Immobilization of Candida bombicola Cells on Free-Standing Organic-Gold Nanoparticle Membranes and Their Use as Enzyme Sources in Biotransformations Sumant Phadtare, † Sachin Shah, ‡ Asmita Prabhune, ‡ Prakash P. Wadgaonkar, § and Murali Sastry* ,† Materials Chemistry, Biochemical Sciences, and Polymer Chemistry Divisions, National Chemical Laboratory, Pune 411008, India Preparation of chemically functionalized biocompatible surfaces is of current interest, with application in the immobilization of various bioactive species such as DNA, enzymes, whole cells, etc. We report herein the one-step synthesis of a self-supporting gold nanoparticle membrane, its surface modification, and application in the im- mobilization of Candida bombicola (yeast) cells. The gold nanoparticle membrane is prepared by the spontaneous reduction of aqueous chloroaurate ions by a diamine at a liquid-liquid interface. The gold nanoparticles in the polymeric membrane may be capped with octadecylamine (ODA) molecules, thereby rendering the nanoparticle membrane hydrophobic. Exposure of the hydrophobized organic-gold nanoparticle membrane to C. bombicola yeast cells results in their binding to the membrane, possibly through nonspecific interactions such as hydrophobic interactions between the yeast cell walls and the ODA molecules. The enzyme cytochrome P450 present in the yeast cells immobilized on the organic-gold nanoparticle membrane was then used in the transformation of the arachidonic acid (AA) to sophorolipids followed by acid hydrolysis to form 20-hydroxyeicosatetraneoic acid (20-HETE). The organic-gold nanoparticle membrane-C. bombicola bioconjugate could be easily separated from the reaction medium and reused a number of times. Introduction Impressive advances are being made in the synthesis of chemically functionalized and patterned biocompatible surfaces for the immobilization of biomolecules such as enzymes and whole cells of microorganisms such as bacteria and yeast (1-4). Such surfaces have important application wherein immobilized bacterial and fungal cells (genetically engineered and otherwise) may be used as “factories” for the production of industrially and medically important enzymes and metabolites (5). The repair or replacement of damaged tissues using in vitro strategies has focused on manipulation of the cell envi- ronment by modulation of cell-extracellular matrix interactions and cell-cell interactions. These methodolo- gies have potential applications in tissue engineering and implant biology (6, 7). Moreover, entrapment, immobili- zation, and spatial control over cell patterns on different surfaces represents an important tool for fundamental studies in cell biology (8, 9), biosensing (10, 11), and studies of cell interactions with different materials (12, 13). Control over cell shape and functions is generally achieved by the immobilization of the cells on spatially controlled (preferably on a submicron to micron scale) designed surfaces of varying “adsorptivity” of the biologi- cal components. Such patterned surfaces for immobiliza- tion of cells have been obtained using microcontact printing (μ-CP) on reactive (8), mixed self-assembled monolayers (SAMs) (14), SAMs made up of alkane- thiolates (15), alkylsilanes (16), by the sol-gel technique (17), nanometer controlled laser ablation (18), and using elastomeric membranes (19). Recently μ-CP of organic monolayers and subsequent polymer functionalization has been used to develop patterns in the seeding of bacterial cells (20). Patterned copolymers on chitosan substrates have been used to form two-dimensional patterns that limit cell attachment and spreading within the unprinted chitosan regions (21). In this laboratory, we have been interested in assembly of specific cells on surfaces from the point of view of using the cells as sources of enzymes for biotransformations and synthesis of new materials. Some of us have recently shown that Yarrowia lipolitica (22) and Candida bom- bicola yeast cells (23) can be immobilized on patterned hydrophobic regions of thermally evaporated fatty lipid films. In the latter case, the enzyme cytochrome P450 present in the yeast cells was used to catalyze in situ ω and ω-1 hydroxylation of arachidonic acid (AA). As part of our search for newer and more versatile materials with tailorable surfaces for cell immobilization, we describe herein the synthesis of a free-standing gold nanoparticle membrane whose surface may readily be modified to render it compatible for a variety of applications. More specifically, we demonstrate the formation of a self- supporting gold nanoparticle membrane at a liquid- liquid interface by the spontaneous reduction of aqueous * To whom correspondence should be addressed. Ph: +91 20 25893044. Fax: +91 20 25893952/25893044. E-mail: sastry@ ems.ncl.res.in. † Materials Chemistry Division. ‡ Biochemical Sciences Division. § Polymer Chemistry Division. 1817 Biotechnol. Prog. 2004, 20, 1817-1824 10.1021/bp049792h CCC: $27.50 © 2004 American Chemical Society and American Institute of Chemical Engineers Published on Web 10/22/2004