Field-Effect Transistor Biosensor By Capture of Nucleic Acid Strands with Isothermal Amplification for RNA Detection
Authors: Sato Yuichi , Hayashi Hiroki , Kataoka Yuka , Kuwahara Masayasu , Momma Toshiyuki 
Published: December 2024

Nucleic acid amplification methods are widely used for sensitive detection of biomolecules utilizing signals derived from amplified DNA strands[1]. Signal amplification by ternary initiation complexes (SATIC) has been attracted attention as high specific RNA detection method, because rolling circle amplification (RCA) by φ29 DNA polymerase requires the formation of ternary initiation complexes with DNA primers, circular DNA templates, and target RNAs[2]. In SATIC system, target RNA was detected via fluorescence derived from the complex of guanine quadruplex (G4) structure, which generated within the amplified DNA strands, and thioflavin T (ThT)[3]. To further simplify RNA detection using the SATIC system, it was expected to be effective to combine it with a field-effect transistor (FET) biosensor. A FET biosensor detects target molecules by altering the interfacial potential through the adsorption of charged molecules, thereby enabling simple label-free detection. Previously, we demonstrated the RNA detection via the negative charges of amplified DNA strands using the SATIC system, which originated from the DNA primer-immobilized FET sensor surface[4]. This approach will be required the immobilization of different DNA primers for the detection of various targets. As the immobilization conditions of the DNA primers needed to be optimized for detection of each target RNA, there was concern that the versatility of the FET biosensor would be reduced. Here, the versatility of the FET biosensor could be reinforced by the capture of nucleic acid strands amplified in the bulk by the sensing interface formed under the same conditions. For the realizing the above approach, the interaction between the G4 structure and ThT was considered to be effective in capturing amplified DNA strands. In this study, we attempted to detect target RNA by capture of the amplified DNA strands containing the G4 structures using ThT-immobilized FET biosensor.

ThT derivatives (ThT-OE11[5]) were immobilized to aminopropylsilane-modified FET gate surface via cross-linking by glutaraldehyde (1 h). After that, residual aldehyde group were capped by the ethanolamine (1 h). Subsequently, amplified DNA strands were generated by mixing target RNAs, DNA primers, circular DNA templates, the four deoxynucleotide triphosphates (dNTPs), and φ29 DNA polymerase at 37°C. Following that, the SATIC reaction solution was added to the ThT-immobilized FET (30 min.). Finally, the gate voltage (V
g)-drain current (I
d) characteristics were measured before and after the addition of the SATIC reaction solution for calculating gate voltage shift (ΔV
g).

The FET response to 100 nM target RNA was measured when the SATIC reaction solution was added ThT-immobilized FET biosensor. As shown in Figure 1(a), ΔV
g in the positive direction was obtained, indicating that the electron concentration in the channel was decreased by electrostatic interaction from negative charges of the amplified DNA strands. To optimize ThT immobilization condition, we evaluated the relationship between concentrations of ThT-OE11 solution and the sensor responses to target RNA. As a result, maximum response obtained at 500 mM ThT-OE11 solution. Subsequently, the sensor signal to 1 base-mismatched RNA as a negative control was almost no response (Figure. 1(b)). This result suggested that the nucleic acid strands were not generated by SATIC system, because the ternary initiation complexes did not form with 1 base-mismatched RNA. Thus, the ThT-immobilized FET with SATIC system specifically detected target RNAs. Finally, the measurement of ΔV
g at different concentrations of target RNAs were conducted using ThT-immobilized FET to examine the quantitative detectability. As a result, ThT-immobilized FET with SATIC system showed a concentration dependence in the range of 1 pM to 100 nM. In addition, we plan to obtain additional data on sensitivity by controlling the reaction time of FET biosensor with SATIC system. From these results, the capture of nucleic acid strands containing G4 structure by ThT-immobilized FET could be effective for RNA detection.

[1] M. Falco et al., TrAC Trends in Anal. Chem., 2022, 148, 3, 116538

[2] H. Fujita et al., Anal. Chem., 2016, 88, 7137−7144

[3] J. Mohanty et al., J. Am. Chem. Soc., 2013, 135, 1, 367–376

[4] H. Hayashi et al., Talanta, 2023, 273, 125846

[5] M. Kuwahara et al., Molecules, 2020, 25, 4936

Figure 1