The overall goal of this video is to present an electro kinetic technique called Isot apheresis, abbreviated ITP, and its microfluidic applications for sensitive detection and separation of ions. First, the physical principles underlying ITP are discussed in addition to the two standard modes of operation, namely peak mode and plateau mode, ITP on-chip ITP is then demonstrated with three experiments, beginning with a simple protocol for sample pre concentration, where Fluor fours are detected by focusing them in a sharp peak. Second, an ITP assay used for on-chip sample preparation is shown where DNA and RNA are selectively extracted from bacterial cell lysate.
Finally, a label-free detection assay is used to purify and quantitate amino acids using plateau mode ITP. Ultimately amino acid purification analyte pre concentration and selective extraction of nucleic acids can be achieved through ITP. Today we will introduce you to the underlying physics and methodology of a versatile electro kinetic technique called Isot Apheresis.
Our group has pioneered ITP methods for label-free detection, sample preparation, and rapid hybridization assays. We will share with you three representative assays that demonstrate the ability of ITP to pre concentrate analytes, extract, lytic acids, and purify and detect amino acids. These assays highlight the versatility of ITP and demonstrate the simple steps needed to ensure repeatable experiments.
In the lab. In ITP sample ions selectively focus at the interface between a leading and trailing electrolyte, abbreviated LE and TE respectively. The sample ions travel through the microchannel at a constant speed determined by the velocity of the leading ions.
Sample ions focus if their effective electrophoretic mobility is bracketed by the mobility of LE and TE ions, where mobility is defined as the ratio of ion drift velocity to the local electric field Sample ions mixed in the TE move faster than the surrounding TE ions and therefore accumulate at the LETE interface. Furthermore, the ITP interface is self sharpening LE ions, which may diffuse into the TE zone experience a strong restoring flux and return to the leading zone. The same is true for TE ions in the LE Zone.
The self sharpening and focusing properties of I two P contribute to this technique's robustness and make it insensitive to disturbances at the interface such as pressure driven flow. In peak mode sample ion concentrations are significantly lower than those of LE and TE ions. Therefore, sample ions contribute negligible to local conductivity under these conditions.
Multiple analytes focus within the ITP interface region mostly is overlapping peaks. Eventually, sample ions may reach sufficiently high concentration to separate and focus in plateau mode. In plateau mode sample ions separate and purify into zones of locally uniform and constant concentration in an order determined by their electrophoretic mobility.
Ions in plateau mode determine local conductivity. Very dilute ions may still focus in peak mode between plateau zones. For the assays presented in this protocol, an isotropically wet etched glass microfluidic chip with a cross channel design is used.
However, ITP can also be performed in simple straight channels or capillaries with no intersections or junctions. The following cleaning procedure should be performed prior to experiments to ensure run to run repeatability and successful application of dynamic coatings used to suppress electro osmotic flow. To decontaminate the channel, fill the north, east and south reservoirs with 10 to 20 microliters of 10%bleach solution.
If using a standard caliper chip caddy, attach the wide end of a 200 microliter pipette tip to the west reservoir of the chip and connect the vacuum line to a two millimeter inner diameter tube. Apply a vacuum for two minutes, then empty the reservoirs clean thoroughly by filling the reservoirs with deionized water and emptying with the vacuum line. Perform the DI wash several times.
Repeat this process with one molar sodium hydroxide. The channel surfaces will become gently etched, yielding a clean bora silicate surface to help establish uniform surface properties. Clean the reservoirs with DI and then rinse the channels with le.
During this period, surface properties and dynamic coatings will equilibrate within the channels. To begin the experiment, prepare one milliliter of leading electrolyte and one milliliter of trailing electrolyte. Then combine 90 microliters of TE with 10 microliters of one micromolar.
Alexa Fluor 4 88. After rinsing with le empty the west reservoir and rinse the reservoir several times with di water to strongly dilute any LE remaining in the reservoir. Finally, fill this reservoir with 20 microliters of TE containing dye.
