The Effects of Applied Local Heat on Transdermal Drug Delivery Systems

Transdermal drug delivery systems have been developed over the past several decades and now include patches for birth control, nicotine addiction, and pain relief. The local application of heat can increase the diffusion coefficient of the drug in the skin and result in faster delivery of the drug and shorter time to reach a steady state concentration of the drug. While this procedure is desirable for some systems where a faster dose will aid in alleviating pain and/or symptoms, it can also be a cause of concern for some drugs. Fentanyl, a chronic pain relief drug, can cause accidental death by overdose. We report herein an analysis of the effects of various heating situations on transdermal fentanyl delivery based upon a model developed using COMSOL Multiphysics. The utilization of such a model allows for the determination of situations which may be potentially dangerous for fentanyl drug users, and enables the development of usage guidelines and safety mechanisms for transdermal delivery systems. Using the computer model, the following cases were simulated: no applied heat, ThermaCare heat pad, fever, and heating blanket. The heating blanket and ThermaCare heat pad simulations showed the most dangerous increases in fentanyl blood concentration above no-heat levels: about 180% and 100%, respectively, over 30 hours; by contrast, the patient fever model reported a 40% increase in fentanyl blood concentration. These simulations demonstrate the dangers of fentanyl transdermal pain patches when skin temperature is increased, and can be used to develop better patient guidelines for patch use and to improve fentanyl transdermal systems. Lastly, this computer model may be used to model other transdermal drug delivery systems for the improvement of patient guidelines and/or the development of new systems, thus decreasing the need for experimentation on subjects

Computational cytometer based on magnetically modulated coherent imaging and deep learning.

Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications

Digital Loop-Mediated Isothermal DNA Amplification

Nucleic acid amplification has applications in diagnostics, sequencing, genetic fingerprinting, among others. Currently nucleic acid amplification is treated as the “gold standard” method for several diagnostics; however, because of the multi-step protocols and the large equipment, these assays are lengthy and require laboratory settings. Digital nucleic acid amplifications assays developed have greatly improved several aspects of nucleic acid amplification by creating a more robust and sensitive assay. This is due to the reduction in background noise and the ability to effectively concentrate target analytes in nano- or picoliter volumes by compartmentalization of these samples. We were able to demonstrate a 69-fold fluorescence change in an isothermal nucleic acid amplification assay, with a >60% increase in fluorescence stability with elevated temperatures over the time course of the reaction, with the use of a unique dye combination of EvaGreen and hydroxynapthol blue (HNB). Due to the improvements in signal, we were able to demonstrate comparable results using a mobile phone based fluorescence plate reader as with that of a benchtop reader. The unique dye combination was then applied to a digital system, demonstrating signal improvements that are crucial to developing a robust assay, giving a higher efficiency (percentage of “on” wells closer to the theoretical value) and a larger difference in fluorescence intensities for “on” versus “off” wells. Lastly, we examined the mechanism of the dye combination to best determine additional ways of improving the signal generation. By sequestering EvaGreen, HNB allows amplification to proceed without interference. Additionally, a F�rster resonance energy transfer (FRET) interaction between the dye molecules, when DNA is absent from the solution, acts to lower the background fluorescence such that a greater fluorescence fold change occurs with DNA amplification. The EvaGreen and HNB have a highly-tuned binding affinity such that prior to DNA amplification, they have FRET interactions, and afterwards in the presence of large amounts of DNA, EvaGreen binding to DNA becomes more favorable. All of these developed technologies and methods work in conjunction to improve upon currently developed techniques for nucleic acid amplification in point of care settings

Ferrodrop Dose-Optimized Digital Quantification of Biomolecules in Low-Volume Samples

We present an approach to estimate the concentration of a biomolecule in a solution by sampling several nanoliter-scale volumes and determining if the volumes contain any biomolecules. In this method, varying volume fractions (nanoliter-scale) of a sample of nucleic acids are introduced to an array of uniform volume reaction wells (100 μL), which are then fluorescently imaged to determine if signal is above a threshold after nucleic acid amplification, all without complex instrumentation. The nanoliter volumes are generated and introduced using the simple positioning of a permanent magnet, and imaging is performed with a cellphone-based fluorescence detection scheme, both methods suitable for limited-resource settings. We use the length of time a magnetic field is applied to generate a calibrated number of nanoliter ferrodrops of sample mixed with ferrofluid at a step emulsification microfluidic junction. Each dose of ferrodrops is then transferred into larger microliter scale reaction wells on chip through a simple shift of the external magnet. Nucleic acid amplification is achieved using loop-mediated isothermal amplification (LAMP). By repeating each nanoliter dosage a number of times to calculate the probability of a positive signal at each dosage, we can use a binomial probability distribution to estimate the sample nucleic acid concentration. Using this approach we demonstrate detection of lambda DNA molecules down to 25 copies per microliter. The ability to dose separate nanoliter-scale volumes of a low-volume sample across wells in this platform is suited for multiplexed assays. This platform has the potential to be applied to a range of diseases by mixing a sample with magnetic nanoparticles

Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings

Computational cytometer based on magnetically modulated coherent imaging and deep learning.

Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications