Elsevier

Analytica Chimica Acta

Volume 1137, 15 November 2020, Pages 1-10
Analytica Chimica Acta

Developing a colorimetric nucleic acid-responsive DNA hydrogel using DNA proximity circuit and catalytic hairpin assembly

https://doi.org/10.1016/j.aca.2020.08.059Get rights and content

Highlights

  • DNA nanotechnology has sparked the construction of the many nanostructures in biosensing platform.

  • The present study enabled fabrication of a colorimetric nucleic acid responsive biosensor via LOD as low as 1 pM.

  • Three amplification strategies are implemented in this assay, namely SPC, CHA, and DNA hydrogel that provide high sensitivity and selectivity.

  • Monitoring of various targets, such as nucleic acid, metabolites, cancer cells, and sites of infection can be considered for this assay.

Abstract

The development of powerful techniques for sensitive detection of nucleic acids has attracted much attention for fabricating accurate biosensors in various fields, such as genomics, clinical diagnostics, and forensic sciences. Up to now, different systems have been introduced, the majority of which are expensive, time-consuming, and relatively low selectivity/limit of detection. These limitations caught our attention to fabricate a nucleic acid responsive system by combining three layers of signal amplification strategy, namely a split proximity circuit (SPC), a catalytic hairpin assembly (CHA), and a DNA hydrogel. Herein, by SPC operation, two initiators and a target strand were assembled and activated the CHA reaction in the presence of three 5′-cytosine (C)-rich hairpins. Then, produced C-rich embedded three-way junction structures could form i-motif structures under acidic environment followed by a transition from sol to gel state. To acquire a quantitative and colorimetric measurement, gold nanoparticles (GNPs) were used that encapsulated and sediment by the gel formation. The resulting platform detected the target with a limit of detection of 1 pM and considerable selectivity.

Introduction

The programmable self–assembly ability of DNA molecule into a variety of dynamical nanoscale structures has moved this molecule forward its biological property for emerging the field of DNA nanotechnology [1]. DNA nanotechnology, owing to the unique properties of nucleic acids (programmability, biocompatibility, stability, and low cost synthesize and purification) has been tailored in different applications, such as tissue engineering, biosensing, drug delivery, and cancer therapy [2]. In particular, dynamic DNA nanotechnology using toehold-mediated strand displacement (TMSD is an enzyme-free and spontaneous reaction based on the toehold exchange that is powered by enthalpy instead of enzymes) has showed powerful capability to develop diverse enzyme-free molecular dynamic systems with moving fragments in time-varying manners [3,4]. Over the past decades, TMSD has enabled the assembly of highly versatile DNA nanostructures, such as smart DNA hydrogels, signal amplifiers, logic gates, DNA circuits, and switches with good stability, programmability, and tunable multifunctionality properties [5]. These nanostructures can autonomously assemble or modify with a variety of functional elements, such as aptamer structures, i-motif assemblies, DNAzymes, and antisense nucleic acids for smart biological functions, especially for biosensing various targets [6,7]. In most of these systems, input stimulus, such as protein, nucleic acid, metabolite, metal ion, pH, and heat can trigger the running of the nanostructure assembly through a series of TMSD reactions in an autonomous paradigm [8].

DNA circuits propose a unique molecular detection gadget for translating the recognition events of specific analytes into a series of TMSD reactions for the following readout signals [9,10]. DNA circuits are significant signal amplifiers, in which the reagents recycling are important to assist the generation of continuous signals [11,12]. Recently, split proximity circuit (SPC) has been utilized for transducing the recognition processes into an amplified signal, using an enhanced association of branch migration and toehold domains to a continuous string [13]. As reported by Soderberg group, an enzyme-free method using proximity-dependent initiation of hybridization chain reaction (HCR) was introduced to image the protein interactions [14]. In another study, Yung group reported a similar system for the detection of cell receptors [15]. In their design, two initiator strands conjugated to antibodies were in close proximity in the presence of the target that exposed the complete trigger strand followed by activating the HCR. Consequently, increased local concentration of initiator strands improved the assembly rate and signal-to-noise, reduced the circuit leakage, and eliminated the washing steps. Moreover, the SPC acted autonomously via a self-contained manner to improve the hybridization kinetics.

Catalytic hairpin assembly (CHA) is one of the most widely used signal amplifiers that exploits kinetically metastable hairpins to assemble stable DNA nanostructures [16,17]. Because of the original CHA possess high background signal, this strategy has been modified to conquer this limitation [18]. For instance, Chen et al. constructed a cascaded amplifier platform for the monitoring of the aflatoxin B1 (AFB1) using cyclic production of three-way junction structures via CHA [19]. Using this process Chen and coworkers reported a label-free G-quadruplex embedded three-way junction structure for the amplified monitoring of the 17β-estradiol [20]. More recently, Hosseinzadeh et al. presented the imaging of the miR-21 based on the CHA-induced three-way junction combining with concatemers functionalized nanocomposites [21].

