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.

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.

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.

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.

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.

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.

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone.

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

Selective and vertical microfabrication of lipid tubule arrays on glass substrates using template-guided gentle hydration.

Generally, asymmetric tubular lipid structures have been formed under the specific condition of gentle hydration or by using hydrodynamic and/or electrical elongation of vesicular lipid structures. Small-size lipid tubes are, however, very difficult to allocate or align in the vertical direction on the specific site of the substrate and, therefore, the ability to produce them selectively and in large quantities as an array form is limited. Herein, we propose an easy and novel method to fabricate selective and vertical lipid tube arrays using template-guided gentle hydration of dried lipid films without any external forces. A lipid solution was drop-dispensed onto a porous membrane and dried to form a lipid film. Then, the lipid-coated porous membrane was transferred to a glass substrate by using a UV-cured polymer layer to achieve tight bonding. Upon swelling with an appropriate buffer, expansion forces due to osmotic pressure during the gentle hydration process were highly constrained to confined pores, thereby resulting in the nucleation of tube-like lipid structures through the pores. Interestingly, according to the aspect ratio of pores (ARpore, pore length/pore diameter), different shapes of lipid structures, including vesicular, oval, and tube-like, were generated, which indicates the importance of the ARpore, as well as the pore diameter, during fabrication of tubular lipid structures. Also, this approach was easily modified with 1% chitosan to enhance the stability of the lipid tubes (>30 min in life time), by lipid coating twice and by using unsaturated lipids to increase tube length (>30 µm in length). Therefore, in the future, the simple but robust template-guided gentle hydration method will be a useful tool for fabricating addressable and engineered lipid tube arrays as a sensory unit.

Droplet-based magnetic bead immunoassay using microchannel-connected multiwell plates (µCHAMPs) for the detection of amyloid beta oligomers.

Multiwell plates are regularly used in analytical research and clinical diagnosis but often require laborious washing steps and large sample or reagent volumes (typically, 100 µL per well). To overcome such drawbacks in the conventional multiwell plate, we present a novel microchannel-connected multiwell plate (µCHAMP) that can be used for automated disease biomarker detection in a small sample volume by performing droplet-based magnetic bead immunoassay inside the plate. In this µCHAMP-based immunoassay platform, small volumes (30-50 µL) of aqueous-phase working droplets are stably confined within each well by the simple microchannel structure (200-300 µm in height and 0.5-1 mm in width), and magnetic beads are exclusively transported into an adjacent droplet through the oil-filled microchannels assisted by a magnet array aligned beneath and controlled by a XY-motorized stage. Using this µCHAMP-based platform, we were able to perform parallel detection of synthetic amyloid beta (Aß) oligomers as a model analyte for the early diagnosis of Alzheimer's disease (AD). This platform easily simplified the laborious and consumptive immunoassay procedure by achieving automated parallel immunoassay (32 assays per operation in 3-well connected 96-well plate) within 1 hour and at low sample consumption (less than 10 µL per assay) with no cumbersome manual washing step. Moreover, it could detect synthetic Aß oligomers even below 10 pg mL(-1) concentration with a calculated detection limit of ∼3 pg mL(-1). Therefore, the µCHAMP and droplet-based magnetic bead immunoassay, with the combination of XY-motorized magnet array, would be a useful platform in the diagnosis of human disease, including AD, which requires low consumption of the patient's body fluid sample and automation of the entire immunoassay procedure for high processing capacity.

Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles.

Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 µm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs).

Age-dependent inverse correlations in CSF and plasma amyloid-ß(1-42) concentrations prior to amyloid plaque deposition in the brain of 3xTg-AD mice.

