Electrochemical Surface Plasmon Resonance Fiber-Optic Sensor: In

Subscriber access provided by UCL Library Services

Article

Electrochemical surface Plasmon resonance fiberoptic sensor: in-situ detection of electroactive biofilms Yong Yuan, Tuan Guo, Xuhui Qiu, Jiahuan Tang, Yunyun Huang, Li Zhuang, Shungui Zhou, Zhaohui Li, BaiOu Guan, Xuming Zhang, and Jacques Albert Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01314 • Publication Date (Web): 23 May 2016 Downloaded from http://pubs.acs.org on May 29, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 10

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

Analytical Chemistry

Electrochemical surface Plasmon resonance fiber-optic sensor: in-situ detection of electroactive biofilms Yong Yuan a, Tuan Guo b,*, Xuhui Qiu b, Jiahuan Tang a, Yunyun Huang b, Li Zhuang a, Shungui Zhou a,*, Zhaohui Li b, Bai-Ou Guan b, Xuming Zhang c, Jacques Albert d Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650, China b Guangdong Key Laboratory of Optical Fiber Sensing and Communications, Institute of Photonics Technology, Jinan University, Guangzhou 510632, China c Department of Applied Physics, Hong Kong Polytechnic University, Hong Kong, People’s Republic of China d Department of Electronics, Carleton University, Ottawa K1S5B6, Canada a

Abstract—Spectroelectrochemistry has been found to be an efficient technique for revealing extracellular electron transfer (EET) mechanism of electroactive biofilms (EABs). Herein, we propose a novel electrochemical surface Plasmon resonance (EC-SPR) optical fiber sensor for insitu monitoring EABs. The sensor uses a tilted fiber Bragg grating (TFBG) imprinted in a commercial single mode fiber and coated with nanoscale gold film for high efficiency SPR excitation. The wavelength shift of the SPR over the fiber surface clearly identifies the electrochemical activity of the surface localized (adjacent to the electrode interface) bacterial cells in EABs, which differs from the “bulk” detections of the conventional electrochemical measurements. A close relationship between the variations of redox state of the EABs and the changes of the SPR under potentiostatic conditions has been achieved, pointing a new way to study the EET mechanism of the EABs. Benefiting from its compact size, high sensitivity, ease of use together with remote operation ability, the proposed sensor opens up a multitude of opportunities for monitoring EABs in various hard-to-reach environments. @ 2015 Elsevier B.V. All rights reserved. Keywords—Spectroelectrochemistry; surface Plasmon resonance; optical fiber sensor; electron transfer; electroactive biofilm

1.

Introduction 1

applications for in-situ monitoring of various hard-to-reach environments such as field wastewater, soils and sediments. Surface Plasmon resonance (SPR) spectroscopies are desirable techniques for in-situ measuring of electronic states of the chemical and biological molecules. When integrated with electrochemistry (EC), electrochemical surface Plasmon resonance (EC-SPR) offers a promising way to simultaneously explore optical and electrochemical properties of chemical and biological matrix accompanying redox reactions. For example, Baba et al. coupled EC with SPR to probe the electrochromic and redox properties of conductive polymer thin films.15 Yao et al. quantified the orientation changes of ferrocenyl alkanethiol self-assembled monolayers during electrochemical oxidation using an EC-SPR sensor.16 Boussaad et al. used EC-SPR to survey conformational and electronic changes of purified c-Cyts protein.17 However, the EC-SPR was constructed with a bulky prism configuration, and has never been applied to measure the optical and electrochemical properties of a living biofilm accompanied by redox transformation in-situ. Herein, we report the first application of EC-SPR for in-situ measuring of the electrochemical activity of EABs. The electrochemical surface Plasmon resonance optical fiber sensor (ECSPR-OFS) proposed here consists of a gold-coated tilted fiber Bragg grating (TFBG) imprinted in a commercial single mode fiber core, in which the nanometric-scale gold-coating over the fiber surface offers the simultaneous detection of both the electrochemical information (electrochemical current) and the optical information (Plasmon wave). Such sensor is a cost- established free-space optics (i.e. bulky prism configurations) for high sensitivity biochemical detections due to the high efficiency light-matter interaction.18 Moreover, its tiny size makes it feasible to be inserted into various hard-to-reach environments for in-situ detection (avoiding the necessary of bringing bio-samples inside an instrument) either as a hand-held probe or as a set of remotely operated devices along a fiber-optic cable, especially for environmental monitoring over the urban and

Electroactive biofilms (EABs) have received considerable attention due to their electrochemical connections with the substrate, which can be employed for harvesting energy from organic waste and converting waste into value-added chemicals.1-3 The electrochemical properties of biofilms primarily contribute to the presence of some specific strains (i.e. Shewanella and Geobacter) that are able to exchange electrons with solid substrate.4,5 So far, two main mechanisms have been distinguished for the electron transfer between EABs and the substrata, including direct electron transfer (DET) via bacterial outer-membrane redox proteins and indirect electron transfer via redox metabolites.6 Nevertheless, present understanding of the specific interactions between electrochemically active microbes and the solid electron acceptors is far from complete. In the case of the DET, available data suggest that surfacelocalized redox proteins in the EABs, such as outer membrane (OM) c-type cytochromes (OM c-Cyts), multicopper proteins and pilin proteins, are responsible for the electron transfer,7-10 but the identity of the redox proteins wiring EABs to electrodes is still challenging. Spectroelectrochemical techniques have been proven to be useful in revealing the microbial electron-transfer process by correlating voltammetry data with spectral characteristics of the EABs. Various spectral techniques such as UV-visible, Raman, and infrared adsorption spectroscopy could be linked with electrochemical analysis to monitor the redox status of redox-active proteins in EABs, from which valuable information about the extracellular electron transfer (EET) process can be obtained.11-14 However, the bulky optics that are necessary for these spectroscopic techniques limit their 1

*Corresponding author. Institute of Photonics Technology, Jinan University, Guangzhou 510632, China. Tel.: +86 20 85221606; fax: +86 20 85222046. E-mail address: [email protected] (T. Guo). *Corresponding author. Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650, China. Tel.: +86 20 37300951; fax: +86 20 87025872. E-mail address: [email protected] (S. Zhou).

