Multi-modality characterization of fluids using a single droplet

Abstract

To analyze and characterize physical properties of fluid when there are only a few micro liters or less of available fluid.SOLUTION: An apparatus for performing a plurality of different measurements on a small specimen sample to enable point-of-care, real-time testing and diagnosis is described. A core of the apparatus includes an ultrasonic resonator cavity that uses acoustic resonance in order to determine the speed of sound and attenuation of sound within a single droplet. Acoustic measurements are performed in a reflection mode using electrical impedance of a small piezoelectric crystal transducer operating in a thickness longitudinal mode. By a combination of this approach with an electromagnetic field, an electric field, and a magnetic field, multiple types of measurements can be performed using the same resonator cavity.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Patent Application No. 16 / 843,669, filed April 8, 2020, by Dipen N. Sihna and Peter G. Espina, for "Multi-Modality Fluid Characterization Using a Single Droplet," and U.S. Provisional Patent Application No. 62 / 833,467, filed April 12, 2019, by Dipen N. Sihna and Peter G. Espina, for "Multi-Modality Fluid Characterization Using a Single Droplet," the entire contents of which are hereby specifically incorporated by reference herein for all that they disclose and teach. [Background technology]

[0002] Few techniques exist for analyzing and characterizing the physical properties of fluids when only a few microliters or less of the fluid is available. Examples of bodily fluids available in small amounts include blood, tears, sweat, saliva, and sputum. Urine is typically available in larger volumes, but it is desirable to use only small samples when possible. Measurements on such fluids are optical or chemical. Summary of the Invention

[0003] Blood samples are commonly analyzed using multiple techniques, namely biochemical analysis, molecular profiling, and cellular evaluation, as exemplified. Each type of analysis uses different fluid samples from the same subject, which are performed in parallel using different instruments. For complete blood counts or bacterial growth analysis, light microscopy is the standard procedure. Other medical diagnostic tests using saliva, for example, are uncommon, with the exception of DNA extraction.

[0004] In accordance with the purposes of the present invention, as shown and generally described herein as an embodiment, one embodiment of an apparatus herein for optically and ultrasonically characterizing a single liquid droplet comprises a piezoelectric crystal effective to operate in a thickness longitudinal mode, the piezoelectric crystal having an electrical impedance responsive to acoustic waves impinging thereon; a metal holder having a front surface with a cavity configured to receive and hold the piezoelectric crystal in electrical contact with the metal holder; and a flat, optically transparent substrate having a first surface and a second surface, the first surface being parallel to and positioned a selected distance from the front surface of the metal holder, thereby forming an acoustically resonant cavity within which a single liquid droplet is formed. The device includes a flat, optically transparent substrate forming an acoustically resonant cavity in which a droplet is held by surface tension; a source of optical radiation that directs optical radiation through a first surface of the substrate, onto a second surface of the substrate, and into the droplet; an acoustic function generator that sweeps a piezoelectric crystal over a selected frequency range, thereby generating a series of equally spaced resonances in the droplet; an impedance analyzer that measures the impedance of the piezoelectric crystal as a function of frequency; and a signal processor that receives the output from the impedance analyzer and determines the spacing between the equally spaced resonances, from which the speed and attenuation of sound within the droplet are determined.

[0005] In accordance with the purposes of the present invention, as shown and generally described herein as an embodiment, one embodiment of an apparatus herein for electrically and ultrasonically characterizing a single liquid droplet comprises a piezoelectric crystal effective to operate in a thickness longitudinal mode, the piezoelectric crystal having an electrical impedance responsive to acoustic waves impinging thereon; a metal holder having a front surface with a cavity configured to receive and hold the piezoelectric crystal in electrical contact with the metal holder; and a flat substrate having a first surface and a second surface, the first surface being parallel to and positioned a selected distance from the front surface of the metal holder, thereby forming an acoustically resonant cavity within which a single liquid droplet is formed. The apparatus includes a flat substrate forming an acoustically resonant cavity in which a droplet is held by surface tension, the first surface further having a plurality of parallel or concentric conductive portions thereon; an acoustic function generator that sweeps a piezoelectric crystal over a selected frequency range, thereby generating a series of evenly spaced resonances in the droplet; a power source that provides an electric field between the plurality of conductive portions; an impedance analyzer that measures the impedance of the piezoelectric crystal as a function of frequency; and a signal processor that receives the output from the impedance analyzer and determines the spacing between the evenly spaced resonances, from which the speed and attenuation of sound within the droplet are determined.

[0006] In accordance with the purposes of the present invention, as shown and generally described herein as an embodiment, one embodiment of an apparatus herein for magnetically and ultrasonically characterizing a single droplet of liquid comprises a piezoelectric crystal effective to operate in a thickness longitudinal mode, the piezoelectric crystal having an electrical impedance responsive to acoustic waves impinging thereon; a metal holder having a front surface with a cavity configured to receive and hold the piezoelectric crystal in electrical contact with the metal holder; a flat substrate having a first surface and a second surface, the first surface being parallel to and positioned a selected distance from the front surface of the metal holder, thereby forming an acoustically resonant cavity within which a single droplet of liquid is held by surface tension; and a method for sweeping the piezoelectric crystal over a selected frequency range, whereby a series of evenly spaced resonances in frequency are generated within the liquid. the signal processor includes an acoustic function generator that generates resonances within the droplet; two flat, parallel, spaced-apart coils, one of which is positioned on or near and parallel to each of the first and second surfaces of the substrate, and each of which has an axis collinear with the axis of the other coil; a current source that supplies current to each of the two coils so that a uniform magnetic field perpendicular to the first and second surfaces of the substrate is generated in the same direction by each coil; an impedance analyzer that measures the impedance of the piezoelectric crystal as a function of frequency; and a signal processor that receives the output from the impedance analyzer and determines the spacing between equally spaced resonances, from which the speed of sound and attenuation of sound within the droplet are determined.

