Beneficial Effects of Deoxyshikonin on Delayed Wound Healing in Diabetic Mice.

Shiunko ointment is composed of five ingredients including Lithospermi Radix (LR), Angelicae Gigantis Radix, sesame seed oil, beeswax, and swine oil. It is externally applied as a treatment for a wide range of skin conditions such as eczema, psoriasis, hair loss, burns, topical wounds, and atopic dermatitis. Deoxyshikonin is the major angiogenic compound extracted from LR. In this study, we investigated the efficacy of LR extract and deoxyshikonin on impaired wound healing in streptozotocin (STZ)-induced diabetic mice. Treatment with LR extract elevated tube formation in human umbilical vein endothelial cells (HUVECs) and exerted antioxidant activity. An open skin wound was produced on the backs of diabetic mice and was then topically treated with deoxyshikonin or vehicle. In addition, deoxyshikonin promoted tube formation in high glucose conditions exposed to HUVECs, and which may be regulated by increased VEGFR2 expression and phosphorylation of Akt and p38. Our results demonstrate that deoxyshikonin application promoted wound repair in STZ-induced diabetic mice. Collectively, these data suggest that deoxyshikonin is an active ingredient of LR, thereby contributing to wound healing in patients with diabetes.

A trachea-inspired bifurcated microfilter capturing viable circulating tumor cells via altered biophysical properties as measured by atomic force microscopy.

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina-inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid-structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead-conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 µL min(-1), an 8 µm filter gap optimizes the recovery rate and purity. MCF-7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re-collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.

Full surface embedding of gold clusters on silicon nanowires for efficient capture and photothermal therapy of circulating tumor cells.

We report on rapid thermal chemical vapor deposition growth of silicon nanowires (Si NWs) that contain a high density of gold nanoclusters (Au NCs) with a uniform coverage over the entire length of the nanowire sidewalls. The Au NC-coated Si NWs with an antibody-coated surface obtain the unique capability to capture breast cancer cells at twice the highest efficiency currently achievable (~88% at 40 min cell incubation time) from a nanostructured substrate. We also found that irradiation of breast cancer cells captured on Au NC-coated Si NWs with a near-infrared light resulted in a high mortality rate of these cancer cells, raising a fine prospect for simultaneous capture and plasmonic photothermal therapy for circulating tumor cells.

Standardized image-based polysomnography database and deep learning algorithm for sleep-stage classification.

STUDY OBJECTIVES: Polysomnography (PSG) scoring is labor-intensive, subjective, and often ambiguous. Recently several deep learning (DL) models for automated sleep scoring have been developed, they are tied to a fixed amount of input channels and resolution. In this study, we constructed a standardized image-based PSG dataset in order to overcome the heterogeneity of raw signal data obtained from various PSG devices and various sleep laboratory environments. METHODS: All individually exported European data format files containing raw signals were converted into images with an annotation file, which contained the demographics, diagnoses, and sleep statistics. An image-based DL model for automatic sleep staging was developed, compared with a signal-based model, and validated in an external dataset. RESULTS: We constructed 10253 image-based PSG datasets using a standardized format. Among these, 7745 diagnostic PSG data were used to develop our DL model. The DL model using the image dataset showed similar performance to the signal-based dataset for the same subject. The overall DL accuracy was greater than 80%, even with severe obstructive sleep apnea. Moreover, for the first time, we showed explainable DL in the field of sleep medicine as visualized key inference regions using Eigen-class activation maps. Furthermore, when a DL model for sleep scoring performs external validation, we achieved a relatively good performance. CONCLUSIONS: Our main contribution demonstrates the availability of a standardized image-based dataset, and highlights that changing the data sampling rate or number of sensors may not require retraining, although performance decreases slightly as the number of sensors decreases.

Highly efficient assay of circulating tumor cells by selective sedimentation with a density gradient medium and microfiltration from whole blood.

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 µm) and DMS-79 small cell lung cancer cells (average diameter, 10 µm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.

Pumpless dispensing of a droplet by breaking up a liquid bridge formed by electric induction.

Dispensing uniform pico-to-nanoliter droplets has become one of essential components in various application fields from high-throughput bio-analysis to printing. In this study, a new method is suggested and demonstrated for dispensing a droplet on the top plate with an inverted geometry by using electric field. The process of dispensing droplets consists of two stages: (i) formation of liquid bridge by moving up the charged fluid mass using the electrostatic force between the charges on the fluid mass and the induced charges on the substrate and (ii) its break-up by the motion of the top plate. Different from conventional electrohydrodynamic methods, electric induction enables the droplets to be dispensed on various surfaces including non-conducting substrate. The use of capillarity with an inverted geometry removes the need of external pumps or elaborates control for constant flow feed. The droplet diameter has been characterized as a function of the nozzle-to-plate distance and the plate moving velocity. The robustness of the present method is shown in terms of nozzle length and applied voltage. Finally, its practical applicability is confirmed by rendering a 19 by 24 array of highly uniform droplets with only 1.8% size variation without use of any active feedback control.

