Phosphopeptide-bridged NH2-TiO2-mediated carbon dots self-enhancing and electrochemiluminescence microsensors for label-free protein kinase A detection

Phosphopeptide-bridged NH2-TiO2-mediated carbon dots self-enhancing and electrochemiluminescence microsensors for label-free protein kinase A detection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Phosphopeptide-bridged NH2-TiO2-mediated carbon dots self-enhancing and electrochemiluminescence microsensors for label-free protein kinase A detection Jianping Guo, Lele Yue, Lingya Ning, Ailing Han, Junping Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4598795/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Sep, 2024 Read the published version in Microchimica Acta → Version 1 posted 10 You are reading this latest preprint version Abstract A novel electrochemiluminescence (ECL) method was developed for analyzing protein kinase A (PKA) ultra-sensitively based on amidated nano-titanium (NH 2 -TiO 2 ) embellished carbon dots (Mg@N-CDs) fluorescent probe, which integrated the target recognition and ECL-signal enhancement. The Cys-labeled kemptides were employed to build a serine-rich synthetic substrate-heptapeptide (Cys-kemptide) on the Au-electrode surface. Then, the PKA-induced biosensor was triggered as a signal switch to introduce the large amounts of TiO 2 decorated Mg@N-CDs nanohybrid (Ti@NMg-CDs) into AuE/Cys-phosphopeptides for signal output. In particular, the presence of PKA could induce the formation of Cys-phosphopeptides by the catalytic reaction between specific substrate (kemptide) and PKA, which could act as an initiator to link the Ti@NMg-CDs according to the bridge interactions Ti-O-P. By this way, multiple Cys-phosphopeptides were adsorbed onto a single Ti@NMg-CDs and the Ti@NMg-CDs not only provided the high specific selectivity but also large surface area, as well as unprecedented high ECL efficiency. Using this PKA-induced enhanced sensor, the limit-of-detection of the PKA was 4.89 × 10 − 4 U/mL (S/N = 3). The proposed ECL biosensor was also universally applicable for the screening of PKA inhibitors and the determining of other kinases activity. Our sensing-system has excellent performance of specificity and the screening of kinase inhibitors, as well as it will inspire future effort on clinical diagnostics and new drugs discovery. protein kinase activity Ti@NMg-CDs Cys-kemptide electrochemiluminescence method inhibitors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction For the important impact of kinases in human health[ 1 ] and drug development[ 2 ], protein kinase has been used as one of the major drug targets against human diseases. Under the catalysis of protein kinase, one or more γ-phosphate groups are transferred to specific amino acids (i.e., serine, tyrosine, and threonine residues) of target proteins, and this phosphorylation process is one of the most important post-translational modifications (PTM) [ 3 , 4 ]. Whereas, the overexpression and mutations of protein kinases and aberrant protein-phosphorylation states can result in cancer, heart dysfunctions and Alzheimer’s disease[ 5 ], and some other diseases, posing huge threats to human health. So, protein kinases are a group of especially significant targets for drug therapy. In this case, sensitive, accurate and widely applicable assays for monitoring protein kinase A (PKA) activity are beneficial to the development of protein kinase-related clinical diagnosis and kinase-targeted drug discovery, as well as for further understanding the signal transduction mechanism of molecular[ 6 ]. Simultaneously, the screening of inhibitors of protein kinases A are currently also important, and several protein kinase inhibitors have been FDA approved for targeted therapy. Real examples of kinase inhibitors (Pazopanib, Nilotinib, Sunitinib) provide a wealth of information about how tumors respond to such targeted therapy[ 7 ]. On the basis of above, it is also urgent that a fast pathway selectivity-based ECL-signal reading out the activity of different inhibitors is constructed. The conventional approaches for the detection of PKA activities are almost based on the expensive and harmful radioactive labels with 32 P[ 8 ]. In this regard, various alternative ways have been recently exploited for PKA analysis and inhibitor screening, such as colorimetric, quartz crystal microbalance (QCM), Raman Spectroscopy and fluorescent immuno-assay[ 9 – 10 ]. Although these designs are smart and antibody-antigen with high affinity have improved the selectivity, however, low accuracy due to false positive results, specialized instruments and tedious operations heavily hinder the real application. Recently, electrochemiluminescence (ECL), as a signal-emission process in a redox reaction of electrogenerated reactants, appears in scientific research. On the one hand, ECL technology is a potential analysis-method on account of its distinct merits lower background noise and cost, higher selectivity and sensitivity, as well as more convenient operation. In addition, ECL platform has also been increasingly employed in biomarkers detection based on the potential for rapid and sensitive diagnosis of human diseases[ 11 ]. Green nano materials have emerged as key components in most, if not all, optical and electrical sensing field. Unfortunately, the expensiveness, toxicity and hard to prepare limit the existing ECL luminophores[ 12 ] applications. Therefore, the synthesis of eco-friendly electro-chemiluminescent (ECL) nanomaterials becomes urgent needs to solve above key issues and relies greatly on advanced luminescent materials as well as the novel amplification technologies of the ECL-signal. In recent years, some carbon-based materials (CMs) and their complexes have been successfully used to sensing-fields because of their high flexibility, good electrical properties and the harmless to environment, e.g., carbon dots (CDs)[ 13 ], carbon nanotube (CNT)[ 14 ], and graphene[ 15 ]. Among them, carbon dots (CDs) are the most popularly used light-responsive nanomaterial material in sensing-fields due to the good biocompatibility, photocatalysis and high energy-conversion efficiency. Fixing CDs directly on Au-electrode (AuE) has problems such as unstable response-signal and low ECL-intensity. Some attempts [ 16 ] to enhance their ECL signal focus on co-reactive reagents (H 2 O 2 ) introduce or structure design. Studies have revealed that the cathodic ECL behavior of TiO 2 is comparable to that of H 2 O 2 [ 17 ]. Therefore, TiO 2 can act as co-reactants for CDs. In addition, TiO 2 -based nanomaterials with good biocompatibility and relative conductivity have been extensively certified to enhance ECL-performances cooperatively (such as TiO 2 @SnS 2 , TiO 2 -CNTs, ZnO/TiO 2 , CeO 2 @TiO 2 , etc.)[ 18 ]. However, only few researches focused on improving the ECL-properties of CDs, in which NH 2 -TiO 2 acted as a signal-carrier for CDs-luminor in biosensor. Semiconductor photocatalyst nano titanium oxide (TiO 2 ) with the large surface area is considered as a promising transducing material due to its high chemical stability, low cost, and non-toxicity[ 19 ]. On the other hand, its affinity toward phosphoric group via Ti-O-P coordination has gained enormous application in phosphoprotein-enrichment. In addition, the lower Lewis acidity and higher hydrophilicity of amino-functionalized TiO 2 (NH 2 -TiO 2 ) than TiO 2 show strong affinity with phosphoproteins, then modifying -NH 2 onto TiO 2 has gained enormous attraction[ 20 ]. Based on the prominent photocatalytic performance and strong specific recognition utility between NH 2 -TiO 2 and phosphate group, if or not a label-free ECL- biosensor may be developed to detect the activity of PKA? In light of these unique and outstanding properties of NH 2 -TiO 2 -doped CDs, herein, a robust ECL platform for the test of PKA activity was reported by using eco-friendly blue-emissive Ti@NMg-CDs. The Ti@NMg-CDs feature both one of the most protentional ECL-luminous known to date and as an ideal PKA-reporter and phosphopeptide-recognizing matrix. It was noteworthy that a single Mg@N-CDs could assemble multiple NH 2 -TiO 2 to form the single-donor/multiple-acceptor configuration with an improved ECL intensity. In this alternative ECL-biosensor, the synthesis of the fluorescent nanoprobes Ti@NMgCDs increases the stability of the CDs and intensity of the ECL-signal. The in-situ phosphorylated Cys-kemptide-sensor could efficiently anchor the Ti@NMgCDs by Ti-O-P matching, achieving highly sensitive detection of PKA. To our knowledge, this is a new report on in-situ construction of Cys-kemptide and Ti@NMgCDs ECL-sensor on Au-electrode for PKA tests. 2. Experimental 2.1. Reagents and chemicals All materials and related instruments used in the experiments have been listed in supporting information. 