In addition to static 3D DNA structures, the concept of DNA assembly has recently been developed to synthesize dynamical 3D nanoassemblies, such as DNA hydrogels [22,23]. DNA hydrogels are 3D network polymers including a large amount of entrapped water molecule with nucleic acids as building blocks [24]. As a particular component in DNA nanotechnology, DNA hydrogel has enabled the production of several gels due to the extravagant mechanical stability, flexibility biocompatibility, and biodegradability [25]. These properties have potentially powered DNA hydrogels as scaffolds in various applications, particularly in drug delivery, biosensing, glues, and tissue engineering [26,27]. Stimuli-responsive DNA hydrogels are intelligent materials within this field that can change their structure in the response to many stimuli, including pH, temperature, metal ions, and light [28]. Based on these principles, many intelligent analyte-responsive DNA hydrogels have been reported by several groups, hitherto. Pioneered by Cheng et al. a pH-responsive DNA hydrogel was reported using pre-formed three-way junction nanostructures and GNPs [2,29]. Indeed, C-rich overhangs conjugated to 5′ ends of three-way junctions that upon acidic pH, self-assembled into i-motif structures. Moreover, Li and co-workers hybridized three-way junction structures with linker strands in response to enzymatic and thermal stimulates [30]. Although the preparation of the three-way junction structure is a time-consuming process and also requires a large amount of nucleic acid monomers, exploring a continuous assembly of nanostructures in an autonomous manner seems worthy of effort.

Following this route of investigations, we explore new dimensions in nucleic acid monitoring through combining different TMSD-based DNA nanostructures. Given the enhanced signal intensity and flexible designing of SPC, target recycling by CHA, and mechanical stability of DNA hydrogel, we fabricated a programmable biosensing platform using a bottom-up approach. With this in mind, two initiator strands were partially hybridized to a target DNA strand (a part of Acinetobacter baumannii structure as an example) and brought in close proximity to assemble the complete trigger strand (Scheme 1). Upon revealing of the trigger strand and in the presence of three hairpin structures, the CHA reaction was performed to periodically catalyze the cyclic formation of hundreds of nanometer-sized three-way junctions and trigger strand. Incorporation of C-rich sequences at 5′ ends of three-way junctions under acidic pH induced the formation of i-motif structures to facilitate the visualization of DNA hydrogel. Utilizing GNPs as the indicator, the colorimetric and quantitative evaluation of the assay was possible that assisted the observation by the naked-eye. By applying three-layer amplification systems, exponential signal intensity was provided that afforded a method with simplicity, generality, and quick response properties. The resulting assay paved the detection of the target strand with a limit of detection (LOD) value of 1 pM.

Section snippets

Materials and reagents

All PAGE-purified oligonucleotides were purchased from Bioneer Co. (South Korea) that dissolved in deionized water and reserved at −20 °C until use. The sequences of the designed oligonucleotides are illustrated in Table 1. Tetrachloroauric (III) acid trihydrate (HAuCl4·3H2O), trisodium citrate, and ethylenediaminetetraacetic acid disodium salt (EDTA) were obtained from Sigma-Aldrich Co. (USA). Hybridization buffer, phosphate buffered saline (PBS), including 1 mM phosphate buffer (from Bio

The Principle and characterization of the designed assay for nucleic acid detection

In this approach, we demonstrate the feasibility of monitoring a nucleic acid target through an enzyme-free, sensitive, and simple method. The detecting system underwent three steps, including the assembly of the trigger strands by SPC, forming three-way junction structures, and visualizing the gel through i-motif organizations. As illustrated in Scheme 1, Initiator strands were consisted of six domains that in the absence of the target strand were separated entities. Upon meeting the target,

Conclusion and perspective

Taken in its entirety, we have successfully fabricated a multi-layer signal amplifier biosensing system for the detection of fuel nucleic acid. The colorimetric sensor was consisted of the formation of the three-way junction structures upon meeting the target strand. In the next step, assembling of i-motif structures in a pH-dependent manner induced the gel state that GNPs as tracer agents were encapsulated in the gel. Owing to the minimal circuit leakage and continuous generation of three-way

CRediT authorship contribution statement

Sima Khajouei: Methodology, Validation, Data curation, Writing - original draft, Visualization, Project administration. Hadi Ravan: Conceptualization, Software, Formal analysis, Writing - review & editing, Supervision, Funding acquisition. Ali Ebrahimi: Investigation, Resources, Writing - original draft.

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.

Acknowledgments

This work was supported by Shahid Bahonar University of Kerman, Kerman, Iran.

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