Dynamic Monitoring of the Oxidation Process of Phosphatidylcholine

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Dynamic monitoring of oxidation process of phosphatidylcholine using SERS analysis Songtao Xiang, Yi Xu, Xin Liao, Xiangquan Zheng, Li Chen, and Shunbo Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04216 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 15, 2018

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Analytical Chemistry

Dynamic monitoring of oxidation process of phosphatidylcholine using SERS analysis Songtao Xiang,1,2,4 Yi Xu*,1,2,3,4 Xin Liao,1,2,4 Xiangquan Zheng,1,2,4 Li Chen*,2,3,4 and Shunbo Li2,3,4 1. Key Disciplines Laboratory of Novel Micro-nano Devices and System Technology & Key Lab for Optoelectronic Technology & Systems of Ministry of Education, Chongqing University, Chongqing 400044, China 2. School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China 3. School of Optoelectronic Engineering, Chongqing University, Chongqing 400044, China 4. International R & D center of Micro-nano Systems and New Materials Technology, Chongqing University, Chongqing 400044, China Correspondence should be addressed to Yi Xu; [email protected]

Phosphatidylcholine oxidation is closely related to many neurodegenerative diseases. In this paper, Raman spectroscopy was proposed to continuously monitor the oxidation of phosphatidylcholine and provided deep understanding in this biochemical process. To increase the detection sensitivity, surface enhanced Raman spectroscopy (SERS) with Micro/nano silver complex substrate was prepared by electrodeposition. The prepared SERS substrate had an enhancement factor as high as 7.8 ⅹ 107, ensuring the detection sensitivity in phosphatidylcholine oxidation process. It was illustruated that the oxidation of phosphatidylcholine in ethanol-water solution under the experimental conditions could be monitored and well described by second order kinetics through continuously measuring and analyzing the SERS spectra of phosphatidylcholine oxidation intermediates in 20 days. Meanwhile, the oxidation products were confirmed by mass spectrometry and the oxidation process was in good concordance with mass spectrometry detection. The use of SERS in following biochemical process has advantages including simple instrumentation, low cost, short detection time and no sample pretreatment. Therefore, as a kind of vibration spectrum, SERS is preferable to tranditional detection approach,such as MS, HPLC, MRI, for dynamic monitoring and analysising of complex biochemical process. Alzheimer's diseases[6-8], atherosclerosis[9], rheumatoid arthritis[10,11], diabetes[12,13], multiple scleaosis[14] and medium chain acyl-CoA dehydrogenase deficiency[15]. Meanwhile, PC is a kind of amphiphilic substance, with the function of surface activity, that makes it widely used in functional food, drugs, medicines’ auxiliary materials, and cosmetics fields. However, PC is easy to generate complex oxides and produces toxicity[16]. This will severely impact the effectiveness and safety of its products. It makes sense, therefore, to develop a detection method of PC oxidation.

1.Introdution Phosphatidylcholine (PC, also known as lecithin) is a significant physiological active substance. It is an important part of the biofilm, and it has the function of regulating a variety of substances in and out of the cell[1,2]. It also plays a variety of health-care effect on human bodies, such as anti-aging[3] anti-thrombotic[4] and regulating immunity[5]. The content of PC in body and its oxidation products are closely related to many diseases, for example

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Currently, PC oxidation degree is traditionally evaluated by peroxide or other oxidation decomposition products. Evaluation indexes include the peroxide value, acid value, anisidine value, thiobarbituric acid reactive substances (TBARS) values and so on. Gas chromatography(GC), high performance liquid chromatography (HPLC) can be used to measure the content of the oxidation of specific product[17,18]. Chemiluminescence is often used to evaluate the oxidation of PC[19,20], and differential scanning calorimetry is also an alternative. It can determine the oxidation induction time and the oxidation induction temperature[21]. However, we are incapable of getting the information in PC oxidation process on molecular level. In recent years, the development of mass spectrometry makes it a powerful tool in structure identification of the PC oxidation products, including GCMS[22], HPLC-MS[23], ES-MS[24], MALDI-MS[25,26], etc. Nevertheless, the PC oxidation will be enforced to a pause in the detection since the instruments used are usually expensive. The operation is always complicated and time consuming. Raman detection, by contrast, has advantages of non-destructive analysis, short testing time (usually few seconds to dozens of seconds) and samples without pre-treatment. In addition, as a kind of vibration spectrum analysis, the number of peaks, the peak displacement, and band intensities directly reflect the information of the extension of chemical molecular bonds and bending vibration modes. Thus, we can be more insight into the molecular structure and conformation of the analyte. Surface-enhanced Raman spectroscopy (SERS) is a detection technology which can greatly enhance the intensity of the Raman signal of the sample with micro/nano materials by electromagnetic and chemical enhancement effect. Its appearance effectively solved the problem of weak signal, and make SERS an ideal means widely used in biochemical sample detection. Although there are still some challenges in bioanalytical SERS, including sensitivity, reproducibility and spatial and time resolution[34]. With the rapid development of SERS enhancement materials, SERS technique has been more and more applied in the detection of complex biological macromolecules such as DNA[27,28], RNA[29] and protein[30]. Various SERS tags[31,32] were prepared to measure biochemical samples, such as, bacteria[33], cells[34] and so on. However, few studies have focused on biochemical reaction process using SERS approach. Moreover, as far as we know, no previous study of PC oxidation process monitoring by SERS has been reported.

