Development of Simple and Portable Surface Acoustic Wave Biosensors for Applications in Biology and Medicine

Abstract

There has been a renewed interest in the development of surface acoustic wave (SAW) biosensors because they hold great promise for opening new frontiers in biology and medicine. The promise of SAW technology is grounded in the advantages SAW devices hold over traditional laboratory techniques used in biological and medical laboratories. These advantages include having smaller sizes to allow greater portability, using smaller sample volumes, requiring lower power requirements, the ability to integrate them into microfluidic platforms, and their compatibility with smart devices such as smartphones. The devices offer high sensitivity and can be designed to allow microfluidic interfacing. Other major advantages of SAW-based technologies include the fact that they can be operated remotely in harsh conditions without the need for an AC power supply. Their compatibility with lab-on-a-chip systems allows the creation of fully integrated devices with the ability to isolate the sample from the operator. In this mini-review, we will discuss SAW devices and their ability to enable a variety of applications in Biology and Medicine. The operating principles of the SAW biosensors will be discussed along with some technological trends and developments.

1. Introduction

Over the past three decades, the emergence of small portable lab-on-a-chip biosensors has developed into an important area in biology and medicine [1]. Lab-on-a-chip devices promise to perform all the functions of a traditional laboratory on a miniature microfluidic platform [2]. These small devices allow full automation of analysis, reduce the sample volumes, and reduce the time of analysis as well [2] SAW devices are ideally suited to provide the sensing element in the Lab-on-a-chip platform [3]. One limitation that has plagued lab-on-a-chip devices has been the lack of a miniaturized sensor that facilitates the development of high-sensitivity assays. Several technologies have been explored including Mass spectrometry field [4, 5], electrochemical sensors field [6], optical detection field [7, 8], surface plasmon resonance spectroscopy field [9], and interferometry [10, 11] but each has significant limitations in complex biological matrices. The SAW biosensor technology is based on Surface acoustic waves (SAW), a type of acoustic wave that propagates along a surface of a solid material [12, 13, 14]. Propagating SAW are impacted by mass loading and changes in viscoelastic properties of the media on the surface of the sensor and compared to a reference, as seen in Figure 1a and b [15]. These waves were first described by John William Strutt, 3rd (Lord Rayleigh), in an article on the propagation of acoustic waves in a piezoelectric material [16]. White and Voltmer introduced the concept of using interdigital transducers (IDTs) as a more efficient method of generating surface acoustic waves [17, 18, 19]. This method is still used today to generate surface acoustic waves on a piezoelectric material [20, 21]. The piezoelectric materials that are typically used for fabricating SAW devices are listed in Table 1 [17].

Figure 1.

Acoustic wave measurement using (a) a delay line SAW device with measurement performed via a two-port vector network analyzer (VNA). Connections are made using a customized printed circuit board with connectors to the VNA. In (b) there is a plot of the change in phase of the reference, the change in phase of the sample and the difference between the two channels.

Piezoelectric substrate	Orientation (axis)	Wave velocity (m/s)	Temperature coefficient (ppm/°C)	Coupling coefficient (%)	Wafer cost (relative cost)
Quartz	32*YX	3159	0	0.16	Low
Quartz	43*	3159	0	0.16	Low
Lithium tantalate	36°-YX	4160	5.0	28–32	Medium
Lithium tantalate	42°-YX	4022	7.6	40	Medium
Lithium niobate	128*YX	3980	75	5.5	High
Lithium niobate	64*YX	4742	70	11.3	High
Langasite	138.5*	2330	38	0.37	High

Table 1.

Properties of common piezoelectric substrates used in the fabrication of surface acoustic wave devices. Courtesy of The Roditi International Corporation Ltd, UK.

