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WO2024229027A2 - Outil portable et simultané de détection optique et de masse de sécurité alimentaire basé sur une microbalance à cristal de quartz basée sur un téléphone intelligent - Google Patents

Outil portable et simultané de détection optique et de masse de sécurité alimentaire basé sur une microbalance à cristal de quartz basée sur un téléphone intelligent Download PDF

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Publication number
WO2024229027A2
WO2024229027A2 PCT/US2024/027060 US2024027060W WO2024229027A2 WO 2024229027 A2 WO2024229027 A2 WO 2024229027A2 US 2024027060 W US2024027060 W US 2024027060W WO 2024229027 A2 WO2024229027 A2 WO 2024229027A2
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pathogen
selective
subsystem
destination
micropump
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WO2024229027A3 (fr
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Joseph Paul ROBINSON
Bartlomiej Rajwa
Euiwon Bae
Jung Joo SONH
Hyun Jung MIN
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Purdue Research Foundation
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Purdue Research Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G01N15/0612—Optical scan of the deposits
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G01N15/0637—Moving support
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/022—Fluid sensors based on microsensors, e.g. quartz crystal-microbalance [QCM], surface acoustic wave [SAW] devices, tuning forks, cantilevers, flexural plate wave [FPW] devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/036—Analysing fluids by measuring frequency or resonance of acoustic waves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
    • G01N5/02—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by absorbing or adsorbing components of a material and determining change of weight of the adsorbent, e.g. determining moisture content
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0255—(Bio)chemical reactions, e.g. on biosensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)

Definitions

  • PRF-70248-02 PORTABLE AND SIMULTANEOUS MASS AND OPTICAL FOOD SAFETY DETECTION TOOL BASED ON SMARTPHONE-BASED QUARTZ CRYSTAL MICROBALANCE CROSS-REFERENCE TO RELATED APPLICATIONS
  • the present non-provisional patent application is related to and claims the priority benefit of U.S. Provisional Patent Application Serial 63463541, filed May 02, 2023, the contents of which are hereby incorporated by reference in its entirety into the present disclosure.
  • STATEMENT REGARDING GOVERNMENT FUNDING [0002] This invention was made with government support under 59-8072-1-002 awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.
  • the present disclosure is generally related to pathogen detection and in particular to a system and method based on mass-change measurement and photonic detection when a fluid having a potential pathogen is studied with pathogen-selective antibodies and pathogen-selective photoluminescent label.
  • BACKGROUND [0004] This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.
  • Foodborne pathogens play havoc in the supply chain of food items every year. These foodborne pathogens often cause acute illnesses when unsuspecting individuals consume tainted food.
  • these foodborne pathogens are introduced at the farm level or at food processing plants, but they are also possible at any point in the supply chain.
  • the supply chain takes PRF-70248-02 painstaking measures to identify lots and batches.
  • the pathogens are only discovered after the consuming public has already become acutely ill, the solution is reactionary only in the form of destroying vast quantities of food, whether the food is tainted or not. Such reactionary measures are economically disastrous for the companies and the economy.
  • One of the challenges in detecting microbial pathogens to prevent foodborne illness is a time-sensitive endeavor. Many such detection schemes provide accurate and specific culture- based, molecular-based, immunological, and microscopy-based detection methods.
  • a pathogen detection system includes a microfluidic channel adapted to transfer a volume of fluid from a source to a destination, the destination defining a target.
  • the system further includes a micropump coupled to the microfluidic channel and configured to force fluid along the microfluidic channel from the source to the destination and a mass measurement subsystem adapted to measure changes in mass of a substance at the destination of at least 1 nanogram.
  • the system further includes a light excitation subsystem adapted to project light onto the target, and an image capture subsystem adapted to monitor the target and obtain images therefrom.
  • the system also includes a processing subsystem executing instructions held in a non-transitory memory.
  • the processing subsystem is adapted to: (i) operate the micropump to thereby force a first fluid hosting a pathogen-selective antibody from the source to the destination, (ii) operate the micropump to thereby force a second fluid potentially hosting the selective pathogen from the source to the destination, (iii) obtain a signal from the mass measurement subsystem indicating a mass change between steps (i) and (ii), (iv) compare the mass change to a predetermined threshold to indicate conditional presence of the pathogen and concentration of the pathogen, (v) operate the micropump to thereby force a third fluid hosting a pathogen-selective photoluminescent label from the source to the destination, (vi) activate the light excitation subsystem, capture one or more images from
  • the mass measurement subsystem includes a quartz crystal microbalance (QCM) adapted to provide a signal corresponding to mass change thereon based on a shift in resonant frequency of the QCM.
