The Development Of Novel Fluorescence Quenching Immu-nochromatographic Strip And Application In The Detection Of Small Molecules

Objective: The establishment of novel immunochromatographic techniques with characters ofhigh-sensitivity and detection signal intensity was positively related to concentration of analyte, andusing this techniques to detdcts small molecules trivalent chromium ion (Cr³⁺) and clenbuterol （CL）.Methods: Anti-Cr³⁺-EDTA monoclonal antibody was labeled with gold nanoparticles （Au-NPs）to prepared fluorescent quencher （mAb-gold Quencher） and then dispensed on the conjugate pad. Byamide reaction, complete antigen Cr³⁺-EDTA-BSA labeled fluorescent nanoparticles （FNPs）(Cr³⁺-EDTA-BSA-FNPs) was prepared. Cr³⁺-EDTA-BSA-FNPs and a certain amount ofCr³⁺-EDTA-BSA were dispended on a specific area of the NC membrane as the test line （T-line）. BSAlabeled FNPs （BSA-FNPs） was prepared and dispended on a specific area of the NC membrane as acalibration line（C-line）. After the fluorescence quenching immunochromatographic strip （ICS） as-sembly was completed a series of process was carried on to optimize the performance of the devel-oped ICS. The prepared fluorescence quenching ICS was used for detected Cr³⁺in water, and the re-sults were analyzed by ImageJ software to obtain quantitative results. The specificity of the fluores-cence quenching ICS was studied. Cr³⁺spiked in tap water and detected by fluorescence quenchingICS, the results were compared with ICP-AES.The fluorescence quenching ICS for detect CL was prepared using the same method. After a se-ries of optimizations, performance of the ICS in porcine urine and specificity of the ICS was studiedResults: The detection of Cr³⁺was completed within15min. Detection signal of fluorescencequenching ICS was positively correlated with Cr³⁺concentration. When Cr³⁺concentration of sam-ples greater than1.56ng/mL, fluorescent signal of T-line was appeared, and with increasing of Cr³⁺concentration, fluorescence signal of T-line was also enhanced. The low detection limit of the ICSwas1.56ng/mL, the detection range of the strip is6.25ng/mL-800ng/mL. The developed ICS exists1.56%,15%and17.5%cross-reactivity with Ca2+, Fe³⁺and Fe2+, and no cross-reactivity with othermetals ion. The recoveries of fluorescence quenching ICS detect Cr³⁺in tap water were between94.7%-101.7%. This result is closed to the result obtained by ICP-AES.The developed fluorescence quenching ICS detects CL in PBS was complete with in7min. Un-der excitation light, the T-line fluorescence signal intensity of the prepared fluorescence quenchingICS was positively correlated and concentration of CL. The low detection limit of the ICS is0.04ng/mL. Under natural light, the prepared ICS could performed as a traditional colloidal gold ICS.Detection signal of T-line was inversely related to CL concentration of CL. The sensitivity of the stripis2.5ng/mL. CL in porcine urine was detected by prepared fluorescence quenching ICS. This detec-tion results were closed to the result performed in PBS. The fluorescence quenching ICS was studiedby detect clenbuterol, ractopamine, phenylethanolamine A, salbutamoland shown good specificity.Conclusions: Fluorescence quenching ICS for detection Cr³⁺and CL was prepared. The detec- tion signal of fluorescence quenching ICS was positively correlated with the concentrations of analyte.Because the characters of high sensitivity, low-cost, easy to operation, the developed fluorescencequenching ICS could be used as an effective means for the small molecules detection.