Potential Anti-Skin Aging Effect of (-)-Catechin Isolated from the Root Bark of Ulmus davidiana var. japonica in Tumor Necrosis Factor-α-Stimulated Normal Human Dermal Fibroblasts.

Reactive oxygen species (ROS) are generated during skin aging, including intrinsic (chronologic aging) and extrinsic aging (photoaging). Therefore, antioxidants that inhibit ROS generation can delay skin aging. In this study, we evaluated the potential anti-skin aging effect of (-)-phenolic compounds isolated from the root bark of Ulmus davidiana var. japonica. We preferentially investigated the possible preventive effects of isolates against the degradation of skin extracellular matrix. Among the isolates, (-)-catechin suppressed the activity of collagenase MMP-1, and reversed the degradation of collagen induced by tumor necrosis factor-α (TNF-α) in normal human dermal fibroblast. This action mechanism of (-)-catechin was validated by the suppression of tumor necrosis factor-α-induced accumulation of ROS and activation of mitogen-activated protein kinases, protein kinase B (Akt), and cyclooxygenase-2 (COX-2). The proinflammatory cytokines upregulate inflammatory reactions, and ultimately promote aging-related reactions. In this milieu, we demonstrated that (-)-catechin decreased the expression and secretion of proinflammatory cytokines, including interleukin (IL)-1ß and IL-6. In conclusion, (-)-catechin is a candidate to ameliorate both intrinsic and extrinsic skin aging.

A fully automated immunoassay from whole blood on a disc.

A portable, disc-based, and fully automated enzyme-linked immuno-sorbent assay (ELISA) system is developed to test infectious diseases from whole blood. The innovative laser irradiated ferrowax microvalves and centrifugal microfluidics were utilized for the full integration of microbead-based suspension ELISA assays on a disc starting from whole blood. The concentrations of the antigen and the antibody of Hepatitis B virus (HBV), HBsAg and Anti-HBs respectively, were measured using the lab-on-a-disc (LOD). All the necessary reagents are preloaded on the disc and the total process of the plasma separation, incubation with target specific antigen or antibody coated microbeads, multiple steps of washing, enzyme reaction with substrates, and the absorbance detection could be finished within 30 minutes. Compared to the conventional ELISA, the operation time was dramatically reduced from over 2 hours to less than 30 minutes while the limit of detection was kept similar; e.g. the limit of detection of Anti-HBs tests were 8.6 mIU mL(-1) and 10 mIU mL(-1) for the disc-based and the conventional ELISA respectively.

Bacteria concentration using a membrane type insulator-based dielectrophoresis in a plastic chip.

We report an insulator-based (or, electrodeless) dielectrophoresis utilizing microfabricated plastic membranes. The membranes with honeycomb-type pores have been fabricated by patterning the SU-8 layer on a substrate which was pretreated with self-assembled monolayer of octadecyltrichlorosilane for the easy release. The fabricated membrane was positioned between two electrodes and alternating current field was applied for the particle trap experiments. The particle could be trapped due to the dielectrophoresis force generated by the non-uniformities of the electric fields applied through the membranes with pores. Simulations using CFD-ACE+(CFD Research, Huntsville, Alabama) suggested that the dielectrophoresis force is stronger in the edge of the pores where the field gradient is highest. The bacteria could be captured on the near edge of the pores when the electric field was turned on and the trapped bacteria could be released when the field was turned off with the release efficiency of more than 93+/-7%. The maximal trapping efficiency of 66+/-7% was obtained under the electric fields (E=128 V/mm and f=300 kHz) when the dilute bacteria solution (Escherichia coli: 9.3 x 10(3) cell/mL, 0.5 mS/m) flowed with a flow rate of 100 microL/min.

Microslit on a chip: A simplified filter to capture circulating tumor cells enlarged with microbeads.

Microchips are widely used to separate circulating tumor cells (CTCs) from whole blood by virtues of sophisticated manipulation for microparticles. Here, we present a chip with an 8 µm high and 27.9 mm wide slit to capture cancer cells bound to 3 µm beads. Apart from a higher purity and recovery rate, the slit design allows for simplified fabrication, easy cell imaging, less clogging, lower chamber pressure and, therefore, higher throughput. The beads were conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) to selectively bind to breast cancer cells (MCF-7) used to spike the whole blood. The diameter of the cell-bead construct was in average 23.1 µm, making them separable from other cells in the blood. As a result, the cancer cells were separated from 5 mL of whole blood with a purity of 52.0% and a recovery rate of 91.1%, and also we confirmed that the device can be applicable to clinical samples of human breast cancer patients. The simple design with microslit, by eliminating any high-aspect ratio features, is expected to reduce possible defects on the chip and, therefore, more suitable for mass production without false separation outputs.

