Surface modification of cerasomes with [email protected](ionic liquid)s for an enhanced stereo biomimetic membrane electrochemical platform

Journal Pre-proofs Surface Modification of Cerasomes with AuNPs@Poly(Ionic Liquid)s for an Enhanced Stereo Biomimetic Membrane Electrochemical Platform Daliang Liu, Qiong Wu, Shun Zou, Feiyun Bao, Jun-ichi Kikuchi, Xi-Ming Song PII: DOI: Reference:

S1567-5394(19)30548-1 https://doi.org/10.1016/j.bioelechem.2019.107411 BIOJEC 107411

To appear in:

Bioelectrochemistry

Received Date: Revised Date: Accepted Date:

12 August 2019 22 October 2019 22 October 2019

Please cite this article as: D. Liu, Q. Wu, S. Zou, F. Bao, J-i. Kikuchi, X-M. Song, Surface Modification of Cerasomes with AuNPs@Poly(Ionic Liquid)s for an Enhanced Stereo Biomimetic Membrane Electrochemical Platform, Bioelectrochemistry (2019), doi: https://doi.org/10.1016/j.bioelechem.2019.107411

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier B.V.

Surface Modification of Cerasomes with AuNPs@Poly(Ionic Liquid)s for an Enhanced Stereo Biomimetic Membrane Electrochemical Platform Daliang Liua,b, Qiong Wua, Shun Zoua, Feiyun Baoa, Jun-ichi Kikuchic, Xi-Ming Songa,b,[email protected] aCollege

of Chemistry, Liaoning University, Shenyang 110036, China

bLiaoning

Key Laboratory for Green Synthesis and Preparative Chemistry of Advanced Materials,

Shenyang 110036, China cDivision

of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama,

Ikoma, Nara 630-0192, Japan

Abstract: A novel liposomal nanocomposite, Au@PIL-cerasome, with biocompatibility and conductivity was fabricated via the self-assembly of cerasomes and gold nanoparticles (AuNPs) stabilized by poly(ionic liquid)s (PILs). The surface charge, morphology and chemical composition of the nanocomposites were characterized by the zeta potential, UV-vis, TEM, SEM and EDS. The nanocomposites exhibited structural stability directly on the surface of solid electrodes, without fusion. Electrochemical impedance experiments demonstrated that the nanocomposites had an enhanced conductivity compared with unmodified cerasomes. Horseradish peroxidase (HRP), as a reporter, was immobilized on the nanocomposites without denaturation or inactivation. The direct electron transfer of HRP was achieved, and the HRP/Au@PIL-cerasome/GCE exhibited an amplified current and improved electrocatalytic activity. Activity towards H2O2 displayed a linear range over 10-70 μM and a detection limit of 3.3 μM. Activity towards NO2- displayed linear ranges over 1-5 mM and 5-1280 mM, and the limit of detection was 0.11 mM. In addition, the electrode was stable and reproducible, with 6% RSD. Such multi-component liposomal nanocomposites with an enhanced electrical performance pave a better way for building novel and straightforward 3D stereo biomimetic electrochemical platforms and even molecular communication systems to investigate information transduction between cells. Keywords: cerasome, gold nanoparticle, horseradish peroxidase, stereo lipid bilayer membrane electrode, signal transduction system

1

1. Introduction Electrochemical platforms have attracted attention in recent years due to their simple instrumentation requirements, high sensitivity and rapid detection speed, and they play a vital role as an analytical device in healthcare[1], drug development[2], food security and environmental monitoring[3]. Additionally, a suitable electrochemical platform has already been used to investigate electron transfer mechanisms by establishing a biomimetic interface on an electrode[4]. As bioactive materials in vivo with excellent selectivity and catalysis, enzymes have been widely used to develop biomimetic electrochemical platforms[5]. However, it is difficult to utilize enzyme bioactivity for normal working electrodes because of the inaccessibility of the electroactive centres buried deep within the enzyme’s structures, as well as the denaturation or unfavourable orientation of enzymes on the surface of hydrophobic working electrodes[6]. Suitable materials, especially those that possess biocompatibility, can efficiently retain the bioactivity of enzymes on the electrode surface[7]. Cell membranes are biological membranes with embedded proteins and are involved in a variety of cellular processes, such ion conductivity, cell recognition and cell signalling[8]. Cell membranes can be used as an ideal electrode material because of their good biocompatibility. Moreover, the investigation of enzyme electrical signals on cell membranes contributes to the fabrication of highly sensitive biomimetic electrodes and can even clarify signal transmission mechanisms in biological systems[9]. Unfortunately, natural cell membranes are hardly used due to their intrinsic complexity and participation in diverse cellular processes. This has led to the development of several simplified membrane models[10]. Planar lipid bilayer membranes are artificial cell membranes that have good biocompatibility due to their similar components and structures with real cell membranes. Thus, they can be used as a matrix and modified with bioactive materials to perform recognized, catalytic functions, making them excellent candidates for a biosensing platform[11]. In addition to planar lipid bilayer membranes, liposomes are another artificial membrane type and are spherical vesicles formed by the lipid bilayer membrane, endowing them with the unique ability to amplify signals by encapsulating signal marker compounds and simulating real cell stereo-structures through separating the inner aqueous cavity from the extracellular environment[12]. A series of liposome electrochemical platforms have been developed to detect glucose[13], mercury(II) ion[14] and pesticides[15]. However, their instability and the fusion process required to form planar lipid bilayer membranes limit the application of liposomes on solid electrode surfaces. To immobilize liposomes on solid electrode surfaces, researchers have employed many compounds such as chitosan[15], cholesterol[16] and polyethylene imine[17] to improve liposome stability. Cerasomes containing an inner aqueous compartment with an artificial vesicular membrane and a silicate surface framework are a novel organic-inorganic hybrid liposomal nanomaterial[18]. Due 2

