Visible-Light Driven Photoelectrochemical Platform Based on the

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Visible-light Driven Photoelectrochemical Platform based on Cyclometalated Iridium(III) Complex with Coumarin 6 for Detection of MicroRNA Chunxiang Li, weisen lu, xiaoming zhou, mengmeng pang, and Xiliang Luo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03246 • Publication Date (Web): 14 Nov 2018 Downloaded from http://pubs.acs.org on November 14, 2018

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Analytical Chemistry

Visible-light Driven Photoelectrochemical Platform based on Cyclometalated Iridium(III) Complex with Coumarin 6 for Detection of MicroRNA Chunxiang Li*, Weisen Lu, Xiaoming Zhou, Mengmeng Pang and Xiliang Luo* Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE; Ministry of Education; Shandong Key Laboratory of Biochemical Analysis; College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China. ABSTRACT: In this study, a novel iridium(III) complex based photoactive species, [(C6)2Ir(dppz)]+PF6− was synthesized

with coumarin 6 (C6) as a cyclometalated ligand and dipyrido[3,2-a:2’,3’-c]phenazine (dppz) as an ancillary ligand. The photophysical and electrochemical properties of the complex have been investigated. It exhibits intense visible-light absorption with a molar extinction coefficient up to 9.8 × 104 M−1 cm−1 at 485 nm. The photoelectrochemical (PEC) properties of [(C6)2Ir(dppz)]+PF6− was also investigated by spin-coating on the ITO electrode. By illumination with 490 nm light, cathodic photocurrent up to 260 nA/cm2 was observed at 0 V bias potential with dissolved O2 as an electron acceptor. On the other hand, an anodic photocurrent generated in the presence of triethanolamine (TEOA) as a sacrificial electron donor. The probable mechanisms for photocurrent generation were deduced by UV−vis absorption spectrum and cyclic voltammetry data. Adopting [(C6)2Ir(dppz)]+PF6− as photoactive intercalator, a visible-light driven PEC detection platform was successfully fabricated for microRNA detection based on an enzyme-free hybridization chain reaction as amplification strategy. Expectedly, the PEC platform for microRNA-122b detection showed excellent linear response with a limit of detection down to 0.23 fM (3σ), comparable or superior to those of the reported analogous approaches. Encouragingly, this study would provide a new approach to exploit efficient photoactive species for PEC bioanalysis.

Photoelectrochemical (PEC) bioanalysis, as a vibrant analytical technology, has attracted increasing attention in view of its unique merits, such as high sensitivity, low background and simple equipment.1-3 PEC analysis utilizes light as input and the transduced electric current as readout signal, which involves the conversion of photoactive materials from photo to electricity. Therefore, the performance of PEC analysis depends intimately on the nature of photoactive materials used. More and more effort was devoted to exploit diverse photoactive materials to meet the needs for bioanalysis.4-9 Among the various photoactive materials studied for PEC bioanalysis, an important branch is organometallic complexes due to their remarkable thermal and photo-stability and fascinating electrochemical and photoelectric properties.10-12 However, so far most of the PEC bioanalysis on organometallic complexes are just focused on Ru complexes. The search for novel PEC materials with superior performance than that of conventional Ru(II) complexes is still a hot topic. Cyclometalated Ir(III) complexes, as a typical type of organometallic complex, has made major breakthroughs in electroluminescence (ECL) field in the past decades, originating from their advantageous and tunable triplet excited states over a wide range.10,13,14 These favorable performances endow Ir(III) complexes tremendous potential in a variety of applications beyond ECL. Recently, the study from Cosnier et al has demonstrated that Ir(III) complexes exhibited analogous

