Evaluation of a biosensor-based graphene oxide-DNA nanohybrid for lung cancer

Lung cancer is nowadays among the most prevalent diseases worldwide and features the highest mortality rate among various cancers, indicating that early diagnosis of the disease is of paramount importance. Given that the conventional methods of cancer detection are expensive and time-consuming, special attention has been paid to the provision of less expensive and faster techniques. In recent years, the dramatic advances in nanotechnology and the development of various nanomaterials have led to activities in this context. Recent studies indicate that the graphene oxide (GO) nanomaterial has high potential in the design of nano biosensors for lung cancer detection owing to its unique properties. In the current article, a nano biosensor based on a DNA-GO nanohybrid is introduced to detect deletion mutations causing lung cancer. In this method, mutations were detected using a FAM-labeled DNA probe with fluorescence spectrometry. GO was synthesized according to Hummers' method and examined and confirmed using Fourier Transform Infrared (FT-IR) Spectrometry and UV-vis spectrometry methods and Transmission Electron Microscopy (TEM) images.


Introduction
Cancer is a genetic disease that results from the uncontrolled growth and division of cells in a part of the body that results from environmental factors and genetic disorders. [1][2][3][4][5] In other words, cancer occurs as a result of a series of mutations in human genes. [6][7][8][9][10] There are more than 200 types of cancer today, one of the most common of which is lung cancer. [11][12][13][14][15] Lung cancer is the second most common cancer in men and women and is one of the most preventable cancers. There are generally two types of lung cancer: [16][17][18][19][20][21] (1) Small cell lung cancer (SCLC) (2) Non-small cell lung cancer (NSCLC) How both grow and spread in the body and how to treat them are different. Lung cancers are classied under the microscope based on the appearance of the cells. Non-small cell lung cancer (NSCLC) is also divided into three categories: [22][23][24][25] (1) Supercial tissue cancer, (2) mucosal and lymph node carcinoma (glandular epithelium), and (3) large cell lung cancer. [26][27][28][29][30] Among people with this type of cancer, about 85-90% of cases are NSCLC and about 10-15% of cases are SCLC.
The most common clinical symptoms of lung cancer include persistent and chronic cough, chest pain, anorexia, weight loss, sputum, shortness of breath, respiratory infections such as bronchitis, the onset of wheezing and . is, which usually does not appear in the early stages of the disease. Therefore, the mortality rate of this type of cancer is very high. [31][32][33][34] The most prevalent symptoms of lung cancer include continuous and chronic cough, thoracic pain, anorexia, weight loss, hemoptysis, dyspnea, and respiratory infections such as bronchitis, the onset of wheezing, etc., which do not typically appear in the early stages of the disease, leading to a high mortality rate in this type of cancer. [35][36][37][38] In the eld of medicine, lung cancer has so far been detected using various methods, including chest radiography (x-ray), computed tomography (CT) scan, magnetic resonance imaging (MRI), bone scan, bronchoscopy, and sputum cytology. [39][40][41][42][43] In recent years, the dramatic advances in nanotechnology and the development of various nanomaterials have facilitated the detection of cancer biomarkers with high accuracy and sensitivity. [44][45][46][47] Nanotechnology has provided faster, less expensive, and easier methods with lower detection limits

Preparation of the Tris-HCl buffer
Tris(hydroxymethyl)amino methane (0.242 g) was rst dissolved in an Erlenemayer containing 100 ml of sterile water, and the solution pH was made to 7.4 using HCl (with a pH meter). Then, the Tris-HCl buffer was obtained with adding 0.5844 g of sodium chloride, 0.0373 g of potassium chloride, and 0.102 g of magnesium chloride to this solution. 64-68

Synthesis of GO
To synthesize GO with Hummer's method, 0.5 g P 2 O 5 , 0.5 g potassium persulfate, and 0.5 g graphite powder were poured into a beaker containing concentrated H 2 SO 4 and incubated at 80°C for 6 h. The solution was then diluted with 50 ml distilled water and ltered aerward. The lter paper content was washed with 50 ml distilled water and fully dried at the ambient temperature overnight. Then, 0.25 g of the resulting powder was poured into a beaker containing 11.5 ml of concentrated H 2 SO 4 in an ice bath, followed with adding 1.5 g of KMnO 4 while stirring continuously. Sodium nitrate was added aer 15 min and stirred at 35°C for 2 h. Next, 25 ml of distilled water was added to the reaction solution and stirred for 15 min. The reaction was stopped with adding 75 ml of distilled water and 2 ml of H 2 O 2 (30%), yielding a yellow solution. This solution was centrifuged and the obtained pellet was washed with HCL (10%) and then with distilled water several times to completely remove the existing acids and metal ions. 69,70,101 The graphite oxide solution was ultrasonicated for 10 min and then centrifuged at 1000 rpm for 10 min, which was repeated several times to obtain the GO solution.

