Electrochemically deposited Ag structure-based ECL sensing platform for KRAS gene detection in the tumor tissues

https://doi.org/10.1016/j.snb.2022.132212Get rights and content

Highlights

  • Chitosan dots were regulated by phenylboronic acid as ECL emitters.

  • The electrochemically deposited Ag structures enhanced ECL intensity largely.

  • The ECL biosensor was used to detect mutant KRAS gene.

Abstract

In this work, a novel electrochemiluminescence (ECL) biosensor was fabricated to detect the mutant KRAS gene in the tumor tissues. Firstly, a new kind of chitosan-based dots was prepared and regulated by phenylboronic acid (PBA) as ECL nanoprobe. Furthermore, the electrochemically deposited Ag structures with controllable morphology have been developed on the electrode. The detailed electrochemical deposition condition of Ag structure has been investigated deeply. Due to the synergistic effect of the large active surface area and the high conductivity, the layered Ag flower-like structure with branches can significantly enhance the ECL intensity of CS-PBA dots by 12.1 times. Finally, the toehold mediated strand displacement strategy was employed in the ECL biosensor to quantitatively detect mutant KRAS gene with the range of 0.01 pM to 10 nM. The limit of detection was 3.3 fM. This biosensor has been used successfully to analyze the target DNA in the tumor tissues of actual cancer patients. The results showed the novel sensing platform possessed great potential for clinical analysis.

Introduction

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system. Colorectal cancer has a high fatality rate and causes more than 880,000 deaths each year [1], [2]. As a proto-oncogene, the mutation and activation of the KRAS gene are closely related to the occurrence and development of colorectal cancer [3]. KRAS gene mutation is an unfavorable factor in the occurrence, development and prognosis of colorectal cancer, which is closely related to the efficacy of targeted therapy. The most common type of KRAS gene mutation occurs in codons 12 and 13 [4]. So, the evaluation of mutant KRAS is critical to the diagnosis and prognosis of patients with colorectal cancer. The expression level of related biomarkers and genes in tumor tissues is closely related to the development, metastasis, and other malignant biological behaviors of cancer [5]. The analytical methods were currently applied in clinical research, including quantitative polymerase chain reaction (qPCR), mutant-enriched PCR and next-generation sequencing (NGS). Despite the advantages of these methods, these methods were constrained by high cost, long turnaround time and complicated steps [6], [7]. Therefore, new sensing methods for the quantification of the KRAS gene in tumor tissue samples are urgently needed.

Electrochemiluminescence (ECL) is a light emission process involving the substance undergoing high energy electron transfer reaction and forming excited state on the surface of electrode, which possesses the advantages of both spectroscopy and electrochemistry [8], [9]. ECL sensing systems have demonstrated excellent analytical performance in biomarker detection and cancer diagnosis due to the low background noise, high sensitivity, and precise process control [10]. Currently, many nanomaterials with excellent optical and electrochemical properties have been employed as ECL emitters, such as semiconductor nanocrystals [11], quantum dots (QDs) [12], gold nanoclusters (Au NCs) [13] and so on. Among these ECL luminophores, carbon-based nanomaterials including carbon dots [14], graphene QDs [15] and polymer dots [16] have received widespread attention. The carbon-based nanomaterials have unique chemical characteristics, including low toxicity and good biocompatibility. The abundant functional groups make the functionalization and modification of the carbon-based nanomaterials more flexible [17]. Meanwhile, the surface states of the carbon-based luminophores have a decisive effect on ECL performance [14]. However, the low ECL efficiency of carbon-based nanomaterials still limited the ECL sensing application. Until now, many studies with nanotechnology and sensing strategies have been explored to improve the ECL intensity of luminophores. On the one hand, new kinds of carbon-based nanomaterials were prepared as ECL luminophores. For example, element doping has been widely used to improve the optical properties of carbon-based nanomaterials. Doping with heteroatoms (such as N, S and P) can effectively increase the electrochemical activity and adjust the band gap of carbon dots [18]. Organic carbon-based nanoparticles with AIE-ECL characteristics were also synthesized with high ECL efficiency [19], [20]. On the other hand, ECL sensing strategies have been reported in the sensing application, such as coreaction accelerator, ECL-RET and surface enhanced ECL [21]. The ECL intensity of luminophores can be significantly enhanced on the modified sensing interface. The sensing interface integrates with signal transduction and specific biorecognition, which provides superior electrochemical features owing to rapid mass transport and excellent conductivity. Recently, gold nanostars, nanocubes and nanodendrites have been reported to be used as electrochemical and ECL enhanced materials [22], [23], [24], [25]. Compared with spherical nanoparticles, these metal nanomaterials with anisotropic structures have strong enhancement ability. The tips and branches of the anisotropic structures have a large active surface area for analyte immobilization, which can increase electron transfer rates and the luminescence signal to improve detection sensitivity [25], [26]. Especially, the noble metal materials grown in situ on the electrode surface were expected to improve the sensing interface performance. However, the precise regulation of the morphology and anisotropic structure of nanomaterials is still an important challenge with significant research value.

