key: cord-1022980-86kwe2mw
authors: Abad-Valle, Patricia; Fernández-Abedul, María Teresa; Costa-García, Agustín
title: Procedure 36 Genosensor on gold thin-films with enzymatic electrochemical detection of a SARS virus sequence
date: 2007-12-31
journal: Comprehensive Analytical Chemistry
DOI: 10.1016/s0166-526x(06)49079-6
sha: a904e453d953be5cdedcbc1afd76c3184bf16245
doc_id: 1022980
cord_uid: 86kwe2mw

Publisher Summary This chapter presents a procedure for the construction of a hybridization-based genosensor for a SARS (severe acute respiratory syndrome) virus sequence on a 100nm sputtered gold film, which works as immobilization and transduction surface. The chapter tests the sensitivity and the selectivity of the SARS genosensor using complementary strands of SARS virus and three-base mismatch strands. Genosensor construction include following steps: a drop of 5 mL of 1.02 mM thiolated probe deposited on the gold film and maintain at 37°C for 20 min or at 41°C for 12 h; it is cleaned with 0.1M Tris-HCl buffer; further a15 mL drop of a 2% 1-hexanethiol solution is deposited on the gold film and maintained for 10 min and again cleaned with a 2×SSC buffer solution pH 7. From the results of hybridization assay and recording of the analytical signal no significant difference found between the analytical signal obtained for a 3.03nM solution of the complementary target strand and three-base mismatch strand. The limit of detection, calculated as the concentration corresponding to a signal which is three times the standard deviation of the intercept, results to be 5 pM. This means an improvement of various orders of magnitude when compared with limits of detection reported in the bibliography for DNA assays.

Genosensor on gold thin-films with enzymatic electrochemical detection of a SARS virus sequence Patricia Abad-Valle, María Teresa Fernández-Abedul and Agustín Costa-García 36.1 OBJECTIVES (a) To construct a hybridisation-based genosensor for a SARS (severe acute respiratory syndrome) virus sequence on a 100 nm sputtered gold film which works as immobilisation and transduction surface. (b) To test the sensitivity and the selectivity of the SARS genosensor using complementary strands of SARS virus and three-base mismatch strands.

A target sequence corresponds to a portion of SARS virus, bases exactly comprise between 29218 and 29247, both included. A three-base mismatch strand with mismatches located in bases number 5, 15 and 26. Both strands are biotinylated at the 3 0 end for allowing hybridisation detection. Ethanol, magnesium chloride, sodium citrate, Trizma base, sodium chloride, EDTA, formamide, hydrochloric and sulphuric (95-97%) acids are of analytical grade. Water is purified employing a Milli-Q plus 185 equip from Millipore.

A 0.125-mm-thick polyimide substrate named Kapton HN s (Goodfellow), an epoxy resin (CW2400) obtained from RS Components and self-adhesive washers of 5 mm internal diameter (19.6 mm 2 of internal area) are used to construct the working electrodes.

Measurements are performed with an Autolab PGSTAT 10 (Eco-Chemie) potentiostat interfaced to an ADL Pentium 120 computer system and controlled by Autolab GPES software version 4.8 for Windows 98.

A Crison Micro-pH 2001 pH-meter and a Sanyo (MIR-162) incubator are also used.

An Emitech sputter coater model K550 is used to deposit the gold thin film working electrode.

The three-electrode potentiostatic system is schematised in Fig. 36.1 . Working electrodes are made on 5 Â 5 cm 2 supports of 0.125-mm-thick polyimide substrate named Kapton HN s (Goodfellow). The kapton slide is cleaned with ethanol and after drying, it is covered with gold by a sputtering process using an Emitech sputter coater model K550. Gold atoms are deposited (from the cathode) over kapton (placed on the anode) in a vacuum chamber filled with argon. Gold layer thickness is controlled by the time and the intensity of the discharge. For a 100-nm-thick layer a 35 mA discharge is applied over 220 s. After that, a conductor wire is attached to the centre of one of the sides by means of an epoxy resin (CW2400) obtained from RS Components, that is cured at room temperature.

The working area is limited by self-adhesive washers of 5 mm internal diameter (19.6 mm 2 of internal area). The total area of the gold surface lets to stick 23 washers approximately. The gold film is placed on a support where a crocodile connection is fixed.

Reference and auxiliary electrodes are coupled in a micropipette tip. The reference electrode consists of an anodised silver wire introduced in a tip through a syringe rubber piston. The tip is filled with saturated KCl solution and contains a low-resistance liquid junction. The platinum wire that acts as auxiliary electrode is fixed with insulating tape. For measurement recording, the tip is fixed on an electrochemical cell Metrohm support allowing horizontal and vertical movement. 

The response curves for different concentrations of complementary target are shown in Fig. 36. 3. There is a notorious difference between the analytical signal obtained for a 3.03 nM solution of the complementary target strand and three-base mismatch strand. In stringent experimental conditions (2 Â SSC containing 50% formamide), the analytical signals are 47.61 and 3.96 mA, respectively, when hybridisation takes place for 1 h. Therefore, a 100% discrimination is achieved.

Repeatability is checked under these experimental conditions, 1 h of hybridisation and a 2 Â SSC buffer containing 50% of formamide. The value of the RSD was 11% for nine measurements. Genosensor on gold thin-films e255 magnitude when compared with limits of detection reported in the bibliography for DNA assays that detect a-naphthol on thick-film gold electrodes 0.25 nM for Carpini et al. [2] and 0.22 nM for Xu et al. [3] . 146 nM is the limit of detection obtained for an AP-based DNA assay that employs p-aminophenylphosphate as substrate [4] . Specific quantification of 1 nM is achieved with other enzymatic schemes [5] . A limit of detection of 0.20 nM [6] and 30 nM [7] are achieved for indirect detection using an electrochemical indicator and for direct electrochemical detection respectively.

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