A novel electrochemiluminescent biosensor based on resonance energy transfer between poly(9,9-di-n-octylfluorenyl-2,7-diyl) and 3,4,9,10-perylenetetracar-boxylic acid for insulin detection

Highlights

• The PFO dots was employed to detection insulin for the first time.

• In this construction strategy, PTCA could quench the ECL intensity of PFO dots by resonance energy transfer.

• The novel donor-accepter pair of PFO-PTCA expand the ECL-RET system and endowed the biosensor a good stability and excellent selectivity.

Abstract

An electrochemiluminescencent (ECL) biosensor was designed for the determination of insulin using a novel ECL resonance energy transfer (ECL-RET) strategy. In this strategy, carboxyl poly(9,9-dioctyfluorenyl-2,7-diyl) dots (PFO dots) were worked as ECL donor and 3,4,9,10-perylenetetracar-boxylic acid (PTCA) exploited as ECL acceptor, and hydrogen peroxide (H₂O₂) employed as the coreactant. The ECL donor and ECL acceptor were separately labeled with primary antibody (Ab₁) and secondary antibody (Ab₂), forming a sensing interface to the analyte target, insulin. In this expected sandwich-type ECL biosensor, PFO dots acted as sensing platform and PTCA employed as labels to quench the ECL emission of PFO dots. During the determination process, ECL signal of PFO dots was decreased in a gradual way by the increase of insulin concentration, and the quenching mechanism was also investigated. Under the optimal experimental conditions, the constructed biosensor exhibited an excellent performance, including a wide linear range from 1.0 × 10⁻⁵ ng/mL to 1.0 × 10² ng/mL, low detection limit of 3.0 × 10⁻⁶ ng/mL, good stability and selectivity for the detection of insulin. This pair of PFO-PTCA, as a new donor-acceptor pair in ECL-RET system, would provide a promising platform for bioanalysis in ECL field.

Introduction

Insulin is a peptide hormone, which is produced by beta cells of the pancreatic islets (Arvinte et al., 2010). As one of the important anabolic hormone of human body, insulin can stimulate the glucose in the blood circulation into the liver cells, muscle cells, adipocytes and other tissue cells, thus regulating the metabolism of carbohydrates, fats and protein (Schirhagl et al., 2012, Xiong et al., 2015, Yu et al., 2016). Insulin hormone controls the glucose level in blood within a narrow concentration range and its disorder causes severe illness and dysfunctions in humans which called diabetes mellitus (Abdollah et al., 2007). The sensitive and fast detection of insulin is of great importance because it not only served as a predictor of diabetes of insulinoma and trauma (Melloul et al., 2002) but also was used as a doping drug in competitive sports for cheating (Schirhagl et al., 2012). There have been massive methods employed for the insulin analysis, such as immunoassays (Neal and Han, 2008), high performance liquid chromatography (HPLC) (Mercolini et al., 2008), surface plasmon resonance (Frasconi et al., 2010), electrochemistry (Wang and Li, 2009) and flow injection analysis (Salimi et al., 2009). Among these reported methods, electrochemiluminescence (ECL), as a novel method integrated electrochemistry and luminescence, has arisen growing attention in recent years (Gao et al., 2016). It has been used in DNA, protein and biomolecules analysis and detection due to its high sensitivity, good facility, wide dynamic range, spatial and temporal control and simple instrumentation (Zhang et al., 2017). Thus, it is worth using this kind of excellent potential method for insulin studying.

Due to high sensitivity, non-background unselective photoexcitation and non-interference scattered light, ECL resonance energy transfer (ECL-RET) has drawn wide concern in biosensor field (He et al., 2013, Wu et al., 2014, Dong et al., 2014, Liu et al., 2015). The precondition of ECL-RET is the good spectra overlaps between the energy donors’ ECL spectra and the energy acceptors’ Uv–vis absorption spectra (Rajapakse et al., 2010, Zhu et al., 2017). While the ECL-RET has been applied into the test of protein (Ji et al., 2014), DNA (Jie et al., 2015) and some ions (Lei et al., 2015), there are still some challenges for searching the corresponding donor-acceptor pair. One of the big challenges is the lack of donor-acceptor system with good spectrum overlap (Ma et al., 2016). Among the reported ECL-RET system, the donor-acceptor pairs are often confined to the following categories, such as quantum dots-Ru(bpy)₃²⁺ (Wu et al., 2012), quantum dots-metal nanoparticles (Zhang et al., 2015), quantum dots-luminol (Zhao et al., 2015), graphene carbonitride (g-C₃N₄)-luminol (Wang et al., 2016) or g-C₃N₄-Ru(bpy)₃²⁺ (Feng et al., 2016a, Feng et al., 2016b). Above donor-acceptor pairs exhibited disadvantages more or less. For example, the quantum dots are commonly poisonous and bad biocompatibility, and the price of Ru(bpy)₃²⁺ is expensive. So, it's significant to exploit efficient, nontoxic and oversimplified ECL-RET system with more types of donor-acceptor collocations.

