Periodic Reporting for period 1 - BioNanoMagnets (Capturing the biomineralization of magnetite nanoparticles with magnetotactic bacteria in vivo using microfluidic conditions and synchrotron-based X-ray spectroscopy)
Periodo di rendicontazione: 2018-09-01 al 2020-08-31
Magnetotactic bacteria (MTB) produce highly organized chains of magnetite nanoparticles within intracellular membranes called magnetosomes. The alignment of these magnetic nanoparticles endows the bacteria with a substantial magnetic dipole, which it can use in relation to the earth’s magnetic field to navigate its environment. Magnetite nanoparticles of consistent size, composition and shape are produced by MTB through a highly controlled biomineralization process. The resultant size and morphology of magnetite nanoparticles provide optimal magnetic properties for a wide-range of biomedical applications from drug delivery to hyperthermia therapy. For these reasons, magnetite biomineralization from MTB has been of interest for several years as scientists try to understand the chemical mechanism behind the highly efficient production of magnetite nanoparticles. Harnessing or adopting chemical pathways similar to the bacteria should benefit the production and utility of magnetite nanoparticles for technological advancements in areas such as medical imaging, diagnostics, drug delivery systems, industrial catalysts and magnetic data storage.
This project aimed to capture and understand the formation and properties of magnetite nanoparticles within living MTB. Previous studies have utilized ex situ methods where sample extraction and preparation for measurements could produce artefacts or disturb the original state of the biomineralization process. To accomplish this objective, a microfluidic device was constructed to host and accommodate the growth environment conditions for MTB and to enable in situ measurement using a combination of X-ray spectroscopy and X-ray microscopy. The microfluidic device limited the liquid layer to a few micrometers and immobilized a single layer of MTB on the substrate. There are further optimizations to be made, but progress has demonstrated the potential to collect X-ray fluorescence image of a single bacterium. On the biomineralization mechanism, an ex situ study was conducted using X-ray fluorescence microscopy to assess the iron composition at varied stages of biomineralization on a single-cell level. This was achieved by performing X-ray absorption spectroscopy mapping over a single cell region and applying statistical methods to interpret the composition of iron species present. By changing the iron concentration experienced by the bacteria and the magnetosome formation induction mechanism, the work conducted sheds light on how these bacteria are able to store iron intracellularly outside of magnetosomes during the biomineralization process. This will have implications on the formation mechanism that has been postulated from previous studies and on the iron biogeochemical cycle in the environment.
This project aimed to capture and understand the formation and properties of magnetite nanoparticles within living MTB. Previous studies have utilized ex situ methods where sample extraction and preparation for measurements could produce artefacts or disturb the original state of the biomineralization process. To accomplish this objective, a microfluidic device was constructed to host and accommodate the growth environment conditions for MTB and to enable in situ measurement using a combination of X-ray spectroscopy and X-ray microscopy. The microfluidic device limited the liquid layer to a few micrometers and immobilized a single layer of MTB on the substrate. There are further optimizations to be made, but progress has demonstrated the potential to collect X-ray fluorescence image of a single bacterium. On the biomineralization mechanism, an ex situ study was conducted using X-ray fluorescence microscopy to assess the iron composition at varied stages of biomineralization on a single-cell level. This was achieved by performing X-ray absorption spectroscopy mapping over a single cell region and applying statistical methods to interpret the composition of iron species present. By changing the iron concentration experienced by the bacteria and the magnetosome formation induction mechanism, the work conducted sheds light on how these bacteria are able to store iron intracellularly outside of magnetosomes during the biomineralization process. This will have implications on the formation mechanism that has been postulated from previous studies and on the iron biogeochemical cycle in the environment.
This multidisciplinary research effort has enabled significant progress towards achieving in situ measurement of biomineralization. Microbiology and microfabrication practices were acquired to create opportunities to cultivate and manipulate bacterial cells for in situ X-ray measurements within microfluidic devices.
The most promising result to come from this action is the customization of a microfluidic device designed for X-ray fluorescence microscopy measurements at a hard X-ray nanoprobe synchrotron beamline. The microfluidic device is composed of a Si3N4 substrate onto which a PDMS layer with imprinted microfluidic channels is bonded. An approach in the construction and assembly of materials enable MTB to be immobilized as a single layer. The X-ray microscopy technique uses a 50-100 nm beam to probe the sample area, which is close the size order of a magnetosome (organelle of MTB composed of a single magnetite nanoparticle protected by phospholipid bilayer). The combined microfluidic design and high-energy X-ray measurement approach allows for a single-layer of MTB to be available for individual measurement while maintaining media conditions in microfluidic channels that will favour the formation of magnetosomes and prevent oxidative damage to other cells in the microfluidic device. As a result, iron X-ray fluorescence was detectable on the single-cell level but with partial interference from a magnetic field generated by the sample stage.
