This application is a national phase application based on PCT Patent Application No. PCT/EP2021/072830 filed on 17 Aug. 2021 and claims benefit of and priority to German Patent Application No 10 2020 121 574.6 filed on 17 Aug. 2020.
The invention relates to a sensor system and a method for sensing dielectric particles of biological materials in fluids.
Bacteria and other forms of biological material are found ubiquitously on earth and are central to major ecological processes. Almost all bacteria are harmless. However, there are a few pathogenic bacteria which are able to cause diseases in humans and animals. The analysis of microbial isolates and communities to determine the identity and quantity of the bacterial is currently time-consuming and requires expensive culture-dependent or molecular biology-dependent methods.
Identification and quantification methods for bacteria are important to medical diagnostics, to food and water safety control measures, and to basic and applied microbiological research. The classical methods for microbiological diagnostics mainly relied on the growth of known bacteria on a solid media for a long period of time, ranging from hours to days. We know now that 95% of bacteria cannot be cultured with these classical methods. Since approximately 25 years culture-independent, molecular biology-based methods have at least partly replaced the classical approach.
In addition to bacteria, many other types of biological material need to be identified and quantified. It is known that bacteria, as well as the other types of biological material have dielectric properties and therefore could in theory be identified using electronic means.
Electrowetting behavior of mercury and other liquids on variably charged surfaces was first explained by Gabriel Lippmann in a classic paper “Beziehungen zwischen den capillaren und elektrischen Erscheinungen”, Annalen der Physik und Chemie, 225, 546-561. doi:10.1002/andp.18732250807. Nowadays this phenomenon is applied in a wide field of applications ranging from μ-fluidics to camera lenses, large screen e-books, and displays.
In terms of biological material, electrowetting systems have been already reported. Firstly in 2004 Electrowetting-On-Dielectric (EWOD) principle was used for the preparation of biological samples, as taught by Belaubre, P., Guirardel, M., Leberre, V., Pourciel, J.-B., & Bergaud, C. (2004, 2), “Cantilever-based microsystem for contact and non-contact deposition of picoliter biological samples,” Sensors and Actuators A: Physical, 110, 130-135. doi:10.1016/j.sna.2003.09.024.
Since then the development of digital microfluidics in electrowetting platforms is utilized for different manipulation and analyzing processes. For example, Berthier et al., “Mechanical behavior of micro-drops in EWOD systems: drop extraction, division, motion and constraining” NSTI Nanotech 2005, NSTI Nanotechnology Conference and Trade Show, Anaheim, Calif., United States, May 8-12, 2005, described droplet based mechanical manipulations. The development of EWOD-microdevices for biological samples including DNA, proteins, and whole cells are under intense investigation and reveal promising applications. These digital microfluidic devices offer multiple possibilities as a lab-on-chip platform for preparation, manipulation, and analysis.
Dielectric particles in fluids, such as biological cells in a medium, are subjected to a force in a nonuniform alternating electric field depending on their dielectric properties. This phenomenon is called dielectrophoresis (DEP) (in contrast to the exposure of charged particles to a uniform constant electric field, named electrophoresis). Two types of DEP behavior are possible, depending on the geometry of the biological cells, the conductivity of the biological cells, the conductivity of the surrounding fluid or medium and the frequency and amplitude of the electric field. A negative DEP force (nDEP) repels the dielectric particles to a local minimum along the negative gradient of the electric field if the polarizability of the dielectric particles is less than the polarizability of the medium. Therefore, the nDEP force is generally used for continuous extraction of the biological cells under high conductivity liquid conditions.
A positive DEP force (pDEP) on the other hand attracts the biological particles to a local maximum along the positive gradient of the electric field if the polarizability of the biological materials is higher than that of the medium. In general, the pDEP force is used to attract the target cells to electrodes and then release the biological materials from the electrodes using a suspension buffer after the DEP force is removed, as described in Yoon, T., Moon, H.-S., Song, J.-W., Hyun, K.-A., & Jung, H.-I. (2019, 10), “Automatically Controlled Microfluidic System for Continuous Separation of Rare Bacteria from Blood,” Cytometry Part A, 95, 1135-1144.doi:10.1002/cyto.a.23909. A variation in the properties of the electric field, the geometry of the electrodes and the medium thus allows selective manipulation of the biological cells.
