Elevated plasma biomarkers of inflammation in acute ischemic stroke patients with underlying dementia.

BACKGROUND: The blood-brain barrier has been a hindrance to developing blood-based diagnostic tests for dementias, as it limits the appearance of brain biomarkers in the blood. Our aim was to see if the natural opening of the blood-brain barrier induced by ischemic stroke would increase serum levels of inflammatory biomarkers known to be elevated in the brains of patients with Alzheimer's disease and other neurodegenerative dementias. METHODS: Forty-three patients with acute ischemic stroke presenting to Stony Brook University Hospital were prospectively enrolled in the study. Eight of these patients were clinically diagnosed as having an underlying neurodegenerative dementia. Blood was drawn acutely within 72 h of stroke symptom onset, and serum levels of the classic inflammatory biomarkers, interleukin-6 (IL-6) and C-reactive protein (CRP) were measured, along with levels of S100B protein (S100B) and complement C3 (CC3). RESULTS: Serum levels of IL-6 and CRP in patients with acute ischemic stroke and underlying dementia (AIS + D) were significantly higher (p = 0.002 and 0.003, respectively) than in patients with acute ischemic stroke alone (AIS). Serum levels of S100B and CC3 did not differ significantly between the groups. CONCLUSIONS: This study supports the possibility that opening of the blood-brain barrier may enhance the blood appearance of brain tissue markers of inflammation associated with neurodegenerative dementia. Further study is warranted to test this possibility, given the recent emergence of methods to open the blood-brain barrier for diagnostic or therapeutic purposes.

Spatiotemporally Defining Biomolecule Preconcentration by Merging Ion Concentration Polarization.

The ion concentration polarization (ICP) phenomenon at micronanofluidic interfaces has been extensively utilized to preconcentrate low-abundance biological samples. Although preconcentration by ICP is robust, its multiphysics phenomenon does not permit a clear prediction of the preconcentration conditions and sites. Here, we present a new method for spatiotemporally defining preconcentration, which can generate target-condensed plugs in a very specific region (<100 µm) regardless of the operating conditions (time, applied voltage, ionic strength, and pH). In contrast to previous devices that use only ion depletion, this device uses merged ICP zones with opposite polarity, i.e., ion depletion and ion enrichment. In this regard, ICP is initiated between two line-patterned cation exchange membranes. When voltage is applied across two membranes, an ion depletion (enrichment) zone occurs on the anodic (cathodic) side of the membranes. Two ICP zones are then merged and confined between the membranes. Consequently, the preconcentration action is also confined between the membranes. We demonstrate that fluorescent dyes are always preconcentrated at the designated location at all lengths of operating time and at broad voltage (0.5-100 V), ionic strength (1-100 mM KCl), and pH (3.7-10.3) ranges. This device successfully condenses proteins up to 10000-fold in a specific region of the channel (100 × 50 × 10 µm(3)) in 10 min. This work not only characterizes the unique scientific phenomenon of ICP overlapping but also opens the possibility of integrating ICP preconcentrators into commercial analysis equipment, which requires a known, stationary preconcentration site.

Control of artificial membrane fusion in physiological ionic solutions beyond the limits of electroformation.

Membrane fusion, merging two lipid bilayers, is crucial for fabricating artificial membrane structures. Over the past 40 years, in contrast to precise and controllable membrane fusion in-vivo through specific molecules such as SNAREs, controlling the fusion in-vitro while fabricating artificial membrane structures in physiological ionic solutions without fusion proteins has been a challenge, becoming a significant obstacle to practical applications. We present an approach consisting of an electric field and a few kPa hydraulic pressure as an additional variable to physically control the fusion, enabling tuning of the shape and size of the 3D freestanding lipid bilayers in physiological ionic solutions. Mechanical model analysis reveals that pressure-induced parallel/normal tensions enhance fusion among membranes in the microwell. In-vitro peptide-membrane assay, mimicking vesicular transport via pressure-assisted fusion, and stability of 38 days with in-chip pressure control via pore size-regulated hydrogel highlight the potential for diverse biological applications.

Tentonin 3 is a pore-forming subunit of a slow inactivation mechanosensitive channel.

