**1. Introduction**

The most common cancers occur in the lungs, breasts, pancreas, colon, skin, and stomach [1]. Lung cancer is the second most common cancer in men and women and the leading cause of cancer deaths in the United States. The two major types of lung cancer are

**Citation:** Sadiq, M.; Pang, L.; Johnson, M.; Sathish, V.; Zhang, Q.; Wang, D. 2D Nanomaterial, Ti3C2 MXene-Based Sensor to Guide Lung Cancer Therapy and Management . *Biosensors* **2021**, *11*, 40. https:// doi.org/10.3390/bios11020040

Received: 1 January 2021 Accepted: 29 January 2021 Published: 4 February 2021

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small cell lung cancer (SCLC, ~15%) [2] and non-small cell lung cancer (NSCLC, ~85%) [3]. The survival rate of both types of lung cancer is very low [4]. According to the American Cancer Society, lung cancer and asbestos-related lung cancer [5] alone were responsible for 142,670 estimated deaths in 2019, making it the number one killer and three times deadlier than breast cancer [6]. This is because most patients (~75%) are diagnosed at a late stage of the disease (stage III or IV) [7]. To increase the survival rate, major advances in lung cancer control or prevention can be facilitated by early detection and effective anti-cancer therapy. In recent years, a variety of therapeutic and adjuvant methods and nutritional approaches have been developed for lung cancer treatment such as chemotherapy, targeted therapy [8,9], cyclooxygenase (COX)-2 inhibition [10], and omega-3 fatty acid dietary manipulation [11,12].

In addition to these methods, many physical "visualization/detection" methods [13] are available for tumor detection and cancer diagnosis [14]. Some of them are positron emission tomography (PET), magnetic resonance imaging (MRI), computerized tomography (CT), ultrasonography, endoscopy, and the gas chromatography method. However, these methods have some major issues for applications in cancer diagnosis. For example, MRI is very expensive and time-consuming. Sometimes it cannot even distinguish between malignant and benign cancer [15]. In the case of PET, radioactive material is used which is combined with glucose and injected into the patient. This process might cause a health concern for diabetic patients [16]. High-dose radiation involved in CT scanning can even increase the risk of cancer [17]. Ultrasound, however, cannot provide accurate diagnosis and frequently has difficulty determining whether a mass is malignant or not [18]. Endoscopy is relatively safer but still has complications such as perforation, infection, bleeding, and pancreatitis [19]. The fundamental limitation of gas chromatography is that the substance must be volatile. It means that a finite portion of the substance needs to be distributed into the gaseous state [20], which could make it problematic to use gas chromatography–mass spectroscopy (GC–MS) in cancer detection because its sampling procedure is very complicated and the results are difficult to interpret. In addition, the GC–MS technique is very expensive and must be operated by very skilled personnel [21]. Therefore, an effective and accurate technique to diagnose cancer and assist in effective treatment is urgently needed.

In particular, studies have confirmed that cyclooxygenase (COX), typically the inducible form of COX-2, is commonly overexpressed in lung cancer and the abundance of its enzymatic product prostaglandin E2 (PGE2) plays an important role in influencing cancer development. Since PGE2 is a deleterious metabolite formed from COX-2-catalyzed peroxidation of an upstream omega-6 ( ω-6) fatty acid called arachidonic acid (AA), PGE2 promotes tumor growth and metastasis [22]. Subsequently, it can be treated as an indicator of local COX activity to regulate or control lung cancer. Many efforts in lung cancer therapy have been focused on the development of COX-2 inhibitors because they can be used to suppress prostaglandinE2 (PGE2) formation from COX-2-catalyzed ω-6 arachidonic acid peroxidation [23]. However, most COX-2 inhibitors can severely injure the gastrointestinal tract, increase the risk of cardiovascular disease, and provide limited clinical responses [22,23]. To seek a safer and more efficient method to treat cancers, a new anti-cancer strategy [24], as shown in Figure 1, has been recently developed. This is a very different approach than the classic COX-2 inhibitors [24–26]. In detail, this is a strategy that adopts more abundant ω-6s such as dihomo-γ-linolenic acid (DGLA) in the daily diet and the commonly high level of COX expressed in most cancers to promote the formation of 8-hydroxyoctanoic acid (8-HOA) using a newly developed inhibitor, delta-5-desaturase inhibitor (D5Di). This is because the D5D is an enzyme that converts an upstream DGLA in a diet to AA. The high expression of COX-2 will promote the conversion of AA to PGE2, while the D5Di will (1) knock down the conversion of DGLA to AA and limit the generation of metabolic product, PGE2; and (2) promote the COX-2-catalyzed DGLA peroxidation to form 8-HOA, a novel anti-cancer free radical by-product. This strategy has proven to produce more effective and safer therapeutic outcomes in cancer treatment and has

