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Renewable electrochemical sensor for PARP-1 activity detection based on host-guest recognition
Biosensors and Bioelectronics 148 (2020) 111810
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: http://www.elsevier.com/locate/bios
Renewable electrochemical sensor for PARP-1 activity detection based on host-guest recognition Xiaoyuan Zhou a, 1, Chenchen Wang a, 1, Zhuang Wang a, Haitang Yang a, Wei Wei a, *, Yong Liu b, **, Songqin Liu a a
Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China College of Chemistry and Chemical Engineering, Henan University, Kaifeng, 475004, PR China
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Poly(ADP-Ribose) polymerase-1 (PARP-1) Renewable Electrochemical sensor Mono-(6-mercapto-6-deoxy)-beta-cyclodextrin (SH-β-CD) Azobenzene (Abz) Host-guest Molybdate (MoO24)
Poly (ADP-ribose) polymerase-1 (PARP-1) was defined as a new biomarker, which has achieved wide attention in recent years. In this work, we designed a renewable electrochemical sensor based on host-guest recognition for the detection of PARP-1 activity. Mono-(6-Mercapto-6-deoxy)-beta-Cyclodextrin (SH-β-CD) was modified on the electrode surface to recognize the trans-azobenzene labeled dsDNA (Abz-dsDNA). In the presence of PARP-1, PAR 23with abundant PO34 was generated and reacted with MoO4 to form PMo12O40, producing strong current. The proposed method avoided the unspecific adsorption effectively and improved the detection accuracy. Under UV irradiation, Abz-dsDNA was removed from the electrode surface because of the configuration change of azo benzene from trans to cis structure, allowing the electrode to be recycled. The sensor realized the linear detection of PARP-1, ranging from 0.01 U to 1.0 U with a detection limit of 0.008 U, which is comparable to results from reported methods. It is expected to be a potential tool for clinical detection because of its high sensitivity and selectivity.
1. Introduction Poly (ADP-ribose) polymer (PAR) was discovered by Chambon et al. (1966) and poly(ADP-ribose) polymerase-1 (PARP-1), the first PAR po lymerase, was discovered a year later (Shimizu et al., 1967). Poly (ADP-ribose) polymerases (PARPs) were a superfamily containing PARP-1, PARP-2 and other enzymes (Pommier et al., 2016), among which PARP-1 was one of the most important family members discov ered so far and widely existed in eukaryotic nucleus. PARP-1 was a tool enzyme which could repair DNA damage (Wang et al., 2009, 2016; David et al., 2009). During repair process, PARP-1 was contributed to preserving the integrity of differentiated chromatin (Ziegler and Oei, 2001). It was worth noting that most of the treatment strategies for malignant tumors were to damage the DNA of tumor cells, such as radiotherapy and chemotherapy (Liu et al., 2008). It was observed that inhibition of PARP-1 activity could prevent the repair of damaged DNA (Ahmad et al., 2011). Importantly, PARP-1 was more abundant in tumor
cells than normal cells (Tang et al., 2015). Therefore, PARP-1 was ex pected to be a new target of tumor therapy. It was of significance to explore more sensitive and accurate method for PARP-1 activity detection. In the past decades, as a new type of tumor target, a large number of researches have been done on PARP-1. Some researches were conducted to research the role of the PARP-1 in some cellular activities such as cell response (Gibson et al., 2016) and apoptosis (Yu et al., 2002). More researches were aimed to detect the PARP-1 activity with various methods including fluorescence (Ahmad et al., 2011), enzyme-labeled immunoassay (ELISA) (Decher et al., 1999), radiolabeled (Smith et al., 2005), colorimetry (Xu et al., 2011) and electrochemistry (Xu et al., 2016). Ahmad et al. proposed a fluorescent method for PARP-1 activity detection based on FRET using cationic conjugated polymer (CCP) as energy donor and green fluorescent protein (scGFP) as energy receptor (Ahmad et al., 2011), and Xu et al. used hexaammineruthenium (III) chloride (RuHex) as electroactive molecule to develop an
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (W. Wei), [email protected] (Y. Liu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bios.2019.111810 Received 27 August 2019; Received in revised form 11 October 2019; Accepted 21 October 2019 Available online 30 October 2019 0956-5663/© 2019 Elsevier B.V. All rights reserved.
