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Using G-quadruplex/hemin to “switch-on” the cathodic photocurrent of p-type PbS quantum dots: Towards a versatile platform for photoelectrochemical aptasensing Guang-Li Wang, Jun-Xian Shu, Yuming Dong, Xiuming Wu, Wei-Wei Zhao, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5043945 • Publication Date (Web): 04 Feb 2015 Downloaded from http://pubs.acs.org on February 6, 2015

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Analytical Chemistry

Using G-quadruplex/hemin to “switch-on” the cathodic photocurrent of p-type PbS quantum dots: Towards a versatile platform for photoelectrochemical aptasensing Guang-Li Wanga,b,*, Jun-Xian Shua, Yu-Ming Donga, Xiu-Ming Wua, Wei-Wei Zhao b,*, Jing-Juan Xub, Hong-Yuan Chenb a

The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, Jiangsu, China.

b

State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, Jiangsu, China.

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Abstract: We present a novel photoelectrochemical (PEC) biosensing platform by taking advantage of the phenomenon that hemin intercalated in G-quadruplex “switched-on” the cathode photocurrent of ptype PbS quantum dots (QDs). Photoinduced electron transfer (PET) between PbS QDs and Gquadruplex/hemin(III) complexes with the subsequent catalytic oxygen reduction by the reduced Gquadruplex/hemin(II) led to an obvious enhancement in the cathodic photocurrent of the PbS QDs. For the detection process, in the presence of hemin, the specific recognition of the targets with the sensing sequence would trigger the formation of a stable G-quadruplex/hemin complex, which will result in reduced charge recombination and hence amplified photocurrent intensity of the PbS QDs. By using different target sequences, the developed system made possible a novel, label-free “switch-on” PEC aptasensor towards versatile biomolecular targets such as DNA and thrombin. Especially, with ambient oxygen to regenerate G-quadruplex/hemin(II) to G-quadruplex/hemin(III), this substrate-free strategy not only promoted the photoelectric effect and thus the enhanced sensitivity of the system, but also avoided the addition of supplementary substrates of G-quadruplex/hemin such as H2O2 and organic substances.

Keywords: Photoelectrochemistry; PbS quantum dots; G-quadruplex/hemin; Aptasensing


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Ever since 2000, photoelectrochemical (PEC) detection has been actively developing and draws growing attention among the analytical community.1-7 Such detection system could provide excellent sensitivity due to the totally different forms of energy for excitation and detection. Due to its attractive potential in future biomolecular detection, currently, enormous effort has been devoted to its exploitation and substantial advances have been achieved.3-17 However, due to the short development time, the study on PEC bioanalysis is still in its inception phase and yet leaves much to be desired. For example, as we recently reviewed,1,2 only a few photoactive materials are exploited in established formats. As known, the properties of photoactive species would essentially determine the analytical performances of the PEC sensors. To date, previous works rely largely on the photoanodes of n-type semiconductors or sensitized n-type semiconductors such as CdS quantum dots (QDs),3,6,7,15 CdSe/ZnS,8 TiO2 or sensitized TiO2,4,5,9-12,14 and so on. Unfortunately, the practical applications of such n-type semiconductors are limited by the interference from the competitive reactions of the reductive components such as ascorbic acid, 9,10 H2O2,11 nicotinamide adenine dinucleotide,12 dopamine13 and thio-compounds3,14 coexisting in the complex biological fluids. Apparently, the selectivity of the photoanode can hardly be improved by simply changing the composition of n-type semiconductors themselves due to the reactive holes present at the n-type semiconductor/electrolyte interface. On the other hand, the present sensing strategies are also rather limited. The introduction of PEC labels/indicators and generating electron donors/acceptors and/or steric hindrance are the widest used approaches available.4-7,9-12,15 Thus, to design novel photoelectrodes for advanced PEC detection with innovative signalling mechanism would obviously be desirable. The true self-operating photocathode of p-type nickel oxide electrode utilizing a freebase porphyrin or erythrosine B as a sensitizer was firstly introduced by Lindquist et al,18 whose pioneering work has generated renewed interest in solar cells19 and PEC water splitting20,21 in recent years. Photocathode may provide a viable strategy for advanced PEC bioanalysis applications. Different from the commonly studied photoanodes based on the use of n-type semiconductors,3-17 photocathodes made by p-type semiconductors could show intriguing and different PEC properties although whose development was ACS Paragon Plus Environment

