scholar which have been tried for coating Pe-NCs, whereby although the water resistance has improved, the water dispersion is still in question. Embedding Pe-NCs into a polymer matrix (3–5 μm)24Zhang H. Wang X. Liao Q. Xu Z. Li H. Zheng L. Fu H. Embedding perovskite nanocrystals into a polymer matrix for tunable luminescence probes in cell imaging.Adv. Funct. Mater. 2017; 27: 1604382Crossref Scopus (259) Google Scholar or encapsulating it with a solid lipid structure (>700 nm)25Gomez L. de Weerd C. Hueso J.L. Gregorkiewicz T. Color-stable water-dispersed cesium lead halide perovskite nanocrystals.Nanoscale. 2017; 9: 631-636Crossref PubMed Google Scholar have also been investigated. The diameter of coated particles is too large for POC tests, and only cell imaging applications have been demonstrated, although it has shed light on prospects in biological applications. Here, the vital issue of the hydrophobicity and poor stability of Pe-NCs was addressed through one-step mechanochemical synthesis by functionalizing with hydrophilic hydroxyl groups. The aqueous solution of Pe-NCs exhibit high PLQY (71.3%), water dispersity of 3.4 mg/mL, good stability, and a narrow emission spectrum (FWHM ∼18 nm), showing huge potential as fluorescent reporters. Here, Pe-antibody probes were built by conjugating between Pe-NCs and specific monoclonal antibody (mAb) due to general electrostatic adsorption. Additionally, the probes maintain excellent the optical properties of pure Pe-NCs with high luminance and narrow FWHM (18 nm), distinctly better than traditional fluorochrome and recent QDs. On these bases, the novel immunoassay methodology, named perovskite nanocrystal fluorescence-linked immunosorbent assay (PFLISA), is successfully established according to the variation in fluorescence value with the concentration of antigen based on Pe-antibody-antigen recognition in both the food-safety (aflatoxin M1 [AFM1]) and clinical (carcinoembryonic antigen [CEA]) fields. Both limits of detection (LODs) satisfy the requirement of rapid screening, and the whole assay time is less than 4 h, which is much shorter than that of the traditional ELISA. This work addressed the water-dispersion issue of Pe-NCs and showed a proof-of-concept trial in POC applications, transitioning perovskite from the lab to practical biological applications. Such water-dispersed Pe-NCs and quantitative PFLISA methodology may speed the development of new immunoassays. As the basis of PFLISA, the water-dispersed Pe-NCs with a similar structure of oil-in-water is synthesized. This approach is inspired by the concept of “mineralization” in nature, whereby dispersed chemical elements are relatively enriched in certain environments and form mineral deposits, which inspired us to make full use of raw materials, interaction, and reaction environments to form water-dispersed “perovskite deposits” with specific surface modification akin to mineralization. Choosing an appropriate long-chain surfactant to coat perovskite and combining hydrophilic groups in the outer layer to form an oil-in-water structure may be effective in achieving water dispersion in one-pot synthesis. To overcome difficulties, oleylamine (OAm) and the hydroxyl group in ambient water were judiciously introduced for surface engineering. The mechanochemical method of ball milling was selected for the following reasons. First, it is an approach similar to “mineralization” in nature in that it makes full use of raw materials, and interaction and reaction environments such as humidity, high temperature, and high pressure. Second, it is not only chemical reaction, but also extra mechanical energy with high temperature and high pressure provided during ball milling, which likely promote the formation of Pe-NCs. In addition, it is an all-solid-state mechanochemical method whereby post-processing is not needed to obtain a powder sample. The whole process takes place in ambient atmosphere without inert gas protection. The resulting Pe-NCs are expected to show affinity to water environment through outer hydrophilic hydroxyl groups and inner protection of long-chain amine.Br−+H2O→HBr+OH−,(Equation 1) OAm→HBrOAm+,(Equation 2) OAm++OH−→OAM+−−OH,(Equation 3) This synthesis process could be divided into several steps. (1) Bromine in CsBr or PbBr2 form an acid environment with hydroxyl group in moist air (Equation 1, obvious