DEP is a technique commonly used in μ-fluidics for particle or cell separation and is considered a useful tool for manipulating cells prior to detection. Conventional microbiological methods are time consuming, mainly because they require several growth-based enrichment and separation steps. Compared to other separation methods, DEP has unique advantages such as being label-free, fast, and accurate and offers a possibility to concentrate cells without being restricted by bacterial growth. It has been widely applied in μ-fluidics for biomolecular diagnostics and in medical and polymer research (see Zhang, H., Chang, H., & Neuzil, P. (2019, 6), “DEP-on-a-Chip: Dielectrophoresis Applied to Microfluidic Platforms,” Micromachines, 10, 423. doi:10.3390/mi10060423.
An Electrolyte-Gated Organic Field-Effect Transistor (EGOFET) has been developed to detect the ion concentration of an electrolyte. Measuring results show an alteration of the electrical current in the organic semiconductor channel according to the ion concentration of the electrolyte.
The concept of a floating gate field-effect transistor (FG-FET) was developed for detecting purposes of bacteria and was described in publication by Thomas, M. S., White, S. P., Dorfman, K. D., & Frisbie, C. D. (2018, 3), “Interfacial Charge Contributions to Chemical Sensing by Electrolyte-Gated Transistors with Floating Gates,” The Journal of Physical Chemistry Letters, 9, 1335-1339. doi:10.1021/acs.jpclett.8b00285). Van der Spiegel et al. published first a concept which separates a sensor unit from the measuring unit (see Van Der Spiegel, J., Lauks, I., Chan, P., Babic, D. (1983), “The extended gate chemically sensitive field effect transistor as multi-species microprobe,” Sensor and Actuators, 4, 291-298.). Van der Spiegel documented a wire connection between sensor and the measuring unit.
A system for conducting quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) on a micro-chip is described in a publication by Prakash, R.; Pabbaraju, K.; Wong, S.; Wong, A.; Tellier, R.; Kaler, K.V.I.S. “Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology”. Micromachines 2015, 6, 63-79. https://doi.org/10.3390/mi6010063. The system of this publication uses a combination of electrostatic and electrowetting droplet actuation and is capable of sensing respiratory viruses, such as Influenza A and Influenza B.
A microchip-based system that is capable of both the extraction and purification of nucleic acids and the conduction of polymerase chain reaction (PCR) is described in a publication by Prakash, R., Pabbaraju, K., Wong, S. et al. “Integrated sample-to-detection chip for nucleic acid test assays”. Biomed Microdevices 18, 44 (2016). https://doi.org/10.1007/s10544-016-0069-8. The microchip-based system uses dielectrophoresis and electrostatic/electrowetting actuation. A droplet-dielectrophoresis (D-DEP) is used for passive, continuous and unidirectional transport of droplets. The dielectrophoresis and the electrostatic/electrowetting are sequentially arranged in the Prakash et al publication which allows droplet-manipulation before and after the dielectrophoresis. The dielectrophoresis is used for immobilizing, concentrating, and sorting of the particles.
An extended-gate type organic field-effect transistor (OFET)-based sensor system for sensing human immunoglobulin A (IgA) is described in a publication by Minamiki, T., Minami, T., Sasaki, Y., Kurita, R., Osamu, N. I., Wakida, & Tokito, S. (2015), “An Organic Field-effect Transistor with an Extended-gate Electrode Capable of Detecting Human Immunoglobulin A,” Analytical Sciences, 31, 725-728. doi:10.2116/analsci.31.725.
This document teaches a sensor and a method which enables quantification, sorting, and characterization of biological materials, such as bacteria, unicellular, or other small cellular organisms, from different sources.
The method and sensor use a combination of electrowetting-based microfluidics in combination with a floating-gate field effect transistor and dielectrophoresis.
It is envisaged that the sensor and method will enable broad applications in medical diagnostics, food and water safety, agriculture, but also in basic microbiological research can be envisaged.
The sensor system for sensing dielectric particles of biological material in fluids comprises a plurality of electrodes arranged on a substrate and, in one aspect, a dielectrophoretic device arranged on the substrate adjacent to one of the plurality of electrodes. The sensor system further comprises at least one floating gate field effect transistor arranged on the substrate and wherein the dielectrophoretic device is connected to the gate electrode of the floating gate field effect transistor. The dielectric particles of biological material are, for example, bacteria, unicellular or other small cellular organisms.
The dielectrophoretic device can be directly or indirectly connected to the gate electrode of the field effect transistor.
