Potassium ion was chosen as the leading ion because it has a higher effective electrophoretic mobility than all other positive ions including the salt ions in the serum sample, including sodium. Hydronium was chosen as the terminating ion because it migrates slower than the protein of interest, cTnI, due to its association with acetic acid. The sample was depleted human serum containing cTnI. In normal serum, albumin can make up as much as 70% of the total protein in human serum59
at an estimated concentration range between 34 and 54 mg mL−160
and was removed in order to significantly improve ITP results. When albumin was not removed from the serum, the cTnI band was very broad compared to when albumin was removed. The authors believe this could be due to 1) the albumin reaching concentrations above the solubility limit and precipitating out, or 2) the lack of separation power due to the short length of the microchannel. For instance, using information from Harrison and Ivory61
to calculate the band length of albumin in an ITP stack results in an albumin band length of ~ 3.4 cm (see Appendix 1
). The total length of the separation channel described here is only 3.3 cm indicating a lack of separation power to fully resolve albumin from any of the other sample components. Therefore, the latter scenario seems more likely to explain the poor results obtained prior to albumin removal. As a result, albumin removal from the human serum was required to obtain satisfactory results. Therefore, future experiments may require an on-chip depletion step to fully automate the ITP protocol and to remove albumin from the sample prior to ITP in the microchip.
An 8% SDS-PAGE gel was prepared and used to analyze the depletion step. shows the results obtained from the SDS-PAGE gel. The lane numbers and lane components are summarized in . It is observed that the depletion step was successful and that albumin was nearly completely removed from the human serum sample (see lanes 2–6). In addition, even at 10x mass load of the depleted serum sample (lane 6) compared to the original human serum sample (lane 1), only a trace amount of albumin could be visualized in the depleted serum sample (lane 6). From the depleted serum samples, we were also able to putatively identify the new major contaminants by comparing the molecular weight of the rainbow markers to other high abundant proteins in serum.62
These proteins are most likely transferrin (4.75 g/100 g of plasma proteins62
) and α1-antitrypsin (2.95 g/100 g of plasma proteins62
). The depleted human serum was spiked with different amounts of fluorescently labeled cTnI to perform ITP on the cascade microchip.
The total amount of depleted human serum sample spiked with fluorescently labeled cTnI was controlled by the addition of a tee channel between the sample reservoir and the anode reservoir. The initial mass load (Mi
) of cTnI was calculated by multiplying the initial concentration (2300, 460, 115, and 46 ng mL−1
) by the volume of the sample loading region (1 µL). Therefore, the total mass loads of cTnI ranged from 2.3 ng to 46 pg. These total mass loads will be used later in determining the final cTnI concentrations calculated from eqn (1)
and will be discussed later. After, loading the microchip with sample and electrolyte solutions, the microchip was mounted on the x–y translation stage of the microscope platform and platinum electrodes were submerged in the anode and cathode reservoirs to produce the electric field required for analyte migration. The cathode was set to ground and the anode was set to 300 V. Each concentration was run in triplicate.
At high concentrations of cTnI (2300 and 460 ng mL−1) the protein began to collect and form a cohesive band just to the right of the anode reservoir. At a concentration of 115 ng mL−1, the cTnI band was not visible until after the tee channel where sufficient mass of cTnI had accumulated. At a concentration of 46 ng mL−1, the cTnI band was not visible until after the 1st reduction in cross-sectional area and was difficult to see until after the 2nd cross-sectional area reduction. depicts the protein migration over time for the case of 460 ng mL−1 where the cTnI band could be easily visualized in all parts of the channel. In this example, the cTnI continued to accumulate mass through the sample loading zone and the fluorescence intensity increased as the protein migrated through the microchannel (). At this point, the cTnI had now formed a tight cohesive band in the microchannel. The fluorescence intensity increased again as the cTnI passed the 10x depth reduction ().
Fig. 3 An example of protein migration of labeled cTnI spiked into depleted human serum at an initial concentration of 460 ng mL−1 through a cascade microchannel (a) cTnI begins to stack at the terminating electrolyte interface but is difficult to visualize. (more ...)
