Highly efficient cellular cloning using Ferro-core Micropallet Arrays.

Advancing knowledge of biological mechanisms has come to depend upon genetic manipulation of cells and organisms, relying upon cellular cloning methods that remain unchanged for decades, are labor and time intensive, often taking many months to come to fruition. Thus, there is a pressing need for more efficient processes. We have adapted a newly developed micropallet array platform, termed the "ferro-core micropallet array", to dramatically improve and accelerate the process of isolating clonal populations of adherent cells from heterogeneous mixtures retaining the flexibility of employing a wide range of cytometric parameters for identifying colonies and cells of interest. Using transfected (retroviral oncogene or fluorescent reporter construct) rat 208 F cells, we demonstrated the capacity to isolate and expand pure populations of genetically manipulated cells via laser release and magnetic recovery of single micropallets carrying adherent microcolonies derived from single cells. This platform can be broadly applied to biological research, across the spectrum of molecular biology to cellular biology, involving fields such as cancer, developmental, and stem cell biology. The ferro-core micropallet array platform provides significant advantages over alternative sorting and cloning methods by eliminating the necessity for repetitive purification steps and increasing throughput by dramatically shortening the time to obtain clonally expanded cell colonies.

Development of a novel completely-in-the-canal direct-drive hearing device.

OBJECTIVES/HYPOTHESIS: To develop a novel completely-in-the-canal device capable of directly driving the tympanic membrane (TM) and ossicular chain from the ear canal. STUDY DESIGN: Development and feasibility study. METHODS: A voice coil actuator design was developed to drive the TM. Bench testing of the device using laser Doppler vibrometry (LDV) and sound recording was performed. Temporal bone studies using LDV were performed using different designs of the contact tip-TM interface to find the most efficient method of sound transmission. Two short-term clinical performance studies were performed using the latest 3-mm-wide device. Comparison was made to natural sound and to the Vibrant SoundBridge floating mass transducer simulator. RESULTS: On bench testing, the device was found to have a low (<0.5%) total harmonic distortion in all frequencies above 400 Hz. Temporal bone studies revealed the device was capable of producing vibrations equivalent to 104 to 120 dB sound across most frequencies. The most efficient method of stimulation was when the device was coupled to the malleus. Short-term clinical performance studies indicated that pure tones and complex sound can be presented with the device. The sound quality of the experimental device was rated as better than the SoundBridge simulator device. CONCLUSIONS: The direct-drive hearing device is capable of producing a wide range of sound frequencies and amplitudes. The device can transmit complex sound with low power requirements. Further work on the development of the device is needed for long-term and wider clinical use. LEVEL OF EVIDENCE: NA Laryngoscope, 127:932-938, 2017.

Skin-mountable stretch sensor for wearable health monitoring.

This work presents a wrinkled Platinum (wPt) strain sensor with tunable strain sensitivity for applications in wearable health monitoring. These stretchable sensors show a dynamic range of up to 185% strain and gauge factor (GF) of 42. This is believed to be the highest reported GF of any metal thin film strain sensor over a physiologically relevant dynamic range to date. Importantly, sensitivity and dynamic range are tunable to the application by adjusting wPt film thickness. Performance is reliable over 1000 cycles with low hysteresis after sensor conditioning. The possibility of using such a sensor for real-time respiratory monitoring by measuring chest wall displacement and correlating with lung volume is demonstrated.

A fluid collection system for dermal wounds in clinical investigations.

In this work, we demonstrate the use of a thin, self adherent, and clinically durable patch device that can collect fluid from a wound site for analysis. This device is manufactured from laminated silicone layers using a novel all-silicone double-molding process. In vitro studies for flow and delivery were followed by a clinical demonstration for exudate collection efficiency from a clinically presented partial thickness burn. The demonstrated utility of this device lends itself for use as a research implement used to clinically sample wound exudate for analysis. This device can serve as a platform for future integration of wearable technology into wound monitoring and care. The demonstrated fabrication method can be used for devices requiring thin membrane construction.

Multicolor Immunofluorescent Imaging of Complex Cellular Mixtures on Micropallet Arrays Enables the Identification of Single Cells of Defined Phenotype.

A Micropallet-Array-based strategy allowing the identification of cells of defined phenotype in complex mixtures, such as would be obtained from a tissue biopsy, is presented. Following the distribution of single adherent cells from the mixture on individual pedestals, termed "micropallets", immunofluorescent confocal imaging is applied to interrogate the expression of five cell surface molecules.

Large area magnetic micropallet arrays for cell colony sorting.

A new micropallet array platform for adherent cell colony sorting has been developed. The platform consisted of thousands of square plastic pallets, 270 µm by 270 µm on each side, large enough to hold a single colony of cells. Each pallet included a magnetic core, allowing them to be collected with a magnet after being released using a microscope mounted laser system. The micropallets were patterned from 1002F epoxy resist and were fabricated on translucent, gold coated microscope slides. The gold layer was used as seed for electroplating the ferromagnetic cores within every individual pallet. The gold layer also facilitated the release of each micropallet during laser release. This array allows for individual observation, sorting and collection of isolated cell colonies for biological cell colony research. In addition to consistent release and recovery of individual colonies, we demonstrated stable biocompatibility and minimal loss in imaging quality compared to previously developed micropallet arrays.

Attitudes and preferences on the use of mobile health technology and health games for self-management: interviews with older adults on anticoagulation therapy.

BACKGROUND: Older adults are at substantial risk for cardiovascular disorders that may require anticoagulation therapy. Those on warfarin therapy report dissatisfaction and reduced quality of life (QOL) resulting from the treatment. Advances in the area of mobile health (mHealth) technology have resulted in the design and development of new patient-centric models for the provision of personalized health care services to improve care delivery. However, there is a paucity of research examining the effectiveness of mHealth tools on knowledge, attitudes, and patient satisfaction with treatment, as well as self-management, adherence to therapy, and QOL in older adults with chronic illness conditions requiring long-term warfarin therapy. OBJECTIVE: The objective of the study was to explore the attitudes and preferences of older adults on warfarin therapy regarding the use of mHealth technology and health games to gain skills for self-management. METHODS: We conducted group and individual interviews with patients (60 years or older) on warfarin therapy at two anticoagulation clinics affiliated with an academic medical center. We held 4 group and 2 individual interviews, resulting in 11 patient participants and 2 family caregiver participants. We used structured questions on three topic areas including medication self-management strategies, mHealth technology use, and health games for exercise. We demonstrated some commercial health apps related to medication management, vitamin K content of food, and a videogame for balance exercise. Discussions were audiotaped and transcribed verbatim. Common themes were drawn using content analysis. RESULTS: The participants reported awareness of the importance of staying on schedule with warfarin therapy. They also acknowledged that negative experiences of friends or family members who were taking warfarin influenced their desire to keep on schedule with warfarin therapy. In addition, the participants expressed that the use of mHealth technology may be helpful for medication management. They also expressed the need for family support in the use of health technology devices. Moreover, the participants discussed concerns and challenges to use health technology and health games, and provided suggestions on ways to make mHealth technology and health games elder-friendly. CONCLUSIONS: These findings indicate that our older adults on warfarin therapy are interested in mHealth technology specific to warfarin medication management and health games. Further research needs to be done to validate these findings. Elder-friendly designs, technology support, and physical safety using mHealth technology may be useful in this population. These findings can be used to inform a larger study to design and test an elder-centered mHealth technology in this target population.

A micro-drive hearing aid: a novel non-invasive hearing prosthesis actuator.

The direct hearing device (DHD) is a new auditory prosthesis that combines conventional hearing aid and middle ear implant technologies into a single device. The DHD is located deep in the ear canal and recreates sounds with mechanical movements of the tympanic membrane. A critical component of the DHD is the microactuator, which must be capable of moving the tympanic membrane at frequencies and magnitudes appropriate for normal hearing, with little distortion. The DHD actuator reported here utilized a voice coil actuator design and was 3.7 mm in diameter. The device has a smoothly varying frequency response and produces a precisely controllable force. The total harmonic distortion between 425 Hz and 10 kHz is below 0.5 % and acoustic noise generation is minimal. The device was tested as a tympanic membrane driver on cadaveric temporal bones where the device was coupled to the umbo of the tympanic membrane. The DHD successfully recreated ossicular chain movements across the frequencies of human hearing while demonstrating controllable magnitude. Moreover, the micro-actuator was validated in a short-term human clinical performance study where sound matching and complex audio waveforms were evaluated by a healthy subject.

The manumeter: a wearable device for monitoring daily use of the wrist and fingers.

Nonobtrusive options for monitoring the wrist and hand movement are needed for stroke rehabilitation and other applications. This paper describes the "manumeter," a device that logs total angular distance travelled by wrist and finger joints using a magnetic ring worn on the index finger and two triaxial magnetometers mounted in a watch-like unit. We describe an approach to estimate the wrist and finger joint angles using a radial basis function network that maps differential magnetometer readings to joint angles. We tested this approach by comparing manumeter estimates of total angular excursion with those from a passive goniometric exoskeleton worn simultaneously as seven participants completed a set of 12 manual tasks at low-, medium-, and high-intensity conditions on a first testing day, 1-2 days later, and 6-8 days later, using only the original calibration from the first testing day. Manumeter estimates scaled proportionally to the intensity of hand activity. Estimates of angular excursion made with the manumeter were 92.5% ± 28.4 (SD), 98.3% ± 23.3, and 94.7% ± 19.3 of the goniometric exoskeleton across the three testing days, respectively. Magnetic sensing of wrist and finger movement is nonobtrusive and can quantify the amount of use of the hand across days.

Retraining and assessing hand movement after stroke using the MusicGlove: comparison with conventional hand therapy and isometric grip training.

BACKGROUND: It is thought that therapy should be functional, be highly repetitive, and promote afferent input to best stimulate hand motor recovery after stroke, yet patients struggle to access such therapy. We developed the MusicGlove, an instrumented glove that requires the user to practice gripping-like movements and thumb-finger opposition to play a highly engaging, music-based, video game. The purpose of this study was to 1) compare the effect of training with MusicGlove to conventional hand therapy 2) determine if MusicGlove training was more effective than a matched form of isometric hand movement training; and 3) determine if MusicGlove game scores predict clinical outcomes. METHODS: 12 chronic stroke survivors with moderate hemiparesis were randomly assigned to receive MusicGlove, isometric, and conventional hand therapy in a within-subjects design. Each subject participated in six one-hour treatment sessions three times per week for two weeks, for each training type, for a total of 18 treatment sessions. A blinded rater assessed hand impairment before and after each training type and at one-month follow-up including the Box and Blocks (B & B) test as the primary outcome measure. Subjects also completed the Intrinsic Motivation Inventory (IMI). RESULTS: Subjects improved hand function related to grasping small objects more after MusicGlove compared to conventional training, as measured by the B & B score (improvement of 3.21±3.82 vs. -0.29±2.27 blocks; P=0.010) and the 9 Hole Peg test (improvement of 2.14±2.98 vs. -0.85±1.29 pegs/minute; P=0.005). There was no significant difference between training types in the broader assessment batteries of hand function. Subjects benefited less from isometric therapy than MusicGlove training, but the difference was not significant (P>0.09). Subjects sustained improvements in hand function at a one month follow-up, and found the MusicGlove more motivating than the other two therapies, as measured by the IMI. MusicGlove games scores correlated strongly with the B & B score. CONCLUSIONS: These results support the hypothesis that hand therapy that is engaging, incorporates high numbers of repetitions of gripping and thumb-finger opposition movements, and promotes afferent input is a promising approach to improving an individual's ability to manipulate small objects. The MusicGlove provides a simple way to access such therapy.

Microfluidic dielectrophoretic sorter using gel vertical electrodes.

We report the development and results of a two-step method for sorting cells and small particles in a microfluidic device. This approach uses a single microfluidic channel that has (1) a microfabricated sieve which efficiently focuses particles into a thin stream, followed by (2) a dielectrophoresis (DEP) section consisting of electrodes along the channel walls for efficient continuous sorting based on dielectric properties of the particles. For our demonstration, the device was constructed of polydimethylsiloxane, bonded to a glass surface, and conductive agarose gel electrodes. Gold traces were used to make electrical connections to the conductive gel. The device had several novel features that aided performance of the sorting. These included a sieving structure that performed continuous displacement of particles into a single stream within the microfluidic channel (improving the performance of downstream DEP, and avoiding the need for additional focusing flow inlets), and DEP electrodes that were the full height of the microfluidic walls ("vertical electrodes"), allowing for improved formation and control of electric field gradients in the microfluidic device. The device was used to sort polymer particles and HeLa cells, demonstrating that this unique combination provides improved capability for continuous DEP sorting of particles in a microfluidic device.

Laminated microfluidic system for small sample protein analysis.

We describe a technology based on lamination that allows for the production of highly integrated 3D devices suitable for performing a wide variety of microfluidic assays. This approach uses a suite of microfluidic coupons ("microfloupons") that are intended to be stacked as needed to produce an assay of interest. Microfloupons may be manufactured in paper, plastic, gels, or other materials, in advance, by different manufacturers, then assembled by the assay designer as needed. To demonstrate this approach, we designed, assembled, and characterized a microfloupon device that performs sodium-dodecyl-sulfate polyacrylamide gel electrophoresis on a small sample of protein. This device allowed for the manipulation and transport of small amounts of protein sample, tight injection into a thin polyacrylamide gel, electrophoretic separation of the proteins into bands, and subsequent removal of the gel from the device for imaging and further analysis. The microfloupons are rugged enough to handle and can be easily aligned and laminated, allowing for a variety of different assays to be designed and configured by selecting appropriate microfloupons. This approach provides a convenient way to perform assays that have multiple steps, relieving the need to design highly sophisticated devices that incorporate all functions in a single unit, while still achieving the benefits of small sample size, automation, and high speed operation.

