WO2014028167A1

WO2014028167A1 - Device for making liquid phase bulk acoustic waves and use for liquid phase for sensing

Info

Publication number: WO2014028167A1
Application number: PCT/US2013/051087
Authority: WO
Inventors: Hsu-Cheng Ou; Yi Chin Catherine LIU; Hsin-Hung Liu; Boh-Shun Chiu
Original assignee: Loc Micro
Priority date: 2012-07-18
Filing date: 2013-07-18
Publication date: 2014-02-20
Also published as: WO2014028167A9

Abstract

Described is a device is for making bulk acoustic waves in a liquid sample. The device includes a piezoelectric substrate. An input interdigital transducer is located in a region on a surface of the piezoelectric substrate. An output interdigital transducer is located in the region on the surface of the piezoelectric substrate. A sensing area is between the output interdigital transducer and the input interdigital transducer. A channel is configured to contain liquid over the region including the sensing area.

Description

DEVICE FOR MAKING LIQUID PHASE BULK ACOUSTIC WAVES AND USE FOR

LIQUID PHASE FOR SENSING

DESCRIPTION OF THE INVENTION

Cross reference to related application

The present application claims benefit of U.S. Provisional Application no. 60/672,937, filed July 18, 2012, which application is incorporated herein by reference in its entirety.

Background

Acoustic-wave sensors are a class of microelectromechanical systems (MEMS) which rely on acoustic waves to sense a physical phenomenon. The sensor transduces an input electrical signal into a mechanical wave which, unlike an electrical signal, can be easily influenced by physical phenomena. The device then translates this wave back into an electrical signal. The device can be used to measure the presence of the desired phenomenon by measuring the changes in amplitude, phase, frequency, or time-delay between the input and output electrical signals.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1A and Figure 1C are a view of a device according to some embodiments.

Figure 2 shows a view of a cross-section of a device having a waveguide layer in some embodiments.

Figure 3 shows a method of detecting an analyte according to some embodiments.

Figure 4 is data representing signal shifts versus different size of nanobeads. Figure 5 is data representing signal shifts versus different size of nanobeads.

DESCRIPTION OF EMBODIMENTS

Reference will now be made in detail to embodiments of invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

The present inventors realized that with the rapid growth in prevalence and severity of infectious diseases, the capacity to detect infectious diseases rapidly and accurately has become an important— though unmet— medical need. Under current schemas of laboratory-based testing, infectious disease diagnoses can take several days; this necessitates quarantine of patients during the extended testing period, which drastically increases administrative costs and resources. In cases when this is unfeasible, the patient may unintentionally infect others with whom he or she contacts, resulting in the widespread disease outbreak. The development of the device described herein makes it possible to address this need by providing an optionally portable platform capable of diagnosing multiple pathogens.

Figures 1A-C are views of a device according to some embodiments. In some

embodiments, the device is for making bulk acoustic waves in a liquid sample. In some embodiments, such as those of Figures 1A-B, the device 100 includes a piezoelectric substrate 110. An input interdigital transducer 120a is located in a region 130a on a surface of the piezoelectric substrate 110. An output interdigital transducer 140a is located in the region 130 on the surface of the piezoelectric substrate. A sensing area 150a is between the output interdigital transducer 140a and the input interdigital transducer 120a. A channel 160a is configured to contain liquid over the region 130a including the sensing area 150a. In some embodiments, the piezoelectric substrate 110 is chosen from Y-cut L1NO₃ crystals. For example, in some embodiments, the Y-cut L1NO₃ crystal is chosen from 128° Y-cut L1NO₃, 64° Y-cut L1NO₃, and 0° Y-cut L1NO₃ crystals. In some embodiments, the Y-cut L1NO₃ crystal is 128° Y-cut L1NO₃ crystal.

The present inventors discovered that the Y-cut L1NO₃ crystal, e.g., 128° Y-cut L1NO₃, is usable to sense surface binding in the liquid phase.

In some embodiments, the piezoelectric substrate has a thickness greater than about 0.5μιη. In some embodiments, the thickness ranges from 100 to 1,000 μιη or from 250 to 750 μιη. The thickness is selectable based on a desired center frequency of the bulk acoustic wave. In some embodiments, the piezoelectric substrate has a substantially planar surface and a substantially rectangular, circular or semicircular shape.

The present inventors report herein the use of bulk acoustic waves (BAW) or acoustic plate mode (APM) to sense surface binding in the liquid phase. As explained herein, sending a signal between the input interdigital transducer 120a and the output interdigital transducer 140a and across sensing area 150a results makes it possible to sense surface binding in a liquid phase using bulk acoustic waves or acoustic plate mode. The present inventors noted that other types of acoustic waves, such as surface acoustic waves, are not usable to detect surface binding in the liquid phase. The present inventors report here their success in the use of bulk acoustic waves or acoustic plate mode to sense surface binding in the liquid phase.

