Mounting proteins on metal nanoparticles: Statistical analysis of AFM images Leonid Gurevich, Peter Fojan, R.K. Saxena, and Steffen B. Petersen* Nanobiotechnology Group, Institute of Physics and Nanotechnology, Aalborg University, Aalborg, Denmark *To whom the correspondence should be addressed: sp@nanobio.aau.dk ABSTRACT A reliable procedure for controlled mounting of proteins on gold nanoparticles has been developed. The procedure is exemplified using Glucose Oxidase (GOX) as a model protein. Complex characterization of protein-nanoparticle conjugates was performed using absorbance spectrometry, dynamic light scattering (DLS), activity assays combined with atomic force microscopy (AFM). Keywords: colloidal nanoparticles, protein, AFM, image analysis INTRODUCTION It has long been recognised that colloidal gold and silver nanoparticles can serve as versatile substrate for immobilisation of biomolecules. Presence of gold colloids gives the conjugates number of interesting properties not existing in proteins. We can roughly divide these new properties into “active” and “passive”. On one hand, nanoparticles can act as a solid support and anchor for an active component therefore immobilizing it a given location. Properly sized nanoparticles (50-100 nm) can be used to deliver and retain attached drug at a predefined location in the organism preventing its fast removal and exertion on the one hand and accumulation in liver, spleen and kidney on the other hand [1]. The same property makes nanoparticle-enzyme conjugates a promising choice in biocatalysis where it could reduce washing out of enzyme. On the other hand gold and silver nanoparticles can play an active role in sensing applications. As those particles support localised plasmon excitations they can interact with each other and incoming light and greatly enhance the electric field on their surface. This has recently lead to the whole range of new applications including colorimetric assays based on localized surface plasmons [2,3], amplification of surface plasmon resonance assays [4], surface enhanced fluorescence and Raman [4]. The effects of the geometry of the conjugates, the degree of aggregation as well as the influence of nanoparticle conjugate formation on biocatalytic activity are of paramount importance for functioning of the nanoparticle- protein conjugates and therefore attract large attention. In the present paper we report an effective and reliable procedure for controlled formation of gold nanoparticle – protein conjugates and discuss initial measurements on conjugate structure and activity. MATERIALS AND METHODS Materials. Glucose Oxidase, 16-Mercaptohexadecanoic acid (MHA), N-Hydroxysuccinimide (NHS), 1-ethyl-3-(3- dymethylaminopropyl)carbodiimide hydrochloride (EDC), Gold (III) chloride trihydrate (AuCl 4 H), trisodium citrate, D-sorbitol, D(+) Glucose, aceton, horseradish peroxidase 50000 units (POD), were purchased from Sigma-Aldrich. Polyoxyethylene (20) sorbitan monolaurate (TWEEN 20) was purchased from Fluka. Colloidal gold 30 nm solution was purchased from British Biocell. All materials were used without further purification. Nanoparticle preparation. Gold nano particles were prepared by dissolving 1mM Au(III) Cl in deionized water. 20ml of the Au solution was brought to boiling and 2ml of a 1% trisodium citrate solution was added. The solution was kept at boiling temperature until the color of the solution changed to a dark red-violet solution. The dark red-violet solution was taken off the heater plate and let to cool to room temperature. During the cooling the colour changed to a ruby red. This procedure yields gold anno particles of a narrow size distribution of around 20nm radius. Gold particle conjugation. Tween 20 was added to the stock solution of gold nanoparticles and left incubating on a shaker for 2 hours for physisorption of Tween 20 to the gold particles. Separately 20mM solution of MHA was activated with a mixture of 20ul of 10mM EDC and 20ul of 10mM NHS and left standing for 2 hours. After 2 hours the solution of activated MHA was diluted by stock solution of MHA to create a mixture of activated and non-activated molecules. Upon addition to gold nanoparticles/Tween 20 solution it displaces physisorbed Tween 20 and forms a mixed self-assembled monolayer. The solution is centrifugated, supernatant discarded and the pellet is resuspended in PBS buffer. GOX is introduced in the solution and left reacting for 2 hours. The mixture is centrifugated 3 times, after each cycle supernatant is discarded and the pellet resuspended in PBS. Substrate preparation. Silicon wafer was cut into small pieces, sonicated in acetone for 60 min, blow dried and exposed to 3% APTES solution in absolute ethanol for 60 minutes. Substrates were soaked in absolute ethanol for another 60 min, blow dried and baked on a hot plate at NSTI-Nanotech 2006, www.nsti.org, ISBN 0-9767985-7-3 Vol. 2, 2006 459