In Vivo Two-Way Redox Cycling System for Independent Duplexed

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An In-vivo Two-way Redox Cycling System for Independent Duplexed Electrochemical Signal Amplification Yuan Yang, Yangyang Yu, Yu-Tong Shi, Jamile Mohammadi Moradian, and Yang-Chun Yong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00053 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 13, 2019

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Analytical Chemistry

An In-vivo Two-way Redox Cycling System for Independent Duplexed Electrochemical Signal Amplification

Yuan Yang1, Yang-Yang Yu1,2, Yu-Tong Shi1, Jamile Mohammadi Moradian1, Yang-Chun Yong1, 2,*

1Biofuels

Institute, School of the Environment, Jiangsu University, 301 Xuefu Road,

Zhenjiang 212013, Jiangsu Province, China 2Zhenjiang

Key Laboratory for Advanced Sensing Materials and Devices, School of

Mechanical Engineering, Jiangsu University, Jiangsu Province, China

* Corresponding author, E-mail: ycyong@ ujs.edu.cn

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ACS Paragon Plus Environment

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

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ABSTRACT An in-vivo two-way redox cycling system based on whole-cell bidirectional electron transfer was developed and applied for independent duplexed electrochemical signal amplification. This duplexed signal amplification system was established by activating the bacterial “inwards” electron transfer at low electrode potential for oxidative cycling, while accomplishing the bacterial “outwards” electron transfer at high electrode potential for reductive cycling. Therefore, with this two-way bio-redox cycling

system,

simultaneously

and

independently

amplification

of

the

electrochemical signals for oxidation and reduction was achieved. More impressively, by using this duplexed signal amplification system, ultrasensitive and simultaneous detection of two different warfare toxins of Pseudomonas aeruginosa was achieved (sensitivity was improved 302 and 579 times, respectively), which makes it possible for double-checking early diagnosis of the P. aeruginosa infections.

KEYWORDS: Signal amplification; Redox cycling; Electron transfer; Pathogen; Pseudomonas aeruginosa

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Signal amplification is an essential strategy to improve the sensitivity of biosensor and fulfill the growing demands of ultrasensitive detection.1,

2

Generally, signal

amplification can be achieved by chemical approach (generate more signal species by introducing additional chemical reactions) or physical approach (amplify the post-reaction physical signal with transducer improvement).2,

3

Chemical approach

substantially enhances the signal amplitude with negligible perturbation on the noise is more attractive to sustain high sensitivity and reliability.2, 4, 5 Therefore, development of new and efficient chemical approach for signal amplification is crucial for biosensor to meet the requirement of practical applications. For electrochemical detection (one of the most widely used analytical methods), chemical

amplification

could

be

achieved

by

chemically/electrochemically/biochemically generating signal species with improved electroactivity from targeting analyte or cycling the redox species.6-11 By integrating redox cycling system to continuously regenerate the analyte or intermediate could significantly amplify the electrochemical signal output.2, 5, 10 Thus, redox cycling was considered as an efficient chemical amplification approach and offered opportunity for ultra-sensitive electrochemical detection. Especially, biochemical redox cycling system, which involved the use of enzyme as the catalyst attracted much attention due to its advantage of high selectivity.2,

12, 13

However, the in-vitro enzymatic redox

cycling required purified enzymes and sophisticated electron transfer strategies,2 which raised the concerns of complex operation, high cost and low efficiency. Billion years evolution endows microorganism unique capability of in-vivo 3



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assembling of enzyme cascades with exceptional efficiency and stability. More impressively, electroactive bacteria (EAB) evolved various electron transfer conduits to electronically bridged intracellular enzyme cascades with extracellular matrix.5, 14 Thus, in-vivo redox cycling systems by using EAB as the whole-cell catalyst and electron transfer conduit has been developed by our group.4,

5, 15

By employing

Shewanella oneidensis MR-1 (well-recognized model EAB) cell as the redox cycling module, analytes (e.g., riboflavin) were efficiently recycled during the electrochemical analysis which directly enabled efficient signal amplification and ultrasensitive detection.4, 5 In contrast to the in-vitro redox cycling system, this in-vivo redox cycling system directly employed naturally evolved elaborate whole-cell system, which excluded the cost of enzymes and avoided complex artificially assembling procedure.5 Thus, in-vivo redox cycling approach holds great potential to achieve simple, cost-effective, and highly efficient signal amplification. Besides, a single in-vitro or in-vivo redox cycling system was usually developed as sole function of reductive cycling (continuously reduce the target with sacrificed electron donor) or oxidative cycling (continuously oxidize the target with sacrificed electron acceptor).15,

16

Duplexed signal amplification is challenging as it is usually

difficult to couple a reductive cycling system with an oxidative cycling system together that is hard to avoid cross-talking. However, microbial cell with sophisticated cellular compartments (periplasmic space, cytoplasmic space, membrane surface etc.) might avoid cross-talking to achieve multiplexed redox cycling in one single cell/in-vivo system. 4


