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.

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.

Interstitial chemotherapy with ricin-loaded thermosensitive hydrogel in pancreatic cancer xenograft.

BACKGROUND: Pancreatic cancer is one of the most aggressive malignancies, and has a poor prognosis. Despite efforts made in multiple fields, there has been little success in improving the disease-free survival rate of patients. This study was undertaken to investigate the effectiveness and feasibility of using intra-tumoral injection of ricin-loaded thermosensitive hydrogel for treatment of pancreatic cancer xenografts, attempting to develop a new treatment for human pancreatic cancer. METHODS: BALB/c-(nu/nu) nude mice were inoculated subcutaneously in the right flank with the human pancreatic cancer cells, SW1990. Fourteen days after inoculation, 32 mice, bearing tumors of volume 1.5-2.0 cm3, were randomly assigned to one of four groups, and given an intra-tumoral injection of: (1) saline; (2) 23% w/w thermosensitive hydrogel alone; (3) ricin, 10 microg/kg; or (4) 10 microg/kg ricin loaded in thermosensitive hydrogel. On day 14 after administration, the tumors were excised to calculate the inhibition rate of tumor growth and perform histopathological examination. Tumor cell apoptosis was detected by flow cytometry, and RT-PCR was performed to evaluate the mRNA expression levels of Bcl2 and Bax. RESULTS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel resulted in remarkable control of tumor growth. The tumor became necrotic by day 14 after administration. The histological results clearly confirmed that the tumor cells were lysed. The percentage of apoptotic cells detected by flow cytometry was higher in the ricin hydrogel group than in the other groups. Semi-quantitative RT-PCR revealed that the mRNA expression level of Bcl2 was down-regulated whereas Bax was upregulated. CONCLUSIONS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel may provide an effective approach for interstitial chemotherapy in pancreatic cancer. Inducing apoptosis by downregulating Bcl2 expression and upregulating Bax expression may be a key molecular mechanism.

[Effects of interstitial chemotherapy using thermosensitive gel-coated ricin on hepatoma H22-bearing mice].

OBJECTIVE: To explore the efficacy and feasibility of interstitial chemotherapy using thermosensitive gel-coated ricin in hepatoma H22-bearing mice. METHODS: Ricin was purified by chromatography method. The purified ricin was identified by Western blot assay and the purity was determined by high-performance liquid chromatography. BALB/c mice were inoculated subcutaneously in right flank with hepatoma H22 cells. When the tumor size reached about 1.0 cm in diameter, 40 mice were randomly divided into untreated group, thermosensitive gel group, ricin group and thermosensitive gel-coated ricin group. Mice in each group were administered different agents by percutaneous intratumoral injection, including normal saline, thermosensitive hydrogel, ricin and thermosensitive gel-coated ricin. Fifteen days after treatment, the tumors were removed to calculate inhibition rate of tumor growth. The tumor tissues were made into pathological sections to perform histopathological examination. The ultrastructure of tumor tissue was examined by electron microscope examination as well. Blood was collected to detect the hepatic and renal functions. The caspase-3 activity of tumor tissue was determined by using zymologic method with a spectrophotometer. RESULTS: After intratumoral therapy, tumor weight in the thermosensitive gel-coated ricin group was lower than that in the untreated group, with a tumor growth inhibition rate of 71.31%. No obvious hepatic or renal toxicities were detected after thermosensitive gel-coated ricin treatment. Histopathologic observation of the tumor tissue showed massive necrosis and typical apoptosis phenomena, including chromatin margination and apoptotic body. Meanwhile, thermosensitive gel-coated ricin resulted in a significant increase in the caspase-3 activity as compared with the untreated group and the ricin group (P<0.01, P<0.05). CONCLUSION: The above findings indicate that intratumoral therapy with thermosensitive gel-coated ricin has strong antitumor effect and can obviously lessen systemic toxicity, which may provide an effective and feasible method for hepatocellular carcinoma treatment.