Electrochemical Surface Plasmon Resonance Fiber-Optic Sensor: InSitu Detection of Electroactive BiofilmsYong Yuan,† Tuan Guo,*,‡ Xuhui Qiu,‡ Jiahuan Tang,† Yunyun Huang,‡ Li Zhuang,† Shungui Zhou,*,†

Zhaohui Li,‡ Bai-Ou Guan,‡ Xuming Zhang,§ and Jacques Albert∥

†Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmentaland Soil Sciences, Guangzhou 510650, China‡Guangdong Key Laboratory of Optical Fiber Sensing and Communications, Institute of Photonics Technology, Jinan University,Guangzhou 510632, China§Department of Applied Physics, Hong Kong Polytechnic University, Hong Kong, People’s Republic of China∥Department of Electronics, Carleton University, Ottawa K1S5B6, Canada

ABSTRACT: Spectroelectrochemistry has been found to be anefficient technique for revealing extracellular electron transfer(EET) mechanism of electroactive biofilms (EABs). Herein, wepropose a novel electrochemical surface plasmon resonance(EC-SPR) optical fiber sensor for monitoring EABs in situ. Thesensor uses a tilted fiber Bragg grating (TFBG) imprinted in acommercial single-mode fiber and coated with nanoscale goldfilm for high-efficiency SPR excitation. The wavelength shift ofthe surface plasmon resonance (SPR) over the fiber surfaceclearly identifies the electrochemical activity of the surfacelocalized (adjacent to the electrode interface) bacterial cells inEABs, which differs from the “bulk” detections of theconventional electrochemical measurements. A close relation-ship between the variations of redox state of the EABs and thechanges of the SPR under potentiostatic conditions has been achieved, pointing to a new way to study the EET mechanism of theEABs. Benefiting from its compact size, high sensitivity, and ease of use, together with remote operation ability, the proposedsensor opens up a multitude of opportunities for monitoring EABs in various hard-to-reach environments.

Electroactive biofilms (EABs) have received considerableattention, because of their electrochemical connections

with the substrate, which can be employed for harvestingenergy from organic waste and converting waste into value-added chemicals.1−3 The electrochemical properties of biofilmsprimarily contribute to the presence of some specific strains(i.e., Shewanella and Geobacter) that are able to exchangeelectrons with a solid substrate.4,5 So far, two main mechanismshave been distinguished for the electron transfer between EABsand the substrata, including direct electron transfer (DET) viabacterial outer-membrane redox proteins and indirect electrontransfer via redox metabolites.6 Nevertheless, present under-standing of the specific interactions between electrochemicallyactive microbes and the solid electron acceptors is far fromcomplete. In the case of the DET, available data suggest thatsurface-localized redox proteins in the EABs, such as outermembrane (OM) c-type cytochromes (OM c-Cyts), multi-copper proteins, and pilin proteins, are responsible for theelectron transfer,7−10 but the identity of the redox proteinswiring EABs to electrodes is still challenging.Spectroelectrochemical techniques have been proven to be

useful in revealing the microbial electron-transfer process bycorrelating voltammetry data with spectral characteristics of the

EABs. Various spectral techniques such as ultraviolet−visiblelight (UV-vis), Raman, and infrared adsorption spectroscopycould be linked with electrochemical analysis to monitor theredox status of redox-active proteins in EABs, from whichvaluable information about the extracellular electron transfer(EET) process can be obtained.11−14 However, the bulky opticsthat are necessary for these spectroscopic techniques limit theirapplications for in situ monitoring of various hard-to-reachenvironments such as field wastewater, soils, and sediments.Surface plasmon resonance (SPR) spectroscopies are

desirable techniques for in situ measurement of electronicstates of the chemical and biological molecules. Whenintegrated with electrochemistry (EC), electrochemical surfaceplasmon resonance (EC-SPR) offers a promising way tosimultaneously explore optical and electrochemical propertiesof chemical and biological matrix accompanying redoxreactions. For example, Baba et al. coupled EC with SPR toprobe the electrochromic and redox properties of conductive

Received: April 5, 2016Accepted: May 23, 2016Published: May 23, 2016

Article

pubs.acs.org/ac

© 2016 American Chemical Society 7609 DOI: 10.1021/acs.analchem.6b01314Anal. Chem. 2016, 88, 7609−7616

polymer thin films.15 Yao et al. quantified the orientationchanges of ferrocenyl alkanethiol self-assembled monolayersduring electrochemical oxidation using an EC-SPR sensor.16

