Quantitation of polynucleotide hybridization detected by piezoelectric resonance

Robert Brooke Towery

Abstract

In recent years, significant time and energy has been focused on research, design, development, and application of analytical biosensors. The present research refines use of the quartz crystal microbalance (QCM) as a hybridization detector and expands the technique to quantitative detection. In this work, the QCM is used to detect hybridization of nucleic acids isolated from both the plant and animal kingdom as well as synthetic nucleic acids. The QCM technique has several inherent advantages over other hybridization detection methods. The primary advantage is that no labels are needed so that the problems associated with radioactive and other labels are avoided. Another important advantage is that a quantitative signal is obtained. Since the method is label-less, either the target or probe may be immobilized on the sensor. Immobilizing the probe rather than the target, as in conventional membrane hybridizations, has practical advantages. It is also anticipated that the method will be lower in cost and much more rapid than other hybrid detection methods. In this research, the frequency of the QCM is shown to be a negative linear function of the mass of dry nucleic acid target captured to the point of probe saturation. When a nucleic acid probe is immobilized on the surface of a QCM, and hybridized with target, both the amount of probe immobilized and the amount of target captured are easily and accurately quantitated. A value for the sensitivity and detection limit in detecting hybridization is established. Capture of radiolabeled target on the QCM is used to evaluate the accuracy of the QCM signal. The binding ratio of poly (A) to poly (U) in solution under conditions identical to those used in piezoelectric detection is investigated. 0Immobilized nucleic acids attached to the QCM via a carbodiimide coupling reaction are shown to attach at multiple sites. The existence of inactivated nucleic loops is demonstrated and an enzyme, RNase, is used to cut these loops, generating more free nucleic acid ends and greater target capture capacity. It is shown that the second order rate constant for hybridization of sheared, genomic DNA is the same for homogenous hybridization in solution and heterogeneous hybridization on the QCM. In solution a second order rate constant of 2.31 ± 0.09 × 10 -6 ml μg -1 sec-1 was measured while on the QCM the constant was 2.2 ± 0.3 × 10-6 ml ug -1 sec-1 . It is shown that the high hybridization rate obtained on the QCM can be explained solely on the basis of high reactant concentrations. It is demonstrated that hybridizations heretofore requiring many hours, sometimes days, can be completed in less than an hour when carried out in a thin liquid film at high concentrations. A nearly forty-fold decrease in the half reaction time is realized relative to that for hybridization in homogenous solution. The first application of a QCM hybridization biosensor to a practical problem is demonstrated. Using the QCM it is found that the percentage of chloroplast DNA in total soybean DNA is 31 ± 2% (n = 3). The result is shown to be in agreement with the value 27.0 ± 2.8% (n = 6) reported in the literature obtained using radiolabels and a more time intensive procedure.