Elsevier

Biosensors and Bioelectronics

Volume 102, 15 April 2018, Pages 624-630
Biosensors and Bioelectronics

Visual electrochemiluminescence ratiometry on bipolar electrode for bioanalysis

https://doi.org/10.1016/j.bios.2017.12.008Get rights and content

Highlights

  • A visual ECL ratiometry based on a closed bipolar electrode was developed.

  • CdTe QDs and luminol were used to emit red and blue visual ECLemission.

  • Au NRs nanocomposite served as the quencher of cathode ECL and promoter of anode ECL.

  • Highly sensitive ratiometric visual detection of PSA was achieved.

  • The proposed strategy could be applied to detect PSA in human serum sample.

Abstract

In this work, we developed a visual ECL ratiometry on a closed bipolar electrode (BPE) for the detection of prostate specific antigen (PSA), prostate cancer biomarker. High efficient CdTe QDs was synthesized which emitted visualized red light at BPE cathode. Integrating with the anodic ECL emitters, luminol, visual emission of red-blue ratiometric ECL was achieved in BPE array chips. As a sensing probe, Au NRs nanocomposite was assembled on the surface of the cathode and acted as both the quencher of the CdTe QDs ECL and the promoter of the luminol ECL. After incubated with PSA, the Au NRs nanocomposite was peeled off from the electrode surface due to the specific recognition between PSA and aptamer. Consequently, the cathode ECL partly recovered and the anode ECL turned off. By measuring the ratio of visual ECL intensity at two poles of BPE, sensitive detection of PSA was achieved with a linear range from 1.0 ng/mL to 1.0 μg/mL and detection limit of 0.5 ng/mL (S/N=3). This strategy combining the BPE-ECL and visual ratiometry provided an accurate and intrinsic way for the sensing of PSA and showed good perspective in the clinical diagnosis.

Introduction

Electrochemiluminescence (ECL) is a light emission process triggered by electrochemical excitation of molecules or quantum dots (QDs) (Ding et al., 2002, Kim et al., 2005, Zanarini et al., 2009). It is remarkably sensitive, which has been widely used for immune assay in the clinical practice. However, due to the influence from environmental interference, false positive or negative might happen in ECL analysis. To increase the detection accuracy and reduce the errors, Xu and coworkers developed the first ECL ratiometric sensing approach using luminol and CdS QDs as the dual-potential ECL emitters for nucleic acid sensing (Zhang et al., 2013). Yuan's group designed a dual-potential ratiometric ECL-RET system for the determination of lead ion (Lei et al., 2015). Lin's Group used the potential-resolved method to detect the proteins and cancer biomarkers (Dai et al., 2015a, Dai et al., 2016, Zhang et al., 2016b). Later, the first dual-wavelength ratiometric ECL approach was reported for the detection of MiRNA which opened a new way for the ratiometric detection (Feng et al., 2016). However, in traditional three-electrode system, dual-potential ratiometric assays need to add two ECL substances with special characters and typical co-reactants in the same solution, which makes the system complicated.

