Acoustic MEMS Transducers for Biomedical Applications

Eun Sok Kim Department of Electrical Engineering-Electrophysics

University of Southern California Los Angeles, CA 90089-0271

eskim@usc.edu

Abstract—A recent acoustic MEMS transducer, a mass sensor based on FBAR (film bulk acoustic resonator), is shown to detect addition of a single DNA base. As the FBAR sensor can detect minute amount of DNA (and/or RNA) markers without the need for optical imaging, it will enable us to develop an “easy to use” and reliable system to detect the presence of pathogen’s nucleic acids in non-invasive fluids, such as urine and saliva, when arrayed on a small chip and coupled with a simple sample preparation and an “on-chip” isothermal nucleic acid amplification techniques. Another recent acoustic MEMS, self-focusing acoustic transducers (SFATs) are ideally suited for on-chip DNA and protein synthesis, high frequency ultrasonic imaging, cell lysis or high-intensity ultrasonic treatment with excellent localization on particular cells over a micron-sized area. The focused acoustic beam generated by SFAT is strong and precise enough to eject micron-sized droplets at any desired direction, activate cell-detaching cavitation, and/or lyse cells.

Keywords—piezoelectric, acoustic MEMS, DNA synthesis, self-focusing acoustic transducer, resonant mass sensor, FBAR, cell lysis, droplet ejection.

I. INTRODUCTION

Acoustic MEMS (microelectromechanical systems) transducers present unprecedented opportunities for healthcare in diagnostics and treatments because of their small size and mass as well as amenability of being formed into an array over a small area. The small size offers high surface-to-volume ratio, which can be exploited in a medical diagnostic tool that relies on surface reaction. Also, the small size and mass mean small amount of required reagents, accurate (and/or digital) control of the reagents, amenability of making acoustic MEMS implantable in human body, superb performance characteristics (e.g., extremely low mass detectability) that are not possible with macro systems, and low power consumption. These advantages have already been explored for healthcare applications through bioMEMS and diagnostic lab-on-chip. Some notable examples include U. of Michigan’s micromachined microelectrodes for detection and stimulation of neuron firing [1], Affimetrix’s GeneChip for sequencing of relatively short DNA’s, USC’s implantable BION neuron stimulator [2], etc.

In recent years, various MEMS resonators have been applied to resonant mass sensing. Among them are cantilever flexural mode resonators [3], contour mode resonators [4], lateral extensional mode (LEM) resonator [5], and thickness mode resonators such as film bulk acoustic resonator (FBAR)

[17]. Flexural mode resonators have inherently low resonant frequency, and require submicron dimensions for a GHz resonant frequency. (A GHz resonant frequency is desirable for the mass sensitivity, which affects the minimum detectable mass). Moreover, flexural vibration is damped by air or liquid much more severely than extensional vibration, thus having a much reduced quality (Q) factor when used for mass sensing. Consequently, the cantilever flexural mode resonators are inherently disadvantaged in comparison to the extensional mode resonators such as the contour mode resonator, LEM resonator, and FBAR, for mass sensing applications. All the transducers described in this paper rely on the resonance of extensional vibration in the thickness direction for generating acoustic waves in the ranges of 20 – 300 MHz and 1 – 5 GHz.

There exist various commercial techniques for producing DNA probes of pre-synthesized sequences on a chip. The Affymetrix’s GeneChip contains hundreds of thousands of different sequences of DNA single strands on a 13 mm by 13 mm glass substrate. The integration of such large number of DNA probes over such a small area is done with the photolithographic fabrication techniques and solid-phase chemical synthesis. To get full length (N-mer) of oligonucleotides on the glass substrate (e.g., 25 nucleotides), an iterating process involving light-directed phosphoramidite chemistry has to be repeated until the probes reach their full length. Thus, 4xN photomasks are required (i.e., 4x25=100 different photomasks are needed for 25-mer DNA probes). The photomasks would not be necessary, if a micromirror array (e.g., Texas Instruments’ Digital Micromirror Device) is used as a virtual photomask, as done by NimbleGen. The oligonucleotides synthesized with the light-directed phosphoramidite chemistry have poorer quality than those synthesized with standard dimethoxytrityl (DMT) blocked phosphoramidite chemistry, due to its lower overall coupling efficiency. MicroFab Technologies, Inc. has used a piezoelectric dispenser to eject liquid droplets of 25-100 m in diameter at 0 - 4 kHz. With five of those dispensers (for four dimethoxytrityl-blocked phosphoramidites and one activating reagent), they have demonstrated in-situ synthesis of up to 80-mer oligonucleotide array on a glass substrate. This method, however, requires a very large number of a computer-controlled X and Y movements of the substrate to position the substrate under the dispenser nozzles; the larger the number DNA array, the larger the number of the mechanical movements. Rosetta and Agilent also have reported in-situ DNA synthesis technique using their nozzle-based ink-jet print

71978-1-4244-6401-2/10/$26.00 ©2010 IEEE

heads [6]. They use standard dimethoxytrityl (DMT) blocked phosphoramidites to construct oligonucleotides, moving a relatively heavy ink cartridge many times and aligning it with the glass substrate each time it is brought to the glass.