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electrochemical biosensor for PARP-1 activity and inhibitor detection (Xu et al., 2016). In our previous work, we have constructed some methods for PARP-1 activity detection (Wu et al., 2018a, 2018b; Liu et al., 2018a, 2018b, 2018c; Yang et al., 2019a, 2019b; Xu et al., 2019; Wang et al., 2019). For example, Liu et al. proposed an electrochemical method based on the hyperbranched-PAR responsive current in anodic aluminum oxide (AAO) membrane to detect PARP-1 activity (Liu et al., 2018b). Yang et al. used quartz crystal microbalance (QCM) to detect PARP-1 activity based on electrostatic interaction of PAR and gold nanorods (GNRs) (Yang et al., 2019b). Besides, Wang et al. designed a photoelectrochemical (PEC) method for PARP-1 activity detection based on the electrostatic interaction of PAR and poly[9,9-bis(60 -N,N,N-tri methylammonium)hexyl]fluorenylene phenylene (PFP) (Wang et al., 2019). However, previous work detected PARP-1 activity mostly based on the electrostatic interaction between negative charges of PAR and positive probes, which has inevitable unspecific adsorption to impair detection. Cyclodextrins (CDs) were oligosaccharides composed of different amounts of glucose units. There were six glucose units in α-CD, seven in β-CD and eight in γ-CD (Xia et al., 2017; Liu et al., 2008; Sun et al., 2016). CDs were funnel-shaped compound with hydrophobic interior and hydrophilic exterior (Kubendhiran et al., 2018; Le Potier et al., 1998; Park et al., 1998). The unique structure of CDs allowed them to bind a variety of objects, such as amantadine, ferrocene (Fc) and azo benzene (Abz) (Wu et al., 2018c, 2019). Abz was trans-structure under visible light and became cis-structure under UV irradiation, so it was considered as an ideal optical switching material (Stricker et al., 2016; Wagner and Theato, 2014; Goulet-Hanssens and Barrett, 2013). In particular, trans-azobenzene and CDs could form host-guest complexes, while cis-azobenzene cannot form complexes with CDs due to the size mismatch (Bian et al., 2016). Inspired of the unique host-guest interaction between CDs and transazobenzene, a renewable electrochemical sensor was constructed based 2on the interaction of PO34 and MoO4 to detect the activity of PARP-1. When trans-azobenzene labeled dsDNA (Abz-dsDNA) were modified on the surface of electrodes by host-guest interaction, PO34 of Abz-dsDNA 3reacted with MoO24 to generate PMo12O40 with background current (Shen et al., 2016; Xie et al., 2017). In the presence of PARP-1, NADþ was catalyzed to produce PAR, containing a large number of PO34 , which combined with more MoO24 . As a result, strong current produced, which could be used to realize the detection of PARP-1 activity. Compared with previous works that monitored PARP-1 based on electrostatic in teractions between negative charges and positively charged probes, the proposed method avoided the unspecific adsorption effectively and improved the detection accuracy. Under UV irradiation, Abz-dsDNA was removed from the electrode surface because of the configuration change of azobenzene from trans to cis structure. Cis-azobenzene cannot insert into the cavity of SH-β-CD due to the size mismatch, as a result, the electrode can be recycled efficiently. The mechanism of electric current generation was as follows (Hu et al., 2017):
0.5 μM of Abz-ssDNA-1 and ssDNA-2 were added into hybridization buffer (see supporting information) and reacted overnight at 37 � C. 2.3. Detection of PARP-1 activity by electrochemical sensor Firstly, 10 μL of 0.5 μM prepared Abz-dsDNA were assembled on the working surface (5 mm � 5 mm) of SH-β-CD/AuNPs/ITO electrodes. After removing unbound Abz-dsDNA, 10 μL of 500 μM NADþ with different concentrations of PARP-1 were dropped on the working surface of electrodes for 1.5 h at 37 � C. Finally, 10 μL of 5 mM Na2MoO4 solution was dropped on and incubated for 1 h before electrochemical analysis in 0.5 M H2SO4. The recycling of electrodes was realized under UV irra diation for 3 min (365 nm, 10.2 mW cm-2). 2.4. Cell culture and PARP-1 extraction Cancer cells (A2780 and MCF-7) and Normal cells (IOSE80) were cultivated in different culture. In brief, A2780 cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) medium, MCF-7 and IOSE80 cells were cultivated in RPMI-1640 medium, with fetal bovine serum (10%), respectively. And then they were incubated in humid environment containing 5% CO2 and 95% air. The extraction of PARP-1 in the above three types of cells was shown in supporting information. 3. Results and discussion 3.1. Principle of the electrochemical sensor The principle of the electrochemical sensor for PARP-1 detection was illustrated in Scheme 1. After the working parts were modified with AuNPs and SH-β-CD, the inactive sites were blocked by MCH solution. Then Abz-dsDNA were modified based on the unique host-guest recog nition between β-CD and Abz. In the absence of PARP-1, PO34 of Abz3dsDNA combined with MoO24 to generate very small PMo12O40. A weak electrochemical current was observed with DPV method. In the
(2)
VI
2e þ 2H þ →H2 PMo2 V Mo10 O340 VI
2.2. Preparation of Abz-dsDNA
(1)
12MoO24 þ 24H þ þ PO34 →PMo12 O340 þ 12H2 O PMo12 O340
electrodes. The pretreated electrodes were dried in the dark and then immersed in 5 mg/mL SH-β-CD solution overnight. The obtained elec trodes were cleaned carefully with ultrapure water and named as SHβ-CD/AuNPs/ITO electrode. Finally, the prepared electrodes were incubated with 6-Mercapto-1-hexanol (MCH) solution (1 mM) to avoid nonspecific adsorption.