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oxidized to be final oxidation products, such as aldehydes and ketones in small molecules[35-38]. All the products have their fingerprint peaks in Raman signals. Therefore, the use of SERS technique could monitor the whole PC oxidation process in a simple and non-destructive fashion. In this paper, we made full use of the advantages of Raman and SERS spectra in biochemical component identification, and applied them into the analysis of PC oxidation which played an important role in human bodies. Micro/nano silver with controlled size and good stability was prepared as SERS substrate by two-steps electrodeposition. Fenton’ s reagent was used to accelerate the oxidation of PC, and the oxidation in 0-20 days was continuously monitored by Raman spectrometer. To deep understand the mechanism of PC oxidation, the change of PC molecular structure and concentration of oxidation end products malonaldehyde(MDA) were analysed.

2. Experimental 2.1. Reagents. Phosphatidylcholine(from soybean,＞98%), glycerol ( ≥ 99.7%) and 1,1,3,3-Tetraethoxypropane ( ＞ 97%) were purchased from Macklin-Reagent Co. Ltd.(Shanghai, China). Silver nitrate (AgNO3, 99.7%) was obtained from Aladdin-Reagent Co. Ltd. (Shanghai, China). ITO glasses were purchased from Shenzhen huanan Technology Co. Ltd. (Shenzhen China). Rhodamine 6G (R6G, AR), hydrogen peroxide (H2O2, 30%), hydrochloric acid (HCl, 37%), Ammonia solution (NH3, 25%), thiobarbituric acid (TBA), ferrous chloride（FeCl2, AR）and ethanol（95%, AR） were acquired from Chongqing Chemical Reagent Co. Ltd. The water used in this work was ultra-pure water (18.2 MΩ cm, produced by a Milli-Q system). 2.2. Instruments. The instruments used in this study include: Raman spectrometer with 40×objective (785nm IDRaman, Ocean Optics, USA), ultraviolet visible spectrophotometer (UV-2450, Shimadzu, Japan), Scanning electron microscope (SEM, Hitachi JSM-7800F system, Japan), atomic force microscope (AFM, Bruker Dimension, Germany), electrochemical workstation (versastat3, Princeton, USA), and mass spectrometer (Waters SQ Detector 2, waters, USA) 2.3. Preparation of micro/nano silver complex. At room temperature, 2% (W/W) of AgNO3 solution was gradually added into 2% (V/V) weak aqua ammonia while stirring until the precipitate dissolved to prepare the silver ammonia solution. The electrodeposition was conducted in a traditional three-electrode electrochemical cell in the aqueous electrolyte solution including of 3 mL silver ammonia, 2 mL deionized water and 1mL 0.01 mol/L KNO3 as the supporting electrolyte at room temperature. The chronoamperometry was performed on VersaSTAT3 electrochemical workstation (Versastat3 Applied Research Princeton, USA). An ITO glass (0.5 × 1 cm2, 10 Ω cm), Pt plate and a saturated calomel electrode (SCE) were used as the working electrode, the counter electrode and the reference electrode respectively. The electrodeposition process was conducted in two steps: In the first step, the potentials was set to be −1.0 V for 10 s. Then, in the second step, the growth process of Ag was extended at

The oxidation of PC is a complex process. Its mechanism is influenced by the environment and oxidation conditions. To the general point of view, the PC molecule would undergo diacylation and generate fatty acid, glycerphosphocholine, glycerol and other intermediate products in the process of oxidation. Ester groups in PC were hydrolysed into carboxylic acid and glycerphosphocholine. The C=C bonds in the unsaturated carboxylic acid were broken into small molecular ones, and glycerphosphocholine might be oxidized into phosphorylcholine and glycerine. Then glycerol further


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R6G were observed on the Ag substrate. In order to reduce the influence of fluorescence, an improved modified multi-polynomial fitting was performed to remove the fluorescence background of the original SERS data[41]. PC and MDA SERS spectra in 0-20 days were the average from 9 sets of data calculated by excel.

potential of −0.2 V for 1200 s. The substrate was rinsed by deionized water twice to wash away electrolyte after preparation. 2.4. Determination of PC oxidation by SERS. The Fenton’s reagent was prepared with 0.05 M FeCl2 and equal volume 0.05 M H2O2 solution. 0.4 mL the Fenton’s reagent was added to the 1.6 mL PC (from soybean, > 98%) ethanol solution (20 mg PC dissolved in 1.6 mL 95% ethanol water solution) and sealed with sealing film. 10 μL mixed solution was dropped on three micro/nano silver SERS substrates separately in 0, 0.25, 0.5, 1, 2, 5, 10, 15, 20 days. The Raman spectra of PC mixed solution on substrates at different times and glycerol standard sample were measured three times respectively by micro Raman spectrometer with 40× objective (785 nm IDRaman micro, Ocean, USA). Henceforth, for all our Raman measurements, the parameters were: 18.8 mW of laser power (λ=785 nm), 10 seconds of acquisition time and spectral range 400–1800 cm1.