been validated in colon and pancreatic cancers [27]. Therefore, detection of the PGE2 and 8-HOA in lung cancer should be an effective method to evaluate the efficiency of the cancer treatment. Furthermore, the relative ratio of PGE2 and 8-HOA concentrations can become a useful adjuvant method to help diagnose cancers at an early stage. Therefore, it is very critical to develop a sensing technique or device that can track/monitor the PGE2 and 8-HOA concentrations in cancer and provide in-time guidance and feedback for cancer treatment and prevention. However, due to the extremely low concentrations of PGE2 and 8-HOA in cancer cells ~ ng/mL or μM, the detection of these components is quite challenging. Traditional methods of measuring low concentrations of compounds, such as PGE2 and 8-HOA, are using gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS). These techniques, as described above, are accurate and sensitive but heavy (not portable), expensive (needing skilled personnel to operate), and time-consuming (complicated sample processing) and cannot provide in-time feedback.

**Figure 1.** New anti-cancer strategy: target but do not inhibit cyclooxygenase-2 (COX-2) in cancer.

Recent advances in nanotechnology have made it possible to synthesize functionalized nanomaterials for applications in electronics, sensing, biomedicines for disease diagnosis and control, drug delivery, and the food industry [28–31]. Due to the increased surface areas and the feasibility of controllable size and surface properties, nanomaterials such as nanofibers, nanowires, and nanoparticles provide grea<sup>t</sup> opportunities for the development of advanced sensing systems and portable device/instrumentation with improved sensitivity and selectivity [32–38]. In particular, the use of structure-directing synthetic approaches in nanomaterials allows the tailoring of the nanomaterial crystalline phase, surface states, morphology, and facets for specific sensing application. Recently, with the development of two-dimensional (2D) nanomaterials such as graphene, these types of materials have gained tremendous attention because of their astonishing electrical and optical properties featured with an "all-surface" nature [39–43]. This all-surface nature can offer grea<sup>t</sup> opportunities to tune material properties through surface treatment for targetable detection.

In 2011 [44], the discovery of MXenes introduced a new family into the two-dimensional (2D) materials and further proves to be promising in the flexible and broad application due to its controllable preparation methods and fascinating properties. In essence, MXenes consist of transition metals (including Ti, V, Nb, Mo, etc.) and carbon or nitrogen, sharing a general formula of *Mn*+1 *Xn* (*n* = 1–3). As a new star of 2D materials, MXenes have the metallic conductivity and hydrophilic nature due to their uncommon surface terminations. Moreover, the unique accordion-like morphology (Figure 2), excellent conductivity, and rich but tailorable surface functional groups endow MXenes with attractive electronic, mechanical, physical, and chemical properties [45] for applications in energy storage [46], environmental science [47], and sensors [48]. The numerous applications of MXene as sensors in various fields are summarized in the paper [49]. In our conference paper [50], we reported that the as-synthesized Ti3C2 MXene-based nanosensor has effectively detected 8-HOA in cancer cells without and with using a D5D inhibitor. In that paper, we described more details about how the new sensor based on the two-dimensional nanomaterial Ti3C2 MXene [51] can facilitate the lung diagnosis and treatment efficiently by using the new D5D inhibitor and 8-HOA therapy on lung cancer. The preliminary data indicate that this new sensor device can sensitively detect PGE2 and 8-HOA levels in healthy and cancerous lung cells (BEAS2B and A549 respectively) with similar accuracy to GC–MS but much faster and in an in-time manner to guide the cancer treatment.

**Figure 2.** Newly synthesized 2D multilayered Ti3C2 MXene nanosheets (**a**) Scanning electron microscope (SEM) image; (**b**) pristine and surface-terminated Ti3C2 MXene with different functional groups.