Preparation of solutions for the uorescence spectrum measurement
The uorescence spectra were drawn in six steps, namely (1) drawing the DNA probe uorescence spectrum, (2) Optimizing DNA probe adsorption time on the GO surface,   Table 2 The nucleotide sequences of DNAs used in this study a Oligonucleotide Sequence Preparation of the third-step solution. In this step, the 10 4 nM solution of the DNA probe was poured into six prepared solutions, each of which received known volumes (10,20,30,35, and 40 ml) of the 1 mg ml −1 GO solution. Each solution was then made into a nal volume of 2 ml using the Tris-HCl buffer, followed with vortexing and undergoing uorescence emission spectral measurements aer the optimal time of the DNA probe adsorption on the GO surface. 80-82 2.3.4. Preparation of the fourth-step solution. First, 10 ml of the 104 nM solution of the DNA probe and 35 ml of a 1 mg ml −1 GO solution were poured into a known amount of the Tris-HCl buffer. Aer the optimal time of the DNA probe adsorption on the GO surface, 10 ml of the 104 nM solution of the target DNA was added to make the solution into a total volume of 2 ml. The solution was vortexed and underwent emission spectrum measurements at 2, 5, 30, 34, 36, and 42 min. [83][84][85][86] 2.3.5. Preparation of the h-step solution. In this step, 10 ml of 10 4 nM solution of the DNA probe and 35 ml of the 1 mg ml −1 GO solution were poured into ve prepared solutions, each of which received a known volume of the Tris-HCl buffer. Aer the optimal time of the DNA probe adsorption on the GO surface, each solution was then made into a nal volume of 2 ml using the 10 4 nM solution of the target DNA at different volumes (2.5, 5, 7.5, 10, and 15 ml). The solutions were vortexed and their uorescence emission spectra were measured aer the optimal hybridization time of the DNA probe and target DNA. 87, 88 2.3.6. Preparation of the sixth-step solutions. First, 10 ml of the 10 4 nM solution of the DNA probe and 35 ml of the 1 mg ml −1 GO solution were poured into a known amount of the Tris-HCl buffer. Aer the optimal time of the DNA probe adsorption on the GO surface, 10 ml of the 10 4 nM solution of mDNA was added to make the solution into a total volume of 2 ml. The solution was then vortexed and underwent emission spectrum measurements aer the optimal hybridization time of the DNA probe and target DNA. 89

Preparation of GO from graphite
GO was synthesized using graphite powder in the presence of concentrated H 2 SO 4 , NaNO 3 , and KMnO 4 according to the Hummers' method ( Fig. 1). 48

Examination of the GO UV-vis spectrum
The structure of the GO synthesized with the Hummers' method was examined and conrmed using the GO absorption UV-vis spectrum (Fig. 2), which corresponds to those reported in previous studies. The strong and weak bands appearing in 230 and 300 nm wavelengths are respectively attributed to p-p* and n-p* transitions of carbonyl groups. 49,50

Interpretation of the GO IR spectrum
The absorption band observed in the 3449 cm −1 region belongs to OH stretching vibrations. The weak absorption bands in 2867 and 2927 cm −1 regions are related to the CH of aldehyde groups. The absorption bands appearing in 1633 and 1065 cm −1 regions correspond to C]C and C-O stretching vibrations, respectively. The absorption band emerged in the 875 cm −1 region belongs to CH Ar out-of-plane bending vibrations. [91][92][93] Since the absorption band of the acidic C]O group is not observed in the 1730 cm −1 region, the synthesized graphene oxide contains lower carboxylic acid groups and possesses mostly alcoholic and aldehyde groups (Fig. 3). Furthermore, Fig. 3(a) illustrates the Raman spectum of graphene oxide includes D and G peaks where the D peak at ∼1350 (cm −1 ) is the result of defects in the Graphene sheets and the G peak at ∼1600 (cm −1 ) is the result of bond stretching of sp 2 hybridized Carbons, respectively. 56,57 Fig. 1 Conversion of graphite to GO.

