Piezoelectric Sensors

Analytical Applications of QCM-based Nucleic Acid Biosensors (p. 211-212)

Abstract Recent advances in nucleic acid-based detection coupled to piezoelectric transduction will be reported here. The main aspects involved in the development of nucleic acid sensors are considered: the immobilization of the probe, the sample pretreatments (DNA extraction, amplification, denaturation of the amplified material), the sensitivity and specificity, etc.

These systems have been applied to different fields from environmental analysis to clinical diagnostics. Examples taken from different analytical problems will be reported. Another nucleic acid sensor, also based on the affinity between the analyte and the receptor immobilized on the surface, is reported as an example of the most recent trend in the field. This receptor, called aptamer, acts as capturing receptor for a molecule in solution, such as a protein. An aptasensor developed for a specific protein will be reported.

Keywords Biosensor · QCM · Nucleic Acids · Hybridization · Aptamers

1 Introduction

The first report on the direct detection of nucleic acid interactions based on the use of acoustic wave devices was provided by Fawcett et al. in 1988 [1]. They described a quartz crystal microbalance (QCM)-based biosensor for DNA detection by immobilizing single-stranded DNA onto quartz crystals and detecting the mass changes after hybridization. Since this early work, a number of articles have appeared employing similar procedures, resulting in microgravimetricmeasurements based on nucleic acids [2-5].

In general, when dealing with biosensing for this kind of application a few considerations are mandatory. First, the system should be specific and sensitive enough for the required application and should provide reproducible results. The system should have short analysis time and easy formats. Moreover, the sample pretreatment should be as little as possible, ideally absent. In this chapter, the main aspects related to piezoelectric sensing based on nucleic acids are considered and examples taken from different fields of application, from environmental analysis to clinical diagnostics, will be considered. A nucleic acid-based sensor consists of a nucleic acid immobilized on the transduction surface, which interacts with the analyte free in solution. Depending on the immobilized nucleic acid sequence and the relative interaction that drives the binding, different sensors can be distinguished. One based on the detection of the hybridization reaction can be developed by immobilizing a nucleic acid probe on the surface of the sensor to recognize the complementary sequence present in the sample solution. The hybridization reaction between the probe and the target sequence drives the biospecific interaction, which results in the formation of a double helix complex on the sensing surface with the detection of the specific sequence of interest. The interaction is based on the specific recognition between the two single-stranded DNA filaments leading to the formation of the DNA helix. The hydrogen bonds stabilize selectively the couples adenine-thymine (AT) and guanine- cytosine (GC).

In QCM-based sensing, the recognition is displaced as ameasurable shift in the resonant frequency. Ideally, the frequency shift is maximum with the fully complementary sequence and it is absent with non-complementary DNA. A typical profile of a QCM-based DNA sensor signal is reported in Fig. 1, where the addition of complementary sequences leads to the formation of the double helix and causes an increase in the mass loading at the sensor surface, recorded as a decrease in the frequency shift of the crystal. With QCM-based sensing it is then possible to detect, in real time and without the use of any label, specific target sequences characteristic of the DNA of interest as well as point mutations. This kind of QCM DNA sensor has been applied by this research group to the analysis of many different target sequences like genetically modified organisms (GMOs) [2] and bacteria detection [6, 7] in the field of food and environmental analysis, respectively. In clinical diagnostics application, the sensor has been used for the detection of point mutations such as the mutation occurring in the p53 oncogene, related to cancer research, diagnosis and prognosis [8].