Nucleic Acid Amplification by Template-Dominated Click Chemistry for Ultrasensitive DNA/RNA Detection on an Electrochemical Readout Platform.

As an enzyme-free exponential nucleic acid amplification method, the click chemistry-mediated ligation chain reaction (ccLCR) has shown great prospects in the molecular diagnosis. However, the current optics-based ccLCR is challenged by remarkable nonspecific amplification, severely hindering its future application. This study demonstrated that the severe nonspecific amplification was generated probably due to high random collision in the high DNA probe concentration (µM level). To solve this hurdle, a nucleic acid template-dominated ccLCR was constructed using nM-level DNA probes and read on an electrochemical platform (cc-eLCR). Under the optimal conditions, the proposed cc-eLCR detected a low-level nucleic acid target (1 fM) with a single-base resolution. Furthermore, this assay was applied to detect the target of interest in cell extracts with a satisfactory result. The proposed cc-eLCR offers huge possibility for click chemistry-mediated enzyme-free exponential nucleic acid amplification in the application of medical diagnosis and biomedical research.

Multimodal Ochratoxin A-Aptasensor Using 3'-FAM-Enhanced Exonuclease I Tool and Magnetic Microbead Carrier.

Thanks to its preparatory ease, close affinity, and low cost, the aptasensor can serve as a promising substitute for antibody-dependent biosensors. However, the available aptasensors are mostly subject to a single-mode readout and the interference of unbound aptamers in solution and non-target-induced transition events. Herein, we proposed a multimodal aptasensor for multimode detection of ochratoxin A (OTA) with cross-validation using the 3'-6-carboxyfluorescein (FAM)-enhanced exonuclease I (Exo I) tool and magnetic microbead carrier. Specifically, the 3'-FAM-labeled aptamer/biotinylated-cDNA hybrids were immobilized onto streptavidin-magnetic microbeads via streptavidin-biotin interaction. With the presence of OTA, an antiparallel G-quadruplex conformation was formed, protecting the 3'-FAM labels from Exo I digestion, and then anti-FAM-horseradish peroxidase (HRP) was bound via specific antigen-antibody affinity; for the aptamers without the protection of OTA, the distal ssDNA was hydrolyzed from 3' → 5', releasing 3'-FAM labels to the solution. Therefore, the OTA was detected by analyzing the "signal-off" fluorescence of the supernatant and two "signal-on" signals in electrochemistry and colorimetry through the detection of the coating magnetic microbeads in HRP's substrate. The results showed that the 3'-FAM labels increased the activity of Exo I, producing a low background due to a more thorough digestion of unbound aptamers. The proposed multimodal aptasensor successfully detected the OTA in actual samples. This work first provides a novel strategy for the development of aptasensors with Exo I and 3'-FAM labels, broadening the application of aptamer in the multimode detection of small molecules.

DNA Nanosieve-Based Regenerative Electrochemical Biosensor Utilizing Nucleic Acid Flexibility for Accurate Allele Typing in Clinical Samples.

Herein, an interface-based DNA nanosieve that has the ability to differentiate ssDNA from dsDNA has been demonstrated for the first time. The DNA nanosieve could be readily built through thiol-DNA's self-assembly on the gold electrode surface, and its cavity size was tunable by varying the concentration of thiol-DNAs. Electrochemical chronocoulometry using [Ru(NH3)6]3+ as redox revealed that the average probe-to-probe separation in the 1 µM thiol-DNA-modified gold electrode was 10.6 ± 0.3 nm so that the rigid dsDNA with a length of ∼17 nm could not permeate the nanosieve, whereas the randomly coiled ssDNA could enter it due to its high flexibility, which has been demonstrated by square wave voltammetry and methylene blue labels through an upside-down hybridization format. After combining the transiently binding characteristic of a short DNA duplex and introducing a regenerative probe (the counterpart of ssDNA), a highly reproducible nanosieve-based E-DNA model was obtained with a relative standard deviation (RSD) as low as 2.7% over seven cycles. Finally, we built a regenerative nanosieve-based E-DNA sensor using a ligation cycle reaction as an ssDNA amplification strategy and realized one-sensor-based continuous measurement to multiple clinical samples with excellent allele-typing performance. This work holds great potential in low-cost and high-throughput analysis between biosensors and biochips and also opens up a new avenue in nucleic acid flexibility-based DNA materials for future applications in DNA origami and molecular logic gates.

