Introduction

As one of the most toxic elements among heavy metals, lead ion (Pb2+) is a major and persistent environmental pollutant that could pose a risk to environment and human health (serious hazards to the brain, kidneys, and nervous system) with a multitude of toxicities causing carcinogenesis, cardiovascular disorders, and even impose a threat to human life [1–4]. To conquer these obstacles, numerous researchers are trying subtle approaches to dig out methods for selective and sensitive on-site detection of it. In recent years, several excellent techniques have been reported for the detection of Pb2+, including fluorescent [5–7], colorimetric [8, 9], photoelectrochemical [1], and surface plasmon resonance [10] methods. Widespread usage of these techniques is likely to be shadowed by possible limitations, such as sophisticated and expensive equipment, as well as complicated and time-consuming procedures. Therefore, exploring new protocols and strategies for monitoring of Pb2+ with improved sensitivity is still a challenge.

Benefiting from the relatively low background signal, high sensitivity, excellent time/spatial controllability, simplified optical setup, and wide dynamic range, electrochemiluminescence (ECL) technique has been widely employed in DNA detection, immunoassay, and cancer screening [11–14]. However, the assays based on conventional luminescent reagents possess some limitations, such as the loss of biological activity of the biomolecules [15], weak signal response in neutral solution [16], and the release of toxic cadmium ion [17]. Hence, searching for environmental-friendly ECL reagents was necessary. Over the past few decades, metal-free graphite-like carbon nitride (g-C3N4) nanosheet with excellent ECL property has attracted great attention with respect to its high water-dispersibility and good biocompatibility [18, 19]. Thus, keen interest has been triggered in g-C3N4 materials based ECL application. For example, Xu et al. developed a dual-signaling ECL ratiometric sensing platform for the sensitive and reliable detection of HL-60 cancer cells employing the prepared g-C3N4 nanosheets [20]. Gold nanoflower@graphitic carbon nitride polymer nanosheet–polyaniline hybrids was prepared by Yuan and co-authors for the ECL detection of dopamine [21]. Different from common prepared g-C3N4 nanosheets, herein fluorescent g-C3N4 quantum dots (QDs) with up-conversion properties were prepared through a thermal-chemical etching route using bulk g-C3N4 as precursor. The as-prepared g-C3N4 QDs with strong blue emission had abundant carboxyl groups at their surfaces, and exhibited stronger ECL activity than thin g-C3N4 nanosheets.

Currently, various Fe3O4 materials have been systematically investigated for their unique magnetic and chemical characteristic, especially the ability to be manipulated by an external magnetic force [22, 23]. Magnet-controlled molecular recognition sensors have become new tools for the sensitive detection of biomolecules (pesticide residue, heavy metal ion, tumor markers, etc.) in food, environmental, and clinical samples, offering the following advantages: (1) selective separation of the immobilized biomolecules from a reaction mixture with the aid of a magnetic field [24]; (2) more specific surface area for binding larger amounts of biomolecules [25]. In contrast with individual Fe3O4, magnetic nanoparticles (NPs) assembled onto the reduced graphene oxide nanosheets (Fe3O4-rGO) offer more significant use for biomagnetic separations as the carriers for targeted objects, since Fe3O4-rGO enables providng larger surface area for immobilization with biocompatibility [22, 26].

To make full use of the unique magnetic property of Fe3O4-rGO materials, a flow injection ECL cell was designed and constructed using indium tin oxide (ITO) glass as working electrode. Furthermore, employing multifunctional nanomaterials as labels is one of the most popular strategies to amplify the signal responses [27]. To be used in signal amplification for higher ECL response, a delicate balance for carrier was required between large immobilizing surfaces to allow more biomolecules (i.e., increase of the particle size) and relative small volume to remain suspended in solution and to form composite structure with acceptable diffusion kinetics (decrease of the particle size). Among the biomaterials commonly used, nanoporous metal materials have seized an ever increasing interest within the domain of chemistry and materials, since their interesting structure (bi-continuous nanoscale skeletons and interconnected hollow channels) enable easy mass transport and high electron conductivity [28, 29]. Notably, nanoporous gold (NPG) obtained by de-alloying has been extensively studied because of its high surface-to-volume, good biocompatibility, and ease of preparation [30]. Taking into account the above advantages, NPG with good biocompatibility could be employed as ideal carriers to load luminescence reagent.

