Highly Sensitive Exosome Detection for Early Diagnosis of Pancreatic Cancer Using Immunoassay Based on Hierarchical Surface‐Enhanced Raman Scattering Substrate

Abstract

5 year survival rate of just 6% and a median survival of fewer than 6 months due to difficulties in the early diagnosis, PaC will be the second leading cause of cancer death by 2030. [3][4][5] Clinical diagnostic methods for PaC mainly include imaging and pathological biopsy. However, the available imaging diagnostic techniques, such as ultrasound (US), computed tomography (CT) scanning, and magnetic resonance imaging (MRI), have low diagnostic sensitivity and high requirements on operating skills. [2,[6][7][8] Normally, histopathological biopsy is a gold standard for disease diagnosis. [9] However, due to the deep position of the pancreas, biopsy will bring great harm to patients, and it needs to be punctured under the guidance of imaging and is not suitable for screening of the general population. [10,11] Moreover, the sensitivity and specificity of the widely used clinical screening biomarker carbohydrate antigen19-9 (CA19-9) are not high, and its serum level is significantly increased in inflammatory diseases of the pancreas and biliary tract, which leads its capacity for early diagnosis is not be evaluated. [12,13] Therefore, it is urgent and necessary to identify new and better biomarkers for the early diagnosis of PaC.Exosomes have emerged as potential biomarkers for pancreatic cancer (PaC). However, it is still challenging to get quantitative detection of exosomes with the specific surface receptors. In this study, a highly sensitive detection system is first constructed for the direct quantitation of specific exosomes in real samples using hierarchical surface-enhanced Raman scattering substrate (H-SERS substrate) and rapid enrichment strategy magnetic beads @ exosomes @ SERS detection probes (MEDP). It is found that the detection system (MEDP @ H-SERS substrate) could provide a 3.5 times higher SERS intensity compared with MEDP sandwich immunocomplex only. Moreover, LRG1-positive exosomes (LRG1-Exos) and GPC1-positive exosomes (GPC1-Exos) are chosen to distinguish PaC through exosome proteomics and database screening. The lower limit of detection (LOD) is 15 particles µL -1 using the MEDP @ H-SERS substrate. Significantly, the detection in clinical samples shows that the innovative combination of LRG1-Exos and GPC1-Exos could improve the diagnostic efficiency of PaC, with an area under the operating characteristic curve (AUC) of 0.95. Even for the early-stage PaC, the diagnostic accuracy is still high (AUC = 0.95). Collectively, the findings indicate that the MEDP @ H-SERS substrate has great potential for the early diagnosis of PaC.The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smtd.202200154.

Recent progress in surface-enhanced Raman spectroscopy-based biosensors for the detection of extracellular vesicles

“…10A). Moreover, Li et al 49 followed a similar approach to achieve an early diagnosis of pancreatic cancer. Among the EV surface proteins, LRG1 and GPC1 are novel biomarkers of EVs, which are identied by proteomics.…”

Cancer-derived small extracellular vesicles: emerging biomarkers and therapies for pancreatic ductal adenocarcinoma diagnosis/prognosis and treatment

“…Serum N = 142 Highly expressed CD44v6 and C1QBP in sEVs were promising biomarkers for predicting prognosis and liver metastasis in patients with PDAC [112] LRG-1, GPC-1 Serum N = 15 Combination of LRG-1 and GPC-1 positive sEVs could improve the diagnostic accuracy of PDAC with AUC of 0.95, even for the early stage PDAC. [113] Integrin α6 Blood N/A The expression of Integrin α6 in sEVs from blood of PDAC patients significantly decreased after surgery and increased several months before clinical recurrence [114] Mucin-4, Mucin-5AC, Mucin-6, Mucin-16, etc. Pancreatic duct fluid proteins were potential biomarkers of patients with different pancreatic diagnoses [116] curves, miR-10b, -21, -30c, -181a, and -let7a in sEVs all have 100% sensitivity and specificity in detecting PDAC from normal controls, while miR-106b and − 483 failed to distinguish these two groups.…”

Serum exosomal and serum glypican-1 are associated with early recurrence of pancreatic ductal adenocarcinoma

“…The diagnostic value of GPC-1 in PDAC remains controversial. In these studies, the most common method used to detect GPC-1+ exosomes was flow cytometry or some new technologies, such as nanosized molecular beacons with high luminescence efficiency and glypican-1-antibody-conjugated Gd-Au nanoclusters (14)(15)(16)(17), which require expensive equipment and skilled labor and are not suitable for wide application in the clinic. Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used serological methods in the clinic owing to its high efficiency, wide availability, and low cost.…”