In some embodiments, such as those of Figure 1A, the device further includes an optional second input interdigital transducer 120b located in a second region 130b on the surface of the piezoelectric substrate 110. The optional second output interdigital transducer 140b is located in the second region 130b on the surface of the piezoelectric substrate 110. An optional second sensing area 150b between the second output interdigital transducer 120b and the second input interdigital transducer 140b. In some embodiments, such as those of Figure IB, a second channel 160b is configured to contain liquid over the second region 130b including the second sensing area 150b. In some embodiments, not shown, the channel 160a is configured to contain liquid over both the first region 130a including the first sensing area 130a and the second region 130b including the second sensing area 150b.

The second channel 160b makes it possible to have a reference channel. The reference channel is usable, e.g., to calibrate for outside effects. For example, 128° Y-cut L1NO₃ is temperature dependent. The second channel 160b, when used as a reference channel in some embodiments, makes it possible to adjust for changes in temperature between runs using the device.

In some embodiments, not shown, the device further includes an optional additional input interdigital transducer located in an additional region on the surface of the piezoelectric substrate. An optional additional output interdigital transducer is also located in the additional region on the surface of the piezoelectric substrate. An optional additional sensing area is between the additional output interdigital transducer and the additional input interdigital transducer. In some embodiments, the channel 160a is configured to contain liquid over the first region 130a including the first sensing area 150a, the second region 130b including the second sensing area 150b and the additional region over the additional sensing area. In some

embodiments, a second channel 160b is configured to contain liquid over the second region 130b including the second sensing area 150b; and an additional channel is configured to contain liquid over the additional region including the additional sensing area. In some embodiments, such as those of Figure 1A, the sensing area 150a is on the same surface of the piezoelectric substrate 110 as the output interdigital transducer 140a and the input interdigital transducer 120a.

In some embodiments, such as those of Figure 1C, the sensing area 120a is on the opposite surface of the piezoelectric substrate 110 as the output interdigital transducer 140a and the input interdigital transducer 120a. Such a top-bottom configuration makes it possible to avoid short circuiting the electronics.

In some embodiments, the piezoelectric substrate 110 includes a waveguide layer on a surface and the sensing area is on the waveguide layer. In some embodiments, the effective range of analyte detection ranges from 10 to 30 nm away from the surface of substrate in some sensing applications. In some embodiments, detection is improvable by incorporating a waveguide layer (e.g., Si0₂ or polyvinylchloride) to compensate the distance between the surface of substrate and effective range of dynamic viscosity. Furthermore, in some embodiments, the waveguide layer makes it possible to reduce the temperature effects to LiNb0₃ substrate. Si0₂ is also media of surface binding in biological application. In some embodiments, the Si0₂ is functionalized to accommodate a substance configured to immobilize an analyte of interest in a liquid phase.

Figure 2 shows a view of a cross-section of a device having a waveguide layer in some embodiments. Waveguide layer 205 is over substrate 210. (In Figure 2, analogous elements to those in Figures 1A-C have the same reference numeral but offset by 100.) The sensing area 230 is on the opposite surface of the piezoelectric substrate 210 as the output interdigital transducer 240 and the input interdigital transducer 220. The channel 260 is over the waveguide layer 205. Although embodiments of Figure 2 have the top-bottom configuration which makes it possible to avoid short circuiting the electronics, in other embodiments, the sensing area 230, the output interdigital transducer 240, and the input interdigital transducer 220 are on the same side of the piezoelectric substrate 210. In some embodiments, the waveguide layer 205 covers the region on the surface of the piezoelectric substrate including the output interdigital transducer 240 and the input interdigital transducer 220. In some embodiments, the waveguide layer 205 is less than 150 nm thick. For example, in some embodiments, the waveguide layer is less than 100 nm thick.

In some embodiments, the waveguide layer 205 comprises a material selected from Si0₂ and polyvinyl chloride. In some embodiments, the waveguide layer 205 is functionalized, e.g., the material is selected from functionalized Si0₂ and polyvinyl chloride.

In some embodiments, the sensing area includes a film including a substance configured to immobilize an analyte of interest in a liquid phase contained in the channel. In some embodiments, the piezoelectric substrate or waveguide layer in the sensing area is coated with a film of molecules that have a specific binding affinity for the analyte, i.e., a substance configured to immobilize an analyte of interest in a liquid phase. When a liquid sample containing the analyte is contacted to the film, the analyte is immobilized on the surface by molecular recognition, chemical reaction, complex formation, and the like. This immobilization, in some embodiments, is accomplished via any specific ligand-receptor combination such as antibody- antigen or other specific binding combination such as complementary sequences of polynucleic acids (DNA/ RNA).