Mechanically activating (MA) channels transduce numerous physiological functions. Tentonin 3/TMEM150C (TTN3) confers MA currents with slow inactivation kinetics in somato- and barosensory neurons. However, questions were raised about its role as a Piezo1 regulator and its potential as a channel pore. Here, we demonstrate that purified TTN3 proteins incorporated into the lipid bilayer displayed spontaneous and pressure-sensitive channel currents. These MA currents were conserved across vertebrates and differ from Piezo1 in activation threshold and pharmacological response. Deep neural network structure prediction programs coupled with mutagenetic analysis predicted a rectangular-shaped, tetrameric structure with six transmembrane helices and a pore at the inter-subunit center. The putative pore aligned with two helices of each subunit and had constriction sites whose mutations changed the MA currents. These findings suggest that TTN3 is a pore-forming subunit of a distinct slow inactivation MA channel, potentially possessing a tetrameric structure.

Multifunctionalized cantilever systems for electronic nose applications.

Multiple target detection using a cantilever is essential for biosensor, chemical sensor, and electronic nose systems. We report a novel microcantilever array chip that includes four microreaction chambers in a chip, which consequently contains four different functionalized surfaces for multitarget detection. For model tests, we designed microcantilever chips and demonstrated the ability of binding of 2,4-dinitrotoluene (DNT) targets onto four different surfaces. We used peptide receptors that are known to have highly selective binding. By simply using four microreaction chambers, we immobilized DNT specific peptide (HPNFSKYILHQRC; SP), DNT nonspecific peptide (TSMLLMSPKHQAC; NSP), and self-assembled monolayer (SAM) as well as a bare cantilever. After flowing DNT gases through the cantilever chip, we could monitor the four different binding signals simultaneously. The shifts in NSP provided information as a negative control because it contained information of temperature fluctuations and mechanical vibration from gas flow. By utilizing the differential signal of the SP and NSP, we acquired 7.5 Hz in resonant responses that corresponds with 160 part per billion (ppb) DNT concentration, showing the exact binding response by eliminating the inevitable thermal noise, vibration noise, as well as humidity effects on the peptide surface.

Tunable and scalable fabrication of block copolymer-based 3D polymorphic artificial cell membrane array.

Owing to their excellent durability, tunable physical properties, and biofunctionality, block copolymer-based membranes provide a platform for various biotechnological applications. However, conventional approaches for fabricating block copolymer membranes produce only planar or suspended polymersome structures, which limits their utilization. This study is the first to demonstrate that an electric-field-assisted self-assembly technique can allow controllable and scalable fabrication of 3-dimensional block copolymer artificial cell membranes (3DBCPMs) immobilized on predefined locations. Topographically and chemically structured microwell array templates facilitate uniform patterning of block copolymers and serve as reactors for the effective growth of 3DBCPMs. Modulating the concentration of the block copolymer and the amplitude/frequency of the electric field generates 3DBCPMs with diverse shapes, controlled sizes, and high stability (100% survival over 50 days). In vitro protein-membrane assays and mimicking of human intestinal organs highlight the potential of 3DBCPMs for a variety of biological applications such as artificial cells, cell-mimetic biosensors, and bioreactors.

Active locomotion of a paddling-based capsule endoscope in an in vitro and in vivo experiment (with videos).

BACKGROUND: Capsule endoscopy that could actively move and approach a specific site might be more valuable for the diagnosis or treatment of GI diseases. OBJECTIVE: We tested the performance of active locomotion of a novel wired capsule endoscope with a paddling-based locomotion mechanism, using 3 models: a silicone tube, an extracted porcine colon, and a living pig. DESIGN: In vitro, ex vivo, and in vivo experiments in a pig model. SETTING: Study in an animal laboratory. INTERVENTIONS: For the in vitro test, the locomotive capsule was controlled to actively move from one side of a silicone tube to the other by a controller-operated automatic traveling program. The velocity was calculated by following a video recording. We performed ex vivo tests by using an extracted porcine colon in the same manner we performed the in vitro test. In in vivo experiments, the capsule was inserted into the rectum of a living pig under anesthesia, and was controlled to move automatically forward. After 8 consecutive trials, the velocity was calculated. MAIN OUTCOME MEASUREMENTS: Elapsed time, velocity, and mucosal damage. RESULTS: The locomotive capsule showed stable and active movement inside the lumen both in vitro and ex vivo. The velocity was 60 cm/min in the silicone tube, and 36.8 and 37.5 cm/min in the extracted porcine colon. In the in vivo experiments, the capsule stably moved forward inside the colon of a living pig without any serious complications. The mean velocity was 17 cm/min over 40 cm length. We noted pinpoint erythematous mucosal injuries in the colon. LIMITATION: Porcine model experiments, wired capsule endoscope. CONCLUSIONS: The novel paddling-based locomotive capsule endoscope performed fast and stable movement in a living pig colon with consistent velocity. Further investigation is necessary for practical use in humans.