Ultrasensitive Detection of RNA with Single-Base Resolution by Coupling Electrochemical Sensing Strategy with Chimeric DNA Probe-Aided Ligase Chain Reaction.

Accurate and sensitive detection of single-base mutations in RNAs is of great value in basic studies of life science and medical diagnostics. However, the current available RNA detection methods are challenged by heterogeneous clinical samples in which trace RNA mutants usually existed in a large pool of normal wild sequences. Thus, there is still great need for developing the highly sensitive and highly specific methods in detecting single-base mutations of RNAs in heterogeneous clinical samples. In the present study, a new chimeric DNA probe-aided ligase chain reaction-based electrochemical method (cmDNA-eLCR) was developed for RNA mutation detection through the BSA-based carrier platform and the horseradish peroxidase-hydrogen peroxide-tetramethylbenzidine (HRP-H2O2-TMB) system. The denaturing polyacrylamide gel electrophoresis and a fluorophore-labeled probe was ingeniously designed to demonstrate the advantage of cmDNA in ligation to normal DNA templated by RNA with the catalysis of T4 RNA ligase 2 as well as its higher selectivity than DNA ligase system. Finally, the proposed cmDNA-eLCR, compared with the traditional eLCR, showed excellent performance in discriminating single base-mismatched sequences, where the signal response for mismatched targets at a high concentration could overlap completely with that for the blank control. Besides, this cmDNA-eLCR assay had a wide linear range crossing six orders of magnitude from 1.0 × 10-15 to1.0 × 10-10 M with a limit of detection as low as 0.6 fM. Furthermore, this assay was applied to detect RNA in real sample with a satisfactory result, thereby demonstrating its great potential in diagnosis of RNA-related diseases.

A novel ligase chain reaction-based electrochemical biosensing strategy for highly sensitive point mutation detection from human whole blood.

Challenged by the detection of trace amounts of mutants and disturbance from endogenous substances in clinical samples, herein, we present a novel electrochemical biosensor based on ligase chain reaction (eLCR) via the thermostable ligase with high mutation recognizing ability. The lengthened double-stranded DNAs exponentially generated via LCR were uniformly distributed on a bovine serum albumin-modified gold electrode, in which the phosphate buffer was tactfully added to remove adsorbed uninterested-probes, and thereafter the amperometry current was collected for the specific binding of streptavidin-poly-HRP and subsequent catalysis in the 3, 3', 5, 5'-tetramethylbenzidine substrate that contained hydrogen peroxide. It found that, under optimized conditions, the proposed biosensor exhibited a high selectivity of mutant targets from the 104-fold excess of co-existent wild targets within a detection limit of 0.5 fM. Impressively, without the involvement of pre-PCR, the homozygous mutants were specifically distinguished from the wild genotype of CYP2C19*2 allele in human whole blood samples. Therefore, the proposed eLCR, due to its advantages in simple primer design, operational ease and ease of miniaturization, has demonstrated its considerable potential for point-of-care testing in the diagnosis of point mutation-related diseases and personalized medicine.

Ultrasensitive Electrochemical Biosensor Developed by Probe Lengthening for Detection of Genomic DNA in Human Serum.

As an alternative to most of the reported nucleic acid amplification-based electrochemical DNA biosensors used for detection of trace levels of genomic DNA, we herein present a novel detection concept. The proposed system involves the conversion of two short double-stranded DNAs (dsDNAs), labeled with a thiol-tag or biotin-tag, into a single integrated dsDNA containing thiol and biotin at both terminals in the presence of target DNA through ligase chain reaction (LCR) and followed by the immobilization of these integrated dsDNAs on a bovine serum albumin (BSA)-modified gold electrode surface. Owing to rapid depletion of the two short dsDNAs via LCR, the integrated dsDNAs were generated in an exponential manner so that this sensoring approach offered a limit of detection of 25 yoctomoles (15 copies in 50 µL sample volumes), a high discrimination of single-base mismatch and a wide linear concentration range (across 6 orders of magnitude) for target DNA. Significantly, the proposed sensor, which has simplicity in operation and ease of miniaturization, detected the target of interest in total nucleic acid extracts derived from clinical serum samples with excellent results, thereby demonstrating its considerable diagnostic potential in fields ranging from virus detection to the diagnosis of genetic diseases.

