Aptamer Immobilization on Transistor-Based Biosensor Via Crosslinker for Amine-to-Thiol Conjugation Toward Cortisol Detection
Authors: Hayashi Hiroki , Ishikawa Akane , Momma Toshiyuki , Uchida Junichi 
Published: December 2024

A Field-effect transistor (FET) biosensor is a sensing device that detect changes in the interfacial potential associated with the adsorption of target molecules. FET biosensor is expected to be a simple measurement method by detecting target biomarkers without any labeling process. Recently, aptamers, single-stranded DNA molecules with specificity and stability, have attracted attention as receptors of FET biosensor [1,2]. Immobilization of aptamer onto metal oxide insulator surface of FET biosensors is often achieved by combining self-assembled monolayer (SAM) with cross-linking agents. Generally, an immobilization method using cross-linking agent, glutaraldehyde, between amino-modified aptamer and aminopropylsilane (APS) monolayer is often employed [3]. However, there is concern that aptamers may be immobilized by non-terminal amino group, because amino groups are also present within the bases constituting DNA structure. Consequently, decrease in affinity of the aptamer to the target molecule by involving collapse of the higher-order structure and inhibition of conformational change. In this study, we attempted immobilization method on the FET gate surface utilizing cross-linking thiol groups of aptamers to self-assembled monolayer instead of that between amino groups. By immobilizing thiol-modified aptamers onto APS monolayer via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), we demonstrated specific immobilization at the aptamer termini for increasing the number of effective aptamers for binding of cortisol, which is stress-related hormone, was selected as a target.
The SiO2 gate insulator of the FET was treated by piranha solution (H2O2 : H2SO4 = 1:4) for introduction of hydroxyl groups. Then, the FET chip was immersed in toluene solvent including 1%(v/w) 3-aminopropyltriethoxysilane in an argon atmosphere (60ºC for 7 min.). The SPDP was reacted with amino group of APS monolayer. After that, dithiothreitol (DTT) was added to the FET gate surface to form thiol group by cleavage of the disulfide bond within the SPDP. 100 nM 3’-thiol modified cortisol aptamer was immobilized to the FET gate surface by forming disulfide bond. Finally, 1 μM 2-mercaptoethanol was reacted with residual thiol sites to prevent non-specific adsorption of contaminating proteins. The FET characteristics were measured before and after the addition of cortisol for obtaining the threshold voltage shift (ΔV
g).
First, we compared the ability of reducing regent for cleavage of disulfide bond within SPDP to form the thiol group on the FET gate surface. A 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), which reacts with thiol group, was added to SPDP-modified surface treated by DTT, ascorbic acid or l-cysteine ethyl ester (LCEE). Following that, the absorbance derived from 5-mercapto-2-nitrobenzoic acid, which was generated from DTNB depending on the amount of thiol groups, was measured. As a result, thiol groups derived from SPDP were formed on the FET gate surface upon treatment with DTT. On the other hand, no absorbance of 5-mercapto-2-nitrobenzoic acid was observed when ascorbic acid or LCEE was used as reducing regents. Additionally, thiol groups were increased with SPDP concentrations in the range from 0.5 to 25 mM by DTNB absorbance measurements. Subsequently, electrical responses to cortisol using aptamer-immobilized FET were measured upon varying SPDP concentrations, with maximum response obtained at 1 mM. It should be noted that the FET response was acquired by increase in charge within the Debye length due to the conformational changes of the aptamer upon cortisol binding [4]. Finally, the sensitivity of aptamer-immobilized FET biosensor, which was fabricated using SPDP, were compared with that obtained using glutaraldehyde as the cross-linking agent. As a result, the gate voltage shift by addition of 1 μM cortisol when SPDP was utilized to fabricate the aptamer-immobilized FET biosensor was greater than the case of glutaraldehyde (Figure 1). These results indicated that cortisol was efficiently captured by the increase in the amount of aptamer, which was immobilized by 3’-terminus via disulfide bond. Therefore, the utilization of cross-linker for amine-to-thiol conjugation to nucleic acid immobilization could be useful for improving the sensitivity of FET biosensors.
[1] H. Chen et al., Analyst, 141, 2335-2346 (2016).
[2] N. Nakatsuka et al., Science, 362, 319-324 (2018).
[3] S. K. Vashist et al., Chem. Rev., 114, 11083−11130 (2014).
[4] H. Hayashi et al., Electrochemistry, 89, (2), 134-137 (2021).

Figure 1