Wiring Bacterial Electron Flow for Sensitive Whole-Cell Amperometric

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Wiring Bacterial Electron Flow for Sensitive Whole-Cell Amperometric Detection of Riboflavin Rong-Wei Si, Yuan Yang, Yangyang Yu, Song Han, Chun-Lian Zhang, De-Zhen Sun, Dan-Dan Zhai, Xiang Liu, and Yang-Chun Yong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03538 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 20, 2016

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Wiring Bacterial Electron Flow for Sensitive Whole-Cell Amperometric Detection of Riboflavin Rong-Wei Si1,2,#, Yuan Yang1,2,#, Yang-Yang Yu1,2, Song Han2, Chun-Lian Zhang1,2, De-Zhen Sun1,2, Dan-Dan Zhai1,2, Xiang Liu1,2, Yang-Chun Yong1,2,*

1

Biofuels Institute and 2School of the Environment, Jiangsu University, 301 Xuefu

Road, Zhenjiang 212013, Jiangsu Province, China

#Equal contribution. * Corresponding author, Email: ycyong@ ujs.edu.cn, Tel: +86-511-8878 6708, Fax: +86-511-8879 0955.

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Abstract A whole-cell bioelectrochemical biosensing system for amperometric detection of riboflavin was developed. A “bioelectrochemical wire” (BW) consists of riboflavin and cytochrome C between S. oneidensis MR-1 and electrode was characterized. Typically, strong electrochemical response was observed when riboflavin was added to reinforce this BW. Impressively, the electrochemical response of riboflavin with this BW was over 200 times higher than that without bacteria. Uniquely, this electron rewiring process enabled the development of a biosensing system for amperometric detection of riboflavin. Remarkably, this amperometric method showed high sensitivity (LOD=2.2 nM, S/N=3), wide linear range (5 nM~10 µM, three orders of magnitude), good selectivity and high resistance to interferences. Additionally, the developed amperometric method featured good stability and reusability, and was applied for accurate and reliable determination of riboflavin in real conditions including food, pharmaceutical, and clinical samples without pretreatment. Both the cost-effectiveness and robustness make this whole-cell amperometric system ideal for practical applications. This work demonstrated the power of bioelectrochemical signal amplification with exoelectrogen and also provided a new idea for development of versatile whole-cell amperometric biosensors.

Key words: Bioelectrochemical system;Biosensor; Riboflavin; Extracellular electron transfer; Shewanella

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Riboflavin (Vitamin B2, VB2) is an essential vitamin for human health1. Because it serves as the exclusive source for coenzymes (flavin mononucleotide and flavin adenine dinucleotide) that catalyzes key cellular metabolisms associated with carbohydrates, fats, and proteins utilization.1,2 Deficiency of riboflavin might cause serious diseases such as metabolism disorder, cataracts, neurodegeneration, or even cancer.1,3 Excess of riboflavin is also detrimental as it is a photosensitizer inducing oxidative damage to light-exposed tissue.4-6 More importantly, riboflavin can not be synthesized in human body, which has to be taken from food, supplements or medicines with strictly controlled dosage1. Thus, riboflavin becomes an important biomarker for clinic diagnosis and food quality control. It is therefore imperative to develop simple, fast and sensitive methods for riboflavin detection in various samples (food, pharmaceutical, clinical samples, etc.).7 Owing to the distinctive fluorescence characteristic of riboflavin, various physicochemical methods coupled with fluorimetry have been developed, e.g., HPLC,8 capillary electrophoresis,9 and fluorescence resonance energy transfer detection.10 HPLC coupled with fluorescence detector is most widely used among these methods due to its high sensitivity, stability and selectivity. Surface plasma resonance based biosensor was also developed for riboflavin detection in food samples.11 However, problems associated with these methods include high cost of the required instrumentation and complicated/time-consuming sample pretreatment. In order to solve these problems, various electrochemical methods based on the unique redox property of riboflavin were developed. Various electrochemical methods 3

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such as cyclic voltammetry (CV),12,13 differential pulse voltammetry (DPV),13,14 and square wave voltammetry (SWV)13,15 have been successfully adapted for riboflavin detection.

