Lab in a tube: a fast-assembled colorimetric sensor for highly sensitive detection of oligonucleotides based on a hybridization chain reaction

Abstract

In this work, a one-pot signal amplified strategy was constructed based on generating a hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme as the color product for the detection of DNA sequences. A DNA sequence associated with the hepatitis B virus (HBV) was selected as a model target. The enzyme-free and label-free biosensor contained three oligonucleotide terms as target DNA, hairpin structures H₁ and H₂ which were partially complementary. It should be noted that the G-quadruplex structure was partially hidden in the hairpin structure H₂. In the absence of T_HBV, hairpins H₁ and H₂ were stable enough due to the complementary sequences at the end of the oligodeoxynucleotides, which made it difficult to complete the self-assembly. Upon addition of T_HBV, self-assembly of T_HBV with H₁ allows the rest of the DNA sequence of H₁ to facilitate H₁–H₂ complex formation and releases T_HBV to the next cycle. In the presence of hemin and K⁺, the G-quadruplex at the end of the H₁–H₂ complex was liberated to form a hemin/G-quadruplex structure, which could catalyze achromatous tetramethylbenzidine (TMB) into a colored product. The color change of the solution could be quantitated by spectrophotometry and the naked eye. We also employed 20% ethanol in the buffer to accelerate H₁–H₂ complex formation in the DNA strand displacement reaction (DSDR), which cut down the reaction time of the detection process from 12 h to 1 h. The detection limit of the colorimetric sensor is 9.5 pM, and the linear range is 50 pM to 100 nM.

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1. Introduction

The approach for sensitive and selective DNA detection is important in mutation identification,¹ clinical diagnostics² and monitoring lethal infectious diseases³ such as human immunodeficiency virus⁴ (HIV), hepatitis B virus⁵ (HBV), human papilloma virus^6,7 (HPV), influenza A virus⁸ (IAV) and varicella-zoster virus⁹ (VZV). For this reason, an increasing number of analytical methods have been developed for DNA detection, including colorimetric,¹⁰ electrochemical,^11,12 fluorescence detection¹³ and so on. Although the electrochemical and fluorescence methods have high sensitivity, they suffer from the inherent drawbacks of high-cost, time-consuming processes of preprocessing and expensive instruments. Thus, it is necessary to develop novel, fast-assembled and low-cost DNA detection methods.

Advances in DNA amplification strategies have boosted many different kinds of DNA detection methods such as Southern blot,¹⁴ the polymerase chain reaction¹⁵ (PCR), rolling circle amplification¹⁶ (RCA), the hybridization chain reaction¹⁷ (HCR), long-range self-assembly,¹⁸ Exonuclease III-aided target recycling¹⁹ and so on. Contemporarily, a new enzyme-free amplified DNA sensor has been reported, in which the hybridization chain reaction^20,21 (HCR) and target-catalyzed DNA self-assembly²² are responsible for amplification. Although the strategy of cyclic amplification has been widely applied in electrochemical²³ and fluorescence detection,²⁴ most hybridization chain reactions need expensive instruments²⁵ and extra chemical modification steps²⁶ for fluorescent or electrochemical signal readout. Moreover, the hybridization chain reaction requires a long reaction time¹⁰ since the complicated DNA structure limits the speed of DNA assembly. Many reported biosensors based on the hybridization chain reaction have these fatal shortcomings, leading the method to become unable to be applied in clinical detection. Fortunately, Professor Xia²⁷ put forward an excellent method of introducing an organic polar solvent to a traditional aqueous buffer which enhanced the reaction rates involved in DNA nanodevices and has been applied in the DNA strand displacement reaction. This gave us a good idea of how to prepare a colorimetric sensor which offers a low-cost and convenient approach that can detect the ssDNA in a short time.