1

ACS Paragon Plus Environment


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

suburban areas.19 By using such a hybrid technique (electrochemical and plasmonic techniques), the different spectral responses of redox species of the EABs, in real time and in-situ, have been achieved, providing a prospect for the use of this technique for monitoring the redox electrochemistry of the EABs.

Page 2 of 10

Bragg grating (TFBG) imprinted in the core of a commercial single mode fiber provided with a nanometric gold coating. TFBGs benefit from two unique features: one is the strong polarization selectivity of the excited cladding modes and other is the high-density comb of narrowband spectral resonances.20,21 When a linearly polarized light is launched with its polarization parallel to the tilt plane of grating (Ppolarization, Fig. 1), the electric field of the cladding modes is nearly 100% polarized in the radial direction, i.e. in the TM polarization relative to the metal coating, so as to ensure the strongest SPR excitation. The whole plasmonic fiber-optic sensing probe is very compact, with a size of 20 mm in length and 0.125 mm in diameter (the left inset of Fig. 1). Measurements of the Plasmon wave properties by the transmission spectrum of the TFBG provide the insitu optical information for EET of EABs. The Plasmon waves of the gold-coated TFBG are hybrid waves that have intense field localization at the outer surface of the metal film (as the red waves shown in the right inset of Fig. 1).

2. Materials and methods 2.1 Sensor configuration and system The EC-SPR-OFS proposed in this work is composed of a compact plasmonic fiber-optic sensing system together with a bioelectrochemical system. The plasmonic optical fiber sensing probe performs the optical detection function (Fig. 1) and it also works as an electrode in the electrochemical system (Fig. 2). The right inset of Fig. 1 presents the detailed configuration of the plasmonic fiber-optic sensing probe, which consists of a tilted fiber

Computer

Electrochemical Workstation

Counter

Working

Fiber probe

OFF ON

BBS

Reference

Polarizer

PC

Fiber

OSA

Electrolyte

Bioelectrochemical cell

Optic fiber sensor a b

SPR

Polarized light

Bacterial cell

Biocurrent

P-polar

125 um

S-polar

Core

TFBG

Cladding

Gold film

Fig. 1. Schematic of plasmonic fiber-optic sensing system (together with the electrochemistry measurement apparatus) for in-situ biofilm monitoring. The left inset (a) shows photographs of the gold-coated optic fiber sensor; the right inset (b) presents the zoomed configuration of a gold-coated TFBG sensor probe with polarization of light oriented for SPR excitation.

a

b

Reference

Gold-coated optical fiber

Counter Gold-coated optical fiber

Fig. 2. (a) Photographic image of the bioelectrochemical cell with EC-SPR-OFS; (b) the zoomed-in image of gold-coated optical fiber used as the working electrode.

2


Page 3 of 10

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


2.2 Bacterial culture

increase the photosensitivity of the fiber core. Secondly, a high energy pulsed excimer laser together with the phase mask technique were used for grating inscription. In detail, a pulsed 193 nm UV light was focused with a cylinder lens and scanned over the fiber axial region through a diffractive phase mask, which splits the incoming light beam into two and thereby producing a sinusoidal intensity distribution along the fiber axis. Finally, induce a proper tilting angle by rotating the phase mask and fiber consistently (phase mask and fiber are kept parallel) around an axis perpendicular to the laser beam. It should be noted that the tilt of the grating is an important parameter that can be used to choose which set of cladding modes is going to be excited. As a result, it is feasible to adjust the operating range of the sensor in order to optimize the response for certain refractive indices. Here, a grating tilt of 20 o was chosen to maximize the sensing response in aqueous solutions with refractive indices around 1.32-1.34. Thin noble metal sheaths (gold used here) have been successfully deposited on the optical fiber by sputtering technology. To achieve a high quality coating, two issues must be addressed. One is to improve the adhesion of the gold to the surface of fiber glass. Here, we used a 2-3 nm thickness of chromium sandwiched between the fiber surface and the gold film. The second is to ensure a uniform thickness of the gold film over the whole fiber surface (this is very important to the SPR excitation). Traditional flat-plane sputtering technique is not suitable for cylindrical fiber because sputtering is a directional process and the target is fixed. Therefore, in order to avoid thickness nonuniformities, the fiber was rotated along its axis during the sputtering process. This design ensures a very uniform nanometric coating over fiber surface. The current work was carried out with a gold thickness around 50 nm.

