WO2020160041A2 - Capteur pour détecter des micro-organismes et procédé correspondant - Google Patents

Capteur pour détecter des micro-organismes et procédé correspondant Download PDF

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WO2020160041A2
WO2020160041A2 PCT/US2020/015478 US2020015478W WO2020160041A2 WO 2020160041 A2 WO2020160041 A2 WO 2020160041A2 US 2020015478 W US2020015478 W US 2020015478W WO 2020160041 A2 WO2020160041 A2 WO 2020160041A2
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anode
cathode
fuel cell
microbes
sensor
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WO2020160041A3 (fr
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Steve Bitterly
Jack Bitterly
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Priority claimed from US16/259,875 external-priority patent/US20190154621A1/en
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M8/00—Fuel cells; Manufacture thereof
    • H01M8/16—Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • a sensor and corresponding process detects microorganisms such as bacteria, molds or viruses and identifies the microorganism.
  • One way to determine the presence of bacteria on or in a patient is through cul turing a sample. Though techniques for aerobic and anaerobic bacteria are different, they are well known. If a physician suspects, for example, that a child has strep throat, a Group A streptococci infection, the physician or nurse takes a mucus sample from the child’s throat with a swab and rubs the swab onto agar in a Petri dish. There, any bacte ria, if present, incubate. After several days, a technician can determine if streptococci are present. One can also culture microorganism from different sites such as around a wound or surgery or in the blood. Similar incubation techniques also work for molds alt hough the culturing techniques may differ. Some bacteria and many molds also resist culturing. But culturing on a medium in a Petri dish is an accepted practice.
  • Delay seems built into culturing. If the physician does not begin treatment until he or she receives positive results, the infection may become serious. Similarly, one sam ple may find one strain of bacteria, but may overlook a more serious, virulent pathogen. Antibiotics administered for the bacteria found in the sample may not be ideal for other strains in adjacent areas. Delaying treatment may allow an infection to grow and cause severe illness or death.
  • Campylobacter jejuni is found in intestinal tracts of animals and birds, raw milk, untreated water and sewage sludge. Most transmission is from contaminated water, raw milk, and raw or under-cooked meat, poultry or shellfish. Campylobacter jejuni is a gram-negative bacterium like bacilli. It uses oxygen as its final electron acceptor.
  • Clostridium botulinum is widely distributed in nature including soil and water, on plants and in intestinal tracts of animals and fish. It grows only in little or no oxygen. Transmission usually is from improperly canned foods, garlic in oil and vacuum- packaged and tightly wrapped food. Infection can be fatal in three to ten days if not treated.
  • Clostridium botulinum is a gram-positive bacterium typically rod-shaped and ar ranged as singles, pairs, or chains. The spores grow under favorable conditions (anaer- obiosis and substrate-rich environments) and produce toxins as they rapidly propagate.
  • Clostridium perfringens is found in soil, dust, sewage and animal and human in testinal tracts. It grows only in little or no oxygen. It is called“the cafeteria germ” be cause many outbreaks result from food left for long periods in steam tables or at room temperature. Cooking destroys the bacteria, but some toxin-producing spores may sur be. Clostridium perfringens are non-motile, rod-shaped, gram-positive bacteria. It is an anaerobic bacterium, which acquires energy by performing anaerobic respiration using nitrate as its electron acceptor.
  • Escherichia coli 0157:H7 is found in the intestinal tracts of some mammals, raw milk and unchlorinated water. It is one of several E. coli strains causing human illness. It usually is transmitted through contaminated water, raw milk, raw or rare ground beef, unpasteurized apple juice or cider and uncooked fruits and vegetables. Person-to- person transmission also can occur.
  • E. coli serotype 0157:H7 is a mesophilic, gram negative rod-shaped (bacilli) bacterium. It can perform complicated metabolism to main tain its cell growth and cell division and possesses operons for transport and utilization of sucrose, urease, and sorbose.
  • Salmonella can be found in the intestinal tract and feces of animals and in raw eggs. Salmonella transmission comes from raw or undercooked eggs, poultry, seafood and meat and from raw milk and dairy products. Salmonella En- teritidis is a rod-shaped, gram-negative, proteobacteria that is non-motile. These facul tative anaerobes are well adapted to survive in conditions with or without oxygen, allow ing them to live in diverse environments. S. Enteritidis require glucose to survive and uses mixed acid heterofermentation of glucose to produce energy. This method of me tabolism releases carbon dioxide and hydrogen gas as bi-products.
  • Streptococcus Group A is found in noses, throats, pus, sputum, blood and stools of humans. It is transmitted from people to food from poor hygiene, ill food handlers or improper food handling. Many outbreaks come from raw milk, ice cream, eggs, lobster, salads, custard and pudding allowed to stand at room temperature for several hours be tween preparation and eating. Streptococcus Group A is a gram-positive bacterium, which is nonmotile and does not form spores. Rather than using aerobic or anaerobic respiration, Group A Streptococcus uses fermentation, a metabolic pathway of lesser efficiency, which makes the cells grow more slowly.
  • Listeria monocytogenes exist in intestinal tracts of humans and animals, milk, soil, leaf vegetables and processed foods. It can grow slowly at refrigerator tempera tures. It is transmitted from soft cheese, raw milk, improperly processed ice cream, raw leafy vegetables, meat and poultry. It causes illness itself instead of from toxins.
  • Listeria monocytogenes are gram-positive rod-shaped bacteria that form single short chains. They do not form spores or branch and are motile via peritrichous flagella at room tem perature (20 Q C - 25 Q C).