Herein we develop a rapid, label-free and visual DNA assay method based on a target DNA assistant hybridization chain reaction circle and ligand-responsive G-quadruplex formation. As a proof of concept, a DNA sequence associated with the hepatitis B virus (HBV) was selected as a model target.²⁸ In this assay, the target (T_HBV) catalyzed the self-assembly of the hairpins H₁ and H₂ and G-quadruplexes were formed after the self-assembly. In the presence of G-quadruplexes/hemin, H₂O₂-mediated oxidation of TMB was speeded up and a blue colored product was produced. The solution turned yellow after adding strong acid to end the reaction.²⁹ Therefore, the concentration of T_HBV could be quantitated by the color change of the solution and a colorimetric sensor was fabricated (Scheme 1). Furthermore, 20% ethanol was added into the buffer to speed up the H₁–H₂ complex formation in the DNA strand displacement reaction (DSDR), which cut down the reaction time of the DNA machine from 12 h to 1 h. Our approach first utilized 20% ethanol buffer to construct a DNA biosensor based on the hybridization chain reaction to achieve the rapid detection of DNA, which overcame the above-mentioned shortcomings and reached modern analysis requirements. The analytical scheme is simple and can be performed in a single centrifugal tube, which avoids complex experimental steps such as separation and labeling.

Scheme 1 Schematic illustration of the hybridization chain reaction amplification for target DNA detection.

2. Experimental

2.1 Chemicals and materials

Ultrapage purified oligonucleotides were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). Hemin, DMSO, MES and Tris were purchased from Aladdin Reagent. Potassium chloride (KCl), magnesium chloride (MgCl₂), potassium acetate (CH₃COOK), hydrogen peroxide (H₂O₂) and hydrochloric acid (HCl) were purchased from Beijing Chemical Reagent Company (Beijing, China).

2.2 Instruments

A Cary 60 UV/vis spectrophotometer (Agilent, USA) was used to quantify the oligonucleotides. Circular dichroism (CD) spectra were measured on a JASCD J-810 spectropolarimeter that was purchased from JASCO Corporation, Japan. The polyacrylamide gel electrophoresis (PAGE) gels were photographed under a UV imaging system (Vilber Lourmat, Marne laVallee, France).

2.3 Sequences and pretreatment of oligonucleotides

The sequence of the oligonucleotides are listed as follows: T_HBV: 5′-AGTTACTC TCTTTTTTG CCTTCTGA-3′. H₁: 5′-TCAGAAGG CAAAAAAGA GAGTAACT CCTTCTGA GGGTAGGGC AGTTACTC TCTTTTTTG-3′. H₂: 5′-GAGTAACT GCCCTACCC TCAGAAGG AGTTACTC TCTTTTTTG CCTTCTGA GGGTAGGGC GGGTTGGG-3′. T_1-mismatch: 5′-AGTTACTC TCTTA̲TTTG CCTTCTGA-3′. T_2-mismatch: 5′-AGTTACTT̲ TCTTTTTTG A̲CTTCTGA-3′. T_3-mismatch: 5′-AGTTACTT̲ TCTTA̲TTTG A̲CTTCTGA-3′. The oligonucleotides were stored at −20 °C and were heated to 95 °C for 5 min and gradually cooled to room temperature before use.

2.4 Preparation of hemin-G-quadruplex complexes

The oligonucleotides were dissolved in 10 mM Tris–HCl buffer containing 5 mM MgCl₂, 15 mM KCl and 20% ethanol (pH = 8.0). Then H₁, H₂ and T_HBV were mixed to assemble the G-quadruplex complexes. The final volume of each sample for colorimetric detection was 65 μL 10 mM Tris–HCl buffer containing 5 mM MgCl₂, 15 mM KCl and 20% ethanol (pH = 8.0).

2.5 Colorimetric detection

In a typical experiment, 5 μL of 10 μM hemin solution was added to the G-quadruplex complexes and incubated for 30 min to form the hemin/G-quadruplex HRP-mimicking DNAzyme in aqueous solution. Then 25 μL of 15 mM TMB solution and 400 μL MES–Ac buffer (25 mM MES–Ac, pH 4.5, 20 mM KAc) were added into a single centrifugal tube. After that, 10 μL of 200 mM H₂O₂ was rapidly added to initiate the TMB–H₂O₂ reaction, allowing this reaction to proceed for 10 min at 37 °C. Finally, 200 μL HCl (1 M) was added to stop the reaction. When the solution turned yellow after adding strong acid, a photograph of the reaction product was taken using a digital camera. All photographs were used without further modification.

