The Promising Anodic Electrochemiluminescence of Non-Toxic and

Feb 8, 2018 - Herein, electrochemiluminescence (ECL) of CIS NCs in aqueous medium is investigated for the first time with L-glutathione and sodium cit...
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Article Cite This: Anal. Chem. 2018, 90, 3563−3569

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Promising Anodic Electrochemiluminescence of Nontoxic Core/Shell CuInS2/ZnS Nanocrystals in Aqueous Medium and Its Biosensing Potential Xiaoyan Long,†,§ Fang Zhang,†,§ Yupeng He,† Shifeng Hou,†,‡ Bin Zhang,† and Guizheng Zou*,† †

School of Chemistry and Chemical Engineering and ‡National Engineering and Technology Research Center for Colloidal Materials, Shandong University, Jinan 250100, China S Supporting Information *

ABSTRACT: Copper indium sulfide (CuInS2, CIS) nanocrystals (NCs) are a promising solution to the toxic issue of Cd- and Pb-based NCs. Herein, electrochemiluminescence (ECL) of CIS NCs in aqueous medium is investigated for the first time with L-glutathione and sodium citrate-stabilized water-soluble CIS/ZnS NCs as model. The CIS/ZnS NCs can be oxidized to hole-injected states via electrochemically injecting holes into valence band at 0.55 and 0.94 V (vs Ag/AgCl), respectively. The hole-injected state around 0.94 V can bring out efficient oxidative-reduction ECL with a similar color to Ru(bpy)32+ in the presence of tri-n-propylamine (TPrA) and enable CIS/ZnS NCs promising ECL tags with L-glutathione as linker for labeling. The ECL of CIS/ZnS NCs/TPrA can be utilized to determine vascular endothelial growth factor (VEGF) from 0.10 to 1000 pM with the limit of detection at 0.050 pM (S/N = 3). Although the holeinjected state around 0.55 V is generated ahead of oxidation of TPrA and fails to bring out coreactant ECL, annihilation ECL proves that both hole-injected states generated, at 0.55 and 0.94 V, can be involved in electrochemical redox-induced radiative charge transfer by directly stepping CIS/ZnS NCs from electron-injecting potential to hole-injecting potential. CIS/ZnS NCs are promising nontoxic electrochemiluminophores with lowered ECL triggering potential around 0.55 V for less electrochemical interference upon the development of coreactant.

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clusters,22,23 carbon quantum dots,24,25 C3N4 NCs,26 MoS2 NCs,27 and so on. Copper indium sulfide (CuInS2, CIS) NCs are a promising solution to the toxic issue of II−VI and II−VI NCs;16 however, investigations on radiative charge transfer of CIS NCs are normally conducted for solar energy conversion28 and photoluminescence (PL)-based sensing.29−32 Recently, our group achieved electrochemical redox-induced radiative charge transfer of core-only CIS NCs in nonaqueous medium by an ECL strategy.33 Core-only CIS NCs can be injected with holes into valence band (VB) via electrochemical oxidation and can be injected with electrons into conduction band (CB) via electrochemical reduction; charge transfer and/or recombination between electrochemically injected CB electrons and VB holes is capable of ECL. Unfortunately, the core-only CIS NCs are insoluble in aqueous medium and cannot be developed as ECL tag for bioassay. Herein, ECL of CIS NCs in aqueous medium and resulting biosensing were achieved for the first time with Lglutathione- and sodium citrate-stabilized core/shell CIS/ZnS NCs as model, because the dual stabilizers L-glutathione and sodium citrate could enable CIS/ZnS NCs to be water-soluble with desired photoluminescence quantum yields (PLQY),34 and especially, L-glutathione could label biomolecules and

