scholarly journals Highly sensitive and multiplexed in situ protein profiling with cleavable fluorescent streptavidin

2019 ◽  
Author(s):  
Renjie Liao ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
Jia Guo
Keyword(s):  
Signal Amplification ◽  
Individual Cell ◽  
Protein Detection ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Layer By Layer ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Wide Range

AbstractThe ability to perform highly sensitive and multiplexed in situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, here we develop an approach using cleavable biotin conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced by at least 10 fold, compared with the existing methods. After imaging, the fluorophores and the biotins unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be unambiguously detected in individual cell in situ.

scholarly journals Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 852 ◽  
Author(s):  
Renjie Liao ◽  
Thai Pham ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
Joshua Labaer ◽  
...  
Keyword(s):  
Optical Resolution ◽  
Protein Detection ◽  
Protein Analysis ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Layer By Layer ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Wide Range

The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.

scholarly journals Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

2021 ◽  
Vol 22 (16) ◽  
pp. 8644
Author(s):  
Thai Pham ◽  
Christopher D. Nazaroff ◽  
Joshua Labaer ◽  
Jia Guo
Keyword(s):  
Single Cell Analysis ◽  
Expression Profiles ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Great Promise ◽  
Specific Cell ◽  
Protein Targets ◽  
Cell Clusters ◽  
Protein Imaging

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

scholarly journals Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3732
Author(s):  
Agnieszka Dabrowska ◽  
Aleksandra Milewska ◽  
Joanna Ner-Kluza ◽  
Piotr Suder ◽  
Krzysztof Pyrc
Keyword(s):  
Mass Spectrometry ◽  
Zika Virus ◽  
Viral Infections ◽  
Protein Detection ◽  
Extensive Discussion ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Infected Cells ◽  
Ns3 Protease ◽  
To Receive

Mass spectrometry (MS) used in proteomic approaches is able to detect hundreds of proteins in a single assay. Although undeniable high analytical power of MS, data acquired sometimes lead to confusing results, especially during a search of very selective, unique interactions in complex biological matrices. Here, we would like to show an example of such confusing data, providing an extensive discussion on the observed phenomenon. Our investigations focus on the interaction between the Zika virus NS3 protease, which is essential for virus replication. This enzyme is known for helping to remodel the microenvironment of the infected cells. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, clearly shown by proteomic techniques, as processed during infection. Unfortunately, we could not confirm the biological relevance of protein targets for viral infections detected by MS. Thus, although mass spectrometry is highly sensitive and useful in many instances, also being able to show directions where cell/virus interaction occurs, we believe that deep recognition of their biological role is essential to receive complete insight into the investigated process.

scholarly journals Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2206
Author(s):  
Thai Pham ◽  
Renjie Liao ◽  
Joshua Labaer ◽  
Jia Guo
Keyword(s):  
High Performance ◽  
Single Cells ◽  
Protein Quantification ◽  
Cellular Systems ◽  
Protein Profiling ◽  
High Signal ◽  
Protein Targets ◽  
Chemical Reagents ◽  
Ffpe Tissues

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.

A supersandwich fluorescence in situ hybridization strategy for highly sensitive and selective mRNA imaging in tumor cells

2016 ◽  
Vol 52 (2) ◽  
pp. 370-373 ◽  
Author(s):  
Jin Huang ◽  
He Wang ◽  
Xiaohai Yang ◽  
Yanjing Yang ◽  
Ke Quan ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Tumor Cells ◽  
Signal Amplification ◽  
Fluorescence Signal ◽  
Target Mrna ◽  
Highly Sensitive ◽  
Fluorescence Signal Amplification

This strategy uses two fluorophore-labeled signal probes to generate a supersandwich product, which in turn generates numerous signal probes located at the target mRNA position, resulting in thein situfluorescence signal amplification.

