Periodic Reporting for period 1 - RhizoSheet (Patterned microfluidic sheets for studies of root exudation profiles)
Reporting period: 2021-10-04 to 2023-10-03
Root exudates have a key role in the life of a plant. They control critical processes, like water retention capability of soil, root penetrability into soil, they can mobilize nutrients and they can influence the microbial environment around the root. Some of the microbial interactions can positively increase the plant's stress resiliency, can improve the nutrient uptake and immune responses of the plant. Using these beneficial interactions could revolutionise our agriculture, leading to a more sustainable eco friendly crop production.
Unfortunately, the available technologies cannot address this problem with sufficient spatio-temporal resolution and in a cost effective way.
The RhizoSheet project addressed the above mentioned challenges. Using cutting edge paper-polymer technologies a microfluidic device was developed to monitor root exudates over time along the root.
The project had four main research objectives:
O1 Identify materials and fabrication techniques for assembling RhizoSheet microfluidic system and sensors.
O2 Achieve, in situ, robust extraction and mapping of exudates along a growing root, over time.
O3 Generate a reference high resolution library of root exudates of tomato plant.
O4 Localise temporally and spatially the place of exudation and interaction with bacteria.
Unfortunately, the available technologies cannot address this problem with sufficient spatio-temporal resolution and in a cost effective way.
The RhizoSheet project addressed the above mentioned challenges. Using cutting edge paper-polymer technologies a microfluidic device was developed to monitor root exudates over time along the root.
The project had four main research objectives:
O1 Identify materials and fabrication techniques for assembling RhizoSheet microfluidic system and sensors.
O2 Achieve, in situ, robust extraction and mapping of exudates along a growing root, over time.
O3 Generate a reference high resolution library of root exudates of tomato plant.
O4 Localise temporally and spatially the place of exudation and interaction with bacteria.
Work Package 1
Design of the hybrid microfluidic device.
A wide range of materials were tested to select the materials suitable for the microfluidic chip. The most important criteria were biocompatibility and easy fabrication possibility. Poly(methyl methacrylate) (PMMA), cyclo olefin polymer (COP), pressure sensitive adhesives (PSA) seemed to be suitable; however, during the fabrication process some difficulties were found using these materials. The bond between these materials were not strong enough under humid conditions or the material itself generated false signal in the integrated sensors. On the other side microscope glass, wax printed filter paper and polydimethylsiloxane (PDMS) found to be perfect candidates for the microfluidic chip. The wax printed filter paper served as horizontal microfluidic channels and growth substrate for the plant root, the glass slide provided a solid carrier for the system, and the PDMS layer which sealed the microfluidic chip provided the necessary air for the root, it was transparent allowed microscopic investigation and it could also help to keep the humidity of the growth environment.
The wax pattern was printed using a commercially available printer. The wax was melted into the fibre structure of the paper creating horizontal fluidic channels. The filter paper with the channels were placed between a PDMS and glass slide. The slides were plasma treated in order to provide a tight covalent bond between them. The created microfluidic chip is capable to extract root exudates spatially and temporally.
Work package 2
Extraction and mapping of root extracts.
To investigate the spatial and temporal root exudate collection capability of the developed microfluidic chip a titanate nanotube-alginate hydrogel hybrid colorimetric sensor was integrated into each of the channels. The exudate selected was glucose since it is one of the most abundant chemicals exuded by plant roots and it attracts microbes, so it has a significant role in root-microbe interactions. The sensors were integrated into the left outlet of each of the channels. The signal was extracted from the chip using evaporation. The chips were inserted horizontally into a Petri-dish with 40ml of water. The evaporation on the left outlets generated a water gradient in the channels which delivered the exuded molecules, towards the sensors, in this case glucose sensor. Using this extraction method, it was possible to repeat the extraction process day after day and it was possible to map the exudation activity of the plant root along the root over time.
1.2.3 Work package 3
Collection of a library of root exudates.
For the collection of a library of root exudates different sampling methods were tested. The main aim was to collect samples for further analysis using HPLC. The first method applied a segmented “cut-off” sampling method in order to provide a time dependent investigation of the exudation activity. The left outlet part of the channels were elongated and segmented with small wax marks. After a sampling period the end part of the paper channel was cut off at the last wax mark. This process was repeated until the last wax mark day after day. Unfortunately, this sampling method could not live up to expectations since the residual traces of exudates in the remaining paper part of the channels. To decresa the residual traces in the remaining paper segments a series octagonal shaped paper parts were added inline to the paper channels. The octagons, due to their higher surface, increased the efficiency of the extraction, but also provided segments with dead spaces where the exudates could accumulate and were not delivered to the last octagonal.
