Arizona State University

University of Washington

Gene Tools, LLC

Deirdre Meldrum

Laimonas Kelbauskas

Yanqing Tian

Fengyu Su

Jeff Houkal

Roger H Johnson

Liqiang Zhang

Hong Wang

Rhett L Martineau

Clifford Anderson

Shih-Hui Joseph Chao

Weimin Gao

Weiwen Zhang

Jakrey Myers

Kristen Lee

Rishabh Shetty

Xiangxing Kong

Shufang Ci

Bo Wang

Iniyan Soundappa Elango

Jacob Messner

Dean Smith

Honor Glenn

Cody Youngbull

David Nielsen

Kimberly Bussey

Benjamin Ueberroth

Brian Ashcroft

Ronald Seubert

Sean Buizer

Ray Kraft

Shih-Hui (Joseph) Chao

Neal A Friedman

Ganquan Song

Babak Amirparviz

Blake Hannaford

Ehsan Saeedi

Donald B Snow

Andrew Hatch

Peter Jan Wiktor

Samuel Kim

David Wayne Richardson

Thai Tran

Mark Holl

Mark Richards

Andrew Shabilla

Pimwadee Limsirichai

Samantha Chan

Wacey Teller

Xianfeng Zhou

Hongguang Lu

Meryl Rodrigues

Yuguang Jin

Maynard V Olson

Vivek Nandakumar

Roger Johnson

A thixotropic and biocompatible gel, formed by combining of 4-arm polyethylene glycol with fumed silica (PEG-silica). The thixotropicity of the gel is affected by the ratio of PEG to silica. To study the gel's biocompatibility, the cell suspension in PBS or another medium was added to the PEG-silica gel via mixing to uniformly disperse the cells in the gel and then placed in an incubator before performing fluorescence live-dead assays. Duration of cell viability (cell life time) in gels was measured to be up to several days, depending on gel composition. Due to their optical and fluidic properties the gels are compatible with live cell imaging including 3D computed tomography and offer a means for moving the cells in a highly controllable manner by applying and removing pressure on the gels.

Thixotropic biocompatible gel for live cell observation in cell computed tomography

A multi-layer sealing structure for sealing a microwell array defined in or on a substrate includes at least one front compliant layer, a back compliant layer, and a flexural layer arranged between the at least one front compliant layer and the back compliant layer, wherein the at least one front compliant layer is closer than the back compliant layer to microwells of the microwell array. One or more front compliant layers may be optically reflective and/or may embody a sensor layer. The back compliant layer may include an adhesive or various types of rubber, and the flexural layer may include a polymeric material or metal. A multi-layer sealing structure may be separated from a microwell array by peeling. A multi-layer sealing structure allows local disruption of sealing where particle contaminants are present without compromising the sealing performance of an entire microwell array, and without requiring a large sealing force.

Layered structure for improved sealing of microwell arrays

A microfluidic device includes a first substrate including at least one microfluidic channel and a plurality of microwells, as well as a cooperating second substrate defining multiple split-walled cell trap structures that are registered with and disposed within the plurality of microwells. A method for performing an assay includes flowing cells and a first aqueous medium into a plurality of microwells of a microfluidic device, wherein each microwell includes a cell trap structure configured to trap at least one cell. The method further comprises flowing a nonpolar fluid with low permeability for analytes of interest through a microfluidic channel to flush a portion of the first aqueous medium from the microfluidic channel while retaining another portion of the first aqueous medium and at least one cell within each microwell. Surface tension at a non-polar/polar medium interface prevents molecule exchange between interior and exterior portions of microwells.

Device and method for high-throughput multiparameter measurements in one or more live and fixed cells

The invention provides methods for detecting specific nucleic acids using a loop-mediated isothermal amplification (LAMP) reaction and providing a digital readout of the results. One method teaches separating target specificity from indicator detection in a two-stage multiple-threshold array (MTA). During the first stage amplicons incorporating both target sequence and an indicator sequence are synthesized. During the second stage of the reaction, the indicator sequence is targeted and amplified to produce visual results that may be digitalized. Another method teaches a competitive MTA, in which a reference sequence is used to compete against target sequence during amplification producing threshold responses, thus enabling digital readout. Using either method, quantitation is achieved without the need for continuous monitoring of the reaction and the end-point readout is amenable to visual inspection. Methods of the invention are especially useful in circumstances where there is lower or intermittent power supply.

Methods for digital readout quantification of nucleic acids

Methods for detecting single nucleotide polymorphisms in nucleotide sequences using LAMP reactions are provided herein. Generally, two sets of LAMP primers, a wild-type primer that matches expected DNA sequences and an SNP primer that matches the expected SNP DNA are provided. One method includes providing the wild-type primer and the SNP primer in separate wells of a multi-well microfluidic array device, adding the sample nucleotide sequence into the wells seeded with the primers, and initiating LAMP reactions within the wells. The method includes observing the reaction differential between the primers and determining the status of the DNA with regard to that particular SNP. A second method includes providing the primers with tags in a mixture, adding the sample nucleotide sequence to the mixture, and initiating LAMP reactions. The method includes providing a different visual indication when the wild-type primer reacts with the sample nucleotide sequence versus when the SNP primer reacts with the sample nucleotide sequence, and determining the status of the DNA with regard to that particular SNP.

Method for detecting nucleotide polymorphisms

Systems and methods of using the same for functional fluorescence imaging of live cells in suspension with isotropic three dimensional (3D) diffraction-limited spatial resolution are disclosed. The method-live cell computed tomography (LCCT)-involves the acquisition of a series of two dimensional (2D) pseudo-projection images from different perspectives of the cell that rotates around an axis that is perpendicular to the optical axis of the imaging system. The volumetric image of the cell is then tomographically reconstructed.