Rapid exchange of oil-phase in microencapsulation chip to enhance cell viability.

This paper describes a microfluidic device for the microencapsulation of cells in alginate beads to enhance cell viability. The alginate droplet including cells was gelified with calcified oleic acid, using two-phase microfluidics. The on-chip gelation had generated monodisperse spherical alginate beads, which could not be readily obtained via conventional external gelation in a calcium chloride bath. However, the prolonged exposure of encapsulated cells to the toxic oil phase caused serious damage to the cells. Therefore, we proposed the formulation of a rapid oil-exchange chip which transforms the toxic oleic acid to harmless mineral oil. The flushing out of oleic acid after the gelation of alginate beads effected a dramatic increase in the viability of P19 embryonic carcinoma cells, up to 90%. The experimental results demonstrated that the cell viability was proportional to the flow rate of squeezing mineral oil.

Oil droplet generation in PDMS microchannel using an amphiphilic continuous phase.

This paper reports an amphiphilic solution can be used as a new continuous phase to generate double droplet emulsions (water/oil/IPA) with neither surface treatment nor surfactant in PDMS microfluidic chip. The affinity of various amphiphilic solutions in the microchannel was influenced by the polarity ratio and the size of molecules. The polarity ratio of isopropyl alcohol (IPA) was closest to that of the recovered PDMS surface and the chain length of IPA was also suitable for high affinity. IPA showed the highest affinity for the recovered PDMS and was selected as the continuous phase to form oil droplets in a PDMS microchannel. With this new continuous phase solution, IPA, we could successfully generate not only oil droplets but also double emulsions in the PDMS microfluidic chips.

Sensitivity enhancement of a dynamic mode microcantilever by stress inducer and mass inducer to detect PSA at low picogram levels.

We report two types of signal enhancement strategy derived from the origin of mechanical response, surface stress and mass, of the dynamic mode microcantilever for the detection of PSA at low picogram scales (low femtomolar concentration). The PSA detection at extremely low concentration levels is crucial to the early detection of relapses of prostate cancer after the radical prostatectomy and the detection of breast cancer in patient's serum. There is a clear need for the ultrasensitive detection of PSA via simple and rapid diagnostic tools. From the motives, to increase the sensitivity of the microcantilever, PSA polyclonal antibody (PSA pAb) as an additional surface stress inducer and PSA polyclonal antibody-conjugated silica nanoparticles (pAb-SiNPs) as a mass inducer have been applied to the PSA-captured microcantilevers. From two types of sandwich assay, we could confirm the sensitivity enhancement effects (2 approximately 4 times enhanced at the same concentrations) enough to detect PSA at low picogram levels (LOD of 1 pg/mL or below). Moreover, surface stress due to steric interactions between epitope-specific monoclonal antibodies was assessed to support a signal amplification strategy by stress inducer, and the reduction of signal enhancement due to stiffness increase by the mass inducer was studied to clarify the sensitivity enhancement of the microcantilever by mass inducer.

Rapid microfluidic separation of magnetic beads through dielectrophoresis and magnetophoresis.

We present the design and fabrication of a new microfluidic device in which the dielectrophoresis and magnetophoresis phenomena were used for the separation of the superparamagnetic microbeads of different sizes. By exploiting the fact that two different particles can exhibit different dielectrophoretic force-frequency spectra, we utilize this device to perform multiplex detection from a single sample solution. We found the transition frequency range for 1, 2.8, and 4.5 microm magnetic beads using our device. Bead-based analysis revealed that a high separation efficiency ( approximately 90%) could be obtained from a single sample solution containing both 4.5 and 2.8 microm beads. The average flow velocity of the beads was maintained at 9.8 mm/s, enabling fast analysis with a smaller amount of reagents. The magnetic field distribution on the beads and the bead flow at the channel cross section for different dielectrophoretic conditions was obtained using CFD-ACE(+) simulation. Issues relating to the fabrication and operation of the device are discussed in detail. Finally, we demonstrated the feasibility of parallel detection/trapping of different beads on the same chip. This separation approach offers the performance of multiplex analysis in lab-on-a-chip devices.