[0007] In accordance with the purposes of the present invention, as shown and generally described herein as an embodiment, one embodiment of an apparatus herein for electromagnetically and ultrasonically characterizing a single droplet of liquid comprises a piezoelectric crystal effective to operate in a thickness longitudinal mode, the piezoelectric crystal having an electrical impedance responsive to acoustic waves impinging thereon; a metal holder having a front surface with a cavity configured to receive and hold the piezoelectric crystal in electrical contact with the metal holder; and a flat substrate having a first surface and a second surface, the first surface being parallel to and positioned a selected distance from the front surface of the metal holder, whereby an acoustically resonant cavity is formed within the single droplet of liquid, whereby the single droplet of liquid is attracted by surface tension. the piezoelectric crystal is held by a flat substrate, forming an acoustically resonant cavity; an acoustic function generator that sweeps the piezoelectric crystal over a selected frequency range, thereby generating a series of evenly spaced resonances in the droplet; a source of millimeter wave electromagnetic radiation, the electromagnetic radiation impinging on a single droplet; a power supply that provides power to the source of millimeter wave electromagnetic radiation; an impedance analyzer that measures the impedance of the piezoelectric crystal as a function of frequency; and a signal processor that receives the output from the impedance analyzer and determines the spacing between the evenly spaced resonances, from which the speed and attenuation of sound in the droplet are determined. [Effects of the Invention]

[0008] Benefits and advantages of the present invention include, but are not limited to, a device that performs multiple different measurements on small droplet-sized specimen samples that may contain fecal material, allowing for point-of-care, real-time testing and diagnosis.

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of a side view of one embodiment of an apparatus for determining the speed of sound within a single droplet using reflection-mode swept-frequency acoustic interferometry (SFAI). [Figure 2A] 1 is a graph of the frequency response of the real part of the electrical impedance of a piezoelectric crystal as a function of frequency in the absence of liquid. [Figure 2B] 1 is a graph of the frequency response of the real part of the electrical impedance of a piezoelectric crystal as a function of frequency when a liquid (water) is present between the crystal and the substrate, where each of the resonance lines shows a periodic spectrum equally spaced in frequency. [Figure 2C] 2C is a graph of the autocorrelation amplitude as a function of frequency spacing of the resonances shown in FIG. 2B herein after the reference line in FIG. 2B has been subtracted. [Figure 2D] 1 is a graph of the amplitude of the Fast Fourier Transform of the data as a function of time, showing a series of equally spaced (in time) peaks that decay exponentially in amplitude. [Figure 3] Schematic diagram of a side view of one embodiment of an apparatus for measuring the effect of optical absorption of specific species contained in a droplet specimen on the speed of sound within the droplet using reflection-mode swept-frequency acoustic interferometry, where the substrate comprises a material that is transparent over a broad optical spectrum, and a fluid droplet specimen positioned above the substrate can be optically stimulated from below the substrate. [Figure 4] 1 is a graph of the heating and cooling of a droplet as a function of time when a light source located below the substrate is pulsed on and off, the rate at which the liquid heats up being related to the thermal conductivity and diffusivity of the liquid. [Figure 5A] FIG. 4 is a schematic diagram of a perspective view of one embodiment of an apparatus that combines electric field with acoustic measurements utilizing an apparatus similar to that of FIG. 3 herein, except that electrodes are formed on or attached to the surface of a substrate and are powered by an AC power source. [Figure 5B] FIG. 1 is a schematic diagram of a top view of an embodiment of an interdigitated electrode, which may also be used when higher field strengths are required. [Figure 5C] FIG. 1 is a schematic diagram of a top view of an embodiment of an interdigitated electrode, which may also be used when higher field strengths are required. [Figure 6] Graph showing three types of bacteria growing in blood: (a) Escherichia coli, (b) Staphylococcus epidermidis, and (c) Pseudomonas aeruginosa. The change in slope indicates nutrient depletion at different growth stages for the three different bacteria. Two different concentrations of each type of bacteria were investigated using SFAI in small spectrophotometric cells, one bacterial sample per time. [Figure 7] FIG. 1 is a schematic diagram of a perspective view of another embodiment of the device, combining electric fields with acoustic measurements, in which various molecules and polar materials exhibit the generation of surface acoustic waves (SAWs) interacting with surface charges propagating on the surface of the substrate. [Figure 8A] FIG. 1 is a schematic diagram of a side view of one embodiment of an apparatus that combines magnetic field with acoustic measurements, showing a DC or pulsed uniform magnetic field generated by placing two flat coils on either side of a substrate. [Figure 8B] FIG. 1 is a schematic diagram of a perspective top view of one of two coils mounted on a substrate. DETAILED DESCRIPTION OF THE INVENTION

[0011] Briefly, embodiments of the present process include a device that performs multiple different measurements on small specimen samples, enabling real-time testing and diagnosis at the point of care. The core of the device includes an ultrasound resonator cavity in which acoustic resonance is used to determine the speed of sound within a single droplet. Acoustic measurements are made in reflection mode using the electrical impedance of a miniature piezoelectric crystal transducer operating in the thickness extensional mode. This approach can be combined with optical, electrical, and magnetic devices to perform multiple types of measurements.