The substrate has a hydrophobic coating to reduce the angle of contact between a surface of the substrate and drops of the fluids to enable the drops of the fluids to move about the substrate using electrowetting techniques. The substrate can have a structured surface to reduce the area of contact between drops of the fluid and the substrate. The electrodes can be arranged as an active matrix and can be independently switchable.
The system can be used in a method sensing dielectric particles of the biological materials in the fluid droplet with at least one other fluid droplet. In this case, the method comprises placing one or more of the fluid droplets with the dielectric particles on one or more of the plurality of electrodes, applying a potential to ones of the plurality of electrodes to move the fluid droplet with the dielectric particles from one of the plurality of electrodes to a dielectrophoretic device. The dielectrophoretic device can be connected to the gate electrode of the field effect transistor. The method can further comprise the steps of applying a potential to the dielectrophoretic device to immobilize the dielectric particles on the dielectrophoretic device and to sort or concentrate the dielectric particles and measuring the current through the channel of the field effect transistor.
The method can further comprise the step of changing a value or frequency of the potential applied to the dielectrophoretic device to sort different ones of the dielectric particles.
The invention will now be described on the basis of the drawings. It will be understood that the embodiments and aspects of the invention described herein are only examples and do not limit the protective scope of the claims in any way. The invention is defined by the claims and their equivalents. It will be understood that features of one aspect or embodiment of the invention can be combined with a feature of a different aspect or aspects and/or embodiments of the invention.
The dielectrophoretic electrodes 65 are made, for example of transparent indium tin oxide, but this is not limiting of the invention and other materials can be used. The arrangement of the dielectrophoretic electrodes 65 shown in
The operation of the sensor system 10 will now be explained with respect to
In a next step 210, the potentials on the plurality of electrodes 50 is changed such that the droplets 35 of the fluid 30 move to the right as is shown in
The potentials on the plurality of the electrodes 50 are then changed so that the droplets 35 of the fluid 30 can move in step 230 further to the right, as is shown in
It will also be seen in
The method of applying a change to the potential of the plurality of the electrodes 50 and thereby moving the droplets of the fluid 30 continues and more and more dielectric particles 20 of biological material will be collected on the interdigital electrodes 65 of the dielectrophoretic device 60. The dielectric particles 20 are polarized which means that the potential at the interdigital electrodes 65 will change due to the charges of the biological materials.
The simple system shown in
A further embodiment of the system is shown in
After staining, the droplet 35 of the fluid 30 (with the dye from the dye droplet 35′) can then be moved back from the bottom electrode 35 to the dielectrophoretic device 60 and washed by using a wash droplet (35″) from the adjacent electrode on the left of the dielectrophoretic device (which has no biological material in it). The potential is applied to the interdigital electrodes 65 and the stained biological materials are collected at the interdigital electrodes 65. The wash droplet 35″ is passed through the dielectrophoretic device 60 and moves from the dielectrophoretic device 60 to the adjacent electrode on the right of the dielectrophoretic device 60. The wash droplet 35″ removes the redundant dye from the dielectrophoretic device 60 and the washed dielectric particles 20 of biological material remain on the dielectrophoretic device 60.
It is possible to use a second dye to stain the biological material if the electrodes 50 above the dielectrophoretic device 60 are used.
It will be appreciated that the use of the bottom electrode 35 to enable staining of the biological material will also enable reagents in reagent droplets 35′ to be applied to the biological materials in the droplet 35 of the fluid. The reagents are applied instead of the dye.
In a further aspect, the droplet 35 of the fluid 30 does not need to be moved from the dielectrophoretic device 60 to the bottom electrode 50. As long as the reagent has a small electric charge, it would be possible to keep the droplet 35 of fluid 30 with the dielectric particles 20 on the surface of the dielectrophoretic device 60 and move the reagent droplet 35′ over the surface of the dielectrophoretic device 60.
In a further aspect of the system, one of the electrodes 50 or the dielectrophoretic device 60 can be connected to the gate of at least one floating gate field effect transistor 80. This dielectric charge in the biological materials changes the potential of the gate electrode and thus the current through the floating gate field effect transistor 80 (as explained in Minamiki, T., Minami, T., Sasaki, Y., Kurita, R., Osamu, N. I., Wakida, & Tokito, S. (2015), “An Organic Field-effect Transistor with an Extended-gate Electrode Capable of Detecting Human Immunoglobulin A,” Analytical Sciences, 31, 725-728. doi:10.2116/analsci.31.725). The concentration of the dielectric particles on the interdigital electrodes 65 of the dielectrophoretic device 60 can be used to change the potential on the gate of the floating gate field effect transistor 80 and thus enable detection of even small amounts of biological material with dielectric polarization. The floating gate field effect transistors 80 are, for example, organic field effect transistors.
