Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells.     

Exponential isothermal amplification coupled MALDI-TOF MS for microRNAs detection

MicroRNAs (miRNAs) have attracted significant attention in biomedical research and clinical diagnosis. However, due to their inherent characteristics of low abundance and the high complexity of corresponding biological matrices, simultaneous detection of multiple miRNAs at low abundance is still a challenge. In this work, a method coupling exponential amplification reaction (EXPAR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is developed for label-free and simultaneous detection of multiple miRNAs. The assay can be performed under isothermal conditions in a single reaction tube, and finished in less than 30 min. It exhibits good quantification ability and with attomolar-level sensitivity for miRNAs detection. It also shows high specificity to distinguish miRNAs at single-nucleotide resolution. We used the method to detect the miRNA-21, let-7a, miRNA-100, and miRNA-125b in samples of spiked human serum and breast cancer cells (i.e., MCF-7, MDA-MB-231 and SK-BR-3). The quantification results were well consistent with the standard real-time fluorescence EXPAR. Consequently, the label-free mass-spectrometric platform could be a potential tool for miRNAs analysis in complex biological samples, and may be used for clinical diagnosis.     

Electrochemical analysis of microRNAs with hybridization chain reaction-based triple signal amplification

Selective and sensitive detection of trace microRNA is important for early diagnosis of diseases due to its expression level related to diseases. Herein, a triple signal amplification strategy is developed for trace microRNA-21 (miRNA-21) detection by combining with target-triggered cyclic strand displacement reaction (TCSDR), hybridization chain reaction (HCR) and enzyme catalytic amplification. Four DNA hairpins (H1, H2, H3, H4) are employed to form an ultralong double-strand DNA (dsDNA) structure, which is initiated by target miRNA-21. As H3 and H4 are labeled with horseradish peroxidase (HRP), numerous HRPs are loaded on the long dsDNA, producing significantly enhanced electrocatalytic signals in the hydrogen peroxide (H₂O₂) and 3,3′,5,5′-tetramethylbenzidine (TMB) reaction strategy. Compared with single signal amplification, the triple signal amplification strategy shows higher electrochemical response, wider dynamic range and lower detection limit for miRNA-21 detection with excellent selectivity, reproducibility and stability. Taking advantage of the triple signal amplification strategy, the proposed electrochemical biosensor can detect miRNA-21 in 10 HeLa cell lysates, suggesting that it is a promising method for fruitful assay in clinical diagnosis.     

Gold nanoparticle enhanced surface plasmon resonance imaging of microRNA-155 using a functional nucleic acid-based amplification machine

MicroRNAs (miRNAs) are considered as being promising biomarkers for hematological malignancies, their aging, progression and prognosis. The authors have developed a method for the detection of miRNA-155 by using surface plasmon resonance (SPR) imaging coupled to a nucleic acid-based amplification strategy using gold nanoparticles (AuNPs). The target miRNA-155 is captured by surface-bound DNA probes. After hybridization, DNA-AuNP are employed for signal amplification via DNA sandwich assembly, resulting in a large increase in the SPR signal. This method can detect miRNA-155 in concentrations down to 45 pM and over dynamic that extends from 50 pM to 5 nM. The assay is highly specific and can discriminate even a single base mismatch. It also is reproducible, precise, and was successfully applied to the determination of miRNA-155 in spiked real samples where it gave recoveries in the range between 86% and 98%. This biosensor provides an alternative approach for miRNA detection in biomedical research and clinical diagnosis, which is highly effective and efficient.

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Graphical abstract Schematic of a surface plasmon resonance imaging biosensor for detection of miRNA-155 using strand displacement amplification and gold nanoparticle.

     

A Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells

Herein, a Janus three-dimensional (3D) DNA nanomachine was constructed for the simultaneous and sensitive fluorescence detection and imaging of dual microRNAs (miRNAs) in cancer cells, which could effectively eliminate signal interference in a homogeneous nanoparticle-based 3D DNA nanostructure caused by the proximity of the two different signal probes to achieve accurate co-location in the same position of living cancer cells. In this system, the Janus nanoparticles were synthesized as the carrier for immobilizing two different oligonucleotides on two different functionalized hemispheres of the nanoparticles to form a Janus 3D DNA nanostructure, which could convert trace amounts of miRNA-21 and miRNA-155 targets into massive FAM and Cy5-labeled duplexes to induce two remarkable fluorescence emissions by the catalytic hairpin assembly (CHA) and 3D DNA walker cascade nucleic acid amplification strategy, realizing sensitive detection and imaging of miRNA targets in cancer cells. Impressively, in comparison with current miRNA imaging methods based on nanoparticle assemblies, the proposed strategy could efficiently eliminate “false positive” results obtained in single type miRNA detection and distinctly increase the immobilization concentration of two different signal probes using Janus nanoparticles as the carrier to further enhance fluorescence intensity, resulting in accurate co-location in the same position of living cells. Meanwhile, the proposed fluorescence imaging technology makes it possible to visualize low concentrations of miRNAs with tiny change associated with some cancers, which could significantly improve the accuracy and precision compared to those of the conventional fluorescence in situ hybridization (FISH) approach. Therefore, it could serve as persuasive evidence for supplying accurate information to better understand biological processes and investigate mechanisms of various biomolecules and subcellular organelles, resulting in the further validation of their function in tumor proliferation and differentiation. This strategy provided an innovative approach to design new generations of nanomachines with ultimate applications in bioanalysis and clinical diagnoses.