Geobacter sulfurreducens strain PCA (ATCC51573) was subcultured using a standard anaerobic medium at 30 oC. This anaerobic medium contains (per 1 L): 0.2 g NH4Cl, 0.38 g KCl, 0.069 g NaH2PO4·H2O, 0.1 g MgSO4·7H2O, 0.04 g CaCl2·2H2O, and 10 mL of a mineral solution. Acetate was used as an electron donor at 1.0 g/L. The medium was adjusted to pH 6.8 prior to addition of 1.8 g/L NaHCO3, and purged with N2/CO2 (80/20, v/v). G. sulfurreducens mutants were cultured under the same conditions as the wild-type strain. Shewanella oneidensis MR-1 was cultured and inoculated to the electrochemical cell as previously reported. 22 A mixed culture obtained from a well maintained bioelectrochemical system was inoculated to the electrochemical cell to form a mixed culture EAB. E. coli was also cultured in 50 mL of LB broth with gentle shaking until the optical density (OD600) reached 2.0. E. coli culture (10 mL) was inoculated to the electrochemical cell with 50 mM phosphate buffer solution and 10 mM glucose to form a biofilm of E. coli. 2.3 Electrochemical measurements Once bacterial cells were injected into reactors, the working electrodes were set at 0 V vs. SCE using a multi-channel potentiostat (CHI1000C, China). Three parallel reactors were constructed to evaluate the reproducibility of the developed EC-SPR-OFS. Chronoamperometry (CA) and cyclic voltammetric (CV) measurements were performed with the gold-coated optical fiber as the working electrode (Surface area: 0.4 cm 2). All of the measurements were conducted in triplicate, and the average values are reported. The CVs were scanned under both non-turnover and turnover conditions with a scan rate of 5 mV/s. A fit of the Nernst equation for the redox equilibrium state of the cytochrome proteins was conducted as previously described. 13 For a two-electron redox reaction, the redox equilibrium state equation is as follows:

A C     Xi  0.5 n1F / RT ( E E f ,1 )  B   0.5 n2F / RT ( E  E f , 2 )  D  e  e 

3.2 SPR excitation and spectral characteristics The Plasmon waves provide the in-situ information for EET of EABs. They are a hybrid waves that have intense field localization at the outer surface of a metal film deposited on optical fiber support. When the refractive index of the outer medium is smaller than that of the solid support, a propagating wave incident from the support side can be totally internally reflected at the support metal boundary but phase-matched one transfer to a quasi-Plasmon excitation at the outer boundary of the metal. In order for this coupling to occur, the incident wave must be radial at the fiber external boundary, i. e. Ppolarization, to transfer energy to a Plasmon wave on the metal surface. Meanwhile, the thickness of the metal layer must be thin enough (50 nm thickness for the optimal thickness for SPR excitation) to let some light tunnel across. Evidence of SPR in the spectrum for this polarization is the disappearance of the cladding mode resonances (as the clear attenuation near 1520 nm of the red transmission shown in Fig. 3) due to the coupling of the cladding mode energy to the very lossy surface Plasmon wave at these wavelengths. When the other direction of the input polarized light is used, i.e. S-polarization, the electric field is tangent to fiber surface and no SPR occurs (as the black transmission shown in Fig. 3).

(1)

Where E1 and E2 are the formal potentials of the cytochrome proteins, respectively. n1 and n2 are the number of electron exchanged in the reaction, respectively. Xi is the reduced or oxidized fraction of the cytochrome proteins. A and B are the maximum difference intensity between the fully oxidized and the fully reduced state, respectively. B and D are the offset, respectively. R and F represent the gas and the Faraday constant, respectively, and T and E are the reaction temperature and the electrode potential, respectively.

3.

Plasmonic TFBG sensor

3.1 TFBG fabrication and nano-metallic coating The TFBG (about 15 mm in length) was manufactured in a commercial single-mode fiber.19 The fabrication process mainly includes three steps. Firstly, the fiber was hydrogen-loaded so as to

3



a T ra n s m is s ion (dB )

P polarization

SPR

0

Radial electric fields

-1 0 -20 -30

P polarization

-40

1 440

b

1 460

1 480 1 500 1 520 Wavelength (nm)

1 540

1 560

S polarization

T ra n s m is s ion (dB )

0

Tangential electric fields

-1 0 -20 -30

S polarization

-40 1 440

1 460

1 480

1 500

1 520

1 540

Wavelength (nm)

1 560

Fig. 3. Left: Transmission spectra of a TFBG coated with 50 nm of gold and immersed in water, for P-polarized incident light ( a) and S-polarized incident light (b). The SPR location corresponds to the spectral notch near 1520 nm in the upper graph. Right: Simulated electric mode field profiles for two neighboring high order cladding modes. P-polarization resonances have electric fields that are predominantly radial at the boundary (top). S-polarization resonances have predominantly tangential electric fields around the fiber cladding boundary (bottom). Therefore, P-polarization resonances can transfer energy to a Plasmon wave on the metal surface (bright fringe around the fiber) but S-polarization resonances cannot.

c

b

a

100 μm

100 μm e

Abiotic fiber

6

omc S omc B E S TZ

3

f

Abiotic fiber

1 .2

2 1

4

om c S om c B E S TZ

2

Abiotic fiber Wild type

Wild type

Wild type

4

1 μm

C u rre n t /  A

5

C u rre n t /  A

d C u rre n t /  A

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 4 of 10

0.8

omc S omc B E S T Z

0.4 0.0

0 0 0

2

4 6 Time (d)

8

10

-0.6

-0.4

-0.2

0.0

-0.4

P otential / V vs . S C E

-0.6

-0.4

-0.2

0.0

Potential / V vs . S C E

Fig. 4. SEM images of the gold-coated optical fibers: (a) abiotic fiber; (b) optical fiber with G. sulfurreducens biofilm; (c) high resolution image of the G. sulfurredences biofilm. Electrochemical measurements of the EABs: (d) I-t curves of the bioelectrochemical systems with various inoculations; (e) CV scans of the EABs on the optical fibers under turnover conditions; (f) CV scans of the EABs on the optical fibers under non-turnover conditions.

