A High-Throughput Circular Tumor Cell Sorting Chip with Trapezoidal Cross Section.

Circulating tumor cells are typically found in the peripheral blood of patients, offering a crucial pathway for the early diagnosis and prediction of cancer. Traditional methods for early cancer diagnosis are inefficient and inaccurate, making it difficult to isolate tumor cells from a large number of cells. In this paper, a new spiral microfluidic chip with asymmetric cross-section is proposed for rapid, high-throughput, label-free enrichment of CTCs in peripheral blood. A mold of the desired flow channel structure was prepared and inverted to make a trapezoidal cross-section using a micro-nanotechnology process of 3D printing. After a systematic study of how flow rate, channel width, and particle concentration affect the performance of the device, we utilized the device to simulate cell sorting of 6 µm, 15 µm, and 25 µm PS (Polystyrene) particles, and the separation efficiency and separation purity of 25 µm PS particles reached 98.3% and 96.4%. On this basis, we realize the enrichment of a large number of CTCs in diluted whole blood (5 mL). The results show that the separation efficiency of A549 was 88.9% and the separation purity was 96.4% at a high throughput of 1400 µL/min. In conclusion, we believe that the developed method is relevant for efficient recovery from whole blood and beneficial for future automated clinical analysis.

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.

Fluorescence intensity coded DNA frameworks based on the FRET effect enable multiplexed miRNA imaging in living cells.

miRNA analysis has played an important role in precise diagnosis, treatment and prognosis of cancer, especially multiplexed miRNA imaging. In this work, a novel fluorescence emission intensity (FEI) encoding strategy was developed based on a tetrahedron DNA framework (TDF) carrier and the FRET effect between Cy3 and Cy5. Six FEI-encoded TDF (FEI-TDF) samples were constructed by tuning the labeling number of Cy3 and Cy5 at the vertexes of the TDF. For fluorescence characterization in vitro, distinct FEIs in the spectra and different colors under ultraviolet (UV) irradiation of FEI-TDF samples were observed. By dividing the ranges of FEIs of samples, the stability of FEIs was highly improved. Based on the ranges of FEIs in each sample, five codes with good discrimination were finally developed. Before the application of intracellular imaging, the excellent biocompatibility of the TDF carrier was proved by CCK-8 assay. The barcode probes based on samples 12, 21 and 11 were designed as example models to realize multiplexed imaging of miRNA-16, miRNA-21 and miRNA-10b in MCF-7 cells with obviously different fluorescence merged colors. FEI-TDFs provide a new research perspective for the development of fluorescence multiplexing strategies in the future.

Reprogramming of human peripheral blood mononuclear cells into induced mesenchymal stromal cells using non-integrating vectors.

Mesenchymal stromal cells (MSCs) have great value in cell therapies. The MSC therapies have many challenges due to its inconsistent potency and limited quantity. Here, we report a strategy to generate induced MSCs (iMSCs) by directly reprogramming human peripheral blood mononuclear cells (PBMCs) with OCT4, SOX9, MYC, KLF4, and BCL-XL using a nonintegrating episomal vector system. While OCT4 was not required to reprogram PBMCs into iMSCs, omission of OCT4 significantly impaired iMSC functionality. The omission of OCT4 resulted in significantly downregulating MSC lineage specific and mesoderm-regulating genes, including SRPX, COL5A1, SOX4, SALL4, TWIST1. When reprogramming PBMCs in the absence of OCT4, 67 genes were significantly hypermethylated with reduced transcriptional expression. These data indicate that transient expression of OCT4 may serve as a universal reprogramming factor by increasing chromatin accessibility and promoting demethylation. Our findings represent an approach to produce functional MSCs, and aid in identifying putative function associated MSC markers.

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.

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.

DNA tetrahedron-mediated immune-sandwich assay for rapid and sensitive detection of PSA through a microfluidic electrochemical detection system.

