Isolation of thermally sensitive protein-binding oligonucleotides on a microchip

Abstract

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.

An Integrated Microfluidic SELEX Approach Using Combined Electrokinetic and Hydrodynamic Manipulation

“…Because this measurement relied on many manual transfers the error increases with higher concentrations of binding aptamer candidates. The data were well represented by a Langmuir monovalent binding model, with an equilibrium dissociation constant ( K D ) of approximately 12 nM (obtained via nonlinear regression) as is consistent with that of existing IgE aptamers 4b, 27a . In contrast, when the enriched pool was incubated with IgA functionalized beads or bare beads, washed, and bound oligonucleotides were eluted and measured, the fluorescently increased very slowly with the oligonucleotide concentration.…”

“…Next, reverse primer-functionalized magnetic beads are introduced into the amplification chamber and held via an external magnet. The oligonucleotides bound to the bead-immobilized target are thermally released from with the selection chamber’s integrated heater, and electrokinetically transferred through the gel-filled channel to the amplification chamber, and captured therein by the bead-bound reverse primer 27a . PCR reagents are then introduced and PCR performed using the amplification chamber’s integrated heater and temperature sensor.…”

“…While capable of performing individual affinity selection and PCR amplification processes on-chip, these devices apparently require an off-chip procedure to retrieve single-stranded DNA from amplified products between successive rounds of SELEX, and do not yet allow full integration of SELEX on a microchip. We have recently demonstrated integration of the entire SELEX process using solid-phase capture and amplification of oligonucleotides on bead surfaces, which are manipulated using either pressure-driven flow or electrokinetics 27 . In these works we found the use of electrokinetic manipulation simplified the device design but involved electric fields that can potentially comprise the integrity of the target or adversely affect the target-aptamer binding.…”

Recent Microdevice-Based Aptamer Sensors

“…In recent years, microfluidic chips have aroused increasing interest for various application because of their desirable features, such as the smaller sample amount needed and lowered reagent consumption. The substrate materials of microfluidic chips such as polymers (e.g., PDMS, PMMA, PS) [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ], ceramics (e.g., glass) [ 6 , 13 , 14 , 16 , 17 , 18 , 19 , 21 , 25 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ], and semiconduct...…”

Integrated Microfluidic Selex Using Free Solution Electrokinetics

“…Microfluidic affinity selection has been performed, in conjunction with off-chip amplification, against targets retained using silica capillary walls [24], microbeads [25–32] or sol-gels [33–35], or against cells in solution [36, 37], to increase selection stringency [25–29, 36, 37], create more favorable biomolecular environments [33–35], or allow simultaneous positive and negative selections [38]. Moreover, microchips have been developed in an attempt to integrate the overall SELEX process using pneumatically based flow control for isolation of aptamers against viruses [39], cells [28, 40, 41] and proteins [30–32, 42, 43]. While capable of isolating aptamers these microfluidic devices have relied on highly complex fabrication techniques not available to most researchers, extensive flow control components (integrated pumps, valves and mixers which all require additional off-chip equipment), sophisticated operating equipment, complicated designs, and integration of unstable gels.…”