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Electrochemical Conversion of Fe3O4 Magnetic Nanoparticles to Electroactive Prussian Blue Analogues for Novel SelfSacrificial-Label Biosensing of Avian Influenza Virus H5N1 Qi Zhang, Lingyan Li, Zhaohui Qiao, Chunyang Lei, Yingchun Fu, Qingji Xie, Shouzhuo Yao, Yanbin Li, and Yibin Ying Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02784 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 21, 2017

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Analytical Chemistry

Electrochemical Conversion of Fe3O4 Magnetic Nanoparticles to Electroactive Prussian Blue Analogues for Novel Self-Sacrificial-Label Biosensing of Avian Influenza Virus H5N1

Qi Zhang,† Lingyan Li,†,‡ Zhaohui Qiao,† Chunyang Lei,† Yingchun Fu,*,† Qingji Xie,‡ Shouzhuo Yao,‡ Yanbin Li,†, § Yibin Ying†

†

College of Biosystems Engineering and Food Science, Zhejiang University,

Hangzhou 310058, China. ‡

Key Laboratory of Chemical Biology and Traditional Chinese Medicine

Research (Ministry of Education of China), Hunan Normal University, Changsha 410081, China. §

Department of Biological and Agricultural Engineering, University of Arkansas,

Fayetteville, AR 72701, USA

Corresponding author: Yingchun Fu. Tel./Fax: +86 571 88982534. E-mail address: [email protected].

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ABSTRACT

A serious impetus always exists to exploit new methods to enrich the prospect of nanomaterials. Here, we report electrochemical conversion (ECC) of magnetic nanoparticles (MNPs) to electroactive Prussian blue (PB) analogues accompanying with three interfacial effects and its exploitation for novel label self-sacrificial biosensing of avian influenza virus H5N1. The ECC method involves a high potential step to create strong acidic condition by splitting H2O to release Fe3+ from the MNPs, and then a low potential step leading to the reduction of co-existing K3Fe(CN)6 and Fe3+ to K4Fe(CN)6 and Fe2+, respectively, which react to form PB analogues. Other than conventional solid/liquid electrochemical interfaces that need supply of reactants by the transportation from bulk solution and require additional template to generate porosity, the proposed method introduces MNPs on the electrode surface and makes them as natural nano-templates and nano-confined-sources of reactants. Therefore, the method presents interesting surficial “templating”, “generation-confinement”, and “refreshing” effects/modes, which benefit the produced PB with higher porosity and electrochemical activity, and three-magnitude-lower requirement on reactant concentration compared with conventional methods. Based on the ECC methods, a sandwich immunosensor is designed for rapid detection of avian influenza virus H5N1 using MNPs as self-sacrificial labels to produce PB for signal amplification. Taking full advantages of the high abundance of Fe in MNPs and three surficial effects, the ECC method endows the biosensing technology with high sensitivity and a limit of detection down to 0.0022 HAU, which is better than those of most reported analogues. The ECC method may lead to a new direction for the application of nanomaterials and new electrochemistry modes.

Keywords:

Magnetic

nanoparticles;

nanoelectrochemistry;

electrochemical interface; avian influenza virus H5N1

2


biosensor;

