Elsevier

Biosensors and Bioelectronics

Volume 150, 15 February 2020, 111936
Biosensors and Bioelectronics

Magnetic bead-gold nanoparticle hybrids probe based on optically countable gold nanoparticles with dark-field microscope for T4 polynucleotide kinase activity assay

https://doi.org/10.1016/j.bios.2019.111936Get rights and content

Highlights

  • A simple, visible, sensitive and specific sensing for T4 PNK activity was proposed.

  • The probe can be applied to screen inhibitors and test activity in cell lysates.

  • The strategy can be easily generalized to other enzymes by replacing the sequences.

Abstract:

T4 polynucleotide kinase (T4 PNK) plays an essential role in DNA phosphorylation during the DNA repair process. Therefore, the sensitive, selective and convenient detection of T4 PNK activity is of great significance. In this work, we proposed a sensitive non-amplification strategy for the sensing of T4 PNK activity via dark field microscope (DFM) based on magnetic bead (MB)-gold nanoparticle (AuNP) hybrids probe, MB-dsDNA-AuNP (MDA). In the presence of T4 PNK, the 5′-OH termini of DNA are phosphorylated and cleaved into oligonucleotides by lambda exonuclease (λexo), resulting in the destruction of the MDA probe and the separation of AuNP from the MB. Through automatic counting of AuNPs from DFM images, T4 PNK activity can be quantitatively measured. This strategy revealed a limit of detection (LOD) as low as 0.0058 U/mL and exhibited a dynamic range from 0.01 to 1 U/mL. The strategy presents an excellent ability to discriminate T4 PNK from the other proteins and enzymes. Notably, this strategy was applied to screen the T4 PNK inhibitors and test the activity in cell lysates, showing great potential for the discovery of new anticancer drugs and other related research field.

Introduction

Maintaining the integrity and stability of our genome, thus health, is largely challenged by unfavorable factors, which may be resulted from pollution and overly modified foods. DNA, as a carrier of human genetic information, may be damaged naturally or via the effect of environment, resulting in alteration of chemical structures, such as base missing (Wiederhold et al., 2004), single strand breaking (Whitehouse et al., 2001), and DNA oxidation (Breslin and Caldecott, 2009). A common form of DNA damage is a DNA strand bearing a 5′-OH terminus (Ma et al., 2016), which is unable to react with a 3′-OH terminus, blocked the formation of a phosphodiester bond even with the action of DNA ligase (Karimi-Busheri et al., 1998). Therefore, 5′-OH phosphorylation is very crucial for repairing such DNA damage. T4 poly nucleotide kinase (T4 PNK) is a typical DNA repair enzyme that specifically catalyzes 5′-OH phosphorylation through the transfer of γ-phosphate from nucleoside triphosphates (ATP) to nucleic acids or oligonucleotides (Lin et al., 2016), which is important in both DNA repair and cellular nucleic acid metabolism. Several human disorders, such as Rothmund-Thomson Syndrome (RTS), Werber Syndrome (WS), and Bloom's Syndrome (BS), have been closely associated to the abnormal activity of T4 PNK (Sharma et al., 2006; Brosh and Bohr, 2007). More importantly, the inhibition of T4 PNK activity could improve the efficiency of γ radiotherapy in somatic cancers treatment, implying that this kinase may be a promising target in drug design and discovery (Freschauf et al., 2009). Accordingly, establishing a highly sensitive and selective detection method for T4 PNK is crucial in inhibitor screening, drug discovery, early clinical diagnosis, and biochemical research.