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With this context, an in-vivo redox cycling system for independent duplexed signal amplification was demonstrated for the first time in this study. By taking the advantage of efficient two-way electron transfer in S. oneidensis MR-1,17,

18

the

reductive redox cycling branch with lactate as the sacrificed electron donor and oxidative cycling branch with fumarate as the sacrificed electron acceptor were activated simultaneously. With this two-way redox cycling system, reductive amplification of the electrochemical signal from pyocyanin (PYO) and oxidative amplification of electrochemical signal from 1-hydroxyphenazine (OHP) were achieved. As a proof of concept, ultrasensitive and simultaneous detection of PYO and OHP (the virulence factors of Pseudomonas aeruginosa19) were accomplished with a single cyclic voltammetry assay, which would be promising for double-checking early diagnosis of P. aeruginosa infection.

RESULTS AND DISCUSSION S. oneidensis MR-1 is a Gram-negative bacterium that could metabolize lactate (electron donor) with tandem enzymes to release electrons and transport to outer cell membrane, which was likely to establish a reductive cycling branch (Figure 1 and Figure S1). Meanwhile, S. oneidensis MR-1 also has various reductases (e.g., fumarate reductase) that could uptake extracellular electrons with the sacrificed electron acceptor, which was likely to establish an oxidative cycling branch (Figure 1 and Figure S1).17 Thus, it is expected to activate these two in-vivo cycling branches simultaneously for duplexed signal amplification. 5



In another aspect, as PYO and OHP were the two main virulence factors and biomarkers secreted by P. aeruginosa (a ubiquitous pathogen with high mortality19) (Figure S2), ultrasensitive detection of these molecules is vital for early diagnosis20. However, their inherited electrochemical activities were extremely low (the anodic peak current for PYO (100 nM) and OHP (100 nM) were only 0.61 A and 0.69 A, respectively) (Figure S3), signal amplification is essential to meet the requirement of ultrasensitive detection. By employing the reductive cycling branch of S. oneidensis MR-1 (cell with lactate), typical S-shape CV curve with enhanced response (increased from 0.61 A to 6.4 A) derived from PYO was achieved (Figure 2a). Meanwhile, the signal from OHP was also amplified (increased from 0.69 A to 2.5 A, Figure 2a). However, the problem associated with signal cross-talking was observed by using this single cycling branch for simultaneous detection of PYO and OHP (Figure 2a). If the electrochemical signal of OHP could be amplified with an independent oxidative cycling, the problem of signal cross-talking would be overcome. For instance, the electrochemical signal of PYO with higher redox potential could be amplified with the reductive cycling branch, while that of OHP with lower redox potential could be amplified with the oxidative cycling branch (Figure 1). With this context, in-vivo oxidative cycling for OHP was then developed. Periplasmic fumarate reductase was selected as the enzyme for in-vivo oxidative cycling system development (Figure 1). As shown in Figure 2b, upon fumarate addition, significant cathodic current in CV curve was observed which might involve 6


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the electrochemical activity of the cell membrane bound riboflavin.5 However, as the riboflavin synthesis under the biosensing conditions was negligible, the cathodic current from riboflavin was relatively stable and could be considered as a baseline response.4, 5 Interestingly, after OHP addition, the cathodic current greatly increased, while the anodic current for OHP disappeared. This switching from anodic reaction to cathodic reaction was similar to flavin mediated redox bifurcation with this strain, where the cathodic condition stabilized its singly reduced semiquinone form and inhibited its anodic reaction.5, 21, 22 The S-shape CV curve observed could be described by a revised Nernst-Monod Equation,4 indicating continuously oxidation of the electrode-reduced OHP was achieved and oxidative recycling was established. As expected, the signal response from OHP was increased to 15.7 A (Figure 2b), which was about 22 times and 5.3 times higher than that without signal amplification (0.69 A) or reductive amplification (2.5 A), respectively. In addition, the signal amplification could be abolished by extracellular electron transfer pathway disruption (Figure S4), confirming the in-vivo oxidative cycling was essential for OHP signal amplification. These results substantiated that the oxidative cycling for OHP was established. Next, the two-way in-vivo redox cycling system was activated and evaluated for duplexed signal amplification. To activate the two-way redox cycling, the electron donor (lactate) for reductive cycling and electron acceptor (fumarate) for oxidative cycling were simultaneously added into the cell suspension (Figure 1). As shown in Figure 2c, there were two S-shape catalytic waves which corresponding to the 7