Examination of the GO TEM image
Sample preparation methods TEM can be divided into two general categories. The rst category are methods that include reducing the thickness of the sample by chemical or mechanical methods until a thin sample remains. The second category are methods that involve cutting the sample along the crystal planes to obtain a very thin section of the sample.
The structure of the synthesized GO was conrmed using the TEM image (Fig. 4), showing the GO layered structure. 94 In addition, 2-7 graphene layers can be clearly seen in the TEM micrographs in Fig. 4(a)-(f). The SAED patterns shown in Fig. 4(g)-(i) are irregular, and the bilayer graphene, trilayer graphene and ve-layer graphene lms cannot be justied based on these patterns. Thus, other characterizations, such as Raman spectroscopy, are crucial to support the TEM results.

Selection of lung cancer biomarker
As one of the most prevalent cancer types worldwide, lung cancer is detected using various biomarkers, including exhaled air volatile organic compounds (VOCs), carcinogenic embryonic antigen (CEA), neuron-specic enolase (NSE), progastrinreleasing peptide (Pro GRP), cytokeratin-19 fragments (Cyfra21-1), squamous cell carcinoma antigen (SCCA), some miRNAs (e.g., miR-155, miR-197, and miR-182), and some genes such as egfr, kras, etc. In addition to disease detection, some of these biomarkers are useful for examining disease progression, patients' response to treatment, and post-treatment disease recurrence. [51][52][53] According to previous studies, lung cancer patients are prone to numerous gene mutations, a few of which are common among most patients.  mutations in the egfr gene; in most cases, deletion mutations are more frequent than the L858R point mutation. Fig. 6 illustrates the frequencies of other mutations. [55][56][57][58] NSCLC accounts for over 80% of lung cancer cases, and mutations in the egfr gene are highly frequent among gene mutations of the lung cancer cause. Among the mutations of this gene, exon 19 deletion mutations account for a high percentage. In this study, therefore, deletion mutations in exon 19 of the egfr gene (including codons 746-752) were selected as lung cancer biomarkers.
3.6. Interpretation of emission spectra 3.6.1. Examination of the DNA probe uorescence spectrum. The DNA probe oligonucleotide was labeled with the FAM uorescent dye (carboxy uorescein) and codons 746-752 in exon 19 of the egfr gene. As shown in the DNA probe uorescence spectrum (Fig. 7), strong uorescence emission is observed in the 520 nm wavelength in the absence of GO; however, the uorescence intensity decreased with adding GO. The DNA probe uorescence emission intensity decreased with >95% in the presence of GO aer 32 min. This process is attributed to the DNA probe adsorption of the GO surface through non-covalent interactions (e.g., p-p stacking) between the ring-type structures of nucleobases and hexagons of the GO aromatic lattice, hydrogen bonds between the -OH groups of GO, the -NH 2 and -OH groups in the DNA probe, and van der Waals force. [59][60][61][98][99][100] 3.6.2. Optimization of the DNA probe adsorption time on the GO surface. According to the DNA probe uorescence spectra in the presence of GO at different times, the uorescence emission intensity decreased gradually with the DNA probe adsorption on the GO surface over time. The DNA probe uorescence intensity reached a constant value aer 32 min, followed with obtaining the optimal time of the DNA probe adsorption on the GO surface. It should be mentioned that the excitation and emission wavelengths were 485 and 520 nm, respectively (Fig. 8). Fig. 9 shows changes in the DNA probe uorescence intensity in the presence of GO at different times. An optimal time of 32 min was obtained for the DNA probe adsorption on the GO surface.
3.6.3. Optimization of the GO dose in the presence of the DNA probe. The effect of the GO dose on the DNA probe uorescence emission intensity was examined in this step ( Fig. 10 and 11). Based on the data, an increase in the GO dose increased the DNA probe adsorption on the GO surface, and the DNA probe uorescence emission intensity decreased gradually in the 520 nm emission wavelength. Finally, an optimal GO dose of 35 mg was obtained per 100 pmol of the DNA probe.
3.6.4. Examination of the DNA probe-GO + target (healthy) DNA uorescence spectra. As shown in Fig. 12, the DNA probe-GO uorescence emission intensity increased in the 520 nm emission wavelength with adding target (healthy) DNA. The DNA probe is hybridized with target DNA, and the resulting double-stranded DNA (dsDNA) is separated from the GO surface. When the DNA probe is hybridized with target DNA, nucleobases are protected in the phosphate backbone of   dsDNA, which mostly negates the possibility of non-covalent interactions (p-p stacking) and the hydrogen bond. Unpaired nucleobases play an important role in DNA adsorption on the GO surface. Thus, dsDNA is adsorbed on the GO surface at a very lower level than single-stranded DNA. 62, 90 3.6.5. Optimization of the target DNA and DNA probe hybridization time in the presence of GO. According to the uorescence spectra of the GO-DNA probe complex in the presence of target (healthy) DNA at different times, the uorescence emission intensity increased gradually with the target DNA and DNA probe hybridization over time and then reached a constant value aer 34 min. Thus, the optimal time of target      DNA and DNA probe hybridization was obtained in the presence of GO (Fig. 13).
The optimal hybridization time of target (healthy) DNA and the DNA probe was determined at 34 min with drawing the curve of changes in the GO-DNA probe uorescence intensity in the presence of target DNA at different times (Fig. 14).
3.6.6. Examination of changes in the GO-DNA probe uorescence intensity at different concentrations of target DNA.
According to the effect of target (healthy) DNA concentrations on the GO-DNA probe uorescence intensity, an increase in the target DNA concentration in the 520 nm emission wavelength led to a gradual increase in uorescence intensity (Fig. 15). Fig. 17 depicts the curve of changes in the GO-DNA probe uorescence emission at different concentrations of target DNA (0-40 pmol). According to Fig. 16 and 17, this method can be used to determine the concentrations of target DNA (from 0 to 40 pmol) in unknown samples.
3.6.7. Examination of the GO-DNA probe uorescence spectra in the presence of (mutated) mDNA. Fig. 18 displays the GO-DNA probe uorescence spectra in the presence of (mutated) mDNA. Since a complementary sequence of the DNA probe is absent in mDNA due to a deletion mutation, it was not hybridized with the DNA probe, leading to no formation of a dsDNA. Thus, the uorescence intensity showed no effective changes in the 520 nm emission wavelength.