acidity of PbBr2 and CsBr were observed in water as shown in Figure S1). (2) OAm is protonated with acid, and some hydroxyl groups in the environment will be attached to the electrophilic C=C of OAm forming “OAm+--OH” (Equations 2 and 3). The connection between OAm+ and OH⁻ is possibly not a chemical bond but similar to affinity-induced adsorption, and a similar structure such as C-O-C might form. Simultaneously, protonated OAm+ and OAm+--OH combined with CsPbBr3 NCs, called the pre-synthesis Pe-NCs. (3) Similar to adsorption bridging as shown in Figure 1, when the pre-synthesis Pe-NCs are exposed in the humid air of Nanjing, several Pe-NCs get together and form larger particles with hydrophobic long chains of carbon. The long-chain molecules act as bridges and bonds, protecting perovskites from water, and the OAm+--OH surround and form the outer hydrophilic layer. Thus, water-dispersed Pe-NCs of oil-in-water structure can be realized. The water dispersity and optical properties of Pe-NCs are then investigated. Figure 2A shows the digital photographs of different dispersions under natural light (upper panel) and UV light (365 nm, lower panel). The aqueous dispersion remains soluble in water without aggregation and displays bright green. The corresponding UV-visible absorption spectra are recorded in Figure 2B. A maximum concentration of 3.4 mg/mL is obtained by weighing dry powder, which illustrates successful water dispersion of Pe-NCs as expected. As shown in Figure 2C, a typical photoluminescence (PL) spectrum of our aqueous dispersion is presented, which is centered at 522 nm with a FWHM of 18 nm. This narrow emission is quite comparable with solution-synthesis Pe-NCs,26Song J. Fang T. Li J. Xu L. Zhang F. Han B. Shan Q. Zeng H. Organic-inorganic hybrid passivation enables perovskite QLEDs with an EQE of 16.48%.Adv. Mater. 2018; 30: e1805409Crossref PubMed Scopus (274) Google Scholar and is obviously narrower than the FWHM of first-generation fluorescein (>50 nm)7Wu Y. Wei P. Pengpumkiat S. Schumacher E.A. Remcho V.T. A novel ratiometric fluorescent immunoassay for human α-fetoprotein based on carbon nanodot-doped silica nanoparticles and FITC.Anal. Methods. 2016; 8: 5398-5406Crossref Google Scholar,8Verma V.K. Tapadia K. Maharana T. Sharma A. Convenient and ultra-sensitive fluorescence detection of bovine serum albumin by using Rhodamine-6G modified gold nanoparticles in biological samples.Luminescence. 2018; 33: 1408-1414Crossref PubMed Scopus (11) Google Scholar and the second-generation QDs (30–140 nm),3Speranskaya E.S. Beloglazova N.V. Abe S. Aubert T. Smet P.F. Poelman D. Goryacheva I.Y. De Saeger S. Hens Z. Hydrophilic, bright CuInS2 quantum dots as Cd-free fluorescent labels in quantitative immunoassay.Langmuir. 2014; 30: 7567-7575Crossref PubMed Scopus (70) Google Scholar,9Tayebi M. Tavakkoli Yaraki M. Ahmadieh M. Tahriri M. Vashaee D. Tayebi L. Determination of total aflatoxin using cysteamine-capped CdS quantum dots as a fluorescence probe.Colloid Polym. Sci. 2016; 294: 1453-1462Crossref Scopus (23) Google Scholar which is suitable for multivariate analysis with small background interference. The PLQY of fresh Pe-NCs dispersion is 71.3%, qualified by using 2,7-dichlorofluorescein (F-27) as reference. Only 2.68%–3.55% attenuation occurs after storage for 10 days using three parallel data, and 9.7% deterioration for 30 days (see Figure S2), which shows impressive stability. The microstructure of Pe-NCs is also analyzed using transmission electron microscopy (TEM) in Figure 2D. The size of Pe-NCs is in the range of 50–100 nm, and clear lattice fringe of 5.8 Å can be seen in Figure S3. A larger selected area is observed in Figure 2E, and a histogram of size distribution is shown in Figure 2F. The average size is 53.26 nm with side length of 45–70 nm, showing good homogeneity. Considering the surface functionalization, the clear cubic shape of CsPbBr3 tends to be a little rounded. The monoclinic phase is then confirmed to be stable by X-ray diffraction (XRD) (see Figure S4). In addition, the powder sample of Pe-NCs exhibits remarkable air stability as the intensity and position of characteristic peaks of XRD spectrum remains almost unchanged after storage in the air for 1 year (see Figure S5). The outstanding performance of narrow, bright emission and excellent stability indicate the success of the experimental