electrochemical response of reductive cycled PYO and oxidative cycled OHP. More impressively, the current outputs obtained with this two-way redox cycling system were in good agreement with that separately obtained with the single redox cycling. In addition, the response from OHP could be fully discriminated from that of PYO with negligible cross-talking (Figure 2c). It indicated that the in-vivo two-way redox cycling system was established and could be applied for independent duplexed electrochemical signal amplification. Before performance evaluation, optimization was conducted by using biofilm mode instead of suspension cell mode (Figure S5). After optimization, the electrochemical response for OHP was increased to about 400 A with a sensitivity of 4.0 A/nM (Figure 3). It was worth to note that the sensitivity was 579 times higher than that without signal amplification (6.9 nA/nM). Meanwhile, the electrochemical signal for PYO was also increased to about 185 A with a sensitivity of 1.85 A/nM, which was about 302 times higher than that without signal amplification (Figure 3). The results further confirmed that the two-way in-vivo redox cycling system was highly efficient for duplexed electrochemical signal amplification. For duplexed signal amplification, possible signal cross-talking should be taken into consideration. It was found that the signal output for targeting molecule (e.g. OHP) was not significantly affected by addition of interfering molecule (e.g., PYO) (Figure S6 and Figure S7) owing to different redox potential and electron flow direction employed for these molecules.22 The results indicated that the signal crosstalk was negligible and sustained that the independent duplexed signal 8


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amplification was achieved. Calibration curve was further determined under the optimized duplexed signal amplification conditions. As shown in Figure 4, the current response to PYO and OHP increased along with the elevated concentrations from 0.5 nM to 500 nM. The relationship between the current increment (i) and analyte concentration was analyzed to establish the calibration curve. It was found that good linear calibration curves for PYO and OHP could be obtained from 0.5 nM to 50 nM (Figure S8). The limit of detection (LOD, S/N=3) determined for PYO was 304±4 pM, while it was 1.5 ± 0.2 nM for OHP. The LODs obtained are 1-3 orders of magnitude lower than conventional electrochemical method23-25 or that with advanced electrodes3. In addition, it is also comparable with that achieved by sophisticated immunochemical determination method20 and is much lower than the concentrations reported in the samples from the P. aeruginosa infected patients (up to 1-100 M)23, 25. The results confirmed its feasibility for early diagnosis of P. aeruginosa infections. In addition, the concentration ratio of PYO and OHP might vary in different samples which increased the difficulty for simultaneous detection. Impressively, good linear calibration curves could also be achieved with varied PYO and OHP ratios (Figures S9 and S10), indicating the feasibility for practical detection. Furthermore, possible interferences (lactic acid, glucose, blood matrix, etc.) might exist in practical samples were evaluated. Although coexistence of these interferences resulted in slight influence on the signal output, the relative biosensor output was retained between 90.5%-109% (Figure S11), suggesting the interferences were marginal which might 9



not significantly affect the analytical performance. More impressively, as S. oneidensis MR-1 was a cold-resistance bacterium,15 good storage stability could be achieved (Figure S12). By using the simulated water samples with exogenously added authentic analytes, high recovery (88%-100%) and low variation (0.78%-9.7%) were obtained (Table S1), indicating the in-vivo duplexed amplification system had good accuracy and reproducibility. To demonstration the possibility for application in clinical samples, a set of simulated blood samples (human blood spiked with PYO and OHP) were analyzed. It was found that the developed system could simultaneously quantified PYO and OHP in the samples with excellent recovery (88%-96.3%) and low variation (0.7%-7.1%) (Tables S2 and S3). The results suggested that the developed system could be applicable for early diagnosis of P. aeruginosa infections. Moreover, two-way electron transfer conduits have been explored or genetically engineered in other bacterial species,26, 27 the idea of in-vivo two-way redox cycling would open up new dimension for signal amplification.

ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China (51578266, 51708254, 31870112), Natural Science Foundation of Jiangsu Province (BK20160015, BK20170545), Fok Ying-Tong Education Foundation (161074), Open Project of Key Laboratory of Environmental Biotechnology (CAS, kf2016008), State Key Laboratory of Microbial Metabolism (Shanghai Jiao Tong University, 10


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MMLKF18-03) and a project funded by Priority Program Development of Jiangsu Higher Education Institutions.

Supporting information. Details of experimental procedures; Molecular structures and CV analyses of PYO and OHP; Amplification system optimization and analytical performance; Water and human blood samples analysis.

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Figure Legends Figure 1. Schematic representation of the in-vivo two-way redox cycling system for independent duplexed electrochemical signal amplification.

Figure 2. CV analysis of PYO (100 nM) and/or OHP (100 nM) with different redox cycling systems (under suspension cell mode), (a) with reductive cycling branch, (b) with oxidative cycling branch and (c) with duplexed redox cycling system. RC: reductive cycling system (cell with 18 mM lactate). OC: oxidative cycling system (cell with 50mM fumarate). Duplexed redox cycling system (cell with 18 mM lactate and 50mM fumarate).

Figure 3. Improvement on electrochemical response to PYO (100 nM) and OHP (100 nM) by duplexed amplification (DA) or optimized DA system.

Figure 4. Analysis of PYO and OHP simultaneously at different concentrations with optimized DA system.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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In Vivo Two-Way Redox Cycling System for Independent Duplexed

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