Scientific report Regarding the implementation of the project184/2011 during the period 1 January- 12 December 2016

Transcription

1 Scientific report Regarding the implementation of the project184/211 during the period 1 January- 12 December 216 This scientific report is presented in corelation with the milestones M4 - M6 and with the activities T9 - T13 from the workplan. Two kind of PON sensors were optimised during this phase for investigation of the PON reaction with myoglobin and for PON determination in meat extract. I. SPCE/CoPc sensor 2 µl of cobalt phthalocyanine (CoPc) dissolved in DMF (1 mg/ml) was drop-casted on a screen printed carbon electrode (SPCE) from DropSens, Spain. Cyclic voltammetry was used to determine the redox process of peroxynitrite (PON), using the SPCE/CoPc electrode. The oxido-reduction process involving peroxynitrite takes place at around.1 V, with Ec =.47 and Ea=.72 ( E= 25 mv), but smaller current in the reduction, suggesting that the reduction reaction is irreversible. The formal potential is.6 V. Also, under another probable mechanism, an irreversible reduction takes place around -.3 V, probably involving also the chemical oxidative reaction of peroxynitrite over the metallic center: the cobalt being chemically oxidized by PON, is being reduced, with a higher current, depending on the concentration of PON. The influence of the scan rate on the the SPCE/CoPc in PON is presented in Figure 1. During this study is also observed that the shift to higher potentials takes place, due to the competition of absortion and diffusional redox processes, suggesting the irreversibility of the process. Greater currents are obtained in the case of PON (PBS ph 12) at -.3 V, than in the case of control experiment (PBS ph 9), sustaining the explanation given above (higher currents at -,3 V, upon the chemical reduction of Co 2+ to Co 1+ due to the presence of PON). Figure 1. Cyclic voltametry of the SPCE/CoPc electrode for 5 um PON, PBS ph 12. For further studies, the electrode was used to study the reaction of metmb with peroxynitrite. As described, this reaction is not so easy to observe, because the formation of the intermediate form of ferryl myoglobin is hard to observe, due to the instability of the intermediary compound. The case is different when oxymb is reacted with peroxynitrite, but the relevance is low, because meat extract presents in its composition mainly metmb. As the spectrophotometric method is inadequate for the determination of PON concentration (overlapping of the 28 nm peak know for proteins with the 1

2 32 nm peak of peroxynitrite), our new method was found to be suitable so study the reaction of PON with metmb (Figure 2). A B Figure 2. Chronoaperomgram using the FIA-EC system for (A) 5 µm and (B) 15 µm PON, in presence and in absence of 15 µm metmb, at different incubation periods The decomposition of PON alone was studied at the same incubation periods, to prove that the recovery of the current is due to the presence of PON in the metmb solution, and not to the metmb itself (PBS ph 9. E=.1 B and flow rate.4 ml/min). The UV-Vis spectra show that no actual changes take place when the methmb (15 um) is incubated with PON 5 um. In the case of PON 15 um, only a small decrease in absorbance was observed for metmb and an appearance of new peak at 71 nm, but no shift of the peaks occurred. The FIA- EC measurements showed, that in this case, the incubation with metmb decreased the concentration of PON bellow 1.7 um, and the scavenging/decomposition of PON seemed to occur totally at this metmb/pon ratio (Figure 3). A B Figure 3. (A) Kinetic study of 15 um PON in presence (black) and in absence (red) of 15 um metmb. (B) UV-Vis spectra of 15 um PON incubated with 15 um metmb, at different incubation periods. 2