2.2. Fabrication of ECL-biosensor (A) Firstly, the Au electrode (AuE) was polished and ultrasonically washed by ethanol and doubly distilled water (DDW). Subsequently, the AuE dried by N 2 was activated with freshly prepared “piranha” solution of H 2 O 2 /H 2 SO 4 (1:3, v/v) for 5 min. (B) Cys-labeled kemptide (CLRRASLG) self-assembly on the AuE: the electrode was dipped into the 10 mmol/L PBS (pH 7.4) solution of 500 µmol/L CLRRASLG for overnight reaction at room temperature. On the surface of the AuE, an achievable serine-peptide chains for phosphorylation [ 21 ] were formed directly through the Au-S bond. (C) After rinsing with PBS, the unbound Au-sites were blocked completely by 1 mmol/L 6-mercaptohexanol (MCH) solution for 30 min. Afterward, the resulting AuE/Cys-kemptides/MCH was dipped in a desired amount of PKA&ATP-incubation PBS-buffer. In a 5 mL phosphorylation reaction system, sterile water, PBS buffer (10 mmol/L), ATP (100 µmol/L), a specific concentration of PKA (0.1 U/mL) were added in order into a 10 mL centrifuge tube; Subsequently, the resulting AuE/Cys-kemptides/MCH electrodes were was dipped in the above 10 mL centrifuge tube. The tube was placed in a pre-heated shaking bath for 60 min (37 ℃, 200 rpm). Above the phosphorylation reaction, in the presence of ATP and PKA, the Cys-labeled kemptide receptor (CLRRASLG) is rapidly phosphorylated on phosphorylation sites (the hydroxyl site of serine) that are located within the C-terminal domain of the receptor. Previous studies [ 22 ] suggested that serine presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. In particular, the presence of PKA could induce the formation of Cys-phosphopeptides by the catalytic reaction between specific substrate (kemptide) and PKA, which could act as an initiator to link the Ti@NMg-CDs according to the bridge interactions Ti-O-P. This could be attributed to the γ-phosphoryl of ATP will be transferred to the hydroxyl group of serine in the CLRRASLG under the catalysis of PKA [ 21 ]. After incubation with the Ti@NMg-CDs, the phosphorylated CLRRASLG-peptides would be rapidly captured on the Ti@NMg-CDs through the high binding affinity between the Ti 2+ ions of Ti@NMg-CDs and the phosphate group. (D) Finally, the electrode (AuE/Cys-phosphopeptides/MCH) obtained above was incubated in Ti@NMg-CDs-solution for 2 h at room temperature, then thoroughly rinsing with water to remove nonspecific Ti@NMg-CDs and drying with nitrogen. The detailed construction process was displayed in Scheme 1 . 3. Results and discussion 3.1. Characterizations of Ti@NMg-CDs nanoprobes Mg@N-CDs, NH 2 -TiO 2 and Ti@NMg-CDs nanocomposites exhibited different morphological characteristics. The TEM observations indicated that the Ti@NMg-CDs was more uniform and denser with nano NH 2 -TiO 2 (Fig. 1 C) nano-crystal doped into carbon dots (Fig. 1 B). After the modification processes of NH 2 -TiO 2 , patches of nano-crystaled particles were observed on the nanocomposites (Fig. 1 A). The TEM images of the Ti@NMg-CDs (Fig. 1 A) nanocomposites also confirmed the presence of NH 2 -TiO 2 nano-crystal compared with only the Mg@N-CDs. Moreover, Fig. 1 D clearly showed well-resolved lattice fringes (0.33 nm) structure of Ti@NMg-CDs nanocomposites, which were assigned to the (021) plane[ 23 ]. And photocatalytic NH 2 -TiO 2 improved the ECL-performance of the Ti@NMg-CDs [ 24 ]. The Ti@NMg-CDs as the substrate provided larger surface areas, which were beneficial for facilely adsorpting the multiple Cys-phosphopeptides. Energy dispersive spectrometer (EDS) spectra were employed to reveal the presence of different components in Ti@N Mg -CDs. As shown in Fig. 1 E, C, N and O atoms occupy most of element mass. Small amounts of Mg atoms coexist with dominant Ti atoms. N and O is derived from the NH 2 -TiO 2 and Mg@N-CDs. Ti and Mg, C only come from the NH 2 -TiO 2 , Mg@N-CDs, respectively. The EDS analysis results further reveal the synthesis of Ti@NMg -CDs successfully. ECL stabilities were studied by measuring and comparing ECL intensity (Fig. S4). With the extending of storage time from 0 to 10 d, the ECL intensity of Mg@N-CDs/AuE dramatically decreases to 206 a.u. (initial value is 3897 a.u.), while that of Ti@NMg -CDs/AuE has negligible changes (more than 0.93). These experimental results suggest that the film-forming/photocatalytic NH 2 -TiO 2 made considerable progress in ECL efficiency and stability of Ti@NMg-CDs/AuE [ 25 ]. The bind of Ti@NMg-CDs on AuE/Cys-phosphopeptide, only nano NH 2 -TiO 2 and Mg@N-CDs were also probed by X-ray diffraction (XRD) (Fig. 2 A). The XRD patterns of the Ti@NMg-CDs and Mg@N-CDs nanocomposites exhibited different characteristics. The characteristic diffraction peak observed in the Ti@NMg-CDs was ascribed to the mixture crystalline of NH 2 -TiO 2 (004) and Mg@N-CDs (002)[ 26 ], whereas the intensive diffraction appearing at 25.5° was similar and the peak intensity increased obviously, suggesting the pre-synthesized Ti@NMgCDs-probe tended to be well crystallized. As a whole, the typical diffraction peaks of both NH 2 -TiO 2 and Mg@N-CDs were observed from the Ti@NMg-CDs, indicating the formation of the Ti@NMg-CDs fluorescent-probe. Fourier transform infrared (FT-IR) spectra was measured to investigate the functional groups of Ti@NMg-CDs with Mg@N-CDs as a contrast. Figure 2 B showed the FT-IR spectras of Mg@N-CDs (curve a), NH 2 -TiO 2 (curve b), and Ti@NMg-CDs (curve c). The O-H at 3400 cm − 1 , 2972 cm − 1 and C-O at 1087 cm − 1 could be observed in the FT-IR spectra, further indicating the strong hydrophilic effect of the nanoparticles. FT-IR absorption bands at 2889 cm − 1 were assigned to the N-H stretching vibrations. The specific bands at 881 cm − 1 were assigned to the C-C vibrations of the benzene-ring structures as well. Ti@NMg-CDs displayed almost the same functional groups with Mg@N-CDs. In spite of the above similarities, the presence of specific bands (Ti-O) at 725 cm − 1 illustrated the successful synthesis of Ti@NMg-CDs doping with TiO 2 . Zeta potentials were also measured to prove the generation Ti@NMg-CDs. Figure 2 C presented the zeta potential changes of nano Mg@N-CDs, NH 2 -TiO 2 and Ti@NMg-CDs (pH = 7.4), respectively. The as-synthesized Mg@N-CDs displayed negative potential (ζ = -11.1mV), and the potential change to positive after the modification of NH 2 -TiO 2 (ζ = + 27.0 mV). After the formation of the Ti@NMg-CDs fluorescent nano-probe (ζ = + 20.6 mV), the charges increased owing the conjugation of positively charged NH 2 -TiO 2 (ζ = + 27.0 mV). The zeta potential results shown above demonstrated the synthesis of Ti@NMg-CDs-probe successfully. 3.2. ECL behaviors of the Ti@NMg-CDs nanoprobes To estimate the ECL behaviors of Ti@NMg-CDs fluorescent probe, different AuE were assembled and characterized by ECL intensity-potential during a potential scan cycle − 1.8 ~ 0 V, including ECL intensity-potential curves of bare-AuE (curve a), AuE/N@Mg-CDs (curve c), AuE/NH 2 -TiO 2 (curve d), AuE/Ti@NMg-CDs (curve e) in PBS-K 2 S 2 O 8 system and AuE/Mg@N-CDs (curve b) in PBS without K 2 S 2 O 8 . As shown in Fig. 2 D, curve a had a weak ECL signal. What’s more, the ECL signal of N@Mg-CDs/AuE was in an "on" state, however, the signal value disappeared in PBS without K 2 S 2 O 8 (curve b). As we all know, oxidation intermediate (SO 4 · − ) can be produced by S 2 O 8 2− under power, which accelerated the formation the excited state NTi@MgCDs*[ 27 ]. Interestingly, when NH 2 -TiO 2 nano-film and Ti@NMg-CDs was separately decorated on AuE, a sharper ECL signal (4501 a.u.) was given on AuE/Ti@NMg-CDs (curve e), which was about 9.2 and 4.3 times than that on AuE/NH 2 -TiO 2 (curve d), AuE/Mg@N-CDs (curve b). Above results implied that the ECL intensity of Mg@N-CDs was significantly enhanced, which could be ascribed to the excellent photocatalytic performance of nano-TiO 2 . In addition, the 3D ECL spectra was carried out to further confirm that the NH 2 -TiO 2 could act as ECL-signal accelerator for Ti@NMg-CDs hybrid. As shown in Fig. 2 E, the ECL spectrum of Mg@N-CDs realized maximum about 791 a.u. at 520 nm. When the AuE/Ti@NMg-CDs was immersed in PBS containing coreactant K 2 S 2 O 8 , the maximum ECL emission wavelength had no significant change (Fig. 2 F), demonstrating that the probe Mg@N-CDs and the aminated nano titanium dioxide could enhance the ECL-intensity synergistically in K 2 S 2 O 8 -PBS. 3.3. Electrochemical characterization Cyclic voltammetry (CV) behaviors of four different Au-electrodes (bare-AuE, NH 2 -TiO 2 /AuE, Mg@N-CDs/AuE, Ti@NMg-CDs/AuE) were studied in PBS (0.2 mol/L, pH = 7.4) containing 0.1 mol/L K 2 S 2 O 8 upon − 1.2 ~ 0 V. On the surface of bare-AuE, reduction peak was detected (curve a) obviously at − 0.97 V in Fig. 3A, which results from the formation of strong oxidation intermediate (SO 4 · − ) at a certain voltage. In addition, a weak negative cathodic peak (-0.89 V) was observed on the NH 2 -TiO 2 -modified AuE (curve b) in K 2 S 2 O 8 -PBS, which was attributed to the conductivity and narrow energy gap of semiconductor nano-titania. Compared with the Mg@N-CDs/AuE (curve c), a more positive potential (-0.83 V) of Ti@NMg-CDs/AuE was observed than peak potential − 1.02 V. This potential-shift in CVs may be related to the photocatalytic sites of the titanium dioxide. As proved, Ti@NMgCDs was successful synthesized and the AuE/Ti@NMgCDs has better CVs behaviors. Furthermore, electrochemical impedance spectroscopy (EIS) was used to characterize the corresponding interface properties (electron-transfer resistance, Ret) of stepwise assembly process. As Fig. 3B depicted, the bare AuE exhibited a minimum Ret (70 Ω). Ret of AuE/Cys-kemptide/MCH (curve b) exhibited the increased Ret to the redox of the probe K 3 [Fe(CN) 6 ] (480 Ω), which was attributed to the low mass-transfer rate of the peptide that formed an additional barrier. As expected, the Ret value was further increased obviously after the phosphorylation of kemptide (763 Ω curve c) by PKA. The reason was mainly attributed to the electrostatic interaction between the PO 4 − -groups and [Fe(CN) 6 ] 3−/4− probes, which made electrons transfer more difficult after the -OH of Cys-kemptide was replaced by PO 4 − -groups. After incubation with Ti@NMg-CDs, Ret of the AuE/Cys-phosphopeptide/Ti@NMgCDs gradually decreased (350 Ω curve d), demonstrating the successful fabrication of the PKA-induced ECL-sensor. The corresponding surface coverage ( θE ) of CLRRASLG peptide on AuE/Cys-phosphopeptide/Ti@NMgCDs was also evaluated. From calculation, the θE -value reached 85%. The calculation equation is as follows: $${\theta E}_{-Cys-kemptide} = (1 - Ret/{Ret}_{Cys-kemptide}) \times 100\text{\%}$$ Where θE (%) is the surface coverage fraction of CLRRASLG peptide; Ret is the electron-transfer resistance of the bare Au-electrode; Ret Cys−kemptide is the electron-transfer resistance of the surface of Au-electrode modified by CLRRASLG peptide. In this design, the ECL intensities from the signal reporter Ti@NMg-CDs were tested under different experimental settings. Figure 3C demonstrated the comparative time-intensity curves of six compounds decorated bare AuE. The ECL signals of AuE/Cys-kemptide/MCH (101 a.u.) and AuE/phosphopeptide/MCH (213 a.u.) were close to that of the bare AuE (148 a.u.). Correspondingly, AuE/phosphopeptide/MCH/Mg@N-CDs (curve d) exhibited weak electrochemiluminescence emission (~ 181 a.u.) without NH 2 -TiO 2 . The minor changes of ECL intensity showed that the nonspecific effect of Mg@N-CDs for the AuE/phosphopeptide/MCH/Mg@N-CDs was nearly negligible. What's more, the AuE/phosphopeptide/MCH/Ti@NMg-CDs (curve f) exhibited much higher ECL intensity when protein kinase existed than the AuE/Cys- kemptide/MCH/Ti@NMg-CDs (curve e) alone did in the same condition. The strong interaction of Ti defect sites with phosphate groups has prompted the specific recognition between Ti@NMg-CDs-probe and phosphopeptide modified on AuE. Meanwhile, it was found that the ECL signals of the bare-AuE, AuE/Cys-kemptide/MCH and AuE/phosphopeptide/MCH were almost undetectable. As a whole, the results confirmed high recognition and signal amplification effect of the Ti@NMg-CDs nanoprobes, which could be applied for the sensitively analysis of kinase activity. 