To date, the primary use of SAW devices has been in the telecommunications industry, specifically as filters in cellular telephones and other smart devices [17]. SAW devices operate in the following manner: a metal IDT deposited on a piezoelectric surface is driven by a sinusoidal power source with a period specific to the IDT design [22]. This causes the electrode to vibrate, generating an acoustic wave that is perpendicular to the direction of the IDTs [23]. The penetration depth of the acoustic wave is relatively shallow. This is because the wave produces an evanescent field that cannot penetrate more than a few nanometers into the substrate from which the sensors are fabricated. If a guiding layer is used (a top layer), the acoustic wave propagation will be confined to the substrate-sample interface [24]. The confinement of the wave via a guiding layer maximizes the energy density of the acoustic wave at the substrate-sample interface.

The velocity of an acoustic wave in a piezoelectric material is an intrinsic property and varies slightly as a function of temperature [25, 26, 27] In the case of lithium tantalate, one of the more popular substrates used for the fabrication of SAW filters and devices, the wave velocity is 4200 m s⁻¹ [28]. Values for the acoustic properties of commonly used piezoelectric substrates are listed in Table 1 below. The acoustic wave velocity is approximately 10⁴–10⁵ times smaller than the velocity of electromagnetic waves [29]. Surface acoustic waves can be generated and detected by spatially periodic, interdigital electrodes that are deposited on the planar surface of a piezoelectric plate. Excitation of the interdigitated electrode with a radio frequency source generates a periodic electric field, thus permitting piezoelectric coupling to a traveling surface wave. The center frequency of the acoustic wave generated by the sensor, fc, is governed by the Rayleigh wave velocity (VR). VR depends on the piezoelectric substrate and the electrode width (a) of a single finger, according to the equation fc = VR/4a [30]. The velocity for the SAW generated by the device depends on the properties of the piezoelectric substrate (crystal) that is used in the fabrication of the sensor and its crystallographic orientation. Computer models have allowed the careful sorting of numerous crystallographic orientations to enable the discovery of different types of acoustic waves. Where vs. is the SAW velocity and f_c is the center frequency of the device. The SAW velocity is an important parameter determining the center frequency. The mass sensitivity is given by Sauerbrey’s equation, where:

$$\Delta f/f_0\approx \Delta V/V_0$$E1

and

$$f_0=\nu /\lambda$$E2

Propagation loss is one of the major factors that determine the insertion loss of a device and is caused by wave scattering at crystalline defects and surface irregularities. Materials that show high electromechanical coupling factors combined with small temperature coefficients of delay are generally preferred. The free surface velocity, V_f, of the material is a function of the cut angle and propagation direction. The TCD is an indication of the frequency shift expected for a transducer due to a temperature change and is also a function of the cut angle and propagation direction. The substrate is chosen based on the device design specifications, which include operating temperature, fractional bandwidth, and insertion loss.

Indeed, the fabrication of SAW devices requires a few critical components including the physical deposition of a metal on the surface of the piezoelectric substrate, etching of that metal deposited on the surface, optional deposition of guiding, and/or sensing layer. SAW devices mainly have two kinds of structures. The first is a design that features two sets of IDTs where a sinusoidal radio frequency (RF) is applied to one side of the IDT structure while the other is connected to the ground. The RF is applied to the first set of IDTs or the input IDTs, which generates an acoustic wave, as is seen in Figure 1a. The wave is transmitted through a delay line and is received by the second set of output IDTs. The signal is then captured and analyzed. The second type of SAW termed a resonator, features one set of IDTs with grating reflectors that can trap the surface wave. The signal from the IDT can be amplified and then fed back to the input IDT.