lectrochemiluminescence (ECL) is radiative charge transfer from excited states, which are formed at the electrode surface via electrochemical redox.1 ECL is superior to fluorescence in terms of sensitivity and signal-to-noise ratio (S/N) due to absence of background from unselective photoexcitation.2 Since the initial research on ECL in 1964,3,4 the design and screen of electrochemiluminophores has always played an important role in the evolution of ECL.5−8 One breakthrough was the discovery of molecule electrochemiluminophore Ru(bpy)32+ in 1972, which eventually brought out a series of Ru(bpy)32+/tri-n-propylamine (TPrA) reagent kits for various biomedical and diagnostic ECL assays.9 The other important breakthrough was the discovery of nanocrystal (NC)-based non-molecule electrochemiluminophores in 2002, in which Ding and Bard and co-workers10 first demonstrated ECL from Si NCs in nonaqueous medium. With the following progress on ECL of CdSe NCs in aqueous medium achieved by Zou and Ju in 2004,11 ECL of III−V and II−VI NCs was extensively conducted for developing electrochemiluminophores with desired performance.12−15 Unfortunately, III−V and II−VI NCs contain either class A elements (Cd, Pb, and Hg) or class B elements (Se and As) whose potential toxicity is a subject of serious concern.16 For example, Cd-based NCs would degrade and release cytotoxic Cd2+ ions in a biological environment.17,18 Tremendous efforts are being carried out on ECL of toxic-element-free NCs and nanoclusters, such as gold nanoclusters,19−21 silver nano© 2018 American Chemical Society

Received: January 1, 2018 Accepted: February 8, 2018 Published: February 8, 2018 3563

DOI: 10.1021/acs.analchem.8b00006 Anal. Chem. 2018, 90, 3563−3569

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Analytical Chemistry

reference electrode. Differential pulse voltammetry (DPV) was recorded on a CHI 822 electrochemical analyzer (Shanghai, China). ECL spectra were obtained on a homemade ECL spectrum analyzer consisting of an Acton SP2300i monochromator equipped with a liquid N2-cooled PyLoN 400BReXcelon digital charge-coupled device (CCD) detector (Princeton Instruments) and a VersaSTAT 3 electrochemical analyzer (Princeton Applied Research).36−38 Preparation of CuInS2/ZnS Nanocrystal-Based Electrochemiluminescent Biosensor. As demonstrated in Scheme 1, the signal-off ECL biosensor was fabricated by immobilizing

enable CIS/ZnS NCs to be promising tags for bioassay. The CIS/ZnS NCs could be oxidized to hole-injected states via electrochemically injecting holes into VB at 0.55 and 0.94 V, respectively. The hole-injected state around 0.94 V was capable of coreactant ECL with a similar color to Ru(bpy)32+ in the presence of TPrA and could be used to selectively determine vascular endothelial growth factor (VEGF) with high sensitivity, as shown in Scheme 1. The hole-injected state around 0.55 V was anticipated for ECL at low triggering potential for less electrochemical interference upon the development of coreactants. This work would lead to a promising advance for CIS NC-based ECL and its sensing applications.

Scheme 1. Schematic Representation for Determining VEGF165 with CIS/ZnS as Electrochemiluminescent Tags

