Paper-based biosensor for noninvasive detection of epidermal growth factor receptor mutations in non-small cell lung cancer patients
Introduction
The morbidity and mortality of lung cancer is increasing rapidly and it severely endangers human health and life. According to the research, approximately 90% of all lung cancer deaths are caused by tumor metastases [1]. Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations accounts for 80% of all lung cancer cases [2]. In 2004, patients with EGFR-mutation have shown a favorable response to EGFR-tyrosine kinase inhibitor (TKI) that prevents tyrosine phosphorylation and inhibits the proliferation of tumor cells by competing with adenosine triphosphate (ATP) for binding to the tyrosine region of the EGFR in the cell membrane [3], and there was a substantive discovery that has confirmed to be effective in patients with lung cancer and has changed the therapeutic approach to lung cancer [4], [5]. With the in-depth research, the relationship between different types of EGFR gene mutation and the efficacy of TKI have received extensive attention [6]. EGFR gene mutations mainly occurred in exon 18–21, and exon 19 deletion as well as exon 21 L858R point mutation accounted for more than 85% of all mutation types, and these two are the most important and sensitive mutations to TKI, which can lead to the enhancement of the activity of tyrosine kinase. Compared with patients with the exon 19 deletion and exon 21 L858R point mutation, patients with other EGFR mutations were less responsived to TKI [7]. Therefore, an accurate detection of exon 19 deletion and exon 21 L858R point mutation is of great significance for diagnosis and treatment guideline in NSCLC.
Traditional method for detecting EGFR mutations is direct DNA sequencing of polymerase chain reaction (PCR)-amplified genomic DNA fragments of cancerous tissues [8], the limitations of this method mainly display in the following aspects: firstly, traditional method required patient tissue specimens, but most patients did not have the conditions to obtain tissue specimens. Secondly, when the proportion of the mutation sequences in the patients was less than 25%, the sequences of mutation could not be detected by direct DNA sequencing. The results showed false negative. Therefore, it is urgent to explore a better detection method.
The identification of biomarkers plays an important role in the early stage of lung cancer. Biomarkers can be derived from saliva, serum, plasma and tissue [9], [10]. Tissue biopsy has been required as a gold standard for tumor genotyping, the examination is all-sided and it can reflects the status of disease more reliable [11]. Yet there are several limitations associated with lung cancer biopsy. For example, the sample must be fresh and active tissue, sometimes it is necessary to make a multi-spot biopsy, furthermore, single tumor biopsy do not always reveal the whole genome of the tumor [12]. The development of analytical tools using biological fluids as the main material has been pursued recently. During the last few years, more and more attention has been paid to EGFR mutations, which can be detected in blood of NSCLC patients [13], [14]. The most recent research found that a variety of protein ingredients in the blood were also present in saliva. As an easily accessible biological fluid, saliva has drawn more attention and concern of scientists [15], [16]. Comparatively speaking, saliva collection is noninvasive, easily obtain and storage than tissue sampling. Besides, saliva can be helpful for studying a large population and is advantageous to children with NSCLC particularly [17]. The analysis of saliva provides a desirable and promising platform for the diagnosis of several diseases such as pancreatic cancer and ovarian cancer [18], [19], [20], [21], and the detection of biomarkers in biological fluids will gradually develop into the core technology in the future.
As a newly developed promising analytical tool for point-of-care testing, microfluidic paper-based analytical device (μ-PAD) possess attractive features consist of cost-effective, easy-to-use, portable, high integration, low consumption of reagents and the porous structure of the paper [22], [23], and μ-PAD has been widely used in various fields such as diagnostic testing and environmental analysis [24], [25], [26], [27], [28].
In the proposed article, we constructed a microfluidic paper-based electrochemical DNA biosensor (μ-PEDB), which could detect whether or not the EGFR mutation occurs in patients with NSCLC through the analysis of DNA hybridization reaction. Herein, the single stranded DNA was adsorbed onto the polypyrrole (PPy) membrane modified gold electrode surface by non-covalent interaction, due to PPy by electrochemical polymerization is positively charged, under the condition of physiological pH, the phosphate group of DNA in solution is negatively charged, the immobilization of DNA on the surface of PPy membrane was achieved by electrostatic force [29]. The horseradish peroxidase (HRP) catalyze the redox reaction between H2O2 and methylene blue (MB) by applying differential pulse voltammetry (DPV). This work explored a noninvasive electrochemical DNA detection method and would provide potential applications for the detection of tumor markers of lung cancer.
Section snippets
Reagents
All oligonucleotides were synthesized and purified by TaKaRa Bio. Inc. (Dalian, China). Their sequences were shown below:
Capture probe 1 (CP1) for exon 19 deletion: 5′-TGT TGC TTC CTT GAT AGC GAC G-3′.
Detector probe 1 (DP1) for exon 19 deletion: 5′-GGA ATT TTA ACT TTC TCA CCT-FITC-3′.
