Single-molecule amplification-free multiplexed detection of circulating microRNA cancer biomarkers from serum

AbstractMicroRNAs (miRNAs) play essential roles in post-transcriptional gene expression and are also found freely circulating in bodily fluids such as blood. Dysregulated miRNA signatures have been associated with many diseases including cancer, and miRNA profiling from liquid biopsies offers a promising strategy for cancer diagnosis, prognosis and monitoring. Here, we develop size-encoded molecular probes that can be used for simultaneous electro-optical nanopore sensing of miRNAs, allowing for ultrasensitive, sequence-specific and multiplexed detection directly in unprocessed human serum, in sample volumes as small as 0.1 μl. We show that this approach allows for femtomolar sensitivity and single-base mismatch selectivity. We demonstrate the ability to simultaneously monitor miRNAs (miR-141-3p and miR-375-3p) from prostate cancer patients with active disease and in remission. This technology can pave the way for next generation of minimally invasive diagnostic and companion diagnostic tests for cancer.

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Simultaneous Discrimination of Single-Base Mismatch and Full Match Using a Label-Free Single-Molecule Strategy

Analytical Chemistry ◽

10.1021/acs.analchem.8b01285 ◽

2018 ◽

Vol 90 (13) ◽

pp. 8102-8107 ◽

Cited By ~ 3

Author(s):

Qiufang Yang ◽

Tingting Ai ◽

You Lv ◽

Yuqin Huang ◽

Jia Geng ◽

...

Keyword(s):

Single Molecule ◽

Simultaneous Discrimination ◽

Label Free ◽

Single Base ◽

Single Base Mismatch ◽

Base Mismatch

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Single-Step FRET-Based Detection of Femtomoles DNA

Sensors ◽

10.3390/s19163495 ◽

2019 ◽

Vol 19 (16) ◽

pp. 3495 ◽

Cited By ~ 7

Author(s):

Sapkota ◽

Kaur ◽

Megalathan ◽

Donkoh-Moore ◽

Dhakal

Keyword(s):

Sample Size ◽

Single Molecule ◽

Single Step ◽

Small Sample ◽

Sensitive Detection ◽

Quantitative Detection ◽

Single Base ◽

Single Base Mismatch ◽

Mechanical Methods ◽

Base Mismatch

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers.

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Overexpression of circulating MiR-30b-5p identifies advanced breast cancer

Journal of Translational Medicine ◽

10.1186/s12967-019-02193-y ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 2

Author(s):

Helena Estevão-Pereira ◽

João Lobo ◽

Sofia Salta ◽

Maria Amorim ◽

Paula Lopes ◽

...

Keyword(s):

Breast Cancer ◽

Validation Cohort ◽

Advanced Disease ◽

Clinical Tool ◽

Liquid Biopsies ◽

Primary Tumors ◽

Bodily Fluids ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Abstract Background Breast cancer (BrC) remains the leading cause of cancer-related death in women, mainly due to recurrent and/or metastatic events, entailing the need for biomarkers predictive of progression to advanced disease. MicroRNAs hold promise as noninvasive cancer biomarkers due to their inherent stability and resilience in tissues and bodily fluids. There is increasing evidence that specific microRNAs play a functional role at different steps of the metastatic cascade, behaving as signaling mediators to enable the colonization of a specific organ. Herein, we aimed to evaluate the biomarker performance of microRNAs previously reported as associated with prognosis for predicting BrC progression in liquid biopsies. Methods Selected microRNAs were assessed using a quantitative reverse transcription-polymerase chain reaction in a testing cohort of formalin-fixed paraffin-embedded primary (n = 16) and metastatic BrC tissues (n = 22). Then, miR-30b-5p and miR-200b-3p were assessed in a validation cohort #1 of formalin-fixed paraffin-embedded primary (n = 82) and metastatic BrC tissues (n = 93), whereas only miR-30b-5p was validated on a validation cohort #2 of liquid biopsies from BrC patients with localized (n = 20) and advanced (n = 25) disease. ROC curve was constructed to evaluate prognostic performance. Results MiR-30b-5p was differentially expressed in primary tumors and paired metastatic lesions, with bone metastases displaying significantly higher miR-30b-5p expression levels, paralleling the corresponding primary tumors. Interestingly, patients with advanced disease disclosed increased circulating miR-30b-5p expression compared to patients with localized BrC. Conclusions MiR-30b-5p might identify BrC patients at higher risk of disease progression, thus, providing a useful clinical tool for patients’ monitoring, entailing earlier and more effective treatment. Nonetheless, validation in larger multicentric cohorts is mandatory to confirm these findings.

