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AUTONOMOUS IMPLANTABLE DEVICE FOR APPLICATION IN LATEPHASE HEMORRHAGIC SHOCK PREVENTION Vlad Oncescu1, Seoho Lee1, Abdurrahman Gumus2, Kolbeinn Karlsson2, David Erickson1* 1 Sibley School of Mechanical and Aerospace Engineering, Cornell University, USA 2 Electrical and Computer Engineering, Cornell University, USA ABSTRACT Hemorrhagic shock (HS) is the leading cause of death for people with traumatic injuries. The onset of HS is correlated with changes in plasma vasopressin levels and some studies indicate that administrating vasopressin in the bloodstream can stabilize the situation. This situation calls for the use of implantable devices for both the monitoring and treatment of HS. In this work, we present a self-powered hemorrhagic-shock autonomous integrated device (hemoAID) that continuously monitors vasopressin levels and releases vasopressin automatically when levels drop below a certain threshold, thus paving the way towards the development of an autonomous implantable device for HS prevention. KEYWORDS: autonomous, implantable, biosensor, hemorrhagic shock INTRODUCTION Over the past decade, implantable autonomous microsystems have been developed to counter life-threatening medical conditions and to improve patient care by replacing cumbersome treatment procedures.[1-3] Although, independently performing sensing[4, 5] and drug delivery[6] has been demonstrated, developing implantable medical devices capable of achieving both of those functions simultaneously would be suitable for a number of applications.[7] One such case is the detection and treatment of traumatic injuries such as hemorrhagic shock (HS). Studies have correlated the onset of HS with marked changes in the plasma vasopressin levels.[8, 9] Morales et al.[8] has reported a set of human cases and canine experiments where vasopressin levels showed a marked increase at the onset of hemorrhage (to as much as 319pg/mL) followed by a fall to well below the normal physiological level (29pg/mL) as hemorrhage progressed. In addition, evidence suggests that replenishment of vasopressin at this stage can reverse hypotension in patients.[10, 11] Therefore, vasopressin can serve as both a biomarker indicative of a critical injury state and a therapeutic agent for treating it. Developing an implantable autonomous device that can provide initial medical treatment when physician care is not readily available is highly suitable in this case. In this paper, we present a self-powered hemorrhagic-shock prevention autonomous integrated device (hemoAID) that continuously monitors vasopressin levels in solution and releases vasopressin automatically when vasopressin levels drop below a certain threshold. We demonstrate that the device works at physiological concentrations of vasopressin in sheep serum. This represents the first steps towards the development of an implantable, self-powered microfluidic device for late-phase HS prevention. In the theory section, we present the integrated device. In the following sections, we present characterization experiments for the sensor and then demonstrate the operation of the entire system in the physiological range of vasopressin concentrations in which we expect the device to operate. THEORY The hemoAID shown in Fig 1a consists of a nanowire biosensor for detecting changes in vasopressin levels, an onboard electrochemically driven drug delivery system for administration of vasopressin, and a nonenzymatic glucose fuel cell for powering the system. Those three main functional components are integrated inside a 2cm3 package. In this prototype version the electronic control system is external to the system and uses the circuit shown in Fig. 1b. External housing of the circuitry was done to facilitate prototype development and could be integrated into the system in the future. The fabrication procedures for the glucose biosensor[12], fuel cell[13] and drug delivery[6] units have been described in detail elseFigure 1: a) Integrated device consisting of a biosensor, where. Here we focus on demonstrating the ability of drug delivery and fuel cell unit b) schematic of the electrical the biosensor to function at physiologically relevant connections between the different components of the vasopressin concentrations and the integration of hemoAID these components in an autonomous device. The non-enzymatic glucose fuel cell is used to continuously charge a small rechargeable battery (Thinergy MEC225) that is then used to power a low power microcontroller (MSP430F2012, Texas