Micromagnetic selection of aptamers in microfluidic channels

Lou, Xinhui; Qian, Jiangrong; Xiao, Yi; Viel, Lisan; Gerdon, Aren E.; Lagally, Eric T.; Atzberger, Paul J.; Tarasow, Theodore M.; Heeger, Alan J.; Soh, H. Tom

doi:10.1073/pnas.0813135106

Cited by 320 publications

(294 citation statements)

References 45 publications

(67 reference statements)

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“…Aptamers have been shown to be capable of binding a wide variety of target molecules [5,17]. The possibility that the genome might encode similar such sequences for mRNAs with the capability to bind promoter sites and perform regulatory functions has also been discussed in [5,19,24].…”

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confidence: 99%

Stochastic reduction method for biological chemical kinetics using time-scale separation

Pahlajani

Atzberger

Khammash

2011

Journal of Theoretical Biology

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Abstract. Many processes in cell biology encode and process information and enact responses by modulating the concentrations of biological molecules. Such modulations serve functions ranging from encoding and transmitting information about external stimuli to regulating internal metabolic states. To understand how such processes operate requires gaining insights into the basic mechanisms by which biochemical species interact and respond to internal and external perturbations. One approach is to model the biochemical species concentrations through the van Kampen Linear Noise Equations, which account for the change in biochemical concentrations from reactions and account for fluctuations in concentrations. For many systems, the Linear Noise Equations exhibit stiffness as a consequence of the chemical reactions occurring at significantly different rates. This presents challenges in the analysis of the kinetics and in performing efficient numerical simulations. To deal with this source of stiffness and to obtain reduced models more amenable to analysis, we present a systematic procedure for obtaining effective stochastic dynamics for the chemical species having relatively slow characteristic time scales while eliminating representations of the chemical species having relatively fast characteristic time scales. To demonstrate the applicability of this multiscale technique in the context of Linear Noise Equations, the reduction is applied to models of gene regulatory networks. Results are presented which compare numerical results for the full system to the reduced descriptions. The presented stochastic reduction procedure provides a potentially versatile tool for systematically obtaining reduced approximations of Linear Noise Equations.

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confidence: 99%

Stochastic reduction method for biological chemical kinetics using time-scale separation

Pahlajani

Atzberger

Khammash

2011

Journal of Theoretical Biology

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“…We performed four rounds of M-SELEX using the micromagnetic separation (MMS) device previously developed by our group (16), which allowed us to uniformly control the selection and washing stringency (15)(16)(17)(18). Although the MMS device is capable of performing highly stringent washing, we used moderate selection conditions for this experiment.…”

Section: Resultsmentioning

confidence: 99%

“…S1). We minimized nonspecific binding of DNA to the bead surface by tuning the hydroxyl passivation layer, as previously described (15).…”

Section: Resultsmentioning

confidence: 99%

“…For example, novel separation methodologies such as capillary electrophoresis (13,14) and microfluidic devices (15)(16)(17) can isolate high-affinity aptamers after a minimal number of selection rounds. This increased efficiency is achieved through tighter control over selection stringency by applying rigorous washing to isolate aptamers with slow off-rates or by using minimal target quantities to establish a highly competitive binding environment.…”

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confidence: 99%

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Quantitative selection and parallel characterization of aptamers

Cho

Nie

et al. 2013

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

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115

114

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Aptamers are promising affinity reagents that are potentially well suited for high-throughput discovery, as they are chemically synthesized and discovered via completely in vitro selection processes. Recent advancements in selection, sequencing, and the use of modified bases have improved aptamer quality, but the overall process of aptamer generation remains laborious and low-throughput. This is because binding characterization remains a critical bottleneck, wherein the affinity and specificity of each candidate aptamer are measured individually in a serial manner. To accelerate aptamer discovery, we devised the Quantitative Parallel Aptamer Selection System (QPASS), which integrates microfluidic selection and nextgeneration sequencing with in situ-synthesized aptamer arrays, enabling simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel. After using QPASS to select aptamers for the human cancer biomarker angiopoietin-2 (Ang2), we in situ synthesized arrays of the selected sequences and obtained equilibrium dissociation constants (K d ) for every aptamer in parallel. We thereby identified over a dozen high-affinity Ang2 aptamers, with K d as low as 20.5 ± 7.3 nM. The same arrays enabled us to quantify binding specificity for these aptamers in parallel by comparing relative binding of differentially labeled target and nontarget proteins, and by measuring their binding affinity directly in complex samples such as undiluted serum. Finally, we show that QPASS offers a compelling avenue for exploring structure−function relationships for large numbers of aptamers in parallel by coupling array-based affinity measurements with next-generation sequencing data to identify nucleotides and motifs within the aptamer that critically affect Ang2 binding.

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“…[22][23][24] Using CE microfluidic chips and sol-gel microfluidic chips, SELEX was successfully performed with purified proteins, however, these systems are not suitable for selections against whole living cells. Compared to the magnetic-bead-based microfluidic chips, which can also be used for cell-SELEX, in our microfluidic chip, the conjugation of magnetic beads to the cells is not required.…”

Section: Cell-selex Experiments With the Developed Microfluidic Chmentioning

confidence: 99%

Microfluidic chip system for the selection and enrichment of cell binding aptamers

Stoll¹,

Kiessling

Stelzle

et al. 2015

Biomicrofluidics

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Aptamers are promising cell targeting ligands for several applications such as for the diagnosis, therapy, and drug delivery. Especially, in the field of regenerative medicine, stem cell specific aptamers have an enormous potential. Using the combinatorial chemistry process SELEX (Systematic Evolution of Ligands by Exponential enrichment), aptamers are selected from a huge oligonucleotide library consisting of approximately 10 15 different oligonucleotides. Here, we developed a microfluidic chip system that can be used for the selection of cell specific aptamers. The major drawbacks of common cell-SELEX methods are the inefficient elimination of the unspecifically bound oligonucleotides from the cell surface and the unspecific binding/uptake of oligonucleotides by dead cells. To overcome these obstacles, a microfluidic device, which enables the simultaneous performance of dielectrophoresis and electrophoresis in the same device, was designed. Using this system, viable cells can be selectively assembled by dielectrophoresis between the electrodes and then incubated with the oligonucleotides. To reduce the rate of unspecifically bound sequences, electrophoretic fields can be applied in order to draw loosely bound oligonucleotides away from the cells. Furthermore, by increasing the flow rate in the chip during the iterative rounds of SELEX, the selection pressure can be improved and aptamers with higher affinities and specificities can be obtained. This new microfluidic device has a tremendous capability to improve the cell-SELEX procedure and to select highly specific aptamers. V C 2015 AIP Publishing LLC. [http://dx

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Micromagnetic selection of aptamers in microfluidic channels

Cited by 320 publications

References 45 publications

Stochastic reduction method for biological chemical kinetics using time-scale separation

Stochastic reduction method for biological chemical kinetics using time-scale separation

Quantitative selection and parallel characterization of aptamers

Microfluidic chip system for the selection and enrichment of cell binding aptamers

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