Gold nanoparticles for one step DNA extraction and real-time PCR of pathogens in a single chamber.

The optothermal properties of nanoparticles are of interest for biosensors and highly sensitive biochip applications. In this respect, the longitudinal resonance of Au nanorods was used to transform near infrared energy into thermal energy in a microfluidic chip. The resulting heat generated effectively caused pathogen lysis. Consequently the DNA was extracted out of the cell body and transferred to a PCR system. This resulted in the successful demonstration of a one step real-time PCR system for pathogen detection without removal or changing of reagents.

Multifunctional microvalves control by optical illumination on nanoheaters and its application in centrifugal microfluidic devices.

Valving is critical in microfluidic systems. Among many innovative microvalves used in lab-on-a-chip applications, phase change based microvalves using paraffin wax are particularly attractive for disposable biochip applications because they are simple to implement, cost-effective and biocompatible. However, previously reported paraffin-based valves require embedded microheaters and therefore multi-step operation of many microvalves was a difficult problem. Besides, the operation time was relatively long, 2-10 s. In this paper, we report a unique phase change based microvalve for rapid and versatile operation of multiple microvalves using a single laser diode. The valve is made of nanocomposite materials in which 10 nm-sized iron oxide nanoparticles are dispersed in paraffin wax and used as nanoheaters when excited by laser irradiation. Laser light of relatively weak intensity was able to melt the paraffin wax with the embedded iron oxide nanoparticles, whereas even a very intense laser beam does not melt wax alone. The microvalves are leak-free up to 403.0 +/- 7.6 kPa and the response times to operate both normally closed and normally opened microvalves are less than 0.5 s. Furthermore, a sequential operation of multiple microvalves on a centrifugal microfluidic device using a single laser diode was demonstrated. It showed that the optical control of multiple microvalves is fast, robust, simple to operate, and requires minimal chip space and thus is well suited for fully integrated lab-on-a-chip applications.

One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device.

We report a fully integrated, pathogen-specific DNA extraction device utilizing centrifugal microfluidics on a polymer based CD platform. By use of the innovative laser irradiated Ferrowax microvalve (LIFM) together with the rapid cell lysis method using laser irradiation on magnetic particles, we could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood on a CD. As a model study, DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) and E.coli were conducted. The total process of the plasma separation, mixing with magnetic beads conjugated with target specific antibodies, removal of plasma residual, washing and DNA extraction was finished within 12 min with only one manual step, the loading of 100 microL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD using a portable sample preparation device was as good as those by conventional bench top protocol. It demonstrates that our novel centrifugal microfluidics platform enables a full integration of complex biological reactions that require multi-step fluidic control.

DNA biosensor based on the electrochemiluminescence of Ru(bpy)3(2+) with DNA-binding intercalators.

This paper reports a novel detection method for DNA hybridization based on the electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) with a DNA-binding intercalator as a reductant of Ru(bpy)(3)(3+). Some ECL-inducible intercalators have been screened in this study using electrochemical methods combined with a chemiluminescent technique. The double-stranded DNA intercalated by doxorubicin, daunorubicin, or 4',6-diamidino-2-phenylindole (DAPI) shows a good ECL with Ru(bpy)(3)(2+) at +1.19 V (versus Ag/AgCl), while the non-intercalated single-stranded DNA does not. In order to stabilize the self-assembled DNA molecules during ECL reaction, we constructed the ECL DNA biosensor separating the ECL working electrode with an immobilized DNA probe. A gold electrode array on a plastic plate was assembled with a thru-hole array where oligonucleotide probes were immobilized in the side wall of thru-hole array. The fabricated ECL DNA biosensor was used to detect several pathogens using ECL technique. A good specificity of single point mutations for hepatitis disease was obtained by using the DAPI-intercalated Ru(bpy)(3)(2+) ECL.

Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification.

Optimal detection of a pathogen present in biological samples depends on the ability to extract DNA molecules rapidly and efficiently. In this paper, we report a novel method for efficient DNA extraction and subsequent real-time detection in a single microchip by combining laser irradiation and magnetic beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli and Gram-positive bacterial cells as well as the hepatitis B virus mixed with human serum. We further demonstrate that the real-time pathogen detection was performed with pre-mixed PCR reagents in a real-time PCR machine using the same microchip, after laser irradiation in a hand-held device equipped with a small laser diode. These results suggest that the new sample preparation method is well suited to be integrated into lab-on-a-chip application of the pathogen detection system.

