FULL PAPER

1901170 (1 of 8) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.small-journal.com

Aggregation-Induced Electrochemiluminescence by Metal-Binding Protein Responsive Hydrogel Scaffolds

Hui Jiang, Zhaojian Qin, Youkun Zheng, Liu Liu, and Xuemei Wang*

Dr. H. Jiang, Z. J. Qin, Y. K. Zheng, L. Liu, Prof. X. M. WangState Key Laboratory of BioelectronicsNational Demonstration Center for Experimental Biomedical Engineering EducationSchool of Biological Science and Medical EngineeringSoutheast UniversityNanjing 210096, P. R. ChinaE-mail: xuewang@seu.edu.cn

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.201901170.

DOI: 10.1002/smll.201901170

In contrast to ACQ agents, the low lumi-nescent AIE molecules, i.e., AIEgens, can be conjugated together, resulting in sig-nificantly enhanced luminescence. Such a phenomenon is initially reported for organic compounds,[8b] complexes,[9] or polymers[10] containing special rotor-like structures, but now has been observed for metallic nanoclusters (NCs). In this aspect, Xie’s group has intensively reported the AIE by different NCs.[11] For example, the mixture of glutathione and chloroaurate, namely, [Au (I)-SG] com-plex, may exhibit a sharp increase in PL with the increasing ratios of ethanol/water.[12] In another work, the AIE mecha-nism was proposed to explain the high PL quantum yield in [Au22(SG)18] NCs.[13] The

AIE by NCs may offer highly bright and stable PL for assays and bioimaging applications.[14]

Electrochemiluminescence (ECL), or so-called electrogen-erated chemiluminescence, is an important technique for the development of highly sensitive sensors, which realizes the light release by the collision of excited species generated during electrochemical processes. Considering the intimate relevance between ECL and PL, it seems plausible that the aggregation events can also induce enhanced ECL, i.e., aggregation-induced ECL (AIECL). This topic, however, does not evoke sufficient concerns until recently. The first paradigm of AIECL was reported for a pyridine-containing Pt (II) complex in 2017.[15] Sequentially, several systems, including cyclometalated-Ir (III) complex,[16] organic nanoparticles,[17] and polymer dots,[18] may cause AIECL on the basis of the large differences of materials in organic and aqueous solubility. To the best of our knowledge, no metallic NC-based AIECL has been reported. In the mean-time, the applications in these previous works are still limited in the assays of common small molecules.

Herein, we have developed a gel-based AIECL system by bivalent metal ion induced NC aggregation. In aqueous buffer, the obtained gel may offer a maximum of 50-fold enhancement in ECL emission, in comparison with that of approximately fivefold in PL emission under same conditions. In virtue of the adsorption process on the electrode, calmodulin (CaM), an important calcium-binding protein, can be used to regulate the ECL dynamics, while no similar effect is observed for PL processes (Scheme 1). Since the proposed system is completely fabricated in aqueous solution, i.e., without the engagement of any organic phases, it may pave a new avenue for facile design of a biomacromolecule-responsive AIECL-type hydrogel with multifunctional purposes.

Functionalized hydrogels have aroused general interest due to their versa-tile applications in biomaterial fields. This work reports a hydrogel network composed of gold nanoclusters linked with bivalent cations such as Ca2+, Mg2+, and Zn2+. The hydrogel exhibits both aggregation-induced emission (AIE) and aggregation-induced electrochemiluminescence (AIECL) effects. Most noteworthy, the AIECL effect (≈50-fold enhancement) is even more significant than the corresponding AIE effect (approximately fivefold enhance-ment). Calmodulin, a Ca2+ binding protein, may efficiently regulate the AIECL dynamics after specific binding of the Ca2+ linker, with the linear range from 0.3 to 50 µg mL−1 and a limit of detection of 0.1 µg mL−1. Considering the important roles of bivalent cations in the life system, these results may pave a new avenue for the design of a biomolecule-responsive AIECL-type hydrogel with multifunctional biomedical purposes.

