A label-free PFP-based photoelectrochemical biosensor for highly sensitive detection of PARP-1 activity

https://doi.org/10.1016/j.bios.2019.05.013Get rights and content

Highlights

  • It is the first time that PEC has been constructed to monitor PARP-1 activity.

  • The method is label-free and based on simple electrostatic interaction between PAR and PFP.

  • The method is applicable for real samples.

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1), as an original tumor marker, has aroused wide attention in recent years. However, only a few researches have been done for PARP-1 activity detection because PARP-1 is lack of optical or electrochemical property. In this work, a label-free and high-sensitive photoelectrochemical (PEC) biosensor for PARP-1 activity detection based on poly[9,9-bis(6′-N,N,N-trimethylammonium)hexyl]fluorenylene phenylene (PFP) has been designed. To the best of our knowledge, it is the first time that PEC has been used to monitor PARP-1 activity. PARP-1 were activated under the function of activated dsDNA, as a result, branched polymers of ADP-ribose (PAR) with plentiful negative charge were formed in the presence of nicotinamide adenine dinucleotide (NAD+). Subsequently, positively charged PFP with good photoelectrochemical properties, were absorbed on PAR via electrostatic interaction. High photocurrent was produced under light induction, which was depended on the PARP-1 activity. The biosensor has a wide linear range from 0.01 to 2 U with a detection limit of 0.007 U. The strategy has been applied in breast and ovarian cancer cells to detection PARP-1 activity with approving results, which signifies that it is a promising tool for clinical diagnosis.

Introduction

Poly(ADP-ribose) polymerases (PARPs) are a superfamily of signaling enzymes which were discovered in 1963 and composed of 18 members (Sodhi et al., 2010; Ame et al., 2004). Among the superfamily, PARP -1 is the most extensively researched and found member which participates in DNA damage and replication. PARP-1 is a ubiquitous multifunctional nuclear protein involving in DNA break caused by stress of oxidation, radiation stimulation and cytotoxic agents. Furthermore, PARP-1 has been implicated in the process of inflammatory signal transduction and regulation of various proteins expressing in transcription (Szabo et al., 1997; Ullrich et al., 2001). In the process of PARP-1 hyperactivation, nicotinamide adenine dinucleotide (NAD+) and ATP are the source, after being activated by damaged dsDNA, PARP-1 catalyzes NAD+ to form linear or branched polymers of ADP-ribose (PAR), which have a lot of negative charges because of luxuriant phosphate groups (Gagne et al., 2006; Meyer-Ficca et al., 2005; Rouleau et al., 2010; Ahmad et al., 2011). Compared to normal cells, PARP-1 is highly expressed in tumor cells, that is why PARP-1 was regarded as an original biomarker for clinical diagnostics particularly for breast and ovarian cancer (Rojo et al., 2012). Therefore, it is urgent to explore more efficient, sensitive and accurate method for detection of PARP-1 activity.

In the past few decades, only a few researches have been constructed for PARP-1 activity detection because PARP-1 is lack of optical, electrochemical or other properties that can be detected easily. The developed techniques included ELISA (Decker et al., 1999), biotin-labeled (Bakondi et al., 2002), radiolabeled (Smith et al., 2005), immunoblotting (Zhang et al., 2013), colorimetry (Xu et al., 2011), fluorescence (Tang et al., 2015; Jiang et al., 2010), electrochemistry (Xu et al., 2016). In our previous work, we have constructed various methods such as UV-vis (Wu et al., 2018b; Liu et al., 2018c), fluorescence (Wu et al., 2018a), electrochemistry (Liu et al., 2018a, 2018b) for PARP-1 detection. Colorimetric method was based on hemin-graphene nanoparticles. A “turn-off-on” biosensor was constructed based on FRET between MnO2 nanosheets and PFP. Besides, an electrochemical method based on the hyperbranched-PAR responsive current in AAO membrane was also fabricated and showed superior sensitivity.