Boussaad et al. used EC-SPR to survey conformational andelectronic changes of purified c-Cyts protein.17 However, theEC-SPR system was constructed with a bulky prismconfiguration, and it has never been applied to measure theoptical and electrochemical properties of a living biofilmaccompanied by redox transformation in situ.Herein, we report the first application of EC-SPR for in situ

measuring of the electrochemical activity of EABs. Theelectrochemical surface plasmon resonance optical fiber sensor(EC-SPR-OFS) proposed here consists of a gold-coated tiltedfiber Bragg grating (TFBG) imprinted in a commercial single-mode fiber core, in which the nanometric-scale gold coatingover the fiber surface offers the simultaneous detection of boththe electrochemical information (electrochemical current) andthe optical information (plasmon wave). Such a 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 sizemakes it feasible to be inserted into various hard-to-reachenvironments for in situ detection (avoiding the need to bringbiosamples inside an instrument) either as a hand-held probe oras a set of remotely operated devices along a fiber-optic cable,especially for environmental monitoring over the urban andsuburban areas.19 By using such a hybrid technique (electro-chemical and plasmonic techniques), the different spectralresponses of redox species of the EABs, in real time and in situ,have been achieved, providing a prospect for the use of thistechnique for monitoring the redox electrochemistry of theEABs.

■ MATERIALS AND METHODSSensor Configuration and System. The EC-SPR-OFS

proposed in this work is composed of a compact plasmonicfiber-optic sensing system combined with a bioelectrochemicalsystem. The plasmonic optical fiber sensing probe performs the

Figure 1. Schematic of plasmonic fiber-optic sensing system (together with the electrochemistry measurement apparatus) for in situ biofilmmonitoring. The left inset (a) shows photographs of the gold-coated optic fiber sensor; the right inset (b) presents an enlargement of thebioelectrochemical cell, showing the configuration of a gold-coated TFBG sensor probe with polarization of light oriented for SPR excitation.

Figure 2. (a) Photographic image of the bioelectrochemical cell with EC-SPR-OFS; (b) enlarged image of the gold-coated optical fiber used as theworking electrode.

Analytical Chemistry Article

DOI: 10.1021/acs.analchem.6b01314Anal. Chem. 2016, 88, 7609−7616

7610

optical detection function (Figure 1) and it also works as anelectrode in the electrochemical system (Figure 2). The rightinset of Figure 1 presents the detailed configuration of theplasmonic fiber-optic sensing probe, which consists of a tiltedfiber Bragg grating (TFBG) imprinted in the core of acommercial single-mode fiber provided with a nanometric goldcoating. TFBGs benefit from two unique features: one is thestrong polarization selectivity of the excited cladding modesand other is the high-density comb of narrowband spectralresonances.20,21 When a linearly polarized light is launched withits polarization parallel to the tilt plane of grating (P-polarization, Figure 1), the electric field of the claddingmodes is ∼100% polarized in the radial direction (i.e., in theTM polarization relative to the metal coating), to ensure thestrongest SPR excitation. The entire plasmonic fiber-opticsensing probe is very compact, with a size of 20 mm in lengthand 0.125 mm in diameter (the left inset of Figure 1).Measurements of the plasmon wave properties by thetransmission spectrum of the TFBG provide the in situ opticalinformation for EET of EABs. The plasmon waves of the gold-coated TFBG are hybrid waves that have intense fieldlocalization at the outer surface of the metal film (representedby the red waves shown in the right inset of Figure 1).Bacterial Culture. Geobacter sulfurreducens strain PCA

(ATCC51573) was subcultured using a standard anaerobicmedium at 30 °C. This anaerobic medium contains (per 1 L):0.2 g of NH4Cl, 0.38 g of KCl, 0.069 g of NaH2PO4·H2O, 0.1 gof MgSO4·7H2O, 0.04 g of CaCl2·2H2O, and 10 mL of amineral solution. Acetate was used as an electron donor at aconcentration of 1.0 g/L. The medium was adjusted to pH 6.8prior to the addition of 1.8 g/L NaHCO3, and purged with N2/CO2 (80/20, v/v). G. sulfurreducens mutants were culturedunder the same conditions as the wild-type strain. Shewanellaoneidensis MR-1 was cultured and inoculated to the electro-chemical cell, as previously reported.22 A mixed cultureobtained from a well-maintained bioelectrochemical systemwas inoculated to the electrochemical cell to form a mixedculture EAB. E. coli was also cultured in 50 mL of LB broth withgentle shaking refractive indices. Here, a grating tilt of 20° waschosen to maximize until the optical density (OD600) reached2.0. E. coli culture (10 mL) was inoculated to the electro-chemical cell with 50 mM phosphate buffer solution and 10mM glucose to form a biofilm of E. coli.Electrochemical Measurements. Once bacterial cells

