Paper-based biosensor for noninvasive detection of epidermal growth factor receptor mutations in non-small cell lung cancer patients

Sensors and Actuators B 251 (2017) 440–445

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Paper-based biosensor for noninvasive detection of epidermal growth factor receptor mutations in non-small cell lung cancer patients Tian Tian a,1 , Haiyun Liu a,1 , Li Li a , Jinghua Yu a , Shenguang Ge a,b , Xianrang Song c,∗ , Mei Yan a,∗ a

Institute for Advanced Interdisciplinary Research, University of Jinan, Jinan 250022, China Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022, China c Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Jinan 250117, China b

a r t i c l e

i n f o

Article history: Received 14 February 2017 Received in revised form 9 May 2017 Accepted 16 May 2017 Available online 17 May 2017 Keywords: EGFR mutations Microfluidic paper-based electrochemical DNA biosensor Saliva

a b s t r a c t In this paper, a microfluidic paper-based electrochemical DNA biosensor was constructed for sensitive detection of EGFR mutations in patients with saliva. In order to achieve the purpose of detection, oligonucleotides were modified on the electrode surface, and the outputs of the electrochemical signal were gained by analyzing DNA hybridization reaction, after that, the horseradish peroxidase (HRP) recognized the indicator labeled on DNA and exhibited excellent electrocatalytic behavior to H2 O2 , bringing the rapid enhancement of current response. Under optimum conditions, the as-prepared biosensor showed a good linear relationship between the current value and logarithm of the target DNA concentration ranging from 0.5 nM to 500.0 nM and a detection limit as low as 0.167 nM. Meanwhile, the DNA biosensor emerged good stability and high specificity in distinguishing single nucleotide polymorphism of target DNA. This work not only opened a different horizon for investigating biomarker in biological fluids but also offered a promising and reliable method in biosensing and clinical diagnosis in general. © 2017 Elsevier B.V. All rights reserved.

1. 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

∗ Corresponding authors. E-mail address: chm [email protected] (M. Yan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.snb.2017.05.082 0925-4005/© 2017 Elsevier B.V. All rights reserved.

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 allsided and it can reflects the status of disease more reliable [11]. Yet

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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 [18–21], 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-ofcare testing, microfluidic paper-based analytical device (␮-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 ␮-PAD has been widely used in various fields such as diagnostic testing and environmental analysis [24–28]. In the proposed article, we constructed a microfluidic paperbased electrochemical DNA biosensor (␮-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 H2 O2 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.

2. Experimental 2.1. 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 sequences complementary to DP1). Deletion tDNA1: 5 -CGT CGC TAT CAA GGA AGC AAC AAG GTG

AGA AAG TTA AAA TTC C-3 (The green letters represent bases for deletion). Point mutation tDNA1: 5 -CGT CGC TAT CAA GGA AGC AAC AAG GTG AGA TAG TTA TTA AAA TTC C-3 (The red letter represents a base for point mutation). Capture probe 2 (CP2) for exon 21 point mutation: 5 -CAG TTT GGC CCG CCC AAA ATC-3 .

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Detector probe 2 (DP2) for exon 21 point mutation: 5 -TTG ACA TGC TGC GGT GTT TTC A-FITC-3 . Template DNA 2 (tDNA2) for exon 21 point mutation: 5 -TGA AAA CAC CGC AGC ATG TCA AGA TTT TGG GCG GGC CAA ACT G-3 (The italic letters indicate the sequences complementary to CP2. The underlined letters indicate the sequences complementary to DP2). Deletion tDNA2: 5 -TGA AAA CAC CGC AGC ATG TCA AGA TTT

TGG GCG GGC CAA ACT G-3 (The green letters represent bases for deletion). Point mutation tDNA2: 5 -TGA AAA CAC CGC AGC ATG TCA AGA