A Janus three-dimensional DNA nanomachine was constructed for the simultaneous and sensitive fluorescent detection and imaging of dual microRNAs in the cancer cells.     

A Target Recycling Amplification Process for the Digital Detection of Exosomal MicroRNAs through Photonic Resonator Absorption Microscopy

Exosomal microRNAs (miRNAs) have considerable potential as pivotal biomarkers to monitor cancer development, dis-ease progression, treatment effects and prognosis. Here, we report an efficient target recycling amplification process (TRAP) for the digital detection of miRNAs using photonic resonator absorption microscopy. We achieve multiplex digital detection with sub-attomolar sensitivity in 20 minutes, robust selectivity for single nucleotide variants, and a broad dynamic range from 1 aM to 1 pM. Compared with traditional qRT-PCR, TRAP showed similar accuracy in profiling exosomal miRNAs derived from cancer cells, but also exhibited at least 31-fold and 61-fold enhancement in the limits of miRNA-375 and miRNA-21 detection, respectively. The TRAP approach is ideal for exosomal or circulating miRNA biomarker quantification, where the miRNAs are present in low concentrations or sample volume, with potentials for frequent, low-cost, and minimally invasive point-of-care testing.     

Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging

Cell status changes are typically accompanied by the simultaneous changes of multiple microRNA (miRNA) levels. Thus, simultaneous and ultrasensitive detection of multiple miRNA biomarkers shows great promise in early cancer diagnosis. Herein, a facile single-molecule fluorescence imaging assay was proposed for the simultaneous and ultrasensitive detection of multiple miRNAs using only one capture anti-DNA/RNA antibody (S9.6 antibody). Two complementary DNAs (cDNAs) designed to hybridize with miRNA-21 and miRNA-122 were labelled with Cy3 (cDNA1) and Cy5 (cDNA2) dyes at their 5′-ends, respectively. After hybridization, both miRNA-21/cDNA1 and miRNA-122/cDNA2 complexes were captured by S9.6 antibodies pre-modified on a coverslip surface. Subsequently, the Cy3 and Cy5 dyes on the coverslip surface were imaged by the single-molecule fluorescence setup. The amount of miRNA-21 and miRNA-122 was quantified by counting the image spots from the Cy3 and Cy5 dye molecules in the green and red channels, respectively. The proposed assay displayed high specificity and sensitivity for singlet miRNA detection both with a detection limit of 5 fM and for multiple miRNA detection both with a detection limit of 20 fM. Moreover, it was also demonstrated that the assay could be used to detect multiple miRNAs simultaneously in human hepatocellular cancer cells (HepG2 cells). The proposed assay provides a novel biosensing platform for the ultrasensitive and simple detection of multiple miRNA expressions and shows great prospects for early cancer diagnosis.

A single-molecule assay for multiple microRNA detection.     

One-step, multiplexed fluorescence detection of microRNAs based on duplex-specific nuclease signal amplification

Traditional molecular beacons, widely applied for detection of nucleic acids, have an intrinsic limitation on sensitivity, as one target molecule converts only one beacon molecule to its fluorescent form. Herein, we take advantage of the duplex-specific nuclease (DSN) to create a new signal-amplifying mechanism, duplex-specific nuclease signal amplification (DSNSA), to increase the detection sensitivity of molecular beacons (Taqman probes). DSN nuclease is employed to recycle the process of target-assisted digestion of Taqman probes, thus, resulting in a significant fluorescence signal amplification through which one target molecule cleaves thousands of probe molecules. We further demonstrate the efficiency of this DSNSA strategy for rapid direct quantification of multiple miRNAs in biological samples. Our experimental results showed a quantitative measurement of sequence-specific miRNAs with the detection limit in the femtomolar range, nearly 5 orders of magnitude lower than that of conventional molecular beacons. This amplification strategy also demonstrated a high selectivity for discriminating differences between miRNA family members. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method promises a great potential of becoming a routine tool for simultaneously quantitative analysis of multiple miRNAs in tissues or cells, and supplies valuable information for biomedical research and clinical early diagnosis.     