the working electrode (Surface area: 0.4 cm 2). Scanning electron microscope (SEM) images confirmed the formation of the biofilm on the optical fiber. As shown in Fig. 4a, a smooth optical fiber surface was observed before inoculation. After 10 days of culturing, microbial cells forming a biofilm were clearly observed on the optical fiber (Fig. 4b and 4c). During the biofilm formation, the electrochemical current was also recorded, which is shown in Fig. 4d. After a lag phase, an

4. Results and discussion

4.1 Biofilm formation and characterization Prior to EC-SPR-OFS measurements, initially well cultured G. sulfurreducens cells were inoculated into the bioelectrochemical cell by polarizing the optical fiber working electrode at 0 V vs. SCE to form a biofilm on the gold-coated optical fiber electrode using a multi-channel potentiostat (CHI1000C, China). CA and CV measurements were performed with the gold-coated optical fiber as

4


Page 5 of 10

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


electrochemical current was observed from the biofilm formed from wild-type G. sulfurreducens. In contrast, almost no current was obtained from the abiotic control. For comparison, mutant strains were also inoculated into the bioelectrochemical cell, which showed a small current generation from the strain with the omcS gene deleted (ΔomcS) and almost no current from the mutant strain with omcB, omcS, omcT, omcE, and omcZ genes deleted (ΔomcBESTZ). Previous studies have demonstrated that omcB, omcS and omcZ are the key redox proteins to relay electron transfer from inside of G. sulfurreducens to outside substrata.23 CVs under turnover and nonturnover conditions further confirmed the electrochemical activity of the EABs with a scan rate of 5 mV/s. As shown in Fig. 4e, sigmoidal CVs were observed within the biofilms resulted from wild-type and omcS deletion mutant in the presence of acetate, indicating the oxidation of acetate by these biofilms. In contrary, almost no catalytic currents were observed from the biofilms resulting from the ΔomcBESTZ mutant and the abiotic optical fiber. Figure 4f presents the CVs of the biofilms under the acetate depleted condition. Redox couples with formal potentials of -0.41 and -0.49 V vs. SCE (-0.17 and -0.25 V vs. Standard hydrogen electrode, SHE) appeared in the CV of the biofilm with the wild-type strain. These redox peaks were assigned to omcB (outer membrane c-type cytochrome B) and omcZ (outer membrane c-type cytorchrome Z), respectively.24 However, only one couple of redox peaks was observed from the biofilm with the omcS deletion mutant, and almost no redox peaks were observed from the biofilm with the ΔomcBESTZ. This confirmed that the presence of OM c-Cyts is important for the EET of the G. sulfurreducens biofilm.

plasmonic resonance in subsequent measurements is enabled by referencing all wavelengths to the location of the core mode reflection resonance (Fig. 5c) which is completely insensitive to the external environment of the fiber. Furthermore, since all resonance wavelengths have the same response to temperature shifts, the aforementioned referencing of wavelengths to the core mode resonance removes any influence of temperature on the measurements. Monitoring of the power level of the transmission spectrum near the core resonance also ensures that light power level fluctuation (from the light source and anywhere else in the optical path) and be identified and eliminated from the measured data, thus maximizing the signal to noise ratio, the stability and the reliability of the proposed sensor. 4.3 SPR responses of the redox properties of the EABs under potentiostatic control To further observe the SPR responses of the redox process in the EABs, detailed changes in the SPR amplitude were measured during the EET. EC-SPR-OFS experiments were performed employing potential jumps from positive potentials (0 V) to the negative potentials (-0.8 V). Figure 6a shows the current response of various biofilms on the potential jumps from 0 and -0.8 V, which showed the decrease in the current generation of the EABs. A current of 4.6 μA was obtained from the wild-type G. sulfurreducens biofilm at the potential of 0 V, which was the highest value among all G. sulfurreducens strains. According to previous studies, the EET ability of the EABs was assigned to the activity of surface-localized redoxactive proteins or bulk redox compounds, such OM c-Cyts, multicopper proteins, pilin proteins and endogenous mediators excreted by bacteria.27-29 It is well known that the electron states of the redox species were associated with the applied potentials at the working electrodes.30,31 When an anodic potential was applied to the EABs, the redox species in the EABs were in their oxidation states. On the contrary, the redox species in the EABs were in their reduction states when a cathodic potential was applied to the EABs. Associated with the potential jumps, we found that the differential SPR amplitude obtained by subtracting the SPR amplitude between the measured potentials and 0 V was correspondingly varied. Notably, using the differential SPR amplitude (instead of absolute amplitude) can eliminate the effect of the bacterial biomass and cell viability on the measurements of the redox activity among different optic fibers and EABs. This approach can also effectively cancel out any unwanted fluctuations originating from the light source or transmission path. As presented in Fig. 6b, the differential SPR amplitudes were inversely related with the current generation. Note that differential SPR amplitude of the EABs with G. sulfurreducens mutants and other biofilms at the potential of -0.8 V (i.e. S. oneidensis MR-1, E. coli, and a mixed culture) was smaller compared to the wild-type G. sulfurreducens biofilm (Table 1), which can identify the lower electroactivities of biofilms. Based on these experimental results, the maximum current density (Imax) of the EABs varied linearly with the measured differential SPR amplitude (ΔSPR) with a linear relationship of [ΔSPR] = 6.97+ 0.74 × [Imax], R2 = 0.998, as shown in the right inset figure in Table 1.