3. Results and discussion Characterisation of micro/nano silver complex micro/nano silver complex SERS substrate was prepared by two-steps chronoamperometry electrodeposition on the ITO glass. A large concentration of silver ammonia solution (about 80 mmol/L) and a long electrodeposition (1200s) made silver particles size on ITO glass up to about 5 microns (Figure 1) that effectively resolved the problems of poor stability of Raman signal from the substrate. The micro/nano silver complex SERS substrate was characterized by SEM and AFM respectively. From the SEM image, the aggregates of silver particles were observed, and which would produce interparticle hot spots[42]. It can be seen from Figure 2, the surface of the substrate was undulations nanostructures, they were the main reasons for producing SERS activity.

2.5. Determination of MDA by SERS. Malondialdehyde(MDA) is the end product in PC oxidation. Since the weak Raman signal of MDA, the interactants of thiobarbituric acid (TBA) and oxidized PC solution were measured by SERS according to the literature of D. Zhang[39] and N. Candan[40]. 0.4 mL Fenton’s reagent mixed PC ethanol solution was added into 2 mL TBA hydrochloric acid solution (0.375g TBA dissolved in 0.25 M hydrochloric acid) in 0, 0.25, 0.5, 1, 2, 5, 10, 15, 20 days. Then, the mixture was heated at 90℃ for 50 min. 10μL mixture was dropped on three substrates separately and each one measured three times by SERS. 2.6. Measurement of thiobarbituric acid reactive substances (TBARS) value by spectrophotometry. TBA reacted with MDA series of standard solution with different concentrations, and thus thiobarbituric acid reactive substances (TBARS) were prepared. Their absorbances were measured using ultraviolet visible spectrophotometer at 456nm wavelength to obtain the calibration curve. So that the concentration of PC-TBA mixed solution at different times could be calculated. The detail procedure was shown in the supporting information. 2.7. Measurement of acid value. Acid value is one of the traditional indexes to evaluate the oxidation degree of phospholipids. For the determination of acid value in the PC oxidation, Fenton’s reagent mixed PC ethanol solution was prepared as previous description in 2.3. The method from Chinese national standard GB/T 55302005/ISO 660:1996 was used to measure the PC solution oxidized by Fenton’s reagent in 0, 0.25, 0.5, 1, 2, 5, 10, 15, 20 days. The details of this method was shown in the supporting information.

FIGURE 1: SEM image of the micro/nano silver complex SERS substrate

2.8. Determination of PC oxidation by MS. PC solution samples oxidized with Fenton’s reagent at different times were measured by mass spectrum. The mass spectrometry parameters: positive ion mode (ESI +), the scanning range: m/Z 400-1100 and 50-1000, capillary voltage 2.50 kV, cone hole voltage 30 V, source temperature 100 ℃, desolvation temperature 350 ℃, cone gas flow 55 L/h, desolvation gas flow 550 L/h. 2.9. Data pre-processing. Spectral pre-processing and analysis were performed using metlab2014a. Before statistical analysis, a Savitsky-Golay filter (5th order, 15 points) was applied to smooth the spectra. Strong fluorescence intensities from TBA, MDA and



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3.3 SERS spectra of PC Oxidation process. The process of PC oxidation in 0,0.25,0.5,1,2,5,10,15,20 days was detected using SERS technique and the corresponding series of spectra was shown in Figure 3. The intensity changes of the peaks at 872, 1266, 1300, 1441, 1651cm-1 were chosen as typical peaks to analyze the oxidation process. The bands at 1266, 1300, 1441, 1651cm-1 could be attributed to the bending vibration of =CH, twist of -CH2, bending vibration of CH2, and stretching vibration of C=C respectively[45,46]. Since the intensity of the peak at 1441cm-1 (bending vibration of CH2) detected was quite stable, it was set as a comparison standard. Therefore, the change of PC concentration could be represented by the relative intensity of I(872)/I(1441), I(1266)/I(1441), I(1300)/I(1441), I(1651)/I(1441). The red curve in Figure 4 depicted the normalized intensity curve of I(872)/I(1441), and we could learn that the curve rose sharply with the help of Fenton’s reagent in the first day and tended to be smooth gradually. In 10 days, I(872)/I(1441) reached a maximum at 0.67, then it showed a decreasing tendency in the next 10 days. It was speculated that the peak at 872 cm-1 was probably attributed to the intermediate of oxidation. According to the literatures[37,38], glycerol was one of the most important intermediate in this process. Then we validated this inference with glycerol SERS spectrum, and the result showed a good agreement (Figure 3). So we drew a conclusion that 872cm-1 was the characteristic peak of glycerol.