Aggregation-Induced Electrochemiluminescence

1. Introduction

Nano or micro-sized hydrogels have been a focus in multi-farious biomedical fields, such as biosensing,[1] antibacte-rial,[2] drug delivery,[3] tissue engineering,[4] and construction of ionotronic devices,[5] due to their excellent biocompatibility, porous polymeric networks, and stretchable mechanistic prop-erties.[6] The introduction of different types of physical and chemical stimuli endows the hydrogels with versatile features, facilitating the fabrication of electric-magnetic, light, thermo, acoustic, pH, and biomolecule-responsive soft materials.[7] To design a stimulus-triggered system, the gel monomers are usually modified with external labels such as electro-active probes or luminophores. For luminophore-linked gel monomers, the assembly to nanogel may accompany with the decrease in spacing of emitters, which may result in the loss in photoluminescence (PL) because of well-known aggrega-tion-caused quenching (ACQ) effect. However, the concept of aggregation-induced emission (AIE) developed in recent years has brought tremendous impact on this traditional view.[8]

Small 2019, 1901170

1901170 (2 of 8)

www.advancedsciencenews.com

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.small-journal.com

2. Results and Discussion

2.1. PL Enhancement of Au NCs by Bivalent Cations

Oligo-DNA chains have been demonstrated as excellent tem-plates for preparation of NCs.[19] Their versatile coordina-tion features make it possible that even single nucleoside/tides can act as effective stabilizers for Au NCs. Lopez et al.[20] used adenosine series compounds for preparation of lumines-cent NCs and larger coordination polymers. We also proposed the effective synthesis of multicolored Au NCs by using cyti-dine (Cyt) series templates.[21] By using an oligonucleotide as the stabilizer, the NCs can be linked to form the hydrogel by using a matched complementary DNA/RNA sequence. How-ever, for NCs coated with a single base, this strategy seems not feasible. An alternative route may be the introduction of metal ions, because nucleotide and metal ions are known to form the hydrogel via strong coordination bonds.[22]

Here we have observed that adenosine phosphate (AXP, where X represents M, D, or T, i.e., adenosine monophosphate, diphos-phate, or triphosphate) coated Au NCs (AuAXP) are sensitive to bivalent cations, such as Ca2+, Mg2+, and Zn2+. In ultrapure water, the PL emission intensity of AuAXP of 10 µg mL−1 significantly increases upon addition of these metal ions with concentrations ranging from 5 to 30 × 10−6 m, while no changes in emission wavelengths are observed (Figure 1 and Figure S1, Supporting Information). The PL enhancement

effect may be caused by the strong binding of these ions to phos-phate group on the ligands. As known, in nucleoside/tide-coated NCs, the metal coordination sites are N atoms from bases, usually N7 position in adenine and N3 in cytosine. At the same time, the hydrophilic and negatively charged phosphate groups are exposed to aqueous environments, making the whole NCs soluble and stable. Thus these phosphate groups are liable to capture certain metal ions and form metal linked networks. In our cases, the PL enhancement ratio is similar for Ca2+, Mg2+, and Zn2+ (Table S1, Supporting Information), with an exception case for the combination of Ca2+ and AuADP. Considering the different ionic radii (Ca 99 pm, Mg 72 pm, and Zn 74 pm), it may be reasonable that Ca2+ gives a rela-tively low PL enhancement due to a mismatched size with AuADP. The average enhancement ratio may be up to ≈1.4, ≈4.7, and ≈10-fold for AuAMP, AuADP, and AuATP, respectively. As to the PL enhance-ment ratio, it shows an order of AuAMP < AuADP < AuATP, perhaps caused by the increasing charges on

the ligands. Accordingly, such PL enhancement may be attrib-uted to the AIE effect, as reported for glutathione-coated Au (I) polymers.

We also attempted to confirm that the PL enhancement belongs to a nondynamic type, in which the small metal ions are unbound to luminophores and can be removed by ultracen-trifuge. First, AuATP has a molecular weight (MW) larger than 3 kDa, because after ultracentrifuge of AuATP sample (black curve, Figure S2, Supporting Information), only high molecular MW part (MW larger than 3 kDa; magenta curve, Figure S2, Supporting Information) still shows significant PL enhance-ment upon addition of Ca2+ (purple curve, Figure S2, Supporting Information). In comparison, when the mixture of AuATP and Ca2+ (red curve, Figure S2, Supporting Information) was sub-jected to ultracentrifuge, very weak PL was found for both high MW part (green curve, Figure S2, Supporting Information) and small MW part (blue curve, Figure S2, Supporting Information). The further addition of Ca2+ of 20 × 10−6 m to high MW part also gives no PL enhancement (cyan curve, Figure S2, Supporting Information). In this case the mixture of AuATP and Ca2+ was actually trapped on the ultracentrifuge film (Figure S2, inset, Sup-porting Information), implying the aggregation of NCs.