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INTRODUCTION Magnetic nanoparticles have been one of most intriguing and promising nanomaterials in the nanotechnology researches and applications.1 For this flourishment, one of the most essential reasons derives from the diversified physical and chemical properties of ferrous element and their compounds. The basic and unique moving behaviors of magnetic nanomaterials in magnetic field are especially useful for “wireless” smart controlling/manipulation.2-6 However, to realize the functions, further discovery and incorporation of other merits are necessary and have been the prevail development strategy, such as magnetic properties (magnetic relaxation,7-9 magnetoresistance10 and nuclear magnetic resonance,11-12) and surficial properties (catalysis).12-16 They endow magnetic nanomaterials with abundant and flexibly extended functions to meet the applications for collection, transportation, imaging, catalysis, and sensing. However, above explorations are mainly based on the physical and chemical manners of magnetic nanomaterials, other methodology (such as electrochemistry) has been rarely adopted. Therefore, a serious impetus exists to exploit new methods to enrich the prospect of MNPs considering the diversified properties and reactivity of ferrous element and its compounds. For solid/liquid electrochemistry, the control of interface is crucial, such as modulating the surficial morphology and activity, as well as mass transportation, which are especially important for electrochemical synthesis/deposition and quantification. Generally, enhancing the roughness/porosity increases the surface area and active sites, such as using templates.17-18 The control of the mass transport, however, is not so easy since it is influenced by diversified factors.19-20 One of the most important reasons might be that the reactants are generated from the bulk solution.19-24 This leads to complicated and susceptible transportation paths, making it hard to be controlled in good space- and time-resolution. Furthermore, the performance of electrochemical quantification is also hindered because the concentration of reactants (targets) decreases due to the unavoidable dilution by a 3



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generally large volume of bulk solution plus the concentration gradient of transportation.25 Therefore, introducing reactants/precursors to the surface of solid may create a new mode for the control of the interface and bring some benefits. The first one is that these species can act as physical templates themselves and “templated” source of reactants, the other is to reduce some unbeneficial dependence on mass transportation from bulk solution. This mode should also provide new capability to control the mass transport in space and time manner, such as by electrochemical manipulation, as well as a confinement effect on reactants in the proximity of electrodes to realize enrichment, which is especially important to electrochemical quantification. Signal amplification is a key and challenge issue in the biosensing field. Nanomaterials have been one of the most common and efficient labels to generate and amplify signals. Cooperating with appropriate signal collection technology, nanomaterials readily take every aspects of the physical/chemical/biological properties in developing novel biosensing methods based on magnetism, optics, catalysis, and so on.26 Besides these common properties, nanomaterials can also devote themselves as self-sacrificial labels, namely, by appropriate chemical/physical treatments, the labels are dissolved to release metal ions, which are further adopted to amplify the signals.21-24 For example, quantum dot labels with different metal components could be readily dissolved by H2O2 or acid to release different metal ions to realize sensitive even simultaneous detection of single or multiple targets through collecting the electrochemical reduction current of metal ions.22-24 This strategy takes advantages of the high abundance of metal element in the nanoparticles themselves, in turn avoids the introduction of other functional materials and facilitates the integration with other properties/functions of nanoparticles. Therefore, this strategy realizes the signal generation and amplification in a rather facile and efficient way. However, although magnetic nanomatrials have been broadly applied in biosensing as separation/concentration matrix or label, their explorations as self-sacrificial labels are

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limited. In this consideration, exploiting magnetic nanomatrials themselves as self-sacrificial label may further simplify the separation/concentration and preparation of labels, as well as amplify signals through integrating the magnetic properties. Herein, we report on an exploitation of electrochemical conversion (ECC) of Fe3O4 magnetic nanoparticles (MNPs) to electrochemically active Prussian blue (PB) analogues and its accompanying electrochemical interfacial modes. As briefly shown in Scheme 1, a method of two potentiostatic processes was developed. The application of a high potential to the Au electrode splits H2O to O2 and H+, the latter dissolves Fe3O4 to continuously release Fe3+ and Fe2+ (electrochemically oxidized to Fe3+ immediately); at the following low potential, electrochemically reduced K4Fe(CN)6 on the surface of the electrode combines with reduced Fe2+ and forms ferrous ferrocyanide (Prussian white, PW, analogue of PB). PW and PB present mutual transformation behavior through switching the potential, therefore, we mentioned them as PB for convenience. This method exploited new properties of MNPs by realizing the conversion of MNPs to functional products through a facile electrochemical process, and presented a unique mass transport mode of surficial “generation-confinement” with much more enhanced production efficiency than conventional ones, as well as interesting interfacial “templating” and “refreshing” effects. As a conceptional application, the ECC method was exploited to develop an electrochemical biosensing technology using MNPs as self-sacrificial labels to ultra-sensitively detect Avian Influenza (AI) virus H5N1, which is a severe threat to the poultry and human health,2 as shown in Scheme 1. EXPERIMENTAL SECTION Materials and Apparatus. All electrochemical experiments were conducted on a CHI660C electrochemical work-station (CH Instrument Co.) with a conventional three-electrode electrolytic cell. The Au electrode with 2.0-mm diameter (area = 0.03 cm2, purchased from Tianjin

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Incole Union Technology Co., Ltd) served as the working electrode, a KCl-saturated calomel electrode (SCE) as the reference electrode, and a carbon rod as the counter electrode. All potentials reported here are cited versus SCE. The FT-IR spectrophotometry was conducted on a Nicolet 6700 FT-IR Spectrometer (Thermo Scientific). UV-vis spectrophotometry was conducted on a UV-2450 UV-vis spectrophotometer (Shimadzu, Japan). The magnetic separator with a magnetic strength of 0.8 T was provided by Aibit Tech (Jiangyin, China). Scanning electronic microscopy (SEM) images were collected on a SIRION-100 (FEI, the Netherlands) or a SU-8010 (Hitachi, Japan) scanning electronic microscope. Magnetic hysteresis loop was collected on a Squid-VSM vibrating sample magnetometer (VSM) (Quantum Design, USA) at 298.15 K between ± 2 T. Transmission electronic microscopy (TEM) image of the MNPs was recorded with a JEOL JEM-1200EX transmission electron microscope (Hitachi, Japan). Dynamic light scattering (DLS) was performed in virtue of ZETASIZER Nano-ZS equipment (Malvern Instruments Ltd., UK). Inactivated AI virus subtypes H5N1

(A/Duck/Guangdong/383/2008, 128

hemagglutination units (HAU) in phosphate buffered saline (PBS, 10 mM, pH 7.4)), the specific antibody and the negative chicken swab samples were provided by College of Veterinary Medicine, South China Agricultural University. The specific antibody have been proved for highly sensitive and specific detection.27-28 PBS, protein

A,

concanavalin

A

(Con

A),

bovine

serum

3-dithiobis-(sulfosuccinimidylpropionate)

albumin

(BSA), (DTSP),

N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide

hydrochloride

(EDC)

and

N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fe3O4 MNPs were from Huier Nano-Technology Co. (Henan, China). A pH 6.0 phosphate buffer solution (PBS, 0.1 M KH2PO4/K2HPO4 + 0.1 M K2SO4) was used for the electrochemical measurement of PB. All other chemicals were of analytical grade or better quality, and used as received. Milli-Q (Millipore, ≥18 MΩ cm) ultra-pure water was used throughout.

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Three types of bacteria and three types of viruses that could be found in poultry were adopted to examine the specificity. The no-target bacteria were E. coli O157:H7, S. aureus and S. Typhimurium all with a concentration of 105 CFU mL-1 in PBS. The interference viruses were as below. Marek's Disease virus (MDV) was from Marek's Disease (Serotype I, III) Bivalent Vaccine (Merial Animal Health Co., Ltd., USA). Newcastle Disease virus (NDV) and Infectious Bronchitis virus (IBV) were from Combined Newcastle Disease and Infectious Bronchitis Vaccine (Strain La Sota+Strain H120) (Qingdao Yebio Biotechnology Engineering, Ltd., China). Infectious Bursal Disease virus (IBDV) was from Infectious Bursal Disease Vaccine (Strain M.B.) (ABIC Biological Laboratories Ltd., Iseral). All vaccines were live (not inactivated). According to the instructions, we prepared the virus solutions with concentrations required for adult chickens. The MDV solution contained virus strain CVI 988/Rispens of 3750 PFU mL-1 and HVT FC-126 of 5000 PFU mL-1. The NDV + IBV solution contained NDV of 4 × 104 EID50 mL-1 and IBV of 126 EID50 mL-1. The IBDV was 31.6 EID50 mL-1. The volume of all solutions for measurements was 6 µL. HAU means hemagglutination units. The haemagglutination (HA) is defined as the agglutination of red blood cells which is caused by the existence of viruses, an antibody or other microbes.29 One HA unit (HAU) in the haemagglutinin titration is the minimum amount of virus that will cause complete agglutination of the red blood cells.30 CFU means colony-forming unit which is used in bacteria tests. PFU means plaque forming unit, which is a measure of the number of particles capable of forming plaques per unit volume, such as virus particles.31 EID50 means 50% egg infectious dose caused by virus. HAU, PFU and EID50 were used in virus tests. Preparation of Con A modified MNPs MNPs suspension of 10 mg mL-1 was washed with water once and then re-dispersed in 0.2 M citric acid solution, followed by ultra-sonication for 10 min and stirred overnight to modify the MNPs with carboxyl groups.32 After washing with

