Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: A method for analyzing cells through measurement of live-cell parameters followed by measurement of gene and protein expression is disclosed herein. The method comprises measuring one or more live-cell parameters for a plurality of cells contained in at least one liquid in a plurality of isolated microchambers of a microarray device. The method further comprises removing a lid bounding the plurality of isolated microchambers. The method further comprises microdispensing a quantity of lysate into each microchamber of the plurality of isolated microchambers. The method further comprises microdispensing a quantity of reverse transcription polymerase chain reaction mix into each microchamber of the plurality of isolated microchambers. The method further comprises microdispensing a quantity of oil into each microchamber of the plurality of isolated microchambers.

Abstract: 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 R1 and R2 are 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: The invention provides methods for detecting specific nucleic acids using a LAMP reaction and providing a digital readout of the results. One method teaches separating target specificity from indicator detection in a two-stage 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.

Abstract: 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 gels 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.

Abstract: 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.

Abstract: The present disclosure relates to real-time quantification using loop-mediated isothermal amplification, in particular real-time colorimetric reverse transcription quantitative loop-mediated isothermal amplification (RT-qLAMP). In some embodiments, RT-qLAMP is used to diagnose the presence of and also quantitate the amount of Zika virus in a sample.

Abstract: 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.

Abstract: 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.

Abstract: A method for analyzing cells through measurement of live-cell parameters followed by measurement of gene and protein expression is disclosed herein. The method comprises measuring one or more live-cell parameters for a plurality of cells contained in at least one liquid in a plurality of isolated microchambers of a microarray device. The method further comprises removing a lid bounding the plurality of isolated microchambers. The method further comprises microdispensing a quantity of lysate into each microchamber of the plurality of isolated microchambers. The method further comprises microdispensing a quantity of reverse transcription polymerase chain reaction mix into each microchamber of the plurality of isolated microchambers. The method further comprises microdispensing a quantity of oil into each microchamber of the plurality of isolated microchambers.

Abstract: The present disclosure relates to an optical luminescence dual sensor comprising a polymerized form of a probe for sensing pH; a polymerized form of a probe for sensing oxygen; a polymerized form of an internal reference probe; and a matrix. The present disclosure also relates to methods of preparing an optical luminescence dual sensor and methods of using them.