In this study, g-C3N4 QDs with attractive up-conversion properties were prepared through a thermal-chemical etching route, and used to construct an ECL sensor for the sensitive monitoring of Pb2+ in real samples using target-dependent DNAzyme as the recognition unit. Functional rGO attached with Fe3O4 NPs that integrate both the electrically conductive property and the superparamagnetism were obtained and employed as carriers of the DNAzyme (S1). Particularly, the preparing method is simple since the reduction of GO to rGO and the in situ formation of Fe3O4 NPs on rGO sheets were accomplished in one step. NPG loaded with g-C3N4 QDs served as nano-carrier to immobilize signal strand (S3), resulting in the formation of g-C3N4 QDs@NPG-S3 bioconjugates. In the presence of Pb2+, S1 was activated and the substrate strand (S2) was cleaved. Then, with the incubation with g-C3N4 QDs@NPG-S3 bioconjugates, ECL signal could be measured. By integrating the g-C3N4 QDs with magnetic Fe3O4-rGO, lead(II) ions in real samples were successfully analyzed by the proposed ECL sensor, which showed a promising application in clinical and environmental analyses.

Experimental

Reagents and apparatus

Poly(diallyldimethylammonium chloride) (PDDA), melamine, triethanolamine (TEA), diethylene glycol (DEG), and ferric chloride (FeCl3) were obtained from Sinopfarm Chemical Reagent Co., Ltd. (Shanghai, China). ITO glass (thickness of ITO layer: 150 nm; resistance < 15 U/square; thickness of glass: 1.1 mm) was bought from Xiamen ITO Photoelectricity Industry (Xiamen, China). Ultrapure water (≥18.25 MΩ∙cm) obtained from a Lichun water purification system (Jinan, China) was used in all assays and solutions. All oligonucleotides used in this study were purchased from Sangon Biotech Co., Ltd. (Shanghai, China), and the sequences of oligonucleotides used in this work were as follows:

  • (S1) DNAzyme strand: 5′-SH-(T)7 CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG AGT-3′

  • (S2) Substrate strand: 5′-ACT CAC TAT rA GGA AGA GAT G-3′

  • (S3) Signal strand: 5′-SH-(CH2)6-AAC TCA CCT GTT AGAACT CAC TAT TTC GA-3′

ECL experiments was performed with a home-made system consisting of a flow injection luminescence analyzer (IFFM-E, Xi’an Remex Analysis instruments Co. Ltd., Xi’an, China) controlled by a personal computer, and combined with a CHI 760 electrochemical workstation (Shanghai Chenghua Instrument Co., China). A conventional three-electrode system was employ, including an ITO working electrode, a platinum counter electrode, and Ag/AgCl (sat. KCl) reference electrode. ECL signals from the flow injection ECL detecting cell were recorded by the photomultiplier tube (PMT) from the monitor window. The photoluminescence characteristic of g-C3N4 QDs was investigated with a rf-5310pc spectrofluorophotometer (Shimadzu, Kyoto, Japan). The morphology of prepared g-C3N4 QDs, Fe3O4-rGO, and NPG were measured with the aid of a QUANTA FEG 250 thermal field emission scanning electron microscopy (FEI Co., Hillsboro, OR, USA) and a JEOL JEM-1400 transmission electron microscopy (TEM) (JEOL, Tokyo, Japan). Powder X-ray diffraction (XRD) patterns were collected on a D8 advance diffractometer system equipped with Cu Kα radiation (Bruker Co., Karlsruhe, Germany). Ultraviolet-visible (UV–vis) absorption spectra were measured using a UV-3101 spectrophotometer (Shimadzu, Kyoto, Japan).

Design of the flow injection ECL detecting cell

To make full use of the superparamagnetism of prepared Fe3O4-rGO materials, a simple method was used to integrate three electrodes in a flow injection ECL detecting cell. As illustrated in Scheme 1, the flow injection ECL detecting cell was constructed with cuboid poly(methylmethacrylate) (PMMA). Different from conventional three-electrode electrochemical system, ITO glass (the diameter was 1 cm) was accepted as working electrode instead of conventional glass carbon electrode. Inlet and outlet were designed on the slide surface of the cell, which were nearly in a straight line with the ITO working electrode. Once the components were assembled by screws, ECL reaction could be carried out and the produced signal could be recorded by the PMT through the optical window.