First clinical trial of the "MiRo" capsule endoscope by using a novel transmission technology: electric-field propagation.

BACKGROUND: We developed a capsule endoscope (CE), "MiRo," with the novel transmission technology of electric-field propagation. The technology uses the human body as a conductive medium for data transmission. Specifications of the prototype include the ability to receive real-time images; size, 10.8 x 24 mm; weight, 3.3 g; field of view, 150 degrees; resolution of power, 320 x 320 pixels; and transmittal speed, 2 frames per second. OBJECTIVE: To evaluate the clinical safety and diagnostic feasibility of the prototype MiRo, we conducted a multicenter clinical trial. DESIGN AND PATIENTS: All volunteers underwent baseline examinations, including EGD and electrocardiography for the screening of GI obstructive and cardiovascular diseases, before the trial. In the first 10 cases, 24-hour Holter monitoring was also performed. To evaluate the diagnostic feasibility, transmission rate of the captured images, inspection rate of the entire small bowel, and quality of transmitted images (graded as outstanding, excellent, good/average, below average, and poor) were analyzed. RESULTS: Of the 49 healthy volunteers, 45 were included in the trial, and 4 were excluded because of baseline abnormalities. No adverse effects were noted. All CEs were expelled within 2 days, and the entire small bowel could be explored in all cases. The transmission rates of the captured image in the stomach, small bowel, and colon were 99.5%, 99.6%, and 97.2%, respectively. The mean total duration of image transmission was 9 hours, 51 minutes, and the mean transit time of the entire small bowel was 4 hours, 33 minutes. Image quality was graded as good or better in 41 cases (91.1%). Details of the villi and vascular structures of the entire small bowel were clearly visualized in 31 cases (68.9%). CONCLUSIONS: MiRo is safe and effective for exploring the entire small bowel, with good image quality and real-time feasibility. This novel transmission technology may have applications beyond the field of capsule endoscopy.

Enhancement of membrane protein reconstitution on 3D free-standing lipid bilayer array in a microfluidic channel.

The bio-sensory organs of living creatures have evolved to have the best sensing performance. They have 3-dimensional protrusions that have large surface areas to accommodate a large number of membrane proteins such as ion channels and G-protein coupled receptors, resulting in high sensitivity and specificity to target molecules. From the perspective of mimicking this system, BLM, which has been used extensively as a platform for a single nanopore-based sensing systems, has some limitations, i.e., some residual solvent, low mechanical stability, small surface area for appropriate stability, and difficulty in high-throughput fabrication. Herein, to eliminate these limitations, a solvent-free, size-controllable, 3-dimensional free-standing lipid bilayer (3DFLB) structure array with high stability (∼130 h) and high density (∼300,000 cm-2) is proposed, and its structural advantages for efficient and rapid protein reconstitution, compared to BLM, is demonstrated by human 5-HT3A receptor assay as well as α-hemolysin assay. A continuous process of 3DFLB array fabrication, 5-HT3A reconstitution, and 5-HT detections in a microfluidic channel proves the applicability of the proposed structures as a highly-sensitive sensing platform mimicking bio-sensory organs.

A serial dilution microfluidic device using a ladder network generating logarithmic or linear concentrations.

In this paper, we propose a serial dilution microfluidic chip which is able to generate logarithmic or linear step-wise concentrations. These concentrations were generated via adjustments in the flow rate of two converging fluids at the channel junctions of the ladder network. The desired dilution ratios are almost independent of the flow rate or diffusion length of molecules, as the dilution device is influenced only by the ratio of volumetric flow rates. Given a set of necessary dilution ratios, whether linear or logarithmic, a serial dilution chip can be constructed via the modification of a microfluidic resistance network. The design principle was suggested and both the logarithmic and linear dilution chips were fabricated in order to verify their performance in accordance with the fluorescence intensity. The diluted concentrations of a fluorescein solution in the microfluidic device evidenced relatively high linearity, and the cytotoxicity test of MCF-7 breast cancer cells via the logarithmic dilution chip was generally consistent with the results generated with manual dilution.