  • Shigella (over 30 Types) is found in human intestinal tract. They are rare in other animals. It is transmitted from person to person by a fecal-oral route and by fecal con tamination of food and water. Most outbreaks result from food, especially salads, pre pared and handled by workers using poor personal hygiene. Shigella is a non-spore forming, gram-negative bacterium that aids in the facilitation of intracellular pathogens. Shigella pathogens use a mixed acid fermentation pathway to metabolize substrates. Products of this anaerobic pathway include ethanol, acetic acid, lactic acid, succinic ac id, formic acid, and carbon dioxide.
  • Staphylococcus aureus is found on humans (skin, infected cuts, pimples, noses, and throats). Most transmission occurs through improper food handling. It multiplies rap idly at room temperature to produce a toxin that causes illness. Staphylococci are spherical gram-positive bacteria, which are immobile and form grape-like clusters. The central routes of glucose metabolism are the Embden-Meyerhof-Parnas (EMP) pathway and the pentose phosphate cycle. Lactate is the end product of anaerobic glucose me tabolism, and acetate and CO2 are the products of aerobic growth conditions.
  • EMP Embden-Meyerhof-Parnas
  • Molds can adversely affect the buildings, e.g., by eating through walls, floors, ceilings and internal supports. Molds also produce spores that may be toxic to people. Pulmonary hemorrhage/hemosiderosis, and aspergillosis are examples of diseases associated with mold exposure.
  • Molds require spores to reproduce and spread. Ambient indoor and outdoor air contains large quantities of mold spores. When they land on damp soil or vegetation outdoors, they grow. However, the applicants’ device and process are concerned pri marily with mold growing on damp indoor surfaces and on food, or spores transported through building air ducts. Bread, especially artisan breads made without preservatives, is attacked by molds such as Neurospora crassa. Usually, mold growth is visible, and the offending food discarded. Other food mold is not as easily detected. Aspergillus fla- vus is a common mold on peanuts and may be present on other legumes and grains. That mold generates the toxin aflatoxin, which is a carcinogenic and which can be dead ly if enough is eaten.
  • Mold detection usually relies on sight and smell. Many molds smell bad, so peo ple often determine molds’ presence by smell. Mold colonies may be visible unless they are behind walls, above ceilings, under flooring or in other inaccessible locations. Occa- sionally, people resort to monitoring building air especially if the occupants are sensitive to small quantities of building mold.
  • Air monitoring requires sampling of indoor and outdoor air simultaneously. Be cause mold spores are everywhere, a high concentration of indoor spores becomes rel evant only if the concurrent outdoor sample concentration is lower. However, most ex perts believe that routine air sampling is not beneficial because visual and scent mold detection usually works. But relying on scent to locate a mold may be difficult. In addi tion, many molds are difficult to detect through standard tests. Therefore, air sampling can yield false negative results.
  • Identifying the particular mold usually is not crucial because remediation tech niques are similar for most molds. However, identifying the particular bacteria infecting a person or animal usually is important because particular antibiotics target particular bac teria.
  • microbial fuel cells for power generation typically concentrate on a limited type of bacte ria.
  • the fuel cells also are costly, rigid, and semi-permanent. Rather than generating electricity for power, applicants rely on microbes producing measurable signals, or characteristic signals indicating known pathogens under controlled conditions such as temperature and the type of carbohydrate food source.
  • Applicants’ sensor uses the metabolic reactions of bacteria or other microbes to produce a quantifiable and measurable voltage signal.
  • An electronic pro cessor, recorder or other device reads and records such signals.
  • the biological sensor is inexpensive and disposable and is useful for detecting living pathogens or other living substances.
  • the sensor or detector can be made small ( ⁇ 1 mm 2 or smaller) to large (> multiple m 2 ) and can be constructed from inexpensive and abundant materials. In formation from the device could lead to determining the presence or the particular type and kind of pathogen(s) present.
  • the operational principals of the biosensor senses electrons transferred during metabolic biochemical reactions resulting from microbial and bacterial metabolism. It is known and well understood that bacteria metabolism involves electron transfers, and bacteria act as the catalyst during metabolic chemical breakdown of carbohydrates. These microbial fuel cells require a positive (cathode) and negative (anode) electrode inside the device, and they can be constructed in a dual- or single-chamber configura tion.
  • the dual-chamber configuration consists of two half-cell reactors separated by a polymeric proton exchange membrane (sometimes called“cation exchange mem brane”).
  • the proton exchange membrane should be hydrated with H2O to function properly.
  • the membrane conducts protons from the anode to the cathode but insulates passage of O2 gases and electrons.
  • the anode chamber contains the negative electrode
  • the cathode chamber contains the positive electrode. Oxidation occurs in the anode chamber (electron loss), and reduction occurs in the cathode chamber (electron gain).
  • the proton exchange membrane separates the two chambers and allows protons to migrate freely by electro static forces from the anode chamber to the cathode chamber. The proton exchange membrane also prevents oxygen from the cathode chamber to migrate back through the proton exchange membrane to the anode chamber.
  • Proton exchange membranes typi cally are made of Nafion with pore sizes about of 1.5 nm (0.015 miti) and made of sul- fonated tetrafluoroethylene-based fluoropolymer-copolymer. Other types of membranes may be acceptable.
  • Proton exchange membranes made of Tetratex (ePTFE) also are cost effective and durable. They have pore hole sizes from 0.05 miti to 7 miti in size.