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still far behind. The p-type semiconductors prone to interact with electron acceptors (e.g. dissolved oxygen) but not electron donors (e.g. H2O2, ascorbic acid, dopamine, cysteine) in electrolyte,22 implying their great potential for anti-interference capability from reductive substances. PbS is an intriging direct band gap (about 0.41 eV) semiconductor and usually exists as a p-type condutivity.23 It has a large Bohr radius, high dielectric constant, very high carrier mobility, as well as strong confinement effects on charge carriers.24 Especially, PbS NPs can exhibit multiple exciton generation (MEG), in which the absorption of one high energy photon can produce multiple electron-hole pairs.25 With MEG, enhanced energy conversion efficiency is expected due to reduction in the thermalization of electron−hole pairs.26 Especially, compared to CdSe or CdS quantum dots (QDs), longer excited state lifetimes (∼2.6 µs) are observed for PbS QDs, which enables photo induced electron transfer (PET) of PbS QDs with redox species that even not adsorbed on the QDs’ surface.27 Though PbS QDs have more fascinatingly photovoltaic or photocatalytic properties than many n-type semiconductors, to the best of our knowledge, PbS QD has not been exploited for PEC analytical application. G-quadruplex/hemin owns some unique characteristics, such as much low cost in production, relatively facile for labling, more resistant to heat treatment or hydrolysis.28 The flexibility to encode in the base sequences of the nucleic acid probes (which can be tailored to comprise the catalytic domain with enzym-like activity and a sensing domain that can hybridizes with the target molecules due to the complementariness), together with the reduced nonspecific binding properties of nucleic acids, making the G-quadruplex/hemin an ideal candidate for developing versatile biosensing platforms.29-35 Specifically, G-quadruplex/hemin-based DNAzyme displays peroxidase-like catalytic activity30-33 and electrocatalytic activity,34 which was widely applied as a label for the colorimetric,30,31 chemiluminescent32,33 or electrochemical34 detection of different targets with amplified signal. Furthermore, another fluorescence aptasensor was also implemented based on the phenomenon that the fluorescence of CdSe/ZnS QDs was quenched by G-quadruplex/hemin due to photo induced electron transfer (PET).35 However, this strategy was based on “turn-off” mechanism. It is believed that “turn-


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on” moitf is preferable than the “turn-off” one in terms of sensitivity and selectivity due to its lower background and reduction in the chance of false positives. Herein, we report a novel and versatile “switched-on” PEC biosensing platform based on the exquisite integration of intriguing photocathode made of p-type PbS QDs with the versatile function of the G-quadruplex/hemin configuration. Specifically, the G-quadruplex/hemin(III) complex accepted electrons from the illuminated PbS QDs and mediated the catalytic reduction of dissolved oxygen, which resulted in improved charge separation and an obviously increased cathodic photocurrent of PbS QDs. The p-type conductivity nature of PbS QDs23 and their longer excited state lifetimes27 enabled the exquisite PET interactions between the illuminated PbS QDs and G-quadruplex/hemin. Through encoding in the base sequences of the nucleic acid probes, a versatile PEC platform could be developed to detect various targets such as DNA and thrombin. The recognition between the target biomolecules and the tailored sensing nucleic acid domain on the capture probe could trigger the in-situ generation of the G-quadruplex/hemin configuration and thus “switch-on” the cathodic photocurrent of PbS QDs. To our knowledge, this work for the first time presents a “switch-on” PEC biosensor based on in-situ PET between PbS QDs-based photocathode and G-quadruplex/hemin structure, which might open a new perspective for the construction of novel PEC sensing platforms. EXPERIMENTAL SECTION Chemicals and Materials. Chemicals and materials used are provided in the supplementary information. Synthesis of PbS QDs and the Fabrication of PbS QDs Modified ITO Electrodes. We prepared thioglycolic acid (TGA)-stabilized PbS quantum dots (QDs) according to a slightly modified procedure.36 A 17 µL of TGA was mixed with 25 mL of 4.0 mmol/L Pb(NO3)2 aqueous solution, followed by dropwise addition of 1.0 mol/L NaOH to adjust its solution pH to 11. Being purged by high pure nitrogen to eliminate oxygen for 30 min, 2.0 mL of 0.015 mol/L Na2S was injected. The solution promptly became dark-brown after the addition of Na2S, indicating the formation of PbS QDs. The ACS Paragon Plus Environment

