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Acoustic biosensors for ultrasound imaging of enzyme activity

Nature Chemical Biology volume 16, pages 988–996 (2020)Cite this article

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A Publisher Correction to this article was published on 23 July 2020

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Abstract

Visualizing biomolecular and cellular processes inside intact living organisms is a major goal of chemical biology. However, existing molecular biosensors, based primarily on fluorescent emission, have limited utility in this context due to the scattering of light by tissue. In contrast, ultrasound can easily image deep tissue with high spatiotemporal resolution, but lacks the biosensors needed to connect its contrast to the activity of specific biomolecules such as enzymes. To overcome this limitation, we introduce the first genetically encodable acoustic biosensors—molecules that ‘light up’ in ultrasound imaging in response to protease activity. These biosensors are based on a unique class of air-filled protein nanostructures called gas vesicles, which we engineered to produce nonlinear ultrasound signals in response to the activity of three different protease enzymes. We demonstrate the ability of these biosensors to be imaged in vitro, inside engineered probiotic bacteria, and in vivo in the mouse gastrointestinal tract.

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Fig. 1: Acoustic biosensor of TEV endopeptidase.
Fig. 2: Acoustic biosensor of calcium-activated calpain protease.
Fig. 3: Acoustic biosensor of ClpXP protease.
Fig. 4: Monitoring intracellular protease activity and circuit-driven gene expression in engineered cells.
Fig. 5: Ultrasound imaging of bacteria expressing ASGs in the gastrointestinal tract of mice.

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Data availability

Data supporting the findings of this study are available within the article and its Supplementary Information. Additional data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

The authors thank Z. Sun, A. Shur and R. Murray for sharing the protocols and reagents used for the cell-free TX–TL system. TEM was done at the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. This research was supported by the National Institutes of Health (NIH) (no. R01-EB018975) and Defense Advanced Research Projects Agency (no. W911NF-14-1-0111). A.L. was supported by a National Science Foundation (NSF) Graduate Research Fellowship (no. 1144469) and the Biotechnology Leadership Pre-doctoral Training Program in Micro/Nanomedicine (Rosen Bioengineering Center and NIH Training Grant no. 5T32GM112592-03/04). D.P.S. was supported by an NSF Graduate Research Fellowship (no. 1745301). D.M. was supported by the Human Frontier Science Program (no. LT000637/2016). Related research in the Shapiro Laboratory is supported by the Heritage Medical Research Institute, Burroughs Wellcome Career Award at the Scientific Interface, Pew Scholarship in the Biomedical Sciences and Packard Fellowship for Science and Engineering.

Author information

Author notes

  1. David Maresca

    Present address: Department of Imaging Physics, Delft University of Technology, Delft, Netherlands

  2. These authors contributed equally: Anupama Lakshmanan, Zhiyang Jin.

Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA

    Anupama Lakshmanan, Suchita P. Nety, Audrey Lee-Gosselin, Dina Malounda, Mararet B. Swift, David Maresca & Mikhail G. Shapiro

  2. Division of Engineering and Applied Sciences, California Institute of Technology, Pasadena, CA, USA

    Zhiyang Jin

  3. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Daniel P. Sawyer

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Contributions

A.L. and M.G.S. conceived the study. A.L., Z.J. and S.P.N. designed and planned the experiments. A.L., Z.J., S.P.N., D.P.S., A.L-G., M.B.S. and D. Malounda conducted the experiments. Z.J., D.P.S. and D. Maresca wrote the MATLAB scripts for ultrasound imaging and data processing. A.L., Z.J. and M.G.S. analyzed the data. A.L., Z.J. and M.G.S. wrote the manuscript with input from all authors. All authors gave approval to the final version of the manuscript.

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Correspondence to Mikhail G. Shapiro.

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Extended data

Extended Data Fig. 1 Engineering an acoustic sensor of TEV endopeptidase activity.

a, Coomassie-stained SDS-PAGE gel of OD500nm-matched samples of GVWT incubated with dTEV and TEV protease, before and after buoyancy purification (labeled pre b.p. and post b.p., respectively). N = 3 biological replicates. b, Scatter plots showing normalized OD500nm of GVSTEV as a function of hydrostatic pressure. (N = 3 biological replicates for GVSTEV + TEV and N = 4 for GVSTEV + dTEV.) c, Scatter plots showing the ratio of nonlinear (x-AM) to linear (B-mode) ultrasound signal as a function of applied acoustic pressure for all the replicate samples used in the x-AM voltage ramp imaging experiments for GVSTEV. N = 3 biological replicates and total number of replicates is 8. d, Scatter plots showing normalized OD500nm of GVWT as a function of hydrostatic pressure. (N = 3 biological replicates for GVWT + dTEV and N = 4 for GVWT + TEV.) e, Representative ultrasound images of agarose phantoms containing GVWT incubated with TEV or dTEV protease at OD500nm 2.2. The B-mode image was acquired at 132kPa and the x-AM image at 569 kPa. Similar images acquired for N = 3 biological replicates, with each N consisting of 3 technical replicates. CNR stands for contrast-to-noise-ratio, and color bars represent relative ultrasound signal intensity on the dB scale. Scale bars represent 1 mm f, Scatter plots showing the ratio of nonlinear (x-AM) to linear (B-mode) ultrasound signal as a function of applied acoustic pressure for all the replicate samples used in the x-AM voltage ramp imaging experiments for GVWT. N = 3 biological replicates, with each N consisting of 3 technical replicates. Solid curve represents the mean of all the replicates.