With

the

progress

on

electrode

modification

such

as

DNA

functionization,12,13 alkanethiols modification,15 screen printing,14 and nanoparticle modification,16 the sensitivity and selectivity of these electrochemical detection methods were greatly improved. Compared with traditional physicochemical methods, these electrochemical methods showed enormous advantages such as low cost, free of sample pretreatment, high sensitivity, and simple procedure. However, all these electrochemical methods are based on voltammetry assay, no amperometric method is available for riboflavin detection. Amperometric assay is the most simple and applicable electrochemical sensing method which gained vast attention during the past 50 years.17,18 Compared with voltammetry assays, it is featured by a fixed potential impressed on the working electrode thereby generating current response related to the electrochemical oxidation or reduction that occurs at the working electrode surface.18 Amperometric assay posed the advantages of low instrument requirement (only simple potentiostat needed for potential control and current measurement), simple operation (no voltammetry) and directly readable output signal (minimized post data processing).18 However, most of the

amperometric

assays

required

sophisticated

catalysts

such

as

rare

metal/nanostructured material or purified enzyme19 to facilitate the electrochemical reaction or amplify the output signal, which largely hindered its practical application. Whole-cell biosensor featured by self-regeneration and self-maintenance has the 4

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unique advantages of low cost and robust. By integrating the advantages of whole-cell and amperometric assay, whole-cell amperometric biosensor became the most attractive

electrochemical

microbial

biosensor.20

The

developed

whole-cell

amperometric biosensor generally can be categorized into two groups which individually uses intracellular enzyme (IE)21 or outer-membrane bound enzyme (OE) as catalyst.22,23 The amperometric signal usually generated from the electroactive metabolite/intermediate or oxygen consumption (detected by oxygen electrode).20,24 The metabolite/intermediate produced by cells usually has higher electroactivity than targeted analyte, which amplifies the amperometric signal and enables high sensitivity. Although riboflavin is an electroactive compound, its amperometric signal is supposed to be weak and unstable owing to its fast oxidation-reduction turnover rate and low concentration in samples. Moreover, no suitable enzyme could be found to catalyze riboflavin transformation to produce metabolite/intermediate with better amperometric response. Therefore, it is quite challenging to detect riboflavin by whole-cell amperometric assay. In this work, a “bioelectrochemical wire” (BW) consists of riboflavin-cytochrome C proteins between cells and electrode was characterized, and a whole-cell amperometric biosensor based on this “wire” was developed for riboflavin detection. In this system, riboflavin shuttled the electron transfer between electrode and cells for intracellular fumarate reduction, which generated an amperometric signal (only negligible amperometric signal was detected with riboflavin alone) and was sued for riboflavin detection. By wiring the electrode with bacteria, the bacterial inwards 5

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electron flow dramatically amplified the electrochemical response of riboflavin. Upon parameters optimization, this biosensor showed low limit of detection (LOD), high selectivity, and high robust. Moreover, it was also applied for food, pharmaceutical and clinical samples. To the best of our knowledge, this is the first ampermetric sensing system developed for riboflavin detection.

EXPERIMENTAL SECTION Bacterial strains and cultivation conditions Shewanella oneidensis MR-1 was routinely cultivated in LB broth (peptone 10g/L, yeast extract 5g/L, NaCl 5g/L, pH 7.0) at 30 oC with shaking (150 rpm).25,26 Mutants of S. oneidensis MR-1 were kindly provided by Prof. Bin Cao (Nanyang Technological University). Pseudomonas aeruginosa PAO1 or Escherichia coli was cultivated in LB broth at 37 oC with shaking (220 rpm). Electrochemical apparatus and measurements All electrochemical measurements were conducted with CHI 660E electrochemical workstation or CHI1000B potentiostat (Chenhua Instruments Co., Ltd., Shanghai, China). A platinum wire electrode and a saturated calomel electrode (SCE) were used as counter electrode and reference electrode, respectively. A carbon cloth electrode (1 cm × 2 cm) was used as the working electrode. A 12 mL cylindrical borosilicate glass bottle was used as the electrochemical cell for all electrochemical tests. All potentials are reported versus SCE. Riboflavin detection with biosensing system 6