Template DNA 1 (tDNA1) for exon 19 deletion: 5′-CGT CGC TAT CAA GGA AGC AAC AAG GTG AGA AAG TTA AAA TTC C-3′ (The italic letters indicate the sequences complementary to CP1. The underlined letters indicate the
Characterization of AuNP-PWE and PPy/AuNP-PWE
Fig. 1A depicts the SEM image of paper fiber of sample zone on the bare PWE. The bare PWE has excellent adsorption effect to provide a good platform for AuNP seeds growth. Visibly, a dense conducting AuNP layer was obtained completely on the fiber surface to enhance the conductivity of sample zone (Fig. 1B). After the polymerization of PPy onto the surface of the AuNP-PWE, it can be seen that an apparent layer of membrane was coated on the surface of AuNP, there is no obvious change of the size
Conclusion
In a word, we have successfully introduced electrochemical DNA biosensor into microfluidic paper-based analytical device for highly sensitive detection of EGFR mutation occurred in patients with low detection limit and high reproducibility. In this paper, DNA without modified derivatization was adsorbed onto the PPy membrane in order to improve the activity of hybridization, on the other hand, the immobilization of DNA without of the application of continuous voltage, which could enhance the
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (21505052, 51473067, 21675064), Natural Science Foundation of Shandong Province, China (ZR2015JL019), and Shandong Distinguished Middle-Aged and Young Scientist Encourage and Reward Foundation (BS2014SW035).
Tian Tian studies in school of chemistry and chemical engineering, University of Jinan as postgraduate student.
References (34)
- et al.
Cell signaling by receptor-tyrosine kinases
Cell
(2010) - et al.
Cigarette smoke extract exposure induces EGFR-TKI resistance in EGFR-mutated NSCLC via mediating Src activation and EMT
Lung Cancer
(2016) - et al.
EGFR tyrosine kinase inhibitor in patients with advanced non-small cell lung cancer harboring uncommon EGFR mutations: a real-world study in China
Lung Cancer
(2016) - et al.
Effectiveness of gefitinib against non-small-cell lung cancer with the uncommon EGFR mutations G719X and L861Q
J. Thorac. Oncol.
(2014) - et al.
Spectral sensing for tissue diagnosis during lung biopsy procedures: the importance of an adequate internal reference and real-time feedback
Lung Cancer
(2016) - et al.
Plasma is a better source of tumor-derived circulating cell-free DNA than serum for the detection of EGFR alterations in lung tumor patients
Lung Cancer
(2013) - et al.
Role of pancreatic cancer-derived exosomes in salivary biomarker development
J. Biol. Chem.
(2013) - et al.
Integration of platinum nanoparticles with a volumetric bar-chart chip for biomarker assays
Angew. Chem. Int. Ed.
(2014) - et al.
In-situ synthesized polypyrrole-cellulose conductive networks for potential-tunable foldable power paper
Nano Energy
(2017) - et al.
Cu@Au alloy nanoparticle as oligonucleotides labels for electrochemical stripping detection of DNA hybridization
Biosens. Bioelectron.
(2003)
Comprehensive detection of diverse exon 19 deletion mutations of EGFR in lung Cancer by a single probe set
Biosens. Bioelectron.
A primer and probe set for detecting multiple types of EGFR exon 19 deletions
Anal. Biochem.
Quantitative proteomic analysis identifies CPNE3 as a novel metastasis-promoting gene in NSCLC
J. Proteome Res.
Comprehensive genomic characterization of squamous cell lung cancers
Nature
Activating mutation in the epidermal growth factor receptor underlying responsiveness of non-small-cell-lung cancer to gefitinib
N. Engl. J. Med.
Sea-urchin-like Au nanocluster with surface-enhanced raman scattering in detecting epidermal growth factor receptor (EGFR) mutation status of malignant pleural effusion
ACS Appl. Mater. Interfaces
Novel glycan biomarkers for the detection of lung cancer
J. Proteome Res.
Cited by (0)
Tian Tian studies in school of chemistry and chemical engineering, University of Jinan as postgraduate student.
Haiyun Liu received his PhD degree in Chemical Engineering and Materials Science from Shandong Normal University in 2014. He then worked in University of Jinan, where currently he is a lecturer of chemistry. His current scientific interests are focused on nucleic acid DNA-based sensor, bio-imaging and biochemical analysis.
Li Li studies in school of chemistry and chemical engineering, University of Jinan as postgraduate student.
Jinghua Yu received her PhD degree in analytical chemistry in 2003 from Lanzhou. Institute of Chemical Physics, China. She is currently positioned as a professor at University of Jinan. She spends most of her time investigating biomedical engineering, especially for the development of biosensor devices and analytical tools.
Shenguang Ge received his PhD degree in Chemistry and Chemical Engineering in 2013 from Shandong University, completed his master degree studies in University of Jinan in 2006. He joined University of Jinan, where currently he is a lecturer of chemistry. His research interests are in the area of biosensor and chemsensor.
Xianrang Song works in Cancer Research Center, Shandong Tumor Hospital in China.
Mei Yan received her BSc in applied chemistry from University of Jinan in 1999, and obtained her PhD in 2005 from Institute of Chemistry Chinese Academy of Sciences. She then joined University of Jinan, as an associate professor working on the synthesis and performance of advanced functional materials.
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These authors contributed equally to this work.