  • QCM quartz crystal microbalance
  • the selective pathogen is Salmonella Typhimurium
  • the pathogen- selective antibody is Anti-Salmonella CSA-1
  • the pathogen-selective photoluminescent label is FITC-labeled anti-Salmonella CSA-1.
  • the light excitation subsystem is adapted to project light in the range from 250 nm to 900 nm.
  • the pathogen-selective photoluminescent label produces a fluorescence signal.
  • the pathogen-selective photoluminescent label produces a phosphorescence signal.
  • the pathogen-selective photoluminescent label is a small-molecule organic fluorophore.
  • the pathogen-selective photoluminescent label is a nano-particle.
  • the nano-particle is one of a semiconductor-type quantum dot or an up-converting nanoparticle.
  • the image capture subsystem is a smartphone.
  • the image capture subsystem includes a complementary metal oxide semiconductor sensor or a charge-coupled device or a charge-coupled device adapted to capture photoluminescence images.
  • the processing subsystem includes a processor operating the smartphone.
  • volume of the second fluid forced by the micropump is based on a predetermined schedule depending on the selected pathogen.
  • Another pathogen detection system is also disclosed.
  • the system includes a microfluidic channel adapted to transfer a volume of fluid from a source to a destination, the destination defining a target, a micropump coupled to the microfluidic channel and configured to force fluid along the microfluidic channel from the source to the destination, a mass measurement PRF-70248-02 subsystem adapted to measure changes in mass of a substance at the destination of at least 1 nanogram, a light excitation subsystem adapted to project light onto the target, an image capture subsystem adapted to monitor the target and obtain images therefrom, and a processing subsystem executing instructions held in a non-transitory memory.
  • the processing subsystem is adapted to: (i) operate the micropump to thereby force a first fluid hosting a pathogen-selective antibody from the source to the destination, (ii) operate the micropump to thereby force a second fluid potentially hosting the selective pathogen from the source to the destination, (iii) obtain a first signal from the mass measurement subsystem indicating a first mass change between steps (i) and (ii), (iv) compare the first mass change to a predetermined threshold to indicate conditional presence of the pathogen and concentration of the pathogen, (v) operate the micropump to thereby force a third fluid hosting a pathogen-selective photoluminescent label from the source to the destination, (vi) activate the light excitation subsystem, (vii) capture one or more images from the target by the image capture subsystem, (viii) analyze the captured one or more images to determine presence of the pathogen-selective photoluminescent label thus double-confirming a) presence of the pathogen, and b) concentration of the pathogen
  • the mass measurement subsystem includes a quartz crystal microbalance (QCM) adapted to provide a signal corresponding to mass change thereon based on a shift in resonant frequency of the QCM.
  • QCM quartz crystal microbalance
  • the selective pathogen is Salmonella Typhimurium
  • the pathogen- selective antibody is Anti-Salmonella CSA-1
  • the pathogen-selective photoluminescent label is FITC-labeled anti-Salmonella CSA-1.
  • the light excitation subsystem is adapted to project light in the range from 250 nm to 900 nm.
  • the pathogen-selective photoluminescent label produces a fluorescence signal.
  • the pathogen-selective photoluminescent label produces a phosphorescence signal.
  • PRF-70248-02 In the above system, the pathogen-selective photoluminescent label is a small-molecule organic fluorophore.
  • the pathogen-selective photoluminescent label is a nano-particle.
  • the nano-particle is one of a semiconductor-type quantum dot or an up-converting nanoparticle.
  • the image capture subsystem is a smartphone.
  • the image capture subsystem includes a complementary metal oxide semiconductor sensor or a charge-coupled device or a charge-coupled device adapted to capture photoluminescence images.
  • the processing subsystem includes a processor operating the smartphone.
  • volume of the second fluid forced by the micropump is based on a predetermined schedule depending on the pathogen.
  • a method of detecting a pathogen is also disclosed.