Prognostic significance of the expression of HER family members in primary osteosarcoma.

The prognosis of patients with metastatic osteosarcoma is poor and has shown no significant improvement in nearly 20 years. The human epidermal growth factor (EGF) receptor (HER) family is frequently overexpressed in the majority of human carcinomas, and is involved in promoting the proliferation and survival of cancer cells. However, the role of EGFR and HER-2 expression in osteosarcoma survival remains controversial and no previous study has simultaneously investigated the association of the expression of all the four HER family members with the prognostic significance of osteosarcoma. Therefore, the present study investigated the expression levels of the complete members of the HER family in osteosarcoma specimens, as well as their associations with the clinicopathological parameters, progression-free survival (PFS) and overall survival (OS) time of patients with osteosarcoma. The expression of HER family members was detected in osteosarcoma tumor specimens from 60 patients using immunohistochemistry. The association of the expression of HER receptors in osteosarcoma with clinicopathological parameters was analyzed using χ2 test and Fishers exact test. Survival analyses were evaluated by Kaplan-Meier method and Cox proportional hazards regression model. Overall, 18 (30%), 13 (22%), 23 (38%) and 19 (32%) patients presented with high expression of EGFR, HER-2, HER-3 and HER-4, respectively, and the co-expression of 2, 3 and all 4 members of the HER family was observed. High expression of EGFR and HER-4 was associated with distant metastasis. High HER-3 expression was significantly associated with an advanced Enneking stage and distant metastasis. Multivariate analysis demonstrated that the expression of EGFR, HER-3, HER-4, EGFR/HER-3, EGFR/HER-4 and HER-3/HER-4 was an independent predictor of poor PFS and OS time in osteosarcoma patients with stage I-IIB disease. In patients with stage IIB osteosarcoma, the expression of HER-4 and EGFR/HER-4 demonstrated a more significant effect on PFS and OS time. In conclusion, therapies targeting EGFR, HER-3 and HER-4 may provide promising strategies for primary osteosarcoma.

The clinical significance of the Ezrin gene and circulating tumor cells in osteosarcoma.

PURPOSE: The aim of this study was to investigate the clinical significance of circulating tumor cells (CTCs) in the peripheral blood of an osteosarcoma and the Ezrin gene expressed in CTCs. PATIENTS AND METHODS: CTC enrichment was done with CanPatrol™ CTC enrichment technique in 41 patients with osteosarcoma. The characterization of CTCs was performed using a multiple messenger RNA in situ analysis (MRIA). The expression of the Ezrin gene in CTCs was detected by RNA probe technology. The correlations of CTC counts, cell type and the expression level of the Ezrin gene with clinical stage and metastasis of osteosarcoma were analyzed using SPSS 16.0 software. RESULTS: The CTC counts correlated significantly with Enneking stage (P<0.001). The ratio of mesenchymal CTCs correlated with the distant metastases (P<0.001). Ezrin gene expression in CTCs correlated significantly with distant metastases (χ2=152.51, P=0.000). CONCLUSION: The ratio of mesenchymal CTCs in the peripheral blood of osteosarcoma correlates with distant metastases. High expression of Ezrin gene in CTCs correlates with distant metastases.

Dual-probe electrochemical DNA biosensor based on the "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification for detection of double-strand DNA of PML/RARα related fusion gene.

Taking advantage of "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification, a dual-probe electrochemical DNA (DE-DNA) biosensor was designed to detect double-stranded DNA (dsDNA) of acute promyelocytic leukemia (APL) related gene. Two groups of detection probes were designed, and each group was composed of a biotinylated capture probe and an assisted probe. They were separately complementary with two strands of target dsDNA in order to prevent the reannealing of the two separate strands from target dsDNA. First, thiol functionalized capture probes (C1 and C2) were severally assembled onto two different gold electrodes, followed by hybridizing with target dsDNA (S1a-S1b) and assistant probes to form two Y-junction-structure ternary complexes. Subsequently, restriction sites on the ternary complexes were digested by Rsa I, which can release S1a, S1b and biotins from the electrode surfaces. Meanwhile, the released S1a and S1b can further hybridize with the unhybridized corresponding detection probes and then initiate another new hybridization-cleavage-separation cycle. Finally, the current signals were produced by the enzyme-catalyzed reaction of streptavidin-horse reddish peroxidase (streptavidin-HRP). The distinct difference in current signals between different sequences allowed detection of target dsDNA down to a low detection limit of 47 fM and presented excellent specificity with discriminating only a single-base mismatched dsDNA sequence. Moreover, this biosensor was also used for assay of polymerase chain reaction (PCR) samples with satisfactory results. According to the results, the power of the DE-DNA biosensor as a promising tool for the detection of APL and other diseases.

