Enzyme-free detection of DNA based on hybridization chain reaction amplification and fluorescence resonance energy transfer
Introduction
It is of great importance for the sensitive detection of DNA in pathogenic bacteria or virus detection and clinical diagnosis. Several DNA amplification techniques have been proposed to achieve a high sensitivity for DNA detection, such as polymerase chain reaction (PCR) [1], rolling circle amplification (RCA) [2], [3], and ligase chain reaction (LCR) [4], strand displacement amplification (SDA) [5]. However, these amplified techniques require protein enzymes, which are cost consuming and sensitive to reaction conditions, such as temperature, pH or toxic chemicals. Compared to these techniques, hybridization chain reaction (HCR), which was first introduced by Dirk and Pierce [6], has become a fascinating and effective technique for the detection of DNA, because it is an enzyme-free amplification technique where DNA self-assembly is triggered by an initiator DNA and leads to the formation of long nicked double-stranded DNA structures at mild conditions. Nowadays, HCR has been extensively utilized to construct various platforms for sensitive detection of DNA [7], [8], microRNA [9], [10], proteins [11], [12], ions [13], [14], small molecules [15], [16], cells [17], [18], [19], etc. Many signal transduction techniques such as colorimetric method [13], [20], [21], [22], electrochemical method [9], [11], [12], [23], [24], fluorescence [7], [8], [10], [14], [15], [16], [19], [25], [26], [27], [28], chemiluminescence [29], bioluminescence [30], surface plasmon resonance (SPR) [31], cytometry [32], have been utilized in HCR amplification methods. Among these techniques, fluorescence is particularly attractive due to its high sensitivity, easy readout, low sample volume, simple operation and feasibility of quantification. However, most HCR-based fluorescent sensors are quantified by the fluorescent intensity at single wavelength, which might suffer from the influence due to the change of probe concentration and excitation intensity.
To address this problem, herein we develop a versatile DNA detection method by combining HCR for enzyme-free signal amplification and fluorescence resonance energy transfer (FRET) for signal transduction. The underlying physical principle of FRET is a nonradiative energy transfer process from an excited state of donor molecule (usually a fluorophore) to a proximal ground state of acceptor molecule via long-distance dipole–dipole interaction. The acceptor absorbs energy at the emission wavelength of the donor. The rate of FRET depends on the extent of spectral overlap between donor emission and acceptor absorption, the relative orientation of the transition dipoles and the distance between the donor and acceptor molecules. The molecular distance to occur FRET is typically in the range of 10–100 Å [33], [34], [35]. The FRET technique is very appealing for bioanalysis by means of ratiometric measurement, which use the ratio of two fluorescent intensities at different wavelengths to quantitatively detect the analytes. Using ratiometric measurement, it allows more precise measurements due to reduce the influence of those external factors in practical application such as excitation source fluctuation and probe concentration [36], [37]. In this study, nucleic acid hairpin probes (H1 and H2), which were labelled with carboxyfluorescein (FAM) as the donor and tetramethylrhodamine (TAMRA) as the acceptor respectively, can coexist metastably in solution. Target DNA triggers the HCR process to form long nicked dsDNA nanowires. The donor and the acceptor are brought in close proximity, resulting in a FRET process between them. The relative fluorescent intensities of the acceptor and donor can be used to quantitatively detect target DNA.
Section snippets
Chemical and materials
All oligonucleotides (Table 1) were synthesized and HPLC-purified by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Tris(hydroxymethyl)aminomethane (Tris, BR), disodium hydrogen phosphate (Na2HPO4, AR), sodium chloride (NaCl, AR) were bought from Sinopharm Group Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade. The water used was purified by Millipore Milli-Q (18.2 MΩ cm).
Detection of target DNA
The stock solutions (10.0 μmol L−1) of hairpin probes were
Principle of detection method
The principle of enzyme-free DNA detection based on HCR and FRET is illustrated in Scheme 1. Hairpin probes H1 and H2 are labelled with FAM and TAMRA, respectively. FAM and TAMRA are a classic example of a FRET pair, where FAM is the donor and TAMRA is the acceptor. The absorption maximum of FAM is 494 nm with an emission peak at 520 nm. TAMRA (acceptor) can absorb light emitted by FAM and emits fluorescence with a peak at 580 nm when FRET occurs. Each hairpin probe has a stem of 18 base pairs
Conclusions
In summary, a versatile sensitive enzyme-free detection of DNA based on HCR and FRET was developed. Target DNA triggers the HCR process to form long nicked dsDNA nanowires and bring the donor and the acceptor fluorophores labelled on the hairpin probes into close proximity, resulting in a FRET process. The ratio of fluorescent intensity of the acceptor and donor was used to quantitatively detect target DNA with a limit of detection of 0.7 nmol L−1. This proposed method shows a promising
Acknowledgements
This work was financially supported by NSFC (21177023), Key Project of Chinese Ministry of Education (211084) and Science Foundation of Fujian Province of China (2013J01044).
Ying Chen obtained her bachelor degree from China Three Gorges University in 2013. Now she is a master degree candidate majoring in pharmaceutical analysis in Fuzhou University. Her current field of interest is chemical sensor and biosensor.
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Ying Chen obtained her bachelor degree from China Three Gorges University in 2013. Now she is a master degree candidate majoring in pharmaceutical analysis in Fuzhou University. Her current field of interest is chemical sensor and biosensor.
Ling Chen obtained her bachelor degree from Fujian Agriculture and Forestry University in 2015. Now she is a master degree candidate majoring in applied chemistry in Fuzhou University. Her current field of interest is chemical sensor and biosensor.
Yidian Ou obtained her bachelor degree from Fuzhou University in 2015.
Liangqia Guo obtained his doctor degree majoring in analytical chemistry from Fuzhou University in 2009. Now he is a professor in college of chemistry of Fuzhou University. His research interest includes chemical sensor and biosensor, food safety and environmental analysis.
Fengfu Fu obtained his doctor degree majoring in environmental chemistry from Tokyo University of Agriculture and Technology in 2002. Now he is a professor in college of chemistry of Fuzhou University. His research interest includes speciation analysis, chromatography, atomic spectroscopy biosensor and chemical sensor.