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water three times, MNPs were dispersed in 10 mM MES solution containing 10 mM EDC and 15 mM NHS and stirred for 2 h to activate the carboxyl groups. After washing with PBS for three times, the activated MNPs (1 mg mL-1) was mixed with 1 mg mL-1 Con A in PBS and stirred for 1 h to conjugate Con A onto MNPs (Con A-MNPs). The Con A-MNPs was stored in 4 oC when not in use. ECC of MNPs The Au electrode with MNPs anchored on the surface using a magnet was immerged in the solution containing 0.4 mM K3Fe(CN)6 and 0.1 M K2SO4. A potential of 1.60 V was then applied to generate O2 bubbles and H+ on the surface for 450 s, which was followed by a potential of 0 V for 300 s to finally yield PB. The reaction equations are given below. 2H2O 4H+ + O2 + 4e-

(1)

8H+ + Fe3O4 Fe2+ + 2Fe3+ + 4H2O

(2)

Fe2+ + Fe(CN)64- + 2K+ K2Fe[Fe(CN)6]

(3)

Fabrication of the biosensor Original Au electrode was carefully polished and cleaned according to a reported method.33 Then, the Au electrode was immediately immerged in 2 mM DTSP (acetone) and kept overnight. After washing with acetone and water thoroughly, DTSP modified Au electrode was casted with 5 µL 0.1 mg mL-1 protein A for 45 min. The protein A modified electrode was then washed with PBS and incubated with 5 µL 0.1 mg mL-1 antibody for 1 h. After blocking with 5% BSA for 0.5 h, the antibody modified electrode was washed with PBS and stored in 4 oC before use. For detection, the antibody modified electrode was casted by 10 µL AI virus H5N1 solution with different concentrations and incubated for 1 h. After carefully washing with PBS three times, 5 µL of Con A-MNPs was dropped onto the surface of electrode and kept for 30 min to conjugate with the virus through the strong interaction of lectin-glycosyl.2 Finally, the biosensor was transferred to the solution containing 0.4 mM K3Fe(CN)6 and 0.1 M K2SO4 and underwent the ECC treatment.

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Finally, cyclic voltammetry (CV) in solution containing 0.1 M HCl and 0.1 M K2SO4 was conducted to obtain the peak current, which is proportional to the concentration of AI virus H5N1. Calculation of the concentrations of iron ions and MNPs. The concentration of 1 ng Fe3O4 MNPs in different volumes of solutions could be calculated according to Eq. (4) as follows (assuming all iron content in Fe3O4 were transferred in to ions). c = mMNPs/(Mr × v)

(4)

where c is the concentration of Fe3O4 in M, mMNPs is the mass of MNPs (1 × 10-9 g), Mr is the molecular weight of Fe3O4 (232 g mol-1) , and v is the volume used for the ECC. If we use the total volume of the ECC solution (200 µL), c is 2.2 × 10-8 M (6.6 × 10-8 M for iron ions). If we consider the volume of a diffusion layer (a cylinder with a diameter of 2 mm and a height of 50 µm (the thickness of the diffusion layer)), v is 1.57 × 10-7 L, and then c is 2.7 × 10-5 M (8.1 × 10-5 M for iron ions). The amount of 1 ng Fe3O4 MNPs was calculated as follows. n = mMNPs/(4/3 × π × r3 × ρ)