The variable relationship between arm and hand use: a rationale for using finger magnetometry to complement wrist accelerometry when measuring daily use of the upper extremity.

Wrist-worn accelerometers are becoming more prevalent as a means to assess use of the impaired upper extremity in daily life after stroke. However, wrist accelerometry does not measure joint movements of the hand, which are integral to functional use of the upper extremity. In this study, we used a custom-built, non-obtrusive device called the manumeter to measure both arm use (via wrist accelerometry) and hand use (via finger magnetometry) of a group of unimpaired subjects while they performed twelve motor tasks at three intensities. We also gave the devices to four stroke subjects and asked them to wear them for six hours a day for one month. From the in-lab testing we found that arm use was a strong predictor of hand use for individual tasks, but that the slope of the relationship varied by up to a factor of ~12 depending on the task being performed. Consistent with this, in the daily use data collected from stroke subjects we found a broad spread in the relationship between arm and hand use. These results suggest that analyzing the spread of the relationship between daily hand and arm use will give more insight into upper extremity recovery than wrist accelerometry or finger magnetometry alone, because the spread reflects the nature of the daily tasks performed as well as the amount of upper extremity use.

The Manumeter: a non-obtrusive wearable device for monitoring spontaneous use of the wrist and fingers.

This paper describes the design and pilot testing of a novel device for unobtrusive monitoring of wrist and hand movement through a sensorized watch and a magnetic ring system called the manumeter. The device senses the magnetic field of the ring through two triaxial magnetometers and records the data to onboard memory which can be analyzed later by connecting the watch unit to a computer. Wrist and finger joint angles are estimated using a radial basis function network. We compared joint angle estimates collected using the manumeter to direct measurements taken using a passive exoskeleton and found that after a 60 minute trial, 95% of the radial/ulnar deviation, wrist flexion/extension and finger flexion/extension estimates were within 2.4, 5.8, and 4.7 degrees of their actual values respectively. The device measured angular distance traveled for these three joints within 10.4%, 4.5%, and 14.3 % of their actual values. The manumeter has potential to improve monitoring of real world use of the hand after stroke and in other applications.

Flexible shrink-induced high surface area electrodes for electrochemiluminescent sensing.

Photolithographically defined metallic thin film on commodity shrink-wrap is leveraged to create robust electrodes. By thermally shrinking the film, electrodes are reduced by 20× in footprint for improved resolution and conductivity with >600% enhancements in electrochemically active surface area; as electrochemiluminescent sensors, they demonstrate improved limits of detection.

Completely-in-the-canal magnet-drive hearing device: a temporal bone study.

The magnet-drive hearing device (MHD) is a small completely-in-the-canal hearing aid prototype that drives the tympanic membrane (TM) through a magnetic interface. A cadaveric temporal bone was prepared. The MHD was coupled to a nickel-epoxy pellet glued to the umbo. Frequency sweeps between 0.3 and 10 kHz were performed, and the MHD was driven with various levels of current. Displacements of the posterior crus of the stapes were measured using a laser Doppler vibrometer and compared with sound-induced displacements. The MHD had a linear frequency response and low total harmonic distortion. The pellet placement altered the stapes movements; however, the changes were statistically insignificant. Inputs of 100 and 300 mV produced displacements equivalent to those of the natural sound at 70- and 80-dB sound pressure level, respectively. The coupling of this novel device using a magnetic interface to the umbo had a frequency output wider than air conduction devices, and its actuator was effective in driving the TM.

Investigation of a novel completely-in-the-canal direct-drive hearing device: a temporal bone study.

