Electromechanical lysis of bacterial pathogens using ion concentration polarization
1. A method of lysing a cell membrane comprising the steps:
- a. directing a fluid stream containing cells in a channel comprising an inlet and an outlet and defined, at least in part, by at least a first ion exchange membrane and at least a second ion exchange membrane, wherein the ion exchange membranes are juxtaposed and characterized by the same charge;
b. applying an electric field across the channel at a voltage and duration sufficient to cause helical electroconvective vortex formation across the channel, thereby lysing the cell membranes of the cells;
c. collecting an output fluid stream comprising lysate from the outlet; and
d. isolating the lysate from the output fluid stream.
Scalable, high throughput and power-efficient electromechanical lysis using low electric potential, which can be used for harvesting valuable intracellular biomolecules (DNA, RNA, and proteins) and metabolites (e.g., biodiesels, bioplastics, antibiotics, and antibodies), and for sterilizing large volume solutions (e.g. disinfection of bacterial contaminated drinking water). The method can be directly integrated with other microfluidic devices for all-in-one, fully integrated total-analysis systems for various bacterial (and cellular) studies and clinical applications.
|Water Desalination/Purification and Bio-Agent Preconcentration by Ion Concentration Polarization|
Patent #US 20140374274A1
Current AssigneeMassachusetts Institute of Technology
Sponsoring EntityMassachusetts Institute of Technology
|PURIFICATION OF ULTRA-HIGH SALINE AND CONTAMINATED WATER BY MULTI-STAGE ION CONCENTRATION POLARIZATION (ICP) DESALINATION|
Patent #US 20160115045A1
Current AssigneeMassachusetts Institute of Technology
Sponsoring EntityMassachusetts Institute of Technology
|Ion concentration polarization-electrocoagulation hybrid water treatment system|
Patent #US 9,850,146 B2
Current AssigneeMassachusetts Institute of Technology
Sponsoring EntityMassachusetts Institute of Technology
- 1. A method of lysing a cell membrane comprising the steps:
a. directing a fluid stream containing cells in a channel comprising an inlet and an outlet and defined, at least in part, by at least a first ion exchange membrane and at least a second ion exchange membrane, wherein the ion exchange membranes are juxtaposed and characterized by the same charge; b. applying an electric field across the channel at a voltage and duration sufficient to cause helical electroconvective vortex formation across the channel, thereby lysing the cell membranes of the cells; c. collecting an output fluid stream comprising lysate from the outlet; and d. isolating the lysate from the output fluid stream.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
This application claims the benefit of U.S. Provisional Application No. 62/523,404, filed on Jun. 22, 2017. The entire teachings of the above application are incorporated herein by reference.
This invention was made with Government support under Grant No. R01 AI117043 awarded by the National Institutes of Health. The Government has certain rights in the invention.
Lysis is the disruption of the cell membrane, which is a standard process for not only eliminating pathogens but also accessing intracellular contents such as nucleic acids, proteins, metabolites, and other organelles. In particular, the extracted biomolecules from mammalian or microbial cells provide essential information about genetic or disease characteristics1. Thus, the cell lysis is the first procedure for various biological and clinical studies, including genomics, proteomics and metabolomics, with a wide range of applications in medicine and pharmacy, water-food-energy industry, agriculture, and for recovering of valuable intracellular products from recombinant cells.
Many conventional techniques have been developed to secure the highest yield and purity of the lysates from various organisms; among them, chemical, mechanical, and other physical methods were commonly employed. Chemical (detergents) or enzymatic permeation of the cell membrane was an attractive way of recovering lysates due to the simple operation and high lysis efficiency2-4, but the added reagents and proteins often hindered particular reactions and/or damaged lysates, resulting in narrow choices for downstream assays5. In addition, the chemical composition and concentration needed to be specifically optimized according to organisms so that it was difficult to be applicable for globally lysing various species in a complex cell mixture. By contrast, mechanical methods such as bead beating6, 7 and sonication8, 9, versatilely lysed any cell types without addictive ingredients10; however, they often require bulky and expensive equipment, and the lysis efficiency and recovery rate of lysates fluctuated greatly due to uncontrollable mechanical shearing of released intracellular biomolecules according to the apparatus and operational conditions8, 11. Moreover, it was often inefficient to apply the conventional mechanical lysis, which required somewhat large volume solutions to operate (>1 mL), using modern biochemical analysis tools (e.g., Nanostring) that only utilized a small quantity of samples (<50 μL) for executing genetic analysis12.
