Electrochemiluminescence of dual excited states modulated with arginine modi cation of black phosphorus quantum dots

Huangxian Ju (  hxju@nju.edu.cn ) Nanjing Universtiy https://orcid.org/0000-0002-6741-5302 Siqi Yu Nanjing University Yu Du Nanjing University Xianghong Niu Nanjing University of Posts and Telecommunications https://orcid.org/0000-0001-6475-8839 Guangming Li Nanjing University Da Zhu Nanjing University Qian Yu Nanjing University Guizheng Zou Shandong University, Jinan, China https://orcid.org/0000-0002-3295-3848


Full Text
The electrochemiluminescence (ECL) is generally emitted via radiative transition of singlet or triplet excited state (S 1 or T 1 ). Herein, an ECL mechanism with the transitions of both S 1 and T 1 of black phosphorus quantum dots (BPQDs) was found for the rst time, and an arginine modi cation strategy was proposed to passivate the surface oxidation defects of BPQDs, which could modulate the excited states for enhancing the ECL of BPQDs. The Arg modi cation led to greater spatial overlap of HOMO with LUMO and spectral shift of radiative transitions, and improved the stability of anion radical of BPQDs. The enhanced cathodic ECL was used to construct a sensitive method for conveniently evaluating the inhibiting e ciency of cyclo(RGDyK) to cell surface integrin by using RRGDS peptide modi ed BPQDs as signal tag. The dual excited states mediated ECL emitters provided a new paradigm for adjustable ECL generation and extended the application of ECL analysis. Electrochemiluminescence (ECL) is a light-emitting process, in which the excited state species (R * ) are generated via exergonic electron transfer and exchange between the electrogenerated intermediates, following the radiative transitions to the ground state (S 0 ) 1 . Generally, the produced R * can be either the lowest excited singlet state (S 1 ) species ( 1 R * ) or the triplet state (T 1 ) species ( 3 R * ), depending on the difference of their relative energy 2,3 . According to the rules of spin statistics, the internal quantum e ciency of the luminophores emitting ECL is limited to 25% due to their 1 R * -to-3 R * ratio of 1:3 and the non-radiative transition from T 1 to S 0 4 , which limits the full utilization of the energy during the ECL process. In this work we found for the rst time a possibility to emit the ECL via both S 1 -to S 0 and T 1 -to-S 0 transitions by using black phosphorus quantum dots (BPQDs) as the emitter.
BPQDs were rstly prepared in 2014 with bulk black phosphorus (BP) 5,6 , which is a metal-free semiconductor and displays tunable bandgap varying from 0.3 eV for bulk BP to 2.0 eV for monolayer BP 7 , and have been extensively applied in photothermal therapy, electrocatalysis and exible devices 5,6,[8][9][10] . However, their optical and electrical performances are greatly limited by the oxidation defects of nanostructure surface due to the extremely easy degradability under ambient conditions [11][12][13] . Great efforts have been made to stabilize BPQDs by either preventing the occurrence of oxidation process via encapsulation with polyethylene glycol, uorine and poly(lactic-co-glycolic acid) 7,9,14 , or passivating the existing oxidation defects with ethanol 15 . Meanwhile, arginine (R or Arg) contained peptides and poly-Llysine have also been used to modify BP nanosheets via electrostatic and/or hydrophobic interaction for the preparation of delivery carrier and the immobilization of protein, respectively 16,17 . Inspired by these interaction mechanisms, this work designed a strategy to passivate the oxidation defects of BPQDs with Arg for enhancing the ECL performance.
Here, the ECL emission of BPQDs can be attributed to the radiative transitions of both 1 R * and 3 R * to S 0 species. Although the radiative T 1 -to-S 0 transition has been observed in the ECL emission of some organic small molecules with phosphorescence (PL) properties, such as benzophenone, platinum-based organometallics and tris(1-phenyl isoqsuinoline-C2, N)iridium (III) 18-20 , the ECL mechanism mediated by dual excited states has not been reported yet. More interestingly, the cathodic and anodic ECL emission upon Arg modi cation of BPQDs could increase dramatically by 25 and 2 folds, respectively, which resulted from the change of the highest occupied molecular orbital (HOMO) from the surface oxidation defects to the central zone of R-BPQDs. This change caused greater spatial overlap of the HOMO with the lowest unoccupied molecular orbital (LUMO), as proved by time-dependent density functional theory (TD-DFT) calculations, and the adjustable transition routes from S 1 and T 1 to S 0 . Thus Arg modi cation could be used to e ciently modulate the excited states for enhancing the ECL emission. Due to the improved stability of anion radical of BPQDs, the cathodic ECL showed greater improvement of performance than anodic ECL. More importantly, the Arg functionalization can improve the solubility and modi ability of BPQDs for extending the bioanalytical applications. As an example, an ECL system was proposed for conveniently evaluating the integrin inhibitor by using RRGDS-BPQDs as signal tag to recognize cell surface integrin. This work demonstrated a novel ECL mechanism and an exciting avenue to modulate the excited states for enhancing the ECL emission of luminophores.

Results
Synthesis and characterization of BPQDs and R-BPQDs. BPQDs were prepared with a solvothermal method 5,6 , and then functionalized with Arg to obtain R-BPQDs (Fig. 1a). From the transmission electron microscopic (TEM) images, their average lateral sizes were measured to be about 4 nm (Figs. 1b,c). The lattice fringes of 0.23 nm could be ascribed to the (041) plane of the BP crystal (Inset in Fig. 1b) 21 . The  24,25 . The Arg modi cation also led to a slight red-shift of three prominent Raman peaks of BPQDs (Fig. 1e), identi ed as one out-of-plane phonon mode (A 1 g ) at 360.1 cm -1 and two in-plane modes (B 2 g and A 2 g ) at 435.5 and 462.7 cm -1 5,6 , which manifested a decrease of the Raman scattering energy due to the interaction between guanidine group and P x O y moiety 26 . The R-BPQDs retained the characteristic P2p 3/2 and P2p 1/2 X-ray photoelectron spectroscopic (XPS) peaks of BP at 128.6 and 129.3 eV ( Fig. 1f and Supplementary Figs. 2a,b), while BPQDs did not show these peaks, demonstrating that the introduction of Arg endowed BPQDs with better stability. The shifts of P−O and P=O XPS peaks from 132.6 and 133.6 eV of BP to 134.8 and 135.9 eV of R-BPQDs could be attributed to the decrease of the outer valence electron density of P atom in the presence of guanidine group ( Fig. 1f and Supplementary Fig. 2b), which did not obviously change the N1s XPS peak due to the delocalized positive charge distribution over the guanidine group Photophysical and electrochemical properties of BPQDs and R-BPQDs. The cathodic process of BPQDs/GCE showed an ambiguous reduction peak of BPQDs at around −1.22 V, which shifted to −1.15 V and became more distinct after introducing the electron-withdrawing guanidine group by Arg modi cation (Fig. 2a). In the presence of K 2 S 2 O 8 , R-BPQDs/GCE showed a cathodic ECL emission at −1.20 V, which was 25 folds stronger than that of BPQDs/GCE (Fig. 2b). The ECL emission followed a general K 2 S 2 O 8 mediated co-reactant ECL mechanism containing the reduction of K 2 S 2 O 8 and BPQDs or R-BPQDs to produce the excited state BPQDs * or R-BPQDs * (Inset in Fig. 2b). The reduction peak of K 2 S 2 O 8 at bare GCE occurred at −0.95 V, and almost disappeared at BPQDs/GCE due to the greatly increased electron transfer impedance (Re) ( Supplementary Fig. 3). The much lower Re than BPQDs/GCE led to obvious reduction peak of K 2 S 2 O 8 at R-BPQDs/GCE, which overlapped with the reduction peak of R-BPQDs ( Fig. 2a). The consistence of onset potentials for electrochemical reduction and cathodic ECL emission of R-BPQDs indicated that the electrogeneration of R-BPQDs •− was necessary for the formation of excited state R-BPQDs *27 . Interestingly, the ECL depended on both Arg concentration and pH for preparation of R-BPQDs ( Supplementary Fig. 4), implying that the amount of Arg assembled on BPQDs affected the ECL performance, though Arg did not participate in the ECL process ( Supplementary Fig. 5).
The FL and PL emission of BPQDs centering at 505 and 580 nm (Fig. 2c) originated from the oxidation defects associated S 1 and T 1 , respectively 8,15 , which was demonstrated by the photoluminescence decay spectrum 15 (PDS) (Supplementary Fig. 6). The PDS of both BPQDs and R-BPQDs showed biexponential functions with lifetime components of 3.08 and 12.27 μs and 1.82 and 13.14 μs, respectively(Supplementary Table 1), indicating the presence of two decay channels assigned to the two excited states for FL and PL emissions, respectively 28 . R-BPQDs displayed 45-nm and 20-nm blue shift and the increased FL and PL emission intensity, respectively. The hypochromic shift and intensity enhancement were related to the change of the two excited states due to the passivation of oxidation defects by Arg. Excitingly, the cathodic ECL spectra of both BPQDs and R-BPQDs displayed two emission peaks at 500 and 580 nm for BPQDs, and 460 and 570 nm for R-BPQDs (Fig. 2d). Compared with the FL and PL spectra of BPQDs and R-BPQDs (Fig. 2c), the ECL emission peaks at 500 and 460 nm could be attributed to the transition from S 1 , while the peaks at 580 and 570 nm originated from T 1 -to-S 0 transition. Thus it could be concluded that ECL emissions of BPQDs and R-BPQDs contained two radiative transitions from both S 1 and T 1 , as shown in Fig. 2e. After Arg modi cation, the hypochromic shifts of both S 1 -to-S 0 and T 1 -to-S 0 transitions were observed in the ECL spectra, which was consistent with larger experimental band gap of R-BPQDs than BPQDs (Supplementary Fig. 7). However, their lifetimes did not obviously change at temperatures from 170 to 310 K (Supplementary Fig. 8 and Table   2), so the possibility of thermally activated delayed uorescence process could be excluded 4,29 , further indicating the existence of two radiative transitions from both S 1 and T 1 for ECL emission.
