Recognition and Determination of Bovine Hemoglobin Using a Gold Electrode Modified With

Electronic Supplementary Material

Recognition and determination of bovine hemoglobin using a gold electrode modified with gold nanoparticles and molecularly imprinted self-polymerized dopamine

Lu Li, Limei Fan, Yunlong Dai, Xianwen Kan* College of Chemistry and Materials Science, Anhui Key Laboratory of Chemo-Biosensing, Anhui Key Laboratory of Functional Molecular Solids, Anhui Normal University, Wuhu 241000, P.R. China *Corresponding author, E-mail: [email protected]; Tel: +86-553-3937135; Fax: +86- 553-3869303

The affect of AuNP sizes on performance of the modified electrode The size of AuNP on electrode surface was investigated by the electrodeposition method within different potential and time. SEM images in Fig. S1 showed the morphologies of AuNP prepared by electrodeposition at -0.19 V, -0.20 V, -0.21 V, -0.22 V, and -0.23 V. We found that the electrode surface can be even covered with small AuNP when the electrodeposition potential was -0.20 V. And the different electrodeposition times including 100 s, 150 s, 200 s, 250 s, and 300 s were used to prepare AuNP under the same electrodeposition potential of -0.20 V. With the subsequent preparation of MIP, the modified electrodes were used to detect

3-/4- -4 -1 the change of current response of Fe(CN)6 in 1.0×10 mg mL BHb. The results have been compared and summarized in Table S1. The modified electrode electrodeposited AuNP at 200 s or more time displayed a large change of current, indicating that 200 s was the optimized electrodeposition time for AuNP preparation. Fig. S1 SEM images of AuNP electrodeposited at -0.19 V (A), -0.20 V (B), -0.21 V (C), -0.22V (D), and -0.23 V (E).

3-/4- -4 -1 Table S1 The change of current response of Fe(CN)6 in 1.0×10 mg mL BHb on MIP/AuNP modified electrode under different electrodeposition time of AuNP.

Electrodeposition time / s 100 150 200 250 300 ∆i/ μA 1.92 2.12 2.57 2.57 2.51

AFM characterization of the modified electrode AFM image was used to characterize the thickness of the modified electrode. The morphology of the poly-DA layer was uneven because of the modification of gold nanoparticles in advance, which has been characterized by SEM (Fig. 1). It’s not suitable for AFM measurement. Therefore, we characterized the poly-DA layer on the surface of ITO directly, as shown in Fig. 2S. The average thickness of poly-DA layer was about 10 nm, which was agreed with other reported articles. Fig. S2 AFM images of the poly-DA layer.

Adsorption kinetics of the modified electrode The adsorption kinetics has been investigated by immersing the modified electrode in BHb

-4 -1 3-/4- solution containing 1.0×10 mg mL Fe(CN)6 . It was found that the change of the current

3-/4- response of Fe(CN)6 increased with the extension of incubation time from 0 to 20 min, as shown in Fig. S3. The change of current response almost kept equilibrium when incubation time was over 20 min. Therefore, the binding time was 20 min. Fig. S3 Adsorption kinetics of the modified electrode. Optimization the preparation conditions of MIP/AuNP modified electrode In order to achieve the optimized conditions for the preparation of MIP/AuNP modified electrode, different influencing factors were studied, such as the concentration of functional monomer, the concentration of template protein, and the polymerization time of the reaction.

3-/4- DPV method was used to record the oxidized peak current of Fe(CN)6 on the modified

3-/4- electrode. The current change of Fe(CN)6 recorded before and after the rebinding of protein molecules was calculated for optimizing the preparation conditions of the modified electrode. The thickness of poly-DA can be controlled by the concentration of DA and the time of DA self-polymerization [1-2]. Different concentrations of DA, ranging from 1.5 mg mL-1 to 3.0 mg mL-1, were used for MIP/AuNP modified electrodes preparation with the constant concentration of BHb of 1mg mL-1. As shown in Fig. S4, with the increase of the

3-/4- concentration of DA, the current response of Fe(CN)6 increased on the modified electrode. It can be explained that the thickness of poly-DA film would increase with the increase of DA concentration during the polymerization process and more template protein would be entrapped into the poly-DA film, leading to the increase of the number of recognition cavities after the extraction process. While the DA concentration was higher than 2.0 mg mL-1, the

3-/4- change of the current response of Fe(CN)6 decreased, which may be that the protein molecules would be embedded deeply in the formed poly-DA film with over-thickness. Thus, the protein molecules can not be removed completely during the extraction process, leading to the decrease of imprinted cavities. So the concentration of DA in the polymerization process was selected as 2.0 mg mL-1. Similarly, the effect of the concentration of template protein was also investigated in a range from 0.5 mg mL-1 to 1.5 mg mL-1, which was shown in Fig. S5. The current response of

3-/4- Fe(CN)6 increased with the increase of the concentration of the template protein due to the increase in the number of recognition cavities. And the current response achieved the highest when the concentration of template protein was 1.0 mg mL-1. The current response was sharply weakened when BHb concentration was over 1.0 mg mL-1, which probably because poly-DA chain can not capture so many protein molecules. Thus, the optimized template protein concentration was 1.0 mg mL-1. Fig. S4 Optimization of the concentration of DA.

