Introduction

The Fe3O4 nanoparticles itself and its nanocomposites [1–4] have gained conspicuous research in biomedical and biotechnological applications [5, 6]. It has been reported that the modified Fe3O4 showed good biocompatibility for immobilizing desired biomolecules such as proteins including hemoglobin (Hb), enzyme, DNA or RNA [7–9]. Recent researches also show that the modified electrode with Fe3O4 nanoparticles exhibited excellent catalytic activity toward the reduction of organic pollutants [9]. The Fe3O4 nanoparticles not only have the advantages as benign biocompatibility and super-paramagnetism but also tend to induce the redox of accessed molecules owing to its surface defects especially for oxygens even with themselves. In order to take full advantage of nanoFe3O4, its nanocomposite with chitosan (CS/Fe3O4) is desired to be prepared. CS is a natural biopolymer, possesses a large amount of positive charges which are beneficial for film forming, and has been widely used to attract biomolecules [10, 11]. Then the unique characteristics of each components of the composite can be associated to trigger new features, such as enlarged surface area and improved electrochemical stability and biocompatibility [12]. As far as we know, based on the modification of CS/Fe3O4 nanocomposites on electrode surface to adsorb the Hb, its electro-catalytic performance is meaningful for analytical applications.

Trichloroacetic acid (TCA) [13], a member of chloroacetic compounds, is the disinfection by-products (DBPs) of chlorination since its wide practices for reducing the incidence of waterborne diseases [14]. It is also found in hematuria as metabolite of trichloroethylene [15], a common commercially industrial solvent [16]. Researches showed that TCA would alter and induce the hepatocellular adenomas/carcinomas [17, 18]. As an environmental contaminant in ground water, considering its high toxicity to humans [19], animals [20], plants, or algae [21], the World Health Organization (WHO) and US Environmental Protection Agency (USEPA) recommended the safety threshold of TCA in drinking water [22, 23]. The rapid, reliable, and accurate analytical methods for its trace detection are also urgently appealed [24]. The detecting and controlling of TCA in various samples have attracted more attention recently [25–27]. Compared to the traditional methods for TCA monitoring as HPLC-MS [28], GC-MS [29], and ion-exchange chromatography [30], with expensive and sophistic equipment, high operating cost, complex pretreatment, and difficulty for on-site or online detecting, the electrochemically modified electrode is a better choice for meeting the abovementioned demands.

Experimental

Reagents and materials

Hb (>90 %, bovine blood) and trichloroacetic acid were purchased from Sigma Co. Ltd. (www.sigmaaldrich.com) and used without further purification. Chitosan (90 % of deacetylation, Sinopharm Chemical Reagent Co., Ltd) and all other chemicals were of analytical grade. A 0.1 M phosphate buffer solution was prepared with Na2HPO4 and NaH2PO4. All solutions were prepared with double distilled water.

Apparatus and instrumentations

Electrochemical measurements were performed on a CHI 660 electrochemical workstation (CH Instruments Co., USA, www.chinstruments.com) with a traditional three-electrode system where the modified glassy carbon electrode (GCE, Φ = 3.0 mm) was used as the working electrode, a platinum wire as a counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. All measurements were carried out at the temperature of 25.0 ± 0.2 °C in a Faraday cage. The amperometric measurements were carried out in stirred condition by exerting a potential of −380 mV on the working electrode. The electrochemical impedance spectroscopy (EIS) was performed in 0.10 M KNO3 solution containing 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) as supporting electrolyte with an Autolab PGSTAT 302N electrochemical workstation (Metrohm Co. Ltd., http://www.metrohm.com/), within the frequency range from 1.0 mHz to 100 kHz. The morphology of CS/Fe3O4 nanocomposites was investigated using a transmission electron microscope (TEM) (Jeol-1210, Japan, www.jeol.com). The X-ray diffraction patterns (XRD) was recorded by D8 Advance (Bruker, Germany, www.bruker.com). The energy dispersive X-ray analysis (EDS) was recorded by EX -250 (HORIBA, Japan, www.horiba.com). The Fourier transform infrared (FT-IR) spectra was recorded on an Avatar 370 instrument (Nicolet, USA, www.thermoscientific.com) with KBr discs.

Synthesis of Fe3O4 nanoparticles and CS/Fe3O4 nanocomposite

Fe3O4 nanoparticles were prepared using co-precipitation method. FeC13·6H2O and FeC12·4H2O (molar ratio 2:1) were thoroughly mixed under magnetic stirring and heated till 50–60 °C. Then the black precipitate was obtained after NH3·H2O was added into dropwise under continuously stirring. The nanoparticles were collected using a magnet and washed several times with ethanol and double distilled water, then dried in a vacuum oven at 50 °C.

