Plasmonics-Based Detection of Virus Using Sialic Acid Functionalized Gold Nanoparticles
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Biosensor for the detection of virus was developed by utilizing plasmonic peak shift phenomenon of the gold nanoparticles and viral infection mechanism of hemagglutinin on virus and sialic acid on animal cells. The plasmonic peak of the colloidal gold nanoparticles changes with the aggregation of the particles due to the plasmonic interaction between nearby particles and the color of the colloidal nanoparticle solution changes from wine red to purple. Sialic acid reduced and stabilized colloidal gold nanoparticle aggregation is induced by the addition of viral particles in the solution due to the hemagglutinin-sialic acid interaction. In this work, sialic acid reduced and stabilized gold nanoparticles (d = 20.1 ± 1.8 nm) were synthesized by a simple one-pot, green method without chemically modifying sialic acid. The gold nanoparticles showed target-specific aggregation with viral particles via hemagglutinin-sialic acid binding. A linear correlation was observed between the change in optical density and dilution of chemically inactivated influenza B virus species. The detection limit of the virus dilution (hemagglutinination assay titer, 512) was shown to be 0.156 vol% and the upper limit of the linearity can be extended with the use of more sialic acid-gold nanoparticles.
Key wordsBiosensor Sialic acid Gold nanoparticle Viral detection Colorimetric measurement
The SPR band position relies strongly on the size and shape of individual nanoparticles and interparticle distance [1, 2]. Gold nanoparticles typically display an SPR band at around 520 nm. The assembly or aggregation of the metallic nanoparticles usually results in a red shift of the plasmonic band. This SPR band shift can be easily observed by naked eyes with the color of the colloidal gold solution changing from red to purple to blue. Kreibig et al. showed the relationship of SPR band position and interparticle distance decades ago . The interparticle distance-dependent SPR band shift is attributed to the electric dipole-dipole interactions and coupling between plasmons of the neighboring particles in the aggregates. When the interparticle distance is greater than the average particle diameter (dispersed state), the SPR band appears to be red, whereas the color turns to blue when the interparticle distance decreases to less than the average particle diameter (aggregated state).
This phenomenon has been well adopted in various detection schemes with specific targeting elements decorated on the nanoparticle surface for the targets of interest, such as polynucleotides [4, 5], enzymes and proteins [6, 7], cells , and heavy metals . In this chapter, we describe a method to develop the gold nanoparticles for the colorimetric detection of influenza virus based on the interaction between hemagglutinin, a protein expressed on the viral surface, and sialic acid, utilized as a surface stabilizing ligand on gold nanoparticles through a simple one-pot synthesis .
IKAMAG® Safety Control heating magnetic stirrer (IKA).
Eppendorf bench-top centrifuge (Model 5424).
Nanotrac particle size analyzer (Microtrac).
Ocean Optics USB 4000 UV-Vis spectrometer.
Millipore Milli-DI System.
2.2 Sialic Acid and Gelatin Gold Nanoparticle Synthesis
1.25 mM N-acetylneuraminic acid (SA) (Catalogue No. A2388, Sigma-Aldrich, St. Louis, MO) was prepared in DI water.
0.1 wt% gelatin (Catalogue No. 53028, Sigma-Aldrich, St. Louis, MO) was prepared in DI water.
1 M sodium hydroxide (Catalogue No. S5881, Sigma-Aldrich, St. Louis, MO) was prepared in DI water.
0.02 M chloroauric acid (HAuCl4) (Catalogue No. 254169, Sigma-Aldrich, St. Louis, MO) was prepared in DI water.
2.3 Influenza Virus Deactivation
Dulbecco’s Modified Eagle Media (DMEM) (Catalogue No. 11965, Life Technology, Grand Island, NY) was used for the culture of virus.
Fetal Bovine Serum (FBS) (Catalogue No. 16000-044, Life Technology, Grand Island, NY) was added to 10% in DMEM.
β-propiolactone (Catalogue No. P5648, Sigma-Aldrich, St. Louis, MO) was added to 0.05% of final concentration in the viral solution.
3.1 Sialic Acid Gold Nanoparticle (SA-AuNP) Synthesis
Ten milliliter of 1.25 mM sialic acid (SA) solution was prepared in DI water at room temperature. The SA solution was mixed with 250 μL of 0.02 M HAuCl4 followed by the addition of 50 μL 1 M NaOH in 20 mL glass vial (see Note 1 ).
The mixture was then stirred and heated for 80 °C at 20 × g for 15 min on a heating magnetic stirrer. The color of the solution changed from yellow to a dark red wine.
After the solution was cooled to room temperature, the gold nanoparticles were washed twice by centrifugation at 4500 × g) using a bench-top centrifuge (Eppendorf 5424) for 20 min. After the centrifugation, supernatant was removed and the dark red pellet was suspended in DI water and stored until further use.
3.2 Influenza Virus Inactivation
Virus solution was provided in 10% FBS containing DMEM and it was inactivated by the addition of β-propiolactone (final concentration of 0.05%) for 1 h at 37 °C.
Inactivated virus was stored at −20 °C until further use.
3.3 Particle Size Analysis
3.4 Determination of SA-AuNP Concentration
(N = number of atoms in a gold nanoparticle, ρ = density of fcc gold (19.3 g/cm3), r = radius of nanoparticle (10.05 nm), NA is Avogadro’s number, and M is gold atom’s atomic weight (196.97 g/mol).
The calculation result yielded ~250,000 gold atoms in 20.1 nm spherical-shaped gold nanoparticles. Since the initial concentration of HAuCl4 is 0.5 mM, the final SA-AuNP concentration is 2 nM when resuspended in the same volume of DI water.
3.5 UV-Vis Measurements
Five hundred microliter of the solution containing SA-AuNP with or without virus was added into the 1 cm path length quartz cuvette and placed in the cuvette holder of the UV-Vis spectrometer (Ocean Optics USB 4000 UV-Vis spectrometer). Then the UV-Vis spectrum was measured immediately. Results are shown in Fig. 3a, b.
3.6 Particle Size Analysis Measurements of SA-AuNP with Virus
Particle size of the SA-AuNP with virus was measured in the same way as described in Subheading 3.3. In short, the solution containing virus incubated SA-AuNP was placed in the built-in liquid sample holder of the particle size analyzer (Microtrac). The sample concentration was adjusted for the optimum result, as shown in Fig. 3d.
3.7 Colorimetric Detection of Viral Particles
Five hundred microliter of SA-AuNP solution was mixed with different dilutions of influenza virus solution and immediately added to a 1-cm path length quartz cuvette.
The cuvette was then placed in UV-Vis spectrometer (USB 4000, Ocean Optics) and absorption spectra were measured every minute until a plateau was reached.
3.8 Increasing the Linear Dynamic Range of Virus Measurements
In conclusion, we demonstrate a simple method to synthesize gold nanoparticles using sialic acid as both the reducing agent and the stabilizing agent. Experimental results support the hypothesis that sialic acid molecules on SA-AuNP surface interact with viral envelope protein hemagglutinin, causing a colorimetric change of the SA-AuNP solution. The resulting SA-AuNPs can readily detect influenza B virus (HA titer of 512) diluted to 0.156 vol%. The method is effective for different influenza B lineages (Victoria and Yamagata) .
The amount of 1 M NaOH can be varied in the range of 20–100 μL to achieve the best results, as the local deionized water may have slightly different pH values.
P.Z. acknowledges support from the National Science Foundation (CBET-0931677/1065633). A.A.W. acknowledges National Institute of Health (R01AI089450 from NIAID) for support.
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