Microsensor for limonin detection: An indicator of citrus greening disease

Highlights

• Abnormally high level of limonin is an indicator of Citrus greening disease.

• Detection performed using silk-ceria modified organic electrochemical transistor.

• Limonin triggers Ce³⁺ → Ce⁴⁺ which increases channel current.

• Specificity test against common interfering species showed no change in current.

• A sensitivity of ∼10 μA/μM and detection limit of 10 nM achieved using this approach.

Abstract

Limonin is a biomolecule which is responsible for the bitter taste in citrus fruits such as oranges, grapes etc. The abnormally high level of limonin is indicative of citrus greening disease which results in stunted tree growth and affects fruit quality in terms of nutritional value, taste, texture and aroma. Therefore, quantification and detection of limonin is crucial for an early management of citrus greening disease to save the multibillion dollar citrus industry. To this end, an organic electrochemical transistor (OECT) functionalized with Ceria Nanoparticles (CNPs) as transducer has been developed to detect ultralow concentration of limonin. The device exhibited high sensitivity (detection limit: 10 nM) and selectivity towards limonin with response time in seconds. The detection is attributed to the switching of Ce³⁺ to Ce⁴⁺ at the gate electrode which decreases the overall effective gate voltage resulting in an increase in the output current. The increase in output current was observed in transfer characteristics as well as time-current curve. In-situ spectro-electrochemical studies were also performed to analyse the change in oxidation state of CNPs in the presence of limonin. This novel biosensor successfully detected the increase in limonin in infected juice samples as compared to healthy ones with a sensitivity of ∼10 μA/μM. A rapid, easy and on-site testing tool to detect and quantify the amount of limonin for an early detection of citrus greening disease has been demonstrated for the first time.

Introduction

Limonin is a white crystalline biomolecule which belongs to the limonoid category and is responsible for imparting bitter taste to many citrus fruits such as oranges, grapes etc. [[1], [2], [3], [4]]. During juice extraction, the limonite β ring lactone tasteless precursor from the juice sacs comes in contact with the acidic component of the juice and is gradually lactonized to form bitter limonin. An abnormally high level of limonin is an indicative of Huanglongbing (HLB) disease also known as “citrus greening disease” [5]. HLB disease results in stunted tree growth which bears small sized, discolored and deformed fruits. The infection is caused by Candidatus Liberibacter asiaticus bacteria which strongly affects the taste and quality of the fruit. The introduction of the diseased fruits into mainstream juice production is directly affecting the multibillion-dollar citrus industry all over the world [[6], [7], [8]]. In Florida, citrus greening has caused a decline in revenue by $4.64 billion over 10 seasons which affected more than 3400 jobs and $1.76 billion in labor income [8,9]. The quality of juice becomes objectionable if limonin exceeds 6 ppm (∼13 μM) [[10], [11], [12]]. Therefore, it is very important to quantify and detect limonin for improvement in the fruit quality and early management of citrus greening disease.

Several techniques and procedures have been developed to quantify and detect limonin in citrus juices [14,15]. Many of the techniques are based on reverse-phase high performance liquid chromatography (HPLC), thin layer chromatography (TLC), gas-liquid chromatography (GLC), radioimmunoassay (RIA) and enzyme immunoassay (EIA) for limonin analysis [[16], [17], [18]]. The major drawback of HPLC technique includes lack of a selective chromophore (lambda max 207 nm) for limonin resulting in low sensitivity and cumbersome sample preparation [15]. All the current techniques have limitations as they alter the chemical composition of the juice affecting the quality in terms of flavor, texture and stability. Moreover, these techniques are also time consuming, expensive and not designed for on-site testing [19].

In order to circumvent these limitations, we have designed a ceria nanoparticle (CNPs) based organic electrochemical transistor (OECT) to detect limonin in nanomolar range with high selectivity. OECTs have been widely used in biological and chemical sensing due to their stable performance in aqueous medium, low operating voltages (<1 V) and signal amplification [[20], [21], [22], [23], [24], [25], [26], [27]]. They have significant advantages such as miniaturization, feasibility, high throughput and portability [28]. The current OECT device uses CNPs as a transducer which is integrated in electrospun silk fibroin, immobilized on the gate electrode. It has been reported that CNPs are redox active in nature and act as antioxidants due to coexistence of its oxidations states, Ce³⁺ and Ce⁴⁺ [[29], [30], [31], [32], [33], [34], [35], [36]]. The shuttling between these oxidation states results in the production/annihilation of oxygen vacancies that act as hot spots for actively reacting with electroactive entities. Limonin being an antioxidant itself possesses an intrinsic electroactive component to its chemical structure [37,38]. Thus, it was hypothesized that the interaction between limonin and CNPs could be used for the detection of limonin using an organic transistor platform.

The CNPs sensor material is uniformly integrated in Silk fibroins (SF) which are a strong natural fiber protein produced by Bombyx mori silkworm. Previously, SF has been shown to be an excellent platform for the immobilization of enzymes/nanomaterials for developing amperometric sensors to detect biomolecules such as glucose, uric acid, hydrogen peroxide etc. [[39], [40], [41], [42], [43], [44], [45], [46], [47], [48]]. Along with the application in biodiagnostic industry, SF has also been used in flexible electronic devices owing to their high thermal stability and robust mechanical properties [[49], [50], [51], [52]]. The shelf life of the electrospun silk fibroin can be extended to years by storing at lower temperature like 4 °C. SF offers highly crystalline microarray of antiparallel β-sheets which leads to less defects and traps improving the quality of dielectric layer which improves the device performance [53,54]. The relative high permeability of SF (>5) along with the highly ordered array structure makes them a promising candidate in OECT which can operate at low operating voltages [55].

Fig. 1C illustrates the schematic of the fabricated CNPs based OECT device which consists of three electrodes labeled as gate, drain and source. The drain and source electrodes are bridged by a semiconducting layer of Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) which is represented as dark blue rectangle in the schematic (Fig. S5, Supplementary information). The highly crystalline electrospun silk fibroins are used as a platform to uniformly immobilize CNPs onto the gate electrode. Phosphate buffer saline (PBS), contained in a PDMS (Polydimethylsiloxane) mold, bridges the active layer and the modified gate electrode. The working of the present device is based on the variation in the output current signal in the presence of limonin. It is hypothesized that CNPs being highly electroactive in nature changes its oxidation state from Ce³⁺ to Ce⁴⁺ in the presence of limonin. The production of faradaic current at the gate electrode decreases the dedoping effect and increases the output current. The present sensor enables the quantification of the bitter taste in oranges via detection of limonin in a highly sensitive, selective and cost-effective manner.