  • the anode and cathode chambers operate in aqueous conditions, but the anode chamber is maintained in a low- or no-oxygen environment.
  • Bacteria metabolizing inside the anode chamber operate in“anaerobic” conditions (i.e., without oxygen).
  • Many bac teria thrive anaerobically including: obligate anaerobe bacteria, facultative anaerobe bacteria, microaerophilic anaerobe bacteria and aerotolerant anaerobe bacteria.
  • the bacteria can transfer electrons to the anode by chemical mediators, direct membrane associated electron transfer, or by“nanowire” linkages ex tending from the bacteria cell wall to the anode.
  • the biosensors may have two“half-cell” reactions that are catalyzed by the met abolic presence of bacteria or other microbes within the sensor. A single-chamber alter native also is contemplated. Inside the sensor, a substrate or carbohydrate material is added that is advantageous to the growth of bacteria or other microbes. The device can use specific carbohydrates if a physician suspects specific microbes.
  • Each bacterium has its own specific growth rate under controlled fuel-cell condi tions and temperatures. Therefore, each produces its own specific voltage versus time growth-plot.
  • biosensors in an array of sen sors may have different substrates and different inhibitors. Inhibitors are weak, specific antibiotics that prevent growth of specific bacteria.
  • a system including the sensors may use algorithms to analyze out puts from an array of micro-sensors. The results can narrow unknown bacteria down to one or more probable candidates.
  • the sensor uses substrate materials that are advantageous to mold.
  • an tibiotics may be added to the substrate to decrease the chance or eliminate false positive detections of bacteria.
  • Initial building construction or later construction can in stall these arrays at predetermined locations in homes or commercial buildings.
  • the arrays could be attached at the baseboard of the walls or on floors below carpet padding to detect if mold occurs due to accidental flooding. In these examples, the sensors remain dormant until they are activated by mold growth.
  • the present device may use partic ular substrates and inhibitors to determine which molds are present.
  • the device may use an array of sensors. Each sensor is designed for a particular mold species. By analyzing the signals from each sensor, the system determines which mold is present by a process of elimination.
  • bandages could include an array of disposable sensors. Sensors may be embedded into a patient during or after surgical procedures. These embedded sensors may be small (needle size), which may facilitate their remov al after healing or after the critical period of concern. Though applicants discuss physi cians and human patients, veterinarians can use similar sensors on animals.
  • biosensor device and system include inexpensive and disposable sensors for the food industry to determine whether meats, cheeses, other dairy products and other food are spoiling. Integrating a small, inexpensive sensor into food packaging could detect bacteria and register a tiny voltage. Applying the voltage to voltage sensitive paper could change the paper’s color (e.g., green to yellow to red). Green might mean that no bacteria are detected; yellow could mean that some detec tion of bacteria is present, and red could signal that the food is spoiled. Such a sensor could save food from early disposal after the“use-by date” when the product remains fresh. The absence of bacteria may not be the only reason to reject food after a date. Conversely, products exposed to lack of refrigeration and then re-refrigerated may spoil even before their printed expiration date.
  • the background signal from normal metabolic processes can be subtracted from the total signal potential when unwanted pathogens are present and multiplying. Further, by categorizing the magnitude of this electrical growth potential for various pathogens in unison and togeth er, the resulting growth potential can predict quantitatively the presence of the particular pathogen(s) present because the growth increase is different and can be known for dif ferent pathogens at different temperatures.
  • the categorized growth rate is also determined relative to the surrounding temperature because temperature affects the pathogen growth rate.
  • the categorical knowledge from the measured output signals strongly indicates the type or kind of pathogen present by a process of elimination when measur- ing the growth rate signal at measured temperatures and comparing these signals to the baseline categorical database signals. In this way, it is possible to predict with higher probability the specific pathogen(s) present by subtracting out the baseline signal at the measured temperature and comparing it to the pathogen databases.
  • the technique for measuring the electrical signal output from the unknown path ogen or microorganism involves strategically placing the sensor(s) in the area of interest and waiting for the random migratory passage of the pathogens from their present loca tion into the sensor(s). Because of this uncertainty when, or whether, a pathogen will encounter the sensor, choosing the size, placement and number of sensors should in crease the probability of detecting any pathogens. Making the sensors of varying sizes also may increase the probability of detection. Therefore, sensors may range in size. Some may cover larger surface areas. This flexibility assists in enhancing the probability of measuring the presence of a pathogen.
  • sensors could be made as small as the diameter of a surgical needle using nanotechnology. Therefore, one or more may be implanted into human or other living tissue as“real-time” incubators with the ability to measure and or detect the growth of bacteria. Similarly, miniaturizing the sensors could allow an array of sensors to remain small and manageable.
  • Each bacterium has its own specific growth rate under controlled fuel-cell condi tions. Therefore, each produces its own specific voltage versus time growth-plot. Sen sors for each microbe could have a different substrate that is advantageous to a specific bacterium. Specific antibiotics act like specific inhibitors to the individual bacteria.
  • the process can use an algorithm that reviews the outputs from a plurality of sensors and eliminates microbes from sensors that generate no signals or signals below a threshold. Therefore, the method narrows the unknown microbe down to the most probable candidates.
  • One proposed sensor involves the utilization of two separate cavities - one con taining an anode (negative terminal) along with the pathogen and a food source (such as carbohydrates), and one containing a cathode (positive terminal) with moisture (wa- ter) - separated by a semipermeable membrane (proton exchange membrane) allowing charge transfer across the semipermeable membrane.
