Fluorescence Biodetection Based On ZnO Nanobrushes Synthesized On Microfluidic Chip

Cancer has become a major threat to people’s health,among which the mortality of lung cancer remains a high level due to poor prognosis and unsignificant symptoms in early stage.Early diagnosis is an important appraoch to improve the survival and prognosis.The combined assay for multiple cancer biomarkers is promising for early diagnosis,and fluorescence immunoassay is widely used for early detection and diagnosis due to the high sensitivity,high specificity and high reliability.At present,nanomaterials with controllable physical and chemical properties and excellent specific surface area hold great potential in the early diagnosis of cancer.They can be integrated with microfluidics to develop highly sensitive biosensors with high integration,low cost and convenient operation to realize the early diagnosis and point-of-care testing(POCT)of lung cancer.ZnO nanorods,as cheap,biocompatible and waveguieding substrates for biodetecion,the performance of fluorenscence biodetection relies on the surface area and the size of waveguides.Therefore,the establishement of ZnO three-dimensioanl(3D)structures with excellent detection performance in geometry confined space is important to further improve the sensitivity of fluorescence biodetection.In this paper,the ZnO nanobrush arrays were prepared in microfluidic channels via two seed layers,and the preparation conditions were optimized and was further utilized in the fluorescence immunoassay of lung cancer biomarkers.The main work is as follows:1.Microfluidic chips with four parallel channels were fabricated by standard photolithographic process.The size and morphology of ZnO nanomaterials,prepared by intermittent method and the secondary nucleation method,were studided both for conventional hydrothermal synthesis and microfluidic synthesis.Meanwhile,the variation of the size and morphology of ZnO nanorods were also studied for the concentration of the first seed solution.The results showed that well-oriented ZnO nanorods with larger space could be obtained with the concentration of 1 m M for the primary seed solution,which promoted the secondary nucleation and branching growth.FITC labeled goat anti-bovine immunoglobulin G(FITC-anti Ig G)was used as a model protein to characterize the fluorescence enhancement performance of the nanobrushes prepared in different grwoth conditions.The nanobrushes prepared in the microfluidic chip showed better fluorescence enhancement performance.2.Nanobrushes were prepared via the secondary nucleation method on microfluidic chip and the effect of secondary growth time and the density of primary seed layer on the morphology of ZnO nanobrushes were investigated.The results showed that the height,diameter and trunk diameter of the nanobrushes initially increased and then decreased with the increase of secondary growth time.Lower density of primary seed layer resulted in the growth of ZnO nanobrushes,whereas higher density resulted in the grwoth of ZnO nanorods.Among them,the largest surface area of single nanobrush could be obtained with the desity of 20 perμm~2 for the primary seed layer and 25 min for the secondary growth.3.The fluorescent detection of FITC-anti Ig G was performed to evaluate the fluorescence amplification of various ZnO nanobrushes arrays obtained in different secondary growth time and the density of primary seed layer.The diameter of the trunk met the requirement for the multi-mode transmission of waveguide and the abundant branches of nanorods located on the side walls of the trunk,which lead to significant enhancement of fluorescence.The fluorescence biodetetion performance of nanobrush arrays were investigated with vaired secondary growth time and varied density of the primary seed layer.The results showed that the nanobrushes array prepared when the secondary growth time was 20 min and the density of primary seed layers was 20 perμm~2 exhibited the strongest fluorescence enhancement performance.Herein,the notably increased surface area provided more biniding sites for target proteins and the enhanced evanescent wave penetated into the air medium when the light propagated in waveguides facilitated the fluorescence excitation.Therefore,the adsorption efficiency of fluorescent protein was improved.The ZnO nanobrushes array prepared under this condition was used to detect the lung cancer biomarker carcinoembryonic antigen(CEA)and cytokeratin 19 fragment antigen(Cyfra 21-1).The detection dynamic range of CEA was 1 pg/m L to 1μg/m L,while the detection limit was as low as 0.2 pg/m L.The detection dynamic range of Cyfra 21-1 was 10 fg/m L to 1μg/m L,while the detection limit was as low as 5.6 fg/m L.In addition,the biological detection had high specificity by using non-target protein to evaluate its detection specificity.Therefore,the microfluidic chip based on ZnO nanobrushes array can provide a reliable scheme for high-throughput,highly sensitive and highly specific detection of biomarkers of lung cancer,which was of great significance for the early diagnosis of lung cancer.