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1.
Chem Biomed Imaging ; 1(9): 817-830, 2023 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-38155726

RESUMEN

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) of around 50 µm × 50 µm at once and more recently up to ∼200 µm × 200 µm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints to the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust single-molecule localization with up to ∼520 µm × 520 µm single FOVs and 25-40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm2 sample areas can be completed in as little as 40 min per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality, particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ∼106 caveolae structures in ∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.

2.
Electrophoresis ; 44(15-16): 1234-1246, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37431197

RESUMEN

Dielectrophoresis (DEP) is a successful method to recover nanoparticles from different types of fluid. The DEP force acting on these particles is created by an electrode microarray that produces a nonuniform electric field. To apply DEP to a highly conducting biological fluid, a protective hydrogel coating over the metal electrodes is required to create a barrier between the electrode and the fluid. This protects the electrodes, reduces the electrolysis of water, and allows the electric field to penetrate into the fluid sample. We observed that the protective hydrogel layer can separate from the electrode and form a closed domed structure and that collection of 100 nm polystyrene beads increased when this occurred. To better understand this collection increase, we used COMSOL Multiphysics software to model the electric field in the presence of the dome filled with different materials ranging from low-conducting gas to high conducting phosphate-buffered saline fluids. The results suggest that as the electrical conductivity of the material inside the dome is reduced, the whole dome acts as an insulator which increases electric field intensity at the electrode edge. This increased intensity widens the high-intensity electric field factor zone resulting in increased collection. This informs how dome formation results in increased particle collection and provides insight into how the electric field can be intensified to the increase collection of particles. These results have important applications for increasing the recovery of biologically-derived nanoparticles from undiluted physiological fluids that have high conductance, including the collection of cancer-derived extracellular vesicles from plasma for liquid biopsy applications.


Asunto(s)
Electricidad , Programas Informáticos , Electroforesis/métodos , Conductividad Eléctrica , Electrodos
3.
Electrophoresis ; 43(12): 1366-1377, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35377504

RESUMEN

Many biomedical analysis applications require trapping and manipulating single cells and cell clusters within microfluidic devices. Dielectrophoresis (DEP) is a label-free technique that can achieve flexible cell trapping, without physical barriers, using electric field gradients created in the device by an electrode microarray. Little is known about how fluid flow forces created by the electrodes, such as thermally driven convection and electroosmosis, affect DEP-based cell capture under high conductance media conditions that simulate physiologically relevant fluids such as blood or plasma. Here, we compare theoretical trajectories of particles under the influence of negative DEP (nDEP) with observed trajectories of real particles in a high conductance buffer. We used 10-µm diameter polystyrene beads as model cells and tracked their trajectories in the DEP microfluidic chip. The theoretical nDEP trajectories were in close agreement with the observed particle behavior. This agreement indicates that the movement of the particles was highly dominated by the DEP force and that contributions from thermal- and electroosmotic-driven flows were negligible under these experimental conditions. The analysis protocol developed here offers a strategy that can be applied to future studies with different applied voltages, frequencies, conductivities, and polarization properties of the targeted particles and surrounding medium. These findings motivate further DEP device development to manipulate particle trajectories for trapping applications.


Asunto(s)
Electroósmosis , Dispositivos Laboratorio en un Chip , Electroforesis/métodos , Microfluídica/métodos , Poliestirenos
4.
Lab Chip ; 21(7): 1318-1332, 2021 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-33877235