(5)

where n is the amount of MNPs, mMNPs is the mass of MNPs (1 × 10-9 g), r is the radius (10 nm), and ρ is the density of Fe3O4 (5.18 g cm-3). Therefore, n was calculated to be 7.7 × 10-20 mol. Biosafety Issue. All of testing samples were prepared using inactivated AI virus H5N1 and all tests were conducted in a biosafety level 2 laboratory. Research people were required to get relevant training to conduct the avian influenza tests. RESULTS AND DISCUSSION The effect of the electrochemically generated H+ was first simulated by externally adding acid to the preparation solution, as recorded by digital photo (Figure 1, insert). The mixtures of K4Fe(CN)6 and HCl (sample 1) or MNPs (sample 2) kept clear and colorless. In contrast, the mixing of HCl with MNPs and K4Fe(CN)6 (sample 3) led to

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gradually deepened blue color, indicating the formation of PB. Clearly in principle, H+ was the trigger to rapidly dissolve Fe3O4 to produce Fe3+ and Fe2+, in turn to form PB (Fe4[Fe(CN)6]3) and Prussian white (K2Fe[Fe(CN)6]), respectively. The PB products of the ECC method were then characterized. After careful investigation and optimization of some key ECC parameters (Figure S1), we obtained PB products presenting strong characteristic peaks located around 0.2 V from cyclic voltammetry (CV) (Figure 1), demonstrating satisfactory electrochemical activity of PB. In contrast, the absence of MNPs led to CV curve without any peak, indicating that MNPs were the self-sacrificial source of Fe3+ for the PB formation. We further characterized and compared the proposed PB film with that prepared through conventional electrochemical deposition using scanning electronic microscopy (SEM) (Figure 2). We observed condense and flat PB particles film prepared by the conventional method. In contrast, PB film prepared by the ECC showed porous structure. The structure might derive from a dual-templating effect. First, MNPs themselves acted as the templates to increase the porosity. Moreover, little oxygen bubbles on the surface of electrode generated in high potential should also be good templates.34 This porous structure should increase effective surface area and mass-transfer efficiency, in turn benefit the electrochemical activity and performance. Besides the structure, some other merits were expected because the iron ions are generated in the proximity of electrode (surficial generation), rather than transported from a homogeneous bulk solution. Therefore, we investigated and compared the production efficiencies of PB from ECC and conventional electrochemical deposition. Surprisingly, the mass of MNPs that could cause distinguishable PB signal was down to 1 ng, corresponding to 0.066 µM Fe3+ in a total volume of 200 µL solution adopted for the ECC (please see the Experimental section for the detailed calculation, same as below), which is almost three-magnitude lower than 50 µM Fe3+ that was necessary in the conventional method, as shown in Figure 3A and 3B. This huge discrepancy directed us to speculate the real concentration on the surface of the electrode. It thus

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turned out the concentration of Fe3+ to be several tens micromolar (such as 81 µM assuming a thickness of 50 µm for the diffusion layer), which was large enough and thus explained for the PB formation. Furthermore, we replaced Fe(CN)64- with Fe(CN)63- (nonreactive to Fe3+), and got the mass of MNPs that could generate distinguishable signal was 20 ng, which is larger than that for Fe(CN)64- but is still much smaller than that of conventional method (Figure 3C and 3D). Imaginably, Fe3+ was in situ generated on the electrode surface and confined in a nano-liter-volume layer (generation-confinement effect), which should significantly increase the local concentration of reactants to benefit the electro-synthesis. In contrast for the conventional mode, the concentration of Fe3+ was decreased due to the dilution in milliliter scale solution and then further decreased after a transportation gradient. Moreover, Fe(CN)64- ions formed a diffusion layer and in turn constructed a confined space for Fe3+ to restrict its diffuse-away by forming PB, which led to enhanced confinement effect and higher production efficiency than that using Fe(CN)63- ions, as illustrated in Scheme 2. The above results thus highlight the superiority of the proposed mode based on the generation and confinement mode over conventional one for better control and enhancing of electrodeposition and quantification. Known the mass limit of the MNPs for the ECC method (1 ng), the amount of the MNPs was calculated to be down to ca. 8 × 10-20 mol, which implies significant potential for ultra-sensitive detection when MNPs were adopted as labels incorporating with the proposed ECC method. Therefore, a sandwich electrochemical biosensing method was developed using MNPs as self-sacrificial labels based on the ECC method. Con A modified MNPs (Con A-MNPs) were prepared and characterized. The load of Con A on MNPs was 0.2 mg per mg MNPs, as characterized by UV-vis and FT-IR spectroscopies (Figure S2). As shown in Figure S3, SEM and TEM images displayed that the Con A-MNPs were nanoparticles of ca. 20 nm. The morphology was similar as those in previous reports.31,

