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A Self-enhanced Ultrasensitive Photoelectrochemical Biosensor based on Nanocapsule Packaging both Donoracceptor Type Photoactive Material and Its Sensitizer Yingning Zheng, Wenbin Liang, Chengyi Xiong, Yali Yuan, Yaqin Chai, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01984 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 11, 2016

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Analytical Chemistry

A Self-enhanced Ultrasensitive Photoelectrochemical Biosensor based on Nanocapsule Packaging both Donor-acceptor Type Photoactive Material and Its Sensitizer Ying-Ning Zheng a, Wen-Bin Liang a, b, Cheng-Yi Xiong a, Ya-Li Yuan a, Ya-Qin Chai * a, Ruo Yuan * a a

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University),

Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China b

Department of Clinical Biochemistry, Laboratory Sciences, Southwest Hospital, Third Military

Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, PR China

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ACS Paragon Plus Environment


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ABSTRACT: In this work, a self-enhanced ultrasensitive photoelectrochemical (PEC) biosensor was established based on functionalized nanocapsule packaging both donor-acceptor type photoactive material and its sensitizer. The functionalized nanocapsule with self-enhanced PEC responses was achieved firstly by packaging both the donor-acceptor type photoactive material (poly{4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl-alt-3fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophene-4,6-diyl}, PTB7-Th) and its sensitizer nano-C60 in polyethylene glycol (PEG) to form nanocapsule, which significantly enhanced PEC signal and stability of the PEC biosensor. Moreover, a quadratic enzymes-assisted target recycling amplification strategy was introduced to the system for ultrasensitive determination. Compared with other established PEC biosensors, our proposed self-enhanced approach showed higher effectivity, accuracy, sensitivity and convenience without any additional coreactant or sensitizers into testing electrolyte for photocurrent amplification, and performed excellent analytical properties for microRNA estimation down to femtomole level with microRNA-141 as a model. Additionally, the proposed PEC biosensor was employed for estimination of microRNA in different cancer cells and pharmacodynamic evaluation in cancer cells. This self-enhanced PEC strategy has laid the foundation for fabrication of simple, effective and ultrasensitive PEC diagnostic devices, leading to the possibility for early diagnosis, timely stage estimination and accurate prognosis judgment of disease. KEYWORDS: photoelectrochemical biosensor, self-enhanced, donor-acceptor type, signal amplification, microRNA-141

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INTRODUCTION Photoelectrochemical (PEC) assay was a newly emerging and promising analytical technique with desirable advantages such as lower background signal and higher sensitivity, which was helpful and essential to explore effective means for early diagnosis.1-3 Recently, great efforts have been made by in situ generating electron donor or electron acceptor of photoactive materials.4-7 Ju’s group fabricated a PEC biosensor by in situ generating electron acceptor O2 through the mimicking catalytic reaction of hemin toward H2O2.4 Chen’s group developed a PEC bioanalysis by generating electron donor ascorbate via enzymatic hydrolysis of ascorbic acid 2phosphate.5 Although the photocurrent intensity of the fabricated biosensor was significantly enhanced by these operations based on the in situ mimicking enzymatic or enzymatic reactions, limitations were still existed such as limited quantity and unevenly distribution of the generated electron donor/acceptor. Besides, these mimicking enzymatic or enzymatic reactions often leaded to unsatisfactory reproducibility, high cost, and instability for long-term experiment or clinical practice. More importantly, the electron transfer among the photoactive materials and electron donor/acceptor was an inter-molecular process, leading to poor stability and low efficiency of electron transfer. The donor-acceptor (D-A) type photoactive materials with an electron-rich monomer (donor) and an electron-deficient monomer (acceptor) in one molecule possessed many excellent properties compared with traditional photoactive materials enabling PEC responses via intermolecular electron transfer.

