A biosensor based on hemoglobin immobilized with magnetic molecularly imprinted nanoparticles and modified on a magnetic electrode for direct electrochemical determination of 3-chloro-1, 2-propandiol

Highlights

• A biosensor for direct electrochemical determination of 3-MCPD for the first time

• The biosensor was developed based on Hb bioelectrocatalysis.

• Magnetic carrier and an external magnetic field improved Hb catalytic activity.

• Hb was concentrated in MMIP NPs to enhance the sensitivity of the biosensor.

Abstract

A biosensor for determination of 3-chloro-1, 2-propandiol (3-MCPD) was developed based on the electrochemically catalytic reduction by hemoglobin (Hb). Hb was concentrated and immobilized with magnetic molecularly imprinted nanoparticles (MMIPs NPs). The MMIP NPs had a core-shell structure, with magnetic Fe₃O₄ NPs as the core and SiO₂ as the shell. MMIPs NPs were modified on the surface of a magnetic electrode to constitute the Hb enzyme-like sensor. The biosensor could catalyze the electrochemical reduction of 3-MCPD, and a direct electrochemical determination of 3-MCPD could be achieved for the first time. The biosensor, where Hb immobilized with MMIPs NPs had high electrochemical response for 3-MCPD, increased by 37.5% compared with Hb immobilized with magnetic non-imprinted nanoparticle (MNIPs NPs). The biosensor increased the response by 19.56% again under an external magnetic field. The reduction peak current had a linear relationship with 3-MCPD concentration ranging from 1.0 mg·L⁻¹ to 160 mg·L⁻¹ with a detection limit (S/N = 3) of 0.25 mg·L⁻¹. The biosensor was applied to the determination of 3-MCPD in soy sauce samples successfully with recoveries between 97.4% and 99.6%.

Introduction

3-Chloro-1, 2-propandiol (3-MCPD) belongs to a group of chloropropanols, the by-products of acid hydrolyzed vegetable protein (HVP) [1]. During acid hydrolysis, the vegetables with abundant proteins, such as soybeans, rice and wheat are treated with hydrochloric acid at high temperature, and coexistent triglycerides are also hydrolyzed to form the chloropropanols [2]. Among the chloropropanols including 1, 3-dichloro-2-propanol (1, 3-DCP), 2, 3-dichloro-1-propanol (2, 3-DCP), 3-MCPD and 2-chloro-1, 3-propanediol (2-MCPD), 3-MCPD is a major pollutant of acid-HVP [3]. Besides, 3-MCPD has been found in many kinds of heat processed foods, such as coffee, toast and biscuits [4].

Lynch found that the high dose of 3-MCPD generated benign renal tumors in both male and female mice, and Leydig-cells and mammary tumors in male mice, which revealed that 3-MCPD was genotoxic in vitro [5]. Later, there were many other reports on the toxicity of 3-MCPD including neurotoxicity [6], nephrotoxicity [7], immunotoxicity [8], genotoxicity [9] and reproductive toxicity [10]. 3-MCPD has been classified as a genotoxic carcinogen by the European Commission's Scientific Committee on Food in 1995 [11]. The European Community regulated the maximum limit of 0.02 mg·kg⁻¹ for 3-MCPD in soy sauce [12]. The restricted value of 3-MCPD in soy sauce was 1 mg·kg⁻¹ in China [13]. The UK Food Advisory Committee (FAC) has proposed that they should continue to take all steps necessary to reduce concentrations of 3-MCPD in foods and food ingredients to the lowest technologically achievable level [14]. The European Commission Scientific Committee on Food has been established 2 μg·kg⁻¹ body weight as an endurable daily intake of 3-MCPD [15]. Therefore, it is imperative to develop a simple and sensitive method for analysis of 3-MCPD.

Many analytical methods such as mass spectrometry (MS) [16], gas chromatography–mass spectrometry (GC–MS) [17], GC–MS/MS [18], and GC with electron capture detection (GC-ECD) [19] had been applied for determination of 3-MCPD. The preconcentration procedures were introduced to improve the sensitivity further. Liquid-phase microextraction combined with magnetic solid-phase extraction (MSPE) was applied to extract 3-MCPD from edible oils, followed by determination of 3-MCPD with GC–MS. The limit of detection was as low as 1.1 ng·g⁻¹ [20]. With headspace on-fiber derivatization solid-phase microextraction combined with GC–MS, the limit of detection for 3-MCPD in soy sauce was 3.91 ng·g⁻¹ [21]. Though the above-mentioned approaches mostly based on GC–MS achieved high sensitivity, they had some defects such as expensive instruments, complicated sample preparation and long time-consuming.

