High-sensitive bioactivity assay for hybrid nanostructured materials with plasmonic properties

J.L. Hernández-López

Centro de Investigación y Desarrollo Tecnológico en Electroquímica, S.C., Parque Tecnológico Querétaro S/N, Pedro Escobedo, Qro., C.P. 76703, México. jhernandez@cideteq.mx

Abstract- The binding of biotin to gold nanorods (Au-NRs) conjugated to neutravidin is accompanied by an increase in the fluorescence intensity of protein's tryptophan residues. Localized surface plasmon resonance (LSPR), combined with the known affinity of streptavidin for biotin, led to the development of a high-sensitive enzyme activity assay.

Keywords- Au-NRs/Neutravidin; LSPR; Enzyme Activity; In-vivo Imaging; Biological Sensing.

I. INTRODUCTION

Localized surface plasmon resonance (LSPR) has been a very ancient studied nanoscale phenomenon (Faraday, 1847; Mie, 1908). These resonances, associated with noble metal nanostructures create sharp spectral absorption and scattering peaks as well as strong electromagnetic near-field enhancements (Maier, 2007). By coupling the strong-SPR-generated evanescent field to a layer of fluorophores or biotin-binding molecules confined near the metallic surface, the fluorescence signal can be pickup by a sensitive detector. These effects can be understood by considering a Jablonski diagram that includes a metal-enhanced fluorescence (MEF) type mechanism (Lakowicz, 2006) (Fig. 1).

Figure 1. Jablonski diagram without (top) and with (bottom) the effects of near metal surfaces. E is the rate of excitation without metal. E_m is the additional excitation in the presence of metal (Lakowicz, 2001).

For simplicity we will only consider radiative decay (G) and non-radiative decay (k_nr). In the absence of metals the quantum yield and lifetimes are given by the following equations:

	(1)
	(2)

Since the radiative decay rate is nearly constant for any fluorophore the quantum yield can only be increased by decreasing the value of k_nr.

Now consider the effect of a metal. If the metal results in an increased rate of excitation (E+E_m) this will result in increased brightness without changing the quantum yield or lifetime. This is a useful effect that can allow decreased incident intensities and decreased background. Metal-enhanced excitation can also result in selective excitation of fluorophores near the metal. Another possible effect is an increase in the radiative decay rate. In this case the quantum yield and lifetime of the fluorophore near the metal surface are given by the following equations:

	(3)
	(4)

These equations result in unusual predictions for a fluorophore near a metal surface. As the value of Γ_m increases the quantum yield increases while the lifetime decreases.

The method presented in this work possesses a valuable biotechnological potential that can be addressed for studying specific biomolecular interactions (viz., biotin/streptavidin, antibody/antigen, sugar/lectin, DNA hybridization, analyte/receptor, etc.) via homogeneous assays, in-vivo imaging and biological sensing. However, in order to realize these endeavours, novel methods that enable to quantificate the bioactivity of the proteic part conjugated to the metallic nanostructure must be developed. Proteins possess three intrinsic fluorophores: phenylalanine, tyrosine and tryptophan. The emission from proteins is dominated by tryptophan because of its longer excitation and emission wavelengths, good quantum yield, and fluorescence resonance energy transfer (FRET) from tyrosine to the tryptophan residues (Lakowicz, 2006).

In this context, a first factor that dictated the choice of gold nanorods (Au-NRs) among others nanostructured materials, available commercially, was its remarkable capability for tuning the peak extinction wavelength towards the visible and near-infrared range. In the last decade, some authors (Weissleder, 2001; Hirsch et al., 2003) have pointed out that the background absorption and scattering of endogeneous chromophores from biological samples (e.g., whole blood and serum) and of water are minimal in these regions of the electromagnetic spectrum.

A second consideration was the use of neutravidin as a biotin-binding molecule. This protein plays a very important role because it contains 16 tryptophan residues and is relatively bright compared with other proteins.

Neutravidin has a mass of 60 kDa and is a deglycosylated form of avidin. As a result, lectin binding is reduced to undetectable levels, yet biotin-binding affinity (K_d = 10^{-15 M}) (Hiller et al., 1987) is retained because the carbohydrate and RYD sequence is not necessary for this activity. This protein offers the advantages of a neutral isoelectric point (pI = 6.3) to minimize nonspecific adsorption, along with lysine residues that remain available for derivatization or conjugation. By other side, it yields the lowest nonspecific binding among the known biotin binding proteins (Table 1).

Table 1. Physicochemical properties of biotin-binding proteins (Pierce, 2009).

*Av-avidin, Sav-streptavidin, Nav-neutravidin

In this technical note a method that exploits intrinsic fluorescence of streptavidin or derivatives for detection of specific binding reactions on metallic nanostructured materials is described.

II. METHODS

A. Reagents

Sodium phosphate buffer (Pierce), 0.15 M, pH 7.2; D-biotin standard (Sigma-Aldrich), ca. 20 μg/mL (80 μM); deionized water, type I (ρ = 18.2 MΩ⋅cm), the total organic (TOC) content was less than 10 ppb (according to the manufacturer); Ntheraphy in-vivo Gold Nanorods, neutravidin-polymer (Product Nr.: 30-PN-750, Nano-partz), 50 OD-mL (1.6 mg/mL).

B. Procedure

All solutions are prepared in 0.15 M sodium phosphate buffer at pH 7.2, ionic strength = 0.35. The fluorescence measurements described were performed with a Horiba-Jobin-Yvon spectrofluorometer (Model FluoroLog 3-22).

Calibration: Emission calibration check was verified accordingly using deionized water, type I, as indicated in the HJY's Operation Manual (2006).

All measurements were made at 25 °C. The excitation monochromator was set at 290 nm using a slit-width of 4 nm, and the fluorescence monitored after each addition of titrant at 350 nm with a slit-width of 12 nm.

C. Assay of enzymatic activity

D-Biotin standard solution. A standard solution of D-biotin is prepared by dissolving 5 mg of solid in 5.0 mL of buffer followed by dilution to 20 μg/mL.

Au-NRs/Neutravidin-polymer standard solution. A standard solution of Ntheraphy in-vivo Gold Nanorods, neutravidin-polymer is prepared by dissolving x μg of the product in 1.0 mL of buffer followed by dilution to ca. 67.5 μg-Au(0)/mL. Two hundred microliters of this solution are pippeted into a 250-μL fluorometer cell (b = 0.5 cm) and titrated with successive 2.0-μL aliquots of the standard 20-μg/mL D-biotin solution using an Eppendorf tip and micropipette (Eppendorf Research). Additions are made until no further increase in fluorescence is observed, indicating the attainment of the equivalence point (Fig. 2).

Figure 2. Typical titration of Au-NRs/Neutravidin-polymer with D-biotin. Approximately 75 μg of neutravidin in 200 μL (6.275 μM) of 0.15 M sodium phosphate buffer, pH 7.2, was titrated with 2.0-μL aliquots of a 80 μM (20 μg/mL) solution of D-biotin. Fluorescence was measured at 350 nm with excitation at 290 nm. Each neutravidin molecule is tetrameric and binds four molecules of biotin.

III. CONCLUSIONS

We have demonstrated one way to quantificate the enzymatic activity present in proteins conjugated to metallic nanostructured materials, fluorescently. As mentioned before, because the assay is based on a simple optical measurement, it is conceivable that robust, portable devices for point-of-care diagnostics (e.g., applied to the early detection of chronico-degenerative diseases) could be developed for the biological sensing of target molecules in complex matrices.

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Received: April 26, 2013.
Accepted: July 31, 2013.
Recommended by Subject Editor: María Luján Ferreira