Zero-Mode Waveguides For Single-molecule Fluorescence Detection

Single-molecule fluorescence microscopy can reveal molecular dynamics and composition so that it has been a powerful tool for single-molecule detection.However,because of the diffraction limit of light,the concentration of fluorescent species is nanomolar or lower.But in many biological and chemical mechanisms,the concentration of reactants is up to micromolar or millimolar concentrations,such as metabolites,neurotransmitters,enzymes,and catalysts,which are required to react at high concentrations.Zero-mode waveguides are nanostructures with the arrays of50-150 nm diameter holes in a 100 nm thick metal film which are fabricated on the silica substrate.Zero-mode waveguides provide the observation volumes on the order of zeptoliter(z L,10^-21L).The diameter of the zero-mode waveguides nanopores is less than the diffraction limit of light,so there are no propagating modes in the waveguides.Because of the structure of zero-mode waveguides,the fluorescent species can be allowed to be observed at high concentrations.In addition,the background fluorescence is greatly reduced because light cannot enter the nanoholes.Zero-mode waveguides have been confirmed that zero-mode waveguides can be successfully applied in the single-molecule fluorescence detection without isothermal nucleic acid amplification technology.In another word,zero-mode waveguides have achieved the truly single-molecule detection.This paper uses zero-mode waveguides chip to achieve single-molecule fluorescence detection,the specific research content is as follows:(1)Based on lithography and etching,we use an improved metal lift-off method to fabricate zero-mode waveguides chips.First,the silica substrate is coated with negative photoresist.After the electron beam exposure,there remains the array of the photoresist nanopillars on the silica substrate.Then a 100 nm gold film is evaporated onto the substrate and the top of the photoresist nanopillars with the electron beam.At last,we use hydrofluoric acid to dissolve the photoresist nanopillars and the gold on the top of photoresist nanopillars is dissolved as well.After etching,we obtain the zero-mode waveguides chip with the array of nanoholes.The images of the scanning electron microscope(SEM)show that we have successfully fabricated a compliant zero-mode waveguides chip.(2)Endonuclease IV(Endo IV)is a DNA repairing enzyme that recognizes the apurinic or apyrimidinic sites(AP sites)of the double-stranded DNA and cleaves the phosphodiester bond.In addition to studying DNA damage and repair,Endo IV can also be used for signal amplification and detection of DNA or RNA targets.In chapter two,we designed and the target and the fluorescently labeled probe with AP site according to the characteristics of Endo IV.First,the target was immobilized at the bottom of the nanohole,when we added the probe,the target and the probe were perfectly matched so that we can use the confocal laser scanning microscope to observe fluorescence in the absence of Endo IV.Then,we added Endo IV into the nanoholes,Endo IV can recognize the AP site on the probe and cleave the phosphodiester bond.As the result,the fluorescence disappeared.If we add the probe into the nanoholes again,the fluorescence can be observed again and then disappeared again due to the existence of Endo IV.The results demonstrate that we can use the zero-mode waveguides chip to achieve real-time single-molecule fluorescence detection.(3)Mutations in the KRAS gene can reduce the activity of KRAS proteins and have an effect on cell-signaling pathways.Many cancers,such as pancreatic,colon,lung and ovarian cancer,are highly likely to be caused by mutations in the KRAS gene.In the third chapter,we chose the KRAS mutation at codon 12 as our target mutation.We designed the target and the short(9-to 10-nucleotide,nt)probe with fluorescent label.The binding between the target and the short probe is unstable.As a result,the fluorescence,which caused by the perfect match of target and probe,would disappear after a moment.First,the chip was incubated with the appropriate target which made each pore only has a single molecule.Then we added different concentrations of probes into the nanopores.The changes of fluorescence intensity in real time can be detected by the confocal laser scanning microscope.As a result,we observed the binding and dissociation of individual molecules.Compared with other methods,using the zero-mode waveguide chip can achieve a true sense of single-molecule sequencing and the single base mismatch.At the same time,the array of zero-mode waveguides consisting of nanopores provides the possibility of high parallel method for single-molecule detection.