to the existence of a surface silicate layer, cerasomes exhibit a higher morphological stability and can maintain their initial spherical shape on a solid surface instead of fusing to form multi-wall planar lipid bilayer membranes, as liposomes do[19]. Cerasomes have already been utilized in many biological fields, such as cancer photodynamic diagnosis and therapy, drug delivery and as gene carriers[20]. Recently, cerasomes have been explored as an efficient biomaterial matrix for constructing several electrochemical platforms by our group. Vitamin B12 was embedded in the bilayer membrane of cerasomes to establish the first cerasome-based electrochemical platform on a glassy carbon electrode that can successfully convert a molecular input signal to an electrical response[21]. Owing to the liposomal structure, cerasomes also provide a suitable microenvironment that is beneficial for enzymes to retain their catalytic activities and served as a soft interface for the immobilization of horseradish peroxidase (HRP)[22] and cholesterol oxidase[23]. Fluorescent cerasomal nanocomposites were prepared via the self-assembly of quantum dots on cerasomes to monitor the immobilization process of redox proteins[24]. Such applications of cerasomal nanocomposites on electrodes exploit a new opportunity to establish electrochemical platforms in the biological information field. An excellent biological electrochemical platform must be able to translate weak biological signals into strong electrical signals. The electron transfer efficiency of electrodes impacts the translation and even the applications of bioelectrodes[25]. To improve the electron transfer efficiency and signal intensity, conductive materials such as metal nanoparticles, metallic oxides, carbon nanotube and conducting polymers have been widely employed to modify electrodes[26, 27]. Gold nanoparticles (AuNPs) that do not cause acute negative effects for redox proteins and enzymes are usually selected[28-31]. However, bare AuNPs are unstable and cannot be connected with cerasomes directly. Poly(ionic liquid)s (PILs) are a subclass of ion-conductive polyelectrolytes featuring an ionic liquid species in each monomer repeating unit; they are good stabilizers for AuNPs and have the ability to tune the surface charge of the AuNPs[32, 33]. Furthermore, PILs can also be used as immobilizing matrixes to immobilize redox proteins and as electron transfer promoters to improve direct electron transfer between the redox proteins and the underlying electrode in electrochemical biosensors[33, 34]. In this article, to solve the low electron transfer efficiency of a cerasome-modified electrode caused by the silicate surface and lipid bilayer of the cerasome, a ceraosomal nanocomposite with biocompatibility and better conductivity, Au@PIL-cerasome, was fabricated. The nanocomposite was utilized as a matrix for the immobilization of HRP, where HRP acted as a biological reporter of

the

direct

electron

transfer

on

a

glassy

carbon

electrode

(GCE).

The

HRP/Au@PIL-cerasome/GCE displayed amplified electrochemical signals and an improved electrocatalytic performance. This novel electrochemistry platform based on Au@PIL-cerasomes is a better way to develop biomimetic membrane electrodes or even a molecular communication system for information transduction.

3

2. Experimental 2.1. Materials 3-(Triethoxysilyl)propylisocyanate, hexadecyl bromide and hexadecylamine were obtained from Sigma-Aldrich (USA). Other chemicals and reagents, including horseradish peroxidase (HRP), chloroauric acid (HAuCl4) and ethyl bromide, were obtained from Sinopharm Chemical Reagent Co. Ltd (China). Double-distilled water was used for all aqueous solutions (18 MOhms·cm). All chemicals and reagents used in the experiments were of analytical grade and used without further purification. The concentration of phosphate-buffered saline (PBS) was 0.1 M and its pH was 8.0. The concentration of citrate buffer solution was 0.1 M and its pH was 5.0. 2.2. Preparation of cerasomes The cerasomes were prepared by the ethanol sol injection method[35]. First, the cerasomes-forming lipid, N,N-dicetyl-N'-[3-(triethoxysilyl)propyl] urea was synthesized from 3-(triethoxysilyl)propylisocyanate and dihexadecylamine and was absolutely dry, according to a previous method[18, 36]. The lipid, diluted hydrochloric acid and ethanol were mixed to form a solution. The molar ratio of lipid / EtOH / HCl / H2O was approximately 1: 200: 0.03: 19. The solution was incubated with vortex mixing for 12 h at 25 °C. The obtained sol (30 μL) was injected into H2O (5 mL) at 40 ◦C. The suspension was incubated at room temperature (25 °C) for 24 h to complete the formation of the siloxane network on the surface of the vesicles. Finally, the cerasomes were obtained, and the final concentration of lipid was 0.5 mM. 2.3. Preparation of Au@PIL Poly (1-vinyl-3-ethyl imidazolium) bromide (PIL), was synthesized as the reference[37]. HAuCl4 (1 mL 0.025 mmol) aqueous solution was added dropwise with vigorous stirring to PIL (0.25 mmol, calculated by monomer) in water (10 mL). To this reaction mixture, freshly prepared NaBH4 solution (1 mL 0.25 mmol) was slowly added dropwise at ambient temperature. The solution was stirred overnight; it changed to pale pink, which indicated the formation of gold nanoparticle (AuNPs). The resulting solution was centrifuged, and the sediment was collected and re-dispersed in water (50 mL). The gold nanoparticles coated by polymeric ionic liquid (Au@PIL) were thus obtained[32]. 2.4. Preparation of Au@PIL-cerasomes Au@PIL-cerasomes were fabricated by the self-assembly method. Cerasome suspension (1 mL 0.5 mM) was mixed with Au@PIL aqueous solution (1 mL) at room temperature. The mixture was stirred constantly for 24 h. The resulting material was centrifuged to remove excess free Au@PIL, and the final homogeneous Au@PIL-cerasome suspension was obtained. 4

2.5. Preparation of HRP/Au@PIL-cerasome/GCE Glassy carbon electrode (GCE) was polished using a polishing cloth with 1.0, 0.3, and 0.05 μm alumina powder and rinsed with double-distilled water, followed by sequential sonication in acetone, ethanol and double-distilled water. The electrode was then allowed to dry under nitrogen gas flow. Au@PIL-cerasome suspension (5 μL) was first deposited on the surface of the pretreated GCE. A beaker was placed over the electrode, ensuring the slow evaporation of the solvent and formation of a uniform film on the electrode at 4 °C. HRP PBS (5 μL 10 mg/mL pH 8.0) was then dripped onto the film, and the electrode was dried at 4 °C while covered with a beaker. HRP that was not absorbed was washed away with double-distilled water. After the water was volatilized at 4 °C, the modified electrode was further coated with 3 wt% polyvinyl alcohol (PVA) aqueous sol (7 μL) and then dried at 4 °C to obtain the modified electrode. As the comparison, HRP/Au@PIL/GCE was prepared with a similar procedure as described above. HRP/cerasome/GCE was prepared by dripping HRP PBS (5 μL 10 mg/mL pH 5.6) onto negatively charged cerasome/GCE. All the electrodes were stored at 4 °C when they were not in use. 2.6. Apparatus and measurements UV-vis experiments were performed using a Perkin Elmer Lambda 35 ultraviolet-visible spectrometer. Transmission electron micrographs (TEM) were recorded on a JEOL JEM-2100 electron microscope operated at an acceleration voltage of 200 kV. Scanning electron microscope (SEM) images were recorded on a Hitachi SU8010 field-emission scanning electron microscope operated at 10 kV without ion sputtering deposition. Semi-quantitative chemical analyses were performed through energy dispersion spectroscopy using an Apollo XL (EDAX Inc., USA) on a silicon wafer. Electrochemical measurements were carried out on a CHI660E electrochemical analyzer (CH Instruments, Shanghai, China) connected with a conventional three-electrode system: a GCE with a diameter of 3 mm and modified with nanomaterials was used as the working electrode, Ag/AgCl electrode was the reference electrode, and Pt wire was the counter electrode. A nitrogen atmosphere was maintained during the entire electrochemical measurements.