Conjugated polymers have seen an ongoing interest due to their high fluorescence brightness and excellent photostability, and several views and perspectives about conjugated polymers have been reported in past decades (Wu et al., 2007, Wu and Chiu, 2013). Barbara and his co-workers investigated the ECL behavior of PFBT CPNs for the first time (Chang et al., 2008). Chi and co-workers studied the MEH-PPV CPNs’ ECL performance in aqueous solution (Dai et al., 2015). Cheng's group prepared a silole-containing polymer dot, and studied its ECL properties with the coreactant TPrA (Feng et al., 2016a, Feng et al., 2016b). And for our group, we mainly studied poly(9,9-dioctyfluorenyl-2,7-diyl) (PFO). As one of the conjugated polymers, PFO has attracted considerable interest in optoelectronic device, ECL sensors, and other biological labels due to its pleased characters such as high electroluminescence quantum yields with bule light emission, high charge carrier and chemical stability (Zhang et al., 2017). In our previous work, the anodic ECL behavior of PFO was explored in the presence of coreactant Na₂C₂O₄. And following we found that H₂O₂ can serve as coreactant to greatly amplify the ECL signal of PFO, thus constructed an oxidase-based biosensor using PFO-H₂O₂ system (Zhang et al., 2017, Chen et al., 2016). We also constructed a ratiometric sensing for detecting organophosphorus pesticides based on carboxyl-PFO dots as anodic luminophore and reduced graphene oxide-CdTe quantum dots as cathodic luminophore (Chen et al., 2017a, Chen et al., 2017b). And all these sensors exhibited a good stability, high sensitivity and excellent selectivity. However, these researches were not mentioning the contents of energy transfer of PFO, and even the application of PFO as energy donor or acceptor. Consequently, investigating the energy transfer performance of PFO was valuable and of great significance since it can extend the application of PFO material in ECL analytical field. Furthermore, it can also expand the range of ECL-RET system, making an addition of energy donor and acceptor pair.

3,4,9,10-perylenetetracar-boxylic acid (PTCA) is the hydrolyzate of perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA). The ECL properties of perylene derivatives have been studied in recent years in our group. It can be exploited as cathodic luminescent material when worked with the co-reactant S₂O₈^2- (Lei et al., 2016), or it worked as the accelerator of coreaction reagent and fixed stabilizer for luminous agent, thus presenting excellent ECL performance (Zhao et al., 2016). However, scanty studies of PTCA was related to the ECL-RET capability. Our group constructed a ratiometric aptasensor based on the energy transfer from peroxydisulfate/oxygen (O₂/S₂O₈^2-) to amino-terminated perylene derivative (PTC-NH₂). It showed that perylene derivatives such as PTCA may be well employed in ECL-RET system.

Encouraged by the excellent performance of above materials, an ECL biosensor was developed for insulin detection based on the energy transfer from PFO (donor) to PTCA (acceptor). In the present work, PFO dots were connected with the primary of insulin antibody (Ab₁) through the amide bond to synthesize the Ab₁-PFO biocomposites, which then could interact with insulin antigen via specific recognition between antibody and antigen. When the biosensor was modified with PTCA labeled secondary antibody (Ab₂-PTCA), the ECL-RET strategy for detecting insulin was completely achieved. Based on the RET from PFO to PTCA, a series quantitative detection of insulin could be well obtained. In this strategy, a new donor-acceptor pair was investigated, and the absorption spectra of PTCA showed great overlaps with the ECL spectra of synthesized PFO dots. Furthermore, the ECL acceptor PTCA exhibited excellent quenching capability to the ECL donor PFO dots. This new pair of donor-acceptor would extend the ECL-RET system and present their admirable potentiality in further ECL analysis.