*2.2. Methodology*

To verify the roles of 8-HOA and PGE2 in cancer development and treatment, the experiments have been designed to do testing in healthy lung cells and A549 lung cancer cells. The 8-HOA and PGE2 concentrations were carefully calculated before applying them onto the sensing films with certain accuracy and consistency. The sensor performance was further verified in comparison with the measurement using traditional GC–MS.

### 2.2.1. Normal Cells

For application to test the effect of 8-HOA and PGE2 in normal lung cells, 10<sup>6</sup> BEAS2B non-tumorigenic epithelial cell lines were collected. 8-HOA, PGE2, and BSA (Bovine Serum Albumin) were applied to the samples right before measuring the resistance change. Once the samples were applied onto the Ti3C2 MXene-based sensors, resistances were measured immediately and repeated at regular time intervals. The experiment is listed in Table 1 and the resistance change of the MXene slides for each of the samples is measured and shown in Figure 4. The resistance increases dramatically when BEAS2B is added with PGE2 but BEAS2B alone and BEAS2B with 8-HOA do not show obvious change of resistance.


**Table 1.** Table showing the composition of each sample for BEAS2B.

**Figure 4.** Resistance change measured using Ti3C2 MXene-based sensors for BEAS2B cells.

2.2.2. A549/H1299 Lung Cancer Cells

A549 and H1299 both are lung cancer epithelial cell lines. To study the effect of 8-HOA, both H1299 and A549 cell lines have been studied on lung cancer cell apoptosis, proliferation, and survival as shown in Figure 5. H1299 cells were treated with 1 μM 8-HOA for 48 h and afterward were subjected to flow cytometry to observe the apoptosis in the staining of Annexin V-FITC/PI. Wound-healing assay of H1299 lung cancer (non-small cell lung carcinoma cell line) treated with 8-HOA was observed and recorded having the relative area of the wound to 0 **h** respectively normalized to 1. Finally, the Colony formation assay of H1299 lung cancer cells treated with 8-HOA was observed. Survival fraction of different treatment groups to control were respectively normalized to 1.

**Figure 5.** Effect of 8-hydroxyoctanoic acid (8-HOA) on lung cancer cell apoptosis, proliferation, and survival. (**A**) Cell apoptosis was determined by flow cytometry. (**B**) Wound-healing assay of H1299 lung cancer cells. (**C**) Colony formation assay of H1299 lung cancer. \* *p* < 0.05 vs. Control group. Data represent mean ± SEM, unpaired t-test.

Figure 6 shows the effect of D5D inhibitor on lung cancer apoptosis. Cell apoptosis was determined by flow cytometry on H1299 lung cancer cells in staining of Annexin V-FITC/PI. H1299 cells were treated with DGLA (100 μM) and D5D inhibitor (10 μM) for 48 h before flow cytometry analysis. Immunofluorescence images of cleaved poly (ADPribose) polymerase (PARP) in lung tumor tissues after 4 weeks of treatment with DGLA (5 mg/mouse) and D5D inhibitor (15 mg/kg) were collected. The expression of cleaved PARP was stained in violet, and cell nuclei were counter-stained with 4,6-diamidino-2- phenylindole (DAPI).

The experimental results showed a similar response to 8-HOA in A549 and H1299 cell lines. In sensing tests, A549 cells were collected after being cultured. The complete design of the experiments to verify the relative concentration of generated 8-HOA and PGE2 with and without using the new cancer treatment via the detection of Ti3C2 Mxene-based sensor are listed in Table 2. Similar to the BEAS-2B cell lines, 8-HOA and PGE2 samples were applied to the A549 cell lines just before conducting the experiment. The sensing tests of these samples are shown in Figure 7. The resistances of A549 cancer cells, A549 cells treated by DGLA, and PGE2 are much higher than the cancer cells treated by adding 8-HOA, applying D5Di, or using the new anti-cancer treatment DGLA + D5Di.

**Figure 6.** Effect of delta-5-desaturase (D5D) inhibitor on lung cancer apoptosis. (**A**) Cell apoptosis determined by flow cytometry. (**B**) Immunofluorescence images of cleaved poly (ADP-ribose) polymerase (PARP) in lung tumor tissues. \* *p* < 0.05 vs. Control group. Data represent mean ± SEM, unpaired t-test.

**Table 2.** The composition of each sample for A549 cells treated by 8-hydroxyoctanoic acid (8-HOA), Prostaglandin E2 (PGE2), dihomo-γ-linolenic acid (DGLA), delta-5-desaturase inhibitor (D5Di), and DGLA + D5Di.


**Figure 7.** Resistance change measured using Ti3C2 MXene-based sensors for A549 cancer cells with and without using the new anti-cancer treatment.

