Enhanced Fluorescence of Proteins and Label-Free Bioassays Using Aluminum Nanostructures

Abstract

We report the enhanced intrinsic fluorescence from several proteins in proximity to aluminum nanostructured surfaces. Intrinsic fluorescence in proteins is dominated by the tryptophan residues. Intensities and lifetimes of several proteins with different numbers of tryptophan residues assembled on the surfaces of quartz or aluminum nanostructured films were measured. Immobilized protein molecules on the surface of an aluminum nanostructured film resulted in a significant fluorescence intensity enhancement (up to 14-fold) and lifetime decrease (up to 6-fold) compared to the quartz substrates. These large spectroscopic changes allow design of label-free bioassays where detection of binding interactions between proteins can be observed in the presence of a bulk sample solution. Binding of streptavidin to the biotinylated aluminum surface was demonstrated in the presence of 100 µg/mL bovine serum albumin in the sample solution by measurements of tryptophan intensity and lifetime changes.Fluorescence detection is a central technology in analytical chemistry, clinical chemistry, drug discovery, proteomics, genomics, and biochemical research. Almost without exception, fluorescence detection is accomplished using extrinsic fluorophores which are used to label the biomolecules. There is an increasing need for rapid and inexpensive detection of a large number of biomolecules. For example, protein arrays can contain tens or hundreds of spots. 1 -3 Testing of thousands of samples are performed for drug discovery using high throughput screening (HTS).4 , 5 Because of the added complexity of labeling on traditional fluorescencebased bioassays, there is a growing interest in optical methods which provide label-free detection (LFD),6 -8 such as surface plasmon resonance (SPR)9 , 10 or Raman scattering.11 , 12 Also, efforts are currently underway for the direct measurement of the native fluorescence of proteins to eliminate the problems of external tagging in many biological applications. [13][14][15] It is difficult to use intrinsic fluorescence of proteins for specific assays because almost all proteins display tryptophan emission. Additionally, there is typically a high background emission due to the UV absorption and emission wavelengths of 280 and 350 nm, respectively.During the past several years, there have been significant efforts in utilizing the metallic nanostructures or nanoparticles for improved detection of fluorescence.16 -22 This approach represents a fundamental change in fluorescence technology because the fluorophores can be excited by the near fields created by plasmons on the metallic structures, rather than freely propagating light. Additionally, the metallic structure can substantially modify the rates of spontaneous emission and the directionality of the emission. It has been shown that the fluorescence intensity of a number of probes can be increased by proximity to metal island films or nanoparticles. We referred to this phenomenon as metal-enhanced fluorescence © XXXX American Chemical Society ...

Surface‐Enhanced Fluorescence from Fluorophore‐Assembled Monolayers by Using Ag@SiO2 Nanoparticles

“…[26] Different nanostructure architectures have also been studied, including islands, [27] colloids, [28] nanotriangles, [29] nanorods, [30] nanoshells, [31,32] as well as fractal-like metalized surfaces, [9] thus greatly extending the breadth of the applications of SEF, which has been shown to be potentially useful in fields such as DNA detection, [8] RNA sensing, [33] immunoassays, [34] and label-free bioassays. [35] However, some potential problems still remain. First, the accuracy of the estimated enhancement factors of those metalized substrates compared with the nonmetalized control substrates is questionable because the increased quantity of attached fluorophores, due to the increased surface area, may also contribute to the measured enhancement.…”

Quantitative Comparison of Protein Surface Coverage on Glass Slides and Silver Island Films in Metal-Enhanced Fluorescence-based Biosensing Applications

“…In the current fluorescence-based biosensing applications, typically fluorophores with high-quantum yields are employed to increase the sensitivity of the bioassays, which results in high background emission and poor fluorophore photostability. The use of metal nanoparticles (such as silver [7], gold [8], copper [9], aluminum [10]) in these applications afford for the use of low-quantum yield fluorophores, which can withstand the prolonged exposure to excitation light for multiple measurements to be done. In this regard, numerous reports on MEF-based biosensing applications can be found in literature [11-15].…”

Strategies for the Development of Metallic‐Nanoparticle‐Based Label‐Free Biosensors and Their Biomedical Applications

“…Ray and co‐workers described the detection of proteins by using aluminium nanostructures . Nearly 99.5 % of human protein comprises at least one tryptophan, which constitutes a significant part of the fluorescence of a protein in UV region .…”

“…Ray and co-workersd escribed the detection of proteins by using aluminium nanostructures. [157] Nearly 99.5 %o fh uman protein comprises at least one tryptophan,w hichc onstitutes a significant part of the fluorescenceo faprotein in UV region. [158] These native fluorescenceintensities of ap rotein are very low for detection, so the MEF protocol has been followed to enhancet he fluorescencei ntensity.…”