Liquid Metal-Based Multifunctional Micropipette for 4D Single Cell Manipulation.

A novel manufacturing approach to fabricate liquid metal-based, multifunctional microcapillary pipettes able to provide electrodes with high electrical conductivity for high-frequency electrical stimulation and measurement is proposed. 4D single cell manipulation is realized by applying multifrequency, multiamplitude, and multiphase electrical signals to the microelectrodes near the pipette tip to create 3D dielectrophoretic trap and 1D electrorotation, simultaneously. Functions such as single cell trapping, patterning, transfer, and rotation are accomplished. Cell viability and multiday proliferation characterization has confirmed the biocompatibility of this approach. This is a simple, low-cost, and fast fabrication process that requires no cleanroom and photolithography step to manufacture 3D microelectrodes and microchannels for easy access to a wide user base for broad applications

Photothermal Intracellular Delivery Platforms

Intracellular delivery of diverse biomolecules, such as protein, nucleic acids, nano-devices, has been of great importance and interest in biomedical fields like cancer therapy, gene editing and intracellular environment probing. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to meet the emerging requirements of the cutting-edge research. In this thesis, I focused on three major hurdles in the current intracellular delivery, which are suspension cell delivery, complexity of incorporating nanotechnology, and large cargo delivery. Photothermal mechanism is the underlying physics throughout all the work to be introduced here. It utilizes the light energy and transforms it into thermal energy and then into mechanical energy, serving for different functions in delivery. Nanosecond laser was chosen as the original power tool due to its high energy density, remote operation capability, and selective absorption. The combination of laser and micro/nano structure has been extensively explored to develop various delivery capabilities.The first problem tackled in this thesis is to deliver materials into suspension cells with high efficiency, viability, and throughput. Suspension cells, especially lymphocytes, which represent 25-30% of immune cells, are of great interest in cancer immunotherapies and known as hard-to-transfect cells. To achieve effective delivery, the microwell structure with metallic sharp tips were designed to provide both cell anchoring and controllable membrane disruption on each cell. Suspension cells self- position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100 000 cells delivered per minute. The bacterial enzyme β-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.The second problem raised from the notice of the huge potential of nanostructures, especially combined with photothermal mechanism, in contrast with their current limited applications in this field. Nanostructures, such as nanoneedle array, have been adopted in the intracellular delivery field due to its unique scale advantages, including minimal damage of the cell membrane and large cargo loading capacity from high surface-to-volume ratio. However, nanotechnologies have suffered from its complexity of high-precision fabrication and are limited to small area. Thus, we demonstrate the fabrication of large-area plasmonic gold (Au) nanodisk arrays that enable photothermal intracellular delivery of biomolecular cargo at high efficiency. The Au nanodisks (350�nm in diameter) were fabricated using chemical lift-off lithography (CLL), a high-throughput and low-cost for nanoscale chemical patterning. This technique is applied to produce Au nanostructures on a variety of substrates (e.g., silicon, glass, and plastic), which facilitate in situ intracellular delivery in laboratory cell culture environments, enabling integration with existing medical devices. Nanosecond laser pulses were used to excite the plasmonic nanostructures, thereby generating transient pores at the outer membranes of targeted cells that enable the delivery of biomolecules via diffusion. We studied nanodisks of various sizes and found that an increase in delivery efficiency correlated with decreasing disk radius, which we attribute to higher density of pores per cell. Delivery efficiencies of >98% were achieved with 1-μm Au plasmonic disk arrays, using the cell impermeable dye Calcein (0.6�kDa) as a model payload, while maintaining cell viabilities at >98%. The highly efficient intracellular delivery approach demonstrated in this work will facilitate translational studies targeting molecular screening and drug testing that bridge laboratory and clinical investigations.Despite that major problems were nicely solved in the prior two projects, an apparent drawback appears, as the delivery efficiency drops significantly when cargo size increases. Photothermal energy was adopted, in both projects, to generate bubble explosion near the adjacent cell membrane so as to disrupt the membrane. Cargoes had to passively diffuse into the membrane, which posed the hardship to large cargoes. Thus, in the third project, the integration of membrane disruption and active pumping was studied to facilitate large cargo delivery with precise control and large-area uniformity. We utilized the high initial pressure of the laser-induced bubbles as the pump source for high-speed fluidic jet, which cuts the cell membrane and delivers cargos into the cytosol and nucleus. The fabrication processes of the devices are designed to be conventional and simple with large-area uniformity. The penetration was demonstrated by injecting 140 nm polystyrene beads into Agarose hydrogel which was prepared to have similar Young’s Modulus as cells. With delicate device designs, we achieved penetration depths from tens of microns to a hundred microns, indicating the capability of three-dimensional tissue delivery and epidermal in vivo delivery, besides intracellular delivery into single layer of cells

