Analytica Chimica Acta 481 (2003) 55–64 Detection of -thalassemia by a DNA piezoelectric biosensor coupled with polymerase chain reaction Maria Minunni a , Sara Tombelli a,∗ , Rosalia Scielzi a , Ilaria Mannelli a , Marco Mascini a , Carlo Gaudiano b a Dipartimento di Chimica, Polo Scientifico, Università degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy b Centro per la Lotta alle Microcitemie, ASL no. 4, Ospedale Madonna delle Grazie, 75100 Matera, Italy Received 4 September 2002; received in revised form 16 December 2002; accepted 14 January 2003 Abstract -Thalassemia is an inherited disorder mainly caused by mutations in the gene of the -globin chain of adult haemoglobin (HbA). Clinically, -thalassemia can be a mild or silent condition, or it can cause severe diseases, leading to transfusion dependence. Studies at the gene level have identified a large number of variations in the -globin gene in different populations. In the Mediterranean area one of the most common mutation is the C → T substitution in the codon 39 of the gene. A new procedure for detecting codon 39 mutation in the -globin gene is reported, based on a DNA piezoelectric biosensor. An oligonucletidic probe (25-mer), specific for the region around codon 39, is immobilised on the gold surface of a piezoelectric quartz crystal. The hybridisation between the immobilised probe and the complementary strand in solution is detected recording the variations of the crystal frequency. Experiments with synthetic oligonucleotides were initially performed. Distinguishable frequency shifts were obtained from the interaction between the immobilised probe and the complementary and the mismatch oligonucleotides. A solution containing 50% of both the oligonucleotides has been also tested and distinguished from the others evaluating the resulting signals. Experiments with non-complementary oligonucleotides gave no signal variation. The biosensor was able to distinguish between sequences differing in only one base also using polymerase chain reaction-amplified samples [771 base pairs (bp)] of DNA extracted from human blood of thalassemic and healthy (normal) patients or patients with -thalassemia traits. The optimised DNA piezoelectric biosensor has been successfully applied to the determination of one of the most frequent mutation characteristic of -thalassemia in the Mediterranean population. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Piezoelectric biosensor; -Thalassemia; Mutation; PCR 1. Introduction Nucleic acid-based biosensors represent a promis- ing tool for gene sequence analysis and for muta- tion detection [1]. Building of such devices involves ∗ Corresponding author. Tel.: +39-0554573323; fax: +39-0554573384. E-mail address: sara.tombelli@unifi.it (S. Tombelli). the immobilisation of single-stranded oligonucleotides (probe) on the surface of the transducing element and recording the variations of the transducer signal caused by the hybridisation between the probe and the com- plementary strand in solution (target). Several types of nucleic acid-based biosensors have been developed over recent years [1–5]. In particular, piezoelectric biosensors offer the possibility of monitoring the hy- bridisation reaction in real time and without the use 0003-2670/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0003-2670(03)00065-5