Precise Differentiation of Wobble-Type Allele via Ratiometric Design of a Ligase Chain Reaction-Based Electrochemical Biosensor for CYP2C19*2 Genotyping of Clinical Samples.

Due to the comparable stability between the perfect-base pair and the wobble-base pair, a precise differentiation of the wobble-type allele has remained a challenge, often leading to false results. Herein, we proposed a ligase chain reaction (LCR)-based ratiometric electrochemical DNA sensor, namely, R-eLCR, for a precise typing of the wobble-type allele, in which the traditionally recognized "negative" signal of wobble-base pair-mediated amplification was fully utilized as a "positive" one and a ratiometric readout mode was employed to ameliorated the underlying potential external influence and improved its detection accuracy in the typing of the wobble-type allele. The results showed that the ratio between current of methylene blue (IMB) and current of ferrocene (IFc) was partitioned in three regions and three types of wobble-type allele were thus precisely differentiated (AA homozygote: IMB/IFc > 2; GG homozygote: IMB/IFc < 1; GA heterozygote: 1 < IMB/IFc < 2); the proposed R-eLCR successfully discriminated the three types of CYP2C19*2 allele in nine cases of human whole blood samples, which was consistent with those of the sequencing method. These results evidence that the proposed R-eLCR can serve as an accurate and robust alternative for the identification of wobble-type allele, which lays a solid foundation and holds great potential for precision medicine.

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.

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.

Genotyping of common EGFR mutations in lung cancer patients by electrochemical biosensor.

In this study, we constructed a sandwich-type biosensor to identify six common types of mutations in exon 19 of the epidermal growth factor receptor (EGFR) gene, and tested them using tissue samples from patients with non-small cell lung carcinomas. Considering the characteristics that different locations of non-complementary in DNA probes resulting in different hybridization efficiency, we investigated the design of DNA capture probes with varying non-complementary sequence locations in an effort to optimize the selectivity of the biosensor. Our results revealed that non-complementary sequences located in the middle of a capture probe allow excellent hybridization specificity and achieve the strongest discrimination between mutations that differ by a single nucleotide. Based on this finding, we designed capture probes to identify six common types of EGFR mutations (del1-del6) successfully. Further, we proposed a grouped testing approach to reduce workload and rapidly identify mutation types. Subsequently, EGFR exon 19 hotspot deletion types in real samples were discriminated by this method. RT-PCR products from lung cancer patients were digested with λ-Exo and analyzed using electrochemical biosensors. The results of our grouped testing approach with optimized biosensors were consistent with that of direct sequencing, suggesting that our proposed protocol can be excellent candidate for genotyping of EGFR mutations in lung cancer patients.

Detection EGFR exon 19 status of lung cancer patients by DNA electrochemical biosensor.

Epidermal growth factor receptor (EGFR) exon 19 mutation status is a very important prediction index for tyrosine kinase inhibitors (TKIs) therapy. In this paper, we constructed a superior selective sandwich-type electrochemical biosensor to detect in-frame deletions in exon 19 of EGFR in real samples of patients with non-small cell lung carcinoma. Based on the characteristics of different hybridization efficiency in different hybridization phase conditions, different region around EGFR exon 19 deletion hotspots was selected to design DNA probes to improve biosensor performance. The results confirm that alteration of deletion location in target deliberately according to different hybridization phase is able to improve selectivity of sandwich-type DNA biosensor. Satisfactory discrimination ability can be achieved when the deletions are located in the capture probe interaction region. In order to improve efficiency of ssDNA generation from dsDNA, we introduce Lambda exonuclease (λ-exo) to sandwich-type biosensor system. EGFR exon 19 statuses of clinical real samples from lung cancer patients can be discriminated successfully by the proposed method. Our research would make the electrochemical biosensor be an excellent candidate for EGFR detection for lung cancer patients.

Antifungal activity of chitooligosaccharides against the dermatophyte Trichophyton rubrum.

Antifungal activity against the dermatophytic fungus Trichophyton rubrum by a well-characterized chitooligosaccharides (COS) sample, hydrolyzed using a recombinant chitosanase, was investigated in vitro and in vivo. The minimum inhibitory concentration (MIC) of COS ranged between 0.25 and 0.50%, which was measured using a microdilution method. Analysis of inhibition rates using an agar diffusion method showed that treatment with 0.5% and 1% COS significantly suppressed T. rubrum cell growth (p<0.05 and p<0.01, respectively, in comparison with untreated control). Morphological changes and structural alterations of cells were observed by TEM. In vivo efficacy of COS in treatment of T. rubrum dermatophytosis was evaluated using a guinea pig model. Skin lesion scores revealed a strong, dose-dependent therapeutic effect of COS. The 5% COS group showed a reduction of skin lesions even greater than that of the positive control group treated with 1% fluconazole (FCZ). Histological analysis revealed no inflammation or tissue destruction in the groups treated with 5% COS or 1% FCZ. Hyperkeratosis was also observed, perhaps resulting from a defensive response of the tissue cells to COS. The findings indicate that COS has excellent potential for development of novel antifungal drugs for clinical treatment/remission of dermatophytoses.

In situ growth of porous platinum nanoparticles on graphene oxide for colorimetric detection of cancer cells.

A green approach is proposed for in situ growth of porous platinum nanoparticles on graphene oxide (PtNPs/GO). The resulting nanocomposite has been proven to function as peroxidase mimetics that can catalyze the reaction of peroxidase substrate in the presence of hydrogen peroxide. On the basis of the peroxidase-like activity, we used the PtNPs/GO as a signal transducer to develop a colorimetric assay for the direct detection of cancer cells. By using folic acid as a recognition element, a total of 125 cancer cells (MCF-7) can be distinguished by naked-eye observation. We envision that this nanomaterial could be used as a power tool for a wide range of potential applications in biotechnology and medicine.

[Surfaced-enhanced Raman spectroscopic study on single living human nasopharyngeal carcinoma cells incubated with colloidal gold].

The surface-enhanced Raman scattering (SERS) spectroscopy and normal Raman spectroscopy of single living human nasopharyngeal carcinoma cells(CNE-1) were tested and analyzed by gold nanoparticles incubation into cells. Six obvious Raman bands (718, 1001, 1123, 1336, 1446 and 1660 cm(-1)) were observed in the normal Raman spectroscopy of living CNE-1 cells. The characteristic Raman bands in the SERS spectra of living cells were tentatively assigned. Colloidal gold particles that were introduced inside cells result in strongly enhanced Raman signals of the native chemical constituents of the cells, and over twenty SERS Raman bands were observed in the SERS spectroscopy of living CNE-1 cells. The Raman lines of 1026, 1097, 1336 and 1585 cm(-1) were assigned to vibrations of the DNA backbone, which confirms that some gold nanoparticles were able to enter the nucleus. The results showed that, based on colloidal gold, the SERS spectroscopy might provide a sensitive and structurally selective detecting method for native chemicals inside a cell, such as DNA and phenylalanine.

[Isolation and structure identification of chemical constituents from Viscum liquidambaricolum].

AIM: To study the chemical constituents of Viscum liquidambaricolum Hayata. METHODS: Various chromatographic techniques were used to separate and purify the compounds. Their spectral data (MS,IR,NMR) were measured for structure identification. RESULTS: Five compounds were isolated from Viscum liquidambaricolum and their structures were identified as trans-cinnamic acid (I), oleanolic acid (II), chrysin (III), eriodictyol (IV) and liquidamboside (V). CONCLUSION: Liquidamboside (V) is a new compound, the known compounds I - IV were isolated from this plant for the first time, I, III, IV were isolated from Loranthaceae for the first time.