Article pubs.acs.org/acssensors
Wireless Passive Stimulation of Engineered Cardiac Tissues Shiyi Liu,† Ali Navaei,‡ Xueling Meng,† Mehdi Nikkhah,*,‡ and Junseok Chae*,† †
School of Electrical, Computer and Energy Engineering and ‡School of Biological and Health Systems Engineering, Arizona State University, Tempe, Arizona 85287, United States S Supporting Information *
ABSTRACT: We present a battery-free radio frequency (RF) microwave activated wireless stimulator, 25 × 42 × 1.6 mm3 on a flexible substrate, featuring high current delivery, up to 60 mA, to stimulate engineered cardiac tissues. An external antenna shines 2.4 GHz microwave, which is modulated by an inverted pulse to directly control the stimulating waveform, to the wireless passive stimulator. The stimulator is equipped with an on-board antenna, multistage diode multipliers, and a control transistor. Rat cardiomyocytes, seeded on electrically conductive gelatin-based hydrogels, demonstrate synchronous contractions and Ca2+ transients immediately upon stimulation. Notably, the stimulator output voltage and current profiles match the tissue contraction frequency within 0.5−2 Hz. Overall, our results indicate the promising potential of the proposed wireless passive stimulator for cardiac stimulation and therapy by induction of precisely controlled and synchronous contractions. KEYWORDS: wireless stimulator, passive, implant, cardiac tissue, contraction
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current demanding applications, such as cardiac tissue stimulation.15 In this work, we report a passive wireless stimulator, activated by radio frequency (RF) microwaves, which delivers current as high as 60 mA to engineered cardiac tissues. The use of microwave eliminates the need for a bulky external inductive coil. The stimulator consists of an on-board miniaturized antenna, multistage diode voltage multipliers, and only one output controlling transistor; therefore, the entire system consumes low power (maximum 18 mW). A small-size external antenna, less than 10 × 10 × 0.9 mm3, irradiates 2.4 GHz modulated RF signals to the stimulator at a distance of up to 1.2 cm. The function of the wireless stimulator was evaluated by electrically stimulating engineered cardiac tissues, inducing synchronous contractions. Electrical stimulation has been shown to enhance the key biological properties in cardiac cells including alignment, protein expression, and contractility.15 Up until now, all stimulation approaches have utilized a wire-connected external electrical instrument with large power supply. Herein, we present a wireless stimulator with the ability to electrically trigger synchronous contractions in cardiac cells, with significant potential for clinical applications. We primarily describe the design of passive wireless stimulator, implementation of the stimulator and external antenna, and characterization of the wireless stimulator using emulated loads. Then we discuss preparation of engineered cardiac tissue constructs, and
lectrical stimulation delivers effective treatment for many clinical fields, such as restoring paralyzed limbs,1 inhibiting epilepsy,2 suppressing pain,3 correcting abnormal heart rhythm,4 and restoring vison.5 In traditional stimulating systems, clinicians place surface or percutaneous electrodes near the malfunctioning nerve or muscle tissue. These electrodes are wired to an external electrical pulse generator.6,7 Such a configuration encumbers the maneuvering capability of patients and clinicians, increases the likelihood of infection, and ultimately fails to provide efficient treatment due to loss of contact of the electrodes.7,8 Wireless implantable stimulating systems are attractive, particularly for long-term implementation, as they can be portably used in patients’ homes without restricting movement.1 To date, numerous clinical wireless stimulators, such as the Freehand system for neural muscular stimulation,1,9 have demonstrated successful clinical evaluation on tetraplegia patients. However, these systems are composed of many unconcealed long electrical leads, which result in a complex and extensive surgical implantation procedure.10 To address these challenges, several miniaturized injectable stimulators have been reported so far.8,11 Nevertheless, such systems require patients to wear bulky external inductive coils to power the implants.8 Moreover, the strong inductive field may induce an unwanted temperature rise within the tissue due to ohmic loss.8,12 Having an implantable rechargeable battery mitigates this drawback,10,13 yet the patients are required to recharge the battery for a few hours every 3 days, and the battery itself needs to be replaced every 10 years. Aside from these challenges, existing wireless stimulators also deliver relatively low current, ranging from 2.5 μA to 20 mA,8,10,13,14 which is insufficient for high© XXXX American Chemical Society
Received: April 26, 2017 Accepted: June 28, 2017 Published: June 28, 2017 A
DOI: 10.1021/acssensors.7b00279 ACS Sens. XXXX, XXX, XXX−XXX
Article
ACS Sensors