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For biosensing assay, S. oneidensis MR-1 cells cultured in LB broth with optimized period (5-18 h) were harvested by centrifugation, washed two times with M9 minimal medium, and re-suspended with fresh electrolyte (5% LB medium and 95% M9 minimal medium, 10 mM lactate, pH=7.0) to the designed cell density (OD600=0.05-2). Next, the cell suspensions (10 mL) were transferred into a three-electrode electrochemical cell (12 mL), and the potential of the working electrode was poised at -0.6V (vs. SCE) using CHI1000B potentiostat or CHI 660E electrochemical workstation. After discharge for about 30 min under the poised potential, fumarate was added to obtain a baseline current. Then, riboflavin or riboflavin contained samples (24-120 µL) without pretreatment was directly injected into the electrochemical cell, and the current response was recorded. Urine used was collected from healthy volunteer. Vitamin tablets and VB complex tablets were purchased from local pharmacy. Milk powder was purchased from local supermarket produced by Beingmate Co. (Hangzhou, China). HPLC analysis HPLC analysis was performed as follows using an Agilent HPLC system equipped with Agilent C18 analytical column (4.6 mm×150 mm, 5 µm particle size) and a fluorescence detector. Riboflavin was eluted with a mobile phase (water with 20% methanol and 1% glacial acetic acid) at the flow rate of 1 ml/min. Samples were filtrated through a 0.22 µm filter before injection. The HPLC profile was monitored at an excitation wavelength of 440 nm and an emission wavelength of 252 nm.27

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RESULTS AND DISCUSSION Wiring bacterial intracellular electron flow to amplify electrochemical response of riboflavin S. oneidensis MR-1 is a model exoelectrogen that is able to exchange electrons with solid electrode and can directly transduce the cell response to electric signal. It was found that cytochrome C proteins (MtrC, MtrA, MtrB, OmcA) and riboflavin were involved in the electron exchange between cells and electrode. Since riboflavin serves as the electron shuttle to facilitate electron transfer between cells and the electrode,26,28 it is potentially applicable to employ S. oneidensis MR-1 cells to develop a whole-cell biosensor system for riboflavin detection. As illustrated in Fig.1A, S. oneidensis MR-1 is capable of bacterial inwards electron transfer (BIET), which uptakes extracellular electrons from the electrode to “power” the intracellular reductive

metabolism.25,29

A “bioelectrochemical

wire”

(BW)

consists

of

riboflavin-cytochrome C was considered to directly wire the electrode with cells.30 Fumarate reductase (catalyzed fumarate reduction to succinate) is the typical reductase that is wired with extracellular electrode by that “BW” (Fig. 1A).29,31 To confirm the riboflavin mediated BIET in S. oneidensis MR-1, CV analysis was first employed. CV curve of fumarate did not show obvious electrochemical response. As expected, riboflavin showed a pairs of symmetric redox peaks but with low response current (~0.4 µA) (Fig. 1B), while addition of fumarate did not affected this CV behavior (data not shown). Cells without riboflavin and fumarate showed no obvious electrochemical response (Fig. 1B). However, once cells were mixed with 8

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fumarate, a sigma shape CV curve with obvious electrochemical response (with the catalytic current ~18 µA). The result is in good agreement with previous reports,29,31 indicating electron exchange between cells and electrode was established. More strikingly, an extremely high catalytic current (~113 µA) was achieved once riboflavin was further added (riboflavin+cell+fumarate) (Fig. 1B). Impressively, the electrochemical response to riboflavin with cells and fumarate (~95 µA, I (riboflavin+cell+fumarate)-I (cell+fumarate)) is about 236 times higher than that without cells and fumarate (~0.4 µA, I (riboflavin)). While the minus current generated means electron flow from the electrode to bacteria, indicating efficient BIET between electrode and cells was established. These results confirmed that riboflavin can efficiently reinforce the BIET of S. oneidensis MR-1.31 More importantly, the high catalytic current generated with this set-up suggested a stable amperometric assay could be adopted. In accordance with previous reports,29,31 fumarate reduction by S. oneidensis MR-1 resulted in a catalytic waveform centered at about -0.6 V (Fig. 1B). Thus, according to previous reports29,31 and the CV results, a potential of -0.6 V was employed to enable fumarate reduction and maintain stable amperometric response. As expected, a stable current response to riboflavin and fumarate (50 mM) addition was obtained (Fig. 1C) under amperometric condition. To further characterize the function of BW, a series of cytochrome C mutants of S. oneidensis MR-1 were analyzed. As shown in Fig. 1C, with the BW (cytochrome C proteins and riboflavin), a high current response of about 77 µA was reached. However, deletion of the key component of cytochrome C (MtrC, MtrA, MtrB, or 9

