Multiplexed Detection of mRNA Using Porosity-Tuned Hydrogel Microparticles

Abstract

Transcriptional profiling, which is directly or indirectly associated with expressed protein levels, has been used in various applications including clinical prognosis and pharmaceutical investigation of drug activities. Although the widely used reverse transcription polymerase chain reaction (RT-PCR) allows for the quantification of absolute amounts of mRNA (mRNA) from inputs as small as a single cell, it is an indirect detection method that requires the amplification of cDNA copies of target mRNAs. Here, we report the quantification of unmodified full-length transcripts, using poly(ethylene) glycol diacrylate (PEGDA) hydrogel microparticles synthesized via stop flow lithography (SFL). We show that PEG600 serves as an effective porogen to allow for the capture of large (∼1000-3700 nt long) mRNAs. Our relatively simple hydrogel-based mRNA detection scheme uses a multibiotinylated universal label probe and provides assay performance (limit of detection of ∼6 amol of an in-vitro-transcribed model target) comparable to an existing commercial bead-based technology that uses branched DNA (bDNA) signal amplification. We also demonstrate a 3-plex mRNA detection, without cross-reactivity, using shape-encoded "intraplex" hydrogel microparticles. Our ability to tune the porosity of encoded hydrogel microparticles expands the utility of this platform to now quantify biomacromolecules ranging in size from large mRNAs to small miRNAs.

Flow lithography in ultraviolet-curable polydimethylsiloxane microfluidic chips

“…3(c)), which can be employed for encoding 10,11 and the multiplexed detection of biomolecules. 7,13 Structural variations of the homo-and heterogeneous microparticles remained minimal with the coefficient of variation (CV) of less than 2% during the entire period of SFL (Figs. 3(b) and 3(c)).…”

“…[1][2][3][4] Owing to this merit of FL, new applications have emerged for in vitro diagnostics, [5][6][7][8][9][10][11][12][13] selfassembly of microparticles, 14,15 tissue engineering, [16][17][18] and drug delivery. 19 Materials that enable microfluidic devices to perform FL must satisfy two major requirements.…”

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

“…As these fluorescent ultra-bright probes for PCR-independent nucleic acid detection develop, they might benefit from new chemical substances, nanotechnology and straightforward biotechnological approaches to enhance the signal [68][69][70]. Among other methods, fluorescence microscopy and SPR are two very promising techniques with a high potential for future biomolecular science and diagnostics.…”

Polyplex Formation Influences Release Mechanism of Mono- and Di-Valent Ions from Phosphorylcholine Group Bearing Hydrogels

“…Swelling%, was determined immediately before and immediately after release of the ionic species at various temperatures by measuring the weight of the hydrogel disc at those times (S b -before and S a -after). The difference in Swelling% as a result of the release was determined from the before and after measurements and was calculated using Equation (8). DoH was calculated using Equation (9):…”

“…A canonical example of a hydrogel, poly(2-hydroxyethyl methacrylate) (pHEMA), was first used in contact lenses for its optical clarity, dimensional stability, mechanical properties and biologically benign nature [6]. Since then, hydrogels have expanded in molecular repeat unit composition, architecture and applications to have technological impact in drug and biomolecule theranostics [7], multiplexed biomolecular detection [8], biosensor-based molecular diagnostics [9], drug free macromolecular therapeutics [10] and tissue engineering and regenerative medicine [11]. Moreover, hydrogels are continually being molecularly engineered to serve as stimuli-responsive polymers [12] within their particular end-use environments by exploiting various sense and respond mechanisms [13][14][15].…”