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EXPERIMENTAL SECTION Chemicals and Materials. All chemicals and reagents were of analytical grade or better, and all aqueous solutions were prepared with doubly distilled water (DDW) (see Supporting Information). HPLC-purified DNA capture probe S1, VEGF165 aptamer, and VEGF165 were purchased from Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China). VEGF165 aptamer was modified with a carboxyl group at the 5′-terminus and its sequence is as follows: 5′-COOH-GGCCCGTCCGTATGGTGGGTGTGCTGGCC3′.35 DNA capture probe S1 was modified at the 5′-terminus with an amino group and its sequence is as follows: 5′-NH2TTTTTTTATGGGTTGGGCGGGATGGGCCAGCACACCCACC-3′.35 All the samples were prepared with 0.10 M phosphate buffer (PB, pH 7.4) and stored at 4 °C before use. CIS/ZnS NCs were prepared by a one-pot synthetic strategy with L-glutathione and sodium citrate as stabilizing agents.34 Briefly, 20 mL of doubly distilled water, 20 μmol of glutathione, 10 μmol of CuCl2·2H2O, 40 μmol of InCl3·4H2O, 160 μmol of sodium citrate, and 62 μmol of Na2S·9H2O were loaded into a three-neck flask with continuous magnetic stirring. The reaction mixture was kept at 95 °C for 40 min to obtain CuInS2 core. Then, 2 mL of ZnS shell stock solution (pH 6.0) containing 0.8 mmol of Zn(OAc)2·2H2O, 1.2 mmol of glutathione, and 0.8 mmol of thiourea was introduced into the CuInS2 core solution; the final mixture was kept at 95 °C for another 30 min to form CIS/ZnS NCs. The obtained CIS/ZnS NCs were precipitated with isopropyl alcohol, purified via centrifugation, and then redispersed in DDW. Apparatus. Ultraviolet−visible (UV−vis) absorption spectra were recorded on a TU-1901 UV−vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.). PL spectra were recorded on an F-320 spectrofluorometer (Tianjin Gangdong Sci&Tech Development Co., Ltd.). Fluorescence lifetime and PLQY were recorded on a fluorescence spectrometer (model FLS920, Edinburgh Instruments). X-ray photoelectron spectra (XPS) were taken from an Escalab 250 XPS using monochromatic Al Kα radiation (Thermo Fisher Scientific Co.). X-ray diffraction (XRD) patterns were recorded on an X-ray diffractometer with Cu Kα radiation (λ = 1.5418 Å) (Bruker AXS D8 Advance). High-resolution transmission electron microscopic (HRTEM) images were taken on a TecnaiG2 F30 transmission electron microscope with an acceleration voltage of 300 kV (Thermo Fisher Scientific Co.). Cyclic voltammetry (CV) and ECL−potential profiles were recorded on a MPI-A ECL analyzer (Xi’an Remex Analytical Instrument Co., Ltd.) with a three-electrode system, including a glassy carbon working electrode (GCE, diameter of 5 mm), a Pt counter electrode, and a Ag/AgCl (saturated KCl)

CIS/ZnS NCs onto GCE, via labeling them to the 5′-terminal carboxyl group of VEGF165 aptamer in hybrids of VEGF165 aptamer and capture probe S1, and then removing CIS/ZnS NCs from GCE via the competitive reaction between VEGF165 and VEGF165 aptamer for ECL assay. GCE was polished with 0.3 μm alumina slurry, washed with copious amounts of DDW, and then electrodeposited with a layer of p-aminobenzoic acid (ABA), via scanning potential between 0.40 and 1.20 V (vs Ag/ AgCl) in 10 mM PB, pH 7.4, containing 1.0 mM ABA and 10 mM KCl at 10 mV/s for two cycles.39 The carboxyl groups of GCE-ABA were activated in 0.10 M PB, pH 6.0, containing 100 mg/mL 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) and 100 mg/mL N-hydroxysuccinimide (NHS), for 30 min and then covalently bound to 10 μL of DNA capture probe S1 (5 μM) at room temperature for 20 h. The obtained GCE-ABA-S1 was incubated with a drop (10 μL) of VEGF165 aptamer (5 μM) to form GCE-ABA-S1-aptamer. CIS/ZnS NCs were labeled onto GCE-ABA-S1-aptamer via the terminal carboxyl groups of aptamers with NHS- and EDCassisted labeling protocol. GCE-ABA-S1-aptamer-CIS/ZnS was incubated with a drop (10 μL) of VEGF165 at various concentrations for 2 h to form GCE-ABA-S1-aptamer-CIS/ ZnS-VEGF165, which was then used for ECL bioassay in 0.10 M PB, pH 7.4, containing 10 mM TPrA.

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RESULTS AND DISCUSSION Optical, Structural, and Morphological Characterization of CuInS2/ZnS Nanocrystals. Absorption of CIS/ ZnS NCs displayed a broad shoulder with a tail in the longwavelength direction (curve a of Figure 1A),29 and no obvious peak related to the first excitonic absorption was observed. PL spectrum of CIS/ZnS NCs displayed a broad peak with maximum emission wavelength around 577 nm and full width at half-maximum around 120 nm (curve b of Figure 1A), similar to previously reported CIS/ZnS NCs with L-glutathione 3564