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resulting solution mixture was kept for reaction under nitrogen atmosphere for 4 h. The as-synthesized TGA-capped PbS QDs were stored at 4 ºC before use. The assembly of PbS QDs on the treated ITO slices to obtain ITO/PbS film was according to our previous procedure.7 Preparation of PbS QDs-based PEC Biosensor and the Detection Procedure. To conjugate hairpin DNA(1) onto the PbS QDs modified ITO electrode, we took advantage of the classic EDC coupling reactions between COOH groups of the TGA-capped PbS QDs and the NH2 groups of the hairpin DNA(1). Firstly, the PbS QDs modified electrode was immersed in 1.0 mL of aqueous solution consisted of 20 mg of EDC and 10 mg of NHS for 1 h at ambient temperature to activate their COOH groups, which was then thoroughly rinsed with the washing buffer to remove excess reactants. After that, 25 µL of 1.0 µmol/L hairpin DNA(1) was dropped onto the above electrode surface and incubated at 4 ºC for 16 h under moisture atmosphere. After rinsing with the washing buffer, the electrode was then blocked with 25 µL of blocking solution to reduce nonspecific adsorption for 1 h at 4 ºC and again washed thoroughly. Then, the photocurrent of the ITO/PbS/DNA(1) electrode was detected (defined as I0) in 0.1 mol/L Tris-HCl (pH 7.4) consisted of 0.05 mol/L KCl and 0.1 mol/L NaCl. Subsequently, 25 µL of target DNA(2) at different concentrations and 25 µL of 1.0 µmol/L hemin were spread onto the hairpin DNA(1) immobilized electrodes for a 50 min incubation at 37 ºC before washing. Finally, the electrode was introduced to the electrolyte for PEC measurements (the photocurrent is defined as I). The detection process for TB was similar to that for target DNA. RESULTS AND DISCUSSION Morphological and Structural Characterization of TGA-PbS QDs. As shown in Figure 1A, the size of the spherical TGA-capped PbS QDs were about 3-5 nm. The inset HRTEM image of individual QDs implied that they had relatively good crystallization. A lattice spacing of ~0.17 nm was observed in HRTEM image, corresponding to t he (222) plane of cubic PbS. The XRD pattern of the TGA-capped PbS QDs showed sharp diffraction peaks at scattering angles (2θ) of 25.94°, 30.02°, 43.02°, 50.86°, 53.44°, 62.38°, 68.80°, 71.02° and 78.84° (Figure 1B), corresponding to scattering from the (111), ACS Paragon Plus Environment

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(200), (220), (311), (222), (400), (331), (420) and (422) planes of the standard cubic PbS [JCPDS File No. 5-592]. The XRD result demonstrated that the product was face-centered cubic PbS. It was observed that all the peaks were very prominent and sharp, indicating that the product had a relatively good crystallinity. A broad absorption range from 400 to 1300 nm was monitored for PbS QDs (Figure 2), indicating its suitability to be a photoactive material under the light (λ≥400 nm) irradiation. The inset image in the absorption spectrum showed a clear absorption peak at around 1192 nm, from which the size of the resulting PbS QDs was estimated37 to be about 4.1 nm. There was consistence for the particle size calculated from the absorption spectrum and that from the TEM image. Figure 1. Figure 2. PEC Response of the ITO/PbS Electrode. Compared to the widely used CdSe or CdS ones, PbS QDs are more fascinating for photocatalytic and photovoltaic applications. This is because (i) they have broad absorption spectrum in the visible to near infrard range,38 (ii) multiple charge carriers can be produced after absorbing a photon, leading to higher photo-to-electron out-put,24 and (iii) owning to their longer exciton lifetime, they have more sufficient time for transferring electrons to redox species.27 In this experiment, when irradiating the PbS QDs electrode, a small cathodic photocurrent was observed in air-saturated Tris-HCl solution (0.1 mol/L, pH 7.4, containing 0.1 mol/L NaCl and 0.05 mol/L KCl) (Figure S1, curve b, see the Supporting Information). It should be reasonable to observe cathodic photocurrent for PbS QDs electrode since PbS is usually a p-type semiconductor.23 The cathodic photocurrent intensity increased when cathodic bias was applied (Figure S2, Supporting Information), which was also a characteristic of p-type semiconductors.39 The photocurrent generation mechanism of the PbS QDs is displayed in Scheme 1A. Electrons/holes were formed on the conduction band (CB)/valence band (VB), respectively, when PbS QDs were exicitated by photons with energies equal to or higher than that of their band gap. The photogenerated electrons transferred from the CB of PbS to the electron acceptors in electrolyte and photogenerated holes were easily captured by the electrons of ITO electrode, generating cathodic photocurrent.20 ACS Paragon Plus Environment