were injected into reactors, the working electrodes were setat 0 V vs SCE using a multichannel potentiostat (ModelCHI1000C, China). Three parallel reactors were constructed toevaluate the reproducibility of the developed EC-SPR-OFS.Chronoamperometry (CA) and cyclic voltammetry (CV)measurements were performed with the gold-coated opticalfiber as the working electrode (surface area = 0.4 cm2). All ofthe measurements were conducted in triplicate, and the averagevalues are reported. The CV measurements were obtainedunder both nonturnover and turnover conditions with a scanrate of 5 mV/s. A fit of the Nernst equation for the redoxequilibrium state of the cytochrome proteins was conducted aspreviously described.13 For a two-electron redox reaction, theredox equilibrium state equation is as follows:

=−

+

+−

+

⎧⎨⎪⎩⎪ ⎡⎣ ⎤⎦

⎫⎬⎪⎭⎪

⎧⎨⎪⎩⎪ ⎡⎣ ⎤⎦

⎫⎬⎪⎭⎪

( )

( )

XA

n E EB

C

n E ED

0.5exp ( )

0.5exp ( )

i FRT f

FRT f

1 ,1

2 ,2 (1)

where E1 and E2 are the formal potentials of the cytochromeproteins, respectively; n1 and n2 are the number of electronexchanged in the reaction, respectively; Xi is the reduced oroxidized fraction of the cytochrome proteins; A and B are themaximum difference intensity between the fully oxidized andthe fully reduced state, respectively; B and D are the respectiveoffsets; R is the gas constant, F represents the Faraday constant,T is the reaction temperature, and E is the electrode potential.

■ PLASMONIC TFBG SENSORTFBG Fabrication and Nanometallic Coating. The

TFBG (∼15 mm in length) was manufactured in a commercialsingle-mode fiber.19 The fabrication process mainly includesthree steps. First, the fiber was hydrogen-loaded, to increase thephotosensitivity of the fiber core. Second, a high-energy pulsedexcimer laser, together with the phase mask technique, wereused for grating inscription. In detail, a pulsed 193 nm UV lightwas focused with a cylinder lens and scanned over the fiber axialregion through a diffractive phase mask, which splits theincoming light beam into two components, thereby producing asinusoidal intensity distribution along the fiber axis. Finally,induce a proper tilting angle by rotating the phase mask andfiber consistently (phase mask and fiber are kept parallel)around an axis perpendicular to the laser beam. Note that thetilt of the grating is an important parameter that can be used tochoose which set of cladding modes is going to be excited. As aresult, it is feasible to adjust the operating range of the sensor inorder to optimize the response for certain refractive indices.Here, a grating tilt of 20° was chosen to maximize the sensingresponse in aqueous solutions with refractive indices of ∼1.32−1.34.Thin noble-metal sheaths (here, gold has been used) have

been successfully deposited on the optical fiber by sputteringtechnology. To achieve a high-quality coating, two issues mustbe addressed. One is to improve the adhesion of the gold to thesurface of fiber glass. Here, we used a 2−3 nm thickness ofchromium sandwiched between the fiber surface and the goldfilm. The second is to ensure a uniform thickness of the goldfilm over the entire fiber surface (this is very important to theSPR excitation). The traditional flat-plane sputtering techniqueis not suitable for cylindrical fibers, because sputtering is adirectional process and the target is fixed. Therefore, in order toavoid thickness nonuniformities, the fiber was rotated along itsaxis during the sputtering process. This design ensures a veryuniform nanometric coating over the fiber surface. The currentwork was carried out with a gold thickness of ∼50 nm.