3.4. Optimization of the detection conditions The prepared method was derived from the specific recognition ability of Cys-phosphopeptides self-assembling on the AuE/Cys-phosphopeptides/MCH to fluorescent-probe Ti@NMg-CDs. Therefore, phosphorylation conditions (times, concentrations of ATP) and fluorescent-probe incubation times were critical for determining the sensitivity of PKA. It is observed in Fig. 4 A, as the phosphorylation time/temperature increase, ECL signal intensities increases. The ECL signal was the highest and reached a steady platform after 60 min at 37℃. At the same time, the concentration of the ATP on the ECL response was optimized due to the important role in PKA-catalyzed phosphorylation. The ECL intensity achieve the excellent sensing performance when the concentration of ATP was 100 µmol/L (Fig. 4 B). Hence, phosphorylation time (60 min)/temperature (37℃) and ATP-concentration (100 µmol/L) were chosen to be phosphorylated conditions. Another crucial factor was the incubation time of Ti@NMg-CDs (Fig. 4 C). The appropriate incubation time not only enhances the ECL signal but also improves the detection efficiency for PKA activity. As a result, the optimal incubation time is 120 min and the ECL intensity is achieved 2243 a.u with 0.1 U/mL PKA and 100 µmol/L ATP. 3.5. Specificity of PKA assay and the stability of the biosensor In this strategy, the level of proteins kinase expression also played an important role in ECL signal in addition to the concentration of PKA. Thus, the specificity of the ECL-sensor was demonstrated by measuring the ECL intensity of Ti@NMg-CDs in the presence/absence(blank) of PKA or in the presence of other enzymes, such as kinase Akt1, glucose oxidase. Also, the concentration of kinase Akt1 and glucose oxidase was 5 times than that of the PKA. In the presence of Akt1 or glucose oxidase, no significant enhancing of ECL signal was saw (Fig. 5 A), suggesting no efficient reaction between Cys-kemptides and Akt1/glucose oxidase. In contrast, a stronger ECL intensity was observed in the presence of PKA. The above analysis obviously signified that the constructed ECL biosensor presented the high specificity toward target PKA. Notably, the stability of the AuE/Cys-phosphopeptides/MCH/Ti@NMg-CDs was not ignored. As shown in Fig. 5 B, the AuE/Cys-phosphopeptides/MCH/Ti@NMg-CDs could keep stable signal after 10 cycles by testing the 0.10 U/mL PKA. Simultaneously, the RSD was calculated by 1.42%, signifing the good stability of the builded ECL-device. 3.6. Inhibition study As we all know, the screening of kinase inhibitor has played a major role not only in drug development but also in the treatment of human diseases. To examine whether this Ti@NMg-CDs-based sensor has universal application in quantitatively assessing kinase inhibitor, inhibited experiment was performed with PKA inhibitor (H-89) at different concentrations. In this regard, 0.1 U/mL of PKA with H-89 (curve a) was chosen to analyze ECL inhibitory behaviors in Fig. 5 D. As expected, the higher concentration of H-89 (0.1 nmol/L ~ 10 6 nmol/L), the lower ECL responses, indicating the phosphorylation reaction on AuE/Cys-kemptides/MCH was inhibited. The calculated half-maximal inhibition value of H-89 (IC 50 : 63.1 nmol/L) was well consistent with the reported value[ 28 ]. To confirm the facts, the control experiment was conducted (Fig. 5 D). The results clearly indicated that the changes of ECL signal were almost unobservable in the presence of tyrphostin AG 1478 and EGCG. The above facts demonstrated that the proposed AuE/Cys-phosphopeptides/MCH/Ti@NMg-CDs biosensor was promising for sensitive, rapid screening kinase inhibitor. 3.7. PKA activity assay PKA activity is a key factor in protein-phosphorylation pathway, which involves in several vital biological processes, including cell apoptosis, growth, and cellular signal communications. Many human diseases were caused by the aberrant expression of PKA activity, such as diabetes, and cardiovascular and cancer. Therefore, monitoring the PKA activity is prospective for fundamental biochemical research, disease diagnosis, and kinase-targeted drug discovery. ECL is a luminous-in-based technique that records the different changes in ECL-signal of a specific recognition layer upon variation of the target concentration in an as-prepared sensor. Under the above-mentioned experimental conditions, the results were achieved in Fig. 5 E-F improving the concentration of PKA from 0.001 to 30 U/mL, the ECL intensity gradually increased. The ECL-signal exhibited a linear correlation with the logarithm of PKA concentration over a range from 0 to 30000 mU/mL (Fig. 5 F, inset). The linear equation was determined to be F (a.u.) = 618.472 log ( C PKA ) + 633.002 (R 2 = 0.993) and the detection limit was 4.89 × 10 − 4 U/mL (S/N = 3). Compared with the published researches summarized in Table 1 , the LOD (4.89 × 10 − 4 U/mL) of the TiO 2 -assisted Mg@N-CDs-enhanced biosensor improved by more than 97.8, 376.2 and 4.89 × 10 3 -folds, which had good application prospects for other protein kinases. Table 1 Comparison of different methods for the determination of PKA. Methods Materials Linear range (U/mL) LOD (U/mL) Ref. ECS Mesoporous-SiO 2 0.1–200 8.3 × 10 − 2 [ 29 ] ECS AuNPs/rGO 0.1–500 5.3 × 10 − 2 [ 30 ] PEC ZrO 2 /CdS 0.001-100 3.5 × 10 − 4 [ 23 ] PEC TiO 2 /g-C 3 N 4 0.1–100 4.8 × 10 − 2 [ 31 ] FL Ti-MIL125-NH 2 0.05-10 3.0 × 10 − 2 [ 32 ] FL UiO-66 0.09-5.0 5.0 × 10 − 2 [ 33 ] ECL AuNPs/g-C 3 N 4 0.02-20 5.0 × 10 − 3 [ 34 ] ECL Ti@NMgCDs 0.001-30 4.89 × 10 − 4 This paper 4. Conclusions In summary, a single environment-friendly Ti@NMgCDs-based ECL sensor was established for sensitive determination of protein kinase A activity. The self-assembly of Cys-phosphopeptides catalyzed via PKA on AuE and the subsequent ECL signal could be easily tested based the Ti@NMgCDs chelated to the phosphate groups. A single Ti@NMgCDs could be anchored by more than one Cys-labeled phospho-kemptide, which consumed only a small amount of sample without the expensive antibodies and instruments for detection. Moreover, this single Ti@NMgCDs-based ECL sensor had several distinct advantages: (a) the record of ECL-signal integrated all step-analysis steps together including washing, separation and detection, making the assay much faster and easily manipulated; (b) the NH 2 -TiO 2 -based photocatalysis and large surface could accelerate the electron transfer on Au-electrode interface, enhancing and stabilizing ECL signal emission; (c) Importantly, the as-prepared fluorescent-probe Ti@NMgCDs was low-cost and had good biocompatibility and high luminous efficiency of 4.5-folds; (d) The developed Ti@NMgCDs/phospho-kemptide-Cys/AuE realized a wider linear range from 0.001 to 30 U/mL, ultra-low f molar detection limit (LOD: 4.89 × 10 − 4 U/mL). Additionally, the developed ECL-sensor was successfully employed to the inhibitor characterizing. This method presents a potential platform in drug-development that is feasible for clinic diagnosis. Declarations Supporting Information The lists of instruments and reagents; Synthesis of Mg@N-CDs and Ti@NMg-CDs; ECL tests; Optimization of the experimental conditions. Fig. S1 . ECL intensity-potential behavior obtained at the bare-AuE in PBS; Fig. S2. (A)/(B) FL emission spectra of Ti-doped Mg@N-CDs under excitation at different wavelengths. (C) Fluorescence spectra (FL, Ex:360 nm) and (D) ECL spectra; Fig. S3. UV–vis absorption spectra of prepared Ti-doped Mg@N-CDs; Fig. S4. ECL intensity of Mg@N-CDs/AuE and Ti-doped Mg@N-CDs/AuE stored in the refrigerator for 10 days (detected every two day); Fig. S5. ECL response of the biosensor for seven parallel detections. The concentration of PKA is about 30 U/mL; Fig. S6. TEM images of carbon dot. Declaration of competing interests The authors declare that they have no conflicts of interest to this work. Funding This work was supported by the Natural Science Foundation of Shanxi Province, China (Grant No. 202203021222222 and 202203021212399). Author Contribution Jianping Guo: Experimental Investigation, Formal analysis, Writing – original draft, Writing – review & editing, Data curation, Validation; Lele Yue: Conceptualization, Resources, Writing – original draft, Writing – review & editing, Formal analysis; Lingya Ning: Writing – original draft; Ailing Hana: Conceptualization; Junping Wang: review & editing, Data curation, Validation. All authors reviewed the manuscript. Acknowledgement Jianping Guo: Experimental Investigation, Formal analysis, Writing – original draft, Writing – review & editing, Data curation, Validation; Lele Yue: Conceptualization, Resources, Writing – original draft, Writing – review & editing, Formal analysis; Lingya Ning: Writing – original draft; Ailing Hana: Conceptualization; Junping Wang: review & editing, Data curation, Validation.This work was supported by the Natural Science Foundation of Shanxi Province, China (Grant No. 202203021222222 and 202203021212399). References Wang, X.; Ogata, A. F.; Walt, D. R. Ultrasensitive detection of enzymatic activity using single molecule arrays. Journal of the American Chemical Society, 2020, 142 (35), 15098-15106, DOI: 10.1021/jacs.0c06599. Li, Z.; Yin, B.; Zhang, S.; Lan, Z.; Zhang, L. Targeting protein kinases for the treatment of Alzheimer's disease: Recent progress and future perspectives. 