Amyloid-ß (Aß) plays a critical role as a biomarker in Alzheimer's disease (AD) diagnosis. In addition to its diagnostic potential in the brain, recent studies have suggested that changes of Aß level in the plasma can possibly indicate AD onset. In this study, we found that plasma Aß(1-42) concentration increases with age, while the concentration of Aß(1-42) in the cerebrospinal fluid (CSF) decreases in APPswe, PS1M146V and TauP301L transgenic (3xTg-AD) mice, if measurements were made before formation of ThS-positive plaques in the brain. Our data suggests that there is an inverse correlations between the plasma and CSF Aß(1-42) levels until plaques form in transgenic mice's brains and that the plasma Aß concentration possesses the diagnostic potential as a biomarker for diagnosis of early AD stages.

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.

Magnetic bead droplet immunoassay of oligomer amyloid ß for the diagnosis of Alzheimer's disease using micro-pillars to enhance the stability of the oil-water interface.

Despite scientific progress in the study of Alzheimer's disease (AD), it is still challenging to develop a robust and sensitive methodology for the early diagnosis of AD due to the lack of a decisive biomarker in blood. Recent reports on the oligomer amyloid ß (Aß) as a biomarker demonstrated its possibility for identifying early onset of AD in patients, but its low concentration in blood requires highly reliable detection techniques. To overcome the low reliability and labor-intensive procedures of conventional enzyme-linked immunosorbent assay (ELISA), we present a magnetic bead-droplet immunoassay platform for simple and highly sensitive detection of oligomer Aß for the diagnosis of AD. This microchip consists of chambers that contain water-based reagents or oil for consecutive assay procedures, and there are arrays of micro-pillars fabricated between the two adjacent chambers to form robust water-oil interfaces. With the aid of these micro-pillars, magnetic beads can stably pass through each chamber by linearly actuating a magnet along the microchip. The robust water-oil interface and simple procedures of the assay make it possible to obtain reliable results from this microchip. The intensity of the fluorescence at the read-out chamber increased quantitatively and linearly, depending on the amount of serially-diluted standard Aß solution. The results of the assay indicated that the limit of detection was about 10 pg/mL even though it was done with manual manipulation of the magnet. This platform simplified the complicated ELISA procedure and achieved high sensitivity that was no lower than that of the conventional magnetic bead immunoassay. The magnetic bead-droplet platform reduced the assay time to 45 min, and it also reduced the amount of antibody usage in a single diagnosis significantly (10-30 ng of antibody per single assay). Consequently, this microfluidic chip has strong potential as a feasible system for use in the diagnosis of AD with a fast and easy immunoassay process, since the suggested platform can be automated with ease for point-of-care testing as well as high-throughput diagnostic equipment.

Correlations of amyloid-ß concentrations between CSF and plasma in acute Alzheimer mouse model.

Amyloid-ß (Aß) is one of the few neuropathological biomarkers associated with transporters of the blood-brain barrier (BBB). Despite the well-characterized clinical indication of decreasing Aß levels in the cerebrospinal fluid (CSF) during the development of Alzheimer's disease (AD), the link between the alternation of Aß level in the blood and the progress of the disorder is still controversial. Here, we report a direct correlation of Aß(1-42) levels between CSF and plasma in AD mouse model. We injected monomeric Aß(1-42) directly into the intracerebroventricular (ICV) region of normal adult mouse brains to induce AD-like phenotypes. Using sandwich enzyme-linked immunosorbent assays, we observed proportional elevation of Aß(1-42) levels in both CSF and plasma in a dose-dependent manner. Our findings that plasma Aß(1-42) reflects the condition of CSF Aß(1-42) warrant further investigation as a biomarker for the blood diagnosis of AD.

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.

Protocol for the use of a silica nanoparticle-enhanced microcantilever sensor-based method to detect HBV at femtomolar concentrations.