Tightly Sealed 3D Lipid Structure Monolithically Generated on Transparent SU-8 Microwell Arrays for Biosensor Applications.

Artificial lipid membranes are excellent candidates for new biosensing platforms because their structures are similar to cell membranes and it is relatively easy to modify the composition of the membrane. The freestanding structure is preferable for this purpose because of the more manageable reconstitution of the membrane protein. Therefore, most of the lipid membranes for biosensing are based on two-dimensional structures that are fixed on a solid substrate (unlike floating liposomes) even though they have some disadvantages, such as low stability, small surface area, and potential retention of solvent in the membrane. In this paper, three-dimensional freestanding lipid bilayer (3D FLB) arrays were fabricated uniformly on SU-8 microwells without any toxic solvent. The 3D FLBs have better stability and larger surface area due to their cell-like structure. In order to improve the sealing characteristics of the 3D FLBs, the applied frequency of the ac field was controlled during the electroformation. The 3D FLBs were observed through transparent SU-8 microwell arrays using confocal microscopy and demonstrated perfect sealing until 5.5 days after the electroformation at more than 1 kHz. Also, the details of the sealing of a fixed 3D freestanding lipid structure were discussed for the first time. The unilamellarity and biofunctionality of the 3D FLBs were verified by a transport protein (α-hemolysin) assay.

Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase.

We report the nanomechanical microcantilevers operated in vibration modes (oscillation) with use of RNA aptamers as receptor molecules for label-free detection of hepatitis C virus (HCV) helicase. The nanomechanical detection principle is that the ligand-receptor binding on the microcantilever surface induces the dynamic response change of microcantilevers. We implemented the label-free detection of HCV helicase in the low concentration as much as 100 pg/ml from measuring the dynamic response change of microcantilevers. Moreover, from the recent studies showing that the ligand-receptor binding generates the surface stress on the microcantilever, we estimate the surface stress, on the oscillating microcantilevers, induced by ligand-receptor binding, i.e. binding between HCV helicase and RNA aptamer. In this article, it is suggested that the oscillating microcantilevers with use of RNA aptamers as receptor molecules may enable one to implement the sensitive label-free detection of very small amount of small-scale proteins.

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip.

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.

Multi-well chip for forming a uniform embryoid body in a tiny droplet with mouse embryonic stem cells.

A multi-well chip (MWC) is described by which mouse embryonic carcinoma (EC) stem cells form a comparatively more rapid and uniform embryoid body (EB) over the conventional hanging drop (HD) method. The newly developed MWC consists of an array of extruded through-holes, each of which holds a droplet of the cell suspension. The study found that the small curvature radius of the droplet in the MWC improved the EB formation rate of a hanging drop from 70% to 98%. Furthermore, the EBs formed by the MWC were uniformly round in shape regardless of the number of suspended cells ranging from 0.5 x 10(3) to 20 x 10(3). The ratio of beating colonies from the MWC was over 2-fold larger than that from HD. The experiments demonstrate that the MWC will be a valuable experimental tool for robust and reproducible EB-based differentiation of a defined number of ES cells.

How the capillary burst microvalve works.

The capillary burst microvalve offers an attractive means to regulate microliquid flow owing to its simple structure and operation process. However, there existed no rigorous theoretical work to elucidate how the valve works and consequently to predict the valve-bursting condition. Therefore, here we report the theoretical investigation of how the capillary burst valve can stop the advancing liquid meniscus and when it bursts. We confirm our theory with experiments using a centrifugal microfluidic valve system fabricated by soft lithography.

[Development of micro/nano devices for cancer diagnosis].

In order to rapidly detect and analyze the presence of a target ligand of interests in environmental or industrial fluids as well as biological samples, numerous test systems have been designed and developed, which have been based on the combination of a test reagent and absorbing paper or membrane. The main limitations of these conventional devices are the difficulty of quantitative or semi-quantitative detection and poor detection limit (around nanogram/ml range detection capability). In this paper, new approaches and research trends which use nano/MEMS (micro electro mechanical systems) technology is introduced, which has a concept of fast, precise, and massive diagnosis and analysis of target molecules using very small amount of samples.