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Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx GraphicalAbstract.pdf Scheme.tif Scheme 1 The fabrication procedure and signal amplification strategy of the Ti@NMg-CDs-based ECL-sensor for PKA activity detection. Cite Share Download PDF Status: Published Journal Publication published 25 Sep, 2024 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 30 Aug, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviews received at journal 08 Jul, 2024 Reviews received at journal 05 Jul, 2024 Reviewers agreed at journal 29 Jun, 2024 Reviewers agreed at journal 29 Jun, 2024 Reviewers invited by journal 29 Jun, 2024 Editor assigned by journal 21 Jun, 2024 Submission checks completed at journal 21 Jun, 2024 First submitted to journal 18 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4598795","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":324361178,"identity":"7d55a819-cacb-4c67-a4c7-38be23c2cfca","order_by":0,"name":"Jianping Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIie3QvWrDMBDAcZkDZZHxKhOSZzgQKEtoX0XB4LyCt7oU1C1eHchDGPICCoJ2EWT1aCh46pAt3fIBmUqRk62D/uOhH3eIkFDoH5YA7DqFp2lyG0TlEEnfdYaHAkRa3kvQOZnWDhaNuZeQVuE41jTa7p38YmQ+aQz0nU9EtVIi1gxkmy8FI7loDJ2hjwBXJos1p7KFjzEj9nIho9xHKF+UNtbIRLXTF3IaJozZ6LV2iiPJrlvMMOEjDeRQGORtnqcbzMTaUuklzzY5/ig0L1XlJP8uniarz7feS351/Sp44H0oFAqF/u4MBWVIBeCzfWUAAAAASUVORK5CYII=","orcid":"","institution":"School of Food Science, Shanxi Normal University","correspondingAuthor":true,"prefix":"","firstName":"Jianping","middleName":"","lastName":"Guo","suffix":""},{"id":324361179,"identity":"f8d1f1fb-388c-4f13-93f0-1878616a34f9","order_by":1,"name":"Lele Yue","email":"","orcid":"","institution":"School of Food Science, Shanxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lele","middleName":"","lastName":"Yue","suffix":""},{"id":324361181,"identity":"2e5798df-d301-4250-be9a-39b668b24ab9","order_by":2,"name":"Lingya Ning","email":"","orcid":"","institution":"Tianjin Economy and Technology Development Area","correspondingAuthor":false,"prefix":"","firstName":"Lingya","middleName":"","lastName":"Ning","suffix":""},{"id":324361183,"identity":"57827c51-a20c-407b-9bb1-73d90f51c3df","order_by":3,"name":"Ailing Han","email":"","orcid":"","institution":"School of Food Science, Shanxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Ailing","middleName":"","lastName":"Han","suffix":""},{"id":324361186,"identity":"68460734-42e4-400c-a2d1-1de8e95e4631","order_by":4,"name":"Junping Wang","email":"","orcid":"","institution":"Tianjin Economy and Technology Development Area","correspondingAuthor":false,"prefix":"","firstName":"Junping","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-06-18 09:11:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4598795/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4598795/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-024-06711-8","type":"published","date":"2024-09-25T15:57:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60059158,"identity":"7bd2df8d-56b2-434c-a490-ad104cc6e3cd","added_by":"auto","created_at":"2024-07-11 08:08:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":959877,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images (A)Ti@NMg-CDs, (B) Mg@N-CDs, (C)NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e, (D) HR-TEM images and (E) EDS spectra of Ti@NMg-CDs.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4598795/v1/e841a6ad4054fb0114592c94.png"},{"id":60058508,"identity":"35bffc46-bd16-4fb5-8f89-8197a7511c5c","added_by":"auto","created_at":"2024-07-11 08:00:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":246455,"visible":true,"origin":"","legend":"\u003cp\u003e(A) XRD patterns; (B) FT-IR; (C) Zeta potentials; (D) ECL-potential curves, bare-AuE in PBS-K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e solution (curve a); AuE/Mg@N-CDs in PBS (curve b); AuE/Mg@N-CDs (curve c), AuE/NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e (curve d) and AuE/Ti@NMg-CDs (curve e) in PBS-K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e system; (E) ECL spectrum of AuE/Mg@N-CDs and (F) AuE/Ti@NMg-CDs in K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e-PBS system.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4598795/v1/41b54fc3378345a3ee70ab18.png"},{"id":60058057,"identity":"02457168-ab65-4c9c-b2a9-4644d20aa614","added_by":"auto","created_at":"2024-07-11 07:52:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130086,"visible":true,"origin":"","legend":"\u003cp\u003e(A) CVs of (curve a) the bare AuE in PBS containing 100 mmol/L K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e, the NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e-modified (curve b) and Mg@N-CDs-modified (curve c) AuE, respectively and (curve d) Ti@NMg-CDs-modified Au electrode; (B) EIS of (a) the bare-AuE; (b) AuE/Cys-kemptide/MCH; (c) AuE/Cys-phosphopetide/MCH and (d) AuE/Cys-phosphopetide/MCH/Ti@NMg-CDs recorded in 5 mmol/L [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e4-/3-\u003c/sup\u003e containing 0.2 mol/L KNO\u003csub\u003e3\u003c/sub\u003e (pH 7.4); (C) ECL curves of (a) the bare-AuE, (b) AuE/Cys-kemptide/MCH, (c) AuE/phosphopeptide/MCH, (d) AuE/phosphopeptide/MCH/Mg@N-CDs, (e) AuE/Cys- kemptide/MCH/Ti@NMg-CDs, and (f) AuE/phosphopeptide/MCH/Ti@NMg-CDs at the concentrations of PKA of 0.1 U/mL.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4598795/v1/cfe110c8452b3e948693712d.png"},{"id":60058510,"identity":"13a75de1-82d7-4008-9d48-42e2317105c7","added_by":"auto","created_at":"2024-07-11 08:00:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73735,"visible":true,"origin":"","legend":"\u003cp\u003eThe optimizations of (A) different phosphorylation times of AuE/Cys-kemptides/MCH caused temperatura-dependent ECL-changes of AuE/Cys-phosphopeptides/MCH/Ti@NMg-CDs; (B) Effect of the concentration of ATP on ECL intensity; (C) Incubation time of the fluorescent-probe Ti@NMg-CDs.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4598795/v1/a7240d1238d3afcf4cf4f114.png"},{"id":60059159,"identity":"00b8d74a-7dcf-412f-b18e-7bb815f33ba2","added_by":"auto","created_at":"2024-07-11 08:08:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":172243,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The interference of AKT1 and glucose oxidase on the determination of PKA; (B) The ECL-time curves of AuE/Cys-phosphopeptides/MCH/Ti@NMg-CDs; (C)The structure of H-89; (D)ECL responses of different concentrations: a: H-89, b: tyrphostin AG 1478, c: EGCG; (E) ECL-time curves in PBS-K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e solution and in the presence of PKA (0.001 ~ 30 U/mL); (F) ECL curves of different PKA concentrations from 0.001 to 30 U/mL. Calibration curve between ECL intensity and PKA concentrations (inset).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4598795/v1/4ac9474e094d3f50d36ddadc.png"},{"id":65628056,"identity":"49a840b6-6184-4698-a749-de81e7125566","added_by":"auto","created_at":"2024-09-30 16:17:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2309254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4598795/v1/8fbf2011-1fbf-4ebc-af59-38f3477d6f24.pdf"},{"id":60058053,"identity":"219eba5c-63b3-4edb-9b14-d55dbb552ad1","added_by":"auto","created_at":"2024-07-11 07:52:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1091900,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4598795/v1/8c3efb4f81813e3d12c4617f.docx"},{"id":60058509,"identity":"063eedae-f3a8-4f49-899b-d36224a0ff8b","added_by":"auto","created_at":"2024-07-11 08:00:22","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":72519,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4598795/v1/576469ffa1904b436f3963d4.pdf"},{"id":60058055,"identity":"3f5c5846-f317-46fd-8acb-74dfc3a678e4","added_by":"auto","created_at":"2024-07-11 07:52:22","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":262354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1 \u003c/strong\u003eThe fabrication procedure and signal amplification strategy of the Ti@NMg-CDs-based ECL-sensor for PKA activity detection.\u003c/p\u003e","description":"","filename":"Scheme.tif","url":"https://assets-eu.researchsquare.com/files/rs-4598795/v1/6e51c1636abaeb87a155394c.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phosphopeptide-bridged NH2-TiO2-mediated carbon dots self-enhancing and electrochemiluminescence microsensors for label-free protein kinase A detection","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFor the important impact of kinases in human health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and drug development[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], protein kinase has been used as one of the major drug targets against human diseases. Under the catalysis of protein kinase, one or more γ-phosphate groups are transferred to specific amino acids (i.e., serine, tyrosine, and threonine residues) of target proteins, and this phosphorylation process is one of the most important post-translational modifications (PTM) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Whereas, the overexpression and mutations of protein kinases and aberrant protein-phosphorylation states can result in cancer, heart dysfunctions and Alzheimer\u0026rsquo;s disease[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and some other diseases, posing huge threats to human health. So, protein kinases are a group of especially significant targets for drug therapy. In this case, sensitive, accurate and widely applicable assays for monitoring protein kinase A (PKA) activity are beneficial to the development of protein kinase-related clinical diagnosis and kinase-targeted drug discovery, as well as for further understanding the signal transduction mechanism of molecular[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Simultaneously, the screening of inhibitors of protein kinases A are currently also important, and several protein kinase inhibitors have been FDA approved for targeted therapy. Real examples of kinase inhibitors (Pazopanib, Nilotinib, Sunitinib) provide a wealth of information about how tumors respond to such targeted therapy[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. On the basis of above, it is also urgent that a fast pathway selectivity-based ECL-signal reading out the activity of different inhibitors is constructed.