Amplified detection of telomerase activity using electrochemical and quartz crystal microbalance measurements

Abstract

Telomerase is considered as an important biomarker for cancer cells. Two different methods for the amplified electrochemical and microgravimetric quartz-crystal-microbalance detection of telomerase activity originating from HeLa cancer cells are described. One method involves the telomerization of a primer (1) linked to the electrode, in the presence of telomerase from HeLa cell extract and dNTP, followed by the hybridization of a biotin-labeled nucleic acid (2) that is complementary to the telomere repeat units. The subsequent binding of an avidin-alkaline phosphatase conjugate (3) that catalyzes the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (4) results in the precipitation of the insoluble product (5) on the electrode. The second method involves the telomerization of the primer (1) associated with the electrode, in the presence of the telomerase-containing HeLa cell extract and the dNTP nucleotide mixture that includes biotin-labeled dUTP. The telomerization leads to the labeling of the telomeres with biotin labels. The association of the avidin-alkaline phosphatase conjugate (3) to the biotin labels results in the biocatalyzed transformation of (4) to (5) and the formation of a precipitate on the electrode or the Au-quartz crystal. As numerous precipitate molecules are formed as a result of the formation of a single telomere, the methods represent routes for the amplified detection of telomerase activity. The formation of the precipitate on the respective transducers is probed by following the changes in the electrode resistance using chronopotentiometry, or by following the frequency changes of the piezoelectric quartz crystals. The amount of precipitate generated on the electrodes is controlled by the concentration of the HeLa cancer cells. The methods enable the detection of telomerase activity that is extracted from 1000 HeLa cancer cells.

Introduction

Substantial recent research efforts are directed to the development of amplified bioelectronic detection schemes for DNA (Wang, 2001, Wang, 2002). Nucleic acid-functionalized metal nanoparticles have been used as labels for the amplified detection of DNA using microgravimetric, quartz crystal microbalance (Patolsky et al., 2000, Zhou et al., 2000), electrochemical (Wang et al., 2001a, Wang et al., 2001b, Wang et al., 2003) or electrical (Park et al., 2002) transduction as the electronic readout signals. The catalytic deposition of metals on the nanoparticle labels in these systems has been used as an amplification path that controls the weight, the voltammetric response of the dissolved metal associated with the DNA, or the conductivity of the resulting metal cluster. The coupling of the DNA recognition events to secondary biocatalytic processes that use the high turnover rate of enzymes as an amplification route was used as a different approach to amplify DNA analysis. DNA binding processes have been amplified by the coupling of the recognition event to a bioelectrocatalytic process using a redox-enzyme (Caruana and Heller, 1999, de Lumley-Woodyear et al., 1999) by the generation of a redox-active replica to the analyzed DNA and its coupling to a bioelectrocatalytic transformation (Patolsky et al., 2002a), by the conjugation of an enzyme that stimulates the biocatalyzed deposition of an insoluble product on the electronic transducer (Patolsky et al., 1999, Patolsky et al., 2001a), or by the application of an enzyme for the electro-generation of chemiluminescence as a result of the DNA analysis (Patolsky et al., 2002b).

Telomerase is a ribonucleoprotein that is responsible for the addition of the telomere repeat units at the ends of human chromosomes (Nakamura et al., 1997), providing cells with a mechanism that prevents the erosion of the chromosomal DNA. The replication of the 3′-ends of linear chromosomes by telomerase originates from its internal RNA component acting as a template, and the reverse transcriptase-type activity of the protein (Feng et al., 1995). Telomerase is responsible for the immortality of cells and for the uncontrolled growth of cancer cells (Harley and Villeponteau, 1995). Nearly all cancer types that were screened reveal a direct relation to the telomerase content in the cells (Kim et al., 1994). Also, it was demonstrated that the increased levels of telomerase in cells correlate with early stages of cancer development (Oshita et al., 2000). Accordingly, telomerase is considered as an important biomarker for cancer cells and malignancy (Schalken, 1998) and as a potential target for anticancer therapy (Lichtsteiner et al., 1999). Several analytical procedures for the determination of telomerase activity were developed, and these are based on the TRAP method (telomeric repeat amplification protocol) (Kim et al., 1994) or on the fluorescence labeling of the telomere units (Schmidt et al., 2002). The TRAP method, however, suffers from severe limitation since it depends on PCR amplification, and thus is susceptible to polymerase inhibition by clinical extracts (thus leading to false negative results). Also, it is difficult to quantify the telomerase in the samples due to the logarithmic amplification of the PCR analysis step. Similarly, the analysis of telomerase by labeling of the telomere with fluorescence units reveals relatively low sensitivity, and the telomerase originating from only 10⁵ to 10⁶ cells could be detected by this method. Recently, the optical detection of telomerase activity was reported by monitoring the fluorescence resonance energy transfer from CdSe/ZnS quantum dots (Patolsky et al., 2003a). Also, surface plasmon resonance, SPR, was successfully applied for telomerase activity detection (Maesawa et al., 2003).

Here we wish to report on the amplified detection of telomerase using the biocatalytic precipitation of an insoluble product as an amplification path. We use chronopotentiometry as the electronic readout signal for the detection process.