The electrochemical method is simple, rapid, low-cost, sensitive and selective for electroactive analytes [[22], [23], [24]]. Based on the electrochemical oxidation of hydroxyl group of 3-MCPD on the copper electrode at alkaline medium, capillary electrophoresis (CE) separation coupled with amperometric detection was applied to determine 3-MCPD in soy sauces [25]. The detection limit of 3-MCPD was 0.13 mg·kg⁻¹, lower than the allowance value of China standard. An electrochemical sensor for 3-MCPD had been developed based on a gold nanoparticle modified glassy carbon electrode coated with 3-MCPD molecular imprinted polymer film [26]. An indirectly electrochemical determination of 3-MCPD was achieved by the redox of [Fe (CN)₆]^3−/4− on the electrochemical sensor. As 3-MCPD occupied the imprinted cavities, the diffusion of [Fe (CN)₆]^3−/4− into the surface of the electrode retarded. Thus, there was a correlation between 3-MCPD concentration and the current reduction of [Fe (CN)₆]^3−/4−. The molecular imprinted sensing was simple and selective without complicated pretreatment, but the imprinting sites were easily blocked by interfering substances in soy sauce. Therefore, the accuracy and reliability of the indirectly electrochemical determination of 3-MCPD would be improved.

Hemoglobin (Hb) is an oxygen-transport metalloprotein in red blood cells, an assembly of four globular protein subunits. Each subunit is composed of a protein chain tightly associated with a non-protein prosthetic heme group [27]. A heme group consists of an Fe(II) ion which is the electron active center in the porphyrin ring. Because the heme group is embedded deeply by the protein chains, the electron transfer is very slow, and the direct redox of Hb on the electrode is difficult [28,29]. Just like Hb, catalase and peroxidase, the common oxidoreductases also possess of the heme cofactors in their active sites, which could catalyze the reduction of hydrogen peroxide. Besides, catalase can catalyze the oxidation of various metabolites and toxins, including formaldehyde, formic acid, phenols, acetaldehyde and alcohols. Since Hb contains the porphyrin ring and Fe(II) electroactive center, it would have an enzyme-like activity. Recently, some biosensors based on Hb enzyme-like activity for electrochemically catalysis of H₂O₂ [30], NO₂⁻ [31] and O₂ [32] have been reported. It is worth mentioning that trichloroacetic acid (TCA) could be electrochemical reduced by Hb bioelectrocatalysis [33]. The mechanism on the electrochemical reduction of trichloroacetic acid by Hb catalysis through reductive dechlorination has been investigated [34]. According to the reference, it was speculated that chloropropanols would be electrochemical reduced by Hb biocatalysis through the reduction of chloralkane to chlorideion.

$$\text{HbFe}\left(\mathrm{III}\right)+{\mathrm{e}}^{-}\to \text{HbFe}\left(\mathrm{II}\right)$$

$$\text{2HbFe}\left(\mathrm{II}\right)+{\text{ClCH}}_2{\text{CHOHCH}}_2\mathrm{OH}+{\mathrm{H}}^{+}\to \text{2HbFe}\left(\mathrm{III}\right)+{\mathrm{CH}}_3{\text{CHOHCH}}_2\mathrm{OH}+{\mathrm{Cl}}^{-}$$

The overall reaction mechanism is:

$${\text{ClCH}}_2{\text{CHOHCH}}_2\mathrm{OH}+{\mathrm{H}}^{+}+{\text{2e}}^{-}\to {\mathrm{CH}}_3{\text{CHOHCH}}_2\mathrm{OH}+{\mathrm{Cl}}^{-}$$

The direct electron transfer between Hb redox centers and the electrode surfaces plays a crucial role for dechlorination of 3-MCPD. In recent years, the great efforts have been made to improve the electron transfer rate of Hb by the use of magnetic nanoparticles as the modified working electrode or under the magnetic field to increase the sensitivity of sensors [35].

In the present work, a biosensor for 3-MCPD was developed based on Hb bioelectrocatalysis, where Hb was immobilized by magnetic molecularly imprinted polymers nanoparticles (MMIPs NPs). The MMIPs NPs had the core-shell structure with the magnetic Fe₃O₄ as the core and SiO₂ as the shell. Hb was imprinted the surface of MMIPs NPs by the surface imprinting technique, therefore Hb could be concentrated and fixed on the surface of the MMIPs NPs. Hb MMIPs NPs assisted by chitosan (CS) membrane were modified on the surface of a magnetic electrode to constitute Hb enzyme-like biosensor to catalytic reduction of 3-MCPD. A magnet in the biosensor made the paramagnetic MMIPs NPs fix and be in order on the surface of the electrode. Furthermore, it could improve the catalytic activity of Hb owing to the superparamagnetism of Hb. In brief, the electrostatic interaction between SiO₂ shell and Hb, and the magnetic force on paramagnetic Hb and paramagnetic MMIPs NPs make Hb electroactive center be exposed. To the best of our knowledge, this is the first report on 3-MCPD biosensor based on the direct electrochemical reduction by Hb bioelectrocatalysis.