Cyclic Enzymatic Amplification Research Articles

Oral Fluid Nanosensor Test (OFNASET) with Advanced Electrochemical‐Based Molecular Analysis Platform

High-impact diseases, including cancer, cardiovascular disease, and neurological disease, are challenging to diagnose without supplementing clinical evaluation with laboratory testing. Even with laboratory tools, definitive diagnosis often remains elusive. The lack of three crucial elements presents a road block to achieving the potential of clinical diagnostic tests: (1) definitive disease-associated protein and genetic markers, (2) easy and inexpensive sampling methods with minimal discomfort for the subject, and (3) an accurate and quantitative diagnostic platform. Our aim is to develop and validate a solution for requirement (3) and also to develop a portable system. Requirements (1) and (2) will be addressed through the utilization of novel and highly specific oral cancer saliva proteomic and genomic biomarkers and the use of saliva as the biofluid of choice, respectively. The Oral Fluid NanoSensor Test (OFNASET) technology platform combines cutting-edge technologies, such as self-assembled monolayers (SAM), bionanotechnology, cyclic enzymatic amplification, and microfluidics, with several well-established techniques including microinjection molding, hybridization-based detection, and molecular purification. The intended use of the OFNASET is for the point of care multiplex detection of salivary biomarkers for oral cancer. We have demonstrated that the combination of two salivary proteomic biomarkers (thioredoxin and IL-8) and four salivary mRNA biomarkers (SAT, ODZ, IL-8, and IL-1b) can detect oral cancer with high specificity and sensitivity. Our preliminary studies have shown compelling results. We sequentially delivered a serial dilution of IL-8 antigen, probe solution, wash, enzyme solution, wash, and mediator solution to sensor reaction chambers housed in a prototype cartridge and demonstrated strong signal separation at 50 pg/mL above a negative control.

Enzynomics moves into Toulouse

MicroRNA (miRNA) is riveting nowadays due to its close relevance to human malignancies. Exploiting a fast and convenient biosensor for sensitive and specific detecting miRNA is necessary and it has significant meaning for oncology. In this work, to understand the relationship between miRNA-21 and gastric cancer, we employ T4 RNA ligase 2 to initiate specific ligation reaction depending on the sequence of target RNA, and T7 exonuclease to catalyze the first stage cyclic enzymatic amplification method (CEAM) specifically, which also resting on the target RNA sequence, and the second stage CEAM to further amplify the response resulting from the initial target RNA. Our two-stage CEAM strategy can detect miRNA-21 as low as 0.36 fM with remarkable specificity, and most importantly, it can be used to inspect the expression level of miRNA-21 in blood serum of gastric cancer patients. This will offer new perspective for figuring out the pathways of miRNA-21 involving in cancer.