Microfluidic magnetic detection system combined with a DNA framework-mediated immune-sandwich assay for rapid and sensitive detection of tumor-derived exosomes.

Tumor-derived circulating exosomes (TDEs) are being pursued as informative and noninvasive biomarkers. However, quantitatively detecting TDEs is still challenging. Herein, we constructed a DNA tetrahedral-structured probe (TSP)-mediated microfluidic magnetic detection system (µFMS) to provide a rapid and sensitive platform for analyzing TDEs. CD63 aptamer-modified Fe3O4 magnetic nanoparticles (MNPs) were constructed to form magnetic nano-report probes (MNRs). The microfluidic chips were fabricated from glass functionalized with DNA TSP-modified aldehyde groups and a PDMS layer designed with serpentine microchannels. An induction coil-based magnetic detector was used to measure the magnetic signal. The linear dynamic range of the µFMS system for TDE assays was 1.98 × 103-1.98 × 107 particles/mL with a limit of detection of 1.98 × 103 particles/mL in PBS. There was no significant difference in TDE detection between the simulated serum and PBS, which indicated the feasibility of the constructed µFMS system for TDE analysis in complex biological systems. In terms of cost, reaction time and operation procedure, this µFMS has the potential to be developed as a clinical point-of-care testing tool for cancer diagnosis and therapeutics.

Tetrahedral DNA framework based CRISPR electrochemical biosensor for amplification-free miRNA detection.

microRNA (miRNA) is a kind of small non-coding RNA that has been regarded as potential biomarkers for cancers. Sensitive and specific detection of miRNA at low expression levels is highly desirable but remains challenging, especially for amplification-free and portable point of care (POC) diagnostics. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a has been recently discovered and used in the field of RNA detection. Nonetheless, most CRISPR/Cas13a-based methods were burdened with expensive equipment, time-consuming procedures, and complicated operations which were not suitable for POC analysis. In this work, we constructed a three-dimensional tetrahedral DNA framework based CRISPR-electrochemical biosensor (CRISPR-E). By combining tetrahedral DNA framework, CRISPR, and electrochemical biosensor, the process of activation, cleavage of Cas13a, and signal readout were all finished on the chip, and a simple, amplification-free and sensitive detection of miRNA-19b was realized. Under the optimal experimental conditions, a linear range from 10 pM to 104 pM with detection limit of 10 pM for miRNA-19b in buffer solution was achieved. Selectivity analysis indicated that our CRISPR-E had good distinguishing ability between miRNA-19b and miRNA-197. The results of miRNA-19b detection in mimic serum samples were consistent with that of the buffer solution. This all-on-chip strategy of our CRISPR-E is very suitable for POC testing.

Designer tetrahedral DNA framework-based microfluidic technology for multivalent capture and release of circulating tumor cells.

Circulating tumor cells (CTCs) have been recognized as a general biomarker for the early detection, diagnosis and therapy monitoring of cancer. Due to their extreme rarity in peripheral blood, the isolation and analysis of CTCs with high efficiency, high purity and high viability remains a tremendous technological challenge. Herein, we combined tetrahedral DNA framework (TDFs), herringbone channel (HB) chip, together with aptamer-triggered hybridization chain reaction (apt-HCR) to develop an efficient microfluidic system (T-µFS) for capture and release of simulated CTCs. The capture efficiency of MCF-7 â€‹cells was from 83.3% to 94.2% when the cell numbers ranged from 10 to 103 using our T-µFS in the whole blood. The release efficiency of the MCF-7 â€‹cells was 96.2% and the MCF-7 â€‹cell viability after release was 94.6% using our T-µFS in PBS buffer. Reculture and RT-qPCR studies showed that there was almost no damage by the capture and release treatment for the MCF-7 â€‹cells viability. These results revealed that our T-µFS could be developed as an integrated and automatic technical platform with great performance for multivalent capture and release of CTCs and have a wide application prospect for tumor liquid biopsy.

A designer DNA tetrahedron-based molecular beacon for tumor-related microRNA fluorescence imaging in living cells.

