## 3 Results and Discussion
### Spectral Observation  
FTIR spectra of mycoplasma-contaminated and -decontaminated HepG2 were acquired in the range of 3600-800 cm\({}^{-1}\) (Figure 2A). The primary FTIR of the two groups displayed similar peak profiles and absorbance intensity. The second derivative spectra were also conducted from the primary FTIR spectra to resolve the overlapping peaks in the primary spectra and to spot differences between spectra of each cell group (Figure 2B). The IR spectral ranges were categorized into three regions by the major biological components presented in each region [19; 20]: (1) the lipid region (3000-2800 cm\({}^{-1}\)) is assigned for the phospholipid bilayer of the cell and organelle membranes. The fatty acid side chains consist of the repeated CH\({}_{2}\)- and CH\({}_{3}\)- moieties that yield the stretching vibration in the lipid region. (2) The protein region (1700-1450 cm\({}^{-1}\)) is assigned for the amide bonds. Amino acids bind to each other to form proteins, with the peptide bond giving two sharp bands near 1600 cm\({}^{-1}\), amide I (stretching vibration) and amide II (bending vibration). (3) The nucleic acid region (1280-900 cm\({}^{-1}\)) is assigned for phosphodieser bonds. Nucleic acids bind to each other to form DNA/RNA through the phosphodieser bond, which yields several peaks around 1200 cm\({}^{-1}\). Figure 2B displays the average of second derivative FTIR spectra of mycoplasma-decontaminated and -contaminated HepG2 cells with biomolecule region assignment as lipid, protein, and nucleic acid regions. The spectral subtraction (Figure 2B) indicates that the differences between mycoplasma-treated cells and-untreated cells occurred mainly in the lipid and protein regions. Notably, the differences in the second derivative spectra between the two groups would be in the opposite direction when compared to the primary spectra. The second derivative spectra results illustrate the change of biomolecule content upon mycoplasma contamination, as reflected in the change in peak intensity and also the molecular bond rearrangements, as indicated by peak broadening or shifting in lipid, protein, and nucleic acid regions. In the lipid region, the alteration of the peak intensities was found at 2923 and 2852 cm\({}^{-1}\), which relate to asymmetric and symmetric stretching of CH\({}_{2}\) in the lipid side chain (Figure 3A). The lipid head group (phospholipid ester; 1743 cm\({}^{-1}\)) was also decreased in the contaminated group. Moreover, the second derivative spectra of contaminated cells exhibited a peak at 3010 cm\({}^{-1}\) (assigned as -HC=CH- stretching in unsaturated fatty acid [21]), which was shifted to 3006 cm\({}^{-1}\) (unsaturated lipid [21; 22]) after removal of mycoplasma contamination (Figure 3A). The peak intensity observed at 3006 and 1743 cm\({}^{-1}\) in the mycoplasma-decontaminated HepG2 cells represents the proportion  
Figure 1: Mycoplasma detection in the HepG2 cells by polymerase chain reaction (PCR) before and after BM-Cyclin treatment (\(n=3\)). glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. bp, base pair. of unsaturated fatty acid and total lipid, respectively [23]. The presence of the peaks at 1477 and 1467 cm\({}^{-1}\) in the decontaminated cells (assigned as the specific peaks for the orthorhombic lipid phase) also indicates the change of polymorphism of the lipid bilayer [24]. It indicates that the structural compactness of the lipid bilayer in host cells is affected by mycoplasma decontamination. The changes in the amide I band, intermolecular \(\beta\)-sheet (1639 cm\({}^{-1}\)), and \(\alpha\)-helix (1650 cm\({}^{-1}\)) also appear, as shown in Figure 3B. The change in amide II displayed a duplex peak at 1544 and 1552 cm\({}^{-1}\) after the HepG2 cells were decontaminated from mycoplasma (Figure 3B). Identified nucleic acid was observed as the PO\({}_{2}\)\({}^{-}\) asymmetric stretching vibrations around 1225 cm\({}^{-1}\) for DNA and the PO\({}_{2}\)\({}^{-}\) symmetric stretching vibrations at 1085 cm\({}^{-1}\) for RNA.