#### **2. Materials and Methods**

*2.1. Sensing Material Synthesis, Sensing Tests, and Cell Lines Preparation*

2.1.1. Ti3C2 Nanomaterial-Based Sensor Preparation

The sensor that we used is based on a new 2D nanomaterial, Ti3C2 MXene. This nanomaterial was prepared using a method developed in our group and named the "hot etching method" [52]. In detail, the synthesis of Ti3C2 MXene followed the steps: (1) Preparing the Ti3AlC2 MAX phase. It was obtained through ball milling TiC, Ti, and Al powders in the molar ratio 2:1:1.2 respectively, for 5 h. Under argon flow, the resulting powder was then pressed into a pellet and sintered at 1350 ◦C for 4 h. The collected pellet after being milled back into powder was sieved through a 160-mesh sieve; (2) etching Al from the MAX phase to form the MXene phase. The as-prepared MAX powder was collected at an elevated temperature for etching through the "hot-etch method". Hydrofluoric (HF) acid in 25 mL Teflon line autoclave at a temperature of 150 ◦C was used in a Thermolyne furnace for 5 h to etch 0.5 g of the MAX phase. To remove Al from the MAX phase, 5%wt of HF was used. Materials after being sonicated for one hour using a sonicating bath were collected through centrifuge; all the materials were dried overnight in a drying oven at 65 ◦C; (3) synthesizing MXene powders for the sensing film. Finally, the synthesized nanomaterial was drop-casted on the gold electrode-patterned glass substrate to form a thin film. The thin film is made by first making a paste for the application of the MXene powder to the substrate. This paste is made using 0.1 g of MXene material in 0.3 mL of ethanol which is then dispersed via mixing on a stir-plate. This paste is then blade-coated onto the sensor substrate at a thickness of 0.05 mm. The morphology of the synthesized 2D multilayered nanomaterial is shown in Figure 2a as the scanning electron microscope (SEM) image, which clearly exhibits multilayered nanosheets and accordion-like morphology. Figure 2b reveals the as-synthesized Ti3C2 material's special surface terminations, which can lead to the unique surface and material properties of Ti3C2.

#### 2.1.2. Ti3C2 MXene Based Sensor Device Fabrication

In order to monitor the promoting 8-HOA formation and the variation of PGE2 in cancer cells during the new anti-cancer treatment, the sensor device, as shown in Figure 3, is fabricated followed by the steps: (1) Ti3C2 MXene sensing film fabrication: the Au electron contact patterned using photolithography and deposited on the glass wafer as the substrate, and then direct drop-casting the as-synthesized Ti3C2 suspension solution onto the patterned substrate to form the sensor slide; (2) the resistance change caused by the exposure of 8-HOA/PGE2 on the sensing film is measured through a Keithley resistance meter and data collected via computer.

**Figure 3.** Sketch of Ti3C2 MXene-based sensing system.

#### 2.1.3. Cancer Cell Lines and Materials

A549 (ATCC®CCL-185™), NCI-H1299 (ATCC® CRL-5803), and BEAS-2B (ATCC®CRL-9609™) were purchased from American Type Culture Collection (ATCC, VA, USA). Iminodibenzyl (CAS Number: 494-19-9) and 8-hydroxyoctanoic acid (8-HOA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). PGE2 and DGLA (for in vitro study) and DGLA ethyl ester (for in vivo study) were acquired from Cayman Chemical (Ann Arbor, MI, USA).

#### 2.1.4. Preparation of Cell Samples

About 3 × 10<sup>5</sup> A549 or BEAS-2B cells were trypsinized and seeded into each well of the 6-well plates. Then, the cells were randomly assigned into different groups for the administration of DGLA (100 μM), iminodibenzyl (10 μM), or their combination accordingly. After 48 h, the cell culture medium was collected. Cells were washed with phosphate buffer solution (PBS) and collected by centrifugation after trypsinization. A 1 mL cell culture medium with collected cells was homogenized and ready for testing. Three different groups of control samples were prepared using the same preparation procedures, including (a) blank group in 1 mL cell homogenate without any treatment; (b) 8-HOA group in 1 mL cell homogenate containing 0.6 ug/mL exogenous 8-HOA; (c) PGE2 group in 1 mL cell homogenate containing 6 ug/mL exogenous PGE2.

#### 2.1.5. Xenografted Lung Tumor Model on Nude Mice

Six-week-old nude mice were purchased from The Jackson Laboratory. The mice were housed in a pathogen-free IVC System with water and food ad libitum. All the animal experiments in this study were approved by the Institutional Animal Care and Use Committees at North Dakota State University. About 2 × 10<sup>6</sup> A549 or H1299 cells were injected

into the hind flank of the nude mouse to induce tumors as we previously described [26]. The mice were randomly assigned to the following treatments: Control (treated with the same volume of the vehicle), DGLA (5 mg/mouse, oral gavage, every day), iminodibenzyl (15 mg/kg, intraperitoneal injection, daily), and DGLA+ iminodibenzyl. The treatment started at two weeks of injection of A549 cells in nude mice. All the administrations lasted for four weeks. At the end of the treatment, mice were sacrificed, and tumors were isolated. Tumor tissues were crushed and homogenized by using a mortar in liquid nitrogen. The blood was centrifuged for 10 min at 2000 rpm for separating serum. The supernatant of tumor tissues and serum was collected for analysis.