Most commercial ink-jet printers (or microfluidic ejectors) are powered by a thermal or piezoelectric actuation, and eject ink (or any fluid) droplets through droplet-defining small nozzles. Since the commercial print heads shoot out droplets through nozzles, the smallest droplet size depends on the nozzle size. Small nozzles are difficult to construct with good uniformity, and are easily clogged due to accumulation of the phosphoramidite precipitates, especially when volatile solvents are used [7]. In addition, it is very difficult to produce directional ejections with a nozzle-based ejection, and there should be many mechanical movements when nozzle-based ejectors are used for DNA synthesis. Moreover, the thermally-actuated ink ejector (which heats ink at localized spots to about 250 °C to form a bubble to push the ink) requires a thermally-stable solution. Thus, nozzleless and heatless fluid ejection is desired.

Nozzleless liquid ejection has been shown with a high intensity focused acoustic beam [8,9]. A high intensity burst of acoustic energy focused to a free-liquid surface through an acoustic lens can result in a droplet ejection from the surface. A focused acoustic beam is capable of ejecting liquid droplets as small as a few m in diameter that are stable in size and directionality. Focusing of acoustic waves can be achieved with a spherical acoustic lens [10] or a Fresnel lens [11,12]. Though the Fresnel lens is easier to fabricate than a spherical lens, the lens geometry is critical for efficient focusing, and thus tight thickness control of the lens elements is needed. Thus, we have come up with a micromachined self-focusing acoustic-wave transducer (SFAT).

The SFAT focuses acoustic waves through near-field wave interference by patterning the electrodes (or air-reflectors) into annular rings [13,14]. The acoustic radiation pressure at the liquid-air interface is raised high enough to eject liquid droplets from the liquid into air. The heatless and nozzleless SFAT has been observed to eject DI water droplets less than 5 µm in diameter with RF pulses of 5 µsec pulsewidth [13]. The SFAT is capable of ejecting droplets in any direction, so that a spot on a chip can be inked by any of multiple such ejectors without mechanical movement and alignment.

This paper presents some selected biomedical applications of FBAR-based mass sensing and SFAT-based microfluidic management.

II. MASS SENSING BASED ON FILM BULK ACOUSTIC

RESONATOR (FBAR)

A. FBAR Mass Sensor

A bulk-micromachined Film Bulk Acoustic Resonator (FBAR) consists of a thin piezoelectric film sandwiched by two metal layers, and is typically built on a micromachined silicon nitride [15]. Such a FBAR is very easy to be adapted for a resonant mass sensing, since one of its two sides is typically inert, and can easily be exposed to sensing environment, while the other side where there are electrodes and piezoelectric film

is well protected from the harsh environment, as shown in Fig. 1. Since a three-layer FBAR (metal/piezo/metal) structure is mechanically weak, a support membrane is used to support a three-layer FBAR, especially for sensing applications.

Fig. 1 Cross section of FBAR used for vapor mass sensing (Left) and for mass sensing in liquid (Right).

Piezoelectric film (e.g., ZnO) in FBAR converts electrical energy into mechanical energy and vice versa. The mechanical energy generated by an RF electric field is in the form of acoustic wave. The acoustic wave is reflected wherever there is an impedance mismatch, and an acoustic resonance (with a standing wave) is obtained if the thickness of the thin film (d) is equal to an integer multiple of a half of the wavelength (res). The fundamental resonant frequency (Fres= Va/res) is then inversely proportional to the thickness of the piezoelectric film, and is equal to Va/2d where Va is an acoustic velocity at the resonant frequency. Thus, for GHz resonant frequency, the piezoelectric film needs to be about a few m thick.