VI
H2 PMo2 V Mo10 O340 þ 2e þ 2H þ →H4 PMo4 V Mo8 O340
(3)
2. Experiments 2.1. Preparation of SH-β-CD/AuNPs/ITO electrodes First, indium tin oxide (ITO) slices (100 mm � 100 mm � 1.1 mm) were cleaned ultrasonically with ethanol and water for 3 times in sequence. Then, the cleaned and dried ITO electrodes were immersed in HAuCl4 solution (2%) and kept at a constant potential of -0.2 V for 60 s to electrodeposit gold nanoparticles (AuNPs) on the surface of ITO
Scheme 1. Schematic illustration of (A) working mechanism of electro chemical sensor for PARP-1 detection based on host-guest recognition and (B) renewable process of electrode. 2
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electrochemical sensor. CV and EIS were performed in 10 mL 0.1 M KCl containing 5 mM [Fe(CN)6]3-/4- at a scan rate of 100 mV/s. As shown in Fig. 2A, there was a reversible redox peak for the AuNPs/ITO, which indicated that AuNPs with good conductivity were modified perfectly (curve a). With the modification of SH-β-CD, Abz-dsDNA and MCH on the surface of AuNPs/ITO, the current was decreased in sequence (curve b, c and d). The current dropped further with the PARP-1 and NADþ added, which was attributed to the electrostatic repulsion between negatively charged products PAR and [Fe(CN)6]3-/4- (curve e). Similarly, as shown in Fig. 2B, the AuNPs/ITO had a low charge transfer resistance because of the good electrical conductivity of AuNPs (curve a). When the nonconductive SH-β-CD and MCH were assembled successfully on the electrode surface, the resistance value (R) increased constantly because they prevent [Fe(CN)6]4-/3- to reach the surface of electrodes (curve b and c). Moreover, with negatively charged Abz-dsDNA was combined with CDs, R had a further increase based on the repulsion between PO34 and [Fe(CN)6]4-/3- (curve d). When PARP-1 and NADþ were added, the hyper-branched products PAR were formed with a mass of PO34 and R increased obviously, which mainly caused by both steric hindrance and bulk charge effect between negatively charged PAR and [Fe(CN)6]4-/3(curve e). The above results proved that the electrochemical sensor was assembled successfully.
Fig. 1. DPV responses of (a) SH-β-CD/AuNPs/ITO, (b) Abz-dsDNA/SH-β-CD/ AuNPs/ITO, (c) PARP-1/Abz-dsDNA/SH-β-CD/AuNPs/ITO, (d) NADþ/AbzdsDNA/SH-β-CD/AuNPs/ITO and (e) PAR/Abz-dsDNA/SH-β-CD/AuNPs/ITO. The measurements were performed in 0.5 M H2SO4.
presence of PARP-1, NADþ was used as substrate to generate PAR con 2taining abundant PO34 to capture a large number of MoO4 , resulted in a strong electrochemical current (Scheme 1A). The electrochemical cur rent was closely related to PARP-1 activity, which could be applied to detect PARP-1 activity. Under UV irradiation, the Abz-dsDNA was removed from the electrode surface because the configuration change of azobenzene from trans-to cis-structure, allowing the recycling use of electrodes (Scheme 1B).
3.4. Condition optimization of the electrochemical sensor In order to ensure the detection of PARP-1 under the best experi mental conditions, the following three conditions were optimized: AbzdsDNA concentration, PAR amplification time and reaction time with MoO24 . The concentration of Abz-dsDNA directly affected the signal-tonoise ratio. As shown in Fig. 3A, the DPV signal increased gradually with the increase of Abz-dsDNA concentrations from 0.05 μM to 1 μM. However, the signal-to-noise ratio was the largest when the Abz-dsDNA concentration was 0.5 μM. According to these results, 0.5 μM was selected as the most optimal concentration of Abz-dsDNA. The time of PAR amplification affected the number of PO34 generated. As shown in Fig. 3B, the DPV signal increased gradually with the increase of ampli fication time of PAR from 0 to 90 min, and there was a slight decrease at 120 min. Hence, 90 min was chosen as the best amplification time. The reaction time with MoO24 was a crucial factor in this electrochemical sensor. As demonstrated in Fig. 3C, the DPV signal tended to be stable when the reaction time with MoO24 was 60 min. Therefore, 60 min was the suitable reaction time.
3.2. Feasibility of the electrochemical sensor The feasibility of the electrochemical sensor for PARP-1 activity detection has been shown in Fig. 1. DPV signal of SH- β-CD/AuNPs/ITO was nearly zero (curve a). After Abz-dsDNA was modified, there was a weak DPV signal because of the exposed PO34 of Abz-dsDNA (curve b). With the addition of PARP-1, no obvious DPV signal change was observed (curve c). However, when 500 μM NADþ were added, there was a slight increase of DPV signal (curve d). When PARP-1 and NADþ were all added, there was a much higher DPV signal because hyper branched PAR that has plenty of PO34 was produced (curve e). The change of electrochemical current mentioned above proved that the increase of DPV signal was resulted from PAR generated by PARP-1. Therefore, it was reasonable to detect PAPR-1 activity by the electro chemical sensor.