Another issue is that although these bivalent cations may be mainly responsible for PL enhancement, the possible influences by ionic strengths of saline cannot be omitted. Several cations (Na+, K+) were tested by using AuATP as a model (Figure S3A, Supporting Information). At 10 × 10−6 m

Small 2019, 1901170

Scheme 1. Illustration of bivalent cations (Ca2+ as an example) induced aggregation of NCs with CaM-regulated ECL.

1901170 (3 of 8)

www.advancedsciencenews.com

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.small-journal.com

level, no PL changes are observed. Note that the anions (Cl−, NO3−)

here also act as counterpart ions in Ca2+, Mg2+, and Zn2+ salts we used. This result can verify the negligible ionic strength effect. Therefore PL changes are only caused by several special cations. We also checked the PL influence by typical heavy metal ions. Fe2+, Hg2+, Cu2+, and Ag+ only cause PL quench at 10 × 10−6 m (Figure S3B, Supporting Information). Slight PL enhancement can be observed for Ni2+ (69 pm, ≈1.5-fold) and Mn2+ (67 pm, ≈2.5-fold) at 10 × 10−6 m, while Cd2+ ions (95 pm), with a similar ionic size and coordination feature of Ca2+ (99 pm), can greatly enhance the PL up to sixfold at the same con-centration. These results indicate that the ionic radius of bivalent metal ions may greatly affect PL enhancement ratio. On the other hand, although citrate is also present on the surface of Au NCs, it may show limited interaction with Ca2+ compared with phos-phate because the dissociation constant for Ca2+-citrate is only 1.7 × 10−3, much larger than Ksp for Ca2+-phosphate (2.0 × 10−29). In the meantime, we observe that ethylenediamine tetraacetic acid, a well-known Ca2+ or Zn2+ chelator, may partially decrease the enhanced PL, suggesting that the PL enhancement is mediated

by bivalent metal ions (Figure S3C,D, Supporting Information). Generally, it can be deduced that only invariably bivalent cations can induce PL enhancement through their solid interaction with phosphate groups.[23] The ionic sizes can affect their binding capa-bility and thus the PL enhancement ratios, similar to those results reported by crown ether cavity based chemosensors.[24]

To give a clear scene of the network, we used transmission electron microscopy (TEM) to check the morphology in the presence of bivalent cations. Take the combination of Ca2+ and AuATP as an example. For AuATP, NCs of around 2 nm can be observed with organic backgrounds. After addition of bivalent ions, it clearly shows the aggregated rod-like AuATP assembly (Figure 2). Au NCs are decorated inside the matrices. The width of these rod-like structures is around 20 nm. The high-resolu-tion TEM shows a crystal lattice of 0.52 nm (Figure S4, Sup-porting Information), similar to (101) facet for hydroxyl apatite (JCPS 86-0740).[25] This clearly shows the formation of a composite structure, namely, Ca2+@AuATP. We may describe the hydrogel as hydroxyl apatite like structure because of the coexistence of calcium, hydroxyl groups (from ribose), and phosphate groups.

Small 2019, 1901170

Figure 1. PL monitoring of titration of A) Ca2+, B) Mg2+, and C) Zn2+ to AuAMP (black), AuADP (red), and AuATP (green), respectively. The concentra-tion of AuAXP NCs is 10 µg mL−1. The final concentration of bivalent cations is 30 × 10−6 m, with an aliquot of 5 × 10−6 m for each addition. Excitation/emission wavelength: 350 nm/480 nm. D) Comparison of PL enhancement ratio of AuAXP in the presence of Ca2+, Mg2+, and Zn2+.

1901170 (4 of 8)

www.advancedsciencenews.com

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.small-journal.com

As comparison, we also checked the bivalent ions effect on cytidine and cytidine phosphate coated Au NCs (Figure S5A, Supporting Information). It is reasonable that no significant PL enhancement is observed for cytidine-coated Au NCs due to the absence of phosphate groups. However, for AuCMP and AuCTP NCs, the PL still shows very limited increase even at a concentration of Ca2+ up to 100 × 10−6 m, Mn2+ and Cd2+ up to 200 × 10−6 m (Figure S5B, Supporting Information). The TEM image also shows that these NCs cannot form aggregate in the presence of Ca2+ (Figure S6, Supporting Information). These results suggest that AXP-coated Au NCs may be unique for their AIE behaviors.