  • a single chamber device also is contemplated.
  • the sensors can use inexpensive, disposable and flexible materials.
  • the bio sensors may be made by laminating flexible, thin, porous plastic or other sheets togeth er with internal electrodes, anode and cathode, separated by a semipermeable mem brane (proton exchange membrane) between the electrodes.
  • the outer, thin sheets surrounding the anode use porous materials to allow pathogens to migrate into the anode to act as the catalyst if patho gens are present to sense the electrical signal occurring as the pathogens metabolize the carbohydrates or glucose fuels seeded in the anode cavity.
  • porous mate rials surrounding the cathode allow the free exchange of gases and oxygen into the cathode chamber.
  • the porous material could be Gore-Tex ® , an expanded and porous polytetrafluo- roethylene (ePTFE), or a similar material.
  • Gore-Tex used for clothing has about 9 billion pores/in 2 or around 1.4 billion pores/cm 2 .
  • the average pore has a length of about 1 1 m-in or 27 m-cm (0.27 m-m).
  • Typical smaller bacteria have dimensions of about 8 m-in or 20 m-cm (0.20 m-m), semipermeable membranes made with ePTFE having pore sizes used in clothing would probably form biofilms and block most all bac teria entrance into the biosensor.
  • ePTFE with a size of 0.5-1.5 m-m can easily infiltrate the pores of ePTFE, which are stretched to 10-30 m-m in size. See Jin YJ, Kang S, Park P, Choi D, Kim DW, Jung D, Koh J, Jeon J, Lee M, Ham J, Seo JH, Jin HR, Lee Y.,“Anti-inflammatory and antibacterial effects of covalently attached biomembrane-mimic polymer grafts on Gore- Tex implants.” ACS Appl Mater Interfaces 9:19161 -19175 (2017). Therefore, ePTFE with large pores could be a suitable option as the outside semipermeable membrane.
  • the top sheet 130 could also be made of a similar porous semipermeable material to allow O2 exchange from the cathode end.
  • a signal processor such as a computer or other electronic device can read, ana lyze and process signals generated by the sensors.
  • the output of the sensor can act on disposable electro-photo-sensitive paper, which can change color based on accumulated charge.
  • the color sensor could be built into the top portion of the sensor itself.
  • the senor could be useful for medical bandages or packaging for perishable foods.
  • color sensors sensitive to different voltages or signals one might use an array of voltage sensitive papers sensitive to different voltages to deter mine which microbe is present.
  • the system also could amplify the output signals into a recording device for data logging and post-processing. This approach could be useful for real-time data collection in Petri-dish laboratory growth samples or recording of data from air conditioner vents in buildings, hospitals or even residences.
  • the sensors may be connected electrically parallel or in series. There may be benefit to series connections for amplifying the output signal by summing the signal from multiple sensors.
  • FIG. 1 is a schematic of a conventional biological fuel cell.
  • Fig. 2 is a side, sectional view of applicants’ two-chamber biological fuel cell sensor.
  • FIG. 3 is a plan view showing an array of applicants’ biological fuel cells.
  • Fig. 4 is a schematic of any array of applicants’ biological fuel cells in electrical series.
  • Fig. 5 is a chart showing the size range for various microbes, other molecules and atoms.
  • FIG. 6A is a perspective view of an adhesive bandage with applicants’ biological fuel cells.
  • Figs. 6B and 6C shows the bandage applied to the back of a person’s hand.
  • Figs. 7A and 7B are figures of a food package containing meat and applicant’s biological fuel cell being exposed to potential contamination from microbes.
  • Fig. 7B shows the food being removed from the package, and a portable device can read the output of the fuel cell.
  • Fig. 8 is a schematic of a one-chamber biological fuel cell.
  • FIGs. 9 and 10 also are schematics of a one-chamber biological fuel cell
  • a microbial fuel cell MFC is a biological reactor that converts chemical energy present in the bonds of organic compounds into electric energy through the catalytic re actions of microorganism, typically in anaerobic conditions. They use biocatalysts for the conversion of chemical energy to electrical energy.
  • Microbial fuel cells share similarities with conventional fuel cells, but instead of relying on inorganic catalysts like platinum or other noble metals, they use biocatalysts such as enzymes or whole living organisms as catalysts for converting chemical energy into electricity.
  • Microbial fuel cells can operate in two ways. They can use biological catalysts - enzymes extracted from biological systems - to oxidize fuel molecules at the anode and to enhance oxygen reduction at the cathode of the fuel cell. Whole microbial cells also can be catalysts in the fuel cells. In both cases, electrical coupling of the biological components of the system with the fuel cell’s electrodes must occur. Molecules known as electron-transfer mediators can provide efficient transport of electrons between the biological components, enzymes or microbial cells, and the anode electrodes of the fuel cell. Further, integrated biocatalytic systems that include biocatalysts, electron-transfer mediators and electrodes have been recently developed and utilized in fuel cells.
  • the typical chemical reactions inside the anode and cathode chambers for anaerobic bacte ria include CxFIyOz + FI2O -> CO2 +e _ + Fl + (anode) and O2 + 4H + + 4e _ -> 2FI2O (cath ode).
  • the Fig. 1 schematic shows two chambers, cathode chamber 12 and anode chamber 14.
  • the cathode chamber contains cathode 18 in a liquid medium such as wa ter or hydrogen peroxide.
