Novel Nanozymes-Based Label-Free Chemiluminescence Imaging Multianalyte Immunoassay Methods

Due to the complexity and dynamics of the human immune system,current clinical cancer screening and disease diagnosis require simultaneous detection of multiple disease markers.Multianalyte chemiluminescence immunoassay based on antibody arrays has become more and more popular due to its ability to achieve high sensitivity,high throughput,wide linear range,real-time,in situ and in vivo detection.However,the current chemiluminescence multianalyte immunoassay has the problems such as requiring labeling,separation and multiple washing steps,time-consuming and cumbersome operation,and large consumption of reagents.Label-free immunoassay require no label process for antigens or antibodies in advance,as well as separation steps,thus effectively avoiding the inactivation of antigens or antibodies due to labeling and separation processes.So this assay mode has the obvious advantages such as less reagent consumption,faster analysis,and easier operation.Compared with the natural enzymes commonly used in chemiluminescence analysis systems,nanomaterials with peroxidase activity have excellent catalytic activity,simple preparation,stable properties,and controllability.Therefore,this paper proposed a new strategy and new method for label-free chemiluminescence imaging multianalyte immunoassay?CLI-MIA?based on a variety of novel nanozymes sensing arrays.Array-based CLI-MIA strategy integrates low cost,stability and controllability of nanozymes,high sensitivity of chemiluminescence detection,high specificity of immunoassays,and high detection throughput achieved by inductively coupled CCD imaging based on antibody arrays.Here,metal hydroxide nanomaterials,hollow bimetallic nanospheres,porous bimetallic core-shell nanomaterials and metal-organic framework derivative nano-octahedrons were synthesized,and their peroxidase activities were explored in detail.And then these nanozymes were applied to the construction of label-free chemiluminescence imaging multianalyte immunosensor arrays for label-free,high-throughput,high-sensitivity,low-consumption,reliable,and rapid detection of multi-tumor markers.The main research is as follows:?1?In the present work,a novel label-free chemiluminescence imaging muitianalyte immunoassay method was designed by employing 3D Cu?OH?₂ supercages?SCs?as peroxidase mimetics.3D Cu?OH?₂ SCs were prepared by self?assembly of ID Cu?OH?₂ nanoribbons through a low-energy green synthesis procedure.3D Cu?OH?₂ SCs dispersed in chitosan were entrapped into the epoxy functionalized microwells array to form a nanozyme sensing interface.The biofunctionalized complex has large reaction surface area and excellent biocompatibility,and thus various biotinylated antibodies can be efficiently immobilized in the streptavidin functionalized sensing array.After online incubation with the corresponding antigens,the formed immune complexes could prevent the diffusion channel of chemiluminescent substrate toward the 3D Cu?OH?₂ SCs nanozyme sensing interface,finally resulting in the decrease of CL signals.Herein,the CLI signals of each of the sensing microwells in the array were collected,thereby enabling multi-component quantitative detection.Using AFP,CEA,CA 125 as model group,the linear ranges of the MIA were 0.0010-100 ng/mL?AFP?,0.0010-75 ng/mL?CEA?,0.0010-200 U/mL?CA125?and the detection limits were down to 0.16 pg/mL?AFP?,0.20 pg/mL?CEA?,and 1.7×10^-4 U/mL,respectively.Novel label-free CLI-MIA strategy based on Cu?OH?