Challenges:
Currently mRNA is detected by making cDNA and running the cDNA through a gel electrophoresis. More specific probing is achieved with a microarray where cDNA is washed over a chip with known DNA probes. cDNA binds to complementary probes. Fluorescent tags are used to mark the hybridized probes and then a special microscope is used to detect the fluorescence.
We have two options for the detection of RNA is this current scheme: optical or electrical. There are several methods that tie fluorescence to reaction kinetics, ie the reaction will glow when product is made, therefore indicating the presence of a particular mRNA. There are challenges ensuring the optics stay in alignment with the reaction and there is a limitation to parallelization, as there are less than 10 fluorescent tags proofed.
The second method senses a change in Resistance and Capacitance of the reaction media as impedance. The change in impedance is due to a reduction of free ions in the reaction. Although electrode sensitivity can be enhanced by direct contact with the media, this causes the corrosion of the electrodes and may impede the reaction do to electrolysis. Instead, capacitively coupled contactless conductivity detection, C4D, allows for detection of impedance outside the reaction, as the name suggests. Unlike the contact method, much higher current frequencies need to be used to overcome the capacitance of the reaction vessel. Shielding is also necessary to prevent current coupling directly between electrodes as well as interference from cosmic rays and other electromagnetic radiation. Also, there maybe a limit as to how small the reaction volume can be, given the significant but small change in voltage that is being detected, 10mV range for a 200 uL reaction.
We are also looking into turbidity sensors as detectors as well.
Currently mRNA is detected by making cDNA and running the cDNA through a gel electrophoresis. More specific probing is achieved with a microarray where cDNA is washed over a chip with known DNA probes. cDNA binds to complementary probes. Fluorescent tags are used to mark the hybridized probes and then a special microscope is used to detect the fluorescence.
We have two options for the detection of RNA is this current scheme: optical or electrical. There are several methods that tie fluorescence to reaction kinetics, ie the reaction will glow when product is made, therefore indicating the presence of a particular mRNA. There are challenges ensuring the optics stay in alignment with the reaction and there is a limitation to parallelization, as there are less than 10 fluorescent tags proofed.
The second method senses a change in Resistance and Capacitance of the reaction media as impedance. The change in impedance is due to a reduction of free ions in the reaction. Although electrode sensitivity can be enhanced by direct contact with the media, this causes the corrosion of the electrodes and may impede the reaction do to electrolysis. Instead, capacitively coupled contactless conductivity detection, C4D, allows for detection of impedance outside the reaction, as the name suggests. Unlike the contact method, much higher current frequencies need to be used to overcome the capacitance of the reaction vessel. Shielding is also necessary to prevent current coupling directly between electrodes as well as interference from cosmic rays and other electromagnetic radiation. Also, there maybe a limit as to how small the reaction volume can be, given the significant but small change in voltage that is being detected, 10mV range for a 200 uL reaction.
We are also looking into turbidity sensors as detectors as well.