Integrated microfluidic bioprocessor for single-cell gene expression analysis

Toriello, Nicholas M.; Douglas, Erik S.; Thaitrong, Numrin; Hsiao, Sonny C.; Francis, Matthew B.; Bertozzi, Carolyn R.; Mathies, Richard A.

doi:10.1073/pnas.0806355106

Cited by 224 publications

(184 citation statements)

References 35 publications

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“…The critical step of integrating all steps of singlecell analysis into a robust system capable of performing measurements on large numbers of cells has yet to be reported. A single demonstration of an integrated device for directly measuring gene expression in single cells was described by Toriello et al, combining all steps of RNA capture, PCR amplification, and end-point detection of amplicons using integrated capillary electrophoresis (17). Despite the engineering complexity of this system, throughput was limited to four cells per run, cell capture required metabolic labeling of the cells, and the analysis was not quantitative.…”

mentioning

confidence: 99%

High-throughput microfluidic single-cell RT-qPCR

White

VanInsberghe

Petriv

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

409

368

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A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells. Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity. We apply this technology to 3,300 single-cell measurements of ( i ) miRNA expression in K562 cells, ( ii ) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and ( iii ) single nucleotide variant detection in primary lobular breast cancer cells. The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed.

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mentioning

confidence: 99%

High-throughput microfluidic single-cell RT-qPCR

White

VanInsberghe

Petriv

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

409

368

View full text Add to dashboard Cite

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“…NCVs made of hybrid glass/PDMS materials [28] have been used for automated and quantitative single cell gene expression analysis [29,30], but are millimeters in size, rendering them non-scalable. NCVs introduced by Devaraju et al [16 ] achieve scalability, but require a flash curable polymer; this technology was used for building static gain valves and consequently for realizing microfluidic logic circuits.…”

Section: Component-level Developmentsmentioning

confidence: 99%

Recent developments in microfluidic large scale integration

Araci

Brisk

2014

Current Opinion in Biotechnology

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In 2002, Thorsen et al. integrated thousands of micromechanical valves on a single microfluidic chip and demonstrated that the control of the fluidic networks can be simplified through multiplexors [1]. This enabled realization of highly parallel and automated fluidic processes with substantial sample economy advantage. Moreover, the fabrication of these devices by multilayer soft lithography was easy and reliable hence contributed to the power of the technology; microfluidic large scale integration (mLSI). Since then, mLSI has found use in wide variety of applications in biology and chemistry. In the meantime, efforts to improve the technology have been ongoing. These efforts mostly focus on; novel materials, components, micromechanical valve actuation methods, and chip architectures for mLSI. In this review, these technological advances are discussed and, recent examples of the mLSI applications are summarized. Recent advancements in microfluidic technologies have created new opportunities in the fields of biology and chemistry through miniaturization and automation of common processes, including mixing, metering, trapping, reaction, filtering, and separation, among others. Microfluidic large scale integration (mLSI) enables the fabrication of microfluidic chips containing hundreds or more of these functions using well-established lithography techniques, similar in principle to electronic LSI in the semiconductor industry [1]. Small feature dimensions, abundant spatial parallelism, and control over fluid flow provide mLSI technology with advantages such as high throughput, precise metering, automation and low reagent consumption. The implications of mLSI technology can especially be seen in recent advances in single cell, genomic, and protein analysis.

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“…37.5 g of IrCl 4 was added to 75 mL of de-ionized water and stirred for 90 min. Next, 125 mg of oxalic acid was added, and the solution was stirred for 3 h. Finally, the solution pH was adjusted to 11 using K 2 CO 3 .…”

Section: Electrode Sensor Fabricationmentioning

confidence: 99%

“…With size and volume scales comparable to those of individual cells, microfluidic devices provide a powerful tool for control of the cellular microenvironment.1 Previously we have demonstrated the use of engineered cell surface DNA (cell adhesion barcodes) for cell capture,2,3 and the use of this capture technique to perform single-cell gene expression analysis in a microfluidic chip. 4 Here we describe the use of DNA barcode cell capture to populate an array of pH-sensitive microelectrodes, enabling the rapid, selective and reversible capture of both adherent and non-adherent single cells on the pH sensor surface. This bifunctional system enables accurate real-time monitoring of single cell metabolism because extracellular acidification is proportional to overall energy usage.…”

Section: Introductionmentioning

confidence: 99%

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array

et al. 2009

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A microdevice is developed for DNA-barcode directed capture of single cells on an array of pHsensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound singlestranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min −1 , while primary T cells exhibited only 2 milli-pH min −1 . This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties.

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Integrated microfluidic bioprocessor for single-cell gene expression analysis

Abstract: lab-on-a-chip ͉ microfabrication ͉ RNAi ͉ stochastic gene expression

Cited by 224 publications

References 35 publications

High-throughput microfluidic single-cell RT-qPCR

High-throughput microfluidic single-cell RT-qPCR

Recent developments in microfluidic large scale integration

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array

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