Elsevier

Biosensors and Bioelectronics

Volume 55, 15 May 2014, Pages 379-385
Biosensors and Bioelectronics

Novel plasmonic field-enhanced nanoassay for trace detection of proteins

https://doi.org/10.1016/j.bios.2013.11.073Get rights and content

Highlights

  • New gated fluorescence resonance energy transfer (GRET) method has been developed.

  • By modulating of plasmonic field-enhanced RET, high sensitivities are attained.

  • Cyt c and other proteins can be detected by GRET using 5 nm gold nanoparticles.

  • Low limit of detection of 370 pM Cyt c has been achieved for dye:Cyt c ratios up to 30:1 (over-labeling).

Abstract

Recently discovered effects of plasmonic field on molecular fluorescence offer new insights into the optical interactions at the nanoscale which can help solving problems encountered in widely applied fluorescent labeling of biomolecules for studying life processes in biomedicine and pharmacy. In this work, we have focused on exploring a novel sensitivity-enhancing phenomenon based on protein modulation of plasmon-controlled fluorescence. We have demonstrated that a protein (cytochrome c (Cytc c) or bovine serum albumin (BSA)) can be employed to gate fluorescence resonance energy transfer occurring from a fluorescein isothiocyanate fluorescent dye to plasmonic citrate-capped gold nanoparticles. By applying plasmonic field gated by protein, facilitated by the formation of multi-shell nanoparticles, (AuNP@Cit/Cytc-FITC or AuNP@Cit/BSA-FITC), low limits of detection for Cyt c (LOD=370 pM) and for BSA (LOD=1.8 nM) have been achieved even for large fluorophore:protein ratios, up to 30:1 (over-labeling), normally plagued with energy migration and background fluorescence problems. Control experiments confirming adsorption of proteins on AuNPs have been performed using light scattering and piezometric techniques. The proposed nanoassay may be applied in microanalysis of trace amounts of proteins, e.g. in microfluidic devices.

Introduction

Biomolecule conjugates with chromophores have been extensively utilized in studies of biochemical reactions and life processes. The utilization of such conjugates has enabled rapid development of fluorescence microscopy and fluorescence-based bioanalysis (Alam et al., 2013, Barczyk et al., 2005, Hepel and Stobiecka, 2011, Song et al., 2009, Stobiecka et al., 2012). Despite of broad applications of fluorescence microscopy, serious challenges concerning rigorous quantitative treatment remain (Hungerford et al., 2007). These challenges are due to under- and over-labeling, energy migration, and autofluorescence. In particular, the fluorescence resonance energy migration (REM) between fluorescent labels occurs when over-labeling (unintentional but common) is encountered, posing a problem in biomolecule quantitation.

There are several mechanisms leading to the diminished intensity of fluorescence signal. The best known (and widely used as an analytical tool itself) is the fluorescence resonance energy transfer (RET) occurring intramolecularly or during molecular collisions.

Recent discoveries of plasmonic enhancement of molecular fluorescence by Lakowicz (2006), Lakowicz et al. (2008) group and plasmon-induced spectral modulation for dyes (Grubisic et al., 2013, Taminiau et al., 2008, Zhao et al., 2011) provide novel venues for sensitive microanalysis, achieving single-molecule detection (Anger et al., 2006). Unfortunately, plasmonic enhancement of fluorescence is difficult to achieve in solution and is thus limited to specially prepared monolayer nanoparticle films (Lakowicz et al., 2008, Ray et al., 2009), unless plasmonic “hotspots” are created in solution by forming plasmonic nanostructures of nanoparticles (the “bowtie” effect (Hao and Schatz, 2004; Kinkhabwala et al., 2009)). Recently, D’Alfonso group (Sironi et al., 2010) has proposed a new method for suppression of background fluorescence appearing at longer excited state lifetimes by limiting the analysis to short lifetime fluorescence bursts found to be proportional to analyte concentration. This method may not be readily applicable in other systems though, since both components of the time-resolved emission decay may be analyte dependent or may be dependent on the under- and over-labeling.