Photothermal Intracellular Delivery Platforms

Intracellular delivery of diverse biomolecules, such as protein, nucleic acids, nano-devices, has been of great importance and interest in biomedical fields like cancer therapy, gene editing and intracellular environment probing. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to meet the emerging requirements of the cutting-edge research. In this thesis, I focused on three major hurdles in the current intracellular delivery, which are suspension cell delivery, complexity of incorporating nanotechnology, and large cargo delivery. Photothermal mechanism is the underlying physics throughout all the work to be introduced here. It utilizes the light energy and transforms it into thermal energy and then into mechanical energy, serving for different functions in delivery. Nanosecond laser was chosen as the original power tool due to its high energy density, remote operation capability, and selective absorption. The combination of laser and micro/nano structure has been extensively explored to develop various delivery capabilities.The first problem tackled in this thesis is to deliver materials into suspension cells with high efficiency, viability, and throughput. Suspension cells, especially lymphocytes, which represent 25-30% of immune cells, are of great interest in cancer immunotherapies and known as hard-to-transfect cells. To achieve effective delivery, the microwell structure with metallic sharp tips were designed to provide both cell anchoring and controllable membrane disruption on each cell. Suspension cells self- position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100 000 cells delivered per minute. The bacterial enzyme β-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.The second problem raised from the notice of the huge potential of nanostructures, especially combined with photothermal mechanism, in contrast with their current limited applications in this field. Nanostructures, such as nanoneedle array, have been adopted in the intracellular delivery field due to its unique scale advantages, including minimal damage of the cell membrane and large cargo loading capacity from high surface-to-volume ratio. However, nanotechnologies have suffered from its complexity of high-precision fabrication and are limited to small area. Thus, we demonstrate the fabrication of large-area plasmonic gold (Au) nanodisk arrays that enable photothermal intracellular delivery of biomolecular cargo at high efficiency. The Au nanodisks (350�nm in diameter) were fabricated using chemical lift-off lithography (CLL), a high-throughput and low-cost for nanoscale chemical patterning. This technique is applied to produce Au nanostructures on a variety of substrates (e.g., silicon, glass, and plastic), which facilitate in situ intracellular delivery in laboratory cell culture environments, enabling integration with existing medical devices. Nanosecond laser pulses were used to excite the plasmonic nanostructures, thereby generating transient pores at the outer membranes of targeted cells that enable the delivery of biomolecules via diffusion. We studied nanodisks of various sizes and found that an increase in delivery efficiency correlated with decreasing disk radius, which we attribute to higher density of pores per cell. Delivery efficiencies of >98% were achieved with 1-μm Au plasmonic disk arrays, using the cell impermeable dye Calcein (0.6�kDa) as a model payload, while maintaining cell viabilities at >98%. The highly efficient intracellular delivery approach demonstrated in this work will facilitate translational studies targeting molecular screening and drug testing that bridge laboratory and clinical investigations.Despite that major problems were nicely solved in the prior two projects, an apparent drawback appears, as the delivery efficiency drops significantly when cargo size increases. Photothermal energy was adopted, in both projects, to generate bubble explosion near the adjacent cell membrane so as to disrupt the membrane. Cargoes had to passively diffuse into the membrane, which posed the hardship to large cargoes. Thus, in the third project, the integration of membrane disruption and active pumping was studied to facilitate large cargo delivery with precise control and large-area uniformity. We utilized the high initial pressure of the laser-induced bubbles as the pump source for high-speed fluidic jet, which cuts the cell membrane and delivers cargos into the cytosol and nucleus. The fabrication processes of the devices are designed to be conventional and simple with large-area uniformity. The penetration was demonstrated by injecting 140 nm polystyrene beads into Agarose hydrogel which was prepared to have similar Young’s Modulus as cells. With delicate device designs, we achieved penetration depths from tens of microns to a hundred microns, indicating the capability of three-dimensional tissue delivery and epidermal in vivo delivery, besides intracellular delivery into single layer of cells

