WO2017087749A1 - Essai rapide de susceptibilité antimicrobienne au moyen d'un capteur piézoélectrique - Google Patents

Essai rapide de susceptibilité antimicrobienne au moyen d'un capteur piézoélectrique Download PDF

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WO2017087749A1
WO2017087749A1 PCT/US2016/062674 US2016062674W WO2017087749A1 WO 2017087749 A1 WO2017087749 A1 WO 2017087749A1 US 2016062674 W US2016062674 W US 2016062674W WO 2017087749 A1 WO2017087749 A1 WO 2017087749A1
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sensor
resonance
phase angle
resonance peak
peak
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Wan Y. Shih
Wei-Heng Shih
Xin Xu
Christopher EMERY
Suresh Joshi
Wei Wu
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Drexel University
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Drexel University
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    • 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
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/01—Indexing codes associated with the measuring variable
    • G01N2291/012—Phase angle
    • 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
    • G01N2291/024—Mixtures
    • G01N2291/02466—Biological material, e.g. blood
    • 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
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/04—Wave modes and trajectories
    • G01N2291/042—Wave modes
    • G01N2291/0422—Shear waves, transverse waves, horizontally polarised waves
    • 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
    • G01N2291/04—Wave modes and trajectories
    • G01N2291/042—Wave modes
    • G01N2291/0426—Bulk waves, e.g. quartz crystal microbalance, torsional waves
    • 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
    • G01N2291/04—Wave modes and trajectories
    • G01N2291/042—Wave modes
    • G01N2291/0427—Flexural waves, plate waves, e.g. Lamb waves, tuning fork, cantilever

Definitions

  • the invention relates to methods and systems for antimicrobial susceptibility testing of microorganisms/microbes.
  • the present invention may be particularly useful in reducing the time period required to determine drug susceptibility of microorganisms and to detect drug resistance in microorganisms, including, for example, bacterial and fungal pathogens.
  • AST Antimicrobial susceptibility testing
  • AST is used to determine whether an organism is susceptible or resistant to one or more antimicrobial agents, and usually phenotypic AST methods are used.
  • AST plays an important role in patient care and the control of antibiotic resistance since such testing can indicate which antibiotics are most likely to cure an infection and eliminate the infecting organism before resistance can develop. Rapid AST results are critical to ensure effective antimicrobials are promptly administered to patients with severe infections, e.g. blood stream infection and sepsis.
  • AST can also reduce the empirical prescription of broad-spectrum antibiotics that are partly responsible for the rapid increase in antibiotic resistance worldwide.
  • AST can also be used to reduce unnecessary prescription of expensive antibiotics (such as potent broad spectrum drugs) to reduce healthcare costs, when a less expensive antibiotic can be used/substituted.
  • AST methods exist to test microbes.
  • popular and widely-used AST methods include a broth dilution test, a disk diffusion test, and an antimicrobial gradient diffusion method.
  • an exemplary broth dilution test for bacteria two-fold dilutions of antibiotics (e.g. 1, 2, 4, 8, and 16 ⁇ g/ml) are prepared in a liquid growth medium dispensed in test tubes. They are then inoculated with a standardized bacterial suspension of 1 -5 * 10 5 CFU/ml. Following overnight incubation at 35°C, the tubes are examined for visible growth by observation of turbidity. The lowest concentration of antibiotic that prevents growth is deemed the minimal inhibitory concentration (MIC).
  • MIC minimal inhibitory concentration
  • microdilution method The miniaturization of the test by use of small, disposable, plastic 96-well trays (microbroth dilution method) has made this test practical and popular. Each tray allows approximately 12 antibiotics to be tested over a range of 8 two-fold dilutions. The cost of pre-prepared panels for this test ranges from proximately $10 to $22 each.
  • the advantages of the microdilution procedure include the quantitative measurement of MICs and the convenience of having pre-prepared panels and the method is adaptable to test fungal organisms.
  • the main disadvantage is the lack of flexibility of drug selections available in standard commercial panels as well as the time required to complete the test.
  • An exemplary disk diffusion test for bacteria is performed by applying a bacterial inoculum of approximately 1-2 x lO 8 CFU/ml to the surface of a Mueller-Hinton agar plate.
  • a bacterial inoculum of approximately 1-2 x lO 8 CFU/ml
  • Commercially-prepared, fixed concentration paper antibiotic disks are placed on the inoculated agar surface.
  • the plates are then incubated for 16-24 hours at 35°C.
  • the zones of growth inhibition around each of the antibiotic disks are measured.
  • the diameter of the zone is related to the susceptibility of the bacterial inoculum and can be interpreted into categories of susceptibility (i.e. susceptible, intermediate, or resistant).
  • the advantages of this method include its simplicity because it does not require special equipment, its flexibility for selection of disks (antibiotics) for testing and it can be adapted to test fungal organisms.
  • This method is generally the least expensive of all susceptibility testing methods (approximately $2.5-$5 per test for materials).
  • the disadvantages of the disk diffusion test include the lack of automation of the test and inaccuracies that arise especially in testing slowly growing bacteria. The test also requires significant time to complete.
  • the antimicrobial gradient diffusion method creates an antimicrobial concentration gradient on a plastic strip or other substrate placed on an agar medium to determine the susceptibility.