The CS was dissolved in 0.05 M HCl to form a 0.2 wt% solution, then filtered through a 0.45-μm syringe filter. Afterward , to adjust its pH value to 5.0 with NaOH, the Fe3O4 nanoparticles were dispersed in to achieve the solid content of 2 mg mL−1, thereafter, to ultrasonically irradiate for 1 h. The chitosan-magnetite nanocomposites were then separated with a magnet and washed several times with ethanol and double distilled water, then dried at 40 °C in a vacuum oven.

Fabrication of Hb/CS/Fe3O4-modified GCE

The glassy carbon electrode (3 mm diameter, CH Instruments, Inc.) was polished with 1.0, 0.3, and 0.05 μm alumina slurry, respectively, then rinsed thoroughly with double distilled water. It was also then cleaned by successive sonication in 1:1 HNO3-H2O, ethanol, and double distilled water.

Ten milligrams of CS/Fe3O4 powder was dispersed into 5 mL of pH 5.0 phosphate buffer solution, with 30 min of ultrasonic agitation. Ten microliters of this obtained dispersion was cast onto the GCE surface. After airing dried, it was washed with double distilled water and then dried again. Finally, the modified electrode was immersed in the Hb solution (10 mg mL−1) for 30 min. In this modification approach, there is a coordinate bond from the N atom of CS with the lone pair electrons to the 3d orbital of Fe atom in Fe3O4 [31] to ensure their binding. Meanwhile, in weak-acid environment, the protonated chitosan tends to form a gelled cationic polyelectrolyte, which can also combine –COOH group of heme [32] with the –NH3 + group via the electrostatic interaction. Thus, it is clear that the CS was the central role to create the deposition film on GCE surface.

Results and discussion

The morphology and characteristics of CS/Fe3O4 nanocomposites

The morphology and average size of the Fe3O4 or CS/Fe3O4 nanocomposite can be represented by TEM, as shown in Fig. 1. These two types of nanoparticles were evenly distributed with an average diameter of about 25 nm (Fig.1a) or 35 nm (Fig.1b), respectively.

Fig. 1
figure 1

The TEM images of a Fe3O4 nanoparticles and b CS/Fe3O4 nanocomposite. c The EDS spectra of CS/Fe3O4 nanocomposite. d The XRD spectra of (a) Fe3O4 and (b) CS/Fe3O4 nanocomposite

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The CS/Fe3O4 nanocomposite film was modified onto the surface of GCE directly. The chitosan, as previously discussed can form strong combination with Fe3O4 nanoparticles and biomolecules [32, 33], is adequate to imbed the Fe3O4 nanoparticles to form a nanocomposite. Also, owing to the high specific surface area and random spatial orientation, CS/Fe3O4 nanocomposites make easier to unfold the electroactive centers of biomolecules [34]. This would be helpful for Hb to expose the iron centers to realize the electron transfer.

The energy-dispersive spectrometry (EDS) was utilized for verification of the presence of CS/Fe3O4 nanocomposite on ITO glass. As presented in Fig. 1c, the characteristic peaks at 0.70 and 6.40 KeV respectively for the CS/Fe3O4 nanocomposite correspond to elemental iron. The peak of carbon element was observed at 0.26 KeV. It is well known that the peak at 0.51 KeV corresponded to the signal of oxygen.

The X-ray diffraction (XRD) analysis was carried out to confirm the structure of Fe3O4 and CS/Fe3O4 nanocomposites. As Fig. 1d showed, the XRD pattern owned well-resolved diffraction peaks which revealed the good crystalline of Fe3O4 pattern, which are located at 2θ of 30.58°, 35.56°, 43.32°, 53.75°, 57.36°, and 62.77°, respectively. These peaks are consistent with the standard sample for crystalline magnetite [35]. The XRD spectrum of the nanocomposite showed two more characteristic peaks at 10.04° and 20.10°. These peaks are occurred due to the hydroxyl (−OH) and amine (−NH2) groups in CS, which can form strong inter- or intra-molecular hydrogen bonds [36].