[0012] Reference will now be made in detail to the present embodiments of the invention, examples of which are illustrated in the accompanying drawings, in which like structures are identified using the same reference characters. It will be understood that the figures are presented for the purpose of illustrating particular embodiments of the invention and are not intended to limit the invention thereto. Turning now to FIG. 1 , there is shown a schematic diagram of a side view of one generalized embodiment of an apparatus 10 for determining the speed and attenuation of sound within a single droplet using swept-frequency acoustic interferometry (SFAI). This measurement provides a foundational measurement for the combined optical / acoustic, electrical / acoustic, and magnetic / acoustic measurements described below.

[0013] Acoustic measurements are performed in reflection mode by measuring the electrical impedance of a single 0.5 mm diameter piezoelectric crystal 12 operating in thickness extensional mode and having a frequency range between 5 MHz and 20 MHz. The 20 MHz piezoelectric crystal is thin and fragile, and physical support is required both for mechanical stability and for bandwidth extension by mechanically loading the backside of the crystal. Otherwise, the crystal will resonate within a very narrow bandwidth, requiring a wider bandwidth.

[0014] Because the surface area of ​​the piezoelectric crystal 12 is small, the resulting signal is similarly small. Therefore, the signal must be protected from external electrical noise pickup. The metal housing 14 serves as an electrical shield (Faraday cage) and also serves as a holder for handling the piezoelectric crystal. Typically, a microminiature coaxial cable is electrically connected to the piezoelectric crystal to further reduce noise pickup. The long metal tube shown is for convenience.

[0015] The piezoelectric crystal 12 occupies a portion of the front surface 16 of the holder 14. The piezoelectric crystal may be embedded in an epoxy material supported by a housing, such as the metal housing 14. Obviously, other housing and crystal mounting configurations can be envisioned. Due to its small size, the piezoelectric crystal 12 acts as a point source over the frequency range of use. This tends to broaden the acoustic beam and create some edge effects. Therefore, a larger front surface 16 of the holder 14 than the crystal embedded therein creates a better resonator cavity. While it is practical to use the crystal face by itself without any extra material around it, commercial crystals are sold with holders for mechanical stability.

[0016] The piezoelectric crystal holder 14 is supported vertically near the top surface 20 of a flat substrate 22. It can be oriented at any angle relative to the substrate 22, or even supported below the substrate if necessary. A droplet 24 (requiring only sub-microliters of fluid) is first placed on the surface 20 of the substrate 22, and then the crystal holder 14 is positioned over the droplet 24 using a simple mechanical loading device (not shown in FIG. 1), where it is held in place by surface tension. Because the droplet is free-standing, no container is required, making sample preparation much simpler.

[0017] An impedance analyzer 26, which may include a return-loss bridge and a matching network (a combination of parallel capacitors and resistors) used to balance the bridge, is connected to the piezoelectric crystal 12 via a miniature coaxial cable 28 (not shown in detail in FIG. 1 ) through a multiplexer 30. Commercial instruments such as the Bode 100 system by Omicron are capable of measuring the electrical impedance of the piezoelectric crystal 12 over a frequency range between a few Hz and 50 MHz. Vector network analyzer versions provide real and imaginary impedance values ​​as a function of frequency, which provides amplitude and phase information. Such instruments offer a tracking bandpass filter function with selectable filter bandwidths, which is important for reducing electrical noise pickup.

[0018] The substrate 22 can be made of any material, but advantageously, a chemically stable material with low optical absorption can be selected. The small sub-millimeter space 32 between the piezoelectric crystal surface, i.e., the front surface 16 of the holder 14, and the substrate 22 forms a resonator cavity. A variable sinusoidal excitation frequency is applied to the piezoelectric crystal 12 from a waveform generator 34 via a multiplexer 30, causing the piezoelectric crystal to generate longitudinal waves that propagate between the crystal and the substrate. The frequency at which a standing wave is generated within the droplet (resonant frequency) is related to the properties of the liquid (speed of sound) and the spacing between the piezoelectric crystal and the substrate. A resonance condition is established whenever an integer multiple of the corresponding wavelength in the fluid is equal to the path length within the fluid. This resonance affects the crystal's electrical impedance, which is detected by an impedance analyzer.

[0019] 2A-2D show data obtained using a broadband small piezoelectric crystal as shown in the apparatus of FIG. 1, where water is the fluid between substrate 22 and piezoelectric crystal 12. FIG.

[0020] Figure 2A is a graph of the frequency response of the real part of the electrical impedance of the piezoelectric crystal 12 as a function of frequency when no liquid is present. The measured impedance is a complex value with both real and imaginary components (equivalent to measuring amplitude and phase). Figure 2B shows a series of periodic resonances equally spaced in frequency when liquid (water) is present between the crystal 12 and the substrate 22. The speed of sound is related to Speed ​​of sound = 2 x liquid path length x frequency interval It is shown as follows.