  • the EtestTM bioMerieux AB BIODISK
  • the EtestTM is a commercially available version of this test. It includes thin plastic test strips with an antibiotic concentration gradient on the underside and a concentration scale marked on the upper surface. The microbial suspension is inoculated on the agar plate and the test strips are placed on the surface, similar to the disk diffusion test. After overnight incubation, the MIC is determined by the intersection of the lower part of the elliptical growth inhibition area with the test strip. EtestTM strips cost approximately $2-$3 each. As a result, this method is best suited to situations in which a MIC for only 1 or 2 drugs is needed, and again, has the disadvantage of requiring at least overnight incubation.
  • the MicroScan WalkAwayTM (Siemens Healthcare Diagnostics) is a large self-contained incubator/reader device that can incubate and analyze 40-96 microdilution trays. It incubates the trays for the appropriate time period and then examines them periodically with either a photometer or fluorometer to determine microbial growth development, for example. It can give the results in 3.5-18 hours.
  • the Vitek 2 SystemTM (bioMerieux) is highly automated and uses very compact plastic reagent cards that contain microliters of antibiotics and test media in a 64-well format. It employs turbidimetry to monitor the microbial growth during incubation.
  • the instrument can accommodate 30-240 simultaneous tests and generate results in 4-12 hours, and the system can also perform yeast susceptibility testing.
  • antimicrobial susceptibility testing method is needed that does not require incubation of the microorganism in the presence and absence of antimicrobials.
  • Piezoelectric sensors can also detect the presence of microbes within a sample solution by allowing the microbes to bind to the sensor and measuring the resonance change as the mass of the sensor changes, as disclosed in, for example, U. S. Patent No. 8,778,446 and "Label-free flow-enhanced specific detection of Bacillus anthracis using a piezoelectric microcantilevers sensor," Analyst. 2008 May, 133(5), pp. 649-654. Piezoelectric sensors with enhanced detection sensitivity are described in U. S. Patent No. 8,741,663, the disclosure of which is hereby incorporated by reference herein.
  • the present invention relates to a method of antimicrobial susceptibility testing including steps of:
  • MIC minimum inhibitory concentration
  • the width of the resonance peak may be determined from any one of: (1) a phase angle versus frequency plot where the phase angle is the phase angle of the electrical impedance of the sensor, (2) a real part of a plot of electrical impedance versus frequency of the sensor, (3) a plot of the magnitude of the electrical impedance versus frequency of the sensor, (4) a phase angle versus frequency plot where the phase angle is the phase angle between the output voltage and the input voltage of the sensor.
  • the top of a resonance peak comprises the portion of the resonance peak that is within a vertical distance of a highest point of said resonance peak that is larger than a standard deviation of phase angles of the electrical impedance, or the real port of the electrical impedance, or the magnitude of the electrical impedance, or the phase angle between the output voltage and the input voltage of said detected resonance peaks and less than about one thousand times the standard deviation.
  • the top of the resonance peak can be defined as the portion of the resonance peak that is within a distance from the highest point of the said resonance peak larger than 0.01% of the total height of the said resonance peak and smaller than 10% of the total height of the said resonance peak.
  • the present invention relates to a system for rapid antimicrobial susceptibility testing comprising: a plurality of sensors having at least one outer surface portion,
  • a processing system configured to:
  • a width of a top of first and second detected resonance peaks of at least one said sensor from one of: (1) a phase angle versus frequency plot where the phase angle is the phase angle of the electrical impedance of the sensor, (2) a real part of a plot of electrical impedance versus frequency of the sensor, (3) a plot of a magnitude of electrical impedance versus frequency of the sensor, and (4) a phase angle versus frequency plot where the phase angle is the phase angle between an output voltage and an input voltage of the sensor, and
  • system and method may employ software that executes algorithms to determine and report the MIC and an antimicrobial interpretive category, e.g. sensitive (S), intermediate (I) or resistant (R), based on guidelines from, for example, the United States Food and Drug Administration (FDA), the Clinical Laboratories Standard
  • S sensitive
  • I intermediate
  • R resistant
  • FDA United States Food and Drug Administration
  • EUCAST EUCAST
  • Software may also be used to access databases and use expert rules to compare microbial identification, microbial phenoiypic biochemical infonnation. known microbial resistance mechanisms, known microbial MIC distributions, and previously defined microbial wild type and resistant phenoiypic information to modify or accept the obtamed MIC value and/or antimicrobial interpretive category of he test microbe Brief Description of the Drawings
  • FIG. 1 A shows a piezoelectric plate sensor (PEPS) that can be used in the methods of the present invention.
  • PEPS piezoelectric plate sensor
  • Figure IB shows a PEPS with a non-piezoelectric layer.
  • Figure 2 shows how a 10 by 6 array of wells 8 within a tray 9 may be used as an AST test.
  • Figures 3(a)-3(b) show an exemplary connection of a computing device to the piezoelectric sensor used in the invention.
  • Figure 4 shows that as bacteria on the sensor are killed with an antibiotic, in this case ampicillin (AMP), stresses produced by the live bacteria cease and the shape of the resonance peak in the phase angle versus frequency plot more closely resembles the sharper peaks of the control sensor without bacteria.
  • an antibiotic in this case ampicillin (AMP)
  • AMP ampicillin
  • Figure 5 shows the relationship of the width of the top of the peak plotted against rising quantities of AMP applied to the PEPS.
  • Figure 6 shows that the noise of the peak frequency of a PEPS increases when it is coated with live bacteria, but that the noise level is reduced with increasing AMP
  • Figure 7 is a photograph of the PZT PEPS used in Example 1.
  • Figure 8 shows the phase angle versus frequency spectrum of the sensor shown in Error! Reference source not found, where the tallest peak at around 1.8 MHz is the first width extension mode (WEM), which was the resonance peak used in this example.