According to the electrostatic and hydrogen bond interactions between Arg and oxidation defects of BPQDs, TD-DFT computation was implemented to rationalize above conclusion. The existence of oxidation defects resulted in the localized HOMO of BPQDs at the defect sites (Fig. 2f), which hindered the charge transfer as a trap state, and thus weakened the FL, PL and ECL intensity. The Arg modi cation passivated the surface oxidation defects, accordingly leading to the delocalization of HOMO of R-BPQDs to the central zone (Fig. 2f), and thus the change of the electron transition channel, which signi cantly improved the emission oscillator strength and the charge transfer capability (Supplementary Fig. 3).
Besides, R-BPQDs exhibited the strongest ECL emission and the most positive reduction potential among the BPQDs modi ed with 20 kinds of amino acids (Supplementary Table 3 The BPQDs/GCE showed two anodic peaks at +0.85 and +1.45 V (Fig. 3a), which were attributed to the electrochemical oxidation of surface groups such as phosphite and hypophosphoric groups 25,32 . These peaks negatively shifted to +0.42 and +0.86 V after Arg modi cation (Fig. 3a) due to the much lower Re ( Supplementary Fig. 3), which decreased the oxidation overpotentials. Although the oxidation of Arg could be observed at bare GCE at +1.62 V (Supplementary Fig. 9), it did not occur at R-BPQDs/GCE in the applied potential range due to the relative higher Re. Considering the low oxidation potential of R-BPQDs,  (Fig. 3c). The anodic ECL peak potential and intensity of R-BPQDs were 0.15 V lower and 2 times higher than those of BPQDs, which could be attributed to the better charge transfer capability, the greater spatial overlap between HOMO and LUMO, and the better stability under ambient conditions ( Supplementary Fig. 10) after Arg modi cation. Similar to the cathodic ECL process, the anodic ECL spectra of BPQDs and R-BPQDs also displayed two emission peaks associating S 1 and T 1 transitions, along with the hypochromic shifts ( Supplementary Fig. 11). Thus the co-reactant ECL mechanisms of R-BPQDs at the cathode and the anode could be illustrated in Fig. 3d.
ECL transient technology was further used to examine the stability of radical intermediates in two ECL processes of R-BPQDs. The ion annihilation ECL intensity at +1.60 V was stronger than that at −1.40 V in the absence of coreactant (Fig. 3e), indicating that the anion radical R-BPQDs •− was more stable than cation radical R-BPQDs •+35 . Thus Arg stabilized the anion radical of BPQDs under ambient conditions, and thus led to the greater enhancement of cathodic ECL intensity than the anodic process.
Evaluation of integrin inhibitor with RRGDS-BPQDs. To implement the application of R-BPQDs/K 2 S 2 O 8 ECL system, this work designed an ECL method to evaluate the inhibiting e ciency of integrin inhibitor, cyclo(RGDyK) 36 , by using Arg containing peptide RRGDS to modify BPQDs (RRGDS-BPQDs). The RRGDS-BPQDs functionalized carboxylated multi-wall carbon nanotubes (MWNTs) were coated on GCE to act as both the recognition unit and signal tag 37 . Compared to Arg-free peptide GGGDS, the presence of RRGDS could greatly enhance the ECL intensity (Fig. 4a), verifying the vital importance of Arg for improving the ECL emission of BPQDs. Upon the speci c recognition of αV/β3 integrin on A549 cell membrane with RRGDS on GCE, the Re increased greatly ( Supplementary Fig. 12), and thus the ECL intensity decreased obviously ( Supplementary Fig. 13). In contrary, MCF-7 cells with low abundance of surface αV/β3 integrin showed little decrease. Under optimal conditions (Supplementary Figs. 14,15), the IC50 of cyclo(RGDyK) for 1 ´ 10 6 A549 cells mL -1 was obtained to be 12.0 nM from the ECL response plot of cyclo(RGDyK) treated A549 cells (Fig. 4b), which was comparable to 20 nM for immobilized αV/β3 integrin 36 and 29.3 nM for B16-F10 cells 38 . Compared to general MTT method, this method was sensitive, simple and convenient. Moreover, this method could be extended for the evaluation of other inhibitors or the detection of cell surface groups by changing the Arg-containing peptide, showing the excellent practicability of the designed ECL modulating strategy.

