Sensitive detection of adenosine triphosphate by exonuclease III-assisted cyclic amplification coupled with surface plasmon resonance enhanced fluorescence based on nanopore
Introduction
Adenosine 5′-triphosphate (ATP) is the major energy currency of cells, playing a critical role in the regulation of cellular metabolism and biochemical pathways in cell physiology [1], [2]. Additionally, intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds [3]. The sensitive and feasible detection of ATP is important for biological study and drug design as well as clinical diagnosis.
The traditional method for ATP detection is luciferase bioluminescence assay involving the measurement of light emitted by the luciferase due to ATP splitting, which suffers from the use of costly and unstable bioluminescence agents [4]. Recently, several novel strategies for ATP detection based on the ATP-dependent ligation reaction [5], various enzymatic reactions [6] and aptamer-based methods [7] are developed. In particular, the aptamer-based biosensing has attracted much attention due to the high binding affinity and specificity. A number of detection techniques such as fluorescence [8], electrochemistry [9], electrochemiluminescence [10], chemiluminescence resonance energy transfer (CRET) [11], fluorescence resonance energy transfer (FRET) [12] have been demonstrated. Signal amplification strategies are often combined with the biosensor to improve detection sensitivity [13]. We have reported a surface plasmon resonance (SPR) detection system based on a hybridization chain reaction (HCR) for amplified detection of ATP with high sensitivity [14]. Very recently, a kind of sensitive methods for ATP detection based on exonuclease-induced amplification reaction were presented [15], [16], which did not require a specific recognition site. These exonuclease-based methods were both performed with fluorescent detection.
Mesoporous silica nanoparticle (MSN) has been a useful material due to its nontoxic nature, high surface area, tunable pore size and chemically modifiable surface, which has been used as a promising carrier system for drug delivery [17] and imaging materials [18]. MSN has been applied in bioanalysis [19], [20]. In all the analysis systems, the fluorescence signal was quenched by encapsulation of fluorescence molecules in the pores. The fluorescence enhancement by MSN for bioanalysis has not been demonstrated yet.
It is now well known that plasmonic excitations in metallic nanostructures allow concentrating optical fields within deep-subwavelength volumes and are able to dramatically enhance molecular fluorescence intensities [21], [22]. Plasmonic nanoantennas could boost the brightness of fluorophores by concentrating the electromagnetic fields into sub-diffraction limited volumes, which even led to single-molecule detection [23], [24]. For example, circular holes of 50- to 200-nm diameter in a metal cladding film deposited on a transparent substrate enable monitoring of enzymatic reactions at high substrate concentration by reducing the observation volumes [25]. Therefore such nanostructures are promising for trace molecular detection and disease diagnoses due to the significant improved quantum efficiency of the probes. Inspired by these discoveries, we developed a sensitive ATP detection strategy based on Exo III-assisted cyclic amplification coupling with surface plasmon resonance enhanced fluorescence localized on nanopore.
Section snippets
Apparatus
Fluorescence emission spectra were recorded on an F-4500 FL spectrophotometer (Tokyo, Japan) equipped with 1 cm quartz cells. The excitation was made at 480 nm with recording emission range of 500–600 nm. All excitation slits were set at 5 nm and emission slits were set at 10 nm. UV/Vis absorption spectra were obtained with a Cary 50 Series Spectrophotometer (Varian, Australia). The sizes of the AuNPs were verified by transmission electron micrograph (TEM) using a JEOL JEM-2100EX microscope (Japan).
Chemicals
Principle of the sensing strategy
The configuration and operation principle of the sensing strategy was depicted in Scheme 1. AuNP@SiO2 mesoporous silica core–shell nanoparticles were synthesized on the basis of the amino acid derived chiral surfactants as the template molecules for shell growth previously reported [26]. There are numbers of pores in the Si shell, pass through which thiolated S3 sequence was immobilized on the AuNP core. The S1 contained an ATP aptamer fragment and was labeled with Black Hole Quencher (BHQ1) at
Conclusion
In summary, we have developed an amplified fluorescence platform for the detection of ATP by coupling of Exo III-assisted cyclic amplification and nanopore-based fluorescence enhancement. The fluorescent intensities were dramatically enhanced by the enhanced localized plasmon field existing in the nanovoid regions and reduced effective observation volumes in the nanopores. Together with the Exo III-assisted target cycling, the present strategy exhibited high sensitivity towards the ATP
Acknowledgments
This work was supported by the National Natural Science Foundation of China (21275083 and 21545004) and the Scientific and Technical Development Project of Linyi (201312023).
Xuemei Li is a professor of Chemistry, School of Chemistry and Chemical Engineering, Linyi University. Her research interests cover biosensor and biochemical analysis.
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Cited by (0)
Xuemei Li is a professor of Chemistry, School of Chemistry and Chemical Engineering, Linyi University. Her research interests cover biosensor and biochemical analysis.
Yan Wang studies in College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology as postgraduate student.
Jie Luo studies in College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology as postgraduate student.
Shiyun Ai is a professor of Chemistry, College of Chemistry and Material Science, Shandong Agricultural University. His research interests cover biosensor and nanomaterials.