Source data

Extended Data Fig. 2 Engineering an acoustic sensor of calpain activity.

a, Individual scatter plots for Fig. 2b. N = 5 biological replicates for +Calp/+Ca2+, 6 for -Calp/+Ca2+ and +Calp/-Ca2+, 7 for -Calp/-Ca2+. b, Coomassie-stained SDS-PAGE gel of OD500nm-matched samples of GVScalp incubated in the presence (+) or absence (-) of calpain (first + /-) and calcium (second + /-), before and after buoyancy purification (labeled pre b.p. and post b.p. respectively). N = 3 biological replicates. c, Representative TEM images of GVScalp after incubations in the presence or absence of calpain and/or calcium. Scale bars represent 100 nm. At least 20 GV particles were imaged for each condition. d, DLS measurements showing the average hydrodynamic diameter of GVScalp and GVWT samples after calpain/calcium incubations (N = 2 biological replicates for GVScalp + /-, +/+, GVWT + / + and 3 for other conditions, individual dots represent each N and horizontal line indicates the mean). Error bars indicate SEM when N = 3. e–g, Individual scatter plots for Fig. 2d, f, h. N = 3 biological replicates with each N consisting of 2 technical replicates (total number of replicates is 18 for + /+ and 6 for each of the remaining conditions). Solid line represents the mean of all the replicates for (a, e–g). h, Scatter plots for Fig. 2i; N = 3 biological replicates, individual dots represent each N and solid blue line showing the fitted curve (a Hill equation with a coefficient of 1, with a half-maximum effective concentration (EC50) of 140 μM).

Source data

Extended Data Fig. 3 Characterization of GVWT sample with calpain protease.

a–c, Representative ultrasound images of agarose phantoms containing GVWT incubated in the presence (+) or absence (-) of calpain (first + /-) and calcium (second + /-), at OD500nm 2.2. The B-mode images were taken at 132 kPa for a, b and c and the x-AM images corresponding to the maximum difference in non-linear contrast between the + /+ sample and the negative controls were taken at 438 kPa for a, b and at 425 kPa for c. CNR stands for contrast-to-noise-ratio and color bars represent ultrasound signal intensity in the dB scale. Scale bars represent 1 mm. N = 2 biological replicates for a–c. d–f, Scatter plots showing the ratio of x-AM to B-mode ultrasound signal as a function of increasing acoustic pressure for GVWT after incubation in the presence or absence of calpain and/or calcium (N = 2 biological replicates). g, Hydrostatic collapse curves of GVWT after incubations in the presence (+) or absence (-) of calpain and/or calcium. The legend lists the midpoint collapse pressure for each condition (±95% confidence interval) determined from fitting a Boltzmann sigmoid function (N = 5 biological replicates for -/+ and N = 6 for other conditions) h, Coomassie-stained SDS-PAGE gel of OD500nm-matched samples of GVWT incubated in the presence (+) or absence (-) of calpain/calcium, before and after buoyancy purification (labeled pre b.p. and post b.p., respectively, N = 1). Individual dots in d–g represent each N and solid line represents the mean of all the replicates.

Source data

Extended Data Fig. 4 Engineering an acoustic sensor of ClpXP proteolytic activity.

a, b, Scatter plots for Fig. 3d, g. N = 5 biological replicates. c, Coomassie-stained SDS-PAGE gel of OD500nm-matched GVWT samples incubated in a reconstituted cell-free transcription-translation (TX-TL) system containing a protease inhibitor cocktail or ClpXP. N = 3 biological replicates. d, Coomassie-stained SDS-PAGE gel of 30x diluted content of TX-TL system containing ClpXP. N = 2 biological replicates(e) DLS measurements showing the average hydrodynamic diameter of GVSClpXP and GVWT samples, after incubations with protease inhibitor or ClpXP (N = 2 biological replicates, individual dots represent each N and horizontal line indicates the mean). f, g, Scatter plots showing the ratio of x-AM to B-mode acoustic signal as a function of applied acoustic pressure for all the replicate samples used in the x-AM voltage ramp experiments for GVSClpXP (f) and GVWT (g). N = 3 biological replicates, with each N consisting of 3 technical replicates. Individual dots represent each N and solid line represents the mean of all the replicates for a, b, f, g.

