Bead-based polymerase chain reaction on a microchip

Hilton, John P.; Nguyen, Thai; Barbu, Mihaela; Pei, Renjun; Stojanović, Milan N.; Lin, Qiao

doi:10.1007/s10404-012-0993-8

Cited by 8 publications

(5 citation statements)

References 42 publications

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“…The amplification chamber was tested for its ability to properly amplify DNA by performing bead-based PCR of a sample of library DNA. We previously characterized bead-based microfluidic PCR in an integrated device (Hilton et al 2012). Sample loss and bubble generation were minimized by coating the chip with Parylene C and degassing reagents prior to temperature cycling.…”

Section: Resultsmentioning

confidence: 99%

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Isolation of thermally sensitive protein-binding oligonucleotides on a microchip

Hilton

Olsen

Kim

et al. 2015

Microfluid Nanofluid

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This paper presents a microfluidic chip capable of isolating thermally sensitive protein-binding aptamer candidates. The chip makes use of bead-immobilized target molecules and DNA (deoxyribonucleic acid) sequences to enable a simplified chip design, in which affinity selection and PCR (polymerase chain reaction) amplification of target-binding sequences occur in temperature-controlled microchambers. Using pressure-driven flow, buffer containing single-stranded DNA molecules with randomized sequences is cycled through a series of affinity selection and PCR amplification steps on microbeads. Successive introduction of the sample to each chamber effects a process of competition whereby DNA strands with weak binding strength to target molecules are rejected in favor of strongly binding sequences. Using bead-based PCR, the amplification step was miniaturized and integrated with affinity selection, resulting in significant reductions in process time and reagent use. As a demonstration, temperature-dependent selection and amplification of single-stranded oligonucleotides that bind to human Immuno-globulin E (IgE) was performed in 4 h, a 20-fold reduction in process time as compared to conventional methods that would require approximately a week. Fluorescent binding assays then demonstrated that the desired temperature specificity was imparted to the aptamer candidates within just one round of selection, and within two rounds the aptamer candidates exhibited enhanced affinity toward IgE.

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Section: Resultsmentioning

confidence: 99%

“…Target-binding oligomers obtained by affinity selection are then chemically amplified by polymerase chain reaction (PCR), using a bead-based protocol described in detail elsewhere (Hilton et al 2012). Briefly, polymeric microbe-ads are functionalized with reverse primers and held in a second chamber of the microchip.…”

Section: Methodsmentioning

confidence: 99%

Isolation of thermally sensitive protein-binding oligonucleotides on a microchip

Hilton

Olsen

Kim

et al. 2015

Microfluid Nanofluid

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“…The target-binding oligonucleotides are then amplified by PCR on microbeads 44 ( Fig. 1d and Supplementary Information Figure S2 ).…”

Section: Methodsmentioning

confidence: 99%

“…The electric field (25 V/cm) for electrophoresis was generated in the interconnection channel by applying a voltage (from the power supply) to platinum (Pt)-wire electrodes inserted into the bead inlets. The temperature in each chamber was controlled within ±0.5 °C using the integrated heater and temperature sensors by a LabVIEW-based proportional-integral-derivative (PID) controller that was connected to a multimeter (34410A, Agilent Technologies) and a power supply (E3631A, Agilent Technologies) ( Supplementary Information, Figure S4 ) 44 .…”

Section: Methodsmentioning

confidence: 99%

Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

Kim

Olsen

Zhu

et al. 2016

Sci Rep

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We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours.

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“…With the optimal concentration, PCR efficiency has been doubled [ 22 ]. Hilton et al [ 111 ] used a Parylene C coating on microchamber surfaces.…”

Section: Amplificationmentioning

confidence: 99%

Towards Multiplex Molecular Diagnosis—A Review of Microfluidic Genomics Technologies

Basha

Yousuff

et al. 2017

Micromachines

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Highly sensitive and specific pathogen diagnosis is essential for correct and timely treatment of infectious diseases, especially virulent strains, in people. Point-of-care pathogen diagnosis can be a tremendous help in managing disease outbreaks as well as in routine healthcare settings. Infectious pathogens can be identified with high specificity using molecular methods. A plethora of microfluidic innovations in recent years have now made it increasingly feasible to develop portable, robust, accurate, and sensitive genomic diagnostic devices for deployment at the point of care. However, improving processing time, multiplexed detection, sensitivity and limit of detection, specificity, and ease of deployment in resource-limited settings are ongoing challenges. This review outlines recent techniques in microfluidic genomic diagnosis and devices with a focus on integrating them into a lab on a chip that will lead towards the development of multiplexed point-of-care devices of high sensitivity and specificity.

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Bead-based polymerase chain reaction on a microchip

Cited by 8 publications

References 42 publications

Isolation of thermally sensitive protein-binding oligonucleotides on a microchip

Isolation of thermally sensitive protein-binding oligonucleotides on a microchip

Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

Towards Multiplex Molecular Diagnosis—A Review of Microfluidic Genomics Technologies

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