3. Results and Discussion 3.1. Preparation of Au@PIL-cerasomes The preparation of Au@PIL-cerasomes is depicted in Scheme 1A. The cerasomes with lipid bilayer structure were synthesized through a combination of self-assembly of the lipidic 5

organotrialkoxysilanes and a sol–gel reaction[18]. They exhibited much higher morphological stability than the conventional liposomes, attributed to the Si-O-Si framework on the surface. The surface charge value of cerasomes was -20.3 mV (Figure 1) due to the Si-OH on their surface. Au@PIL nanoparticles were synthesized by an in situ chemical reduction in the presence of HAuCl4 and PILs[32]. They showed good stability in aqueous solution (Figure S1). Their zeta potential was +69.1 mV in neutral aqueous solution due to the existence of PILs on the surface of the AuNPs (Figure 1). When the cerasomes and excess Au@PIL nanoparticles were simply mixed together, Au@PIL nanoparticles were assembled on the cerasome surface, forming Au@PIL-cerasome nanocomposites via self-assembly. After removing excess Au@PIL nanoparticles, the zeta potential of the Au@PIL-cerasomes was approximately +28.5 mV (Figure 1).

3.2. UV-Vis spectra and biocompatibility of Au@PIL-cerasomes UV-vis spectroscopy was used to monitor the formation of Au@PIL-cerasomes and their interaction with HRP. As shown in Figure 2, no obvious adsorption was present in the range of 300-800 nm for the cerasomes (a), and a peak at about 525 nm in curve b indicates the UV-vis absorption of Au@PIL (b). After Au@PIL was assembled on the cerasome surface, Au@PIL-cerasomes (c) exhibited a peak at approximately 525 nm, confirming the successful modification of Au@PIL on the surface of the cerasomes. UV-vis spectroscopy is an effective method to probe the structural change of enzymes. The Soret band of HRP is sensitive to the variation of the microenvironment around the heme group; this can provide information about the native structure of the enzyme, especially regarding possible denaturation or conformational changes[38]. The spectrum of free HRP solution (Figure 2d) had a strong absorbance at 403 nm, which is the Soret band of HRP[39]. HRP-Au@PIL-cerasomes (Figure 2e) exhibited a peak at 403 nm, which is at exactly the same wavelength as the Soret band of free HRP. The two UV-vis spectra confirmed that the native secondary structure of HRP had not changed. The Au@PIL-cerasomes had good biocompatibility, and the microenvironment provided by Au@PIL-cerasomes is suitable for supporting HRP.

3.3. Morphological characterization of Au@PIL-cerasomes Morphological characterization was employed to confirm the formation of Au@PIL-cerasomes. Figure 3a displays a TEM image of the cerasomes. The spherical cerasomes could be clearly observed after they were stained with 2 wt% phosphotungstic acid. The cerasomes that appeared on the grid were approximately 300 nm in size. Figure 3b shows a TEM image of Au@PIL-cerasomes without any staining. Because of the low electron density of cerasomes, their 6

morphology could not be well observed without stained, so only AuNPs are observed in the image. In Figure 3b, Au@PIL nanoparticles are displayed as a ring. The diameter of the ring is approximately 300 nm, which is consistent with the diameter of cerasomes, indicating that Au@PIL nanoparticles had been successfully absorbed onto the surface of the cerasomes. SEM was employed to directly explore the morphological structure information of Au@PIL-cerasomes on the electrode. As shown in Figure 3d, Au@PIL-cerasomes exhibited a spherical structure on the surface of the electrode, which is similar to the cerasomes (Figure 3c). EDS microanalysis (Figure 3e) confirmed the presence of strong peaks, indicating elemental gold (14.21 wt%), silicon (25.09 wt%), oxygen (32.88 wt%) and carbon (27.83 wt%).

3.4. Fabrication of HRP/Au@PIL-cerasome/GCE The fabrication of HRP/Au@PIL-cerasome/GCE is depicted in Scheme 1B. Au@PIL-cerasome nanoparticles were deposited on the surface of pretreated and polished GCE, making the surface charge of the electrode become positive. HRP is an amphoteric protein (the pH value at the isoelectric point is 7.0), which has a net negatively charged surface at pH values above its isoelectric point. When the HRP solution (pH=8.0) was dripped onto the modified electrode, the HRP became immobilized on the electrode surface via electrostatic self-assembly. Since the Au@PIL/GCE was unstable because of the solvation of Au@PIL in PBS, 3 wt% PVA aqueous sol was used to cover all the electrodes. 3.5.

EIS

spectroscopy

and

direct

electrochemical

properties

of

HRP/Au@PIL-cerasome/GCE The conductivity, as the greatest property metric of electrodes, was investigated with electrochemical impedance spectroscopy. The redox couples of Fe(CN)63−/4− were applied as the probes. The Nyquist plots of cerasome/GCE (a), Au@PIL/GCE (b), Au@PIL-cerasome/GCE (c) and HRP/Au@PIL-cerasome/GCE (d) are shown in Figure 4. A semicircle portion at higher frequencies and a linear portion at low frequencies were observed, indicating an electron transfer-limited process and a diffusion-controlled process, respectively. The charge transfer resistance (Ret) of an electrode can be obtained from the diameter of the semicircle. The curve of cerasome/GCE (a) exhibited a larger semicircle radius compared with the other electrodes, which showed that the cerasomes have the weakest electron transfer efficiency. In sharp contrast, the curves of Au@PIL/GCE (b) and Au@PIL-cerasome/GCE (c) exhibited much smaller semicircle radii, indicating that the Au@PIL-cerasome nanocomposites have an excellent conductivity and fast electron transfer efficiency on the electrode surface as Au@PIL nanoparticles. The modification of Au@PIL nanoparticles efficiently improved the conductivity of the individual cerasomes. The Au@PIL-cerasomes can provide a biocompatible microenvironment for HRP and also supply a good electron transfer pathway between HRP and the electrode, improving the 7

performance of the cerasome electrode. The marked increase of the semicircle diameter of HRP/Au@PIL-cerasome/GCE (d) indicates the successful immobilization of HRP on the electrode.

