Revolutionizing Surface Immobilization Techniques for Extended Gate Electric-Double-Layer (EDL) Bio-FETs
Authors: Lin Chia Kai , Lee Chee-Seng , Chen Heuy-Ling , Hou Yueh-Ju , Huang Hsuan-Wei , Zeng Guan-Cheng , Kalaimani Vishal Mani , Wang Yu-Lin 
Published: August 2024

The development of biosensors, known for their high sensitivity and specificity, has sparked high interest in semiconductor-related studies and bioanalysis. Within this domain, the establishment of immobilization techniques has become significant. However, conventional antibody immobilization on gold surface techniques using PEG linkers, Traut’s reagent, and 2-Mercaptoethylamine (2-MEA) have some drawbacks such as long debye length, may interact with other amino groups on the antibody, impacting the antibodies' binding affinity, and half-antibody will reduce antibody activity respectively. In this study, we present a novel surface immobilization strategy by using a short DNA sequence. Through the characteristics of synthetic DNA, we can modify its 3’ and 5’ ends with different functional group, enabling effective immobilization of antibodies across diverse sensor surfaces. By doing so, we can match the specific requirements of various materials and test subjects. Also, we can verify the successful surface immobilization by fluorescent labeling. The length of short DNA sequence is only about 1 nanometer, which is much shorter than PEG Linker (> 4.8 nm), enabling a reduced debye length.

Firstly, aptamer was immobilized on the surface as the first layer. This modified aptamer carries a disulfide bond at its 5' end, a fluorescent dye (FAM) at its 3' terminal, and an amino group (-NH2) at its center, as shown as Figure 1(A). The amino group served as a functional linker facilitating the attachment of the antibody onto the gold extended gate, providing a stable linkage. After the surface treatment with ATA, the sensor surface was incubated again with EDC/NHS antibody solution. This particular antibody was labeled using the CF® Dye Antibody Label (ROX) conjugate, which responds to excitation by light wavelengths falling within the range of 580 to 610 nanometers (nm). The distinctive red color serves as an indicator to confirm the successful immobilization of the antibody, as shown as Figure 1(B).

The process began with the cleaning of the electrode surface using oxygen plasma. Subsequently, the probe was immobilized onto the cleaned surface. To prepare the ATA aptamer, it was mixed with TCEP for 30 minutes at room temperature. The solution mixture was then applied onto the sensor surface and left to immobilize at 24°C for 24 hours. To eliminate unbound aptamers, the sensor underwent a washing step using 1× PBS before applying the antibody solution. We clearly notice a substantial increase in fluorescent intensity following the implementation of the new strategy, as shown in Figure 1.

After checking the immobilization of ATA and antibody, the electrical signal will be tested by dropping different concentrations of target antigen. The system we developed is using N-Channel Enhancement-Mode DMOS FET (VN10LP). For the measurement setting, a constant drain voltage (Vd) was applied at 3.5 V. The signals (changes of drain currents, ΔId) were measured when applying gate biases (Vg) at -2 V and 3 V. A signal was read out every 20 seconds, and 10 measurements were taken for each concentration. Real-time data recorded the signal change under different concentrations of target protein from 0, 0.1, 10, 25, 100 ng/mL, as shown as Figure 1(C).

Comparing the fluorescent and electrical outcomes between ATA with a PEG linker and Traut’s reagent allows us to address shortcomings encountered in conventional techniques, offering promising solutions for enhanced specificity, improved uniformity, and minimized interference with antibody binding affinity. The successful integration of this novel immobilization strategy into the EDL-FET detection platform marks a significant stride forward in advancing biosensing technologies, promising heightened accuracy and efficiency in bioanalytical applications.

Figure 1