Introduction Electrochemiluminescence (ECL) method is attractive in terms of high sensitivity and low background. In ELISA method, ECL sensor uses antibody as recognition element and utilize enzyme reaction as enhancer of ECL, but in this study, we use new enzyme-like catalytic reaction of gold nanoparticle (AuNP) instead of enzyme. New catalytic reaction of AuNP produces hydrogen peroxide from dissolved oxygen in presence of amine. We can utilize this reaction for ECL measurement because hydrogen peroxide enhances ECL intensity of luminol. AuNP can be modified with antibody easily and more resistant to pH change and high temperature than enzyme, so it’s expected for this method to be simpler and more stable ECL measurement. In addition, we fabricate the electrode which has a lot of microwells above anode and cathode, and conduct this ECL measurement in microwells. If the AuNP concentration is the same, the smaller sample solution is, the fewer AuNPs exist. When sample volume is small enough, we can obtain ECL signal from just one AuNP theoretically. In that case, the number of AuNP can be directly counted from the presence or absence of light emission from each well, and concentration of target substance can be calculated without calibration curve. We define this sensing method as “digital sensing”. In this study, we try to obtain ECL signal from smaller sample volume and fewer AuNPs for digital sensing. Experiment The electrode is made by photolithography and metal sputtering. Positive photoresist (AZ P4330-RS, Merck) patterning is made on the glass (Matsunami) by photolithography and Titanium and Gold are sputtered on the patterned glass. Titanium serves to bond glass and gold. After washing with acetone(Fujifilm), only electrode pattern remains. Further, negative photoresist (SU-8, KAYAKU MICROCHEM) is spin-coated on the substrate, and photolithography develops microwells. Completed electrode is put under the microscope (MVX10, OLYMPUS) and CCD camera (ImagEM, Hamamatsu). It is connected to a potentiostat (µPMT-STAT, BioDeviceTechnology) and applied with various type of voltage. All equipment for this measurement is covered with a black curtain. The sample solution is made of AuNP (Sigma Aldrich) and tris(hydroxymethyl)aminomethane (Tris, Wako) buffer and luminol (Wako). Tris is added as not only buffer but also amine co-reactant, and luminol is added as luminophore. 10 ~ 20 µL of sample solution is dropped onto the substrate and cover glass is put on it and pressed lightly. Then the solution fills the microwells and excessive solution is removed. We have optimized various conditions of measurement such as thickness of electrode, applied potential, design of electrode and so on in order to make the electrode more sensitive to hydrogen peroxide produced by AuNP. Further we have made the volume of microwells smaller as possible as we can. Result & Discussion We’ve succeeded the fabrication of the electrode which has multiple microwell structures and we could obtain ECL signal from that device. About 13nL is the smallest volume from which we obtain ECL signal and 87.5 NPs/nL is the lowest AuNP concentration we could detect the production of hydrogen peroxide. This result means we can detect the production of hydrogen peroxide from one AuNP if we fabricate wells whose volume is about 10 pL. Further, we found this electrode emits ECL when we applied chronoamperometry (CA), and 500mV is the best. By modifying antibodies to gold nanoparticles and wells, application to various target substances can be expected. Figure 1