\u003c/p\u003e \u003cp\u003eThe conventional approaches for the detection of PKA activities are almost based on the expensive and harmful radioactive labels with \u003csup\u003e32\u003c/sup\u003eP[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In this regard, various alternative ways have been recently exploited for PKA analysis and inhibitor screening, such as colorimetric, quartz crystal microbalance (QCM), Raman Spectroscopy and fluorescent immuno-assay[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although these designs are smart and antibody-antigen with high affinity have improved the selectivity, however, low accuracy due to false positive results, specialized instruments and tedious operations heavily hinder the real application. Recently, electrochemiluminescence (ECL), as a signal-emission process in a redox reaction of electrogenerated reactants, appears in scientific research. On the one hand, ECL technology is a potential analysis-method on account of its distinct merits lower background noise and cost, higher selectivity and sensitivity, as well as more convenient operation. In addition, ECL platform has also been increasingly employed in biomarkers detection based on the potential for rapid and sensitive diagnosis of human diseases[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Green nano materials have emerged as key components in most, if not all, optical and electrical sensing field. Unfortunately, the expensiveness, toxicity and hard to prepare limit the existing ECL luminophores[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] applications. Therefore, the synthesis of eco-friendly electro-chemiluminescent (ECL) nanomaterials becomes urgent needs to solve above key issues and relies greatly on advanced luminescent materials as well as the novel amplification technologies of the ECL-signal.\u003c/p\u003e \u003cp\u003eIn recent years, some carbon-based materials (CMs) and their complexes have been successfully used to sensing-fields because of their high flexibility, good electrical properties and the harmless to environment, e.g., carbon dots (CDs)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], carbon nanotube (CNT)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and graphene[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among them, carbon dots (CDs) are the most popularly used light-responsive nanomaterial material in sensing-fields due to the good biocompatibility, photocatalysis and high energy-conversion efficiency. Fixing CDs directly on Au-electrode (AuE) has problems such as unstable response-signal and low ECL-intensity. Some attempts [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] to enhance their ECL signal focus on co-reactive reagents (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) introduce or structure design. Studies have revealed that the cathodic ECL behavior of TiO\u003csub\u003e2\u003c/sub\u003e is comparable to that of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, TiO\u003csub\u003e2\u003c/sub\u003e can act as co-reactants for CDs. In addition, TiO\u003csub\u003e2\u003c/sub\u003e-based nanomaterials with good biocompatibility and relative conductivity have been extensively certified to enhance ECL-performances cooperatively (such as TiO\u003csub\u003e2\u003c/sub\u003e@SnS\u003csub\u003e2\u003c/sub\u003e, TiO\u003csub\u003e2\u003c/sub\u003e-CNTs, ZnO/TiO\u003csub\u003e2\u003c/sub\u003e, CeO\u003csub\u003e2\u003c/sub\u003e@TiO\u003csub\u003e2\u003c/sub\u003e, etc.)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, only few researches focused on improving the ECL-properties of CDs, in which NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e acted as a signal-carrier for CDs-luminor in biosensor.\u003c/p\u003e \u003cp\u003eSemiconductor photocatalyst nano titanium oxide (TiO\u003csub\u003e2\u003c/sub\u003e) with the large surface area is considered as a promising transducing material due to its high chemical stability, low cost, and non-toxicity[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. On the other hand, its affinity toward phosphoric group via Ti-O-P coordination has gained enormous application in phosphoprotein-enrichment. In addition, the lower Lewis acidity and higher hydrophilicity of amino-functionalized TiO\u003csub\u003e2\u003c/sub\u003e (NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e) than TiO\u003csub\u003e2\u003c/sub\u003e show strong affinity with phosphoproteins, then modifying -NH\u003csub\u003e2\u003c/sub\u003e onto TiO\u003csub\u003e2\u003c/sub\u003e has gained enormous attraction[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Based on the prominent photocatalytic performance and strong specific recognition utility between NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e and phosphate group, if or not a label-free ECL- biosensor may be developed to detect the activity of PKA?\u003c/p\u003e \u003cp\u003eIn light of these unique and outstanding properties of NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e-doped CDs, herein, a robust ECL platform for the test of PKA activity was reported by using eco-friendly blue-emissive Ti@NMg-CDs. The Ti@NMg-CDs feature both one of the most protentional ECL-luminous known to date and as an ideal PKA-reporter and phosphopeptide-recognizing matrix. It was noteworthy that a single Mg@N-CDs could assemble multiple NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e to form the single-donor/multiple-acceptor configuration with an improved ECL intensity. In this alternative ECL-biosensor, the synthesis of the fluorescent nanoprobes Ti@NMgCDs increases the stability of the CDs and intensity of the ECL-signal. The in-situ phosphorylated Cys-kemptide-sensor could efficiently anchor the Ti@NMgCDs by Ti-O-P matching, achieving highly sensitive detection of PKA. To our knowledge, this is a new report on in-situ construction of Cys-kemptide and Ti@NMgCDs ECL-sensor on Au-electrode for PKA tests.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents and chemicals\u003c/h2\u003e \u003cp\u003eAll materials and related instruments used in the experiments have been listed in supporting information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Fabrication of ECL-biosensor\u003c/h2\u003e \u003cp\u003e(A) Firstly, the Au electrode (AuE) was polished and ultrasonically washed by ethanol and doubly distilled water (DDW). Subsequently, the AuE dried by N\u003csub\u003e2\u003c/sub\u003e was activated with freshly prepared \u0026ldquo;piranha\u0026rdquo; solution of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (1:3, v/v) for 5 min. (B) Cys-labeled kemptide (CLRRASLG) self-assembly on the AuE: the electrode was dipped into the 10 mmol/L PBS (pH 7.4) solution of 500 \u0026micro;mol/L CLRRASLG for overnight reaction at room temperature. On the surface of the AuE, an achievable serine-peptide chains for phosphorylation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] were formed directly through the Au-S bond.\u003c/p\u003e \u003cp\u003e(C) After rinsing with PBS, the unbound Au-sites were blocked completely by 1 mmol/L 6-mercaptohexanol (MCH) solution for 30 min. Afterward, the resulting AuE/Cys-kemptides/MCH was dipped in a desired amount of PKA\u0026amp;ATP-incubation PBS-buffer.\u003c/p\u003e \u003cp\u003eIn a 5 mL phosphorylation reaction system, sterile water, PBS buffer (10 mmol/L), ATP (100 \u0026micro;mol/L), a specific concentration of PKA (0.1 U/mL) were added in order into a 10 mL centrifuge tube; Subsequently, the resulting AuE/Cys-kemptides/MCH electrodes were was dipped in the above 10 mL centrifuge tube. The tube was placed in a pre-heated shaking bath for 60 min (37 ℃, 200 rpm).\u003c/p\u003e \u003cp\u003eAbove the phosphorylation reaction, in the presence of ATP and PKA, the Cys-labeled kemptide receptor (CLRRASLG) is rapidly phosphorylated on phosphorylation sites (the hydroxyl site of serine) that are located within the C-terminal domain of the receptor. Previous studies [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] suggested that serine presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. In particular, the presence of PKA could induce the formation of Cys-phosphopeptides by the catalytic reaction between specific substrate (kemptide) and PKA, which could act as an initiator to link the Ti@NMg-CDs according to the bridge interactions Ti-O-P. This could be attributed to the γ-phosphoryl of ATP will be transferred to the hydroxyl group of serine in the CLRRASLG under the catalysis of PKA [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. After incubation with the Ti@NMg-CDs, the phosphorylated CLRRASLG-peptides would be rapidly captured on the Ti@NMg-CDs through the high binding affinity between the Ti\u003csup\u003e2+\u003c/sup\u003e ions of Ti@NMg-CDs and the phosphate group.