design, and foreshadows the potential applications for fluorescent labeling material. Several reports about water-dispersed or water-resistant perovskites have been compared, and the summary of the optical properties and stability in water is presented in Table 1. PbBrOH was used to protect CsPbBr327Lin H. Zhang X. Cai L. Lao J. Qi R. Luo C. Chen S. Peng H. Huang R. Duan C. High-stability fluorescent perovskites embedded in PbBrOH triggered by imidazole derivatives in water.J. Mater. Chem. C. 2020; https://doi.org/10.1039/D0TC00939CCrossref Google Scholar and can maintain 60% of initial PL intensity after storage in water for 8 days. In addition, the maximum PLQY is only 36%. Cobalt-doped CsPbBr3/Cs4PbBr6 NCs28Mu Y.F. Zhang W. Guo X.X. Dong G.X. Zhang M. Lu T.B. Water-tolerant lead halide perovskite nanocrystals as efficient photocatalysts for visible-light-driven CO2 reduction in pure water.ChemSusChem. 2019; 12: 4769-4774Crossref PubMed Scopus (57) Google Scholar was reported to have a PLQY of 80% but with 10% deterioration after only 100 h. Larger molecules such as solid lipid nanoparticles25Gomez L. de Weerd C. Hueso J.L. Gregorkiewicz T. Color-stable water-dispersed cesium lead halide perovskite nanocrystals.Nanoscale. 2017; 9: 631-636Crossref PubMed Google Scholar and microhemispheres24Zhang H. Wang X. Liao Q. Xu Z. Li H. Zheng L. Fu H. Embedding perovskite nanocrystals into a polymer matrix for tunable luminescence probes in cell imaging.Adv. Funct. Mater. 2017; 27: 1604382Crossref Scopus (259) Google Scholar have also been chosen, but these particles are either too large for immunoassay or present low PLQY. By comparison, our results are competitive or better.Table 1Comparison of the Optical Properties and Stability in WaterStructureSizeMax. PLQYStability in WaterReferenceCsPbBr3/PbBrOHirregular microsized particles36%60% after 8 daysLin et al.27Lin H. Zhang X. Cai L. Lao J. Qi R. Luo C. Chen S. Peng H. Huang R. Duan C. High-stability fluorescent perovskites embedded in PbBrOH triggered by imidazole derivatives in water.J. Mater. Chem. C. 2020; https://doi.org/10.1039/D0TC00939CCrossref Google Scholar[email protected]3/Cs4PbBr6∼20 nm∼80%90% after 100 hMu et al.28Mu Y.F. Zhang W. Guo X.X. Dong G.X. Zhang M. Lu T.B. Water-tolerant lead halide perovskite nanocrystals as efficient photocatalysts for visible-light-driven CO2 reduction in pure water.ChemSusChem. 2019; 12: 4769-4774Crossref PubMed Scopus (57) Google ScholarCsPbX3/solid lipid nanoparticles700 ± 240 nm33%–85%<50% after 3 weeksGomez et al.25Gomez L. de Weerd C. Hueso J.L. Gregorkiewicz T. Color-stable water-dispersed cesium lead halide perovskite nanocrystals.Nanoscale. 2017; 9: 631-636Crossref PubMed Google ScholarCsPbX3 NCs/microhemisphere3–5 mm16%–27%water resistantZhang et al.24Zhang H. Wang X. Liao Q. Xu Z. Li H. Zheng L. Fu H. Embedding perovskite nanocrystals into a polymer matrix for tunable luminescence probes in cell imaging.Adv. Funct. Mater. 2017; 27: 1604382Crossref Scopus (259) Google ScholarCsPbBr3 NCs∼50 nm71.3%90.3% after 30 daysThis work Open table in a new tab The mechanism of water dispersion is further confirmed by a series of measurements. The hydroxyl group is expected to play the key role in water dispersion, and is firstly confirmed by Fourier transform infrared (FTIR) spectra as shown in Figure 3A. A strong band of -OH (∼3,500 cm−1) was observed29Xu J. Sun Y. Chen J. Zhong S. Novel application of amphiphilic block copolymers in Pickering emulsions and selective recognition of proteins.New J. Chem. 2018; 42: 3028-3034Crossref Google Scholar in the spectrum of Pe-NCs, while pure CsPbBr3 (without OAm) showed a weaker and broader band nearby, which is related to adsorbed water molecules on the surface. X-ray photoelectron spectroscopy (XPS) analysis is further performed to unveil the surface microstructure (see Figure S6). The typical survey spectrum of Pe-NCs and high-resolution Cs 3s, Pb 4d, and Br 3d have binding energy positions similar to those reported previously,30Yuan S. Wang Z.K. Zhuo M.P. Tian Q.S. Jin Y. Liao L.S. Self-assembled high quality CsPbBr3 quantum dot films toward highly efficient light-emitting diodes.ACS Nano. 2018; 12: 9541-9548Crossref PubMed Scopus (115) Google Scholar and only a small binding energy observed in the Pb 4f XPS spectrum of Pe-NCs, attributed to Pb-OAm species.31Pan J. Quan L.N. Zhao Y. Peng W. Murali B. Sarmah S.P. Yuan M. Sinatra L. Alyami N.M. Liu J. et al.Highly efficient perovskite-quantum-dot light-emitting diodes by surface engineering.Adv. Mater. 