PEC properties and comparable photon-to-current conversion efficiency as those of Ru(II) complexes.15 Just recently, our group exploited a Ir(III) complex, [(ppy)2Ir(dppz)]+PF6− as photoactive species, and realized sensitive determination of target DNA combined with DNA amplification strategy.16 Although cyclometalated Ir(III) complexes are just a nascent species in PEC field, they are garnering increasing attention in view of some advantages compared to usual Ru(II) complexes. For example, Ir(III) complexes possess higher stability, a longer state lifetime, a higher degree of charge transfer and a greater variety of excited state including the singlet and triplet ligand-centered (1LC and 3LC) transitions, metal-to-ligand charge-transfer (1MLCT and 3MLCT) and intraligand chargetransfer (ILCT) transition, which facilitate the improvement of the conversion efficiency.17-19 In particular, the wider tunability of the excited state energies and redox potentials for Ir(III) complexes overmatch the limited controllability of Ru(II) complexes. However, conventional iridium complexes, designed for ECL field, suffered from weak absorption in the visible region, which hinder the efficient conversion from photo to electricity. Improving the visible absorption properties of Ir(III) complexes to enhance light-harvesting natures is a key point in the PEC field.20,21 To achieve the strong visible absorption for Ir(III) complexes, a straightforward method is to attach covalently a visible light-harvesting chromophore, such as rhodamine, boron dipyrromethene (BODIPY) and coumarin, to the traditional coordinated ligands. Draper et al. have

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reported BODIPY-appended Ir(III)-dipyridyl complex, which absorbs visible light strongly with ε = 8.3 × 104 M−1 cm−1 at λ = 527 nm.22 By integrating coumarin into the diimine ligand, Zhao et al. developed a Ir(III) complex with intense visible absorption (ε = 7.1× 104 M−1 cm−1 at 466 nm).23 Another effective approach is to elaborately choose the opportune light-harvesting chromophores as the ligand. Coumarin 6 is just suitable due to perfect coordinated sites with iridium center. Adopting Coumarin 6 (C6) as cyclometalated ligand, Crutchley et al. synthesized a neutral complex Ir(C6)2(vacac) (vacac = allylacetoacetate) as fluorescent material for oxygen sense, which displays strong visible-light absorption with ε = 8.7 × 104 M−1 cm−1 at 474 nm.24 Murata et al. have investigated a series of C6 based cyclometalated Ir(III) complexes with various substituted bipyridine as sensitizers for the visible-light-driven H2 generation.25 Their work demonstrated that the properties of excited-state are tuned selectively through modification of the ancillary ligand, indicating the potential for more application fields. Elliott et al. have developed a complex [Ir(C6)2(dcbH2)]+PF6− with strong visible absorption, as sensitizer for dye-sensitised solar cells (DSSCs).26 Moreover, our group have exploited the Ir(III) complex as novel PEC material and successfully applied it for thrombin detection.27 Inspired by these pioneering works, herein we tentatively designed a novel cationic complex, [(C6)2Ir(dppz]+PF6− (depicted in Scheme 1), where C6 as the cyclometalated ligand, and the choice of ancillary ligand dppz lies in its ability of intercalating into DNA helix with extremely high affinity. The ability of its visible light-absorption, the electrochemical and PEC performances were investigated systematically. Employing [(C6)2Ir(dppz]+PF6− as intercalated photoelectric transducer, a PEC platform was fabricated based on an enzyme-free amplification strategy of hybridization chain reaction (HCR) to demonstrate the applicability of the complex for PEC bioanalysis. It is anticipated that the exploratory work might stimulate more attempt to design and exploit remarkable photoactive materials for PEC bioanalysis.

Scheme 1 The structure of [(C6)2Ir(dppz)]+PF6−

EXPERIMENTAL SECTION Synthesis of the Complex [(C6)2Ir(dppz]+PF6−. The preparation and characterization of the ligand C6 and dppz was reported in our previous work.27 The synthetic procedure and characterization of [(C6)2Ir(dppz]+PF6− were depicted in the Supporting Information. Preparation of [(C6)2Ir(dppz]+PF6− Attached ITO Electrodes. The ITO electrode was sonicated respectively in ultrapure water, acetone, ethanol and ultrapure water for 15 min, following dried at room temperature. Subsequently, 10 μL of [(C6)2Ir(dppz]+PF6− solution (5 × 10-5 M) in CH3CN was spread dropwise on the cleaned electrodes and dried at ambient temperature.

