| Acute kidney injury(AKI)is one of the most common renal emergencies,with a high misdiagnosis rate and high mortality rate,and inadequate diagnosis,which can delay the early treatment and prognosis and lead to chronic kidney disease(CKD).Early detection of AKI through biomarker identification is an important way to improve patient health and reduce healthcare costs.In this study,we developed a paper-based colloidal gold multichannel vertical flow assay(VFA)for the rapid detection of three AKI biomarkers in a test kit,This lipid carrier protein includes neutrophil gelatinase-associated lipid carrier protein(NGAL),Cystain C(CysC)and retinol binding protein4(RBP4).In this system,specific capture antibodies were pre-immobilized on the sensing membrane at four spatially isolated immunoreactive sites.After the antigen solutions for NGAL,CysC,and RBP4 were added and inhaled into the central reaction area of the cassette,the gold nanoparticles were conjugated with detection antibodies as the colorimetrical labels,and the gold nanoparticle(Au NPs)antibody conjugate solution was added to the sample wells.After the reaction,The resultant images could be read out qualitatively by the naked eyes,or the detection result was imaged using a smartphone-based reader device,and the colorimetric signal was quantitatively evaluated.The calibration curves were fitted by a four-parameter logistic equation,y represented the colorimetric intensity at the test points,and x represented the corresponding concentration of the biomarkers.The limits of detection(LODs)for NGAL,CysC,and RBP4,which were estimated from the sigmoidal fitting curves on the basis of three times the standard deviation(SD)of the blank control signals,were1.04,0.96,and 1.17 ng/m.In this study,a single VFA test kit can detect three AKI biomarkers simultaneously in 10-15 minutes with a sensitivity of 5 ng/ml.Several parameters of this kit were optimized,such as the composition of the resuspension buffer,the size of colloidal gold particles,and the order of detection and capture antibodies.In addition,we evaluated and validated the reliability of the VFA kit in clinical serum samples in this study.Fifty clinical patients were selected and divided into two groups,namely 25 patients with renal failure and 25 healthy people,and their serum samples were detected.ROC curve was drawn for the test results,and the area under the ROC curve(AUC)of NGAL,CysC and RBP4 markers was 0.89,0.92 and 0.86,respectively.The cutoff values for NGAL,CysC,and RBP4 were determined as 82.95,86.20,and 114.50,respectively.The detection sensitivity and specificity determined by a single biomarker were 88% and 84% for NGAL,88% and 88% for CysC,and 76%and 80% for RBP4.The combined sensitivity and specificity of the three biomarkers were 92.00% and 88.00%,respectively.In general,the VFA detection system established in this experiment has high sensitivity and specificity for the detection of NGAL,CysC and RBP4.The VFA test system developed in this study can significantly distinguish between healthy individuals and patients with AKI.In conclusion,our paper-based colloidal gold multichannel VFA can provide a supportive basis for the diagnosis of AKI and contribute to the early detection of AKI to improve patient outcomes.We believe this is a promising diagnostic tool for multiple tests of disease biomarkers at the point of care.This vertical flow assay platform is easy to use,rapid,and low cost.It is a promising diagnostic tool for multiplex detection of disease biomarkers at Point of care testing(PDCT). |