Very little, if any, band distortion was observed through the depth reduction; however, as the protein began to migrate through the 10x width reduction, a slight distortion was observed (). The authors believe that the distortion occurs in both cases, but is only visible at the width reduction because we can only see distortions that occur in the x-y plane and cannot see distortions that may occur in the z-direction. Distortion through cross-sectional area reductions are common;51,63
however these distortions are quickly eliminated by ITP’s self-sharpening effect as the band migrates into the last leg of the microchannel (). The cTnI band has clearly stacked into a relatively pure ITP zone. Each picture in the bottom row of was modified using the brightness/contrast function in Adobe Photoshop to more easily visualize the cTnI band formation and progression through the cascade microchannel; the pictures in the middle row are untouched. Experiments could be performed in ~ 10 min. The captured images were compiled for each trial using Windows Movie Maker 2.6. An example video for each concentration can be viewed as a movie file found in the supplementary information
Electropherograms for three trials at each concentration were obtained using ImageJ software. Each electropherogram was obtained from images of the cTnI located in the last leg of the microchannel where the fluorescence intensity was at a maximum. The resulting electropherograms yielded distance (mm) relative to the field of view of the camera versus average intensity over the entire width of the channel for the fluorescent protein, cTnI. The raw data was then transferred to Microsoft Excel and plotted for each case. Due to the dynamic nature of ITP and the capturing of images at different positions in the channel, the peak heights for each trial did not align. Therefore, the plots were translated along the x-axis (distance (mm) relative to the field of view of the camera) so that maximum peak heights occurred at the same x-position. The intensity plots were then averaged for each concentration and the average intensity as a function of position is shown in . The signal intensity was proportional to initial cTnI concentration.
Fig. 4 This is the average of the electropherograms extracted from ImageJ software for each of the four concentrations. The electropherograms were obtained by plotting distance (mm) relative to the field of view of the camera versus average intensity over the (more ...)
The electropherograms for all three trials at each concentration were needed to perform a statistical analysis of band attributes such as peak width and to sequentially determine final protein concentration and concentration factors obtained by ITP in the cascade microchip. This was done by using moment analysis as previously described.53
Briefly, the nth
moment for discrete position values using the trapezoidal rule64
is given by
) is the intensity signal, assumed to be proportional to the concentration,65
is the spatial position. The variance (σ2
) and the resulting peak width (Wi
) can then be derived using nth
moments to the following equations
where σ is the standard deviation. The final concentrations for each trial are calculated from eqn (1)
is the peak width of each protein calculated from eqn (4)
is the cross-sectional area of the last leg in the microfluidic chip (0.001 mm2
), and Mi
is the initial mass load of cTnI ranging from 2.3 ng to 46 pg. The final concentrations calculated from eqn (1)
range from 13.7 ± 0.24 to 0.42 ± 0.02 mg mL−1
and show a linear relationship with initial concentration (). This is consistent with results obtained by Kaigala and co-workers65
who observed a linear response with initial analyte concentration in the nanomolar range, i.e.
, “peak mode” using ITP on fluorescently labeled dye molecules.
Fig. 5 Final concentrations and concentration factors as a function of initial concentration. There is a linear relationship between final concentration and initial concentration; indicating that the ITP is running in “peak mode.” The concentration (more ...)
ITP in “peak mode” indicates a near-Gaussian peak shape which is consistent with most of the results obtained here and indicates that further preconcentration is possible. At the highest initial concentration (2300 ng mL−1
), we were able to concentrate cTnI to its plateau concentration of 13.7 ± 0.24 mg mL−1
which is the maximum allowable concentration for this particular analyte. Although not apparent from the electropherogram due to intensity saturation of the ImageJ software, the resulting peak from an initial concentration of 2300 ng mL−1
was plateau shaped. This was only apparent after decreasing the signal intensity in Adobe Photoshop using the brightness function to values that did not saturate the ImageJ software. This is shown in the inset of where the peaks are not Gaussian, but rather plateau shaped at this concentration. The plateau concentration is derived from the Kohlrausch regulating function,66
and converting to units of mg mL−1
, is shown below as
are the concentrations of the leading electrolyte (40 mM) and the plateau concentration of cTnI (13.7 mg mL−1
(76.2 × 10−9
(−42.4 × 10−9
are the electrophoretic mobilities of the cTnI, leading electrolyte, and counterion, respectively, zLE
(+1) and zcTnI
are the charge on the leading electrolyte and cTnI, respectively and MWcTnI
is the molecular weight of cTnI (24.1kDa). The theoretical charge of cTnI was +29.4 in the leading electrolyte at pH 4.4 and was determined by inserting the amino acid sequence of cTnI68
into the Protein Calculator V3.3 (http://www.scripps.edu/~cdputnam/protcalc.html
). After rearrangement of eqn (5)
, the electrophoretic mobility of labeled cTnI at our experimental conditions can be calculated. The electrophoretic mobility of labeled cTnI was ~ 15.6 × 10−9
The concentration factors were determined by dividing the final concentration from the initial concentration and the results are shown in . At lower initial concentrations, a higher concentration factor was observed. At the low cTnI concentration we observe a concentration factor increase of ~ 9000. The average peak widths, final concentrations, and concentration factors for each initial concentration are shown in .
