ThaiHuu Nguyen

Specific capture and temperature-mediated release of cells in an aptamer-based microfluidic device

Isolation of cells from heterogeneous mixtures is critically important in both basic cell biology studies and clinical diagnostics. Cell isolation can be realized based on physical properties such as size, density and electrical properties. Alternatively, affinity binding of target cells by surface-immobilized ligands, such as antibodies, can be used to achieve specific cell isolation. Microfluidics technology has recently been used in conjunction with antibody-based affinity isolation methods to capture, purify and isolate cells with higher yield rates, better efficiencies and lower costs. However, a method that allows easy release and collection of live cells from affinity surfaces for subsequent analysis and detection has yet to be developed. This paper presents a microfluidic device that not only achieves specific affinity capture and enrichment, but also enables non-destructive, temperature-mediated release and retrieval of cells. Specific cell capture is achieved using surface-immobilized aptamers in a microchamber. Release of the captured cells is realized by a moderate temperature change, effected via integrated heaters and a temperature sensor, to reversibly disrupt the cell-aptamer interaction. Experimental results with CCRF-CEM cells have demonstrated that the device is capable of specific capture and temperature-mediated release of cells, that the released cells remain viable and that the aptamer-functionalized surface is regenerable.

An Aptameric Microfluidic System for Specific Purification, Enrichment, and Mass Spectrometric Detection of Biomolecules

We present an innovative microfluidic system that accomplishes specific capture, enrichment, and isocratic elution of biomolecular analytes with coupling to label-free mass spectrometric detection. Analytes in a liquid phase are specifically captured and enriched via their affinity binding to aptamers, which are immobilized on microbeads packed inside a microchamber. Exploiting thermally induced reversible disruption of aptamer-analyte binding via on-chip temperature control with an integrated heater and temperature sensor, the captured analytes are released into the liquid phase and then isocratically eluted and transferred via a microfluidic flow gate for detection by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The utility of the device is demonstrated using adenosine monophosphate (AMP) as a model analyte. Experimental results indicate that the device is capable of purifying and enriching the analyte from a sample mixed with nonspecific analytes and contaminated with salts. In addition, thermally induced analyte release is performed at modest temperatures (45degC), and mass spectra obtained from MALDI-MS demonstrate successful detection of AMP at concentrations as low as 10 nM following enrichment by consecutive infusion of a diluted sample.

Label-free microfluidic characterization of temperature-dependent biomolecular interactions

We present a microfluidic approach to characterizing temperature-dependent biomolecular interactions. Solvated L-arginine vasopressin (AVP) and its immobilized RNA aptamer (spiegelmer) were allowed to achieve equilibrium binding in a microchip at a series of selected temperatures. Unbound AVP were collected and analyzed with matrix-assisted laser desorption∕ionization mass spectrometry (MALDI-MS), yielding melting curves that reveal highly temperature-dependent zones in which affinity binding (36-45 °C) or dissociation (25-33 °C and 50-65 °C) occurs. Additionally, temperature-dependent binding isotherms were constructed; from these, thermodynamic quantities involved in binding were extracted. The results illustrated a strong change in heat capacity of interaction for this system, suggesting a considerable thermodynamic influence controlling vasopressin-spiegelmer interaction.

A microfluidic device for multiplex single-nucleotide polymorphism genotyping

Single-nucleotide polymorphisms (SNPs) are the most abundant type of genetic variations; they provide the genetic fingerprint of individuals and are essential for genetic biomarker discoveries. Accurate detection of SNPs is of great significance for disease prevention, diagnosis and prognosis, and for prediction of drug response and clinical outcomes in patients. Nevertheless, conventional SNP genotyping methods are still limited by insufficient accuracy or labor-, time-, and resource-intensive procedures. Microfluidics has been increasingly utilized to improve efficiency; however, the currently available microfluidic genotyping systems still have shortcomings in accuracy, sensitivity, throughput and multiplexing capability. To address these challenges, we developed a multi-step SNP genotyping microfluidic device, which performs single-base extension of SNP specific primers and solid-phase purification of the extension products on a temperature-controlled chip. The products are ready for immediate detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), providing identification of the alleles at the target loci. The integrated device enables efficient and automated operation, while maintaining the high accuracy and sensitivity provided by MS. The multiplex genotyping capability was validated by performing rapid, accurate and simultaneous detection of 4 loci on a synthetic template. The microfluidic device has the potential to perform automatic, accurate, quantitative and high-throughput assays covering a broad spectrum of applications in biological and clinical research, drug development and forensics.