The accurate and effective imaging of tumor-related miRNA in living cells has been playing an increasingly important role in cancer imaging. However, due to the low miRNA content and complex intracellular microenvironment, the current imaging methods of miRNAs in living cells still have some limitations. In this work, we developed a designer nanoprobe of tetrahedral DNA framework (TDF) combined with MB (termed TDFM nanoprobe) for the efficient fluorescence imaging of tumor-related miRNA-214 in living cells. In cell-free experiments, we demonstrated that the TDFM nanoprobe has sensitive detection and good specificity by fluorescence measurements. Before the TDFM nanoprobe was used for intracellular miRNA-214 fluorescence imaging, we confirmed its intracellular stability and negligible cytotoxicity by a standard MTT assay. In intracellular imaging experiments, we observed the strong fluorescence signal exhibited by the cells incubated with the TDFM nanoprobe using confocal fluorescence microscopy, which indicated that the TDFM nanoprobe was suitable for detecting and imaging tumor-related miRNA-214 in living cells. Furthermore, under the optimal incubation conditions, we employed the TDFM nanoprobe to study differences in the expression levels of tumor-related miRNA-214 in human breast cancer cells (MCF-7) and human umbilical vein endothelial cells (HUVEC). The TDFM nanoprobe we designed shows great potential to be applied in the development of DNA nanodevices, providing an improved strategy for the fluorescence imaging of miRNAs in living cells.

Tetrahedral DNA Framework-Programmed Electrochemical Biosenors with Gold Nanoparticles for Ultrasensitive Cell-Free DNA Detection.

Tumor-associated cell-free DNA (cfDNA) is a dynamic biomarker for genetic analysis, early diagnosis and clinical treatment of cancers. However, its detection has limitations because of its low abundance in blood or other complex bodily fluids. Herein, we developed an ultrasensitive cfDNA electrochemical biosensor (E-cfDNA sensor) based on tetrahedral DNA framework (TDF)-modified gold nanoparticles (Au NPs) with an interface for cfDNA detection. By accurately controlling the numbers of base pairs on each DNA framework, three types of TDFs were programmed: 26 base pairs of TDF; 17 base pairs of TDF; and 7 base pairs of TDF (TDF-26, TDF-16 and TDF-7, respectively). We also combined the TDF with hybridization chain reaction (HCR) to achieve signal amplification. Under optimal conditions, we detected the breast cancer susceptibility gene 1 (BRCA-1), a representative cfDNA closely related to breast cancer. An ultra-low detection limit of 1 aM with a linear range from 1 aM to 1 pM by TDF-26 was obtained, which was superior to the existing methods. Each type of TDF has excellent discrimination ability, which can distinguish single mismatch. More significantly, we also detected BRCA-1 in mimic serum samples, demonstrating that the E-cfDNA sensor has potential use in clinical research.

Cubic DNA nanocage-based three-dimensional molecular beacon for accurate detection of exosomal miRNAs in confined spaces.

In situ nondestructive bioanalysis of targets in nanoscale confined space, e.g. exosomes, poses a high challenge to analytical technologies, especially to molecular fluorescent probes, because it is required to enter the confined space to recognize the target, and maintain independent and stable signal output. The unexpected fluorescence quenching and fluorescence resonance energy transfer (FRET) caused by high-frequency Brownian motion and collision in confined space are the main limiting factors. Herein, we constructed a well-defined and programmable cubic DNA nanocage-based three-dimensional molecular beacon (ncMB), which successfully broke through the above dilemma, and realized the detection of miRNA in exosomes. Specifically, steric hindrance and electrostatic repulsion derived from the unique three-dimensional structure of ncMB result in a barrier between fluorescent probes, thus eliminating unexpected fluorescence quenching during single exosomal miRNA detection and unexpected FRET during dual exosomal miRNA detection. Benefiting from the excellent anti-fluorescence and anti-FRET performance of ncMB, compared with traditional molecular beacons (MB), the detected fluorescence signal in exosomes can be improved by an order of magnitude. Moreover, ncMB is proven to have powerful programmability and anti-interference capability. Overall, it is believed that the ncMB can eliminate the signal distortion that was usually associated with commonly used MB, especially in the confined space. The ncMB is considered as a powerful and versatile tool for accurate in situ signal output in exosomes and maybe other confined spaces.

Poly-adenine-mediated spherical nucleic acid probes for live cell fluorescence imaging of tumor-related microRNAs.