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Scheme 1. It was found that ambient dissolved oxygen could affect the cathodic photocurrent intensity of PbS QDs greatly. When the air-saturated electrolyte was deoxygented by purging with high pure nitrogen, a great inhibition was found in the cathodic photocurrent of the PbS QDs modified electrode (Figure S1, curve a, see the Supporting Information). The decreased cathodic photocurrent of ITO/PbS might be attributed to the fact that the dissolved oxygen accepted electrons from the illuminated PbS QDs. As is observed, a suitable electron acceptor could capture photogenerated electrons and enhance charge separation of p-type seminconductors effectively, leading to the greatly enhanced cathodic photocurrent.40 In addition, we also observed that the subsequent addition of hemin in the air-saturated electrolyte solution further led to obviously increased photocurrent of PbS QDs and the cathodic photocurrent enlarged progressively with an increment in the concentration of hemin (Figure S1, curve c-e, see the Supporting Information). This phenomenon inspired us to explore the effect of intercalated hemin in G-quadruplex on the PEC property of PbS QDs. Excitingly, we found that even if hemin was conjugated in G-quadruplex, the enhanced photocurrent of PbS QDs by hemin was also observed (Figure 3, curve b and d). The enhancement effect of G-quadruplex/hemin for the PbS QDs was influenced by dissolved oxygen. If dissolved oxygen was removed, the enhancement effect of hemin in G-quadruplex for the PbS QDs still existed, indicating that there must be a direct interaction between illuminated PbS QDs and hemin. However, the photocurrent enhancement effect of hemin in Gquadruplex for PbS QDs in aerated solutions was larger than that in deaerated solutions (Figure 3), demonstrating that oxygen also participated in the PEC interaction between PbS QDs and hemin. Figure 3. We ascribed this photocurrent enhancement to the PET from the CB of PbS QDs to hemin in Gquadruplex/hemin complex, which greatly inhibiting the electron-hole recombination in illuminated PbS QDs, leading to enhanced photocurrent. To confirm that the increased photocurrent of PbS QDs by hemin proceeded via a PET mechanism, electrochemical measurements41 were employed to study the CB and VB edge of PbS QDs. The results showed that PbS had a CB edge at −0.83 V and a VB edge at ACS Paragon Plus Environment

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0.34 V vs Ag/AgCl (saturated KCl) (Figure S3, see the Supporting Information). That is, the CB and VB potential of PbS QDs was -0.63 and 0.54 V vs normal hydrogen electrode (NHE), respectively. The hemin in G-quadruplex complex revealed a reduction potential of −0.378 V vs Ag/AgCl (i.e., −0.178 V vs NHE) (Figure 4, curve b) corresponding to the FeIII/FeII-protoporphyrin IX couple, whose potential was approximately in accordance with the previously reported result.42 By comparison, it can be found that the reduction potential of the G-quadruplex/hemin complex was more positive than the conduction band levels of the PbS QDs (Scheme 1B), implying that there was a potential gradient that drove the electron transfer from the excited electrons (-0.63 V vs NHE) of the PbS QDs to hemin in the Gquadruplex/hemin complex (−0.178 V vs NHE). PET between Ag nanoclusters or CdSe/ZnS QDs and G-quadruplex/hemin were also observed previously, which caused serious fluorescence quenching of the nanomaterials.35,42 However, the G-quadruplex/hemin was found to hardly affect the anodic photocurrent of n-type CdS QDs.43 In our experiment, the PET between PbS QDs and Gquadruplex/hemin occurred and was reflected successfully by the cathodic photocurrent measurements. This was because p-type PbS QDs with faster electron transfer towards the electrolyte than that of holes enabled them to interact with electron acceptors rather than electron donors in the electrolyte.21 While for n-type semiconductors, holes transfer faster towards the surface of the semiconductors than electrons made them prone to interact with electron donors in the electrolyte.21 Especially, the long exciton lifetime of PbS QDs (∼2.6 µs)27 allowed them more time to PET with redox partners in solution, a property that facilitated the interaction between redox partners and PbS QDs. Figure 4. It should be pointed out that the enhancement of G-quadruplex/hemin for PbS QDs in air-saturated solution was more obvious than that in deaerated solutions (Figure 3). This may indicate that Gquadruplex/hemin not only acted as an electron acceptor of PbS, but also behaved as a mediator toward the catalytic reduction of oxygen. After accepting electrons from illuminated PbS QDs, hemin(III) in Gquadruplex/hemin was reduced to hemin(II). The hemin(II) could easily react with oxygen to regenerate hemin(III) through the reduction of oxygen.44 The cyclic voltammetry （ CV ） ACS Paragon Plus Environment

