Human genes contain small codes called nucleotides which are the main building blocks of DNA. These nucleotides can repeat themselves in 2-10 nucleotides combinations at different locations in the gene. When these repetitive codes expand in an uncontrollable manner, a type of genetic mutation, it is described as a ‘DNA repeat expansion’. There are now almost 50 neurodegenerative disorders associated with these repeat sequences, such as Huntington’s disease (HD) and Amyotrophic Lateral Sclerosis (ALS).
There is still no sensitive and reliable method available to detect these mutations at point-of-care, especially when it comes to long sequences. Therefore, it is of great importance to find new ways to detect DNA repeat expansions, to help predict neurodegenerative disorders before the symptoms appear. Such technology will help healthcare providers to set treatment procedures through timely diagnosis. Dr Mohtashim Shamsi and his lab undertook this challenge to develop new biosensors based on the electrochemical properties of DNA and nanomaterials.
Label free
Testing methods for repeat expansion detection have numerous pros and cons. For instance, state-of-the-art nanopore sequencing by Oxford Nanopore Technologies is low-cost and is able to read long repeat sequences, but the flow-cells are prone to clogging and have a limited shelf-life.
Single molecule real-time (SMRT) sequencing by PacBio is also highly accurate, but relies on fluorescent labels for detection. DNA microarray technology relies on fluorescence labels and repeat primed PCR for signal amplification. DNA microarrays also have critical limitations because disease-associated repeats are by nature highly repetitive.
Shamsi’s group demonstrated that their microprobe-based strategy can potentially be evolved into a simple platform requiring a very low sample amount and volume for discriminations of abnormal repeat expansions, which may facilitate rapid testing of a wide range of repeat-associated neurodegenerative disorders. Therefore, Shamsi’s lab proposed to use a new low-cost and miniaturisable biosensor which does not require a label, but relies solely on the interfacial electrochemical properties of DNA sequences with 2D nanomaterials, eg, graphene and molybdenum disulfide (MoS2).
How it works
2D nanomaterials are a single layer of atoms arranged just like a thin sheet. DNA and 2D nanomaterials can interact with these materials through a variety of weak bonding mechanisms. The interaction causes a change in the electronic structure of these materials, which consequently improves the conductivity. Conductivity changes can be easily measured with miniaturisable instruments called potentiostats. 2D nanomaterials can also be easily transformed into an ink, which can be printed on a variety of flexible surfaces such as plastics by inkjet printing.
“These chips could be incorporated in the clinic and mark a new beginning for the diagnosis of neurodegenerative diseases.”
For point-of-care diagnostics, Shamsi’s group has pioneered a new class of flexible devices known as wax-on-plastic platforms, which involve simple patterning of non-toxic and hydrophobic wax layers on top of a flexible plastic substrate. The wax-on-plastic platforms can be applied in a variety of formats such as microwells, microchannels, electrical and electrochemical chips. Shamsi has also developed a graphene-based ink which is partially coated with oxygen groups and an MoS2 ink to fabricate electrodes in the wax-on-plastic chips. In the absence of the DNA sample, the unmodified electrodes show low current when an electroactive species was measured on the surface. However, when DNA is adsorbed on the electrodes, it leads to a higher current response, which support the previous theories highlighting the improved electronic properties of such DNA-2D nanomaterials interface.
The findings
The biosensing approach developed in Shamsi’s group detects not only the presence of a DNA sample, but it could also provide information regarding its sequence, conformation, concentration, and length characteristics, all of which are crucial when it comes to identifying mutations causing neurodegenerative diseases. Making use of that, Shamsi and his group tried to study DNA samples which contain trinucleotides repeats, associated with two different diseases: Fragile X syndrome (GAA repeats) and Huntington’s disease (CAG repeats). The TNR-2D nanomaterials interface shows enhanced current signal with regard to length of repeats and double-stranded conformation, and can detect target sequences down to a few strands on the surface.
