Electronic Supplementary Material
Amperometric oxygen biosensor resulting from hemoglobin encapsulated in nanosized grafted starch particles
Xiaojun Liua1, Zhongqin Pana1, Zenglin Donga, Yannan Lua, Qiaoling Sunc, Tingting Wua, Ning Baoa, Hong Heb[(], Haiying Gua[(]
a School of Public Health, Institute of Analytical Chemistry for Life Science, Nantong University, Nantong 226019, China
b Affiliated Hospital, Nantong University, Nantong 226001, China
c Department of Clinical Laboratory, Nantong Tumor Hospital, Nantong 226361, China
Preparation of the starch-oleic acid grafted polymer
The fatty acids grafted starch was prepared according to Simi's method with some modifications [1]. Briefly, an appropriate amount of starch soluble was dissolved in 10 mL DMSO. After introduced into 5.2 g oleic acid and 2.0 g K2S2O8, the solution was stirred at 100 ºC for 8 h. The reacted solution was then mixed with 50 mL absolute ethyl alcohol and broke the emulsion. When two layers separated, the lower layer was evaporated into a semisolid under 0.1 MPa at room temperature. To remove the redundant reagents, the mixture was washed for three times by absolute ethyl alcohol and then dried into a solid mixture at room temperature. The final product was the starch-oleic acid grafted polymer.
Figure S1 The reaction of starch-oleic acid grafted polymer
Preparation of grafted starch-encapsulated hemoglobin
Under nitrogen atmosphere, the starch-oleic acid grafted polymer was dissolved in 3 mL DMSO in the ice-bath. Then 20 mg bovine Hb was dissolved into 10 mL 0.9% physiological saline followed by adding 0.3 mL Tween™ 20. After strong stirring for 30 min, the above solution was dropped into starch-oleic acid grafted polymer mixed liquor at the speed of 15 drops per minute. After 30 min ultrasonic vibration, dialyzed membrane (MW, 12000 g∙mol-1) and double-distilled water were used to remove the uncombined materials every 6 h, and repeated for four times. Then the reaction mixture was filtered using a 0.22 μm syringe filter, the unencapsulated Hb and DMSO were removed by high-speed centrifugation at 20000 r∙min-1. After removing the supernatant, the residual products were dialyzed 3 times against physiological saline for 3 h, and then physiological saline was exchanged at intervals of 3-4 h over 24 h to remove the organic solvent. The resulting product was used for immediate analysis or freeze-dried.
The vertical view of grafted starch-encapsulated hemoglobin (GS-Hb)
The vertical view of GS-Hb was shown in Figure S2. The middle of GS-Hb was Hb nanoparticles, and the surface was packaged by starch-oleic acid grafted polymer.
Figure S2 Schematic representation of GS-Hb (the vertical view).
Zeta potential of grafted starch-encapsulated hemoglobin (GS-Hb)
Zeta potential distribution of GS-Hb was measured by Malvern Nano-ZS 90. As shown in the following Figure S3, the zeta potential of GS-Hb was -0.60 ± 0.08 mV, which indicated that GS-Hb took negative charge. It was assembled on the CS modified GCE through electrostatic attraction
Figure S3 Zeta potential distribution of GS-Hb.
Effect of solution pH and temperature
It is observed that the formal potential (E0′) shifts negatively with increasing pH in the range of 3.0 – 9.0 in 0.10 M pH 7.4 phosphate buffer (Figure S4A). The linear regression equation is E0′(V) = –0.0396 – 0.0448 pH (R2=0.9913) (Figure S4B). The slope of –45 mV∙pH-1 is less than the theoretical value (–59 mV∙pH-1) at 25 ºC for reversible proton-coupled electron transfer [2]. This might be due to the influence of the protonation of the water molecules coordinated with the central iron [3]. Therefore, this reaction scheme of one electron and one proton transfer can be expressed as follows:
Hb hemeFe(III) + H+ + e− ⇋ Hb hemeFe(II)
As reported [4], with the increase of temperature, ΔEp decreased while the peak currents increased owing to the thermally facilitated interfacial electron transfer process. Figure S4C shows the cyclic voltammetrys of GS-Hb at various temperatures, the value of E0′ decreased linearly with the increase of temperature (Figure S4D) and the linear regression equation is E0′ (V) = – 0.14 – 6.52 × 10-4 [temperature] (K) with the correlation coefficient (R2) of 0.996 (n=3). According to the below equation:
(3)
The standard entropy of the immobilized GS-Hb (ΔS0′) was − 60.41J∙mol−1∙K−1, which was a little larger than that of RBCs on chitosan modified glassy carbon electrode (– 41.9 J∙mol−1∙K−1) [5]. Such relationship showed that GS-Hb was more stable than native RBCs.
Figure S4 (A) Cyclic voltammetrys of GS-Hb/chitosan/glassy carbon electrode in 0.10 M pH 7.4 phosphate buffer at 100 mV∙s−1 with various pH values (a) 10.0, (b) 9.0, (c) 8.0, (d) 7.0, (e) 6.0, (f) 5.0, (g) 4.0 and (h) 3.0 of the solution. (B) Plots of formal potential of GS-Hb vs. pH. (C) Cyclic voltammetrys of GS-Hb/chitosan/glassy carbon electrode at 293, 298, 303, 308, 313, 318, 323, 328, 333 K (a-i) in 0.10 M pH 7.4 phosphate buffer at 100 mV∙s−1. (D) Plots of formal potential of GS-Hb vs. temperature.
References
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4. Gavioli G, Borsari M, Cannio M, Ranieri A, Volponi G (2004) Redox thermodynamics of cytochrome c adsorbed on mercaptoundecanol monolayer electrodes. J Electroanal Chem 564:45-52. doi:10.1016/j.jelechem.2003.10.033
5. Pan ZQ, Xie J, Liu XJ, Bao N, Gu HY (2014) Direct electron transfer from native human hemoglobin using a glassy carbon electrode modified with chitosan and a poly(N,N-diethylacrylamide) hydrogel containing red blood cells. Microchim Acta 181 (11-12):1215-1221. doi:10.1007/s00604-014-1222-9
[(]* Corresponding author: Tel/fax: +86 513 85012913. E-mail addresses: , .
1Xiaojun Liu and Zhongqin Pan have equal contribution to this work.
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