Recently, we developed several ECL ratiometric sensing platforms based on bipolar electrode (BPE) (Feng et al., 2014, Wu et al., 2012, Wu et al., 2013). BPE was an electrically conductive material that immersed in electrolyte solutions (Arora et al., 2001, Xing et al., 2017). Electrochemical reactions occurred at the two poles of the BPE when external voltage was sufficiently high (Chow et al., 2009). The system was named as “open” BPE with the existence of both electronic and ionic current paths (Fosdick and Crooks, 2012, Sentic et al., 2012). On the other hand, a“closed” BPE system physically separates the solutions contacting the two poles of the BPE, with only a current path between the two half-cells (Wu et al., 2015, Zhai et al., 2016). Such BPE structure hold promise for the fabrication of ratiometric ECL biosensor since different ECL reactions could process in separated cells, which was defined as “spatial-resolved” ECL (Wang et al., 2016). In 2016, we proposed a visual ECL ratiometric sensor based on a dual-bipolar electrode (D-BPE) array chip for the determination of tumor cells (Zhang et al., 2016a). Conventional ECL emitters, Ru(bpy)32+ and luminol were added in different arrays of reservoirs and presented turn-on and turn-off phenomenon after addition of tumor cell lysate. Combining the merit of BPE and the visual detection, ratiometric ECL shows perspective in high-throughput bioanalysis. Nevertheless, most electrochemiluminophores with high efficiency are excited at positive potential (Chow et al., 2008, Wang et al., 2017, Zhou et al., 2015). Therefore, the complicated multichannel BPEs arrays were needed. QDs, the regular cathodic electrochemiluminophores, are good candidates for the development of visualized BPE-ECL system. However, most reported QDs such as CdS, CdSe suffered from low ECL efficiency (Huang et al., 2015, Ke et al., 2015, Zhang et al., 2015b, Zhang et al., 2015c). Recently, Ding's group reported a high efficient CdTe QDs, which could be visualized at FTO electrode with K2S2O8 as the coreactant (Zhang and Ding, 2016, Zhang and Ding, 2017). Using such cathodic material, visualized ECL emissions could be observed at both anode and cathode of the BPE. Through modulating the faradic reactions at one pole, ratiometric determination could be achieved in a visual way.

In this paper, we report a visual ECL ratiometry on a closed BPE. CdTe QDs and luminol were used as the cathode and anode ECL emitters. As a sensing probe, Au NRs nanocomposite was introduced to the surface of cathode via DNA hybridization with PSA aptamer. The nanocomposite acted as the quencher of the CdTe QDs ECL because of the spectrum overlap between CdTe and Au NRs nanocomposite and the promoter of the anode luminol ECL due to the increase electrochemical current of BPE caused by catalysis of Au NRs nanocomposite to the reaction of CdTe/S2O82-. Therefore, the red ECL emission of CdTe QDs was quenched and luminol ECL was enhanced. After incubated with PSA, the Au NRs nanocomposite was disassembled from the cathode because of the binding of PSA and aptamer. Consequently, the cathode ECL was partly recovered and the anode ECL decreased. By analyzing the intensity value of visual ECL images of CdTe QDs and luminol via Image-Pro 6.0 software and subsequently calculating the ratio of the anode ECL to the cathode ECL, sensitive detection of PSA was achieved.

Section snippets

Materials and reagents

CdCl2·2.5H2O, telluruim powder, sodium borohydride (NaBH4), 3-mercaptopropionic acid (MPA), luminol, hydrogen peroxide (H2O2) (37%), chitosan (CS), N-[3-(Dimethylamino) propyl]-N’-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), mPEG-SH, Tween 20 and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Au NRs were purchased from the Nanoseedz Co (NR-60–630, 10 O.D.). Fluoride doped tin oxide (FTO)-coated (thickness ~110 nm, resistance ~10 Ω/square) aluminosilicate glass

Characterization of CdTe quantum dots and Au NRs

Fig. 1A shows the TEM image of the MPA-capped CdTe QDs. The average diameter of QDs was 3.8 nm with uniform morphology. It was found that the QDs solutions remained stable for several months. ECL of the QDs was measured in a three-electrode system. As shown in Fig. 1B, driving by CV from 0~ −1.6 V, the ECL emission of the CdTe QDs reached the peak at the potential of −1.55 V. Here, CdTe and K2S2O8 were firstly reduced to (CdTe)∙− and SO4∙−. The generated (CdTe)∙− was then oxidized to excited state

Conclusions

In summary, we developed a visual ECL ratiometry based on a closed BPE for the detection of PSA. Visualized red-emitting CdTe QDs was synthesized and integrated with luminol in a single BPE device as cathodic and anodic emitters, respectively. Under sufficient driving voltage, anodic and cathodic emissions could be observed simultaneously, and quickly recorded by CCD camera. The images were analyzed with Image-Pro software. Compared with PMT recording, this strategy provided high throughput

Acknowledgement

This work was supported by the Science and Technology Ministry of China (Grant no. 2016YFA0201200), the National Natural Science Foundation of China (Grants 21475058). This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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