Electrical cell lysis would be a preferred method for microfluidic systems because the operational setting was simple without lytic additives and enabled prompt lysis using a sub-microliter solution with a wide range of cellular density (1-107 cells/mL)5, 13. Furthermore, the miniaturized electrical lysis module was directly integrated with post-processing elements, resulting in on-line, all-in-one, in-situ, and accurate analysis of the lysates14. However, for small cells such as bacteria (approximately 1 μm long and 0.5 μm thick), the required electric field was extremely high (>15 kV/cm)15 in order to satisfy the transmembrane potential for lysis (˜1.5 V)16, which might induce negative effects associated with high electric power, including biomolecule degradation, Joule heating, and water dissociation.
To alleviate the issues, the bacterial lysis by an electric field was only performed either in a low salinity solution (e.g., distilled water)17 for minimizing the current density or using a pinched microchannel (25 μm width)18 and small electrode gap (10-20 μm)19, 20 to reduce the electric potential. This eventually required additional steps to exchange buffers, and resulted in extremely low lysis throughput (<1 μL/min)18, 20-22. In this context, it was difficult for electrical lysis to produce enough quantity of lysates to implement off-chip post processing and analysis such as mass spectrometry or capillary electrophoresis, which generally required at least 100 μL solutions for handling. Recently, electrical cell permeation that also takes advantage of mechanical agitation (vortex) was reported to minimize the required electric field for permeating the membrane of mammalian cells23. However, challenges still exist in achieving reliable bacterial lysis that can be versatile and yet generally applicable to a wide range of bacterial pathogens, utilizing a low electric field and providing high throughput.
Here we disclose a novel bacterial lysis mechanism to take advantages of both mechanical shearing and electrical permeation, so called “electromechanical lysis”, enabling a rapid, continuous, versatile, and high-throughput lysis of hard-to-lysis bacterial pathogens by only applying substantially low electrical potentials (a few tens volts). Our invention involves the following specific innovations and technological advances:
- 1. Demonstration that Ion Concentration Polarization (ICP) generated near Ion Selective Membranes (ISMs)24, 25 facilitates electrical lysis of bacterial cells, owing to formation of anomalously fast electroconvective vortexes25-27. The electroconvective vortex concentrated and agitated bacterial cells toward the ISM walls where electric fields and ionic concentrations were spatiotemporally enhanced and fluctuated26-28, inducing additional mechanical shearing and bombardment by the ISMs.
- 2. Characterization of the bacterial lysis in various electrical and ionic conditions, and demonstration that a low electric field (20-60 V) enables bacterial lysis, resulting in not only minimization field-associated negative effects but also utilization of highly salted solutions (e.g., 150 mM buffer).
- 3. Operation of the electromechanical lysis in a continuous and programmed manner to achieve the higher lysis throughput and lysate yield.
- 4. Successful recovery of intracellular biomaterials such as proteins and Ribonucleic acids (RNAs) from both easy-to-lysis (Escherichia coli, E. coli) and hard-to-lysis (Mycobacterium smegmatis, M smeg) bacterial pathogens that typically required extremely high electric potentials (e.g., 10-20 KV/cm)15, 17.
- 5. Demonstration that a microfluidic lysis device can be highly scaled-up due to the simple operational principle that only required a fluidic channel between two ISMs, resulting in ultra-high-throughput (>1 mL/min) electrical lysis of bacterial pathogens in a power-efficient and portable manner.
The invention encompasses a method of lysing a cell membrane comprising the steps:
- c. collecting a fluid stream comprising lysate; and
- d. isolating or collecting the lysate.
Since lysis is the starting point of various post analysis of bacterial cells, the invention would have a wide impact on not only fundamental studies on biomolecular studies but also industrial applications including, but not limited to, water disinfection, wastewater treatment, aquarium cleaning, food/beverage sterilization and recovery of valuable metabolites in biorefinery and pharmaceutical industries.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
A description of preferred embodiments of the invention follows.