DNA sensors that are capable of detecting specific DNA sequences in a bio-sample have recently been highlighted as a powerful and sensitive approach to detect infectious diseases caused by pathogens such as viruses and bacteria. Generally, DNA samples extracted from biological fluids are amplified by PCR prior to analysis by DNA sensors or directly analyzed by DNA sensors equipped with a signal amplification process. Nanoparticles have recently been used to amplify the sensor signal and have been shown to play an important role in improving the sensitivity of mechanical resonating sensors. This is because the weight of the nanoparticle can increase the change in the resonance response of the mechanical sensor since this signal change is closely related to mass. Here, we introduce an experimental method to detect HBV at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever resonating sensor. This method includes the preparation of detection probe-conjugated silica nanoparticles, immobilization of capture probe on the microcantilever sensor and sandwich type detection of HBV DNA.

Peptide receptor-based selective dinitrotoluene detection using a microcantilever sensor.

We reported that peptide could be utilized as receptor molecule in the gas phase for application in micro/nano sensors by using a specific peptide that recognizes 2,4-dinitrotoluene at room temperature and in an atmospheric environment and measuring changes in the resonant frequency of the peptide immobilized microcantilevers. By using these peptides as receptors on a microcantilever sensor, we were able to experimentally detect 2,4-dinitrotoluene (DNT) vapor at concentrations as low as parts per billion (ppb) in the gas phase. While resonant frequency changes after binding between 2,4-DNT and the specific peptide receptor that was immobilized on microcantilevers were observed, the resonant frequency of DNT nonspecific peptide immobilized microcantilever did not change when exposed to 2,4-DNT vapor. The limit of detection (LOD) was calculated to be 431 ppt of limit of detection is numerically expected by experimental based on an equation that describes the relationship between the noise-equivalent analyte concentration. These results indicate that the peptide receptors hold great promise for use in the development of an artificial olfactory system and electronic nose based on micro/nanotechnology for monitoring various chemical vapors in the gas phase such as explosive mixtures of chemicals and/or volatile organic compounds.

The Microscopic Origin of Residual Stress for Flat Self-Actuating Piezoelectric Cantilevers.

In this study, flat piezoelectric microcantilevers were fabricated under low-stress Pb(Zr0.52Ti0.48)O3 (PZT) film conditions. They were analyzed using the Raman spectrum and wafer curvature methods. Based on the residual stress analysis, we found that a thickness of 1 µm was critical, since stress relaxation starts to occur at greater thicknesses, due to surface roughening. The (111) preferred orientation started to decrease when the film thickness was greater than 1 µm. The d33 value was closely related to the stress relaxation associated with the preferred orientation changes. We examined the harmonic response at different PZT cantilever lengths and obtained a 9.4-µm tip displacement at 3 Vp-p at 1 kHz. These analyses can provide a platform for the reliable operation of piezoelectric microdevices, potentially nanodevice when one needs to have simultaneous control of the residual stress and the piezoelectric properties.

Analysis of DNA hybridization regarding the conformation of molecular layer with piezoelectric microcantilevers.

Lead Zirconate Titanate (PZT)-embedded microcantilevers were fabricated with dimensions of 30 × 90 × 3 µm(3) (width × length × thickness). A thicker PZT layer improved the actuation and enabled long-term data acquisition in common aqueous buffers with a frequency resolution of 20 Hz. A quantitative assay was conducted in the range of 1-20 µM and the resonant frequency was found to increase with the concentration of target DNAs and the probe DNAs were almost saturated at 20 µM. Back-filling with ethyleneglycol-modified alkanethiol was shown to facilitate the hybridization efficiency and stabilize the surface reaction, resulting in a signal enhancement of 40%. We report for the first time how secondary structures in oligonucleotide monolayer change the surface property of a dynamic mode microcantilever and subsequently affect its oscillating behavior. Using fabricated microcantilevers, the real time changes in resonant frequency upon hybridization were measured by utilizing different probe and target sets. The results revealed that the microcantilevers experienced a resonant frequency upshift during the hybridization with complementary DNAs if a dimer structure was present between DNA probes. A resonant frequency downshift was observed for DNA probes that did not contain any complex secondary structures. In addition, the results demonstrate the potential of using these microcantilevers to extract structural information of oligonucleotides.