Electrochemical immunosensor for detection of topoisomerase based on graphene-gold nanocomposites.

A facile electrochemical immunosensor based on graphene-three dimensional nanostructure gold nanocomposites (G-3D Au) using simple and rapid one-step electrochemical co-reduction technique was developed for sensitive detection of topoisomerase. The resultant G-3D Au nanocomposites were characterized by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy, and then were used as a substrate for construction of the "sandwich-type" immunosensor. Amperometric current-time curve was employed to monitor the immunoreaction on the protein modified electrode. The proposed method could respond to topoisomerase with a linear calibration range from 0.5 ng mL(-1) to 50 ng mL(-1) with a detection limit of 10 pg mL(-1). This new biosensor exhibited a fast amperometric response, high sensitivity and selectivity, and was successfully used in determining the topoisomerase which was added in human serum with a relative standard deviation (n=5)<5%. The immunosensor served as a significant step toward the practical application of the immunosensor in clinical diagnosis and prognosis monitor.

Overexpressing neuroglobin improves functional recovery by inhibiting neuronal apoptosis after spinal cord injury.

The current study was performed to evaluate the mechanisms and therapeutic effects of overexpressing neuroglobin (Ngb) on spinal cord injury (SCI). Adeno-associated virus (AAV) was injected in the T12 section 7 days before SCI. Animals were randomly divided into four groups: a sham group, a vehicle group, an AAV-EGFP group and an AAV-Ngb group. Recovery of hind limb locomotor function was determined during the 3-week post operation period by the Basso, Beattie and Bresnahan locomotor rating scale. At 24 h after SCI and at the end of the study, the segments of spinal cord, centered with the lesion site were harvested for histopathological analysis. Immunofluorescence was performed using antibodies to recognize neuN in the lesion sections. At 24 h after SCI, the spinal cord tissue samples were removed to analyze tissue concentrations of superoxide dismutase (SOD) and malondialdehyde (MDA). Apoptotic cells were assessed using a terminal deoxynucleotidyl transferase, dUTP nick end labeling (TUNEL) kit. The expression of bcl-2, bax, cytochrome c, and cleaved caspase-3, were determined by Western blot assay and immunostaining analysis. The results showed that animals overexpressing Ngb had significantly greater recovery of locomotor function, less neuronal loss and fewer apoptotic cells. In addition, overexpressing Ngb significantly increased bcl-2 expression and SOD level, decreased bax expression, attenuated the release of cytochrome c from mitochondria to the cytosol fraction, and reduced the activity of caspase-3 and MDA level after SCI. These findings suggest, that overexpressing Ngb can significantly improve the recovery of locomotor function. This neuroprotective effect may be associated with the inhibition of neural apoptosis via the mitochondrial pathway.

A sandwich-type DNA biosensor based on electrochemical co-reduction synthesis of graphene-three dimensional nanostructure gold nanocomposite films.

A novel electrochemical DNA biosensor based on graphene-three dimensional nanostructure gold nanocomposite modified glassy carbon electrode (G-3D Au/GCE) was fabricated for detection of survivin gene which was correlated with osteosarcoma. The G-3D Au film was prepared with one-step electrochemical coreduction with graphite oxide and HAuCl4 at cathodic potentials. The active surface area of G-3D Au/GCE was 2.629cm(2), which was about 3.8 times compared to that of a Au-coated GCE under the same experimental conditions, and 8.8 times compared to a planar gold electrode with a similar geometric area. The resultant nanocomposites with high conductivity, electrocatalysis and biocompatibility were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). A "sandwich-type" detection strategy was employed in this electrochemical DNA biosensor and the response of this DNA biosensor was measured by CV and amperometric current-time curve detection. Under optimum conditions, there was a good linear relationship between the current signal and the logarithmic function of complementary DNA concentration in a range of 50-5000fM with a detection limit of 3.4fM. This new biosensor exhibited a fast amperometric response, high sensitivity and selectivity and has been used in a polymerase chain reaction assay of real-life sample with a satisfactory result.