Construction and characterization of the Hb/CS/Fe3O4-modified GCE

The stepwise assembly of Hb/CS/Fe3O4 film on GCE was confirmed by electrochemical impendence spectroscopy (EIS) (Fig. 2a) [37], using Fe(CN)6 3−/Fe(CN)6 4− redox couple as the electrochemical probe. The Nyquist plots for bare GCE (curve a), CS/Fe3O4-modified GCE (curve b), or Hb/CS/Fe3O4-modified GCE (curve c) are displayed respectively. A bare GCE, showed a small semicircle, implying a low electron transfer resistance of 676 ± 2 Ω. While CS/Fe3O4 nanocomposite was deposited onto the surface of GCE, the resistance increased to 1036 ± 4 Ω, suggesting the insulation of CS to block the direct electron transfer with Fe(CN)6 3−/Fe(CN)6 4− [38]. After the loading of Hb, the interfacial resistance further increased obviously to as high as 4327 ± 5 Ω, suggests that the presence of Hb molecules greatly obstructed the electron transfer. This demonstrated that the Hb had been successfully immobilized on the CS/Fe3O4-modified GCE.

Fig. 2
figure 2

a The EIS spectra of (a) GCE, (b) CS/Fe3O4-modified GCE, (c) Hb/CS/Fe3O4 modified GCE, in 0.1 M KNO3 containing [Fe(CN)6]3−/4-. b The cyclic voltammograms of (a) bare GCE, (b) CS/Fe3O4-modified GCE, (c) Hb/CS/Fe3O4-modified GCE, in 0.1 M pH 7.0 phosphate buffer solution. Scan rate 100 mV s−1. c The cyclic voltammograms of (a) GCE, (b) CS/Fe3O4-modified GCE, (c) Hb/CS/Fe3O4-modified GCE in 0.1 M KCl solution, containing K3Fe(CN)6 3−/4-. Scan rate 100 mV s−1

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During the modification procedure, the cyclic voltammograms of different electrodes in pH 5.0 phosphate buffer solution was also studied, as shown in Fig. 2b. There is no any redox current on either bare GCE (curve a) or CS/Fe3O4/GCE (curve b). When Hb was attached onto the CS/Fe3O4-modified electrode, a pair of redox peaks appeared at −0.31/−0.41 V (curve c and inset C). According to reference [39], the emerged peaks can be attributed to the Fe(III)/Fe(II) redox couple of the heme protein.

Also, the CVs in Fig. 2c illustrated the electrochemical properties of modified electrode. With Fe(CN)6 3−/Fe(CN)6 4− as electrochemically active probe in the solution, its CV performance can be employed to demonstrate the characteristics of modified electrode. As was expected here, a basically symmetric redox couple that appeared on CV curve of bare GCE (curve a) means a reversible and diffusion controlled redox of probe on GCE. With slight shift of peak separation but greatly enlarged current, the CV fashion of probe on CS/Fe3O4 nanocomposites-modified GCE (curve b) demonstrates that the nanocomposite worked as the electroconducting tunnel to provide larger electroactive surface but did not change the reaction route.

Behaviors of immobilized Hb on CS/Fe3O4 nanoparticles-modified GCE

Here, we recommended the FT-IR spectra to demonstrate the coalescence of Hb onto the CS/Fe3O4 nanocomposites and its succedent function. Figure 3 has confirmed there were no obvious altered wavenumbers of absorption bands when they were immobilized. Here, the amide I band located at 1655 cm−1 was ascribed to the stretching of carbonyl vibrations of peptide linkages in the heme protein backbone, as well as the amide II band at 1538 and 1391 cm−1 attributed to the C–N bond and N–H bond stretching in Hb skeleton (Fig. 3a). These peaks hold the position at same sites after they were immobilized onto nanocomposite (Fig. 3b), indicating that heme protein maintained its original structure; thus, it is expectable to keep its intrinsic features.

Fig. 3
figure 3

The FT-IR spectra of a Hb and b Hb bonded on CS/Fe3O4 nanocomposite

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The CV performance of Fe(CN)6 3−/Fe(CN)6 4− on Hb/CS/Fe3O4-modified GCE is displayed as curve c in Fig. 2c. Here, the CV redox peaks further enlarged, but the peak form appeared some variation, means a different electrochemical mechanism. By the analysis of peak currents (I pa, I pc) toward the scan rate, they are all linearly related to the scan rate within the range of 50 to 400 mV s−1 (Fig. 4) with the correlation coefficients of at least 0.997 (see Fig. 4b), it can be identified as a surface controlled procedure [24, 40]. According to Laviron’s equations [41, 42], the electron transfer rate constant (k s) was calculated for 3.12 s−1. The value is not only higher than our previous work (0.357 s−1 [12] and 2.90 s−1 [43]) but also than the value of 2.58 s−1 obtained by Li et al. [44] and 0.3 s−1 acquired by Wang et al. [45], suggesting that the Hb might expose its iron center to accelerate the electron transfer on modified electrode.

