Jeffrey D. Zahn

A microfluidic device for continuous, real time blood plasma separation

A microfluidic device for continuous, real time blood plasma separation is introduced. The principle of the blood plasma separation from blood cells is supported by the Zweifach-Fung effect and was experimentally demonstrated using simple microchannels. The blood plasma separation device is composed of a blood inlet, a bifurcating region which leads to a purified plasma outlet, and a concentrated blood cell outlet. It was designed to separate blood plasma from an initial blood sample of up to 45% inlet hematocrit (volume percentage of cells). The microfluidic network was designed using an analogous electrical circuit, as well as analytical and numerical studies. The functionality of this device was demonstrated using defibrinated sheep blood. During 30 minutes of continuous blood infusion through the device, all the erythrocytes (red blood cells) traveled through the device toward the concentrated blood outlet while only the plasma was separated at the bifurcating regions and flowed towards the plasma outlet. The device has been operated continuously without any clogging or hemolysis of cells. The experimentally determined plasma selectivity with respect to blood hematocrit level was almost 100% regardless of the inlet hematocrit. The total plasma separation volume percent varied from 15% to 25% with increasing inlet hematocrit. Due to the devices simple structure and control mechanism, this microdevice is expected to be used for highly efficient continuous, real time cell-free blood plasma separation from blood samples for use in lab on a chip applications.

Microfabricated Polysilicon Microneedles for Minimally Invasive Biomedical Devices

A two-wafer polysilicon micromolding process has been developed for the fabrication of hollow tubes useful for microfluidic applications. These small tubes can be fabricated with a pointed end, resulting in a micro hypodermic injection needle. Microneedles are desired because they reduce both insertion pain and tissue damage in the patient. Such microneedles may be used for low flow rate, continuous drug delivery, such as the continuous delivery of insulin to a diabetic patient. The needles would be integrated into a short term drug delivery device capable of delivering therapeutics intradermally for about 24 hours. In addition, microneedles can be used for sample collection for biological analysis, delivery of cell or cellular extract based vaccines, and sample handling providing interconnection between the microscopic and macroscopic world.The strength of microneedles was examined analytically, experimentally and by finite element analysis. Metal coatings provide significant increases in the achievable bending moments before failure in the needles. For example, a 10 μ m platinum coating increased the median bending moment of a 160 μ m wide, 110 μ m high microneedle with a 20 μ m wall from 0.25 to 0.43 mNm. In addition, fluid flow in microneedles was studied experimentally. Microneedles 192 μ m wide, 110 μ m high and 7 mm long have flow rates of 0.7 ml/sec under a 138 kPa inlet pressure. This flow capacity exceeds previous microneedle capacities by an order of magnitude.

Continuous On-Chip Micropumping for Microneedle Enhanced Drug Delivery

Microneedles are promising microfabricated devices for minimally invasive drug delivery applications. Needles can be integrated into a variety of devices. However, any portable drug delivery device with integrated microneedles will need an equally compact means to deliver therapeutics. This work presents microneedles integrated with an on-chip MEMS positive displacement micropump for continuous drug delivery applications. The generation and collapse of thermally generated bubbles with flow rectified by directional check valves are used to achieve net pumping through the device. Visualization methods have observed net flow rates of water out of a microneedle at approximately 2.0 nl/s with a pressure of 3.9 kPa. In addition, continuous pumping was achieved for more than 6 hours with the heaters actuating for over 18 hours (15,000 cycles) without failing.

Noninvasive Glucose Monitoring: A Novel Approach:

Background: The main concern in noninvasive (NI) glucose measurement is achieving high accuracy readings, although no blood (or other fluid) is involved in the process. Using methods based on different physical properties of a measured object can ensure the independence of each of the readings and therefore improve the validity of the end result. By using a combination of (three) independent technologies—ultrasonic, electromagnetic, and thermal—GlucoTrack™ presents a unique approach for a real-time, truly NI blood glucose spot measurement. Methods: Clinical trials were performed in two stages. Stage 1 was an initial method validation and performance verification of the device. In this stage, 50 type 1 and 2 diabetic patients, as well as healthy subjects, were evaluated with GlucoTrack against Ascensia Elite® (Bayer). In the second stage, 85 additional diabetic subjects were evaluated in half and full daytime sessions using a GlucoTrack comparison with HemoCue® (Glucose 201+). Results: A total of 135 subjects were tested during the trial period, producing 793 data pairs. Using Clarke error grid analysis, 92% of the readings fell in the clinically acceptable zones A and B, with 50% in the A zone. Mean and median relative absolute differences were 29.9 and 19.9%, respectively. Conclusions: Integrating several modalities for NI assessment of glucose level enables more accurate readings, while a possible aberration in one modality is bypassed by the others. The present generation of GlucoTrack gives promising results; however, further improvement of the accuracy of the device is needed.

Microfiltration platform for continuous blood plasma protein extraction from whole blood during cardiac surgery.

