Protein Counting in Single Cancer Cells

Schubert, Stephanie; Walter, Stephanie R.; Manesse, Maël; Walt, David R.

doi:10.1021/acs.analchem.6b00146

Cited by 39 publications

(39 citation statements)

References 41 publications

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“…21 Recently, single molecule assay (SiMoA), an ultrasensitive bead-based assay for protein quantification, has been developed and employed to detect PSA secretion at the single-cell level. 22 SiMoA fails to achieve a large number of single-cell measurements with multiplexed protein detection. In this study, we describe a BOBarray-based method that enables thousands of single-cell multiplexed protein measurements.…”

Section: Discussionmentioning

confidence: 99%

Single-Cell, Multiplexed Protein Detection of Rare Tumor Cells Based on a Beads-on-Barcode Antibody Microarray

Liu¹,

Wang²,

Deng³

et al. 2016

Anal. Chem.

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Circulating tumor cells (CTCs) shed from tumor sites and represent the molecular characteristics of the tumor. Besides genetic and transcriptional characterization, it is important to profile a panel of proteins with single-cell precision for resolving CTCs’ phenotype, organ-of-origin, and drug targets. We describe a new technology that enables profiling multiple protein markers of extraordinarily rare tumor cells at the single- cell level. This technology integrates a microchip consisting of 15000 60 pL-sized microwells and a novel beads-on-barcode antibody microarray (BOBarray). The BOBarray allows for multiplexed protein detection by assigning two independent identifiers (bead size and fluorescent color) of the beads to each protein. Four bead sizes (1.75, 3, 4.5, and 6 μm) and three colors (blue, green, and yellow) are utilized to encode up to 12 different proteins. The miniaturized BOBarray can fit an array of 60 pL- sized microwells that isolate single cells for cell lysis and the subsequent detection of protein markers. An enclosed 60 pL-sized microchamber defines a high concentration of proteins released from lysed single cells, leading to single-cell resolution of protein detection. The protein markers assayed in this study include organ-specific markers and drug targets that help to characterize the organ-of-origin and drug targets of isolated rare tumor cells from blood samples. This new approach enables handling a very small number of cells and achieves single-cell, multiplexed protein detection without loss of rare but clinically important tumor cells.

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Section: Discussionmentioning

confidence: 99%

Single-Cell, Multiplexed Protein Detection of Rare Tumor Cells Based on a Beads-on-Barcode Antibody Microarray

Liu¹,

Wang²,

Deng³

et al. 2016

Anal. Chem.

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“…To detect biomarkers at ultra-low concentrations for early diagnosis in asymptomatic patients of diseases such as Parkinson's disease, Alzheimer's disease, cancer, and malaria, new strategies need to be combined with microfluidic PoC devices. One such strategy includes magnetic nanoparticles for signal amplification and capture of analytes [23,24], which also opens up the possibility for multiplex biomarker detection [25,26]. For example, Reboud et al [23] combined magnetic nanoparticles, for lysis and analyte extraction, with a loop-mediated isothermal amplification (LAMP) [24] method for DNA diagnostics of malaria in rural communities.…”

Section: Recent Trends In Microfluidic Device Fabricationmentioning

confidence: 99%

“…Multiplexed detection, i.e., quantifying several analytes at the same time, is another requirement to be fulfilled. Signal amplification for detection of analytes at ultra-low concentrations is another important point, which is being mostly addressed through the use of nanoparticles to capture the analytes for improved signals [24][25][26]. A major challenge in microfluidic PoC devices is the detection and separation of chiral enantiomers, which exhibit the same physical and chemical properties in achiral environments.…”

Section: Introductionmentioning

confidence: 99%

Microfluidic Point-of-Care Devices: New Trends and Future Prospects for eHealth Diagnostics

Mejía‐Salazar

Cruz

Vásques

et al. 2020

Sensors

145

111

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Point-of-care (PoC) diagnostics is promising for early detection of a number of diseases, including cancer, diabetes, and cardiovascular diseases, in addition to serving for monitoring health conditions. To be efficient and cost-effective, portable PoC devices are made with microfluidic technologies, with which laboratory analysis can be made with small-volume samples. Recent years have witnessed considerable progress in this area with “epidermal electronics”, including miniaturized wearable diagnosis devices. These wearable devices allow for continuous real-time transmission of biological data to the Internet for further processing and transformation into clinical knowledge. Other approaches include bluetooth and WiFi technology for data transmission from portable (non-wearable) diagnosis devices to cellphones or computers, and then to the Internet for communication with centralized healthcare structures. There are, however, considerable challenges to be faced before PoC devices become routine in the clinical practice. For instance, the implementation of this technology requires integration of detection components with other fluid regulatory elements at the microscale, where fluid-flow properties become increasingly controlled by viscous forces rather than inertial forces. Another challenge is to develop new materials for environmentally friendly, cheap, and portable microfluidic devices. In this review paper, we first revisit the progress made in the last few years and discuss trends and strategies for the fabrication of microfluidic devices. Then, we discuss the challenges in lab-on-a-chip biosensing devices, including colorimetric sensors coupled to smartphones, plasmonic sensors, and electronic tongues. The latter ones use statistical and big data analysis for proper classification. The increasing use of big data and artificial intelligence methods is then commented upon in the context of wearable and handled biosensing platforms for the Internet of things and futuristic healthcare systems.

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“…Currently, this is achieved primarily using protein-specific antibodies that are conjugated to various kinds of reporters. While there are advances in single-cell multiomics (Macaulay, Ponting, & Voet, 2017), such as performing cytometry and RNA sequencing simultaneously by using oligonucleotides conjugated to protein-specific antibodies (Stoeckius et al, 2017), and even in single-cell proteomics (Schubert, Walter, Manesse, & Walt, 2016), these are still far from applicable in the context of high-throughput assays for complex biological systems. Fluorescence flow cytometry (FCM) has been the standard single-cell measurement system since the 1970s.…”

Section: Challenges Of Multi-parametric Single Cell Analysis: Flow Anmentioning

confidence: 99%

High‐Dimensional Fluorescence Cytometry

2017

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The immune system consists of a complex network of cells, all expressing a wide range of surface and/or intracellular proteins. Using flow cytometry, these cells can be analyzed by labeling with fluorophore-conjugated antibodies. The recent expansion of fluorescence flow cytometry technology, in conjunction with the ever-expanding understanding of the complexity of the immune system, has led to the generation of larger high-dimensional fluorescence flow cytometry panels. However, as panel size and complexity increases, so too does the difficulty involved in constructing high-quality panels, in addition to the challenges of analyzing such high-dimensional datasets. As such, this unit seeks to review the key principles involved in building high-dimensional panels, as well as to guide users through the process of building and analyzing quality panels. Here, cytometer configuration, fluorophore brightness, spreading error, antigen density, choosing the best conjugates, titration, optimization, and data analysis will all be addressed. © 2017 by John Wiley & Sons, Inc.

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Protein Counting in Single Cancer Cells

Cited by 39 publications

References 41 publications

Single-Cell, Multiplexed Protein Detection of Rare Tumor Cells Based on a Beads-on-Barcode Antibody Microarray

Single-Cell, Multiplexed Protein Detection of Rare Tumor Cells Based on a Beads-on-Barcode Antibody Microarray

Microfluidic Point-of-Care Devices: New Trends and Future Prospects for eHealth Diagnostics

High‐Dimensional Fluorescence Cytometry

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