Two-Photon–NIR-II Antimicrobial Graphene-Nanoagent for Ultraviolet–NIR Imaging and Photoinactivation

Nitrogen doping and amino-group functionalization, which result in strong electron donation, can be achieved through chemical modication. Large π-conjugated systems of graphene quantum dot (GQD)-based materials acting as electron donors can be chemically manipulated with low two-photon excitation energy in a short photoexcitation time for improving the charge transfer eciency of sorted nitrogen-doped amino acid–functionalized GQDs (sorted amino-N-GQDs). In this study, a self-developed femtosecond Ti-sapphire laser optical system (222.7 nJ pixel −1 with 100-170 scans, approximately 0.65-1.11 s of total effective exposure times; excitation wavelength: 960 nm in the near-infrared II region) was used for chemical modication. The sorted amino-N-GQDs exhibited enhanced two-photon absorption, post-two-photon excitation stability, two-photon excitation cross-section, and two-photon luminescence through the radiative pathway. The lifetime and quantum yield of the sorted amino-N-GQDs decreased and increased, respectively. Furthermore, the sorted amino-N-GQDs exhibited excitation-wavelength-independent photoluminescence in the near-infrared region and generated reactive oxygen species after two-photon excitation. An increase in the size of the sorted amino-N-GQDs boosted photochemical and electrochemical ecacy and resulted in high photoluminescence quantum yield and highly ecient two-photon photodynamic therapy. sorted two-photon contrast probes localizing two-photon conducting therapy for eliminating


Background
Graphene quantum dot (GQD)-based materials with π-π con guration and surface groups exhibit a high surface area, large diameter, and excellent surface grafting. These materials may contribute to intrinsicstate and defect-state emissions to achieve photoluminescence (PL). Intrinsic-state emissions are induced by the quantum size effect, zigzag edge sites, or recombination of localized electron-hole pairs, whereas defect-state emission originates from the defect effect (energy traps) [1,2]. The PL emission of a material determines its suitability for imaging and photochemistry [3]. GQDs can be bonded with nitrogen atoms (N-GQDs) to alter their chemical composition and modulate their band gap, thereby enhancing photochemical properties and facilitating tunable luminescence in bioimaging and photodynamic (or photoinactivation) applications [4,5]. Furthermore, primary amine molecules (also known as amino-group functionalization) can be chemically modi ed to realize strong electron donation for considerably altering the electronic properties of nitrogen-doped amino acid-functionalized GQD (amino-N-GQDs) materials, thereby augmenting electrochemical and photochemical activities [6].
An effective approach can be realized for investigating photoexcitation by combining multiphoton and near-infrared (NIR) excitations. This approach has a lower absorption and shorter photoexcitation period than other types of excitations. Furthermore, this approach exhibits ultralow energy consumption.
Because of these attributes, deep penetration of biological specimens and effective observation are possible. This study used a novel femtosecond Ti-sapphire laser optically inverted microscopy system (a repetition rate of 80 MHz; Mai Tai with the optical parametric oscillators; Spectra-Physics, USA; Scheme 1). Moreover, given the possible excitation-wavelength-dependent PL phenomenon caused by the derived amino-N-GQDs featuring quantum con nement in the sp 2 domains and intrinsic-state and defectstate emissions, this study revealed that sorted amino-N-GQDs sieved by membranes with the pores of various sizes bearing homogeneous atom dopant functionalities enabled investigation of the electronic and intrinsic properties related to the optical behavior with the quantum-con nement effect [7]. This phenomenon resulted in excitation-wavelength-independent two-photon luminescence (EWI-TPL) emission under the two-photon excitation (TPE) wavelength extending to 960 nm in the NIR-II region [8,9]. X-ray photoelectron spectroscopy (XPS) revealed that with the enlargement of sorted amino-N-GQDs, the numbers of the C-N group, pyridinic-, amino-, and pyrrolic-N functionalities increased. This increase can induce a radiative recombination of localized electron-hole pairs, resulting in conspicuous two-photon properties, including favorable two-photon absorption (TPA), high TPL emission, excellent absolute TPE cross-sections, short lifetime, high ratio of radiative-to-nonradiative decay rates, and high post-TPE stability. Moreover, an increase in the mean lateral size results in a high PL quantum yield (QY) and highly e cient photodynamic therapy (PDT, or photoinactivation) action under TPE (Ex: 960 nm, an ultralow energy of 222.7 nJ pixel − 1 with the short photoexcitation period of 100-170 scans, total effective exposure times of approximately 0.65-1.11 s). The current results indicated that sorted amino-N-GQDs can function as a promising two-photon contrast probe in tracking and localizing analytes with in-depth two-photon imaging of a biological environment on two-photon-PDT to eliminate infectious microbes easily.