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Gel Electrophoresis. The 17.5% polyacrylamide gel was prepared by successively adding 40% gel solution (39:1) (3.5 mL), deionized H2O (4256 µL), 10% APS (80 µL), 50 × TAE Buffer (160 µL) and TEMED (4 µL). A sample containing 5 µL of different reaction products were injected into the prepared polyacrylamide gel. The electrophoresis was run in 1 × TAE buffer at 180 V for 3 min, and then at 135 V for 90 min at room temperature. After staining in EB dye solution for 15 min, the gel was imaged with a digital camera system. DNA Emission Titrations Assay. Different concentrations of [(C6)2Ir(dppz]+PF6− solution in a mixture of tris-HCl buffer and DMF (5:1, V:V) was cumulatively added to the mixture of calf thymus DNA (100 μM) and EB (10 μM) for 30 min. And then the emission spectra of the mixture were measured. Fabrication of PEC Detection Platform. After cleaning according to the method above, the ITO electrodes were immersed in a mixture solution containing 30% H2O2, 30% NH4OH and H2O (1:1:5) for 15 min. After rinsing with ultrapure water and then dried under nitrogen stream, the electrodes were immersed in 5% APTMS ethanol solution overnight to acquire a NH2-rich self-assembled layer. Then, the electrodes were washed thoroughly with ethanol and dried at 110 °C for 15 min. Next, the electrodes were decorated with AuNPs by incubating in AuNPs solution for 12 h, and then rinsing with ultrapure water and dried under nitrogen stream. 20 μL of capture-H1 (2 μΜ) in SPSC buffer containing 10 mM TCEP was dropped on the electrode, and the immobilization reaction was incubated overnight at room temperature in humidity. After carefully rinsing with washing buffer solution, a droplet of 20 μL MCH solution (1 mM) was added for 2 h to minimize the nonspecific adsorption. After carefully rinsing with washing buffer solution, 20 μL different concentration of miRNA-122b was dropped on the capture-H1 modified electrode for 2 h. Unreacted oligonucleotides were removed by gently washing the electrodes three times with washing buffer solution. 20 μL mixture of H1(1 μM) and H2 (1 μM) was added and incubated at 37°C for 2 h. After that, 10 μL of [(C6)2Ir(dppz)]+PF6− (0.01 mM) solution was placed on the modified electrode and incubated for 40 min. Finally, the electrodes were rinsed with washing buffer solution to remove the nonspecifically adsorbed complexes. PEC Measurement. The photocurrent intensity of the resulting functionalized electrode was recorded in 0.01 M PBS solution (pH = 5.5) irradiated with 490 nm light. The light was switched on and off every 20 s, and the applied potential was −0.1 V.

RESULTS AND DISCUSSION Electronic Absorption Spectroscopy. The electronic absorption spectrum for [(C6)2Ir(dppz]+PF6− was measured in DMF solution at room temperature, as depicted in Figure 1. Like other C6-based iridium complexes reported previously,2830 the complex exhibits notably intense absorption in visible region, with ε = 9.8 × 104 M−1 cm−1 at λ = 485 nm and discernible absorption out to λ ∼540 nm, which is assigned to coumarin 6-centred intraligand charge transfer (ILCT) along with weaker MLCT-based transitions. It is heartening that the ε value is 5 times more than that of a frequently used photoactive species, Ru(bpy)32+ at 450 nm (ε = 1.8 × 104 M−1 cm−1), indicating viable candidates for photoactive material. In the UV region, the absorption at λ = 275, 365 and 385 nm are


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dominated by spin-allowed ligand-centered (LC) π → π* transitions.