SPR Excitation and Spectral Characteristics. Theplasmon waves provide the in situ information for EET ofEABs. They are hybrid waves that have intense field localizationat the outer surface of a metal film deposited on an optical fibersupport. When the refractive index of the outer medium issmaller than that of the solid support, a propagating waveincident from the support side can be totally reflected internallyat the support metal boundary but phase-matched one transfer

Analytical Chemistry Article

DOI: 10.1021/acs.analchem.6b01314Anal. Chem. 2016, 88, 7609−7616

7611

to a quasi-Plasmon excitation at the outer boundary of themetal. In order for this coupling to occur, the incident wavemust be radial at the fiber external boundary (i.e., P-polarization), to transfer energy to a plasmon wave on themetal surface. Meanwhile, the thickness of the metal layer mustbe thin enough (50 nm thickness for the optimal thickness forSPR excitation) to let some light tunnel across. Evidence ofSPR in the spectrum for this polarization is the disappearanceof the cladding mode resonances (as the clear attenuation near1520 nm of the red transmission shown in Figure 3) due to thecoupling of the cladding mode energy to the very lossy surfaceplasmon wave at these wavelengths. When the other directionof the input polarized light is used, i.e., S-polarization, theelectric field is tangent to fiber surface and no SPR occurs (asthe black transmission shown in Figure 3).

■ RESULTS AND DISCUSSIONBiofilm Formation and Characterization. Prior to EC-

SPR-OFS measurements, initially well-cultured G. sulfurreducenscells were inoculated into the bioelectrochemical cell bypolarizing the optical fiber working electrode at 0 V vs SCEto form a biofilm on the gold-coated optical fiber electrode,using a multichannel potentiostat (Model CHI1000C, China).CA and CV measurements were performed with the gold-coated optical fiber as the working electrode (surface area = 0.4cm2). Scanning electron microscopy (SEM) images confirmedthe formation of the biofilm on the optical fiber. As shown inFigure 4a, a smooth optical fiber surface was observed beforeinoculation. After 10 days of culturing, microbial cells forming abiofilm were clearly observed on the optical fiber (see Figures4b and 4c).During the biofilm formation, the electrochemical current

was also recorded, which is shown in Figure 4d. After a lagphase, an electrochemical current was observed from thebiofilm formed from wild-type G. sulfurreducens. In contrast,almost no current was obtained from the abiotic control. Forcomparison, mutant strains were also inoculated into the

bioelectrochemical cell, which showed a small currentgeneration from the strain with the omcS gene deleted(ΔomcS) and almost no current from the mutant strain withomcB, omcS, omcT, omcE, and omcZ genes deleted(ΔomcBESTZ). Previous studies have demonstrated thatomcB, omcS, and omcZ are the key redox proteins to relayelectron transfer from inside the G. sulfurreducens to outsidesubstrata.23 CVs under turnover and nonturnover conditionsfurther confirmed the electrochemical activity of the EABs witha scan rate of 5 mV/s. As shown in Figure 4e, sigmoidal CVswere observed within the biofilms that resulted from wild-typeand omcS deletion mutant in the presence of acetate, indicatingthe oxidation of acetate by these biofilms. In contrary, almostno catalytic currents were observed from the biofilms resultingfrom the ΔomcBESTZ mutant and the abiotic optical fiber.Figure 4f presents the CVs of the biofilms under the acetatedepleted condition. Redox couples with formal potentials of−0.41 and −0.49 V vs SCE (−0.17 and −0.25 V vs standardhydrogen electrode (SHE)) appeared in the CV of the biofilmwith the wild-type strain. These redox peaks were assigned toomcB (outer membrane c-type Cytochrome b) and omcZ(outer membrane c-type Cytochrome Z), respectively.24

However, only one couple of redox peaks was observed fromthe biofilm with the omcS deletion mutant, and almost noredox peaks were observed from the biofilm with theΔomcBESTZ. This confirmed that the presence of OM c-Cyts is important for the EET of the G. sulfurreducens biofilm.

Optical Response of the EABs. Accompanying theelectrochemical measurements, the optical spectral responsesof the plasmonic fiber-optic sensor are recorded. As shown inFigure 5a, the transmission spectra of the EAB-attachedplasmonic fiber-optic sensor (inscribed with a TFBG) presentsa fine comb of narrowband resonances that overlaps thespectral region where the plasmon wave absorbs some of thelight (this spectral region is colored orange). These fine spectralresonances provide a unique tool to measure small shifts of theplasmon with high accuracy: when the plasmon wave spectral

Figure 3. (Left) Transmission spectra of a TFBG coated with 50 nm of gold and immersed in water, for (a) P-polarized incident light and (b) S-polarized incident light. The SPR location corresponds to the spectral notch near 1520 nm in the upper graph. (Right) Simulated electric mode fieldprofiles for two neighboring high order cladding modes. P-polarization resonances have electric fields that are predominantly radial at the boundary(panel a). S-polarization resonances have predominantly tangential electric fields around the fiber cladding boundary (panel b). Therefore, P-polarization resonances can transfer energy to a plasmon wave on the metal surface (bright fringe around the fiber) but S-polarization resonancescannot.