Figure 5 displays the cyclic voltammograms (CVs) for HRP/Au@PIL-cerasome/GCE (a), Au@PIL-cerasome/GCE (b), HRP/Au@PIL/GCE (c) and HRP/cerasome/GCE (d) in 0.1 M PBS (pH 8.0) over the potential range from 0 ~ -0.6 V at scan rate of 200 mV s-1. No redox peaks were observed for the Au@PIL-cerasome/GCE (b) in the absence of HRP; however, redox peaks were observed for HRP/Au@PIL-cerasome/GCE (a), HRP/Au@PIL/GCE (c) and HRP/cerasome/GCE (d). The formal potential (Ep) calculated from the average value of the cathodic and anodic peak potentials was -0.36 V, which is characteristic of the HRP heme FeIII/FeII redox couple[40-42], demonstrating that the immobilized HRP retained its electrochemical activity. Reversible redox reactions of heme groups inside HRP can be revealed from the 1:1 ratio of the oxidation and reduction peak current intensities of the electrodes. However, it is obvious that HRP/Au@PIL/GCE and HRP/cerasome/GCE exhibited smaller redox peaks compared with the HRP/Au@PIL-cerasome/GCE. Taking the intensity of the cathodic peak current as an example, the values of the peak currents of HRP/Au@PIL-cerasome/GCE, HRP/Au@PIL/GCE and HRP/cerasome/GCE were 0.20 μA, 0.08 μA and 0.04 μA, respectively. The weaker current responses of HRP/Au@PIL/GCE may have been due to a smaller amount of immobilized enzyme. The maximum differential value of the cathodic and anodic peak potentials and the minimum redox current values of HRP/cerasome/GCE result from the weak interaction between HRP and the cerasomes in a basic solution and the weak conductivity of the lipid bilayer membranes. These experiment results demonstrate that Au@PIL-cerasomes are much more efficient than Au@PIL and cerasomes in the direct electron transfer between immobilized HRP and the surface of the glassy carbon electrode. This ability of the Au@PIL-cerasomes is due to integrating the biocompatibility of cerasomes and the conductivity of Au@PIL. The kinetic parameters of HRP/Au@PIL-cerasome/GCE were investigated by scan rate experiments. CVs of the HRP/Au@PIL-cerasme/GCE at various scan rates and the plots of peak currents versus scan rate are shown in Figure 6. The reduction and oxidation peak currents increased linearly with increasing scan rate, as shown in the inset, which demonstrates that the redox process of HRP immobilized on Au@PIL-cerasome is a surface-confined redox process[24]. According to Faraday’s law, Q = nFAΓ*, where Q is the total amount of charge, n is the number of electrons transferred, F is Faraday's constant, and A is the electrode area, the average surface coverage concentration of electroactive HRP (Γ*) on HRP/Au@PIL-cerasome/GCE is calculated as 1.84×10-11 mol cm-2. This value is almost the same as the theoretical monolayer coverage value (~2 ×10-11 mol cm-2) of HRP on the bare glassy carbon electrode surface, suggesting that a monolayer of HRP participates in the direct electron transfer[43]. This result is also consistent 8

with our previous work[22]. By comparison, such value is higher than that of HRP/Au@PIL/GCE (4.01×10-12 mol cm-2) and HRP/cerasome/GCE (1.98×10-12 mol cm-2). The markedly improved portion of electroactive protein is probably due to two reasons: 1) the stereo structure of Au@PIL-cerasomes, which have a larger surface area on which to immobilize active HRP molecules, and 2) the good electron conductivity of Au@PIL-cerasomes due to the presence of Au@PIL.

3.6. Electrocatalytic properties of HRP/Au@PIL-cerasome/GCE To evaluate the effect of combining Au@PIL with cerasomes on signal transduction, HRP/Au@PIL-cerasome/GCE, HRP/Au@PIL/GCE and HRP/cerasome/GCE in different concentrations of H2O2 were investigated by cyclic voltammetry in N2-saturated 0.1 M PBS (pH 8.0) at a scan rate of 200 mV s-1. As shown in Figure 7A, when H2O2 was added to the electrolyte solution, the reduction peak currents of HRP/Au@PIL-cerasome/GCE increased markedly and the oxidation peak currents decreased, indicating a typical electrocatalytic reduction process of H2O2. The mechanism can be expressed as follows[22]: HRP (FeIII) + H2O2 → Compound I +H2O Compound I +H2O2 → HRP

(FeIII)

(1)

+ O2

(2)

HRP (FeIII) + e- → HRP (FeII)

(3)

HRP (FeII) + O2 → HRP (FeII)-O2

(4)

HRP (FeII)-O2 + e- + 2H+ → HRP (FeIII) +H2O2

(5)

It is known that enhanced faradic responses are very important in cyclic voltammetry investigations of interfacial electron transfer for redox proteins and are also highly desired for the establishment of sensitive biomimetic systems. From the calibration curves of the reduction peak currents versus H2O2 concentrations for the protein electrodes (Figure 7B), it can be observed that the

electrocatalytic

current

of

HRP/Au@PIL-cerasome/GCE

is

larger

than

that

of

HRP/Au@PIL/GCE and HRP/cerasome/GCE at each H2O2 concentration. The currents of HRP/Au@PIL-cerasome/GCE had a good linear relationship with the H2O2 concentration over the range of 10-70 μM. The detection limit for HRP/Au@PIL-cerasome/GCE was 3.3 μM based on S/N = 3, and its linear regression equation was y = 0.027 x + 0.021 (R = 0.9996, n = 7), where y is the peak current (μA) and x is the concentration (μM) of H2O2. The sensitivity value of the HRP/Au@PIL-cerasome/GCE was 0.39 μA cm-2 μM-1. In contrast, HRP/Au@PIL/GCE had a linear relationship with the H2O2 concentration over the range of 10-40 μM, which is much smaller than that of HRP/Au@PIL-cerasome/GCE. The linear regression equation of the HRP/Au@PIL/GCE was y = 0.021 x + 0.024 (R = 0.9916, n = 4), and the sensitivity value was only 0.30 μA cm-2 μM-1. HRP/cerasome/GCE had a linear relationship with the H2O2 concentration over the range of 10-60 μM. The linear regression equation was y = 0.016 x - 0.031 (R = 0.9922, n = 6), and the sensitivity value was 0.23 μA cm-2 μM-1. 9

