Development Of Multianalyte Immunoassay On Paper-based Immunochipand The Validation Of Smartphone-based Portable Device

Development of multianalyte immunoassay to detect contaminants in water is of great significance.In this study,the multianalyte detection mode was developedon96well plates and the immunoassay was developed onpaper-based immunochip.The accuracy of smartphone on the signal obtaining and analyzing was initially explored.The results showed that the combination of paper-based immunosensors with smartphone-based portable device to achieve the rapid,on spot,multi-detection has great possibilities.?1?Panning of antibodies and coating antigensBased on possibly existent contaminants in water,such as MC-LR,and other antibiotics,corresponding 10 coating antigens and 11 antibodies?both ployclonal antibodies and monoclonal antibodies?were collected.After deliberate panning,three combinations without interference were selected to achieve the subsequent multi-detection.The selected three targets are MC-LR,furaltadone,and pazufloxacin.The coating antigens and antibodies used in multi-detection have a low shared-reactivity,which is below 15%;and the target molecules could only inhibit the binding of its own coating antigen and antibody,while have no influence on other combinations.?2?Development of multianalyte detection mode on 96well platesCalibrate curves of the three target molecules both in separate detection and mixed detection forms have been plotted,then the sensitivity under two forms were compared.As for MC-LR,the limit of detection is 3 pg/mL in separate detection form and 1.68 pg/mL in mixed detection form;IC₅₀ is 58.73 pg/mL and 57.38 pg/mL,respectively;the testing range is from 9.01 to 382.99 pg/mL,which is narrower than the one obtained in mixed form ranging between 4.43-742.61 pg/mL.As for furaltadone,the limit of detection is 11.17ng/mL in separate detection form and 6.34 ng/mL in mixed detection form;IC₅₀ is 118.55ng/mL and 73.29 ng/mL,respectively;the testing range is 26.76-525.21 ng/mL in separate form,and 15.6-344.21 ng/mL in mixed form.The sensitivity value is not so similar,however,it is in the accepted range for variation.As for pazufloxacin,the limit of detection is quite similar,showing 0.29 ng/mL;the IC₅₀ is 4.80 ng/mL and 3.92 ng/mL,respectively;the detection range is 0.81-28.56 ng/mL in separate form,which is very closed to the one in mixed form demonstrating 0.76-20.14 ng/mL.The results on 96 well plates demonstrated that the selected three combinations of coating antigens and antibodies co uld satisfy the requirement of multi-detection,thus accurate quantitative analysis for each target molecule can be obtained by using such multianalyte immunoassay.?3?Development of immunoassay on paper-based immunochipUsing MC-LR as the model,a series of factors that influence the performance of paper-based immunochip were optimized.The results showed that the 0.22?m pore of NC membrane,carbonate buffer as the spotting buffer is in favour of the fixing of coating antigen;5%non-fat milk can be used as block buffer whose blocking effect can match the market ones;the best reacting time is fixing for 1 hour,blocking for 30min,incubation with antibodies for 15min,and incubation with second antibody for 15 min;the reactant volume is 1mL;the spotting formats have no influence on the color of final spot.Then the specificity between three combinations were assessed again,no obvious cross-reaction was observed,which is consistent with expectation.Finally,the working concentration of coating antigen and antibody forthe three analytes was optimized,threestandard curves were obtained.Thelimit of detection for each analyte is 0.493 ng/mL,0.105 ng/mL,0.706ng/mL,respectively,satisfying the requirement of detection.?4?Exploration on the accuracy of smartphoneThe paper-based immunochip was imaged by the smartphone-based portable device,then the signal was analyzed using the smartphone APP.The standard curve plotted by the smartphone quite fit the one plotted by the scanner,with LOD achieving 0.778 ng/mL and0.706 ng/mL,respectively.Then 36 unknown samples were measured by the standard curves on both smartphone and scanner,and the correlation coefficient is as high as 0.90.Based on the current result,The combination of paper-based immunochip with smartphone-based portable device to analyze the world live water samples is foreseen to be accomplished.