[0021] There are two common procedures for determining this frequency interval. One is to perform an autocorrelation of the spectrum, as shown in Figure 2B, after subtracting a baseline from the spectrum. The results of the autocorrelation are shown in the graph of Figure 2C, where the amplitude of the autocorrelation is plotted versus the frequency interval. The location of the first peak provides the information necessary to calculate the speed of sound. Analysis of the reflection mode spectrum can also be achieved by performing a fast Fourier transform (FFT) of the data, converting the measurement from the frequency domain to the time domain. The results of the FFT are shown in Figure 2D, where the FFT amplitude is plotted against time in μs. Figure 2D shows a feature not present in the autocorrelation plot: a series of equally spaced (in time) peaks whose amplitudes decay exponentially. This plot is equivalent to a pulse reflecting back and forth between the piezoelectric crystal 12 and the substrate 22 through the liquid 24. Here, the first peak represents the propagation of the first pulse from the piezoelectric crystal to the substrate and back to the crystal. If the path length is known, measuring the time to the first peak provides the speed of sound. The remaining peaks are subsequent echoes. Due to sound absorption in the liquid, energy is lost and the amplitude appears to decay exponentially for each echo, providing a measure of sound absorption from the same measurement. The same measurement can also be made directly in the time domain by sending a short pulse (<1 μs) and observing the returning echo on an ultra-fast digitizer. Unless a digitizer with a multi-GHz sampling rate is used, the accuracy of the measurement suffers. Furthermore, electrical noise from the entire bandwidth of the piezoelectric crystal is recorded along with the signal.

[0022] Since typical commercial equipment does not provide either FFT or autocorrelation functions, these data manipulations are performed in a digital signal processor (DSP) or microprocessor, or by a laptop with appropriate software. The apparatus in Figure 3 below illustrates the use of this hardware.

[0023] Because piezoelectric crystals are typically small in size, the actual signal is small and requires wideband amplification and extensive averaging to recover the echo signal shown in Figure 2D. The processing gain (gain = frequency bandwidth x duration of excitation) is small for direct pulse measurements in the time domain, where the excitation pulse is approximately 1 μs or less. In contrast, frequency sweeps are typically performed over a period longer than 1 s, resulting in a processing gain of approximately 10 6 Furthermore, at each frequency step in a frequency sweep measurement, the bandwidth of the measurement may be limited to between 100 Hz and 1 kHz, which is equivalent to using a tracking filter where a fixed narrow bandpass filter follows each frequency measurement throughout the frequency sweep. Assuming a 1 kHz bandwidth for each frequency step measurement in a frequency sweep over a bandwidth of 10 MHz, the signal-to-noise ratio gain is 10 MHz / 1 kHz, or 10 5 Therefore, the frequency sweep approach plays a major role in detecting small signals that are relevant to the measurement.

[0024] A. Combining optical and acoustic measurements Figure 3 shows an apparatus for measuring the effect of optical absorption of specific species in a droplet specimen on the sound velocity within the droplet. If the substrate 22 comprises a material that is transparent across a wide spectrum of light, a fluid droplet specimen 24 positioned above the substrate can be optically stimulated from below. Examples of such substrates include, but are not limited to, Infrasil™ Quartz, which has an effective range of 220 nm to 3800 nm. Infrared (IR) and near-IR radiation penetrate most glasses. The light source 36 can be a tunable diode laser or a high-power multi-wavelength LED, such as those available from LED Engine, Inc., powered by a driver 40 and controlled by a digital signal processor and controller 42 and processor 44, which also provides data processing. One such LED light source, when driven with 700 mA of current, provides the following wavelengths: red (80 lumens), green (140 lumens), blue (33 lumens), white (210 lumens), amber (70 lumens), cyan (95 lumens), and violet (0.90 W). If necessary, multiple LEDs can be combined to increase light power. Wavelengths can be individually selected and pulsed, or some or all of the wavelengths may be combined together. Such an LED light source can be used to excite any biological fluid containing various proteins and molecules, or any material with an absorption band within the excitation range. A lens 38 may be used to focus the light into the droplet 24.

[0025] The XY positioning device 48 allows the holder 14 carrying the piezoelectric crystal 12 to be precisely positioned above the sample droplet 24, and the Z positioning device 50 allows the distance 32 between the front surface 16 of the holder 14 and the upper surface 20 of the substrate 22 to be precisely selected.

[0026] When any component of a fluid is selectively excited by a particular wavelength, the absorbed energy heats the fluid, which changes the speed of sound within it. This change can be detected using reflection-mode acoustic measurements with a sensitivity of greater than one part in a million in sound speed measurements. Selective excitation can therefore detect the presence of molecules or components of interest.

[0027] Figure 4 shows the results of turning a white pulsed light source on and off. When the light is on, the liquid (water) heats up and the speed of sound changes, and then cools down when the light is turned off. The rate at which the liquid heats up is related to the thermal conductivity and thermal diffusivity of the liquid.