Finally, a separate sampling paper was developed to provide a secured sampling from each of the channels. This sampling paper has six sampling octagons in the distance the paper fluidic channels. For the extractions the sampling paper is attached to the paper channels and due to capillary and evaporation the sample flows to the octagons. Later the octagons can be separated and the exudates can be washed port for HPLC analysis. For time resolved sampling another sampling paper with the octagons can be inserted to the paper channels.
Design of the hybrid microfluidic device.
A wide range of materials were tested to select the materials suitable for the microfluidic chip. The most important criteria were biocompatibility and easy fabrication possibility. Poly(methyl methacrylate) (PMMA), cyclo olefin polymer (COP), pressure sensitive adhesives (PSA) seemed to be suitable; however, during the fabrication process some difficulties were found using these materials. The bond between these materials were not strong enough under humid conditions or the material itself generated false signal in the integrated sensors. On the other side microscope glass, wax printed filter paper and polydimethylsiloxane (PDMS) found to be perfect candidates for the microfluidic chip. The wax printed filter paper served as horizontal microfluidic channels and growth substrate for the plant root, the glass slide provided a solid carrier for the system, and the PDMS layer which sealed the microfluidic chip provided the necessary air for the root, it was transparent allowed microscopic investigation and it could also help to keep the humidity of the growth environment.
The wax pattern was printed using a commercially available printer. The wax was melted into the fibre structure of the paper creating horizontal fluidic channels. The filter paper with the channels were placed between a PDMS and glass slide. The slides were plasma treated in order to provide a tight covalent bond between them. The created microfluidic chip is capable to extract root exudates spatially and temporally.
Work package 2
Extraction and mapping of root extracts.
To investigate the spatial and temporal root exudate collection capability of the developed microfluidic chip a titanate nanotube-alginate hydrogel hybrid colorimetric sensor was integrated into each of the channels. The exudate selected was glucose since it is one of the most abundant chemicals exuded by plant roots and it attracts microbes, so it has a significant role in root-microbe interactions. The sensors were integrated into the left outlet of each of the channels. The signal was extracted from the chip using evaporation. The chips were inserted horizontally into a Petri-dish with 40ml of water. The evaporation on the left outlets generated a water gradient in the channels which delivered the exuded molecules, towards the sensors, in this case glucose sensor. Using this extraction method, it was possible to repeat the extraction process day after day and it was possible to map the exudation activity of the plant root along the root over time.
1.2.3 Work package 3
Collection of a library of root exudates.
For the collection of a library of root exudates different sampling methods were tested. The main aim was to collect samples for further analysis using HPLC. The first method applied a segmented “cut-off” sampling method in order to provide a time dependent investigation of the exudation activity. The left outlet part of the channels were elongated and segmented with small wax marks. After a sampling period the end part of the paper channel was cut off at the last wax mark. This process was repeated until the last wax mark day after day. Unfortunately, this sampling method could not live up to expectations since the residual traces of exudates in the remaining paper part of the channels. To decresa the residual traces in the remaining paper segments a series octagonal shaped paper parts were added inline to the paper channels. The octagons, due to their higher surface, increased the efficiency of the extraction, but also provided segments with dead spaces where the exudates could accumulate and were not delivered to the last octagonal.
Finally, a separate sampling paper was developed to provide a secured sampling from each of the channels. This sampling paper has six sampling octagons in the distance the paper fluidic channels. For the extractions the sampling paper is attached to the paper channels and due to capillary and evaporation the sample flows to the octagons. Later the octagons can be separated and the exudates can be washed port for HPLC analysis. For time resolved sampling another sampling paper with the octagons can be inserted to the paper channels.
The developed hybrid microfluidic chip can reveal exudation differences spatially and temporally. Additionally it can reveal phenotypic between varieties of the same species. The easy, cost effective fabrication method, the easy usability makes the developed system ideal for fundamental researches to reveal the exudation mechanisms, to investigate root-microbe interactions. It is also beneficial for plant breeders, since it can show the phenotypic differences without costly gene activity tests, so an industrial application is highly possible. A patent claim was submitted to protect the intellectual rights of the developed chip (EP23382947.2).