8-10

By combining the electron-rich monomer and the electron-

deficient monomer in one molecule, it could be obtained that an intra-molecular electron transfer process with the significantly shorted electron transfer distance, reduced energy loss, and

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enhanced photo-to-current efficiency.11-14 Recently, massive types of D-A type photoactive materials have been synthesized and researched in the field of polymer solar cells. However, as a newly developed photoactive material, their poor water solubility and biocompatibility seriously restricted the application in PEC biosensors. Further modifications for these materials were still necessary to improve their photo-to-current transfer efficiencies and expanded their applications in PEC biosensors. The sensitization strategy was an ideal and fascinating choice to improve photoelectric transformation efficiency of the photoactive materials in the PEC biosensors.15 The light energy utilization and charge separation efficiency were remarkably enhanced by coupling the narrow band gap semiconductors with wide band gap ones in sensitization strategy.16 Currently, great attentions have been focused on fabricating PEC biosensor based on the sensitization strategy. Zhu's group reported a cosensitized structure with two kinds of quantum dots for improving the photoelectric transformation efficiency of the PEC biosensor.17 These systems with sensitization strategy showed improved sensitivity, but challenged with efficient immobilization strategy, which was normally involved DNA or antibody labeling, leading to limited immobilization amount, complicated operation and high cost. In order to enhance PEC signal and improve detection sensitivity, a self-enhanced photoactive materials was designed for the first time by utilizing polyethylene glycol (PEG), a versatile synthetic polymer with excellent biocompatibility and membrane-forming ability, to form nanocapsule with packaging both the D-A type photoactive materials and its sensitizer.18, 19 The poly{4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl-alt-3-fluoro2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophene-4,6-diyl}

(PTB7-Th)

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and

its

sensitizer

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fullerene (nano-C60) were served as model in this work. There are several advantages for the nanocapsule structure: (1) the loading amount of D-A type photoactive material and its sensitizer could be largely improved by being packaged in the nanocapsule; (2) the poor water solubility and biocompatibility of both the D-A type photoactive material and its sensitizer could be efficiently improved with the hydrophilic nanocapsule; (3) the electron transmission distance among photoactive material and its sensitizer could be reduced and controlled into one nanocapsule, leading to a more efficient and stable photocurrent response. Herein, a self-enhanced PEC biosensor by utilizing the PEG to package both intra-molecular self-enhanced photoactive material (PTB7-Th) and its sensitizer nano-C60 in one nanocapsule (PCP nanocapsule) was proposed for ultrasensitive detection of microRNA-141. The PCP nanocapsule can be successfully immobilized on the electrode due to the excellent membraneforming ability of PEG shell, which not only avoided the traditional fussy operations for immobilizing both photoactive materials and sensitizers, leading to a significantly reduced background signal, but also provided a highly initial photocurrent signal. Then, the streptavidin coated magnetic beads (STV-MB)/biotin-primer DNA bioconjugates (MB-primer) were hybridized with the padlock to initiate rolling cycle amplification (RCA). By controlling the sequence of padlock in the RCA reaction, the primer labeled on MB could be extended from a short specific nucleotide sequence to a long repeated nucleotide sequence. Each repeated nucleotide sequence in the extended primer could hybridize with the target microRNA for participating in the further enzymes-assisted target quadratic recycling strategy. Furthermore, the duplex-specific nuclease (DSN) and exonuclease III (Exo III) enzymes-assisted target quadratic recycling strategy was introduced for signal amplification, leading to improved detection

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sensitivity. In the existence of the target, the hairpin DNA on the SiO2 NPs could be cleaved by target quadratic recycling strategy, and then hybridized with the capture DNA on the modified electrode surface. The initial photocurrent signal could be efficiently decreased by the SiO2 NPs via increasing steric hindrance of electrode and depressing the transference of electron.17 Thus, the reduced photocurrent signal was related with the concentration of target microRNA and could be successfully employed for the quantitative detection of this microRNA. Due to the selfenhanced photoactive materials PCP nanocapsule and the target induced enzymes-assisted double cycling strategy, an ultrasensitive and highly selective photocurrent response for the target microRNA could be obtained based on this PEC biosensor, leading to the possibility for early diagnosis, timely stage estimation and accurate prognosis judgment of disease.