From the input impedance between the two parallel electrodes, we obtain [17]

m

m

l

l

f

f

pp

dd

(Eq. 1)

where d and d are the mass density and thickness of the support layer (or added layer), respectively, while p and lp are those of the piezoelectric layer. This is one form of the famous Sauerbrey equation that was derived for quartz crystal microbalance (QCM) in 1959, which indicates that the resonant frequency of a QCM is linearly related to the mass of a material absorbed by the QCM. Here it is shown to be valid also in case of FBAR, when the added mass on FBAR is small. From Eq. 1, we can obtain an equation for the minimum detectable mass as follows.

ff

mm (Eq. 2)

As can be seen in Eq. 2, the minimum detectable mass ( m ) is smaller (i.e., better) for smaller minimum measurable frequency shift ( f ), which depends on the Q of FBAR, the

measurement instrument noise, the TCF of FBAR, etc. Also, the minimum detectable mass depends on the ratio between the mass and resonant frequency of FBAR, which is one main reason for reducing the mass and increasing the resonant frequency. However, for a given resonator, the f·Q product is usually constant, and the reduction of the m/f ratio does not always lead to reduced minimum detectable mass, as it affects the Q and consequently the minimum measurable frequency shift.

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The FBAR’s resonant frequency is linearly related to the mass of a material absorbed by the FBAR, when the added mass on FBAR is small [17]. A 1.4 GHz FBAR (0.1 x 0.1 mm2) with Al/ZnO/Al/SiN (0.2m/2.0m/0.2m/0.2m) has an expected mass sensitivity of 726 cm2/g, more than 50 times that of a QCM (5 x 5 x 0.5 mm3) operating at 6 MHz [16], mainly due to the GHz resonant frequency. However, the FBAR’s relatively low Q makes its minimum detectable mass to be comparable to that of the QCM. The FBAR has been measured to be able to detect as small as 1 and 10 ng/cm2 in air and liquid, respectively [17].

B. Sequence Specific Label-Free DNA Detection with FBAR

Recently, we introduced FBAR as a novel MEMS sensor for sequence specific and label-free detection of DNA molecules from the resonant frequency shift measurement [18]. A very thin gold layer (~ 800 Å) was deposited on the FBAR’s SiN, and on the gold a 15-mer probe nucleotide was immobilized. Selected targets were complementary, single-mismatched and more than one mismatched.

For hybridization, 1 µM target DNA solution was prepared. To observe real-time hybridization, the FBAR sensor was exposed to target DNA solution and the resonant frequency shift was recorded in real-time using a desktop computer connected to the network analyzer (that measured the impedance of the FBAR, giving the information on the resonant frequency shift). The resonant frequency dropped significantly (~ 70 kHz) when the FBAR was exposed to the target complementary sequence as shown in Fig. 2. With the single-mismatch sequence, the resonant frequency shift was ~ 35 % (~ 25 kHz) of that (~ 70 kHz) obtained with the complementary sequence. Therefore, the FBAR is capable of distinguishing the complementary DNA from a single-nucleotide mismatch DNA sequence. It was possible that the single-mismatch sequence could hybridize with the probe to a certain extent using one of the two regions bordering the mismatched nucleoside base. On the other hand, with two or more mismatches, no noticeable resonance shift was observed from the base line resonant frequency (Fig. 2). Consequently, using a FBAR with an immobilized 15-mer oligonucleotide probe, we have demonstrated selective, label-free and real-time detection of oligonucleotides sequences and DNA match/mismatched determination, in liquid.

Fig. 2 Measured resonant frequency shifts of the FBAR which has immobilized 15-mer probe DNA on its surface, when DNA’s of various sequences are brought to the FBAR for hybridization. From [8].

In addition to applying FBAR to detection of oligonucleotides sequences, we have demonstrated the following FBAR-based sensors to be highly sensitive and selective: a mercuric ion sensor with gold-coated FBAR [19], an explosive-vapor-trace detector with FBAR coated with anti-TNT [20], a neuron-firing sensor with an FBAR sensor on a 1.5mm long, 250m wide and 15m thick polymer probe [21], and a protein-ligand binding sensor.

III. APPLICATIONS OF SELF-FOCUSING ACOUSTIC

TRANSDUCERS (SFAT)

A. Self-Focusing Acoustic Transducers (SFAT)

One type of SFAT ejectors is consisted of complete annular electrode rings (Fig. 3) forming half-wave band sources (we call this the ring SFAT). The ring SFAT is usually designed to give a large focused acoustic pressure directed perpendicular to the plane of the annular rings, and is excellent for liquid droplet ejection. When the circular rings are sectored, the acoustic radiation pressure at the focal point is unbalanced in the plane of the liquid top surface, and the droplet ejection happens in a direction that is oblique to the liquid surface plane [13].

