Magnetic bead-gold nanoparticle hybrids probe based on optically countable gold nanoparticles with dark-field microscope for T4 polynucleotide kinase activity assay
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).
References (44)
- et al.
Mol. Cell
(2005) - et al.
Talanta
(2018) - et al.
Biosens. Bioelectron.
(2015) - et al.
Biosens. Bioelectron.
(2018) - et al.
Sens. Actuators, B
(2015) - et al.
Anal. Chim. Acta
(2013) - et al.
Sens. Actuators, B
(2018) - et al.
J. Biol. Chem.
(1999) - et al.
Biosens. Bioelectron.
(2018) - et al.
Biosens. Bioelectron.
(2017)
Biosens. Bioelectron.
Colloids Surf., B
Anal. Biochem.
J. Biol. Chem.
Cell
Mol. Cell
Biosens. Bioelectron.
Sens. Actuators, B
Talanta
Chin. J. Anal. Chem.
Mol. Cell. Biol.
Nucleic Acids Res.
Cited by (23)
Single-molecule detection-based super-resolution imaging in single-cell analysis: Inspiring progress and future prospects
2023, TrAC - Trends in Analytical ChemistryTtAgo sensor for the sensitive and rapid detection of T4 polynucleotide kinase activity
2023, Sensors and Actuators B: ChemicalVersatile fluorescence detection of T4 PNK and mRNA based on unique DNA nanomachine amplification
2023, Analytica Chimica ActaA multiple primers-mediated exponential rolling circle amplification strategy for highly sensitive detection of T4 polynucleotide kinase and T4 DNA ligase activity
2022, Microchemical JournalCitation Excerpt :The electrochemical method exhibits improved sensitivity, but requires the immobilization of DNA probes on the electrode surface, which is complicated and time-consuming. Fluorescent method, including nanomaterial-based method [25–28] and DNA-based biosensor [29–42], exhibits simplicity and sensitivity and has attracted attention in the field of repair enzyme activity detection. Nanomaterial-based method involves the complicated nanomaterial preparation and/or functionalization.
Plasmonic biosensor for the highly sensitive detection of microRNA-21 via the chemical etching of gold nanorods under a dark-field microscope
2022, Biosensors and BioelectronicsCitation Excerpt :Furthermore, the dark-filed signal output presents in the form of light scattering intensity (Yan et al., 2021), color (Liu et al., 2020a) or counting (Tian et al., 2021; Li et al., 2017) of nanomaterials, which could be adjusted by means of assembly (Wang et al., 2021a), etching (Li et al., 2020; Wang et al., 2019b), and growth (Li et al., 2019) of nanomaterials. Vigorous research efforts have advanced the state-of-the-art plasmonic nanomaterial with ultrahigh sensitivity for bioanalysis (Jin et al., 2020). Among them, AuNRs, as a fascinating light scattering optical probe have attracted increasing attention due to their unique localized surface plasmon resonance (LSPR) properties (Xianyu et al., 2021).