Scheme 1
scheme 1

Schematic presentation of the constructed flow injection ECL cell

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One-pot synthesis of Fe3O4-rGO hybrid

Magnetic Fe3O4-rGO materials were prepared with the reduction of GO to rGO and the in situ formation of Fe3O4 NPs on rGO sheets in one step [23, 31]. Prior to the experiment, a NaOH/DEG stock solution was initially prepared by adding 200 mg NaOH into DEG (20 mL) and heated at 120 °C for 1 h under nitrogen atmosphere, and cooled down to 70 °C. GO was synthesized from natural graphite powder according to our previously reported article [32], and redispersed in 20 mL DEG by sonication for 1 h. Subsequently, 120 mg FeCl3 was added and stirred for 1 h. The above mixture was heated to 220 °C for 30 min under constant stirring and nitrogen flow protection. Then, 5 mL prepared NaOH/DEG stock solution (70 °C) was injected rapidly into the above hot mixture, and further heated at 220 °C for another 1 h. In the end, magnetic rGO nanosheets attached with Fe3O4 NPs were obtained after washing with ethanol several times. To get dry Fe3O4-rGO hybrid materials, the residue was paced in vacuum at 60 °C.

To provide loading site for target dependent DNAzyme, Au NPs were coated on the prepared Fe3O4-rGO hybrid materials with the aid of PDDA. Briefly, the homogeneous Fe3O4-rGO dispersion (10 mL) was mixed with 50 μL PDDA solution and stirred for 50 min. Subsequently, 10 mL prepared AuNPs solution [14] was added and sonicated for 30 min. After centrifugation, the supernatant was casted and the obtained Fe3O4-rGO-Au composites were further washed with water and redispersed in 10 mL water for further use.

Preparation of g-C3N4 QDs functionalized NPG

Bulk g-C3N4 were prepared by one-step thermal-induced self-condensation of melamine under atmospheric condition [18, 19]. Then, 100 mg of bulk g-C3N4 powder was ground well with a mortar and a pestle, followed by oxidation in concentrated H2SO4 (10 mL) and HNO3 (30 mL) for 16 h under mild ultrasonication (500 W, 40 kHz). Afterwards, a clear solution was formed, which was then diluted with ultrapure water to produce a colloidal suspension. To remove the acids, the colloidal suspension was filtered with microporous membrane. The obtained product was carefully redispersed in water under ultrasonication and transferred to a Teflon-sealed autoclave and maintained for 10 h at 200 °C. Finally, the blue g-C3N4 QDs were obtained after filtering through a 0.22 μm microporous membrane.

NPG was synthesized via selective dealloying of silver from Ag/Au alloy according to the previously reported article [32]. To make the g-C3N4 QDs functionalized NPG bioconjugates, g-C3N4 QDs with carboxyl groups [see Electronic Supplementary Material (ESM) Fig. S1] were modified with (3-aminopro-pyl)-triethoxy-silane through the formation of acrylamide bond with the aid of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide- hydrochloride. Then the amino-group functionalized g-C3N4 QDs were mixed with NPG and stirred for 5 h. Subsequently, the products were centrifuged and washed with water several times. In the end, the g-C3N4 QDs coated NPG composites (g-C3N4 QDs@NPG) were obtained.

To generate g-C3N4 QDs@NPG labeled S3, 30 μL of 50 μM HS-modified signal strand and 15 μL of 10 mM tris(2-carboxyethyl)-phosphine hydrochloride were mixed and incubated for 2 h at room temperature to cut any possible disulfide bonds. Then, the required amount of g-C3N4 QDs@NPG was added into the above solution and incubated for 16 h at room temperature under gentle shaking in a dark environment. The mixture was then washed and centrifuged to remove the nonconjugated oligonucleotides, followed by adding with 6-mercapto-1-hexanol (MCH) to block the nonspecific active binding sites. Finally, after washing with buffer solution, the obtained g-C3N4 QDs@NPG-S3 conjugate was dispersed in Tris-HCl buffer and stored at 4 °C until use.

