Enzyme Responsive Biosensors Fabrication And Disease Diagnosis Applications Thereof

Point-of-care testing（POCT）is a diagnostic technique that allows for rapid testing at the site of sample collection,using portable analytical instruments and corresponding reagents to obtain immediate results without time or location constraints.Recently,this method has played a key role in medical diagnostics,food safety,environmental monitoring and public safety due to its miniaturisation,ease of use and timely presentation of results.To meet the growing demand for immediate detection,this thesis constructs enzyme-responsive immediate detection nanosensors and uses them for early non-invasive detection of major diseases and infectious diseases,with two main components of work:Part I:Point-of-care real-time detection of transplant rejections based on Granzyme B responsive nanosensors.Here,a size-transformable nanosensor was constructed for colorimetric detection of organ transplant rejection.The designed nanosensor comprises a ternary system:i)a protein carrier neutravidin for enlongating blood circulation time;ii)a peroxidase-like Au NCs for signal amplification;iii)link two of which with a Granzyme B-responsive peptide.In animals where no transplant rejection occurs,the large-sized nanosensor（～11nm）cannot be excreted with urine（kidney filtration size～5.5 nm）.When transplant rejection occurs,massive Granzyme B will be produced by effective T-cells,which can specifically cleavage the linker peptide and release the ultra-small Au NCs（～2 nm）.The Au NCs was then filtered through the glomerulus into the urine.The peroxidase-like Au NCs can catalyse H₂O₂ to form hydroxyl radicals,resulting in the oxidation of3,3’,5,5’-Tetramethylbenzidine（TMB）to produce a blue product.This strategy transformed the molecular signal into color readout by using the highly specificity of enzyme recognition.Moreover,the color readout can be amplified by the artificial enzyme Au NCs,ganranteed a promoted sensitivity.This real-time detection technique offers several advantages,including simple operation,reliable results,and non-invasive testing without the need for large instruments.We envision that it can be widely applied for on-site testing in regions and countries with poor medical conditions,providing a rapid and convenient diagnostic tool for monitoring post-transplant progress.Part II:Smartphone-based analysis of nanosensors for the POCT of the novel coronavirus SARS-Co V-2.Based on the first work,we consider that naked-eye visualisation detection mainly relies on the human eye for observation,which leads to low accuracy due to subjective factors of the observer.To address these issues,we designed and constructed a smartphone-based analysis nanosensor for instant detection of SARS-Co V-2.In this study,the 3CL^pro protein was used as a specific biomarker for active virus replication.We designed a peptide sequence that can be specifically cleaved by the 3CL^pro protein,and constructed the nanosensor on a 96-microtiter plate.When a patient is infected with the SARS-Co V-2 virus,the 3CL^pro protein in their saliva can specifically cleave the peptide sequence on the nanosensor,releasing Au NCs that exhibit peroxidase-like activity and oxidize TMB to produce a blue colorimetric reaction.By using a smartphone app to analyze the colorimetric reaction,this method enables accurate and easy-to-read results without requiring complex optical equipment.Furthermore,the visual test reading allows for semi-quantitative analysis of assay results.The development of a smartphone-based biosensor analysis platform has accelerated testing and reduced costs,resulting in better accuracy.This portable and affordable technology can be designed for POCT,home testing,or community high-throughput screening,which is particularly relevant for outbreak prevention and control in resource-poor areas.