Table 1 Summary of average experimental peak widths determined from moment analysis, final concentrations from eqn (1), and concentration factors for cTnI with standard deviations
This demonstrates the detection of cTnI in depleted human serum at clinically relevant concentrations without the use of antibodies and relying solely on ITP in a cascade microchip. The authors acknowledge that increased sensitivity is required to stand up to current point-of-care instruments for cTnI detection which have a detection limit of ~ 0.02 ng mL−1
and use antibody amplification. Antibody signal amplification can lower limits of detection by more than 1000 fold69
meaning that, by incorporating antibodies into our assay, we can reduce our limit of detection to below 0.05 ng mL−1
which puts us in a position to compete with current point-of-care instruments. Therefore, we are in the process of incorporating antibodies for immobilization of the cTnI molecule to significantly increase the sensitivity of our assay. In addition, sensitivity can be enhanced further using only ITP by using an infinite sample loading technique65
or stationary ITP with unlimited volume stacking,70
or by fabricating further cross-sectional area reductions in the microchannel, but these techniques were not analyzed here.
In a separate experiment, labeled PKA phosphorylated cTnI and labeled cTnI were both spiked into leading electrolyte at concentrations of 585 ng mL−1
and 920 ng mL−1
, respectively. ITP was performed in the same manner on a similar, but different, microchip. All geometrical attributes of the microchip were identical except that the side channel for these experiments was 1 mm in width (). Experiments were performed in triplicate and representative images for each trial in the last section of the microchannel and corresponding electropherograms are shown in . The cTnI (blue) ran in front of the PKA phosphorylated cTnI (green) indicating that the electrophoretic mobility decreases with increasing phosphorylation and is consistent with results obtained by non-equilibrium isoelectric focusing gel electrophoresis.37
In addition, our preliminary results indicate the formation of two distinct ITP zones. An example video showing the migration of the two different phosphorylation states of cTnI in distinct zones can be viewed as a movie file found in the supplementary information
Fig. 6 (a) Representative images of ITP for each trial taken in the small-cross sectional area portion of the channel just before the cathode reservoir. Labeled cTnI (blue) migrates in front of the labeled PKA phosphorylated cTnI (green) indicating that the (more ...)
The technique described here, ITP as a preconcentration platform, could be implemented as a 1st dimension to preconcentrate the different forms of cTnI and transfer them via
ITP to a 2nd dimension that has the ability to capture, distinguish, and quantify different phosphorylation states that all current point-of-care assays lack. In addition, Kaigala and co-workers65
have recently demonstrated how ITP systems can be miniaturized to device designs as small as a cell phone for portable low-cost ITP instruments demonstrating that the technique described here is a promising approach for point-of-care applications.
The authors’ on-going research includes creating an on-chip ITP immunoassay that includes both capture antibodies and secondary detection antibodies to increase limits of detection by immobilizing the cTnI with the former and amplifying the signal with the latter. The capture antibodies will need to be specific to the phosphorylation state of cTnI allowing differentiation of phosphorylated and unphosphorylated cTnI. The detection antibodies, which only need to be cTnI specific, can then quantify the amount of each cTnI form present. We believe that by delivering ITP bands to an immunoassay we can lower the limit of detection, increase sensitivity, and differentiate and quantify different phosphorylation states of cTnI. Also, the authors are attempting to perform the removal of albumin on-chip so that this approach could potentially be used to concentrate and fractionate cTnI in clinical applications. This would be an invaluable tool for attending physicians to quickly monitor cTnI levels and correctly diagnose patients. Future experimentation will need to be carried out to test these hypotheses.