A possible reason lies in that the negative bias potential accelerates the electron transfer from the conduction band of ITO to the HOMO of [(C6)2Ir(dppz]+PF6−, and then to the electron acceptor, accordingly inhibits the recombination of electron-hole pairs. Deaeration induces a remarkable reduction of the cathodic photocurrent by about 85% (Figure S4), indicating that the dissolved O2 in the electrolyte solution acts as an electron acceptor via the formation of a superoxide anion.34 When triethanolamine (TEOA), a common electron donor, was added to the electrolyte solution, the photocurrent decreases obviously, as shown in Figure S5. Besides when O2 was degased by bubbling N2 for 30 min, the direction of the photocurrent was changed and anodic photocurrent was observed.

Figure 1. Absorption spectra of [(C6)2Ir(dppz]+PF6− and Ru(bpy)32+ (1.0 × 105 M) in DMF.

Electrochemical Properties. The electrochemical behavior for [(C6)2Ir(dppz]+PF6− was evaluated by cyclic voltammetry (CV). As shown in Figure S1, the complex exhibits a series of oxidation and reduction waves. At positive potential, it shows a quasi-reversible single electron oxidation peak at E1/2,ox = +1.28 V vs. Ag/ AgCl, which is assigned to IrIV/IrIII metal oxidation process and mainly influenced by iridium-centered orbitals. In addition, the complex displays relatively complex reduction couples at negative potential ranging from −0.8 to −2.3 V with a first reduction couple at E1/2,red = −1.11 V. As opposed to the oxidation process, the reduction may associate with the low lying π* orbitals involving the ligands together with only a minor contribution from the iridium center.31-33 Photoelectrochemical Behaviors. PEC characteristics for [(C6)2Ir(dppz]+PF6− were studied through coating it onto ITO substrates. Through converting the illumination wavelength from 200 to 800 nm, the action spectrum on photocurrents of [(C6)2Ir(dppz]+PF6− films on ITO was recorded in PBS solution. As shown in Figure S2, normalized photocurrent response curve corresponds to the absorption spectrum for [(C6)2Ir(dppz]+PF6− (Figure 1) with a maximum photocurrent at 490 nm. While negligible response is shown on a bare ITO electrode at the same experiment condition. Thus, [(C6)2Ir(dppz]+PF6− coated on the electrodes is responsible for the photocurrent generation. Upon illumination by 490 nm light with a 0.08 mW/cm2 intensity, a cathodic photocurrent up to 260 nA/cm2 is observed from the [(C6)2Ir(dppz]+PF6− modified electrode at 0 V bias voltage in 0.01 M PBS solution, as shown in Figure 2(A). The photocurrent response is rapid and reproducible with only 3.7% decay under 50 on/off cycles of irradiation. The maximum incident photon to current conversion efficiency (IPCE) at 0 V bias voltage was improved by about 8.5 times compared to our previously reported [(ppy)2Ir(dppz]+PF6−, as depicted in Figure S3. To gain insight into the electron-transfer process, the effect of bias voltage on photocurrent was investigated. As depicted in Figure 2(B), it can be seen that the negative bias voltages have a great effect on photocurrent. The cathodic photocurrent increases along with the increasing of the negative bias of the electrode, whereas positive bias voltages do the opposite, which attenuate the cathodic photocurrent.

Figure 2 (A) Photocurrent generation from [(C6)2Ir(dppz]+PF6− modified ITO electrode by switching on and off the illumination (490 nm) in 0.01 M PBS solution (pH = 7.4); (B) Photocurrent response at different bias voltage (vs Ag/AgCl) of (a) 0.3, (b) 0.2, (c) 0.1, (d) 0,(e) −0.1, (f) −0.2, (g) −0.3 V.

