Highly Magnetized Encoded Hydrogel Microparticles with Enhanced Rinsing Capabilities for Efficient microRNA Detection

Abstract

Encoded hydrogel microparticles mounting DNA probes are powerful tools for high-performance microRNA (miRNA) detection in terms of sensitivity, specificity, and multiplex detection capability. However, several particle rinsing steps in the assay procedure present challenges for rapid and efficient detection. To overcome this limitation, we encapsulated dense magnetic nanoparticles to reduce the rinsing steps and duration via magnetic separation. A large number of magnetic nanoparticles were encapsulated into hydrogel microparticles based on a discontinuous dewetting technique combined with degassed micromolding lithography. In addition, we attached DNA probes targeting three types of miRNAs related to preeclampsia to magnetically encoded hydrogel microparticles by post-synthesis conjugation and achieved sensitivity comparable to that of conventional nonmagnetic encoded hydrogel microparticles. To demonstrate the multiplex capability of magnetically encoded hydrogel microparticles while maintaining the advantages of the simplified rinsing process when addressing multiple samples, we conducted a triplex detection of preeclampsia-related miRNAs. In conclusion, the introduction of magnetically encoded hydrogel microparticles not only allowed efficient miRNA detection but also provided comparable sensitivity and multiplexed detectability to conventional nonmagnetic encoded hydrogel microparticles.

Keywords: magnetic hydrogel; microRNA detection; microparticles; preeclampsia.

Figure 1

Overview of magnetic hydrogel microparticle synthesis and post-synthesis probe conjugation. (a) Scheme of discontinuous dewetting on a degassed PDMS mold. UV light is exposed in a vacuum chamber to offset the oxygen inhibition effect. (b) Bright-field images of synthesized magnetic microparticles. (c) Conjugation of thiolated FAM probes to magnetic microparticles via post-synthesis conjugation. (d) Fluorescence intensities of microparticles depending on blocking treatment for unreacted double bonds. Scale bars are 100 μm.

Figure 2

Magnetization value of magnetic hydrogel particles according to the intensities of an applied magnetic field. The inset is a linearized graph using the reciprocal of the magnetic field as the x-axis.

Figure 3

Probe loading densities in microparticles depending on UV exposure time.

Figure 4

miRNA singleplex detection results using magnetic hydrogel microparticles. (a) Grayscale fluorescence images of particles observed during singleplex detection. Scale bars are 20 μm. (b) Background-subtracted fluorescence intensities of the particles. Inset is signal-to-noise ratio, signal values divided by corresponding standard deviations. LOD is the x value where each line intersects with y = 3.

Figure 5

Multiplexed detection of three pre-eclampsia related miRNAs (miR-18a, miR-29a, and miR-210). The multiplexed detection was conducted for eight combinations of samples depending on the presence (+) or absence (−) of each target. The vertical bar graph indicates average fluorescence intensities of each target miRNA.