HYPOTHESIS: Whether a prototype direct-drive hearing device (DHD) is effective in driving the tympanic membrane (TM) in a temporal bone specimen to enable it to potentially treat moderate-to-severe hearing loss. BACKGROUND: Patient satisfaction with air conduction hearing aids has been low because of sound distortion, occlusion effect, and feedback issues. Implantable hearing aids provide a higher quality sound but require surgery for placement. The DHD was designed to combine the ability of driving the ossicular chain with placement in the external auditory canal. METHODS: DHD is a 3.5-mm wide device that could fit entirely into the bony ear canal and directly drive the TM rather than use a speaker. A cadaveric temporal bone was prepared. The device developed in our laboratory was coupled to the external surface of the TM and against the malleus. Frequency sweeps between 300 Hz to 12 kHz were performed in 2 different coupling methods at 104 and 120 dB, and the DHD was driven with various levels of current. Displacements of the posterior crus of the stapes were measured using a laser Doppler vibrometer. RESULTS: The DHD showed a linear frequency response from 300 Hz to 12 kHz. Placement against the malleus showed higher amplitudes and lower power requirements than when the device was placed on the TM. CONCLUSION: DHD is a small completely-in-the-canal device that mechanically drives the TM. This novel device has a frequency output wider than most air conduction devices. Findings of the current study demonstrated that the DHD had the potential of being incorporated into a hearing aid in the future.

Integrated printed circuit board device for cell lysis and nucleic acid extraction.

Preparation of raw, untreated biological samples remains a major challenge in microfluidics. We present a novel microfluidic device based on the integration of printed circuit boards and an isotachophoresis assay for sample preparation of nucleic acids from biological samples. The device has integrated resistive heaters and temperature sensors as well as a 70 µm × 300 µm × 3.7 cm microfluidic channel connecting two 15 µL reservoirs. We demonstrated this device by extracting pathogenic nucleic acids from 1 µL dispensed volume of whole blood spiked with Plasmodium falciparum. We dispensed whole blood directly onto an on-chip reservoir, and the system's integrated heaters simultaneously lysed and mixed the sample. We used isotachophoresis to extract the nucleic acids into a secondary buffer via isotachophoresis. We analyzed the convective mixing action with micro particle image velocimetry (micro-PIV) and verified the purity and amount of extracted nucleic acids using off-chip quantitative polymerase chain reaction (PCR). We achieved a clinically relevant limit of detection of 500 parasites per microliter. The system has no moving parts, and the process is potentially compatible with a wide range of on-chip hybridization or amplification assays.

Microelectrode arrays fabricated using a novel hybrid microfabrication method.

We present novel hybrid microfabrication methods for microelectrode arrays that combine microwire assembly, microelectromechanical systems (MEMS) manufacturing techniques and precision tool-based micromachining. This combination enables hybrid microfabrication to produce complex geometries and structures, increase material selection, and improve integration. A 32-channel shank microelectrode array was fabricated to highlight the hybrid microfabrication techniques. The electrode shank was 130 µm at its narrowest, had a 127 µm thickness and had iridium oxide electrode sites that were 25 µm in diameter with 150 µm spacing. Techniques used to fabricate this electrode include microassembly of insulated gold wires into a micromold, micromolding the microelectrode shank, post molding machining, sacrificial release of the microelectrode and electrodeposition of iridium oxide onto the microelectrode sites. Electrode site position accuracy was shown to have a standard deviation of less than 4 µm. Acute in vivo recordings with the 32-channel shank microelectrode array demonstrated comparable performance to that obtained with commercial microelectrode arrays. This new approach to microelectrode array fabrication will enable new microelectrodes, such as multi-sided arrays, drug eluding electrodes and biodegradable shanks.

Intensity coding in electric hearing: effects of electrode configurations and stimulation waveforms.

OBJECTIVES: Current cochlear implants typically stimulate the auditory nerve with biphasic pulses and monopolar electrode configurations. Tripolar stimulation can increase spatial selectivity and potentially improve place pitch related perception but requires higher current levels to elicit the same loudness as monopolar stimulation. The present study combined delayed pseudomonophonasic pulses, which produce lower thresholds, with tripolar stimulation in an attempt to solve the power-performance tradeoff problem. DESIGN: The present study systematically measured thresholds, dynamic range, loudness growth, and intensity discrimination using either biphasic or delayed pseudomonophonasic pulses under both monopolar and tripolar stimulation. Participants were five Clarion cochlear implant users. For each subject, data from apical, middle, and basal electrode positions were collected when possible. RESULTS: Compared with biphasic pulses, delayed pseudomonophonasic pulses increased the dynamic range by lowering thresholds while maintaining comparable maximum allowable levels under both electrode configurations. However, delayed pseudomonophonasic pulses did not change the shape of loudness growth function and actually increased intensity discrimination limens, especially at lower current levels. CONCLUSIONS: The present results indicate that delayed pseudomonophonasic pulses coupled with tripolar stimulation cannot provide significant power savings nor can it increase the functional dynamic range. Whether this combined stimulation could improve functional spectral resolution remains to be seen.