This report describes the design, fabrication, and testing of a cross-flow filtration microdevice, for the continuous extraction of blood plasma from a circulating whole blood sample in a clinically relevant environment to assist in continuous monitoring of a patients inflammatory response during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures (about 400,000 adult and 20,000 pediatric patients in the United States per year). The microfiltration system consists of a two-compartment mass exchanger with two aligned sets of PDMS microchannels, separated by a porous polycarbonate (PCTE) membrane. Using this microdevice, blood plasma has been continuously separated from blood cells in a real-time manner with no evidence of bio-fouling or cell lysis. The technology is designed to continuously extract plasma containing diagnostic plasma proteins such as complements and cytokines using a significantly smaller blood volume as compared to traditional blood collection techniques. The microfiltration device has been tested using a simulated CPB circulation loop primed with donor human blood, in a manner identical to a clinical surgical setup, to collect plasma fractions in order to study the effects of CPB system components and circulation on immune activation during extracorporeal circulatory support. The microdevice, with 200 nm membrane pore size, was connected to a simulated CPB circuit, and was able to continuously extract ~15% pure plasma volume (100% cell-free) with high sampling frequencies which could be analyzed directly following collection with no need to further centrifuge or modify the fraction. Less than 2.5 ml total plasma volume was collected over a 4 h sampling period (less than one Vacutainer blood collection tube volume). The results tracked cytokine concentrations collected from both the reservoir and filtrate samples which were comparable to those from direct blood draws, indicating very high protein recovery of the microdevice. Additionally, the cytokine concentration increased significantly compared to baseline values over the circulation time for all cytokines analyzed. The high plasma protein recovery (over 80%), no indication of hemolysis and low level of biofouling on the membrane surface during the experimental period (over 4 h) were all indications of effective and reliable device performance for future clinical applications. The simple and robust design and operation of these devices allow operation over a wide range of experimental flow conditions and blood hematocrit levels to allow surgeons and clinicians autonomous usage in a clinical environment to better understand the mechanisms of injury resulting from cardiac surgery, and allow early interventions in patients with excessive postoperative complications to improve surgical outcomes. Ultimately, monolithic integration of this microfiltration device with a continuous microimmunoassay would create an integrated microanalysis system for tracking inflammation biomarkers concentrations in patients for point-of-care diagnostics, reducing blood analysis times, costs and volume of blood samples required for repeated assays.

Blood plasma separation in microfluidic channels using flow rate control.

Several studies have clearly shown that cardiac surgery induces systemic inflammatory responses, particularly when cardiopulmonary bypass (CPB) is used. CPB induces complex inflammatory responses. Considerable evidence suggests that systemic inflammation causes many postoperative complications. Currently, there is no effective method to prevent this systemic inflammatory response syndrome in patients undergoing CPB. The ability to clinically intervene in inflammation, or even study the inflammatory response to CPB, is limited by the lack of timely measurements of inflammatory responses. In this study, a microfluidic device for continuous, real-time blood plasma separation, which may be integrated with downstream plasma analysis device, is introduced. This device is designed to have a whole blood inlet, a purified plasma outlet, and a concentrated blood cell outlet. The device is designed to separate plasma with up to 45% hematocrit of the inlet blood and is analyzed using computational fluid dynamics simulation. The simulation results show that 27% and 25% of plasma can be collected from the total inlet blood volume for 45% and 39% hematocrit, respectively. The device’s functionality was demonstrated using defibrinated sheep blood (hematocrit = 39%). During the experiment, all the blood cells traveled through the device toward the concentrated blood outlet while only the plasma flowed towards the plasma outlet without any clogging or lysis of cells. Because of its simple structure and control mechanism, this microdevice is expected to be used for highly efficient, real-time, continuous cell-free plasma separation.

Design of a Side-View Particle Imaging Velocimetry Flow System for Cell-Substrate Adhesion Studies

Experimental models that mimic the flow conditions in microcapillaries have suggested that the local shear stresses and shear rates can mediate tumor cell and leukocyte arrest on the endothelium and subsequent sustained adhesion. However, further investigation has been limited by the lack of experimental models that allow quantitative measurement of the hydrodynamic environment over adherent cells. The purpose of this study was to develop a system capable of acquiring quantitative flow profiles over adherent cells. By combining the techniques of side-view imaging and particle image velocimetry (PIV), an in vitro model was constructed that is capable of obtaining quantitative flow data over cells adhering to the endothelium. The velocity over an adherent leukocyte was measured and the shear rate was calculated under low and high upstream wall shear. The microcapillary channel was modeled using computational fluid dynamics (CFD) and the calculated velocity profiles over cells under the low and high shear rates were compared to experimental results. The drag force applied to each cell by the fluid was then computed. This system provides a means for future study of the forces underlying adhesion by permitting characterization of the local hydrodynamic conditions over adherent cells.

Continuous on-chip micropumping through a microneedle

Microneedles are promising microfabricated devices for minimally invasive drug delivery applications. Microneedles can be integrated into a variety of devices. However, any portable drug delivery device with integrated microneedles will need an equally compact means to deliver the therapeutics. This work presents microneedles integrated with an on-chip MEMS positive displacement micropump for continuous drug delivery applications. The generation and collapse of thermally generated bubbles with flow rectified by directional check valves are used to achieve net pumping. Visualization methods have observed net flow rates of water out of a microneedle at approximately 1.0 nl/s with a pressure of 3.9 kPa. In addition, continuous pumping was achieved for more than 6 hours. The heaters operated for over 18 hours (15,000 cycles) without failing.

Microfabricated microdialysis microneedles for continuous medical monitoring

Enzyme based biosensors suffer from loss of activity and sensitivity through a variety of processes. One major reason for the loss is through large molecular weight proteins settling onto the sensor and affecting sensor signal stability and disrupting enzyme function. One way to minimize loss of sensor activity is to filter out large molecular weight compounds before sensing small biochemicals such as glucose. A novel microdialysis microneedle is introduced that is capable of excluding large MW compounds based on size. Preliminary experimental evidence of membrane permeability is shown, as well as diffusion and permeability modeling. Solutions should be able to equilibrate across the microdialysis membrane in a few seconds, as opposed to a few minutes with existing technologies. Microdialysis microneedles present an attractive first step towards decreasing size, patient discomfort and energy consumption of portable medical monitors over existing technologies.

Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery

Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein-Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.