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Herein, an innovative glyphosate imprinted poly(hydroxyethyl methacrylate-N-methacroyl-(L)-phenylalanine methyl ester nanoparticles (MIP@NPs) based plasmonic nanosensor featured with high sensitivity and selectivity was constructed by using the molecular imprinting technique and used for real-time glyphosate detection. The characterization of nanoparticles was performed by the nano Zetasizer and scanning electron microscopy (SEM), while nanosensors were characterized by the Fourier transform infrared-attenuated total reflection (FTIR-ATR) and contact angle measurement. Control experiments were conducted to evaluate the imprinting efficiency on the signal response using a non-imprinted surface plasmon resonance (NIP SPR) nanosensor prepared without adding glyphosate pesticide into the polymerization mixture. The MIP@NPs integrated molecularly imprinted surface plasmon resonance (MIP SPR) nanosensor having synthetic molecular recognition elements yielded a novel biosensing platform for label-free detection and real-time monitoring of glyphosate pesticide. The MIP SPR nanosensor detected the target glyphosate molecule 4.950 times more selectively than the competitor molecule malathion while 3.918 times more selectively than the competitor molecule malaoxon. In addition, the imprinting efficiency factor was found to be 6.76, indicating that the molecular imprinting process was successful. In addition, the imprinting factor was found to be 6.76. Kinetic studies and adsorption characteristics of glycosate adsorption were carried out to assess adsorption dynamics. The linear concentration range for glyphosate detection was 0.001 ppm–10.000 ppm of pesticide, and the detection limit was found to be 0.120 ppb. Studies on the repeatability of the MIP SPR nanosensor revealed that even after five cycles, the signal response for glyphosate detection did not change significantly with relative standard deviation, RSD<1.5 value. The artificial urine selected as the real sample was spiked with glyphosate at a final concentration of 10.000 ppm to evaluate the matrix effect, and the glyphosate amount was reported.
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