RESUMEN

Tumor-secreted exosomes and other extracellular vesicles (EVs) in circulation contain valuable biomarkers for early cancer detection and screening. We have previously demonstrated collection of cancer-derived nanoparticles (NPs) directly from whole blood and plasma with a chip-based technique that uses a microelectrode array to generate dielectrophoretic (DEP) forces. This technique enables direct recovery of NPs from whole blood and plasma. The biomarker payloads associated with collected particles can be detected and quantified with immunostaining. Accurately separating the fluorescence intensity of stained biomarkers from background (BG) levels becomes a challenge when analyzing the blood from early-stage cancer patients in which biomarker concentrations are low. To address this challenge, we developed two complementary techniques to standardize the quantification of fluorescently immunolabeled biomarkers collected and concentrated at predictable locations within microfluidic chips. The first technique was an automated algorithm for the quantitative analysis of fluorescence intensity at collection regions within the chip compared to levels at adjacent regions. The algorithm used predictable locations of particle collection within the chip geometry to differentiate regions of collection and BG. We successfully automated the identification and removal of optical artifacts from quantitative calculations. We demonstrated that the automated system performs nearly the same as a human user following a standard protocol for manual artifact removal with Pearson's r-values of 0.999 and 0.998 for two different biomarkers (n = 36 patients). We defined a usable dynamic range of fluorescence intensities corresponding to 1 to 2000 arbitrary units (a.u.). Fluorescence intensities within the dynamic range increased linearly with respect to exposure time and particle concentration. The second technique was the implementation of an internal standard to adjust levels of biomarker fluorescence based on the relative collection efficiency of the chip. Use of the internal standard reduced variability in measured biomarker levels due to differences in chip-to-chip collection efficiency, especially at low biomarker concentrations. The internal standard did not affect linear trends between fluorescence intensity and exposure time. Adjustments using the internal standard improved linear trends between fluorescence intensity and particle concentration. The optical quantification techniques described in this paper can be easily adapted for other lab-on-a-chip platforms that have predefined regions of biomarker or particle collection and that rely on fluorescence detection.


Asunto(s)
Exosomas , Vesículas Extracelulares , Humanos , Dispositivos Laboratorio en un Chip , Microfluídica , Plasma
5.
Electrophoresis ; 42(5): 539-564, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33191521

RESUMEN

The 20th century has seen tremendous innovation of dielectrophoresis (DEP) technologies, with applications being developed in areas ranging from industrial processing to micro- and nanoscale biotechnology. From 2010 to present day, there have been 981 publications about DEP. Of over 2600 DEP patents held by the United States Patent and Trademark Office, 106 were filed in 2019 alone. This review focuses on DEP-based technologies and application developments between 2010 and 2020, with an aim to highlight the progress and to identify potential areas for future research. A major trend over the last 10 years has been the use of DEP techniques for biological and clinical applications. It has been used in various forms on a diverse array of biologically derived molecules and particles to manipulate and study them including proteins, exosomes, bacteria, yeast, stem cells, cancer cells, and blood cells. DEP has also been used to manipulate nano- and micron-sized particles in order to fabricate different structures. The next 10 years are likely to see the increase in DEP-related patent applications begin to result in a greater level of technology commercialization. Also during this time, innovations in DEP technology will likely be leveraged to continue the existing trend to further biological and medical-focused applications as well as applications in microfabrication. As a tool leveraged by engineering and imaginative scientific design, DEP offers unique capabilities to manipulate small particles in precise ways that can help solve problems and enable scientific inquiry that cannot be addressed using conventional methods.


Asunto(s)
Biotecnología , Electroforesis , Nanotecnología , Animales , Separación Celular , Células Cultivadas , Humanos , Ratones , Tamaño de la Partícula
6.
Anal Bioanal Chem ; 412(16): 3871-3880, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32277243

RESUMEN

Though the advances in microelectronic device fabrication have realized new capabilities in integrated analytical and diagnostic platforms, there are still notable limitations in point-of-care sample preparation. AC electrokinetic devices, especially those leveraging dielectrophoresis (DEP), have shown potential to solve these limitations and allow for sample-to-answer in a single point-of-care device. However, when working directly with whole blood or other high conductance (~ 1 S/m) biological fluids, the aggressive electrochemical conditions created by the electrode can fundamentally limit the device operation. In this study, platinum wire-based electrode devices spanning circular polytetrafluorethylene (PTFE) wells and a planar microarray device with sputtered platinum electrodes were tested in plasma and PBS buffers of differing concentration across a wide range of frequencies and electric field intensities (AC voltages) to determine their respective safe regions of operation and to gain an understanding about the failure mechanisms of this class of device. At frequencies of 10 kHz and below, the upper bound of operation is the degradation of electrodes due to electrochemical attack by chlorine overcoming the native platinum oxide passivation. At higher frequencies, 100 kHz and above, the dielectric loss and subsequent heating of the buffer will boil before the electrodes suffer observable damage, due to the slow irreversible reaction kinetics. Effective dielectrophoretic capture of small biological particles at these frequencies is limited, and heat/oxidative denaturation of target material are a major concern. A new class of smaller devices, ones capable of high throughput at voltages low enough to maintain the integrity of the platinum passivation layer, is needed to mitigate these fundamental limitations.