3.5. Detection performance of the electrochemical sensor
3.3. Characterization of the electrochemical sensor
PARP-1 was quantitatively detected under the optimal conditions. As shown in Fig. 4, the peak current at 0.22 V increased linearly with PARP1 concentrations from 0.01 U to 1.0 U. Moreover, there was a linear relationship between DPV signal and PARP-1 concentrations. The linear
In Fig. 2, cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) were used to demonstrate the stepwise modification of the
Fig. 2. CV (A) and EIS (B) responses corresponding to (a) AuNPs/ITO, (b) SH-β-CD/AuNPs/ITO, (c) MCH/SH-β-CD/AuNPs/ITO, (d) Abz-dsDNA/MCH/SH-β-CD/ AuNPs/ITO and (e) PAR/MCH/SH-β-CD/AuNPs/ITO. 10 mL 0.1 M KCl containing 5 mM [Fe(CN)6]3-/4- was performed as detection buffer. 3
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Fig. 3. Effects of Abz-dsDNA concentration (A), PAR amplification time (B) and Reaction time of PO-4 with MoO24 (C) on the DPV current of the electro chemical sensor.
Fig. 4. (A) DPV responses of this sensor incubated with various PARP-1 concentrations: 0 U, 0.01 U, 0.05 U, 0.1 U, 0.3 U, 0.5 U, 0.75 U, and 1.0 U. (B) Plot of current change versus concentration of PARP-1. Inset: the calibration curve for PARP-1 detection. The measurement was performed in 0.5 M H2SO4.
Fig. 5. (A) Selectivity of this sensor for different targets: PARP-1, BSA, GOX, Telomerase, and HRP. (B) The relative DPV intensity for target determination for 3 cycles under UV irradiation.
equation for PARP-1 was described as I ¼ -0.003-2.249 CPARP-1 (R2 ¼ 0.9963), where I and CPARP-1 represented current intensity and PARP-1 concentration, respectively. The detection limit was 0.008 U (S/ N ¼ 3), which showed a better sensitivity for PARP-1 detection compared with other reported methods. Five samples for each concen tration were detected. The relative standard deviations (RSD) for the detection of different concentrations of PARP-1 were from 0.22% to 6.32%, indicating the acceptable reproducibility of the method. We also validated the selectivity of the electrochemical sensor. As illustrated in Fig. 5A, telomerase, BSA, GOX and HRP were chosen as interferes and they all had a very weak peak current compared with PARP-1. These results demonstrated that the electrochemical sensor had an outstanding selectivity for PARP-1 activity detection. On the other hand, under UV irradiation, Abz-dsDNA were released from the elec trode surface, leading to the recycling use of the electrode. As shown in
Table 1 Detection results of PARP-1 in 50 cancer cells and normal cells. Cells
Found (U) in Nuclei
Found (U) in Cytoplasm
A2780 MCF-7 IOSE80
0.410 0.423 0.086
0.250 0.242 0.040
Fig. 5B, the DPV intensity basically kept same during 3 cycles under UV irradiation, suggesting the cycle of the detection was efficient. 3.6. Applicability of the electrochemical sensor for PARP-1 assay in cancer cells and normal cells A2780 (ovarian cancer cells), MCF-7 (breast cancer cells) and 4
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IOSE80 (normal ovarian cells) were used in this experiment. The extracted nucleus and cytoplasm was studied. As shown in Table 1, 0.410 U in the nucleus and 0.250 U in the cytoplasm were found in 50 A2780 cells. 0.423 U and 0.242 U PARP-1 in the nucleus and cytoplasm of 50 MCF-7 cells were found. However, for IOSE80, the amount of PARP-1 in the nucleus and cytoplasm were 0.086 U/50 cells and 0.040 U/50 cells respectively. The results were acceptable compared to those carried out by Human PARP ELISA Kit (Table S2). The above results indicated that the quantification of PARP-1 from cancer cells had a key potential for clinical diagnosis of cancers.
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4. Conclusions In summary, on the basis of the reaction of masses of PO34 of PAR with MoO24 , a renewable electrochemical sensor based on β-CD and Abz recognition for PARP-1 activity detection was constructed. Compared with previous works based on electrostatic interactions between nega tive charges and positively charged probes, the proposed method avoi ded the unspecific adsorption and improved the accuracy greatly. It was also demonstrated that the proposed electrochemical sensor detected PARP-1 activity has a good linear range from 0.01 U to 1.0 U and a low detection limit of 0.008 U. On the other hand, under UV irradiation, Abz-dsDNA were released from the electrode, resulted in 3 times cycle use of the electrode, which achieved fast and efficient detection compared to previously reported methods. These results indicated that the electrochemical sensor provided a useful tool in clinical diagnosis and cancer drug development. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Xiaoyuan Zhou: Methodology, Data curation, Writing - original draft. Chenchen Wang: Methodology. Zhuang Wang: Validation. Haitang Yang: Formal analysis. Wei Wei: Conceptualization, Funding acquisition, Project administration, Writing - review & editing. Yong Liu: Methodology, Writing - review & editing. Songqin Liu: Supervision. Acknowledgements We gratefully appreciate the support from National Natural Science Foundation of China (21775019, 21635004 and 81730087), Funda mental Research Funds for the Central Universities and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Ed ucation Institutions (Grant Nos. 2242018K3DN04), The Open Project of The Key Laboratory of Modern Toxicology of Ministry of Education, Nanjing Medical University (NMUMT201804). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.