3 I / A II. Poly(2,6-DHN)/SPCE sensor For the first time the electropolymerization of 2,6-dihydroxynaphthalene (2,6-DHN) on a screen printed carbon electrode (SPCE) was investigated and evaluated for peroxynitrite (PON) detection. Cyclic voltammetry was used to electrodeposit the poly(2,6-dhn) on the carbon electrode surface (Figure 4). 4 3 scan 1 2 scan 1 1 scan scan E / V A B Figure 4. (A) 1 successive CVs of SPCE in 2 mm 2,6-DHN at scan rate 5 mv s 1.; (B) Aspect of the modified SPCE with poly(2,6-dhn). The surface morphology and structure of poly(2,6-dhn) film were investigated by SEM and FTIR analysis, and the electrochemical features by cyclic voltammetry. The poly(2,6-dhn)/spce sensor showed excellent electrocatalytic activity for PON oxidation in alkaline solutions at very low potentials (-1 mv vs Ag/AgCl pseudoreference). An amperometric FIA (flow injection analysis) system based on the developed sensor was optimized for PON measurements and a linear concentration range from 2 to 3 M PON, with a LOD of.2 M, was achieved. The optimized sensor inserted in the FIA system exhibited good sensitivity (4.12 na M 1 ), selectivity, stability and intra-/inter-electrode reproducibility for PON determination. The electrocatalytic behavior of the poly(2,6-dhn)/spces was also evaluated by cyclic voltammetry in the presence and absence of 5 M PON in PBS ph 9.. The response of the sensor prepared with a scan rate of 5 mv s 1, presented in Figure 5, shows a significant increase of the anodic and cathodic peaks characteristic to the poly(2,6-dhn) film in the presence of PON. PON induced potentials shift of both anodic and cathodic peak to lower values. One may also observe that in the presence of PON the anodic peak is higher than the cathodic peak. These results indicate that the poly(2,6-dhn)/spce sensor has electrocatalytic activity towards the PON oxidation. The Flow Injection Analysis (FIA) technique was used for the amperometric detection of PON using the developed poly(2,6-dhn)/spce sensor. A very simple single-line FIA system based on injection of 1 L sample in a flow of PBS ph 9. pumped with an optimized flow rate of.38 ml min 1 was used. When the samples reach the electroactive surface of the sensor which is inserted in a special flow cell for screen printed electrodes characteristic FIA peaks are recorded such as shown in Figure 9a. The height of the FIA peaks is proportional to the concentration of PON solution injected. 3

4 I / na I / A I / A 4 PBS ph 9. PON 5 M E / V Figure 5. Cyclic voltammograms of poly(2,6-dhn)/spce in.1 M PBS ph 9., in the presence (bue line) and absence (black line) of 5 M PON. Potential range:.3 V to +.5 V; scan rate: 1 mv s 1. The influence of the applied potential on the height of the FIA signals for different PON concentrations as well as on the background noise was investigated for E of, 5, 75 and 1 mv vs Ag/AgCl pseudoreference. In Figure 6 the plots representing the dependence of FIA peaks height on PON concentration for the investigated applied potentials are shown. The optimum applied potential was found to be 75 mv vs Ag/AgCl pseudoreference due to the broadest concentration range of PON (2 3 M) and a sensitivity of 4.12 na M 1. The formula LOD = 3 b /m was used to calculate the limit of detection, were b represents the standard deviation of the background and m is the slope of the calibration graph. For the optimum applied potential of 75 mv a LOD of.2 M PON was achieved. Similarly, a LQD (limit of quantification) of.7 M was calculated according to the formula LQD = 1 b /m M 1 2 M E= V E=5 mv E=75 mv E=1 mv M M 1 M 12.5 M 16.5 M 25 M 5 M Time / s PON / M (a) (b) Figure 6. (a) FIA amperogram for different PON concentration solutions. E = +75 mv; flow rate =.38 ml min 1 ; (b) Dependence of FIA height on the applied potential for different concentration of PON; flow rate =.38 ml min 1 ; In order to test the applicability of the developed sensor for PON detection in real samples, some possibly coexisting interferents in meat extracts, such as ascorbic acid and nitrite, were investigated. It is evident that nitrite does not interfere in PON determination and for a 2 times higher concentrated solution of ascorbic acid than that of PON, the interference was low (around 7%). The near zero operation voltage of the poly(2,6-dhn)/spce succeeds to eliminate the interference of these two important compounds which create major difficulties in PON detection with sensors operating at high potentials. 4