DOI: 10.1021/acs.analchem.8b00006 Anal. Chem. 2018, 90, 3563−3569

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Figure 1. (A) UV−vis absorption (a, black line) and PL (b, red line) spectra, (B) PL decay curve, (C) XRD pattern, and (D) HRTEM image of CIS/ZnS NCs.

and sodium citrate as stabilizers.34 PLQY of CIS/ZnS NCs was determined to be ∼26%. PL decay of CIS/ZnS NCs was wellfitted with a triexponential model with PL lifetime of 1029.6 ns (Figure 1B and Table S1). The broad PL, large Stokes shift, and long PL lifetime indicate that as-prepared CIS/ZnS NCs are typical I−III−VI NCs (Figure 1A,B).40 It is well-known that photoexcitation of CIS NCs can promote electrons from VB into CB to produce both CB-like electrons (e−CB) and VB holes41 and then give off PL via charge transfer and recombination.42 PL dynamics contains information about both radiative and nonradiative relaxation processes; long-timescale decay components in PL of CIS NCs are often related to intrinsic defects and surface defects.43 The triexponential PL decay indicates that some traps (or defects) at the surface of CIS/ZnS NCs were involved in radiative charge transfer.44 XPS patterns proved that CIS/ZnS NCs were composed of Cu, In, S, and Zn elements (Figure S1). The XRD pattern showed three major peaks at 2θ values of 28.6°, 46.8°, and 55.3° (Figure 1C), which are intermediate between CuInS2 and ZnS phases and are consistent with previously reported CIS/ZnS NCs.34 The HRTEM pattern demonstrated that the CIS/ZnS NCs were nearly monodispersed with an average size of ∼3 nm (Figure 1D).34 Electrochemistry and Tri-n-propylamine Coreactant Electrochemiluminescence of CuInS2/ZnS Nanocrystals in Aqueous Medium. To avoid the possible interference of dissolved oxygen, ECL of monodispersed CIS/ZnS NCs in aqueous medium was carried out at anode with TPrA as coreactant (Figure 2), as TPrA can be electrochemically oxidized to produce strongly reducing radical species TPrA• (E° = ∼−1.7 V vs saturated calomel electrode, SCE),9 and then bring out oxidative-reduction ECL of NCs.10 No obvious electrochemical oxidizing process was detected with CIS/ZnS NCs in PB containing 15 mg/mL CIS/ZnS NCs and 0.10 M KCl (Figure 2A, curve a), indicating the electrochemical redoxinduced hole-injecting processes of CIS/ZnS NCs are weak.

Figure 2. (A) CV and (B) ECL−potential profiles of 15 mg/mL CIS/ ZnS NCs in 0.10 M PB, pH 7.4, containing 0.10 M KCl and (a, black line) 0.0 or (b, red line) 10 mM TPrA at 100 mV/s. (Insets) Anodic (A) CV and (B) DPV profiles of bare GCE in 0.10 M PB, pH 7.4, containing 0.10 M KCl and (a, black line) 0.0 or (b, red line) 15 mg/ mL CIS/ZnS NCs.

Introducing TPrA into the aforementioned PB not only brought out a strong oxidative process with onset around 0.66 V and maximum intensity around 0.90 V (Figure 2A, curve b) but also enabled efficient ECL with onset around 0.66 V and maximum emission around 0.94 V (Figure 2B, curve b). The anodic ECL was initiated with onset of oxidizing TPrA, which is similar to the previously reported TPrA coreactant ECL.45 It is clear that TPrA can be electrochemically oxidized to tremendous TPrA• radicals for injecting electrons into CB of CIS/ZnS NCs at large scale. The electrochemical oxidationinduced hole-injecting process is the rate-determining step for ECL of CIS/ZnS NCs/TPrA. 3565