  • WEM first width extension mode
  • Figure 9 shows the peak frequency of PZT PEPS monitored over time as different volumes of deionized water was added to the sensor.
  • Figure 10 shows the peak frequency of PZT sensor monitored over time as different volumes of ampicillin solutions (lmg/ml, 5, 5, 10, 20, 40, and 120 ⁇ ) were added to make the final concentrations of ampicillin 1, 2, 4, 8, 16, and 32 ⁇ g/ml.
  • Figure 11A shows the peak frequency in antimicrobial susceptibility Test 1 and Figure 11B shows the standard deviation (SD) of fitted peak frequency and that of raw peak frequency in Test 1.
  • SD standard deviation
  • Figure 12A shows the peak frequency in antimicrobial susceptibility Test 2 and Figure
  • Figure 13A shows the peak frequency in antimicrobial susceptibility Test 3 and Fig. 13B shows the standard deviation (SD) of fitted peak frequency and that of raw peak frequency in Test 3.
  • SD standard deviation
  • Figure 14 shows the peak shapes of the PZT sensor in DI water (red), after adding E. coli (blue), and after adding ampicillin ⁇ g/ml).
  • Figure 25 is an illustration showing how to deduce the width of the top of the peak from the raw data to characterize the roundness of the peak.
  • Figures 16A, 16B and 16C show the width of the top of the peak for the PZT sensor in Test 1, Test 2 and Test 3, respectively.
  • Figure 17A shows the peak frequency of PZT sensor monitored over time and Fig. 17B shows the standard deviation of the peak frequency, when pH adjusted ampicillin solutions were added to it to make the final concentration of ampicillin 1, 2, 4, 8, 16, and 32 ⁇ g/ml.
  • Figure 18 A shows the peak frequency in antimicrobial susceptibility test 4 and Fig. 17B shows the width of the top of the peak of the PZT sensor in Test 4.
  • the detection sensitivity of certain piezoelectric sensors is such that they may measure minute stresses produced by live microbes which have been bound to the surface of the sensor. Live microbes generate random stresses through metabolic functions. In turn, these random stresses function to blunt the peaks of frequency resonance intensities. As shown by Figure 4, as microbes on the sensor are killed with an antibiotic, in this case ampicillin (AMP), stresses produced by the live microbes cease and the shape of the resonance peak more closely resemble the sharper peaks of the control sensor without live microbes.
  • an antibiotic in this case ampicillin (AMP)
  • the data plotted in Figure 5 confirms the ability of the present method to sense and determine the minimal inhibitory concentration (MIC) of a microbe, because the known MIC of the E. coli that was tested is 8 ⁇ g/ml for ampicillin (AMP) [M. Goswami and N. Jawali, "Glutathi one-mediated augmentation of beta-lactam antibacterial activity against Escherichia coli,” J Antimicrob Chemother, vol. 60, pp. 184-5, Jul 2007].
  • MIC minimal inhibitory concentration
  • the width and/or shape of the top of a resonance peak can signify and determine the presence of live microbes.
  • the sensor can be operated in width extension mode, which is a type of linearextensional resonance mode, but it may also be operated in other resonance modes such as a length extension mode, a thickness extension mode, a flexural mode, a width shear mode, a length shear mode, a thickness shear mode or any other suitable resonance mode which is known to the art.
  • Width mode enables more sensitive detection with high peak frequency intensities and minimized damping effects.
  • the invention also includes a method of antimicrobial susceptibility testing including steps of:
  • MIC minimum inhibitory concentration
  • the width of the resonance peak may be determined from any one of: (1) a phase angle versus frequency plot where the phase angle is the phase angle of the electrical impedance of the sensor, (2) a real part of an electrical impedance versus frequency plot of the sensor, (3) a plot of the magnitude of the electrical impedance versus frequency of the sensor, (4) a phase angle versus frequency plot where the phase angle is the phase angle between the output voltage and the input voltage of the sensor.
  • the top of a resonance peak comprises the portion of the resonance peak that is within a vertical distance of a highest point of said resonance peak that is larger than a standard deviation of phase angles of the electrical impedance, or the real port of the electrical impedance, or the magnitude of the electrical impedance, or the phase angle between the output voltage and the input voltage of said detected resonance peaks and less than about one thousand times of the standard deviation.
  • the top of the resonance peak can be defined as the portion of the resonance peak that is within a distance from the highest point of the said resonance peak larger than 0.01 % of the total height of the said resonance peak and smaller than 10% of the total height of the said resonance peak.
  • the present invention employs a determination and comparison of the standard deviation of the frequency of said resonance peak before and after the application of said substance instead of comparing the widths of the resonance peaks as discussed above.
  • the peak frequency can be either the frequency with the raw maximum phase angle or the frequency of the fitted maximum phase angle. Even though the top of a peak may be flattened the raw data can still be fitted to a peak form. Either way, the peak frequency is monitored over a period of time such as 1 -10 minutes, or about 5 minutes. Upon completion of the time period, the standard deviation of the peak frequency is determined from the collected data. If the top of the peak is flattened, the standard deviation of the peak frequency will be significantly larger than if the top of the peak is not flattened, due to the uncertainty of the peak position created by the flattening of the peak. However, in practice this is not a problem since peak fitting involves portions of the peak that do not change and thus uncertainty was reduced to within acceptable limits by this factor.
