In this work, we designed a new immunodevice that combines competitive immunoreactions on the microparticles, accumulation of these particles by negative dielectrophoresis (n-DEP), and their subsequent capture through hybridization among single-stranded DNAs (ssDNAs). Two widely used pesticides, atrazine and bromopropylate, were used as target molecules to test the resulting simultaneous detection system. For sensing, we prepared two different sets of microparticles: one modified with atrazine-conjugated bovine serum albumin (BSA-2d) and ssDNA-J1up and the other with bromopropylate-conjugated aminodextran (AD-155) and ssDNA-J2up. The microparticles were incubated in a mixture of analyte-specific antibody and analyte at different concentrations to trap the unreacted antibodies prior to being labeled with antibodies conjugated with a fluorescence molecule. A suspension containing both types of microparticles was introduced into an n-DEP device consisting of an interdigitated microarray (IDA) electrode and channel modified with ssDNA-J1down and ssDNA-J2down, which are complementary to ssDNA-J1up and ssDNA-J2up, respectively. The n-DEP force generated by applying AC voltage to the IDA electrode displaced the microparticles toward the encoded areas, causing them to rapidly accumulate on the upper surfaces. Hybridization allowed us to distinguish the microparticles and sense multiple analytes by spatial recognition in the DNA-encoded areas. The fluorescence intensity of the captured particles, which depends on analyte concentrations, was measured selectively by focusing on specific areas. The strategy is advantageous for sensitivity due to the equivalent trapping efficiency by DNA hybridization and large surface area of the microparticle for immunoreactions. The rapidity and simplicity were still supported by particle manipulation. Using this concept, we detect atrazine and bromopropylate simultaneously with limits of detection (LODs) of 0.2 μg· L -1, which covered the maximum residue level (MRL) in food samples established the European Union (EU) and Japan Ministry of Health, Labor and Welfare (MHLW).
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