TTT TGG GCC GGC CAA ACT G-3 (The red letter represents a base for point mutation). Before use, the oligonucleotides were incubated at 95 ◦ C for 5 min and then slowly cooled to room temperature over 30 min. To identify anti-fluorescein antibody (Ab), DPs were labeled with fluorescein isothiocyanate (FITC) at 3 . FITC was obtained based on fluorescein through increased thiocyanate groups, thiourea bond was formed by thiocyanate groups react with primary amine groups on bioactive substances. Tetrachloroauric acid (HAuCl4 ), potassium ferricyanide. Phosphate buffered solutions (PBS, 0.01 M) with different pH values were prepared with KH2 PO4 and Na2 HPO4 . Pyrrole was distilled repeatedly by distillation under vacuum until a colorless liquid was obtained, and kept under the protection of high purity nitrogen in darkness at 4 ◦ C in refrigerator before use. 30% H2 O2 , MB, 0.05 M Tris–HCl (pH 8.0), HRP. Anti-fluorescein antibody was purchased from Shanghai Kemin Biological Technology Co., Ltd. All reagents were analytical grade and were used without further purification and modification. Ultrapure water obtained from a Millipore water purification system (>18.2 M, Milli-Q, Millipore) was used in all assays and solutions. 2.2. Apparatus

All electrochemical experiments were performed on a CHI760D electrochemistry work station (Chenhua, Shanghai, China) with a conventional three electrode system: the modified paper work electrode (PWE) with a diameter of 6.0 mm was used as the working electrode, screen-printed carbon electrode and Ag/AgCl electrode were used as the counter electrode and the reference electrode, respectively. Scanning electron microscopy (SEM) images were obtained using a QUANTA FEG 250 thermal field emission scanning electron microscope (FEI Co., USA). Energy dispersive X-ray spectroscopy (EDS) was carried out using an X-MAX50 energy dispersive spectrometer (Oxford, UK). All experiments were carried out at room temperature. 2.3. Measurement and fabrication of the biosensor The fabrication process of ␮-PEDB shows in Scheme 1. In the first place, the sample zone of AuNP-PWE was fabricated through growth of an AuNP layer to enhance the conductivity and enlarge the effective surface area of the bare PWE [30]. The next, 5.0 ␮L 100.0 nM CP was copolymerized with 5.0 ␮L 0.1 M pyrrole onto the AuNP-PWE by applying a continuous cyclic voltammetric (CV) scanning for 20 cycles (between −0.1 and +0.7 V; 50 mV/s). This result from the fact that the strong electrostatic effect of PPy attraction with the oligonucleotide probes, the oligonucleotides were incorporated into the polymer matrix in the process of the growth of conducting polymer [31]. It is worth mentioning that the solution was deoxidized by bubbling nitrogen gas for 15 min before applying electric field. Then the modified surface was rinsed with ultrapure water. Once more, 10.0 ␮L 100.0 nM tDNA was mixed with 5.0 ␮L 100.0 nM DP and transferred onto the PWE after polymerization. The hybridization reaction was performed by applying a continuous

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metric responses of the ␮-PEDB were recorded by DPV with a pulse amplitude of 50 mV and a pulse width of 50 ms from −0.6 to 0.0 V. 3. Results and discussion 3.1. 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 of AuNP after the polymerization of PPy (Fig. 1C). The electric resistence of the PPy/AuNP-PWE was measured to be 0.8 /mm, indicating that the electrical conductive property of the biosensor was excellent. In addition, EDS was also used to prove the successful preparation of pyrrole as shown in Fig. 1D, it clearly confirms the presence of C, N, and Au elements (other peaks originate from the substrate used). Scheme 1. Illustration for the synthesis of the ␮-PEDB and measurement protocol.

CV scanning for 20 cycles (between −0.1 and +0.7 V; 50 mV/s). Upon hybridization, 5.0 ␮L 150.0 U/mL Ab was immobilized onto the DP/tDNA/CP/PPy/AuNP-PWE. Approximately 30 min later, 5.0 ␮L HRP (2.0 mg/mL) were applied into Ab/DP/tDNA/CP/PPy/AuNPPWE. Ultimately, the electrode was rinsed with ultrapure water and dried at room temperature.