  • the present invention relates to a system for rapid antimicrobial susceptibility testing comprising:
  • a processing system configured to:
  • a width of a top of detected resonance peaks of at least one said sensor from one of: (1) a phase angle versus frequency plot where the phase angle is the phase angle of the electrical impedance of the sensor, (2) a real part of an electrical impedance versus frequency plot of the sensor, (3) a plot of a magnitude of electrical impedance versus frequency of the sensor, and (4) a phase angle versus frequency plot where the phase angle is the phase angle between an output voltage and an input voltage of the sensor, and
  • Sensors useful for the present invention can be a piezoelectric plate sensor (PEPS), a piezoelectric microcantilever sensor (PEMS), a quartz crystal microbalance (QCM), a surface acoustic wave (SAW) device, or any other device consisting of a piezoelectric or piezo- resistive material that would allow measurement of the resonance peak using electrical means are useful for detecting the presence and/or mass of various compounds and molecules.
  • PEPS piezoelectric plate sensor
  • PEMS piezoelectric microcantilever sensor
  • QCM quartz crystal microbalance
  • SAW surface acoustic wave
  • Sensors may be fabricated by bonding a layer of a piezoelectric material, such as commercial lead zirconate titanate (PZT), to a non-piezoelectric substrate, such as stainless steel, titanium or glass, and have a number of advantageous properties, such as the capability of electrical self-excitation and self-sensing. Furthermore, piezoelectric sensors that include an insulation layer are capable of preventing conduction in liquid media, rendering them promising for biological in-situ electrical detection.
  • PZT commercial lead zirconate titanate
  • the detection sensitivity of piezoelectric cantilever sensors which may be viewed as harmonic oscillators, is correlated to the resonance frequency shift capability of the sensor.
  • the resonance frequency shift capability in turn is dependent upon the ability to detect changes in the effective spring constant and effective mass of the sensor.
  • Current cantilever sensor technologies such as non-piezoelectric microcantilevers and piezoresistive microcantilevers are only useful for methods which detect changes in mass of the sensor. As such, they are not effective for the detection of the presence of living microbes.
  • Fig. 1 A shows an exemplary structure of a type of piezoelectric sensor, a piezoelectric plate sensor (PEPS), which may be used in the present invention.
  • PEPS piezoelectric plate sensor
  • the present invention provides a piezoelectric plate sensor (PEPS) 100, comprising a piezoelectric layer 1, two electrodes 2 positioned one on each side of the piezoelectric layer 1, an insulation layer 3 encompassing the piezoelectric layer 1 and two electrodes 2, and a binding/receptor layer 4 bound to the surface of the insulation layer 3 of the PEPS 100 for binding a biomolecule or a microbe of interest.
  • Piezoelectric layer 1 is positioned between electrodes 2, functioning to enable electrical detection and actuation within the PEPS 100.
  • Piezoelectric layer 1 may function as a driving element, vibrating element, sensing element, or a combination thereof. Applying an alternating current (AC) voltage across piezoelectric layer 1 as an input induces bending and vibration of piezoelectric layer 1, which in turn induces a change in an output voltage that provides readily detectable changes in the magnitude and phase of the output voltage as well as the magnitude, phase, and real part of the electrical impedance of the sensor.
  • the resonance frequency of the PEPS 100 may be obtained, for example, by monitoring the maximum of the phase shift of the output voltage relative to the input voltage or the phase of the electrical impedance of the sensor. This measurement may be accomplished ail- electrically, i.e. using both electrical actuation and electrical sensing.
  • Piezoelectric layer 1 may be fabricated from any piezoelectric material, such as (Nao.5Ko .5 V 945 Lio .055 Nbo .96 Sbo .04 O 3 (hereinafter "Sb— NKNLN”), Sb— (Nao.sKo ⁇ NbOs— LiTa0 3 (hereinafter "Sb— NKNLT”), Sr— (Nao. 5 Ko.5)Nb0 3 — LiTa0 3 (Sr— NKNLN), Sr— Nao.5Ko.5)Nb0 3 — LiTaOs (Sr— NKNLT), SbSr— (Nao. 5 Ko.5)Nb0 3 — LiTa0 3 (SrSb— — LiTa0 3 (SrSb—
  • BNKT (Bii/ 2 Ki /2 )Ti0 3
  • BNBZT (Bii/ 2 Nai/ 2 )Ti0 3 — BaTi0 3 — (Bii /2 Ki /2 )Ti0 3
  • BNBK (Bii/ 2 Nai/ 2 )Ti0 3 — BaTi0 3 — (Bii /2 Ki /2 )Ti0 3
  • the piezoelectric layer 1 is fabricated from highly piezoelectric lead magnesium niobate-lead titanate films (hereinafter "PMN-PT”), such as
  • piezoelectric layer 1 may be fabricated from any highly piezoelectric material with a high -d 3 i coefficient in the range of from about 20 pm/V to about 5000 pm/V, or from about 200 pm/V to about 5000 pm/V, or from about 500 pm/V to about 5000 pm/V, or from about 2000 pm/V to about 5000 pm/V.
  • the -d 31 coefficient may be greater than about 20 p m/V.
  • piezoelectric layer 1 may have a piezoelectric coefficient d33 greater than about 40 pm/V.
  • piezoelectric layer 1 is made from highly piezoelectric lead magnesium niobate-lead titanate films, e.g. (Pb(Mgn3Nb273)03)o 65- (PbTi03)o 35 (PMN065- PTo 35) (PMN-PT), highly piezoelectric lead zirconate titanate (PZT) films or high piezoelectric lead-free films.
  • highly piezoelectric lead magnesium niobate-lead titanate films e.g. (Pb(Mgn3Nb273)03)o 65- (PbTi03)o 35 (PMN065- PTo 35) (PMN-PT), highly piezoelectric lead zirconate titanate (PZT) films or high piezoelectric lead-free films.