2.2. ECL Enhancement of Au NCs by Bivalent Cations

Although a lot of works have attempted to construct a frame-work between PL and ECL, their detailed relationships are still not unveiled.[26] Since the first work around ECL of fluorescent Ag NCs reported in 2009,[27] an increasing number of works have been focused on this field for various sensing applications, including both anodic and cathodic ECL.[28] These works pro-vided more possibility in the discovery of novel highly efficient ECL systems.

Inspired by these backgrounds, we further investigated the anodic ECL features of AuAXP NCs before and after biva-lent cations induced aggregation. Because AuATP shows the most sensitive response to cations, we use AuATP as a repre-sentative in the following experiments. Although AuATP NCs show typical PL features, their ECL emission is rather weak on glass carbon electrode (GCE) at a constant potential of +1.4 V (black curve, Figure 3). The ECL increases significantly in the 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (i.e., 10 × 10−3 m HEPES of pH 7.0 containing 10 × 10−3 m NaCl and 10 × 10−3 m triethylamine) with Ca2+ up to 500 × 10−6 m, indicating the occurrence of AIECL behaviors. The dynamic ECL may reach maximum in 1 min or so under bubbling with N2, since the ECL emission will decay to baseline level in 5 s in the static state (Figure S7A, Supporting Information). After the 1 min treatment at the constant potential, the cyclic voltam-metric mode was used to check the ECL co-reaction. An electro - chemical oxidation peak is observed around +1.4 V after addition of Ca2+ (Figure 3B, inset), implying the enhanced co-reaction by NC species and triethylamine, as the proposed processes shown in Scheme S1 (Supporting Information). This process results in the AIECL with a peak potential of around +1.4 V and a steady peak intensity of 50-fold larger than that for AuATP NCs only. Note that this enhancement effect is much larger than the PL enhancement

Small 2019, 1901170

Figure 2. TEM images of AuATP NCs A) before and B) after addition of Ca2+.

Figure 3. A) Dynamic ECL by AuATP NCs of 0.1 mg mL−1 and different concentrations of Ca2+ (from bottom to top: 0, 100, 200, 300, 400, and 500 × 10−6 m) in HEPES buffer. Applied potential: +1.4 V versus Ag/AgCl. B) Potential-dependent ECL emission by AuATP NCs of 0.1 mg mL−1 and different concentrations of Ca2+ (from left to right: 0, 100, 200, 300, 400, and 500 × 10−6 m) using cyclic voltammetric scans. Inset: The corresponding cyclic voltammetric curves.

1901170 (5 of 8)

www.advancedsciencenews.com

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.small-journal.com

ratio (approximately fivefold for AuATP in HEPES buffer; Figure S8, Supporting Information). Generally, besides the instinct AIE effects, the main reason for more significant AIECL emission may be attributed to the improved accessibility of electrode surface to metal-linked gel structures than ultrasmall NCs. After treatment in HEPES buffer containing Ca2+@AuATP at +1.4 V for 1 min, the GCE still shows an observable ECL signal in Ca2+@AuATP-free HEPES buffer, indicating the adsorption of Ca2+@AuATP on GCE (Figure S7B, Supporting Information). Moreover, the scanning electron microscopy (SEM) images on the GCE also show the adsorption of Ca2+@AuATP gel and the X-ray energy dispersive spectrometer (EDS) spectrum validates the presence of Au and P (Figure S9, Supporting Information). Sequentially, the Ca2+@AuATP gel was used for the following applications.

Here the AIECL is selective to electrodes, as no ECL signals are observed for Ca2+@AuATP gel modified on common gold, platinum, and indium-tin oxide electrodes (Figure S10B, Sup-porting Information). The different ECL behaviors on different electrodes are quite common. For example, we have noted the ECL only on glass carbon electrode for glutathione-capped Au NCs[29] and CdSe quantum dots.[30] The ECL inertness may be attributed to the inefficient electron exchanges on these elec-trodes, evidenced by the absence of above-mentioned electro-chemical oxidation peak of triethylamine around +1.4 V on GCE (Figure S10A, Supporting Information).