The Michaelis–Menten model of enzyme kinetics assumes that the first step is a reversible formation of the complex between the enzyme and a reactant, the complex then irreversibly breaks up to give a product and the regenerated enzyme. For a film of immobilized enzyme on the electrode surface, the electrochemical version of the Michaelis–Menten equation becomes[44]: 𝐼𝑐𝑎𝑡 =

nFAΓ ∗ 𝑘𝑐𝑎𝑡𝑐𝑠

(6)

𝑐𝑠 + 𝐾𝑀

where 𝐼𝑐𝑎𝑡 is the catalytic peak current when the concentration of substrate is at 𝑐𝑠; 𝐾𝑀 is the Michaelis constant of the enzyme–substrate complex; and 𝑘𝑐𝑎𝑡 is the turnover rate constant. Eq. (6) can be linearized to Eq. (7) to estimation the apparent 𝐾𝑀 and 𝑘𝑐𝑎𝑡. 1 𝐼𝑐𝑎𝑡

=

𝐾𝑀

1 nFAΓ ∗ 𝑘𝑐𝑎𝑡

(7)

+ nFAΓ ∗ 𝑘

𝑐𝑎𝑡𝑐𝑠

The plot of 1/𝐼𝑐𝑎𝑡 versus 1/𝑐𝑠 provides K𝑀/(nFAΓ∗𝑘𝑐𝑎𝑡) as the slope and 1/(nFAΓ∗𝑘𝑐𝑎𝑡) as the intercept on the 1/𝐼𝑐𝑎𝑡 axis. Good linearity of 1/𝐼𝑐𝑎𝑡 versus 1/𝑐𝑠 was obtained for the system. The apparent K𝑀 value was then estimated to be 0.96 mM, and the turnover rate constant 𝑘𝑐𝑎𝑡 was 110 s−1. The 𝑘𝑐𝑎𝑡 value was smaller than that of free HRP (167 s-1) but was much larger than the values of HRP encapsulated in lithocholic acid nanotubes (52 s-1) [45], immobilized on carboxylated sphere-on-sphere silica microspheres (7 s-1)[46] or immobilized on TiO2 nanoparticle films (15 s-1)[44]. This is likely due to the synergistic effect of cerasomes and Au@PIL, which accelerates the direct electron transfer and improves the turnover rate of HRP. Since the catalytic reduction of nitrite also depends on the bioactivity of HRP[47, 48], another sensing

target,

nitrite,

was

HRP/Au@PIL-cerasome/GCE.

chosen Figure

to 8A

confirm shows

the the

electrocatalytic cyclic

signals

of

voltammograms

of

HRP/Au@PIL-cerasome/GCE with different concentrations of NaNO2 in 0.1 M citrate buffer (pH 5.0) at a scan rate of 200 mV s-1. Well-defined cathodic peaks for the reduction of NO2- were observed with the addition of NaNO2. The reduction product near -0.7 V was most likely N2O, and the mechanism of the electrocatalytic reduction of nitrite on the HRP-based electrode can be proposed as follows[48]: HRP (FeIII) + e- → HRP (FeII) (8) HRP (FeII) + HNO2 + H+ → [HRP (FeII)-NO]+ + H2O (9) [HRP (FeII)-NO]+ + 2e- → [HRP (FeII)-NO]- (10) [HRP (FeII)-NO]- + H+ → HRP (FeII) + HNO (11) 2HNO → N2O + H2O (12) The electrocatalytic cathodic currents were enhanced significantly by further increases of the NO2concentration in the buffer solution (Figure 8B), indicating that the HRP/Au@PIL-cerasomes could act as an effective catalyst toward the reduction of NO2-. Even when the concentration of NO2- was 1280 mM no current plateau appeared. A linear relationship between the reduction peak current (the observed peak at -0.75 V) and the NO2- concentration was obtained in the range from 1 mM to 5 mM, with a linear regression equation of y = 0.544 x + 1.89 (R=0.990, n=5), and from 10

5 mM to 1280 mM, with a linear regression equation of y = 0.131 x + 7.33 (R=0.998, n=22), where y is the peak current (μA) and x is the concentration (mM) of NO2-. The latter range is much wider than 2.5-325 mM, which was previously reported for HRP/cerasome/GCE[22]. The sensitivity values were 7.77 μA cm-2 mM-1 and 1.96 μA cm-2 mM-1, for the two ranges, respectively. The detection limit of HRP/Au@PIL-cerasome/GCE toward NO2- was 0.11 mM (S/N =3), which was lower than that for HRP/cerasome/GCE (0.83 mM).

Because there are few studies on constructing HRP-based electrodes with lipid bilayer membranes for the detection of H2O2 and NO2-, the performance of some representative HRP-based electrodes that use gold nanoparticles, magnetic nanoparticles, membrane structures, or silica materials are shown in Table S1 and Table S2 for comparison with HRP/Au@PIL-cerasome/GCE. From the tables it can be seen that the electrodes constructed with high conductivity materials, such as graphene, quantum dots and gold nanoparticles, have high sensitivity and low detection levels. Because of the non-conductive lipid bilayer, the electrochemical performance of the cerasome electrodes were generally not as good as the reported non-lipid bilayer membrane electrodes constructed

with

better

conductivity

materials,

as

expected.