In this work, a novel electrochemically deposited Ag structure-based ECL sensing platform was developed for mutant KRAS gene detection in tumor tissues. A novel chitosan-based dots were synthesized as ECL probes via microwave assisted hydrothermal synthesis method. With the regulation of phenylboronic acid (PBA), the luminescence efficiency of chitosan-based dots (CS-PBA dots) was significantly improved. Furthermore, as shown in Scheme 1, Ag structures with different morphologies were prepared and investigated by the electrochemical deposition process on the electrode surface. Due to the synergistic effect of the large active surface area for the analyte immobilization and the high conductivity, the layered Ag flower-like structures (Ag FS) enhanced the ECL intensity of CS-PBA dots by 12.1-fold. Based on the electrochemically deposited Ag FS on the electrode, the ECL biosensor was constructed to detect the mutant KRAS gene. In the sensing system, toehold-mediated strand displacement (TMSD) was employed as the fast enzyme-free nucleic acid amplification strategy to reduce background noise and improve detection sensitivity [27], [28]. The formed duplex DNA structures based on the template DNA, assisted DNA and protected DNA were immobilized on the Ag FS with Ag-S bond. The first TMSD was initiated when target DNA was added to this sensing system. Then, assisted DNA was displaced because of the hybridization of target DNA in the toehold region. Another toehold region in the middle of the template DNA was exposed and hybridized with CS-PBA dots/probe DNA to complete the second TMSD. The released target DNA can be hybridized with the first toehold region again and achieve the recycling process. The feasibility of the ECL biosensor was further verified by detecting target DNA in the patients' actual tumor tissue lysate with satisfactory results.

Section snippets

Materials

Carboxymethyl chitosan, PBA, N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS) were purchased from Aladdin Reagent Company (Shanghai, China). Silver nitrate (AgNO3), citric acid, sodium dihydrogen phosphate (NaH2PO4), hydrogen diphosphate sodium (Na2HPO4), sodium phosphate (Na3PO4) and sodium thiosulfate (K2S2O8) were from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Dithiothreitol (DTT) was obtained from BBI Life Sciences (Shanghai,

Characterization of CS-PBA dots

The TEM image in Fig. 1A displayed that the monodisperse CS-PBA dots had spherical characteristics and a uniform diameter of ca. 20 nm. FT-IR spectra were used to characterize the functional groups of CS dots and CS-PBA dots. The results in Fig. 1B proved that PBA successfully participated in the structure of CS dots during the synthesis process. It can be clearly observed that CS-PBA dots and CS dots had similar functional groups. The characteristic peak in the range of 3200 cm−1 to 3500 cm−1

Conclusions

In this work, the ECL biosensor has been constructed with CS-PBA dots and Ag FS. CS-PBA dots were synthesized with microwave assisted hydrothermal method as ideal ECL emitters. Ag FS were electrochemically deposited on the electrode surface. The Ag FS with layered structure and branches provided the synergistic effect of the large active surface area and the high conductivity. It can amplify the ECL intensity of CS-PBA dots by 12.1 times. The sensing system has been employed in the detection of

CRediT authorship contribution statement

Peilin Wang: Investigation, Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing. Zizhun Wang: Investigation, Supervision, Resources. Zhenrun Li: Visualization, Investigation. Yuan Wang: Methodology, Visualization, Formal analysis. Qiang Ma: Supervision, Project administration, Resources, 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.

Acknowledgment

The present work was supported by the National Natural Science Foundation of China (No. 22174051), the science research project (No. JJKH20211050KJ) in the Education Department of Jilin Province, China and the Graduate Innovation Fund of Jilin University.

Statement on ethical approval

The use of human tumor tissue samples has been approved by the ethics committee of the First Hospital of Jilin University (No. 2021-162).

Peilin Wang is a postgraduate student in Jilin University. Her research interests are largely focused on electrochemiluminescence and surface plasmon coupling electrochemiluminescence.

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  • Cited by (0)

    Peilin Wang is a postgraduate student in Jilin University. Her research interests are largely focused on electrochemiluminescence and surface plasmon coupling electrochemiluminescence.

    Zizhun Wang works in Electron Microscopy Center, Jilin University. His main research direction is the transmission electron microscopy characterization method of nanomaterials.

    Zhenrun Li is an undergraduate student in Jilin University. Her current scientific interests are electrochemiluminescence and novel electrochemiluminescence nanomaterials.

    Yuan Wang is an undergraduate student in Jilin University. His research interest is on the preparation of novel nanomaterials.

    Qiang Ma is an Associate Professor of Jilin University. His research interests cover the novel electrochemiluminescence nanomaterials, electrochemiluminescence biosensors, surface plasmon coupling electrochemiluminescence and their applications in cancer diagnosis.

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