Currently, cardiac troponin I (cTnI) is the most sensitive and preferred biomarker in determining myocardial damage,⁶ in part, because cTnI is specific to only the heart muscle. Current assays measure cTnI concentration in the blood using immunoassay based principles.^6–21 The i-STAT by Abbott Diagnostics is the preferred point-of-care technique in detecting cTnI levels in hospital facilities^18,19,21 and requires 16 µL of blood at a concentration above ~ 0.02 ng mL⁻¹ to accurately detect cTnI.²² Typically, cTnI concentrations above 0.08 ng mL⁻¹ are considered elevated.²² In healthy individuals, cTnI levels are usually negligible and are at concentrations lower than the limits of detection of the diagnostic test.²³ In addition, these assays only measure total cTnI concentration. However, total cTnI concentration may not be the best indicator of overall heart health and functionality. For example, there is growing evidence in the literature that up or down regulation of the phosphorylation state of the cTnI molecule is a better indicator of the overall health and functionality of the heart muscle.^24–29 Recently, Lin and co-workers³⁰ developed the first immunoassay to sequentially detect phosphorylated and unphosphorylated cTnI in real time, however, these results were only qualitative. Further, a recent study by Messer et al.³¹ showed a 6x higher phosphorylation state of cTnI in healthy heart samples compared to failing heart samples. More importantly, Solaro and van der Velden³² indicate that protein kinase A (PKA) phosphorylation at Ser23/Ser24 residues are perhaps the only phosphorylated sites relevant to control cardiac function. If this is the case, then only two different forms of the cTnI phosphorylated state would need to be monitored. However, further research needs to be performed to validate this.

cTnI concentrations can also be elevated in the blood of elderly individuals (> 65 yrs of age),³³ patients with renal failure,³⁴ and critically ill patients³⁵ without having any actual heart related illness. Further, diagnosing myocardial infarction in intensive care unit patients is difficult because cTnI elevation may occur for alternative reasons.³⁵ Therefore, a better indicator for overall heart health for these individuals would be an assay that measured both phosphorylated and unphosphorylated cTnI in the blood. Thus, there is a need for an assay that can differentiate and quantify phosphorylated and unphosphorylated cTnI in blood samples to determine whether elevated troponin levels are a result of cardiac events or such factors as age, sex, race, and medical history.

Current detection strategies for measuring phosphorylation levels in cTnI include non-equilibrium isoelectric focusing,^36,37 mass spectrometry,^38,39 and phosphate affinity SDS-PAGE;^29,31 however these techniques are generally slow, cumbersome, have low throughput, and are not compatible with point-of-care instrumentation. Thus, there are currently no point-of-care instruments available that differentiate and quantify the phosphorylation states of cTnI in symptomatic patients.

The current limitation in detecting differences in phosphorylation states using point-of-care instrumentation is that cTnI is a low abundant protein in blood with concentrations in the sub ng mL⁻¹ range. Thus, quantifying different phosphorylation states of an already low abundant protein becomes more challenging than quantifying the total concentration of all the states. As a result, lower limits of detection are necessary to not only quantify different phosphorylation states, but, also, to quantify cTnI in individuals that have concentrations below current limits of detection. For instance, an increase in analytical and diagnostic sensitivity should allow more accurate early diagnosis.⁴⁰ This could provide tending physicians with baseline concentrations and, more importantly, the ratio of phosphorylated cTnI to unphosphorylated cTnI regardless of age, sex, race, and medical history etc., to determine during routine office visits whether or not myocardial damage has occurred. Monoclonal antibodies⁴¹ have been developed that recognize unphosphorylated cTnI and PKA phosphorylated cTnI and are also commercially available through Abcam and other vendors. A preconcentration technique coupled to an immunoassay with specific antibodies can be used in combination with preconcentration methods to lower detection limits, increase sensitivity, and quantify the phosphorylation states of cTnI.