Besides electrodes, we also noted that the ECL enhance-ment is also dependent on buffer saline. Compared with that in HEPES buffer, both PL and ECL enhancement by Ca2+ was not significant in 0.1 m phosphate buffer saline (PBS) or 50 × 10−3 m tris(hydroxymethyl) aminomethane (tris) buffer at pH 7.4 (Figures S11 and S12, Supporting Information). This may be relevant with the binding of phosphate or chelation of tris with bivalent cations. So in all experiments, we select HEPES buffer, in which the AIECL effect can be well maintained. In the mean-time, the AIECL intensity is relevant with the concentrations of Ca2+@AuATP gel (Figure S13, Supporting Information).

The ECL spectrum for Ca2+@AuATP was also analyzed through different cut-off filters placed on the top of the photomultiplier tube in the chemiluminescence detector (Figure 4). A typical ECL peak around 500 nm is similar with the PL emission,

while another shoulder emission appears at ≈560 nm. The maximum ECL intensity for this shoulder is about one third of that located at 500 nm. We have also checked the ECL spec-trum for AuATP NCs, which shows a similar spectrum to that for Ca2+@AuATP. In our previous work, a similar ECL emis-sion with a redshift of 50 nm in wavelength is observed for thioglycol attached glutathione coated Au NCs.[29] Ding’s group has also inspected the ECL mechanistic details of thiolate NCs in organic solvent such as acetonitrile.[31] The changes in ECL emission are not rare for NCs with different charges and different concentrations of co-reactants (tri-n-propylamine in most works).[32] In this case, compared with PL emission, the redshift in ECL emission should be caused by the dif-ferent charged NC species generated by hole/electron injec-tion during electrochemical processes. Moreover, the changes in valence states inside NCs can regulate the ECL efficiency.[33] So here the shoulder ECL peak indicates the presence of some unique species that may be diverse from the light-excited species. Nevertheless, the following ECL assays will not be affected by the various mechanisms because the ECL signal includes the integrated luminescence within a wide range of wavelengths to eliminate the influence.

2.3. AIECL Regulated by CaM

As a Ca2+ binding messenger protein of 16.7 kDa, CaM has four Ca2+ binding sites and may sense Ca2+ selectively. For Ca2+@AuATP with abundant Ca2+ linkers in the gel structure, this small protein may also conjugate to these linkers. Although this process cannot be monitored by PL probably because CaM may not reverse the aggregation state (Figure S14, Sup-porting Information), it may affect the AIECL dynamics by Ca2+@AuATP. Upon addition of CaM of 10 µg mL−1 to buffer containing Ca2+@AuATP, the stable ECL signal decreases by ≈65% (Figure 5A), mostly because the binding of CaM to Ca2+@AuATP may retard the adsorption of Ca2+@AuATP. As a comparison, bovine serum albumin (BSA) of same concentrations shows little influence on the dynamic ECL of Ca2+@AuATP, indicating that the ECL changes originate from

Small 2019, 1901170

Figure 4. ECL spectra of Ca2+@AuAXP. A) The ECL intensity obtained by using 11 pieces of light filters with band cut-off wavelengths from 400 to 650 nm. B) The spectrum obtained by the difference between ECL intensity obtained by two adjacent light filters.

1901170 (6 of 8)

www.advancedsciencenews.com

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.small-journal.com

the specific binding of CaM and Ca2+@AuATP. We also use an indirect method to show the specific binding of CaM and Ca2+@AuATP in virtue of two control groups, i.e., CaM with Mg2+@AuATP and CaM with Zn2+@AuATP. Undoubtably, Mg2+@AuATP and Zn2+@AuATP also show AIECL behav-iors (Figure S15, Supporting Information). However, the ECL system using Mg2+@AuATP or Zn2+@AuATP as emitters also hardly changes in the presence of CaM (Figure 5B). These results support that the binding of protein to nanogel is deter-mined by the Ca2+ induced ECL dynamic changes.

