Smartphone-Based Fluorescent Profiling of Quaternary MicroRNAs in Urine for Rapid Diagnosis of Urological Cancers Using a Multiplexed Isothermal Exponential Amplification Reaction.

Urological cancers such as bladder or prostate cancer represent one of the most malignant tumors that accounts for an extremely high mortality. However, conventionally standard diagnostics for urological cancers are hardly available in low-resource settings. We developed herein a hand-held fluorescent imaging platform by integrating a multiplexed isothermal exponential amplification reaction (EXPAR) with a microgel-enriched methodology for sensitive profiling of quaternary microRNAs (miRNAs) in urine and quick diagnosis of urological cancers at the early stage. The target miRNA mixtures in the urine underwent four parallel EXPARs without cross-reactivity, followed by surface concentration and hybridization by the encoded polyacrylamide microgels. This mix-and-read strategy allowed for one-pot analysis of several key miRNAs simultaneously and provided 5-fold enhancement in fluorescent detection sensitivities compared to the individual EXPAR-based assays. Four urinary miRNAs (let-7a, miRNA-155, -223, and -143) could be quantitatively determined in a wide linear range from 50 fM to 30 nM, with the limits of detection at femtomolar levels. Using a smartphone-based imaging microreader, healthy and cancerous cohorts with prostate, bladder, and renal cell cancers could be discriminated in 30 min with the accuracy >83% using linear discriminant analysis. The developed detection platform has proven to be a portable, noninvasive, and useful complement to the toolbox for miRNA-based liquid biopsies, which holds immense potential and advantage for regular and large-scale applications in early cancer diagnosis.

Interfacial Profiling of MicroRNAs at Patterned Nanogaps for an Integrated Microfluidic-SERS Liquid Biopsy.

A versatile microfluidic-SERS barcoding system is developed for sensitive and multiplexed imaging of circulating microRNAs through interfacial probing of encoded nanorod aggregates at diverse patterned nanogaps. The use of a single-layer, vertically oriented nanorod array creates a plasmonic coupling-based electromagnetic field with enormously enhanced Raman outputs. The introduction of the herringbone micromixer with circulated microflow sampling accelerates the hybridization and capture of nanorod aggregates on the plasmonic substrate. The method is able to achieve ideal sensitivities at subfemtomolar levels for four miRNAs, with multiplexed assay capability for an integrated liquid biopsy. The on-chip digital profiling of serum miRNAs in mapping and barcoding formats enable both clear discrimination of untreated cancer patients from the healthy cohort and precise classification of tumor stages, metastatic conditions, and subtypes, with an overall accuracy of 94%. The SERS-based microfluidic barcoding system therefore holds great promise in early cancer screening, diagnosis, and prognosis.

Plasmon-enhanced biosensors for microRNA analysis and cancer diagnosis.

MicroRNAs are a class of reliable biomarkers for noninvasive detection of a variety of diseases, including cancers. This is because miRNA, especially exosome miRNAs, can stably circulate in the blood and are consequently indicative of the development and progression of typical cancer cells. Among a variety of tools for miRNA analysis, plasmon-enhanced biosensors have attracted special interests due to their remarkable sensing properties. It originates from the principle that local surface plasmon resonance occurs when the dimensions of a metallic nanostructure are shorter than the wavelength of the incident light, leading to collective but non-propagating oscillations of free electrons that generates intriguing optoelectronic properties. This article presents a review of recent progress in miRNA detection based on plasmon-enhanced optical sensing, including surface enhanced Raman scattering, plasmon-enhanced fluorescence, and plasmon-enhanced electrochemiluminescence. The article is focused on the molecular sensing mechanisms and the assembling strategies of the nanomaterial substrates to plasmonically enhance optical outputs of miRNAs. In particular, this paper discusses different methods of enzyme-mediated or enzyme-free amplification and substrate-enhanced sensing, and highlights the potential of plasmon-enhanced optical sensors for multiplexed analysis of complex biological samples, as well as for point-of-care testing for the onset of typical cancers.

Alkaline lysis-recombinase polymerase amplification combined with CRISPR/Cas12a assay for the ultrafast visual identification of pork in meat products.

The "Horsemeat Scandal" makes people pay more attention to the meat authenticity. However, expensive equipment, complicated operations, and professional personnel of current methods limit their field testing. In this study, CRISPR/Cas12a combined with recombinase polymerase amplification was used to establish a sensitive and rapid detection method for pig-derived component. The detection limit can reach to 10-3 ng for pig DNA and be completed within 30 min. Beef and pork binary mixture models under different processing conditions of raw meat, boiled, and high-pressure were tested. Combining with two different DNA extraction methods, the detection limits of pork are as low as 0.1% and 0.001% (w/w), respectively. After 125 commercial products are tested, the results are completely consistent with the Chinese national standard real-time PCR method. This method not only has better detectability, but also can quickly and conveniently realize the visual identification of pig-derived ingredients, thus is suitable for on-site detection.