Fabrication and analysis procedure of the ECL sensor

The construction and principle of the ECL sensing system for Pb2+ ion is depicted in Scheme 2. First, the resulting Fe3O4-rGO-Au composites were coated with DNAzyme strands via incubation with 10.0 μL S1 (1.0 mM) for 6 h at 37 °C. Unbound oligonucleotides were removed by discarding the supernatants with the aid of a permanent magnet. Subsequently, the obtained product was washed with Tris-HCl buffer solution, and 0.2 mg MCH was added and incubated for 30 min to resist the nonspecific adsorption. After washing with buffer solution, the functionalized magnetic composite was mixed with 10 μL substrate strand (1.0 mM) and incubated in a 65 °C water bath for 10 min. After that, the reaction solution gradually cooled down to room temperature. Then, the prepared sensor was incubated with different concentrations of Pb2+ to react for 1 h at ambient temperature. In the presence of target, the DNAzyme was activated and cleaved the substrate strand, resulting in free DNAzyme strand and two fragments, respectively. After thorough rinsing, the modified magnetic rGO was incubated with synthesized g-C3N4 QDs@NPG-S3 conjugates. After hybridization, the product was extensively rinsed with Tris–HCl buffer and transported into the designed ECL detecting cell with a flow injection system. With the aid of an external magnet, the formed conjugates were accumulated on the surface of the ITO working electrode. After washing with buffer solution, ECL detection was performed with the injection of Tris–HCl buffer solution containing 0.1 M KCl and 20 mM TEA. After determination, the ITO electrode was washed with the aid of flow injection system using buffer solution and ultrapure water. Then the ITO electrode could be cyclic utilized.

Scheme 2
scheme 2

Fabrication and analysis protocol of the proposed Pb2+ sensor

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Results and discussion

The properties of flow injection ECL detecting cell

Exploiting the ubiquity of magnetic rGO for quantitative chemical sensing of lead(II) ions imposes strong demands on detecting devices. The desired products should be autonomous, disposable, and integrate enrichment and separation functions. As expected, we demonstrated such a concept and its implementation as flow injection ECL detecting cell (Scheme 1). Compared with traditional detecting cells, the proposed device could make full use of the magnetic characteristics of prepared Fe3O4-rGO materials with apparent advantages as follows:

  1. 1.

    The low-cost PMMA makes it competitive to commercial detecting cells.

  2. 2.

    The self-designed detecting cell could meet more requirements of customers.

  3. 3.

    Relative smaller dead volume was obtained. Since the inlet and outlet were designed on the slide surface of the cell, which were nearly in a straight line with the ITO working electrode.

  4. 4.

    Employing ITO glass slide as working electrode not only reduced the cost but also increased the effective working area. Furthermore, putting ITO working electrode opposite the transparent window resulted in nearly 100 % of the ECL signals generated at the ITO surface that could be recorded by the PMT.

Structure characterization

It is well-known that graphene is seldom employed as a direct precursor material to obtain graphene-based materials since the solubility of pure graphene in solvents is poor owing to the absence of functional moieties [22, 31]. Thus, GO is always used as a suitable substitute to synthesize graphene-based hybrids because it possesses plentiful oxygen-containing functional groups and can be steadily suspended in water and polar organic solvents. Although the preparation and reduction of GO is feasible, once GO has been integrated with other components, it is difficult to be completely reduced as graphene without causing aggregation of rGO nanosheets. This indicates that it is difficult to achieve well-dispersed Fe3O4-rGO sheet. Thus, if GO could be reduced during the synthesis process of composites, the protocol will be magnificent. Herein, Fe3O4 NPs were attached on the surface of rGO nanosheets by in situ precipitation method. The results confirmed that the one-step approach is superior indeed. Compared with pure GO (Fig. 1a), it could be seen that the surface of rGO has been densely covered by narrowly distributed Fe3O4 NPs (Fig. 1b) with an average diameter of 12.1 nm (Fig. 1c). It may be attributed to the oxygen-containing functional groups on GO surface. In the reaction, iron ions were first interacted with oxygen-containing functional groups on GO surface, which acted as the nucleation center. Then, it was transformed into Fe3O4 NPs in the presence of reducing solvent of DEG with the addition of NaOH at elevated temperature in situ, giving rise to Fe3O4-rGO hybrid nanosheets. Moreover, the rGO nanosheets provided a large surface area for the assembly of the magnetic nanoparticles at the top and the bottom of nanosheets.

