Electric-Double-Layer (EDL)-Gated FET Aptasensors for Highly Sensitive MMP7 Detection
Authors: Lin Chia Kai , Wu Chia-Che , Paulose Akhil Kavanal , Hou Yueh-Ju , Lee Chee-Seng , Chen Heuy-Ling , Wang Yu-Lin 
Published: September 2023

ABSTRACT

Biliary Atresia (BA) is a severe liver disease that affects newborns. This disease may cause cholestasis and progressive hepatic failure or even death if not treated immediately. ELISA blood tests for metabolic screening are the current method of diagnosing BA. However, newborns need rapid BA detection for treatment in the future. The increased matrix metalloproteinase-7 (MMP7) cause BA-related liver fibrosis and newborns diagnosed with BA show higher MMP7 levels than those diagnosed with cholestasis. Several platforms have been developed for the fast detection of Biliary Atresia (BA). Here, we developed an electric-double-layer (EDL)-gated field-effect transistor (FET) platform for Biliary Atresia (BA) detection. The core idea of this sensor array is to use a uniquely designed DNA probe to capture MMP7 proteins and monitor the change in drain current caused by the target capturing. Different concentrations of MMP7 spiked and tested over a functionalized sensor. The simple and cost-effective fabrication and ease of use make this sensor a promising tool as a point care detection device.

Keywords: EDL, FET, Biliary atresia, MMP7, Biosensors

INTRODUCTION

Due to the symptomatic similarity to cholestatic liver diseases, it is challenging to diagnose a precise BA for newborns. False diagnosis may delay timely treatment (e.g., surgery) and worsens the prognosis. However, BA's underlying causes are still unclear. A level increase of intrahepatic matrix metalloproteinase-7 (MMP7) signifies BA-related liver fibrosis. BA cases found in newborns revealed higher MMP7 levels than cholestasis. MMP7 is a protease that contributes to tissue remodeling and breaks down extracellular matrix through a signal pathway. Based on the clinical results, MMP7 has emerged as a novel biomarker for BA diagnosis. ELISA was employed as the current method for detecting Biliary Atresia (BA). However, the procedure of ELISA has shortcomings, such as being time-consuming, high cost, and complicated operation. Hence, rapid and precise diagnostic methods need to be developed.

In this work, we propose to detect MMP7 proteins using an electric-double-layer (EDL)-gated field-effect transistor (FET). As shown in Figure 1, The aptamer of MMP7 was immobilized on sensor electrodes, allowing a binding with the protein. As the number of the captured analytes increases; the sensor signals, amplified by a FET, will respond to it. As such, we look forward to detecting MMP7 in serum samples collected from newborns and helping the clinical diagnostics by this EDL-gated FET biosensor.

MATERIALS AND METHODS

An extended gate chip was employed in the sensor array. The electrode surface was cleaned using oxygen plasma, following which, the probe was immobilized on it. The MMP7 aptamer is mixed with TCEP for 30 minutes at room temperature, TCEP was used as a reducing agent which helps in the formation of dithiol bonds (SS), making the attachment of the probe easier. The mixture solution is then dropped and cast over the sensor surface and immobilized at 24 ℃ for 24 hr. The sensor is washed with 0.01× PBS to remove unbound aptamers and was verified with fluorescent imaging.

ELECTRICAL MEASUREMENT

After the aptamer was immobilized over the sensor surface, a baseline measurement was carried out. The drain current was measured at different voltage biases (Vg=-2V and 3V, at Vd=3.5V). different concentration of MMP7 protein was spiked in 0.01x PBS and tested. A signal was read out every 1min, and 20 measurements were taken for each concentration of the target for 20 minutes.

RESULTS AND CONCLUSIONS

The platform established is to detect MMP7 protein precisely and further identify BA. Owning a characteristic of electrical amplification, the EDL-gated FET will be utilized to test MMP7 proteins with concentrations ranging from 10 ng/mL to 100 ng/mL, as shown in Figure 2. When testing proteins level at 100 ng/mL, the signal can increase by around 3.5 mA. The tests using EDL-FET were successfully validated for MMP7 detection in serum and 0.01x PBS and the results indicate that the proposed platform had a high sensitivity. The calibration curve (signal vs. analyte's concentration) will be established to quantify the unknown sample. Conclusively, the sensor developed in this study has the potential to be developed as a point-of-care testing device and is suitable for clinical diagnosis.

Figure 1