  • the anode chamber contains anode 20 suspended in a liquid medium.
  • Bacterium 16 is suspended in the anode chamber’s medium.
  • a con ductor 22 electrically connects the anode and cathode through a load (resistor). Elec tronics or other devices (not shown) can read the current or voltage.
  • Nicotinamide adenine dinucleotide is an important cell coenzyme.
  • NAD oxidation-reduction
  • NADFI reduced nico tinamide adenine dinucleotide
  • NAD+ oxidized
  • the removal of hy- drogen atoms accomplishes most oxidation.
  • the reaction is NAD + + 2H -> NADH + H + .
  • Fig. 1 shows the reaction schematically including the Fl + ions 24.
  • Electron-transfer mediators MED may also be present to assist the reaction. Some microbes are more resistant than others are to the redox reaction and may need mediators to help strip off electrons.
  • NAD also is involved with glycolysis. Glucose oxidizes to pyruvic acid. NAD + is the oxidizing agent, which reduces to NADFI. Pyruvate oxidizes further to CO2, 26 in Fig. 1.
  • the redox reaction varies in different microbes. Though the redox mechanism is similar, the rate of the reactions varies and depends on the cell’s metabolism. There fore, the voltage between the anode and cathode varies depending upon microbe type, temperature and growth time.
  • the dual-chamber sensor works by forming two half-cell chambers separated by a semipermeable membrane (proton exchange membrane).
  • the reaction can occur in applicants’ sensor such as sensor 100 (Fig. 2).
  • This sensor has two, half-cell chambers, anode chamber 1 10 and cathode chamber 130.
  • the anode chamber has an outer sem ipermeable wall 1 12 that may be formed of flexible material, allowing free passage of microbes and moisture into the anode chamber.
  • the cathode chamber in cludes a semipermeable outer wall 132, allowing passage of oxygen into the cathode chamber.
  • Semipermeable membrane 150 (proton exchange membrane) separates the anode and cathode chambers.
  • Attached applicants mean that the ends attach directly to each other or that the ends of semipermeable membrane 150 may be intermediate at the ends. Those ends may be heat-sealed or otherwise attached to semipermeable membrane 150.
  • sensor 100 can be made thin, flexible, inexpensively and disposable, features that may be useful for many applications.
  • Anode 1 18 is in anode chamber 1 10. It may extend through outer wall 1 12 in Fig. 2.
  • a wire or other electrical connecter may extend through outer wall 1 12 and contact the anode.
  • the anode is shown planar, but it can curve, especially near outer wall end 1 16.
  • cathode 138 in cathode chamber 130 may extend through outer wall 132 and may attached to a wire or other electrical connecter through outer wall 132 to con tact the cathode.
  • the cathode also can curve, especially near outer wall end 136.
  • the anode and cathode connect to signal processor 140, which may be a computer, special electronics or another device for indicating current or voltage between the anode and cathode.
  • anode 1 18 and cathode 138 When choosing the type and kind of material for anode 1 18 and cathode 138, satisfactory attributes may include good conductivity, acceptable physical strength, high surface area, favorable surface properties, good chemical and electrochemical stability and low cost.
  • Graphite fiber cloth, graphite fiber, felt and carbon fiber paper are candi dates for the materials.
  • a graphite powder could be sprayed or brushed onto a semi- permeable membrane for the anode or cathode.
  • Permanent anodes and cathodes used in biochemical fuel cells, which are designed for generating electricity are usually de signed to be permanent. Therefore, their design must account for longer-term use. Ap plicants’ anodes and cathodes are in disposable devices that may operate for a few days more or less.
  • current output is a function of the surface area of the anode and cathode. Because the sensors can be made small, and the output voltage is small, the anode and cathode have a relatively large surface area in the small space of the sen sors’ chambers 1 10 and 130.
  • At least a portion 120 of outer wall 120 of the anode chamber 1 10 is a porous semipermeable membrane having pores large enough for ingress and egress of mois ture and those microbes of interest such as bacteria 122.
  • Fig. 5 shows the sizes of vari- ous microbes. Knowing the sizes is a useful guide for choosing the minimum semiper- meable membrane pore sizes.
  • Bacteria are prokaryotes because they contain no cell nucleus, or any other membrane-bound organelles.
  • the semipermeable membrane may be Gore-Tex ® or similar material. Using wo ven fabric treated to be hydrophilic on the outer surface and a hydrophobic on the inside surface is another possibility, but may not be necessary provided the anode chamber is maintained moist by the source.
  • the semipermeable membrane holds liquid within chamber 1 10.
  • Anode chamber may contain fluid passed through the semipermeable membrane from the source, or it may contain fluid before use.
  • Membrane 120 provides the free-flow of fluid and microbes into the anode chamber.
  • Anode chamber 1 10 also may contain glucose or carbohydrate 124 to attract (chemotaxis) and feed the microbes’ metabolism.
  • outer wall 132 of cathode chamber 130 also could have a porous semipermeable membrane.
  • Cathode chamber also contains liquid, preferably water or hydrogen peroxide, H2O2.
  • liquid preferably water or hydrogen peroxide, H2O2.
  • aerobes organisms that require oxygen or that live in the presence of oxygen, produce hydrogen peroxide naturally as a by-product of me tabolism. Aerobes have enzymes that can decompose low concentrations of h C o water H2O and oxygen O2.
  • Semipermeable membrane 150 is a proton exchange membrane. They usually are made from ionomers. They conduct protons but do not allow gases to permeate. DuPont’s National is a commonly used proton exchange membrane. Gelatinous agar with salt also acts as a proton exchange membrane. Its low cost may make it suitable for applicants’ sensor.