Fig. 1
figure 1

TEM images of prepared (a) GO and (b) Fe3O4-rGO materials. (c) Size distribution analysis of Fe3O4 nanoparticles

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As shown in Fig. 2a, the NPG obtained by selective dealloying of silver from Ag/Au alloy displayed a type of sponge-like pore structure. Benefitting from its unique nanoporous structure, NPG possess high specific area and good biocompatibility, which enhances the immobilized amount of targets. Furthermore, wire-like electron transfer rate in the ECL reaction is achieved because of the high conductivity in NPG. The morphology of bulk g-C3N4 was observed by SEM and TEM. As shown in Fig. 2b, the bulk g-C3N4 were solid agglomerates about several micrometers in size. Figure 2c shows the representative TEM image of as-prepared fake-like bulk g-C3N4 with average sizes around several microns after manual milling by an agate mortar, which was the analogue of wrinkled rGO. As expected, the size of bulk g-C3N4 decreased dramatically after hydrothermal treatment. The morphologies of g-C3N4 QDs are depicted in Fig. 2d with dots well separated from each other.

Fig. 2
figure 2

Representative SEM images of prepared NPG (a) and bulk g-C3N4 (b). Representative TEM images of bulk g-C3N4 (c) and g-C3N4 QDs (d)

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To further confirm the formation of g-C3N4 QDs, XRD patterns of bulk g-C3N4 and g-C3N4 QDs were also carried out for comparison. As shown in Fig. 3a, two characteristic peaks commonly found in carbon nitride, one attributed to the typical interplanar stacking peak of conjugated aromatic systems at 27.21°, the other one belonging to an in-planar structural packing motif at 13.02°, were observed in bulk g-C3N4 [33]. After treatment with acid and hydrothermal approach, the strong diffraction peak at 27.21° still existed. However, the other peak was weakened, indicating that the g-C3N4 QDs was achieved. Figure 3b and c show the UV-vis absorption spectrogram and fluorescence (FL) emission spectrum of prepared g-C3N4 QDs. As shown in the inset of Fig. 3c, the as-synthesized g-C3N4 QDs emit bright blue luminescence under UV excitation, suggesting significant potential as applications in bioimaging and biolabeling. Interestingly, it was found that the prepared g-C3N4 QDs has obvious up-conversion characteristics when irradiated with long-wavelength light. As shown in Fig. 3d, once g-C3N4 QDs aqueous solution was excited by red light (643, 680, and 700 nm), up-conversion emissions in visible-light region (400 to 550 nm) were generated. Based on previous reported literature, the up-conversion emission of g-C3N4 QDs might be ascribed to the multiphoton active process [34, 35].

Fig. 3
figure 3

(a) XRD spectra of bulk g-C3N4 and g-C3N4 QDs. (b) UV-vis absorption spectrum, (c) FL emission spectrum (λEX = 365 nm), and (d) the upconversion FL spectra of the prepared g-C3N4 QDs, curve a (λEX = 643 nm), curve b (λEX = 680 nm), curve a (λEX = 700 nm). The inset in (c): photograph of the g-C3N4 QDs aqueous solution irradiated by 365 nm UV light

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Possible mechanism for the ECL behavior of g-C3N4 QDs

According to previously reported anodic and cathodic ECL behaviors of various semiconductors [13, 19, 32], the possible ECL mechanism of g-C3N4 QDs-TEA system could be inferred. When the electrode was scanned from 0 to 1.9 V, the g-C3N4 QDs immobilized on magnetic rGO were oxidized to positively charged g-C3N4 (g-C3N4 •+) (Eq. 1), since the applied potential was more positive than the valence band of g-C3N4 [19, 36]. Meanwhile, when the potential was positive enough, co-reactant TEA employed in the present system could be electro-oxidized to a cation (TEA•+), as shown in Eq. 2, and then, upon deprotonation of an α-carbon from one of the hydroxyethyl groups, TEA•+ would subsequently decompose to produce a radical of powerful reductive properties (TEA•) [37], as shown in Eq. 3, which could react with the positively charged g-C3N4 (g-C3N4 •+) to obtain the excited state g-C3N4 (g-C3N4*) through electron transfer in the aqueous solution (Eq. 4). In the end, an intense emission was obtained when g-C3N4* fell from the excited state to the ground state g-C3N4 (Eq. 5). Corresponding ECL mechanisms were presented as follows:

$$ \mathrm{g}\hbox{-} {\mathrm{C}}_3{\mathrm{N}}_4-{\mathrm{e}}^{-}\to \mathrm{g}\hbox{-} {\mathrm{C}}_3{{\mathrm{N}}_4}^{\bullet +} $$
(1)
$$ \mathrm{TEA}-{\mathrm{e}}^{-}\to {\mathrm{TEA}}^{\bullet +} $$
(2)
$$ {\mathrm{TEA}}^{\bullet +}\hbox{-} {\mathrm{H}}^{+}\to {\mathrm{TEA}}^{\bullet } $$
(3)
$$ \mathrm{g}\hbox{-} {\mathrm{C}}_3{{\mathrm{N}}_4}^{\bullet +}+{\mathrm{TEA}}^{\bullet}\to \mathrm{g}\hbox{-} {\mathrm{C}}_3{\mathrm{N}}_4* $$
(4)
$$ \mathrm{g}\hbox{-} {\mathrm{C}}_3{\mathrm{N}}_4*\to \mathrm{g}\hbox{-} {\mathrm{C}}_3{\mathrm{N}}_4+\mathrm{h}v $$
(5)

Analytical characteristics

To achieve the best analytical result for our designed system, two key experimental parameters were investigated, including pH value and TEA concentration. As shown in Fig. 4a, it was obvious that the ECL response was dependent on the solution value. The signal intensity increased with the increase of buffer solution pH value up to 7.4, and then began to drop. The performance could be ascribed to two reasons: (1) highly acidic or alkaline value would affect the activity of oligonucleotides; (2) the hydrogen ion or hydroxyl concentration influenced the ECL reaction process. In the acid solution, it was difficult for the TEA radical cation (TEA·+) to deprotonate to produce a radical of powerful reductive properties (TEA·), which played a significant role in the ECL intensity. Meanwhile, in the alkaline environment, hydroxyl would compete with TEA· to react with g-C3N4·+, inhibiting the formation of excited-state g-C3N4 (g-C3N4*). A similar performance had been reported in the previous anodic ECL system [19, 38]. Thus, pH 7.4 buffer solution was chosen for subsequent experiments. Figure 4b illustrates the influence of co-reaction regent (TEA) concentration on the ECL response. It was found that the ECL intensity was improved with the increase of TEA concentration up to 20 mM, since more excited g-C3N4 (g-C3N4*) would be achieved from electron-injection into the positively charged g-C3N4 (g-C3N4·+) by further generated radical TEA·, as described in Eqs. 2–4. Meanwhile, once the TEA concentration was higher than 20 mM, the ECL intensity was weakened, which was the result of excess co-reactant reacting readily with the positively charged g-C3N4 and inhibiting the formation of excited-state g-C3N4 (g-C3N4*). Therefore, 20 mM TEA was employed in this work.

Fig. 4
figure 4

The influence of the pH (a) and TEA concentration (b) on ECL intensity

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Under optimum experimental conditions, the proposed ECL sensor was employed for the detection of Pb2+ at various concentrations. As described in Fig. 5a, ECL intensity increased accordingly upon increasing the Pb2+ concentration in the range from 0.05 to 20 nM. Figure 5b illustrates the corresponding calibration plots of the ECL intensity with the Pb2+ concentration, whereas the inset shows a good linear relationship between ECL intensity and the logarithm of Pb2+ concentration. It was found that the regression equation is I = 2569.94logc + 3376.23 with a correlation coefficient of 0.996, where I and c represent the ECL signal intensity and the Pb2+ concentration, respectively. The detection limit was 0.02 nM (S/N = 3), which is adequate for the analysis of drinking water and environmental samples [39]. Compared with previously reported analytical methods (Table 1), the proposed sensor showed a better sensitivity, which might be ascribed to the unique separation and enrichment properties of magnetic Fe3O4-rGO materials as well as wire-like conductivity of NPG. Furthermore, it is obvious that target-dependent DNAzyme was necessary for all of the sensitive detecting methods for Pb2+, just like in Refs. 4 and 39. Different from previous articles, magnetic characteristics were used to enrich the low concentration target.