Fig. 4
figure 4

a The cyclic voltammograms of Hb/CS/Fe3O4-modified electrode in 0.1 M pH 7.0 phosphate buffer solution with the scan rates from inner to outer as 50, 100, 150, 200, 250, 300, 350, and 400 mV s−1, respectively. b The plots of cathodic and anodic current vs. scan rate

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Electrocatalysis of Hb/CS/Fe3O4 modified GCE to the reduction of TCA

Trace detection of TCA is important in environmental monitoring, clinical diagnosis, and food safety testing [46–48]. As Fig. 5a showed, when a certain amount of TCA was injected into the PBS solution, the reduction peak current at about −0.40 V increased significantly, which implied a typical electrocatalytic reduction of TCA. It can be attributed to the dechlorination by the iron center of Hb [49, 50].

Fig. 5
figure 5

a The cyclic voltammograms of Hb/CS/Fe3O4 modified electrode in pH 7.0 phosphate buffer solution containing (a) 0, (b) 6.8 × 10−5, (c) 1.4 × 10−4, and (d) 2.3 × 10−4 M of TCA. Scan rate 50 mV s−1. b The amperometric response of Hb/CS/Fe3O4-modified electrode upon successive injection of 5.7 μM TCA into 5 mL phosphate buffer (0.1 M, pH 7.0). c The calibration curve of amperometric current vs. concentration of TCA

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The possible reaction mechanism can be deduced as shown in Scheme 1 [4].

Scheme 1
scheme 1

The schematic illustration of the electrochemically catalytic reduction of TCA on Hb/CS/Fe3O4-modified electrode

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Figure 5b shows the typical amperometric response of TCA on Hb/CS/Fe3O4-modified GCE at an applied potential of −0.38 V, the current increased stepping with each increment of TCA within a linear range from 5.70 × 10−6 to 2.05 × 10−4 M. Figure 5c displays a second-order nonlinear regression between current and TCA concentration. Also, the detection limit was estimated using a 3σ/slope rule for 1.9 × 10−6 M, where σ is the standard deviation of 10 paralleled tests of blank solutions, determined according to the IUPAC recommendation [51].

Analytical performance, stability, and reproducibility of Hb/CS/Fe3O4-modified GCE

The stability was studied after the Hb/CS/Fe3O4-modified GCE was stored in phosphate buffer solution for 3 weeks in a 4 °C refrigerator. The results showed that the Hb still had reduction ability to TCA and kept 96 % of its original response in the same condition. The repeatability was also investigated for 10 successive measurements at TCA concentration of 8.5 × 10−5 mol L−1; it exhibited a good result with the RSD of 1.5 %.

Table 1 lists the results about the comparison for the determination of TCA by different modified electrode. The linear range and detection limit of the constructed modified electrode for TCA determination are comparable or better than other reports. It can also be found that the constructed electrochemical modified electrode exhibited better reproducibility and stability when compared with previous works.

Table 1 Comparison of the proposed method for TCA with others
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Effect of chemical interferences

The interference of various substances as potential disturbance was investigated under the optimum conditions with TCA concentration of 50 μM. The ions of K+, Na+, Cu2+, Cl−, SO4 2−, NO3 −, dopamine, and ascorbic acid were investigated as the interference substance, in pH 5.0 phosphate buffer solutions. It showed that there was almost no interference from 25 times of K+, Na+, and Cl−, 50 times of Cu2+ and SO4 2−, 5 times of dopamine, and 4 times of ascorbic acid, respectively. The above experimental results suggested that this developed electrochemical electrode owned good selectivity for TCA trace detecting. Also in Table 2, the comparison of detection results with different methods are listed.

Table 2 The anti-interference possibility of the resultant electrode and the result comparison with other methods
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Analytical application of Hb/CS/Fe3O4-modified GCE for real samples

The developed modified electrode had been applied to detect TCA in several different water samples. According to the results, showed in Table 3, the added TCA in real water samples can be quantitatively recovered. The results showed that the presented TCA detecting method in this work can be valid for the analysis of TCA in real samples.

Table 3 Determination of TCA in real samples, pH 5.0, at 25 ± 0.2 °C
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Conclusion

A fast, easy, and efficient electrode was exploited by immobilizing CS/Fe3O4 nanocomposite and Hb onto the GCE surface in performing the modification. The results suggest that the Hb on CS/Fe3O4 maintained its native biological activity and electro-catalysis toward the reduction of TCA. High sensitivity, rapid response, broad linear range, low detection limit, good reproducibility, and long-term stability are achieved. All achievements could be ascribed to the significant characteristics of CS/Fe3O4 nanocomposite; it provides favorable stretch space for Hb molecules to facilitate its electron transference.