208 Sensors and Actuators B, 6 (1992) 208-210 Langmuir-Blodgett film based membrane for DNA-probe biosensor M. A. Karymov, A. A. Kruchinin, Yu. A. Tarantov, I. A. Balova, L. A. Remisova, N. G. Sukhodolov and A. I. Yanklovich Department of Chemistry, St. Petersburg University, Peterhof, St. Petersburg 198904 (Russia) A. M. Yorkin Department of Genetics, St. Petersburg University, Peterhof, St. Petersburg 198904 (Russia) Abstract Covalent attachment of DNA to diacetylene film produced on the surface of oxidized silicon by the Langmuir- Blodgett technique has been studied. Coupling of phage M13 DNA with polymer film was carried out by the carbodiimide method and by the formation of linker structures. Sorption capacity and hybridization efficiency determined using the radioactive label 32p were about 10-20 pg mm -2 and near 100%, respectively. 1. Introduction There is a growing interest in the development of microelectronic sensors for detecting biological substrata and antigens; the DNA-probe based sen- sor is one of them [ 1]. This sensor can be used for DNA detection in express diagnostics of gene deaths and for specific bacterial gene (e.g. toxin genes) disclosure. It should comprise a solid-state transducer and a membrane with an immobilized DNA-probe, the DNA having a specific nucleotide sequence. Hybridization of a DNA-probe with analyzed DNA will lead to a change of mass, electrical charge or optical properties of the mem- brane to be detected by gravimetric, potentiomet- ric or optical transducers. Methods for the membrane deposition on the surface of the solid substrate should be compatible with microelec- tronic technology, as well as allowing DNA immo- bilization and hybridizatio n . It must also be mechanically and electrically stable. In this work a membrane for a DNA-probe biosensor based on Langmuir-Blodgett (LB) di- acetylene film with covalently bonded DNA has been developed. 2. Experimental Replicative and single-stranded forms of bacte- riophage M 13 DNA with 6400 nucleotides pre- pared by the routine method [2] were used in experiments. The replicative form was labelled by nick translation with [0~-32p] dNTP and had a specific activity 3 x 109 cpm Ixg -1. An LS Beckman 100C counter was used to determine the DNA sorption capacity of the diacetylene film and im- mobilized DNA hybridization efficiency. Diacetylene films were produced on the surface of oxidized silicon wafers by the conventional LB technique [3]. For this purpose the amphiphilic diacetylene acids, 7,9-dokozadiynic CI2H25C=--C- C-C-(CH2)sCOOH (C22 acid) and 3,5-octa- decadiynic C~0H21C-C-C-C-(CH2)3COOH (C18 acid), were synthesized according to ref. 4, with prototropic isomerization of diacetylene hydro- carbons by lithium 2-aminoethylamide as a princi- pal stage. The formed terminal isomer was alkylated by to-Br alkanols with a covered hydroxyl group. Using this method it was possible to obtain amphiphilic diacetylene acids with a fixed number of methylene groups between triple bonds and a functional group from accessible initial reagents by three stages of synthesis in one flask. Polished silicon wafers were thermally oxidized at 1100 °C in dry oxygen. The silicon dioxide film thickness was 100 nm. The hydrophobization pro- cedure included treatment in a 10% solution of dimethyldichlorosilane in chloroform (20min at room temperature) and subsequent annealing for 2 h at 180 °C in air. Previously hydrophobization samples were boiled for 8 h in distilled water. 0925-4005/92/$5.00 © 1992 - - Elsevier Sequoia. All rights reserved