An In-Situ Oblique-Incidence Optical Reflectance Difference Study of Co Electrodeposition on a Polycrystalline Au(111) Surface

2002 ◽  
Vol 721 ◽  
Author(s):  
J. Gray ◽  
W. Schwarzacher ◽  
X.D. Zhu
Keyword(s):  
Oblique Incidence ◽  
Three Dimensional ◽  
Oscillatory Behavior ◽  
Layer Growth ◽  
Deposition Condition ◽  
Layer By Layer ◽  
Optical Reflectance ◽  
Growth Modes ◽  
Highly Sensitive

AbstractWe studied submonolayer and multilayer deposition of Co on Au(111) using in-situ oblique-incidence optical reflectance difference (OI-RD). We show that the optical technique is highly sensitive and accurate in determining the electrodeposited film thickness and growth mode. We found that the optically determined thickness of the ultrathin Co film is in very good agreement with that deduced from the integration of the anodic current during cyclic voltammetry (CV). From a weak oscillatory behavior of the optical reflectance difference signal, it seems that the growth of electrodeposited Co on Au(111) under pulsed deposition condition proceeds by a combination of three dimensional island and quasi layer-by-layer growth modes.

scholarly journals Lateral Flow Immunoassay Coupled with Copper Enhancement for Rapid and Sensitive SARS-CoV-2 Nucleocapsid Protein Detection

Biosensors ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 13
Author(s):  
Tao Peng ◽  
Xueshima Jiao ◽  
Zhanwei Liang ◽  
Hongwei Zhao ◽  
Yang Zhao ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Nucleocapsid Protein ◽  
High Efficiency ◽  
Protein Detection ◽  
Antigen Detection ◽  
Lateral Flow ◽  
Copper Deposition ◽  
Detection Sensitivity ◽  
Rapid Screening ◽  
Lateral Flow Immunoassay

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) is still raging all over the world. Hence, the rapid and sensitive screening of the suspected population is in high demand. The nucleocapsid protein (NP) of SARS-CoV-2 has been selected as an ideal marker for viral antigen detection. This study describes a lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles for rapid NP antigen detection, in which sensitivity was improved through copper deposition-induced signal amplification. The detection sensitivity of the developed LFIA for NP antigen detection (using certified reference materials) under the optimized parameters was 0.01 μg/mL and was promoted by three orders of magnitude to 10 pg/mL after copper deposition signal amplification. The LFIA coupled with the copper enhancement technique has many merits such as low cost, high efficiency, and high sensitivity. It provides an effective approach to the rapid screening, diagnosis, and monitoring of the suspected population in the COVID-19 outbreak.

scholarly journals Highly Sensitive Formaldehyde Detection Using Well-Aligned Zinc Oxide Nanosheets Synthesized by Chemical Bath Deposition Technique

2018 ◽  
Author(s):  
Eun-Bi Kim ◽  
Hyung Kee Seo
Keyword(s):  
Chemical Sensor ◽  
In Situ Deposition ◽  
Highly Sensitive ◽  
Wide Range ◽  
Zno Nanosheets ◽  
Effect Transistor ◽  
Serious Injury ◽  
Synthesized Materials ◽  
Zinc Oxide Nanosheets

Detection of formaldehyde is very important in terms of life protection, as it can cause serious injury to eyes, skin, mouth and gastrointestinal function if indirectly inhaled and hence researchers are putting effort in developing novel and sensitive device. In this work, we have fabricated an electro-chemical sensor in the form of a field effect transistor (FET) to detect formaldehyde over wide range (10 nM to 1 mM). For this, ZnO nanosheets (NS) were first synthesized by hydrothermal method with in-situ deposition on cleaned SiO2 coated Si (100) substrate. The synthesized materials were characterized for morphology and purity and surface area (31.718 m2/g). The developed device was tested for formaldehyde detection at room temperature that resulted in a linear response with concentration (96%), sensitivity value of 0.27 mA/M/cm2 and a low detection limit of 210 nM, and a high 0.93194 return.

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