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Single-molecule FRET for Ultrasensitive Detection of Biomolecules

NanoBioImaging ◽

10.2478/nbi-2013-0002 ◽

2014 ◽

Vol 1 (1) ◽

Cited By ~ 5

Author(s):

Kun Yang ◽

Yong Yang ◽

Chun-yang Zhang

Keyword(s):

Single Molecule ◽

New Technologies ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Molecular Probes ◽

Ultrasensitive Detection ◽

Single Molecule Fret ◽

Biological Reaction ◽

Spatio Temporal ◽

Detection Of Biomolecules

AbstractSingle-molecule Förster resonance energy transfer (sm- FRET) has been widely employed to detect biomarkers and to probe the structure and dynamics of biomolecules. By monitoring the biological reaction in a spatio-temporal manner, smFRET can reveal the transient intermediates of biological processes that cannot be obtained by conventional ensemble measurements. This review provides an overview of singlemolecule FRET and its applications in ultrasensitive detection of biomolecules, including the major techniques and the molecular probes used for smFRET as well as the biomedical applications of smFRET. Especially, the combination of sm- FRET with new technologies might expand its applications in clinical diagnosis and biomedical research

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Advance in Pancreatic Cancer Diagnosis and Therapy

10.5772/intechopen.94413 ◽

2020 ◽

Author(s):

Xiaojie Cai ◽

Jie Gao ◽

Yanfang Liu ◽

Ming Wang ◽

Qiulian Ma ◽

...

Keyword(s):

Pancreatic Cancer ◽

Stem Cells ◽

Survival Rate ◽

Tumor Microenvironment ◽

Cancer Diagnosis ◽

Treatment Resistance ◽

Serum Markers ◽

Molecular Probes ◽

Cancer Death ◽

Cancer Diagnosis And Therapy

Pancreatic carcinoma is the fourth leading cause of cancer death in the word wild. Although the advance in treatment this disease, the 5-years survival rate is still rather low. In the recent year, many new therapy and treatment avenues have been developed for pancreatic cancer. In this chapter, we mainly focus on the following aspect: 1) the treatment modality in pancreatic cancer, including chemotherapy, radiotherapy, and immunotherapy; 2) the mechanism of pancreatic cancer treatment resistance, especially in cancer stem cells and tumor microenvironment; 3) the diagnosis tools in pancreatic cancer, including serum markers, imaging methods and endoscopic ultrasonography. Novel molecular probes based on the nanotechnology in the diagnosis of pancreatic cancer are also discussed.

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Multiplexed Detection of COVID-19 with Single-Molecule Technology

10.1101/2021.05.25.21257501 ◽

2021 ◽

Author(s):

Noa Furth ◽

Shay Shilo ◽

Niv Cohen ◽

Nir Erez ◽

Vadim Fedyuk ◽

...

Keyword(s):

Immune Response ◽

Viral Infection ◽

Single Molecule ◽

Direct Detection ◽

Genetic Material ◽

Diagnostic Tools ◽

Proof Of Concept ◽

Multiplexed Detection ◽

Diagnostic Approaches ◽

Quantitative Nature

The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for multiplexed detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.

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Facile profiling of molecular heterogeneity by microfluidic digital melt

Science Advances ◽

10.1126/sciadv.aat6459 ◽

2018 ◽

Vol 4 (9) ◽

pp. eaat6459 ◽

Cited By ~ 14

Author(s):

Christine M. O’Keefe ◽

Thomas R. Pisanic ◽

Helena Zec ◽

Michael J. Overman ◽

James G. Herman ◽

...

Keyword(s):

Colorectal Cancer ◽

Tumor Suppressor ◽

Tumor Suppressor Gene ◽

Single Molecule ◽

Rapid Assessment ◽

Suppressor Gene ◽

Molecular Heterogeneity ◽

Liquid Biopsies ◽

Digital Microfluidic ◽

Colorectal Cancer Patients

This work presents a digital microfluidic platform called HYPER-Melt (high-density profiling and enumeration by melt) for highly parallelized copy-by-copy DNA molecular profiling. HYPER-Melt provides a facile means of detecting and assessing sequence variations of thousands of individual DNA molecules through digitization in a nanowell microchip array, allowing amplification and interrogation of individual template molecules by detecting HRM fluorescence changes due to sequence-dependent denaturation. As a model application, HYPER-Melt is used here for the detection and assessment of intermolecular heterogeneity of DNA methylation within the promoters of classical tumor suppressor genes. The capabilities of this platform are validated through serial dilutions of mixed epialleles, with demonstrated detection limits as low as 1 methylated variant in 2 million unmethylated templates (0.00005%) of a classic tumor suppressor gene,CDKN2A(p14ARF). The clinical potential of the platform is demonstrated using a digital assay forNDRG4, a tumor suppressor gene that is commonly methylated in colorectal cancer, in liquid biopsies of healthy and colorectal cancer patients. Overall, the platform provides the depth of information, simplicity of use, and single-molecule sensitivity necessary for rapid assessment of intermolecular variation contributing to genetic and epigenetic heterogeneity for challenging applications in embryogenesis, carcinogenesis, and rare biomarker detection.

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Magnetic-capture-based SERS detection of multiple serum microRNA biomarkers for cancer diagnosis

Analytical Methods ◽

10.1039/c8ay02423e ◽

2019 ◽

Vol 11 (6) ◽

pp. 783-793 ◽

Cited By ~ 4

Author(s):

Hao Zhang ◽

Chaopeng Fu ◽

Shutao Wu ◽

Youqing Shen ◽

Chunhui Zhou ◽

...

Keyword(s):

Cancer Diagnosis ◽

Multiplexed Detection ◽

Serum Microrna ◽

Magnetic Capture ◽

Highly Sensitive ◽

Sers Detection

A highly sensitive and reproducible magnetic-capture SERS assay was developed for multiplexed detection of cancer-related microRNAs in serum solution.

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