2.4. Electrochemical detection The measurements of the biosensor was carried out in 10.0 mL of PBS (pH 7.4) containing 1.0 mM MB and 15.0 mM H2 O2 , the ampero-

3.2. Electrochemical behavior of the biosensor As an effective and convenient tool for the characterization of the interfacial properties of electrodes, electrochemical impedance spectroscopy (EIS) was used to get the information of the interface structure of the electrode and monitor modification process of the biosensor. The EIS of layer-by-layer modified steps was monitored in a solution of 2.5 mM [Fe(CN)6 ]3−/4− in the frequency range 100 MHz to 10 kHz. Fig. 2A shows the EIS of different surface conditions of the fibers in the PWE, and the diameter of semicircle was increased along with the increase of the interfacial electron transfer resistance (Ret ). At bare PWE (curve a), it was observed that a relatively small semicircular domain. Opposite, after AuNP layer was modified on the bare PWE, a much lower resistance was observed

Fig. 1. SEM images of (A) the bare paper sample zone of the PWE; (B) the AuNP-PWE; (C) magnification SEM image of the PPy/AuNP-PWE (insert: the PPy/AuNP-PWE); (D) EDS of the PPy/AuNP-PWE.

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Fig. 2. (A) EIS and (B) the corresponding CV curve of different electrodes: (a) bare PWE; (b) AuNP-PWE; (c) PPy and CP modified AuNP-PWE; (d) DP and tDNA modified CP/PPy/AuNP-PWE; (e) Ab modified DP/tDNA/CP/PPy/AuNP-PWE; (f) HRP modified Ab/DP/tDNA/CP/PPy/AuNP-PWE in solution of 2.5 mM [Fe(CN)6 ]3−/4− . (C) Current response of the biosensor: in the absence and presence of tDNA.

Fig. 3. (A) Fluorescent microscopic imaging of ␮-PEDB has been modified without DP and (B) with DP.

(curve b), explaining that the AuNP had been successfully deposited in the surface of fibers, meanwhile, it shows a high electronic conductivity and conducive to electron transfer. Subsequently, PPy as a fixed-interface of DNA probe is further immobilized on the electrode (curve c), the Ret value increases greatly, which was attributed of the electrostatic repulsion between anionic [Fe(CN)6 ]3−/4− and the negatively charged phosphate backbone of the DNA [32]. Similarly, the Ret increased when DP and tDNA were applied on the electrode surface after the hybridization between DNAs (curve d), the resistance of charge exchange also increased due to the negative charge of double strands DNA increased, implying DNA played the part of a kinetic barrier for the charge transfer. The Ab and HRP were immobilized on DP/tDNA/CP/PPy/AuNP-PWE in turn. Compared with curve d, it turned out that the impedance increased significantly (curve e). However, the impedance was decreased apparently in curve f illustrated that the biosensor was effectively constructed. To further confirm the fabrication process of the biosensor, CV was employed to characterize the stepwise assembly process. As shown in Fig. 2B, the bare PWE exhibited a good redox peaks (curve a). After the growth of an AuNP layer, the redox peak currents was shown an obvious increase, explaining that the AuNP-PWE possessed excellent conductivity and a large surface area (curve b). Subsequently, an obvious decrease in CV response was observed when AuNP-PWE was incubated with PPy and CP (curve c). The redox peak currents further decreased after DP and tDNA were applied on the electrode surface (curve d). Eventually, the redox peak currents increased when modified with HRP (curve f) compared with the modified with Ab (curve e). These results demonstrated the biosensor was fabricated successfully as expected.

3.3. Feasibility of the biosensor The proof-of-concept experiments were carried out to test the feasibility of the proposed method through fluorescent microscopic imaging. After PPy was immobilized on AuNP-PWE, the CP was attached to PPy through electrostatic repulsion between anionic [Fe(CN)6 ]3−/4− and the negatively charged phosphate backbone of the DNA [31]. Part of tDNA hybridized with CP, and another part of tDNA hybridized with FITC-labeled DP. From Fig. 3B we can see the result of fluorescent microscopic imaging of DP, explaining that the hybridization between DNAs were happened and DP was successfully fixed on the electrode. The feasibility was also characterized through the DPV recorded for the biosensor platform in response to tDNA and in the control experiments, respectively (Fig. 2C). As shown in red curve, a very low current response was observed in the absence of tDNA, illustrating that Ab were not attached to DP. However, it exhibited an apparent current response in the present of tDNA. This might be attributed to the fact that the reduction reaction of H2 O2 was catalyzed by HRP, thus accelerating the electron transfer.