For the T4 PNK activity assay, some conventional methods, including radioisotope 32P-labeling, autoradiography, and polyacrylamide gel electrophoresis (Bernstein et al., 2005; Jilani et al., 1999; Karimi-Busheri et al., 1998; Rasouli-Nia et al., 2004; Wang and Shuman, 2001), are relatively unsafe, time-consuming, laborious, and require expensive reagents and instruments. In recent years, a variety of novel and convenient strategies have emerged to overcome the drawbacks of the above-mentioned methods, including fluorescent (Li et al., 2017; Jiang et al., 2018), luminescent (Du et al., 2014), electrochemical (Cui et al., 2018), colorimetric (Jiang et al., 2013), and nanomaterial-based methods (Cen et al., 2018). Among these, nanomaterial-based methods have attracted more attention due to the excellent optical properties, unique structures, and good biocompatibility of nanomaterials, such as quantum dots, copper nanoclusters, silver nanoclusters, carbon nanotubes, and AuNPs. Undoubtedly, AuNPs are the most popular type of nanoparticles used in biosystems owing to their overwhelming advantages, including easy preparation and modification, large surface area, and outstanding optical performance of localized surface plasmon resonance (LSPR) (Kaewwonglom et al., 2019; Giljohann et al., 2010; Jain et al., 2008). AuNPs larger than 40 nm in diameter can be easily detected by DFM under a common white light illumination (Liu et al., 2014). The AuNPs-based DFM detection only requires a universal white light source and has the advantage of low cost, thereby offering an optical detection technology with broad application prospects (Huang et al., 2006; Gu et al., 2015). With the development of DFM technology, detection at the single-particle level has become increasingly essential. However, it is difficult to promote single-particle level detection due to the necessity of expensive optical devices and instruments (Lee et al., 2014; Chen et al., 2015; Guo et al., 2013). The combination of automatic counting technology with AuNPs-based DFM allows the automatic counting of individual AuNP without any amplification design, and enables the significant improvement of detection sensitivity. (Poon et al., 2016). Since the number of AuNPs correlates to the contents of a target via a specific reaction, it provides a fast, convenient, low cost, highly sensitive, and visible detection for targets, such as DNA, antigens, miRNA, and proteins (Li et al., 2018; Wu et al., 2017). Herein, we developed a MDA probe consisting of MB and AuNP hybrids for the sensitive, selective, and visible detection of T4 PNK activity. In addition, this strategy can be easily generalized to detect other biological enzymes. Moreover, this strategy was effectively applied to screen inhibitors and test the activity in cell lysates, suggesting its potential as a powerful tool to discover new anti-tumor drugs and in other related fields.

Section snippets

Material and reagents

All of the HPLC-purified DNA oligonucleotides in this work were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The sequence of SH-DNA1 was 5′-GTACACAGACTCAGCTCGTTTTTTTTTT–SH–3′. The sequence of biotin-DNA2 was 5′-CGAGCTGAGTCTGTGTACTTTTTTTTTT-biotin-3′. The streptavidin-modified magnetic beads (DynabeadsTM M-280 Streptavidin 10 mg/mL) were purchased from Invitrogen (Carlsbad, CA). The AuNPs with a diameter of 60 nm were purchased from NanoSeedz Ltd (Hong Kong, China). The T4 PNK, T4

Principle of the strategy

As shown in Scheme 1, the MDA probe, denoted MB-dsDNA-AuNP, consists of AuNPs modified with DNA through Au–S bonding and magnetic bead modified with biotin-DNA through a streptavidin-biotin interaction. AuNP and MB are linked via DNA hybridization, forming a short double-strand DNA (dsDNA) with two 5′-OH termini, which can be phosphorylated by T4 PNK under ATP existence. 5′- phosphorylation DNA is the substrate of λexo that cleaves dsDNA into DNA oligonucleotides, resulting in the separation of

Conclusions

In conclusion, a sensitive strategy is proposed for the detection of T4 PNK activity based on MDA probe and AuNPs counting using DFM. Practically, the MDA probe can be prepared in advance and no amplification step is involved in this method. Following magnetic separation, only a common white-light source is required for DFM imaging, thus providing a convenient and cost-effective technique. Selective and sensitive sensing of T4 PNK activity has been achieved with the detection limit of 0.0058

CRediT authorship contribution statement

Tian Jin: Conceptualization, Methodology, Writing - original draft, Writing - review & editing. Jiewen Zhang: Investigation, Visualization, Data curation. Yuanfang Zhao: Software, Validation, Resources. Xiaoting Huang: Formal analysis, Investigation. Chunyan Tan: Investigation, Resources. Shuqing Sun: Resources, Project administration. Ying Tan: Funding acquisition, Supervision, Project administration.

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.

Acknowledgements

This work is supported by grants from Shenzhen Municipal government (JCYJ20160301153959476 and JCYJ20160324163734374) and Shenzhen Reform Commission (Disciplinary Development Program for Chemical Biology).

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