Simultaneous photoelectrochemical detection of dual microRNAs by capturing CdS quantum dots and methylene blue based on target-initiated strand displaced amplification

Herein,we propose a novel photoelectrochemical(PEC) biosensor for dual microRNAs(miRNAs) highly sensitive and simultaneous biosensing based on strand displaced amplification(SDA) reaction.The recognition of H_(miR-21) and H_(let-7 a) by microRNA-21 and let-7 a leads to their change in hairpin structures,subsequently initiating the immobilization of abundant CdS quantum dots(CdS QD s) and methylene blue(MB) based on SDA reaction.The immobilized CdS QDs and MB produce both high PEC currents under430 nm light and 627 nm light illumination,respectively,and the generated PEC currents are closely relied on target miRNAs amounts.Thus,highly sensitive and simultaneous detection of microRNA-21 and let-7 a was readily achieved with detection limit at 6.6 fmol/L and 15.4 fmol/L based on 3σ,respectively.Further,this PEC biosensor was applied in simultaneous analysis of miRNA-21 and let-7 a in breast cancer patient's serum with acceptable results.We expect this biosensor will find more useful application in diagnosis of miRNA-related diseases.     

Entropy-driven reactions in living cells for assay let-7a microRNA

Imaging of microRNA (miRNA) in living cells could facilitate the monitoring of the expression and distribution of miRNA and research on miRNA-related diseases. Given the low expression levels and even down-regulation of cellular miRNA that is associated with some diseases, enzyme-free amplification strategies are imperative for intracellular miRNA assay. In this work, we report an entropy-driven reaction for amplification assay miRNA with a detection limit of 0.27 pM. The resulting signal amplification provides excellent recognition and signal enhancement of specific miRNAs in living cells. This method supplies accurate information regarding cellular miRNA-related biological events and provides a new tool for highly sensitive and simultaneous imaging of multiple low-level biomarkers, thereby improving the accuracy of early disease diagnosis.     

Signal Amplification Technologies for the Detection of Nucleic Acids: from Cell-Free Analysis to Live-Cell Imaging

Due to their unique properties, such as programmability, ligand-binding capability, and flexibility, nucleic acids can serve as analytes and/or recognition elements for biosensing. To improve the sensitivity of nucleic acid-based biosensing and hence the detection of a few copies of target molecule, different modern amplification methodologies, namely target-and-signal-based amplification strategies, have already been developed. These recent signal amplification technologies, which are capable of amplifying the signal intensity without changing the targets’ copy number, have resulted in fast, reliable, and sensitive methods for nucleic acid detection. Working in cell-free settings, researchers have been able to optimize a variety of complex and quantitative methods suitable for deploying in live-cell conditions. In this study, a comprehensive review of the signal amplification technologies for the detection of nucleic acids is provided. We classify the signal amplification methodologies into enzymatic and non-enzymatic strategies with a primary focus on the methods that enable us to shift away from in vitro detecting to in vivo imaging. Finally, the future challenges and limitations of detection for cellular conditions are discussed.     

Dumbbell probe-mediated cascade isothermal amplification: A novel strategy for label-free detection of microRNAs and its application to real sample assay

Considering the great significance of microRNAs (miRNAs) in cancer detection and typing, the development of sensitive, specific, quantitative, and low-cost methods for the assay of expression levels of miRNAs is desirable. We describe a highly efficient amplification platform for ultrasensitive analysis of miRNA (taking let-7a miRNA as a model analyte) based on a dumbbell probe-mediated cascade isothermal amplification (DP-CIA) strategy. The method relies on the circularization of dumbbell probe by binding target miRNA, followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking/polymerization/displacement cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal. In terms of the high sensitivity (as low as 1 zmol), wide dynamic range (covering 9 orders of magnitude), good specificity (even single-base difference) and easy operation (one probe and three enzymes), the proposed label-free assay is successfully applied to direct detection of let-7a miRNA in real sample (total RNA extracted from human lung tissue), demonstrating an attractive alternative for miRNA analysis for gene expression profiling and molecular diagnostics, particularly for early cancer diagnosis.     

Amplified electrochemical detection of nucleic acid hybridization via selective preconcentration of unmodified gold nanoparticles

The common drawback of optical methods for rapid detection of nucleic acid by exploiting the differential affinity of single-/double-stranded nucleic acids for unmodified gold nanoparticles (AuNPs) is its relatively low sensitivity. In this article, on the basis of selective preconcentration of AuNPs unprotected by single-stranded DNA (ssDNA) binding, a novel electrochemical strategy for nucleic acid sequence identification assay has been developed. Through detecting the redox signal mediated by AuNPs on 1, 6-hexanedithiol blocked gold electrode, the proposed method is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the target microRNA (let-7a) in human breast adenocarcinoma cells, and a detection limit of 16 fM is readily achieved with desirable specificity and sensitivity. These results indicate that the selective preconcentration of AuNPs for electrochemical signal readout can offer a promising platform for the detection of specific nucleic acid sequence.     

Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels

This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro-channel delivers fresh analyte solution to the reaction site which maintains a high concentration gradient differential to enhance mass transport. Based on the dual signal amplification strategy, the developed microfluidic bead-based nucleic acid sensor could discriminate as low as 5 fM (signal-to-noise (S/N) 3) of synthesized carcinoembryonic antigen (CEA) gene fragments and showed a 1000-fold increase in detection limit compared to the off-chip test. In addition, using spiked colorectal cancer cell lines (HT29) in the blood as a model system, the detection limit of this chip-based approach was found to be as low as 1 HT29 in 1 mL blood sample. This microfluidic bead-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence.     

Nucleic Acid-to-Small Molecule Converter through Amplified Hairpin DNA Circuits

Many microRNAs (miRNAs) are characteristically found in cancer cells, making miRNAs promising marker biomolecules for cancer diagnosis and therapeutics. However, it is challenging to use miRNA as a cancer signature because it is difficult to convert the nucleic acid sequence information into molecular functionality. To address this challenge, we realize nucleic acid-to-small molecule converters using hairpin DNA circuits. Harnessing a Staudinger reduction as a trigger for the conversion, we constructed hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) circuits that respond to oncogenic miR-21. Fluorophore and dye molecules were released in response to miR-21 through the HCR, providing fluorogenic and chromogenic readouts. Selective cytotoxicity in miR-21-abundant cells was realized by the CHA to release the anticancer drug SN-38. This would be the first example of selective activation of a small-molecule prodrug triggered by oncogenic miRNA in human living cells.     

单细胞核酸编码扩增成像分析

单细胞成像可在单细胞水平观测目标物位置、 确定目标物含量, 在生命科学与临床医学研究领域应用广泛. 核酸编码扩增技术利用特定分子反应将待测目标识别转化为核酸条码的扩增, 具有探针种类多、 易编程、 反应条件温和及信号放大效率高等特点, 在单细胞低丰度、 高灵敏、 多目标物成像中优势显著, 为理解细胞状态、 探索生命过程提供了新思路. 本文综合评述了核酸编码扩增在单细胞荧光成像领域的研究进展, 以目标物的编码方式为分类依据, 系统阐述了固定细胞原位成像和活细胞成像中不同目标物编码与扩增成像方式的区别, 并对活细胞成像中多重检测面临的问题以及未来发展前景进行了展望.     

Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification

MicroRNAs (miRNAs) play an important regulatory role in cells and dysregulation of miRNA has been associated with a variety of diseases, making them a promising biomarker. In this work, a novel biosensing strategy has been developed for label-free detection of miRNA using surface plasmon resonance (SPR) coupled with DNA super-sandwich assemblies and biotin–strepavidin based amplification. The target miRNA is selectively captured by surface-bound DNA probes. After hybridization, streptavidin is employed for signal amplification via binding with biotin on the long DNA super-sandwich assemblies, resulting in a large increase of the SPR signal. The method shows very high sensitivity, capable of detecting miRNA at the concentration down to 9 pM with a wide dynamic range of 6 orders of magnitude (from 1 × 10⁻¹¹ M to 1 × 10⁻⁶ M) in 30 min, and excellent specificity with discriminating a single base mismatched miRNA sequence. This biosensor exhibits good reproducibility and precision, and has been successfully applied to the detection of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells. It, therefore, offers a highly effective alternative approach for miRNA detection in biomedical research and clinical diagnosis.     

An electrochemical microRNAs biosensor with the signal amplification of alkaline phosphatase and electrochemical–chemical–chemical redox cycling

MicroRNAs (MiRNAs) have been regarded as clinically important biomarkers and drug discovery targets. In this work, we reported a simple and ultrasensitive electrochemical method for miRNAs detection based on single enzyme amplification and electrochemical–chemical–chemical (ECC) redox cycling. Specifically, upon contact with the target miRNAs, the hairpin structure of biotinylated DNA immobilized on gold electrode was destroyed and the biotin group in DNA was forced away from the electrode surface, allowing for the coupling of streptavidin-conjugated alkaline phosphatase (SA-ALP). Then, ascorbic acid (AA, the enzymatic product of ALP) triggered the ECC redox cycling with ferrocene methanol (FcM) and tris(2-carboxyethyl)phosphine (TCEP) as the redox mediator and the chemical reducing reagent, respectively. The method was more sensitive than that with horseradish peroxidase (HRP) or glucose oxidase (GOx) triggered recycling since one ALP molecule captured by one target miRNA molecule promoted the production of thousands of AA. Analytical merits (e.g., detection limit, dynamic range, specificity, regeneration and reproducibility) were evaluated. The feasibility of the method for analysis of miRNA-21 in human serum has also been demonstrated.