FIGURE 2: AFM image of the SERS substrate surface 3.1. SERS Activity test of the prepared substrate. To evaluate the SERS activity of the micro/nano silver complex SERS substrate, apparent enhancement factor (AEF) was calculated by comparing the intensity of the 1360 cm−1peak in the SERS spectrum with that in the normal Raman spectrum of R6G. R6G with the concentration as low as 10-9 M could be detected on the micro/nano silver complex SERS substrate, while the lowest detectable concentration was 10-2 M on normal silicon substrate ( Figure S1, Figure S2). The apparent enhancement factor (AEF) was calculated according to the formula: 𝐈𝐒𝐄𝐑𝐒

The peak at 1266 cm-1 represented the bending vibration of = CH, which could be found in linoleic acid, linoleic acid ester and other unsaturated metabolites. As can be seen from the blue curve in Figure 4, it dropped rapidly in the first day and declined very slowly in the remaining 19 days. From the curve, it could be inferred that a large number of C = C bonds in PC molecules had broken under the catalytic oxidation of Fenton’s reagent in the first day. The C = C bonds contained the structure of = CH, and the content of = CH in PC consequent decreased. After the first day, Fenton’s reagent was consumed and no longer produced OH·, then the oxidation process of PC was spontaneous under the experimental condition, so the curve of I(1266)/I(1441) tend to be smooth in the remaining 19 days .

𝐂𝐑𝐒

AEF=𝐂𝐒𝐄𝐑𝐒 × 𝐈𝐑𝐒 [43,44], where ISERS and IRS were peak intensities of the peak at 1360 cm-1 from R6G on the SERS substrate and silicon slice, respectively. CSERS and CRS were the concentrations of R6G which could been detected on the two substrates. As a result, the AEF was calculated to be 7.8 ⅹ 107. The substrate prepared showed a good SERS activity. 3.2. Homogeneity and reproducibility test of SERS substrate. As we all know, poor reproducibility of Raman signals in conventional SERS analysis in the biochemical sample is the main obstacle to use SERS as a routine analytical technology. As a good substrate for SERS detection, the characteristics of high enhancement ability and good reproducibility are essential. Therefore, to investigate the homogeneity of the micro/nano silver complex SERS substrate, SERS spectra of PC ethanol solution (20 mg PC dissolved in 2 mL 75% ethanol water solution) were collected from 5 spots selected randomly on single substrate under the same laser power, and the Raman spectra were shown in Figure S3 (A). The differences among the five SERS signal intensities were small with relative standard deviation (RSD) of 7.4% at the 1441 cm−1. To evaluate the reproducibility of the micro/nano silver complex SERS substrate, five substrates were prepared under identical experimental conditions. The SERS spectra of PC ethanol solution on these five substrates were shown in Figure S3(B). The RSD were 11.7% for substrate-to-substrate. The results indicated that the substrates had excellent homogeneity and reproducibility. Consequently, they are quite suitable for SERS detection of PC oxidation.

The peak at 1300 cm-1 represents -CH2 twist, as can be seen from the green curve in Figure 4. It was stable and stabilised in 12h. The bond of -CH2 in PC molecules mainly existed in the aliphatic chain linked to two ester group. After oxidation, the ester group would break and aliphatic chain would turn into fatty acids. These fatty acids might be difficult to be further oxidized under the condition of this experiment, so the total content of -CH2 was relatively stable. The normalized intensity curve of I(1300)/I(1441) showed a good agreement with the theoretical analysis. The peak at 1651 cm-1 was assigned to C=C stretching vibration, and the tendency of I(1651)/I(1441) curve was similar to the one of I(1266)/I(1441) (the black curve in Figure 4). There are two C=C bonds in a PC molecule, and PC might turn into lysophosphatidylcholine(LPC) and phosphatidyl choline peroxide (PC-OOH), then they might be oxidized into short chain fatty acids and other dicarboxylic acids[47]. So the decrease of I(1651)/I(1441) could directly reflect the rupture of C=C bonds in PC molecule. Compared to the curve of I(1266)/I(1441), the one of I(1651)/I(1441) showed a long-term downtrend. So we concluded that =CH bonds in PC molecule were more vulnerable than C=C bonds in the process of oxidation.


