Potassium ion was chosen as the leading ion because it has a higher effective electrophoretic mobility than the protein samples. Hydronium ion was the terminating ion even though it has a higher electrophoretic mobility than the protein samples because its net electrophoretic mobility was sufficiently reduced by its association with the acetic acid. The total protein loaded onto the microfluidic chip was controlled by the addition of a tee channel between the sample reservoir and the anode reservoir. Therefore, the initial mass load (Mi
) was calculated from the following equation:
is the initial concentration of protein i
(4.0 μg mL−1
and 2.3 μg mL−1
for PE and cTnI, respectively), L
is the length of the sample loading zone (11 mm), and A
is the cross-sectional area of the channel (0.1 mm2
). Thus, the total mass loaded for PE and cTnI was 4.40 and 2.53 ng, respectively. The loaded chip was mounted on the microscope platform, platinum electrodes were immersed in the anode and cathode reservoirs, power was applied, and protein migration was observed through the microfluidic chip. Both proteins were positively charged at the running pH and migrated from the anode to the cathode.
The protein migration was initially slow because the current density in the large cross-sectional area of the microfluidic channel was low. The proteins were not visualized until just before the T-junction where sufficient mass had accumulated in the zone to detect both PE and labeled cTnI (). At the running pH, cTnI ran ahead of PE because it had a higher effective electrophoretic mobility. The proteins continued to collect mass through the sample loading zone and the intensity of the fluorescence increased as they migrated through the 1st reducing union (). Prior to the 1st reducing union, the voltage on the cathode was reduced from 400 V to 100 V. Voltage reduction was performed to slow down the migrating proteins to collect more images to be obtained over the course of the ITP experiment. Therefore, voltage reduction was not required, but performed in order to record protein migration through the two reductions. Still, the entire experiment could be completed in less than 20 min.
Fig. 3 Experimental ITP stacking of labeled cTnI and PE at different locations in the microfluidic chip. (a) The proteins begin to stack into their respective zones but are difficult to see until just before the tee junction. (b) The proteins have migrated past (more ...)
No distortion is observed through the 1st
reduction (5× depth change), however; as the proteins migrate through the 2nd
reduction (10× width change) a slight distortion of the protein bands occurred where the protein concentration is higher at the walls than at the center of the channel (). Distortion through reducing unions is normal;44,50
however, this distortion is quickly eliminated by ITP’s self-sharpening effect (). No discernible differences in protein band attributes are observed as the proteins migrate through the last leg of the microfluidic channel (). A final image was collected prior to protein bands migration into the cathode reservoir clearly demonstrating ITP stacking of labeled cTnI and PE into nearly pure and distinct zones (). The images illustrated in are raw images collected directly from the digital color camera and have not been modified in any way.
Over 60 images were acquired for each successive trial and compiled into a movie using Windows Movie Maker 2.6. A representative video of the protein migration through the microfluidic channel can be viewed as a movie file found in the ESI†
. Final images from three successive trials were modified for further analysis using Adobe Photoshop 5.5 as previously described to remove background noise (). However, the raw data pictures are also shown in . After background noise removal, the images were transferred to ImageJ software where electropherograms were produced. Electropherograms were obtained by plotting distance (mm) relative to the field of view of the camera versus
average intensity over the entire width of the channel for each protein. An example electropherogram is shown in including the raw data and modified data. The inset in is a blow-up of the protein peaks and reveals that the protein peaks are more Gaussian than plateau shaped. This indicates that the proteins have not reached their maximum allowable concentration, i.e.
, plateau concentration (eqn (8)
), derived from the Kohlrausch regulating function.51
The electropherograms for all three trials were necessary to perform a statistical analysis of band attributes such as peak width. Moment analysis obtained from the data of the electropherogram was used to obtain peak width information for each trial and is described below.
Fig. 4 (a) Representative images of three subsequent trials of ITP performed in the microfluidic chip. The images are taken in the small-cross-sectional area portion of the channel just before the cathode reservoir. The images are the raw figures collected from (more ...)