2.6 Non-denaturing polyacrylamide gel electrophoresis

H₁, H₂ and T_HBV were kept in 20% ethanol buffer (5 mM MgCl₂, 15 mM KCl, 10 mM Tris–HCl, pH = 8.0) for 1 h. By contrast, the mixture (H₁, H₂ and T_HBV) was also kept in a buffer solution free from ethanol for 12 h. We also analyzed H₁, H₂ and the mixture (H₁ and H₂) in 20% ethanol buffer and in buffer solution. Non-denaturing polyacrylamide gels were prepared in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3). 10 μL of each sample was mixed with loading buffer and loaded into the gels. The electrophoresis was run under a constant voltage of 110 V for 2 h. The gels were stained with Gel-Dye Super Buffer Mix, and then photographed under a UV imaging system (Vilber Lourmat, Marne lavallee, France).

3. Results and discussion

3.1 Principle of the colorimetric strategy for DNA detection

The principle of the colorimetric biosensor is depicted in Scheme 1. The DNA detection system consisted of hemin, H₂O₂, tetramethylbenzidine (TMB), the target (T_HBV) and hairpin structures H₁ and H₂ which were partially complementary. The G-quadruplex structure was partially hidden at the end of hairpin structure H₂ to avoid complicated chemical labeling. In order to explain the process of hybridization chain reaction amplification clearly, the sequences of the target sequence T_HBV and the hairpin structures H₁ and H₂ are divided into different segments (Scheme 1). Segments D and E in H₂ are able to form G-quadruplex. When H₂ is in its hairpin structure, segment D is hidden in the skeleton of H₂ and G-quadruplex cannot form at this point. The A segment in T_HBV is initiated by hybridization with the A* segment in H₁, which pushes forward further the hybridization of segments B and C in T_HBV to B* and C* in the H₁ skeleton. As a consequence, hairpin H₁ with segment C is exposed, which can function as a new initiator to nucleate between segments C and C* in hairpin H₂. Since the H₁–H₂ complex is more stable than the T_HBV–H₁ duplex, segments D, A, C*, B* and A* gradually hybridize with segments D*, A*, C, B and A in H₂, which results in the release of T_HBV from the complex. Segments D and E in H₂ are liberated and can form G-quadruplex. The process of the hybridization chain reaction was conducted in 20% ethanol buffer to speed up the rate of DNA self-assembly. In the presence of hemin and K⁺, G-quadruplex at the end of the H₁–H₂ complex was liberated to form the hemin/G-quadruplex structure. As the previous research reported,³⁰ hemin is a porphyrin characterized by a special structural selectivity for G-quadruplexes from other DNA structures to form a kind of DNAzyme which will mimic the catalytic activity of horseradish peroxidase (HRP).³¹ This G-quadruplex-based DNAzyme can catalyze its colorless substrate TMB into a blue colored product with the help of H₂O₂ and the solution will turn yellow after adding 1 M HCl to end the reaction. The released T_HBV will start a new cycle to continuously generate H₁–H₂ complexes containing G-quadruplex, leading to an amplified optical signal with the aid of hemin, K⁺, H₂O₂ and TMB.

3.2 UV-vis spectra of the system

UV-vis spectra were used to investigate the feasibility of our method, as shown in Fig. 1. The system only containing H₁ or H₂ exhibited very weak visible region absorption (Fig. 1, curves b and c), because G-quadruplex was difficult to form when the sequence was partly hidden in the skeleton of H₂. The system containing the mixture of H₁ and H₂ exhibited slightly enhanced visible region absorption (Fig. 1, curve d), indicating that both of the hairpins were stable enough to coexist in solution without the target. Upon addition of the target T_HBV, significant enhanced absorbance was observed (Fig. 1, curve e). This is because a large amount of self-assembly product had formed via hybridization chain reaction amplification, confirming the viability of our approach.

Fig. 1 The absorbance response of the system under different conditions. (A) UV-vis spectra of the system after mixing for 1 h under different conditions: (a) hemin + H₂O₂ + TMB; (b) H₁ + hemin + H₂O₂ + TMB; (c) H₂ + hemin + H₂O₂ + TMB; (d) H₁ + H₂ + hemin + H₂O₂ + TMB; (e) H₁ + H₂ + T_HBV + hemin + H₂O₂ + TMB. [H₁] = 100 nM, [H₂] = 100 nM, [T_HBV] = 50 nM. (B) Photographs of the color intensity of the system after mixing for 1 h under the corresponding conditions.