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FIGURE 4 ： Temporal evolution of relative intensities of Raman peaks in the Raman spectra of phosphatidylcholine at different oxidation time (n=9)

FIGURE 3：Temporal variation of the averaged Raman spectra from phosphatidylcholine at different oxidation time (n=9) FIGURE 5 ： Temporal variation of the averaged SERS spectra of aldehydes (the final product of the oxidation of phosphatidylcholine) and TBA reaction mixtures at different oxidation time (n=9),and the SERS spectrum of the mixture of TBA and 1μg/mL MDA standard solution 3.5. SERS spectra of MDA. To measure MDA produced in PC oxidation indirectly by SERS, the interactants of oxidized PC in 020 days and TBA were prepared, and their SERS spectra were shown in Figure 5. 1272 cm-1 was assigned to the characteristic peak of MDA, and 1178 cm-1 for the characteristic peak of TBA from the literature. The SERS spectrum of the mixture of TBA and 1μg/mL MDA standard solution was also measured. The test method was shown in 2.4, and the preparation method of 1 μ g/mL MDA standard solution was depicted in the supporting information. The chemical reaction equation of TBA and MDA was shown in Figure 6. 5.2μmol TBA was added in the PC and alcohol mixture solution, and the amount of substance of TBA was far more than the MDA produced in PC oxidation. So the consumption of TBA in the reaction was very small, and its concentration could be regarded approximate constant. According to Figure 7, the peak at 1178 cm-1 is much stable, so I(1272)/I(1178) was used to represent the concentration of MDA. As shown in Figure 7, PC was oxidized into MDA under the action of Fenton reagent at a high speed in the first day, and slowed down in the later four days. In the tenth day, the concentration of MDA reached the maximum, and decreased gradually in the following ten days. The tendency of the curve was quite similar to that of oxidation intermediate glycerol. It also



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illustrated that MDA was the further oxidation product of glycerol. Here, we proposed two speculations for this phenomenon. Initially, as the oxidation process went on, further oxidation of the glycerphosphocholine was gradually decreased. Furthermore, MDA was likely to be slowly reduced due to the inevitable volatilization. The combination of these two factors might give rise to a decline in the overall level of MDA.

FIGURE 6：Chemical reaction equation of TBA and MDA FIGURE 7：Temporal evolution of relative intensities of the interactant of MDA and TBA from the Raman spectra of mixtures at different oxidation time

3.6. Measurement of TBARS. Figure 8 was the standard curve of TBARS. The horizontal coordinate was the concentration of interactants of MDA standard from 0.1-1.0 μg/mL and TBA. The vertical one is absorbance. It showed that the TBARS concentration exhibited a good linear behavior versus the absorbance. The linear equation was y=0.415x+0.001, R2 = 0.993. The absorbance of interactants of oxidized PC in 0-20 days and TBA were obtained by spectrophotometre, thus TBARS values at all stages of PC oxidation were calculate from the standard curve. The results were shown in Table 1. It revealed that PC stored in 4 ℃ refrigerator had also been oxidized slightly. The concentration of aldehyde/MDA reached a maximum in the tenth day of PC oxidation, and reduced gradually after following 10 days. The result was consistent with the SERS spectra of MDA. FIGURE 8：Standard curve of TBARS TABLE 1: Relative intensities of Raman peaks at different oxidation time and TBARS values (n=9)

Time(d) 0 0.25 0.5 1 2 5 10 15 20

TBARS (μg/mL) 0.87 1.33 2.19 2.39 2.87 3.22 5.16 3.97 3.04

I(1272)/I(1178)

I(1651)/I(1441)

0.17 0.31 0.41 0.44 0.45 0.47 0.78 0.58 0.45

0.85 0.81 0.74 0.70 0.64 0.54 0.47 0.44 0.38

I(1266)/I(1441)

I(1300)/I(1441)

I(872)/I(1441)

0.74 0.58 0.19 0.63 0.56 0.29 0.59 0.62 0.43 0.56 0.57 0.46 0.57 0.64 0.51 0.55 0.57 0.55 0.54 0.59 0.67 0.52 0.60 0.40 0.47 0.58 0.45 PC(MW=758) with the action of Fenton’ s reagent, generated lysophospholipids (LP, MW=762), phosphatidyl choline peroxide (PC-OOH, MW=790) and other primary products at first[47]. Figure 9 was the mass spectrum of PC oxidized with Fenton’s reagent at 10d. According to the SERS spectra(Figure 7),

3.7. Analysis of structure change of PC in oxidization stage. The mass spectrum of PC oxidized with Fenton’s reagent at 0.25 d was shown in Figure S5. It represented the initial oxidation stage of PC.


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maximum amount of malondialdehyde metabolites was detected at this time. It was also detected in the mass spectrum with the fragment ion peak of m/Z= 184, and its structure was shown in Figure 10. Fragment ion peaks (m/Z=780, 782, 784) were the fragment ion ones for PC oxidation products, and they might be the mother ion of m/Z=184[40]. Unsaturated fatty acids such as linoleic acid and their esters also might be generated, and the PC eventually metabolism substance.