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Scheme 1. Schematic diagram of (A) quadratic enzymes-assisted target recycling amplification strategy for the biosensor; (B) fabrication of the biosensor; (C) enlarged view of PCP nanocapsule and (D) the electron transfer

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route in PCP nanocapsule. (The energy levels of the conduction band (CB) and valence band (VB) for PTB7Th and nano-C60 originated from the literatures.20, 21)

EXPERIMENTAL METHODS Reagents and Materials. 2,6-bis(trimethytin)-4,8-bis(5-(2-ethylhexyl)thiophen-2-yl)benzo[1,2b:4,5-b’]dithiophene (the electron-rich monomer, donor materials, D type), 2-ethylhexyl 4,6dibromothieno[3,4-b]thiophene-2-carboxylate

(the

electron-deficient

monomer,

acceptor

materials, A type) and PTB7-Th (the D-A type photoactive materials) were purchased from Luminescence Technology Corp. (Taiwan, China). Fullerene (C60), gold chloride (HAuCl4) and 6-mercaptohexanol (MCH) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). The polyethylene glycol (PEG) was purchased from Kelong Chemical Inc. (Chengdu, China). Streptavidin-coated magnetic dynabeads (STV-MB, MyOneTM Streptavidin C1, diameter of 1.0 µm) were obtained from Invitrogen Corp. (Oslo, Norway). The T4 DNA ligase and phi29 DNA polymerase were obtained from Vazyme Biotech Co. (Nanjing, China). Deoxynucleotides (dNTPs) was purchased from Genview scientific Inc. (EI Monte, CA, USA). DSN and 10×DSN master buffer were obtained from Axxora, LLC (San Diego, CA, USA). Exo III and exonuclease I (Exo I) were obtained from Thermo Fisher Scientific Inc. (Shanghai, China). Ferricyanide/ferrocyanide mixed solution ([Fe(CN)6]3-/4-, 5.0 mM) was obtained by dissolving potassium ferricyanide and potassium ferrocyanide with PBS (pH 7.4) solution. The microRNA-141 was custom-synthesized by TaKaRa (Dalian, China). All DNA oligonucleotides were bought from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). The sequences were listed as following: Table 1. The sequences used in the experiment.

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Apparatus. The PEC measurement during the experiment was performed by a PEC workstation (Ivium, Netherlands). A three-electrode system was used for photocurrent measurement, in which a platinum wire as the counter electrode, a saturated calomel electrode as the reference electrode and a bare/modified glassy carbon electrode (GCE) with 4 mm diameter as working electrode, respectively. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and depositions were performed with a CHI 660e electrochemical workstation (Shanghai Chenhua Instrument, Shanghai, China). The morphologies characterization of the prepared nanomaterials were performed on a scanning electron microscopy (SEM, S-4800, Hitachi, Japan). The laser confocal microscopy measurement was characterized by a laser confocal microscope (Leica Microsystem Inc., Heidelbery, Germany). Cell Culture and Total RNA Extraction. The five kinds of human cancer cell lines including the human prostate carcinoma cell lines (22Rvl), human cervical cancer cell lines (HeLa), human breast cancer cell lines (MB231 and MCF-7) and human lung cancer cell lines (A549) were obtained from the Cell bank of type culture collection of the Chinese Academy of Science (Shanghai, China). The cells were grown in a humidified atmosphere with 5% CO2 at 37 °C, 9



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using Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS, 100 U/mL penifcillin and 100 U/mL streptomycin. The cells were collected and re-suspended by trypsinization and fresh medium, respectively. The cellular extracts were obtained using RNAprep pure Cell/Bacteria Kit according to the manufacturer’s protocol. Finally, the obtained cellular extracts was diluted and stored at -20 ºC when no use. Gel Electrophoresis. The polyacrylamide gel electrophoresis (PAGE) and agarose gel electrophoresis were used for characterizing the enzymes-assisted target quadratic recycling strategy in our experiment. Briefly, 10 µL different DNA samples were mixed with 2 µL loading buffer solution, followed by injecting into the grooves of freshly prepared polyacrylamide gel (16%) system or agarose gel electrophoresis system (10%). The electrophoresis was performed in 1×TBE buffer at 120 V constant voltage. Then the gels were carefully washed with ultrapure water and stained by ethidium bromide for 30 min. Finally, the image of the gel was taken by a camera (EOS 550D, Canon, Japan) under UV light illumination. Synthesis of PCP Nanocapsule. For preparation of PCP nanocapsule, briefly, 3.5 g PEG was added to the 50 mL ultrapure water containing 1.2 g ammonium sulfate, 0.1 g potassium ferricyanide and 0.1 g potassium ferrocyanide to obtain solution A. At the same time, proper amount of nano-C60 and PTB7-Th were dissolved by cyclohexane with continual ultrasonication to obtain solution B. Under continuous stirring, the solution B and 0.1 µL Triton X-100 were added to the solution A. The obtained mixture was stirred for 7 days to get a homogeneous solution with obtained PCP nanocapsule. The obtained product was stored in dark place. Amplified Procedure for MicroRNA Detection. The rolling circle amplification (RCA) was performed by mixing MB-primer DNA bioconjugates (preparation process in Supporting 10


