Amplification of aptamer sensor signals by four orders of magnitude via interdigitated organic electrochemical transistors

https://doi.org/10.1016/j.bios.2019.111668Get rights and content

Highlights

  • Novel iOECT-based aptasensor with ultralow ATP detection limit of 10 pM.

  • Detection limit is four orders of magnitude lower than that of a comparable amperometric sensor.

  • The potentiometric detection platform possessed high selectivity and reusability for ATP detection.

Abstract

Electrochemical aptamer receptor/transducer systems are key elements of emerging E-AB sensors (aptasensor) used for the detection of various kinds of targets. However, the performance of these amperometric sensors is often limited by the low density of receptors attached to the sensor surface and high background signals. In the present work, interdigitated organic electrochemical transistors (iOECT) were used as a transducer to enhance the sensitivity and dynamic detection range of aptasensors. Therefore, the electrode of an amperometric sensor was utilized as gate electrode to operate the iOECT. This device was used to detect the low weight target molecule adenosine triphosphate (ATP), a common biomarker, which plays an important role for cardiovascular, neurodegenerative, and immune deficiency diseases. The novel aptasensor can selectively detect ATP with ultrahigh sensitivity down to the concentration of 10 pM, which is four orders of magnitude lower than the detection limit of the same aptasensor using an amperometric transducer principle (limit-of-detection of 106 nM) and most other previously reported electrochemical sensors. Furthermore, sensor regeneration was demonstrated, which facilitates reusability of OECT aptasensors. The small device size in combination with high transconductances paves the way for the development of highly sensitive integrated micro-biosensors for point-of-care applications.

Introduction

The detection of disease-related biomarkers is a primary goal of bioanalytical chemistry, which seeks to determine a certain analyte at very low concentrations to enable an early diagnosis. For the development of highly sensitive and selective biosensors, two functional components are crucial. One is the target recognition element (receptor) and the other one is the signal conversion unit (transducer). Aptamer receptors-based biosensors (aptasensors) have rapidly gained in importance because of their high affinity, selectivity, low cost, and easy-fabrication (Tan et al., 2013) (Privett et al., 2010). Various types of transduction principles have been exploited for aptasensors determining mass changes (e.g. quartz crystal microbalance) (Gründler, 2007), optical (e.g. fluorimetric) (E Wang et al., 2011), and electrochemical signals (Feng et al., 2015b; Lai et al., 2007; Xiao et al., 2005a). Especially amperometric aptasensors are of interest since they are easy to assemble, compatible to point of care applications, reusable (Feng et al., 2016), and sensitive in a wide range of concentrations (Lubin and Plaxco, 2010). However, the low density of oligonucleotide receptors on the electrode surface significantly limits the detectable electrochemical signal since each receptor contributes with only a specific number of exchanged charge carriers per redox probe (n) to the current signal and the sensors usually do not support an intrinsic signal amplification (White et al., 2008). A few strategies have been proposed to enhance the sensor signals by exploiting nanomaterials, enzymes, or electrocatalytic redox tags (Feng et al. 2011, 2015b; Li et al., 2013b). For example, nanomaterials can amplify the signal by increasing the load with receptor molecules (Palchetti and Mascini, 2012). Alternatively, electrochemical current rectification enhances the amperometric signal remarkably by introducing solution-phase redox molecules that reactivate the aptamer bound redox probes (Feng et al. 2015a, 2015b). However, these approaches require complicated receptor modifications or detection processes. Consequently, developing simple signal amplification strategies remains a challenge to improve sensor performance and to meet the requirements of early disease diagnosis.

A promising alternative electrochemical transducer principle utilizes organic electrochemical transistors (OECTs) as an intrinsically amplifying unit to bust the sensitivity of the biosensor. OECTs possess additional benefits of device properties such as biocompatibility, simple processability, and straightforward operation in aqueous solution as an ion-to-electron converter (Bernards and Malliaras, 2007; Zeglio and Inganäs, 2018). These exceptional properties, together with simple signal readout, the inherent amplification function, and small device dimensions makes OECTs attractive in particular for lab-on-a-chip applications in chemical and biological sensing (Fu et al., 2017; Khodagholy et al., 2013a). Compared to conventional electrochemical approaches, the OECT-based biosensors have proven to outperform other state-of-the-art devices (Khodagholy et al., 2013a) and exhibit higher sensitivity for a wide range of targets including proteins (Fu et al., 2017; Kim et al., 2010) or even cells (Lin et al., 2010b) due to their high current modulation in response to changes of the gate potential. For example, Fu et al. fabricated an OECT-based biosensor for human epidermal growth factor receptor 2 by nanoprobe modified antibody receptors, which showed several orders of magnitude lower detection limit than previously reported electrochemical transducers (Fu et al., 2017). Braendlein et al. developed a Wheatstone bridge OECT circuit for ex vivo detection of lactate concentration (Braendlein et al., 2017b). Lin et al. used OECTs integrated into a flexible microfluidic system to detect complementary DNA targets (Lin et al., 2011). Saraf et al. introduced aptamer to selectively bind to epinephrine molecule whose oxidation caused the decrease of the channel current of OECT (Saraf et al., 2018). Chen et al. chose aptamer-modified gold nanoparticles to form a specific probe on the gate electrode of an OECT for the detection of glycan expression on living cells (Chen et al., 2018). However, these OECT-based sensors are complicated, expensive, and mainly used to detect heavy molecules, like proteins, while rapid, convenient, inexpensive, and OECT-based aptasensors detecting light molecules, to our knowledge, are still lacking mainly because light molecules have only little impact on the potential of gate electrodes at low concentrations.