To deduce the route for cathodic and anodic photocurrent generation, excited-state redox potentials of [(C6)2Ir(dppz]+PF6− were estimated according to the following equations:

E0-0 = 1240/abs(onset) III*/IV

(1)

III/IV

E (Ir ) = Eonset(Ir )  E0-0 (2) E (IrIII,L*/III,L ) = Eonset(IrIII,L/III,L )  E0-0 (3) Where λabs(onset), Eonset(IrIII/IV) and Eonset(IrIII,L/III,L-) is 540 nm, 1.18 V and −1.26 V, respectively, according to UV−vis absorption spectrum and cyclic voltammetry data. Thus, the excited state potentials of IrIII*/IV and IrIII,L*/III,L- was determined to be −1.12 and 1.04 V, respectively. Employing estimated energy level values, the speculated photocurrent generation



process is depicted in Scheme 2. The process was initiated by excitation of [(C6)2Ir(dppz]+PF6− (IrIII) to the formation of the excited state (IrIII*). The cathodic photocurrent indicates that electrons flow from the electrode to the electrolyte (solid line in Scheme 2), which involves an electron transfer from the IrIII* moiety to the dissolved O2 in the electrolyte solution with a subsequent electron transfer from the ITO electrode to the IrIII. However, in the case of electron donors such as TEOA, the IrIII* was reduced by TEOA to form IrIII,L- where the ligand (L) of the IrIII complex accepts an electron to form L− due to inaccessible electrochemical reduction for metal Ir(III)31,35,36. Subsequently, the IrIII,L- injects an electron into the electrode resulting in an anodic photocurrent (dashed line in Scheme 2).

Scheme 2 Proposed Mechanism for cathodic (solid lines) and anodic (dashed lines) photocurrent generation. All potentials are given vs Ag/AgCl (sat. KCl) DNA Binding Studies. To explore the binding ability of the as-prepared complex with ds-DNA, competitive fluorescent EB displacement assay was performed between EB and [(C6)2Ir(dppz]+PF6− with calf thymus DNA. It is known to all that fluorescence intensity of EB increases when it binds with ds-DNA.37 Competitive binding of other compounds to DNA could displace EB from the EB−DNA system due to its weaker intercalating ability, resulting in the decrease of the fluorescence intensity, which could verify the binding mode of the compound with DNA. The effects of [(C6)2Ir(dppz]+PF6− on the fluorescence intensity of the EB−DNA system are shown in Figure S6. Apparently, the fluorescence intensity of EB−DNA experienced an appreciable decrease with increasing concentrations of [(C6)2Ir(dppz]+PF6−, suggesting [(C6)2Ir(dppz]+PF6− could displace EB from the EB−DNA system and strongly interact with ds-DNA. So [(C6)2Ir(dppz]+PF6− could be adopted as efficient intercalator for the following biochemical analysis. Fabrication of the PEC Platform Based on HCR Amplification. To demonstrate the applicability of the assynthesized [(C6)2Ir(dppz]+PF6− in bioanalysis, a PEC platform was fabricated based on an enzyme-free hybridization chain reaction (HCR) as amplification strategy. MicroRNAs (miRNAs) are an important kind of nonproteincoding RNA molecules with 19-23 nucleotides, which regulate the expression of mRNA, as well as the transcriptional and post-transcriptional gene expression.38 It has been demonstrated that miRNAs was associated with various key biological processes, and could serve as important diagnostic biomarkers for tumor.39,40 However, at present accurate detection and quantification of miRNAs remains a major

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challenge due to their unique characteristics, such as small size, low abundance, and sequence similarity.41 In this study, combining superior PEC analytical technique with Ir(III) complex based photoactive species, a PEC detection platform was present with miRNA-122b as the target model, as depicted in Scheme 3. The presented HCR amplified strategy involves the self-assembly of the thiolated DNA hairpin capture-H1 which is partly complementary to miRNA-122b, onto the ITO electrode decorated with AuNPs. In the presence of target, hairpin capture-H1 modified on the electrode was unfolded by virtue of its hybridization with target miRNA, leading to the newly exposed stem section as toehold initiator. Interaction of the initiator with a mixture of the hairpins H1 and H2 initiates the HCR process. Alternating addition of hairpins H1 and H2 result in the formation of dsDNA polymers for intercalation of abundant [(C6)2Ir(dppz]+PF6−. Thus, an increased photocurrent was realized. While, in the absence of the target miRNA, hairpins capture-H1, H1 and H2 maintain their respective hairpin structures in the solution, leading to a comparatively low background signal.42,43 By recording photocurrent intensities, the detection of the target miRNA can be achieved. Products of miRNA-triggered HCR were demonstrated by gel electrophoresis. As depicted in Figure 3, Lane 1-4 denotes target miRNA-122b, hairpin H1, hairpin H2 and a mixture of H1 and H2, respectively, no secondary structure is observed, indicating HCR couldn’t be initiated in the absence of target RNA. However, long duplex concatamers with high molecular weight are produced in the presence of different concentrations of target (Lane 5 and 6), suggesting the successful initiation of HCR and efficient signal amplification effect.