[0028] Light from an LED or any other suitable light source can be excited with short pulses of a selected wavelength or wavelength range, resulting in photoacoustic measurements. The LED can be pulsed with current pulses from a pulse amplifier, ranging from tens of picoseconds to tens of nanoseconds. These pulses are typically repeated at a constant rate of several kHz to tens of kHz, allowing for signal averaging and better signal quality in terms of signal-to-noise ratio. When light is absorbed by a fluid or any of its internal components (e.g., biomolecules, proteins, bound antigens, etc.), the liquid rapidly heats and expands, generating a shock wave. This shock wave is a pressure pulse generated within the fluid droplet that bounces back and forth within the resonator cavity formed by the piezoelectric crystal, or the front surface of the holder, and the substrate. Resonance (standing waves) occurs within a cavity or any system when an external excitation coincides with the acoustic signal bouncing back and forth within the cavity. Once a photoacoustic signal is generated in the fluid by light absorption, it travels in all directions. However, signals traveling perpendicular to the two walls of the resonator cavity (the crystal face and the substrate) increase in intensity as they bounce back and forth multiple times before decaying. The quality factor (Q) is a measure of cavity quality and determines the number of bounces a signal undergoes before it is completely decayed. However, each time the signal completes a cycle, it becomes stronger, and then the signal steadily increases in amplitude without decaying—i.e., the signal continues to increase as it decays, and the signal is amplified to a certain maximum value until an equilibrium exists. The Q of a cavity can be up to 100, meaning that the signal can be amplified by at least 100 times. Therefore, by timing the repetition rate of the LED pulses, the gain of this mechanical cavity can be utilized; i.e., the repetition rate can be timed to enhance the small signal and coincide with any resonant vibrations of the intrinsic substrate. The amplified small signal is then further amplified by amplifier 52 and digitized by high-speed digitizer 54 (see FIG. 3). The repetition rate of the LED pulses can be synchronized to provide further signal amplification.In this case of repeating pulses and resonances, a much slower digitizer can be used to capture the signal because the signal is constructed from multiple samples.

[0029] This combination of optical and acoustic methods (photoacoustic measurements) can have a sensitivity of parts per billion.

[0030] For some acoustic measurements, better measurement accuracy can be obtained by comparing the measurement to that of a droplet of distilled water. Thus, the apparatus of FIG. 3 may include a protocol for first performing measurements on a droplet of distilled water placed near, but not in contact with, the sample droplet to be investigated. A first measurement, such as sound attenuation as a function of frequency, may be performed on the distilled water droplet, followed by measurements on the test sample. The distilled water provides a known reference, since both the water droplet and the test sample are at the same temperature. In this way, overattenuation may be measured as a function of frequency with greater resolution than by making absolute measurements. All other measurements described in this disclosure also benefit from having a reference medium.

[0031] B. Combining Electrical and Acoustic Measurements (i) Figure 5A shows an apparatus similar to that of Figure 3, combining electric field and acoustic measurements. Two spaced parallel conductors or electrodes 56a, 56b are deposited on or attached to a substrate 24 and are powered using an AC power supply 58 to apply an alternating electric field to the surface 20 of the substrate 22. A droplet 24 and a holder 14 carrying a piezoelectric crystal 12 are shown positioned on or between the conductors 56a, 56b. Note that the electrodes 56a and 56b act as capacitors, so grounding their open ends is not necessary. What is important is the electric field generated between the electrodes. If higher field strengths are required, interdigitated electrodes (IDTs) 60 and 62, shown in Figures 5B and 5C, respectively, can also be used. The AC power supply 58 is controlled by a driver 64 and controller 42. With the exception of the driver 64 and AC power supply 58, electronic control and measurement is performed using the equipment described in FIG. 3 above.

[0032] Electrodes 56a and 56b (as well as 60 and 62) can be formed to be optically transparent. Thin films of materials such as indium tin oxide (ITO) are typically deposited on surfaces by physical vapor deposition to provide optically transparent electrodes. The parallel electrodes shown in FIG. 5A can also be formed as transmission lines that can be excited at frequencies up to 20 GHz. Typically, three parallel lines are used, with the two outer lines grounded and the center conductor used as the signal line. Many polymer and protein structures are strongly affected by GHz electromagnetic waves, and energy can be absorbed from the transmission lines, resulting in changes in the speed of sound and ultrasonic absorption in fluids at specific frequencies, similar to the ultrasonic measurements for optical absorption described above.

[0033] Compact millimeter-wave sources are now commercially available. Millimeter waves are defined as electromagnetic waves between 30 GHz and 300 GHz, corresponding to wavelengths of 10 mm to 1 mm in air. For example, high-speed wireless communications according to the 802.11 Wi-Fi standard operate at 60 GHz. Powered by a power supply 67 and controlled by a digital signal processor and controller 42 and a processor 44, millimeter-wave sources 65 are extremely small and can be used to directly irradiate droplets 24, as shown in FIG. 5A. Such sources are available in a variety of frequency ranges, and some may be tuned over a frequency range of several GHz. Electromagnetic energy may pass through a compact waveguide as part of the millimeter-wave source 65. Higher frequency ranges interact favorably with biological cells, macromolecules, and protein structures. Many biological cells, including bacteria, decrease in size and change their metabolism, both of which affect the acoustic properties of droplets containing these cells.

[0034] Electrodes 56a and 56b (and 60 and 62) allow for dielectrophoretic measurements and can induce microflow within the droplet sample. Dielectrophoresis (DEP) occurs due to forces acting on dielectric particles when exposed to a non-uniform electric field, and the DEP response of cells is determined by their intrinsic dielectric properties or, more broadly, their electrical properties. This force does not require the particles to be charged; all particles exhibit dielectrophoretic activity in the presence of an electric field.

[0035] Because the force depends on the particle's shape, size, and frequency, fields with specific frequencies can manipulate particles with great selectivity. DEP can be used to separate cells, such as red blood cells from white blood cells. The dielectric properties of a cell are related to the physicochemical properties of its plasma membrane and are an important consideration in applying DEP to drug discovery. A potentially useful application of DEP is as a method for evaluating agents aimed at inducing changes in cellular state, such as activation, apoptosis (a form of programmed cell death that occurs in multicellular organisms), and necrosis (induced by factors external to the cell or tissue, such as infection, toxins, or trauma, which results in unregulated digestion of cellular components). Thus, DEP is an electrokinetic technique capable of both selectively sorting cells and monitoring their physiological state without the need for biomarkers such as fluorescent labels or antibody-coated magnetic beads. Furthermore, the AC electric fields and protocols typically used in DEP experiments are not known to damage cells.