  • Piezoelectric layer 1 may be in any form.
  • piezoelectric layer 1 is fabricated from a free standing film for enhancing domain wall motion and piezoelectric performance.
  • piezoelectric layer 1 may be fabricated using a precursor-suspension method.
  • Submicron crystalline PMN powder is first prepared by dispersing Mg(OH)2- coated Nb20s particles in a lead acetate/ethylene glycol solution followed by calcination at about 800 C. The crystalline PMN powder is
  • a lead titanate (PT) precursor solution containing lead acetate and titanium isopropoxide in ethylene glycol subsequently suspended in a lead titanate (PT) precursor solution containing lead acetate and titanium isopropoxide in ethylene glycol to form a PMN-PT precursor powder, which can be sintered at a temperature as low as about 900 C.
  • PT lead titanate
  • Piezoelectric layer 1 may have any structural configuration or dimensions. Thus, piezoelectric layer 1 may be rectangular, triangular, circular, elliptical, or any other geometric shape. Piezoelectric layer 1 may have a thickness of from about 0.5 ⁇ to about 127 ⁇ , or from about 0.5 ⁇ to about 100 ⁇ , or from about 0.5 ⁇ to about 70 ⁇ , or from about 0.5 ⁇ to about 50 ⁇ , or from about 1 ⁇ to about 30 ⁇ . Piezoelectric layer 1 may have a length of from about 1 ⁇ to about 5 mm and a width of from about 1 ⁇ to about 5 mm. Piezoelectric layer 1 may have a length of from about 10 ⁇ to about 5 mm and a width of from about 0.5 ⁇ to about 5 mm.
  • Electrodes 2 of the PEPS 100 may be manufactured from a material capable of conducting an electrical signal from the piezoelectric layer 1 to a device for detecting that signal.
  • electrodes 2 are constructed from a conductive material selected from Ag, Au, Cu, Pt, Ir, Al, Fe, Cr, Ni, C, In, C, Sn, Ti and an alloy of these metals.
  • one electrode 2 is constructed from Au/Cr or Pt/Ti and subsequently patterned in several regions.
  • electrode 2 may be constructed from Pt/Ti0 2 on S1O 2 or Pt/Ti or Au/Cr on a metal substrate or non-piezoelectric layer. One or both of electrodes 2 may also be patterned.
  • Electrodes 2 may be a thin layer of conductive material with a thickness of less than about 6000 nm, or less than about 300 nm, or less than about 200 nm, or less than about 100 nm, or less than about 90 nm, or less than about 80 nm.
  • a non-piezoelectric layer 5 is included in the PEPS 100 as shown in Figure IB.
  • Non-piezoelectric layer 5 may be bonded to piezoelectric layer 1.
  • Non- piezoelectric layer 5 may be made from any compatible material, including ceramic, polymeric, plastic, and/or metallic materials or any combination thereof.
  • Non-piezoelectric layer 5 may be made from silicon dioxide (SiC ), silicon nitride (S13N4), a metal such as Cu, Sn, Ni, Ti, and stainless steel, or any combination thereof. Non-piezoelectric layer 5 may also have any structural configuration or dimension. Non-piezoelectric layer 5 may be rectangular, triangular, circular, elliptical, or have any other geometric shape.
  • Non-piezoelectric layer 5 may have a length of from about 1 ⁇ to about 5 mm, or from about 5 ⁇ to about 5 mm, a width of from about 1 ⁇ to about 5 mm, or from about 5 ⁇ to about 5 mm, and a thickness of from about 0.05 ⁇ to about 100 ⁇ , or from about 0.1 ⁇ to about 80 ⁇ , or from about 1 ⁇ to about 60 ⁇ .
  • PEPS 100 may have a wide variety of structural configurations. Piezoelectric layer 1 may be bonded to a non-piezoelectric layer 5 that is shorter, longer or equal in length, or width.
  • Insulation layer 3 of the PEPS 100 may be made from
  • the insulation layer 3 can electrically insulate the PEPS 100 when the sensor is used for detection in a salty biological fluid such as a serum or culture medium.
  • electrodes 2 may be patterned slightly smaller than piezoelectric layer 1 to ensure complete insulation of the edges and comers of electrodes 2.
  • insulating layer 3 may comprise a 1.5 ⁇ thick parylene (poly- para-xylylene) coating deposited on a conductive element 2 by chemical vapor deposition.
  • a parylene insulating layer 3 When placed in static and 1 ml/min flow rate of PBS solution, a parylene insulating layer 3 essentially prevents background resonance frequency shifts greater than 30 Hz and 60 Hz, respectively, over a period of 30 minutes. As a result, insulating layer 3 can enable complete submersion of the sensor for in situ or in-liquid detection.
  • a sensor may be insulated using self-assembled monolayers with hydrophobic properties, preferably methyltrimethoxysilane (MTMS) or a combination of MTMS with parylene coatings of varying thicknesses, may also be used.
  • MTMS methyltrimethoxysilane
  • parylene coatings of varying thicknesses
  • insulation materials may include AI2O 3 , S1O2 and any functional hydrophobic silane, having a hydrophobic group selected from the group consisting of alkyl, phenyl, alkyl halide, alkene, alkyne, and sulfhydryl.
  • the insulation material is mercaptopropylsilane (MPTS), which can also function to immobilize a receptor on the cantilever.