LiNb0₃ is, in some embodiments, functionalized to accommodate substance configured to immobilize an analyte of interest in a liquid phase contained in the channel. As noted above, the waveguide layer is, in some embodiments, functionalized to accommodate substance configured to immobilize an analyte of interest in a liquid phase contained in the channel. In some embodiments, a holding layer is applied over the LiNb0₃ or, when present, the waveguide layer. The holding layer serves to accommodate the accommodate substance configured to immobilize an analyte of interest in a liquid phase contained in the channel. LiNb0₃ is a piezoelectric material. Sending a signal via the input interdigital transducer makes the LiNb0₃ crystal structure vibrate and generates acoustic waves propagating on the surface (surface acoustic waves or SAW) or in the bulk substrate (bulk acoustic waves or BAW). The BAW will propagate in the substrate and reflect on the top of bottom as shown by the saw-tooth dotted line in Figure 2. The waveguide layer makes it possible to enhance the propagation of BAW signals. When the device merely has the substance configured to immobilize an analyte of interest in a liquid phase contained in the channel (i.e., the substance free of analyte), the device has one reflection profile of BAW signals detectable at the output interdigital transducer. When an analyte is immobilized on the substance configured to immobilize an analyte of interest in a liquid phase contained in the channel, in some embodiments, the device has another profile of BAW signals detectable at the output interdigital transducer.

In some embodiments, the analyte is chosen from antibodies, antigens, viruses, pathogens, parasites, bacteria, proteins, polypeptides, and nucleic acids. In some embodiment, a holding layer serves to accommodate the substance configured to immobilize the analyte of interest in a liquid phase contained in the channel. In some embodiments, the LiNb0₃ or waveguide layer is coated with one or more avidins so that a desired analyte, e.g., antibody, proteins, etc., can bind to the one or more avidins and become immobilized for a period of time sufficient for detection.

In some embodiments, the virus is chosen from rabies, e.g., Rabie virus; Herpes, e.g., Herpes-simplex-Virus Type 1 & Type 2; measles, e.g., measles virus; mumps, e.g., mumps virus, parainfluenza, rhinitis, bronchiolitis, pneumonia, e.g., respiratory syncytial virus; and German measles, e.g., Rubella Virus.

In some embodiments, the pathogen is chosen from Acanthamebiasis, Australian bat lyssa virus, Babesia (atypical or typical), Bartonella henselae, Ehrlichiosis, Encephalitozoon cuniculi, Encephalitozoon hellem, Enterocytozoon bieneusi, Helicobacter pylori, Hendra morbilli virus, equine morbilli virus, Hepatitis C, Hepatitis E, Human herpesvirus 8, Human herpesvirus 6, Lyme borreliosis, and Parvovirus B19. In some embodiments, the pathogen is chosen from Enterovirus 71, Clostridium difficile, Mumps virus, Streptococcus (Group A), and

Staphylococcus aureus.

In some embodiments, the parasite is chosen from visceral leishmanioses (Kala-azar), e.g., Leishmania infantum and Leishmania donovani; Cutaneous leishmanioses (oriental sore), e.g., Leishmania major, Mucocutaneous leishmanioses, e.g., Leishmania brasiliensis; and Cutaneous leishmanioses, e.g., Leishmania Mexicana.

In some embodiments, the bacteria is chosen from whooping cough, e.g., Bordetella pertussis; lyme disease, e.g., Borreliose; trachoma, e.g., C. trachomatis; and Diphtheria, e.g., Corynebacterium diphtheria; Q-Feve, e.g., Coxiella burnetii; Tetanus, e.g., Clostridium tetani; and Syphilis, e.g., Treponema pallidum.

By way of analogy, binding assays such as immunoassays, DNA hybridization assays, and receptor-based assays have been used to detect trace quantities of specific analyte molecules contained in a sample. Such techniques are, in some embodiments, usable to form the film sufficient to detect proteins, polypeptides and nucleic acids.

Referring to Figure 1A, in some embodiments, the input interdigital transducer 120a and the output interdigital transducer 140a have a pitch ranging from 10 to 100 μιη. In some embodiments, the range is from 20 to 50 μιη. The range facilitates the attributes of a mechanical wave induced in the substrate when a signal is applied to the input interdigital transducer 120a.

The input interdigital transducer 120a and the output interdigital transducer 140a are made of or comprise any conductive material. The input interdigital transducer 120a and the output interdigital transducer 140a, in some embodiments, are made of a thin film of aluminum or an aluminum material. In some embodiments, the input interdigital transducer 120a and the output interdigital transducer 140a is made of one or more materials chosen from gold, platinum, cupper, and titanium. These metals are obtainable easily, e.g., in clean room or semiconductor processes.

In some embodiments, the device 100 further includes input pads 115a/l 15b configured to connect to a signal generator (not shown) and output pads 125a/125b configured to connect to a signal detector (not shown). In the embodiment depicted in FIG. 1A and 1C, input pads 115a/l 15b and output pads 125a/125b are on bottom side of the device. In some embodiments, placing interdigital transducers on one side of device 100 and input and output pads on a different side of device 100 allows sufficient space for arranging input interdigital transducers 120a/120b and output interdigital transducers 140a/140b on opposite ends of corresponding sensing areas 150a/150b.

