Novel probes for label-free detection of neurodegenerative GGGGCC repeats associated with amyotrophic lateral sclerosis
DNA repeat expansion sequences cause a myriad of neurological diseases when they expand beyond a critical threshold. Previous electrochemical approaches focused on the detection of trinucleotide repeats (CAG, CGG, and GAA) and relied on labeling of the probe and/or target strands or enzyme-linked assays. However, detection of expanded GC-rich sequences is challenging because they are prone to forming secondary structures such as cruciforms and quadruplexes. Here, we present label-free detection of hexanucleotide GGGGCC repeat sequences, which cause the leading genetic form of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The approach relies on capturing targets by surface-bound oligonucleotide probes with a different number of complementary repeats, which proportionately translates the length of the target strands into charge transfer resistance (RCT) signal measured by electrochemical impedance spectroscopy. The probe carrying three tandem repeats transduces the number of repeats into RCT with a 3× higher calibration sensitivity and detection limit. Chronocoulometric measurements show a decrease in surface density with increasing repeat length, which is opposite of the impedance trend. This implies that the length of the target itself can contribute to amplification of the impedance signal independent of the surface density. Moreover, the probe can distinguish between a control and patient sequences while remaining insensitive to non-specific Huntington’s disease (CAG) repeats in the presence of a complementary target. This label-free strategy might be applied to detect the length of other neurodegenerative repeat sequences using short probes with a few complementary repeats.
KeywordsNucleic acid biosensing Electrochemical biosensors Repeat expansion disorders Amyotrophic lateral sclerosis Electrochemical impedance spectroscopy
M.H. Shamsi acknowledges SIUC startup funds for the research. L.M. Ellerby acknowledges R01 NS100529 grant. Funding to K.T.G. was provided by an Amyotrophic Lateral Sclerosis Research Program (ALSRP) grant from the US Department of Defense.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 1.The Genomes Project C, McVean GA e a. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012;491:56.Google Scholar
- 12.Hubers A, Marroquin N, Schmoll B, Vielhaber S, Just M, Mayer B, et al. Polymerase chain reaction and southern blot-based analysis of the C9ORF72 hexanucleotide repeat in different motor neuron diseases. Neurobiol Aging. 2014;35(5):1214 e1–6.Google Scholar
- 13.Narzisi G, Schatz MC. The challenge of small-scale repeats for indel discovery. Front Bioeng Biotech. 2015;3:8.Google Scholar
- 14.Akimoto C, Volk AE, van Blitterswijk M, Van den Broeck M, Leblond CS, Lumbroso S, et al. A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories. J Med Genet. 2014;51(6):419–24.PubMedPubMedCentralGoogle Scholar
- 15.Buchman VL, Cooper-Knock J, Connor-Robson N, Higginbottom A, Kirby J, Razinskaya OD, et al. Simultaneous and independent detection of C9ORF72 alleles with low and high number of GGGGCC repeats using an optimised protocol of Southern blot hybridisation. Mol Neurodegener. 2013;8(1):12.PubMedPubMedCentralGoogle Scholar
- 21.Liu YL, Li J, Chang G, Zhu RZ, He HP, Zhang XH, et al. A novel electrochemical method based on screen-printed electrodes and magnetic beads for detection of trinucleotide repeat sequence d(CAG)(n). New J Chem. 2018;42(12):9757–63.Google Scholar
- 22.Zhu XQ, Li J, Lv HH, He HP, Liu H, Zhang XH, et al. Synthesis and characterization of a bifunctional nanoprobe for CGG trinucleotide repeat detection. RSC Adv. 2017;7(57):36124–31.Google Scholar
- 27.Miroslav F, Petra B, Kateřina C, Petr P. A single-surface electrochemical biosensor for the detection of DNA triplet repeat expansion. Electroanalysis. 2006;18(2):141–51.Google Scholar
- 34.Ge B, Huang Y-C, Sen D, Yu H-Z. Electrochemical investigation of DNA-modified surfaces: from quantitation methods to experimental conditions. J Electroanal Chem. 2007;602(2):156–62.Google Scholar
- 35.Capaldo P, Alfarano SR, Ianeselli L, Zilio SD, Bosco A, Parisse P, et al. Circulating disease biomarker detection in complex matrices: real-time, in situ measurements of DNA/miRNA hybridization via electrochemical impedance spectroscopy. ACS Sensors. 2016;1(8):1003–10.Google Scholar
- 36.Bertok T, Lorencova L, Chocholova E, Jane E, Vikartovska A, Kasak P, et al. Electrochemical impedance spectroscopy based biosensors: mechanistic principles, analytical examples and challenges towards commercialization for assays of protein cancer biomarkers. ChemElectroChem. 2019;6(4):989–1003.Google Scholar
- 37.Yang J, Jiao K, Yang T. A DNA electrochemical sensor prepared by electrodepositing zirconia on composite films of single-walled carbon nanotubes and poly(2,6-pyridinedicarboxylic acid), and its application to detection of the PAT gene fragment. Anal Bioanal Chem. 2007;389(3):913–21.PubMedGoogle Scholar