Ex situ samples were prepared for X-ray nanoprobe measurements (the same technique used for microfluidic device experiments). A preliminary series of samples measured the progression of magnetosome chain formation using X-ray fluorescence to determine the detection limit of iron in the cell, as this is important for monitoring early stages of biomineralization. It was found that around two to three hours after inducing magnetosome formation an appreciable iron X-ray fluorescence (XRF) signal could be detected. With this information a time-series of samples were measured along with bacteria grown under different conditions. X-ray absorption spectroscopy (XAS) was combined with XRF mapping to generate 2D datasets, where each pixel in the mapped image contains an XAS spectrum. These large datasets were treated with automated statistical analysis (principal components and cluster analysis) and with linear combination fitting to extract the iron composition within individual cells. By changing the iron concentration and the magnetosome formation induction mechanism (via use of a genetic variant), the work conducted sheds light on how these bacteria are able to store iron intracellularly outside of magnetosomes during the biomineralization process, though it is not directly linked to magnetite formation. This will have implications on the formation mechanism that has been postulated from previous studies and on the Fe biogeochemical cycle in the environment.
Overall, this work and these main results have achieved marked progress in creating a novel method to measure in vivo biomineralization by combining X-ray fluorescence microscopy and spectroscopy of a single cell in a microfluidic environment.
As a result, the design and implementation of microfluidic device and the single-cell spectroscopy ex situ study are currently two manuscripts to be soon submitted for publication.
The most promising result to come from this action is the customization of a microfluidic device designed for X-ray fluorescence microscopy measurements at a hard X-ray nanoprobe synchrotron beamline. The microfluidic device is composed of a Si3N4 substrate onto which a PDMS layer with imprinted microfluidic channels is bonded. An approach in the construction and assembly of materials enable MTB to be immobilized as a single layer. The X-ray microscopy technique uses a 50-100 nm beam to probe the sample area, which is close the size order of a magnetosome (organelle of MTB composed of a single magnetite nanoparticle protected by phospholipid bilayer). The combined microfluidic design and high-energy X-ray measurement approach allows for a single-layer of MTB to be available for individual measurement while maintaining media conditions in microfluidic channels that will favour the formation of magnetosomes and prevent oxidative damage to other cells in the microfluidic device. As a result, iron X-ray fluorescence was detectable on the single-cell level but with partial interference from a magnetic field generated by the sample stage.
Ex situ samples were prepared for X-ray nanoprobe measurements (the same technique used for microfluidic device experiments). A preliminary series of samples measured the progression of magnetosome chain formation using X-ray fluorescence to determine the detection limit of iron in the cell, as this is important for monitoring early stages of biomineralization. It was found that around two to three hours after inducing magnetosome formation an appreciable iron X-ray fluorescence (XRF) signal could be detected. With this information a time-series of samples were measured along with bacteria grown under different conditions. X-ray absorption spectroscopy (XAS) was combined with XRF mapping to generate 2D datasets, where each pixel in the mapped image contains an XAS spectrum. These large datasets were treated with automated statistical analysis (principal components and cluster analysis) and with linear combination fitting to extract the iron composition within individual cells. By changing the iron concentration and the magnetosome formation induction mechanism (via use of a genetic variant), the work conducted sheds light on how these bacteria are able to store iron intracellularly outside of magnetosomes during the biomineralization process, though it is not directly linked to magnetite formation. This will have implications on the formation mechanism that has been postulated from previous studies and on the Fe biogeochemical cycle in the environment.
Overall, this work and these main results have achieved marked progress in creating a novel method to measure in vivo biomineralization by combining X-ray fluorescence microscopy and spectroscopy of a single cell in a microfluidic environment.
As a result, the design and implementation of microfluidic device and the single-cell spectroscopy ex situ study are currently two manuscripts to be soon submitted for publication.
There are two main impacts that stem from the activities and research from this action. One being the fundamental scientific question regarding the uptake and storage of iron, and the formation of magnetite nanoparticles in MTB. From ex situ studies, the findings will contribute to the understanding of the magnetite formation mechanism that MTB use. The scientific community in biomineralization and biogeochemical sciences will benefit from this report. Regarding the second impact, the research on testing and designing microfluidic devices with X-ray microscopy techniques will contribute to advancements in the synchrotron science community and will influence researchers who are studying metal uptake and sequestration by microorganisms, as the research activities and development will be translatable to other systems. In particular, the microfluidic device constructed in this project combined with X-ray fluorescence microscopy is easily adaptable to other microorganisms to study metal sequestration. Already this work is being adapted for calcium carbonate-producing microalgae, where important scientific concerns regarding adaptation to a changing sea water chemistry (i.e. ocean acidification) can be addressed.