Simultaneous Detection Of Multiple miRNAs Research Articles

Amplification-Free Analysis of Bladder Cancer MicroRNAs on Wrinkled Silica Nanoparticles with DNA-Functionalized Quantum Dots.

Bladder cancer (BC) occurrence and progression are accompanied by alterations in microRNAs (miRNAs) expression levels. Simultaneous detection of multiple miRNAs contributes to the accuracy and reliability of the BC diagnosis. In this work, wrinkled silica nanoparticles (WSNs) were applied as the microreactor for multiplex miRNAs analysis without enzymes or nucleic acid amplification. Conjugated on the surface of WSNs, the S9.6 antibody was adopted as the universal module for binding DNA/miRNA duplexes, regardless of their sequence. Furthermore, single-stranded DNA (ssDNA) was labeled with quantum dots (QDs) for identifying a given miRNA to form QDs-ssDNA/miRNA, which enabled the specific capture of the corresponding QDs on the wrinkled surface of WSNs. Based on the detection of fluorescence signals that were ultimately focused on WSNs, target miRNAs could be sensitively identified to a femtomolar level (5 fM) with a wide dynamic range of up to 6 orders of magnitude. The proposed strategy achieved high specificity to obviously distinguish single-base mutation sequences and possessed multiplex assay capability. Moreover, the assay exhibited excellent practicability in the multiplex detection of miRNAs in clinical serum specimens.

Cas-Rainbow: Cas12a-driven single-reaction multiplex detection system

Recently, CRISPR/Cas system has garnered considerable attention owing to its high cleavage efficiency. However, it lacks the ability to differentiate between signal sources. To address this, we have developed a multiplex detection system called Cas-Rainbow. This system combines CRISPR/Cas with rolling circle amplification (RCA) and hybridization chain reaction (HCR) to enable the detection of multiple miRNAs using reporter probes labeled with different fluorescent groups. The RCA product can trigger the formation of HCR and activate the Cas12a/crRNA complex, achieving the simultaneous detection of multiple miRNAs. Detection limits of three miRNAs (miR-155, miR-10b, and miR-21) were 33 fM, 41 fM, and 79 fM, respectively. Cas-Rainbow system could effectively identify these three miRNAs in serum, distinguishing breast cancer patients from healthy donors with accuracy of 100 %. Moreover, this system exhibited exceptional performance in logic gate circuits, showcasing its remarkable versatility and robustness. In summary, our proposed method offers a new approach for multiplex detection based on the CRISPR/Cas system.

Triple ligation-based formation of a G-quadruplex for simultaneous detection of multiple miRNAs.

The simultaneous detection of multiple microRNAs (miRNA) is of great necessity but has not been extensively studied. This prompted our study, which involved the development of a triple ligation-based system for detecting three miRNAs at the same time. We designed a multi-ligation-padlock (MLP) probe that consists of three parts, each of which is complementary to two different miRNAs at the same time. In the presence of all three miRNAs, the probe becomes circularized, but in the absence of even one target, the probe remains linear. The first part of the MLP probe (MLP1) contains a T7 promoter part that can initiate RNA synthesis for any given target condition. However, it also includes a G-quadruplex complementary segment, which can only form a parallel RNA G-quadruplex through rolling circle transcription by the circularized template in the presence of all three targets. In this case, the application of our parallel G-quadruplex sensing fluorescent probe lutidine DESA (LutD) produces a strong signal. However, in the absence of any one of the targets, the RNA G-quadruplex cannot be formed and ultimately the LutD probe does not generate any signal. This difference in the signal intensity represents the presence or absence of all the target miRNAs. With our system, we were able to detect miRNA 21 at levels as low as 1.13 fM, miRNA 146a as low as 1.37 fM, and miRNA 25b as low as 1.51 fM within 45 minutes, confirming that our novel system can selectively and sensitively diagnose triple miRNAs.