The connection of one of the electrodes 50 or the dielectrophoretic device 60 to the gate of the at least one floating gate field effect transistor 80 enables the electrode 50 to be used as a multifunctional electrode. The electrode 50 can thus be used for sorting/moving droplets 35 of the fluid 30, for collecting the biological material and as well for detection of the biological material with one single electrode 50. It is therefore not necessary to transport the droplets 35 of the fluid 30 from the electrode 50 that is used for sorting/moving the droplets or for collecting biological material to another electrode 50 that is used for detecting biological material.
On one aspect, the electrodes of the sensor unit 90 are functionalized with modified porphyrins, as explained below, to link bacteria, unicellular, or other small cellular organisms (a biological material) on the electrodes' surfaces. The trapped bacteria, unicellular, or small cellular objects shift the potential at the electrode/suspension interface which, as noted above, affects the voltage on the gate 85 and has an impact on the electrical conductivity of the FG-FET 80 (see Minamiki, T., Minami, T., Sasaki, Y., Kurita, R., Osamu, N. I., Wakida, S.-i., & Tokito, S. (2015), “An Organic Field-effect Transistor with an Extended-gate Electrode Capable of Detecting Human Immunoglobulin A,” Analytical Sciences, 31, 725-728. doi:10.2116/analsci.31.725). The linkage of the bacteria or the unicellular or other small cellular organisms is therefore reflected in changes of the directly measurable current between drain and source electrode of the FG-FET 80.
One example of the functionalization of the gate electrode 85 is a porphyrin structure as self-assembled monolayer, as shown in
If the dielectric particles 20 (in this case bacteria) are linked through the linkers shown in
In order to only focus on the impact of the modification on the electrical behavior of the FG-FET 85 and to exclude other influences resulting from the potential use of non-standard thin-film devices, a hybrid setup using standard SMD-FETs and a thin-film sensor unit is first created. Different modifications of the functionalized porphyrin were analyzed with different characterization methods as UV/V is spectroscopy, infrared reflection absorption spectroscopy (IRRAS), drop shape analysis (DSA), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).
The impact of the functionalized gate electrode 85 on the electrical characteristic of the FG-FET 80 were verified and is visualized in
The gate electrode 85 can also be functionalized by the synthesis of the porphyrin structure, as shown in
The impact of the functionalized gate electrode 85 on the electrical characteristic of the FG-FET 80 were verified using the equivalent circuit according to
In a further aspect of the system, the interdigital electrodes 60 are structured to allow patterns of biological material to grow on the surface of the substrate 40. The surface 40 can be cleaned by merely turning off the potential from the voltage source 70.
As noted above, the dielectric particles 20 are biological materials and include, but are not limited to, bacteria, unicellular and other small cellular organisms (e.g., yeasts, and other unicellular fungi).
The movement of the droplets of fluid 30 is dependent on the properties of the surface 40. The surface 40 can have a hydrophobic coating 42, such as but not limited to, parylene, applied to reduce the angle of contact between a surface of the substrate 40 and the droplets of the fluids 30. This enables the droplets of the fluid 30 to move easily between the plurality of the electrodes 30 and the dielectrophoretic device 60.
In a further aspect, the substrate 40 has a structured surface 44 to reduce the area of contact between drops of the fluid 30 and the substrate 40. This reduces the transfer of thermal energy between the droplets of the fluid 30 and the surface 40.
The electrodes 50 in the sensor system are arranged in a matrix-fashion and are independently switchable. The matrix of the electrodes 50 can be programmed as appropriate
10 Sensor system
20 Dielectric particles of biological material
30 Fluid with dielectric particles
35 Droplet
35′ Other fluid droplet
35″ Wash droplet
40 Substrate
42 Hydrophobic coating
44 Structured surface
50 Electrodes
60 Dielectrophoretic device
65 Dielectrophoretic electrodes
70 Voltage source
80 Floating gate field effect transistor
85 Gate
90 Sensor unit
92 Reference electrode
94 Suspension of biological material
Number | Date | Country | Kind |
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10 2020 121 574.6 | Aug 2020 | DE | national |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2021/072830 | 8/17/2021 | WO |