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1.
Anal Chem ; 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39405519

RESUMO

Planar or chip microresonators decrease the sample volume required for magnetic resonance spectroscopies to the nanoliter scale. However, the interrogation of nanoliter-scale solution samples on planar sensors is hindered by the lack of microfluidic devices that can simultaneously provide a small total volume and long-term sample stability. Here, we report microfluidic devices that decrease the total required sample volume to the submicroliter scale and also provide long-term physical stability and storability. We also report a 3D-printed microfluidic with a self-contained actuation mechanism, which allows the sample to be retracted from the microresonator surface for storage. The microfluidic devices are fabricated easily by laser cutting or 3D printing and are integrable with a broad range of planar sensors. We use planar inverse anapole (PIA) microresonators to obtain continuous wave (CW) electron paramagnetic resonance (EPR) spectra of natural-isotopic-abundance nitroxide radicals, which are ubiquitously used as reporters of biomolecular dynamics. We provide experimental evidence for a concentration sensitivity of 330 ± 40 nmol L-1, a concentration sensitivity limit of 800 ± 100 nmol L-1/mT√Hz, and an active volume no greater than 30 nL. Together, these developments represent an advance not only in the sensitivity of EPR spectroscopy but also in the design of microfluidics for stable, dead-volume-free placement of nanoliter-scale volumes of solutions on planar sensors.

2.
Anal Chem ; 91(16): 10713-10722, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31393105

RESUMO

The ultimate performance of flow-based measurements in microfluidic systems is currently limited by their accuracy at the nanoliter-per-minute scale. Improving such measurements (especially in contexts that require continuous monitoring) is challenging because of constraints associated with shrinking system geometries and limitations imposed by making precise measurements of smaller quantities in real time. A particularly interesting limit is the relative uncertainty as flow approaches zero, which diverges for most measurement methods. To address these problems, we have developed an optofluidic measurement system that can deliver and record light in a precise interrogation region of a microfluidic channel. The system utilizes photobleaching of fluorophore dyes in the bulk flow and can identify zero flow to better than 1 nL/min absolute accuracy. The technique also provides an independent method for determining nonzero flow rates based on a robust scaling relationship between the fluorescence emission and flow. Together, these two independent approaches enable precise measurement of flow to within 5% accuracy down to 10 nL/min and validation of flow control to within 5% uncertainty down to 2 nL/min. We also demonstrate that our technique can be used to extend a calibrated flow meter well below its specified range (e.g., 500 nL/min) and to make dynamic measurements of similar relative uncertainties to the calibrated meter, which would have otherwise expanded significantly in this regime.

3.
Cytometry A ; 85(11): 978-85, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25132217

RESUMO

Widefield fluorescence microscopy is a highly used tool for visually assessing biological samples and for quantifying cell responses. Despite its widespread use in high content analysis and other imaging applications, few published methods exist for evaluating and benchmarking the analytical performance of a microscope. Easy-to-use benchmarking methods would facilitate the use of fluorescence imaging as a quantitative analytical tool in research applications, and would aid the determination of instrumental method validation for commercial product development applications. We describe and evaluate an automated method to characterize a fluorescence imaging system's performance by benchmarking the detection threshold, saturation, and linear dynamic range to a reference material. The benchmarking procedure is demonstrated using two different materials as the reference material, uranyl-ion-doped glass and Schott 475 GG filter glass. Both are suitable candidate reference materials that are homogeneously fluorescent and highly photostable, and the Schott 475 GG filter glass is currently commercially available. In addition to benchmarking the analytical performance, we also demonstrate that the reference materials provide for accurate day to day intensity calibration. Published 2014 Wiley Periodicals Inc.


Assuntos
Benchmarking , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Automação , Calibragem , Citometria de Fluxo
4.
PLoS One ; 18(12): e0295502, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38134031

RESUMO

Signals analysis for cytometry remains a challenging task that has a significant impact on uncertainty. Conventional cytometers assume that individual measurements are well characterized by simple properties such as the signal area, width, and height. However, these approaches have difficulty distinguishing inherent biological variability from instrument artifacts and operating conditions. As a result, it is challenging to quantify uncertainty in the properties of individual cells and perform tasks such as doublet deconvolution. We address these problems via signals analysis techniques that use scale transformations to: (I) separate variation in biomarker expression from effects due to flow conditions and particle size; (II) quantify reproducibility associated with a given laser interrogation region; (III) estimate uncertainty in measurement values on a per-event basis; and (IV) extract the singlets that make up a multiplet. The key idea behind this approach is to model how variable operating conditions deform the signal shape and then use constrained optimization to "undo" these deformations for measured signals; residuals to this process characterize reproducibility. Using a recently developed microfluidic cytometer, we demonstrate that these techniques can account for instrument and measurand induced variability with a residual uncertainty of less than 2.5% in the signal shape and less than 1% in integrated area.