Figure 3 Polyacrylamide gel electrophoresis demonstration of the target miRNA-122b initiated HCR: lane 1, 2 μM target RNA; lane 2, 1 μM H1; lane 3, 1 μM H2; lane 4, a 1 μM mixture of H1 and H2; lane 5 and 6, in the presence of the target RNA (0.1 μM and 0.5 μM) with a 1 μM mixture of H1 and H2; lane 7, DNA ladder markers.

Electrochemical Characterization for the Fabrication of the PEC Platform The stepwise assembly procedures of the PEC platform were monitored by electrochemical impedance spectroscopy(EIS), which is an effective means for probing electrode surfaces. Nyquist plots for the electrode of different assembly stages are illustrated in Figure 4, the semicircular portion of the impedance spectrum in the high-frequency range represents the electron-transfer resistance (Ret), corresponding to the limited electron transfer process. For a bare ITO electrode, a large Ret was presented (curve a). Monolayer of APTMS assembled on the ITO electrode


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resulted in a relatively small Ret (curve b) due to formation of amino ends, which might facilitate the redox probe [Fe(CN)6]4−/3− to trans

Scheme 3 Schematic illustration of PEC detection platform for miRNA based on HCR amplification strategy fer onto the electrode surface. Assembly of Au NPs on the electrode surface led to a further decrease in Ret value (curve c) because of the good conductivity of Au NPs. Subsequently, the immobilization of thiol-modified capture-H1 gave rise to a large increase in Ret value (curve d) on account of the repulsion between phosphate backbone with negative charges and redox probe, and also suppression of interfacial electron transfer. After hybridization of target RNA with capture-H1 on the electrode surface, the Ret value was further increased (curve e) due to the increase of negative charges. A remarkable increase in Ret value was observed in the presence of hairpin H1 and H2 (curve f), ascribing to the formation of a long duplex concatamers from HCR. Accordingly, all the results indicate that the PEC platform was fabricated successfully as expected.

Figure 4 Nyquist plots for electrode of different assembly stages (a) the bare ITO electrode; (b) ITO/APTMS; (c) ITO/APTMS/AuNPs; (d) ITO/APTMS/AuNPs/Capture-H1; (e) ITO/APTMS/AuNPs/Capture-H1/miRNA; (f) ITO/APTMS/ AuNPs/Capture-H1/miRNA/H1+H2

Optimal Conditions for the PEC Assay. To achieve a superior analytical performance of the PEC platform, some experimental conditions, such as hybridization time of HCR,

concentration of [(C6)2Ir(dppz]+PF6−, incubation time for the intercalation of [(C6)2Ir(dppz]+PF6− and pH value of detection solution were optimized by evaluating the changes of the pho tocurrent intensities. Hybridization time of HCR played a vital role which could form long duplex concatamers and then directly influence the amount of signal species, so the effect of the HCR hybridization time on the photocurrent was investigated. As shown in Figure 5(A), the photocurrent enhanced with the prolonging of hybridization time up to 2 h and then levels off after 2 h. Thus 2 h was selected as the optimum time

Figure 5 Effects of (A) hybridization time of HCR, (B) concentration of [(C6)2Ir(dppz]+PF6− in the absence (red round symbol) and presence (black square symbol) of target, (C) incubation time for the intercalation of [(C6)2Ir(dppz]+PF6− in the absence (red round symbol) and presence (black square symbol) of target and (D) pH value of detection solution on photocurrent. The error bars signify the standard deviation of three replicate measurements.