3.3 Non-denaturing polyacrylamide gel electrophoresis characterization

We also used non-denaturing polyacrylamide gel electrophoresis (PAGE) to inquire into the self-assembly of hairpins H₁ and H₂ and the acceleration effect upon addition of 20% ethanol to the buffer solution (Fig. 2). As shown in lane a and lane b, the base number of H₂ was higher than that of H₁, which was consistent with our design. Significantly, after the hairpins H₁ and H₂ were mixed in 20% ethanol buffer for 1 h or in pure aqueous buffer for 12 h, there was few H₁–H₂ complex formed (lane c), which showed that the two hairpins H₁ and H₂ could coexist in the buffer. After adding sequence T_HBV, the DNA belt (H₁–H₂ complex) could be clearly seen with the background intensity (lane d), and it can be concluded that the hybridization with H₁ and H₂ took place. The results indicate that sequence T_HBV could give rise to the self-assembly of hairpins H₁ and H₂, which was in agreement with the UV-vis spectra of the system. Furthermore, the brightness of the product band upon the addition of 20% ethanol was greater than the one reacting in pure aqueous buffer even after a shorter period of time. By comparing lane d (1 μM H₁ + 1 μM H₂ + 100 nM T_HIV in 20% ethanol buffer after 1 h reaction) and lane f (1 μM H₁ + 1 μM H₂ + 100 nM in pure aqueous buffer after 12 h reaction), it can be concluded that adding organic polar solvent has an obvious accelerating effect on DNA self-assembly.

Fig. 2 Native polyacrylamide gel (10%) electrophoresis characterization of the formation of the H₁–H₂ complex: (a) 1 μM H₁ in 20% ethanol buffer; (b) 1 μM H₂ in 20% ethanol buffer; (c) 1 μM H₁ + 1 μM H₂ in 20% ethanol buffer after 1 h reaction; (d) 1 μM H₁ + 1 μM H₂ + 100 nM T_HIV in 20% ethanol buffer after 1 h reaction; (e) 1 μM H₁ + 1 μM H₂ in pure aqueous buffer after 12 h reaction; (f) 1 μM H₁ + 1 μM H₂ + 100 nM in pure aqueous buffer after 12 h reaction.

3.4 CD spectra for analyzing the G-quadruplex structure

Circular dichroism (CD) spectroscopy was used to confirm the formation of the G-quadruplex composed of domains d and e. As shown in Fig. 3, the circular dichroism spectra of the random single stranded DNA, H₁, H₂ and T_HBV, present low amplitude. The CD signal intensities of the H₁/H₂ mixture without T_HBV was also weak, indicating the mixture was stable enough to coexist in solution without the target and could not be folded into the G-quadruplex structure in 3 h after the addition of K⁺. As the previous literature reported,³² the typical CD spectrum of parallel G-quadruplex has a positive ellipticity maximum at 264 nm and a negative maximum at 240 nm. After the addition of T_HBV to the H₁/H₂ mixture, the CD spectrum has an obvious negative peak at 245 nm and a positive peak at 268 nm, indicating the formation of parallel G-quadruplex with the aid of K⁺ in the solution.

Fig. 3 CD spectra of the products via the hybridization chain reaction after 3 h, demonstrating G-quadruplex formation. The concentrations of fuel strands (H₁ and H₂) and T_HBV were 1 μM, 1 μM and 0.5 μM, respectively.

3.5 Reason for the accelerating effect of ethanol

As shown in Fig. S1 (ESI†), the speed of the hybridization chain reaction was controlled by the process of the T_HBV–H₁ strand exchanging with H₂. As the previous research paper discussed,²⁷ a higher ethanol concentration resulted in a lower melting temperature T_m (T_HBV–H₁ and H₁–H₂). The results suggested that the multivalent and cooperative DNA binding weakened in the presence of the ethanol buffer. The activation energies of the T_HBV–H₁ strand exchanging with H₂ (33.46 kJ mol⁻¹ in pure aqueous buffer and 20.941 kJ mol⁻¹ in 20% ethanol buffer) deduced from the Arrhenius plots also suggested that the activation energies reduced upon the addition of 20% ethanol. It was kinetically favorable for the hybridization chain reaction process (Fig. S2–S4 in ESI†). Since the structure T_HBV–H₁ was destabilized upon addition of 20% ethanol based on the decrease of T_m, the complex would provide more free sites for H₂ and facilitate the rest of the sequence of H₁ hybridization with H₂ to form the H₁–H₂ complex. So, the hybridization chain reaction will be accelerated upon the addition of 20% ethanol.