Instruments, TX, USA). This set-up allows the device to operate steadily even with significant fluctuations in the power output of the fuel cell unit. During device operation, the sensor is driven by an adjustable 2-terminal current source (LT3092, Linear Technology, Milpitas, 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1839 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany CA, USA) at 60 mA. The resistivity of the sensor changes with the amount vasopressin in solution, which results in detectable voltage change over the biosensor. In order to improve sensitivity, the voltage is amplified using a non-inverting amplifier (Maxim Integrated, CA) before being detected using a low power microcontroller’s analog to digital converter (ADC). If the voltage is lower than the pre-determined threshold, the microcontroller enables the corresponding output pin to activate the drug delivery system. In order to increase the discharge rate of vasopressin in the drug delivery device, the base voltage (3V) is converted to 35V with a micropower step-up DC/DC converter (LT3464, Linear Technology, Milpitas, CA, USA). EXPERIMENTAL The nanowire biosensor we have developed and integrated in the hemoAID allows for the detection of pM changes in vasopressin concentration. Immobilized aptamer molecules on the nanowire biosensor act as electrical bioreceptors for the surrounding vasopressin. In order to demonstrate the ability of the biosensor (shown if Fig. 2a) to detect gradual changes in vasopressin levels we have performed several characterization experiments. First we show the reversibility of our sensor’s detection mechanism by monitoring the sensor response to alternating vasopressin concentrations in a single experiment. The biosensor was placed into its custom packaging with micro-channels as shown if Fig. 2b and 0.01M PBS solution was introduced through one inlet until the output current was stabilized. The PBS injection was stopped and replaced Figure 2: a) Schematic of the nanowire biosensor and picture with 10µM vasopressin injection through the other inof the flow through system b) demonstration of sensor reverslet, which was again replaced by a PBS injection after ibility c) current change due to change in vasopressin con30s. As shown in Fig. 2c, the initial introduction of centration in sheep serum. At t=0 100pM vasopressin solu10µM vasopressin results in a current decrease through tion is introduces followed by 50 pM, 10pM and sheep serum the sensor almost instantaneously which is a consewith no additional vasopressin d) Percentage decrease in quence of depletion of charge carriers on the carbon current from the initial no vasopressin case due to different nanotubes due to vasopressin to aptamer binding. The vasopressin concentration in sheep serum (average of 5 exrecovery of current upon PBS injection at t= 50s sugperiments) gests the release of vasopressin molecules from the immobilized aptamer which demonstrates the reversible nature of the binding events. The slower rate of current change during the dilution could be attributed to the low dissociation constant of aptamer-vasopressin complex, confirming the high affinity of aptamer molecules to vasopressin.[14] As another extension of our previous work which monitored vasopressin in serum samples ranging from 100nM to 1mM, the sensor in this work was tested in serum with more physiologically relevant range of 10 to 100pM. As shown in Fig. 2d, the biosensor was subjected to sheep serum (Valley Biomedicals Inc.) with 100pM, 50pM, 10pM and no added vasopressin in the respective order. Duration for each injection was 150s. With each dilution, the current through the sensor increased as expected from vasopressin releasing from the aptamer. The sensor was not cleaned in between different serum injections in order to mimic the intended operation conditions. This however signifies that the actual vasopressin concentration in exposure was the weighted sum of those of previous injections. RESULTS AND DISCUSSION The biosensor, drug delivery unit and fuel cell stack were assembled into a single device. The integrated device was placed inside a closed chamber within a fluid network for the integrated testing which was designed to resemble the physiological vasopressin states of late-phase HS patients. According to the clinical study by Jochberger et al.