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OmcA) or without riboflavin substantially reduced the current response. It was found that electrochemical response of single gene mutation on the outer surface cytochrome C (MtrC or OmcA) was much higher than that of the double mutant (∆MtrC∆OmcA), which indicates MtrC or OmcA can separately interact with riboflavin and form active BW. Due to these two proteins showed different affinity to riboflavin,30 obvious difference on current response between ∆MtrC and ∆OmcA was observed. The results confirmed that riboflavin and cytochrome C proteins are the key component of BW. It also implied that fluctuation of riboflavin concentration might greatly affect the amperometric current output, suggesting riboflavin biosensor could be developed based BW of S. oneidensis MR-1.

Biosensing system development and optimization To develop an amperometric sensing system, a stable baseline current should be first established. As illustrated in Fig. 1C, it is interesting that fumarate addition into the S. oneidensis suspension produced a stable amperometric current. More importantly, the magnitude of the current is independent of fumarate concentration (when the concentration over 50 mM) which excluded the interference from fumarate. Fumarate is essential for this sensing system as it served as the final electron acceptor of BIET. It was confirmed by the observation that there is no amperometric response to riboflavin once no fumarate was added (Fig. S1). In addition, the stable current output could last over 30 hours (50 mM fumarate) which promised successive and long-term amperometric assay. Thus, the biosensing system was proposed, i.e., a three-electrode 10

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system with a poised potential of -0.6 V on the working electrode and electrolyte contained S. oneidensis MR-1 cell suspension in 50 mM fumarate. Then, different concentration of riboflavin was added into the proposed sensing system to test its feasibility for riboflavin detection. Once a stable baseline current was established after fumarate addition, sample with riboflavin was injected into the sensing system and a sharp current increase could be observed immediately (Fig. 2a). More impressively, the increased current quickly reached the constant level and formed new stable current output, which implied successive detection might be achieved in a single system (Fig.2). This phenomenon is reasonable as riboflavin was just used as the electron shuttle32, which was not depleted. It is also worth to note that S. oneidensis MR-1 also can produce low level of riboflavin.28 However, no significant riboflavin production was detected under the biosensing condition during the bioassay process. This might be due to the poor nutrition condition (M9 mineral solution) and the room temperature used here are unfavorable for riboflavin biosynthesis. More importantly, as the bioassay process was fast which usually completed within 2 hours, the riboflavin (a secondary metabolite) biosynthesis was negligible and did not affect the biosensing. As S. oneidensis cells in this proposed biosensing system served as a bifunctional module for analyte recognition and signal generation, the cell physiology might largely affect the signal output of the biosensor. Thus, cell cultivation time and cell density were optimized to maximize the signal output of the biosensing system (Fig. S2). It was found that both cultivation time and final cell density significantly affected 11

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the signal output. The optimized cultivation time and cell density is 16 hours and OD600=1.5 (~1.5±0.1×108 CFU/mL), respectively (Fig. S2). Next, biosensing response to different concentration of riboflavin was determined under the optimized conditions. As shown in Fig. 2a, with successive addition of riboflavin, a step-wise current increase was obtained. It suggested that the developed biosensing system is possible to be used for online/continuous monitoring. Moreover, the magnitude of the increased current is proportional to the concentration of riboflavin. Thus, the relationship between riboflavin concentration and current response was analyzed and calibration curve with good linear relationship was obtained. As shown in Fig. 2b, the linear part of the calibration curves covered a wide range concentration of riboflavin from 5 nM to 10 µM (over three orders of magnitudes). The limit of detection (LOD, defined as 3 times of baseline noise) determined is 2.2 nM (S/N=3), while the limit of quantification (LOQ, defined as 10 time of baseline noise) determined is 11 nM (S/N=10). Compared with conventional electrochemical assay for riboflavin reported recently, the method developed here showed lower LOD and much wider linear range (three orders of magnitude) (Table S1). It is also worth to note that the analytical performance obtained in this study just based on raw electrode material and sample without pretreatment, which might be further improved with sophisticated electrode surface engineering. More importantly, this is the first amperometric method developed for riboflavin detection which holds great promise for practical and continuous/online applications.

