scholarly journals Optimization of Rolling-Circle Amplified Protein Microarrays for Multiplexed Protein Profiling

2003 ◽  
Vol 2003 (5) ◽  
pp. 299-307 ◽  
Author(s):  
Weiping Shao ◽  
Zhimin Zhou ◽  
Isabelle Laroche ◽  
Hong Lu ◽  
Qiuling Zong ◽  
...  
Keyword(s):  
High Throughput ◽  
Signal Amplification ◽  
Protein Microarray ◽  
Cost Effective ◽  
Protein Microarrays ◽  
Clinical Applications ◽  
Protein Profiling ◽  
Antibody Microarray ◽  
High Throughput Analysis ◽  
Rolling Circle

Protein microarray-based approaches are increasingly being used in research and clinical applications to either profile the expression of proteins or screen molecular interactions. The development of high-throughput, sensitive, convenient, and cost-effective formats for detecting proteins is a necessity for the effective advancement of understanding disease processes. In this paper, we describe the generation of highly multiplexed, antibody-based, specific, and sensitive protein microarrays coupled with rolling-circle signal amplification (RCA) technology. A total of 150 cytokines were simultaneously detected in an RCA sandwich immunoassay format. Greater than half of these proteins have detection sensitivities in the pg/ml range. The validation of antibody microarray with human serum indicated that RCA-based protein microarrays are a powerful tool for high-throughput analysis of protein expression and molecular diagnostics.

A critical comparison of protein microarray fabrication technologies

The Analyst ◽  
2014 ◽  
Vol 139 (6) ◽  
pp. 1303-1326 ◽  
Author(s):  
Valentin Romanov ◽  
S. Nikki Davidoff ◽  
Adam R. Miles ◽  
David W. Grainger ◽  
Bruce K. Gale ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Microarray ◽  
Protein Microarrays ◽  
Antibody Affinity ◽  
High Throughput Analysis ◽  
Critical Comparison ◽  
Binding Partners ◽  
Fundamental Information ◽  
Microarray Fabrication ◽  
Analytical Tools

Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis.

scholarly journals Next-Generation Phage Display: Integrating and Comparing Available Molecular Tools to Enable Cost-Effective High-Throughput Analysis

PLoS ONE ◽  
2009 ◽  
Vol 4 (12) ◽  
pp. e8338 ◽  
Author(s):  
Emmanuel Dias-Neto ◽  
Diana N. Nunes ◽  
Ricardo J. Giordano ◽  
Jessica Sun ◽  
Gregory H. Botz ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
Cost Effective ◽  
Next Generation ◽  
Molecular Tools ◽  
High Throughput Analysis ◽  
Throughput Analysis

scholarly journals High-Throughput Screening of Novel Peptide Inhibitors of an Integrin Receptor from the Hexapeptide Library by Using a Protein Microarray Chip

2004 ◽  
Vol 9 (8) ◽  
pp. 687-694 ◽  
Author(s):  
Yoonsuk Lee ◽  
Dong-Ku Kang ◽  
Soo-Ik Chang ◽  
Moon Hi Han ◽  
In-Cheol Kang
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Competitive Inhibition ◽  
Protein Microarray ◽  
Dependent Manner ◽  
High Throughput Analysis ◽  
Microarray Chip ◽  
New Drug

Protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, Proteo Chip, in new drug discovery. Integrin αvβ3 microarray immobilized on the Proteo Chip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin αvβ3-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner andwas inhibited not only by the syntheticRGDpeptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and highthroughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the Proteo Chip is a promising tool for highthroughput screening of lead molecules in new drug development.

scholarly journals High-Throughput Genomic and Proteomic Analysis Using Microarray Technology

2001 ◽  
Vol 47 (10) ◽  
pp. 1912-1916 ◽  
Author(s):  
Joe Xi Huang ◽  
Dorothy Mehrens ◽  
Rick Wiese ◽  
Sandy Lee ◽  
Sun W Tam ◽  
...  
Keyword(s):  
High Throughput ◽  
Disease Risk ◽  
Protein Microarray ◽  
Correlation Coefficients ◽  
Rflp Analysis ◽  
Protein Profiling ◽  
Microarray Technology ◽  
Nucleotide Polymorphisms ◽  
Technology Platform ◽  
Nat2 Gene

Abstract Background: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication. Methods: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000–24 000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel. Results: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82–0.99 (depending on analyte) compared with ELISAs. Conclusions: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.

scholarly journals Novel Autoantibody Signatures in Sera of Patients with Pancreatic Cancer, Chronic Pancreatitis and Autoimmune Pancreatitis: A Protein Microarray Profiling Approach

2020 ◽  
Vol 21 (7) ◽  
pp. 2403
Author(s):  
Sahar Ghassem-Zadeh ◽  
Katrin Hufnagel ◽  
Andrea Bauer ◽  
Jean-Louis Frossard ◽  
Masaru Yoshida ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Chronic Pancreatitis ◽  
Autoimmune Pancreatitis ◽  
Large Scale ◽  
Gastrointestinal Disease ◽  
Protein Microarray ◽  
Cost Effective ◽  
Protein Microarrays ◽  
Large Scale Analysis ◽  
Diagnostic Modalities

