Immunosensor Employing Stable, Solid 1-Amino-2-naphthyl Phosphate and Ammonia-Borane toward Ultrasensitive and Simple Point-of-Care Testing

Biosensors for ultrasensitive point-of-care testing require dried reagents with long-term stability and a high signal-to-background ratio. Although ortho-substituted diaromatic dihydroxy and aminohydroxy compounds undergo fast redox reactions, they are not used as electrochemical signaling species because they are readily oxidized and polymerized by dissolved oxygen. In this report, stable, solid 1-amino-2-naphthyl phosphate (1A2N-P) and ammonia-borane (H3N-BH3) are respectively employed as a substrate for alkaline phosphatase (ALP) and a reductant for electrochemical-chemical (EC) redox cycling. ALP converts 1A2N-P to 1-amino-2-naphthol (1A2N), which is then employed in EC redox cycling using H3N-BH3. The oxidation and polymerization of 1A2N by dissolved oxygen is significantly prevented in the presence of H3NBH3. The electrochemical measurement is performed without modification of indium tin oxide (ITO) electrodes with electrocatalytic materials. For comparison, nine aromatic dihydroxy and aminohydroxy compounds, including 1A2N, are evaluated to achieve fast EC redox cycling, and four strong reductants, including H3N-BH3, are evaluated to achieve a low background level. The combination of 1A2N and H3N-BH3 allows the achievement of a very high signal-to-background ratio. When the newly developed combination is applied to the detection of creatine kinase-MB (CK-MB), the detection limit for CK-MB is similar to 80 fg/mL, indicating that the combination allows ultrasensitive detection. The concentrations of CK-MB in clinical serum samples, determined using the developed system, are in good agreement with the concentrations obtained using a commercial instrument. Thus, the use of stable, solid IA2N-P and H3N-BH3 along with bare ITO electrodes is highly promising for ultrasensitive and simple point-of-care testing.