  • H + ions are generated. That gener ates an electrical potential between anode 1 18 and cathode 138, which causes a cur rent to flow from the anode to the cathode.
  • the sensors can be small. That creates a potential issue. If one sensor or a small array of sensors is placed on a wound, a portion of a wound could be badly infected while the area under the sensor could be relatively free of microbes. Similarly, one area could be infected with one pathogen, but the region near the sensor could have a differ ent pathogen. The different pathogens might have dissimilar reactions to antibiotics a physician might prescribe.
  • FIG. 3 shows such an array 200 of sensors 210.
  • the sensors can be held together with adhesive.
  • the array is on tissue 214.
  • the drawing shows 55 sensors in the array. Applicants expect that the array could have more or less sensors.
  • the larger array could be cut into smaller arrays. Cut ting could damage sensors along the cut, but enough sensors will remain to provide suf ficient readings.
  • the electronics could ignore any sensors along the periphery of the ar ray.
  • a large array could have spaces between some sensors so cuts of larger arrays into smaller ones would be along the spaces.
  • Cover 218 (Fig. 3) can connect electrically to each sensor, or the connection can be elsewhere.
  • the electrical output can be a sum of all the signals generated by the sensors 210.
  • the electronics also could provide a readout for each sensor or for a group of adjacent sensors.
  • the sensors could be numbered 1 , 2, 3 ... for the first row along the top in Fig. 3.
  • the next row could be 7, 8, 9 ....
  • the sensors can be linked.
  • the signal from adjacent sensors 1 , 2, 3, 7, 8 and 9 could be combined, and the signals from other groups of sensors also could be combined sepa rately. Combining the output of six sensors is an example and may not be ideal. Using an array of adjacent sensor may help to locate an infected area.
  • Signals from non-adjacent sensors could be combined. If any group shows sig nals indicating infection, the physician would start an antibiotic regimen for the identified infection.
  • the sensors may be connected in parallel or in series. Connecting them in series may be preferable because the tiny voltages from each will add. Connecting the sensors in parallel yields more cur rent, but a parallel connection probably is less desirable because the individual sensors can have mismatched voltages.
  • the process used to manufacture the sensors should insure that no sensor is an open circuit. Even if a few sensors become open cir cuits, applicants believe that with sufficient sensors, the system can have enough re dundancy to account for an occasional open circuit.
  • Fig. 4 shows a schematic of an array 300 of sensors in two rows. Sensors 310 are in one row, and sensors 312 or in the second row. In this arrangement, the voltage between the negative electrode 320 and the positive electrode is the sum of the voltag es from all the sensors in array 300.
  • the sensors are round (shown as circular).
  • An oblong shape may be desirable. Triangular, rectangular, hexagonal and other shapes can pack more tightly, but they have corners in which the reaction may not work as well. Therefore, circular or other rounded shapes may prove beneficial.
  • the two outer material sheets may be fabricated from a semi-porous material to allow the free egress of pathogens to pass into the anode chamber (where the carbohy drate food source is located) and allow fluids to pass freely through the device walls.
  • the sensor or array of sensors could be mounted in or on a bandage.
  • Fig. 6A shows an array of sensors 410 mounted on an otherwise conventional adhesive band age 400.
  • the sensors in the array connect electrically, and the signal can be read at pads 412 and 414.
  • the pads are shown on the inside of the bandage, but the bandage material would have openings (not shown) on the top side.
  • Fig. 6B shows such a bandage 400 on the back of a hand.
  • electrical ac tivity could change the color of the top surface 402 of the bandage. See Fig. 6C.
  • Fig. 7 shows the use of the sensor to monitor food spoilage using applicants’ sensor.
  • Meat, for example steak 500 is contained within a top and bottom sheet of ma terial. Only a top sheet 510 is visible. A single sheet also may be satisfactory. The steak and sheet are within sealable container 530.
  • the food could be monitored constantly. That is, the sensors could be electronically connected to electronic monitors. They also could use color change material. Continuous monitoring of hundreds of food packages in a supermarket likely is impractical. Instead of wired connections, the sensors could connect to an RFID chip that could transmit the sensor signals to a signal processor such as a computer or other local monitor.
  • RFID chips could also monitor mold sensors in regions of buildings by relaying measured signals from the mold sensors short distances back to a nearby staged con trol center.
  • the control center may relay the aggregate room data back to a master con trol station for an entire building or group of buildings. If unknown water damage pro prises mold growth within a room or rooms of a home or commercial building, such an arrangement could pinpoint the infected location for quicker remediation and removal.
  • Another process uses a separate reader 520.
  • Film 510 has two electrodes 512 and 514. When the sensors first contact the food, the sensors act like an uncharged battery because they lack microbes or pathogens within them that have passed into the anode chamber to“activate” the sensor and begin the metabolic action inside the anode chamber.
  • Reader 520 has two probes 522 and 524 that can contact the electrodes on the film.
  • the reader may have on-off and function buttons 526.
  • Fig 7B. Function buttons allow for selecting the food being monitored based upon the signal strengths from the bacteria that predominate on different foods. For example, E-Coli is a more common contaminate in meats, raw milk, uncooked fruits and vegeta bles, and Salmonella is less prevalent in vegetables but still found in meats and dairy products.
  • Screen 528 on the reader has a display for showing what microbe is moni tored. Metabolic activity also is displayed.