Prostate-specific antigen (PSA) is the most widely used biomarker for the early diagnosis of prostate cancer. Existing methods for PSA detection are burdened with some limitations and require improvement. Herein, we developed a novel microfluidic-electrochemical (µFEC) detection system for PSA detection. First, we constructed an electrochemical biosensor based on screen-printed electrodes (SPEs) with modification of gold nanoflowers (Au NFs) and DNA tetrahedron structural probes (TSPs), which showed great detection performance. Second, we fabricated microfluidic chips by DNA TSP-Au NF-modified SPEs and a PDMS layer with designed dense meandering microchannels. Finally, the µFEC detection system was achieved based on microfluidic chips integrated with the liquid automatic conveying unit and electrochemical detection platform. The µFEC system we developed acquired great detection performance for PSA detection in PBS solution. For PSA assays in spiked serum samples of the µFEC system, we obtained a linear dynamic range of 1-100 ng/mL with a limit of detection of 0.2 ng/mL and a total reaction time <25 min. Real serum samples of prostate cancer patients presented a strong correlation between the "gold-standard" chemiluminescence assays and the µFEC system. In terms of operation procedure, cost, and reaction time, our method was superior to the current methods for PSA detection and shows great potential for practical clinical application in the future.

Bio-Separated and Gate-Free 2D MoS2 Biosensor Array for Ultrasensitive Detection of BRCA1.

2D molybdenum disulfide (MoS2)-based thin film transistors are widely used in biosensing, and many efforts have been made to improve the detection limit and linear range. However, in addition to the complexity of device technology and biological modification, the compatibility of the physical device with biological solutions and device reusability have rarely been considered. Herein, we designed and synthesized an array of MoS2 by employing a simple-patterned chemical vapor deposition growth method and meanwhile exploited a one-step biomodification in a sensing pad based on DNA tetrahedron probes to form a bio-separated sensing part. This solves the signal interference, solution erosion, and instability of semiconductor-based biosensors after contacting biological solutions, and also allows physical devices to be reused. Furthermore, the gate-free detection structure that we first proposed for DNA (BRCA1) detection demonstrates ultrasensitive detection over a broad range of 1 fM to 1 µM with a good linear response of R2 = 0.98. Our findings provide a practical solution for high-performance, low-cost, biocompatible, reusable, and bio-separated biosensor platforms.

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.

Poly-adenine-mediated spherical nucleic acids for strand displacement-based DNA/RNA detection.

DNA-gold nanoparticles (AuNPs) conjugate is one of the most versatile bionanomaterials for biomedical and clinical diagnosis. However, to finely tune the hybridization ability and precisely control the orientation and conformation of surface-tethered oligonucleotides on AuNPs remains a hurdle. In this work, we developed a poly adenine-mediated spherical nucleic acid (polyA-mediated SNA) strategy by assembling di-block DNA probes on gold nanoparticles (AuNPs) to spatially control interdistance and hybridization ability of oligonucleotides on AuNPs. By modulating length of poly A bound on the SNA with different degrees of constructing, we presented significant improved biosensing performance including high hybridization efficiency, and expanded dynamic range of analytes with more sensitive detection limit. Furthermore, this polyA design could facilitate the programmable detection for DNA in serum environment and simultaneous multicolor detection of three different microRNAs associated with pancreatic carcinoma. The demonstration of the link between modulation of SNA assembly strategy and biodetection capability will increase the development of high performance diagnostic tools for translational biomedicine.

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.

A pillar[5]arene and crown ether fused bicyclic host: synthesis, guest discrimination and simultaneous binding of two guests with different shapes, sizes and electronic constitutions.

A bicyclic host molecule 1 consisting of a pillar[5]arene and a 1,5-dioxynaphthalene-based crown ether unit has been synthesized, and the two cyclic subunits in 1 were found to recognize two different guest molecules (1,4-dicyanobutane and paraquat) selectively or take up the two guest molecules simultaneously.

3'UTR shortening identifies high-risk cancers with targeted dysregulation of the ceRNA network.

