Competitive multi-immunosensing of pesticides based on the particle manipulation with negative dielectrophoresis.

In this work, we have applied particle manipulation based on negative dielectrophoresis (n-DEP) to develop rapid and separation-free immunosensing systems. Two widely used pesticides, atrazine and bromopropylate, were used as target molecules to demonstrate competitive immunosensing based on the rapid manipulation of microparticles. A suspension of the fluorescence microparticles modified with a specific antibody was injected into the n-DEP device consisting of the interdigitated microarray (IDA) electrode and indium-tin-oxide (ITO) substrate immobilized by protein conjugation with antigen. The application of 2 MHz AC voltage (16 V peak-to-peak) to the IDA forced most of the particles to form a line pattern on the upper ITO over the gaps of IDA within 60s. In the absence of analytes, patterned microparticles were irreversibly captured on the ITO by the construction of immuno-complexes. When the microparticles bearing anti-atrazine IgG antibody were suspended in an analyte (atrazine) solution, irreversible capturing of microparticles on the ITO was inhibited because of the occupation of the binding sites of the antibodies with free-atrazine. As a result, the analyte molecules were re-dispersed from the ITO to disintegrate the line formation after turning off the voltage. We could discriminatively detect the fluorescence intensity of the captured microparticles at the designated areas from that of the uncaptured microparticles suspended in the solution. Thus, the separation steps usually required for conventional immunoassay are eliminated in the present procedure. A pre-incubation of microparticles for 3 min in an orange juice solution containing analyte allowed for the determination of the atrazine and bromopropylate concentrations with a limit of detection of 4 and 1.5 microg L(-1), respectively, providing sufficient detectability to achieve international regulations regarding pesticide residues in food samples. The assay was significantly accelerated by the rapid particle manipulation with n-DEP and totally accomplished within 5 min. We also demonstrated the possibility of the simultaneous determination of two pesticide residues by using the DEP devices with two channels modified with specific competitors for atrazine and bromopropylate.