10,000-fold concentration increase of the biomarker cardiac troponin I in a reducing union microfluidic chip using cationic isotachophoresis

Abstract

This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10,000. The starting concentration of cTnI was 2.3 μg mL⁻¹ and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL⁻¹. To the author's knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.

Fig. 1

A 2-D step reducing microfluidic chip.

Fig. 2

Schematic of PMMA microfluidic chip geometry showing two reductions in the cross-sectional area. The total channel length is 3.3 cm. The reservoirs are 3 mm in diameter. The channel dimensions vary from 1 mm wide and 100 μm deep, to 1 mm wide and 20 μm deep, to 100 μm wide and 20 μm deep. The depth change begins 1.3 cm from the anode reservoir and the width change begins 1.6 cm from the anode reservoir. Both cross-sectional area changes occur over a distance of ~1 mm. The microchip includes a tee channel between the sample reservoir and the anode reservoir in order to control the initial mass load of the two proteins. Thus, the sample loading zone length is 11 mm, and the sample loading volume is 1.1 μL.

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 the 1^st cross-sectional area reduction (5× depth change) and have stacked into relatively distinct zones that are easily visualized. (c) The proteins are entering the 2^nd cross-sectional area reduction (10× width change) where slight distortion occurs; however, the proteins become much brighter and easier to visualize. (d) The proteins have now migrated into the final leg of the microfluidic channel and have concentrated into pure zones. In addition, the slight distortion seen in Fig. 3(c) is gone as a result of ITP’s self-sharpening effect. (e) The proteins have migrated further into the final leg of the microfluidic channel and are approaching the cathode reservoir. It is assumed at this point that all sample mass loaded into the microfluidic chip has concentrated into each respective protein band. These images have not been altered in any way to subtract background noise and are the raw images collected directly from the digital color camera.

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 the camera. Labeled cTnI (blue) migrates in front of the PE (red) because at the running pH the effective electrophoretic mobility of cTnI is higher. (b) The images have been modified using the crop and brightness/contrast function in Adobe Photoshop 5.5 to subtract out background noise.

Fig. 5

This is a representative electropherogram obtained from the middle figures in Fig. 4(a) and (b). 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. Using moment analysis, the peak width of each protein can be determined from the data. Subsequent concentrations and concentration factors for each protein are then calculated based on the electropherograms taken from the modified pictures. The final results are tabulated in Table 1. The inset shows an expanded view of the two proteins indicating a Gaussian peak shape.