\u003c/p\u003e \u003cp\u003e(D) Finally, the electrode (AuE/Cys-phosphopeptides/MCH) obtained above was incubated in Ti@NMg-CDs-solution for 2 h at room temperature, then thoroughly rinsing with water to remove nonspecific Ti@NMg-CDs and drying with nitrogen. The detailed construction process was displayed in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterizations of Ti@NMg-CDs nanoprobes\u003c/h2\u003e \u003cp\u003eMg@N-CDs, NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e and Ti@NMg-CDs nanocomposites exhibited different morphological characteristics. The TEM observations indicated that the Ti@NMg-CDs was more uniform and denser with nano NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) nano-crystal doped into carbon dots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). After the modification processes of NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e, patches of nano-crystaled particles were observed on the nanocomposites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The TEM images of the Ti@NMg-CDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) nanocomposites also confirmed the presence of NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e nano-crystal compared with only the Mg@N-CDs. Moreover, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD clearly showed well-resolved lattice fringes (0.33 nm) structure of Ti@NMg-CDs nanocomposites, which were assigned to the (021) plane[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. And photocatalytic NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e improved the ECL-performance of the Ti@NMg-CDs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The Ti@NMg-CDs as the substrate provided larger surface areas, which were beneficial for facilely adsorpting the multiple Cys-phosphopeptides. Energy dispersive spectrometer (EDS) spectra were employed to reveal the presence of different components in Ti@N Mg -CDs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, C, N and O atoms occupy most of element mass. Small amounts of Mg atoms coexist with dominant Ti atoms. N and O is derived from the NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e and Mg@N-CDs. Ti and Mg, C only come from the NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e, Mg@N-CDs, respectively. The EDS analysis results further reveal the synthesis of Ti@NMg -CDs successfully. ECL stabilities were studied by measuring and comparing ECL intensity (Fig. S4). With the extending of storage time from 0 to 10 d, the ECL intensity of Mg@N-CDs/AuE dramatically decreases to 206 a.u. (initial value is 3897 a.u.), while that of Ti@NMg -CDs/AuE has negligible changes (more than 0.93). These experimental results suggest that the film-forming/photocatalytic NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e made considerable progress in ECL efficiency and stability of Ti@NMg-CDs/AuE [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe bind of Ti@NMg-CDs on AuE/Cys-phosphopeptide, only nano NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e and Mg@N-CDs were also probed by X-ray diffraction (XRD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The XRD patterns of the Ti@NMg-CDs and Mg@N-CDs nanocomposites exhibited different characteristics. The characteristic diffraction peak observed in the Ti@NMg-CDs was ascribed to the mixture crystalline of NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e (004) and Mg@N-CDs (002)[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], whereas the intensive diffraction appearing at 25.5\u0026deg; was similar and the peak intensity increased obviously, suggesting the pre-synthesized Ti@NMgCDs-probe tended to be well crystallized. As a whole, the typical diffraction peaks of both NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e and Mg@N-CDs were observed from the Ti@NMg-CDs, indicating the formation of the Ti@NMg-CDs fluorescent-probe. Fourier transform infrared (FT-IR) spectra was measured to investigate the functional groups of Ti@NMg-CDs with Mg@N-CDs as a contrast. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB showed the FT-IR spectras of Mg@N-CDs (curve a), NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e (curve b), and Ti@NMg-CDs (curve c). The O-H at 3400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2972 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and C-O at 1087 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could be observed in the FT-IR spectra, further indicating the strong hydrophilic effect of the nanoparticles. FT-IR absorption bands at 2889 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned to the N-H stretching vibrations. The specific bands at 881 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned to the C-C vibrations of the benzene-ring structures as well. Ti@NMg-CDs displayed almost the same functional groups with Mg@N-CDs. In spite of the above similarities, the presence of specific bands (Ti-O) at 725 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e illustrated the successful synthesis of Ti@NMg-CDs doping with TiO\u003csub\u003e2\u003c/sub\u003e. Zeta potentials were also measured to prove the generation Ti@NMg-CDs. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC presented the zeta potential changes of nano Mg@N-CDs, NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e and Ti@NMg-CDs (pH\u0026thinsp;=\u0026thinsp;7.4), respectively. The as-synthesized Mg@N-CDs displayed negative potential (ζ = -11.1mV), and the potential change to positive after the modification of NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e (ζ = + 27.0 mV). After the formation of the Ti@NMg-CDs fluorescent nano-probe (ζ = + 20.6 mV), the charges increased owing the conjugation of positively charged NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e (ζ = + 27.0 mV). The zeta potential results shown above demonstrated the synthesis of Ti@NMg-CDs-probe successfully.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. ECL behaviors of the Ti@NMg-CDs nanoprobes\u003c/h2\u003e \u003cp\u003eTo estimate the ECL behaviors of Ti@NMg-CDs fluorescent probe, different AuE were assembled and characterized by ECL intensity-potential during a potential scan cycle \u0026minus;\u0026thinsp;1.8\u0026thinsp;~\u0026thinsp;0 V, including ECL intensity-potential curves of bare-AuE (curve a), AuE/N@Mg-CDs (curve c), AuE/NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e (curve d), AuE/Ti@NMg-CDs (curve e) in PBS-K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e system and AuE/Mg@N-CDs (curve b) in PBS without K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, curve a had a weak ECL signal. What\u0026rsquo;s more, the ECL signal of N@Mg-CDs/AuE was in an \"on\" state, however, the signal value disappeared in PBS without K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e (curve b). As we all know, oxidation intermediate (SO\u003csub\u003e4\u003c/sub\u003e\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e) can be produced by S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e under power, which accelerated the formation the excited state NTi@MgCDs*[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Interestingly, when NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e nano-film and Ti@NMg-CDs was separately decorated on AuE, a sharper ECL signal (4501 a.u.) was given on AuE/Ti@NMg-CDs (curve e), which was about 9.2 and 4.3 times than that on AuE/NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e (curve d), AuE/Mg@N-CDs (curve b). Above results implied that the ECL intensity of Mg@N-CDs was significantly enhanced, which could be ascribed to the excellent photocatalytic performance of nano-TiO\u003csub\u003e2\u003c/sub\u003e. In addition, the 3D ECL spectra was carried out to further confirm that the NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e could act as ECL-signal accelerator for Ti@NMg-CDs hybrid. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, the ECL spectrum of Mg@N-CDs realized maximum about 791 a.u. at 520 nm. When the AuE/Ti@NMg-CDs was immersed in PBS containing coreactant K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e, the maximum ECL emission wavelength had no significant change (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), demonstrating that the probe Mg@N-CDs and the aminated nano titanium dioxide could enhance the ECL-intensity synergistically in K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e-PBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Electrochemical characterization\u003c/h2\u003e \u003cp\u003eCyclic voltammetry (CV) behaviors of four different Au-electrodes (bare-AuE, NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e/AuE, Mg@N-CDs/AuE, Ti@NMg-CDs/AuE) were studied in PBS (0.2 mol/L, pH\u0026thinsp;=\u0026thinsp;7.4) containing 0.1 mol/L K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e upon \u0026minus;\u0026thinsp;1.2\u0026thinsp;~\u0026thinsp;0 V. On the surface of bare-AuE, reduction peak was detected (curve a) obviously at \u0026minus;\u0026thinsp;0.97 V in Fig.\u0026nbsp;3A, which results from the formation of strong oxidation intermediate (SO\u003csub\u003e4\u003c/sub\u003e\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e) at a certain voltage. In addition, a weak negative cathodic peak (-0.89 V) was observed on the NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e-modified AuE (curve b) in K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e-PBS, which was attributed to the conductivity and narrow energy gap of semiconductor nano-titania. Compared with the Mg@N-CDs/AuE (curve c), a more positive potential (-0.83 V) of Ti@NMg-CDs/AuE was observed than peak potential \u0026minus;\u0026thinsp;1.02 V. This potential-shift in CVs may be related to the photocatalytic sites of the titanium dioxide. As proved, Ti@NMgCDs was successful synthesized and the AuE/Ti@NMgCDs has better CVs behaviors.\u003c/p\u003e \u003cp\u003eFurthermore, electrochemical impedance spectroscopy (EIS) was used to characterize the corresponding interface properties (electron-transfer resistance, Ret) of stepwise assembly process. As Fig.