Place the positive electrode in the east reservoir and the ground or negative electrode in the west reservoir and apply two microamps of constant current. The sample peak will migrate at a constant velocity and the voltage between the reservoirs will increase as the lower conductivity TE fills. The channel Nucleic acids from untreated cell lysate can be purified using ITP by selecting a trailing anion with an electrophoretic mobility magnitude lower than the target nucleic acid, but higher than onic PCR inhibitors.
Onic PCR inhibitors migrate in the opposite direction and so are also left behind. To begin resuspend, the e coli cell pellet contending 10 to the eighth colony forming units in 80 microliters of RNAs free water and add 10 microliters of lysing agent mix gently and incubate for five minutes at room temperature following incubation, add 10 microliters of one moer sodium hydroxide to raise the pH of the lysate to approximately 12.5. Gently actuate the pipette up and down until the solution becomes clear at which point lysing is complete.
Next, add 10 microliters of lysate into 90 microliters of 50 millimolar Racine and 100 millimolar bis triss. This solution can now be used as the te also prepare one milliliter of LE containing cyber green dye as described in the written protocol, use the LE to fill the microfluidic chip as demonstrated earlier. After rinsing, replace the contents of the east reservoir with a PCR compatible.
LE rinse the west reservoir with DI and fill with 20 microliters of te. Apply 1000 volts between the east and west reservoirs to begin the experiment. The current between these reservoirs will decrease at the end of the experiment.
The sample eludes into the LA reservoir coincident with this solution. The current versus time for the system typically reaches a plateau value. Gently mix the reservoir contents by actuating the pipette up and down a few times.
Then extract the five microliter volume for analysis by quantitative. R-T-P-C-R-I-T-P can also be used to separate and focus small ions into adjoining and detectable plateaus between the TE and LE allowing detection based on physiochemical properties demonstrated. Here is a non focusing tracer assay where a fluorescent onic species is added to the le.
The fluorescent species does not focus, but its concentration adapts to a local electric field and thereby enables visualization of purified plateau zones. Begin by preparing one milliliter of leading electrolyte containing the cationic Fluor four rod Domine six G and one milliliter of trailing electrolyte. Then combine 90 microliters of TE with 10 microliters, each of 50 millimolar arginine and 50 millimolar lysine to load the sample for finite injection.
Dispense 20 microliters of Ellie in the west and north reservoirs dispense the amino acid sample in the east reservoir. Next, apply a vacuum at the South reservoir for one minute. After rinsing the east reservoir with DI water, replace contents with te.
As a final step, apply 500 volts between the east and west reservoirs in peak mode experiments with the fluorescent reporter Alexa Fluor 4 88. The overall fluorescence intensity can be integrated and compared against a calibration curve to obtain quantitative concentration. Information shown here are experiments over a range of applied currents revealing that sample.
Peak width is inversely proportional to current. This is an example of nucleic acid extraction from whole cell lysate with peak mode ITP. The total nucleic acid ITP peak often takes on a non-ideal shape.
In nucleic acid extraction experiments, sample is allowed to elute into the LE Reservoir and is extracted with a pipette for analysis by quantitative R-T-P-C-R results of quantitative reverse transcription. PCR verify successful purification of 16 s ribosomal, RNA and 16 s ribosomal, DNA from bacterial culture negative control threshold cycles for 16 s ribosomal, RNA and 16 s ribosomal, DNA were each above 30 cycles as expected. Plateau mode ITP was performed for two amino acids, arginine and lysine using the NFT assay with RUMINE six G as the under speeding fluorescent tracer.
Here, fluorescence intensity relative to LE or TE zone intensity can be used for zone identification while zone widths enable quantitation. After watching this video, you should have a good physical understanding of ITP and of the methodology behind three distinct ITP based assays. In particular, we have shown how to use ITP to pur amino acids, pre concentrate analytes, and selectively extract nucleic acids.
Don't forget that working with high voltage could be extremely hazardous. You should always take precautions such as using equipment which limits current and maintaining sufficient distance from the erodes.