Analytical Chemistry Article

DOI: 10.1021/acs.analchem.6b01314Anal. Chem. 2016, 88, 7609−7616

7612

maximum moves in response to changes in the immediatevicinity of the metal surface, the amplitudes of the individualresonances of the TFBG either grow or decrease, dependingwhether the plasmon peak (SPR) loss gets closer or farther.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 beenobserved and their response varies in accordance with theapplied potentials. The most accurate measure of the SPRwavelength shift is obtained by monitoring the amplitudechange of a select resonance located on the short wavelengthside of the SPR maximum (this resonance is identified by a redasterisk (*) and is labeled “plasmonic” in Figure 5b). Theamplitude of this resonance increases when the SPR loss movestoward longer wavelengths, in response to surroundingrefractive index or applied potential increases.25,26 Here, inour experiment, the amplitude of the “SPR” resonance increases∼15 dB when the applied potential (at the gold-coated fiberworking electrode) changes from 0 V to −0.8 V. Theidentification of the plasmonic resonance in subsequentmeasurements is enabled by referencing all wavelengths tothe location of the core mode reflection resonance (Figure 5c),which is completely insensitive to the external environment ofthe fiber. Furthermore, since all resonance wavelengths havethe same response to temperature shifts, the aforementionedreferencing of wavelengths to the core mode resonanceremoves any influence of temperature on the measurements.Monitoring of the power level of the transmission spectrumnear the core resonance also ensures that fluctuation of the lightpower level (from the light source and anywhere else in theoptical path) is identified and eliminated from the measureddata, thus maximizing the signal-to-noise ratio, the stability, andthe reliability of the proposed sensor.SPR Responses of the Redox Properties of the EABs

under Potentiostatic Control. To further observe the SPRresponses of the redox process in the EABs, detailed changes in

the SPR amplitude were measured during the EET. EC-SPR-OFS experiments were performed by employing potentialjumps from the positive potentials (0 V) to the negativepotentials (−0.8 V). Figure 6a shows the current response ofvarious biofilms on the potential jumps from 0 and −0.8 V,which showed the decrease in the current generation of theEABs. A current of 4.6 μA was obtained from the wild-type G.sulfurreducens biofilm at the potential of 0 V, which was the

Figure 4. (a−c) SEM images of the gold-coated optical fibers ((a) abiotic fiber, (b) optical fiber with G. sulfurreducens biofilm, and (c) high-resolution image of the G. sulfurreducens biofilm). Electrochemical measurements of the EABs: (d) Current−time (I−t) curves of thebioelectrochemical systems with various inoculations; (e) CV scans of the EABs on the optical fibers under turnover conditions; (f) CV scans of theEABs on the optical fibers under nonturnover conditions.

Figure 5. Optical spectral responses of EAB measurement: (a) spectralresponse of gold-coated 20° TFBG with SPR excitations underdifferent electrode polarizations; (b) enlarged view of the spectralchange of SPR modulated cladding modes (note that the “plasmonic”cladding mode is marked by a red asterisk “*”); and (c) enlarged viewof the spectrum near the core mode reference resonance.

Analytical Chemistry Article

DOI: 10.1021/acs.analchem.6b01314Anal. Chem. 2016, 88, 7609−7616

7613

highest value among all G. sulfurreducens strains. According toprevious studies, the EET ability of the EABs was assigned tothe activity of surface-localized redox-active proteins or bulkredox compounds, such OM c-Cyts, multicopper proteins, pilinproteins, and endogenous mediators excreted by bacteria.27−29

It is well-known that the electron states of the redox specieswere associated with the applied potentials at the workingelectrodes.30,31 When an anodic potential was applied to theEABs, the redox species in the EABs were in their oxidationstates. In contrast, the redox species in the EABs were in theirreduction states when a cathodic potential was applied to theEABs. Associated with the potential jumps, we found that thedifferential SPR amplitude obtained by subtracting the SPRamplitude between the measured potentials and 0 V wascorrespondingly varied. Notably, using the differential SPRamplitude (instead of absolute amplitude) can eliminate theeffect of the bacterial biomass and cell viability on themeasurements of the redox activity among different opticfibers and EABs. This approach can also effectively cancel outany unwanted fluctuations originating from the light source ortransmission path. As presented in Figure 6b, the differentialSPR amplitudes were inversely related to the currentgeneration. Note that differential SPR amplitude of the EABswith G. sulfurreducens mutants and other biofilms at thepotential 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 lowerelectroactivities of biofilms. Based on these experimentalresults, the maximum current density (Imax) of the EABs variedlinearly with the measured differential SPR amplitude (ΔSPR),