In this work, we have focused on exploring new sensitivity-enhancing paradigm based on gating of plasmon-controlled fluorescence by protein, which enables reducing or eliminating the background fluorescence and overcoming the energy migration problem. The principle of this new method, called protein gated resonance energy transfer (gated RET, or: GRET), is illustrated in Scheme 1. The method is based on the strong plasmonic field enhancement of resonance energy transfer observed between a fluorescent probe (donor dye D) and an AuNP acceptor. A protein P adsorbing on the surface of AuNP reduces strongly the rate of RET, practically acting as a blocking agent. An efficient RET is then taking place only through spaces (voids) between adsorbed protein molecules creating the gates. Thus, the net efficiency of the RET depends on the gate area 1−θ, where θ is the partial surface coverage of AuNP's by protein molecules.

This paradigm enables sensitive detection of proteins, even at very high over-labeling with considerable REM loss. This effect is achieved by introducing plasmonic field to the medium and measuring the reduction in RET efficiency rather than directly the biomolecule or bioconjugate emission. We show that GRET offers the sensitivity enhancement of 2.4×103 with respect to RET in absence of plasmonic field for Cyt c and 4.7×102 for BSA. The high GRET enhancement is achieved in solution. We have investigated this new paradigm using RET gated by protein, with cytochrome c (Cyt c) or bovine serum albumin (BSA) as the gating protein, fluorescein isothiocyanate (FITC) as the chromophore (donor), and multi-shell gold nanoparticles (AuNP@Cit/Cytc or AuNP@Cit/BSA) as the acceptors.

Cytochrome c (Cyt c), used in this study as a model protein, is a small mobile protein (12,327 Da) participating in the electron transfer chain in mitochondria in living cells in all vertebrates, enabling efficient energy production. It plays also a well-established role as a mediator of apoptotic cell death (Barczyk et al., 2005, Platoshyn et al., 2002, Renz et al., 2001). The pre-apoptotic release of Cyt c from mitochondria in response to hypoxia is key process to understand the events in congestive heart failure and stroke. Another model protein used in this study, bovine serum albumin (BSA), as a common blood serum protein. While Cyt c is positively charged at neutral pH, BSA is negatively charged. Hence, these two model proteins can help evaluating the effects of different surface interactions in the GRET system.

In the paper, we describe first the quenching of FITC by an excess of Cyt c which is needed for later evaluation of the GRET enhancement, and show strong quenching of FITC fluorescence by gold nanoparticles. Then, we demonstrate for the first time the gating of RET in plasmon-controlled fluorescence of FITC by Cyt c in which an enhancement of RET sensitivity to Cyt c is observed and a limit of detection of 370 pM Cyt c by GRET is achieved. Control experiments confirming adsorption of proteins on AuNPs have also been performed using light scattering and piezometric techniques. The GRET experiments for BSA are described in SD.

Section snippets

Chemicals

Bovine heart cytochrome c (Cyt c) and fluorescein isothiocyanate (FITC) were obtained from Sigma-Aldrich Chemical Company (St. Louis, Missouri, USA). All chemicals used for investigations were of analytical grade purity. Other chemicals with their sources are listed in Supplementary Data (SD).

Apparatus

A Spectrometer model LS55 (Perkin Elmer, Waltham, MA, USA) was used for fluorimetric measurements. The RELS spectra were obtained at 90° angle from the incident (excitation) light beam, at 1% T

Direct energy transfer from a fluorescent dye to a biomolecule

Fig. 1 presents the characteristics of the FITC-Cyt c system for the case of high under-labeling. As seen, the addition of Cyt c to a solution of FITC leads to considerable quenching of FITC fluorescence. The quenching efficiency E is calculated from the dependence:E=1IFL/IFL,0where IFL,0 is the fluorescence emission intensity for donor (FITC) alone and IFL is the fluorescence emission intensity for donor in the presence of an acceptor (Cyt c). The plot of quenching efficiency E vs. CCyt c, is

Conclusions

In summary, we have demonstrated that the plasmonic quenching of FITC fluorescence by AuNP can be controlled by proteins, Cyt c and BSA. The control of plasmonic fluorescence quenching is achieved by forming multi-shell nanostructures (AuNP@Cit/Cytc-FITC or AuNP@Cit/BSA-FITC). Due to the strong acceleration of the FRET from FITC by plasmonic fields of AuNP, the quenching control by Cyt c or BSA is amplified and results in highly sensitive gating of the resonance energy transfer (GRET) from FITC

Acknowledgements

This research was partially supported by funding provided by the Program SONATA of the National Science Center, Grant no. DEC-2012/05/D/ST4/00320 and Grant Iuventus Plus, No. IP2012058072 awarded by the Ministry of Science and Higher Education.

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