3D printed microfluidic devices for integrated solid-phase extraction and microchip electrophoresis of preterm birth biomarkers

Abstract

Background: Preterm birth (PTB) is a leading cause of neonatal mortality, such that the need for a rapid and accurate assessment for PTB risk is critical. Here, we developed a 3D printed microfluidic system that integrated solid-phase extraction (SPE) and microchip electrophoresis (μCE) of PTB biomarkers, enabling the combination of biomarker enrichment and labeling with μCE separation and fluorescence detection.

Results: Reversed-phase SPE monoliths were photopolymerized in 3D printed devices. Microvalves in the device directed sample between the SPE monolith and the injection cross-channel in the serpentine μCE channel. Successful on-chip preconcentration, labeling and μCE separation of four PTB-related polypeptides were demonstrated in these integrated microfluidic devices. We further show the ability of these devices to handle complex sample matrices through the successful analysis of labeled PTB biomarkers spiked into maternal blood serum. The detection limit was 7 nM for the PTB biomarker, corticotropin releasing factor, in 3D printed SPE-μCE integrated devices.

Significance: This work represents the first successful demonstration of integration of SPE and μCE separation of disease-linked biomarkers in 3D printed microfluidic devices. These studies open up promising possibilities for rapid bioanalysis of medically relevant analytes.

Keywords: 3D printing; Microchip electrophoresis; Microfluidics; Preterm birth biomarkers; Sample preparation; Solid-phase extraction.

Figure 1.

3D printed, valve-integrated SPE-μCE devices. (A) OpenSCAD design with channels (cyan or red), reservoirs (green), valves (yellow), and pneumatic lines with connections to the side of the device (gray). Gnd: ground, v: valve, MW: monolith window, VP: vacuum port, and HV: high voltage (B) SEM image of a SPE monolith in a 3D printed microfluidic device. (C) Device photograph.

Figure 2.

Device operation for SPE-μCE. (A) Elution: valves 1 and 4 are closed, and valves 2 and 3 are opened when sample (green shading) is eluted through the reversed-phase SPE monolith (blue shading) into the intersection with the aid of vacuum. (B) Sample plug capture: valves 2 and 3 are closed. (C) Electrophoresis: valves 1 and 4 are opened and sample is electrophoretically separated.

Figure 3.

Photographs of reversed-phase monoliths and injection cross-channels during SPE-μCE. (A) Image of a monolith showing formation and containment in the polymerization window, and as well as an injection cross-channel with valves. (B) Fluorescence image of the injection step during μCE of Alexa Fluor. (C) Fluorescence images of successive steps during SPE experiments, including equilibration, sample loading and labeling, BCB rinse, 30% ACN elution, and 90% ACN elution.

Figure 4.

SPE elution profiles of on-chip-labeled PTB biomarkers in 3D printed microfluidic devices. (A) 30 nM P1. (B) 600 nM P3. (C) 250 nM CRF. (D) 100 nM Fer. (E) 7.5 nM P1, 150 nM P3, 75 nM CRF, and 25 nM Fer.

Figure 5.

Microchip electrophoresis of PTB biomarkers in 3D printed SPE-μCE integrated microfluidic devices. (A) Electropherogram of off-chip-labeled 34 nM P1, 680 nM P3, 340 nM CRF, and 110 nM Fer, in a 3D printed microfluidic device with a reversed-phase SPE monolith. (B) Electropherogram of on-chip-labeled and enriched 7.5 nM P1, 150 nM P3, 75 nM CRF, and 25 nM Fer, in a 3D printed microfluidic device with a reversed-phase SPE monolith.

Figure 6.

Microchip electrophoresis of PTB biomarkers in 3D printed integrated SPE-μCE devices. Electropherogram of off-chip-labeled 34 nM P1, 680 nM P3, 340 nM CRF, and 110 nM Fer in 50% maternal blood serum, in 3D printed microfluidic devices with (A) a reversed-phase SPE monolith or (B) no reversed-phase SPE monolith.

Figure 7.

Calibration plot for SPE-μCE of CRF in 3D printed microfluidic devices.