4.2 Optical response of the EABs Accompanying the electrochemical measurements, the optical spectral responses of the plasmonic fiber-optic sensor are recorded. As shown in Fig. 5a, the transmission spectra of the EAB-attached plasmonic fiber-optic sensor (inscribed with a TFBG) presents a fine comb of narrowband resonances that overlaps the spectral region where the Plasmon wave absorbs some of the light (this spectral region is colored orange). These fine spectral resonances provide a unique tool to measure small shifts of the Plasmon with high accuracy: when the Plasmon wave spectral maximum moves in response to changes in the immediate vicinity of the metal surface, the amplitudes of the individual resonances of the TFBG either grow or decrease depending whether the Plasmon peak (SPR) loss gets closer or further. With the variation of the redox state of the EAB (0, -0.4 V and -0.8 V vs. SCE), strong SPR wavelength shifts have been observed and their response varies in accordance with the applied potentials. The most accurate measure of the SPR wavelength shift is obtained by monitoring the amplitude change of a select resonance located on the short wavelength side of the SPR maximum (this resonance is identified by a red asterisk "*" and labeled "plasmonic" in Fig. 5b). The amplitude of this resonance increases when the SPR loss moves towards longer wavelengths in response to surrounding refractive index or applied potential increases.25,26 Here in our experiment, the amplitude of the “SPR” resonance increases about 15 dB when the applied potential (at the gold-coated fiber working electrode) changes from 0 V to -0.8 V. The identification of the

5


a

Page 6 of 10

SPR shift

0 -1 0 -20

1 440

Amplitude change (∆RI≠0 )

*

0 V -0.4 V -0.8 V

-30 -40

1 460

1 480

1 500

Core fixed (∆T=0 )

1 520

1 540

Wa vele n gth (nm )

c

0

Tran s m is s io n (d B )

T ra n s m is s io n (dB )

b

-1 0

-20

0V -0.4 V -0.8 V

*

Plasmonic ∆RI resonance

-30 1 490

1 492

1 494

0

-1 0

-20

0V -0.4 V -0.8 V

-30

Core resonance

1 547 1 548 1 549 1 550 1 551 Wavelength (nm)

1 496

Wavelength (nm )

Fig. 5. Optical spectral responses of EAB measurement: (a) spectral response of gold-coated 20 o TFBG with SPR excitations under different electrode polarizations; (b) zoomed-in spectral change of SPR modulated cladding modes (note: the “plasmonic” cladding mode is marked by a red asterisk “*”); (c) zoomed-in spectrum near the core mode reference resonance.

a

b

5

C u rre n t /  A

3 2 1 0 -0.8

-0.4

-0.2

0.0

12 8 4 0

-0.8

-0.6

R educ ed

Oxidized

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

d

1 .0

-0.8

-0.6 -0.4 -0.2 P otential / V vs . S C E

0.0

D e riva tive d (dB ) / d E

1 .0 0.8

-0.4

-0.2

0.0


Potential / V vs . S C E

O xid ize d fra c tio n

c

-0.6

Abiotic fiber Wild-Type omc S omc B E S TZ

16

D iffe re n tia l in te n s ity (d B )


4

R e du c e d fra c tio n

0 -1 0 -20 -30

-50 -60

0.0


-40

-0.8

-0.6

-0.4

-0.2

0.0


Fig. 6. (a) Current-potential curves of the EABs under turnover conditions; (b) potential-dependent background-subtracted SPR intensity of the EABs; (c) fraction of C-cyts in the biofilm as a function of the potential, as determined by EC-SPR-OFS spectroscopy (the dash lines indicate the ﬁt to a model); (d) the 1st derivative of the SPR intensity curve over the potential. Table 1 Relationship between the maximum current density (Imax) and the corresponding differential SPR amplitudes (ΔSPR) of various EABs (subtraction of the SPR amplitudes between the applied potential of -0.8 and 0 V) is presented both in the table (left) and plot (not including the non-EABs, ΔomcBESTZ and E. coli).

Biofilms G. sulfurredences (wild-type) G. sulfurredences (ΔomcS) G. sulfurredences (ΔomcBESTZ) S. oneidensis MR-1 E. coli A mixed culture

Imax (μA/cm2) 11.4 ± 0.5 2.6 ± 0.4 0.9 ± 0.2 5.9 ± 0.4 0.5± 0.3 8.9 ± 0.7

Differential SPR amplitude (dB) 15.5± 1.0 9.0± 0.4 1.4± 0.2 11.2 ± 0.7 1.2± 0.3 13.6 ± 0.8

D iffe re n tia l SaPm Rp litu d e (d B )

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

T ra n s m is s io n (d B )


6


18

 S PR =6.97+0.74Im ax 15

R 2=0.998

12

9

6

2

4

6 8 2 Im ax (A/c m )

10

12

Page 7 of 10

b

5

a

C u re rre n t /  A

4 3

b

2 Medium reple nis hment

1

c 0

5

10

15

20

Time / d

c

a b c

12

8

4

-0.8

-0.6

-0.4

2 1

-0.8

-0.2

0.0


-0.6

-0.4

-0.2

0.0


d

16

0

3

0

25

D iffe re n tia l S P R a m p litu d e (d B )

0

a b c

5 4

C u rre n t /  A

a

D iffe re n tia l S P R a m p litu d e (d B )

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


16

Day 26 Day 45 Day 87

12

8

4

0

-0.8

-0.6

-0.4

-0.2

0.0


Fig. 7. (a) I-t curves of the bioelectrochemical systems with G. sulfurreduences for a long-term operation; (b) current-potential curves of the G. sulfurreduences biofilm obtained at point a, b, and c in the panel (a), respectively; (c) potential-dependent background-subtracted SPR amplitude of the biofilm at point a, b, and c in the panel (a), respectively; (d) potential-dependent background-subtracted SPR amplitude of the biofilm under non-turnover condition at different time intervals, showing the remarkable stability of the system for a long-term operation.