In some embodiments, input and output pads 115a/115b and 125a/125b are electrically coupled to external circuitry by using a bonding process or structure. In some embodiments, example bonding process or structure includes using a bond wire, a solder ball, or a metallic stud. In some embodiments, input pads 115a/l 15b and output pads 125a/125b have a size sufficient for attaching a bond wire, a solder ball, or a metallic stud thereto. In some

embodiments, input and output pads 115a/l 15b and 125a/125b are electrically coupled to external circuitry without using any bonding process or structure. In some embodiments, input pads 115a/l 15b and output pads 125a/125b are electrically coupled to external circuitry through detachable conductive pins, such as spring-loaded pins. In some embodiments, input pads 115a/l 15b and output pads 125a/125b have a size sufficient to receive a corresponding detachable conductive pin.

In some embodiments, the signal generator generates a signal in the form of a pulsed waveform having an amplitude, phase, and frequency. In some embodiments, the pulsed waveforms are chosen from sinusoidal, square wave, and saw tooth wave. In some

embodiments, the center frequency ranges from 9.0 to 11.5 MHz. In some embodiments, the range is from 9.5 MHz to 11.0 MHz.

The device 100 makes it possible to generate surface acoustic wave (SAW) and bulk acoustic waves (BAW) at the same time. In some embodiments, the operation of device 100 relies primarily on BAW signals.

The inventors realized that there are actually bulk signals in, e.g., the 128° LiNb0₃. The interdigital transducers generate base and harmonics frequency of BAW signal. These frequencies, as noted above, are scalable based on the thickness of piezoelectric substrate.

By way of a non-limiting example, assume the piezoelectric substrate has a thickness of 0.45 mm. A base frequency is about 10.7MHz and the first harmonic frequency is about 16MHz ( 10MHz x ( 2x1+1 ) 12 ), the second harmonic frequency is about 26.75MHz. (10MHz x ( 2x2+1 ) 12 ), etc. The formula of nth harmonic frequency is as follows:

harmonic f = base f x ( 2n+l ) / 2 In some embodiments, one detects the base, I^s , 2d, 3^r , or 4th harmonic frequency, although any detectable harmonic is usable. In some embodiments, multiple harmonics are detectable. In general, the higher order of harmonic frequency, the less power the signal has.

These harmonic and base frequencies (10.7MHz ) are all BAW signals, and, e.g., one uses the base frequency to detect surface binding.

In some embodiments, the range of base frequency is about 10.5MHz to 11MHz, e.g., when the thickness of substrate in the market is from 0.45mm to 0.55mm.

In some embodiments, the signal detector detects one or more signal properties chosen from amplitude, phase, and frequency. In some embodiments, the signal detector detects the time-delay between the time the signal is arrives at the input interdigital transducer and the time the signal arrives at the output interdigital transducer.

The device is makeable by routine steps. In some embodiments, e.g., the piezoelectric substrate is modifiable by multiple techniques to make the interdigital transducers. For example, in some embodiments, the interdigital transducers are made by screen printing of electrodes with silver and silver-palladium inks. In some embodiments, the interdigital transducers are made by an electron beam thermal evaporation process followed by a conventional mask etching. In some embodiments, the interdigital transducers are made of a material chosen from aluminum, gold, platinum, silver, copper, and NiCr. In some embodiments, the interdigital transducers are made of a material chosen from conductive materials.

The signals sent by input interdigital transducer are converted into mechanical waves and propagate through sensing area. Due to properties of piezoelectric substrate, those waves propagating through sensing area are translated into electric signals in the output interdigital transducer. By detecting the output signal from the interdigital transducer, one determines information about the sensing area and the substances in the vicinity of the sensing area.

The center frequency detected in the output interdigital transducer is affected by different fluid or particles on the top of sensing area. This effect is interpreted into frequency shifts, phase shifts, and/or amplitude shifts versus different concentrations of fluid or particles. While the different fluids or particles are filled in the channel, the change in fluids or particles causes a change of dynamic viscosity on the top of sensing area. The change produces a different reflecting angle of bulk acoustic wave and frequency shift of signals in the output interdigital transducer.

In some embodiments, the signal in the output interdigital transducer has a single-pole center frequency and high Q factor (>2000). The definition of a Q factor is as follows:

Q = Center frequency / Bandwidth.

In the other words, e.g., Q is inversely proportional to the bandwidth. If Q is high, the bandwidth is narrow. High Q devices facilitate detecting frequency shifts of the bulk acoustic wave, because the signal shift will be clearer and larger if bandwidth is narrower and the device is high Q. Since signal shift is relative to sensitivity, it has higher sensitivity if signal shift is more clear and larger.

Figure 3 shows a method of detecting an analyte according to some embodiments. In some embodiments, the method of detecting an analyte 300, includes, in the device described herein, placing a sample in the channel 310. And afterwards, a signal is sent to the input interdigital transducer 320. The signal from the output interdigital transducer is detected 330.