  • Reader 520 is a small electrical device that reads the electrical voltage output from the sensor films. The reader allows quick reads after microbes in the sensors have had time to incubate and generate an electrical potential. One could connect the reader temporarily connected to the output electrodes 512 and 514 to read any metabolic sig nal. The reader also could be clipped onto the packaging so electrodes 512 and 514 remain connected to the reader’s electrodes 522 and 524.
  • Fig. 2 showed a dual chamber MFC.
  • Fig. 8 shows the construction of a single chamber MFC 600.
  • Anode 610 and cathode 630 may be made of conductive, semiper- meable material. For example they may be carbon paper or carbon fiber cloth that also can be coated with graphite powder spray to facilitate the conduction of current.
  • the anode and cathode should allow liquids to flow through them to permeate the MFC bio sensor.
  • the anode could also be coated with a carbohydrate substance as a fuel source and accelerator to attract the microbes (chemotaxis) to the anode if bacteria 612 are present.
  • Proton exchange membrane 620 may be a coating or film of salt and agar. The spacing between the anode and cathode may vary. It is exaggerated in Fig. 8. There may be a thin structural open-cell material (like foam) placed between the anode 610 and proton exchange membrane 620 to hold the two regions apart.
  • Metabolizing and reproducing microbes generate H+ ions, which produces an electrical potential between anode 610 and cathode 630.
  • the protons, H+ ions pass through the open-cell matrix and through the proton exchange membrane 620 to the cathode 630 and react with O2 from the air to form H2O.
  • the reaction generates a po tential between the anode and cathode.
  • Current flows between the anode and cathode though load 650.
  • the semipermeable membranes forming the anode and cathode allow water and air to pass from the outside into the MFC biosensor.
  • a tougher open-cell screen or permeable material may cover the outside of the anode and cathode to protect them. This tougher material also would be semipermeable to allow bacteria and water to reach the anode easily and air (oxygen) to enter the cath ode, creating water on the cathode surface by the redox reaction with FI+ ions.
  • An alternative single chamber biosensor could have the anode and cathode made of semipermeable material coated with graphite powder spray to facilitate the conduction of current and moisture effusion into the biosensor from the source contain ing the unknown microbes.
  • the anode could also be coated with a carbohydrate sub stance to facilitate a fuel source (chemotaxis) for microbe catalysts.
  • Figs. 9 and 10 are another way of looking at the single chamber MFC.
  • a semipermeable cover 728 allows easy migration of microbes and water into the biosen sor but also protects anode 726 on one side of chamber 720.
  • Proton exchange mem brane 722 is on the other side of the chamber, and cathode 724 is adjacent membrane 722.
  • the proton exchange membrane may be Nation.
  • the anode may be formed of carbon paper, and the cathode may be carbon cloth.
  • An opening 730 into chamber 720 might be provided to monitor the process.
  • chamber 700 is on a source 702 of bacteria such as meat, other food or a wound.
  • semipermeable membrane 712 is adapted to conform to source 702, and anode 710 mounts above the membrane and conforms to the membrane.
  • Coarse foam separator 708 separates the anode from proton exchange membrane 706.
  • Cathode 704 is above the proton exchange membrane.
  • the single chamber biosensor may be cost effective because it is thin, flexible, and inexpensive, and, therefore, disposable.
  • the layers are flexible, laminar layers and not bulky rigid plastic like state of the art MFC fuel cells.
  • the two electrodes 704 and 710 in Fig. 10 are typically made of carbon paper or carbon cloth connected to electrical wires or contact points 714 and 716 (thickness ranges: « 0.004” to « 0.1”). Coatings may be applied to the electrodes to enhance electrical sensitivity.
  • the anode is coated with a thin film of carbohydrate (glucose or other sugar) on the side touching the source.
  • a permeable membrane 712 covers the anode, but it may not be needed in all configurations (thickness ranges: « 0.004” to « 0.05” if used).
  • the pore size of this per meable membrane is unimportant as long as it allows free migration of bacteria from the source to the sugarcoated anode and allows water (moisture) to transfer from the source to the anode and throughout the inside of the single chamber.
  • the permeable membrane primarily provides physical protection of the anode. It could be made from similar screen material used to cover building windows, which may have square mesh sizes of about 0.05 inches. This mesh size easily passes bacteria and moisture into the single chamber.
  • Separator 708 between the anode and cathode prevents them from touching and electrically shorting.
  • the separator could be a thin, coarse (open-cell) foam. Ideally, it is advantageous to make the separator thin to minimize the size of the single chamber bi osensor while still electrically separating the two electrodes. Size ranges could be from « 0.05” to « 0.25” depending upon application and usage. If no proton exchange mem brane 706 is used, the foam separator may need to be thicker.
  • Cathode 704 is exposed to air on one side and may have a thin proton exchange membrane 706 (typically 12 miti to 250 miti thick with pore holes about 0.015 miti to 1 miti) attached to the opposite side of the cathode, i.e., inside the single chamber, to wards the anode.
  • the proton exchange membrane has a small pore hole size, which allows protons to migrate across the boundary but prevents O2 from migrating from the cathode to the anode.
  • the source is a steak or other flat food
  • flexibility of the course membrane 712 and anode 710 may not be crucial because the biosensor can lie flat on the flat food forming a partial gas boundary between the biosensor and the source. This reduces the permeation of O2 onto the anode and creates anaerobic conditions inside the anode chamber side. Flexibility is more important if the source is not flat to allow the mem brane and anode to conform to the source’s surface. If the sources are identically shaped, the biosensor can be designed to conform to the shape.