  • the method includes (i) operating a micropump to thereby force a first fluid hosting a pathogen-selective antibody from a source to a mass measurement subsystem (destination), defining a target, via a microfluidic channel, (ii) operating the micropump to thereby force a second fluid potentially hosting the selective pathogen from the source to the destination, (iii) obtaining a signal from the mass measurement subsystem indicating a mass change between steps (i) and (ii), (iv) comparing the mass change to a predetermined threshold to indicate conditional presence of the pathogen and concentration of the pathogen, (v) operating the micropump to thereby force a third fluid hosting a pathogen- selective photoluminescent label from the source to the destination, (vi) activating a light excitation subsystem, (vii)capturing one or more images from the target by an image capture subsystem, and (vii) analyzing the one or more captured images to determine presence of the pathogen-selective photoluminescent label thus double-confirming
  • the mass measurement subsystem includes a quartz crystal microbalance (QCM) adapted to provide a signal corresponding to mass change thereon based on a shift in resonant frequency of the QCM.
  • QCM quartz crystal microbalance
  • PRF-70248-02 the selective pathogen is Salmonella Typhimurium
  • the pathogen- selective antibody is Anti-Salmonella CSA-1
  • the pathogen-selective photoluminescent label is FITC-labeled anti-Salmonella CSA-1.
  • the light excitation subsystem is adapted to project light in the range from 250 nm to 900 nm.
  • the pathogen-selective photoluminescent label produces a fluorescence signal.
  • the pathogen-selective photoluminescent label produces a phosphorescence signal.
  • the pathogen-selective photoluminescent label is a small-molecule organic fluorophore.
  • the pathogen-selective photoluminescent label is a nano- particle.
  • the nano-particle is one of a semiconductor-type quantum dot or an up-converting nanoparticle.
  • the image capture subsystem is a smartphone.
  • the image capture subsystem includes a complementary metal oxide semiconductor sensor or a charge-coupled device or a charge-coupled device adapted to capture photoluminescence images.
  • the processing subsystem includes a processor operating the smartphone.
  • volume of the second fluid forced by the micropump is based on a predetermined schedule depending on the pathogen.
  • Another method of detecting a pathogen is also disclosed.
  • the method includes (i) operating a micropump to thereby force a first fluid hosting a pathogen-selective antibody from a source to a mass measurement subsystem (destination), defining a target, via a microfluidic channel, (ii) operating the micropump to thereby force a second fluid potentially hosting the selective pathogen from the source to the destination, (iii) obtaining a signal from the mass measurement subsystem indicating a mass change between steps (i) and (ii), (iv) comparing the mass change to a predetermined threshold to indicate conditional presence of the pathogen and concentration of the pathogen, (v) operating the micropump to thereby force a third fluid hosting PRF-70248-02 a pathogen-selective photoluminescent label from the source to the destination, (vi) activating a light excitation subsystem, (vii) capturing one or more images from the target by an image capture subsystem, (viii) analyzing the one or more captured images to determine presence of the pathogen-select
  • the mass measurement subsystem includes a quartz crystal microbalance (QCM) adapted to provide a signal corresponding to mass change thereon based on a shift in resonant frequency of the QCM.
  • QCM quartz crystal microbalance
  • the selective pathogen is Salmonella Typhimurium
  • the pathogen- selective antibody is Anti-Salmonella CSA-1
  • the pathogen-selective photoluminescent label is FITC-labeled anti-Salmonella CSA-1.
  • the light excitation subsystem is adapted to project light in the range from 250 nm to 900 nm.
  • the pathogen-selective photoluminescent label produces a fluorescence signal.
  • the pathogen-selective photoluminescent label produces a phosphorescence signal.
  • the pathogen-selective photoluminescent label is a small-molecule organic fluorophore.
  • the pathogen-selective photoluminescent label is a nano-particle.
  • the nano-particle is one of a semiconductor-type quantum dot or an up-converting nanoparticle.
  • the image capture subsystem is a smartphone.
  • the image capture subsystem includes a complementary metal oxide semiconductor sensor or a charge-coupled device or a charge-coupled device adapted to capture photoluminescence images.
  • the processing subsystem includes a processor operating the smartphone. PRF-70248-02 [0059] In the above method, volume of the second fluid forced by the micropump is based on a predetermined schedule depending on the pathogen. BRIEF DESCRIPTION OF FIGURES [0060]
  • FIG.1 is a simple schematic of a Quartz Crystal Microbalance (QCM) system to illustrate the major components thereof.
  • FIG.2 is graph of admittance (1/impedance) vs.
  • FIG.3 is a schematic which demonstrates application of nanoparticles in connection with a QCM system.