₂ SCs nanozymes has the advantages such as high throughput,ultra-sensitivity,low consumption,etc.This work provides new idea and platform for large-scale cancer screening and joint accurate diagnosis of cancer.?2?This study proposed a new label-free chemiluminescence imaging multianalyte immunoassay method based on porous PtAg bimetallic nanozymes for simultaneous detection of multi-tumor markers with excellent sensitivity,high detection throughput and acceptable stability.PtAg nanoparticles?PtAgNPs?are porous core-shell nano-octahedral structures with intrinsic peroxidase and oxidase bifunctional enzyme activities and are capable of maintaining long-term catalytic activity.The porous PtAgNPs were entrapped into an epoxy-silanized microwell array,and various biotinylated antibodies were specifically immobilized on streptavidin-functionalized sensor array.After incubation of the corresponding antigens,the formed immunocomplexes hindered the catalytic reaction of the porous PtAgNPs nanozyme toward the chemiluminescent substrate.The chemiluminescence brightness of different regions in the array was integrated by inductively coupled CCD camera integration,thereby enabling simultaneous detection of CA 125,CEA and AFP.The label-free CLI-MIA strategy based on porous PtAg bimetallic nanozymes shows good linearity in the concentration range of 0.0010-80 U/mL?CA 125?,0.0010-75 ng/mL?CEA?,0.0010-80 ng/mL?AFP?and the detection limits were 1.3×10^-4 U/mL?CA 125?,0.10 pg/mL?CEA?,0.13 pg/mL?AFP?,respectively.The label-free CLI-MIA array has practical application potential in detection of real samples and exhibits excellent stability and specificity.?3?This study proposed a novel label-free chemiluminescence imaging multianalyte immunoassay?CLI-MIA?for simultaneous detection of multi-tumor markers in breast cancer based on hollow PdNi nanospheres?PdNi hNPs?.PdNi hNPs have dual enzyme activities of oxidase and peroxidase.Using the excellent peroxidase activity of PdNi hNPs,nanozymes sensing array was prepared by embedding PdNi hNPs into epoxy-silanized microwell array.Various biotinylated antibodies were then immobilized on sensing array by the streptavidin-biotin system.After blocking the non-specific site by bovine serum albumin,various antigens were added to the corresponding sites for online incubation.The immunocomplexes produced by the specific reaction can hinder the catalysis of the PdNi hNPs nanozymes toward the chemiluminescent substrate.The inductively-coupled CCD camera integrates the chemiluminescence brightness of different regions to achieve label-free,high-throughput,and rapid detection of multi-tumor markers.The signal values of chemiluminescence imaging collected from the microwell region were plotted against the logarithmic values of the tumor marker concentration,and the working curves for detecting various markers can be obtained.The CLI-MIA strategy based on PdNi hNPs nanozyme sensing array enables simultaneous detection of CEAS CA 125,and CA 15-3 in the range of 0.0050-75 ng/mL,0.0010-100 U/mL and 0.0010-240 U/mL.and can be successfully applied for real sample detection.The proposed label-free CLI-MIA can be used for early screening and clinical diagnosis of cancer,and has promising clinical application potential.?4?In this work,Cu/CuO/Cu₂O nano-octahedral metal organic framework materials?MOFs?derivative was synthesized by utilizing Cu-BTC as precursors.It was firstly found that Cu/CuO/Cu₂O has excellent peroxidase activity and applied to construct label-free chemiluminescence imaging immunoassay for label-free,ultrasensitive and high-throughput detection of multi-tumor markers.The chitosan-dispersed Cu/CuO/Cu₂O nanozyme was embedded in epoxy-silanized microwell array,and various biotinylated antibodies were specifically immobilized into the microwell sensing array by streptavidin-biotin system.After online incubation with the corresponding antigens,the formed antibody-antigen complexes can prevent the diffusion channel of chemiluminescent substrate toward the Cu/CuO/Cu₂O nanozyme sensing interface,finally resulting in the decrease of CLI signals.Herein,the luminescence brightness of each of the sensing microwells in the array were collected,thereby achieving high-throughput,ultra-sensitive,low-consumption detection of multiple biomarkers.CEA,CA 125 and CA 19-9 as model the CLI-MIA shows linear ranges of 0.0010-75 ng/mL?CEA?,0.0010-500 U/mL?CA 125?,0.0010-240 U/mL?CA 19-9?and the detection limits down to 0.25 pg/mL?CEA?,2.0×10^-4 U/mL?CA 125?,1.4×10^-4 U/mL?CA 19-9?.The MOFs derivative nanozyme-based label-free immunosensing array has the advantages such as reliability,sensitivity,simplicity,and high throughput,and provides new idea and platform for the development of multiple biosensing assay.