Microfluidic Device for Aptamer-Based Cancer Cell Capture and Genetic Mutation Detection.

We present a microfluidic device for specifically capturing cancer cells and isolating their genomic DNA (gDNA) for specific amplification and sequence analysis. To capture cancer cells within the device, nucleic acid aptamers that specifically bind to cancer cells were immobilized within a channel containing micropillars designed to increase capture efficiency. The captured cells were lysed in situ, and their gDNA was isolated by physical entanglement within a second smaller-dimensioned micropillar array. This type of isolation allows the gDNA to be retained and purified within the channel and enables amplification and analysis to be performed on the gDNA without the loss of the original template. We developed a technique for selectively amplifying genes from whole gDNA using multiple displacement amplification. The amplified gene samples were sequenced, and the resulting sequence information was compared against the known wild-type gene to identify any mutations. We have tested cervical and ovarian cancer cells for mutations in the TP53 gene using this technology. This approach offers a way to monitor multiple genetic mutations in the same small population of cells, which is beneficial given the wide diversity in cancer cells, and therefore it requires very few cells to be extracted from a patient sample.

Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device.

We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA's reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles. Several aptamers were discovered with nanomolar dissociation constants. Furthermore, aptamers were identified that not only bound with high affinity, but also acted as inhibitors to significantly reduce the activity of their target protein, mouse decapping exoribonuclease (DXO). The aptamers resisted DXO's exoribonuclease activity, and in studies monitoring DXO's degradation of a 30-nucleotide substrate, less than 1 µM of aptamer demonstrated significant inhibition of DXO activity. This aptamer selection method using MEDUSA helps to overcome some of the major challenges with traditional aptamer selections, and provides a platform for high-throughput selections that lends itself to process automation.

Microfluidic isolation of nucleic acids.

The detection of nucleic acids (NAs) within micro total analysis systems (µTASs) for point-of-care use is a rapidly developing research area. The efficient isolation of NAs from a raw sample is crucial for these systems to be maximally effective. The use of microfluidics assists in reducing sample sizes and reagent consumption, increases speed, avoids contamination, and enables automation. Through miniaturization into microchips, new techniques have been realized that would be unfavorable and inconvenient to use on a macroscopic scale, but provide an excellent platform for the purification of NAs on a microscopic scale. This Review considers the complexities of NA isolation with miniaturized and microfluidic devices, as well as the considerations when choosing a technique for microfluidic NA isolation, along with their advantages, disadvantages, and potential applications. The techniques presented include using silica-based surfaces, functionalized paramagnetic beads, oligonucleotide-modified polymer surfaces, pH-dependent charged surfaces, Al2O3 membranes, and liquid-phase isolation. This Review provides a basis to develop the chemistry to improve NA isolation and move it toward achieving 100% efficiencies.

High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device.

We describe a versatile 96-well microplate-based device that utilizes affinity microcolumn chromatography to complement downstream plate-based processing in aptamer selections. This device is reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We demonstrate the utility of this device by simultaneously performing characterizations of target binding using five RNA aptamers and a random library. This was accomplished through 96 total selection tests. Three sets of selections tested the effects of target concentration on aptamer binding compared to the random RNA library using aptamers to the proteins green fluorescent protein (GFP), human heat shock factor 1 (hHSF1), and negative elongation factor E (NELF-E). For all three targets, we found significant effects consistent with steric hindrance with optimum enrichments at predictable target concentrations. In a fourth selection set, we tested the partitioning efficiency and binding specificity of our three proteins' aptamers, as well as two suspected background binding sequences, to eight targets running serially. The targets included an empty microcolumn, three affinity resins, three specific proteins, and a non-specific protein control. The aptamers showed significant enrichments only on their intended targets. Specifically, the hHSF1 and NELF-E aptamers enriched over 200-fold on their protein targets, and the GFP aptamer enriched 750-fold. By utilizing our device's plate-based format with other complementary plate-based systems for all downstream biochemical processes and analysis, high-throughput selections, characterizations, and optimization were performed to significantly reduce the time and cost for completing large-scale aptamer selections.

Isolation and amplification of mRNA within a simple microfluidic lab on a chip.

The major modules for realizing molecular biological assays in a micro-total analysis system (µTAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic mRNA within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid nonspecific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 10(15) fold and still result in successful on-chip reamplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to benchtop devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes.