A rapid and economical method for profiling feature heights during microfabrication

Yen, Gloria S.; Fujimoto, Bryant S.; Schneider, Thomas; Huynh, Desmond T.K.; Jeffries, Gavin D. M.; Chiu, Daniel T.

doi:10.1039/c0lc00470g

Cited by 12 publications

(10 citation statements)

References 15 publications

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“…After curing, non-exposed SU-8 was dissolved in PGMEA and the wafer was cleaned with isopropyl alcohol and hard baked for 10 minutes at 155 °C. The height of the positive features in the master was measured in a home-built interferometer 33 to be around 75 μm. To avoid sticking of PDMS onto the wafer during replication, the wafer was coated with (tridecafluoro)-1,1,2,2-tetrahydrooctyl) trichlorosilane (Gelest, Morrisville, PA, USA) by gas-phase deposition.…”

Section: Methodsmentioning

confidence: 99%

Digital LAMP in a sample self-digitization (SD) chip

et al. 2012

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This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource—limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we showed the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings.

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Section: Methodsmentioning

confidence: 99%

Digital LAMP in a sample self-digitization (SD) chip

et al. 2012

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“…After developing the photoresist, the wafers were etched to a depth of 61 m using a Bosche deep reactive ion etch process (Oxford Instruments ICP 380). The etch depth of 61 m was characterized using an optical profilometer (41). The wafers were diced and mounted in aluminum molds.…”

Section: Methodsmentioning

confidence: 99%

Clonal Variants of Plasmodium falciparum Exhibit a Narrow Range of Rolling Velocities to Host Receptor CD36 under Dynamic Flow Conditions

et al. 2013

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c Cytoadhesion of Plasmodium falciparum parasitized red blood cells (pRBCs) has been implicated in the virulence of malaria infection. Cytoadhesive interactions are mediated by the protein family of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). The PfEMP1 family is under strong antibody and binding selection, resulting in extensive sequence and size variation of the extracellular domains. Here, we investigated cytoadhesion of pRBCs to CD36, a common receptor of P. falciparum field isolates, under dynamic flow conditions. Isogeneic parasites, predominantly expressing single PfEMP1 variants, were evaluated for binding to recombinant CD36 under dynamic flow conditions using microfluidic devices. We tested if PfEMP1 size (number of extracellular domains) or sequence variation affected the pRBC-CD36 interaction. Our analysis showed that clonal parasite variants varied ϳ5-fold in CD36 rolling velocity despite extensive PfEMP1 sequence polymorphism. In addition, adherent pRBCs exhibited a characteristic hysteresis in rolling velocity at microvascular flow rates, which was accompanied by changes in pRBC shape and may represent important adaptations that favor stable binding.

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“…The main channel height was measured to be 25 ± 1 μm, and the chamber height was 104 ± 3 μm for the serpentine design and 113 ± 5 μm for the bifurcated design as determined by a custom-built white-light interferometer. 20 The serpentine device, used in single-cell experiments, contained 1020 chambers, while a bifurcated main channel device, used in dilution series experiments, contained 1024 channels. Details of the device dimensions and assembly are available as Figure S-2 and S-3 ( Supporting Information ).…”

Section: Methodsmentioning

confidence: 99%

Self-Digitization Microfluidic Chip for Absolute Quantification of mRNA in Single Cells

et al. 2014

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Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

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A rapid and economical method for profiling feature heights during microfabrication

Cited by 12 publications

References 15 publications

Digital LAMP in a sample self-digitization (SD) chip

Digital LAMP in a sample self-digitization (SD) chip

Clonal Variants of Plasmodium falciparum Exhibit a Narrow Range of Rolling Velocities to Host Receptor CD36 under Dynamic Flow Conditions

Self-Digitization Microfluidic Chip for Absolute Quantification of mRNA in Single Cells

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