MIPs as antibody mimics in SPE and automated on-line fluorescent competitive assays of penicillins

Aim

MIPs can be engineered to exhibit good sensitivity and specificity for various analytes of medical, environmental and industrial interest. They are also highly robust, showing excellent operational stability under a wide variety of conditions.
The aim of the project is to develop an SPE method and an automated MIA for `β`-Lactam antibiotics compatible with aqueous samples.

Approach

An SPE method and a flow-through solid-phase competitive assay were developed by the use of a stoichiometrically imprinted polymer showing good target binding in aqueous media. This polymer was prepared using Penicillin G procaine salt (PenG) as the template molecule and a stoichiometric quantity of a recently developed urea-based functional monomer to target the single oxyanionic species in the template molecule. Ethyleneglycoldimethacrylate served as crosslinker and methacrylamide as an additional hydrogen bonding comonomer.

Results

As a proof of concept, the optimized fluorescent MIA has been applied to the analysis of PenG in human urine samples. With this aim, a calibration curve was prepared by spiking the urine samples with increasing concentrations of PenG potassium salt in the range of 10^-8-10^-3 M. The analytical characteristics of the assay were: dynamic range 7.87*10^-7 to 1.71*10^-5 M, detection limit 2.97*10^-7 M and EC50 4.00*10^-6 M.
Urine samples were fortified with increasing concentrations (4*10^-6 and 8*10^-6 M) of PenG and analyzed using the optimized method and HPLC-DAD as an alternative technique for validation purposes. No significant differences at a 95% confidence limit were observed between the results obtained by both methods. The LOD (Limit of detection) for the analysis of PenG is much lower than that obtained using the same labels and a methacrylic acid-based molecularly imprinted polymer, and only thirty times higher than that obtained using anti-PenG antibodies as the recognition element. Nevertheless, the MIA provides shorter analysis times and the cost per analysis is drastically reduced in comparison to immunoassays.

Figure 22: Dose-response calibration curves (n = 3) for PENG (from 0.0013 to 890 ``8``M), for the MIP (...), the NIP (...), and in urine samples for the MIP (...)

Funding

Joint project: Sellergren, INFU and Prof. Moreno Bondi, University of Madrid.

Contact

Priv.-Doz. Dr. Börje Sellergren
B.Sellergren@infu.uni-dortmund.de
Phone: +49-(0)231-755-4082

Paper

1. Urraca, J. L.; Moreno-Bondi, M. C.; Sellergren, B. (2007):
   "Direct extraction of penicillin and derivatives from aqueous samples using a stoichiometrically imprinted polymer" Anal. Chem., 79 (2), 695-701.

2. Urraca, J. L.;Hall, A.; Moreno-Bondi, M. C.; Sellergren, B. (2006):
   "A Stoichiometric Molecularly Imprinted Polymer for the Class-Selective Recognition of Antibiotics in Aqueous Media" Angew. Chem. Int. Ed., 45, 1-5.