Lung cancer detection
In the general procedure of lung cancer detection using the nanobiosensor (Fig. 19), the DNA probe is adsorbed on the GO surface, and the uorescence intensity increases with adding target (healthy) DNA. However, the uorescence intensity shows     no changes with adding (mutated) mDNA. In other words, the designed nanobiosensor responds differently to the two (healthy and mutated) DNAs, making it possible to detect mDNA (in cancer patients). Fig. 20 shows the GO-DNA probe uorescence spectra, indicating the different DNA probe uorescence intensity in the presence of two (healthy and mutated) DNAs. Accordingly, the incidence of deletion mutations in codons 746-752 of the egfr gene as the lung cancer biomarker is examined and detected in the DNA of interest.

Conclusion and outlook
1. Since graphene oxide (GO) is readily available and exhibits exceptional optical, electrical, mechanical and chemical properties, it has attracted increasing interests for use in GO-DNA based sensors. In solution, graphene oxide is as an excellent acceptor of uorescence resonance energy transfer (FRET) to quench the uorescence in dye labeled DNA sequences. The application of the electrochemical GO-DNA based sensors is also summarized because graphene oxide possesses exceptional electrochemical properties. GO-DNA based sensors perform well at low cost, and high sensitivity, and provide low detection limits. Additionally, GO-DNA based sensors should appear in the near future as scientists explore their usefulness and properties. Finally, future perspectives and possible challenges in this area are outlined. The results of these recent research studies exhibit the outstanding performance of graphene oxide compared with current techniques. However, some challenges related to DNA sensors-based graphene oxide remain and need to be resolved. Because ssDNA is adsorbed on the surface of graphene oxide, not all dsDNA can detach from the surface of graphene oxide aer the complementary ssDNA, protein or other molecules combine to ssDNA. This hinders the further improvement of the sensitivity of reported DNA sensors based on most of the recent publications reviewed, although a few authors have reported some methods for solving this problem. Currently, most published literature reports that only one target can be detected for one DNA-based sensor using graphene oxide in the liquid phase. Graphene oxide in the solid phase was scarcely explored. If more targets can be detected with one sensor, the throughput of detection will be improved. Graphene oxide bears oxygen functional groups on its basal planes and edges. Therefore, graphene oxide in the solid phase can also be used to make devices for sensing without chemical modication on the surface of graphene oxide. The devices are made using lithography, thermal evaporation and other micro-nano related scientic technology. If one GO-DNA based sensor is like an array with different DNA elements, many targets will be detected.
2. In the present research, a GO-DNA-based Nano biosensor was proposed for lung cancer detection. Graphene oxide was synthesized using the Hummers' method, and its structure was examined and conrmed using FT-IR, UV-vis, and TEM images. The adsorption of a FAM-labeled DNA probe on graphene oxide in the presence of target (healthy) DNA and (mutated) mDNA was then investigated using uorescence spectroscopy. The different responses of the nano biosensor to healthy and mutant DNAs allowed for lung cancer detection. Relying on nanotechnology, therefore, lung cancer can be detected through fast, easy, and cost-effective methods.

Conflicts of interest
The author declare that they have no competing interests.