Most protein microarrays based on the theory of immobilized metal affinity chromatography (IMAC) were fabricated using the chelator or compound of mono-metallic ion, such as Ni(2+), to capture the histidine-tagged protein. In this study, a novel protein chip with Ni-Co alloy layer fabricated on the substrate of printed circuit board by electrodeposition was developed. It is an innovative microarray surface with bi-metallic elements, i.e. by means of adding cobalt to enhance the specific binding capability to immobilize functional proteins with His-tag attached. Taguchi method was adopted to determine the optimal electrodeposition condition using L18 orthogonal array. Then, the immunoassay was utilized to investigate the properties of the protein chip, and the signal was detected by confocal scanner. The results show that the alloy coating belongs to codeposition of Ni-Co alloy. Due to the affinity adsorption between intermediate metal and protein, this chip provides specific binding. Compared with Ni-coated chip and nitrocellulose (NC) chip, this Ni-Co alloy chip is a high sensitive protein microarray because of high detected fluorescence intensity with low fluorescent background. The proportion of Ni-Co composition will affect the affinity of electroplated layer with proteins and, consequentially, influence the results of immunoassay. Moreover, the test of long-term sensibility of Ni-Co chip showed that it had better binding capability in the first two weeks for coating buffers of pH 7.4 and pH 9 and the immobilization ability began to decay after the third week. The advantages of this novel protein chip include good performance, high repeatability, and inexpensive.
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