Preparation of amino-N-GQDs and sorted amino-N-GQDs
Amino-N-GQDs: graphene oxide was prepared from a natural graphite powder (Bay carbon Inc., USA) using a modi ed Hummers' method [10]. Graphite (8.5 M) and NaNO 3  O were performed several times, and the graphene oxide was collected. The as-prepared graphene oxide was placed in a tube furnace and heated to 400-600°C in the presence of NH 3(g) for 4-6 h; it was subsequently introduced to concentrated HNO 3 (16.0 M; Sigma Aldrich Co., USA) and stirred for 2 days. The mixture was placed in a sonicator for 2 days and subsequently placed in an oven at 160°C for 1 day to vaporize all the liquid. Washing and centrifugation (83000 rpm; Optima TLX Ultracentrifuge, Optima TLX Ultracentrifuge, Beckman Coulter Inc., USA) with ddH 2 O were conducted several times. The supernatant was ltered through a 0.22 µm microporous membrane. The pH of the resulting black suspension was tuned to 7.4 with NaOH (1M; Sigma Aldrich Co., USA), and it was stayed in a dialysis bag (retained molecular weight: 300 kDa) > 12 h, and N-GQDs were obtained. The as-prepared N-GQDs were mixed with NH 3(aq) (28 %; Sigma Aldrich Co., USA), stored in a Te on-lined stainless steel autoclave, and reacted at 180°C for 5 h. The resulting mixture was washed with ddH 2 O, centrifuged several times, and subsequently dried in an oven at 50°C overnight. Eventually, amino-N-GQDs were obtained (Additional le, Scheme S1a).
All Materials section for this article can be found in the Additional le.
Recombination of zigzag edge sites, localized electron-hole pairs, and quantum effect are used to induce intrinsic-state emission of GQD-based materials, whereas the defect effect (energy traps) triggers defectstate emission [2,5,6]. To demonstrate the effect, Fig. 1a displays sorted amino-N-GQDs dispersions, various levels of PL emission (gray-level images), dots with slight variation in sizes, and wavelengths encompassing the NIR-I window at 630 nm. The laser system's x-y focal point and z-axis resolution (full width at half maximum, FWHM) were set at approximately 0.45 and 0.90 µm, respectively (Fig. 1b). Satisfactory TPA in the NIR-II window was measured using a self-developed femtosecond Ti-sapphire laser optical system, as displayed in Scheme 1; for details of the system, please refer to the Materials section), with an approximately 960 nm extension in subsequent experiments (Fig. 1c). With the application of the most e cient excitation wavelength, the materials can considerably advance relevant two-photon properties applied in bioimaging with TPE [11]. Figure 2a displays the TPL spectra of sorted amino-N-GQDs, with peaks of amino-N-GQD 9.1, amino-N-GQD 9.9, amino-N-GQD 11.1, and amino-N-GQD 12.0 at approximately 719, 772, 810, and 862 nm in the NIR region under TPE (222.7 nJ pixel − 1 with 20 or 170 scans, approximately 0.13 or 1.11 s of total effective exposure times; Ex: 960 nm). XPS revealed that as the number of carbonyl groups increased (Additional le, Fig. S3), larger electron redistribution appeared, which eventually decreased energy gaps and TPL red shifts [12]. Given that the quadratic dependence of TPL intensi es with the TPE power in the process [13], Fig. 2b con rms the existence of a two-photon process with exponent 2.00 ± 0.02 for sorted dots and conventional uorophore (e.g., Rhodamine B and Fluorescein; Fig. 2b).

