Electronic modulation of biochemical signal generation

Journal name:
Nature Nanotechnology
Volume:
9,
Pages:
605–610
Year published:
DOI:
doi:10.1038/nnano.2014.151
Received
Accepted
Published online

Microelectronic devices that contain biological components are typically used to interrogate biology1, 2 rather than control biological function. Patterned assemblies of proteins and cells have, however, been used for in vitro metabolic engineering3, 4, 5, 6, 7, where coordinated biochemical pathways allow cell metabolism to be characterized and potentially controlled8 on a chip. Such devices form part of technologies that attempt to recreate animal and human physiological functions on a chip9 and could be used to revolutionize drug development10. These ambitious goals will, however, require new biofabrication methodologies that help connect microelectronics and biological systems11, 12 and yield new approaches to device assembly and communication. Here, we report the electrically mediated assembly, interrogation and control of a multi-domain fusion protein that produces a bacterial signalling molecule. The biological system can be electrically tuned using a natural redox molecule, and its biochemical response is shown to provide the signalling cues to drive bacterial population behaviour. We show that the biochemical output of the system correlates with the electrical input charge, which suggests that electrical inputs could be used to control complex on-chip biological processes.

At a glance

Figures

  1. Schematic of the biohybrid device controlled by electronic signals.
    Figure 1: Schematic of the biohybrid device controlled by electronic signals.

    a, Schematic of the biohybrid device receiving both chemical (enzyme reaction precursor) and electronic inputs, and, through biochemical intermediates, translating them to both electrochemical signals and biological cell responses. b, Representation of the components of the multidomain fusion protein (HLPT) used in the study. c, Experimental concept. By varying the electronic inputs through the electrodes on which the HLPT is attached, the attenuation of HLPT activity can be varied, thus affecting the electrochemical and biological responses in proportion to the input. Purple rectangles, silicon wafer; gold rectangles, patterned gold electrodes; semitransparent turquoise rectangles, biocompatible chitosan scaffold. Hcy, homocysteine; AI-2, autoinducer-2; His, histidine; Tyr, tyrosine. LuxS and Pfs are enzymes within HLPT.

  2. Electronically driven HLPT attenuation by natural mediator acetosyringone (AS).
    Figure 2: Electronically driven HLPT attenuation by natural mediator acetosyringone (AS).

    a, Schematic of electrochemical oxidation of AS(R) in solution, followed by its addition to HLPT, where it oxidizes and attenuates HLPT activity and is reduced back to AS(R) in the process. b, Spectrophotometric measurements of AS(R) and AS(O). As AS is oxidized, it turns a brownish-orange colour, detectable at 490 nm. c, Oxidation of HLPT by AS(O) can be detected with the electron paramagnetic resonance (EPR) probe CPH, which is oxidized by the AS(O)-oxidized protein. A higher EPR intensity is seen when the protein is treated with AS(O). The EPR spectra show samples measured after 2 min of CPH reaction with protein. The bar graph represents an average of several normalized measurements (Supplementary Section 8). d, Sulfhydryl groups detected through Ellman's assay after treatment of the proteins with AS(O) or AS(R). e, HLPT activity calculated from electrochemical measurements after incubation with AS(O) or AS(R). Inset: Reaction of Hcy as it is oxidized at the electrode. Measurements in c were performed as described in Supplementary Section 8, in duplicate. Measurements in d and e were performed in triplicate. All error bars indicate s.d. Two-tailed, unequal variance student t-tests were run on data in ce. *P  <  0.05, **P  <  0.01, ***P  <  0.001, ****P  <  0.0001. AS(R) and AS(O) are reduced or oxidized acetosyringone, respectively. CPH is the EPR spin probe 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine-HCl. HLPT, fusion protein. LuxS and Pfs are the catalytic enzymes within HLPT.

  3. On-chip enzyme activity is linear with input charge.
    Figure 3: On-chip enzyme activity is linear with input charge.

    a, Immobilization of enzyme onto a silicon chip involves chitosan electrodeposition as a thin film followed by enzymatic assembly of HLPT. Fluorescent pictures of red-labelled chitosan and blue-labelled HLPT show film and enzyme co-localization onto the gold-patterned electrode. b, Schematic depicting in situ enzyme attenuation. The same electrode on which the HLPT is attached is used to oxidize the AS (grey to brown hexagons) in the vicinity of the protein and leads to activity attenuation. Activity is then measured by electrochemical detection of Hcy (green hexagon) as described in the main text. c, Correlation between input charge applied for in situ AS oxidation as in b and the Hcy measured from HLPT thus attenuated at the end of 3.5 h incubation. Three series are depicted, with different initial enzymatic activities. There are two activity and four input charge measurements per data point. Error bars indicate s.d. R2 values indicate Pearson correlation coefficients for linearity for the displayed averaged data. Values for Pearson and Spearman rank coefficients for monotonic correlation for all non-averaged data are, respectively, –0.81 and –0.91 for activity level 1, 0.92 and –0.93 for activity level 2, and 0.92 and –0.96 for series 3. Hcy, homocysteine; mC, millicoulombs; HLPT, fusion protein.

  4. In situ enzyme attenuation mediates biological signalling.
    Figure 4: In situ enzyme attenuation mediates biological signalling.

    a, Schematic of experiment: HLPT is attenuated in situ as in Fig. 3. The generated solution with AI-2 is added to reporter cells, which fluoresce blue. A bright-field image of the cells is overlaid with the blue fluorescent image, showing co-localization of cells and fluorescence. b, Histograms from FACS (measuring blue DAPI fluorescence) run on AI-2 reporter cells to which the products of differentially attenuated HLPT-immobilized electrodes were added. c, Comparison of the Hcy measured electrochemically and the average blue fluorescence of AI-2 reporter cells from HLPT immobilized on an electrode and attenuated with the indicated input charges. Cell fluorescence averages correspond to those in the histograms in b. Three measurements were taken for the activity in c, and error bars indicate s.d. The Pearson correlation coefficient for linearity calculated for cell fluorescence versus enzyme activity averages in c yielded an R2 value of 0.98. AI-2, autoinducer-2; Hcy, homocysteine; HLPT, electrically attenuated fusion enzyme; mC, millicoulombs.

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Author information

Affiliations

  1. Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, USA

    • Tanya Gordonov,
    • Gregory F. Payne &
    • William E. Bentley
  2. Institute for Bioscience & Biotechnology Research, University of Maryland, College Park, Maryland 20742, USA

    • Tanya Gordonov,
    • Eunkyoung Kim,
    • Gregory F. Payne &
    • William E. Bentley
  3. Institute for Systems Research, University of Maryland, College Park, Maryland 20742, USA

    • Yi Cheng,
    • Hadar Ben-Yoav,
    • Reza Ghodssi &
    • Gary Rubloff
  4. Department of Materials Science and Engineering, University of Maryland, College Park, Maryland 20742, USA

    • Yi Cheng &
    • Gary Rubloff
  5. Department of Electrical and Computer Engineering, University of Maryland, College Park, Maryland 20742, USA

    • Hadar Ben-Yoav &
    • Reza Ghodssi
  6. Division of Analytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, Maryland 20740, USA

    • Jun-Jie Yin

Contributions

T.G., E.K., G.F.P. and W.E.B. developed the concepts and planned and designed the experiments. T.G., E.K., H.B. and Y.C. fabricated components and performed the experiments and data analysis. J.J.Y., G.F.P., W.E.B. and G.R. supervised the work. T.G., E.K., G.F.P. and W.E.B. wrote and edited the manuscript.

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The authors declare no competing financial interests.

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