Protein Quantification Using Resonance Energy Transfer between Donor Nanoparticles and Acceptor Quantum Dots
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- 作者：Harri H盲rm盲 ; Sari Pihlasalo ; Piotr J. Cywinski ; Piia Mikkonen ; Tommy Hammann ; Hans-Gerd L枚hmannsr枚ben ; Pekka H盲nninen
- 刊名：Analytical Chemistry
- 出版年：2013
- 出版时间：March 5, 2013
- 年：2013
- 卷：85
- 期：5
- 页码：2921-2926
- 全文大小：300K
- 年卷期：v.85,no.5(March 5, 2013)
- ISSN：1520-6882
文摘
A homogeneous time-resolved luminescence resonance energy transfer (TR-LRET) assay has been developed to quantify proteins. The competitive assay is based on resonance energy transfer (RET) between two luminescent nanosized particles. Polystyrene nanoparticles loaded with Eu3+ chelates (EuNPs) act as donors, while protein-coated quantum dots (QDs), either CdSe/ZnS emitting at 655 nm (QD655-strep) or CdSeTe/ZnS with emission wavelength at 705 nm (QD705-strep), are acceptors. In the absence of analyte protein, in our case bovine serum albumin (BSA), the protein-coated QDs bind nonspecifically to the EuNPs, leading to RET. In the presence of analyte proteins, the binding of the QDs to the EuNPs is prevented and the RET signal decreases. RET from the EuNPs to the QDs was confirmed and characterized with steady-state and time-resolved luminescence spectroscopy. In accordance with the F枚rster theory, the approximate average donor鈥揳cceptor distance is around 15 nm at RET efficiencies, equal to 15% for QD655 and 13% for QD705 acceptor, respectively. The limits of detection are below 10 ng of BSA with less than a 10% average coefficient of variation. The assay sensitivity is improved, when compared to the most sensitive commercial methods. The presented mix-and-measure method has potential to be implemented into routine protein quantification in biological laboratories.