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quadruplex/hemin-coated electrode in deaerated solution (Figure 4, curve b) demonstrated quasireversible redox peaks resulting from the direct electrochemistry of hemin, indicating the successful assembly of G-quadruplex/hemin on the electrode after the DNA recognition event. In aerated solution, the oxidation peak of hemin in G-quadruplex decreased greatly and a prominently enhanced reduction peak was observed (Figure 4, curve c), which was much stronger than that of hemin in deaerated solution. This phenomenon confirmed the catalytic reduction of dissolved oxygen by hemin(II) in the Gquadruplex/hemin complex. In the PEC detection process, due to the regeneration of the reduced hemin(II) to hemin(III) by oxygen, a much enhanced photoelectrochemical response of PbS QDs to hemin intercalated in G-quadruplex was observed in the presence of oxygen. Through the electron accepting by G-quadruplex/hemin(FeIII) and its subsequent catalyzed reduction of oxygen, the electronhole recombination of the illuminated PbS QDs was inhibited and a greatly enhanced photocurrent intensity was attained. PEC Biosensor. Based on the greatly enhanced photocurrent of PbS QDs by hemin intercalated in the G-quadruplex, we used the hybrid systems by coupling the G-quadruplex/hemin to PbS QDs to construct versatile biosensing platforms by taking DNA and an aptamer substrate-thrombin (TB) as examples. The detection of a nucleic acid utilizing the PbS QDs G-quadruplex/hemin hybrid is schematically depicted as Scheme 2A. The surface of the PbS QDs were conjugated by an aminated hairpin DNA(1) that included domain α, which hybridized with the target analyte DNA(2), and domain β that consisted of the nucleic acid sequence to form G-quadruplex. At first, the nucleic acid sequence for yielding of the G-quadruplex was caged in the stem region of the hairpin, and the formation of the active G-quadruplex/hemin structure was avoided.42 After hybridizing with the target DNA by the domian α of DNA(1), the hairpin structure was opened, which released the sequence for G-quadruplex, allowing the formation of the G-quadruplex/hemin on the QDs. Once the G-quadruplex/hemin was introduced, PET from the CB of PbS QDs to hemin embeded in G-quadruplex and then to dissolved oxygen occurred, leading to increased cathodic photocurrent of the PbS QDs. Scheme 2. ACS Paragon Plus Environment

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The construction of the DNA sensor was characterized by electrophoresis, UV-vis absorption spectra, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry measurements (CVs). Polyacrylamide gel electrophoresis (PAGE) assays and UV-vis absorption spectra were used to confirm nucleic acid hybridization and the formation of G-quadruplex/hemin structure. The gel electrophoresis result is indicated in the inset image in Figure S4. Lane 1 displayed a band for hairpin DNA(1). A faint and fast migration band was observed in lane 2 due to the single-stranded structure and the short sequences of the target DNA(2). After the hairpin DNA(1) and the target DNA(2) were mixed with 1:1 molar ratio, a new band with lower mobility appeared (lane 3), demonstrating the hybridization of the hairpin DNA(1) with the target DNA(2).45 Compared to lane 3, the was no obvious change after the further addition of hemin to the mixture of hairpin DNA(1) and the target DNA(2) (lane 4), probably due to the small molecular weight of hemin. Subsequently, we used UV-vis absorption spectra (Figure S4) to characterize the formation of G-quadruplex/hemin structure after the hybridization between hairpin DNA(1) and the target DNA(2). Free hemin had an absorption band centered at around 395 nm (curve a). No change was observed after the addition of hairpin DNA(1) or target DNA(2) to hemin (data not shown). However, after the incubation of hemin with hairpin DNA(1) and target DNA(2), a red shift of the absorption peak from 395 to 404 nm with enhanced absorption intensity was observed (curve b), indicating the formation of a G-quadruplex/hemin structure.46 The semicircle diameter in the impedance spectrum reflects the electron-transfer resistance (Ret) of the redox probe on the surface of the electrode.7 Bare ITO electrode displayed a small semicircle (Figure S5, curve a, the Supporting Information, 25 Ω) due to that it was well conductive. The subsequent adsorption of negatively charged TGA-capped PbS QDs blocked the access of the Fe(CN)63-/4- molecules to the electrode surface, resulted in an increase of the Ret for ITO/PbS (curve b, 43 Ω). The addition of the hairpin DNA(1) layer further suppressed charge transfer at the interface (curve c), mainly because of the large stereotically hinder effect of the stem-loop structure,47 as well as the electrostatic repulsion between the DNA backbone and the redox probe (Fe(CN)63-/4-) with the same negative charges. Upon hybridization with the target DNA(2) and then treated with hemin, the Ret dramatically increased (curve d). It was expected that the ACS Paragon Plus Environment