35

Some Con A-MNPs were presented as clusters,

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which might be due to the accumulation during the drying process in the sample preparation.36 To find out the actual dispersion status of the Con A-MNPs in solutions, DLS and digital inspection of the Con A-MNPs suspensions were conducted, as shown in Figures S4 and S5 (the inserted pictures). The DLS gave an average hydrodynamic diameter of 79 nm and a particle dispersion index (PDI) of 0.12. Generally, the hydrodynamic diameter is larger than that in dry state (such as that observed in SEM or TEM).31, 36 Therefore, considering the diameter of ca. 20 nm and the hydrodynamic effect, we speculated the Con A-MNPs were dispersed as clusters of two or three nanoparticles in solution. The low PDI value indicated good uniformity of these kinds of clusters. The suspension of the Con A-MNPs was also inspected to be very clear (sample 1, Figure S5) and could rapidly recover its initial dispersion after the magnetic collection (sample 3, Figure S5). This inspection also proved the satisfactory dispersion. Therefore, the Con A-MNPs nanoparticles/clusters with good dispersibility and uniformity should act as satisfactory labels for signal readout. The magnetic performance was also examined by VSM. As shown in Figure S5, the saturation magnetization and the coercivity of the Con A-MNPs were 57.2 emu g-1 and 29.1 Oe, respectively. These results were comparable with those of previous reports.35, 37 The Con A-MNPs could be rapidly collected in magnetic field (sample 2, Figure S5) and recovered its dispersion immediately through simple vortex. Above results should prove the satisfactory magnetic performance for magnetic manipulation. The conjugation of the Con A-MNPs to H5N1 virus-modified electrode was realized

through the Con A-glycosyl interaction.2 The modifications were monitored

by CV and electrochemical impedance spectroscopy (EIS) (Figure S6). We observed H5N1 virus particles of ca. 50 nm diameter after the capturing (Figure 4A, indicated by arrows), and plenty of MNPs particles after the labeling (Figure 4B). After the application of the ECC treatment, PB particles were observed on the electrode surface

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(Figure 4C), as well as peaks of CV curves (as detailed below). Interestingly, after the same ECC treatment except the presence of K4Fe(CN)6 to the MNPs-conjugated electrode, we obtained a significant recovery of 82% of the charge transfer resistance (Rct) from the EIS characterization, as well as well-recovered current peaks of CV (Figures 5A and 5B). The SEM image also proved smoother surface (Figure 4D). The results indicate to a refreshment effect of the ECC method due to high potential treatment on the electrode surface, which has well adopted to clean electrodes.33 As well known, the conductivity of the modified materials on the surface of an electrode is crucial for amperometric electrochemistry, especially for the electrochemical biosensing that use electrochemically active labels. However for general methods, the modification of capture probe (antibody, DNA, etc.) and then the capture of the targets (protein, virus, bacteria, etc.), which are all highly insulating, significantly hamper the amperometric signal readout. Therefore, the proposed ECC method may bring a new merit of refreshment effect and benefit the electrochemistry. Under above understanding and careful optimization of various parameters, the crucial performance of the ECC-based biosensing method was estimated, as shown in Figure 6. The reduction peak current was adopted as the analytical signal that was proportional to the concentration of H5N1 virus. The biosensor exhibited a linear detection range from 0.0025 to 0.16 HAU, a limit of detection (S/N = 3) of 0.0022 HAU in 6 µL, which is comparable with our previous study2 but is much lower than the others,17, 27-28, 38-39 as listed in Table S1. Clearly, above beneficial effects of the ECC method and the high abundance of Fe in MNPs should well explain the high biosensing performance. Note that the MNPs labels just needed simple modification of Con A which was also facilitated by magnetic separation during the preparation, thus significantly simplifying the construction of the biosensor. Note that the CV curves in Figure 6 were slightly different in shape from that in Figure 1, which might be due the different surface between bare (Figure 1) and modified (Figure 6) electrode.