More biosensors developed
It is important to highlight that this is the first comprehensive study of the electrochemical behaviour of DNA adsorbed on the 2D nanomaterials surface. Shamsi’s group has also pioneered the development of many more biosensing platforms aiming to detect potential pathogenic DNA repeats. One example is the successful development of short nucleic acid probes (known as microprobes), that contain a specific sequence of nucleotides, to detect frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The proof of concept is based on the binding sites of these probes with the biological samples, considering certain nucleotides bind with specific others. These microprobes, once they bind with the sample, cause a change in resistance of the biosensing interface, which is detected by a sophisticated technique called electrochemical impedance spectroscopy (EIS).
A chip for tomorrow’s diagnosis
Shamsi’s work vitally contributes to a thorough understanding of the electrochemical behaviour of DNA when adsorbed on 2D nanomaterials, such as oxidised graphene and molybdenum disulfide. His achievements help in the development of tools that could act as biosensors to diagnose a myriad of neurodegenerative diseases, using a simple, ultrasensitive, low cost, and sophisticated method. A sensing system such as the one devised by Shamsi could be translated into the creation of flexible printable chips: a wax-on-plastic electrochemical device. These chips could then be incorporated into the clinic and could mark a new beginning for the diagnosis of neurodegenerative diseases, initiating a new field of chip-based diagnostics.
The wax-on-plastic chips can also be used to detect single nucleotide polymorphism, or SNP, which is a variation at a single position in a DNA sequence. SNPs may lead to variations in the amino acid sequence. Some SNPs are associated with certain diseases. Detection of SNPs may help predict an individual’s response to certain drugs, susceptibility to environmental factors such as toxins, and risk of developing diseases. The SNP-containing targets have usually lower binding affinity for the probe on chip surface, therefore, their weak current response can be easily distinguishable from a normal sequence.
References
- Shamsi, Mohtashim, et al, (2022) PNA microprobe for label-free detection of expanded trinucleotide repeats. RSC Advances, 12, 7757–7761, doi.org/10.1039/D2RA00230B
- Shamsi et al, (2021) Sequence-independent DNA adsorption on few-layered oxygen-functionalized graphene electrodes: an electrochemical study for biosensing application. Biosensors, 11(8) 273. doi.org/10.3390/bios11080273
- Asefifeyzabadi, Narges, et al, (2020) Label-free electrochemical detection of CGG repeats on inkjet printable 2D layers of MoS2. ACS Applied Materials & Interface, 12(46), 52156–52165. doi.org/10.1021/acsami.0c14912
- Asefifeyzabadi, Narges, et al, (2020) Unique sequence-dependent properties of trinucleotide repeat monolayers: electrochemical, electrical, and topographic characterization. Journal of Materials Chemistry B 8(24), 5225–5233. doi.org/10.1039/D0TB00507J
- Taki, Motahareh, et al, (2019) Novel probes for label-free detection of neurodegenerative GGGGCC repeats associated with amyotrophic lateral sclerosis. Analytical and Bioanalytical Chemistry 411(26) 6995–7003. doi.org/10.1007/s00216-019-02075-8
10.26904/RF-142-2829633104
Research Objectives
Dr Mohtashim Shamsi researches new electrochemical biosensing approaches for the detection of DNA repeat expansions.
Funding
- National Science Foundation
- National Institute of Health
Collaborators
- Lisa M Ellerby
- Keith T Gagnon
Bio
Mohtashim Shamsi is an associate professor in the School of Chemical and Biomolecular Sciences at the Southern Illinois University Carbondale. He earned his PhD and postdoctoral fellowship from the University of Toronto. He has many scientific publications and patents in peer-reviewed journal with an impact factor of more than 200. He has received several awards including the National Science Foundation EAGER Award, President’s Award for Distinguished International Alumni, Gwangju Institute of Science & Technology.
Contact
Chemical and Biomolecular Sciences
Southern Illinois University at Carbondale
1245 Lincoln Dr, Carbondale IL, 62901, USA