In some embodiments, the method of detecting an analyte 300, optionally includes placing a reference sample in the second channel 315. In some embodiments, the reference sample is a control sample. The signal to the input interdigital transducer and to the second input interdigital transducer 325 are sent. The signal from the output interdigital transducer and the second output interdigital transducer 335 are detected.

In some embodiments, the detecting is for changes in amplitude, phase, frequency, or time-delay between the input signal to the input interdigital transducer and the output electrical signals from the output interdigital transducer.

By way of a non-limiting example, consider embodiments of the device having two or more channels, one of which is referred to as a sample channel and the other is referred to as a reference channel. When a sample is injected into the sample channel, the bulk acoustic wave signal of sample channel will shift due to the immobilized analyte and, e.g., and for purposes of explanation, temperature effects caused, e.g., by the sample's having a different temperature than the environment and/or device. In a scenario in which the analyte and temperature affect the device at the same time, it will be most difficult to determine the real frequency shift attributable to the analyte.

The temperature difference in sample channel will also affect the reference channel, and if the sample channel and reference channel are next to each other (or nearby in proximity), the temperature difference effects will exist in both the sample channel and the reference channel.

A control sample is prepared. The control sample is substantially the same as the sample but the control sample lacks the analyte. The control sample is added to the reference channel. In the reference channel, the bulk acoustic wave signal of sample channel will not shift due to the immobilized analyte, because the control sample lacks the analyte. Thus, the signal from the reference channel acts as a control to determine, in this example, the effects of temperature on the signal, because the frequency shift in reference channel must from temperature effects. In this example, the sample channel has a frequency shift from the analyte and temperature effects, and the reference channel has a frequency shift from temperature.

If Sample channel subtracts Reference channel, one arrives at a first order approximation of the frequency shift from particles.

In this way, a reference channel is useable to calibrate and optionally compensate for the temperature effects.

In some embodiments, the signal shifts due to immobilized analyte is equal to signal difference between sample channel and reference channel:

f analyte— f sample channel ~ f reference channel

In some embodiments, data are stored and values calculated using a general purpose computer system having a computer readable storage medium. A computer readable storage medium is an electronic, magnetic, optical, electromagnetic, infrared, and/or a semiconductor system (or apparatus or device). For example, the computer readable storage medium includes a semiconductor or solid-state memory, a magnetic tape, a removable computer diskette, a random access memory (RAM), a read-only memory (ROM), a rigid magnetic disk, and/or an optical disk. In some embodiments using optical disks, the computer readable storage medium includes a compact disk-read only memory (CD-ROM), a compact disk-read/write (CD-R/W), and/or a digital video disc (DVD).

The general purpose computer system includes, in at least some embodiments, an input/output interface and a display unit. The input/output interface is coupled to a controller and allows the circuit designer to manipulate the general purpose computer system. In at least some embodiments, the display unit displays the status of executing and in some embodiments provides a Graphical User Interface (GUI). In at least some embodiments, the input/output interface and the display allow an operator to operate the computer system in an interactive manner.

In some embodiments, the reader is configured to communicate with devices chosen from smart phones and other computers/laptops. In some embodiments, the reader is configured to upload data to a database or the cloud.

The method is, in some embodiments usable to approximate other effects, e.g., differences in solute (surfactant, etc.) pH, etc. between the control sample and the sample.

Example of Applications

In some embodiments, the analyte is from a human sample. In some embodiments, the human samples are chosen from plasma, serum, urine, saliva breast milk, tears. Although applications are discussed based these different human samples, the applications are not limited to human samples.

Samples are collected under the circumstances: Sample providers are asked avoid alcohol consumption for 12 hours prior to sample collection, and sample providers are asked to abstain from major meals 60 minutes before sample collection. Sample providers are also asked to avoid airy foods, high sugar foods, and high caffeine content foods.

Plasma: Method 1 - Blood is collected and use EDTA or herapin as anticoagulant. The blood is centrifuged at 1000-2000x g for 10-15 minutes using a mini-portable centrifuge and the liquid supernatant is collected as plasma.

Method 2 - A Blood sample is collected in commercially available EDTA, herapin treated tubes (lavender or green top). The blood is processed in a centrifuge-free filtration device to obtain the plasma. Serum: Blood is collected into serum separator tubes. The blood sample is at room temperature for at least about 30 minutes or a period of time sufficient for the blood will clot. The blood is thereafter centrifuged at 1000 x g with the mini-portable centrifuge for about 15 minutes. The liquid supernatant is collected as serum.

Urine: Void urine (mid-stream to reduce microbial or cellular contamination) to sterile container. Centrifuge at 10,000 x g at 4oC for minutes to remove particles.