Fig. 3 Cross-sectional view and the electrode pattern of SFAT. A 300 MHz acoustic beam is focused on a 5-m-diameter spot through constructive interference of acoustic waves generated by piezoelectric ZnO film and annular electrode rings. From [13].

B. On-chip DNA Synthesis with SFAT Array

To synthesize DNA sequence from four DNA bases, we integrated a two-dimensional array of 2 x 2 directional ejectors and reservoirs on a single chip so that the ejector array would not have to be moved and aligned to spot one point (on a glass substrate) with different DNA bases [22,24]. This greatly reduced the needed control circuitry and automation. The photos of the fabricated array of PZT directional ejectors and silicon chambers before they were adhesively bonded together are shown in Fig. 4. The reservoirs were used to store DNA bases, and were directly connected to the ejection chambers. The fabricated device was packaged in a dual-in-line (DIP) package, and the package was placed on a mounting station with high frequency microstrips.

We used standard cyanoethyl phosphoramidite chemistry [23] to synthesize any oligonucleotide sequence on a poly-l-lysine coated glass slide. Since phosphoramidite chemistry can be used for DNA synthesis also on silicon [25] and plastic [26] surfaces that are modified with poly-l-lysine, this technique is also suitable for DNA synthesis on plastic or silicon substrates. Four activated monomers initially contained in four on-chip reservoirs were brought to the ejection chambers through the

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embedded microchannels and ejected by the directional ejectors. A pulse of 18-MHz sinusoidal wave was applied to eject droplets of DNA bases onto a glass slide placed above the device. Each ejector was individually actuated by a computer program. The 3’-phosphorous of the ejected monomer was linked by trivalent phosphite bond to the free 5’-hydroxyl on the poly-l-lysine-coated glass slide. The glass slide, attached to a motorized rotation stage, was then rotated by 180 to a wash/dry area to be treated with capping solution, which blocked out the nucleotides that had failed to couple. An oxidation step was then performed to stabilize the coupling, which was followed by deblocking process to remove dimethoxytrityl (DMT) protecting group at 5’-end for incoming oligonucleotides. Next, the glass slide was rotated back without any alignment with the ejector array to the synthesis area at the starting step of next iterating cycle.

Fig. 4 (a) Cross-sectional view of SFAT built on a 191 m thick PZT sheet. (b) Annular electrode rings for the SFAT that produce acoustic waves that interfere constructively at the focal point on the liquid top surface. From [24].

This oligonucleotide synthesis is a cyclic sequence of reactions, adding one nucleotide to the growing oligonucleotide chain and proceeds in four main steps during each cycle: activation/coupling, capping, oxidation, and deblocking [27]. The glass slide was rotated between the synthesis and wash areas repeatedly.

For automated DNA synthesis, a computer program was used to control the power delivery to each ejector and to decide the synthesis sequence. We synthesized a 15-mer 5’-CGCCAAGCAGTTCGT-3’, using a pulsewidth of 30 µs and 10-Hz PRF for 3 seconds for each DNA base. The substrate was placed at the height of 3 mm from the array, predetermined by the characterization tests.

To test whether the DNA sequence was properly synthesized, hybridization was performed using complementary DNA probes with FITC fluorescence tagging. Fluorescence microscopy was carried out, and fluorescence images on a control spot and the spot on which the DNA droplets had repeatedly been ejected were obtained and analyzed. The observation of fluorescence only at the spot where the droplets had been ejected confirmed that we had the correct sequence synthesized [24].

C. Microreaction Technology Based on Nanoliter Droplets

With an array of directional SFAT ejectors, we have demonstrated a microreaction technology for biochemical assay using nanoliter droplets encapsulated inside oil droplets [28]. Microreaction chambers are constructed on a glass substrate by accumulating oil droplets that are dispensed by the

directional droplet ejector, as illustrated in Fig. 5. Droplets of different aqueous reagents are then directionally ejected into the oil microchambers for parallel and combinatory analysis. Because the reagents are encapsulated in oil, the evaporation rate is greatly reduced, and only small amount of reagents are required. The microchamber size and reagent amount are digitally controlled by the number of ejected oil and reagent droplets, respectively. Ejectors for oil and reagents have been integrated on a single chip so that each assay is performed efficiently without any mechanical movement and alignment. We have carried out both physical and chemical microreactions with this method, and observed negligible difference (in response) from conventional macroreactions.

