The Design And Application Of Paper-based Sensors Based On Upconversion Fluorescence Resonance Energy Transfer

Paper-based analytical devices (PADs), which use paper as the substrate material, are a technology for fabricating simple, tow-cost, portable and disposable analytical devices for many areas including clinical diagnosis, environmental monnitoring and food quality control. Microfluidic paper-based analytical devices (?PADs). which combine some of the capabilities of conventional micro fluidic chips (miniaturization, high-throughput, integration) with the advantages of paper substrate (inexpensive, biocompatibility, pump ing-free), have extended from fabrication methods into practical applications. A series of detection methods such as colorimetric detection, electrochemical detection, chemiluminescence detection, electrochemical luminescence detection and fluorescence detection have been applied in ?PADs. Fluorescence resonance energy transfer (FRET), an important fluorescence assay method, is considered as a reliable "spectroscopic molecular ruler" over the distances of 1-10 nm. FRET, which is simple, fast and sensitive, has been widely used in homogeneous detection system and successfully extended to paper-based analytical devices to detect different kinds of target such as metal ion, small molecule, biological macro molecule, cell and pathogen. Nevertheless, the existing paper-based analytical methods, which use conventional down conversion fluorescent materials such as organic dyes, quantum dots and photoluminescent carbon nanoparticles as energy donors, still suffer from some disadvantages. These materials were excited with ultraviolet or visble light. In this excitation condition, the autofluorescence and scattering light arises from paper substrate and biomolecules in biological sample matrixes arise. Besides, the energy acceptor is often coexcited with energy donor because of the spectra overlap of their excitation spectra, resulting in false positive signals. The above-mentioned interference factors limit the application of paper-based FRET sensors, especially in complicated biological matrixes.Upconversion nanoparticles (UCNPs), a kind of rear-earth doped anti-Stokes fluorescent material, can be excited with near-infrared (NIR) light or infrared light and exhibit visible emission. The NIR-excitation nature endows UCNPs with the ability to eliminate interferences come from biological sample matrixe and the energy acceptor. Hence, UCNPs is a kind of promising energy donor, and the homogeneous UC-FRET technique has been widely used in nucleic acid hybridization, immunoassay, and other bio medical analysis. Delightful achievements have made in homogeneous UC-FRET assay. Nevertheless, the paper-based UC-FRET system, which using UCNPs as the energy donor in paper-based FRET, is still a completely new research direction. When used in paper-based analytical devices, UCNPs can effectively eliminate the interferences arising from fluorescent additives in paper substrates and scatter light. Paper-based UC-FRET system, which combines the capabilities of homogeneous UC-FRET assay with the simplicity of PADs, is expected to develop in complicated biological matrixes detection and bring new opportunities for point-of-care testing (POCT). Therefore, aming to solve the background interference arise from complicated biological matrixes, this dissertation proposes the strategy to use UCNPs as the energy donor of paper-based FRET to construct novel paper-based UC-FRET analytical methods. Several energy acceptors including organic dyes, carbon nanomaterials and gold nanoparticles and different kind of recognition mechanism such as hydrolytic cleavage of peptide substrate, aptamer recognition and immune response were used to construct paper-based UC-FRET sensors, which were applicable to complicated biological matrixes assay. Utilizing the unique optical properties of UCNPs, the testing equipment was simplified and portable sensor was developed, realizing the visualization of targets in complicated biological matrixes.The main contents were as follows:1. Paper-based UC-FRET sensor using UCNPs as the energy donor and organic dye TAMRA as the energy acceptor was designed. MMP-2 was detected based on the hydrolytic cleavage of the peptide substrate. Paper-based test zones were prepared on the surface of printing paper by on-step plotting method and the photostability and immolization of UCNPs on the surface of test zones were investigated. TAMRA modified peptide substrate was covalently labeled to the surface of UCNPs, which brought