The invention encompasses a method of lysing a cell membrane and methods of removing bacteria from a fluid stream, including, for example, a method of disinfecting water and a method of treating wastewater. The methods comprise the use of Ion Concentration Polarization (ICP) generated near Ion Selective Membranes (ISMs) to induce electrical lysis of bacterial cells. Ion Concentration Polarization has been described, for example, in Kim et al., Nat Nanotechnol 5, 297 (2010) and U.S. Pat. App. Pub. No. 20140374274 A1 (entitled “WATER DESALINATION/PURIFICATION AND BIO-AGENT PRECONCENTRATION BY ION CONCENTRATION POLARIZATION”), the contents of each of which are expressly incorporated by reference herein. Ion exchange membranes (IEMs) act as an ion filter by allowing only cations or anions to pass through. This selective ion transport initiates a unique phenomenon called ion concentration polarization (ICP) near the membranes, which is characterized by significant, dynamic perturbation in ion concentrations (also known as ion depletion and ion enrichment).
The ion exchange membranes are cationic or anionic exchange membranes. The two membranes can be the same or different. Strong anion or cation exchange membranes, as those products are generally sold in the art, are preferred. NAFION™ membranes, FUMASEP® FTAM-E and FTCM-E (FuMATech CmbH, Germany) are suitable membranes. However, others can also be used. In particular, the term “ion exchange membrane” is intended to include not only porous, microporous or nanoporous films, but also resins or materials through which ions can pass. Thus, in one embodiment, an ion exchange resin can be entrapped by one or more meshes (or porous membranes) in lieu of or in addition to one or more of the ion exchange membranes.
The ion exchange membranes can be placed into a support, such as glass, polydimethylsiloxane (PDMS), or other inert material. Thus, the support can also contribute to the formation of the channels.
The first channel (defined by the ISMs) can, for example, be a microchannel.
The electric field can be created by an electrode and a ground each located external and parallel to the channel. In general, the electrode forms a second channel with the first of said two juxtaposed ion exchange membranes and the ground forms a third channel with the second of said two juxtaposed ion exchange membranes. These channels are generally filled with an electrolyte solution, for example, phosphate buffered saline (PBS).
The lysis can be conducted in a batch, continuous, or semi-continuous manner. In certain aspects, the lysis is conducted in continuous manner. In certain additional aspects, the lysis is a semi-continuous electromechanical lysis process. For example, the fluid stream comprising the cells can be directed to the first channel at a semi-continuous flow.
The methods can be used to isolate intracellular biomolecules, including, but not limited to, DNA, RNA, and proteins, and/or to isolate or harvest bacterial metabolites including, for example, biodiesels, bioplastics, antibiotics, and antibodies. The method can further comprise isolating a bacterial protein and/or a bacterial nucleic acid (for example, RNA) from the lysate.
The invention also encompasses a method comprising the use of a plurality or channels in parallel, each defined by an ion exchange membrane.
Working Principle of Electromechanical Lysis
Consequently, the bacterial cells were exposed to spatiotemporally unstable electrical and fluidic fields, resulting in bacterial lysis by the electrical and mechanical synergic forces.
Visualization and Quantification of the Lysis Performance
A bacterial solution (˜5 μL) was introduced to the lysis channel and a DC electric potential (20-60 V) was applied across the electrolyte and lysis channels (i). After lysis, the lysis channel was flushed with an elution buffer (100 μL, 1×PBS) (ii). Lysates and remaining bacterial cells at the outlet reservoirs were then collected for quantification (˜105 μL) (iii). Both bacterial population and background GFP intensity were measured before and after the lysis process by simple image processing. The un-lysed bacterial cells showed higher GFP intensity than the background (red spots in
Distinguishingly, bacterial lysis was enabled within a few minutes by applying only 20-60 volts across the electrolyte and lysis channels, which is an extremely small electric field strength (100-300 V/cm) compared to that required for previous electrical lysis techniques for bacterial cells (10-20 kV/cm)15. It is noted that the electric field of the electromechanical lysis was estimated by assuming no potential loss at the electrode-electrolyte interfaces and along the electrolyte channel, so that the actual portion consumed for the bacterial lysis would be lower than the calculated value. In this context, our electromechanical approach enabled bacterial lysis by only applying a small electric field (<100 V/cm), which was at least 100 times lower electric field than previous bacterial lysis using an electric field. This may be attributed to the additional mechanical shearing applied to bacterial cells by the electroconvective vortices in addition to the applied electric effect that was efficiently focused on the tip of concentrated bacterial plug by the ICP phenomena. Furthermore, as shown in
Continuous and Programmed Electromechanical Lysis
One limitation of the previously described batch-type lysis operation is the requirement of an elution step, leading to unavoidable lysate dilution and discretized operation. To address this, a continuous electromechanical lysis process was developed, which maintains a balance between applied electric fields and bacterial flow rates.