Source data

Extended Data Fig. 5 Constructing intracellular acoustic sensor genes for dynamic monitoring of protease activity and circuit-driven gene expression.

a, Normalized pressure-sensitive optical density at 600 nm of WT Nissle cells expressing either ARGWT or ASGClpXP. The legend lists the midpoint collapse pressure for each cell type (±95% confidence interval) determined from fitting a Boltzmann sigmoid function (N = 5 biological replicates and 8 total replicates for ASGClpXP; N = 3 biological replicates for ARGWT and 6 total replicates). b, Representative ultrasound images of WT Nissle cells expressing either ARGWT or ASGClpXP at OD600nm 1.5 (N = 4 biological replicates and the number of total replicates is 10). c, Scatter plots showing x-AM/B-mode ratio as a function of applied acoustic pressure for WT Nissle cells expressing either ARGWT or ASGClpXP at OD600nm 1.5 (N = 4 biological replicates and the number of total replicates is 10). d, Scatter plots for Fig. 4b, N = 3 biological replicates. e, f, Scatter plots showing the ratio of x-AM to B-mode acoustic signal as a function of acoustic pressure for all the replicate samples used in the x-AM voltage ramp experiments for ΔclpXP Nissle cells expressing ASGClpXP and araBAD driven clpXP, with or without L-arabinose induction (e) and WT Nissle cells expressing ASGClpXP and pTet-TetO driven WT gvpC, with or without aTc induction (f). N = 3 biological replicates, with each N having 3 technical replicates for (e) and N = 5 biological replicates for (f). Individual dots represent each N and solid line represents the mean of all the replicates for a, c–f.

Extended Data Fig. 6 Schematic illustrating the in vivo ultrasound imaging experiment.

Cells in cylindrical hydrogel with the indicated cross-sectional arrangements were injected into the GI tract of mice and imaged with ultrasound.

Extended Data Fig. 7 Ultrasound imaging of bacteria expressing acoustic sensor genes in the gastrointestinal tract of mice.

a, Schematic illustrating two orientations of the wild type (WT) E. coli Nissle cells expressing ARGWT or ASGClpXP introduced into the mouse colon as a hydrogel. b, c, Representative transverse ultrasound images of the colon for two mice used in the in vivo imaging experiments, with orientation #1 (b) and with orientation #2. (c). Cells are injected at a final concentration of 1.5E9 cells ml-1. B-mode signal is displayed using the bone colormap and x-AM signal is shown using the hot colormap. Color bars represent B-mode and x-AM ultrasound signal intensity in the dB scale. Scale bars represent 2 mm. d, e, B-mode and xAM contrast-to-noise ratio (CNR) in vivo, for WT Nissle cells expressing ARGWT or ASGClpXP in orientation #1 (d) and orientation #2. (e). N = 5 mice for orientation #1 (b, d) and N = 4 mice for orientation #2 (c, e). Error bars indicate SEM. P = 0.0014 for x-AM signal from cells expressing ASGClpXP versus the ARGWT control in orientation #1, and P = 0.0016 for that in orientation #2. P = 0.0570 for B-mode signal in orientation #1 and P = 0.3445 in orientation #2. P-values were calculated using a two-tailed paired t-test. Individual dots represent each N and horizontal line indicates the mean.

Extended Data Fig. 8 ASGClpXP -expressing cells showed higher contrast to tissue with nonlinear imaging.

B-mode and xAM contrast-to-tissue ratio (CTR) in vivo, for WT Nissle cells expressing ARGWT or ASGClpXP in both orientations. P = 7.8E-5 for the CTR from xAM imaging of cells expressing ASGClpXP versus CTR from xAM imaging of cells expressing ARGWT. P = 1.4E-6 for the CTR from xAM imaging of cells expressing ASGClpXP versus CTR from B-mode imaging of cells expressing ASGClpXP and P = 4.9E-7 for the CTR from xAM imaging of cells expressing ASGClpXP versus CTR from B-mode imaging of cells expressing ARGWT. Individual dots represent each N, and the thick horizontal line indicates the mean. Error bars indicate SEM. N = 9 mice. P-values were calculated using a two-tailed paired t-test for each comparison independently. Individual dots represent each N and horizontal line indicates the mean.

Extended Data Fig. 9 Absence of memory effect from imaging at sequentially increasing acoustic pressure.

Ratio of sensor-specific signal (xAM/B-mode) acquired at the indicated acoustic pressures in the process of voltage ramping (comprising 36 points from 458 kPa to 1.6 MPa) or stepping the transducer output directly to corresponding pressure in a single step, for WT Nissle cells expressing either ARGWT or ASGClpXP. N = 3 biological replicates, with each N having 3 technical replicates. Individual dots represent each replicate, and the thick horizontal line indicates the mean. Error bars indicate SEM derived from biological replicates (see Online Methods).

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Unprocessed gels for Extended Data Fig. 4(c,d)

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Lakshmanan, A., Jin, Z., Nety, S.P. et al. Acoustic biosensors for ultrasound imaging of enzyme activity. Nat Chem Biol 16, 988–996 (2020). https://doi.org/10.1038/s41589-020-0591-0

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