Assuntos
Reprodutibilidade dos Testes , Incerteza , Tamanho da Partícula , Citometria de Fluxo/métodos
5.
J Biomed Opt ; 27(1)2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35102729

RESUMO

SIGNIFICANCE: Performance improvements in microfluidic systems depend on accurate measurement and fluid control on the micro- and nanoscales. New applications are continuously leading to lower volumetric flow rates. AIM: We focus on improving an optofluidic system for measuring and calibrating microflows to the sub-nanoliter per minute range. APPROACH: Measurements rely on an optofluidic system that delivers excitation light and records fluorescence in a precise interrogation region of a microfluidic channel. Exploiting a scaling relationship between the flow rate and fluorescence emission after photobleaching, the system enables real-time determination of flow rates. RESULTS: Here, we demonstrate improved calibration of a flow controller to 1% uncertainty. Further, the resolution of the optofluidic flow meter improved to less than 1 nL / min with 5% uncertainty using a molecule with a 14-fold smaller diffusion coefficient than our previous report. CONCLUSIONS: We demonstrate new capabilities in sub-nanoliter per minute flow control and measurement that are generalizable to cutting-edge light-material interaction and molecular diffusion for chemical and biomedical industries.


Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica
6.
Lab Chip ; 22(17): 3217-3228, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35856829

RESUMO

Flow cytometry is an invaluable technology in biomedical research, but confidence in single-cell measurements remains limited due to a lack of appropriate techniques for uncertainty quantification (UQ). It is particularly challenging to evaluate the potential for different instrumentation designs or operating parameters to influence the measurement physics in ways that change measurement repeatability. Here, we report a direct experimental approach to UQ using a serial flow cytometer that measured each particle more than once along a flow path. The instrument was automated for real-time characterization of measurement precision and operated with particle velocities exceeding 1 m s-1, throughputs above 100 s-1, and analysis yields better than 99.9%. These achievements were enabled by a novel hybrid inertial and hydrodynamic particle focuser to tightly control particle positions and velocities. The cytometer identified ideal flow conditions with fluorescence area measurement precision on the order of 1% and characterized tradeoffs between precision, throughput, and analysis yield. The serial cytometer is anticipated to improve single-cell measurements through estimation (and subsequent control) of uncertainty contributions from various other instrument parameters leading to overall improvements in the ability to better classify sample composition and to find rare events.


Assuntos
Hidrodinâmica , Citometria de Fluxo
7.
Anal Chem ; 83(10): 3890-6, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21506521

RESUMO

Numerous opportunities exist to apply microfluidic technology to high-throughput and high-content cell-based assays. However, maximizing the value of microfluidic assays for applications such as drug discovery, screening, or toxicity evaluation will require assurance of within-device repeatability, day-to-day reproducibility, and robustness to variations in conditions that might occur from laboratory to laboratory. This report describes a study of the performance and variability of a cell-based toxicity assay in microfluidic devices made of poly(dimethylsiloxane) (PDMS). The assay involves expression of destabilized green fluorescent protein (GFP) as a reporter of intracellular protein synthesis and degradation. Reduction in cellular GFP due to inhibition of ribosome activity by cycloheximide (CHX) was quantified with real-time quantitative fluorescence imaging. Assay repeatability was measured within a 64-chamber microfluidic device. Assay performance across a range of cell loading densities within a single device was assessed, as was replication of measurements in microfluidic devices prepared on different days. Assay robustness was tested using different fluorescence illumination sources and reservoir-to-device tubing choices. Both microfluidic and larger scale assay conditions showed comparable GFP decay rates upon CHX exposure, but the microfluidic data provided the higher level of confidence.