Competing endogenous RNA (ceRNA) interactions form a multilayered network that regulates gene expression in various biological pathways. Recent studies have demonstrated novel roles of ceRNA interactions in tumorigenesis, but the dynamics of the ceRNA network in cancer remain unexplored. Here, we examine ceRNA network dynamics in prostate cancer from the perspective of alternative cleavage and polyadenylation (APA) and reveal the principles of such changes. Analysis of exon array data revealed that both shortened and lengthened 3'UTRs are abundant. Consensus clustering with APA data stratified cancers into groups with differing risks of biochemical relapse and revealed that a ceRNA subnetwork enriched with cancer genes was specifically dysregulated in high-risk cancers. The novel connection between 3'UTR shortening and ceRNA network dysregulation was supported by the unusually high number of microRNA response elements (MREs) shared by the dysregulated ceRNA interactions and the significantly altered 3'UTRs. The dysregulation followed a fundamental principle in that ceRNA interactions connecting genes that show opposite trends in expression change are preferentially dysregulated. This targeted dysregulation is responsible for the majority of the observed expression changes in genes with significant ceRNA dysregulation and represents a novel mechanism underlying aberrant oncogenic expression.

In vitro effects of helium-neon laser irradiation on human blood: blood viscosity and deformability of erythrocytes.

OBJECTIVE: The purpose of this study was to investigate the in vitro effects of He-Ne laser irradiation on some rheological factors of human blood, such as blood viscosity, erythrocyte deformability, and sedimentation rate. BACKGROUND DATA: The intravascular irradiation of low power laser has been applied in pre-clinical and clinical to treat various pathological processes. However, the mechanism is not fully understood so far. Especially the interaction and related mechanism between the laser and blood are unclear. In this work, by measuring the change of the main rheological factors after laser irradiation, the interaction and mechanism were explored. METHODS: A30-mW He-Ne laser was used for irradiation with a 4-5-mm-diameter beam spot on blood samples, with a fluence rate of about 150 mW/cm.(2) The irradiation time was 60 min, so the total dose of irradiation was 540 J/cm.(2) The pathological samples of blood were obtained from patients (volunteers), and each sample was divided into two tubes for irradiation and control. The blood viscosity, erythrocyte deformability, and sedimentation rate were measured after laser irradiation and compared with un-irradiated control. The blood samples with poor erythrocyte deformability were prepared by adding Ca(2+) to the normal erythrocytes of a healthy person for investigating the laser effect on erythrocyte deformability further. RESULTS: Laser irradiation reduced the erythrocyte sedimentation rate of blood samples, which had a hyper-sedimentation rate originally. The blood viscosity of samples in hyper-values was lowered by laser irradiation in all shear rates measured (10-110 S(-1)), with a relative variation of approximately 10%. The deformability of erythrocytes from pathological samples and Ca(2+)-treated samples was improved after laser irradiation. CONCLUSIONS: The positive effects of laser irradiation on improving the rheological properties of blood were demonstrated in vitro.

ATP-sensitive potassium channels mediate hyperosmotic stimulation of NKCC in slow-twitch muscle.

In mildly hyperosmotic medium, activation of the Na+ -K+ -2Cl- cotransporter (NKCC) counteracts skeletal muscle cell water loss, and compounds that stimulate protein kinase A (PKA) activity inhibit the activation of the NKCC. The aim of this study was to determine the mechanism for PKA inhibition of NKCC activity in resting skeletal muscle. Incubation of rat slow-twitch soleus and fast-twitch plantaris muscles in isosmotic medium with the PKA inhibitors H-89 and KT-5720 caused activation of the NKCC only in the soleus muscle. NKCC activation caused by PKA inhibition was insensitive to MEK MAPK inhibitors and to insulin but was abolished by the PKA stimulators isoproterenol and forskolin. Furthermore, pinacidil [an ATP-sensitive potassium (KATP) channel opener] or inhibition of glycolysis increased NKCC activity in the soleus muscle but not in the plantaris muscle. Preincubation of the soleus muscle with glibenclamide (a KATP channel inhibitor) prevented the NKCC activation by hyperosmolarity, PKA inhibition, pinacidil, and glycolysis inhibitors. In contrast, glibenclamide stimulated NKCC activity in the plantaris muscle. In cells stably transfected with the Kir6.2 subunit of the of KATP channel, inhibition of glycolysis activated potassium current and NKCC activity. We conclude that activation of KATP channels in slow-twitch muscle is necessary for activation of the NKCC and cell volume restoration in hyperosmotic conditions.