DOI: 10.1021/acs.analchem.8b00006 Anal. Chem. 2018, 90, 3563−3569

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Analytical Chemistry DPV was adopted to verify the exact hole-injecting processes of CIS/ZnS NCs. The DPV profile of CIS/ZnS NCs displayed two hole-injecting processes around 0.55 and 0.94 V (inset, Figure 2B), indicating CIS/ZnS NCs can be electrochemically oxidized to two hole-charged states at 0.55 V (Ox1) and 0.94 V (Ox2). Compared with the four hole-injecting processes of core-only CIS NCs that occurred at anode,33 the limited two hole-injecting processes of CIS/ZnS NCs indicate that forming a wide-band-gap ZnS shell around the CIS core would make the CIS core less sensitive to external environment. As displayed in Scheme 2, both the hole-injecting processes of CIS/ZnS NCs and the electrochemical oxidation of Scheme 2. Schematic Illustration for Coreactant Electrochemiluminescence of CIS/ZnS Nanocrystals

Figure 3. (a) PL spectrum of CIS/ZnS NCs; (b, c) ECL spectra of (b) 15 mg/mL CIS/ZnS NCs and (c) GCE-ABA-S1-aptamer-CIS/ZnSVEGF165 in 0.10 M PB, pH 7.4, containing 0.10 M KCl and 10 mM TPrA. GCE-ABA-S1-aptamer-CIS/ZnS-VEGF165 was formed with 10 μL of sample containing 10 pM VEGF165. ECL spectra resulted from the total photons generated by scanning the potential from 0 to 1.6 V for one cycle at 100 mV/s.

tioned PL decay of CIS/ZnS NCs (Figure 1B), which confirmed the existence of surface defects on CIS/ZnS NCs. Importantly, the ECL spectrum of surface-confined CIS/ZnS NCs on GCE-ABA-S1-aptamer-CIS/ZnS-VEGF165 was almost the same as that of monodispersed CIS/ZnS NCs, with maximum emission around 642 nm (curves b and c of Figure 3), indicating the L-glutathione- and sodium citrate-stabilized CIS/ZnS NCs preserved their surface states very well via complicated labeling and bioreaction processes. CIS/ZnS NCs are promising ECL tags for bioassay. Annihilation Electrochemiluminescence of CuInS2/ ZnS Nanocrystals in Aqueous Medium. As coreactant ECL of CIS/ZnS NCs/TPrA mainly derived from charge transfer between Ox2 and TPrA• (curve b of Figure 2B), annihilation ECL was conducted to verify the ECL activity of Ox1 (Figure 4). Annihilation ECL was conducted in N2saturated PB to avoid the possible interference of dissovled oxygen. CIS/ZnS NCs demonstrated obvious annihilation ECL upon stepping potential from −1.50 V to +0.55 V and from −1.50 V to +0.94 V. The annihilation ECL upon stepping potential from −1.50 V to +0.94 V indicated that CIS/ZnS NCs could be electrochemically reduced to electron-injected state, via injecting electrons into CB at −1.50 V, and then bring out radiative charge transfer between the electron-injected state and the hole-injected state Ox2. The annihilation ECL upon stepping potential from −1.50 V to +0.55 V confirms that radiative charge transfer in CIS/ZnS NCs can also be achieved between the electron-injected state and Ox1 via electrochemical redox. Importantly, stepping potential from positive holeinjecting potentials (both +0.55 V and +0.94 V) to negative electron-injecting potential (−1.50 V) could also bring out efficient annihilation ECL for CIS/ZnS NCs in the followed potential stepping procedures, which further confirmed the ECL activity of hole-injected states Ox1. Possibility for Improved Coreactant Electrochemiluminescent Performance of CuInS2/ZnS Nanocrystals. Figure S2 displays the oxidative-reduction ECL of CIS/ZnS NCs in the presence of several common coreactants in the same concentration to TPrA in Figure 2. 2-(Dibutylamino)-

coreactant played important roles in the generation of oxidative-reduction ECL. The oxidative potential of TPrA (0.66 V) was obviously higher than the hole-injecting potential for Ox1 (0.55 V); hole-injected-state Ox1 failed to take part in TPrA coreactant ECL, and no ECL was detected around +0.55 V (Figure 2B, curve b). TPrA coreactant ECL of CIS/ZnS NCs was mainly achieved via charge transfer between the holeinjected state Ox2 and TPrA• radicals, with maximum emission around +0.94 V. The mechanism for ECL of CIS/ZnS NCs/TPrA is proposed in eqs 1−6: +• CIS/ZnS NCs → nR (CIS/ZnS) + ne−

