Quantum dot-based FRET for sensitive determination of hydrogen peroxide and glucose using tyramide reaction
Highlights
► A strategy is developed for detection of H2O2 and glucose using QD-based FRET. ► The principle of FRET is based on highly sensitive tyramide reaction. ► Emissions of energy donors and acceptors both appeared on the spectra. ► This method is successfully used for determination of glucose levels in human sera. ► This method is 1 order of magnitude more sensitive than in the commercially method.
Introduction
Recently, hydrogen peroxide (H2O2) has attracted growing attentions in biochemical fields owing to its important role in biological systems. H2O2 is an important intermediate product of pathological processes in various diseases, such as cancer, cardiovascular disorders, and Alzheimer's disease [1]. It is of great importance to develop a simple and highly sensitive method for the determination of H2O2. Many methods are now available for the assay of H2O2 in biological samples including spectrophotometry, fluorometry, chemiluminescence and electrochemistry [2], [3], [4], [5]. Among these methods, certain new probes [5], [6], [7], [8] and nanoparticles [9], [10], [11] have been widely used for the assay of H2O2 because of their simplicity and high sensitivity. Fluorescence resonance energy transfer (FRET)-based approaches for the ratiometric fluorescence detections of H2O2 have been reported recently [12], [13], [14], while the synthesis of ratiometric fluorescence reporter is needed and the detection limit is usually about μmol/L level [13].
FRET is a powerful technique for probing very small changes in the distance between donor and acceptor fluorophores. In recent years, QDs have been favorably adopted in the FRET-based studies due to large Stokes shift, high quantum yield, good photo stability, and size-dependent emission-wavelength tunability. QDs are widely applied to FRET for probing DNA replication and telomerization [15], [16], pH and ion sensing [17], [18], photochromic switching [19], photodynamic medical therapy [20], [21], sensing enzymatic activity [22], [23], and single molecule fluorescence energy transfer [24].
Recently, we reported a new strategy for highly sensitive determination of H2O2 based on FRET using gold nanoparticles and tyramide reaction [25]. In the AuNPs-based FRET modes, only the FL emission of the donors appeared on the spectra. Since the acceptors have no emission, applications of these methods were limited in monitoring molecular interactions and measuring the conformational changes of biomolecules. In this study, we present a new FRET system for the high sensitive assay of H2O2 and glucose, and in this case the emissions of energy donors and acceptors both appeared on the spectra. The 605 QDs were used as fluorescent donors and tyramide labeled Cy5 were used as acceptors. Usually, tyramide labeled with fluorescent probe is utilized as reporter fluorescent substrate for HRP-catalyzed deposition that is signal amplification technique in immunoassay [26], [27] and in situ hybridization of nucleic acids [28], [29]. In the labeling process, tyramide generates 2, 2’-dihydroxydiphenyl derivatives via tyramide radical in the HRP-catalyzed oxidation with H2O2 at the high concentration of tyramide. However, in the presence of lower tyramide concentration, tyramide radical binds to the electron-rich moieties of protein such as a tyrosine residue, to generate tyramide-labeled protein [30]. Upon excitation with a wavelength of 488 nm, FRET occurred between 605 QDs and Cy5 in the assemblies due to the tyramide reaction. The fluorescence signals of 605QD and Cy5 were observed simultaneously. The 605 QD is an excellent energy donor with Cy5 for several reasons: no cross-talk between the emission spectra of 605 QD and that of Cy5, no direct excitation of Cy5 at the wavelength of 488 nm and multiple Cy5 efficiently coupling to a single 605QD, and this couple had been used in many FRET-based studies [24], [31], [32], [33], [34].
Under the optimized conditions, the fluorescence signal of Cy5 depended linearly on the H2O2 concentration within a range from 10 to 100 nM and the detection limit of this assay was 10 nM. In addition, H2O2 is generated in the oxidation reaction catalyzed by almost all oxidases. So the activity of oxidases, or the enzyme substrates, such as glucose, lactate, glutamate, urate, xanthine, choline, cholesterol and NADPH can be quantitatively assayed by the determination of the H2O2 concentration produced in the system. By coupling with glucose oxidase-mediated reaction, the present method was applicable to selective assay of glucose. The method was successfully used for determination of glucose levels in human sera, and the results obtained were in good agreement with commercially available methods.
Section snippets
Chemicals and materials
QDs were obtained from Invitrogen (USA). Tyramide signal amplification (TSA) Plus Cy5 Kit was obtained from Perkin–Elmer (USA). Glucose was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glucose oxidase (GOx) was from BBI (UK). HRP, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and all other reagents were acquired from Sigma–Aldrich Chemical Co. (Milwaukee, USA). A glucose assay kit (hexokinase method) was purchased from Shanghai Kehua Bio-engineering Co., Ltd.
Principle of detection for H2O2
In this study, the design for determination of H2O2 is based on FRET principle. As shown in Scheme 1, in the design of FRET system, we choose HRP to catalyze the deposition of a tyramide labeled Cy5 amplification reagent onto QD surfaces that have been previously conjugated with HRP. This reaction is quick (usually 3–7 min) and results in the deposition of numerous Cy5 labels immediately adjacent to the immobilized HRP enzyme. Because the added labels are deposited proximal to the initial
Conclusion
In summary, we described a new method for the determination of hydrogen peroxide and glucose based on FRET system using 605 QD as energy donor and Cy5 as energy acceptor, and demonstrated an efficient FRET between HRP conjugated 605 QDs (as energy donors) and Tyramide labeled Cy5 (as energy acceptors) based on the HRP-catalyzed deposition, tyramide radical binds to an electron-rich moieties of HRP such as a tyrosine residue. The tyramide reaction initiated the conjugation between the HRP
Acknowledgments
This work was financially supported by the NSFC (20705019, 21075081 and 21135004), the National Basic Research Program of China (2009CB930400), and the Nano-Science Foundation of Shanghai (1052nm04000).
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