[0036] The combination of acoustic scattering, and therefore DEP, provides a powerful means for determining the state of these cells and their interactions with drugs with high sensitivity and precision. That is, acoustic scattering is sensitive to the properties of the scatterer, including its physical composition and shape. Therefore, when acoustic transducers are used as both transmitters and receivers, as in a pulse-echo system, and a frequency chirp (short burst of a linearly swept frequency signal) signal is used, a lot of information can be obtained about the scatterer (e.g., blood cells, or whatever they may be). In contrast, DEP can separate solids from fluids, allowing these patterns to grow on a substrate. Furthermore, acoustic scattering from these sharp patterns can provide information about the system.

[0037] The generated microfluidic flow can increase bacterial growth due to the motion it can introduce into the fluid. This is called AC electrokinetic flow (ACEKF) for flow-induced vortices, which may provide a solution for improving the efficiency of the immune response. The electrode pattern can take many different shapes beyond those shown in Figures 5A-5C.

[0038] Typically, bacterial growth rates are controlled by the type of bacteria and the nutrient medium, with growth slowing as nutrients are depleted. Microflow can bring new nutrients to the vicinity of the bacteria, thereby increasing growth, which can be observed via sound velocity measurements using reflection-mode acoustic measurements above the electrodes. Many such possibilities exist with the combination of acoustic and electric fields. The small spacing of the electrodes in an IDT (circular or linear) can generate very high field strengths even with small voltages applied between the electrodes.

[0039] Figure 6 shows three types of bacteria growing in blood: (a) Escherichia coli, (b) Staphylococcus epidermidis, and (c) Pseudomonas aeruginosa. The change in slope indicates nutrient depletion at different growth stages for the three different bacteria. Two different concentrations of each bacterial type were investigated using swept-frequency acoustic interferometry (SFAI) in small spectrophotometric cells, one bacterial sample per run. While measurements were not performed using a single droplet, such measurements can be performed with the device shown in Figures 5A-5C.

[0040] As bacteria consume nutrients from their surroundings, the speed of sound in the surrounding fluid is affected. Bacterial growth can then be measured by this sound speed variation using reflectance-mode SFAI. Each bacterial species observed has a different growth rate, which can be used as a signature to identify the bacteria, assuming only one type is present.

[0041] Measurements were performed under the following conditions: a temperature-controlled circulating water bath was maintained at a nominal temperature of 37°C, and the bacterial container was placed in the water bath and gently agitated. Signal processing was performed using a signal source (digital function generator), a pair of acoustic transducers (one transmitting and one receiving), and a receiver (lock-in amplifier) ​​phase-locked to the signal generator. The signal generator provided a 3.8 volt RMS sinusoidal output to the transmitting acoustic transducer, swept at frequencies from 11 MHz to 16 MHz. The receiver, phase-locked to the signal source, received the signal from the transmitting transducer on the opposite side of the container and measured the signal's phase and amplitude relative to a reference. A standard desktop PC controlled the signal source output in 200 Hz increments and recorded amplitude and phase data from the receiver as a function of frequency. In operation, the system performed baseline acoustic measurements of the growth media at each bacterial inoculation and every few minutes for the duration of the test, which lasted several hours. The data presented in Figure 6 show the change in the acoustic properties of the growth medium over time as metabolic by-products of bacterial growth accumulate within the medium, and when plotted, follows the typical shape of a bacterial growth curve.

[0042] The change in sound speed within the medium was derived from the cross-correlation method, in which a baseline spectrum was cross-correlated with future measurements to determine a shift in the SFAI spectrum. This shift indicates a change in sound speed. The change in coefficient means that the change in coefficient is 1 minus the correlation coefficient. For each bacterium, two concentrations (colony forming units - CFU) were measured to demonstrate that the characteristics remained essentially the same.

[0043] (ii) Another implementation is the combination of reflection-mode SFAI with surface acoustic waves (SAWs). Surface acoustic waves (SAWs) are acoustic waves (Rayleigh or Love waves) traveling parallel to the surface of an elastic material, and their displacement amplitudes are attenuated within the material so that they are limited to approximately one wavelength at the surface. In piezoelectric materials such as gallium arsenide, quartz, or lithium niobate, the mechanical deformation associated with the SAW generates an electric field. Since this electric field does not affect the propagation of the mechanical wave, the result is a variation in electrostatic potential traveling along the SAW. For use as a biosensor, the surface is coated with a biospecific layer corresponding to the analyte. The surface plays a major role, as such devices rely on mass changes on the surface due to the attachment of various molecules and biomolecules, which are detected by the frequency characteristics of the received signal by the receiver IDT 60b. Changes in thin layers of biomaterials are detected by reflection-mode SFAI, which relaxes the stringent requirements for specially treated surfaces for typical SAW devices used as biosensors.

[0044] FIG. 7 illustrates one embodiment of a SAW device of the present invention, in which various molecular and polar materials interact with surface charges propagating on the surface of a substrate. The SAW IDTs are shown as a pair, one acting as a transmitter and the other as a receiver. However, a single IDT is required to generate a surface wave. The transmitter IDT 60a is driven by a high-frequency sinusoidal voltage source 58 and controlled by the driver 64 and controller 42 via an impedance matching network 66. The receiver IDT 60b induces an electrical signal to an amplifier 68, and the amplified signal is received by a digital signal processor 70 and directed to the controller 42 and processor 44.