  • FIG.4 is a schematic showing steps involved in testing for presence of a pathogen using the system of the present disclosure.
  • FIG.5 provides chemical structures involved in the steps shown in FIG.4.
  • FIG.6A is a partial perspective view of one of the modules of the system of the present disclosure.
  • FIG.6B is another partial perspective view of another of the modules of the system of the present disclosure.
  • FIG.6C is a perspective view of yet another of the modules of the system of the present disclosure including a flowcell but also including other modules.
  • FIG.7 is an operational schematic of Module 1 shown in FIG.6A.
  • FIG.8 is a schematic of the flowcell of FIG.6C.
  • FIG.9 is a schematic of the flowcell of FIG.8 over the quartz crystal providing the orientation of the inlet and outlet of the flowcell over the quartz crystal.
  • FIG.10 is a graph of frequency vs.
  • FIG.11 is a graph of frequency shift vs. time, where a close look at frequency changes is provided to clearly show the effect of the different concentrations of a pathogen (Salmonella Typhimurium) in terms of frequency shift.
  • FIGs.12A, 12B, 12C, 12D, 12E, 12F, 12G, and 12H are photographs showing Fluorescein isothiocyanate (FITC) beads that were dropped on the gold surface of the quartz crystal at different concentrations to show the viability of fluorescence imaging system of the present disclosure.
  • FIGs.13A, 13B, 13C, and 13D are photographs showing FITC-labeled anti-Salmonella CSA-1 antibodies bound to Salmonella Typhimurium. DETAILED DESCRIPTION [0075]
  • the term “about” can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.
  • the term “substantially” can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range.
  • a dual- and triple- modality Quartz Crystal Microbalance (QCM) system combining a smartphone, an in-situ optical imaging subsystem, and a flow injection component is disclosed.
  • This system enables a smartphone to receive real-time frequency data via Bluetooth, while a camera detects the presence of bacteria on the quartz crystal surface of the QCM using a pathogen-selective photoluminescent label.
  • the imaging subsystem utilizes a camera to capture the bacteria optical signal
  • the flow injection subsystem employs a mini peristaltic pump and controller to introduce biochemical solutions, antibodies, and bacteria. All components are contained package that is portable.
  • Fluorescein isothiocyanate (FITC) images were captured with 5 MHz quartz crystals when a prototype system was tested.
  • PRF-70248-02 [0079]
  • the present disclosure provides a smartphone-based biosensor for detecting a variety of pathogens, e.g., Salmonella Typhimurium, using a combination of a small flow injection system and an imaging system integrated with a QCM circuit.
  • the biosensor system of the present disclosure is adapted for dual- and triple- modalities of near-simultaneous detection of mass change and optical imaging, e.g., fluorescent imaging, providing a comprehensive approach to pathogen detection.
  • the QCM circuit equipped with a Bluetooth module, sends frequency and temperature data to the smartphone for real-time monitoring.
  • the biosensor also includes an LED-based optical excitation source and a peristaltic pump which are designed to work together.
  • the flow injection system introduces solutions containing pathogen-specific antibodies, pathogen-selective photoluminescent label, and solutions that are potentially containing the pathogens to the surface of the quartz crystal. The frequency changes are measured throughout the entire process, from immobilizing antibodies to detecting the pathogen.
  • photoluminescence images including fluorescence images are captured using the smartphone's camera to confirm the pathogen's presence.
  • a QCM is a mass-based biosensor that quantifies a change in mass resulting from bio- molecular interactions occurring on the quartz crystal surface. It comprises a quartz crystal, a bioreceptor, and a transducer to record frequency signals.
  • FIG.1 a simple schematic of a QCM is provided to illustrate the major components of the QCM.
  • the QCM sensor’s functioning can be categorized into three main stages: bioreceptor immobilization techniques, antibody-antigen reaction, and signal acquisition. Initially, the driving circuit stimulates the quartz crystal, causing it to oscillate at a precise resonant frequency.
  • biological recognition molecules such as antibodies, aptamers, or engineered protein scaffolds
  • biological recognition molecules such as antibodies, aptamers, or engineered protein scaffolds
  • the quartz crystal vibrates at a resonant frequency when a voltage is applied across the two electrodes, shown in FIG.1.