It should be noted that the biocurrent flowing on the fibers may potentially influence the SPR signals. To demonstrate the relationship between the biocurrent flowing and the SPR signal of the EABs, we measured the SPR of the same mature EABs under both turnover and non-turnover conditions. As shown in Fig. 7a, ECSPR measurements were performed at three time points of the same EAB. Current generation properties of the EAB were recorded at these three time points, which showed great variations (Fig. 7b). The generated currents of 3.7, 2.4 and 0.1 μA were achieved at point a, b and c, respectively, which represented different redox state of the EAB. However, the changes in the differential SPR amplitudes were almost identical (Fig. 7c). This means that the variations in the differential SPR amplitudes were associated with the redox activities of the biofilms rather than the current flowing through the gold coating over the fiber surface. Note that almost identical SPR responses were achieved from the biofilm over a long time span, suggesting the remarkable stability of the constructed EC-SPR-OFS for monitoring the EABs (Fig. 7d). According to previous studies, the SPR angular shift from the immobilized redox metalloprotein in the EC-SPR system is mainly caused by the conformation changes of the redox protein (c-Cyts) in the EABs. These are in turn induced by the changes in the valent state of iron in heme during the redox process. 17,32 In addition, the SPR angular shift can result from the refractive index difference between the reduced and oxidized forms of the bulk redox species.33,34 Thus, the EC-SPR-OFS mainly probes the presence and redox states of the redox species in the EABs, which can be further used to indirectly predict the EET ability of the EAB. As shown in Fig. 6c, the contribution of the reduced components increased with the decrease of the electrode potential, and vice versa, which was obtained by normalizing the differential SPR amplitude by the differential SPR amplitude at -0.8 V (assumed a completely reduced state), respectively. The resultant potential-dependent changes of the redox states of the EABs were then analyzed in terms of the Nernst equation.11 A fit of the Nernst equation for independent redox couples to the data in Fig. 6c afforded a two-electron transfer

process with apparent formal potentials of -0.15 V and -0.25 V vs. SHE for the EAB with the wild-type strain, which are in very good agreement with the CV results. The electron transfer from G. sulfurreducens to solid electron acceptors is accomplished by a wide variety of OM c-Cyts with broad potential windows, such as omcS (from – 0.36 to – 0.04 V vs. SHE), and OmcZ (from – 0.42 to – 0.06 V vs. SHE).35 Interestingly, the broad shape of the redox potential window of the EAB with the wild-type strain is observed on the firstdeviation curve of the differential SPR amplitude curve over the potential (Fig. 6d), whereas a narrow shape of redox potential window was obtained from the EAB with the ΔomcS mutant, and no signals were observed from the EAB with the ΔomcBESTZ mutant. It is worth mentioning that the EET process of G. sulfurreducens biofilms was complicated and has not been fully understood yet. In addition to OM c-Cyts, Okamoto et al. revealed that efficient bacterial EET involved OM c-Cyts-bound flavins that function as a redox cofactor in the scaffold of OM c-Cyts.36 The formal potential of OM c-Cyts-bound flavins was observed at -0.20 V vs. SHE, which should also be included in the redox potential window of the EAB with the wild-type strain in Fig. 6d. On the other hand, other surfacelocalized redox proteins that are also involved in the EET, such as multicopper proteins and pilin proteins, might also contribute to SPR signals. Shewanella biofilms are also well studied EABs, which showed a similar SPR response as the Geobacter biofilms in the ECSPR-OFS system (Table 1). It has been revealed that OM c-Cyts (OmcA and MtrC), protein-bound and bulk endogenous mediators excreted by bacteria contribute to the EET of Shewanella biofilms.37,38 Therefore, the SPR signal of this biofilm can be assigned to surfacelocalized redox proteins and bound or bulk endogenous mediators. The EABs commonly vary in thickness from a few micrometers to several millimeters, which involves complex long-distance electron transfer.39,40 Two layer bacterial aggregations have been identified in the thick EABs, including a live outer-layer (nearer the solution interface) and a dead inner-layer of biofilm (nearer the electrode interface).41 The live outer-layer of bioﬁlm has been considered to be responsible for current production and the dead inner-layer has

7



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

been suggested to function as an electrically conductive matrix. However, investigations are still insufficient to provide insight into the biofilm structure relative to its electrochemical properties. To date, electrochemical techniques have been primarily applied to study the EET process in EABs, yielding important insights into their redox process.30,42 However, electrochemical techniques cannot provide structural and spatial information because they only perform a "bulk" detection in which the current or change information originates from the whole electrode surface.43 The EC-SPR method proposed here provides a very "localized" and "surface" detection of the inner-layer of biofilm (adjacent to the electrode interface) because the SPR signals are mainly confined to about 1 μm spatial thickness above the gold film surface. To be more specific, in this case, the Plasmon waves mainly propagate in the inner-layer of multilayered biofilm. In this regard, the EC-SPR-OFS measurements revealed that the inner-layer of the EAB was electrochemically active during the EET, which is consistent with previous surveys.41 In addition, as discussed earlier, the combinations of UV-vis, Raman and FTIR spectroscopies with electrochemistry offer good opportunities for revealing the structure information the EABs. However, free-space configurations of these conventional spectroscopies required the measurements to be mainly conducted in laboratories. The inherent advantages of the proposed fiber-optic sensors include their low cost, light weight, small size, ruggedness and remote operation ability, making them possible to fully integrate with various in-field environments such as soil, sediments, and wastewaters, especially for those hard to reach places.