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Three types of bacteria and three types of viruses that could be found in poultry were adopted to examine the specificity. After the measurements, we found that the responses of the interference bacteria and viruses were lower than 17% of that for 0.16 HAU, as shown in Figure S7. Above result should prove the satisfactory specificity of the proposed biosensor. Furthermore, we applied the biosensor for the detection of AI virus H5N1 in chicken swab sample using the standard addition method. By spiking 0.16, 0.12, 0.08, 0.05, 0.03 and 0.01 HAU H5N1 virus in negative swab samples, we prepared a series of solutions to simulate the detection in real samples. As listed in Table S2, the recovery values were ranged from 92.5% to 120% with RSD lower than 20%, indicating the good potential of this biosensor for detection in real samples. CONCLUSIONS We have developed the ECC of MNPs to be functional PB and its application for biosensing of AI virus H5N1 in poultry. The ECC was readily realized by controlling two potentials processes. The method enriched the functionalization of MNPs besides the magnetic properties, and presented series of interesting surficial “templating”, “generation-confinement”, and “refreshing” effects/modes, which significantly benefited the production, properties and performance of PB. The ECC have endowed the biosensing technology using MNPs as self-sacrificial labels with superior sensitivity and simplicity, as well as satisfactory specificity and feasibility for real sample detection of AI virus H5N1. The ECC method may create a new direction for the application of magnetic materials and other nanomaterials. The novel surficial modes of ECC may also inspire new modes for electrochemical deposition and detection.

ACKNOWLEDGEMENTS

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This work was supported by National Natural Science Foundation of China (Grants 21475041, 21505120, 21775137), the Research Foundation of Education Bureau of Zhejiang

Province

(2014QNA6014),

and

the

State

Key

Laboratory

of

Chemo/Biosensing and Chemometrics. Q. Zhang and L. Li contributed equally to this work.

Supporting Information Optimization of the ECC parameters; FT-IR spectra for the modification and characterization of Con A-modified MNPs; TEM, SEM, DLS, VSM characterizations of Con A-MNPs; CV and EIS characterization of the modification of electrodes; Specificity tests; Comparison of performance of the proposed and reported biosensors; Detection results of H5N1 in spiked chicken swab samples. These data can be found in the online version.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Con A-MNP H2O

+1.6 V

Page 18 of 26

H+

0V

H+ Fe3+

Fe2+

Virus

K4Fe(CN)6

K4Fe(CN)6 Antibody

O2

B

Fe3O4

Protein A DTSP

PW

PW Au

Au

Scheme 1. Illustration of the ECC and ECC-based immuno-biosensor.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


III

II K4Fe(CN)6

I K3Fe(CN)6 3+ Fe

K3Fe(CN)6

Fe3+

Fe3+

Fe3O4

Fe3O4

PW Au

Scheme 2. Illustration of three modes of PB production based on different interfacial mass transport modes of conventional one (I), ECC using K4Fe(CN)6 (II) or K3Fe(CN)6 (III).