Δ = + × =⎡⎣ ⎤⎦I R[ SPR] 6.97 0.74 0.998max2

as shown in the right inset figure in Table 1.Note that the biocurrent flowing on the fibers may

potentially influence the SPR signals. To demonstrate therelationship between the biocurrent flowing and the SPR signalof the EABs, we measured the SPR of the same mature EABsunder both turnover and nonturnover conditions. As shown inFigure 7a, EC-SPR measurements were performed at three time

points of the same EAB. Current generation properties of theEAB were recorded at these three time points, which showedgreat variations (Figure 7b). The generated currents of 3.7, 2.4,and 0.1 μA were achieved at points a, b and c, respectively,which represented different redox state of the EAB. However,the changes in the differential SPR amplitudes were almostidentical (Figure 7c). This means that the variations in the

Figure 6. (a) Current−potential curves of the EABs under turnoverconditions; (b) potential-dependent background-subtracted SPRintensity of the EABs; (c) fraction of c-Cyts in the biofilm as afunction of the potential, as determined by EC-SPR-OFS spectroscopy(the dashed lines indicate the fit to a model); and (d) the firstderivative 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 V and 0 V) Is PresentedBoth in the Table (Left) and the Plot (Right) (Not Including the Non-EABs, ΔomcBESTZ, and E. coli)

Figure 7. (a) Current−time (I−t) curves of the bioelectrochemicalsystems with G. sulfurreducens for a long-term operation; (b) current−potential (I−V) curves of the G. sulfurreducens biofilm obtained atpoints a, b, and c in panel (a), respectively; (c) potential-dependentbackground-subtracted SPR amplitude of the biofilm at points a, b, andc in panel (a), respectively; and (d) potential-dependent background-subtracted SPR amplitude of the biofilm under nonturnover conditionat different time intervals, showing the remarkable stability of thesystem for long-term operation.

Analytical Chemistry Article

DOI: 10.1021/acs.analchem.6b01314Anal. Chem. 2016, 88, 7609−7616

7614

differential SPR amplitudes were associated with the redoxactivities of the biofilms rather than the current flowing throughthe gold coating over the fiber surface. Note that almostidentical SPR responses were achieved from the biofilm over along time span, suggesting the remarkable stability of theconstructed EC-SPR-OFS for monitoring the EABs (Figure7d).According to previous studies, the SPR angular shift from the

immobilized redox metalloprotein in the EC-SPR system ismainly caused by the conformation changes of the redoxprotein (c-Cyts) in the EABs. These are, in turn, induced by thechanges in the valency state of iron in heme during the redoxprocess.17,32 In addition, the SPR angular shift can result fromthe refractive index difference between the reduced andoxidized forms of the bulk redox species.33,34 Thus, the EC-SPR-OFS mainly probes the presence and redox states of theredox species in the EABs, which can be further used toindirectly predict the EET ability of the EAB. As shown inFigure 6c, the contribution of the reduced componentsincreased with the decrease of the electrode potential, andvice versa, which was obtained by normalizing the differentialSPR amplitude by the differential SPR amplitude at −0.8 V(assuming a completely reduced state), respectively. Theresultant potential-dependent changes of the redox states ofthe EABs were then analyzed in terms of the Nernst equation.11