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Renewable electrochemical sensor for PARP-1 activity detection based on host-guest recognition Xiaoyuan Zhou a, 1, Chenchen Wang a, 1, Zhuang Wang a, Haitang Yang a, Wei Wei a, *, Yong Liu b, **, Songqin Liu a a
Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China College of Chemistry and Chemical Engineering, Henan University, Kaifeng, 475004, PR China
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Poly(ADP-Ribose) polymerase-1 (PARP-1) Renewable Electrochemical sensor Mono-(6-mercapto-6-deoxy)-beta-cyclodextrin (SH-β-CD) Azobenzene (Abz) Host-guest Molybdate (MoO24)
Poly (ADP-ribose) polymerase-1 (PARP-1) was defined as a new biomarker, which has achieved wide attention in recent years. In this work, we designed a renewable electrochemical sensor based on host-guest recognition for the detection of PARP-1 activity. Mono-(6-Mercapto-6-deoxy)-beta-Cyclodextrin (SH-β-CD) was modified on the electrode surface to recognize the trans-azobenzene labeled dsDNA (Abz-dsDNA). In the presence of PARP-1, PAR 23with abundant PO34 was generated and reacted with MoO4 to form PMo12O40, producing strong current. The proposed method avoided the unspecific adsorption effectively and improved the detection accuracy. Under UV irradiation, Abz-dsDNA was removed from the electrode surface because of the configuration change of azo benzene from trans to cis structure, allowing the electrode to be recycled. The sensor realized the linear detection of PARP-1, ranging from 0.01 U to 1.0 U with a detection limit of 0.008 U, which is comparable to results from reported methods. It is expected to be a potential tool for clinical detection because of its high sensitivity and selectivity.
1. Introduction Poly (ADP-ribose) polymer (PAR) was discovered by Chambon et al. (1966) and poly(ADP-ribose) polymerase-1 (PARP-1), the first PAR po lymerase, was discovered a year later (Shimizu et al., 1967). Poly (ADP-ribose) polymerases (PARPs) were a superfamily containing PARP-1, PARP-2 and other enzymes (Pommier et al., 2016), among which PARP-1 was one of the most important family members discov ered so far and widely existed in eukaryotic nucleus. PARP-1 was a tool enzyme which could repair DNA damage (Wang et al., 2009, 2016; David et al., 2009). During repair process, PARP-1 was contributed to preserving the integrity of differentiated chromatin (Ziegler and Oei, 2001). It was worth noting that most of the treatment strategies for malignant tumors were to damage the DNA of tumor cells, such as radiotherapy and chemotherapy (Liu et al., 2008). It was observed that inhibition of PARP-1 activity could prevent the repair of damaged DNA (Ahmad et al., 2011). Importantly, PARP-1 was more abundant in tumor
cells than normal cells (Tang et al., 2015). Therefore, PARP-1 was ex pected to be a new target of tumor therapy. It was of significance to explore more sensitive and accurate method for PARP-1 activity detection. In the past decades, as a new type of tumor target, a large number of researches have been done on PARP-1. Some researches were conducted to research the role of the PARP-1 in some cellular activities such as cell response (Gibson et al., 2016) and apoptosis (Yu et al., 2002). More researches were aimed to detect the PARP-1 activity with various methods including fluorescence (Ahmad et al., 2011), enzyme-labeled immunoassay (ELISA) (Decher et al., 1999), radiolabeled (Smith et al., 2005), colorimetry (Xu et al., 2011) and electrochemistry (Xu et al., 2016). Ahmad et al. proposed a fluorescent method for PARP-1 activity detection based on FRET using cationic conjugated polymer (CCP) as energy donor and green fluorescent protein (scGFP) as energy receptor (Ahmad et al., 2011), and Xu et al. used hexaammineruthenium (III) chloride (RuHex) as electroactive molecule to develop an
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (W. Wei), [email protected] (Y. Liu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bios.2019.111810 Received 27 August 2019; Received in revised form 11 October 2019; Accepted 21 October 2019 Available online 30 October 2019 0956-5663/© 2019 Elsevier B.V. All rights reserved.