#### **3. Results and Discussion**

#### *3.1. Observation from the Non-Tumorigenic Sample Graph*

In a healthy subject, both the concentration of PGE2 and 8-HOA should be low. The sensing test is conducted on the normal lung cell, BEAS2B, without extra treatment and BEAS2B by treating with extra PGE2 or 8-HOA. A significant resistance increase is observed in BEAS2B by adding 10 μM PGE2, while the untreated normal cells and cells treated by 8-HOA do not show obvious resistance change. This result indicates a unique role of PGE2 in healthy cells through the change of the electrical property of sensing material. Considering the elevated concentration of PGE2 can indicate a cancer development, such a sensitive response to PGE2 using Ti3C2 MXene-based sensor can be potentially used to diagnose cancer even at a very early stage.

#### *3.2. Observation from the CARCINOGENIC Samples*

As we have discussed in this paper previously, D5D inhibitor (D5Di) is used for preventing the conversion of DGLA to AA and ultimately limiting the formation of PGE2. According to the main mechanism of the new anti-cancer strategy, D5Di along with DGLA can effectively limit the formation of PGE2 but promote the formation of 8-HOA. The sensing test using the newly developed Ti3C2 MXene-based sensor, as shown in Figure 6, exhibits an interesting trend of resistance change. Similar to showing high resistance for A549, A549 with adding 10 uM PGE2, and A549 treated by DGLA both show high resistance in the sensing test. The results indicate a higher concentration of PGE2 generated in A549 cells while the high resistance in the sample only treated by DGLA confirms that omega-6 (DGLA) are pro-inflammatory and promote the formation of PGE2. However, the new anti-cancer treatment using DGLA and D5Di to treat A549 cells shows a similar low resistance level to that of A549 cells with 8-HOA. This result indicates promising information: the Ti3C2 MXene-based sensor can be used to monitor or validate the anticancer effect of the new strategy: DGLA + D5Di, which should be an effective anti-cancer effect because of the generation of 8-HOA.

#### *3.3. Correlation between the Sensing Test Results and GC–MS Results*

To verify the Ti3C2-based sensor for PGE2 and 8-HOA detection, both sensor and GC–MS have been used to detect very low concentrations of 8-HOA and PGE2 in A495 lung cancer cells. The Ti3C2 Mxene sensors can provide the information of concentration of 8-HOA via the value of resistance while GC–MS can quantitatively provide the exact concentration of 8-HOA. As shown in Figure 8, an obvious correlation is obtained between the GC–MS measurement and resistances that the Ti3C2 MXene-based sensor measured. This correlation further confirmed the capability of the Ti3C2 MXene-based sensor to detect trace concentrations of 8-HOA. It can be a convenient, fast, and low-cost tool to help the anti-cancer strategy in lung cancer treatment.

**Figure 8.** Correlation between different concentration of 8-HOA detected by gas chromatography– mass spectroscopy (GC–MS) and resistance measured by Ti3C2 MXene sensor using the same sampling conditions.

#### **4. Conclusions and Discussion**

A new sensor based on 2D nanosheets, Ti3C2 MXene, has been designed and used for the sensing response to 8-HOA and PGE2 in lung cancer cells. The preliminary results indicate an important conclusion: this new Ti3C2-based sensor can provide a convenient and simple method for anti-cancer treatment guidance. In addition, the high sensitivity of this new sensor opens a potential application for early-stage cancer detection via monitoring variation of PGE2 and 8-HOA in cells. Instead of using heavy, expensive, and timeconsuming GC–MS to assist the anti-cancer treatment, the Ti3C2 MXene-based sensor can provide a fast, simple, low-cost, highly efficient, and much less invasive assistant tool to detect and cure cancer.

**Author Contributions:** M.S. initially wrote the paper, performed sensing experiments, and analyzed experimental data; L.P. prepared cancer cells and data analysis; M.J. performed all XRD, FT-IR, and Raman spectroscopy experiments, analyzed data, and synthesized the materials; V.S. designed cancer testing; Q.Z. designed material synthesis procedures; D.W. revised and finalized the paper and led the research team. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is supported in part by the Offerdahl Seed Grant, NDSU Centennial Endowment Award, FAR0029296; and ND NASA EPSCoR research grant, FAR0030154, and ND EPSCoR seed award, FAR0030452.

**Institutional Review Board Statement:** All the animal studies were approved by the IACUC (Institutional Animal Care and Use Committees) at the North Dakota State University (protocol code A18031 and date of approval: 7 February 2018).

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We wish to acknowledge the NDSU Core Research Facilities for providing access to microfabrication tools and materials characterization instruments.

**Conflicts of Interest:** The authors declare no conflict of interest.