  • a baseline for natural frequency is first established of the mass with the biological recognition molecules that bind to one of the two electrodes. Once the pathogen is captured by the biological recognition molecules, the mass changes slightly, thus shifting the natural frequency towards lower frequencies. This shift is shown in PRF-70248-02 FIG.2 which is a graph of admittance (1/impedance) vs.
  • Nanoparticles are crucial in analyzing target analytes, enhancing both the quantification and sensitivity of biosensors. This increased sensitivity is vital in food safety, as even trace amounts of pathogens can cause severe issues.
  • Extensive research on biologically- functionalized nanoparticles has been conducted to improve the performance of both biological recognition molecules and transducers. Nanoparticles exhibit excellent biocompatibility, unique optical properties, and a large specific surface area. Nanoparticles have accelerated the detection of viruses and bacteria in the field of food safety, where they serve as both labels and signal amplifiers in immunoassays.
  • nanoparticles come in various types depending on their adjustable size and shape, including nanorods, nanowires, nanotubes, and nanobelts, all of which are employed in biosensor platforms.
  • metal nanoparticles are AuNPs.
  • CFU colony forming unit
  • a primary pathogen specific antibody, aptamer or other biological recognition system binds to the surface of the electrode and is capable of attracting and binding to the pathogen.
  • a secondary pathogen-specific antibody or other biological recognition molecule with nanoparticles binds to the pathogen.
  • Nanomaterials exhibit unique optical, electrical, or catalytic properties.
  • AuNPs gold nanoparticles
  • FIG.4 is a schematic showing the steps involved in testing for the presence of a pathogen. These steps were taken as parts of two separate experiments but combined here as one experiment description. The chemical structures of each step are shown in FIG.5.
  • a 5 MHz crystal (AT5- 14-12-AU-WRAP, openQCM, Italy) was used which has gold electrodes on the top surface of PRF-70248-02 the quartz crystal surface. Each chemical was transferred to the gold surface using a micropump (a peristaltic pump).
  • 11-Mercaptoundecanoic acid (11-MUA) was channeled to the quartz crystal to create thiol groups on the gold surface, producing a monolayer of MUA with COOH groups. Once this 11-MUA was transferred to the gold surface, the pump was stopped, and the reaction was allowed to proceed for 1 hour.
  • 250 ⁇ L of 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 250 ⁇ L of N-hydroxysuccinimide (NHS) were mixed, injected, and allowed to react for 1 hour to form NHS esters.
  • EDC 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • a pathogen e.g., Salmonella Typhimurium
  • BSA-PBS a 0.01 M phosphate-buffered saline solution (PBS) containing 0.138 M NaCl and 0.0027 M KCl at pH 7.4, injected and the pump was stopped for 1 hour, resulting in their immobilization on the surface.
  • the antibody concentration was 200 ⁇ g/mL.
  • the Anti- Salmonella CSA-1 antibody as described below the FITC-labeled anti-Salmonella CSA-1 antibody are commercially available.
  • FITC-labeled anti-Salmonella CSA-1 was injected for binding to the Salmonella Typhimurium for optical and mass measurements. Again PBS was injected to rinse and eliminate any non-specific binding.
  • the FITC-labeled anti- Salmonella CSA-1 allows for optical measurements which requires a light source and an image capture device. The optical measurements is used to double-confirm the results of the mass measurements as far as i) presence of the pathogen and ii) concentration of the pathogen based on the optical measurements and subsequent analysis.
  • the mass measurement approach can be used again after the binding of the FITC-labeled anti-Salmonella CSA-1 to Salmonella PRF-70248-02 Typhimurium, by measuring another shift in natural frequency, to triple-confirm i) presence of the pathogen and ii) concentration of the pathogen.
  • a smartphone-based QCM system was developed.
  • a smartphone SAMSUNG S22 Ultra
  • the smartphone was combined with the QCM system and fluorescence imaging system.
  • the smartphone plays a pivotal role in the system, serving both as a data acquisition tool and an imaging device.
  • the system also includes a Pierce oscillator, known to a person having ordinary skill in the art, for its frequency stability and minimal components.
  • This quartz crystal oscillator was incorporated into the openQCM Teensy shield (openQCM-TEENSY-2 from openQCM, Italy) which was specifically designed to be compatible with a Teensy 3.2 microcontroller (PJRC, USA).
  • the Teensy microcontroller, equipped with openQCM firmware demonstrates versatility, allowing precise frequency measurements ranging from 1 kHz up to 65 MHz while monitoring the temperature as well.