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formed G-quadruplex/hemin structure was a more orderly and dense configuration with negative charges on the interface, which had stronger repelling ability for the negatively charged redox species. What′s more, the CV curve of the ITO electrode in deaerated solution showed no obvious redox peak (Figure 4, curve a). After dropping the DNA(1), target DNA(2) and hemin on the electrode surface, a quasireversible redox wave occurred (Figure 4, curve b), also indicating that the G-quadruplex/hemin complexe was successfully formed on the electrode. The factors including the concentrations of hairpin DNA and hemin, incubation time and applied potential were examined on the performance of the PEC DNA sensor. The photocurrent change was defined as ∆I=I-I0, where I and I0 were the photocurrent intensity of the PbS QDs modified electrode with and without target DNA, respectively. As shown in Figure S6A, the biosensor with the hairpin DNA concentration of 1.0 µmol/L showed the highest photocurrent enlargement efficiency. There existed a larger steric obstacle effect at higher concentrations (>1.0 µmol/L) of immobilized hairpin DNA, while there would be more space to unfold of the hairpin probe to form G-quadruplex structure at low concentrations. Thus, an optimal hairpin DNA concentration of 1.0 µmol/L was chosen for the immobilization. The photocurrent enlarged with the increasing concentration of hemin added and attained the highest response at 1.0 µmol/L of hemin (Figure S6B，see the Supporting Information). From Figure S6C, we could know that the PEC responses increased with the extension of DNA incubation time and achieved its maximum at 50 min and then a platform was observed, mainly because that the hybridization reaction completed. In addition, we also investigated the effect of applied potential (-0.3 to 0 V) on the photocurrent responses (∆I/I0) of the ITO/PbS electrodes to G-quadruplex/hemin complex (Figure S6D). It was found that the maximum photocurrent response (∆I/I0) reached when the applied potential was -0.1 V. So, -0.1 V (vs saturated Ag/AgCl) was employed to detect target DNA(2). Once the target DNA(2) and hemin were introduced, the G-quadruplex/hemin configuration came into being stabilized by K+. The G-quadruplex/hemin accepted electrons from illuminated PbS QDs and catalytically reduced dissolved oxygen in solution, thus the cathodic photocurrent of the ITO/PbS


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electrode was increased. Based on this principle, a PEC DNA sensor was developed. Under the optimized experimental conditions, the intensity of the cathodic photocurrent of PbS QDs was highly sensitive to target DNA(2) with increased concentrations (as displayed in Figure 5A). The photocurrent increment (∆I/I0) was linear with the logarithmical concentration of target DNA(2) ranging from 0.8 fM to 10 pM (R=0.992, the inset in Figure 5A). The detection limit was estimated as 0.2 fM (S/N=3). Moreover, the result of this methodology for DNA sensing was compared with other sensors (Table S1, the Supporting Information). A lower detection limit and wider linear range was acquired by comparing the proposed method with currently developed PEC methods6,48,49 or other different measurement protocols including chemiluminescence resonance energy transfer (CRET),32 electrochemiluminescence (ECL),44 chemiluminescence (CL),33 chronocoulometric DNA sensor (CDS)50 and fluorescence resonance energy transfer (FRET).51 There are also some advantages for this method, such as it avoided the process of labeling with markers (such as enzymes), which makes the detection procedure facile and cost-effective. What′s more, unlike other methods utilizing the peroxidase-like activity of the Gquadruplex/hemin, the in-situ generated G-quadruplex/hemin could be directly used for signal production. Supplementary chemical reagents such as substrates of peroxidase including H2O2 and ABTS30,31 or luminol32,33 were not used in our experiment, which indicated that this method was substrate-free. Also, it avoided a catalytic reaction time for the reaction between G-quadruplex/hemin and the substrates (H2O2 and ABTS or luminol). Previously, the electrochemical methods based on the differential pulse voltammogram of the G-quadruplex/hemin52 or the electrocatalytic activity of the Gquadruplex/hemin toward H2O234 was also used to fabricate biosensors. However, these detections should be conducted at nitrogen atmosphere to eliminate the interference from dissolved oxygen. In our experiment, the presence of dissolved oxygen in the solution had a positive effect for the PEC detection and there was no need for the deoxygenation process. Finally, the detection strategy was versatile, which can be extended to multiple targets detection by changing the sensing sequence slightly. The responses from other DNA strands with one-base (T1), two-base (T2), or four-base (T3) mismatched nucleotides (Table S2, see the Supporting Information) were also assessed to examine the 13 ACS Paragon Plus Environment