In the meantime, the circuits charge the capacitor C7 through multistage diodes until the voltage across C7 reaches 8−20 V. In the discharging state, the transistor turns “on”, and C7 discharges its accumulated charges. The stimulating current is determined by the voltage across C7 and the resistance of the cardiac tissue. The “on” state of the circuits is controlled to maintain only a few milliseconds at a frequency of only a few hertz. The total power consumption of the circuits is mainly contributed by the heat dissipation of resistors R1 and R2, which are 22 and 300 kΩ, respectively, contributing approximately 2.9−18 mW. In our experimental settings, 2.9 mW is sufficient to excite cardiac tissue contraction. It should be noted that increasing the values of R1 and R2 can significantly decrease the power consumption of the stimulator. For example, increasing R1 and R2 by 10 times will reduce to a power consumption of merely 1.8 mW. The two states of the circuits are controlled by the external RF interrogator. Two popular methods exist for wireless power delivery: inductive coupling and electromagnetic (RF) coupling. A comparison of the two methods is well studied.20 Generally speaking, inductive coupling performs better in close distance and high power requirement settings whereas RF coupling is better suited for power-limited, longrange applications. At a given transmitting power, RF coupling delivers higher DC voltage than inductive coupling does.20 Additionally, inductive coupling features that the external coil scales dramatically large as the working distance increases,21 whereas RF coupling demonstrates that the size of the external antenna is rather independent upon the working distance. A comparison of the efficiency of inductive and RF coupling is provided in the Supporting Information, demonstrating that RF coupling fits better in our target application (Figure S-3). Figure 1b shows the photograph of the designed passive wireless stimulator, including the antenna and circuits. The wireless stimulation circuit was fabricated on a 25-μm-thick polyimide film. All the metal traces were formed by a thin chrome-gold-chrome film (Cr/ Au/Cr, 20/200/20 nm).22,23 Discrete components were mounted using biocompatible conductive sliver paste (ED21TDCSMED, Masterbond). The total size of the stimulator is 25 × 42 × 1.6 mm3, including the on-board antenna. The RF carrier frequency was chosen to be 2.4 GHz through the trade-off between the size of the on-board antenna and the skin depth penetration into the body. A high frequency RF is preferred to minimize the size of antenna. On the other hand, a high frequency RF results in a significant loss within tissues.24 Based on our previous work,25−27 2.4 GHz was found to be the optimized frequency for the on-board antenna. We designed the antenna using the finite element method (FEM) of high frequency structure simulator (HFSS) (Figure S-1). We used PDMS for the antenna dielectric material as PDMS is a well-known flexible and biocompatible material, and has a relatively low dielectric loss (δ = 0.0015−0.0035) (Figures S-1, S-2). External Transmitter. Figure 1c illustrates the schematic of external transmitter. An inverted pulse modulates 2.4 GHz RF carrier through amplitude modulation. The modulated signal is amplified, then radiated from the external antenna. The frequency and pulse width of the inverted pulse were set to be 0.5−2 Hz and 2 ms, whereas the high/low levels of the inverted pulse were set at 2.8 and 0 V, respectively. The pulse and RF carrier were generated by an arbitrary waveform (Agilent 33250A) and RF signal generators (Agilent E4432B), respectively. Modulation depth of RF generator was set at 32.5%. The modulated signal was amplified by a low-noise power amplifier (MPA-24-20, RF bay). The radiating external antenna is a 2.4 GHz 10 × 10 mm2 ceramic chip antenna (A10194, Antenova) (Figure S-6). We set the maximum power at the RF signal generator to be 30 dBm, guided by Federal Communication Commission (FCC) regulation.28 The actual radiating power from the external antenna is less than 30 dBm. Development of Cardiac Tissue Model. The design and fabrication procedure of the gold nanorod (GNR)-embedded gelatin hydrogel cardiac constructs are fully described in the Supporting Information.29
fabrication of a custom-made bioreactor. Finally, we present the measured results of wireless stimulations on the engineered cardiac tissues, side by side compared to the wired configuration.
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MATERIALS AND METHODS
Passive Wireless Stimulator. The preferred electrical stimulation signal parameters, of engineered cardiac tissue, have been described by a published protocol.15 Briefly, a monophasic square wave pulse with relatively short duration is sufficient to excite heart tissue; the stimulating pulse frequency is in the range of several hertz, depending on heart beating rate; large stimulating current, at least 60 mA,16,17 is required. While a wire connected electrical stimulator system can easily achieve such high current, however, it is a challenging specification for a wireless system. In addition, for an implantable device, the maximum power dissipation of the device and the maximum allowable external RF energy need to be within the safety regulations.18 Table 1 summarizes our target design parameters of the flexible wireless stimulator for engineered cardiac tissue.
Table 1. Design Requirement of the Flexible Wireless Stimulator design parameters
requirements
Stimulating current Frequency External RF power Channel number Heat dissipation Battery
>60 mA 0.5−2 Hz