RNA Recovery from Various Bacterial Strains
After achieving the recovery of readily detectable lysates such as GFP, we performed bacterial lysis to secure more challenging and invisible lysates such as RNAs that can play an important role in clinical and cellular studies1.
Parallelization for High-throughput Operation
Another unique advantage of the electromechanical lysis is the scalability by laterally parallelizing the single lysis unit, CEM and lysis channel. This is achieved by employing a unipolar ISM (cation exchange) so that the system is symmetrical and has low complexity, thereby enabling simple stacking and/or parallelization. To demonstrate the scalability, a laterally-arrayed electromechanical lysis device was constructed, consisting of six CEMs and five individual lysis channels, all connected by a common inlet and outlet (
Ultra-high-throughput Device for Water Disinfection
This large-scale device may require further engineering for accurate biomolecular applications but would be highly attractive to secure non-sensitive intracellular metabolites and/or to sterilize large-volume solutions in a cost-effective, energy-efficient, portable, and ultra-high-throughput manner. The throughput (over mL/min) and power consumption (˜0.5 Wh/L in 1.5 mM PBS) obtained in this work is highly competitive to not only other electrical lysis techniques that required extremely high electric fields (˜10-20 kV/cm), but also other sterilization techniques such as ultraviolet light irradiation (˜0.2 Wh/L)35 or heat treatment (e.g., autoclave). Other potential uses include water disinfection, wastewater treatment, aquarium cleaning and food/beverage sterilization.
A novel electromechanical lysis mechanism was presented that can be versatilely available for various bacterial cells, providing highly efficient collection of lysates in a rapid, continuous, and programmed manner by only applying a small electrical field (<100 V/cm). In this work, it was demonstrated for the first time that the ICP phenomena near ISMs facilitated electrical lysis of bacterial cells due to formation of anomalously strong electroconvective vortexes. The vortex can be spontaneously generated without additional treatments by applying an overlimiting potential, and contributed to concentrating and agitating bacterial cells toward the ISM walls where the cell underwent additional mechanical shearing and bombardment by the membrane. Lysis of bacterial cells was achieved by only applying a few tens of volts in a highly salted buffer (e.g., 150 mM) and by maximizing the mechanical and electrical synergic effects. This enabled high recovery rates of valuable intracellular biomaterials such as proteins (>75% yield) and RNAs (>5% yield). The electromechanical lysis operated in a continuous and programmed fashion, which seems to be highly advantageous for integration with other microfluidic modules for on-line downstream assays. It was also demonstrated that the microfluidic lysis device can be highly scaled-up toward multiscale fluidic platforms using layer-by-layer assemble of fluidic channels and ISMs, resulting in ultra-high-throughput electromechanical water disinfection (>99% removal rate) in a power-efficient and portable manner. It is believed that the proposed novel lysis mechanisms will facilitate not only fundamental studies in microbiology due to efficient recovery of intracellular contents (e.g., proteins, nucleic acids, metabolites, drugs, antibodies, bioplastics, and biofuels), but also industrial applications for ultra-high-throughput and portable water disinfection for pathogenic wastewater treatment, aquarium sanitation, and food/water sterilization.
Fabrication and preparation of microfluidic devices. The ISM-integrated microfluidic device was fabricated and prepared according to our previous protocols25, 29. Briefly, a 3-dimensional (3D) master mold was prepared using a stereolithographic technique (3D Systems Inc., Rock Hill, S.C., USA), followed by a standard soft lithography process using PDMS elastomer kits (Dow Corning, Midland, Mich., USA). The PDMS prepolymer mixed with the curing agent in 10:1 (w/w) ratio, which was poured onto the 3D printed master mold and cured in a 65° C. convection oven over 4 h. After curing, the top PDMS replica was integrated with two CEMs (Fumasep FTCM-E, FuMA-Tech GmbH, Germany) by inserting the membrane into the membrane slots, followed by irreversible bonding with the bottom PDMS block by an oxygen plasma (Harrick Plasma, Ithaca, N.Y., USA). Then, the electrolyte and lysis channels were filled with deionized water over 48 h at room temperature. This allowed volume expansion of the ISMs by swelling, forming a tight seal between ISMs and the membrane slots. All lysis channels were coated with 0.01% Pluronic surfactant (F-127, Sigma-Aldrich, Natick, Mass., USA) to minimize non-specific binding between the cells and PDMS surfaces. Then, the channels were flushed again with 1×PBS to remove the residual chemicals and impurity before loading the cells.