A Knowledge-based Recommendation System for Time Series Classification

Time series data sets reflect the state and extent of things as they change over time. Information extraction based on such data plays an important role in many fields. The time series classification is a typical supervised learning problem, which is applied in speech recognition, image processing and so on. However, because the attributes of time series data don't make sense and the feature dimensions are particularly large, people can't treat them as general machine learning classification problems. Currently, many different time series classification problems have been proposed. But how to choose and use these methods is still a huge problem for non-computer professional researchers. This article uses the ontology technology to build a recommendation system that contains the details and features of such algorithms. When the users input the characteristics of the data and the task requirements, they can get reasonable suggestions and a description of the workflow of the algorithm. Such a system saves the user a lot of analysis and comparison time. It also makes such problems easier to understand

An Ontology of Machine Learning Algorithms for Human Activity Data Processing

Machine learning algorithms are the main tools in the field of data analysis. However, extracting knowledge from data sets originating in real life requires complex data processing. Obtaining the available tidy data sets and selecting the appropriate analysis algorithm are important issues for data analysts. Because of the complexity of the dataset and the diversity of the algorithms the researchers take too much time in selecting and comparing these algorithms. Human Activity Recognition is a typical example in Internet of Things. Its principle is to identify human behavior by analyzing the coordinate data from the sensors on the human body so that we can achieve remote monitoring. A precise Human Activity Recognition application can serve as a real-time monitoring of the elderly or vulnerable behavior. However, due to the unpredictability of human behavior, these sensor data require relatively complex processing. Therefore, we propose an ontology-based algorithm recommendation system. It consists of several parts: algorithm pool, data features, model features, and mathematical theory. The framework provides data researchers with reasonable solutions based on the characteristics of the data set and the task requirements. Especially for the Internet of Things data such as Human Activity Recognition data set, its recommendations can save users much time for analysis and comparison

Urban intelligent assistant on the example of the escalator passenger safety management at the subway stations

Abstract Intelligent assistants often struggle with the complexity of spatiotemporal models used for understanding objects and environments. The construction and usage of such models demand significant computational resources. This article introduces a novel multilevel spatiotemporal model and a computationally efficient construction method. To facilitate model construction on different levels, we employ a meta-mining technique. Furthermore, the proposed model is specifically designed to excel in foggy environments. As a practical application, we develop an intelligent assistant focused on enhancing subway passenger safety. We present case examples involving jammed objects, such as shoes, in escalator combs. Our results demonstrate the effectiveness of the proposed model and method. Specifically, the accuracy of breakdown detection has improved by 10% compared to existing information systems used in subways. Moreover, the time required to build a spatiotemporal model is reduced by 2.3 times, further highlighting the efficiency of our approach. Our research offers a promising solution for intelligent assistants dealing with complex spatiotemporal modeling, with practical applications in ensuring subway passenger safety

Graphene woven fabric as high-resolution sensing element of contact-lens tonometer

In our work, the graphene woven fabrics (GWFs) are investigated as the sensing element of the contact-lens tonometer, which enables precisely monitoring IOP all the daytime. The current-voltage relationship of the device was tested under voltage sweep and the relationships between resistance change and deformation were calculated. Eight devices with GWF in different sizes and CVD conditions were fabricated and the relationship between the current changes of each device and effective IOP increasing, when keeping the voltage constant, was obtained. Combining the highly strain sensing sensitivity and transparency, the contact-lens tonometers with GWF as high-resolution sensing element have a promising prospective. ? 2014 IEEE.EICPCI-S(ISTP)

