Electrochemical Detection of NT-Probnp Using High Performance Fully Inkjet-Printed Organic Electrochemical Transistors

Abstract

Cardiovascular diseases are the number one cause of death worldwide, with Heart Failure (HF) accounting for a third of these fatalities. Therefore, there is a growing need for a point-of-care (POC) device capable of providing timely and accurate information on HF to minimize its impact on patients. N-terminal pro-B-type natriuretic peptide (NT-proBNP), released by myocardial cells in the blood in response to stress or strain, is regarded as the gold standard biomarker for HF management given its superior clinical relevance in diagnosis, prognosis, and therapy. Electrochemical potentiometric biosensors are promising alternatives to traditional analytical methods for biomarker detection. In particular, organic electrochemical transistors (OECTs) have garnered increased attention due to their ability to interface bodily fluids by simply operating inside an ionic environment. Additionally, they possess inherent transistor-specific amplification capabilities represented by the transconductance (gm), shown to scale with the thickness of the semiconducting channel, yielding values that are orders of magnitudes higher than those of field effect transistors (FETs). This feature enables OECTs to detect and amplify the presence of the target molecule without relying on complementary methodologies.HF patients lack self-operated and reliable POC devices that can provide immediate information about their health ensuring clinical accuracy for diagnosis and intervention. While several methods have been proposed, they fail to combine self-operation, speed, low-cost, and clinical accuracy. In this work, we aim to develop a POC HF sensor that can detect and quantify NT-proBNP levels in a finger prick blood volume, all while being cheap to fabricate, easy to operate, and rapid in providing results.OECTs were fabricated using inkjet-printing. First, the source, drain, and gate (2.5x2.5 mm) were patterned using a conductive gold nanoparticle ink. Subsequently the leads were insulated with polyimide, after which PEDOT:PSS was deposited to form the semi-conducting channel (15x400µm). Antibody immobilization for NT-proBNP detection was achieved by chemical functionalization of either the channel or the gate. To functionalize the channel, the PEDOT:PSS surface was treated with oxygen plasma to introduce hydroxyl groups. The following steps included silanization, then streptavidin grafting by immersion, and finally, antibody immobilization using biotinylated anti-NT-proBNP antibodies. To functionalize the gate, a quick three step protocol was adapted by employing thiolated-biotinylated-Polyethylene Glycol followed by streptavidin and then biotinylated antibodies. Surface functionalization, in both cases, was verified using electrochemical impedance spectroscopy (EIS). To detect the presence and quantify the levels of NT-proBNP using the realized OECT, several concentrations of NT-proBNP were prepared in PBS and tris-base buffer (pH=8.5) and introduced into the device sensing area. The different concentrations were distinguished through scanning of the transfer characteristic at drain-source voltages (VDS)=-0.05V or -0.5V and gate-source voltages (VGS)=0 to 0.8V.The developed biosensor has a miniaturized active recording area, enabling the use of minimal volumes to operate and detect NT-proBNP. The functionalization of either the in-plane gold gate or the PEDOT:PSS channel was validated by an increase in the electric impedance of the system at every step. Repetitive transfer characteristics scans revealed better stability at -0.05 VDS compared to -0.5 VDS, which is attributed to decreased channel current, and subsequently, preserved channel integrity in solution at the lower bias. The latter increases channel stability, but decreases gm, yet remains sufficiently high to successfully detect NT-proBNP at clinically relevant concentrations (0 to 1000 pg/ml). Functionalizing the gate led to a linearity of 95%, and a decrease in variability across trials compared to functionalizing the channel which can be attributed to the smaller detection area and better reactivity. The sensor also demonstrated selectivity by exhibiting a stronger response to NT-proBNP compared to BSA (a non-specific protein) proving the effectiveness of the chemical functionalization, which is particularly important when using the sensor with a complex solution like blood.In this work, we have developed an electrochemical potentiometric solution towards HF POC applications, capable of quantifying NT-proBNP in microvolumes at clinically relevant concentrations with high sensitivity, specificity, and repeatability. The adapted microfabrication methodology provides a cheap and quick approach for producing high performing sensors, rendered sensitive to HF biomarkers by chemical modification. The functionalization was found to be efficient and faster when applied at the gate instead of the channel given the versatile nature of the in-plane gate and the size-dependent performance limitations of the channel. The successful bedside integration of such technology holds promise in reducing HF mortality by enabling early diagnosis and immediate intervention, improving disease progression and management, and facilitating personalized strategies for prognosis, therapy, and prevention.