for the HCR hybridization. Figure 5(B) illustrates the influences of [(C6)2Ir(dppz]+PF6− concentration on the photocurrent in the presence and absence of target RNA, respectively. Obviously, a peak photocurrent was obtained when the concentration is 1.0 × 10−5 M. Also, excessive concentration gives rise to increased blank current due to the stronger diffusion process. So a concentration of 1.0 × 10−5 M was chosen as the optimum. The intercalation time for signal species into DNA helix was also studied, as illustrated in Figure 5(C). With increasing incubation time, the photocurrent increased significantly due to the augment of signal species loaded on the working electrode in the presence of target, and it reaches a plateau after 30 min probably ascribing to saturation of DNA helix. Simultaneously, the background photocurrent increased slowly with the increment of incubation time. So, 30 min was adopted as the optimum incubation time for the intercalation process due to the maximum signal-background ratio. The pH value of detection solution was also assessed since the acidity or alkalinity of the electrolyte solution has a great impact on the photocurrent generation. As depicted in Figure 5(D), in acid medium with pH value from 3.0 to 5.5, the photocurrent has a negligible variation. While it is rapidly decreased with the increasing pH value in alkaline range, indicating that an acidic electrolyte is favorable for the PEC platform. In view of favorable environment for bioanalysis and excellent photocurrent response, a pH value of 5.5 was selected for the following measurement.

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photocurrent was observed in the absence of target miRNA, which ascribe to background current from nonspecific adsorption and interaction with capture hairpins on the electrode. When target was introduced into this assay, photocurrent monotonically increased along with the increasing miRNA-122b concentration, which ascribes to the fact that HCR was initiated by target and the increased amount of signal species were loaded on the resulting long duplex concatamers for signal generation. And a signal plateau was found when concentrations of the target exceed 1000 fM. As illustrated in Figure 6(B), an excellent linear function was achieved between photocurrent intensity and logarithm of miRNA concentration over the range from 1 to 1000 fM with R2 = 0.998. The limit of detection was estimated to be 0.23 fM based on 3σ (n = 7), indicating a competitive detection sensitivity superior or compared to those of previous reports (Table S2).44-46 A relative standard deviation of 6.2% is obtained by detection target miRNA at 10 fM from five parallel assays, indicating the fine reproducibility of the strategy. The stability of the detection platform was investigated by recording its photocurrent every day over 7 days (Figure S7 in the Supporting Information), the photocurrent intensity decay by 17.6% after 7 days, indicating good stability. The impressive analytical performance could be attributed to the combination of perfect PEC behavior of the [(C6)2Ir(dppz]+PF6− complex with efficient signal amplification of HCR.

Figure 7 Specificity investigation of the PEC platform for miRNA-122b against miRNA-122, miRNA-21, miRNA-144, miRNA-199 and 1-mismatch-m. All the measurements were performed three times independently.

Figure 6 (A) Photocurrent response of the PEC platform toward varying concentrations of microRNA; (B) Dependence of photocurrent intensity (I) on concentration of microRNA. Inset shows the linear fit plots of I vs logarithm of microRNA concentrations. The error bars represent the standard deviation of three parallel assays.

Analytical Performance of the PEC Platform. The analytical performance of the proposed PEC platform was evaluated under optimized conditions discussed above. The photocurrent responses vs. varying concentrations of miRNA122b were shown in Figure 6(A). It can be seen that a lower

The specificity of the proposed detection platform for miRNA122b was further evaluated by comparing photocurrent response to other control miRNAs, including miRNA-122, miRNA-21, miRNA-144, miRNA-199 and single mismatched nucleotide in the middle of the strand (1-mismatch-m). As depicted in Figure 7, signal responses from miRNA-21, miRNA-144 and miRNA-199 were negligible compared to the blank control. In contrast, a higher nonspecific signal, acquired from miRNA-122 with just single nucleotide deficiency at the 3’ end compared to the target, could be clearly discriminated from that of target miRNA-122b. While for the synthesized RNA with 1-mismatch-m, signal response is almost the same as the blank control. The above results indicate that the proposed detection platform possesses satisfactory specificity and it is capable of discriminating quite similar miRNAs even single-nucleotide mismatches.