TPrA → TPrA• + + e−

( +0.94 V)

( +0.66 V)

TPrA• + → TPrA• + H+ •

nTPrA + CIS/ZnS NCs →

(1) (2) (3)

−• nR (CIS/ZnS)

(4)

+• −• R (CIS/ZnS) + R (CIS/ZnS) → R *(CIS/ZnS)

(5)

R *(CIS/ZnS) → CIS/ZnS + hv

(6)

(∼642 nm)

The ECL spectrum of CIS/ZnS NCs/TPrA displayed a broad peak with maximum wavelength at 642 nm (curve b of Figure 3), indicating ECL of CIS/ZnS NCs/TPrA was redshifted by ∼65 nm with respect to CIS/ZnS NC PL spectrum (curve a of Figure 3) and had a similar color to the ECL of Ru(bpy)32+/TPrA.9,46 Previous research demonstrated that maximum emission for ECL and PL of core-only CIS NCs was located at ∼775 and ∼670 nm, respectively.33 The red shift of ECL to PL for core-only CIS NCs was more than 100 nm. The less red-shifted ECL to PL for CIS/ZnS NCs than that for core-only CIS NCs not only proved that CIS/ZnS NCs were partially passivated by forming ZnS shell around CIS core47 but also indicated that surface defects still play an important role in the electrochemical redox-induced radiative charge transfer of CIS/ZnS NCs. These results are consistent with aforemen3566

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Figure 5. ECL−potential profiles of GCE-ABA-S1-aptamer-CIS/ZnSVEGF165 formed with a drop (10 μL) of sample containing (a) 0.10, (b) 0.50, (c) 1.0, (d) 5.0, (e) 10.0, (f) 50.0, (g) 100.0, (h) 500.0, or (i) 1000.0 pM VEGF165. ECL measurements were conducted in 0.10 M PB, pH 7.4, containing 0.10 M KCl and 10 mM TPrA at 100 mV/s. (Inset) Calibration curve for determining VEGF165.

Figure 4. ECL transients of bare GCE in N2-saturated 0.10 M PB, pH 7.4, containing 0.10 M KCl and (a, black line) 0.0 or (b, red line) 15 mg/mL CIS/ZnS NCs by stepping potential between (A) −1.50 and 0.94 V or (B) −1.50 and 0.55 V at 1 Hz. Blue dotted lines indicate applied potential steps.

ethanol (DBAE) brought out efficient ECL around +0.8 V for CIS/ZnS NCs, with the maximum intensity much lower than that for ECL of CIS/ZnS NCs/TPrA, while both potassium oxalate monohydrate (K2C2O4·H2O) and H2O2 merely brought out weak ECL for CIS/ZnS NCs around +0.90 V. The potentials for maximum emission of DBAE, K2C2O4, and H2O2 coreactant ECL were different from that of the CIS/ZnS NCs/ TPrA system, which was due to the different oxidative potentials for these coreactants to take part in oxidativereduction ECL. Similar to the TPrA coreactant ECL (Figure 2B, curve b), no obvious oxidative-reduction ECL related to Ox1 was observed around 0.55 V with DBAE, K2C2O4, and H2O2, because these coreactants were oxidized at potentials higher than that required for forming hole-injected state Ox1. Anyhow, the hole-injecting potential of Ox1 was obviously lower than the oxidizing potential of Ru(bpy)32+ and some reported NCs with promising low ECL triggering potential.48−51 Coreactant ECL from Ox1 could be anticipated to efficiently avoid undesired electrochemical interference upon the evolution of desired coreactants (Scheme 2). Bioassay Performance of Electrochemiluminescence from CuInS2/ZnS Nanocrystals/Tri-n-propylamine. It is well-known that TPrA is a traditional coreactant and has been extensively used in various ECL bioassay procedures.9 TPrA coreactant ECL from Ox2 of CIS/ZnS NCs was eventually adopted for determining VEGF165 in this case, because TPrA can bring out obviously stronger oxidative-reduction ECL for Ox2 than other coreactants such as DBAE, K2C2O4, and H2O2 (Figure S2). The fabrication procedure of ECL sensor for VEGF165, that is, GCE-ABA-S1-aptamer-CIS/ZnS, was examined by Fe(CN)63−/Fe(CN)64− redox couple in 0.10 M PB, pH 7.4 (Figure S3), which confirmed that CIS/ZnS NCs could be immobilized onto GCE surface via designed hybridization processes and then removed from GCE surface by VEGF165 for signal-off ECL sensing. Figure 5 displays the potential-resolved ECL profiles of GCE-ABA-S1-aptamer-CIS/ZnS-VEGF165, which was formed with 10 μL VEGF165 samples at various concentrations. The onset potential for ECL of GCE-ABA-S1-aptamer-CIS/ZnS-