2016; 28: 8718-8725Crossref PubMed Scopus (719) Google Scholar However, the Pe-NCs show increased intensity of O 1s peaks compared with pure CsPbBr3 as shown in Figure 3B. The single peak at 531.5 eV is assigned to -OH,32Tan B.J. Klabunde K.J. Sherwood P.M.A. XPS studies of solvated metal atom dispersed catalysts. Evidence for layered cobalt-manganese particles on alumina and silica.J. Am. Chem. Soc. 1991; 113: 855-861Crossref Scopus (840) Google Scholar,33Xia H. Lai M. Lu L. 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The 1H nuclear magnetic resonance (NMR) spectrum (see Figure S7) is also established to explore the changes in OAm during reaction. No new peaks occur with Pe-NCs by both hand grinding and mechanochemically, confirming that no extra hydrogen bonding formed. We then transfer our attention to the reaction between CsBr, PbBr2, and OAm. Further investigation through FTIR spectra is displayed in Figure 3D. The typical peak of hydroxyl group only exists in the spectrum of “PbBr2 + OAm,” indicating that the key role of water dispersion can be focused on PbBr2 and OAm, whereas hygroscopic CsBr just plays the role of capturing water molecules. Considering water molecules, all possible combinations are analyzed using FTIR. As shown in Figure S8, the characteristic peaks occur only in spectrum of PbBr2 + OAm (+H2O). We hypothesized that hydroxyl groups are held to the surface of Pe-NCs by OAm. Thus, the FTIR spectra of OAm and PbBr2 + OAm are carefully analyzed in Figure 3E. Despite the band of hydroxyl group (∼3,500 cm−1), both characteristic bands of N–H stretching vibrations (3,190 cm−1) and N+–H formation vibration (1,580 cm−1) are observed in spectrum of PbBr2 + OAm, indicating successful protonation of OAm. The C=C bond of OAm is not broken during synthesis, as can be inferred from the emergence of typical C=C stretching vibration bands at 3,009 and 1,621 cm−1 in the FTIR spectra of PbBr2 + OAm.35Peng B. Lu X. Chen S. Huan C.H.A. Xiong Q. Mutlugun E. Demir H.V. Yu S.F. Exciton dynamics in luminescent carbon nanodots: electron-hole exchange interaction.Nano Res. 2015; 9: 549-559Crossref Scopus (7) Google Scholar Furthermore, the weak double absorption bands at 1,137 and 1,254 cm−1 referring to extension vibrations of the C-O-C group are observed.29Xu J. Sun Y. Chen J. Zhong S. Novel application of amphiphilic block copolymers in Pickering emulsions and selective recognition of proteins.New J. Chem. 2018; 42: 3028-3034Crossref Google Scholar The band of C-O-C is possibly assigned to the connection of C=C bonds with hydroxyl groups, i.e., the hydroxyl group adsorbed by electrophilic C=C bonds at a very close distance, forming a similar structure of C-O-C but not a new hydrogen bond, as we cannot find evidence in H1 NMR spectra. To shed more light on this adsorption, we quantify the XPS spectra in of the corresponding samples (Figure 3F). Different from other samples, the O 1s core level for PbBr2 + OAm is fitted with two components at 530.8 and 532.45 eV. The dominant peak at 532.3–532.6 eV corresponds to water,36Andersson K. Gómez A. Glover C. Nordlund D. Öström H. Schiros T. Takahashi O. Ogasawara H. Pettersson L.G.M. Nilsson A. Molecularly intact and dissociative adsorption of water on clean Cu(110): a comparison with the water/Ru(001) system.Surf. 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Coat. 2006; 55: 231-243Crossref Scopus (258) Google Scholar which correspond to FTIR analysis. Additional dispersion in oil-phase solution (toluene) is observed to confirm the importance of humidity. As seen in Figure S10, smaller particles (10–20 nm) with regular cubic shape are obtained. The larger particles separated into smaller ones because of the oil solution, and the initial OAm-OH is no longer wrapped around Pe-NCs. Conversely, when Pe-NCs are firstly dispersed in water they become larger (50–100 nm) with a relatively irregular boundary, as seen in Figure 2. In addition, if the outer hydroxyl groups and larger particles are not formed, the powder samples cannot be stored for such a long time (1 year). Thus, the larger particles are not initially formed when dispersed in water but in the humid environment during synthesis, and Pe-NCs