On the basis of this principle, the ECL decreased with the increasing concentration of CaM (Figure 6A). The calibration curve shows a linear range from 0.3 to 50 µg mL−1 (Figure 6B) according to the calibration equation of log I (a.u.) = 3.69–0.727 × log c (µg mL−1) (R = 0.989). The limit of detec-tion is 0.1 µg mL−1. This method also allows the assays of CaM spiked in four groups of serum samples, with an acceptable recovery of 92%–117.5% (Table S2, Supporting Information). Note that although phosphate ions from PBS can affect the ECL emission because they can form precipitate with bivalent metal ions, the interference from common serum matrix pro-teins such as albumins on ECL signals is limited. Besides the

detection purposes, it can be deduced that this NC gel can be applied for calmodulin delivery. Considering the important roles of bivalent metal ions in life system, like Ca in neuro-signal transportation in synapse, Mg in chloroplast, and Zn in zinc-finger motif, this biomacromolecule-responsive AIECL-type hydrogel may be potentially used for the design of bivalent metal ion relevant events.

3. Conclusion

Generally, we have developed a novel AIECL system on the basis of bivalent cation mediated hydrogel networks, with up to 50-fold ECL enhancement after aggregation. Most impor-tantly, the ECL is mediated by CaM after its specific binding to Ca2+@AuATP hydrogel, thus enabling the effective assay of CaM, even in the complicated matrices such as serum sam-ples. By using the important roles of other bivalent cations, such as Mg in chloroplast, and Zn in zinc-finger motif, this biomacromolecule-responsive AIECL-type hydrogel may be further extended to more biological systems for versatile purposes.

Small 2019, 1901170

Figure 5. A) ECL responses of Ca2+@AuATP (black) to BSA of 10 µg mL−1 (red), and CaM of 10 µg mL−1 (green). B) ECL response of Mg2+@AuATP (red) or Zn2+@AuATP (blue) to CaM of 10 µg mL−1 (addition indicated by black arrow). The concentration of Mg2+@AuAXP or Zn2+@AuAXP is 10 µg mL−1.

Figure 6. A) The relationship between ECL intensity and concentration of CaM. From left to right: 0, 0.1, 0.3, 0.5, 1.0, 2.0, 4.0, 8.0, 12, 20, 30, 40, and 50 µg mL−1. B) Linear plot of ECL intensity against concentration of CaM.

1901170 (7 of 8)

www.advancedsciencenews.com

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.small-journal.com

Small 2019, 1901170

4. Experimental SectionMaterials and Apparatus: The ligands of adenosine mono-/di-/tri-

phosphate, i.e., AXP (X represents M, D, or T, respectively), Cyt, cytidine monophosphate, cytidine triphosphate, and CaM, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroauric acid (HAuCl4 3H2O), sodium citrate, HEPES, and triethylamine were products of Sinoreagent Co. Ltd. (Shanghai, China). All other reagents were of analytical grade. The Milli-Q ultrapure water (18.2 MΩ cm−1, Millipore, USA) was used throughout. The Amicon Ultra centrifugal filters with molecular weight cut-off of 3 kDa (Millipore, USA) were used for ultracentrifuge.

The PL spectra were obtained on an RF-5301 PC fluorescence spectrometer (Shimadzu, Japan). The TEM images were shot on a JEM-2100 Electron Microscope (JEOL, Japan). The SEM images were recorded on a Zeiss Ultra Plus field emission SEM (Zeiss, Germany) equipped with an EDS system (Oxford, England).

Synthesis of Bivalent Cation Linked AuAXP NC Gel: AXP was used as a template for preparation of fluorescent Au NCs according to a previous work.[20] Briefly, AXP and HAuCl4 were mixed in ultrapure water at room temperature, with a final concentration of 2 × 10−3 m and 1 × 10−3 m (i.e., a molar ratio of 2:1), respectively. After 30 min, the light yellow mixture was added with sodium citrate buffer of pH 7.0 to reach a final concentration of 50 × 10−3 m. The color rapidly faded. The colorless solution was kept in dark overnight. For all cases using different ligands, the emission at ≈480 nm could be observed after 30 min incubation, which normally shows a maximum intensity in 12 h. Then, the AXP-stabilized Au NCs were precipitated with absolute ethanol of 1:1 v/v and centrifuged at 10 000 rpm for 15 min. The pale-gray solid was dried in a desiccator. The sample could be easily redispersed in ultrapure water at a concentration of 10 mg mL−1. In control experiments, cytidine and cytidine phosphate-coated Au NCs were prepared according to our previous report.[21]

To prepare bivalent cation linked AuAXP NC gel, the cations were directly added to AuAXP NCs of 10 mg mL−1. White turbid might form and precipitate after centrifugation at 10 000 rpm for 15 min. The precipitate was collected and dried in a desiccator. The as-prepared sample could be redispersed in ultrapure water to obtain the hydrogel.