Page 8 of 10

Financial support was provided by the National Natural Science Foundation of China (Nos. 21277035, 61225023 and 51403077), the Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2014A030306033), the Guangdong Youth Science and Technology Innovation Talents of China (No. 2014TQ01X539), the Guangdong Natural Science Foundation of China (No. 2014A030313387), the Guangdong Innovation Foundation of China (No. 2015KTSCX016), the Guangzhou Key Collaborative Innovation Foundation of China (No. 201504290942056) and the Fundamental Research Funds for Central Universities of China (No. 21615446). J. Albert acknowledges the support of the Natural Sciences and Engineering Research Council of Canada (No. RGPIN 2014-05612) and the Canada Research Chairs Program (No. 950-217783). Prof. D. R. Lovley at the University of Massachusetts is acknowledged for his kind provision of Geobacter sulfurreducens mutants.

References [1] [2] [3] [4] [5]

5. Conclusions

[6]

In summary, we have demonstrated the first EC-SPR optical absorption spectrum of EABs during metabolism. By implementing such hybrid technique (electrochemical and plasmonic methods) over one compact optical fiber sensor, further understanding on how electron transfer occurs in EABs, in real time, has been achieved. This powerful technique associated with mutant analysis, reveals the important role of the redox species in the EET as well as the redox states of these redox species during the metabolism. Benefiting from the advantages of compact size, cost-effectiveness, simple implementation and the possibility of remote operation, the proposed EC-SPR-OFS is an appealing solution for observing the electrochemical activity of biofilms developed in various natural environments.

[7]

[8] [9] [10] [11]

Acknowledgments

[12] [13] [14] [15] [16] [17] [18]

Biffinger, J. C.; Pietron, J.; Ray, R.; Little, B.; Ringeisen, B. R. Biosens. Bioelectron. 2007, 22, 1672–1679. Logan, B. E.; Hamelers, B.; Rozendal, R.; Schröder, U.; Keller, J.; Freguiac, S.; Verstraete, W.; Rabaey, K. Environ. Sci. Technol. 2006, 40, 5181–5192. Marshall, C. W.; Ross, D. E.; Fichot, E. B.; Norman, R. S.; May, H. D. Environ. Sci. Technol. 2013, 47, 6023–6029. Bond, D. R.; Lovley, D. R. Appl. Environ. Microbiol. 2003, 69, 1548–1555. Yong, Y. C.; Yu, Y. Y.; Zhang, X. H.; Song, H. Angew. Chem. Int. Ed. 2014, 53, 4480–4483. Borole, A. P.; Reguera, G.; Ringeisen, B.; Wang, Z. W.; Feng, Y. J.; Kim, B. H. Energy Environ. Sci. 2011, 4, 4813–4834. Reardon, C. L.; Dohnalkova, A. C.; Nachimuthu, P.; Kennedy, D. W.; Saffarini, D. A.; Arey, B. W.; Shi, L.; Wang, Z.; Moore, D.; McLean, J. S.; Moyles, D.; Marshall, M. J.; Zachara, J. M.; Fredrickson, J. K.; Beliaev, A. S. Geobiol. 2010, 8, 56–58. Xiong, Y.; Shi, L.; Chen, B.; Mayer, M. U.; Lower, B. H.; Londer, Y.; Bose, S.; Hochella, M. F.; Fredrickson, J. K.; Squier, T. C. J. Am. Chem. Soc. 2006, 128, 139781–3979. Holmes, D. E., Mester, T.; O’Neil, R. A.; Perpetua, L. A., Larrahondo, M. J.; Glaven, R.; Sharma, M. L.; Ward, J. E.; Nevin, K. P.; Lovley, D. R. Microbiol. 2008, 154, 1422–1435. Filzgerald, L. A.; Petersen, E. R.; Ray, R. I.; Little, B. J.; Cooper, C. J.; Howard, E. C.; Ringeisen, B. R.; Biffinger, J. C. Process Biochem. 2012, 47, 170–174. Nakamura, R.; Ishii, K.; Hashimoto, K. Angew. Chem. Int. Ed. 2009, 48, 1606–1609. Liu, Y.; Kim, H.; Franklin, R. R.; Bond, D. R. Chem. Phys. Chem. 2011, 12, 2235–2241. Millo, D.; Harnisch, F.; Patil, S. A.; Ly, H. K.; Schröder, U.; Hildebrandt, P. Angew. Chem. Int. Ed. 2011, 50, 2625 –2627. Busalmen, J.; Esteve-Núñez, A.; Berná, A.; Feliu, J. M. Angew. Chem. Int. Ed. 2008, 47, 4874–4877. Baba, A.; Taranekar, P.; Ponnapati, R. R.; Knoll, W.; Advincula, R. C. ACS Appl. Mater. Interfaces 2010, 2, 2347–2354. Yao, X.; Wang, J.; Zhou, F.; Wang, J.; Tao, N. J. J. Phys. Chem. B 2004, 108, 7206–7212. Boussaad, S.; Pean, J.; Tao, N. J. Anal. Chem. 2000, 72, 222– 226. Leung, A.; Shankar, P. M.; Mutharasan, R. Sens. Actuators B: Chem 2007, 123, 688–703.