Assuntos
Técnicas Analíticas Microfluídicas/métodos , Testes de Toxicidade/métodos , Animais , Chlorocebus aethiops , Cicloeximida/química , Cicloeximida/toxicidade , Dimetilpolisiloxanos/química , Proteínas de Fluorescência Verde/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência/métodos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Células Vero
8.
Lab Chip ; 10(9): 1120-7, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20390129

RESUMO

The mammalian olfactory system is able to discriminate among tens of thousands of odorant molecules. In mice, each odorant is sensed by a small subset of the approximately 1000 odorant receptor (OR) types, with one OR gene expressed by each olfactory sensory neuron (OSN). However, the sum of the large repertoire of OR-OSN types and difficulties with heterologous expression have made it almost impossible to analyze odorant-responsiveness across all OR-OSN types. We have developed a microfluidic approach that allowed us to screen over 20,000 single cells at once in microwells. By using calcium imaging, we were able to detect and analyze odorant responses of about 2900 OSNs simultaneously. Importantly, this technique allows for both the detection of rare responding OSNs as well as the identification of OSN populations broadly responsive to odorants of unrelated structures. This technique is generally applicable for screening large numbers of single cells and should help to characterize rare cell behaviors in fields such as toxicology, pharmacology, and cancer research.


Assuntos
Bioensaio/instrumentação , Separação Celular/instrumentação , Análise em Microsséries/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Odorantes , Neurônios Receptores Olfatórios/fisiologia , Olfato/fisiologia , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Neurônios Receptores Olfatórios/efeitos dos fármacos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Olfato/efeitos dos fármacos
9.
Lab Chip ; 10(2): 246-9, 2010 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-20066254

RESUMO

We present a new type of microfluidic connector that employs a ring magnet on one side of the microfluidic chip and a disc magnet on the other side to produce a sealed connection between external tubing and inlets or outlets of microfluidic devices. The connectors are low-cost, simple to use and assemble, and reusable. We used numerical (finite element) simulations in order to optimize their geometry. Configurations that achieve interfacial forces in the range of 2 N to 15 N are discussed. Several types of gasket materials were explored. Finally, we demonstrate an application of these connectors in a microfluidic device used to generate liposomes.

10.
Langmuir ; 26(17): 14111-7, 2010 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-20666411

RESUMO

Type I collagen fibrillar thin films have been prepared on hydrophobic recovered poly(dimethylsiloxane) (PDMS) surfaces and inside of irreversibly sealed PDMS microfluidic devices. Fibrillar films prepared on PDMS surfaces have been characterized with optical microscopy and atomic force microscopy and compared with films prepared using more traditional bulk methods on thiol-coated gold substrates. Collagen fibril films formed after 18 h of incubation on PDMS surfaces were observed to have similar underlying film thicknesses (15 nm), fibril size (67 nm), fibril coverage (45%), and physiologically supermolecular structure when compared to films on gold substrates. Collagen fibrils formed within devices were also determined to be usable across physiologically relevant cell perfusion rates. To validate the utility of these collagen fibril thin films for cell culture applications, vascular smooth muscle cells are shown to attach to collagen fibrils and exhibit cell spread areas equivalent to those seen on collagen fibrils created via bulk cell culture methods on thiol-coated gold substrates. These results extend the use and benefits of collagen fibril thin films into microfluidic-based cellular studies.


Assuntos
Colágeno Tipo I/química , Dimetilpolisiloxanos/química , Membranas Artificiais , Técnicas Analíticas Microfluídicas/métodos , Tamanho da Partícula , Propriedades de Superfície
11.
Lab Chip ; 9(9): 1298-300, 2009 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-19370253

RESUMO

The lack of simple interfaces for microfluidic devices with a large number of inlets significantly limits production and utilization of these devices. In this article, we describe the fabrication of a reusable manifold that provides rapid world-to-chip connectivity. A vacuum network milled into a rigid manifold holds microdevices and prevents leakage of fluids injected into the device from ports in the manifold. A number of different manifold designs were explored, and all performed similarly, yielding an average of 100 kPa (15 psi) fluid holding pressure. The wide applicability of this manifold concept is demonstrated by interfacing with a 51-inlet microfluidic chip containing 144 chambers and hundreds of embedded pneumatic valves. Due to the speed of connectivity, the manifolds are ideal for rapid prototyping and are well suited to serve as "universal" interfaces.


