(174au) Peptide Biosensing for Minimally Invasive Diagnosis of Melanoma Skin Cancer

Skin cancer represents one of the most commonly manifested malignancies worldwide. In 2020, over 1.5 million cases were diagnosed and by 2040, the number of skin cancer cases is estimated to surpass 2.3 million ^[1]. Alterations in the stratospheric ozone layer and global climate change lead to the augmentation of the ultraviolet (UV) radiation levels sustained by ecosystems, which is the major etiologic factor for cutaneous malignancies affecting most skin cancer cases ^[2]. Cutaneous melanoma is the most severe and hazardous type of skin malignancy, being responsible for 80% of skin cancer deaths with a remarkably elevated risk of metastasis ^[3]. Advancements in understanding skin tumour pathophysiology have led to improved methods for identifying and treating cutaneous malignancies. Initially, melanoma diagnosis relied on morphological assessments, progressing to light-based visual technologies and digital analyses. While non-invasive digital technologies enhance accuracy, they are primarily clinical decision-support tools rather than standalone diagnostics ^[4]. Despite numerous apps aiming to enhance melanoma diagnosis, their clinical efficacy remains limited, leaving deployment to individual discretion ^[5]. Biopsy remains the gold standard but is costly, painful, and labour-intensive. The need for timely, accurate, and accessible cancer diagnosis calls for minimally invasive, rapid point-of-care (POC) analytical methods ^[6].

Clinical evidence supports the diagnostic and prognostic potential of S100B, a promising melanoma biomarker present in the interstitial fluid (ISF) of the dermis^[7]. S100B has shown correlation with the efficacy of dabrafenib and trametinib in melanoma treatment, making it valuable for monitoring patient response. This work aims to monitor the concentrations and variations of S100B in ISF by a melanoma-tailored fluorescently labelled peptide beacon, functioning as a highly specific bioreceptor. Molecular beacons, with a fluorophore-quencher mode of action, efficiently detect specific sequences of proteins through fluorescence modulation. Peptide nucleic acid (PNA) beacons offer the advantage of high affinity while minimising the likelihood of adverse immune responses in biological samples or in vivo settings.

This advanced design leverages the dimeric nature of the S100B protein, which provides two binding sites, ultimately enhancing the detection sensitivity ^[8]. The fluorescently labelled PNA beacon consists of two arms, each carrying a peptide sequence that binds to one of the subunits of the homodimeric protein S100B. Integration of PNA bases promotes intramolecular interactions that enable spatial proximity of the arms, causing donor fluorescence quenching via Förster resonance energy transfer (FRET), while binding to S100B positions the peptide arms distantly, leading to increased donor fluorescence. The two arms of the beacon were synthesised separately through solid-phase peptide synthesis (SPPS) while optimizing the deployed protocols^[9]. Once synthesized, both hybrid peptides were cleaved from the resins independently, deprotected, purified and then inter-molecularly linked with click chemistry.

The comprehensive evaluation of the bioreceptor’s performance is tailored to melanoma detection. The sensing evaluation criteria will include response time, sensitivity, stability, susceptibility to interference, and binding efficiency. We have concurrently developed hydrogel-based microneedle arrays with superior mechanical properties, allowing for efficient sampling and recovery of artificial ISF through porcine skin. These patches have been explored as a potential platform for integrating the S100B sensing system onto the needles. Our approach aims to create a point-of-care analysis and employs an optimized synthetic method that could contribute to enhancing the effectiveness of the diagnostic tool.

In summary, this technology utilizes peptide-based bioreceptors for comprehensive diagnosis and monitoring of melanoma skin cancer. Coupling wearable microneedle sensors with peptide biosensors provides a rapid and accurate alternative to traditional biopsies, enhancing disease management and treatment efficacy assessment due to their high specificity, customization potential, and reduced immunogenicity.

References

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[2] D'Orazio, J.; Jarrett, S.; Amaro-Ortiz, A.; Scott, T. UV radiation and the skin. Int. J. Mol. Sci. 2013, 14 (6), 12222-12248. DOI: 10.3390/ijms140612222.

[3] Davis, L. E.; Shalin, S. C.; Tackett, A. J. Current state of melanoma diagnosis and treatment. Cancer Biol. Ther. 2019, 20 (11), 1366-1379. DOI: 10.1080/15384047.2019.1640032.

[4] Chatzilakou, E.; Hu, Y.; Jiang, N.; Yetisen, A. K. Biosensors for melanoma skin cancer diagnostics. Biosens. Bioelectron. 2024, 250, 116045. DOI: https://doi.org/10.1016/j.bios.2024.116045.

[5] Young, A. T.; Vora, N. B.; Cortez, J.; Tam, A.; Yeniay, Y.; Afifi, L.; Yan, D.; Nosrati, A.; Wong, A.; Johal, A.; et al. The role of technology in melanoma screening and diagnosis. Pigment Cell Melanoma Res 2021, 34 (2), 288-300. DOI: 10.1111/pcmr.12907.

[6] Linares, M. A.; Zakaria, A.; Nizran, P. Skin Cancer. Prim. Care. 2015, 42 (4), 645-659. DOI: 10.1016/j.pop.2015.07.006.

[7] Eftekhari, A.; Ahmadian, E.; Salatin, S.; Sharifi, S.; Dizaj, S. M.; Khalilov, R.; Hasanzadeh, M. Current analytical approaches in diagnosis of melanoma. TrAC, Trends Anal. Chem. 2019, 116, 122-135. DOI: 10.1016/j.trac.2019.05.004.

[8] Dhar, A.; Ahmed, I.; Mallick, S.; Roy, S. A Peptide-PNA Hybrid Beacon for Sensitive Detection of Protein Biomarkers in Biological Fluids. ChemBioChem 2020, 21 (15), 2121-2125. DOI: 10.1002/cbic.202000097.

[9] Al Musaimi, O.; Morse, S. V.; Lombardi, L.; Serban, S.; Basso, A.; Williams, D. R. Successful synthesis of a glial-specific blood–brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin. Journal of Peptide Science 2023, 29 (2), e3448. DOI: https://doi.org/10.1002/psc.3448.