Oil

Glass

(a)

Reagent A

Glass

(b)

Reagent CReagent B

Glass

(c)

Oil

Glass

(a)

Reagent A

Glass

(b)

Reagent CReagent B Reagent CReagent B

Glass

(c)

Fig. 5 Sequence of microreactions using nanoliter droplets. (a) Formation of oil microreaction chamber. (b) Dispensation of reagent A. (c) Microreaction inside oil with concurrent or sequential dispensing of reagents B and C from the two ejectors in the back. From [28].

D. Applications of High Frequency Focused Ultrasound

For cell lysis, there are many methods that have been applied successfully, such as thermal lysis, electronic lysis, mechanical lysis and chemical lysis. Though most of them are practical for lyzing a cell by breaking the cellular membrane, they have their own drawbacks. For example, thermal lysis and chemical lysis always induce the physical or chemical actions which would change the nature of the cells. Acoustic cavitation has long been explored for cell lysis and tumor treatment [29]. However, conventional acoustic devices are working on a large area with low acoustic frequency at tens of kHz, which would affect healthy cells inevitably. Recently, we demonstrated a localized cell detachment with SFAT operating at tens of MHz [30]. The acoustic waves generated by SFATs focused at a spot of around 100 μm in diameter with the peak intensity of 25 W/cm2. These focused high intensity acoustic waves produced

2mm

Base T

Reservoir for base T

Base A

Base GBase C

2mm

1.4mm

PZT

LWAR Electrode

Ejector 1

Ejector 3 Ejector 4

Ejector 2

(a) (b)Reservoir for base G

2mm

Base T

Reservoir for base T

Base A

Base GBase C

2mm

1.4mm

PZT

LWAR Electrode

Ejector 1

Ejector 3 Ejector 4

Ejector 2

(a) (b)Reservoir for base G

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ultrasonic cavitation to break and detach a few hundred cells in that tiny area, without harming other ones outside.

An experiment for cell detachment was carried out with an SFAT working at 18 MHz. The SFAT was attached to the 1100 μm deep chamber with cells grown on a 10 μm thick parylene. The acoustic waves were focused at the focal point (less than 100 micron in diameter) where the cells were grown. With peak acoustic intensity of 25 W/cm2, cavitation was generated. After 40 pulses, the cavitation broke and detached the cells in the focal point within the area of around 160 μm by 200 μm, which was very close to the focal area of the transducer with the diameter of around 100 μm. Images of the cell monolayer before and after lyzing effect are shown in Fig. 6. Thus, the SFAT would be ideal for localized cell lysis and tumor treatment.

PZT Ejector Silicon wafer

Culture Media 1100 m

191 m

10um Parylene

Fig. 6 Cell detachment experiment with an SFAT (top) and the cells on parylene diaphragm before (bottom left) and after (bottom right) a focused acoustic beam from the SFAT was imparted. From [30].

IV. SUMMARY

Described in this paper are two major acoustic MEMS transducers that are poised to make unprecedented contribution to biomedical field: film bulk acoustic resonator (FBAR) and self-focusing acoustic transducer (SFAT). A 1.4 GHz FBAR is shown to possess a mass sensitivity of 726 cm2/g with a minimum detectable mass of 1 ng/cm2 in air (where the Q is about 250) and 10 ng/cm2 in water (where the Q is about 15). The FBAR sensor is capable of distinguishing a complementary DNA that is mismatched to a 15-mer probe DNA by a single nucleotide. In case of SFAT, an array of 2x2 directional SFAT ejectors has been used to synthesize 15-mer DNA sequence. Thus, SFAT is shown to be ideally suited to eject micron-sized droplets at any desired angle either simultaneously (from multiple SFATs integrated on a single chip) or sequentially (either from multiple SFATs or single SFAT into multiple directions), for genetics and proteomics. Also demonstrated with directional SFAT ejectors is a new biochemical microreaction platform where reagent evaporation rate is greatly reduced by oil encapsulation and the required reagent amount is minimized. Finally, SFAT is shown to produce a high intensity, high frequency, focused acoustic beam that can potentially be applied to cell lysis and/or tissue cutting. Both FBAR and SFAT are powered by a piezoelectric film or sheet, and are inherently fast, consume low power, and require no heat.

ACKNOWLEDGMENT

The following former and current students of the author produced the results presented in this paper: Sanat Kamal-Bahl (Intel), Jaewan Kwon (U. of Missouri, Columbia), Chuang-Yuan Lee (Touchdown Tech.), Hongyu Yu (Arizona State U.), and Hao Zhang (Tianjin U.). The following funding agencies supported most of the works presented in this paper: National Science Foundation under grant ECS-0310622, Defense Advanced Research Projects Agency under contracts #N66001-00-C-8094, and Office of Naval Research under award #N00014-01-1-0479.

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