Surface-enhanced Raman Scattering Biosensor Combined With Nucleic Acid Signal Amplification Strategy For The Early Diagnosis Of Oral Cancer

Ⅰ.Background and PurposeAs one of the most common malignant tumours in humans,oral cancer is a serious threat to human life,and early screening and diagnosis can effectively reduce mortality,which is a factor affecting the prognosis of oral cancer.At present,excisional tissue biopsy is still the main tool for early oral cancer diagnosis,but it is invasive and has potential complications,making it impractical to perform large-scale screening on patients with suspicious lesions.Most traditional detection methods rely on the experience,knowledge and other factors of the practitioner,which makes the identification of early-stage tumours difficult.MicroRNAs(micro RNAs)can be found stably in human serum,urine,saliva and other body fluids.Their expression levels are closely related to the development of cancer and are considered to be tumour biomarkers,which are currently a hot research topic.However,the sensitivity of diagnosing oral cancer is poor due to the low levels of miRNAs in the early stages of the disease.Therefore,there is an urgent need to develop a new technique that is more sensitive,specific and efficient for oral cancer diagnosis.Surface enhanced Raman scattering(SERS)is widely used in the field of disease diagnosis and biomedicine because of its high specificity,high sensitivity and nondestructive detection.Hybridisation chain reaction(HCR)is a simple and effective signal amplification technique that can be performed at room temperature without the need for enzymatic reactions.In addition,HCR is compatible with other detection techniques and can improve the sensitivity of biomolecules such as nucleic acids and proteins without the involvement of enzymes,which can also improve the accuracy of the assay.The aim of this study is to investigate and propose a HCR nucleic acid signal amplification strategy combined with surface Raman scattering technology using miR-31 and miR-21 as detectors in oral squamous cell carcinoma,which can be used to develop a novel biosensor with high sensitivity and specificity,providing a new diagnostic method for early screening of oral cancer.Ⅱ.Experimental methods1.30 patients with squamous cell carcinoma who attended the Department of Oral andMaxillofacial Surgery of the Affiliated Hospital of Yangzhou University from October2021 to October 2022 with complete data and 30 healthy volunteers were selected for this study.2.Gold nanohexagons with regular shape and uniform particle size were prepared by reduction reaction.The gold nanohexagons arrays were then self-assembled at the oilwater interface,and SERS capture substrates were obtained by modifying hairpin DNA on their surfaces.Similarly,gold and silver core shells were prepared by an electrocoupling reaction,and 4-MBA,DTNB and hairpin DNA were linked to the gold and silver core shells to form SERS probes.3.In the presence of the target miRNAs,the SERS probe is added and the HCR process is triggered and a long DNA hybrid strand is formed on the capture substrate.Quantification of miR-31 and miR-21 is achieved by detecting the SERS signal of the Raman reporter molecule.Ⅲ.Experimental results1.This study did not find that oral squamous cell carcinoma had a unique SERS Raman peak,but there was a difference in SERS intensity between oral squamous cell carcinoma patients and healthy people,which was statistically significant(P<0.05).SERS intensity in oral squamous cell carcinoma was significantly enhanced,indicating abnormal expression of miR-21 and miR-31.2.The detection limits of miR-31 and miR-21 in phosphate buffer(PBS)are 6.21 aM and6.29 aM,respectively,in the linear range of 10 aM-10 pM.3.The detection limits for miR-31 and miR-21 in saliva in the linear range of 10 aM-10 pM are 6.51 aM and 6.52 aM,respectively.Ⅳ.Conclusion1.This study confirmed that there was no significant difference in the SERS spectral characteristics of oral squamous cell carcinoma patients with different clinical stages,while significant differences in SERS intensity were seen between healthy volunteers and cancer patients.2.This study confirms that the SERS biosensor and quantitative reverse transcription polymerase chain reaction(qRT-PCR)assays are consistent in practice.3.This study demonstrates that this biosensor can provide a more efficient,sensitive,convenient and reliable method for the diagnosis of oral squamous cell carcinoma.