BACKGROUND: Accurately detecting and quantifying tumor-related microRNAs (miRNAs) in living cells is of great value for early cancer diagnosis. Herein, we present poly-adenine (polyA)-mediated spherical nucleic acid (SNA) nanoprobes for intracellular miRNA imaging in living cells. METHODS AND RESULTS: polyA-mediated spherical nucleic acid (pASNA) nanoprobes consist of gold nanoparticles (AuNPs) anchored with fluorophore-labeled DNA molecules pre-hybridized with recognition sequences and polyA tails. The detection performance for miRNAs in vitro was studied to confirm the feasibility of pASNA nanoprobes for imaging live cell miRNAs. Before the pASNA nanoprobes were used for imaging intracellular miRNAs in MCF-7, HeLa, and LO2 cells, the stability and non-cytotoxicity were investigated using Dnase I and a standard colorimetric CCK8 assay. Flow cytometry, qRT-PCR analyses were conducted to confirm the different expression levels of miR-155 in live cells. Results showed that the pASNA nanoprobes had good detection sensitivity and specificity, excellent stability, and low toxicity. After incubating with pASNA nanoprobes, noticeable fluorescence signal enhancement could be clearly observed in MCF-7 and HeLa cells but not LO2 cells by confocal microscopy. Flow cytometry analysis and qRT-PCR indicated that MCF-7 and HeLa cells had higher miR-155 expression levels compared to LO2 cells. CONCLUSIONS: The pASNA nanoprobes we developed had good sensitivity and specificity, excellent nuclease stability and low toxicity, thus representing a new approach to exquisitely reveal the distribution of endogenous miRNAs in live cells.

A double-tetrahedral DNA framework based electrochemical biosensor for ultrasensitive detection and release of circulating tumor cells.

Detecting circulating tumor cells (CTCs) in patients' blood is essential for early diagnosis, precise treatment and prognosis of cancer. Yet due to CTCs being extremely rare in the peripheral blood of patients, it is still a challenge to detect CTCs with high sensitivity and high selectivity. Here, we developed a double-tetrahedral DNA framework (DTDF) based electrochemical biosensor system (E-CTC sensor system) for ultrasensitive detection and release of CTCs. In this work, an upright tetrahedral DNA framework (UTDF) was used as a rigid scaffold to modify a screen-printed gold electrode (SPGE), and an inverted tetrahedral DNA framework (ITDF) provided three vertex chains to multivalently bind with aptamers. Meanwhile, a streptavidin tagged horseradish peroxidase homopolymer (SA-polyHRP) was linked to biotin-modified aptamers to significantly amplify the signal. Moreover, the captured CTCs could be effectively released via benzonase nuclease with little cell damage. Our E-CTC sensor system achieved a linear range from 1 to 105 MCF-7 cells with an ultralow detection limit of 1 cell. The release efficiency reached 88.1%-97.6% and the viability of the released cells reached up to 98%. We also detected the MCF-7 cells in mimic whole blood samples, suggesting that the E-CTC sensor system shows promise for use in clinical research.

Electrochemical DNA Sensor for Sensitive BRCA1 Detection Based on DNA Tetrahedral-Structured Probe and Poly-Adenine Mediated Gold Nanoparticles.

BRCA1 is the biomarker for the early diagnosis of breast cancer. Detection of BRCA1 has great significance for the genetic analysis, early diagnosis and clinical treatment of breast cancer. In this work, we developed a simple electrochemical DNA sensor based on a DNA tetrahedral-structured probe (TSP) and poly-adenine (polyA) mediated gold nanoparticles (AuNPs) for the sensitive detection of BRCA1. A thiol-modified TSP was used as the scaffold on the surface of the screen-printed AuNPs electrode. The capture DNA (TSP) and reporter DNA were hybridized to the target DNA (BRCA1), respectively, to form the typical sandwich system. The nanocomposites of reporter DNA (polyA at the 5' end) combined with AuNPs were employed for signal amplification which can capture multiple enzymes by the specificity between biotin and streptavidin. Measurements were completed in the electrochemical workstation by cyclic voltammetry and amperometry and we obtained the low limit of detection of 0.1 fM with the linear range from 1 fM to 1 nM. High sensitivity and good specificity of the proposed electrochemical DNA sensor showed potential applications in clinical early diagnosis for breast cancer.