A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells

Haider, Michaela; Ji, Bozhi; Haselgrübler, Thomas; Sonnleitner, Alois; Aberger, Fritz; Hesse, Jan

doi:10.1016/j.bios.2016.06.019

Cited by 6 publications

(2 citation statements)

References 38 publications

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“…Ottesen used microfluidic digital PCR to amplify multiple, various genes from bacterial cells and used a gene involved in termites mutualistic symbiosis to discover the unknown ribosomal RNA‐based species identity of several symbionts . Enzyme‐free detection of mRNA using specific staining and immobilization of the target molecules via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification from very few cells was also realized by Haider . Zhang reviewed the advances and challenges on continuous‐flow microfluidic PCR in droplets .…”

Section: Advanced Microfluidic Applicationsmentioning

confidence: 99%

Advances in Microfluidics Applied to Single Cell Operation

Zhu

Chu

Wang

2018

Biotechnology Journal

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The field of microbiology have traditionally been concerned with and focused on studies at the population level. Microfluidic platforms have emerged as important tools for biology research at a small scale, even down to a single cell level. The spatial and temporal control of cells and stimuli transported by microfluidic channels in well-designed microsystems realized the studies of specific cells in a controlled microenvironment. The true cellular physiology responses, which are obtained mostly by inference from population-level data, could be revealed in this way. Nowadays, significant applications like cell culture, analysis, sorting, genomics, and proteomics at the single cell level have been achieved in microfluidic chips. Highly integrated microfluidic systems with complete bio-analytic functions are also coming forth and of great promise for single cell related physiology, biomedical, and high throughput screening research. Herein, the leads of technologies applied to single cell operation are reviewed. Challenges and potentials of these works are also summarized, to highlight fields for further research.

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Section: Advanced Microfluidic Applicationsmentioning

confidence: 99%

Advances in Microfluidics Applied to Single Cell Operation

Zhu

Chu

Wang

2018

Biotechnology Journal

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“…This enormous discrepancy between theoretical and practical performance in microarray readout technology stems from the physical and optical requirements for single-fluorophore detection. Enumerating single molecules with fluorescence labeling is now a routine process and has been used extensively to develop ultrasensitive biosensors for a wide variety of applications, for example, single-molecule pull-down (SIMPull) co-immunoprecipitation for studying protein–protein interactions, Förster resonance energy-transfer measurements of individual molecular interactions, , single-molecule counting protein microarrays for protein expression analysis, − and single-molecule DNA microarrays for quantification of low-abundance RNA and DNA. , These techniques do indeed close the performance gap described earlier. Combining single-molecule counting with total fluorescence measurements has been shown to provide a usable dynamic range as high as 1 million and a detection limit near or below 1 femtomolar. , …”

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confidence: 99%

Digital Microarrays: Single-Molecule Readout with Interferometric Detection of Plasmonic Nanorod Labels

et al. 2018

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DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology's Achilles' heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ("digital") regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique's simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.

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