Opal Photonic Crystal-enhanced Upconversion Turn-off Fluorescent Immunoassay For Salivary CEA With Oral Cancer

Background:Globally,oral cancer is the sixth most common type of cancer,in which oral squamous cell carcinoma dominated about 90%aggressive cases.Although modern medical strategies have made excellent progress in prevention and treatment,the overall 5-years survival rate of oral cancers has not significant improved in the last decades due to diagnostic delay,which would make the patients miss the optimal timing of professional treatment.Early and accurate diagnosis of oral cancer can be a more effective means of improving survival rates,reducing the high mortality and economic burden of health care.Currently,the most common procedures for detecting precancerous lesions and cancers of the oral cavity include visualization and palpation,followed by confirmation by tissue biopsy.Recently,biomarkers have been widely used for the detection of cancer.Carcinoembryonic antigen（CEA）,a broad-spectrum cancer biomarker present in serum,has been positively correlated in concentration with the degree of occurrence and progression of oral cancer.However,diagnostic aids by CEA in serum for the detection of oral cancer has unsolved problems on detective sensitivity and accuracy.The numerous interfering substances in blood would hinder the precise detection of CEA molecules.In addition,high CEA content can also be served as related markers of other types of tumors.Thus,the determination of CEA in serum will not definitively and accurately support the diagnosis of oral cancers.Saliva is more suitable for point-of-care testing（POCT）than blood due to its advantage of painless and non-invasive sampling.Compared with traditional diagnostic tools,saliva testing reduces the cost of time and resources and will most likely take the place of traditional blood diagnosis.Furthermore,in the clinical situation of oral cancer,the location of the tumor in close proximity to the oral cavity makes it easier for tumor markers to enter the saliva directly,in addition to the blood and lymphatic circulatory systems.Therefore,rapid and continuous monitoring of tumor marker（e.g.,CEA）levels in saliva will lead to important breakthroughs in oral cancer diagnosis.Among the various methods for detecting biomarkers,optical sensing based on upconversion fluorescence is widely used for its low toxicity,low damage to biological samples,and low background noise.While photonic crystals provide significant advantages for upconversion fluorescence detection,the combination builds a chip-based sensing platform that is easy to operate and very portable,which will greatly advance the development of oral cancer diagnosis.Objective:The turn-off biosensor based on opal photonic crystal（OPC）enhanced upconversion fluorescence is proposed to detect the carcinoembryonic antigen（CEA）in saliva by applying fluorescence resonance energy transfer sensing strategy.Methods:1.Na YF₄:Yb³⁺,Er³⁺upconversion nanoparticles were synthesized by the oil thermal method,and the oleic acid（OA）ligands formed on the surface of UCNPs by the oil thermal method were washed off and replaced with hydrophilic PEI ligands using suitable concentrations of hydrochloric acid.2.The UCNPs were characterized and determined by transmission electron microscopy（TEM）,Fourier infrared spectroscopy（FTIR）,and fluorescence spectroscopy.3.980 nm PMMA microspheres were synthesized by hydrothermal method,and the formed PMMA films were treated for water resistance.The UCNPs were modified on the OPC film after growing a PDA film on top of the OPC film.4.The OPC/UCNPs films were characterized by scanning electron microscopy（SEM）,FTIR,transmission spectroscopy,and element mapping.The fluorescence intensity of OPC/UCNPs films and glass substrates modified with UCNPs were compared to verify the amplification effect of OPC on upconversion fluorescence.5.Water stability test,acid-base stability test and storage stability test were performed on OPC/UCNPs films to verify that the biosensing films meet the requirements of POCT,respectively.The OPC/UCNPs films modified with captured CEA antibodies were tested for anti-interference and proved to be highly specific for the detection of CEA.6.The sensitivity of the synthesized sensor was tested by adding different concentration gradients of CEA in PBS buffer.The sensitivity of the sensor for saliva detection was verified by adding the same concentration gradient of CEA to artificial saliva.Saliva from healthy individuals and oral cancer patients was collected to verify the feasibility of the sensor for application in real sample detection.Results:1.Na YF₄:Yb³⁺,Er³⁺were successfully synthesized by oil thermal method,and the PEI-UCNPs with the replaced ligand had better dispersion in water.The fluorescence intensity of UCNPs can still meet the requirements of the test after the replacement of PEI ligand.2.PMMA microspheres with the band gap at 980 nm were successfully prepared.A dense PDA film was grown on the surface of the PMMA film after waterproof treatment,and PEI-UCNPs were attached to the PDA film by chemical bonding.3.The treated OPC/UCNPs film enhanced the upconversion fluorescence of Na YF₄:Yb³⁺,Er³⁺itself by about 66 times.The fluorescence film also passed various stability tests,with good water stability,acid-base stability,storage stability,and excellent specificity for the target to be measured.4.The sensor was sensitive to different concentration of CEA dissolved in PBS buffer,and the intensity of fluorescence quenching increased with the increase of CEA concentration,and showed a linear trend in the range of 0.1-2.5 ng m L^-1 and>2.5 ng m L^-1,respectively,with the detection limit as low as 0.1 ng m L^-1.The same trend was observed for CEA dissolved in artificial saliva.5.The sensor was tested on actual clinical samples collected using the sensor.The test results clearly distinguish between healthy individuals and oral cancer patients.Conclusion:The CEA biosensor based on upconversion fluorescence resonance energy transfer designed and developed in this work enhanced the fluorescence intensity of UCNPs by using OPC structure as the detection base to address the problem of low efficiency of upconversion fluorescence.By optimizing the formation of OPC/UCNPs films,the upconversion fluorescence was enhanced by 66-fold,exhibiting higher detection sensitivity for the substances to be measured.The biosensor showed good linearity in the ranges of 0.1-2.5 ng m L^-1 and 2.5-20 ng m L^-1,respectively,with a detection limit of 0.1 ng m L^-1,which is sufficient to achieve the goal of detecting CEA in saliva.The entire assay can be completed within 90 minutes with easy operation,which is very beneficial for the diagnosis of oral cancer.