Glycerol phosphatidylcholine (MW=257) was detected, the saturated fatty acid and its derivatives might be produced at the same time. Combined with the determination of acid value (Figure S4), it depicted that with the extension of oxidation of PC, large amounts of acid was produced. The acid value after 10 d was stable at around 120 mg/g. The change of PC (m/Z=759) and MDA (m/Z=73) concentration over time was reflected in Figure 11. The abscissa was time, and the ordinate was the intensities of m/Z=759 and m/Z=73 peaks, which were measured with the same intensity of ion source. PC was oxidized violently in the first day and then levelled off. The mass spectrometry results were consistent with SERS detection.

Malondialdehyde (MW=72) was also detected at the same time. Figure S6 was the mass spectrum of PC oxidized with Fenton’

s reagent at 20 d. It represented the end oxidization stage of PC. According to the mass spectrum ，the PC ion peak strength was very weak, and it suggested that plenty of PC had been oxidized.

FIGURE 9：Mass spectrum of PC oxidized with Fenton’s reagent at 10 d

FIGURE 11：The change of PC (m/Z=759) and MDA (m/Z=73) peaks intensity over time (n=3).

FIGURE 10：The decomposition product in PC oxidation

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3.8. Kinetic analysis of PC oxidation process. Chemical kinetics is very important to study the reaction mechanism and control the reaction process. Determining the chemical reaction order is the primary issue in the study of reaction rate, which requires examining the relationship between concentration and time. Here, Kinetic analysis by SERS could be carried out using the proportional relation between the intensity of the Raman signal and the concentration of the sample[48-51]. As a kind of vibration spectrum, SERS spectra reflect the change of groups in molecules directly. The relative peak intensities can be regarded as an index to reflect the biochemical reaction process. In this experiment, the peak at 1651 cm-1 was characterised as the stretching vibration of C=C bonds. The intensity of I(1651)/I(1441) decreased gradually with the breakage of C=C bonds

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in PC oxidation (Figure 7). Therefore, I(1651/I(1441) can represent the concentration of C=C directly, and to some degree, it also can represent the concentration of unoxidized PC. Therefore, the change of I(1651)/I(1441) was monitored to reflect the process of PC oxidization in 20 days. The Raman intensity of I(1272)/I(1178) from MDA and the thiobarbituric acid reactive substances (TBARS) value were used to get linear curve fitting (Figure 12), and it showed a good linear relationship. The calibration curve validated that there was a linear relationship between the relative intensity in a Raman spectrum and sample concentration. So the relative peak intensity of I(1651)/I(1441) was used for subsequent kinetic analysis. To eliminate the influence of Fenton’s reagent, 2-20d time points were selected to determine the reaction order of PC oxidation by graphing method. The relationship between 1/ (I(1651)/I(1441)) versus time was ploted, and linear regression analysis method was used to fit experimental data. It was approximated to be a straight line (Figure 13). This suggested that PC auto-oxidation process in PC ethanol solution in centrifuge tube under the condition of normal light could be well described by second order kinetics of the same two molecules, namely: 2PC → MDA. Its rate equation was: 2

= 𝑘 ⋅ [𝐶] , finite integral

FIGURE 13：Fitting curve of PC (I(1651)/I(1441)) versus time

4.Conclusions This article described the use of SERS technique to follow the dynamics of biochemical reactions - PC oxidation as an example. An effective and stable micro/nano silver complex SERS substrate was prepared and the oxidation process was continuously monitored within 20 days. The amounts of oxidation intermediate glycerol and end product MDA were successfully detected. The SERS spectra showed a high degree of consistency with the PC mass spectrometry detection. Chemical dynamics analysis indicated that the PC spontaneous oxidation in ethanol solution could be well described by second order kinetics, and dynamics equation y=0.0551 + 1.5165x (R2 = 0.9752) was obtained. In summary, the oxidation of PC could be monitored using SERS, which was a complicated biochemical reaction. Our results demonstrated a broad application prospect of studying a complex reaction process by SERS.

―ⅆ[𝐶]

ⅆ𝑡 [𝐶]𝑡 𝑡 expression: ∫[𝐶]0.ⅆ[𝐶] = ∫0𝑘 ⋅ ⅆ𝑡, and 1 1 1

converted into linear equation:[𝐶]𝑡 = 𝑘 ∙ 𝑡+[𝐶]0, drawing with

[𝐶]𝑡~t,

namely: 1/(I(1651)/I(1441) ～ t. The fitting equation was obtained as follows: y = 0.0551 + 1.5165 x, R2 = 0.9752 (Figure 13).