Flexible and Implantable Polyimide Aptamer-Field-Effect Transistor Biosensors

Monitoring neurochemical signaling across time scales is critical to understanding how brains encode and store information. Flexible (vs stiff) devices have been shown to improve in vivo monitoring, particularly over longer times, by reducing tissue damage and immunological responses. Here, we report our initial steps toward developing flexible and implantable neuroprobes with aptamer-field-effect transistor (FET) biosensors for neurotransmitter monitoring. A high-throughput process was developed to fabricate thin, flexible polyimide probes using microelectromechanical-system (MEMS) technologies, where 150 flexible probes were fabricated on each 4 in. Si wafer. Probes were 150 μm wide and 7 μm thick with two FETs per tip. The bending stiffness was 1.2 × 10-11 N·m2. Semiconductor thin films (3 nm In2O3) were functionalized with DNA aptamers for target recognition, which produces aptamer conformational rearrangements detected via changes in FET conductance. Flexible aptamer-FET neuroprobes detected serotonin at femtomolar concentrations in high-ionic strength artificial cerebrospinal fluid. A straightforward implantation process was developed, where microfabricated Si carrier devices assisted with implantation such that flexible neuroprobes detected physiological relevant serotonin in a tissue-hydrogel brain mimic

Photothermal Intracellular Delivery Using Gold Nanodisk Arrays

Local heating using pulsed laser-induced photothermal effects on plasmonic nanostructured substrates can be used for intracellular delivery applications. However, the fabrication of plasmonic nanostructured interfaces is hampered by complex nanomanufacturing schemes. Here, we demonstrate the fabrication of large-area plasmonic gold (Au) nanodisk arrays that enable photothermal intracellular delivery of biomolecular cargo at high efficiency. The Au nanodisks (350 nm in diameter) were fabricated using chemical lift-off lithography (CLL). Nanosecond laser pulses were used to excite the plasmonic nanostructures, thereby generating transient pores at the outer membranes of targeted cells that enable the delivery of biomolecules via diffusion. Delivery efficiencies of >98% were achieved using the cell impermeable dye calcein (0.6 kDa) as a model payload, while maintaining cell viabilities at >98%. The highly efficient intracellular delivery approach demonstrated in this work will facilitate translational studies targeting molecular screening and drug testing that bridge laboratory and clinical investigations

Narrower Nanoribbon Biosensors Fabricated by Chemical Lift-off Lithography Show Higher Sensitivity

Wafer-scale nanoribbon field-effect transistor (FET) biosensors fabricated by straightforward top-down processes are demonstrated as sensing platforms with high sensitivity to a broad range of biological targets. Nanoribbons with 350 nm widths (700 nm pitch) were patterned by chemical lift-off lithography using high-throughput, low-cost commercial digital versatile disks (DVDs) as masters. Lift-off lithography was also used to pattern ribbons with 2 μm or 20 μm widths (4 or 40 μm pitches, respectively) using masters fabricated by photolithography. For all widths, highly aligned, quasi-one-dimensional (1D) ribbon arrays were produced over centimeter length scales by sputtering to deposit 20 nm thin-film In2O3 as the semiconductor. Compared to 20 μm wide microribbons, FET sensors with 350 nm wide nanoribbons showed higher sensitivity to pH over a broad range (pH 5 to 10). Nanoribbon FETs functionalized with a serotonin-specific aptamer demonstrated larger responses to equimolar serotonin in high ionic strength buffer than those of microribbon FETs. Field-effect transistors with 350 nm wide nanoribbons functionalized with single-stranded DNA showed greater sensitivity to detecting complementary DNA hybridization vs 20 μm microribbon FETs. In all, we illustrate facile fabrication and use of large-area, uniform In2O3 nanoribbon FETs for ion, small-molecule, and oligonucleotide detection where higher surface-to-volume ratios translate to better detection sensitivities