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electrochemical biosensor for PARP-1 activity and inhibitor detection (Xu et al., 2016). In our previous work, we have constructed some methods for PARP-1 activity detection (Wu et al., 2018a, 2018b; Liu et al., 2018a, 2018b, 2018c; Yang et al., 2019a, 2019b; Xu et al., 2019; Wang et al., 2019). For example, Liu et al. proposed an electrochemical method based on the hyperbranched-PAR responsive current in anodic aluminum oxide (AAO) membrane to detect PARP-1 activity (Liu et al., 2018b). Yang et al. used quartz crystal microbalance (QCM) to detect PARP-1 activity based on electrostatic interaction of PAR and gold nanorods (GNRs) (Yang et al., 2019b). Besides, Wang et al. designed a photoelectrochemical (PEC) method for PARP-1 activity detection based on the electrostatic interaction of PAR and poly[9,9-bis(60 -N,N,N-tri methylammonium)hexyl]fluorenylene phenylene (PFP) (Wang et al., 2019). However, previous work detected PARP-1 activity mostly based on the electrostatic interaction between negative charges of PAR and positive probes, which has inevitable unspecific adsorption to impair detection. Cyclodextrins (CDs) were oligosaccharides composed of different amounts of glucose units. There were six glucose units in α-CD, seven in β-CD and eight in γ-CD (Xia et al., 2017; Liu et al., 2008; Sun et al., 2016). CDs were funnel-shaped compound with hydrophobic interior and hydrophilic exterior (Kubendhiran et al., 2018; Le Potier et al., 1998; Park et al., 1998). The unique structure of CDs allowed them to bind a variety of objects, such as amantadine, ferrocene (Fc) and azo benzene (Abz) (Wu et al., 2018c, 2019). Abz was trans-structure under visible light and became cis-structure under UV irradiation, so it was considered as an ideal optical switching material (Stricker et al., 2016; Wagner and Theato, 2014; Goulet-Hanssens and Barrett, 2013). In particular, trans-azobenzene and CDs could form host-guest complexes, while cis-azobenzene cannot form complexes with CDs due to the size mismatch (Bian et al., 2016). Inspired of the unique host-guest interaction between CDs and transazobenzene, a renewable electrochemical sensor was constructed based 2on the interaction of PO34 and MoO4 to detect the activity of PARP-1. When trans-azobenzene labeled dsDNA (Abz-dsDNA) were modified on the surface of electrodes by host-guest interaction, PO34 of Abz-dsDNA 3reacted with MoO24 to generate PMo12O40 with background current (Shen et al., 2016; Xie et al., 2017). In the presence of PARP-1, NADþ was catalyzed to produce PAR, containing a large number of PO34 , which combined with more MoO24 . As a result, strong current produced, which could be used to realize the detection of PARP-1 activity. Compared with previous works that monitored PARP-1 based on electrostatic in teractions between negative charges and positively charged probes, the proposed method avoided the unspecific adsorption effectively and improved the detection accuracy. Under UV irradiation, Abz-dsDNA was removed from the electrode surface because of the configuration change of azobenzene from trans to cis structure. Cis-azobenzene cannot insert into the cavity of SH-β-CD due to the size mismatch, as a result, the electrode can be recycled efficiently. The mechanism of electric current generation was as follows (Hu et al., 2017):
0.5 μM of Abz-ssDNA-1 and ssDNA-2 were added into hybridization buffer (see supporting information) and reacted overnight at 37 � C. 2.3. Detection of PARP-1 activity by electrochemical sensor Firstly, 10 μL of 0.5 μM prepared Abz-dsDNA were assembled on the working surface (5 mm � 5 mm) of SH-β-CD/AuNPs/ITO electrodes. After removing unbound Abz-dsDNA, 10 μL of 500 μM NADþ with different concentrations of PARP-1 were dropped on the working surface of electrodes for 1.5 h at 37 � C. Finally, 10 μL of 5 mM Na2MoO4 solution was dropped on and incubated for 1 h before electrochemical analysis in 0.5 M H2SO4. The recycling of electrodes was realized under UV irra diation for 3 min (365 nm, 10.2 mW cm-2). 2.4. Cell culture and PARP-1 extraction Cancer cells (A2780 and MCF-7) and Normal cells (IOSE80) were cultivated in different culture. In brief, A2780 cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) medium, MCF-7 and IOSE80 cells were cultivated in RPMI-1640 medium, with fetal bovine serum (10%), respectively. And then they were incubated in humid environment containing 5% CO2 and 95% air. The extraction of PARP-1 in the above three types of cells was shown in supporting information. 3. Results and discussion 3.1. Principle of the electrochemical sensor The principle of the electrochemical sensor for PARP-1 detection was illustrated in Scheme 1. After the working parts were modified with AuNPs and SH-β-CD, the inactive sites were blocked by MCH solution. Then Abz-dsDNA were modified based on the unique host-guest recog nition between β-CD and Abz. In the absence of PARP-1, PO34 of Abz3dsDNA combined with MoO24 to generate very small PMo12O40. A weak electrochemical current was observed with DPV method. In the
(2)
VI
2e þ 2H þ →H2 PMo2 V Mo10 O340 VI
2.2. Preparation of Abz-dsDNA
(1)
12MoO24 þ 24H þ þ PO34 →PMo12 O340 þ 12H2 O PMo12 O340
electrodes. The pretreated electrodes were dried in the dark and then immersed in 5 mg/mL SH-β-CD solution overnight. The obtained elec trodes were cleaned carefully with ultrapure water and named as SHβ-CD/AuNPs/ITO electrode. Finally, the prepared electrodes were incubated with 6-Mercapto-1-hexanol (MCH) solution (1 mM) to avoid nonspecific adsorption.