This work describes the preconcentration of clinically relevant concentrations of cTnI (ng mL⁻¹) spiked into depleted human serum using cationic isotachophoresis (ITP) in a cascade microchip to demonstrate the potential of this approach to preconcentrate cardiac biomarkers. The high abundant proteins, albumin and IgG, were removed from the human serum prior to experimentation using a commercially available depletion column.⁴² The resulting serum sample will be referred to as depleted human serum in the remainder of this paper. In addition, preliminary experiments using both cTnI and PKA phosphorylated cTnI were spiked into leading electrolyte at high initial concentrations to easily visualize the ITP of both labeled proteins. The two cTnI states formed distinct and separated ITP zones between the leading and terminating electrolyte. Work in progress includes coupling this preconcentration technique with an immunoassay to increase sensitivity, and capture and differentiate phosphorylation states of cTnI at different positions in the cascade microchip.

ITP is a nonlinear, electrophoresis technique that concentrates sample components into nearly pure zones between a leading electrolyte and a terminating electrolyte in order of their electrophoretic mobilities. The leading and terminating electrolyte are chosen so that their electrophoretic mobilities are faster and slower with respect to the sample components of interest, in this case cTnI. In addition, ITP has the power to concentrate sample components by several orders of magnitude^43–45 and to be coupled to other analytical techniques such as capillary zone electrophoresis,^44–46 isoelectric focusing,⁴⁷ gel electrophoresis,⁴⁸ mass spectrometry,⁴⁹ or an on-chip sandwich immunoassay.⁵⁰ This work uses ITP in combination with reductions in cross-sectional area across the axial length of a microchannel to increase concentration.

Dolnik and co-workers⁵¹ first demonstrated how low abundant analytes could be concentrated by ITP in microfluidic capillaries that included reductions in cross-sectional area. Building off of this work, our group has demonstrated the concentration of proteins in buffers by over four orders of magnitude using ITP in microchips.^52,53 More recently, Bahga and co-workers have used ITP in a variable cross-section geometry to increase sensitivity, detect small molecules at concentrations as low as 100 nM, and to decrease assay time compared to uniform cross-section channels.⁵⁴ This work uses microchips that have a 10x depth and a 10x width reduction across the axial length of the microchannel to concentrate and quantify cTnI spiked into depleted human serum.

An 8% SDS-PAGE gel was prepared and used to analyze the depletion step. Fig. 2 shows the results obtained from the SDS-PAGE gel. The lane numbers and lane components are summarized in Fig. 2. 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 (M_i) 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. Fig. 3 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 (Fig. 3(a) and Fig. 3(b)). 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(c)).

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 (Fig. 3(d)). 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 (Fig. 3 (e)). The cTnI band has clearly stacked into a relatively pure ITP zone. Each picture in the bottom row of Fig. 3 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 Fig. 4. The signal intensity was proportional to initial cTnI concentration.

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 n^th moment for discrete position values using the trapezoidal rule⁶⁴ is given by where I(x) is the intensity signal, assumed to be proportional to the concentration,⁶⁵ and x is the spatial position. The variance (σ²) and the resulting peak width (W_i) can then be derived using n^th moments to the following equations where σ is the standard deviation. The final concentrations for each trial are calculated from eqn (1) where W_i is the peak width of each protein calculated from eqn (4), A is the cross-sectional area of the last leg in the microfluidic chip (0.001 mm²), and M_i 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 (Fig. 5). 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.

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 Fig. 4 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 where c_LE and c_cTnI are the concentrations of the leading electrolyte (40 mM) and the plateau concentration of cTnI (13.7 mg mL⁻¹), μ_cTnI, μ_LE (76.2 × 10⁻⁹ m²/(V s)),⁶⁷ and μ_C (−42.4 × 10⁻⁹ m²/(V s))⁶⁷ are the electrophoretic mobilities of the cTnI, leading electrolyte, and counterion, respectively, z_LE (+1) and z_cTnI are the charge on the leading electrolyte and cTnI, respectively and MW_cTnI 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 m²/(V s)).

The concentration factors were determined by dividing the final concentration from the initial concentration and the results are shown in Fig. 5. 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.

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 (Fig. 1). Experiments were performed in triplicate and representative images for each trial in the last section of the microchannel and corresponding electropherograms are shown in Fig. 6. 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.

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.

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