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Real Sample Analysis. To further evaluate the practical application of the PEC detection platform in a complex biological matrix, cell lysate samples from human breast adenocarcinoma cells (MCF-7 with high miRNA-122b expression) and human liver carcinoma cells (HEPG-2 with low miRNA-122b expression) were chosen to execute the assay. As depicted in Figure 8, total RNA extractions from an increasing number of MCF-7 and HEPG-2 cells (from 300 to 3000 to 30000) led to an increased photocurrent response. The lysates from HEPG-2 cells caused weaker photocurrent enhancement, while the MCF-7 cells exhibited significantly stronger photocurrent response, suggesting the lower expression of miRNA-122b in HEPG-2 cells compared to the MCF-7 cells. These results are in accordance with the previous reports.47,48 For comparison, the detection of miRNA-122b from cell lysates was also carried out employing our asproposed method and previously reported [(ppy)2Ir(dppz)]+PF6− as photoactive species. As depicted in Figure S8, the lysates from both MCF-7 and HEPG-2 cells (300 and 3000 cells) led to negligible photocurrent responses compared to the blank control. Only MCF-7 with high miRNA-122b expression in the presence of 30000 cells caused obvious signal response. The comparison results demonstrated the as-proposed strategy possessed higher sensitivity employing [(C6)2Ir(dppz)]+PF6− as photoactive species rather than [(ppy)2Ir(dppz)]+PF6−, which is probably attributed to the high photon-to-current conversion efficiency of [(C6)2Ir(dppz)]+PF6− and low damage to biomolecules using visible light as excited light source.

Figure 8 Detection of miRNA-122b from cancer cell lysates of MCF-7 and HEPG-2. CONCLUSIONS In summary, a novel cationic Ir(III) complex, [(C6)2Ir(dppz)]+PF6− was successfully prepared and characterized through absorption and redox and PEC behavior. It was found to possess excellent light-harvesting property with a molar extinction coefficient up to 9.8 × 104 M−1 cm−1 at 485 nm, indicating enormous potential for photoactive species. As proved in this study, the as-prepared complex showed rapid and reproducible photocurrent response illuminated by visible light, and cathodic photocurrent up to 260 nA/cm2 was observed in the presence of dissolved O2 as an electron acceptor. On the other hand, an anodic photocurrent was obtained with TEOA as a sacrificial electron donor. The direction of the photocurrent depends on the nature of the redox couple in the electrolyte. Also, the complex [(C6)2Ir(dppz)]+PF6− was verified to exhibit fine binding affinities to ds-DNA by DNA emission titrations assay. In view of the outstanding PEC behavior and fine binding affinities to ds-DNA of the complex, a PEC platform was developed uniquely employing it as intercalated signal

reporter and enzyme-free HCR as amplification strategy. As expected, the PEC platform exhibited an approving performance for the detection of miRNA with a detection limit down to 0.23 fM (3σ) and high specificity. The results demonstrated that Ir(III) complex was the attractive candidates as photoactive species for PEC bioanalysis, as well as it will be valuable for the design of Ir(III) complex based photoactive species with red-shift visible absorption to improve light harvesting properties and thereby increase photon-to-current conversion efficiency.

ASSOCIATEDCONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details for materials and reagents, apparatus, procedure for preparation of the complex [(C6)2Ir(dppz]+PF6−, Table S1-2, Figure S1-5 and reference(PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (C. Li); [email protected] (X. Luo); phone: +86-532-84022681.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the Natural Science Foundation of China (21775079), Shandong Provincial Natural Science Foundation (ZR2017MB010), the Project of Shandong Province Higher Educational Science and Technology Program (J17KA106), and the Taishan Scholar Program of Shandong Province, China (ts20110829).

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