OBJECTIVETo develop a quartz crystal microbalance (QCM) immunosensor with high sensitivity and selectivity for the rapid detection of diethylstilbestrol.

METHODSDextran was used as reducing agent for preparing gold nanoparticles (AuNPs) with the size of 40 nm. The AuNPs were coupled with anti-DES antibody after amination. A monolayer was generated after immersing the quartz crystal into the solution of 5 mmol/L 11-mercaptoundecanoic acid(MUA) for 16 hours. After the monolayer was activated by 1-ethyl-3-(3-dimethylaminopropry) carbodiimide hydrochloride (EDC·HCl) and N-hydrosuccinimide (NHS), 20 μl of 2.2 mg/ml DES-HS-BSA was dropped onto the surface of crystal to prepare a sensitive membrane which can recognize DES specifically. Then, 50 μl of 1 mol/L ethanolamine (pH 8.5) was used to seal the carboxylic groups to make the sensitive membrane which could identify DES specifically. QCM immunosensor was used as detection platform to optimize the reaction conditions. Under the optimized conditions, 10 μl of 28 μg/ml AuNPs-antibody was mixed with 10 μl of 0.03-2.5 μg/ml DES, and the mixture was added on the sensitive membrane. QCM immunosensor was used to detect the signals and the standard curve was obtained at the same time. The detection limit was calculated based on the standard curve. The specificity was evaluated by testing DES and its analogues with the same concentration.

RESULTSThe optimized concentration for the immobilization of DES-HS-BSA on the surface of QCM was 2.2 mg/ml. The optimized concentration for coupling anti-DES antibody with AuNPs was 7 μg/ml and 15 nmol/L, respectively. The optimized concentration of AuNPs-antibody was 14 μg/ml. The logarithm of DES concentration was proportional to the frequency shift in the range of 0.16-500 ng/ml, Δf=-24.170 lgCDES+69.71, R(2)=0.998. The detection limit of this method was 0.13 ng/ml. DES analogues could not influence the detection of DES obviously, so the sensor had good specificity.

CONCLUSIONThe quartz crystal microbalance immunosensor with gold nanoparticals amplification could detect DES sensitively and rapidly.