  • MPTS mercaptopropylsilane
  • an outer coating 4 of a material with suitably-arranged positive charges, suitably-mixed positive charges and negative charges, or microbe specific antibodies can be applied to the PEPS 100.
  • One suitable material with mixed positive and negative charges for use as outer coating 4 is (3- aminopropyl) trimethoxysilane (APS) where the amine group provides the positive charge and the hydrosol of the silanol produced by hydrolysis of the silane group provides the negative charge.
  • Examples of possible positively charged materials include poly-L lysine and polyethyleneimine (PEI) that may be coated and suitably arranged on the outer surface of the PEPS 100 to form the outer binding/receptor coating.
  • PEI polyethyleneimine
  • a coating of antibodies specific to the microbe may be another embodiment of the outer binding/receptor coating.
  • the frequency of a resonance peak such as a length-extension-mode (LEM) resonance peak or a width-extension-mode (WEM) resonance peak of sensor such as a PEPS can change as a result of binding of a target species to the sensor.
  • LEM length-extension-mode
  • WEM width-extension-mode
  • a layer of (3- Aminopropyl) trimethoxysilane (APS) can be coated onto the MPS insulation layer 3 to create a mixed-charge outer coating 4 to the sensor surface.
  • the negative electric charge of the microbe facilitates bonding of the microbe to the positively charged amine group on the sensor surface.
  • the outer coating layer can comprise an antibody specific to the microbe, suitably-arranged, positively-charged molecules such as poly-L lysine, PEI, or other materials suitable for binding the live microbes.
  • bonding microbes to a piezoelectric sensor requires several steps.
  • the microbes to be tested should be isolated, such as with a centrifuge or other separation method, and the microbes may be suspended in deionized water, phosphate buffered saline (PBS) solution, growth broth, or any other suitable medium.
  • PBS phosphate buffered saline
  • the PEPS should subsequently be submerged in the microbe suspension, such as for 1 -10 minutes, or else a small quantity (5 ⁇ to 30 ⁇ ) of the microbe suspension should be placed on the PEPS.
  • the microbe e.g. bacteria, parasites, or fungi
  • the PEPS device may be washed, preferably multiple times, with additional deionized water, PBS, or any buffer solution with a pH around 7, while the microbes remain bonded to the PEPS device.
  • a piezoelectric sensor may be chemically inert, thermally stable and preferably miniaturized and properly fabricated to enhance sensitivity.
  • the piezoelectric sensor has a high detection sensitivity of about 1 x 10 "11 g/Hz or better, more preferably 1 x 10 "16 g/Hz or better and most preferably 1 x 10 "19 g/Hz or better.
  • the piezoelectric sensor has a detection sensitivity of about 1 x 10 " 23 g/Hz or better.
  • an alternating voltage may be applied to conductive element 2 to drive piezoelectric layer 1 of a self-actuating sensor and a conductive element 2 may be used to detect a shift in the mechanical resonance frequency of the sensor due to the binding of the microbes.
  • a positive or negative change is introduced in the Young's modulus of the piezoelectric layer.
  • the change in the Young's modulus may be up to about 70%.
  • the change in the Young's modulus of the piezoelectric layer is preferably greater than about 0.001 %. Most preferably, the change in the Young's modulus may be about 0.001 % to about 70%.
  • One of the factors that induces a change in the Young's modulus is non-180 0 polarization domain switching. By inducing and/or enhancing non-180 0 polarization domain switching, it may be possible to further increase the detection sensitivity of the PEPS.
  • the piezoelectric sensor may be operated in width extension mode, which is a type of linear extensional resonance mode, but it may also be operated in other resonance modes such as a length extension mode, a thickness extension mode, a flexural mode, a width shear mode, a length shear mode, a thickness shear mode or any other suitable resonance mode which is known to the art. Width mode enables more sensitive detection with high peak frequency intensities and minimized damping effects.
  • the piezoelectric sensor may be used at resonance frequencies within the range of about 1 kHz to about 10 GHz.
  • system and method may employ software that executes algorithms to determine and report the MIC and an antimicrobial interpretive category, e.g. sensitive (S), intermediate (I) or resistant (R), based on guidelines from, for example, the United States Food and Drug Administration (FDA), the Clinical Laboratories Standard Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing
  • Suitable software will determine the MIC based on analysis of the top of the resonance peak as described above or analysis of the standard deviation.
  • an effective antimicrobial susceptibility test will employ an array of piezoelectric sensors which measure the effectiveness of various antibiotics and concentrations thereof. As they are simultaneously tested or tested in a series, each piezoelectric sensor within the array will demonstrate the effectiveness (e.g. antimicrobial activity) of the tested antimicrobial solution on the target microbes. While the number and arrangement of piezoelectric sensors within each array is largely immaterial to the invention, standard, rectangular 96-well plates may be used in one embodiment of the invention. In one embodiment, several sensors are coated with the same live microorganism and a number of different materials are tested, one or more with each sensor. In this manner, a plurality of different antibiotics or other antimicrobials can be screened simultaneously and/or a plurality different concentrations of antibiotics/antimicrobials can be tested simultaneously using, for example, an array of serial dilutions of the antibiotic.
  • FIG. 2 shows how a 10 by 6 array of wells 8 within a tray 9 may be used as an AST test.
  • Each well 8 in the X dimension (rows) contains a separate type of antimicrobial substance.
  • Each well 8 in the Y dimension (columns) varies by the concentration of the antimicrobial substance within it.
  • a single type of antimicrobial substance does not need to be used for the entirety of a row, but rather a row may be split between multiple antimicrobial substances.
  • a single antimicrobial substance may be tested with multiple rows.