Acknowledgements The work was financially supported by National Natural Science Foundation of China ( No.21375156), National High Technology Research and Development Program of China (Ministry of Science and Technology 863 Plan ) (2015AA021104), and Fundamental Research Funds for the Central Universities (Fund for Brain Science) (No.10611CDJXZ238826). And the authors gratefully acknowledge the Fundamental Research Funds for the Central Universities (Project No.106112017CDJPT120002), Open Project(No.SKT1705) from the State Key Laboratory of Transducer Technology, China and major subject project of artificial intelligence technology innovation, development and industrialization of MEMS integrated pressure sensor (cstc2017rgzn-zdyfX0019).

FIGURE 12：Fitting curve of MDA (I(1272)/I(1178)) versus TBARS values

Supporting Information Available This material is available free of charge via the Internet at http://pubs.acs.org.

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(27) Coluccio, M. L.; Gentile, F.; Das, G.; Perozziello, G.; Malara, N.; Alrasheed, S.; Candeloro, P.; Fabrizio, E. D. J. Opt. 2015, 17, 114021. (28) Wang, X.; Cui, M.; Zhou, H.; Zhang, S. Chem. Commun. 2015, 51, 13983-13985. (29) Chen, Y.; Chen, G.; Zheng, X.; He, C.; Feng, S.; Chen, Y.; Lin, X.; Chen, R.; Zeng, H. Med. Phys. 2012, 39, 5664. (30) Zhou, X.; Xu, W.; Wang, Y.; Kuang, Q.; Shi, Y.; Zhong, L.; Zhang, Q. J. Phys. Chem. C 2010, 114, 19607-19613. (31) Lin, D.; Qin, T.; Wang, Y.; Sun, X.; Chen, L. ACS Appl. Mat. Interfaces 2014, 6, 1320-1329. (32) Lee, D.; Choe, Y. J.; Lee, M.; Jeong, D. H.; Paik, S. R. Langmuir. 2011, 27, 12782. (33) Wang, Y.; Yan, B.; Chen, L. Chem. Rev. 2013, 113, 1391. (34) Zong, C.; Xu, M.; Xu, L. J.; Wei, T.; Ma, X.; Zheng, X. S.; Hu, R.; Ren, B. Chem. Rev. 2018, 139, 272. (35) Wang, H.; Liu, Y.; Liu, C.; Huang, J.; Yang, P.; Liu, B. Electrochem. Commun. 2010, 12, 258-261. (36) Fernández-Murray, J. P.; Mcmaster, C. R. Biochim. Biophys. Acta, Mol. Cell. Biol. Lipids 2007, 1771, 331-336. (37) Adachi, J.; Yoshioka, N.; Funae, R.; Nushida, H.; Asano, M.; Ueno, Y. J. Chromatogr. B 2004, 806, 41-46. (38) Volinsky, R.; Kinnunen, P. K. FEBS J. 2013, 280, 2806-2816. (39) Zhang, D.; Haputhanthri, R.; Ansar, S. M.; Vangala, K.; De, H. S.; Sygula, A.; Saebo, S.; Pittman, J. C. Anal. Bioanal. Chem. 2010, 398, 3193-3201. (40) Candan, N.; Tuzmen, N. Neurotoxicology 2008, 29, 708-713. (41) Feng, S.; Chen, R.; Lin, J.; Pan, J.; Chen, G.; Li, Y.; Cheng, M.; Huang, Z.; Chen, J.; Zeng, H. Biosens. Bioelectron. 2010, 25, 2414-2419. (42) Liu, B.; Han, G.; Zhang, Z.; Liu, R.; Jiang, C.; Wang, S.; Han, M. Y. Anal. Chem. 2012, 84, 255. (43) Hsu, K. C.; Chen, D. H. Nanoscale Res. Lett. 2014, 9, 193. (44) Mei, Q.; Jiang, C.; Guan, G.; Zhang, K.; Liu, B.; Liu, R.; Zhang, Z. Chem. Commun. 2012, 48, 7468-7470. (45) Czamara, K.; Majzner, K.; Pacia, M. Z.; Kochan, K.; Kaczor, A.; Baranska, M. J. Raman Spectrosc. 2015, 46, 4-20. (46) Chang, W. T.; Lin, H. L.; Chen, H. C.; Wu, Y. M.; Chen, W. J.; Lee, Y. T.; Liau, I. J. Raman Spectrosc. 2010, 40, 1194-1199. (47) Miyazawa, T. Acta Biophy. Sin. 2012. (48) Shadi, I. T.; Cheung, W.; Goodacre, R. Anal. Bioanal. Chem. 2009, 394, 1833-1838. (49) Zhang, Y.; Mcgeorge, G. J. Pharm. Innov. 2015, 10, 1-12. (50) Xie, W.; Herrmann, C.; Kömpe, K.; Haase, M.; Schlücker, S. J. Am. Chem. Soc. 2011, 133, 19302-19305. (51) Zachhuber, B.; Ramer, G.; Hobro, A.; Chrysostom, E. T. H.; Lendl, B. Anal. Bioanal. Chem. 2011, 400, 2439-2447.