3.4. Calibration curve Under optimal conditions, the biosensor was used to detect different concentrations of tDNAs. The current signal increased with the concentration of tDNAs, and exhibited a good linear relationship with logarithm of tDNA concentration in the range from 0.5 to 500.0 nM. The equation of the calibration curve was I1 = 17.5294 lg c1 + 26.04119 (R2 = 0.9970) and the detection limit of tDNA1 was calculated to be 0.167 nM (Fig. 4A). In addition, the equation of the calibration curve was I2 = 18.34394 lg c2 + 28.10103

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Fig. 4. The relationship between the current signal and the concentration of tDNA1 (A), tDNA2 (B). The concentration of tDNAs from 0.5 nM to 500.0 nM, each point is the average of three measurements (insert: logarithmic calibration curve for tDNA).

(R2 = 0.9934) and the detection limit of tDNA2 was also calculated to be 0.167 nM (Fig. 4B), illustrating the favorable result of measure and monitor can be got using the proposed sensor [33,34]. The relative standard deviation (RSD) of 3.50% and 6.81% were acquired (by 8 replicate determinations) that demonstrated the preferable reproducibility of the proposed biosensor. 3.5. Reproducibility, stability and specificity The reproducibility of the proposed ␮-PEDB was evaluated by using the RSD of intra-assay and inter-assay. Under the identical experimental conditions, the RSD of intra-assay and inter-assay were 5.7% and 6.3%, respectively. Both of them were estimated from the determination of 10.0 nM target DNA for five replicate measurements. In addition, to demonstrate the effect of background on experimental results in real samples, the solvent of saliva and aqueous solvent were analyzed here to compare their current intensity because of the complexity of the solvent in saliva samples (Fig. S6B). These results indicated an acceptable precision and reproducibility of the as-prepared biosensor. As a key factor, the stability of the above biosensor was also examined. The biosensor was stored at 4 ◦ C for different periods (from 0 to 4 weeks). The current response decreased gradually with the storage time increased. After 4 weeks of storage, the current response of the proposed biosensor decreased 9.3%, indicating that the biosensor could keep its performance in a long time. The exquisite specificity of the proposed strategy was further investigated by perfect matched tDNAs, point mutation tDNAs, and deletion tDNAs (Fig. S6A). Compared with the blank control (in the absence of tDNAs), the signal responses of point mutation tDNAs and deletion tDNAs showed no obvious difference. We discovered that mismatched DNAs were more difficult to initialize the hybridization reaction, which resulted no electrical signal output. Only perfect matched tDNAs can trigger remarkable current response. Thus, our strategy exhibited good performance in the discrimination of mismatched sequences. 3.6. Analysis in saliva sample So as to verify the applicability of proposed method for real sample analysis, the developed biosensor for the detection of the target DNA extracted from saliva samples was evaluated by performing the standard addition method using synthetic target DNA as the standard. 10.0 ␮L saliva samples were spiked in standard solutions containing synthetic target DNA at concentrations of 0.0 nM, 1.0 nM, 2.0 nM, 3.0 nM, 4.0 nM, 5.0 nM, 6.0 nM, respectively. Then, the electrochemical detection were performed under the optimal conditions. According to the experimental results shown in Fig. 5, the linear equation was I1 = 3.39815c1 + 11.93677 (R2 = 0.98747) and the content of tDNA1 in human saliva sample was calculated

Fig. 5. Standard addition method used for the determination of tDNAs extracted from human saliva samples. The error bar represents the standard deviation of three measurements.

as 3.15127 nM, the linear equation was I2 = 3.21783c2 + 7.89897 (R2 = 0.99268) and the content of tDNA2 in the human saliva sample was calculated as 2.45475 nM, indicating that the proposed biosensor can be applied to monitor the target DNA in saliva samples.

4. 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 stability of immobilized DNA. Simultaneously, the proposed ␮-PEDB exhibited the characteristic of cost-effective, easy-to-use, low consumption of reagents. From the perspective of detection method, saliva collection is safe and convenient, noninvasive, no risk of spread of blood-transmitted diseases, and it is painless for patients as well as easily accepted and can provide real-time information of EGFR mutation status. This work not only can be used to predict the effect of targeted therapy, provide a good reference for the clinical use of drugs, and it can also improve the accuracy and precision of targeted therapy.

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).

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Biographies 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.