Biotin-β-Cyclodextrin: A New Host-Guest System for the Immobilization of Biomolecules Michael Holzinger, Meenakshi Singh, and Serge Cosnier* Dé partement de Chimie Mole ́ culaire, UMR-5250, ICMG FR-2607, CNRS, Universite ́ Joseph Fourier BP 53, 38041 Grenoble Cé dex 9, France ABSTRACT: The formation of stable supramolecular interactions between biotin and β- cyclodextrin was studied. An association constant of 3 × 10 2 M -1 could be determined by NMR measurements by mapping the high field shift differences of the β-cyclodextrin protons (H-3) at different biotin concentrations. With the aim to demonstrate a new alternative for the immobilization of bioreceptors, biotin and β-cyclodextrin tagged biomolecules were immobilized on transducer surfaces, which were functionalized with the correspondent host- guest partner. The reliability of this new affinity system was investigated using two enzymes (glucose oxidase and polyphenol oxidase) as biomolecule models. This supramolecular inclusion complex shows clear advantages to the classic biotin-(strept)avidin-biotin system due to a detrimental effect of the additional avidin layer reducing the transduction efficiency. A 7-fold increase in the maximum current density and an almost 20 times higher sensitivity were exhibited by the immobilized biological layer obtained using this new host-guest system. ■ INTRODUCTION Cyclodextrins (CD) are cyclic oligosaccharides composed by usually more than six α-D-glucopyranoside units linked in 1,4- position. The most prominent examples are α-CD (6 units), β- CD (7 units), and γ-CD (8 units). This particular confirmation leads to a shallow truncated cone structure where the smaller rim is seamed with primary hydroxyl groups and the larger rim bears secondary hydroxyl groups. Against these highly hydro- philic rims, the “wall” of the cone is constituted by the six membered core ring of the glucopyranoside units containing C-H groups and glycosidic oxygen 1 that results in a more hydrophobic character. Due to this particular structural property, cyclodextrins became prominent for their capacity to form stable supramolecular complexes with a large variety of molecules. Cyclodextrins are primarily used for solubilization, masking of compounds, chromatography, and many more. Their biocompatibility and their nontoxicity led furthermore to applications in cosmetics and pharmaceutics. 2,3 In particular, β- cyclodexrin (β-CD) provides an ideal geometry for acting as host for small hydrophobic molecules such as adamantane or pyrene. 2,3 These moieties served for the efficient immobiliza- tion of β-CD tagged biomolecules for biosensing applications. Against hydrocarbons like adamantane and pyrene, biotin is the most used functionality to immobilize biotin tagged bioreceptor units due to its high affinity to form inclusion complexes with the glucoproteins avidin and streptavidin. 4 Even when biotin is considered as a water-soluble vitamin, only concentrations below 220 μg mL -1 were reported. 5 There are several examples where biotin and β-CD were combined in order to form, e.g., pseudo rotaxanes using biotin terminated PEG and fluorophore modifies β-CD. 6 Biotinylated β-CDs were synthesized to capture fluorophores inside the cyclo- dextrin unit immobilized via avidin bridges 7 and were also patented as skin cosmetics. 8 Furthermore, a bis(adamantyl)- biotin compound was used as cross-linker to detect the Fc fragment of a human immunoglobin G via streptavidin bridges immobilizing the biotinylated protein A as the receptor unit. 9,10 None of these studies evoked the possibility of an inclusion complex between biotin and β-CD. Still, such a host-guest system could represent a significant advantage where the sensor construction procedure steps can be reduced, but also could provoke interferences or parasite reactions. In this context, we constantly investigate and evaluate new immobilization techniques for bioreceptor molecules in order to improve our sensor setups and performances. In particular, immobilization via supramolecular interactions using biotin as anchor molecule has the advantage that biotinylated bioreceptors and biotin- modified surfaces are easily available and even, to a certain extent, commercialized. We recently reported that biotin coordinates with copper-NTA complexes that were allowed to omit the disadvantageous (strept)avidin layer for the biosensor construction. 11 Along this vein, we studied the supramolecular interaction between biotin and β-CD, repre- senting a new affinity system for bioreceptor immobilization. ■ EXPERIMENTAL SECTION Materials and Methods. Streptavidin, glucose, and biotin-labeled glucose oxidase (GOx, 120 Umg -1 ) were purchased from Sigma. TBAP (tetra-n-butyl ammonium perchlorate), acetonitrile, mono-, and dibasic phosphates were purchased from Sigma-Aldrich. All other reagents and chemicals products were purchased from Aldrich in reagent grade quality and used as received unless it is mentioned. Received: May 14, 2012 Revised: June 14, 2012 Published: August 3, 2012 Article pubs.acs.org/Langmuir © 2012 American Chemical Society 12569 dx.doi.org/10.1021/la301963v | Langmuir 2012, 28, 12569-12574