Fig. 5
figure 5

(a) ECL response of the proposed sensor for Pb2+ at various concentrations. From 1 to 7: 0.05, 0.10, 0.50, 1.00, 2.00, 10.00, 20.00 nM. (b) The relationship between ECL intensity and different concentrations of lead ions. (c) Selectivity of the proposed method for Pb2+ ions over other metal ions. The concentration of Pb2+ was 5 nM. The concentration of Hg2+, Cd2+, Cu2+, Zn2+, Ni2+, Co2+, Ca2+, Mg2+, Mn2+, Fe2+, Al3+, and K+ was 100 nM)

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Table 1 Comparison of different methods for Pb2+ detection
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The stability of the sensor was investigated. The as-prepared Pb2+ sensor was stored in the refrigerator (4 °C) for 1 wk, and almost no signal intensity changed significantly. Moreover, the storage time was prolonged to 2 wk Over 94.2 % of original responses were retained. Consequently, the proposed immunoassay system possessed perfect stabilization in practical applications.

Furthermore, specificity was another important issue to assess the performance of a newly proposed sensor. Thus, ECL signal intensity was measured in the presence of Pb2+ (5 nM) and various other metal ions (100 nM), such as Hg2+, Cd2+, Cu2+, Zn2+, Ni2+, Co2+, Ca2+, Mg2+, Mn2+, Fe2+, Al3+, and K+. As depicted in Fig. 5c, the influences of other metal ions were negligible, even though the concentration of other metal ions was up to 100 nM, which was 20-fold higher than that of the Pb2+ ions. The satisfactory results were ascribed to high binding selectivity of the DNAzyme for Pb2+. In summary, the proposed sensor exhibited favorable selectivity for the determination of Pb2+.

As the reproducibility is a very important feature for sensors, the reproducibility of the developed sensor was evaluated with intra- and inter-assay precision. Intra-assay and inter-assay precision of the sensors were carried out by assessment of two concentration levels (100 and 500 nM) for seven sensors at the same and different batches, respectively. As shown in ESM Table S1, both the intra- and the inter-assay RSDs were in the range of 3.7 to 8.4 %, which indicated an acceptable reproducibility of the proposed approach.

Screening of real samples

To evaluate the reliability of the proposed methodology, real environmental samples, including mineral water, pure drinking water, and snow water were used for the practical assay (Table 2). No Pb2+ was detected in all water samples. Therefore, the standard addition method was used to evaluate the practicality of the developed approach. When spiked with 0.5, 1.0, and 5.0 nM Pb2+ standard solutions, satisfied recoveries of 94.0–106.0 % for mineral water, 95.0–108.0 % for pure drinking water, and 92–105.4 % for snow water were obtained. The relative standard deviations (RSDs) for the three samples were all less than 8.22 %, indicating an acceptable accuracy. Furthermore, environmental water samples obtained from local rivers were detected by the proposed sensor and ICP-MS method (ESM Table S2). It was found that lead concentration of the samples determined by the proposed sensor coincided quite well with that determined by ICP-MS. The results confirmed that the g-C3N4 QDs based ECL sensing system has potential as an application for the measurement of lead(II) ions in real environment samples.

Table 2 Recovery experiments of Pb2+ in real water samples
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Conclusions

Fluorescence g-C3N4 QDs with attractive up-conversion properties were prepared through a thermal-chemical etching route, and used to construct an ECL sensor for the sensitive monitoring of Pb2+ in real samples using target-dependent DNAzyme as the recognition unit. To achieve high sensitivity, magnetic Fe3O4 functionalized rGO with excellent conductivity were prepared and used as carriers for DNAzyme. Benefits from its superparamagnetism characteristics, rapid separation, and enrichment of targets were easily realized. Furthermore, NPG with sponge-like pore structure were used to immobilize g-C3N4 QDs to amplify ECL signal. Employing self-designed ECL detecting cell, the proposed sensor showed high sensitivity and selectivity for the determination of Pb2+ in real water samples. The present work not only may enrich the foundation study about the ECL characteristic of g-C3N4 QDs but also would promote its practical application.