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Analytical performance Before evaluating its feasibility for application in real samples, the analytical performance related to selectivity, accuracy, stability and reusability were determined. As indicated in Fig. 3a, interferences that might coexist with riboflavin in real samples did not show any significant current response. In addition, after these interferences was added into this biosensing system, addition of 1 µM riboflavin resulted in a 52±1.3 µA current increase, which showed no significant difference from that before interferences addition (50±1.7 µA). This result indicated that coexistence of these interfering compounds did not affect the analytical performance of this biosensing system, which sustained its good selectivity and interference resistance. Bacterial contamination is another concern for biosensing. Here, several model bacteria including E. coli and P. aeruginosa were selected and added into the biosensing system to simulate the contamination, and the effect on signal output was determined. As shown in Fig. 3b, no obvious interference on signal output of the biosensing system was observed under bacterial contamination with E. coli or P. aeruginosa. The results demonstrated high robustness of this developed biosensing system, and implied its feasibility for practical application. Storage stability and reusability are essential for real application of biosensor. Thus, stability of the developed biosensing system was analyzed here by evaluating the signal output decaying under storage. As S. oneidensis MR-1 had good low temperature resistance, it may render this biosensor excellent storage stability.33 As shown in Fig. 4a, the output signal did not dramatically affected by low temperature 13

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storage, and over 85% output was retained after one week storage, which indicated good storage stability. The reusability was determined by analyze the signal output fluctuation (the magnitude of the current response) with successive detection. It was found that over 85% of biosensor output could be retained after 20 times of detection demonstrating the possibility for reuse of this biosensor (Fig. 4b). Next, the accuracy and reproducibility were tested by using synthetic riboflavin water samples to establish the creditability of this newly developed biosensing method. It was found that the riboflavin concentrations quantified by this biosensing system were in good agreement with that spiked in the samples (Table 1). The recovery of the samples (ranging from 8 nM to 5 µM) was between 93% and 106%, which indicated good accuracy of this biosensing system. The coefficient of variation was between 2.3% and 8.7%, which indicated good reproducibility was achieved. These results suggested that this newly developed biosensing system is highly reliable for riboflavin quantification in aqueous samples.

Application to real samples As riboflavin quantification is of great importance for food industry, pharmaceutical and diagnosis, commercial milk powder, commercial pharmaceutical formulation samples and urine samples were selected as the model real samples. To evaluate the reliability of the developed biosensing system, the results obtained from the biosensing system were compared with those determined by HPLC. As shown in Table 2, the results obtained from the biosensing method developed here were in good 14

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agreement with those of HPLC as well as the values claimed by the manufacturer. The accuracy of the developed method is comparable with that of HPLC. Moreover, although these samples contain complicated interferences for riboflavin analysis, the quantification results by the developed biosensing method without sample pretreatment showed high recovery (91.8%~98.4%) and low variation (2%~5.6%), which indicated high reliability for real application. For simulated clinic sample analysis, different amounts of authentic riboflavin were spiked into the urine samples. The samples were then directly used for riboflavin detection without any pretreatment. It was found that possible interferences in urine did not significantly affect the detection accuracy (Table 3), which implied its feasibility for analysis of clinic samples and the possibility for diagnosis application.

CONCLUSIONS An amperometric biosensing system based on S. oneidensis MR-1 was developed by wiring the bacterial electron transfer with electrode and was applied for riboflavin detection. By using fumarate as the electron acceptor and applying a poised potential of -0.6 V on working electrode under the optimized conditions, electrons from electrode was successfully wired into S. oneidensis MR-1 cells with the cytochrome C and riboflavin mediated electron transfer conduit. Interestingly, the current generated on working electrode is in proportional to the concentration of riboflavin. A linear calibration curve with extremely wide riboflavin concentration (5 nM to 10 µM) was obtained. The biosensing method developed showed much low LOD (2.2 nM) with 15

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high selectivity and good reliability. It was also applied to analyze different real samples without sample pretreatment and showed high resistant to various interferences. To the best of our knowledge, this is the first amperometric method for riboflavin detection. The idea of wiring the bacterial intracellular electron flow with electrode might provide powerful way for signal amplification in biosensing system and add new dimension for versatile biosensor design.

ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge on the ACS Publication website. Current response of S. oneidensis MR-1 at different conditions; optimization of bacteria cultivation time and cell density.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (NSFC 51578266), Natural Science Foundation of Jiangsu Province for Distinguished Young Scholars (BK20160015), and a project funded by the Priority Program 16

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Development of Jiangsu Higher Education Institutions. We also appreciate the donation of mutants by Prof. Bin Cao from Nanyang Technological University.