DNA chip replication for a personalized DNA chip.

We report the replication technology of DNA chip using by sequence specific localization of nucleic acids via hybridization and electric transfer of the nucleic acids onto a new substrate without losing their array information. The denatured DNA fragments are first spotted and UV-cross-linked on a nylon membrane. The membrane is then immersed and hybridized in a DNA mixture solution that contains all complementary sequences of the nucleic acids to be hybridized with the DNA fragments on the membrane. The hybridized DNA fragments are transferred to another membrane at the denatured condition. After separating two membranes, the transferred membrane contains a complementary array of DNA fragments. This method can be used for the replication of the same copy of DNA chip repeatedly and moreover could be applied for a personalized DNA chip fabrication, where specific information of each spot of DNA chip is originated from the genetic information of a personal sample.

Electrohydrodynamic (EHD) dispensing of nanoliter DNA droplets for microarrays.

In the present paper, we first demonstrated the possibility of electrohydrodynamic (EHD) dispensing method for preparing nanoliter probe DNA droplets on surfaces in DNA microarrays. To study the effect of an electric field on the dynamic behavior of pendent DNA droplet, visualization experiments with three kinds of electrode shapes are performed. In the early stage of droplet dispensing, it is shown that applied electric field assists a gravitational force exerted on DNA droplet. The pendent droplet is elongated in the parallel direction of applied electric field. However, after making fluid bridge between electrodes, it is shown that the electric force accelerates the capillary breaking of droplet by assisting a surface tension force exerted on droplet surface. Specifically, nanoliter dispensing volume (2 nL) is obtained in the needle-type electrode configuration. In addition, for the case of hydrophobic electrode surface, it is shown that the dispensing volume and spot size are remarkably decreased. Under the high relative humidity condition, it is observed that spot size is rapidly increased because of reduction in evaporation rate on droplet surface during the dispensing procedure. On the other hand, it is obtained that the spot size is not changed significantly in the wide range of DNA concentration from 1 to 10,000 nM. To monitor the influence of high electric voltage on DNA stability, we prepared a silicon-based chip with five capture probes for pathogens related with respiratory infectious diseases by EHD dispensing method. From the examination, it is clearly confirmed that pathogens are detected and the effective signal levels of pathogenic bacteria after hybridization are retained. Consequently, it is found that EHD dispensing method can be used to make cost-effective DNA microarrays with no thermal and electrical influences on DNA properties.

Drop formation via breakup of a liquid bridge in an AC electric field.

Experimental results are presented for the study of drop formation mechanism in a newly proposed electrohydrodynamic (EHD) method of drop generation in an AC electric field. In the method, a small drop is generated in two stages. A pendant drop is elongated with large oscillation by an electric force in the first stage. Then, it undergoes formation and breakup of a liquid bridge between the upper nozzle and the insulator-coated lower flat plate in the second stage. It is found that there exists a resonant frequency for maximum oscillation, which leads to an efficient drop formation in the latter stage. It is also found that breakup of liquid bridge is accelerated by the electrowetting tension acting on the drop perimeter contacting the insulator-coated flat plate. Thus the whole procedure of drop formation depends heavily on the frequency of AC field and the properties of the insulator such as hydrophilicity, thickness, and the dielectric constant. It is demonstrated that a wide range of drop size, from picoliter to nanoliter, can be obtained by controlling such key parameters without changing the nozzle diameter.

A microchip filter device incorporating slit arrays and 3-D flow for detection of circulating tumor cells using CAV1-EpCAM conjugated microbeads.

Circulating tumor cells (CTCs) are rare cells and the presence of these cells may indicate a poor prognosis and a high potential for metastasis. Despite highly promising clinical applications, CTCs have not been investigated thoroughly, due to many technical limitations faced in their isolation and identification. Current CTC detection techniques mostly take the epithelial marker epithelial cell adhesion molecule (EpCAM), however, accumulating evidence suggests that CTCs show heterogeneous EpCAM expression due to the epithelial-to-mesenchymal transition (EMT). In this study, we report that a microchip filter device incorporating slit arrays and 3-dimensional flow that can separate heterogeneous population of cells with marker for CTCs. To select target we cultured breast cancer cells under prolonged mammosphere culture conditions which induced EMT phenotype. Under these conditions, cells show upregulation of caveolin1 (CAV1) but down-regulation of EpCAM expression. The proposed device which contains CAV1-EpCAM conjugated bead has several tens of times increased throughput. More importantly, this platform enables the enhanced capture yield from metastatic breast cancer patients and obtained cells that expressed various EMT markers. Further understanding of these EMT-related phenotypes will lead to improved detection techniques and may provide an opportunity to develop therapeutic strategies for effective treatment and prevention of cancer metastasis.