A microfluidic-based SERS biosensor with multifunctional nanosurface immobilized nanoparticles for sensitive detection of MicroRNA

Developing sensitive and miniaturized biosensors for the detection of microRNAs (miRNAs) is highly desirable due to their association with early cancer diagnosis and prognosis. Here, a new microfluidic-based biosensor, combined with multifunctional nanosurface and DSN-assisted target recycle amplification strategy, is designed for the detection of miRNA-21. The design of nanosurface includes gold nanoparticles on porous anodic aluminum oxide (AAO) for surface enhanced Raman scattering (SERS) substrate, AuMBA@Ag core-shell nanoparticles for SERS nanotags and single-stranded DNA (ssDNA) in between for miRNA capture and nanotags immobilization. When the target miRNA is present near the nanosurface, it will be captured by ssDNA via hybridization reaction. Then, triggered by the DSN-assisted target recycle process, the freshly formed DNA/miRNA heteroduplexes are cleaved by DSN enzyme into DNA fragments and single-strand miRNA. The SERS nanotags are also dissociated from the nanosurface, leading to decrease of SERS signal. The cleaved target miRNA can be captured and SERS nanotags are released again in the next cycle, resulting in amplification of detection signal. To improve the accuracy of this biosensor, the functionalized AAO membrane is subdivided into two groups - AAO/Au array linked with encoded core-shell SERS nanotags acting as a reactor and primary detector and AAO/Au@Ag array serving as a collector and secondary detector for the dissociative SERS nanotags from the reactor. The decrease of SERS signal in primary detector and increase of signal in secondary detector ensures the accuracy and it is called dual-SERS detection strategy. The detection of miRNA-21 can be achieved with only 30 μL sample and 10 μL enzyme and a wide linear range of 10 fM∼10 nM is obtained. In addition, the microfluidic dual-SERS detection strategy can greatly reduce the possibility of false positive or false negative in single detection mode and it can be applied to the simultaneous detection of multiple miRNAs via integrating different probes.

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.

Cotton thread-based multi-channel photothermal biosensor for simultaneous detection of multiple microRNAs

The abnormal expression of microRNAs (miRNAs) is associated with various diseases. Developing simple and portable methods for sensitive, rapid and simultaneous detection of multiple miRNAs is critical to achieve accurate and timely diagnosis. Herein, a cotton thread-based multi-channel photothermal biosensor was proposed for simultaneous detection of three breast cancer-related miRNAs including miRNA-10b, miRNA-27a and miRNA-let-7a. Three cotton thread-based channels with one input were designed and the capture probes for detecting different miRNAs were immobilized on the test zones of the corresponding channels. Cu2-xS nanostrings prepared on the basis of hybridization chain reaction (HCR) were taken as the photothermal agents for signal transduction and amplification. The formation of a sandwich structure among the capture probe, target miRNA, and Cu2-xS nanostrings led to the accumulation of the Cu2-xS nanostrings on the test zones and transformed the concentration of miRNA into temperature signal under 808 nm laser irradiation. The temperature changes were quantified by a portable thermal camera and directly reflected the concentration of miRNAs. Under the optimal conditions, the developed multi-channel photothermal biosensor showed excellent specificity and sensitivity with the detection limits of 37 pM, 38 pM and 38 pM for miRNA-10b, miRNA-27a and miRNA-let-7a, respectively. Furthermore, a simultaneous detection of the three miRNAs in cell lysates were achieved and the results were in accordance with that obtained by the quantitative reverse transcription polymerase chain reaction (qRT-PCR), indicating its excellent capacity for practical applications. The developed biosensor provided an important tool for analysis of multiple targets and showed great potential in clinical diagnosis.

DNA functionalized double quantum dots-based fluorescence biosensor for one-step simultaneous detection of multiple microRNAs

The disease diagnosis by detecting single microRNAs (miRNAs) can produce high false positive rate. Herein, a novel fluorescence biosensor method for one-step simultaneous detection of multiple miRNAs was proposed by using single-stranded DNA (ssDNA) functionalized double quantum dots (QDs) and black hole quencher (BHQ)-decorated magnetic nanobeads (MNs). MNs were linked with two black hole quenchers (BHQ1 and BHQ3) via a complementary DNA (cDNA). The ssDNA/cDNA hybridization contributed to the fluorescence quenching of double QDs due to the fluorescence resonance energy transfer (FRET) between double QDs and BHQ. In the presence of target miRNA-33 (miR-33) and miRNA-125b (miR-125b), the ssDNA1 and ssDNA2 were respectively hybridized with miR-33 and miR-125b to form more stable duplexes. Thus, the double QDs were released into supernatant after the magnetic separation, leading to the fluorescence signals recovery at 537 nm and 647 nm. A wide linear range (0.5 nM–320 nM for miR-33 and 0.1 nM–250 nM for miR-125b) and low limits of detection (0.09 nM for miR-33 and 0.02 nM for miR-125b) were achieved. Moreover, our approach has been demonstrated to simultaneously detect miR-33 and miR-125b in cell extracts. With advantages of high sensitivity, strong specificity, low background and low cost, the strategies show great potentials for the detection of various targets in bioanalysis and disease diagnosis.

NEase-based amplification for detection of miRNA, multiple miRNAs and circRNA

MiRNAs are particularly desirable biomarkers for the diagnosis of many cancers, but the methods for simultaneous detection of multiple miRNAs are rarely published. In addition, circRNAs are a novel class of non-coding RNAs and it has been accepted that circRNAs participate in regulating various biological processes. Herein, we design a method using NEase which has highly selectivity and activity to amplify fluorescence signal, so single miRNA, circRNA and multiple miRNAs could be simply quantified through our proposed assay. Furthermore, this method also can detect miRNA level in different cell lines.

A pico-HPLC-LIF system for the amplification-free determination of multiple miRNAs in cells

MicroRNAs are a class of important biomarkers, and the simultaneous detection of multiple miRNAs can provide valuable information about many diseases and biological processes. Amplification-free determination has been developed for the analysis of multiple miRNAs because of its characteristic low cost and high fidelity. Herein, a method for the amplification-free analysis and simultaneous detection of multiple miRNAs based on a so-called pico-HPLC-LIF system is described. In this process, a bare open capillary with an inner diameter of 680nm is used as a separation column for a sample volume of several hundreds of femtoliters (300fL), followed by separation and detection. The technique has a zeptomolar limit of detection. The method was applied to detect cellular miRNA from adenocarcinomic human alveolar basal epithelial (A549) cell extracts, and the simultaneous detection of the mir-182, miR-155, and let-7a was achieved. The results showed that the expression of mir-182 and miR-155 was up-regulated and that of let-7a was down-regulated in A549 cells. This method for multiple miRNAs detection is expected to have broad applications in miRNA-based disease diagnosis, prognosis, treatment, and monitoring.

In situ multiplex detection of serum exosomal microRNAs using an all-in-one biosensor for breast cancer diagnosis.

Herein, a simple all-in-one biosensor based on a DNA three-way junction has been constructed for in situ simultaneous detection of multiple miRNAs by competitive strand displacement. In our design, three oligonucleotides (Y1, Y2 and Y3) of a Y-type scaffold were extended at their 5' ends by introducing three single-stranded recognition sequences with quenchers (BHQ1, BHQ2 and BHQ2), respectively. Subsequently, three reporter sequences labeled with different fluorophores (FAM, Cy3 and Cy5) were bound to the corresponding recognition sequences to form a multicolour DNA biosensor that gives self-quenched fluorescence. The biosensor can effectively enter into exosomes and then hybridize to the complementary miRNA targets to form longer duplexes and release the reporter sequences, thus activating the readable fluorescence signals for the simultaneous detection of multiple miRNAs in exosomes. As a proof of principle, miR-21, miR-27a and miR-375 were chosen as model targets because of their high expressions in breast cancer cells (MCF-7). Fluorescence signals of MCF-7 exosomes after being treated with the biosensor exhibited positive correlations to their concentrations and the limits of detection were determined to be 0.116 μg mL-1, 0.125 μg mL-1 and 0.287 μg mL-1 for exosomes by detecting three exosomal miRNAs (miR-21, miR-27a and miR-375), respectively. In contrast, there were no obvious correlations between fluorescence intensities and control MCF-10A exosome concentrations. Importantly, by testing multiple exosomal miRNAs using the biosensor in clinical serum samples, breast cancer patients can be effectively differentiated from healthy donors. Consequently, the developed biosensor demonstrates high potential as a routine bioassay for the multiplex quantification of exosomal miRNAs in clinical diagnosis.

Intelligent Detection Platform for Simultaneous Detection of Multiple MiRNAs Based on Smartphone.

As a marker of malignant tumors, miRNA is closely related to the occurrence and metastasis of tumors. How to achieve rapid and sensitive real-time detection is important for clinical prevention and treatment of cancer. In this study, an intelligent detection platform based on smartphone image processing technology made point-of-care testing a reality. This new smart approach could detect multiple targets simultaneously and sensitively. Hydrogel microparticles of different coding modes (shapes, numbers) were prepared by flow lithography to detect different miRNAs. After sandwich immunoassays, different shapes of hydrogels showed different fluorescence intensities depending on their targets. Images were captured by a smartphone and then analyzed by image recognition processing software installed on the smartphone. The concentration of miRNA was obtained within 10 s. The entire reaction process did not exceed 2 h. This intelligent and portable detection platform for miRNAs was reliable and the limit of detection reached the femtomole level. This work provided a demonstration of intelligent, portable, real-time detection of tumor markers.