References (1) Qurat-Ul-Ain; Zia, K. M.; Zia, F.; Ali, M.; Rehman, S.; Zuber, M. Int. J. Biol. Macromol. 2016, 93, 1057. (2) Thiam, A. R.; Jr, R. V. F.; Walther, T. C. Nat. Rev. Mol. Cell Biol. 2013, 14, 775-786. (3) Manca, M. L. Ind. Crop. Prod. 2016, 83, 561-567. (4) Wójcicki, J.; Pawlik, A.; Samochowiec, L.; Kaldoǹska, M.; Myśliwiec, Z. Phytother. Res. 2010, 9, 597-599. (5) Cheung, M. C.; Vaisar, T.; Han, X.; Heinecke, J. W.; Albers, J. J. Biochem. 2010, 49, 7314-7322. (6) Luke, W.; Arundhuti, S.; James, H.; Petroula, P.; Diego, G. G.; Rufina, L.; Norman, S.; Madhav, T.; Iwona, K.; Patrizia, M. Neurobiol. Aging 2014, 35, 271-278. (7) Klavins, K.; Koal, T.; Dallmann, G.; Marksteiner, J.; Kemmler, G.; Humpel, C. Alzheimers Dement. 2015, 1, 295-302. (8) Wurtman, R. J. J. Alzheimers Dis. 2015, 46, 983. (9) Berliner, J. A.; Watson, A. D. J. Lipid Res. 2009, 50, 9. (10) Choy, E.; Sattar, N. Ann. Rheum. Dis. 2009, 68, 460. (11) W, Ł.; Gindzienska-Sieskiewicz, E.; Jarocka-Karpowicz, I.; Andrisic, L.; Sierakowski, S.; Zarkovic, N.; Waeg, G.; Skrzydlewska, E. Free Radic. Res. 2016, 1. (12) Floegel, A.; Stefan, N.; Yu, Z.; Mühlenbruch, K.; Drogan, D.; Joost, H. G.; Fritsche, A.; Häring, H. U.; Angelis, M. H. D.; Peters, A. Diabetes 2013, 62, 639-648. (13) Ried, J. S.; Baurecht, H.; Stückler, F.; Krumsiek, J.; Gieger, C.; Heinrich, J.; Kabesch, M.; Prehn, C.; Peters, A.; Rodriguez, E. Allergy 2013, 68, 629-636. (14) Qin, J.; Goswami, R.; Balabanov, R.; Dawson, G. J. Neurosci. Res. 2010, 85, 977-984. (15) Najdekr, L.; Gardlo, A.; Mádrová, L.; Friedecký, D.; Janečková, H.; Correa, E. S.; Goodacre, R.; Adam, T. Talanta 2015, 139, 62-66. (16) Ramprecht, C.; Jaritz, H.; Streith, I.; Zenzmaier, E.; Köfeler, H.; Hofmannwellenhof, R.; Schaider, H.; Hermetter, A. Chem. Phys. Lipids 2015, 189, 39-47. (17) Fu, Y.; Klonis, N.; Suarna, C.; Maghzal, G. J.; Stocker, R.; Tilley, L. Cytom. Part A 2010, 75A, 390-404. (18) Hui, S. P.; Chiba, H.; Sakurai, T.; Asakawa, C.; Nagasaka, H.; Murai, T.; Ide, H.; Kurosawa, T. J. Chromatogr. B 2007, 857, 158-163. (19) Volkova, P. O.; Alekseev, A. V.; Dzhatdoeva, A. A.; Proskurnina, E. V.; Vladimirov, Y. A. Mosc. Univ. Chem. Bull. 2016, 71, 87-96. (20) Rolewski, P.; Siger, A.; Nogalakalucka, M.; Polewski, K. Food Res. Int. 2009, 42, 165-170. (21) Joanne, P.; Galanth, C.; Goasdoué, N.; Nicolas, P.; Sagan, S.; Lavielle, S.; Chassaing, G.; Amri, C. E.; Alves, I. D. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 1772-1781. (22) Clark, A. J.; Calvillo, J. L.; Roosa, M. S.; Green, D. B.; Ganske, J. Anal. Bioanal. Chem. 2011, 399, 3589-3600. (23) Reis, A.; Domingues, P.; Domingues, M. R. J. Mass Spectrom. 2013, 48, 1207-1216. (24) Reis, A.; Domingues, P.; Ferrercorreia, A. J.; Domingues, M. R. J. Mass Spectrom. 2004, 39, 1513–1522. (25) Fuchs, B.; Bresler, K.; Schiller, J. Chem. Phys. Lipids 2011, 164, 782795. (26) Stutts, W. L.; Menger, R. F.; Kiss, A.; Heeren, R. M.; Yost, R. A. Anal. Chem. 2013, 85, 11410-11419.

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Dynamic Monitoring of the Oxidation Process of Phosphatidylcholine

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