ECL Measurement: The ECL experiments were performed on an IFFM-E chemiluminescent analyzer (Remax, Xi’an, China) equipped with an electrochemical station (CHI 660C, CH Instruments, Austin, USA). The three-electrode system contains a GCE as working electrode, a Ag/AgCl wire in saturated KCl as reference electrode, and a Pt wire as counter electrode. The bias potential was −1000 V. The buffer was comprised of 10 × 10−3 m HEPES (pH 7.0) containing 10 × 10−3 m NaCl and 10 × 10−3 m triethylamine. The ultrapure N2 flow was bubbling throughout the dynamic ECL monitoring and the applied potential was +1.4 V (vs Ag/AgCl). For the measurement of recovery in real samples, the CaM-spiked serum samples were 100-fold diluted in the HEPES buffer.

The ECL spectrum was evaluated by calculation of ECL intensity difference (ΔIn = In − In−1) obtained by two adjacent light filters. The band pass cut-off wavelengths of the light filters ranged from 400 to 650 nm (15 or 20 nm interval between two adjacent filters). Since the transparency efficiency was same (90%) for all light filters, no further calibration steps were required.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsThis work was financially supported by the National Natural Science Foundation of China (21675023, 91753106, and 21828501), the National Key Research and Development Program of China (2017YFA0205300), the Natural Science Foundation of Jiangsu Province (BK20161413),

and the Southeast University-Nanjing Medical University joint project (2242017K3DN29).

Conflict of InterestThe authors declare no conflict of interest.

Keywordsaggregation-induced emission, bivalent metals, calmodulin, electrochemiluminescence, nanoclusters

Received: March 4, 2019Published online:

[1] H. R. Culver, J. R. Clegg, N. A. Peppas, Acc. Chem. Res. 2017, 50, 170.

[2] S. Q. Li, S. J. Dong, W. G. Xu, S. C. Tu, L. S. Yan, C. W. Zhao, J. X. Ding, X. S. Chen, Adv. Sci. 2018, 5, 1700527.

[3] O. S. Fenton, K. N. Olafson, P. S. Pillai, M. J. Mitchell, R. Langer, Adv. Mater. 2018, 30, 1705328.

[4] a) S. T. You, J. W. Li, W. Zhu, C. Yu, D. Q. Mei, S. C. Chen, J. Mater. Chem. B 2018, 6, 2187; b) H. W. Ooi, S. Hafeez, C. A. van Blitterswijk, L. Moroni, M. B. Baker, Mater. Horiz. 2017, 4, 1020.

[5] C. H. Yang, Z. G. Suo, Nat. Rev. Mater. 2018, 3, 125.[6] M. Sepantafar, R. Maheronnaghsh, H. Mohammadi, F. Radmanesh,

M. M. Hasani-Sadrabadi, M. Ebrahimi, H. Baharvand, Trends Biotechnol. 2017, 35, 1074.

[7] a) G. Sharifzadeh, H. Hosseinkhani, Adv. Healthcare Mater. 2017, 6, 1700801; b) Z. Y. Jiang, J. J. Chen, L. G. Cui, X. L. Zhuang, J. X. Ding, X. S. Chen, Small Methods 2018, 2, 1700307.

[8] a) J. Qian, B. Z. Tang, Chem 2017, 3, 56; b) H. Wang, E. G. Zhao, J. W. Y. Lam, B. Z. Tang, Mater. Today 2015, 18, 365; c) J. Mei, N. L. C. Leung, R. T. K. Kwok, J. W. Y. Lam, B. Z. Tang, Chem. Rev. 2015, 115, 11718; d) G. X. Feng, B. Liu, Small 2016, 12, 6528.

[9] S. Maji, P. Alam, G. S. Kumar, S. Biswas, P. K. Sarkar, B. Das, I. Rehman, B. B. Das, N. R. Jana, I. R. Laskar, S. Acharya, Small 2017, 13, 1603780.