\u0026nbsp;3B depicted, the bare AuE exhibited a minimum Ret (70 Ω). Ret of AuE/Cys-kemptide/MCH (curve b) exhibited the increased Ret to the redox of the probe K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e] (480 Ω), which was attributed to the low mass-transfer rate of the peptide that formed an additional barrier. As expected, the Ret value was further increased obviously after the phosphorylation of kemptide (763 Ω curve c) by PKA. The reason was mainly attributed to the electrostatic interaction between the PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-groups and [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e probes, which made electrons transfer more difficult after the -OH of Cys-kemptide was replaced by PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-groups. After incubation with Ti@NMg-CDs, Ret of the AuE/Cys-phosphopeptide/Ti@NMgCDs gradually decreased (350 Ω curve d), demonstrating the successful fabrication of the PKA-induced ECL-sensor. The corresponding surface coverage (\u003cem\u003eθE\u003c/em\u003e) of CLRRASLG peptide on AuE/Cys-phosphopeptide/Ti@NMgCDs was also evaluated. From calculation, the \u003cem\u003eθE\u003c/em\u003e-value reached 85%. The calculation equation is as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$${\\theta E}_{-Cys-kemptide} = (1 - Ret/{Ret}_{Cys-kemptide}) \\times 100\\text{\\%}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003eθE\u003c/em\u003e (%) is the surface coverage fraction of CLRRASLG peptide; \u003cem\u003eRet\u003c/em\u003e is the electron-transfer resistance of the bare Au-electrode; \u003cem\u003eRet\u003c/em\u003e\u003csub\u003e\u003cem\u003eCys\u0026minus;kemptide\u003c/em\u003e\u003c/sub\u003e is the electron-transfer resistance of the surface of Au-electrode modified by CLRRASLG peptide.\u003c/p\u003e \u003cp\u003eIn this design, the ECL intensities from the signal reporter Ti@NMg-CDs were tested under different experimental settings. Figure\u0026nbsp;3C demonstrated the comparative time-intensity curves of six compounds decorated bare AuE. The ECL signals of AuE/Cys-kemptide/MCH (101 a.u.) and AuE/phosphopeptide/MCH (213 a.u.) were close to that of the bare AuE (148 a.u.). Correspondingly, AuE/phosphopeptide/MCH/Mg@N-CDs (curve d) exhibited weak electrochemiluminescence emission (~\u0026thinsp;181 a.u.) without NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e. The minor changes of ECL intensity showed that the nonspecific effect of Mg@N-CDs for the AuE/phosphopeptide/MCH/Mg@N-CDs was nearly negligible. What's more, the AuE/phosphopeptide/MCH/Ti@NMg-CDs (curve f) exhibited much higher ECL intensity when protein kinase existed than the AuE/Cys- kemptide/MCH/Ti@NMg-CDs (curve e) alone did in the same condition. The strong interaction of Ti defect sites with phosphate groups has prompted the specific recognition between Ti@NMg-CDs-probe and phosphopeptide modified on AuE. Meanwhile, it was found that the ECL signals of the bare-AuE, AuE/Cys-kemptide/MCH and AuE/phosphopeptide/MCH were almost undetectable. As a whole, the results confirmed high recognition and signal amplification effect of the Ti@NMg-CDs nanoprobes, which could be applied for the sensitively analysis of kinase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Optimization of the detection conditions\u003c/h2\u003e \u003cp\u003eThe prepared method was derived from the specific recognition ability of Cys-phosphopeptides self-assembling on the AuE/Cys-phosphopeptides/MCH to fluorescent-probe Ti@NMg-CDs. Therefore, phosphorylation conditions (times, concentrations of ATP) and fluorescent-probe incubation times were critical for determining the sensitivity of PKA. It is observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, as the phosphorylation time/temperature increase, ECL signal intensities increases. The ECL signal was the highest and reached a steady platform after 60 min at 37℃. At the same time, the concentration of the ATP on the ECL response was optimized due to the important role in PKA-catalyzed phosphorylation. The ECL intensity achieve the excellent sensing performance when the concentration of ATP was 100 \u0026micro;mol/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Hence, phosphorylation time (60 min)/temperature (37℃) and ATP-concentration (100 \u0026micro;mol/L) were chosen to be phosphorylated conditions. Another crucial factor was the incubation time of Ti@NMg-CDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The appropriate incubation time not only enhances the ECL signal but also improves the detection efficiency for PKA activity. As a result, the optimal incubation time is 120 min and the ECL intensity is achieved 2243 a.u with 0.1 U/mL PKA and 100 \u0026micro;mol/L ATP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Specificity of PKA assay and the stability of the biosensor\u003c/h2\u003e \u003cp\u003eIn this strategy, the level of proteins kinase expression also played an important role in ECL signal in addition to the concentration of PKA. Thus, the specificity of the ECL-sensor was demonstrated by measuring the ECL intensity of Ti@NMg-CDs in the presence/absence(blank) of PKA or in the presence of other enzymes, such as kinase Akt1, glucose oxidase. Also, the concentration of kinase Akt1 and glucose oxidase was 5 times than that of the PKA. In the presence of Akt1 or glucose oxidase, no significant enhancing of ECL signal was saw (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), suggesting no efficient reaction between Cys-kemptides and Akt1/glucose oxidase. In contrast, a stronger ECL intensity was observed in the presence of PKA. The above analysis obviously signified that the constructed ECL biosensor presented the high specificity toward target PKA. Notably, the stability of the AuE/Cys-phosphopeptides/MCH/Ti@NMg-CDs was not ignored. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the AuE/Cys-phosphopeptides/MCH/Ti@NMg-CDs could keep stable signal after 10 cycles by testing the 0.10 U/mL PKA. Simultaneously, the RSD was calculated by 1.42%, signifing the good stability of the builded ECL-device.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Inhibition study\u003c/h2\u003e \u003cp\u003eAs we all know, the screening of kinase inhibitor has played a major role not only in drug development but also in the treatment of human diseases. To examine whether this Ti@NMg-CDs-based sensor has universal application in quantitatively assessing kinase inhibitor, inhibited experiment was performed with PKA inhibitor (H-89) at different concentrations. In this regard, 0.1 U/mL of PKA with H-89 (curve a) was chosen to analyze ECL inhibitory behaviors in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD. As expected, the higher concentration of H-89 (0.1 nmol/L\u0026thinsp;~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e nmol/L), the lower ECL responses, indicating the phosphorylation reaction on AuE/Cys-kemptides/MCH was inhibited. The calculated half-maximal inhibition value of H-89 (IC\u003csub\u003e50\u003c/sub\u003e: 63.1 nmol/L) was well consistent with the reported value[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To confirm the facts, the control experiment was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The results clearly indicated that the changes of ECL signal were almost unobservable in the presence of tyrphostin AG 1478 and EGCG. The above facts demonstrated that the proposed AuE/Cys-phosphopeptides/MCH/Ti@NMg-CDs biosensor was promising for sensitive, rapid screening kinase inhibitor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.7. PKA activity assay\u003c/h2\u003e \u003cp\u003ePKA activity is a key factor in protein-phosphorylation pathway, which involves in several vital biological processes, including cell apoptosis, growth, and cellular signal communications. Many human diseases were caused by the aberrant expression of PKA activity, such as diabetes, and cardiovascular and cancer. Therefore, monitoring the PKA activity is prospective for fundamental biochemical research, disease diagnosis, and kinase-targeted drug discovery. ECL is a luminous-in-based technique that records the different changes in ECL-signal of a specific recognition layer upon variation of the target concentration in an as-prepared sensor. Under the above-mentioned experimental conditions, the results were achieved in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F improving the concentration of PKA from 0.001 to 30 U/mL, the ECL intensity gradually increased. The ECL-signal exhibited a linear correlation with the logarithm of PKA concentration over a range from 0 to 30000 mU/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, inset). The linear equation was determined to be \u003cem\u003eF\u003c/em\u003e(a.u.)\u0026thinsp;=\u0026thinsp;618.472 log (\u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003ePKA\u003c/em\u003e\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;633.002 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.993) and the detection limit was 4.89 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e U/mL (S/N\u0026thinsp;=\u0026thinsp;3). Compared with the published researches summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the LOD (4.89 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e U/mL) of the TiO\u003csub\u003e2\u003c/sub\u003e-assisted Mg@N-CDs-enhanced biosensor improved by more than 97.8, 376.2 and 4.89 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e-folds, which had good application prospects for other protein kinases.