  • the concentrations of the various antimicrobial substances across a given column need not be the same, but may vary according to the range of concentrations of the antimicrobial substances to be tested. Different types of antimicrobial substances at different concentrations may be combined within a well 8, for example for antimicrobial synergy testing.
  • the types of antimicrobial substances which can be tested by the present invention extend to any microbial substance known to the art, but for example may include amoxicillin, ampicillin, cefotetan, cefoxitin, chloramphenicol, clindamycin, imipenem, meropenem, metronidazole, mezlocillin, fluconazole, voriconazole, caspofungin, and amphotericin B.
  • Concentrations of each substance tested may include a broad range of concentrations which can determine the MIC of each substance. For example, because AMP has a MIC with E.
  • concentrations of AMP in an array of piezoelectric sensors with a target might include 0 ⁇ g/ml, 1 ⁇ g/ml, 2 ⁇ g/ml, 4 ⁇ g/ml, 8 ⁇ g/ml, and 16 ⁇ g/ml.
  • a limited range of antimicrobial concentrations e.g. breakpoint concentrations
  • concentration of the antimicrobial substances may be useful to effectively and efficiently determine the MIC of an antimicrobial substance in relation to a target microbe.
  • Piezoelectric sensors 10 to which the target microbes have been bound are submerged within each well 8. For accuracy in testing, more than one piezoelectric sensor may optionally be submerged within each well. The stresses produced by the live microbes may then be measured according to the disclosed methods, and the effectiveness of each concentration of each antimicrobial substance or antimicrobial combination may be determined.
  • Apparatus 11 holds the piezoelectric sensors 10 to submerge them within the wells 8, and also comprises the electrical connections to the piezoelectric sensors 10 which enable the resonances of the piezoelectric sensors to be determined, measured, and recorded by a computing device 12. The connection of the computing device 12 to the piezoelectric sensors is shown by Figs. 3a and 3b. Apparatus 11 can be of any configuration, such as holding a single piezoelectric sensor 10, an entire row of sensors 10 (as shown), an entire column of sensors 10, or a set of sensors which corresponds to the entire array of wells.
  • An alternative embodiment would not require wells, but rather the AST could be performed as different types and concentrations of antimicrobial substances are dropped, sprayed, or deposited on an array or sequence of piezoelectric sensors.
  • the stresses produced by the live microbes on the piezoelectric sensors in such an embodiment could be measured in the same way and would also determine the effectiveness of each concentration of each antimicrobial substance. Examples
  • PZT sheets Commercially available 127 ⁇ thick PZT sheets (PSI-5H4E, Piezo Systems, INC., MA) were cut into 2.5 mm ⁇ 1 mm squares using wire saw. They were soaked in acetone for 10 min to remove the wax and grease on the surface. Then both surfaces were connected with gold wires using conductive epoxy. The strip was fixed on a glass slide using non-conductive epoxy. The gold wires were connected to regular wires using the same conductive epoxy and they were wrapped in non-conductive epoxy for protection.
  • PSI-5H4E Piezo Systems, INC., MA
  • MPS (3-Mercaptopropyl) trimethoxysilane
  • APS (3-Aminopropyl) trimethoxysilane
  • step 3 To minimize the possible MPS cross-linking in the solution, after each 12 h MPS solution soaking, repeat step 3 and replenish the MPS solution with a fresh MPS solution of the same MPS concentration, water content, and pH.
  • the PZT sensor was insulated with MPS and coated with APS, it was immersed in 5 ml of DI water to monitor the stability of width mode frequency peak for more than 30 min. If the peak was symmetric and stable, the sensor could be used for the antimicrobial testing.
  • E. coli suspension in broth was prepared. The concentration was about 10 9 cells/ml. The following procedure was used to replace the broth with DI water, since the ions in the broth might affect the spectrum of the sensor. The suspension was centrifuged at 3000 rpm/min for 5 min and the supernatant was removed. 5mL of DI water was then added to the tube and the E. coli was re-suspended in DI water.
  • Tests 1-3 For the first a few tests (Tests 1-3), a drop of E. coli suspension (20 ⁇ ) was put on the sensor surface. After 10 min, the sensor was washed with 400 ml of DI water in a glass beaker 3 times to remove the unbound bacteria. The sensor was then returned to 5 ml of DI water and its width mode peak was monitored. In Test 4, instead of putting a drop of suspension on the sensor, the sensor was soaked in the E. coli suspension for 10 min. The washing step for Test 4 was the same as mentioned above.
  • FIG. 7 A picture of the sensor used in these examples is shown in Figure 7.
  • the spectrum of the sensor is shown in Figure 8.
  • the length mode of the sensor consisted of multiple peaks and it was difficult to determine the peak position.
  • the width mode of the sensor was very tall (30- 40 degrees). So we used the width mode of the sensor for monitoring.
  • the PZT sensor without any bacteria was monitored in DI water. Instead of ampicillin solution, different amounts of DI water were added. As shown in Error! Reference source not found., although the frequency changed after adding the water every time (especially after adding 40 ⁇ of water), the standard deviation of peak frequency, both for the fitted data and raw data, did not significantly change as illustrated in Table 1. Therefore, in this case, the value of peak frequency cannot be directly used to determine whether adding antibiotics would affect the E. coli. Instead, it was necessary to use the standard deviation of the peak frequency to characterize it.
  • a negative control was carried out in which the PZT sensor without any bacteria on it was monitored in DI water.