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specificity of this sensor. Figure 5B clearly demonstrated that this approach was highly selective for the target DNA(2), which could distinguish nucleotide with even one mismatch. Figure 5. The variation coefficients (PEC signal) of intra- and inter-assays were used to evaluate the reproducibility of the proposed biosensor. The intra-assay precision was 3.48%, which was evaluated from the response to 0.1 pmol/L target DNA(2) at five different electrodes fabricated in the same batch. While an inter-assay precision of 2.73% was obtained by assaying 0.1 pmol/L target DNA with five proposed biosensors made using the same ITO with different batches. Figure S7 (see the Supporting Information) confirmed the stability of the photocurrent response of the PbS QDs under repeated irradiation cycles of more than 30 times. After a storage period of two months at 4 ℃, no obvious decline in the photocurrent intensity of the QDs modified electrode was detected, further indicating that the PDDA/PbS film possessed high stability and was appropriate for constructing PEC detection platforms. The Feasibility and Universality of the Experimental Principle. To further verify that the present proof-of-concept was feasible and versatile, thrombin (TB) was chosen as another model target of this sensing system by minor changing the sensing sequence of probe DNA. As a specific serine protease, TB is involved in the coagulation cascade, which catalyzes many coagulation-related reactions.53 On one hand, it plays crucial role in physiological and pathological coagulation; on the other hand, it regulates many processes in inflammation and tissue repair at the vessel wall.53 As shown in Scheme 2B, the TB aptamer (TBA) possessed the structure composed of the G-quadruplex sequence and the aptamer sequence specific for target TB. The TBA sequence is 5’-G1G2T3T4G5G6T7G8T9G10G11T12T13G14G15-3’ which binds to the TB using residues 4 through 12, while residues 1, 2, 3, 13, 14, and 15 locate at the exposed side of the aptamer.54,55 TBA is also known to adopt a stable G-quadruplex structure, therefore, hemin can intercalate into TBA to form a hemin/G-quadruplex structure.28 It has been proved that the addition of TB promotes the loosely binding of TBA to hemin through the formation of supramolecular TB/G-quadruplex/hemin complex.56 Indeed, different TB detection assays were developed based on TB ACS Paragon Plus Environment

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promoted binding of hemin to TBA.56-58 Because the band resulted from the recognition of TB by TBA was difficult to be identified by electrophoresis,59 we employed UV-vis absorption and CD spectra to measure the structure and the binding of hemin and TB to TBA.60,61,62 As shown in Figure S8A, when mixing TBA and hemin, the typical absorption of hemin became stronger with the absorption peak red shifted from 395 to 404 nm (curve a and b), indicating the formation of a G-quadruplex/hemin structure.46 The presence of TB caused a further hyperchromicity of the hemin (curve c), which demonstrated that TB promoted binding of hemin in G-quadruplex.46,56 In the presence of K+ (50 mmol/L), TBA exhibited a typical CD spectra of an antiparallel form with positive bands at approximately 246 and 292 nm and a negative band at approximately 266 nm (Figure S8B, curve a). Addition of hemin to TBA caused signal increase at 246 and 292 nm and a decrease at 266 nm (curve b), demonstrating G-quadruplex/hemin formation.61 In the presence of TB, the peak intensity of Gquadruplex/hemin increased at 292 nm (curve c). This implied that the pattern of the G-quadruplex structure did not change, but the antiparallel structure became more compact by TB.62 In our PEC experiment, the background photocurrent (Figure 6A) for thrombin detection was higher than that for DNA detection (Figure 5A), indicating that there was loosely bound hemin by TBA in the absence of TB. However, in the presence of TB, enhanced photocurrent was observed due to the promoted binding of hemin with TBA by TB. Due to that TB promoted binding of hemin to TBA, a sensitive PEC sensor for TB would be expected. As the concentration of TB increased, the photocurrent intensity increased accordingly (Figure 6A). The relative photocurrent change was linearly related to the logarithm concentration of TB from 0.1 pM to 10 nM (the correlation coefficient, R=0.987). The detection limit for TB was found to be 15 fM. This detection limit was lower than or comparable to the mostly previous reports involving TB aptasensing (Table S3, see the Supporting Information), such as PEC,63,64 electrochemistry (EC),28 ECL,65 and FRET.66 To testify the specificity of the proposed aptasensor for TB detection, the responses to other potential interferents, including L-Cys, BSA and IgG were also recorded. As indicated in Figure 6B, no significant responses was found in the presence of the interferents other than the target TB, demonstrating that it was selective for TB detection. ACS Paragon Plus Environment

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Figure 6. CONCLUSIONS In summary, we have introduced a novel concept involving PET between PbS QDs and hemin intercalated in G-quadruplex with the contaminant catalytic dissolved oxygen reduction that enabled a PEC platform for the detection of versatile biomolecules with good sensitivity and selectivity by taking DNA and thrombin as examples. Merits of the system are ultrasensitive, versatile, label-free, and substrate-free. We believe this work not only presents a successful paradigm for exploration of the intriguing properties of PbS QDs and its novel PEC applications, but also underlies a new and general PEC biosensing protocol that could be extended for probing numerous of other biological targets. ASSOCIATED CONTENT Supporting Information Supporting Information is available from the http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Fax: +86-510-85917763. E-mail: [email protected], [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by The National Natural Science Foundation of China (Nos. 21275065, 21005031, 21327902 and 21305063), the Fundamental Research Funds for the Central Universities (JUSRP51314B and 20620140158), the MOE & SAFEA for the 111 Project (B13025), and the Opening Foundation of KLACLS (1314).