VEGF165 was around +0.66 V, indicating coreactant ECL was mainly related to Ox2. The maximum intensity for ECL of GCE-ABA-S1-aptamer-CIS/ZnS-VEGF165 decreased obviously with increasing concentration of VEGF165. ECL decrement (ΔI = I0 − I, where I0 is the maximum ECL intensity of GCE-ABAS1-aptamer-CIS/ZnS and I is the maximum ECL intensity of GCE-ABA-S1-aptamer-CIS/ZnS-VEGF165) increased linearly with the logarithmically increasing concentration of VEGF165 in a range from 0.10 to 1000 pM (inset, Figure 5, R = 0.99) with a limit of detection of 0.050 pM (S/N = 3). GCE-ABA-S1aptamer-CIS/ZnS-VEGF165 not only displayed comparable or even better sensitivity than previously reported VEGF165 determining strategies (Tables S2)35,52−54 but also demonstrated acceptable selectivity to common interfering substances such as BSA, ascorbic acid, and thrombin (Figure S4). The desired selectivity of the proposed ECL sensor for VEGF165 was derived from the bioselective reaction between DNA capture probe S1, VEGF165 aptamer, and VEGF165.35

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CONCLUSIONS ECL of nontoxic CIS/ZnS NCs in aqueous medium was investigated for the first time with L-glutathione- and sodium citrate-stabilized, water-soluble CIS/ZnS NCs as model. CIS/ ZnS NCs were efficient electrochemiluminophores for ECL labeling and bioassay with capping agent L-glutathione as linker. Importantly, CIS/ZnS NCs could be electrochemically oxidized to hole-injected states at 0.55 V for Ox1 and 0.94 V for Ox2, respectively; both hole-charged states could bring out annihilation ECL, along with the generation of electroninjected state via electrochemically reducing the CIS/ZnS NCs. Hole-charged state Ox2 was capable of efficient oxidativereduction ECL with a similar color to the Ru(bpy)32+ in the presence of TPrA, and it could be utilized for ECL bioassay with high sensitivity. The hole-injected state Ox1 was promising for novel ECL sensing strategies at low ECL triggering potential to avoid undesired electrochemical interference upon the development of coreactants. 3567

DOI: 10.1021/acs.analchem.8b00006 Anal. Chem. 2018, 90, 3563−3569

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b00006. Additional text, two tables, and four figures describing chemicals and reagents, PL lifetime parameters, XPS spectra, oxidative-reduction ECL, electrochemical characterization, sensing performance of other strategies for determining VEGF165, and specificity of the proposed sensor (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; tel +86-531-88361326. ORCID

Xiaoyan Long: 0000-0003-1788-7317 Yupeng He: 0000-0002-4582-3129 Shifeng Hou: 0000-0001-7486-9153 Bin Zhang: 0000-0002-1529-6356 Guizheng Zou: 0000-0002-3295-3848 Author Contributions §

X.L. and F.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation of China (Grants 21427808, 21375077, and 21475076), and the Fundamental Research Foundation of Shandong University (Grant 2015JC037)

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DOI: 10.1021/acs.analchem.8b00006 Anal. Chem. 2018, 90, 3563−3569

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DOI: 10.1021/acs.analchem.8b00006 Anal. Chem. 2018, 90, 3563−3569