3.6 Optimization of the concentration of ethanol and reaction time

Since ethanol can speed up the rate of DNA self-assembly, the concentration of ethanol was optimized successively in order to shorten the reaction time. As shown in Fig. 4A, time-dependent absorbance changes were monitored. The results indicated that the rate of DNA self-assembly was gradually increased as the ethanol concentration increased. When the hybridization chain reaction (HCR) was run after 1 h in 20% or 30% ethanol buffer, DNA self-assembly was almost completed. Since a higher concentration of ethanol cannot accelerate the reaction more efficiently (Fig. 4B), a concentration higher than 20% (v/v%) was not necessary. According to these results, we selected 20% (v/v%) ethanol content and 1 h reaction time in the subsequent experiments. As shown in Table 1, the experimental period for our approach was much shorter than similar methods.

Fig. 4 Optimization of ethanol percentage and reaction time. (A) UV-vis absorbance of the hybridization chain reaction for the system at 452 nm in different contents of ethanol with 0% in 10 h and [10%, 20%, and 30% (v/v%)] in 5 h. (B) UV-vis absorbance of the hybridization chain reaction for the system at 452 nm in different contents of ethanol [0%, 10%, 20% and 30% (v/v%)] after 1 h. The error bars represent the standard deviation of three independent measurements. [H₁] = 100 nM, [H₂] = 100 nM, [T_HBV] = 50 nM.

Table 1 Comparison of reaction times and detection limits of various amplified DNA sensors

Analytical method	Reaction time	Detection limit
Colorimetric detection of DNA based on DNAzyme^33	12 h	10 nM
Fluorescence detection of DNA based on the hybridization chain reaction^13	10 h	0.2 nM
Electrochemical detection of DNA based on RCA^34	5 h	0.1 nM
Electrochemical supersandwich assay for DNA detection^35	3 h	10 fM
Amplified fluorescence DNA detection based on Exonuclease III-aided target recycling^36	24 h	10 pM
Autonomous assembly of HRP-mimicking DNAzyme nanowires^10	4–6 h	0.1 pM
Our approach	1 h	9.5 pM

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3.7 Quantitative detection of T_HBV

To estimate the sensitivity and linear range of the colorimetric method for T_HBV detection, various concentrations of T_HBV (0, 0.05, 0.1, 1, 10, 50, 100 nM) were examined. Fig. 5A shows that the absorbance responses increased with the increase of T_HBV concentration from 0 nM to 100 nM in the presence of H₁ and H₂. To evaluate the reproducibility of the sensing system the error bars represent the standard deviation of three independent measurements of absorbance against the concentration of T_HBV. The inset in Fig. 5A displays the linear response between absorbance and the logarithm of T_HBV concentration ranging from 0.05 nM to 100 nM. The linear regression equation is Abs = 0.5185 + 0.2175 log C_THBV (R² = 0.9903). Error bars were calculated from three experimental measurements. The limit of detection (LOD) of 9.5 pM was calculated according to the equation LOD = 3σ/slope. The limit of detection (LOD) of the proposed sensor is superior over many existing enzyme assisted amplified sensors (Table 1). Meanwhile, it is easy to distinguish the solution containing 1 nM T_HBV from the solution without T_HBV from the solution color by the naked eye (Fig. 5B). Therefore, the LOD for visual detection was set to 1 nM.

Fig. 5 Sensitivity for T_HBV detection. (A) The responses of absorbance plots at 452 nm to the different concentrations of sequence T_HBV ranging from 0 nM to 100 nM in the presence of 100 nM H₁ and 100 nM H₂. The inset displays the calibration curve of absorbance at 452 nm vs. the logarithm of T_HBV concentration. (B) Photographs of the color intensity with different concentrations of sequence T_HBV (0, 0.05, 0.1, 1, 10, 50, 100 nM).