the energy donor and the acceptor close proximity, resulting in the quenching of the fluorescence of UCNPs. UCNPs-peptide-TAMRA was dropped on paper-based test zones and the photostability was investigated. When MMP-2 was added to the test zone, peptide substrate was cleavged, resulting in the recovery of the fluorescence of UCNPs. The increase of UCNPs fluorescence intensity was proportional to MMP-2 concentration within the range from 50 pg/mL to 5000 pg/mL in aqueous buffer. The limit of detection (LOD) was 8.3 pg/mL. The MMP-2 sensor was applied to real human serum and whole blood samples. Compared to homogeneous UC-FRET assay, the constructed sensor required a tiny amount of samples and reagents and coud finish detection process fasterly. More importantly, the detection sensitivity was corresponded to homogeneous assay. The feasibility of paper-based UC-FRET system was well verified by this MMP-2 sensor.2. Carbon nanomaterials were used as the energy acceptors of paper-based UC-FRET sensors and the quenching capacities on the surface of paper were explored. Carbon nanoparticles (CNPs) and graphene oxide (GO) were used to detect immunoglobulin E (IgE) and carcinoembryonic antigen (CEA) on the basis of aptamer recognization, respectively. UCNPs were covalently tagged with amino group modified aptamer and dropped on the surface of paper-based test zones. The aptamer could bind to the surface of carbon nanomaterials through ?-? stacking interaction, resulting in the fluorescence quenching of UCNPs. When target was added to the test zone, the interaction between aptamer and target changed the conformation of aptamer, which enlarged the distance between energy donor and energy acceptor. The fluorescence of UCNPs was recovered in a target concentration-dependent manner, which built the foundation of target quantifacation. In the concentration range from 0.5 ng/mL to 80 ng/mL, the fluorescence recovery was linear related to the concentration of IgE in buffer solution. This paper-based UC-FRET sensor could be used to detect IgE concentation in human serum samples and the detection results matached well with clinical test results. The applications of paper-based UC-FRET system were further expanded by this aptasensor.3. To develop our paper-based UC-FRET system to immunoassay, gold nanoparticles were used as the energy acceptor and a paper-based UC-FRET immunosensor based on the specific recognition between antigen and antibody was constructed to detection CEA. UCNPs and AuNPs were covalently tagged with CEA coating antibody and labelling antibody respectively, and the former was dropped on the surface of paper-based test zones. When CEA and AuNPs conjugates were presented in test zone, the coating antibody and labelling antibody could recognize CEA simultaneously. As a result, the energy donor and acceptor were taken into closely proximity, resulting in the fluorescence quenching of UCNPs. The quenching efficiency of UCNPs was proportional to CEA concentration, and the quantitative detection of CEA was realized accordingly with the linear range from 0.5 ng/mL to 30 ng/mL in buffer solution.This sensor could realize the quantitative detection of CEA in human serum samples and the qualitative judgment of critical concentration CEA by nake eye, which was of great importance in the early screening and clinical diagnosis of disease.4. On the basis of our preliminary work, we further simplified the testing equipment and constructed portable sensor. A visible paper-based UC-FRET sensor, which used AuNPs as energy acceptor and smartphone as the apparatus, was constructed for cocaine detection. The target recognition was realized by using an anticocaine aptamer (ACA), which was rationally cut into two flexible ssDNApieces, namely, ACA-1 and ACA-2. UCNPs and ACA-1 were covalently coupled on the surface of paper, and ACA-2 was covalently bound to AuNPs. In presence of cocaine, the two piece of ssDNA reassemble into the intact aptamer tertiary, resulting in the fluorescence quenching of UCNPs by AuNPs. The fluoreccence change could be recorded by a smartphone camera and easily transferred to intensity for cacaine quantification. The sensor provided a linear range from 0.01 to 5 ?M for cocaine in buffer solution and 0.05 to 5 ?M in human saliva. This sensor could be used to detect cocaine in human saliva and rat plasma. Compared to physical adsorption, the covalent coupling method used in this work could improve the stability of detection results. The constructed sensor is simple, sensitive and portable, which show great potential in field testing.