Molecularly Imprinted Nanoparticles Assay (MINA) in Pseudo ELISA: An Alternative to Detect and Quantify Octopamine in Water and Human Urine Samples

Abstract

In 2004, octopamine was added to the list of drugs banned by the world anti-doping agency (WADA) and prohibited in any sport competition. This work aims to develop a new analytical method to detect octopamine in water and human urine samples. We proposed a pseudo-enzyme-linked immunosorbent assay (pseudo-ELISA) by replacing traditional monoclonal antibodies with molecularly imprinted polymer nanoparticles (nanoMIPs). NanoMIPs were synthesised by a solid-phase approach using a persulfate initiated polymerisation in water. Their performance was analysed in pseudo competitive ELISA based on the competition between free octopamine and octopamine-HRP conjugated. The final assay was able to detect octopamine in water within the range 1 nmol·L^-1-0.1 mol·L^-1 with a detection limit of 0.047 ± 0.00231 µg·mL^-1 and in human urine samples within the range 1 nmol·L^-1-0.0001 mol·L^-1 with a detection limit of 0.059 ± 0.00281 µg·mL^-1. In all experiments, nanoMIPs presented high affinity to the target molecules and almost no cross-reactivity with analogues of octopamine such as pseudophedrine or l-Tyrosine. Only slight interference was observed from the human urine matrix. The high affinity and specificity of nanoMIPs and no need to maintain a cold chain logistics makes the nanoMIPs a competitive alternative to antibodies. Furthermore, this work is the first attempt to use nanoMIPs in pseudo-ELISA assays to detect octopamine.

Keywords: ELISA; doping; molecularly imprinted nanoparticles assay; molecularly imprinted polymers; octopamine.

Figure 1

(A) Procedure for the immobilisation of octopamine on the surface of the solid support (glass beads). (B) Scheme of the synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) made for octopamine. Low-affinity particles and oligomers of the synthesis were washed at 4 °C. High-affinity nanoMIPs were collected at 60 °C.

Figure 2

TEM image of nanoMIPs for octopamine.

Figure 3

Binding of the horseradish peroxidase-octopamine (HRP-O) conjugate to the nanoMIPs immobilised in a microplate and uncoated microplates. Error bars represent the standard deviation for experiments performed in triplicate.

Figure 4

Calibration curve for MINA assay. Light blue line indicates binding of octopamine to octopamine specific nanoMIPs (circles). Purple line indicates binding of octopamine with labetalol specific nanoNIPs (squares). Dark blue line indicates binding of octopamine to blank, uncoated microplates. Error bars represent the standard deviation for experiments performed in triplicate.

Figure 5

(A). Optimisation of the MINA assay in human urine. Urine was separated into two groups; filtrated and unfiltrated samples. Then the samples were separated into four different dilutions. (B) MINA calibration curve for the best two dilutions of filtrated human urine (1:100 and 1:1000). Error bars represent the standard deviation for experiments performed in triplicate.

Figure 6

(A) Calibration curve for MINA performed in microplates with immobilised nanoMIPs for octopamine. Competitive assay performed with different concentrations of; octopamine, labetalol, pseudoephedrine and l-Tyrosine solutions in human urine samples. Error bars represent the standard deviation for experiments performed in triplicate. (B) MINA assay with nanoNIPs immobilised made for Atenolol. (C) Chemical structures of four drugs used in the cross reactivity assay.