  • the firmware was modified to integrate the shield, with the inclusion of a Bluetooth module for enhanced connectivity in this study.
  • This integrated shield offers essential features, including a 3V3 voltage regulator, a signal conditioning circuit, an Inter- Integrated Circuit (I2C) temperature sensor, and a crystal oscillator driver (SN74LVC1GX04) that generates a square wave output.
  • I2C Inter- Integrated Circuit
  • SN74LVC1GX04 crystal oscillator driver
  • Module 1 primarily focuses on the fluorescence imaging system. A smartphone holder was purposefully designed to securely hold a smartphone in a horizontal orientation.
  • the PRF-70248-02 smartphone holder incorporates the smartphone, a plano-convex lens (PCX) (#37-783, Edmund, USA), enclosed in an optical holder (LMR05, Thorlabs, USA), and an emission filter (2020OFS- 525 Bin 4.G.8, Optical filter shop, USA), aligned to optimize optical performance.
  • PCX plano-convex lens
  • LMR05 optical holder
  • SMR05 Long-convex lens
  • 2020OFS- 525 Bin 4.G.8, Optical filter shop, USA aligned to optimize optical performance.
  • Two excitation filters 2020OFS-480 Bin 4.H.8, Optical Filter Shop, USA
  • two 490 nm LEDs (Fedy, China) were also secured with the holders.
  • the LEDs and excitation filters form an integral part of the flowcell holder in Module 2 and are positioned at an angle of 24 degrees in front of the quartz crystal.
  • Module 1 is adapted to capture the emission light from Salmonella Typhimurium reacted to the FITC-labeled antibody on the quartz crystal enclosed in the flowcell.
  • Two 490 nm LEDs with excitation filters excite the FITC-labeled antibody, while the smartphone’s camera acquires the intensities from Salmonella Typhimurium cells reacted to the FITC-labeled antibody through the emission filter as shown in FIG.7, which is an operational schematic of Module 1.
  • the excitation filters and 490 nm LEDs were tilted by 24 degrees based on the smartphone’s [0089] positioning.
  • Module 2 represents the primary oscillation circuit system. It features a flowcell holder designed to house LEDs, excitation filters, and a flowcell. The flowcell connects to the Teensy shield via a USB connection, which interfaces with both a Bluetooth module and a Teensy microcontroller. The quartz crystal will be positioned on the flowcell and then inserted into the flowcell holder.
  • Module 2 is adapted for maintaining quartz crystal oscillation and facilitating resonant frequency measurements through smartphone connectivity. Module 2 includes Circuit Boards 1 and 2.
  • Circuit Board 1 was combined with Circuit Board 2 to incorporate the Teensy 3.2 microcontroller and a Bluetooth module HC-05 (AMAZON, USA).
  • the Bluetooth module ensures communication and data exchange with the smartphone.
  • Circuit Board 1 and Circuit Board 2 were designed together to optimize space utilization and functionality. Since the Teensy 3.2 microcontroller with pre-installed openQCM firmware, the code was modified to integrate the Bluetooth HC-05 module. Consequently, this enabled the smartphone to obtain real-time frequency and temperature data via the Bluetooth interface.
  • Circuit Board 3 which is in Module 3, includes power management circuits, an LED driver, LEDs, capacitors, a voltage regulator, a PRF-70248-02 heat sink, two switches, and power sources for both the microcontroller and the peristaltic pump.
  • Module 3 encompasses the entire system, inclusive of a microfluidic system.
  • the housing for the peristaltic pump, located under the peristaltic pump, is designed to align with the outlet port of the flowcell at a corresponding level. Additionally, an enclosure of a waste beaker was made, reducing the risk of contamination.
  • a switch holder accommodates two switches, one for the LEDs and the other for the power source.
  • Module 3 serves as a pivotal component of the microfluidic system, facilitating the introduction of liquid samples onto the quartz crystal at a consistent flow rate.
  • the microfluidic system includes the pump controller, the mini peristaltic pump, and a waste beaker.
  • a pump controller (CECS-0100, TAKASAGO FLUIDIC SYSTEMS, Japan) accommodates a wide range of lower flow rates spanning from 0.23 to 350 ⁇ l/min. The controller was manually adjusted, and the rate was fixed for the entire experiment.
  • a versatile 6-channel peristaltic pump (RP-6R01S- 3P6A-DC10VS, TAKASAGO FLUIDIC SYSTEMS, Japan) is positioned near the outlet port to minimize any potential disruptions caused by its operation.