[10] a) R. R. Hu, Y. Kang, B. Z. Tang, Polym. J. 2016, 48, 359; b) S. S. Liow, Q. Q. Dou, D. Kai, Z. B. A. Li, S. Sugiarto, C. Y. Y. Yu, R. T. K. Kwok, X. H. Chen, Y. L. Wu, S. T. Ong, A. Kizhakeyil, N. K. Verma, B. Z. Tang, X. J. Loh, Small 2017, 13, 1603404.

[11] N. Goswami, Q. Yao, Z. Luo, J. Li, T. Chen, J. Xie, J. Phys. Chem. Lett. 2016, 7, 962.

[12] Z. T. Luo, X. Yuan, Y. Yu, Q. B. Zhang, D. T. Leong, J. Y. Lee, J. P. Xie, J. Am. Chem. Soc. 2012, 134, 16662.

[13] Y. Yu, Z. Luo, D. M. Chevrier, D. T. Leong, P. Zhang, D.-e. Jiang, J. Xie, J. Am. Chem. Soc. 2014, 136, 1246.

[14] a) G. X. Feng, B. Liu, Acc. Chem. Res. 2018, 51, 1404; b) S. J. Chen, H. Wang, Y. N. Hong, B. Z. Tang, Mater. Horiz. 2016, 3, 283.

[15] S. Carrara, A. Aliprandi, C. F. Hogan, L. De Cola, J. Am. Chem. Soc. 2017, 139, 14605.

[16] T. B. Gao, J. J. Zhang, R. Q. Yan, D. K. Cao, D. C. Jiang, D. J. Ye, Inorg. Chem. 2018, 57, 4310.

[17] H. W. Liu, L. F. Wang, H. F. Gao, H. L. Qi, Q. Gao, C. X. Zhang, ACS Appl. Mater. Interfaces 2017, 9, 44324.

[18] F. Sun, Z. Y. Wang, Y. Q. Feng, Y. X. Cheng, H. X. Ju, Y. W. Quan, Biosens. Bioelectron. 2018, 100, 28.

1901170 (8 of 8)

www.advancedsciencenews.com

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.small-journal.com

Small 2019, 1901170

[19] S. M. Copp, D. E. Schultz, S. Swasey, E. G. Gwinn, ACS Nano 2015, 9, 2303.

[20] A. Lopez, J. Liu, J. Phys. Chem. C 2013, 117, 3653.[21] H. Jiang, Y. Zhang, X. Wang, Nanoscale 2014, 6, 10355.[22] H. Liang, Z. J. Zhang, Q. P. Yuan, J. W. Liu, Chem. Commun. 2015,

51, 15196.[23] X. Yu, J. Liu, H. W. Li, Y. Q. Wu, Nanoscale 2018, 10, 14563.[24] J. D. Blakemore, R. Chitta, F. D’Souza, Tetrahedron Lett. 2007,

48, 1977.[25] H. J. Wang, L. Yuan, J. L. An, Crystals 2017, 7, 103.[26] Z. Y. Liu, W. J. Qi, G. B. Xu, Chem. Soc. Rev. 2015, 44, 3117.

[27] I. Diez, M. Pusa, S. Kulmala, H. Jiang, A. Walther, A. S. Goldmann, A. H. E. Muller, O. Ikkala, R. H. A. Ras, Angew. Chem., Int. Ed. 2009, 48, 2122.

[28] H. Jiang, X. M. Wang, Chinese J. Anal. Chem. 2017, 45, 1776.[29] H. Jiang, L. Liu, X. Wang, Nanoscale 2017, 9, 9792.[30] H. Jiang, H. P. Wang, X. M. Wang, Electrochim. Acta 2010, 56, 553.[31] M. Hesari, Z. Ding, Acc. Chem. Res. 2017, 50, 218.[32] a) M. Hesari, M. S. Workentin, Z. Ding, Chem. - Eur. J. 2014, 20, 15116;

b) M. Hesari, M. S. Workentin, Z. Ding, Chem. Sci. 2014, 5, 3814.[33] H. Peng, M. Jian, H. Deng, W. Wang, Z. Huang, K. Huang, A. Liu,

W. Chen, ACS Appl. Mater. Interfaces 2017, 9, 14929.