There are several factors that affect the transmission of the acoustic wave in a SAW device and have to be closely monitored or controlled in order to generate repeatable results. These include temperature, pressure, humidity, and mass loading. Indeed, SAW devices can be operated as sensors for temperature, pressure, humidity and mass loading. Temperature effects are the most challenging when trying to perform sensitive acoustic-wave sensing in liquid media. This is controlled in part by including a reference delay line. The reference is functionalized with a passivating agent to minimize nonspecific binding (e.g., PEG). For biological or medical applications, the active sensing line is functionalized with capturing agents that interact specifically with the analyte being targeted. An Analyte can be quantitatively detected by monitoring changes in gas pressure, liquid pressure or from the increase mass due to binding of a biological molecule to a targeting molecule or the increase in the fluid density or mass increases due to absorption of the target. The sensitivity of SAW devices increases as the square of the frequency; therefore, higher frequencies lead to smaller, more sensitive instruments. However, the frequency also determines the depth within the sample that the device can probe; for example, for a solution placed atop a sensor, the higher frequencies will examine a shallower depth than lower frequencies. Thus, the operating frequency of the SAW must be considered when selecting targets, probes, and conjugation schemes to functionalize the active sensing region of a SAW device.

Aside from the layer of targeting molecules that is typically used to decorate a SAW device, an additional guiding layer is often deposited to enhance the sensitivity of the device. The guiding layer traps the energy of the acoustic wave near the surface of the device to increase sensitivity to surface perturbations. The SAW sensors are inherently capable of detecting analytes in solution concentrations on the order of parts-per-billion (ppb) by mass, through the use of higher frequencies >300 MHz Ideally, the guiding layer needs to have a lower density and lower acoustic velocity than the piezoelectric substrate. Materials that have been utilized in past include polymers such as poly-methyl-methacrylate and Novolac and oxides including silicon dioxide and silicon monoxide. Changes in the mass loading of the surface, if all else is kept constant, affect the acoustic wave velocity as it travels across the delay line from the input IDT to the output IDT. The time delay is a result of the interactions between any adsorbed mass, i.e. the analyte, and results in a phase shift between the applied and the detected sinusoidal wave. In the case where there is only a single IDT, the round-trip time is measured by the applied signal. In recent years, there has been an increasing demand for portable, disposable and inexpensive sensors for biological and medical applications. Due to these increasing demands for miniature sensors, SAW devices have received renewed interest for use as sensors in biochemical assays and as detectors in microfluidic biosensors, particularly since it is a label-free technique. In this review, we will outline the use of SAW biosensors in biology and medicine.

2. Principles for SAW biosensors

Surface acoustic waves are generated in a SAW device with the application of a sinusoidal RF to one side of the IDT patterned on the piezoelectric material, while the other side is connected to the ground. An image of a typical measurement scheme is shown in Figure 1a with a custom-built printed circuit board and connections with a vector network analyzer (VNA). The wavelength of the acoustic wave generated will be a function of the material properties, the shape, and layout of the IDTs, and the material deposited as the guiding layer. The main parameter utilized in data analysis is the velocity of the acoustic wave. Each piezoelectric material will allow an acoustic wave to propagate at different velocities for a given wavelength. The larger the wave velocity, the smaller time needed to have the acoustic wave travel from the input IDT to the output IDT. Another important parameter for SAW devices is the frequency that it operates. Only when the wavelength of the applied RF is equal to the intrinsic wavelength of the SAW, i.e. the period of the IDT, can the SAW be stimulated to produce an acoustic wave described by Eq. (2). Measurements of acoustic wave velocity is often reported as a phase shift between a reference channel and a sample channel. An image of a typical measurement is seen in Figure 1a while the plot of phase shift is seen in Figure 1b.

The IDTs can be fabricated as periodic bars with uniform lengths, widths, and gap spacing. They can be designed to give bi-directional or unidirectional acoustic wave propagation. Small IDT gap spacing and widths and thus smaller wavelengths often result in a higher frequency. In some cases, different IDT width/gap ratios can generate higher harmonic waves. The velocity of the wave that is generated can be influenced by several factors such as temperature and the analyte’s concentration change. The temperature has a significant effect on the wave velocity. This effect can be described as the temperature coefficient of the frequency (TCF). TCF is described as a relative change in frequency with the temperature. Other factors that influence the device’s sensitivity include humidity and pressure. Any slight fluctuations in the pressure will impact the mass loading of the device. Any change in the humidity results in changes to the electric field. The electric field strength is also impacted by charged particles that are suspended in the solution with comparatively high dipole moments (Figure 2).