  • Different volumes of ampicillin solutions (lmg/ml, 5, 5, 10, 20, 40, and 120 ⁇ ) were added to the DI water to make the final concentration of ampicillin to 1 , 2, 4, 8, 16, and 32 ⁇ g/ml.
  • the peak frequency of the sensor over time is shown in Figure 10. It can be seen from the figure that after adding ampicillin solution, the peak frequency may change, which was consistent with the finding for the blank control.
  • the ampicillin concentration was low (i.e. 1 and 2 ⁇ g/ml)
  • the standard deviation of the peak frequency was similar to that in DI water.
  • the peak frequency was noisier and the standard deviation became getting larger and larger. This was true for both fitted peak frequency and the raw peak frequency as listed in Table 2.
  • Tests 1 -3 Three repeated Tests 1 -3 were done for the antimicrobial susceptibility testing. The same sensor was used for all three tests.
  • the peak frequency in Test 1 and its standard deviation (SD) change are shown in Figure 1 1.
  • SD standard deviation
  • the raw data was smoothed first (as shown in Figure 15.
  • the amount of variation of the raw data from the smoothed data was calculated as the standard deviation (SD) of the raw data.
  • SD standard deviation
  • the width of the top of the peak could be found when the phase angle was 1 times SD (or 2 times SD) smaller than the maximum phase angle in smoothed data. The more round the peak was, the smaller the width of the top of the peak would be. On the other hand, the more flat the peak was, the larger the width of the top of the top of the peak would be.
  • the widths of the top of the peak of the sensor in three AST tests are plotted in Figure 16. With E. coli on the sensor, the width of the top of the peak increased significantly. After adding ampicillin, the width of the top of the peak started to decrease. When the ampicillin concentration was 4 or 8 ⁇ g/ml, the width of the top of the peak reached its minimum value. This was consistent with the fact that the MIC of ampicillin for that particular bacterial strain is 8 ⁇ g/ml. After that, adding more ampicillin would only cause a slightly change in the width of the top of the peak.
  • Negative control 2 no E. coli, with Ampicillin
  • Antimicrobial susceptibility test (Test 4)
  • the antimicrobial susceptibility test was done using a different sensor.
  • pH adjusted ampicillin was added to the sensor.
  • the peak frequency of the sensor in the test was shown in Figure 18 A. After adding ampicillin, the peak frequency was less noisy. After more pH adjusted ampicillin was added, the noise did not increase.
  • the width of the top of the peak is plotted in Figure 18B. The width of the top of the peak increased as the E. coli was added the sensor surface and it decreased after ampicillin was added to the sensor. Adding more ampicillin did not further affect the width of the top of the peak.

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Abstract

L'invention concerne un système et un procédé d'essai de susceptibilité antimicrobienne qui consiste à détecter une crête de résonance d'un capteur dont la surface comporte des microbes vivants ; à appliquer la substance aux microbes vivants ; à détecter une crête de résonance dudit capteur après l'application de ladite substance ; à déterminer une largeur d'un sommet de chacune desdites crêtes de résonance avant et après l'application de la substance à partir d'un élément parmi : (1) un angle de phase par rapport au report de fréquence lorsque l'angle de phase est l'angle de phase de l'impédance électrique dudit capteur, (2) une partie réelle d'un report d'une impédance électrique par rapport à la fréquence dudit capteur, (3) un report d'une amplitude d'impédance électrique par rapport à la fréquence dudit capteur, et (4) un angle de phase par rapport au report de fréquence, l'angle de phase étant l'angle de phase entre une tension de sortie et une tension d'entrée dudit capteur, et à comparer les largeurs de sommets déterminées desdites crêtes de résonance ou des écarts-types desdites crêtes de résonance pour déterminer la susceptibilité antimicrobienne incluant la concentration inhibitoire minimale (MIC).
PCT/US2016/062674 2015-11-20 2016-11-18 Essai rapide de susceptibilité antimicrobienne au moyen d'un capteur piézoélectrique Ceased WO2017087749A1 (fr)

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US8778446B2 (en) * 2006-08-09 2014-07-15 Drexel University Flow cells for piezoelectric cantilever sensors
WO2015107200A1 (fr) * 2014-01-17 2015-07-23 Dublin City University Evaluation à base de résonateur des propriétés viscoélastiques d'un fluide
US20150301021A1 (en) * 2012-10-29 2015-10-22 Technion Research And Development Foundation Ltd. Sensor Technology for Diagnosing Tuberculosis

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US5856175A (en) * 1988-03-15 1999-01-05 Akzo Nobel N.V. Device for detecting microorganisms
US8778446B2 (en) * 2006-08-09 2014-07-15 Drexel University Flow cells for piezoelectric cantilever sensors
US8741663B2 (en) * 2008-03-11 2014-06-03 Drexel University Enhanced detection sensitivity with piezoelectric sensors
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CN112272586A (zh) * 2018-03-27 2021-01-26 赛录科试诊断公司 用于平行处理使用不同样品的抗微生物剂敏感性测试的系统、方法和界面
EP3774025A4 (fr) * 2018-03-27 2021-12-29 Selux Diagnostics, Inc. Système, procédé et interface pour le traitement en parallèle d'essais de susceptibilité antimicrobienne à l'aide de différents échantillons
US11268126B2 (en) 2018-03-27 2022-03-08 SeLux Diagnostics, Inc. System, method and interface for parallel processing of antimicrobial susceptibility tests using different samples
CN112272586B (zh) * 2018-03-27 2024-05-24 赛录科试诊断公司 用于平行处理使用不同样品的抗微生物剂敏感性测试的系统、方法和界面

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