Assuntos
Dimetilpolisiloxanos/química , Reutilização de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Vácuo
12.
Lab Chip ; 9(3): 417-26, 2009 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-19156291

RESUMO

Microfluidic perfusion systems, characterized by deterministic flow, low reagent consumption, small dead volumes, large integration in small footprints, high-throughput operation, and low-cost fabrication, are being increasingly used for cell culture studies in applications such as basic cell biology, molecular biological assays, tissue engineering, and systems biology. We report a multipurpose, pressure-driven and computer-controlled microfluidic perfusion device containing sixteen inlets and a large cell culture chamber. The user can choose, with sub-second temporal resolution, (a) to feed the chamber with one of 16 inlets, all 16 inlets, or one of 64 combinations of 2, 4, or 8 inlets using a binary multiplexer; (b) to introduce into the chamber a heterogeneous laminar flow of the inlets, a smoothened gradient, or a fully homogenized mixture; (c) to bypass the chamber in order to purge the inlet lines so as to minimize the dead volume; (d) to generate asymmetrical and curvilinear flow patterns within the chamber by opening side outlets; and (e) to slow down the flow by combinatorially adding segments of high fluid resistance (sixteen different levels of flow rates are possible using only four valves). All functionalities are combined to create complex gradient patterns and sequential perfusions within the central chamber.


Assuntos
Técnicas de Cultura de Células , Técnicas Analíticas Microfluídicas/instrumentação , Perfusão/instrumentação , Algoritmos , Técnicas de Cultura de Células/instrumentação , Dimetilpolisiloxanos/química , Desenho de Equipamento , Fenômenos Físicos
13.
Lab Chip ; 22(15): 2786-2788, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35713939
14.
Lab Chip ; 20(15): 2604-2606, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32662472
15.
Lab Chip ; 19(12): 2058-2059, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31090772
16.
Lab Chip ; 14(10): 1665-8, 2014 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-24691845

RESUMO

We present a rapid prototyping technique that expands elastomeric valving capabilities to devices made from thin materials such as plastic films and tapes. The time required from conception to full fabrication of functional devices is within a few hours. A key characteristic of this technology is that devices are thin (typically less than 0.5 mm in thickness), which allows for the fabrication of devices with many layers. This feature also permits folding of devices into 3D structures having fully functional valves. We illustrate this concept with the fabrication of a 25 mm-per-side cube whose walls contain microfluidic channels and valves. Control of liquid delivery through the faces of the cube is demonstrated with a chemotaxis experiment of C. elegans migrating within the enclosed volume of the cube as stimuli are delivered through the walls of the cube to the interior faces.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Plásticos/química , Animais , Caenorhabditis elegans/fisiologia , Quimiotaxia/fisiologia , Elasticidade , Desenho de Equipamento
17.
Lab Chip ; 12(2): 309-16, 2012 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-22113489

RESUMO

This manuscript describes a new method to generate purely diffusive chemical gradients that can be modified in time. The device is simple in its design and easy to use, which makes it amenable to study biological processes that involve static or dynamic chemical gradients such as chemotaxis. We describe the theory underlying the convection-free gradient generator, illustrate the design to implement the theory, and present a protocol to align multiple layers of double sided tape and laminates to fabricate the device. Using this device, a population of mammalian cells was exposed to different concentrations of a toxin within a concentration gradient in a 48 h experiment. Cells were probed dynamically by cycling the gradient on and off, and cell response was monitored using time-lapse fluorescence microscopy. The experiment and results illustrate the type of applications involving dynamic cell behavior that can be targeted with this type of gradient generator.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Animais , Movimento Celular , Proliferação de Células , Chlorocebus aethiops , Cicloeximida/toxicidade , Difusão , Desenho de Equipamento , Membranas Artificiais , Microscopia de Fluorescência , Poliestirenos/química , Fatores de Tempo , Células Vero
18.
Bioanalysis ; 4(15): 1849-54, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22943616

RESUMO

The National Institute of Standards and Technology (NIST) is the National Metrology Institute for the USA. Our mission is to advance measurement science, standards and technology in ways that enhance economic security and improve quality of life in the USA. Due to the increased need for technologies that advance biological research and the many new and exciting innovations in microfluidics, our projects are aimed at engineering well-controlled microenvironments for quantitative measurements of cell behavior in microfluidic systems. Cell-based microfluidics at NIST is a highly multidisciplinary activity and is greatly influenced by NIST programs in biochemical sciences, materials science, engineering and information technology. Although there are many microfluidic-related activities ongoing at NIST, we will focus on projects related to cell-based measurements in this article.


Assuntos
Bioensaio/instrumentação , Técnicas de Cultura de Células/instrumentação , Microfluídica/instrumentação , Bioensaio/métodos , Bioensaio/tendências , Técnicas de Cultura de Células/métodos , Humanos , Leucócitos/imunologia , Microfluídica/métodos , Células Neoplásicas Circulantes/imunologia , Reprodutibilidade dos Testes , Estados Unidos , United States Government Agencies , Estudos de Validação como Assunto
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