[15] vasopressin concentration for patients with hemorrhagic shock rises from the normal 6±3pM range to 52±30pM as the body tries to reverse hypotension by releasing vasopressin. Late-phase HS is correlated Figure 3: a) setup for integrated device testing b) experiment showing voltage decrease due to decrease in vasopressin levels (with 100pM initial concentration and gradual decrease due to flow of 10pM solution). The inset shows the voltage drop during the initial 720s before the drug delivery is activated 1840 to the decrease of vasopressin from this initially high value which indicates the failure for reversal. Therefore in the final experiment to demonstrate the operation of the hemoAID at physiologically relevant vasopressin concentrations, the chamber housing the integrated device (Fig. 3a) was initially filled with 100pM vasopressin solution which is in the order of the high initial value (52±30pM) for patients. The vasopressin concentration was then lowered by introducing 10pM solution into the chamber using a VWR Mini-Pump Variable Flow. The flow-rate of 0.115mL/s was in balance with the rate of gravity-driven discharge and maintained the solution level in the chamber relatively constant. As shown in Fig. 3b, the sensor’s voltage decreased over the 14 minutes in response to the decrease in vasopressin levels. It is expected that the release of vasopressin from the aptamer by dilution increased the conductivity of the CNT platform (decreased the resistivity), causing the sensor’s voltage output to decrease at constant current. Upon reaching the pre-determined threshold of 0.25% voltage decrease ratio for more than 10s, the drug delivery was activated autonomously by 35V application between the top and bottom electrodes. 16μL of highly concentrated vasopressin at 200μM was released, which was detected successfully by the sensor with 3.88% voltage ratio increase. CONCLUSION In this paper, we have demonstrated the operation of an integrated autonomous device for the monitoring and delivery of vasopressin. The final integrated experiment shows how the hemoAID device can be used to detect a gradual drop in vasopressin between physiologically relevant concentrations (from 100pM to 10pM) and deliver a highly concentrated does of vasopressin to stabilize the situation. In addition we have shown the sensor’s ability to operate in a complex medium such as sheep serum thus opening the door to the development of an implantable device for late phase hemorrhagic shock prevention. ACKNOWLEDGEMENTS The authors wish to acknowledge the support of the Office of Naval Research (ONR) under award number N000141010115 “Autonomous Microfluidic Devices for Battlefield Health Assessment and Treatment”. In addition, V.O. wishes to acknowledge the support of the Canadian institution NSERC through a fellowship. Some elements of the integration were also carried out with the support of NSF grant #1014891. The fabrication steps described in this paper were carried out at the Cornell Nanoscale Facility (CNF) and the Nanobiotechnology Center at Cornell University (NBTC). REFERENCES [1] R.A.M. Receveur, F.W. Lindemans, N.F. de Rooij, J Micromech Microeng, 17 (2007) R50. [2] K. Bazaka, M. Jacob, Electronics, 2 (2012) 1. [3] Y. Onuki, U. Bhardwaj, F. Papadimitrakopoulos, D.J. Burgess, Journal of diabetes science and technology, 2 (2008) 1003. [4] M. Fojtik, D. Kim, G. Chen, Y.-S. Lin, D. Fick, J. Park, M. Seok, M.-T. Chen, Z. Foo, D. Blaauw, (2013). [5] H. Li, X. Mu, Z. Wang, X. Liu, M. Guo, R. Jin, X. Zeng, A.J. Mason, Engineering in Medicine and Biology Society (EMBC), 2012 Annual International Conference of the IEEE, IEEE, 2012, p. 503. [6] A.J. Chung, Y.S. Huh, D. Erickson, D - 100887374 (2009) T [7] J.P. Esquivel, J. Colomer-Farrarons, M. Castellarnau, M. Salleras, F.J. del Campo, J. Samitier, P. MiribelCatala, N. Sabate, Lab Chip, 12 (2012) 4232. [8] D. Morales, J. Madigan, S. Cullinane, J. Chen, M. Heath, M. Oz, J.A. Oliver, D.W. Landry, Circulation, 100 (1999) 226. [9] W.G. Voelckel, V.A. Convertino, K.G. Lurie, A. Karlbauer, H. Schochl, K.H. Lindner, H. Trimmel, J Trauma, 69 (2010). [10] T. Anand, R. Skinner, J Surg Res, 178 (2012) 321. [11] V. Wenzel, H. Raab, M.W. Dunser, Best Pract Res Clin Anaesthesiol, 22 (2008) 299. [12] P. He, V. Oncescu, S. Lee, I. Choi, D. Erickson, Anal Chim Acta, 759 (2013) 74. [13] V. Oncescu, D. Erickson, Scientific reports, 3 (2013). [14] K. Maehashi, K. Matsumoto, Y. Takamura, E. Tamiya, Electroanal, 21 (2009) 1285. [15] S. Jochberger, N.G. Morgenthaler, V.D. Mayr, G. Luckner, V. Wenzel, H. Ulmer, S. Schwarz, W.R. Hasibeder, B.E. Friesenecker, M.W. Dunser, The Journal of clinical endocrinology and metabolism, 91 (2006) 4381. CONTACT * D. Erickson; tel: (607) 255-4861; [email protected] 1841