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(111)

20

(422)

40

(400) (331) (420)

60

(311) (222)

80 (220)

Intensity (a.u.)

100

(200)

B 120

0 20

40

60

80

100

2θ (degree) Figure 1. (A) HRTEM image and (B) XRD pattern of the as synthesized PbS QDs. 1.2 1.0

0.05

Abs (a.u.)

Abs (a.u.)

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0.8 0.6

0.04

0.4 1100

1200

1300

Wavelength (nm)

0.2 0.0 400

600

800

1000

1200

Wavelength (nm) Figure 2. UV-vis-NIR absorption spectrum of the as synthesized PbS QDs.


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A

B light E/V vs NHE

ITO

e-

electron acceptor

e

CB

VB

h+

-1.0 V

-

hν

product

PbS e-

FeIII/FeII

h+ PbS QDs

0V

+1.0 V

Scheme 1. (A) The Photocurrent Generation Mechanism of the PbS QDs. (B) Schematic Comparison for the Reduction Potential of Hemin in G-quadruplex and the Conduction/Valence Band Edge of PbS QDs.

0

Current (nA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a b c

-10

-20

d -30 0

5

10

15

Time (sec) Figure 3. The photocurrents of the hairpin DNA(1) conjugated PbS QDs electrode in deaerated (a) or aerated (b) solutions and the photocurrents of this electrode after its subsequent recognition of 0.1 pmol/L target DNA(2) to form G-quadruplex/hemin in deaerated (c) or aerated (d) solutions.


21


10

Current (µA)

b 0

a

-10 -20

c

-30 -0.5

0.0

0.5

E (V vs. Ag/AgCl) Figure 4. CVs of bare electrode in the deaerated (a), and G-quadruplex/hemin-coated electrode in the deaerated (b) or aerated (c) Tris-HCl solution (0.1 mol/L, pH 7.4).

O2 PET

A PbS

PbS

β DNA(1)

Reduction product

PbS

α

PbS

target DNA(2) PbS

hemin

ITO

ITO

PbS

PbS

PbS

PbS

PbS

O2 PET

B

Reduction product

PbS

PbS

NH2-TBA PbS

TB

PbS

hemin

PbS

ITO

ITO

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PbS

PbS

PbS

PbS

PbS

Scheme 2. Schematic Representation of PEC Detection of DNA (A) and TB (B) Based on GQuadruplex/Hemin Conjugated PbS QDs. ACS Paragon Plus Environment

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B4

4

-80

2

-60

0

3

I (nA)

∆I / I 0

6

∆I / I0

A

2

-40 -15

-14

-11 lgCDNA ( mol/L )

10

-14

10

-13

10

-12

0

-11

10

DNA concentration (mol/L)

T3

-15

1 -20

T2

10

-12

T1

0

-13

et rg Ta

Figure 5. (A) Photocurrent responses of PbS QDs in the presence of different concentrations of DNA. The target DNA(2) of elevated concentrations corresponding to 0, 10−15, 10−14, 10−13, 10-12 and 10-11 mol/L (from left to right), respectively. Inset: calibration curve relative to the relative photocurrent change (∆I/I0) and target DNA concentration in logarithmic scale. (B) Selectivity for target DNA (0.1 pmol/L) analysis in the presence of the same concentration of mismatched nucleotides. Error bars were

A2

-240

∆I / I 0

obtained from four repeated determinations.

-180

B

0

-120 -13 -12 -11 -10 -9 lgC TB ( mol/L )

-8

1.0

∆I / I0

I (nA)

1

0.5

-60

0.0 -9

-8

A

G

TB concentration (mol/L)

ys C L-

-10

BS

-11

-12 10 10 10 10 10

Ig

-13

0 10

TB

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. (A) Photocurrent responses of PbS QDs to TB at different concentrations. The target TB of elevated concentrations corresponding to 0, 10−13, 10−12, 10−11, 10−10, 10-9 and 10-8 mol/L (from left to right), respectively. (B) Selectivity for target TB (0.1 nmol/L) analysis in the presence different interferents: IgG (0.1 nmol/L), BSA (0.1 nmol/L) or L-Cys (1.0×10-4 mol/L). ACS Paragon Plus Environment

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For TOC Only

O2

PET

α PbS

PbS

β

Reduction product

PbS

target DNA(2)

hemin

ITO

DNA(1)

ITO

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PbS


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Hemin To - Open Repository of National Natural

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