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of different methods for the determination of PKA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaterials\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLinear range\u003c/p\u003e \u003cp\u003e(U/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLOD\u003c/p\u003e \u003cp\u003e(U/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMesoporous-SiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026ndash;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e8.3 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAuNPs/rGO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026ndash;500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e5.3 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZrO\u003csub\u003e2\u003c/sub\u003e/CdS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.001-100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e3.5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026ndash;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e4.8 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTi-MIL125-NH\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.05-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e3.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUiO-66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.09-5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e5.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAuNPs/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e5.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTi@NMgCDs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.001-30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e4.89 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis paper\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn summary, a single environment-friendly Ti@NMgCDs-based ECL sensor was established for sensitive determination of protein kinase A activity. The self-assembly of Cys-phosphopeptides catalyzed via PKA on AuE and the subsequent ECL signal could be easily tested based the Ti@NMgCDs chelated to the phosphate groups. A single Ti@NMgCDs could be anchored by more than one Cys-labeled phospho-kemptide, which consumed only a small amount of sample without the expensive antibodies and instruments for detection. Moreover, this single Ti@NMgCDs-based ECL sensor had several distinct advantages: (a) the record of ECL-signal integrated all step-analysis steps together including washing, separation and detection, making the assay much faster and easily manipulated; (b) the NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e-based photocatalysis and large surface could accelerate the electron transfer on Au-electrode interface, enhancing and stabilizing ECL signal emission; (c) Importantly, the as-prepared fluorescent-probe Ti@NMgCDs was low-cost and had good biocompatibility and high luminous efficiency of 4.5-folds; (d) The developed Ti@NMgCDs/phospho-kemptide-Cys/AuE realized a wider linear range from 0.001 to 30 U/mL, ultra-low f molar detection limit (LOD: 4.89 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e U/mL). Additionally, the developed ECL-sensor was successfully employed to the inhibitor characterizing. This method presents a potential platform in drug-development that is feasible for clinic diagnosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupporting Information\u003c/h2\u003e \u003cp\u003eThe lists of instruments and reagents; Synthesis of Mg@N-CDs and Ti@NMg-CDs; ECL tests; Optimization of the experimental conditions. Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. ECL intensity-potential behavior obtained at the bare-AuE in PBS; Fig. S2. (A)/(B) FL emission spectra of Ti-doped Mg@N-CDs under excitation at different wavelengths. (C) Fluorescence spectra (FL, Ex:360 nm) and (D) ECL spectra; Fig. S3. UV\u0026ndash;vis absorption spectra of prepared Ti-doped Mg@N-CDs; Fig. S4. ECL intensity of Mg@N-CDs/AuE and Ti-doped Mg@N-CDs/AuE stored in the refrigerator for 10 days (detected every two day); Fig. S5. ECL response of the biosensor for seven parallel detections. The concentration of PKA is about 30 U/mL; Fig. S6. TEM images of carbon dot.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest to this work.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Natural Science Foundation of Shanxi Province, China (Grant No. 202203021222222 and 202203021212399).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJianping Guo: Experimental Investigation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Data curation, Validation; Lele Yue: Conceptualization, Resources, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Formal analysis; Lingya Ning: Writing \u0026ndash; original draft; Ailing Hana: Conceptualization; Junping Wang: review \u0026amp; editing, Data curation, Validation. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eJianping Guo: Experimental Investigation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Data curation, Validation; Lele Yue: Conceptualization, Resources, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Formal analysis; Lingya Ning: Writing \u0026ndash; original draft; Ailing Hana: Conceptualization; Junping Wang: review \u0026amp; editing, Data curation, Validation.This work was supported by the Natural Science Foundation of Shanxi Province, China (Grant No. 202203021222222 and 202203021212399).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang, X.; Ogata, A. F.; Walt, D. R. Ultrasensitive detection of enzymatic activity using single molecule arrays. 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Integrating highly efficient recognition and signal transition of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e embellished Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e mXene hybrid nanosheets for electrogenerated chemiluminescence analysis of protein kinase activity. Analytical chemistry,\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e 92 (15), 10668-10676, DOI: 10.1021/acs.analchem.0c01776.\u003c/li\u003e\n\u003cli\u003eLuo, Q.-X.; Li, Y.; Liang, R.-P.; Cao, S.-P.; Jin, H.-J.; Qiu, J.-D. Gold nanoclusters enhanced electrochemiluminescence of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e for protein kinase activity analysis and inhibition. Journal of Electroanalytical Chemistry\u003cstrong\u003e, 2020,\u003c/strong\u003e 856, 113706, DOI: 10.1016/j.jelechem.2019.113706.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"protein kinase activity, Ti@NMg-CDs, Cys-kemptide, electrochemiluminescence method, inhibitors","lastPublishedDoi":"10.21203/rs.3.rs-4598795/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4598795/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel electrochemiluminescence (ECL) method was developed for analyzing protein kinase A (PKA) ultra-sensitively based on amidated nano-titanium (NH\u003csub\u003e2\u003c/sub\u003e-TiO\u003csub\u003e2\u003c/sub\u003e) embellished carbon dots (Mg@N-CDs) fluorescent probe, which integrated the target recognition and ECL-signal enhancement. The Cys-labeled kemptides were employed to build a serine-rich synthetic substrate-heptapeptide (Cys-kemptide) on the Au-electrode surface. Then, the PKA-induced biosensor was triggered as a signal switch to introduce the large amounts of TiO\u003csub\u003e2\u003c/sub\u003e decorated Mg@N-CDs nanohybrid (Ti@NMg-CDs) into AuE/Cys-phosphopeptides for signal output. In particular, the presence of PKA could induce the formation of Cys-phosphopeptides by the catalytic reaction between specific substrate (kemptide) and PKA, which could act as an initiator to link the Ti@NMg-CDs according to the bridge interactions Ti-O-P. By this way, multiple Cys-phosphopeptides were adsorbed onto a single Ti@NMg-CDs and the Ti@NMg-CDs not only provided the high specific selectivity but also large surface area, as well as unprecedented high ECL efficiency. Using this PKA-induced enhanced sensor, the limit-of-detection of the PKA was 4.89 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e U/mL (S/N\u0026thinsp;=\u0026thinsp;3). The proposed ECL biosensor was also universally applicable for the screening of PKA inhibitors and the determining of other kinases activity. Our sensing-system has excellent performance of specificity and the screening of kinase inhibitors, as well as it will inspire future effort on clinical diagnostics and new drugs discovery.\u003c/p\u003e","manuscriptTitle":"Phosphopeptide-bridged NH2-TiO2-mediated carbon dots self-enhancing and electrochemiluminescence microsensors for label-free protein kinase A detection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-11 07:52:17","doi":"10.21203/rs.3.rs-4598795/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-30T05:20:35+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"80477827187504777851192681745261920506","date":"2024-07-12T05:35:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-08T19:49:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-05T10:33:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"241081917031838758565917992545574700632","date":"2024-06-29T11:44:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320919434665574051842308301601570273105","date":"2024-06-29T10:31:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-29T07:15:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-21T07:48:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-21T07:48:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2024-06-18T09:09:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"26d25f96-f6b4-4548-917a-cc4e911de020","owner":[],"postedDate":"July 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:10:16+00:00","versionOfRecord":{"articleIdentity":"rs-4598795","link":"https://doi.org/10.1007/s00604-024-06711-8","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2024-09-25 15:57:31","publishedOnDateReadable":"September 25th, 2024"},"versionCreatedAt":"2024-07-11 07:52:17","video":"","vorDoi":"10.1007/s00604-024-06711-8","vorDoiUrl":"https://doi.org/10.1007/s00604-024-06711-8","workflowStages":[]},"version":"v1","identity":"rs-4598795","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4598795","identity":"rs-4598795","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}