VI
H2 PMo2 V Mo10 O340 þ 2e þ 2H þ →H4 PMo4 V Mo8 O340
(3)
2. Experiments 2.1. Preparation of SH-β-CD/AuNPs/ITO electrodes First, indium tin oxide (ITO) slices (100 mm � 100 mm � 1.1 mm) were cleaned ultrasonically with ethanol and water for 3 times in sequence. Then, the cleaned and dried ITO electrodes were immersed in HAuCl4 solution (2%) and kept at a constant potential of -0.2 V for 60 s to electrodeposit gold nanoparticles (AuNPs) on the surface of ITO
Scheme 1. Schematic illustration of (A) working mechanism of electro chemical sensor for PARP-1 detection based on host-guest recognition and (B) renewable process of electrode. 2
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electrochemical sensor. CV and EIS were performed in 10 mL 0.1 M KCl containing 5 mM [Fe(CN)6]3-/4- at a scan rate of 100 mV/s. As shown in Fig. 2A, there was a reversible redox peak for the AuNPs/ITO, which indicated that AuNPs with good conductivity were modified perfectly (curve a). With the modification of SH-β-CD, Abz-dsDNA and MCH on the surface of AuNPs/ITO, the current was decreased in sequence (curve b, c and d). The current dropped further with the PARP-1 and NADþ added, which was attributed to the electrostatic repulsion between negatively charged products PAR and [Fe(CN)6]3-/4- (curve e). Similarly, as shown in Fig. 2B, the AuNPs/ITO had a low charge transfer resistance because of the good electrical conductivity of AuNPs (curve a). When the nonconductive SH-β-CD and MCH were assembled successfully on the electrode surface, the resistance value (R) increased constantly because they prevent [Fe(CN)6]4-/3- to reach the surface of electrodes (curve b and c). Moreover, with negatively charged Abz-dsDNA was combined with CDs, R had a further increase based on the repulsion between PO34 and [Fe(CN)6]4-/3- (curve d). When PARP-1 and NADþ were added, the hyper-branched products PAR were formed with a mass of PO34 and R increased obviously, which mainly caused by both steric hindrance and bulk charge effect between negatively charged PAR and [Fe(CN)6]4-/3(curve e). The above results proved that the electrochemical sensor was assembled successfully.
Fig. 1. DPV responses of (a) SH-β-CD/AuNPs/ITO, (b) Abz-dsDNA/SH-β-CD/ AuNPs/ITO, (c) PARP-1/Abz-dsDNA/SH-β-CD/AuNPs/ITO, (d) NADþ/AbzdsDNA/SH-β-CD/AuNPs/ITO and (e) PAR/Abz-dsDNA/SH-β-CD/AuNPs/ITO. The measurements were performed in 0.5 M H2SO4.
presence of PARP-1, NADþ was used as substrate to generate PAR con 2taining abundant PO34 to capture a large number of MoO4 , resulted in a strong electrochemical current (Scheme 1A). The electrochemical cur rent was closely related to PARP-1 activity, which could be applied to detect PARP-1 activity. Under UV irradiation, the Abz-dsDNA was removed from the electrode surface because the configuration change of azobenzene from trans-to cis-structure, allowing the recycling use of electrodes (Scheme 1B).
3.4. Condition optimization of the electrochemical sensor In order to ensure the detection of PARP-1 under the best experi mental conditions, the following three conditions were optimized: AbzdsDNA concentration, PAR amplification time and reaction time with MoO24 . The concentration of Abz-dsDNA directly affected the signal-tonoise ratio. As shown in Fig. 3A, the DPV signal increased gradually with the increase of Abz-dsDNA concentrations from 0.05 μM to 1 μM. However, the signal-to-noise ratio was the largest when the Abz-dsDNA concentration was 0.5 μM. According to these results, 0.5 μM was selected as the most optimal concentration of Abz-dsDNA. The time of PAR amplification affected the number of PO34 generated. As shown in Fig. 3B, the DPV signal increased gradually with the increase of ampli fication time of PAR from 0 to 90 min, and there was a slight decrease at 120 min. Hence, 90 min was chosen as the best amplification time. The reaction time with MoO24 was a crucial factor in this electrochemical sensor. As demonstrated in Fig. 3C, the DPV signal tended to be stable when the reaction time with MoO24 was 60 min. Therefore, 60 min was the suitable reaction time.
3.2. Feasibility of the electrochemical sensor The feasibility of the electrochemical sensor for PARP-1 activity detection has been shown in Fig. 1. DPV signal of SH- β-CD/AuNPs/ITO was nearly zero (curve a). After Abz-dsDNA was modified, there was a weak DPV signal because of the exposed PO34 of Abz-dsDNA (curve b). With the addition of PARP-1, no obvious DPV signal change was observed (curve c). However, when 500 μM NADþ were added, there was a slight increase of DPV signal (curve d). When PARP-1 and NADþ were all added, there was a much higher DPV signal because hyper branched PAR that has plenty of PO34 was produced (curve e). The change of electrochemical current mentioned above proved that the increase of DPV signal was resulted from PAR generated by PARP-1. Therefore, it was reasonable to detect PAPR-1 activity by the electro chemical sensor.
