Amplified detection of telomerase activity using electrochemical and quartz crystal microbalance measurements
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.
Section snippets
Chemicals
Avidin-alkaline phosphatase, TRIZMA® hydrochloride, hexaamine Ru(II) chloride, 6-mercapto-1-hexanol, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), phenylmethylsulfonyl fluoride (PMSF), 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), 5-bromo-4-chloro-3-indolyl phosphate, glycerol, 2′-deoxyadenosine-5′-triphosphate (dATP), 2′-deoxyguanosine-5′-triphosphate (dGTP), thimidine-5′-triphosphate (dTTP), oligonucleotides—-(CH2)6-AATCCGTCGAGCAGAGTT (1
Results and discussion
The modification of electrode surfaces with nucleic acids, and the subsequent formation of nucleic acid-DNA complexes on surfaces alters the interfacial properties of the electrodes due to the negative charges that are accumulated on the electrode surfaces. Also, the coupling of proteins such as avidin-enzyme conjugates, to nucleic acid-DNA complexes associated with electrodes, or the biocatalyzed precipitation of insoluble products on electrode surfaces following the formation of nucleic
Conclusions
The present article describes the first electrochemical assay of telomerase activity. We have employed the biocatalytic precipitation of an insoluble product on the electrode surface as an amplification path to detect the existence of telomerase in the respective samples. The methods were successfully applied to analyze telomerase with a detection limit of 1000 to 5000 HeLa cells. This sensitivity range is comparable, but slightly lower, than the TRAP method. Considering, however, that the
Acknowledgements
The support of this research by the Prostate Cancer Charitable Trust (PCCT, UK) is gratefully acknowledged.
References (33)
- et al.
Chronopotentiometry and Faradaic impedance spectroscopy as signal transduction methods for the biocatalytic precipitation of an insoluble product on electrode supports: routes for enzyme sensors, immunosensors and DNA sensors
Biosens. Bioelectron
(2001) - et al.
Telomeres and telomerase in aging and cancer
Curr. Opin. Genet. Devel
(1995) - et al.
Chronopotentiometry and Faradaic impedance spectroscopy as methods for signal transduction in immunosensors
Sens. Actuators B
(2001) - et al.
Probing of bioaffinity interactions at interfaces using impedance spectroscopy and chronopotentiometry
J. Electroanal. Chem
(2000) - et al.
Detection of activity of telomerase in tumor cells using fiber optical biosensors
Biosens. Bioelectron
(2002) Electrochemical nucleic acid biosensors
Anal. Chim. Acta
(2002)- et al.
Particle-based detection of DNA hybridization
Anal. Chim. Acta
(2003) - et al.
Electrochemical and quartz crystal microbalance detection of the cholera toxin employing horseradish peroxidase and GM1-functionalized liposomes
Anal. Chem
(2001) - et al.
Probing antigen–antibody interactions on electrode supports by the biocatalyzed precipitation of an insoluble product
Electroanalysis
(2000) - et al.
Enzyme-amplified amperometric detection of hybridization and of a single base pair mutation in an 18-base oligonucleotide on a 7-μm-diameter microelectrode
J. Am. Chem. Soc
(1999)
Rapid amperometric verification of PCR amplification of DNA
Anal. Chem
The RNA component of human telomerase
Science
Characterization of the swelling of acrylamidophenylboronic acid-acrylamide hydrogels upon interaction with glucose by Faradaic impedance spectroscopy, chronopotentiometry, quartz-crystal microbalance (QCM), and surface plasmon resonance (SPR) experiments
J. Phys. Chem. B
Specific association of human telomerase activity with immortal cells and cancer
Science
Telomerase: a target for anticancer therapy
Ann. N. Y. Acad. Sci
A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance
Nucl. Acids Res
Cited by (49)
An enzyme-free and PCR-free biosensing platform for accurate monitoring of telomerase activity
2023, Colloids and Surfaces A: Physicochemical and Engineering AspectsElectrochemical, Electrochemiluminescent and Photoelectrochemical Methods for Detection of Telomerase Activity: A Review
2020, International Journal of Electrochemical ScienceAnalysis of telomerase activity based on a spired DNA tetrahedron TS primer
2015, Biosensors and BioelectronicsCitation Excerpt :The detection limit was calculated to be lower than 10 HeLa cells (>3 standard deviations (SDs)), and the dynamic range covered 4 orders of magnitude (0–50,000 cells). The sensitivity of STTS sensor is at least 2 magnitudes lower than other surface extension-based electrochemical telomerase sensors without amplification (Pavlov et al., 2004; Shao et al., 2008; Li et al., 2011; Yang et al., 2011; Kim et al., 2013), and even comparable with to those strategies combined complex amplification to the surface primer strategies (Zheng et al., 2008; Li et al., 2010). To ensure the practicability, we challenged our system with different cell lines.
A novel electrochemical biosensor for sensitive detection of telomerase activity based on structure-switching DNA
2014, Biosensors and BioelectronicsCitation Excerpt :However, the TRAP method has some limitations, it is inappropriate for the determination of telomerase inhibition (Krupp et al., 1997; Cian et al., 2007), requires expensive equipment and reagents, and is time-consuming. To overcome these shortcomings, several alternative, PCR-free assays for telomerase activity techniques have been developed, such as colorimetric strategy (Xiao et al., 2004b; Fu et al., 2009; Freeman et al., 2010), fluorescence method (Zuo et al., 2011; Ding et al., 2010; Wang et al., 2012), chemiluminescence (Li et al., 2011; Niazov et al., 2004; Pavlov et al., 2004b), electrochemiluminescence (Zhou et al., 2009; Wu et al., 2012), surface Plasmon resonance (Sharon et al., 2010; Maesawa et al., 2003), quartz crystal microbalance (Pavlov et al., 2004a), and electrochemical method (Xiao et al., 2004a; Sato et al., 2005; Eskiocak et al., 2007; Sato and Takenaka, 2012; Shao et al., 2008). These developed approaches provide useful platform for telomerase assay and its related biological research.
Label-free, PCR-free chip-based detection of telomerase activity in bladder cancer cells
2013, Biosensors and Bioelectronics