“Signal-on” molecularly imprinting-aptamer electrochemiluminescence platform for ultrasensitive detection of thrombin

https://doi.org/10.1016/j.snb.2021.129870Get rights and content

Highlights

  • A “signal-on” ECL ultrasensitive sensor for thrombin is designed.

  • Molecular imprinting-aptamer improves the selectivity of this sensor.

  • RuNP/PEI-GO as signal label has better ECL efficiency and stability.

  • This sensor shows satisfactory sensitivity with a low detection limit for thrombin.

Abstract

A novel “signal-on” molecular imprinting-aptamer electrochemiluminescence (ECL) platform is designed for ultrasensitive bioanalysis of thrombin (TB) based on the SiO2 loaded with Ru(bpy)32+/polyetherimide-graphene oxide (RuNP/PEI-GO) nanocomposite as signal labels. Specifically, “signal-on” ECL sensing strategy and molecular imprinting technology is combined together to fabricate sensing platform. The nanocavity corresponding to TB produced by molecular imprinting can be used for multiple specific recognition of TB together with aptamers. Moreover, high-quality ECL nanocomposite luminophore is achieved by means of RuNP/PEI-GO nanocomposite with significant ECL efficiency and stability. Simultaneously, PEI, as a co-reaction, can shorten the electron-transfer path of this ECL system to reduce the energy loss, resulting in the enhancement of ECL signal effectively. ECL signal strength of RuNP/PEI-GO is about 7 times higher than that of RuNP due to the synergy effect of PEI, GO and RuNP, thus further expanding the detection range of TB. This ECL sensing platform for TB detection shows satisfactory sensitivity with a low detection limit of 28.73 fM in the concentration range from 10−13 M to 10−8 M. This developed “signal-on” ECL platform with RuNP/PEI-GO nanocomposite is expected to become a promising tool for detecting other biomarkers in the future.

Introduction

Thrombin (TB) plays an important role in some blood-related diseases, such as thromboembolic disease leukemia and Alzheimer’s disease [[1], [2], [3]]. Accordingly, the detection of TB is of great significance for the diagnosis, treatment of related diseases, and the evaluation of drug efficacy [4]. Although TB recognition has been studied for many years, there are still many obstacles in actual clinical application, such as high detection costs and high sensitivity to environmental conditions [5,6]. Therefore, it is extremely important to develop powerful sensing platform for detecting TB stably and effectively.

It is well-known that electrochemiluminescence (ECL) sensors have been widely concerned due to unique advantages such as simple structure and low detection limit [7,8]. With development of ECL sensors, the requirements on the specificity and sensitivity of sensors are constantly increasing [9]. Molecular imprinting method can bind target molecules selectively through unique “lock-key” mechanism based on the specific memory recognition of three-dimensional structure, which becomes one promising method for small molecules detection in the future [[10], [11], [12]]. At present, most reported molecular imprinting methods are used in non-biologically substances, and there are only few reports on the detection of biomarkers [[13], [14], [15]]. Aptamer can be specifically bound to protein due to single-stranded nucleic acid structure [16]. The combination of molecular imprinting method and aptamer detection can bring some advantages to obtain better specificity [17]. However, molecularly imprinting-aptamers sensors are all of the “signal-off” type, which usually has the disadvantages of narrow detection range and low detection sensitivity [[18], [19], [20]]. The “signal-on” ECL sensor can recognize the target through signal label, resulting in “signal-on” to improve the sensitivity and expand the detection range [21,22]. Based on these considerations, it will be interesting if the integration of molecular imprinting-aptamers and ECL “signal-on” method can provide a new detecting platform with high specificity and sensitivity for small biomolecules and biomarkers detection.

In the fabrication of ECL sensing platform, it is important to choose appropriate signal labels to achieve high sensitivity and stability. Ru(bpy)32+ and SiO2 nanocomposite have both excellent biocompatibility of silicon and high ECL efficiency of Ru(bpy)32+ [23]. However, researchers still have higher expectations for enhancing ECL signal of RuNP. Finding effective co-reactants and increasing the loading of luminophore are effective solutions to increase the ECL intensity. Several reports have pointed out that amine compounds can be used as co-reactants to increase the ECL signal of Ru(bpy)32+ [24,25]. Polyetherimide (PEI) has excellent stability and may be an interesting co-reactant to improve the luminous efficiency of RuNP. Simultaneously, graphene oxide (GO) is a good material for fixing lumiphores with abundant carboxyl groups on the large specific surface area [26,27]. As an ECL lumiphore, RuNP can be more loaded on GO due to the abundant functional groups (carboxyl groups) on the GO large specific surface, thereby improving the sensitivity of the designed ECL sensor. On the other hand, these functional groups are removed after reduction of GO to rGO or erGO [26]. Thus, it will be interesting if we fabricate RuNP and PEI onto GO through amide bonds to obtain the RuNP/PEI-GO nanocomposite. RuNP/PEI-GO nanocomposite with PEI as co-reactant can shorten the electron-transfer path and reduce energy loss, resulting in a significant increase of the strength for ECL signal.

