10 results
Search Results
2. Digital detection of proteins.
- Author
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Duffy, David C.
- Subjects
- *
SINGLE molecule detection , *SINGLE molecules , *NUCLEIC acids , *ALZHEIMER'S disease , *BLOOD proteins , *PROTEINS , *MOLECULAR switches - Abstract
This paper reviews methods for detecting proteins based on molecular digitization, i.e., the isolation and detection of single protein molecules or singulated ensembles of protein molecules. The single molecule resolution of these methods has resulted in significant improvements in the sensitivity of immunoassays beyond what was possible using traditional "analog" methods: the sensitivity of some digital immunoassays approach those of methods for measuring nucleic acids, such as the polymerase chain reaction (PCR). The greater sensitivity of digital protein detection has resulted in immuno-diagnostics with high potential societal impact, e.g., the early diagnosis and therapeutic intervention of Alzheimer's Disease. In this review, we will first provide the motivation for developing digital protein detection methods given the limitations in the sensitivity of analog methods. We will describe the paradigm shift catalyzed by single molecule detection, and will describe in detail one digital approach – which we call digital bead assays (DBA) – based on the capture and labeling of proteins on beads, identifying "on" and "off" beads, and quantification using Poisson statistics. DBA based on the single molecule array (Simoa) technology have sensitivities down to attomolar concentrations, equating to ∼10 proteins in a 200 μL sample. We will describe the concept behind DBA, the different single molecule labels used, the ways of analyzing beads (imaging of arrays and flow), the binding reagents and substrates used, and integration of these technologies into fully automated and miniaturized systems. We provide an overview of emerging approaches to digital protein detection, including those based on digital detection of nucleic acids labels, single nanoparticle detection, measurements using nanopores, and methods that exploit the kinetics of single molecule binding. We outline the initial impact of digital protein detection on clinical measurements, highlighting the importance of customized assay development and translational clinical research. We highlight the use of DBA in the measurement of neurological protein biomarkers in blood, and how these higher sensitivity methods are changing the diagnosis and treatment of neurological diseases. We conclude by summarizing the status of digital protein detection and suggest how the lab-on-a-chip community might drive future innovations in this field. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
3. An osmolyte-based micro-volume ultrafiltration technique.
- Author
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Ghosh, Raja
- Subjects
- *
ULTRAFILTRATION , *MACROMOLECULES , *PROTEINS , *OSMOSIS , *BUFFER solutions - Abstract
This paper discusses a novel, simple, and inexpensive micro-volume ultrafiltration technique for protein concentration, desalting, buffer exchange, and size-based protein purification. The technique is suitable for processing protein samples in a high-throughput mode. It utilizes a combination of capillary action, and osmosis for drawing water and other permeable species from a micro-volume sample droplet applied on the surface of an ultrafiltration membrane. A macromolecule coated on the permeate side of the membrane functions as the osmolyte. The action of the osmolyte could, if required, be augmented by adding a supersorbent polymer layer over the osmolyte. The mildly hydrophobic surface of the polymeric ultrafiltration membrane used in this study minimized sample droplet spreading, thus making it easy to recover the retained material after separation, without sample interference and cross-contamination. High protein recoveries were observed in the micro-volume ultrafiltration experiments described in the paper. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
4. A droplet microfluidic system for sequential generation of lipid bilayers and transmembrane electrical recordings.
- Author
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Czekalska, Magdalena A., Kaminski, Tomasz S., Jakiela, Slawomir, Sapra, K. Tanuj, Bayley, Hagan, and Garstecki, Piotr
- Subjects
- *
MICROFLUIDICS , *BILAYER lipid membranes , *ELECTRODES , *HEMOLYSIS & hemolysins , *PROTEINS , *DROPLETS - Abstract
This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
5. An automated integrated platform for rapid and sensitive multiplexed protein profiling using human saliva samples.
- Author
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Nie, Shuai, Henley, W. Hampton, Miller, Scott E., Zhang, Huaibin, Mayer, Kathryn M., Dennis, Patty J., Oblath, Emily A., Alarie, Jean Pierre, Wu, Yue, Oppenheim, Frank G., Little, Frédéric F., Uluer, Ahmet Z., Wang, Peidong, Ramsey, J. Michael, and Walt, David R.
- Subjects
- *
PROTEINS , *SALIVA , *IMMUNOASSAY , *MICROFLUIDIC analytical techniques , *BIOMARKERS - Abstract
During the last decade, saliva has emerged as a potentially ideal diagnostic biofluid for noninvasive testing. In this paper, we present an automated, integrated platform useable by minimally trained personnel in the field for the diagnosis of respiratory diseases using human saliva as a sample specimen. In this platform, a saliva sample is loaded onto a disposable microfluidic chip containing all the necessary reagents and components required for saliva analysis. The chip is then inserted into the automated analyzer, the SDReader, where multiple potential protein biomarkers for respiratory diseases are measured simultaneously using a microsphere-based array via fluorescence sandwich immunoassays. The results are read optically, and the images are analyzed by a custom-designed algorithm. The fully automated assay requires as little as 10 μL of saliva sample, and the results are reported in 70 min. The performance of the platform was characterized by testing protein standard solutions, and the results were comparable to those from the 3.5 h lab bench assay that we have previously reported. The device was also deployed in two clinical environments where 273 human saliva samples collected from different subjects were successfully tested, demonstrating the device's potential to assist clinicians with the diagnosis of respiratory diseases by providing timely protein biomarker profiling information. This platform, which combines noninvasive sample collection and fully automated analysis, can also be utilized in point-of-care diagnostics. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
6. Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis.