3.8 Selectivity of the colorimetric sensor toward T_HBV

Precise recognition of a few base mismatches in DNA sequences having very high sequence similarity is important because it provides information on genetic mutation, DNA damage and single-nucleotide polymorphism (SNP). Since the technique was based on DNA hybridization, the competitive selectivity of our experiments was excellent. The selectivity of this colorimetric sensor was tested with different mutants of the analyte. Various mutations of the target were taken into consideration such as one-base mismatch sequence (T₁), two-base mismatch sequence (T₂) and three-base mismatch (T₃). 50 nM concentration of T_HBV and mutations was chosen. As expected, the mismatch sequences (T₁, T₂, T₃) showed lower absorbance than the perfectly matched target in the competitive selectivity experiments. For the purpose of quantifying the discriminating ability, the ratio (Abs(H₁ + H₂ + different targets) − Abs(H₁ + H₂))/(Abs(H₁ + H₂ + T_HBV) − Abs(H₁ + H₂)) was used to analyze the results (Fig. 6). In particular, one-base mismatch in the middle of the target DNA (T₁) causes a decrease of the ratio from 100% to 63.5% compared with the target DNA. The ratio decreased to 23.9% and 19% upon addition of the two base mismatch sequence (T₂) and three base mismatch sequence (T₃), respectively. These results show that our approach exhibits a high selectivity for T_HBV detection.

Fig. 6 Selectivity for T_HBV detection. (A) The ratio of (Abs(H₁ + H₂ + different targets) − Abs (H₁ + H₂))/(Abs (H₁ + H₂ + T_HBV) − Abs (H₁ + H₂)) under different conditions. [H₁] = 100 nM, [H₂] = 100 nM, [T_HBV] = 50 nM, [different targets] = 50 nM. (B) Photographs of the solution color change with different mutants of the analyte.

3.9 Analysis of T_HBV in a real sample

In order to prove this method can be used in real samples, the biosensor was employed to detect target DNA in a human urine sample. As shown in Fig. 7, the biosensor performed well when used in the human urine sample. The urine sample had only a little effect on the analysis, as indicated by a slight variation in the absorbance value (approximately 10%), which probably resulted from other nonspecific interference. However, if 50 nM T_HBV was present in the solution, an approximate 76% increase of the UV-vis absorbance at 452 nm was observed. Undoubtedly, the results show that the proposed method can be used to detect target DNA in real samples sensitively and specifically.

Fig. 7 Determination of T_HBV in human urine. (A) Histogram of UV-vis absorbance of the hybridization chain reaction for the system at 452 nm in the presence of 100 nM H₁ and 100 nM H₂ in buffer, 50 nM T_HBV, 100 nM H₁ and 100 nM H₂ in buffer, 100 nM H₁ and 100 nM H₂ in buffer containing 1% urine, 50 nM T_HBV, 100 nM H₁ and 100 nM H₂ in buffer containing 1% urine, respectively. (B) Photographs of the solution color in the presence of different matrices.

3.10 Application of techniques in the presence of bio-chemicals

The target DNA can be changed into ATP and thrombin aptamers, which could bind ATP and thrombin to form ATP–aptamer complexes or thrombin–aptamer complexes. Thus, the hybridization chain reaction circle cannot run upon addition of ATP and thrombin. So the method can be applied to detect ATP and thrombin in biological samples. It is well known that a DNA duplex containing thymine–thymine (T–T) mismatches shows high selectivity for Hg²⁺ against other metal ions, owing to the formation of T–Hg²⁺–T base pairs. So the method can also be applied to detect Hg²⁺ in environmental samples. We believe the fast-assembled biosensor can detect more biomolecules in the presence of bio-chemicals.

4. Conclusions

In conclusion, a rapid, label-free and visual DNA biosensor based on a hybridization chain reaction circle was developed. The key point of this strategy lies in that the target DNA (T_HBV) can assist the hybridization chain reaction circle and form G-quadruplex with the aid of K⁺ and hemin in the solution. By monitoring the color change after the addition of TMB, H₂O₂ and HCl, we can detect target DNA as low as 9.5 pM, which is superior over many reported enzyme assisted amplified sensors for DNA detection. 20% ethanol is added to the buffer to speed up the H₁–H₂ complex formation and the experiment can be conducted in a centrifuge tube. This scenario can be expanded to analyze other targets since there is potential for lot of studies regarding the interaction between DNA (aptamer) and other molecules in future research.

Acknowledgements

The project was partly sponsored by the Fundamental Research Funds for the Central Universities of China (grant no. N130605001), and by the National Natural Science Foundation of China (no. 21477082).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra04613k

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