5 I / na I / na For evaluation of the applicability of the developed sensor in real samples, a calibration graph was constructed based on the FIA peaks recorded for samples consisting of meat extract (diluted 1 times with PBS ph.9) spiked with certain amounts of PON in order to reach concentrations between 5 and 2 μm (Figure 7). The samples were injected 15 s after spiking. In Figure 1 it can be observed that the meat extract itself without added PON gave a very small reverse FIA peak. The calibration graph for PON added in meat extract showed a good linearity for the range 5 2 μm. The sensitivity of 3.45 na μm 1 represents ~84% from the sensitivity recorded for PON determination in PBS ph 9., meaning that the meat matrix has a reduced influence on PON determination with the poly(2,6-dhn)/spce sensor. The PON recovery in meat extract is 96% 99% for added PON concentration below 5 μm and 93% 96% for higher concentration. 8 2 M I = 3.45 c PON R 2 = M meat extract (diluted 1/1) M added PON / M 1 M 25 M 37.5 M 5 M Time / s Figure 7. FIA amperogram for PON added in meat extract (diluted 1/1 with PBS ph 9.). Inside: calibration graph for PON in meat extract. E = +75 mv; flow rate =.38 ml min 1 III. Conclusions The both optimized sensors, based on CoPc and poly(2,6-dhn) and the FIA systems exhibited good selectivity, sensitivity, reproducibility and stability for PON determination, as well as low LODs for PON determination. These sensors allow the quantifying of PON with oxymyoglobin in minced beef muscle. All these analytical features of these new sensors represent good premises for a future project related to the integration of this kind of sensors in smart labels for detecting the meat freshness. The objectives of this phase of the project were successfuly reached. 5

6 Results planned for 216: 2 works presented at international conference/ symposium; 1 article published in an international journal; Results achieved 216: 5 works presented at international conferences/symposium/workshop; 1 ISI article. ISI papers 1. I.S. Hosu, D. Constantinescu-Aruxandei, M.-L. Jecu, F. Oancea, M. Badea Doni, Peroxynitrite Sensor Based on a Screen Printed Carbon Electrode Modified with a Poly(2,6- dihydroxynaphthalene) Film, Sensors, 16(11), , 216. Participations at international scientific meetings: 1. M. Badea Doni, M.-L. Arsene, M.-L. Jecu, Sensors based on screen printed electrodes for nitrite and peroxynitrite determination in meet, Invited lecture at International Workshop 'New methodologies and advanced nanomaterials for electrochemical biosensors', Bucharest, Romania, 2 May M. Badea Doni, I.S. Hosu, D. Constantinescu-Aruxandei, M.-L. Arsene, F. Oancea, M.-L. Jecu, Flow injection system based on a modified screen printed electrode for peroxynitrite determination in meat extracts, Poster at RO-ICAC'16-3rd International Conference on Analytical Chemistry, Iasi, Romania, August M. Badea Doni, G. Petcu, M. Patrascu, M.-L. Arsene, V. Fruth, M-L Jecu, Screen printed carbon electrode modified with copper oxide nanoparticles for glucose determination, Poster at RO-ICAC'16-3rd International Conference on Analytical Chemistry, Iasi, Romania, August M. Badea Doni, I.S. Hosu, D. Constantinescu-Aruxandei, M.-L. Arsene, M.-L. Jecu, F. Oancea, Peroxynitrite sensor based on a modified screen printed carbon electrode, Poster at The 16 th International Conference of Physical Chemistry ROMPHYSCHEM, Galati, Romania, September I.S. Hosu, D. Constantinescu-Aruxandei, M.-L. Jecu, M.-L. Arsene, F. Oancea, M. Badea Doni, Screen printed carbon electrodes modified with cobalt phthalocyanine vs poly(2,6 dihydroxynapthalene) for peroxynitrite determination, International Symposium 'Priorities of Chemistry for a Sustainable Development' - PRIOCHEM 12nd Edition, Bucharest, Romania, October 216. Project Manager, Dr. Mihaela (Badea) Doni 6