A universal multicolor immunosensor for semiquantitative visual detection of biomarkers with the naked eyes

3, 3', 5, 5'-tetramethylbenzidine (TMB) has been widely used as a chromogenic substrate for colorimetric immunoassays. Normally, the colorless TMB is oxidized into yellow TMB²⁺ (in acidic solution) to indicate the presence of the target molecules. However, this kind of monochromic intensity changes seriously confine the accuracy of visual inspection. Herein, we demonstrate for the first time that TMB²⁺ can quantitatively and efficiently etch gold nanorods (AuNRs). The addition of AuNRs into a solution containing different amount of TMB²⁺ generates vivid color responses as colorful as a rainbow, and the etching process can be finished within 90s. As a result, the exact concentration of TMB²⁺ can be easily distinguished with the naked eyes by the corresponding solution color. Based on this finding, we incorporate AuNRs into the well-developed, commercially available horseradish peroxidase (HRP)-TMB immunoassay system, so that it can be utilized for semiquantitative detection of a broad range of disease biomarkers with the naked eyes (termed 'NEQ-IA'). Carcinoembryonic antigen (CEA) and Prostate specific antigen (PSA) had been chosen as example targets to test the feasibility of the proposed biosensor. The results showed good accordance with the conventional methods. Because no sophisticated apparatus but human eyes are used as the readout, the proposed NEQ-IA could be a good supplementary to current state-of-the-art immunoassay methods for those applications that require the use of portable and affordable devices, for example, for the detection of disease biomarkers at home and in the field.