Fig. S5 Optimization of the concentration of BHb.

In order to create more recognition cavities and achieve fast adsorption, the poly-DA thickness was investigated by controlling the polymerization time of DA self-polymerization

3-/4- process. As shown in Fig. S6, the current response of Fe(CN)6 increased remarkably with the increase of the polymerization time, which may be the formation of more recognition cavities. However, longer polymerization time, over 10 h, led to a lower current response, which may be that the longer time would increase the thickness of the poly-DA film. And the protein molecules haven’t been absolutely removed from the over-thickness film, resulting in the poor site accessibility of imprinted cavities for template protein. Therefore, the optimized polymerization time of 10 h was selected.

Fig. S6 Optimization of the self-polymerization time of DA. Table S2 Comparison of other methods for BHb detection with our work. Material/ Effects General advantages and Applicability to specific Linear range LODs Interferences Reference Method used of pH disadvantages samples/problems 1.0×10-10 ~ fast rebinding dynamics, bovine blood sample, MIPs/ILa/GR 3.09×10-11 1.0×10-2 - No obvious interference high selectivity, a good average recovery [3] DPV mg mL-1 mg mL-1 excellent sensitivity of 100.2% BSA, Trp, Crp, Glu, Dop, MIP/QDs– 2.49×10-8 ~ No sample pretreatment, 7.08×10-9 Cys, AA, Ins, and their Human blood, a good MWCNTs/PGEb 4.547×10-7 - easy handling, [4] mg mL-1 mixture. recovery of 98.4-100%. DPSCV mg mL-1 good for clinical analysis no interference 1.0×10−9~ Low detection limit, GR-MIP 2.0×10−10 no significantly Bovine blood plasma, 1.0×10−1 - wide linear concentration, [5] DPV mg mL-1 interference good recovery mg mL-1 fast response Most substances such as high selectivity, but couldn’t Hb–H O –Na CO , 2.6×10-6~10-2 1.2×10-6 anions, sodium citrate, Human blood, 2 2 2 3 - omit the inference from the [6] Chemiluminescence mg mL-1 mg mL-1 EDTA, heparin. no good recovery transition metal ions interference. G-quadruplex/hemin complex, 5~200 no significantly low detection limit, 2 nmol L-1 pH 8.1 [7] fluorescence nmol L-1 interference high selectivity - quenching Citric acid, EDTA, heparin, the metal ion no Cu(II)-pts c 1.2×10-5~ 8.13×10-6 interference; Albumin had high selectivity, Human blood, ,Spectrophotometric 2.58×10-4 pH 2.0 [8] mg mL-1 some Influence when it poor linear range RSD 1.37%-5.2% method mg mL-1 was permitted at 6 times the weight of hemoglobin. CdHgTe NP, 6×10−5~ 2.7×10-5 Mostly mental ions no reliable, fluorescence 6.1×10−3 pH 5.56 [9] mg mL-1 interference sensitive - quenching mg mL-1 1.0×10-11~ MIP/AuNP easy preparation, 1.0×10-2 - pH 7.0 No obvious interference - This work DPV sensitive mg mL-1 a IL: ionic liquid. b PGE: pencil graphite electrode. c pt: phthalocyanine References 1. Zhou WH, Tang SF, Yao QH, Chen FR, Yang HH, Wang XR (2010) A quartz crystal microbalance sensor based on mussel-inspired molecularly imprinted polymer. Biosens Bioelectron 26:585-589 2. Liu Y L, Ai K L, Lu L H (2014) Polydopamine and Its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chem Rev 114:5057-5115 3. Wang ZH, Li F, Xia JF, Xia L, Zhang FF, Bi S, Shi GY, Xia YZ, Liu JQ, YH Li, Xia LH (2014) An ionic liquid-modified graphene based molecular imprinting electrochemical sensor for sensitive detection of bovine hemoglobin. Biosens Bioelectro 61: 391-396 4. Prasad BB, Prasad A, Tiwari MP (2013) Quantum dots-multiwalled carbon nanotubes nanoconjugate-modiﬁed pencil graphite electrode for ultratrace analysis of hemoglobin in dilute human blood samples. Talanta 109: 52-60 5. Luo J, Jiang SS, Liu XY (2014) Electrochemical sensor for bovine hemoglobin based on a novel graphene-molecular imprinted polymers composite as recognition element. Sens Actuators B 203:782-789

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