Asunto(s)
Corrosión , Electrodos , Electroforesis/instrumentación , Platino (Metal)/química , Sistemas de Atención de Punto
7.
Expert Rev Mol Diagn ; 15(9): 1187-200, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26189641

RESUMEN

As we move into the era of individualized cancer treatment, the need for more sophisticated cancer diagnostics has emerged. Cell-free (cf) nucleic acids (cf-DNA, cf-RNA) and other cellular nanoparticulates are now considered important and selective biomarkers. There is great hope that blood-borne cf-nucleic acids can be used for 'liquid biopsies', replacing more invasive tissue biopsies to analyze cancer mutations and monitor therapy. Conventional techniques for cf-nucleic acid biomarker isolation from blood are generally time-consuming, complicated and expensive. They require relatively large blood samples, which must be processed to serum or plasma before isolation of biomarkers can proceed. Such cumbersome sample preparation also limits the widespread use of powerful, downstream genomic analyses, including PCR and DNA sequencing. These limitations also preclude rapid, point-of-care diagnostic applications. Thus, new technologies that allow rapid isolation of biomarkers directly from blood will permit seamless sample-to-answer solutions that enable next-generation point-of-care molecular diagnostics.


Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias/sangre , Neoplasias/diagnóstico , Humanos , Técnicas de Diagnóstico Molecular , Ácidos Nucleicos/sangre , Sistemas de Atención de Punto
8.
Nano Lett ; 13(4): 1440-5, 2013 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-23517010

RESUMEN

We experimentally demonstrate that plasmonic nanoparticles embedded in the evanescent field of subwavelength optical waveguides (WGs) are highly sensitive to distances normal to the propagation of light, showing an ~10× increase in spatial resolution compared to the optical field decay of the WG. The scattering cross-section of the Au nanoparticle is increased by the plasmon-dielectric coupling interaction when the nanoparticle is placed near the dielectric surface of the WG, and the decay of the scattering signal is enhanced, showing angstrom level distance sensitivity within 10 nm from the WG. Numerical studies with the finite-difference time-domain (FDTD) method correlate well with the experimental results. To demonstrate real-time monitoring of a single molecule stretching in the evanescent field, we linked individual single-stranded DNA molecules between the WG and plasmonic nanoparticles and pushed on the nanoparticles with fluidic forces. The simple design and ease of obtaining optical feedback on molecular displacements makes our approach ideal for new in situ force sensing devices, imaging technologies, and high-throughput molecular analysis.


Asunto(s)
ADN de Cadena Simple/química , Nanofibras/química , Nanopartículas/química , ADN de Cadena Simple/aislamiento & purificación , Tecnología de Fibra Óptica , Oro/química , Luz , Dispersión de Radiación
9.
Nano Lett ; 12(4): 1905-11, 2012 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-22449127

RESUMEN

We report a systematic study of light coupling at junctions of overlapping SnO(2) nanofiber waveguides (WGs) as a function of gap separation and guided wavelength. The junctions were assembled on silica substrates using micromanipulation techniques and the gap separation was controlled by depositing thin self-assembled polyelectrolyte coatings at the fiber junctions. We demonstrate that the coupling efficiency is strongly dependent on the gap separation, showing strong fluctuations (0.1 dB/nm) in the power transfer when the separation between nanofibers changes by as little as 2 nm. Experimental results correlate well with numerical simulations using three-dimensional finite-difference time-domain techniques. To demonstrate the feasibility of using coupled nanofiber WGs to modulate light, we encased the junctions in an environment-responsive matrix and exposed the junctions to gaseous vapor. The nanofiber junctions show an ~95% (or ~80%) modulation of the guided 450 nm (or 510 nm) light upon interaction with the gaseous molecules. The results reveal a unique nanofiber-based sensing scheme that does not require a change in the refractive index to detect stimuli, suggesting these structures could play important roles in localized sensing devices including force-based measurements or novel chemically induced light modulators.


Asunto(s)
Luz , Nanofibras/química , Compuestos de Estaño/química , Dimetilpolisiloxanos/química , Dióxido de Silicio/química
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