Enzyme-free detection of DNA based on hybridization chain reaction amplification and fluorescence resonance energy transfer

Abstract

A versatile enzyme-free deoxyribonucleic acid (DNA) detection method based on hybridization chain reaction (HCR) and fluorescence resonance energy transfer (FRET) was developed. Hairpin H1 and H2 are labelled with carboxyfluorescein (FAM) as the donor and tetramethylrhodamine (TAMRA) as the acceptor, respectively. In the presence of target DNA, long nicked dsDNA nanowires are self-assembled through HCR process. The donor and the acceptor are brought in close proximity, resulting in a FRET process between them. The ratio of fluorescent intensities of the acceptor and donor can be used to quantitatively detect the target DNA with a limit of detection 0.7 nmol L⁻¹. This proposed method was applied to detect DNA in biologic samples with satisfactory results.

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.