Discussion
The BPQDs showed the lattice fringes ascribed to the (041) plane of the BP crystal with 2-4 layers 5,6 , and could be conveniently modi ed with Arg via the electrostatic and hydrogen bond interactions (Fig. 1d).
The interactions led to decreased Raman scattering energy 26 and outer valence electron density of P atom (Fig. 1f). The presence of Arg on BPQDs could passivate the oxidation defects of BPQDs (Fig. 1g), and thus endowed BPQDs with better stability, solubility and modi ability for extending the bioanalysis application.
The ECL emission of BPQDs showed a novel mechanism containing two radiative transitions from both S1 and T1 to S 0 (Fig. 2e), which were demonstrated by the ECL spectra with two emission peaks (Fig. 2d) as observed from the FL and PL emission of BPQDs (Fig. 2c). Thus a dual excited states mediated ECL emitter was found for the rst time. The modi cation of Arg led to the hypochromic shifts of both S 1 -to-S 0 and T 1 -to-S 0 transitions due to its passivation against the surface oxidation defects (Fig. 2d) to produce the delocalization of HOMO of R-BPQDs to the central zone, which changed the electron transition channel and was demonstrated by TD-DFT computation (Fig. 2f). By comparing the ECL change upon amino acid modi cation (Supplementary Table 3), it was concluded that the ECL enhancement of R-BPQDs was attributed to the presence of electron-withdrawing guanidine group, which could stabilize the adjacent R-BPQDs•− anion radical after electrochemically injecting electron into LUMO of R-BPQDs.
The Arg modi cation could be used to modulate the dual excited states mediated ECL emission due to the change of charge transfer capability and the spatial overlap between HOMO and LUMO, which led to enhanced ECL emission of BPQDs. Moreover, the enhancement of cathodic ECL emission was much greater than anodic ECL emission. This appearance was ascribed to the more stable anion radical R-BPQDs •− than cation radical R-BPQDs •+ (Fig. 3e) 35 .
To demonstrate the application of R-BPQDs in ECL bioanalysis, the Arg attached on BPQDs was used to conjugate RGDS, a peptide speci c to integrin 36 . By coating RRGDS-BPQDs on MWNTs modi ed GCE, the integrin-rich cells could be bound to the electrode surface via the recognition of RGDS to integrin 39 , which led to a sensitive ECL method for the evaluation of integrin inhibitor. The obtained inhibiting e ciency to integrin on A549 cells demonstrated the practicability of the ECL of BPQDs and the modulating strategy.
In summary, both S 1 -to-S 0 and T 1 -to-S 0 radiative transitions have been found in both cathodic and anodic ECL emission of BPQDs. Arg modi cation e ciently passivates the oxidation defects of BPQDs and changes the HOMO from surface defects to the central zone, thus leading to hypochromic shifts and intensity enhancement of ECL emission. The introduction of Arg changes the electron transition channel, and endows BPQDs with better stability, stronger charge transfer capability and greater spatial overlap between HOMO and LUMO. The presence of electron-withdrawing guanidine group greatly stabilizes the anion radical intermediates, and thus leads to greater enhancement of the cathodic ECL intensity. The proposed modulating strategy can be conveniently applied in biosensing by using different Argcontaining recognition units to modify BPQDs, which has been demonstrated by using RRGDS-BPQDs to evaluate the inhibiting e ciency of cyclo(RGDyK) to cell surface integrin. The discovery of dual excitedstates mediated ECL mechanism and the modulation strategy via Arg modi cation open a new avenue to decipher more ECL systems for broadening the ECL applications of nanoemitters.  Calculation method. All calculations were carried out using the Amsterdam Density Functional program package (ADF) 40 . The time-dependent density functional theory (TD-DFT) and DFT calculations were performed by applying the Perdew-Burke-Ernzerhof (PBE) 41 exchange-correlation functional with the triple-zata plus polarization (TZP) basis set 42 . The ground state con guration, S 0 , of the oxidized BPQDs and R-BPQDs, was rst optimized by DFT calculation. To simulate the emission, the con gurations of the lowest singlet state, S 1 , and the lowest triplet state, T 1 , were relaxed using TD-DFT starting from the ground state con guration. We computed the vertical electronic transitions from the excited state relaxed con gurations S 1 and T 1 to the ground-state, yielding the uorescence and phosphorescence, respectively. The Van der Waals interaction was taken into account by the semi-empirical D3 method proposed by Grimme et al 43 . All optimizations were done without any symmetry constraint.

Data availability
The data that support the ndings of this study are available within the article and supplementary information les, or from the corresponding author upon request.

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