  • the pump was linked to the openQCM sensor module (openQCM-SENS-MOD-03, openQCM, Italy) via tubes that housed the quartz crystal.
  • This sensor module is also referred to herein as the flowcell.
  • a schematic of the flowcell is shown in FIG.8.
  • This sensor module includes a transparent fluidic cover, an O- ring, a temperature sensor, and a USB connection.
  • the flowcell is strategically oriented in a vertical configuration to minimize the risk of air bubbles interfering with measurements, with liquid introduced from the bottom.
  • the inlet port is positioned at a lower elevation than the outlet port, ensuring a smooth flow trajectory.
  • FIG.9 a schematic of the flowcell over the quartz crystal is shown providing the orientation of the inlet and outlet of the flowcell over the quartz crystal. Once the quartz crystal is correctly positioned, the flowcell is sealed by the cover, which includes inlet and PRF-70248-02 outlet tube holes. Next, the tubes are inserted into those holes to allow for ingress and egress of fluid. [0095] The experiment's frequency shifts are illustrated in FIG.10, which is a graph of frequency vs. time, where the Y-axis represents the resonant frequency of the 5 MHz quartz crystal.
  • the frequency changed, indicating a reaction between the QCM surface and the molecules.
  • the frequency shift observed before and after the injection of 10 9 CFU/mL of Salmonella Typhimurium was -6.7 Hz with a standard deviation of 0.69 Hz.
  • the frequency shift observed before and after the FITC-labeled antibody was -59.7 Hz with a standard deviation of 0.2 Hz.
  • the combined frequency shift was about -66.4 Hz, indicating that the presence of Salmonella Typhimurium caused the frequency shift, which was improved by the FITC-labeled anti-Salmonella CSA-1 antibodies.
  • the arrows represent the starting point when the solution was injected.
  • FIG.11 is a graph of frequency shift vs. time
  • a close look at frequency changes is provided to clearly show the effect of the different concentrations of Salmonella Typhimurium.
  • This figure presents frequency recordings after the FITC-labeled antibody and the subsequent washing steps.
  • FIGs. 12A-12H which provide images captured by the smartphone, displays 4 ⁇ m FITC beads that were dropped on the gold surface of the quartz crystal to test the fluorescence imaging system.
  • the concentrations used were 1 mg/mL (FIG. 12A), 0.1 mg/mL (FIG. 12B), 0.01 mg/mL (FIG. 12C), and 0.001 mg/mL (FIG. 12D).
  • FIGs. 12E, 12F, 12G, and 12H are the magnified images of FIGs.
  • FIGs. 13A, 13B, 13C, and 13D photographs of Salmonella Typhimurium labeled with the FITC-labeled anti-Salmonella CSA-1 antibodies at different concentrations, representing 10 9 CFU/mL in FIG. 13A, 10 7 CFU/mL in FIG. 13B, 10 5 CFU/mL in FIG. 13C and 0 CFU/mL in FIG.

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Abstract

Un procédé de détection de pathogènes est également divulgué et comprend (i) le fonctionnement d'une micropompe forçant un premier fluide hébergeant un anticorps sélectif vis-à-vis d'un pathogène d'une source à une destination, la définition d'une cible, par l'intermédiaire d'un canal microfluidique, (ii) le fonctionnement de la micropompe pour forcer ainsi un deuxième fluide hébergeant potentiellement le pathogène sélectif de la source à la destination, l'obtention d'un signal à partir de la destination indiquant un changement de masse ; (iii) la comparaison du changement de masse à un seuil prédéterminé pour indiquer la présence conditionnelle du pathogène et la concentration du pathogène, (iv) le fonctionnement de la micropompe forçant un troisième fluide hébergeant un marqueur photoluminescent sélectif d'un pathogène de la source à la destination, (v) l'activation d'un sous-système d'excitation de lumière, (vi) la capture d'images à partir de la cible ; et (vii) l'analyse des images pour déterminer la présence du marqueur, fournissant ainsi la double confirmation de a) la présence du pathogène, et b) la concentration du pathogène.
PCT/US2024/027060 2023-05-02 2024-04-30 Outil portable et simultané de détection optique et de masse de sécurité alimentaire basé sur une microbalance à cristal de quartz basée sur un téléphone intelligent Pending WO2024229027A2 (fr)

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