Live-cell computed tomography

Systems and methods of using the same for functional fluorescence imaging of live cells in suspension with isotropic three dimensional (3D) diffraction-limited spatial resolution are disclosed. The method-live cell computed tomography (LCCT)-in-volves the acquisition of a series of two dimensional (2D) pseudo-projection images from different perspectives of the cell that rotates around an axis that is perpendicular to the optical axis of the imaging system. The volumetric image of the cell is then tomographically reconstructed.

Fluorescent pH sensors are provided. The fluorescent pH sensor comprises a copolymer for sensing pH and a polymerized form of N-(2-hydroxypropyl)methacrylamide (HPMA) or 2-hydroxyethyl methacrylate (HEMA). The probe for sensing pH has formula (I): wherein Rand Rare as defined herein. The fluorescent pH sensors may be used in determining the pH of a sample and detecting extracellular pH in a sample. Methods for preparing the fluorescent pH sensors and the probe for sensing pH are also provided.

Fluorescent pH sensors and methods of preparing them

Methods for seeding live cells onto spatially defined regions of a substrate including multiple features (e.g., microwells or other microenvironments) utilize a stencil embodied in a hole-defining sacrificial film. A sacrificial film devoid of holes may be applied over features of a substrate, and a hole generating mechanism (e.g., hot needle or laser) aligned with features may be used to define holes in the film. Alternatively, holes may be predefined in a sacrificial film to form a stencil, and the stencil may be assembled to the substrate with the holes registered with features thereof. Thereafter, cells are seeded through holes in the film. Seeded cells are subject to incubation, further processing, and/or performance of one or more assays, and the hole-defining sacrificial film (stencil) may be removed.

Live-cell seeding method for microarrays

An integrated technological platform enabling real-time quantitative multiparameter metabolic profiling, utilizing either or both of extra and intracellular optical sensors, individually or simultaneously. A scalable embedded micropocket array structure, generally fabricated on fused silica substrates, facilitates the integration of multiple, spatially separated extracellular sensors for multiparameter analysis in a container formed with the use of an activation mechanism forming part of a device configured to hold the container during the measurements. The creation of hermetically sealed microchambers is carried out with a pneumatically and/or mechanically and/or electromechanically driven device that is 'floating' within the holding device and that is optionally equipped with a vacuum/suction mechanism to hold a component of the container at its surface.

Integrated platform for characterization of single cells or small cell clusters

A device for high-throughput multi-parameter functional profiling of the same cells in multicellular settings and in isolation is provided. In certain aspects, an integrated microfluidic device for multi-parameter metabolic and other phenotypic profiling of live biological cells is useable with: 1) multicellular clusters or small biopsy tissue samples, 2) cultures of the constituent cells obtained after cluster/tissue dissociation, and 3) the same constituent single cells in isolation. The approach enables study of the effects of multicellular complexity, such as in response to treatment, pathogens, stress, or other factors concerning disease origination and progression. Measurements may be performed on single cells or multicellular populations or tissues in the same assay at the same time.

Device for high-throughput multi-parameter functional profiling of the same cells in multicellular settings and in isolation

Mitochondria-targeting potassium sensors and method(s) for making such sensors. The sensor shows a response to potassium and displays a 130-fold dynamic range of fluorescence intensity and high brightness. The sensors response to potassium concentrations was demonstrated to be unaffected by cellular pH value and/or concentrations of other ions. The sensors can be used for monitoring the mitochondrial potassium efflux/influx.

Mitochondria-targeting fluorescent potassium+ sensor and method of making the same

The invention relates to microfluidic devices and array disks for 'one-pot' isolated chemical reactions. The array disks comprise a plurality of sectors in which each sector comprises one microfluidic device. The microfluidic devices comprise a fluid delivery channel and an array of wells wherein the fluid delivery channel delivery fluid into the wells in a serpentine arrangement. In some embodiments, the fluid delivery channel is directly above the array of wells. In other embodiments, the fluid delivery channel is offset from the array of wells so that side channels branching from the fluid delivery channel delivers fluid into the wells. The well of the microfluidic device comprises a gas-permeable membrane that forms the floor, well, or at least a portion of the floor or wall of the well. In preferred embodiments, the well is cylindrical.

Microfluidic device and array disk

A microfluidic device useable for performing live cell computed tomography imaging is fabricated with a cover portion including a first wafer with at least one metal patterned thereon, a base portion including a second wafer with at least one metal patterned thereon and negative photoresist defining recesses therein, and a diffusive bonding layer including a negative photoresist arranged to join the cover portion and the base portion. A composition useful in live cell computer topography includes a long-chain polysaccharide at a concentration of from about 0.01% to about 10.0% in cell culture medium for supporting cell life while enabling cell rotation rate to be slowed to a speed commensurate with low light level imaging.

Microfluidic electrocage device and cell medium for trapping and rotating cells for live-cell computed tomography (CT)

Methods of pre-methylation of foreign DNA to improve genetic transformation in cyanobacterium. Two Type II methyltransferase-encoding genes, i.e., M (sll0729) and C (slr0214), were cloned from the chromosome ofsp. PCC 6803 (hereafter6803) and expressed inthat harbors the integrative plasmid pBS-SPtK or pJU105. After pre-methylation in, the integrative plasmids were extracted and used for transformation of6803. The results showed that expression of slr0214 in the integrative-plasmid-harboringcells before DNA preparation resulted in orders of magnitude higher efficiency in the following integrative transformation of6803.

Premethylation of DNA for high efficiency transformation of cyanobacteria

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Phoenix, AZ, United States of America

Deirdre Meldrum