[0045] Another application of SAW devices is to concentrate fluorescently labeled materials onto a surface, thereby increasing the intensity of the fluorescent manifolds in a regular (periodic) pattern that can be optically observed using a microscope placed under the underside of the substrate, or under a second side. As an example, a simple 10x-500x digital microscope with a USB output may be used so that images can be displayed or captured on a computer screen. The periodic pattern also affects the diffraction of the acoustic beam from the transducer in reflection-mode SFAI measurements, which can be monitored via changes in acoustic reflection properties. SAW devices operate at much higher frequencies than SFAI systems and are particularly suitable for liquid biopsies.

[0046] C. Combining magnetic and acoustic measurements Figure 8A shows a uniform magnetic field 72 generated by two flat coils 74a, 74b positioned on either side of the substrate 22. The magnetic field 72 can be a DC field or a pulsed field generated by a current source 76. The reflection-mode swept-frequency acoustic interferometry apparatus is similar to that shown in Figure 3 herein. The two flat coils function as Helmholtz coils to generate a uniform magnetic field perpendicular to the substrate surface. These coils are electrically connected so that the magnetic fields generated by both coils are in the same direction. There are different types of coils that can be used; this is only an example. The coils are driven by a current that can be pulsed. The droplet is placed in the center of the coil as shown, and a piezoelectric transducer is placed above it. The remaining functionality of the apparatus is represented in the configuration shown in Figures 3 and 5A. The acoustic portion of the measurement is performed slightly differently. For slow-response systems with times greater than a few milliseconds, measurements are performed using frequency chirps with durations of 10 μs to 1 ms. The transducer is excited, and the signal is detected and monitored over several milliseconds to extract multiple reflections of the signal within the fluid cavity formed between the top surface of the substrate and the crystal surface. Once the received signal is processed, a curve similar to that shown in Figure 2D can be generated. Signal processing can be performed using one of two procedures, depending on the time scale involved. For very short times (1-100 μs), processing involves cross-correlation of the excitation signal and the amplified received signal. However, more common processing involves frequency dechirping to determine the time-of-flight. Both procedures result in a curve similar to that shown in Figure 2D.

[0047] For fast dynamics requiring continuous measurements, a single-frequency approach is preferred. Any arbitrary liquid resonance peak is selected, as shown in Figure 2B, and both its amplitude and phase are monitored by the liquid's electrical impedance. The change in magnetic orientation can be recorded as a function of time. For the above measurements, observation begins when a current signal is applied to a coil that reacts to the fluid system, eventually establishing an equilibrium state. The current is then switched off, and the decay of the system is observed to the original randomized state of the magnetic moment.

[0048] Magnetic biosensing is concerned with understanding the interactions between biological systems and magnetic nanoparticles (MNPs). MNPs make it possible to diagnose the state and content of complex media, both inorganic and biological, given that the particles do not chemically react with the medium under investigation, thereby allowing for a specific "non-destructive test". The advantage of magneto-nanosensors is that they allow the analysis of remote motional excitations with the aid of an applied magnetic field.

[0049] Due to their small size, ferromagnetic nanoparticles have a single domain and therefore a magnetic moment of a certain magnitude. Such particles are suspended in an aqueous solution of polymers, some of which have an affinity for the particle surface and adsorb thereon, thereby transforming the particle environment from a Newtonian liquid to a viscoelastic polymer gel. In the absence of a magnetic field, Brownian motion randomizes the magnetization direction, and the overall net magnetization is zero. However, when a uniform magnetic field is applied, the magnetic moments of the particles are oriented in the same direction. When the field is quickly turned off, the magnetic orientation decays back to random behavior. This change causes a change in the viscosity of the fluid, which can be ultrasonically detected using a reflection-mode SFAI sensor. For example, a decrease in the amplitude of the subsequent echo can be observed, as shown in Figure 2D. This attenuation is due to the attenuation of sound in the fluid, which is related to the fluid's viscosity.

[0050] Magnetic beads (MBs) are a group of magnetic nanoparticles. For biosensing applications, these beads generally consist of a magnetic core, a surface coating, and specific binding ligands on the surface of the beads. For magnetic core materials, magnetite (Fe3O4) and maghemite (γ-Fe2O3) are considered attractive candidates for biological applications due to their ferromagnetic and biocompatible properties. Several ligands, namely antibodies (ABs), aptamers, and peptides, are available for analytical purposes. Aptamers are synthetic single-stranded DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) molecules that specifically bind to their target molecules with high affinity. Therefore, aptamers serve as attractive bioreceptors. The orientation of aptamers can be easily controlled during chemical synthesis, and various modifications can be made at defined positions within the aptamer sequence.

[0051] MBs can be used to capture bacteria (e.g., Salmonella), leukemia cells, and various pathogens. To detect the presence of pathogens and other biological entities, the device shown in Figure 8 can be used. Appropriate functionalized MBs must be added to the fluid, where they are introduced in small amounts into the device.

[0052] In addition to attenuation, the SFAI method reduces the 7 It is also possible to monitor changes in the speed of sound in a sensitive manner that can be as sensitive as one-times that of QCM devices used for such detection, due to changes in the mass of a thin film of material on a piezoelectric plate.

[0053] The magnetic state of blood fluctuates based on its oxidation level. There are other biopolymers that are also affected by magnetic fields. Changes in a fluid made of biomaterials placed within this field can be monitored using reflectance-mode SFAI techniques.