Identification of disease-associated autoantibodies is of high importance. Their assessment could complement current diagnostic modalities and assist the clinical management of patients. We aimed at developing and validating high-throughput protein microarrays able to screen patients’ sera to determine disease-specific autoantibody-signatures for pancreatic cancer (PDAC), chronic pancreatitis (CP), autoimmune pancreatitis and their subtypes (AIP-1 and AIP-2). In-house manufactured microarrays were used for autoantibody-profiling of IgG-enriched preoperative sera from PDAC-, CP-, AIP-1-, AIP-2-, other gastrointestinal disease (GID) patients and healthy controls. As a top-down strategy, three different fluorescence detection-based protein-microarrays were used: large with 6400, intermediate with 345, and small with 36 full-length human recombinant proteins. Large-scale analysis revealed 89 PDAC, 98 CP and 104 AIP immunogenic antigens. Narrowing the selection to 29 autoantigens using pooled sera first and individual sera afterwards allowed a discrimination of CP and AIP from PDAC. For validation, predictive models based on the identified antigens were generated which enabled discrimination between PDAC and AIP-1 or AIP-2 yielded high AUC values of 0.940 and 0.925, respectively. A new repertoire of autoantigens was identified and their assembly as a multiplex test will provide a fast and cost-effective tool for differential diagnosis of pancreatic diseases with high clinical relevance.

scholarly journals High-throughput, cost-effective verification of structural DNA assembly

2013 ◽  
Vol 42 (4) ◽  
pp. e22-e22 ◽  
Author(s):  
Yandi Dharmadi ◽  
Kedar Patel ◽  
Elaine Shapland ◽  
Daniel Hollis ◽  
Todd Slaby ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Rolling Circle Amplification ◽  
Cost Effective ◽  
Building Blocks ◽  
Pathway Engineering ◽  
Dna Assembly ◽  
Rolling Circle ◽  
Enzyme Screening ◽  
First Time

Abstract DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (∼1 kb), pathway engineering (∼10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.

Base excision repair initiated rolling circle amplification-based fluorescent assay for screening uracil-DNA glycosylase activity using Endo IV-assisted cleavage of AP probes

The Analyst ◽  
2018 ◽  
Vol 143 (16) ◽  
pp. 3951-3958 ◽  
Author(s):  
Jingfeng Wang ◽  
Yu Wang ◽  
Su Liu ◽  
Haiwang Wang ◽  
Xue Zhang ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Signal Amplification ◽  
Excision Repair ◽  
Rolling Circle Amplification ◽  
Cost Effective ◽  
Ultrasensitive Detection ◽  
Fluorescent Assay ◽  
Uracil Dna Glycosylase ◽  
Rolling Circle ◽  
Base Excision

A simple, robust and cost effective biosensing platform for the ultrasensitive detection of UDG activity was established based on base excision repair-initiated primer generation for RCA with Endo IV-assisted signal amplification.

scholarly journals Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray

2020 ◽  
Author(s):  
Tate Oulton ◽  
Joshua Obiero ◽  
Isabel Rodriguez ◽  
Isaac Ssewanyana ◽  
Rebecca A Dabbs ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Microarray ◽  
Expression System ◽  
Protein Microarrays ◽  
In Vitro Transcription ◽  
Antibody Responses ◽  
Dependent Manner ◽  
Expression Systems ◽  
Protein Antigens

The evaluation of protein antigens as putative serologic biomarkers of infection has increasingly shifted to high-throughput, multiplex approaches such as the protein microarray. In vitro Transcription/Translation (IVTT) systems (a similarly high-throughput protein expression method) are already widely utilised in the production of protein microarrays, though purified recombinant proteins derived from more traditional whole cell based expression systems also play an important role in biomarker characterisation. Here we have performed a side-by-side comparison of antigen-matched protein targets from an IVTT and purified recombinant system, on a protein microarray. The magnitude and range of antibody responses to purified recombinants was found to be greater than that of IVTT proteins, and responses between targets from opposing expression systems did not clearly correlate. However, responses between amino acid sequence-matched targets from each expression system were more closely correlated. Despite the lack of a clearly defined relationship between antigen-matched targets produced in each expression system, our data indicate that protein microarrays produced using either method can be used confidently, in a context dependent manner, though care should be taken when comparing data derived from contrasting approaches.

scholarly journals Microfluidic flow cytometry: The role of microfabrication methodologies, performance and functional specification

TECHNOLOGY ◽  
2018 ◽  
Vol 06 (01) ◽  
pp. 1-23 ◽  
Author(s):  
Anil B. Shrirao ◽  
Zachary Fritz ◽  
Eric M. Novik ◽  
Gabriel M. Yarmush ◽  
Rene S. Schloss ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Point Of Care ◽  
Limited Resource ◽  
Cost Effective ◽  
Particle Analysis ◽  
High Throughput Analysis ◽  
Current State ◽  
Trade Offs ◽  
Microfluidic Flow

Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers.

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