However,

for

HRP/Au@PIL-cerasome/GCE, the poly(ionic liquid)s enhanced the interactions between HRP and the electrode material, ensuring that both electrostatic interactions and π-π interactions occurred between the enzyme and the electrode. Due to the multiple interactions, compared with the cerasome only-modified electrode, the Au@PIL-cerasome-modified electrode can be used at different pH values. Additionally, the gold nanoparticles improved the conductivity of the modified electrode. These synergistic effects enable the Au@PIL-cerasome-modified electrode to have a better electrochemical performance than that of the cerasome only-modified electrode in the same pH. These results indicate that modifications on the surface of cerasomes can improve their electrochemical performance as an electrode material. 3.7. Selectivity, stability, reproducibility and reliability of HRP/Au@PIL-cerasome/GCE To appraise the selectivity of the HRP/Au@PIL-cerasome/GCE, a variety of relevant interfering species in the biological system, including dopamine (DA), uric acid (UA) and ascorbic acid (AA), were investigated as control experiments (Figure S4). The as-prepared electrode showed no response to high concentrations of DA, UA and AA, indicating that the electrode has good selectivity. Figure S5 shows the CV currents of HRP/Au@PIL-cerasome/GCE with continuous scanning for 1000 cycles. During continuous CV measurement, two redox peaks were constantly observed, indicating that Au@PIL-cerasome nanocomposites could realize the direct electron transfer between HRP and the electrode from beginning to end. The cathodic peak currents of the electrode decreased by only 15.9%, demonstrating that the electrode has a good operational 11

stability depending on the favorable biocompatibility and stability of the Au@PIL-cerasomes[41]. Furthermore, after storage at 4°C for 2 weeks, 5% of the initial current response of HRP/Au@PIL-cerasome/GCE was lost. In addition, five HRP/Au@PIL-cerasome/GCEs prepared by the same procedure, independently, were employed to test the 20 μM H2O2. All of the electrodes exhibited similar amperometric responses, and a relative standard deviation (RSD) of 6% was obtained. Compared with other HRP biosensors[42, 49, 50], the above results demonstrate the acceptable reproducibility of HRP/Au@PIL-cerasome/GCE. The reliability of HRP/Au@PIL-cerasome/GCE was investigated to demonstrate its feasibility for unknown sample analysis. The values obtained by the electrochemical method were consistent with those obtained by conventional spectroscopy and titration methods (Table S3), indicating that this electrode might be reliable and effective in further applications.

4. Conclusions AuNPs were modified on the surface of cerasomes by using PILs as a stabilizer and coupling agent. The as-prepared Au@PIL-cerasome nanocomposites showed both good biocompatibility and excellent conductivity on glassy carbon electrode. Immobilized horseradish peroxidase on the stable 3D stereo lipid bilayer membrane exhibited an amplified electrical signal and electrocatalytic ability towards hydrogen peroxide and nitrite due to the synergistic effect of the cerasomes, gold nanoparticles and polymeric ionic liquid. This successful strategy suggests that such multi-component nanocomposites can be used as a novel and straightforward 3D stereo biomimetic membrane electrochemical platform to develop molecular communication systems and investigate the mechanisms of biological signal delivery and information transduction.

Acknowledgements The authors gratefully acknowledge the financial support of this study by the National Natural Science Foundation of China (No. 51773085), the Graduate Student Innovation Program of Liaoning University (No. x201810140198 and x201810140199), the Undergraduate Teaching Reform Projects of Liaoning University(JG2018).

References [1] A. Salek-Maghsoudi, F. Vakhshiteh, R. Torabi, S. Hassani, M.R. Ganjali, P. Norouzi, M. Hosseini, M. Abdollahi, Recent advances in biosensor technology in assessment of early diabetes biomarkers, Biosens. Bioelectron. 99 (2018) 122-135. [2] U. Jurva, L. Weidolf, Electrochemical generation of drug metabolites with applications in drug discovery and development, TrAC, Trends Anal. Chem. 70 (2015) 92-99. [3] A. Amine, H. Mohammadi, I. Bourais, G. Palleschi, Enzyme inhibition-based biosensors for food 12

safety and environmental monitoring, Biosens. Bioelectron. 21 (2006) 1405-1423. [4] W. Ma, Y.L. Ying, L.X. Qin, Z. Gu, H. Zhou, D.W. Li, T.C. Sutherland, H.Y. Chen, Y.T. Long, Investigating electron-transfer processes using a biomimetic hybrid bilayer membrane system, Nat. Protoc. 8 (2013) 439-450. [5] Y. Murakami, J. Kikuchi, Y. Hisaeda, O. Hayashida, Artificial enzymes, Chem. Rev. 96 (1996) 721-758. [6] Y. Wang, C.Y. Bi, A novel nitrite biosensor based on direct electron transfer of hemoglobin immobilized on a graphene oxide/Au nanoparticles/multiwalled carbon nanotubes nanocomposite film, RSC Adv. 4 (2014) 31573-31580. [7] S.J. Guo, D. Wen, Y.M. Zhai, S.J. Dong, E.K. Wang, Platinum nanoparticle ensemble-on-graphene hybrid nanosheet: One-pot, rapid synthesis, and used as new electrode material for electrochemical sensing, ACS Nano 4 (2010) 3959-3968. [8] A.G. Pakhomov, A.M. Bowman, B.L. Ibey, F.M. Andre, O.N. Pakhomova, K.H. Schoenbach, Lipid nanopores can form a stable, ion channel-like conduction pathway in cell membrane, Biochem. Biophys. Res. Commun. 385 (2009) 181-186. [9] W. Ma, D.W. Li, T.C. Sutherland, Y. Li, Y.T. Long, H.Y. Chen, Reversible redox of NADH and NAD+ at a hybrid lipid bilayer membrane using ubiquinone, J. Am. Chem. Soc. 133 (2011) 12366-12369. [10] T. Osaki, S. Takeuchi, Artificial cell membrane systems for biosensing applications, Anal. Chem. 89 (2017) 216-231. [11] D.P. Nikolelis, M.G. Tzanelis, U.J. Krull, The bilayer lipid membrane as a generic electrochemical transducer of hydrolytic enzyme reactions, Biosens. Bioelectron. 9 (1994) xxii-xxxvii. [12] Y.T. Zhao, D. Du, Y.H. Lin, Glucose encapsulating liposome for signal amplification for quantitative detection of biomarkers with glucometer readout, Biosens. Bioelectron. 72 (2015) 348-354. [13] M.A. Taylor, M.N. Jones, P.M. Vadgama, S.P.J. Higson, The characterization of liposomal glucose oxidase electrodes for the measurement of glucose, Biosens. Bioelectron. 10 (1995) 251-260. [14] J. Yu, H.N. Guan, D.F. Chi, An amperometric glucose oxidase biosensor based on liposome microreactor-chitosan nanocomposite-modified electrode for determination of trace mercury, J. Solid State Electrochem. 21 (2017) 1175-1183. [15] H.N. Guan, F.L. Zhang, J. Yu, D.F. Chi, The novel acetylcholinesterase biosensors based on liposome bioreactors–chitosan nanocomposite film for detection of organophosphates pesticides, Food Res. Int. 49 (2012) 15-21. [16] H.N. Guan, X.F. Liu, W. Wang, Encapsulation of tyrosinase within liposome bioreactors for developing an amperometric phenolic compounds biosensor, J. Solid State Electrochem. 17 (2013) 2887-2893. [17] J.S. Graça, R.F. de Oliveira, M.L. de Moraes, M. Ferreira, Amperometric glucose biosensor based on layer-by-layer films of microperoxidase-11 and liposome-encapsulated glucose oxidase, Bioelectrochemistry 96 (2014) 37-42. [18] K. Katagiri, K. Ariga, J. Kikuchi, Preparation of organic-inorganic hybrid vesicle "cerasome" derived from artificial lipid with alkoxysilyl head, Chem. Lett. (7) (1999) 661-662. [19] K. Katagiri, R. Hamasaki, K. Ariga, J. Kikuchi, Layered paving of vesicular nanoparticles formed 13