Isolation of thermally sensitive aptamers on a microchip

We present a microchip for isolation of aptamers that bind to target ligands at prespecified temperatures. The device uses integrated resistive heaters and sensors to control the temperatures of (poly)dimethylsiloxane (PDMS) microchambers for temperature-specific selection and bead-based amplification of aptamers. Aptamers are isolated from a randomized DNA library at specified temperatures, and amplified onto microbeads using bead-based polymerase chain reaction (PCR). As a proof of concept, the device was used to isolate aptamers for human immunoglobulin E (IgE), with the enriched pool of candidate aptamers exhibiting a much higher and temperature-dependent affinity for the target protein. The procedure was performed with significantly less reagent use in a much shorter time period (4 hours) than with conventional devices (up to 2 days).

An integrated microfluidic system for affinity extraction and concentration of biomolecules coupled to MALDI-MS

We present an innovative microfluidic device that accomplishes integrated, all-aqueous realization of specific extraction, concentration, and coupling to mass spectrometric detection of biomolecular analytes. The device uses an aptamer (i.e., oligonucleotide that binds specifically to an analyte via affinity coupling) immobilized on microbeads to achieve highly selective analyte capture and concentration. Here, we demonstrate specific extraction and concentration of adenosine monophosphate (model analyte) by approximately 100x, while providing a reusable platform via sufficiently low temperature release and regeneration of the aptamer surface at 38 degC.

Pathogen detection using microfluidic bead-based polymerase chain reaction

This paper presents a microfluidic device which uses bead-based polymerase chain reaction (PCR) to amplify and detect genomic DNA of Bordetella Pertussis. PCR is a biochemical amplification process in which template DNA is duplicated by repeated thermal cycling and enzymatic amplification. The device uses an integrated resistive heater and temperature sensor beneath a (poly) dimethylsiloxane microfluidic chamber to control the temperature of a solution containing the template DNA and PCR reagents, including bead-based primers. The PCR reaction results in fluorescently tagged double-stranded DNA immobilized on microbeads. The combination of chemical amplification and fluorescent signal concentration on microbeads allows sensitive detection of template DNA at a concentration of 1 pM in only 10 PCR cycles within 13 minutes.

Microcantilever-based label-free thermal characterization of biomolecular affinity binding

We present a microfluidic polymer cantilever array device integrating on-chip temperature control for label-free, temperature-dependent characterization of ligand-protein binding. The microfluidic device features a parylene microcantilever array within a microchamber formed by a PDMS spacer layer and a glass slide including a patterned ITO temperature sensor. The temperature is accurately controlled by using a Peltier device combined with the in situ ITO temperature sensor following a closed-loop control algorithm. For the first time, we use this cantilever-based microfluidic device to systematically characterize the temperature-dependent binding kinetics of an aptamer sensitive to platelet-derived growth factor (PDGF) at temperatures between 19 and 37 °C. Quantitative binding properties including the association and dissociation rate constants (kon and koff) and equilibrium dissociation constant (Kd) are derived, which indicate significant dependencies on temperature.

A Microfluidic Affinity Cocaine Sensor

We present a novel microfluidic sensor that is capable of detecting trace cocaine concentrations and is fully regenerable at modest temperatures. The sensor exploits affinity aptamers (synthetic DNA/RNA oligonucleotides), labeled with a fluorophore and immobilized onto polymer microbeads as a highly sensitive cocaine receptor medium. The device demonstrates the capability of detecting native cocaine concentrations as low as 100 pM, with an eight-orders-of-magnutide dynamic range. By way of enriching rarefied cocaine samples, this detection limit is further improved to 10 pM. Additionally, the device utilizes temperature-dependent reversibility of aptamer-analyte binding to regenerate the sensor surface at a relatively modest temperature (40°C). This feature allows repeated reuse of the sensor without loss in functionality.

Integrating aptamers and microfluidics for biological manipulation and sensing

We present an overview of our efforts to integrate aptamers and microfluidic devices, including manipulation of biomolecules and cells using aptamers, and isolation of target-binding nucleic acids. The aptamer-based devices for target manipulation are capable of specific analyte extraction and enrichment as well as isocratic elution, and can be coupled to biodetection systems for highly sensitive analyte detection. The microfluidic devices for isolation of target-binding nucleic acids demonstrate the potential for integrated selection of aptamers having predefined binding characteristics against a broad spectrum of practically important biological analytes.