Determination of EWI-TPL phenomenon
The sorted amino-N-GQDs with homogeneous O and N functionalities can be used for investigating intrinsic electronic properties related to optical behavior with quantum con nement, leading to EWI-TPL under TPE. Furthermore, sorted amino-N-GQDs exhibited two-photon stability, which could be attributed to limited photobleaching because of dots' post-TPE TPL intensity (Fig. 2c), whereas that of Rhodamine B and Fluorescein's uorescence demonstrated poor robustness against photobleaching on TPE exposure (222.7 nJ pixel − 1 with 20, 100, or 170 scans, approximately 0.13, 0.65, or 1.11 s of total effective exposure times). Furthermore, ultraviolet photoelectron spectroscopy revealed that n-state levels were xed at almost the same energetic positions (6.6-6.8 eV; Additional le, Fig. S6), irrespective of the size determined through TEM and Raman spectroscopy (Additional le, Figs. S2 and S5), which con rmed the highest occupied orbital level of sorted dots. The quantum con nement resulting from the particle size regulated the wavelengths of radiative transitions. Furthermore, EWI-TPL emissions from sorted amino-N-GQDs implied the absence of trap states between the n-state and π* energy levels. A change in the particle size did not cause any disturbance in the n-state level. The EWI-TPL of the sorted dots could be attributed to π*→n recombination triggering electron transition and phonon scattering. Measurements revealed that the absolute uorescence QY [14] of the materials ranged from approximately 0.39 (for amino-N-GQD 9.1) to 0.48 (for amino-N-GQD 12.0). Desirable yields were achieved because of the electron-donating species of the sorted amino-N-GQDs structure. XPS revealed that the high percentage of C-N con gurations functioned as electron-donation species and improved QY through the inhibition of nonradiative transitions (Additional le, Fig. S3). By contrast, low QY was due to the presence of a large amount of electron-withdrawing carbonyl functional groups acting as nonradiative trap centers (Additional le, Fig. S3). Characterization of sorted amino-N-GQDs revealed successful preparation causes the GQDs to exhibit EWI-TPL characteristics. However, a large cross-section is preferred in the monitoring of molecular actions. Sorted amino-N-GQDs exhibited large absolute TPE cross-section ranging approximately from 55946 to 60728 Goeppert-Mayer (GM; with 1 GM = 10 − 50 cm 4 s photon − 1 ), which was more than 2900 times the magnitude of the Fluorescein (~ 19.2 GM; Table 1). The absolute TPE cross section for amino-N-GQD 9.1, amino-N-GQD 9.9, amino-N-GQD11.1, and amino-N-GQD12.0 were approximately 55946, 57332, 59051, and 60728 GM, respectively (Table 2). For detailed calculation, refer to the Materials section. This difference indicates that a high ratio of the energy absorption to the energy input in biospecimens. This phenomenon is highly favorable in two-photon imaging (TPI) [15].
Observation of EM and TPI images during the process of two-photon PDT in the NIR-II window Because the self-developed optically inverted microscopy system is not suitable for investigating in vivo assay processes, the biological environment was mimicked by embedding the Escherichia coli (E. coli.   (Figs. 3g). However, the images captured at depth > 180 µm contained spherical aberrations, which severely degraded image quality. Such aberrations were caused by the mismatch between the refractive indexes of the aqueous sample and maximal z depth of the optical laser system, in addition to the in uence of the set objective, detection e ciency, and maximal z depth of the optical laser system used [17]. Therefore, TPI was not detected at a 200-µm depth for all the sorted dots (Fig. 4). In this study, the maximum z depth for the detection of TPL emissions with the speci c laser optical system was 180 µm because of the detection e ciency and set objective, which was, therefore, set as the optimal depth affording the best resolution for the examination of amino-N-GQDs used as a two-photon contrast probe, particularly for sorted amino-N-GQDs with large lateral size.
Changes in the bacterial cell walls or oxidation were detected. The deterioration of surrounding biological surface substrates was attributable to the reactive oxygen species (ROS), which were through PDT under TPE. These changes may cause bacterial atrophy, morphological damage, and distortion (inset images of Figs. 3c and d) due to amino-N-GQD desorption from the surface of the bacteria (Figs. 3c and d). The LIVE/DEAD kit [18] was used to investigate the green uorescence of living bacteria (the selected amino-N-GQD 12.0 was used to conduct this experiment; viability > 99%; Additional le, Fig. S8a).The results revealed that the bacteria were almost completely undamaged by exposure to laser treatment (222.7nJ  Figs. S9c and d). However, under TPE, sorted amino-N-GQDs displayed excellent bactericidal capability (approximately 89%, 93%, 98%, and 100% elimination for the amino-N-GQD 9.1-Ab LPS , amino-N-GQD 9.9-Ab LPS , amino-N-GQD 11.1-Ab LPS , and amino-N-GQD 12.0-Ab LPS , respectively), amounting to approximately 7.73-7.75 log 10 reduction (Additional le, Figs. S9c and d; corresponding to Fig. 3c). By contrast, bacterial viability was higher for materials without antibody coating (over 98% viability) than that for materials with the coating (Additional le, Figs. S9c and d). However, although antimicrobial capabilities were still not apparent (approximately 6%,8%, 9%, and 11% elimination for the amino-N-GQD 9.1-, amino-N-GQD 9.9-, amino-N-GQD 11.1-, and amino-N-GQD 12.0-without coating antibody, respectively) sorted dots exhibited 100% antimicrobial e cacy with the number of scans increasing to 170 for all the sorted dots-Ab LPS -treated E. coli under TPE (Additional le, Figs. S9e and f; corresponding to Fig. 3d). The results were attributed to sorted amino-N-GQDs functioning as two-photon photosensitizer (PS) to generate ROS that was involved in PDT action. The results also revealed the effectiveness of antibody coating in enhancing the functions of materials.