Figure 2.

Image of a surface acoustic wave biosensor where (a) is an image of new unused sensors while (b) shows an image of used sensors and (c) is a high magnification image of one of the sample channels and the reference channels from (b).

3. Biological and medical application of SAW devices

Changes in the surface mass can attenuate the wave velocity. These minute changes are measured by either measuring the changes in the resonance frequency of the piezoelectric material or by measuring the time the SAW travels from the input IDT to the output IDT. In the case when there is only a single IDT, then we measure the round-trip time.

The temperature dependence of the SAW measurements is a property that can be used in temperature sensors. Borrero et al. report a SAW resonator-based temperature sensor that was fabricated from 128^o Y-X LiNbO₃ crystal. The device operated at a frequency of 65 MHz and was also capable of measuring pressure and impedance. The one-port SAW resonator had an IDT width of 15 μm, 20 IDT finger pairs, and an acoustic aperture of 15λ. There were also 100 electrodes on each side of the IDT pairs. A linear dynamic range was established between 50 and 200°C, while the frequency had a linear response with temperature.

Wireless temperature sensing is possible on SAW devices. This technique takes advantage of the round-trip flight time of travel. Wireless SAWs are made not to require any power supply so they can be used for remote sensing. Reindl et al. designed a delay line wireless SAW temperature sensor. The device operates by sending a VHF/UHF band RF burst delivered by a radar transceiver. The SAW then performed a measurement. Since the temperature affects the changes affect the wave velocity, measurement of the response pattern can be used to determine the temperature. The resolution of this system was ±0.2°C. This type of system could be used for biological applications in extremely isolated locations or for patients who have been isolated from the general population due to a high communicable infection.

SAW sensors can be fabricated and implanted in the body to monitor core-body temperatures in real-time. Martin and colleagues packaged a single-port resonator (single IDT) SAW temperature sensor, in a ceramic and connected it to a small antenna. In vivo tests in dogs demonstrated the capacity to perform wireless interrogation of samples. SAW devices fabricated from materials having large TCF are well suited for fabricating temperature sensors but are not well suited to fabricating other types of sensors. To perform other types of sensing, methods should be developed to compensate for the effects of TCF. One solution is to introduce a guiding layer of a material that reduces the TCF such as SiO₂. SiO₂ is often used to compensate for the negative TCF for most piezoelectric materials used to fabricate SAW devices. Zhang et al., published a report that experimentally verified that a SiO₂ layer of a thickness of 0.3λ gives a TCF of zero for LiNbO₃. A large electro-mechanical coupling coefficient of 7.92% was also observed when the thick SiO₂ guiding layer was used. Another method for performing temperature compensation would be to add a second SAW device and a mixer cell [1, 31]. In such a configuration, one SAW acts as a reference while the other SAW acts as a sensing unit [1, 31]. If the two SAW sensors are placed in such a manner that both devices experience the same temperature with only one sensor actually sensing changes, then any interference occurring to both systems would be canceled out after the mixer [1, 31]. The SAW velocity is strongly affected by the pressure applied to the piezoelectric material. Therefore, a SAW pressure sensor would be a device that exploits this pressure-frequency relationship [32]. To enhance the sensitivity, often a method similar to that of Grousset et al. is followed where the area below the sensing area is etched [8]. In that report, they used an AT-cut quartz film, operated at 430 MHz that was etched by Deep Reactive Ion Etching (DRIE) to expose the sensing area. The resonance frequency showed a linear relationship with the applied pressure and had a sensitivity of 25.8 kHz/bar from 0 to 4.8 bar.