Saliva: The sample provider is asked to refrain from brushing teeth for 45 minutes before collection to avoid blood contamination. Saliva is collected with a salivette or similar material, e.g., cotton to collect saliva. The salivette or similar material is placed into a tube for centrifugation to collect the liquid. The salivette or similar material is centrifuge at 10,000 x g at 4°C for 2 minutes to remove particles.

Breast Milk: After collection, the breast milk is centrifuged at 10,000x g at 4°C for 15 minutes to remove particles. The aqueous fraction is collection and the centrifuging is optionally repeated on the fraction one or two more times, each time the supernatant is collected.

Tears: Tears are collected by applying the glass capillary tubes to lower lid margin tear film meniscus without touching the eye. Tears are alternatively collected with commercial Schirmer strips. The sample processing of tears is consistent from batch-to-batch to avoid process dependent effects on the protein make-up of the sample.

Example Urinalysis

A urinalysis, also known as routine and microscopy (R&M), is an array of tests performed on urine, and one of the most common methods of medical diagnosis. Urinalysis can reveal diseases that have gone unnoticed because they do not produce striking signs or symptoms. In some embodiments, a complete urinalysis is used to diagnose a disease selected from kidney diseases, urinary tract infections, cancers, reactions to medication, prostate infections, liver diseases, viral infections, yeast infections, aparasitic infections.

Epithelial (flat cells) and red and white blood cells are seen in the urine. Sometimes cells, cellular debris, and casts are seen in the microscopic urinalysis. Epithelial cells sometimes suggest inflammation within the bladder. Casts and cellular debris originate from higher up in the urinary tract, such as in the kidneys. These materials are shed from kidney cell lining due to injury or inflammation and travel down through the urinary tubes. These usually suggest an injury to the kidney from an inflammation or lack of blood flow to the kidneys.

A high count of red blood cells in the urine sometimes indicates infection, trauma, tumors, and/or kidney stones. If red blood cells are seen under microscopy to look distorted, the appearance suggests the kidney as the possible source and arises due to kidney inflammation (glomerulonephritis).

Urine is a generally thought of as a sterile body fluid, therefore, evidence of white blood cells or bacteria in the urine is considered abnormal and may suggest a urinary tract infection such as, bladder infection (cystitis), infection of kidney (pyelonephritis). White blood cells may be detected in the urine through a microscopic examination (pyuria or leukocytes in the blood).

In some embodiments, red blood cells, white blood cells and bacteria, such as chlamydia are detected using a device described herein.

A prepared sample of urine is placed in one channel of a device having two channels. A reference sample is placed in the second channel. A signal is sent and the frequency shift is detected. The frequency shift is interpreted to indicate the presence of chlamydia in the urine.

Example: Diagnosis for Diabetes Diabetes is a medical disorder that causes an abnormal amount of sugar in the blood. A doctor is trained to spot signs of the diabetes through urinalysis for diabetes. The doctor sometimes performs glucose tests, ketone tests, and microalbuminuria tests on the urine of person suspected of having diabetes. This analysis helps a doctor diagnose the disease, spot a complication of existing disease, and potentially prevent any further damage.

Glucose is the manner in which sugar is transported around the body through the blood. Diabetes affects a hormone called insulin, which normally regulates the amount of sugar in the bloodstream. Insulin carries the glucose from the blood into the cells that use it. A common sign of diabetes is this abnormal glucose level, and an important form of urinalysis for diabetes is the glucose test. A healthy person's urine does not usually contain any glucose, and presence of the sugar can indicate the presence of diabetes.

A prepared sample of urine is placed in one channel of a device having two channels. A reference sample is placed in the second channel. A signal is sent and the frequency shift is detected. The frequency shift is interpreted to indicate the presence of glucose in the urine.

Early Diagnosis for Disease from Saliva

A simple saliva sample sometimes provides relevant data. This technique facilitates early diagnosis, the only option to control the effects of some diseases and enable a better quality of life. The saliva test is quick, painless and accurate. This diagnostic test is noninvasive, potentially a much more accessible, less expensive and easier to perform procedure than other medical procedures. Above all, saliva tests sometimes help to detect diseases, such as osteoporosis.

In some embodiments, the saliva sample is used to detect the following diseases: Periodontal diseases such as dry mouth of saliva depends in a very broad sense, our oral health. Saliva protects the inner surface of your mouth and teeth naturally and plays a defensive role against caries, as saliva dilutes and removes sugars and maintains a constant pH of our mouth. Saliva even helps diagnose certain diseases such as diabetes, periodontal disease or oral cancer through the analysis of microorganisms.

Oral cancer. Saliva is sometimes used to help detect oral cancer. One study found that saliva contains at least 50 microRNAs (molecules that control the activity and assess the behavior of genes), which would help in detecting oral cancer.

Familial hypercholesterolemia. This silent disease becomes visible through its most drastic consequences: heart attack, stroke or thrombosis. Saliva is used to detect warning signs before the secondary indications arrive.