[0054] Another application utilizes the dynamic symmetry-breaking concept, in which a pulsed magnetic field (e.g., a few cycles of sine waves, or any type of pulse) is synchronized with an acoustic field. This synchronization ensures that the pulsed magnetic field is only excited during half a cycle of the acoustic field. The acoustic field is a pressure field that alternates between compression and rarefaction. Thus, the magnetic field is synchronized with every compression portion of the cycle or every rarefaction portion of the cycle. This is called symmetry breaking because the two halves of the cycle are no longer symmetric. Therefore, if cells are attached to magnetic nanoparticles coated with antibodies against a specific antigen, they can be rapidly separated. This separation combination is faster than acoustic separation alone. SAW waves can also be used in this way in combination with a periodic or pulsed magnetic field. In this situation, the separation is monitored by an acoustic probe. The separation can also be observed optically using a microscope.

[0055] D. Combination of optical, electrical, magnetic, and acoustic measurements The bottom surface of the substrate is accessible for simultaneous optical observation, allowing for the combination of photoacoustic and magnetic observations. Fast light pulses absorbed by the fluid can slightly increase the temperature of the fluid, which also affects the decay time. This allows for the investigation of other physical properties of the fluid, such as specific heat, thermal diffusivity, etc. These types of simultaneous measurements are not available in any of the current standard measurement systems.

[0056] The coil does not need to be glued or attached to the system; a small gap can exist between the coil and the substrate. This allows for the placement of interdigitated electrodes (IDTs) on the substrate (Figure 7, and Figures 5B and 5C). Therefore, it is also possible to simultaneously stimulate and measure surface acoustic waves (SAWs). If the substrate is a piezoelectric material, the surface waves generate an oscillating electric field on the substrate surface, which can interact with various functionalized magnetic beads, affecting the orientation of their magnetic moments, thereby allowing for the simultaneous investigation of the complex interplay of electric and magnetic fields. SAW waves can be continuous or modulated in amplitude and frequency. Such combined effects cannot be investigated by any currently available existing biosensing instrument. Thus, embodiments of the present invention enable new diagnostic modalities for biosensing.

[0057] Optical excitation may be applied from below the substrate 22, and magnetic coils, which may be positioned above and below the substrate 22, may be added to the apparatus of Figure 7, as described above. This allows for the use of a combination of optical, electric, and magnetic fields, along with reflection-mode swept-frequency acoustic interferometry, to perform various measurements. By placing an ITO transparent electrode on the substrate within the magnetic field region, it is possible to combine all four excitation fields. These fields may be independent or synchronized as needed. Thus, embodiments of the present invention enable multimodality single-droplet excitation and measurement. As with the photoacoustic measurements described above, an acoustic sensor (pinducer) may be used as a passive receiver to passively detect and monitor the effects of magnetic, electric, or SAW pulse excitation.

[0058] Embodiments of the present invention can be used with thin films, including tissue samples, or polymers, or almost any other material, as well as with liquid droplets. Thus, embodiments of the device can be adapted for many applications, including the observation of thin film materials on various types of surfaces, although not all modalities can be used in all situations.

[0059] Embodiments of the device provide a versatile tool that is not limited to biomedical measurements but serves as a platform for performing different types of measurements on small samples using the same device. All that is needed to create this low-cost measurement system is a laptop, a multi-channel high-speed data acquisition system, and a mechanical micrometer for adjustments. The ultimate goal is to be able to operate the device on a smartphone or smart pad. Using the same platform device, the ability to perform four major types of blood tests—immunoassays, general chemistry assays, hematology tests, and DNA amplification-based assays—in a faster, more simplified manner is now possible.

[0060] The foregoing description of the present invention has been presented for purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously, many modifications and variations are possible in light of the above teachings. These embodiments have been chosen and described to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to best utilize the invention in various embodiments and with various modifications to suit the particular uses contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.

Claims

1. 1. An apparatus for electrically and ultrasonically characterizing a single droplet, comprising: a piezoelectric crystal effective to operate in a thickness extensional mode, the piezoelectric crystal having an electrical impedance responsive to an acoustic wave impinging thereon; a metal holder having a front surface with a cavity configured to receive and hold the piezoelectric crystal in electrical contact with the metal holder; a flat substrate having a first surface and a second surface, the first surface positioned parallel to and a selected distance from the front surface of the metal holder, thereby forming an acoustically resonant cavity within which the single droplet is held by surface tension, the first surface further having a plurality of parallel or concentric conductive portions thereon; an acoustic function generator that sweeps the piezoelectric crystal over a selected frequency range, thereby generating a series of evenly spaced resonances in the droplet; a power source that supplies an electric field between the plurality of conductive portions; an impedance analyzer that measures the impedance of the piezoelectric crystal as a function of frequency; a signal processor that receives an output from the impedance analyzer and determines spacing between equally spaced resonances from which the speed of sound and attenuation of sound within the droplet are determined; and An apparatus comprising:

2. 10. The apparatus of claim 1, further comprising a mechanical loading device that positions the front surface of the metal holder proximate the first surface of the substrate on which the droplet is disposed.

3. 10. The apparatus of claim 1, wherein the piezoelectric crystal is swept between about 5 MHz and 20 MHz by the acoustic function generator.

4. The device of claim 1 , wherein the power source comprises an AC power source.

5. The device of claim 1 , wherein the plurality of conductive portions comprises interdigitated conductive portions.

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