Photoelectrochemical (PEC) biosensors have received growing attention in recent years (Dai et al., 2016; Li et al., 2016a, Li et al., 2016b; Zhao et al., 2015a, Zhao et al., 2015b). The PEC biosensor is composed of two relatively independent parts, the PEC active materials (organic/inorganic photoelectric materials) (Ji et al., 2017; Zhang et al., 2016; Zhu et al., 2016; Pang et al., 2018; Han et al., 2018a, 2018b) and the biometric elements (enzymes, antibodies, nucleic acids, etc). The PEC active materials are used to produce the signal of photocurrent and the biometric elements are applied to connect with the transducer (Zhao et al., 2015a, Zhao et al., 2015b). Under illumination, the transducers transform the interaction between biometric elements and PEC active materials to photoelectrochemical signals. Such separation of excitation source and detection signal cause higher sensitivity than other detection methods (Shen et al., 2015). In addition, the PEC method is inexpensive and simple, resulting in broad application for detection of biomarkers (such as DNA, enzyme, MicroRNA, etc) (Shi et al., 2016; Liu et al., 2017; Yu et al., 2015; Ma et al., 2016).

Conjugated polymers (CPs) have attracted more attention in biological detection because of their unique optical and electronic properties (Zhu et al., 2012). CPs are composed of π-conjugated backbones and charged side-chains, which make them play a crucial role in bioimaging (Chen et al., 2017), photocatalytic water splitting (Li et al., 2016a, Li et al., 2016b) and disease therapy (Sun et al., 2015). Apart from the common advantages of other CPs, as a kind of conjugated polymers (CPs), poly[9,9-bis(6′-N,N,N-trimethylammonium)hexyl]fluorenylene phenylene (PFP) also has stable photoelectric property. After light illumination, PFP generates electron-hole pairs, electron acceptors capture electrons migrating along the backbone and then the holes disappear together with photocurrent (Liu et al., 2018).

Inspired by the unique property of PFP and PAR, we constructed a PEC biosensor for PARP-1 activity detection based on the electrostatic attraction of PAR and PFP with high sensitivity and wide detection range. To the best of our knowledge, it is the first time to employ photoelectrochemical method for PARP-1 activity assay. As shown in Fig. S1, the PFP contains abundant positive charge, and poly(ADPribose) (PAR) consists of a large number of ADPs containing abundant phosphate groups which results much more negative charge than dsDNA. Therefore, in the absence of PARP-1, the negatively charged active dsDNA combined only a little PFP, which resulted in weak photocurrent. However, in the presence of PARP-1, hyper-branched PAR were generated under the activation of dsDNA by using NAD+ as substrate, which subsequently combined much more PFP and led to a higher photocurrent. Thus, a label-free and high-sensitive method to measure PARP-1 activity was constructed.

Section snippets

Electrode pretreatment

First, indium tin oxide (ITO) (Ren et al., 2017) slices (25 mm × 5 mm × 1.1 mm) were cleaned ultrasonically with ethanol and water for 3 times in sequence. Then, the cleaned and dried slices were immersed into 1 M NaOH solution for 1 h at room temperature. At last, after being washed and dried again, the hydroxylated slices were immersed into ethanol solution (GR) containing 5% (3-aminopropyl)triethoxysilane (APTES) with gently shaking overnight by shaking table (IKA, KS 260 basic, Germany).

Principle of the assay

The principle of photoelectrochemical detection of PARP-1 activity was illustrated in Scheme 1. After hydroxylation and amination, the ITO electrodes were modified with dsDNA by Schiff Base reaction and the inactive sites were blocked by benzaldehyde (BzH) (Scheme 1A). In the absence of PARP-1, the negatively charged dsDNA combined with PFP through electrostatic interaction when the positively charged PFP was added, which resulted in weak photocurrent induced by excitation light source. In the

Conclusions

In summary, on basis of the electrostatic interaction of PAR and PFP, a label-free and high sensitivity PEC method with wide linear range was designed. The strategy avoided complex operations, label procedures and expensive instruments. Importantly, no nanomaterial was necessary, which avoided complex preparation of nanomaterial. The linear range was wide enough from 0.01 to 2 U and the detection limit was as low as 0.007 U, which was comparable to most previously reported methods. The method

CRediT authorship contribution statement

Chenchen Wang: Data curation, Writing - original draft. Ying Li: Conceptualization. Ensheng Xu: Methodology. Qing Zhou: Methodology. Jin Chen: Writing - review & editing. Wei Wei: Funding acquisition, Project administration, Writing - review & editing. Yong Liu: Formal analysis, Software. Songqin Liu: Supervision.

Acknowledgements

We gratefully appreciate the support from National Natural Science Foundation of China (21775019 and 21635004), Fundamental Research Funds for the Central Universities and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant Nos. 2242018K3DN04), The Open Project of The Key Laboratory of Modern Toxicology of Ministry of Education, Nanjing Medical University (NMUMT201804).

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