8


Page 9 of 10

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


[19] Albert, J.; Shao, L. Y.; Caucheteur, C. Laser Photon. Rev. 2013, 7, 83–108. [20] Albert, J.; Lepinay, S.; Caucheteur, C.; DeRosa, M. C. Methods, 2013, 63, 239–254. [21] Caucheteur, C.; Guo, T.; Albert, J. Anal. Bioanal. Chem. 2015, 407, 3883–3897. [22] Zhou, S.; Tang, J.; Yuan, Y. Bioelectrochem. 2015, 102, 29 –34. [23] Voordeckers, J. W.; Kim, B. C.; Izallalen, M.; Lovley, D. R. Appl. Environ. Microbiol. 2010, 76, 2371–2375. [24] Liu, Y.; Harnisch, F.; Fricke, K.; Sietmann, R.; Schröder, U. Biosens. Bioelectron. 2008, 24, 1012–1017. [25] Caucheteur, C.; Voisin, V.; Albert, J. Opt. Express 2013, 21, 3055–3066. [26] Guo, T.; Liu F.; Liang, X.; Qiu, X. H.; Huang Y. Y.; Xie C.; Xu,P.; Mao, W.; Guan, B. O.; Albert, J. Biosens. Bioelectron. 2016, 78, 221–228. [27] Virdis, B.; Millo, D.; Donose, B. C.; Batstone, D. J. PLoS ONE 2014, 9, e89918. [28] Zhou, S.; Wen, J.; Chen, J.; Lu. Q. Environ. Sci. Technol. Lett. 2015, 2, 26–30. [29] Marsili, E.; Baron, D. B.; Shikhare, I. D.; Coursolle, D.; Gralnick, J. A.; Bond, D. R. Proc. Natl. Acad. Sci. USA 2008, 105, 3968– 3973. [30] Fricke, K.; Harnisch, F.; Schröder, U. Energy Environ. Sci. 2008, 1, 144–147. [31] Jain, A.; Gazzola, G.; Panzera, A.; Zanoni, M.; Marshili, E. Electrochim. Acta 2011, 56, 10776–10785. [32] Zhai, P.; Guo, J.; Xiang, J.; Zhou, F. J. Phys. Chem. C Nanomater.

Interfaces 2007, 11, 981–986. [33] Wang, S P.; Huang, X. P.; Shan, X. N.; Foley, K. J.; Tao, N. J. Anal. Chem. 2010, 82, 935–941. [34] Huang, X. P.; Wang, S. P.; Shan, X. N.; Chang, X. J.; Tao, N. J. J. Electroanal. Chem. 2010, 649, 37–41. [35] Qian, X.; Mester, T.; Morgado, L.; Arakawa, T.; Sharma, M. L.; Inoue, K.; Joseph, C.; Salgueiro, C. A.; Maroney, M. J.; Lovley, D. R. Biochim. Biophys. Acta 2011, 1807, 404–412. [36] Okamoto, A.; Saito, K.; Inoue, K.; Nealson, K. H.; Hashimoto, K.; Nakamura, R. Energy Environ. Sci. 2014, 7, 1357–1361. [37] Shi, L.; Richardson, D. J.; Wang, Z.; Kerisit, S. N.; Rosso, K. M.; Zachara, J. M.; Fredrickson, J. K. Environ. Microbiol. Rep. 2009, 1, 220-227. [38] Okamoto, A.; Hashimoto, K.; Nealson, K. H.; Nakamura, R. Proc. Natl. Acad. Sci. USA 2013, 110, 7856–7861. [39] Strycharz-Glaven, S. M.; Snider, R. M.; Guiseppi-Elie, A.; Tender, L. M. Energy Environ. Sci. 2011, 4, 4366−4379. [40] Malvankar, N. S.; Vargas, M.; Nevin, K. P.; Franks, A. E.; Leang, C.; Kim, B. C.; Inoue, K.; Mester, T.; Covalla, S. F.; Johnson, J. P.; Rotello, V. M.; Tuominen, M. T.; Lovley, D. R. Nat. Nanotechnol. 2011, 6, 573−579. [41] Sun, D.; Cheng, S.; Wang, A.; Li, F.; Logan, B. E.; Cen, K. Environ. Sci. Technol. 2015, 49, 5227−5235. [42] Zhao, F.; Slade, R. C. T.; Varcoe, J. R. Chem. Soc. Rev. 2009, 38, 1926-1939. [43] Wang, W.; Foley, K.; Shan, X.; Wang, S.; Eaton, S.; Nagaraj, V. J.; Wikto, P.; Patel, U.; Tao, N. J. Nat. Chem. 2011, 3, 249-255.

9



Table of Contents

SPR Biocurrent

Optical fiber

Electrochemical SPR optical fiber sensor

Table 1 (editable) Biofilms

a m plitu de (dB )

G. sulfurredences (wild-type) G. sulfurredences (ΔomcS) G. sulfurredences (ΔomcBESTZ) S. oneidensis MR-1 E. coli A mixed culture

D iffe re n tia l S P R

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 10 of 10

Imax (μA/cm2) 11.4 ± 0.5 2.6 ± 0.4 0.9 ± 0.2 5.9 ± 0.4 0.5± 0.3 8.9 ± 0.7

Differential SPR amplitude (dB) 15.5± 1.0 9.0± 0.4 1.4± 0.2 11.2 ± 0.7 1.2± 0.3 13.6 ± 0.8

18

S PR =6.97+0.74Imax 15

R 2 =0.998

12

9

6

2

4

6 8 2 Imax ( A/c m )

10

12

10


Electrochemical Surface Plasmon Resonance Fiber-Optic Sensor: In

Recommend Documents