A fit of the Nernst equation for independent redox couples tothe data in Figure 6c afforded a two-electron transfer processwith apparent formal potentials of −0.15 and −0.25 V vs SHEfor the EAB with the wild-type strain, which are in very goodagreement with the CV results. The electron transfer from G.sulfurreducens to solid electron acceptors is accomplished by awide variety of OM c-Cyts with broad potential windows, suchas 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 ofthe redox potential window of the EAB with the wild-typestrain is observed on the first-deviation curve of the differentialSPR amplitude curve over the potential (Figure 6d), whereas anarrow shape of redox potential window was obtained from theEAB with the ΔomcS mutant, and no signals were observedfrom the EAB with the ΔomcBESTZ mutant. It is worthmentioning that the EET process of G. sulfurreducens biofilmswas complicated and has not been fully understood yet. Inaddition to OM c-Cyts, Okamoto et al. revealed that efficientbacterial EET involved OM c-Cyts-bound flavins that functionas a redox cofactor in the scaffold of OM c-Cyts.36 The formalpotential of OM c-Cyts-bound flavins was observed at −0.20 Vvs SHE, which should also be included in the redox potentialwindow of the EAB with the wild-type strain in Figure 6d. Onthe other hand, other surface-localized redox proteins that arealso involved in the EET, such as multicopper proteins andpilin proteins, might also contribute to SPR signals. Shewanellabiofilms are also well-studied EABs, which showed a similarSPR response as the Geobacter biofilms in the EC-SPR-OFSsystem (Table 1). It has been revealed that OM c-Cyts (OmcAand MtrC), protein-bound and bulk endogenous mediatorsexcreted by bacteria contribute to the EET of Shewanellabiofilms.37,38 Therefore, the SPR signal of this biofilm can beassigned to surface-localized redox proteins and bound or bulkendogenous mediators.The EABs commonly vary in thickness from a few

micrometers to several millimeters, which involves complexlong-distance electron transfer.39,40 Two-layer bacterial aggre-gations have been identified in the thick EABs, including a live

outer layer (nearer the solution interface) and a dead innerlayer of biofilm (nearer the electrode interface).41 The liveouter layer of biofilm has been considered to be responsible forcurrent production and the dead inner layer has been suggestedto function as an electrically conductive matrix. However,investigations are still insufficient to provide insight into thebiofilm structure relative to its electrochemical properties. Todate, electrochemical techniques have been primarily applied tostudy the EET process in EABs, yielding important insights intotheir redox process.30,42 However, electrochemical techniquescannot provide structural and spatial information, because theyonly perform a “bulk” detection in which the current or changeinformation originates from the entire electrode surface.43 TheEC-SPR method proposed here provides a very “localized” and“surface” detection of the inner layer of biofilm (adjacent to theelectrode interface) because the SPR signals are mainlyconfined to a spatial thickness of ∼1 μm above the surface ofthe gold film. To be more specific, in this case, the plasmonwaves mainly propagate in the inner layer of a multilayeredbiofilm. In this regard, the EC-SPR-OFS measurementsrevealed that the inner layer of the EAB was electrochemicallyactive during the EET, which is consistent with previoussurveys.41 In addition, as discussed earlier, the combinations ofUV-vis, Raman, and Fourier transform infrared (FTIR)spectroscopies with electrochemistry offer good opportunitiesfor revealing the structure information of the EABs. However,free-space configurations of these conventional spectroscopiesrequired the measurements to be performed mainly inlaboratories. The inherent advantages of the proposed fiber-optic sensors include their low cost, lightweight, small size,ruggedness, and remote operation ability, making it possible forthem to fully integrate with various in-field environments suchas soil, sediments, and wastewaters, especially for those hard-to-reach places.

■ CONCLUSIONS

In summary, we have demonstrated the first electrochemicalsurface plasmon resonance (EC-SPR) optical absorptionspectrum of electroactive biofilms (EABs) during metabolism.By implementing such a hybrid technique (electrochemical andplasmonic 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, whichis associated with mutant analysis, reveals the important role ofthe redox species in the extracellular electron transfer (EET), aswell as the redox states of these redox species duringmetabolism. Benefiting from the advantages of compact size,cost-effectiveness, and simple implementation, as well as thepossibility of remote operation, the proposed electrochemicalsurface plasmon resonance optical fiber sensor (EC-SPR-OFS)is an appealing solution for observing the electrochemicalactivity of biofilms developed in various natural environments.

■ AUTHOR INFORMATION

Corresponding Authors*Tel.: +86 20 85221606. Fax: +86 20 85222046. E-mail:tuanguo@jnu.edu.cn (T. Guo).*Tel.: +86 20 37300951. Fax: +86 20 87025872. E-mail:sgzhou@soil.gd.cn (S. Zhou).

NotesThe authors declare no competing financial interest.