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(14) Kadara, R. O.; Haggett, B. G. D.; Birch, B. J. J Agr Food Chem 2006, 54, 4921-4924. (15) Blanco, E.; Dominguez, C. S. H.; Hernandez, P.; Hernandez, J. V.; Quintana, C.; Hernandez, L. Electroanalysis 2009, 21, 495-500. (16) Mehmeti, E.; Stankovic, D. M.; Chaiyo, S.; Svorc, L.; Kalcher, K. Microchim Acta 2016, 183, 1619-1624. (17) Leland, C. C.; Sachs, G. Ann New York Acad Sci 1968, 148, 133-153. (18) Kotanen, C. N.; Moussy, F. G.; Carrara, S.; Guiseppi-Elie, A. Biosens Bioelectron 2012, 35, 14-26. (19) Kalimuthu, P.; Leimkuhler, S.; Bernhardt, P. V. Anal Chem 2012, 84, 10359-10365. (20) Su, L. A.; Jia, W. Z.; Hou, C. J.; Lei, Y. Biosens Bioelectron 2011, 26, 1788-1799. (21) Schenkmayerova, A.; Bucko, M.; Gemeiner, P.; Katrlik, J. Biosens Bioelectron 2013, 50, 235-238. (22) Liang, B.; Li, L.; Tang, X.; Lang, Q.; Wang, H.; Li, F.; Shi, J.; Shen, W.; Palchetti, I.; Mascini, M.; Liu, A. Biosens Bioelectron 2013, 45, 19-24. (23) Liang, B.; Zhang, S.; Lang, Q. L.; Song, J. X.; Han, L. H.; Liu, A. H. Anal Chim Acta 2015, 884, 83-89. (24) Yoetz-Kopelman, T.; Porat-Ophir, C.; Shacham-Diamand, Y.; Freeman, A. Sensor Actuat B-Chem 2016, 223, 392-399. (25) Yong, Y. C.; Yu, Y. Y.; Zhang, X. H.; Song, H. Angew Chem Int Edit 2014, 53, 4480-4483. 18

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(26) Sun, D.-Z.; Yu, Y.-Y.; Xie, R.-R.; Zhang, C.-L.; Yang, Y.; Zhai, D.-D.; Yang, G.; Liu, L.; Yong, Y.-C. Biosens Bioelectron 2017, 87, 195-202. (27) Covington, E. D.; Gelbmann, C. B.; Kotloski, N. J.; Gralnick, J. A. Mol Microbiol 2010, 78, 519-532. (28) Yong, Y. C.; Cai, Z.; Yu, Y. Y.; Chen, P.; Jiang, R. R.; Cao, B.; Sun, J. Z.; Wang, J. Y.; Song, H. Bioresource Technol 2013, 130, 763-768. (29) Si, R. W.; Zhai, D. D.; Liao, Z. H.; Gao, L.; Yong, Y. C. Biosens Bioelectron 2015, 68, 34-40. (30) Okamoto, A.; Hashimoto, K.; Nealson, K. H. Angew Chem Int Ed 2014, 53, 10988-10991. (31) Ross, D. E.; Flynn, J. M.; Baron, D. B.; Gralnick, J. A.; Bond, D. R. PLoS One 2011, 6, e16649. (32) 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. (33) Abboud, R.; Popa, R.; Souza-Egipsy, V.; Giometti, C. S.; Tollaksen, S.; Mosher, J. J.; Findlay, R. H.; Nealson, K. H. Appl Environ Microbiol 2005, 71, 811-816.

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Table 1. Detection of riboflavin in water samples by the developed biosensing system.

a

Riboflavin spiked (nM)

Determined by biosensor (nM)

COVa(%)

Recovery(%)

8

8.45

6.9

106

30

29.6

3.0

98.7

80

79.1

2.3

98.9

150

152

3.9

101

350

367

8.7

105

900

840

8.4

93.3

5000

5016

3.8

100

COV, coefficient of variation (n=3).

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Analytical Chemistry

Table 2. Detection of riboflavin in urine samples by the developed biosensing system. Sample No.

Riboflavin spiked(nM)

Determined by biosensor (nM)

COVa(%)

Recovery (%)

1

0