SAW pressure sensors can be implanted in the human body. Liang et al. reported a blood pressure sensor where they amplified the signal from a SAW device by using a Colpitts oscillator. A static test showed a 1.75 kHz/mmHg sensitivity with a standard deviation less than 1 mmHg. Another wireless in vivo SAW device was reported by Murphy et al. that was designed to monitor blood pressure remotely from inside the left ventricle of the heart of a living porcine subject. A prototype of the device was able to monitor changes in blood pressure around the clock and then compared the results with a commercial catheter-tip transducer. The primary challenge to building in vivo SAW devices is to have a well-designed antenna to deliver the RF signal and to receive the data with minimal signal loss.

4. Molecular biosensors

Due to their small sizes, ability to monitor label-free, and high accuracy, SAW devices are ideally suited to function as biosensors. They can be operated remotely, they can be used as implantable devices and they facilitate real-time measurements for patients remotely. The high accuracy of the SAW supports it to use as a bacterial cell monitor, viral particle monitoring, and a DNA detection system. Cai et al. used a SAW device to detect DNA sequences and cells. As is frequently done for SAW devices, gold was deposited on the guiding layer to form a sensing layer for DNA detection. The hybridization of the target single-stranded DNA target molecules (ssDNA) with the DNA probe resulted in a frequency shift of the SAW resonator and could be recorded and measured. DNA detection using this device achieved a sensitivity of 6.7 x 10⁻¹⁶ g/cm² per Hz. The device was also capable of detecting a single EMT6 and 3 T3 cancer cell. Biochemical assays need to be able to monitor in liquid environments to known concentrations, however, the immersion of a traditional Rayleigh SAW tends to radiate the acoustic energy into the liquid because the displacement component is perpendicular to the surface. A type of SAW called the Love mode SAW is capable of performing analysis in liquid environments. Love-mode SAW devices guided acoustic modes which propagate in a thin layer deposited on a substrate. The acoustic energy is focused in the guiding layer where the displacement component propagates parallel to the surface. When using traditional piezoelectric substrates, SiO₂ and PMMA are frequently used as the waveguiding layer. The soft polymer poly (dimethylsiloxane) (PDMS) is often used to fabricate the channels in the device. Zhang et al. has reported a prostate-specific antigen (PSA) biosensor based on a love mode device. The sensor used LiTaO₃ with aluminum IDTs which were coated with a SiO₂ guiding layer and then gold forming the sensing layer. A PDMS microfluidic channel was subsequently added to the device to ensure that liquid can flow between the IDTs. The detection limit of this system was 10 mg/ml. The images and mass loading effects of a similar Love wave SAW device was used to measure the mass loading resulting from the covalent attachment of streptavidin-coated one-micron magnetic beads are shown in Figure 3.

Figure 3.

Confirmation of the mass-loading correlation between magnetic nanoparticles. (a) Shows covalently bonded to the surface of the SAW sensor and (b) shows the resulting phase shifts. The semi-log plot illustrates the expected direct proportionality between mass and phase shift.

