Effect of alkanethiol molecular structure on sensitivity of surface plasmon resonance sensor

• Immunosensing of clenbuterol was achieved using surface plasmon resonance.
• Molecular structure and orientation of alkanethiol sensor surfaces determined sensitivity of an immunoassay.
• The best LOD for clenbuterol was 10 ppt.
• Immunosurface could be used for >100 immunoreactions.

This work aims to find the key factor determining the detection limit for an indirect competitive inhibition immunoassay using surface plasmon resonance (SPR) sensing. In our previous work, the thiol solution concentration used in a self-assembly process highly affected the alkanethiol monolayer structure on the sensor surface (Suherman et al., 2014). It was noticed that the monolayer structure determined the immunoassay sensitivity due to the orientation and the surface concentration of antigen in domain structure of monolayer. To study the effect of orientation of antigen, we examined here three types of alkanethiol compounds: dithiobis(succinimidyl undecanoate) (DSU; straight-chain alkanethiol), carboxy-EG6-undecanethiol (CEG6; flexible-chain alkanethiol), and 3,3′-dithiobis[N-(5-amino-5-carboxypentyl)propionamide-N,N′-diacetic acid] (C2-NTA; three-branched alkanethiol). By electrochemical reductive desorption, the surface concentration of DSU, CEG6, and C2-NTA were estimated to be 6.6 × 10−10, 8.5 × 10−10, 4.6 × 10−10 mol cm−2, respectively. Subsequently, SPR suggested that the ratios of immobilized antigen (clenbuterol) per thiol were 0.13, 0.16, and 0.15 for DSU, CEG6, and C2-NTA, respectively. This suggests that the amount of immobilized clenbuterol was defined by the molecular size of clenbuterol. Sensor surface structures on a molecular scale were evaluated using high-resolution scanning tunneling microscopy (STM), which showed tilted clenbuterol with θDSU > θCEG6 > θC2-NTA. For SPR sensing of clenbuterol, C2-NTA showed the highest sensitivity (LOD = 10 ppt) among the examined alkanethiols, because clenbuterol formed a laid-on structure. Based on kinetics, the key factor for sensing performance is discussed.