Effects of nitrogen dopant and amino functionalization
Amino-N-GQDs exhibited remarkable quantum con nement, and their edge effect could be altered to enhance their electrochemical, electrocatalytic, and photochemical activities [4,6]. The effect of strong electron donation and large π-conjugated system reportedly improves charge transfer e ciency in amino-N-GQDs [20], which resulted in favorable TPA, post-TPE stability, TPE cross-sections, and TPL, as well as a higher ratio radiative and nonradiative decay rates (amino-N-GQD 9.1: 0.64; amino-N-GQD 9.9: 0.69; amino-N-GQD 11.1: 0.82; amino-N-GQD 12.0: 0.92; for calculation, refer the Materials section; Table 2). The results indicate that the material passed mainly through the radiative pathway, as uorescence QY increased (amino-N-GQD 9.1: 0.39; amino-N-GQD 9.9: 0.41; amino-N-GQD 11.1: 0.45; and amino-N-GQD 12.0: 0.48) and lifetime decreased (from 1.13 to 0.93 ns; Fig. 6, Tables 2 and 3). Radiative electron-hole pair recombination was induced by N dopants and amino groups on the surface of GQD-based material, which boosted intrinsic-state emission. However, as suggested previously, for N dopants and amino groups, the maximum occupied molecular orbital energy of graphene akes can be increased with the presence of edge amine groups [21]. Thus, the narrowing of orbital band gap boosting PL QY may be caused by resonance between delocalized π orbital and the primary amine's molecular orbital. Furthermore, XPS revealed that C-O, C = O, and amide groups, which induced localized electron-hole pairs' nonradiative recombination and prevented intrinsic-state emission [22], were favorable for small materials (Additional le, Figs. S3 and S4). As the particle size increased, so did PL QY. Moreover, chemical modi cations strongly affected the electronic properties of amino-N-GQDs, enabling strong electron donation in primary amine molecules known as amino-group functionalization. Singlet-triplet splitting of amino-N-GQDs results in intersystem crossing and a high triplet state yield. The e ciency of this splitting is su cient for it to compete with the internal conversion between multiplicity-identical states, which results in the creation of ROS for involvement in PDT [4,20]. Furthermore, as the number of edge sites increased, the amount of C-N, pyridinic-, amino-, and pyrrolic-N groups increased (Additional le, Figs. S3 and S4). Similarly, as the size of the amino-N-GQDs (Additional le, Figs. S3 and S4) enlarged (Additional le, Figs. S2 and S5), their antibacterial ability increased and so did the amount of generated ROS (Fig. 5; Additional le, Tables S2-S7), which lead to highly e cient PDT action.

Conclusions
In this study, given the high edge and quantum-con nement effect of GQDs, their intrinsic properties are typically modi ed through nitrogen functionalization and doping. After TPE, sorted amino-N-GQDs with high crystallinity and uniformity exhibited EWI-TPL in the NIR region as well as favorable electrochemical and photochemical activities. An increase in the mean lateral size, with the increased numbers of the C-N group and enhanced pyridinic-, amino-, and pyrrolic-nitrogen functionalities, induced the radiative recombination of localized electron-hole pairs, leading to favorable TPA and luminescence, absolute cross-section, QY, radiative decay rate, stability and decrease of lifetime. The amounts of ROS generated by and associated with two-photon PDT enhanced the antimicrobial e cacy of a self-developed femtosecond Ti-sapphire laser optical system with low TPE energy and short photoexcitation time     Table 3 The lifetime data and the parameter generated using a time-correlated single-photon counting technique involving a triple-exponential tting function while monitoring the emission under TPE.  Table 4 Stability of the newly prepared and as-prepared sorted-amino-N-GQD for 3 months in physiological environment of the culture medium for E. coli determined by Raman (Additional le, Equations S9-S10) and zeta potential spectroscopy.     Phosphorescence spectra of the sorted amino-N-GQDs (obtained at 1270 nm). Delivered dose: 0.75 μg mL−1 material.