- Author
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Danny Bottenus, Mohammad Robiul Hossan, Yexin Ouyang, Wen-Ji Dong, Prashanta Dutta, and Cornelius F. Ivory
- Subjects
- *
PHOSPHORYLATION , *INTEGRATED circuits , *CATIONS , *ISOTACHOPHORESIS , *DETECTORS , *BIOMARKERS , *SERUM , *PROTEINS - Abstract
This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic channel. The microfluidic chip incorporates a 100× cross-sectional area reduction, including a 10× depth reduction and a 10× width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both “peak mode” and “plateau mode” and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL−1in the presence of human serum proteins and obtain cTnI concentrations factors as high as ∼ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms of cTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
7. Shrink film patterning by craft cutter: complete plastic chips with high resolution/high-aspect ratio channel.
- Author
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Douglas Taylor, David Dyer, Valerie Lew, and Michelle Khine
- Subjects
- *
MICROFLUIDIC devices , *POLYOLEFINS , *OPTICAL properties , *PROTEINS , *MICROBIOLOGICAL assay , *CHEMICAL bonds - Abstract
This paper presents a rapid, ultra-low-cost approach to fabricate microfluidic devices using a polyolefin shrink film and a digital craft cutter. The shrinking process (with a 95% reduction in area) results in relatively uniform and consistent microfluidic channels with smooth surfaces, vertical sidewalls, and high aspect ratio channels with lateral resolutions well beyond the tool used to cut them. The thermal bonding of the layers results in strongly bonded devices. Complex microfluidic designs are easily designed on the fly and protein assays are also readily integrated into the device. Full device characterization including channel consistency, optical properties, and bonding strength are assessed in this technical note. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
8. Shrink film patterning by craft cutter: complete plastic chips with high resolution/high-aspect ratio channelPublished as part of a special issue dedicated to Emerging Investigators: Guest Editors: Aaron Wheeler and Amy Herr.Electronic supplementary information (ESI) available: Optical data (transmission and autofluorescence measurements). See DOI: 10.1039/c004737f
- Author
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Douglas Taylor, David Dyer, Valerie Lew, and Michelle Khine
- Subjects
- *
MICROFLUIDIC devices , *POLYOLEFINS , *OPTICAL properties , *PROTEINS , *MICROBIOLOGICAL assay , *CHEMICAL bonds - Abstract
This paper presents a rapid, ultra-low-cost approach to fabricate microfluidic devices using a polyolefin shrink film and a digital craft cutter. The shrinking process (with a 95% reduction in area) results in relatively uniform and consistent microfluidic channels with smooth surfaces, vertical sidewalls, and high aspect ratio channels with lateral resolutions well beyond the tool used to cut them. The thermal bonding of the layers results in strongly bonded devices. Complex microfluidic designs are easily designed on the fly and protein assays are also readily integrated into the device. Full device characterization including channel consistency, optical properties, and bonding strength are assessed in this technical note. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
9. Handheld mechanical cell lysis chip with ultra-sharp silicon nano-blade arrays for rapid intracellular protein extraction.
- Author
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Sung-Sik Yun, Sang Youl Yoon, Min-Kyung Song, Sin-Hyeog Im, Sohee Kim, Jong-Hyun Lee, and Sung Yang
- Subjects
- *
CELLULAR mechanics , *INTEGRATED circuits , *NANOSILICON , *EXTRACTION (Chemistry) , *PROTEINS , *MICROFABRICATION , *COST effectiveness , *CHEMICAL reagents , *QUANTITATIVE research - Abstract
This paper presents a handheld mechanical cell lysis chip with ultra-sharp nano-blade arrays fabricated by simple and cost effective crystalline wet etching of (110) silicon. The ultra-sharp nano-blade array is simply formed by the undercutting of (110) silicon during the crystalline wet etching process. Cells can be easily disrupted by the silicon nano-blade array without the help of additional reagents or electrical sources. Based on the bench-top test of the proposed device, a handheld mechanical cell lysis chip with the nano-blade arrays is designed and fabricated for direct connection to a commercial syringe. The direct connection to a syringe provides rapid cell lysis, easy handling, and minimization of the lysate dead volume. The protein concentration in the cell lysate obtained by the proposed lysis chip is quantitatively comparable to the one prepared by a conventional chemical lysis method. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
10. Multiplexed proteomic sample preconcentration device using surface-patterned ion-selective membraneElectronic supplementary information (ESI) available: Supplementary movies 1 and 2. See DOI: 10.1039/b717900f.
- Author
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Jeong Hoon LeeThese two authors contributed to the work equally., Yong-Ak Song, and Jongyoon Han
- Subjects
- *
PROTEOMICS , *IONS , *PROTEINS , *MICROFLUIDICS - Abstract
In this paper, we report a new method of fabricating a high-throughput protein preconcentrator in poly(dimethylsiloxane) (PDMS) microfluidic chip format. We print a submicron thick ion-selective membrane on the glass substrate by using standard patterning techniques. By simply plasma-bonding a PDMS microfluidic device on top of the printed glass substrate, we can integrate the ion-selective membrane into the device and rapidly prototype a PDMS preconcentrator without complicated microfabrication and cumbersome integration processes. The PDMS preconcentrator shows a concentration factor as high as ∼104 in 5 min. This printing method even allows fabricating a parallel array of preconcentrators to increase the concentrated sample volume, which can facilitate an integration of our microfluidic preconcentrator chip as a signal enhancing tool to various detectors such as a mass spectrometer. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
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