In the present work, an interdigitated OECT (iOECT) was used to amplify the electrochemical signals of an aptasensor for ATP without utilizing any catalytic active material. ATP is the energy currency for all kinds of cells and therefore involved in multifarious biochemical processes. A deviation of the regular ATP level is often a side effect of an emerging disease. Therefore, ATP levels can provide adjuvant information for health examinations in addition to regular biomarkers and examination methods. Furthermore, monitoring ATP as co-substrate is crucial for process control in biosynthesis. Given the importance of ATP for healthcare, biology, and biotechnology, it was chosen as the model target molecule in this study and for comparing the performance of conventional amperometric transducer with iOECT aptasensors. The general binding process of the analyte to the aptamer receptor is practically the same for both sensor types. Once applying ATP to the respective sensor, the specific target-binding alters the conformation of aptamer, resulting in the changes of the aptamer conformation at the electrode surface, Fig. 1a. In the case of the amperometric transducer, this rearrangement leads to an alteration of the distance between a terminal redox group (ferrocene) attached at the distal end of the aptamer (the yellow dot at the aptamer, Fig. 1) and the electrode surface, which alters the charge transfer and generates a detectable electrochemical signal (Xiao et al., 2007).

The iOECT-transducer system comprises an ionic and an electronic circuit, Fig. 1b. The target-binding event occurs in the ionic part at the gate electrode based on the same recognition process described above for the amperometric transducer. This binding event alters the gate potential and thus causes changes of corresponding source-drain current in the electronic circuit. We found that a slight change of gate potential induced by ATP-binding can result in a distinct variation of transfer characteristics, which facilitates detection limits as small as 10 pM. This sensitivity is superior to most of the previously reported biosensors for ATP, which is a very light molecule and therefore difficult to detect in ultralow concentrations. Furthermore, the selectivity and regeneration performance of the aptasensor were evaluated and demonstrated.

Section snippets

Reagents

An ATP aptamer with the sequence of 5′-Ferrocene–(CH2)6–ACC TGG GGG AGT ATT GCG GAG GAA GGT–(CH2)6–SH-3′ was purchased from FRIZ Biochem (Neuried, Germany). The DNA probes were received as lyophilized powders and stored at −20 °C. The stock solution of DNA probes was prepared with 10 mM tris-EDTA buffer (pH 8.0). The concentration of aptamer in the stock solution was determined by using UV–vis spectroscopy to obtain the average absorbance value at 260 nm. Tris-(2-carboxyethyl) phosphine

Macroelectrode as aptasensor

The surface area of the macro-electrodes (ME) was determined by cyclic voltammetry in 50 mM H2SO4 electrolyte to be approximately 2.2 mm2. The ATP aptamer was covalently attached on the gold electrode via a thiol-gold bond (Fig. S5 and Fig. S7) with a surface density of approximately 1.78 × 1013 molecules/cm2 (Fig. S6). For the target detection, an ATP incubation time of 30 min was chosen, which was reported to provide stable, time-independent redox signals for ME (Feng et al., 2015b). In the

Conclusions

In this work, a novel strategy to detect a very light molecule (ATP) without involving any kind of catalytical amplification is proposed. The signal of a conventional amperometric aptamer sensor is converted and compared to responses of a potentiometric iOECT transducer. Therefore, amperometric biosensor, composed of an aptamer-modified electrode is linked as gate to an interdigitated OECT circuit. The actual binding process of ATP to the aptamer receptors is practically the same for both

CRediT authorship contribution statement

Yuanying Liang: Investigation, Formal analysis, Writing - original draft. Changtong Wu: Investigation. Gabriela Figueroa-Miranda: Investigation. Andreas Offenhäusser: Conceptualization, Funding acquisition, Project administration. Dirk Mayer: Conceptualization, Data curation, Formal analysis, Validation, Supervision, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The devices were fabricated in the Helmholtz Nano Facility of Forschungszentrum Jülich. We thank the Federal Ministry of Education and Research (Germany), funding code 031A095B and Yuanying Liang is grateful for the financial support from the China Scholarship Council (201506240059). Gabriela Figueroa-Miranda is grateful for the scholarship of the Mexican National Council for Science and Technology (CONACyT) and the German Academic Exchange Service (DAAD, grant number: 448904).

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