Development Of An Up-Converting Phosphor Technology-Based Immunochromatographic Chip And Its Application In The Detection Of Biomarkers For Infection

Objective:For multiple and rapid analysis,as well as providing an alternative method for the multiple immunochromatography,an up-converting phosphor technology-based immunochromatographic chip(UPT-chip)for simultaneous detection of C-reactive protein(CRP)and serum amyloid A(SAA)in serum was developed.Methods:Standards was used to evaluate the detection performance of up-converting phosphor technology-based lateral flow assay(UPT-LFA)for CRP or SAA.SAA-UPTLFA was used to establish a microarray horizontal four-dots UPT-chip model,and the stability of the model was tested with a single target detection instrument.Cooperated with a company to develop the biosensor of UPT-chip.CRP-UPT-LFA and SAA-UPTLFA were used to establish the UPT-chip for simultaneous detection of CRP and SAA,and the arrangement of microarray,the number of channels,the use volume of samples and the reaction time of UPT-chip were screened to complete the preliminary optimization.Finally,the detection performance of UPT-chip was evaluated with standards and serum samples,including detection range,cross-reaction and method comparison.Results:The linear ranges of CRP-UPT-LFA and SAA-UPT-LFA were both 1.56 mg/L to150 mg/L.On the basis of the SAA-UPT-LFA,a 10 mm wide UPT-chip model with four horizontal dots(four-channels)was established,and each channel showed good stability(CV ≤ 18.8%)for detecting the standard substance of SAA.The biosensor of UPT-chip was developed to correctly collect the signal values on each channel.UPTchip used for simultaneous detection of CRP and SAA with three horizontal dots was successfully established and preliminary optimized.The technical parameters of the optimized UPT-chip were as follows.The width of the chip was 10 mm;the number of channels was three(for CRP detection,SAA detection and quality control,respectively);the antibody volume for each channel on the nitrocellulose membrane was 80 nl;the use volume of samples was 225 μl during detection;and the reaction time was 15 min.The linear ranges of UPT-chip for the detection of CRP or SAA were both6.25 mg/L to 75 mg/L.Using the UPT-LFA(CRP-UPT-LFA or SAA-UPT-LFA)as a compared method,52 serum samples were quantitatively detected by UPT-chip for analysis of CRP and SAA.A strong correlation between the two methods on the quantitative results of CRP or SAA(CRP: R = 0.9117,P < 0.001.SAA: R = 0.8282,P< 0.001)were found.If the concentrations of CRP and SAA of the sample were in the range of 0 mg/L to 75 mg/L,CRP had little influence for the detection result of UPTchip for SAA,however high concentrations of SAA(75 mg/L)had slight influence for the detection result of UPT-chip for CRP.Conclusions:In this study,the UPT-chip with three horizontal dots with a specific biosensor was successfully established for simultaneous quantitative detection of serum CRP and SAA within 15 min,providing an alternative method for the multiple immunochromatography,as well as a novel idea for the development of new multiple immunoassay technology.The UPT-chip can be also applied for other field,such as multiple detection of food-borne pathogens or toxins related to food safety,quarantine of imported and exported infectious diseases,etc.