Hybridization-initiated exonuclease resistance strategy for simultaneous detection of multiple microRNAs

Multiple miRNA detection is often limited by sample, time-consuming, and complicated procedures. To address such challenges, we present a relatively simple and amplification-free fluorescent strategy based on hybridization-initiated exonuclease resistance for simultaneous detection of multiple miRNAs in a single tube. Single-stranded linear DNA probes were designed with dual roles of capture and reporter DNA, i.e. each DNA probe was labeled with biotin at the 3′-end for signal readout and amino at the 5′-end for probe immobilization. After target miRNAs specifically hybridized with the corresponding probes, the formation of double-stranded probe-target duplex protected the biotin on the probe from Exonuclease I digestion, thus resulting in high fluorescent intensities through the reaction between biotin and streptavidin-phycoerythrin. Coupling with the 3-plex fluorescent microsphere-based assay system, herein we successfully demonstrated the simultaneous and quantitative measurement of three sequence-specific miRNAs at concentration range of 2.5 pM to 1.25 nM and detection limits of 2 pM. To meet high throughput and rapid turnaround time requirements, we have also exemplified that, even shortening the total reaction time within 1 h, wider linear response range and lower detection limit were guaranteed. We further applied this assay to detect endogenous target miRNA levels from five kinds of cancer cell line and one normal cell line HEK 293T. This simple and rapid strategy will hold great potential for monitoring of multiple miRNAs biomarkers in biomedical research and early clinical diagnosis.

Feasibility study for combination of field-flow fractionation (FFF)-based separation of size-coded particle probes with amplified surface enhanced Raman scattering (SERS) tagging for simultaneous detection of multiple miRNAs

We propose a new analytical scheme in which field-flow fractionation (FFF)-based separation of target-specific polystyrene (PS) particle probes of different sizes are incorporated with amplified surface-enhanced Raman scattering (SERS) tagging for the simultaneous and sensitive detection of multiple microRNAs (miRNAs). For multiplexed detection, PS particles of three different diameters (15, 10, 5 μm) were used for the size-coding, and a probe single stranded DNA (ssDNA) complementary to a target miRNA was conjugated on an intended PS particle. After binding of a target miRNA on PS probe, polyadenylation reaction was executed to generate a long tail composed of adenine (A) serving as a binding site to thymine (T) conjugated Au nanoparticles (T-AuNPs) to increase SERS intensity. The three size-coded PS probes bound with T-AuNPs were then separated in a FFF channel. With the observation of extinction-based fractograms, separation of three size-coded PS probes was clearly confirmed, thereby enabling of measuring three miRNAs simultaneously. Raman intensities of FFF fractions collected at the peak maximum of 15, 10 and 5 μm PS probes varied fairy quantitatively with the change of miRNA concentrations, and the reproducibility of measurement was acceptable. The proposed method is potentially useful for simultaneous detection of multiple miRNAs with high sensitivity.

DNA Tetrahedral Nanostructure-Based Electrochemical miRNA Biosensor for Simultaneous Detection of Multiple miRNAs in Pancreatic Carcinoma.

Specific and sensitive biomarker detection is essential to early cancer diagnosis. In this study, we demonstrate an ultrasensitive electrochemical biosensor with the ability to detect multiple pancreatic carcinoma (PC)-related microRNA biomarkers. By employing DNA tetrahedral nanostructure capture probes to enhance the detection sensitivity as well as a disposable 16-channel screen-printed gold electrode (SPGE) detection platform to enhance the detection efficiency, we were able to simultaneously detect four PC-related miRNAs: miRNA21, miRNA155, miRNA196a, and miRNA210. The detection sensitivity reached to as low as 10 fM. We then profiled the serum levels of the four miRNAs for PC patients and healthy individuals with our multiplexing electrochemical biosensor. Through the combined analyses of the four miRNAs, our results showed that PC patients could be discriminated from healthy controls with fairly high sensitivity. This multiplexing PCR-free miRNA detection sensor shows promising applications in early diagnosis of PC disease.

Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor.

We report an ultra-low fouling surface plasmon resonance imaging (SPRi) biosensor for the rapid simultaneous detection of multiple miRNAs in erythrocyte lysate (EL) at subpicomolar levels without need of RNA extraction. The SPRi chips were coated with ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes having optimized thicknesses and directly functionalized with amino-modified oligonucleotide probes. We have characterized the effect of the brush thickness on the probe loading capacity: a loading capacity of ~9.8×10(12) probes/cm(2) was achieved for pCBAA having a thickness of ~40 nm. The probe-functionalized sensor also exhibited a high resistance to fouling from ~90% EL samples (<2 ng/cm(2)). A two-step detection assay was employed for multiplexed miRNA detection in EL. Specifically, the assay consisted of (i) a sandwich-type hybridization of the probe-functionalized pCBAA with target miRNA in EL (bound to biotinylated oligonucleotides) and (ii) the capture of streptavidin-functionalized gold nanoparticles to the aforementioned biotinylated probes. We have demonstrated that this approach enables the detection of miRNAs in EL at concentrations as low as 0.5 pM. Finally, we have confirmed the detection of four endogenous miRNAs representing a set of potential miRNA biomarkers of myelodysplastic syndrome (MDS) in clinical EL samples (miR-16, miR-181, miR-34a, and miR-125b). The results revealed significantly higher levels of miR-16 in all the clinical EL samples compared to the other measured miRNAs.

Rapid Simultaneous Detection of Multiple Micro-Ribonucleic Acids By Surface Plasmon Resonance Array in Cell Lysates of Myelodysplastic Syndromes Patients

IntroductionMicroRNAs (miRNAs) are involved in the control of hematopoiesis, their deregulation also appears to play role in the pathogenesis of several hematopoietic diseases. Multiple miRNAs have been reported to be abnormally expressed in hematologic cancers; moreover, specific miRNA expression profiles have been proposed as diagnostic and prognostic markers in various hematologic malignancies, including Myelodysplastic Syndromes (MDS). MDS are a heterogeneous family of clonal disorders of hematopoietic stem cells. They are characterized by ineffective hematopoiesis and frequent leukemic progression.It has been shown that the mature erythrocytes are rich in diverse miRNAs species, even though they lack ribosomal and other large-size RNAs. miRNAs are expressed during erythrocyte differentiation and have an important role in erythropoiesis and mRNA degradation.MethodsFour miRNAs, i.e. miR-16, miR-181, miR-34a, and miR-125b were selected as a model set of potential MDS biomarkers and evaluated using a surface plasmon resonance imaging (SPRi) biosensor and real-time PCR. The sensor calibration curves were measured with miRNAs spiked in a complex erythrocyte lysate. The total RNA was extracted from erythrocyte lysate using the acid guanidinium-thiocyanate-phenol-chloroform method. The levels of selected miRNAs were measured in all samples by quantitative reverse transcription - real-time polymerase chain reaction.ResultsA high-capacity array SPRi system for rapid simultaneous detection of multiple miRNAs in erythrocyte lysate was developed and demonstrated at the Institute of Photonics and Electronics. The ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes-coated gold surfaces of the SPR sensor were shown to reduce the non-specific interaction between surface of the sensor and sample. A two-step miRNA detection assay for multiplexed miRNA detection in erythrocyte lysate was demonstrated in which hybridization of probe-functionalized pCBAA with target miRNA bound to biotinylated oligonucleotide probes was followed with capture of streptavidin-functionalized gold nanoparticles to biotinylated probes. In preliminary experiments, the array was shown to be capable of detecting multiple miRNAs spiked in erythrocyte lysate without the need for complex lysate sample pretreatment at concentrations as low as 0.5 pM in less than 45 minutes.ConclusionsUsing the newly developed high-capacity array SPRi system we have found significantly increased levels of miR-16 in erythrocyte lysate samples (at the concentration range of ~10-100 pM) compared to other miRNAs tested within this study. The results were confirmed by a reference real-time polymerase chain reaction method. In addition, our results indicate an over-expression of miR-16 in erythrocyte lysate samples of MDS patients with both Refractory Cytopenia with Multilineage Dysplasia (RCMD)and Refractory Anemia with Excess Blasts (RAEB) diagnosis. This clearly demonstrates the potential of SPRi array technology for clinical applications. DisclosuresNo relevant conflicts of interest to declare.