  • a thin sugar or carbohydrate coating may be added on the anode.
  • Bacteria pre sent on the source can migrate into the biosensor and attach to the anode by chemotac- tic mechanisms. (“Chemotaxis is the directional locomotion of cells towards a source of a chemical gradient.”). When the bacteria reach the sugarcoated anode, the bacteria metabolize the sugars and activate or start the electron generation process of the MFC.
  • the semipermeable membrane surrounding the sugarcoated anode may not be necessary, other than for added physical protection of the anode.
  • the pore size could be made much larger than the specified 10 miti to 30 miti reducing the impedance of the bacteria to migrate onto the anode.
  • the entire single chamber may be laminated on the edges or closed on the edg es (as shown in the figure below) to hold the pieces together. Allowing the free migra tion of bacteria and fluids from the source to the anode and inside the single chamber is important.
  • the anode region is placed on the source to minimize oxygen (air flow) be tween the source and anode-side of the single chamber.
  • the opposite side of the single chamber (cathode side) is exposed to air for free-flow of oxygen.
  • the biosensor may be a one-shot device that stores the fuel (carbohydrate) in side and adjacent to the anode. It is not“loaded” with the catalyst (the microbes or pathogens). Instead, bacteria must be able to ingress into the biosensor to start the metabolic reactions.
  • the outer membrane 712 must allow pathogens and some liquid to pass freely into the biosensor. Options for hydrophobic coatings on the inside surface of membrane 712 may be useful to prevent or limit fluids from passing back out. Other wise, the fluids could contaminate food or wound.
  • the anode and cathode must conduct electricity, so graphite cloth is a possible membrane. Making the anode and cathode out of porous graphite paper or porous pa per with graphite sprayed onto the surfaces may be a cost-effective alternative.
  • the benefits of the single chamber MFC biosensor include: a) reduced material, parts, and fabrication cost; b) higher cathode efficiency; c) higher power densities ( « 500 mW/m 2 ).
  • Single chamber MFC’s with air cathodes can operate with or without a proton exchange membrane. Depending upon separation distance between the anode and cathode, performance can be increased without a proton exchange membrane. The maximum power density and generated voltage increases without the proton exchange membrane because of reduced internal resistance.
  • a single chamber biosensor also can be thinner, may produce a larger output signal for the same surface area sensor due to lower internal resistance, and may cost less.
  • “Plurality” means two or more. A“set” of items may include one or more of such items.
  • the terms“comprising,”“including,”“carrying,”“having,”“containing,”“involving,” and the like in the written description or the claims are open-ended, i.e., each means, “including but not limited to.” Only the transitional phrases“consisting of” and“consist ing essentially of” are closed or semi-closed transitional phrases regarding claims.
  • the ordinal terms such as“first,”“second,”“third,” etc., in the claims to modify a claim ele ment do not by themselves connote any priority, precedence, or order of one claim ele ment over another or the temporal order in which acts of a method are performed. In stead, they are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term).
  • Al ternatives such as“or” include any combination of the listed items.

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Abstract

La présente invention concerne de piles à combustible microbiennes qui produisent un signal électrique lorsque des microbes pénètrent dans les cellules par l'intermédiaire d'une membrane semi-perméable. La pile à combustible microbienne à chambre unique comprend une anode conçue pour être positionnée à proximité d'une source potentiellement contaminée pour recevoir des microbes sur la source, une cathode espacée de l'anode et un glucide positionné entre l'anode et la cathode. L'anode est souple de sorte à s'adapter à une source qui pourrait comporter des microbes. Lorsque les microbes pénètrent dans la pile à combustible et atteignent l'anode, la pile à combustible produit un signal électrique. La lecture et l'analyse du signal émanant d'un ou de plusieurs biocapteurs de pile à combustible peuvent indiquer une infection chez des personnes ou des animaux, indiquer des pathogènes se développant dans des aliments ou montrer un développement de moisissures. Dans la mesure où différents microbes ont des métabolismes spécifiques, le signal peut être utilisé pour déterminer quel microbe est présent.
PCT/US2020/015478 2019-01-28 2020-01-28 Capteur pour détecter des micro-organismes et procédé correspondant Ceased WO2020160041A2 (fr)

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CN115469086A (zh) * 2021-06-11 2022-12-13 广东美的白色家电技术创新中心有限公司 一种电器设备及其微生物检测模组和微生物检测方法

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US8277984B2 (en) * 2006-05-02 2012-10-02 The Penn State Research Foundation Substrate-enhanced microbial fuel cells
WO2006044954A2 (fr) * 2004-10-20 2006-04-27 University Of Florida Research Foundation, Inc. Contact electrique ameliore pour microbes dans des piles a combustible microbiennes
US8192854B2 (en) * 2009-02-06 2012-06-05 Ut-Battelle, Llc Microbial fuel cell treatment of ethanol fermentation process water
US20120231492A1 (en) * 2011-03-08 2012-09-13 Steve Bitterly Sensor for detecting microorganisms and corresponding process
US20190154621A1 (en) * 2011-03-08 2019-05-23 Innovative Sciences, Inc. Sensor for detecting microorganisms and corresponding process

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115469086A (zh) * 2021-06-11 2022-12-13 广东美的白色家电技术创新中心有限公司 一种电器设备及其微生物检测模组和微生物检测方法

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