Isolation and Amplification of mRNA within a Simple Microfluidic Lab on a Chip

Abstract

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 messenger RNA (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 non-specific 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 1015 fold and still result in successful on-chip re-amplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to bench-top 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.

Looking into Living Cell Systems: Planar Waveguide Microfluidic NMR Detector for in Vitro Metabolomics of Tumor Spheroids

“…Miniaturized devices not only minimize the sample volume, but also reduce the analysis time and cost, enable high-throughput analysis, allow portability and on site detection. [19][20][21][22][23][24] Despite this progress in nanotechnology, detection is one of the main challenges for the miniaturization because of the ultra-small sample sizes. Many analytical tools like MS and optical methods have been integrated to LOC devices for the detection of samples in the nanoliter range.…”

Microfluidic Isolation of Nucleic Acids

“…The oligo‐dT will hybridize with the poly‐A tail of all the mRNA molecules, while other types of NAs do not bind. An additional bonus of mRNA detection is the fact that only viable cells will have these present, in contrast to the detection of a general DNA sequence 74. Specific mRNA extraction is also advantageous, since the quantity of mRNA is very small compared to the total RNA, yet multiple copies exist for each sequence.…”

“…In contrast, our research group developed a method for modifying microchannels with high immobilization efficiency, without the need for the synthesis of a porous matrix. Oligo‐dT probes were immobilized onto the periphery of 5th generation polyamidoamine dendrimers to achieve a high immobilization efficiency, which resulted in a high capture efficiency 74. Figure 5 illustrates this isolation concept and shows how the microdevices are fabricated.…”

CMOS Enabled Microfluidic Systems for Healthcare Based Applications

“…Hybrid technologies are used to incorporate additional parts, such as syringe pumps, control valves, and microscopes, which not only increase the complexity and cost of the system but also create a dependence of the system on a laboratory‐based environment. Technology has now advanced so much that there are fully integrated sample‐in‐results‐out chips for genetic analysis, sensors that can detect a handful of molecules, and many other similar systems . Complementary metal oxide semiconductor (CMOS) integrated LOC systems can be developed using CMOS technologies as sensors, signal conditioners, data processing circuitry, actuators, and wireless communication capabilities .…”