Cardiovascular diseases. Saliva is used to serve as an indication if there is a genetic predisposition to cardiovascular diseases. That is, whether a person is more likely to have problems in the coronary arteries, have a heart attack, suffer a cardiac arrhythmia or peripheral arterial disease can be determined.

Gastrointestinal disorders. Saliva is used to help in the digestion process. Before food reaches your stomach, saliva starts to decompose while still in your mouth. Saliva moistens food and makes swallowing easier.

Bone diseases. Diseases of the bones are typically detected by densitometry, but several studies have identified some indicators in saliva samples that follow the same pattern as those identified in urine or serum. Therefore, assessing bone diseases by saliva is just as reliable it through the blood. Emotional conflicts. High levels of Cortisol in saliva may indicate the person's exposure to stressors, emotional conflicts or situations that create stress. The researchers found that during puberty and adolescence, when a girl argues with her parents, has a bad relationship or grows in a hostile environment, her saliva has higher level of Cortisol, known as the stress hormone.

A prepared sample of saliva is placed in one channel of a device having two channels. A reference sample is placed in the second channel. A signal is sent and the frequency shift is detected. The frequency shift is interpreted to indicate the presence of Cortisol in the saliva.

Samples and Reference samples

Samples, reference samples, and control samples are liquid phase. In some embodiments, the analyte is in suspension. In some embodiments, the analyte is solvated. In some

embodiments, the liquid phase is or comprises water or alcohol, e.g., ethanol. Other additives, e.g., pH buffers/adjusters, surfactants, and the like may be added to the samples and control samples. In some embodiments, saline water is usable as the reference sample.

Examples

In order to verify the functionality of MEMS biosensors, nanobeads were used to test signal shifts. Figures 4 and 5 show the different signal shifts versus different size of nanobeads. The smaller size of the nanobeads causes a more dynamic change on the surface of biosensor and bulk acoustic wave signal shifts. Clearly, different sizes of nanobead particles on the sensing area cause different signal shifts.

A device for making bulk acoustic waves in a liquid sample included a piezoelectric substrate (128° Y-cut L1NO₃ crystal). The input interdigital transducer (IDT) is located in a region on a surface of the piezoelectric substrate, and the output IDT is located in the region on the surface of the piezoelectric substrate. The IDTs were made of a conductive material. The sensing area between the output IDT and the input IDT. The channel configured to contain liquid is over the region comprising the sensing area.

In Figure 4 and 5, different nanobeads were tested. In these experiments, three steps verified positive and negative controls. In step 1, the BAW signal was detected with deionized water before immobilizing the nanobeads. In step 2, the nanobeads were introduced and immobilized; thereafter the signal was detected. In step 3, the nanobeads were removed by adding excess deionized water; and thereafter the signal was detected. In figure 4, deionized water and nanobeads were introduced. The nanobeads in these experiments were non-adhesive so that they were washed off by using deionized water. So the positive control in these experiments is based on step 1 and 2. The negative control in these experiments is based on step 1 and step 3. The true positive is that the signal shifted after the nanobeads were introduced in the channel. The true negative is that the signals in the step 3 returned to similar level in step 1. Step 3 is to remove beads from cartridge. So the signal should shift back to those levels of Step 1. These results are shown in Figure 4.

The smaller size of particles (<200nm) will case more frequency shift.

Figure 4 shows the effect of size of naonbeads versus signal shifts. The signal shift is the center frequency relative to a reference channel. Using a control sample without an analyte (nanobeads). These data show that the smaller size of particle on the surface cause larger signal shifts. These data also show the sensing applications to detect a virus or a protein.

Data for the non-biotinylated and biotinylated micro beads on the surface of biosensor before/after washing steps (not shown) show a good adhesion for biotinylated micro beads or any other label free sensing application. Based on these results, the device is directly useable for diagnostic platforms that will be used in field settings where practitioners using the device might not have extensive medical background required for its use.

The present inventors made it possible to produce a high-Q bulk acoustic wave sensor with or without a waveguide layer, which device is shown to be able to detect molecules or different viscosities on the surface. These changes in frequency or phase are convertible into electrical signals and read at the output interdigital transducers.

In some embodiments, the described device expands the fluidic sensing applications into many areas including uncoated, or coated with biological/enzymatic films. In some

embodiments, expansion is accomplished by building a device having a variety of channels sufficient to handle a variety of fluids uniquely and optionally substantially simultaneously. In some embodiments, the device has a reference channel which makes it possible to calibrate and optionally compensate for the temperature difference from samples, e.g., human samples. In some embodiments, a waveguide layer, e.g., the Si0₂ or PVC, is deposited on the sensing area which makes it possible in some embodiments not only to increase the sensitivities and changes of dynamic viscosities but also to reduce the temperature effects of a piezoelectric substrate like Y-cut LiNb0₃. In some embodiments, the Si0₂ is also media of surface binding in biological applications. BAW or APM on the 128° Y-Cut LiNb0₃ and other piezoelectric substrate in this invention has high-Q responses which yields a high sensitivity in sensors applications.

In one aspect, the invention is a device is for making bulk acoustic waves in a liquid sample. The device includes a piezoelectric substrate. An input interdigital transducer is located in a region on a surface of the piezoelectric substrate. An output interdigital transducer is located in the region on the surface of the piezoelectric substrate. A sensing area is between the output interdigital transducer and the input interdigital transducer. A channel is configured to contain liquid over the region including the sensing area.

In one aspect, the invention is a device is for making bulk acoustic waves in a liquid sample. The device includes a piezoelectric substrate. An input interdigital transducer is located in a region on a surface of the piezoelectric substrate. An output interdigital transducer is located in the region on the surface of the piezoelectric substrate. On the opposite side of the

piezoelectric substrate is a sensing area between the output interdigital transducer and the input interdigital transducer. A channel is configured to contain liquid over the region including the sensing area.

In one aspect, the invention is a use of the device for making bulk acoustic waves in a liquid sample. The use includes placing a sample in the channel. Thereafter, the use includes sending a signal to the input interdigital transducer and detecting the signal from the output interdigital transducer.

In one aspect, the invention is a use of the device for making bulk acoustic waves in a liquid sample. The use includes, in some embodiments, placing a sample in the channel and placing a reference sample in the second channel. Thereafter the use includes sending a signal to the input interdigital transducer and to the second input interdigital transducer and detecting the signal from the output interdigital transducer and the second output interdigital transducer. The use optionally further includes subtracting, from the detected signal from the output interdigital transducer, the signal from second output interdigital transducer.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A device for making bulk acoustic waves in a liquid sample, comprising:

a piezoelectric substrate;

an input interdigital transducer located in a region on a surface of the piezoelectric substrate;

an output interdigital transducer located in the region on the surface of the piezoelectric substrate;

a sensing area between the output interdigital transducer and the input interdigital transducer; and

a channel configured to contain liquid over the region comprising the sensing area.

2. The device of claim 1, wherein the piezoelectric substrate is chosen from Y-cut L1NO₃ crystals.

3. The device of claim 2, wherein the Y-cut L1NO₃ crystal is 128° Y-cut L1NO₃ crystal.

4. The device of claim 1, further comprising

a second input interdigital transducer located in a second region on the surface of the piezoelectric substrate;

a second output interdigital transducer located in the second region on the surface of the piezoelectric substrate; and a second sensing area between the second output interdigital transducer and the second input interdigital transducer.

5. The device of claim 4, further comprising

an additional input interdigital transducer located in an additional region on the surface of the piezoelectric substrate;

an additional output interdigital transducer located in the additional region on the surface of the piezoelectric substrate; and

an additional sensing area between the additional output interdigital transducer and the additional input interdigital transducer.

6. The device of claim 1, wherein the sensing area is on the same surface of the

piezoelectric substrate as the output interdigital transducer and the input interdigital transducer.

7. The device of claim 1, wherein the sensing area is on the opposite surface of the piezoelectric substrate as the output interdigital transducer and the input interdigital transducer.

8. The device of claim 1, wherein the piezoelectric substrate comprises a waveguide layer on a surface and the sensing area is on the waveguide layer.

9. The device of claim 8, wherein the waveguide layer is less than 150 nm thick.

10. The device of claim 8, wherein the waveguide layer is less than 100 nm thick.

11. The device of claim 8, wherein the waveguide layer comprises a material selected from SiC"2 and polyvinyl chloride.

12. The device of claim 1, wherein the sensing area comprises a film comprising a substance configured to immobilize an analyte of interest in a liquid phase contained in the channel.

13. The device of claim 1, wherein the input interdigital transducer and the output interdigital transducer has a pitch ranging from 10 to 100 μιη.

14. The device of claim 1, further comprising an input pad configured to connect to a signal generator.

15. The device of claim 1, further comprising an output pad configured to connect to a reader.

16. A method of detecting an analyte, comprising, in the device of claim 1:

placing a sample in the channel; and thereafter

sending a signal to the input interdigital transducer; and

detecting the signal from the output interdigital transducer.

17. The method of claim 16, wherein the detecting is for changes in amplitude, phase, frequency, or time-delay between the input signal to the input interdigital transducer and the output electrical signals from the output interdigital transducer.

18. A method of detecting an analyte, comprising, in the device of claim 4:

placing a sample in the channel;

placing a reference sample in the second channel; and thereafter

sending a signal to the input interdigital transducer and to the second input interdigital transducer; and

detecting the signal from the output interdigital transducer and the second output interdigital transducer.

19. The method of claim 18, wherein the detecting is for changes in amplitude, phase, frequency, or time-delay between the input signal to the input interdigital transducer and the output electrical signals from the output interdigital transducer.

20. The method of claim 18, further comprising subtracting, from the detected signal from the output interdigital transducer, the signal from second output interdigital transducer.