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Informination), circular DNA templates (preparation process in Supporting Informination), dNTP, phi29 polymerase and 1×phi29 buffer at 37 °C for 6 h. Then the solutions were heated to 90 °C for 10 min to denature the enzymes. For the first enzyme-assisted target recycling, the obtained RCA products were mixed with the target microRNA-141, 1×DSN master buffer and DSN enzyme (1 U), and incubated at 65 °C; then the enzyme was denatured by adding 5 µL DSN stop solution and incubating at 65 °C for 10 min; subsequently, the obtained solution was separated by magnet and the supernatant was collected for the second cycle. For the second enzyme-assisted target recycling, the collected supernatant was mixed with 20 µL SiO2 NPs/hairpin DNA bioconjugates (preparation process in Supporting Informination), Exo III (1 U) and Exo III buffer (5 µL), and incubated at 37 °C; then, the solution was heated to 90 °C for 10 min to denature the enzymes. Finally, the obtained solution was collected for next incubation on the modified electrode surface to generate the hybridization between the cut SiO2 NPs/hairpin DNA bioconjugates and the capture DNA on the electrode, leading to a signal-off photocurrent response related with the concentration of target microRNA-141. Fabrication of the Self-enhanced PEC Biosensor. First, the GCE was polished alumina powder and ultrasonicated in distilled water. Then the prepared PCP nanocapsule solution was dropped onto the electrode, followed by drying in air. To attach capture DNA, the modified electrode was immersed in 1% HAuCl4 aqueous solution under -0.2 V to modify gold nanoparticles (depAu) onto the GCE. Then the depAu/PCP functionalized electrode was incubated with capture DNA (2.5 µM, 20 µL) at room temperature for 16 h to immobilize capture DNA on the modified electrode. Subsequently, MCH solution (1 mM, 20 µL) was dropped onto the electrode for blocking non-specific binding sites.

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PEC Measurement. The PEC measurement was performed in 6 mL PBS solution (0.1 M, pH 7.0). The excitation light source were provided by the LED lamp and switched off-on-off for 10 s-20 s-10 s under 0.0 V potential. RESULTS AND DISCUSSION Characterizations of the Synthesized Nanomaterials. The morphologies of the synthesized nanomaterials were characterized by SEM. The nano-C60 showed globular structures at approximate 50 nm (Figure 1A). Figure 1B showed the PTB7-Th image, which displayed a flat layer structure. After nano-C60 and PTB7-Th were packaged by the PEG to form nanocapsule, a spherical structure with diameter of 2.5 µm containing multiple sphere could be observed clearly in Figure 1C. The SEM image for the inside structure of PCP nanocapsule and its partially enlarged image obtained under the strong electron beam puncture were showed in the left and right insets of Figure 1C, respectively, which verified the successful synthesization of the PCP nanocapsule. Figure 1D demonstrated the SEM image of the SiO2 NPs with global structure around 100 nm of diameter, indicating the successful synthesis of SiO2 NPs.

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Figure 1. SEM images of (A) nano-C60, (B) PTB7-Th layer, (C) PCP nanocapsule (the left and right insets of (C) showed the inside SEM image of PCP nanocapsule and a partially enlarged SEM image inside PCP nanocapsule, respectively) and (D) SiO2 NPs nanomaterials.

