Isolation Reaction Research Articles

Development of an ultra-sensitive multiplex cytokine assay utilizing a novel digital multi-omics detection technology

Abstract Demand for rapid, highly sensitive measurement of proteins and nucleic acids has increased dramatically over the past several years to support biomarker research, drug development and diagnostics. In addition, the ability to interrogate both protein and nucleic acids (multi-omics) in a single sample allows critical information to be obtained relating the transcriptome to expressed protein levels. Despite this, current platforms for multiplex measurement of circulating biomarkers can lack sensitivity and specificity required for analytical and clinical validation of low abundance proteins and nucleic acids. We report the development of an automated microfluidic-based technology capable of multiplexed measurement of protein and nucleic acid biomarkers utilizing a novel approach combining digital and analog detection modalities. Significant improvements in sensitivity and specificity are achieved through use of a bead-based microfluidic array resulting in a spatially-resolved series of single-plex signal amplification reactions. Critically, the isolation of reactions allows multiplex panels to be constructed in a modular manner while eliminating interference that often limits specificity of traditional multiplex biomarker detection technologies. A specific application of this technology we report here is a multiplexed assay measuring low abundance cytokine biomarkers from serum and plasma samples with utility in a broad range of research and clinical studies.

The nucleotide sequence in the promoter region of the fene for an Escherichia coli tyrosine transfer ribonucleic acid.

The sequence of 29 nucleotides in the region preceding the initiation of the transcription of the Escherichia coli tyrosine tRNA gene has been determined. This is: (5') ---GGGGCGCATCATATCAAATGACGCGCCGC--- (3') (3') ---CCCCGCGTAGTATAGTTTACTGCGGCGGCG--- (5'). The general approach used for the sequence determination involved the DNA polymerase I-catalyzed elongation of suitable deoxyribopolynucleotide primers when hybridized to the 1-strand of phi80psuIII plus DNA at the appropriate site. Sequences of the newly grown oligonucleotide chains were determined by a combination of two-dimensional fingerprinting following partial exonucleolytic degradation, nearst neighbor analyses, and determination of pyrimidine tracts. Primer elongations were carried out in a controlled and stepwise manner and the newly grown oligonucleotide chains were kept short by incorporating the following features into the method: (a) the insertion of a ribonucleotide unit at or near the 3' terminus of the primers; (b) the use of a maximum of three nucleoside 5'-triphosphates in the first stage of the elongation reaction isolation of the elongated primer, and its reuse in a second step together with different sets of deoxy-nucleoside triphosphates; and (c) elongation of the primer using all of the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.