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.

2'-Fluoro ribonucleic acid modified DNA dual-probe sensing strategy for enzyme-amplified electrochemical detection of double-strand DNA of PML/RARα related fusion gene.

In the study, a novel sensing strategy based on dual-probe mode, which involved two groups of 2'-fluoro ribonucleic acid (2'-F RNA) modified probes, was designed for the detection of synthetic target double-strand DNA (dsDNA) of PML/RARα fusion genes in APL. And each pair of probes contained a thiolated capture probe (C1 or C2) immobilized on one of electrode surfaces in the dual-channel electrochemical biosensor and a biotinylated reporter probe (R1 or R2). The two groups of 2'-F RNA modified probes were separately complementary with the corresponding strand (Sa or Sb) from target dsDNA in order to prevent renaturation of target dsDNA. Through flanking target dsDNA, two "sandwitch" complexes (C1/Sa/R1 and C2/Sb/R2) were separately shaped by capture probes (C1 and C2) and free reporter probes (R1 and R2) in hybridization solution on the surfaces of different electrodes after the thermal denaturation. The biotin-modified enzyme which produced the measurable electrochemical current signal was localized to the surface by affinity binding between biotin with streptavidin. Under the optimal condition, the biosensor was able to detect 84 fM target dsDNA and showed a good specificity in PBS hybridization solution. Otherwise, the investigations of the specificity and sensitivity of the biosensor were carried out further in the mixed hybridization solution containing different kinds of mismatch sequences as interference background. It can be seen that under a certain interference background, the method still exhibited excellent selectivity and specificity for the discrimination between the fully-complementary and the mismatch sequences. The results of our research laid a good basis of further detection research in practical samples.

[Study on the Identification of Geographical Indication Wuchang Rice Based on the Content of Inorganic Elements].

Wuchang rice is a geographical indication product in China. Due to its high quality and low production, the phenome- non of fake is more and more serious. An effective identification method of Wuchang rice is urgent needed, for the maintenance of its brand image and interest of consumers. Base on the content of inorganic elements which are analyzed by ICP-AES and ICP-MS in rice, the identification model of Wuchang rice is studied combining with principal component analysis (PCA), Fisher discrimination and artificial neural network (ANN) in this paper. The effect on the identification of samples is poor through PCA, while the samples from Wuchang area and other areas can be identified accurately through Fisher discrimination and ANN. The average accurate identification ratio of training and verification set through Fisher discrimination is 93.5%, while the average accurate identification ratio through ANN is 96.4%. The ability to identify of ANN is better than Fisher discrimination. Wuchang rice can be identified accurately through the result of this research which provides a technology for the protection of geographical indications of this product.

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.

Preparation and application of poly(dimethylsiloxane)/beta-cyclodextrin solid-phase microextraction fibers.

A novel poly(dimethylsiloxane)/beta-cyclodextrin (PDMS/beta-CD) coating was prepared for solid-phase microextraction (SPME). The PDMS/beta-CD coating proved to have a porous structure, providing high surface areas and allowing for high extraction efficiency. The coating had a high thermal stability (340 degrees C) and a long lifetime due to its chemical binding to the fiber surface. Polar phenols and amines were used to evaluate the character of the coating fiber by headspace (HS) extraction and thermal desorption, followed by GC-FID analysis. Parameters that affected the extraction process were investigated; these include extraction time and temperature, desorption time, pH, and ionic strength of the solution. For phenols, the range of linearity of the method was 4-500 microg/L and the LOD was 1.3-2.1 microg/L. For amines, the range of linearity was 1-1000 microg/L and the LOD was 1.2-2.8 microg/L. The presence of beta-CD not only increases the thermal stability of the fiber coating, but also enhances its selectivity. Compared with commercially available SPME fibers, the new phases show better selectivity and sensitivity towards polar compounds.