5. Pathogen detection

SAW devices have seen applications in biology and medicine in the past. Unfortunately, strong radiation losses are observed for Rayleigh surface waves and most Lamb-mode surface waves. These types of devices have surface displacement for propagation modes have displacements normal on the surface. In liquid environments, we need to use surface waves that have the particle displacement parallel to the device surface and normal to the Love-mode SAW biosensors that have also been developed for the detection of microbial species. A series of devices have been developed by Sandia National Laboratory in collaboration with the University of New Mexico’s Health Science Center. Researchers Branch and Brozik at Sandia National Laboratory reported Low-level detection of endospores from Bacillus Antracis simulants using a love mode biosensor based on a 36^o YX LiTaO₃ substrate. When using a polyimide guiding layer, this system was capable of detecting Bacillus thuringiensis B8 endospores at a level between 1 and 2 ng/cm². Larson and Baca from the University of New Mexico reported the benefits of using a Love-mode SAW device for viral and bacterial detection for clinical applications. Figure 4 shows unpublished data from measurement of human cardiac troponin complex from serum samples. The values were measured on a lithium tantalate biosensor and coated with monoclonal antibodies from Hytest, Finland, as seen in Figure 4. In this report, they disused their findings on SAW devices that were in commercial development. In another report, Bisoffi et al. at the University of New Mexico reported using a Love-mode SAW device to detect a series of different viral particles and viral particles complex solution. In one experiment, the authors compare the detection of solutions containing sewage and other waste material. Bisoffi et al. reported developing the detection of HIV virus type 1 and type 2 using a Love-mode SAW device. In this study, three commonly occurring viral particles were detected from a complex matrix; river and sewage effluent. The SAW sensors were first treated with an organo-silane, 10% 3-glycidyloxypropyl trimethoxysilane (GPTMS), and then functionalized with an antibody. The device allowed multiplexed detection that was specific for HIV-1 and HIV-2 were introduced. The report not only confirm that the SAW could detect viral particles at a level below the standard ELISA and PCR methods but also demonstrated that the Love-mode SAW device could distinguish between HIV type 1 and type 2. Branch and Thayne reported the development of a Love-mode acoustic array biosensor platform that allowed autonomous detection of pathogenic microbes that are critical for human health and safety. Branch and Thayne reported antigen-capture of the targeted pathogens with a mass sensitivity of 7.19 ± 0.74 mm²/ng with a detection limit of 6.7 ± 0.40 pg./mm². In yet another report, Baca et al. report the detection of fragmented Ebola antigens at the point of care without the need for added reagents, sample processing, or specialized personnel to run the test. The test could be performed by first responders. The limit of detection for this methodology was below the average level of viremia detected on the first day of symptoms by PCR. Baca and colleagues from the University of New Mexico observed a semi-log sensor response for highly fragmented Ebola viral particles with a detection limit of 1.9 x 10⁴ PFU/ml. The devices used by both the researchers at Fisk University are fabricated at Sandia National Laboratory and are similar to devices used at the University of New Mexico.

Figure 4.

The plot of injected mass versus phase shift. This investigation of rapid human cardiac troponin ITC complex (cTn-ITC) detected from human blood, which is an indication of heart muscle damage following a myocardial infarction, using a SAW biosensor. The region in green represents the clinically relevant region.

6. Trends and future directions

The small size, high sensitivity, and potential low-cost nature of SAW devices make them attractive for large-scale biosensor applications. The detection is primarily focused on monitoring changes in the acoustic wave velocity due to attenuation by a surface mass loading on the surface of a piezoelectric material. The development of clinical biosensors will likely require the use of bodily fluids, which would require a primary focus will on Love-mode SAW devices that are able to operate in liquid environments. Although there are a limited number of SAW biosensors on the market, there are no commercially available devices approved by the United States Food and Drug Administration (FDA) for widespread usage. There is, however, a critical need for a portable rapid screening tool to monitor food safety and screen for infectious diseases. Therefore, point-of-care diagnostic tools are in strong demand in both biological and medical facilities. Currently, there is only one company attempting to commercialize SAW biosensors for biological and medical applications and that is TST Biomedical Company from Taiwan. This company is a subsidiary of TaiSAW.

7. Conclusion

The combination of Love-mode SAW devices with Lab-on-a-chip technology will create a new and exciting area of research. The development of SAW sensors has been welcomed by researchers working on point-of-care diagnostic tools that incorporate microfluidics for sample manipulation. These fully-integrated devices will offer tremendous capabilities since they can replace a traditional Biology or medical laboratory with highly sensitive and accurate devices that are completely portable. The added convenience of a wireless device will make monitoring remote areas or high infectious patients significantly easier.

Funding

Publication funded by NSF 181782 to Fisk University.