Hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease.

Quantitative polymerase chain reaction (qPCR) renders profiling of genes of interest less time-consuming and cost-effective. Recently, multiplex profiling of miRNAs has enabled identifying or investigating predominant miRNAs for various diseases such as cancers and neurodegenerative diseases. Conventional multiplex qPCR technologies mostly use colorimetric measurements in solution phase, yet not only suffer from limited multiplexing capacity but also require target-screening processes due to non-specific binding between targets and primers. Here, we present hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease (AD). Our methodology promises two key advantages compared with the conventional solution-based PCR: 1) nearly no non-specific crosstalks between targets and primers, and 2) practically valuable multiplexing by spatial encoding within a single microchamber. Specifically, we immobilized hydrogel microposts (~ 400µm in diameter) within commercially available polycarbonate PCR chips by multi-step ultraviolet (UV, 365nm) exposure. We optimized this photoimmobilization for thermal cycles of PCR as well. Acrylated forward primers incorporated in polyethylene glycol diacrylate (PEGDA) posts played a crucial role to confine fluorescent signal of cDNA amplification within the PEGDA hydrogel. To demonstrate the potential of our platform, we successfully verified multiplex detection of five miRNAs, which were reported to be highly correlated with AD, from a complex buffer of human plasma.

Multiplexed Detection of Epigenetic Markers Using Quantum Dot (QD)-Encoded Hydrogel Microparticles.

Epigenetic alterations in gene expression are influenced by experiences and environment, resulting in significant variation of epigenetic markers from individual to individual. Therefore, it is imperative to measure various epigenetic markers simultaneously from samples of individual subjects to accurately analyze the epigenetic markers in biological samples. Moreover, the individualized genome-wide analysis has become a critical technology for recent trends in clinical applications such as early diagnosis and personalized medicine screening of numerous diseases. The array-based detection of modified histones, conventionally used for multiplexed analysis of epigenetic changes, requires pooling of samples from many subjects to analyze population-wise differences in the expression of histone markers and does not permit individualized analysis. Here, we report multiplexed detection of genome-wide changes in various histone modifications at a single-residue resolution using quantum dot (QD)-encoded polyethylene glycol diacrylate (PEGDA) hydrogel microparticles. To demonstrate the potential of our methodology, we present the simultaneous detection of (1) acetylation of lysine 9 of histone 3 (Ac-H3K9), (2) dimethylation of H3K9 (2Me-H3K9), and (3) trimethylation of H3K9 (3Me-H3K9) from three distinct regions in the brain [nucleus accumbens (NAc), dorsal striatum (DSt), and cerebellum (Cbl)] of cocaine-exposed mice. Our hydrogel-based epigenetic assay enabled relative quantification of the three histone variants from only 10 µL of each brain lysate (protein content = ∼ 1 µg/µL) per mouse. We verified that the exposure to cocaine induced a significant increase of acetylation while a notable decrease in methylation in NAc.