An enzyme-free and amplified colorimetric detection strategy via target–aptamer binding triggered catalyzed hairpin assemblyby Ke Quan, Jin Huang, Xiaohai Yang, Yanjing Yang, Le Ying, He Wang, Yong He, Kemin Wang

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This journal is©The Royal Society of Chemistry 2014 Chem. Commun.

Cite this:DOI: 10.1039/c4cc08488h

An enzyme-free and amplified colorimetric detection strategy via target–aptamer binding triggered catalyzed hairpin assembly†

Ke Quan,‡ Jin Huang,‡ Xiaohai Yang, Yanjing Yang, Le Ying, He Wang, Yong He and Kemin Wang*

Here we introduce an enzyme-free and colorimetric detection strategy for small molecule adenosine. The approach is based on the adenosine–aptamer binding triggered liberation of an initiator strand that consecutively catalyzes DNA hairpins hybridized from singles to couples. These couples induce gold nanoparticles assembled via crosslinking, which could be visualized by a color change.

Nucleic acid-based signal amplification strategies are frequently utilized for improving detection sensitivity in the design of sensors, such as the polymerase chain reaction (PCR),1 ligase chain reaction (LCR),2 rolling circle amplification (RCA)3 and polymerization/scission-based strand-displacement amplification (SDA).4 Of late, enzyme-free nucleic acid-based signal amplification has been especially appealing due to the low cost and simplicity. Previously, our group developed a series of sensitive detection methods based on the hybridization chain reaction (HCR), in which a pyrene-excimer,5 graphene oxide,6 and gold nanoparticles (AuNPs)7 were engaged for the signal switch, respectively. In these studies, the HCR is an enzyme-free nucleic acid-based signal amplification in which only one target

DNA strand could trigger a cascade of hybridization events to form a long nicked duplex, amplifying the signal of target binding. Alternatively, catalyzed hairpin assembly (CHA) is another robust enzyme-free nucleic acid-based signal amplification in which a pair of hairpins can be designed so that the two hairpins do not initially interact with each other but can catalytically form a duplex in the presence of an initiator strand input.8 Fluorescence and electrochemical signals have been used to combine CHA for sensitive detection of nucleic acids.9

However, these methods need tedious labels and dedicated instrumentation. In the year 1996, two leading groups pioneered the utilization of DNA mediating the assembly of AuNPs.10 Since then, the combination of the programmable capability of DNA and the distinguished assembly performance of AuNPs has found practical applications in terms of colorimetric-based detection.

However, the insufficient detection limit of this strategy restricts its practical application, partly attributed to the absence of signal amplification. Due to the low cost, simplicity, and practicality, in our opinion, the visualization of assembly of AuNPs could combine the signal amplification of CHA to develop an enzymefree and amplified colorimetric detection method.

In the present work, adenosine and its aptamer were chosen as the target and its recognition molecule for demonstration, respectively. Adenosine, which is an important small molecule playing an important role in biochemistry,11 and its aptamer were selected exhibiting relatively good binding properties.12

Fig. 1(a) shows the working mechanism that is based on the assembly of DNA–AuNPs driven by adenosine-triggered CHA.

It involved the design and synthesis of five types of oligonucleotides (see DNA sequences: Apt-T, Inh, H1, H2, and L*, ESI†).

In principle, Apt-T (containing an anti-adenosine aptamer and a trigger) is inhibited by a short strand, Inh. When Apt-T is bound to Inh (Apt-T–Inh duplex), it cannot catalyze the hybridization of H1 and H2. In contrast, free Apt-T can do so. As a result, when the target (adenosine) binds to the aptamer, the

Apt-T–Inh duplex is destabilized and T is available to activate

CHA (step 1). Two hairpin species (H1 and H2) are employed in the system, which can potentially hybridize to form a H1H2 duplex, since H1 contains a segment that is complementary to a segment of hairpin H2. However, the spontaneous hybridization of the two hairpins is kinetically hindered by occluding complementary regions within intramolecular hairpin secondary structures. In order to illustrate the CHA process, we marked letters with ‘*’ complementary to the corresponding unmarked letters. When the T (a*–b*–c*) segment is liberated by adenosine, it nucleates with hairpin H1 via base pairing to a single-stranded

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular

Engineering of Hunan Province, Hunan University, Changsha 410082, China.

E-mail: † Electronic supplementary information (ESI) available: DNA sequences; preparation of DNA–AuNPs; gel electrophoresis, TEM and an analytical protocol; UV-vis spectra for T-triggered CHA; and optimization of inhibitor lengths and concentrations. See DOI: 10.1039/c4cc08488h ‡ K. Quan and J. Huang contributed equally to this work.

Received 28th October 2014,

Accepted 24th November 2014

DOI: 10.1039/c4cc08488h



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Chem. Commun. This journal is©The Royal Society of Chemistry 2014 toehold (a), mediating a branch migration that opens the hairpin to form complex TH1 containing the single-stranded segment (d–c*–b*) (step 2). This complex nucleates with hairpin H2 by means of base pairing to a toehold (c), mediating a branch migration that opens the hairpin to form complex TH1H2 (step 3). This complex is inherently unstable, and T dissociates from the H1H2 complex, completing the reaction and allowing T to act as a catalyst to trigger the hybridization of additional pairs of H1 and H2 hairpins (step 4). The process makes more hairpins from singles to couples (H1H2), resulting in AuNP aggregation through L binding L*-AuNPs (step 5). Therefore, the target detection could be visualized by a color change and also the surface plasmon absorption from AuNP aggregation.