Fig. 5 This is a representative electropherogram obtained from the middle figures in . Electropherograms were obtained by plotting distance (mm) relative to the field of view of the camera versus average intensity over the entire width of the (more ...)
moment converted from temporal to spatial moments52,53
is given by
) is the intensity value, x
is the spatial position, a
are the limits of integration based on the field of view of the camera, and mn,i
was calculated using the trapezoidal rule54
at distinct position values such that
The variance (σ2) is defined by the following relationship
is the mean location of mass. The variance can then be derived using nth
moments to the following equation
where the complete derivation from eqn (5)
to eqn (6)
is shown in Appendix 1
. The resulting peak width (Wi
) for each peak is then given by
is the standard deviation. The peak widths at the end of the ITP experiments for labeled cTnI and PE are 49.66 ± 2.37 μm and 31.68 ± 3.23 μm, respectively. The final concentration can then be calculated from eqn (1)
is the peak width of each protein calculated from eqn (7)
is the cross-sectional area of the last leg in the microfluidic chip (0.002 mm2
), and Mi
is the initial mass load calculated from eqn (2)
(2.53 ng for cTnI and 4.40 ng for PE). The authors assume that all of the protein loaded onto the microfluidic chip collects into the bands presented in . There is no evidence of protein being lost during the experiment, such as by protein adsorption on the channel walls. Using eqn (7)
, the final protein concentrations were 25.52 ± 1.25 mg mL−1
and 69.91 ± 6.76 mg mL−1
for cTnI and PE, respectively. The concentration factors were determined by dividing the final concentration by the initial concentration. For cTnI the concentration factor was 11 094 ± 545 and for PE the concentration factor was 17 477 ± 1689. The peak widths, final concentrations, and concentration factors of each protein for each trial are summarized in .
Table 1 Summary of experimental peak widths determined from moment analysis, final concentrations from eqn (1), and concentration factors for PE and cTnI
The plateau concentration (ci
) or maximum concentration of proteins that can be obtained using ITP can be derived from the Kohlrausch regulating function51
is the concentration of the leading electrolyte (20 mM), μS
, and μC
are the electrophoretic mobilities of the sample, leading electrolyte, and counterion, respectively, and zLE
(+1) and zS
are the charge on the leading electrolyte and sample, respectively. A direct comparison of plateau concentrations for cTnI and PE could not be performed because mobility and charge data could not readily be obtained for cTnI and PE without further experimentation. In addition, mobility and charge data were not found in the literature. As a result, the authors used the protein bovine serum albumin (BSA), whose mobility and charge data were available from the literature,35
as a surrogate protein to compare the plateau concentration of BSA to the experimentally determined concentrations of cTnI and PE.
The mobility and charge of BSA at pH 4.0 are ~2.00 × 10−8
and +13, respectively.35
The mobilities of the leading electrolyte (potassium) and counterion (acetate) are 7.62 × 10−8
and −4.24 × 10−8
By plugging these values into eqn (8)
and modifying eqn (8)
from a molar concentration to a mass concentration by using the molecular weight of BSA (66 kDa), a plateau concentration of 50.65 mg mL−1
was calculated for BSA. As a result, the BSA based plateau concentration is much higher than the experimentally determined concentration for cTnI but slightly lower than the PE concentration. One possible explanation for the PE concentration being slightly higher is that the molecular weight of PE (240 kDa) is significantly higher than the Mw
of BSA so when eqn (8)
is calculated based on the mass concentration, one would expect the plateau concentration to be directly proportional to the ratio of molecular weights, assuming all other values in eqn (8)
are nearly identical for the two proteins. Therefore, it is safe to assume that the plateau concentration calculated from eqn (8)
would be higher for PE which is the case described here.
The authors are aware that the initial concentration of cTnI is significantly higher (10 000×) than clinical levels; however, this paper addresses a potential solution in current technologies that are unable to detect cTnI levels in some patients. In addition, the authors on-going research includes performing cationic ITP at pH 8 of cTnI in serum samples to determine the limit of detection and the potential use of ITP to concentrate and fractionate cTnI for clinical applications. We believe that by incorporating a pre-concentration step prior to an immunoassay, the limits of detection will be lowered, sensitivity will be increased, and different phosphorylation states of cTnI can then be detected and quantified. Future experimentation will need to be performed to test this hypothesis.