with cerasome as a bioinspired organic−inorganic hybrid, J. Am. Chem. Soc. 124 (2002) 7892-7893. [20] Y. Sasaki, K. Matsui, Y. Aoyama, J. Kikuchi, Cerasome as an infusible and cell-friendly gene carrier: Synthesis of cerasome-forming lipids and transfection using cerasome, Nat. Protoc. 1 (2006) 1227-1234. [21] Y. Qiao, K. Tahara, Q. Zhang, X.M. Song, Y. Hisaeda, J. Kikuchi, Construction of molecular communication interface formed from cerasome and hydrophobic vitamin B12 on glassy carbon electrode, Chem. Lett. 43 (2014) 684-686. [22] Y. Qiao, K. Tahara, Q. Zhang, X.M. Song, J. Kikuchi, Cerasomes: Soft interface for redox enzyme electrochemical signal transmission, Chem. Eur. J. 22 (2016) 1340-1348. [23] S.Y. Wu, J.L. Chen, D.L. Liu, Q. Zhuang, Q. Pei, L.X. Xia, Q. Zhang, J. Kikuchi, Y. Hisaeda, X.M. Song, A biocompatible cerasome based platform for direct electrochemistry of cholesterol oxidase and cholesterol sensing, RSC Adv. 6 (2016) 70781-70790. [24] D.L. Liu, Q. Zhuang, L. Zhang, H. Zhang, S.Y. Wu, J. Kikuchi, Z.B. Han, Q. Zhang, X.M. Song, Self-assembly of novel fluorescent quantum dot-cerasome hybrid for bioelectrochemistry, Talanta 154 (2016) 31–37. [25] Y. Liu, M.K. Wang, F. Zhao, Z.A. Xu, S.J. Dong, The direct electron transfer of glucose oxidase and glucose biosensor based on carbon nanotubes/chitosan matrix, Biosens. Bioelectron. 21 (2005) 984-988. [26] L. Murphy, Biosensors and bioelectrochemistry, Curr. Opin. Chem. Biol. 10 (2006) 177-184. [27] H. Song, Y. Ni, S. Kokot, Investigations of an electrochemical platform based on the layered MoS2-graphene and horseradish peroxidase nanocomposite for direct electrochemistry and electrocatalysis, Biosens. Bioelectron. 56 (2014) 137-143. [28] E.E. Connor, J. Mwamuka, A. Gole, C.J. Murphy, M.D. Wyatt, Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity, Small 1 (2005) 325-327. [29] C.S. Shan, H.F. Yang, D.X. Han, Q.X. Zhang, A. Ivaska, L. Niu, Graphene/AuNPs/chitosan nanocomposites film for glucose biosensing, Biosens. Bioelectron. 25 (2010) 1070-1074. [30] K.J. Huang, Y.J. Liu, H.B. Wang, Y.Y. Wang, Y.M. Liu, Sub-femtomolar DNA detection based on layered molybdenum disulfide/multi-walled carbon nanotube composites, Au nanoparticle and enzyme multiple signal amplification, Biosens. Bioelectron. 55 (2014) 195-202. [31] K. Saha, S.S. Agasti, C. Kim, X. Li, V.M. Rotello, Gold nanoparticles in chemical and biological sensing, Chem. Rev. 112 (2012) 2739-2779. [32] K.T.P. Charan, N. Pothanagandhi, K. Vijayakrishna, A. Sivaramakrishna, D. Mecerreyes, B. Sreedhar, Poly(ionic liquids) as “smart” stabilizers for metal nanoparticles, Eur. Polym. J. 60 (2014) 114-122. [33] S. Lee, M.D. Cummins, G.A. Willing, M.A. Firestone, Conductivity of ionic liquid-derived polymers with internal gold nanoparticle conduits, J. Mater. Chem. 19 (2009) 8092-8101. [34] C.S. Shan, H.F. Yang, J.F. Song, D.X. Han, A. Ivaska, L. Niu, Direct electrochemistry of glucose oxidase and biosensing for glucose based on graphene, Anal. Chem. 81 (2013) 2378-2382. [35] K. Katagiri, R. Hamasaki, K. Ariga, J. Kikuchi, Preparation and surface modification of novel vesicular nano-particle "cerasome" with liposomal bilayer and silicate surface, J. Sol-Gel Sci. Technol. 26 (2003) 393-396. [36] K. Ariga, K. Katagiri, J. Kikuchi, Preparation condition of a novel organic-inorganic hybrid vesicle "cerasome", Kobunshi Ronbunshu 57 (2000) 251-253. 14

[37] R. Marcilla, J.A. Blazquez, J. Rodriguez, J.A. Pomposo, D. Mecerreyes, Tuning the solubility of polymerized ionic liquids by simple anion‐exchange reactions, J. Polym. Sci. Pol. Chem. 42 (2004) 208-212. [38] E.A. Theorell H, Olsen E., Spectrophotometric, magnetic and titrimetric studies on the heme-linked groups in myoglobin, Acta Chem. Scand. 5 (1951) 823-848. [39] S. Mao, Y. Long, W. Li, Y. Tu, A. Deng, Core–shell structured Ag@C for direct electrochemistry and hydrogen peroxide biosensor applications, Biosens. Bioelectron. 48 (2013) 258-262. [40] X.S. Yang, X. Chen, L. Yang, W.S. Yang, Direct electrochemistry and electrocatalysis of horseradish peroxidase in α-zirconium phosphate nanosheet film, Bioelectrochemistry 74 (2008) 90-95. [41] M.G. Li, S.D. Xu, M. Tang, L. Liu, F. Gao, Y.L. Wang, Direct electrochemistry of horseradish peroxidase on graphene-modified electrode for electrocatalytic reduction towards H2O2, Electrochim. Acta 56 (2011) 1144-1149. [42] G.M. Bai, X. Xu, Q.M. Dai, Q.Q. Zheng, Y.W. Yao, S.Q. Liu, C. Yao, An electrochemical enzymatic nanoreactor based on dendritic mesoporous silica nanoparticles for living cell H2O2 detection, Analyst 144(2) (2019) 481-487. [43] X.M. Sun, Y. Zhang, H.B. Shen, N.Q. Jia, Direct electrochemistry and electrocatalysis of horseradish peroxidase based on halloysite nanotubes/chitosan nanocomposite film, Electrochim. Acta 56 (2010) 700-705. [44] Y. Zhang, P.L. He, N.F. Hu, Horseradish peroxidase immobilized in TiO2 nanoparticle films on pyrolytic graphite electrodes: Direct electrochemistry and bioelectrocatalysis, Electrochim. Acta 49 (2004) 1981-1988. [45] Q. Lu, Y.C. Kim, N. Bassim, N. Raman, G.E. Collins, Catalytic activity and thermal stability of horseradish peroxidase encapsulated in self-assembled organic nanotubes, Analyst 141 (2016) 2191-2198. [46] Z. Lei, X. Liu, L.N. Ma, D.J. Liu, H.F. Zhang, Z.X. Wang, Spheres-on-sphere silica microspheres as matrix for horseradish peroxidase immobilization and detection of hydrogen peroxide, RSC Adv. 5 (2015) 38665-38672. [47] H. Liu, C.Y. Duan, C.H. Yang, W.Q. Shen, F. Wang, Z.F. Zhu, A novel nitrite biosensor based on the direct electrochemistry of hemoglobin immobilized on Mxene-Ti3C2, Sensors Actuators B: Chem. 218 (2015) 60-66. [48] H. Liu, K. Guo, J. Lv, Y. Gao, C.Y. Duan, L. Deng, Z.F. Zhu, A novel nitrite biosensor based on the direct electrochemistry of horseradish peroxidase immobilized on porous Co3O4 nanosheets and reduced graphene oxide composite modified electrode, Sensors Actuators B: Chem. 238 (2017) 249-256. [49] J. Jia, B. Wang, A. Wu, G. Cheng, Z. Li, S. Dong, A method to construct a third-generation horseradish peroxidase biosensor:  Self-assembling gold nanoparticles to three-dimensional sol−gel network, Anal. Chem. 74 (2002) 2217-2223. [50] Q. Xu, C. Mao, N.N. Liu, J.J. Zhu, J. Sheng, Direct electrochemistry of horseradish peroxidase based on biocompatible carboxymethyl chitosan–gold nanoparticle nanocomposite, Biosens. Bioelectron. 22 (2006) 768-773.

15

Fig. 1. Zeta potentials of cerasomes (A), Au@PIL (B) and Au@PIL-cerasomes (C). Fig. 2. UV-vis absorption spectra of cerasomes (a), Au@PIL (b), Au@PIL-cerasomes (c), HRP (d) and HRP-Au@PIL-cerasomes (e). Fig. 3. TEM images of negatively stained cerasomes (a) and Au@PIL-cerasomes (b). SEM images of cerasomes (c) and Au@PIL-cerasomes (d). EDS spectrum of Au@PIL-cerasomes (d). Fig. 4. Electrochemical impedance spectra of different modified electrodes in a 5 mM [Fe(CN)6]3−/4− and 0.5 M KCl mixed solution, with frequencies ranging from 10−1 to 105 Hz. Open circuit potentials: 0.24 V. Electrodes: cerasome/GCE (a), Au@PIL/GCE (b), Au@PIL-cerasome/GCE (c) and HRP/Au@PIL-cerasome/GCE (d). Figure 5. Cyclic voltammograms of HRP/Au@PIL-cerasome/GCE (a), Au@PIL-cerasome/GCE (b) HRP/Au@PIL/GCE (c) and HRP/cerasome/GCE (d) in 0.1 M PBS, pH 8.0. Scan rate, 200 mV s-1. Fig. 6. Cyclic voltammograms of the HRP/Au@PIL-cerasome/GCE in 0.1 M PBS (pH 8.0) at different scan rates (from a to j): 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 mV s-1. Plots of peak current versus scan rate for HRP/Au@PIL-cerasome/GCE (inset). Fig. 7. (A) Cyclic voltammograms of HRP/Au@PIL-cerasome/GCE with 0 μM (a), 20 μM (b), 40 μM (c), 60 μM (d), 80 μM (e), 100 μM (f), 120 μM (g) H2O2 in 0.1 M PBS (pH 8.0) at a scan rate of 200 mV s-1. Inset: Cyclic voltammograms of HRP/Au@PIL/GCE (i) and HRP/cerasome/GCE (ii) (B) Plots of the electrocatalytic currents versus H2O2 concentration for HRP/Au@PIL-cerasome/GCE (a), HRP/Au@PIL/GCE (b) and HRP/cerasome/GCE (c). Fig. 8. (A) Cyclic voltammograms of HRP/Au@PIL-cerasome/GCE in 0.1 M citrate buffer (pH 5.0) with 0 mM (a), 5 mM (b), 25 mM (c), 75 mM (d), 170 mM (e), 450 mM (f), 800 mM (g), and 1280 mM (h) NaNO2 at a scan rate of 200 mV s-1. (B) Plots of the electrocatalytic currents versus NaNO2 concentration for HRP/Au@PIL-cerasome/GCE. Inset: a partial enlargement detail in 0-5 mM nitrite. Scheme 1. Process of fabricating Au@PIL-cerasomes (A) and HRP/Au@PIL-cerasome/GCE (B)

16