19



20 0

i / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

-20 -40 w/o MNPs w/ MNPs

-60 -.2

0.0

.2

.4

E / V (vs SCE)

Figure 1. CV curves of Au electrodes treated through ECC in the absence (brown) and presence (yellow) of MNPs in an aqueous solution containing 0.4 mM K4Fe(CN)6 and 0.1 M K2SO4. Scan rate: 30 mV s-1. The insert shows the digital photo of the mixture solutions of HCl + K4Fe(CN)6 (1), MNPs + K4Fe(CN)6 (2) and HCl + MNPs + K4Fe(CN)6 (3). Concentrations: 0.1 M HCl, 0.4 mM K4Fe(CN)6, 1 mg mL-1 MNPs.

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A

B

Figure 2. SEM images of PB films prepared by conventional electrochemical deposition (A) and ECC method (B). Scale bar: 500 nm.

21



60

-10

20

40

ipc / µA

ipc / µA

-20

15

-30 -.1 0.0 .1 .2 .3 .4 .5

E / V vs SCE

10

y = 0.22x - 0.16 r2 = 0.9924

5

20 0 -20 -40 -60

50

i / µA

0

A

30

B i / µA

10

25

-.1 0.0 .1 .2 .3 .4 .5

E / V vs SCE 20

y = 53x - 1.4 r2 = 0.9903

10 0

0 0

20

40

60

80

0.0

100

.2

.4

cMNPs / µg mL 5 -5

C

2

-10

i / µA

-.1 0.0 .1 .2 .3 .4 .5

E / V vs SCE

5

1.0

D

0

-15

10

.8

i / µA

0

15

.6

cFe3+ / mM

-1

ipc / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 26

y = 0.18x - 2.55 r2 = 0.9812

1 2

-2

-4 3

0

-6

0

20

40

60

80

cMNPs / µg mL

-.1

100

0.0

.1

.2

.3

.4

.5

E / V vs SCE

-1

Figure 3. The CV responses (the insert) and the calibration curves of reduction peak currents of PB-modified Au electrodes prepared at different concentrations of MNPs in the presence of K4Fe(CN)6 (A) or K3Fe(CN)6 (C) through ECC method, as well as Fe3+ through conventional potential static method (B). CV responses (D) of PB modified electrodes prepared from 20 ng MNPs-modified electrodes in the presence of K3Fe(CN)6 (2) or K4Fe(CN)6 (3) through ECC method, curve 1 was the control without MNPs.

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A

C

Figure 4. SEM images of Au electrodes after the capturing of AI virus H5N1 (A), labeling of Con A-MNPs (B) and the following ECC treatment in the presence (C) and absence (D) of K3Fe(CN)6. Scale bar: 500 nm.

23



20 10

A

1 3

16

B

2 -Z'' / kΩ

5

i / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 -5

12 8

2

4

3 1

0

-10 -.1

0.0

.1

.2

.3

.4

.5

0

10

E / V (vs SCE)

20

30

40

Z' / kΩ

Figure 5. CV (A) and EIS (B) curves of Au electrodes successively modified with DTSP (1), Con A-MNPs (2) and the Con A-MNPs-labeled Au electrodes treated by the ECC in the absence of K3Fe(CN)6 (3).

24


Page 25 of 26

4

A

2.0

B

2 1.5 0

ipc / µA

i / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-2

ipc=0.75lgc+2.35 r2=0.9742

.5

-4 -6 -.4

1.0

0.0 -.2

0.0

.2

.4

.6

-2.5

E / V (vs SCE)

-2.0

-1.5

-1.0

cH5N1 / log HAU

Figure 6. CV responses (A) of the biosensor to AI virus H5N1 of 0.16, 0.04, 0.01, 0.005, 0.0025 and 0 HAU (from bottom to top for the reduction peaks) in 6 µL PBS. The calibration curve is also given (B).

25



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Page 26 of 26

TOC Graphic A

Con A-MNP H2O

+1.6 V

H+

0V

H+ Fe3+

Fe2+

Virus

K4Fe(CN)6

K4Fe(CN)6

Antibody O2

B

Fe3O4

Protein A DTSP

PW

PW Au

Au

26


Electrochemical Conversion of Fe3O4 Magnetic ... - ACS Publications

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