Supercolor Coding Methods for Large-Scale Multiplexing of Biochemical Assays

Rajagopal, Aditya; Scherer, Axel; Homyk, Andrew; Kartalov, Emil P.

doi:10.1021/ac401304t

Cited by 15 publications

(16 citation statements)

References 36 publications

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“…The most common approach is performing PCR with oligonucleotides attached to probes with different emission wavelengths and detect them using several fluorescence channels 17 . The number of genes identifiable per fluorescence channel can be further increased by creating colour codes 18 for the different genes and extracting information on amplicon concentrations via linear combinations on the individual fluorescence channels 19 . Theoretically, the number of genes concurrently detectable is unrestricted, but there are various limits imposed through the optical filter system required and through the dye assays.…”

mentioning

confidence: 99%

Doubling Throughput of a Real-Time PCR

Ahrberg

Neužil

2015

Sci Rep

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The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one was without a probe. The PCR was performed in the presence of the intercalating dye SYBR Green I. We collected the fluorescence amplitude at two points per PCR cycle, at the denaturation and extension steps. The signal at denaturation is related only to the amplicon with the FAM probe while the amplitude at the extension contained information from both amplicons. We thus detected two genes within the same well using a single fluorescent channel. Any commercial real-time PCR systems can use this method doubling the number of detected genes. The method can be used for absolute quantification of DNA using a known concentration of housekeeping gene at one fluorescent channel.

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confidence: 99%

Doubling Throughput of a Real-Time PCR

Ahrberg

Neužil

2015

Sci Rep

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“…As a recent example, an OR gate-based immunoassay was developed for the simultaneous detection of seven protein components of B. cereus toxins [120]. In addition, a novel mathematical theory has been proposed to encode and decode multiplexed assays, which enables a theoretically unlimited number of independent targets to be detected and identified in any combination in the same sample [130]. …”

Section: New Materials and Methodsmentioning

confidence: 99%

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

Zhu

Dietrich

Didier

et al. 2014

Toxins

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Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

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“…The multiplex assays require complex technology such as PCR, ELISA, microarrays, gel electrophoresis, capillary electrophoresis, Sanger sequencing, etc. The fluorescence spectroscopy measurements are important due to its compatibility with biochemical assays, small size of sample, ease of conjugation to potential molecules, affordability, stability, robustness and detection with less expensive optical instruments [7]. Mass spectrometry (MS) can identify molecules without separation.…”

Section: Principle Of Multiplex Assaysmentioning

confidence: 99%

Multiplex Technology for Biomarker Immunoassays

Ahsan¹,

Ahmad²

2021

Innate Immunity in Health and Disease

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The simultaneous measurement of different substances from a single sample is an emerging area for achieving efficient and high-throughput detection in several applications. Although immunoanalytical techniques are established and advantageous over alternative screening analytical platforms, one of the challenges for immunoassays is multiplexing. While ELISA is still commonly used to characterise a single analyte, laboratories and organisations are moving towards multiplex immunoassays. The validation of novel biomarkers and their amalgamation into multiplex immunoassays confers the prospects of simultaneous measurement of multiple analytes in a single sample, thereby minimising cost, time and sample. Therefore, the technological advancement in clinical sciences is helpful in the identification of analytes or biomarkers in test samples. However, the analytical bioanalysers are expensive and capable of detecting only a small amount or type of analytes. The simultaneous measurement of different substances from a single sample called multiplexing has become increasingly important for the quantification of pathological or toxicological samples. Although multiplex assays have many advantages over conventional assays, there are also problems that may cause apprehension among clinicians and researchers. Hence, many challenges still remain for these multiplexing systems which are at early stages of development.

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Supercolor Coding Methods for Large-Scale Multiplexing of Biochemical Assays

Cited by 15 publications

References 36 publications

Doubling Throughput of a Real-Time PCR

Doubling Throughput of a Real-Time PCR

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

Multiplex Technology for Biomarker Immunoassays

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