Target-driven rolling walker based electrochemical biosensor for ultrasensitive detection of circulating tumor DNA using doxorubicin@tetrahedron-Au tags

Highlights

• A novel electrochemical biosensor was developed based on target-driven rolling walker for detection of circulating tumor DNA.

• The rolling walker didn’t need external strands to drive walking, for all walker components were immobilized on the sensing surface.

• The movement of the multiple-legged walker was guided by strand displacement reactions.

• Doxorubicin@tetrahedron-Au was used as electrochemical signal tags.

• The multiple-legged walker was performed in clinical samples with credible results.

Abstract

In this study, a multiple-legged and highly integrated DNA rolling walker based electrochemical biosensor was developed for ultrasensitive ctDNA analysis through rolling circle amplification (RCA) with doxorubicin@tetrahedron-Au (DOX@TDN-Au) as electrochemical indicator. Upon target-driven RCA, the multiple-legged walker could move along with the predesigned track by strand displacement reactions, resulting in numbers of legs binding irreversibly to iStep probes. The binding of massive legs to iStep probes could effectively impede DOX@TDN-Au tags binding on the surface of sensor and then reached a “signal off” state. Benefiting from the highly amplified efficiency of rolling walker machine and DOX@TDN-Au tags, the established biosensor performed high sensitivity for target detection with a low limit of detection down to 0.29 fM. Moreover, the target ctDNA could hybridize with the ring and capture probe simultaneously, greatly enhancing the specificity of the developed biosensing method. Thus, this biosensing method is a promising tool for detection of ctDNA in the field of clinical diagnostic and tumor progression assessment.

Introduction

Circulating tumor DNA (ctDNA) is a single-stranded or double-stranded gene fragments originating from tumor or circulating tumor cells, and subsequently released to peripheral blood (Chan et al., 2013; Speicher and Pantel, 2014). Studies have shown that ctDNA is a potential tumor biomarker which has gained increasing attentions in term of cancer assessment (Koldby et al., 2019; Sumbal et al., 2018). For example, kirsten rat sarcoma-2 virus (kras) point mutations are closely related to different cancers including lung cancer, colorectal cancer, and ovarian cancer (Kamerkar et al., 2017). Thus, quantitative analysis of ctDNA plays a vital role in clinical diagnosis, tumor progress and cancer recurrence (Gorgannezhad et al., 2018; Li et al., 2019).

Currently, the routine methods for ctDNA analysis are polymerase chain reaction (PCR) and DNA sequencing (Das et al., 2016; Hu et al., 2018). PCR-based approaches including digital PCR and droplet digital PCR (ddPCR) have been developed to detect ctDNA (Siravegna et al., 2017), however these methods are susceptible to false negatives and positives produced by interference from chemical species. Alternatively, DNA sequencing is a powerful tool for getting information of mutant status, but it requires expensive instrument and long turnaround time (Dewey et al., 2012). Therefore, developing inexpensive, quick and highly sensitive method for ctDNA mutation analysis remains a great challenge.

DNA as an attractive building material has been constructed various dynamic nanodevices including walkers (Chai; Miao, 2019; Jung et al., 2016), tweezers (Li et al., 2016; Peng et al., 2019), and nanorobots (Kopperger et al., 2018; Torelli et al., 2018) due to its programmable, addressable, and accurate Watson-Crick base pairing. Among them, intelligent DNA walkers have been extensively applied for biosensing (Yang et al., 2019; Zheng et al., 2018a, Zheng et al., 2018b; He et al., 2017), bioimaging (Peng et al., 2017), and drug-delivery (Li et al., 2018a). In most case, the operation of DNA walkers is triggered by strand displacement reactions (Zheng et al., 2018a, Zheng et al., 2018b), nicking enzymes (Yang et al., 2016), and DNAzyme hydrolysis (Cha et al., 2015; Yang et al., 2018; Wang et al., 2018). For example, Liang developed entropy-driven DNA walker onto Au nanoparticle surfaces which can be actuated by additional fuel strand substrate (Liang et al., 2017). Fan et al. designed a stochastic DNA walker propelled by exonuclease III, which can selectively catalytic the removal of hybridized oligonucleotides (Qu et al., 2017). However, these walkers may confront with some challenges. First, DNA walkers needing supplement external substrates for continuous reaction tend to untimely stop operation in a local region of exhausted substrates (Jung et al., 2017). Second, leg DNA of the walkers may be prone to divorce from the preset track before hybridization with next neighboring substrate (Xu et al., 2019). Last, protein enzymes powered DNA walkers are hard to access substrates immobilized on nanoparticles or other sensing surfaces due to steric hindrance effect.

To address these difficulties, we successfully constructed a highly powered rolling walker for sensitive cfDNA analysis based on rolling circle amplification (RCA) with doxorubicin@tetrahedron functionalized gold nanoparticle (AuNP or Au) tags. Owing to ease of synthesis and high loading capacity, AuNPs are widely used to develop various biosensing platforms. However, the undesired aggregation or precipitation of AuNPs always happens in the test (Li et al., 2018b). DNA tetrahedron (TDN), an excellent nanoarchitecture with programmability and high loading capacity, is recently engineered as a significant paradigm for carrying numerous biomolecules (Wiraja et al., 2019). Thus, TDN is here properly employed as the nanocarrier to load doxorubicin (DOX) for forming DOX@TDN complex, which can further immobilize on AuNPs based on Au–S bond. The formed DOX@TDN-Au can not only enhance the dispersibility and solubility of AuNPs, but also offer an electrochemical indicator.

RCA is a powerful isothermal DNA replication tool which can autonomously synthesize a long single-stranded sequence with tandem repeats (Wang et al., 2015; Valero et al., 2018). By designing the circular template, thousands of periodic user-specified sequences can be produced in a single strand. Along with the operating progress, our multiple-legged walker system utilized the products of RCA as leg DNA, which gradually increased the binding power of the legs and the preset path to prevent legs divorcing from the predefined track, because more and more legs irreversibly bind to substrates. In addition, all substrates of the rolling walker are immobilized on the sensing surface, without need of external dissociative strands for driving further walker locomotion. Moreover, the walker relies on strand displacement reactions rather than enzyme's cleavage to move, which avoided the steric hindrance effect. As we can see in Scheme 1, Target binds to capture probe (CP) and ring, triggering RCA. The replicated DNA legs displace continuously the protruding domain (c) of iStep probes by strand displacement reactions, forming more stable dsDNA and guiding the motion of the walker along the path. As replication proceeds, the protruding domain (c) of iStep probes decrease gradually, which prevent DOX@TDN-Au tags from binding to sensing surfaces and producing electrochemical signal. On the contrary, there is a huge signal generated by the tags in the absence of the target.