Fabrication of the large-scale device. The large-scale plastic-based device was made of the CEMs, carbon electrodes (Fuel Cell Store, Inc., Boulder, Colo., USA), silicon rubber (3.8 mm thickness) and acrylic sheets (5 mm thickness, McMaster-Carr, Elmhurst, Ill., USA). First, the electrolyte and lysis channels and holes were formed in the plastic and rubber sheets by a laser and manual cutting, respectively. Then each layer including CEMs was assembled by a mechanical clapping. After fabrication, all membranes were filled with demineralized water over 48 h and coated with Pluronic surfactant in the same manner with experiments on the microdevices.
Preparation of bacterial cells. We used constitutively GFP-expressing recombinant E. coli, K12 strain to characterize the lysis performances by fluorescent measurement. The wild-type pathogenic strains, E. coli and M smeg, were used for the RNA recovery experiments. The same culture and preparation protocols were used for all the cells36. Shortly, a single colony grown on a lysogeny broth (LB) agar plate was inoculated in a 5 ml LB medium (Sigma-Aldrich, Natick, Mass., USA), and grown to mid-log phase in a rotary-shaking incubator (200 rpm, 36° C.). The culture was centrifuged at 5000×g for 3 min and diluted into 1× or 0.1×PBS solutions at the appropriate concentration based on optical density measurements of the mid-log cultures. We note that all the cells were carefully handled and incubated to protect cellular contamination.
RNA Extraction by Bead Beating and RNA analysis. The cell culture was centrifuged at 13,000×g for 10 min, were then resuspended in 800 μL of Buffer RLT with 1% Beta-mercaptoethanol (Sigma-Aldrich, Natick, Mass., USA). The mixture was then transferred into a lysing matrix in a bead beating tube on ice and shaken vigorously. Bead beating using Mini-beadbeater-16 (BioSpec Products Inc, Bartlesville, Okla., USA) was conducted 10 times (10×60 s), with a 1 min rest time in between on ice. The mixture was centrifuged at 13,000×g for 15 min at 4° C., and the top aqueous layer was collected. 80 μL of aqueous layer was transfer to new tube and mixed with 160 μL of Ampure RNAclean SPRI bead solution (Beckman Coulter Inc, Indianapolis, Ind., USA). Per the manufacture'"'"'s protocol, the RNA sample was washed with 70% ethanol twice and finally eluded in 40 μL of Nuclease-free water. The eluate of RNA sample from SPRI beads was analyzed using on-chip gel electrophoresis with RNA Pico Chip (2100 Bioanalyzer, Agilent technologies, Santa Clara, Calif., USA) and NanoDrop assay (ND-1000 Spectrophotometer, NanoDrop Technologies Inc, Wilmington, Del., USA). The eluate from the microfluidic lysis channel was purified and analyzed by the same RNA handling protocol without the RLT treatment and bead beating process.
Experimental setup and data analysis. An inverted fluorescence microscope (IX71, Olympus, Tokyo, Japan) equipped with a CCD camera (ORCA-ER, Hamamatsu Photonics, Shizuoka, Japan) was used to obtain the optical microscopic and fluorescent images using an open source software Micromanager (NIH, Bethesda, Md., USA). A pressure-driven flow was generated by a syringe pump (PHD Ultra, Harvard apparatus, Holliston, Mass., USA) and constant current and voltage were applied and measured by current-voltage source measurement unit (Keithley 236, Keithley Instruments, Ohio, USA). Platinum electrodes (Sigma-Aldrich, Natick, Mass., USA) were used to exclude electrode and reaction overpotential occurred in the electrolyte rinsing channels. For data analysis and post processing of microscopic images, Image J (NIH, Bethesda, Md., USA) and OriginPro 8 (OriginLab, Wheeling, Ill., USA) were used.
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While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. Unless otherwise indicated, all numbers, for example, expressing quantities and so forth, as used in this specification and the claims are to be understood as being modified in all instances by the term “about.”