(J-400) Hydrogel-based hybridization chain reaction (HCR) for detection of urinary exosomal miRNAs as a diagnostic tool of prostate cancer

Introduction::

Although urinary exosomal microRNAs (miRNAs) have recently emerged as potential biomarkers, clinical applications are still limited due to their low concentration in small volumes of clinical samples. Therefore, the development of a non-invasive, specific diagnostic tool, along with profiling exosomal miRNA markers from urine, remains a significant challenge.

Here, we present hydrogel-based hybridization chain reaction (HCR) for multiplex signal amplification to detect urinary exosomal miRNAs from human clinical samples. We succeeded in identifying small amounts (~amol) of exosomal miRNAs from 600 μL of urine with up to ~35-fold amplification and enhanced detection limits by over an order of magnitude for two miRNA biomarker candidates, hsa-miR-6090 and hsa-miR-3665. Furthermore, we proposed ratiometric analysis without requiring normalization to a reference miRNA and validated the clinical diagnostic potential toward differentiating prostate cancer patients from healthy controls.

Our hydrogel-based HCR could serve as a new diagnostic platform for a non-invasive liquid biopsy before burdensome tissue biopsy of various diseases, including prostate cancer screening, complementing the PSA test.

Materials and Methods::

The method employed hydrogel-based hybridization chain reaction (HCR) for signal amplification and subsequent image analysis. The steps involved in the HCR method are as follows:

 

1. Target hybridization: Urinary exosomal total RNAs or synthetic miRNAs were mixed with hydrogel microparticles in a microtube. The hybridization was carried out at 55 °C for 90 minutes. After hybridization, the microparticles were rinsed.

 

2. Initiation: A ligation mix containing NEBuffer, TET buffer, ATP, biotinylated universal adapter, and T4 DNA ligase was added to the microparticles. The microparticles were rinsed again. Neutravidin was added to the particles, followed by another round of rinsing.

 

3. HCR amplification: Biotinylated universal hairpins 1 and 2 were added to the particles. The hairpins were pre-heated and then cooled down. The HCR amplification was performed at 37 °C for 4 hours. After amplification, the particles were rinsed.

 

4. Signal detection: Streptavidin-r-phycoerythrin (SA-PE) was added to the particles and incubated. The particles were rinsed again before imaging.

 

5. Image acquisition and analysis: The SA-PE treated particles were placed between glass slides and imaged using an inverted fluorescence microscope. The signals of the urinary exosomal miRNA targets were estimated by subtracting the background fluorescence intensity. Ratiometric analysis was performed by dividing the background-subtracted fluorescence intensity of one miRNA target by that of another.

Results, Conclusions, and Discussions:: The hydrogel microparticles were designed with probe regions containing target-specific DNA oligomers and a fluorescent code, spatially compartmentalized within the microparticles. The HCR process involved the hybridization of target miRNAs with the probe regions, followed by initiation using a biotinylated universal adapter and neutravidin as a bridge. The amplification step employed a universal pair of hairpins and biotinylated hairpins to trigger a chain reaction, leading to the availability of multiple biotins. Finally, the fluorescent reporter, streptavidin conjugated with phycoerythrin (SA-PE), was added for signal amplification.

Optimization of the hydrogel-based HCR involved determining the temperature and time parameters for the amplification step. The optimal conditions were found to be 37 °C for 4 hours, resulting in approximately 35-fold amplification. The introduction of neutravidin as a bridge enhanced the HCR efficiency by about 80%. The study selected two miRNA targets, hsa-miR-3665 and hsa-miR-6090, as potential urinary exosomal biomarkers for prostate cancer based on their relative expression levels. The hydrogel-based HCR was characterized for performance, demonstrating superior sensitivity and detection limits ranging from 1 to 10 amol for miR-6090 and 10 to 100 amol for miR-3665.

The hydrogel-based HCR method was successfully applied to human clinical urine samples obtained from healthy controls and prostate cancer patients. The results showed the up-regulation of miR-6090 and the down-regulation of miR-3665 in prostate cancer patients compared to healthy controls, consistent with microarray data. Importantly, the hydrogel-based HCR enabled the reliable detection and quantification of the urinary exosomal miRNA biomarkers, surpassing the limit of detection and quantitation.

Overall, the study demonstrates the effectiveness of the hydrogel-based HCR method for the amplification and detection of urinary exosomal miRNAs, specifically targeting prostate cancer biomarkers. The method offers improved sensitivity, reduced assay time, and reliable analytical signals. The findings suggest that this approach has potential as a clinically relevant diagnostic tool for the detection and monitoring of prostate cancer using non-invasive urine samples.

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