Speaker
Description
"INTRODUCTION
Cancer early detection is pivotal to patient survival. The small non-coding nucleic acid sequences, microRNA (miRNA) are a captivating molecular target for cancer early detection. miRNA are dysregulated during the early stages of cancer1, it is found in stable amounts in blood plasma and serum. Therefore, a minimally invasive liquid biopsy screening device would allow for point of care diagnostics. Current miRNAs detection methods are cumbersome and lack reproducibility along with poor sensitivity and low accuracy. To overcome these challenges we aim to develop a new diagnostic platform using a functional 3D peptide hydrogels for sequence specific2, 3, PCR-free, fluorescent detection of miRNAs in a “one-pot” assay. This work will assess the suitability of the novel hydrogel-based technology for rapid, robust and reliable screening of the unique miRNA fingerprint of difficult to detect cancers.
EXPERIMENTAL METHODS
Using a split probe system with a FRET pair conjugated to an anti-parallel β-sheet peptide hydrogel, can be formulated that allow complementary strands of cancer miRNA biomarkers to be identified via fluorescence.
Diffusion characteristics were evaluated via plate reader and Fluoroblok well insert. Allowing to measure the rate at which fluorescently label DNA analogue of miR-21 diffuse into peptide hydrogels.
Cell culture of Panc-1, MIA PaCa-2, LNCaP and PC-3 cells in 2D and 3D (multicellular tumour spheroids) forms was undertaken to validate the sensor. The hydrogels under investigation were also evaluated in their ability to support the four cancer cell lines spheroid structure.
RESULTS & DISCUSSION
Four de novo designed self-assemble peptide hydrogels (SAPH) were tested to understand the diffusion characteristics of miRNA and select system that allow fast trapping and detection of miRNA. MiRNA being negatively charge it was found that positively charted hydrogels promoted miRNA trapping. The mesh size of the hydrogel used (<40nm) also allowed to filter and avoid interference from larger cell debris usually present in biological samples.
The bio-compatibility of the four peptide hydrogels provides a 3D platform for cancer cell culture, and in situ bio-sensing.
CONCLUSIONS
The self-assemble peptide hydrogel is an extremely versatile material. Has the potential to harbour fluorescent properties, to allow for biosensor application in the early detection of cancer.
Future work on 3D culturing of other pancreatic and prostate cancer cells for quantification of the key secreted biomarkers linked to the two cancers that require early detection.
ACKNOWLEDGEMENTS
The work was funded in part by DTA EPSRC Funding from the Department of Materials at University of Manchester, MERCARDO associated with Cancer Research UK Manchester Centre, and ACED.
REFERENCES
[1] Kosaka, Nobuyoshi, Haruhisa Iguchi, and Takahiro Ochiya. ""Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis."" Cancer science 101.10 (2010): 2087-2092.
[2] Yousaf, Sameen, et al. ""Sequence-Specific Detection of Unlabeled Nucleic Acid Biomarkers Using a “One-Pot” 3D Molecular Sensor."" Analytical chemistry 91.15 (2019): 10016-10025.
[3] King, Patrick JS, et al. ""A de novo self-assembling peptide hydrogel biosensor with covalently immobilised DNA-recognising motifs."" Chemical Communications 52.40 (2016): 6697-6700."
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