3.5. Detection performance of the electrochemical sensor
3.3. Characterization of the electrochemical sensor
PARP-1 was quantitatively detected under the optimal conditions. As shown in Fig. 4, the peak current at 0.22 V increased linearly with PARP1 concentrations from 0.01 U to 1.0 U. Moreover, there was a linear relationship between DPV signal and PARP-1 concentrations. The linear
In Fig. 2, cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) were used to demonstrate the stepwise modification of the
Fig. 2. CV (A) and EIS (B) responses corresponding to (a) AuNPs/ITO, (b) SH-β-CD/AuNPs/ITO, (c) MCH/SH-β-CD/AuNPs/ITO, (d) Abz-dsDNA/MCH/SH-β-CD/ AuNPs/ITO and (e) PAR/MCH/SH-β-CD/AuNPs/ITO. 10 mL 0.1 M KCl containing 5 mM [Fe(CN)6]3-/4- was performed as detection buffer. 3
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Fig. 3. Effects of Abz-dsDNA concentration (A), PAR amplification time (B) and Reaction time of PO-4 with MoO24 (C) on the DPV current of the electro chemical sensor.
Fig. 4. (A) DPV responses of this sensor incubated with various PARP-1 concentrations: 0 U, 0.01 U, 0.05 U, 0.1 U, 0.3 U, 0.5 U, 0.75 U, and 1.0 U. (B) Plot of current change versus concentration of PARP-1. Inset: the calibration curve for PARP-1 detection. The measurement was performed in 0.5 M H2SO4.
Fig. 5. (A) Selectivity of this sensor for different targets: PARP-1, BSA, GOX, Telomerase, and HRP. (B) The relative DPV intensity for target determination for 3 cycles under UV irradiation.
equation for PARP-1 was described as I ¼ -0.003-2.249 CPARP-1 (R2 ¼ 0.9963), where I and CPARP-1 represented current intensity and PARP-1 concentration, respectively. The detection limit was 0.008 U (S/ N ¼ 3), which showed a better sensitivity for PARP-1 detection compared with other reported methods. Five samples for each concen tration were detected. The relative standard deviations (RSD) for the detection of different concentrations of PARP-1 were from 0.22% to 6.32%, indicating the acceptable reproducibility of the method. We also validated the selectivity of the electrochemical sensor. As illustrated in Fig. 5A, telomerase, BSA, GOX and HRP were chosen as interferes and they all had a very weak peak current compared with PARP-1. These results demonstrated that the electrochemical sensor had an outstanding selectivity for PARP-1 activity detection. On the other hand, under UV irradiation, Abz-dsDNA were released from the elec trode surface, leading to the recycling use of the electrode. As shown in
Table 1 Detection results of PARP-1 in 50 cancer cells and normal cells. Cells
Found (U) in Nuclei
Found (U) in Cytoplasm
A2780 MCF-7 IOSE80
0.410 0.423 0.086
0.250 0.242 0.040
Fig. 5B, the DPV intensity basically kept same during 3 cycles under UV irradiation, suggesting the cycle of the detection was efficient. 3.6. Applicability of the electrochemical sensor for PARP-1 assay in cancer cells and normal cells A2780 (ovarian cancer cells), MCF-7 (breast cancer cells) and 4
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IOSE80 (normal ovarian cells) were used in this experiment. The extracted nucleus and cytoplasm was studied. As shown in Table 1, 0.410 U in the nucleus and 0.250 U in the cytoplasm were found in 50 A2780 cells. 0.423 U and 0.242 U PARP-1 in the nucleus and cytoplasm of 50 MCF-7 cells were found. However, for IOSE80, the amount of PARP-1 in the nucleus and cytoplasm were 0.086 U/50 cells and 0.040 U/50 cells respectively. The results were acceptable compared to those carried out by Human PARP ELISA Kit (Table S2). The above results indicated that the quantification of PARP-1 from cancer cells had a key potential for clinical diagnosis of cancers.
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4. Conclusions In summary, on the basis of the reaction of masses of PO34 of PAR with MoO24 , a renewable electrochemical sensor based on β-CD and Abz recognition for PARP-1 activity detection was constructed. Compared with previous works based on electrostatic interactions between nega tive charges and positively charged probes, the proposed method avoi ded the unspecific adsorption and improved the accuracy greatly. It was also demonstrated that the proposed electrochemical sensor detected PARP-1 activity has a good linear range from 0.01 U to 1.0 U and a low detection limit of 0.008 U. On the other hand, under UV irradiation, Abz-dsDNA were released from the electrode, resulted in 3 times cycle use of the electrode, which achieved fast and efficient detection compared to previously reported methods. These results indicated that the electrochemical sensor provided a useful tool in clinical diagnosis and cancer drug development. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Xiaoyuan Zhou: Methodology, Data curation, Writing - original draft. Chenchen Wang: Methodology. Zhuang Wang: Validation. Haitang Yang: Formal analysis. Wei Wei: Conceptualization, Funding acquisition, Project administration, Writing - review & editing. Yong Liu: Methodology, Writing - review & editing. Songqin Liu: Supervision. Acknowledgements We gratefully appreciate the support from National Natural Science Foundation of China (21775019, 21635004 and 81730087), Funda mental Research Funds for the Central Universities and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Ed ucation Institutions (Grant Nos. 2242018K3DN04), The Open Project of The Key Laboratory of Modern Toxicology of Ministry of Education, Nanjing Medical University (NMUMT201804). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.
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