Analytical Chemistry Article

DOI: 10.1021/acs.analchem.6b01314Anal. Chem. 2016, 88, 7609−7616

7615

■ ACKNOWLEDGMENTS

Financial support was provided by the National Natural ScienceFoundation of China (Nos. 21277035, 61225023, and51403077), the Guangdong Natural Science Funds forDistinguished Young Scholar (No. 2014A030306033), theGuangdong Youth Science and Technology Innovation Talentsof China (No. 2014TQ01X539), the Guangdong NaturalScience Foundation of China (No. 2014A030313387), theGuangdong Innovation Foundation of China (No.2015KTSCX016), the Guangzhou Key Collaborative Innova-tion Foundation of China (No. 2016201604030084) and theFundamental Research Funds for Central Universities of China(No. 21615446). J.A. acknowledges the support of the NaturalSciences 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 ofMassachusetts is acknowledged for his kind provision ofGeobacter sulfurreducens mutants.

■ REFERENCES(1) Biffinger, J. C.; Pietron, J.; Ray, R.; Little, B.; Ringeisen, B. R.Biosens. Bioelectron. 2007, 22, 1672−1679.(2) Logan, B. E.; Hamelers, B.; Rozendal, R.; Schroder, U.; Keller, J.;Freguia, S.; Aelterman, P.; Verstraete, W.; Rabaey, K. Environ. Sci.Technol. 2006, 40, 5181−5192.(3) Marshall, C. W.; Ross, D. E.; Fichot, E. B.; Norman, R. S.; May,H. D. Environ. Sci. Technol. 2013, 47, 6023−6029.(4) Bond, D. R.; Lovley, D. R. Appl. Environ. Microbiol. 2003, 69,1548−1555.(5) Yong, Y. C.; Yu, Y. Y.; Zhang, X. H.; Song, H. Angew. Chem., Int.Ed. 2014, 53, 4480−4483.(6) Borole, A. P.; Reguera, G.; Ringeisen, B.; Wang, Z. W.; Feng, Y.J.; Kim, B. H. Energy Environ. Sci. 2011, 4, 4813−4834.(7) 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. Geobiology 2010, 8, 56−58.(8) 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, 13978−13979.(9) 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. Microbiology 2008, 154, 1422−1435.(10) Fitzgerald, 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.(11) Nakamura, R.; Ishii, K.; Hashimoto, K. Angew. Chem., Int. Ed.2009, 48, 1606−1609.(12) Liu, Y.; Kim, H.; Franklin, R. R.; Bond, D. R. ChemPhysChem2011, 12, 2235−2241.(13) Millo, D.; Harnisch, F.; Patil, S. A.; Ly, H. K.; Schroder, U.;Hildebrandt, P. Angew. Chem., Int. Ed. 2011, 50, 2625−2627.(14) Busalmen, J.; Esteve-Nunez, A.; Berna, A.; Feliu, J. M. Angew.Chem., Int. Ed. 2008, 47, 4874−4877.(15) Baba, A.; Taranekar, P.; Ponnapati, R. R.; Knoll, W.; Advincula,R. C. ACS Appl. Mater. Interfaces 2010, 2, 2347−2354.(16) Yao, X.; Wang, J.; Zhou, F.; Wang, J.; Tao, N. J. J. Phys. Chem. B2004, 108, 7206−7212.(17) Boussaad, S.; Pean, J.; Tao, N. J. Anal. Chem. 2000, 72, 222−226.(18) Leung, A.; Shankar, P. M.; Mutharasan, R. Sens. Actuators, B2007, 125, 688−703.(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. Methods2013, 63, 239−254.

(21) Caucheteur, C.; Guo, T.; Albert, J. Anal. Bioanal. Chem. 2015,407, 3883−3897.(22) Zhou, S.; Tang, J.; Yuan, Y. Bioelectrochemistry 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.; Schroder, U.Biosens. Bioelectron. 2008, 24, 1006−1011.(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 One2014, 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. U. S. A. 2008, 105, 3968−3973.(30) Fricke, K.; Harnisch, F.; Schroder, U. Energy Environ. Sci. 2008,1, 144−147.(31) Jain, A.; Gazzola, G.; Panzera, A.; Zanoni, M.; Marsili, E.Electrochim. Acta 2011, 56, 10776−10785.(32) Zhai, P.; Guo, J.; Xiang, J.; Zhou, F. J. Phys. Chem. C 2007, 111,981−986.(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, Bioenerg. 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. U. S. A. 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.; Wiktor, P.; Patel, U.; Tao, N. J. Nat. Chem. 2011, 3, 249−255.

Analytical Chemistry Article

DOI: 10.1021/acs.analchem.6b01314Anal. Chem. 2016, 88, 7609−7616

7616