To our knowledge, this novel ECL “signal-on” strategy based on molecularly imprinting-aptamers and RuNP/PEI-GO nanocomposite is firstly proposed and used for the ultrasensitive detection of TB. The specificity of this ECL sensing platform can be effectively improved due to the unique nanocavity generated by molecular imprinting and the specific aptamers. High-performance RuNP/PEI-GO nanocomposite increases the ECL luminous intensity under the synergy effect of PEI, GO and RuNP, thus further expanding the detection range of TB. This work brings a new prospect for the preparation of effective and applicable molecular imprinting sensing ECL “signal-on” platform for ultrasensitive analysis of biomarkers and clinical medical diagnosis.

Section snippets

Reagents and apparatus

The reagents and apparatus used in this experiment are all described in the Supporting Information.

Preparation of molecular imprinting membrane

The preparation process of molecular imprinting membrane was shown in Scheme 1. Firstly, the PI5CA/Au composite modified electrode was prepared (the details of the preparation can be found in Supporting Information). Then, 8 μl EDC (3 mM) and NHS (5 mM) mixture (mixing ratio 1:1) was uniformly applied onto the surface of PI5CA/Au complex modified GCE, and the carboxyl group of PI5CA was activated

Characterizations of molecularly imprinted membrane

As shown in Fig. 1A, Au electrodeposited on GCE exhibited regular granular structure with rough surface. And the size of the structure was mainly distributed in 1.2–1.8 μm (the inset of Fig. 1A). When PI5CA was deposited on Au (Fig. 1B), the composite structure showed folded fiber structure with dense micropores. This structure further increased the specific surface area of the modified electrode. The abundant carboxyl groups on the surface of PI5CA can connect a large number of TBA1 through

Conclusions

An ultrasensitive detection strategy for TB based on ECL “signal-on” and molecular imprinting-aptamer is proposed. RuNP/PEI-GO nanocomposite is assembled on the modified electrode to achieve “signal-on”. Benefitting from multiple recognitions of molecular imprinting-aptamers to TB, the sensitivity of this ECL sensing platform has been effectively improved. In particular, RuNP/PEI-GO, formed by PEI as a co-reactant and RuNP fixed to GO, not only effectively shorten the distance of electron

CRediT authorship contribution statement

Chaonan Yang: Conceptualization, Data curation, Writing - original draft. Ya Tian: Formal analysis, Data curation. Baoying Wang: Investigation, Validation. Qingfu Guo: Methodology, Formal analysis. Guangming Nie: Funding acquisition, Project administration, Resources, 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.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (51973102), Natural Science Foundation of Shandong (ZR2019MB067), Qinghai Provincial Basic Research Program (2021-ZJ-710) and Talent Fund of QUST (2019).

Chaonan Yang is a graduate student in the College Chemistry and Molecular Engineering at Qingdao University of Science and Technology, China. Her current research interests focus on the design and application of electrochemiluminescence sensors.

References (36)

  • T.B. Gao et al.

    Single-molecule microrna electrochemiluminescence detection using cyclometalated dinuclear Ir(III) complex with synergistic effect

    Anal. Chem.

    (2020)
  • M. Yoshikawa et al.

    Molecularly imprinted membranes: past, present, and future

    Chem. Rev.

    (2016)
  • J.J. BelBruno

    Molecularly imprinted polymers

    Chem. Rev.

    (2018)
  • X. Guo et al.

    Molecular-imprinting-based surface-enhanced raman scattering sensors

    ACS Sens.

    (2020)
  • Q. Zhao et al.

    Single-template molecularly imprinted chiral sensor for simultaneous recognition of alanine and tyrosine enantiomers

    Anal. Chem.

    (2019)
  • Z. Li et al.

    Molecularly imprinted sites translate into macroscopic shape- memory properties of hydrogels

    ACS Appl. Mater. Interface

    (2019)
  • S. Shinde et al.

    Urea-based imprinted polymer hosts with switchable anion preference

    J. Am. Chem. Soc.

    (2020)
  • M. Panigaj et al.

    Aptamers as modular components of therapeutic nucleic acid nanotechnology

    ACS Nano.

    (2019)
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    Chaonan Yang is a graduate student in the College Chemistry and Molecular Engineering at Qingdao University of Science and Technology, China. Her current research interests focus on the design and application of electrochemiluminescence sensors.

    Ya Tian is a graduate student in the College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, China. Her current research interests focus on the preparation of novel nanomaterials and their application.

    Baoying Wang is a graduate student in the College of Chemistry and Molecular Engineering at Qingdao University of Science and Technology, China. Her current research interests focus on the field of design and application of nanocomposite and polymer materials.

    Qingfu Guo is a Ph.D. of Applied Chemistry in the college of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, China. His current research interests focus on the preparation of conductive polymer nanocomposites.

    Guangming Nie is currently a professor in College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, China. He received his Ph.D. in Applied Chemistry. Afterwards he worked as a postdoctor in Technical Institute of Physics and Chemistry, Chinese Academy of Sciences and as a visiting scientist in Nanyang Technological University. His current research interests focus on nano-electrochemistry, including electrochemical sensors, electrochemiluminescence sensors and photoelectrochemical sensors based on nano-conducting polymers.

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