Comparison of Different PEC Biosensors. To investigate the photocurrent efficiency of PCP nanocapsule, the comparison study was conducted to contrast the photocurrent response of the biosensor modified with different photocurrent materials under the same conditions. Four kinds of photoactive materials including (A) separated donor and acceptor materials (D, A type; intermolecular molecules); (B) PTB7-Th material (D-A type; intramolecular self-enhanced materials); (C) PTB7-Th and nano-C60 materials (D-A type and its sensitizer); (D) PCP nanocapsule (D-A type/sensitizer) were modified on the electrode and the results were illustrated in Figure 2. For the electrode modified with separated D, A type materials (Figure 2A), a photocurrent response of 8 nA could be obtained. About 319 nA photocurrent response could be observed for the electrode modified with intramolecular self-enhanced materials (PTB7-Th, Figure 2B), in which photocurrent response efficiency was up to 40 times greater than that of D,

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A type materials. The large enhancement could be attributed to intramolecular self-enhancement effect of the D-A type materials. A further enhanced photocurrent response about 460 nA was produced by the biosensor modified with PTB7-Th and nano-C60 materials (Figure 2C), in which photocurrent efficiency is about 1.5 times greater than that of individual D-A type materials. For the electrode modified with PCP nanocapsule, the obtained photocurrent response was noticeably raised to 2368 nA, which was about 5 times greater than that in Figure 2C, indicating the large signal amplification efficiency of the self-enhanced PCP nanocapsule. The comparison results adequately indicated that the self-enhanced PCP nanocapsule could be used for ultrasensitive detection in PEC biosensors with high signal amplification efficiency of the photocurrent signal.

Figure 2. Photocurrent responses of the modified electrode immobilized with various photoactive materials: (A) donor and acceptor materials (D, A type; intermolecular moleculars); (B) PTB7-Th material (D-A type; intramolecular self-enhanced materials); (C) PTB7-Th and nano-C60 materials (D-A type and its sensitizer); (D) PCP nanocapsule (D-A type/sensitizer) in PBS solution (pH 7.0).

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Analytical Performance of the Self-enhanced PEC Biosensor. The proposed biosensor was explored to detect microRNA-141 with different concentration to estimate its analytical performance. In Figure 3, photocurrent response decreased accordingly as the concentration of microRNA-141 raised from 0.25 fM to 0.25 nM with a detection limit of 83.3 aM (S/N = 3). The linear equation was I = 92.240 + 158.96 lgc (where I and c were the photocurrent intensity and the concentration of microRNA-141, respectively). These results demonstrated great potential for ultrasensitive detection of microRNA-141. Furthermore, the analytical performance for our PEC biosensor was compared with other reported methods for microRNA detection. As shown in Table 2, the proposed PEC biosensor showed an enhanced sensitivity and much wider linear range, which may be contributed to the high photocurrent intensity and low background signal of the self-enhanced approach in this work.

Figure 3. (A) Photocurrent response of the biosensor incubated with microRNA-141 of different concentrations in 0.1 M PBS (pH 7.0). (B) The linear relationship between photocurrent response and the concentration of microRNA-141.

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Table 2. Comparison of our PEC biosensor with other reported detection strategies for microRNA. Analytical method Linear range Detection limit Ref. 22 fluorescence 20 pM~10 nM 2.0 nM 23 SWV 1 fM ~1 nM --24 fluorescence 0.1 pM~10 pM 80 fM 25 ECL 10 fM~1 pM 10 fM 26 fluorescence 100 fM~100 nM 100 fM 27 fluorescence 2 fM~2 nM 0.8 fM 28 EIS 2.0 fM~2.0 pM 1.0 fM 29 chemiluminescence 20 fM~5.0 pM 10 fM PEC 0.25 fM~0.25 nM 83 aM Our work Abbreviation: square-wave voltammetry (SWV); electrochemiluminescence (ECL); electrochemical impedance spectroscopic (EIS).

Selectivity and Stability of the PEC Biosensor. The selectivity of the present biosensor was estimated by incubating the biosensor with different interferences, including microRNA-21, microRNA-155 and microRNA-199a. As shown in Figure 4A, compared to the big photocurrent response (~1000 nA) obtained from 25 pM microRNA-141, no obvious photocurrent responses (

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