Analytical Biochemistry

Exonuclease-iii -propelled DNAzyme cascade for sensitive and reliable cervical cancer related miRNA analysis

MicroRNAs (miRNAs) can serve as biomarkers for early-diagnosis, therapy, and postoperative care of cervical cancer. Sensitive and reliable quantification of miRNA remains a huge challenge due to its low expressing levels and background interference. Herein, we propose a novel exonuclease-III (Exo-III)-propelled DNAzyme cascade for sensitive and high-efficient miRNA analysis. This method involves the engineering of compact DNAzyme hairpin probes, including the H1 probe and H2 probe. The H1 probe is designed with exposed analyte recognition subunits that can specifically recognize target miRNA. This recognition triggers two processes: Exo-iii-assisted target regeneration and successive substrate cleavage catalyzed by DNAzyme. The unique character of Exo-III that catalyzes removal of mononucleotides from the blunt or recessed 3'-OH termini of dsDNA confers the approach with a minimal background signal. The multiple signal cycles provided an abundant signal amplification and consequently, the method exhibited a low limit of detection of 3.12 fM, and a better specificity over several homologous miRNAs. In summary, this powerful Exo-III driven DNAzyme cascaded system offers broader and more adaptable methods for comprehending the activities of miRNA in various biological occurrences.

Multiplex analysis of ovarian cancer patients using glycan microarray

Investigation of tumor-associated glycan antigens (TAGs) could be helpful for the development of sensitive cancer diagnostics and novel therapies. Glycan microarrays are effective methods for analyzing glycans and anti-glycan antibodies, which are immobilized arrays of oligo- or poly-saccharides on different substrates, making them a promising class of oncological biomarkers. Blood serum samples from patients (n = 203) with ovarian cancer (OvaCan) and healthy volunteers were analyzed using a glycan microarray containing 63 immobilized glycans to determine changes in anti-glycan IgG and IgM antibody profiles in OvaCan. Levels of anti-glycan IgG and IgM antibodies in OvaCan statistically differed from levels in healthy donors: the most prominent statistically significant difference for anti-glycan IgG antibodies was found for 6-O-su-Le

Isothermal detection of lncRNA using T7 RNA polymerase mediated amplification coupled with fluorescence-based sensor

In this study, the isothermal detection of a cervical cancer-associated long non-coding RNA (lncRNA), namely, lncRNA-ATB, was performed for the first time with high selectivity and sensitivity via a T7 RNA polymerase transcription-mediated amplification system combined with a graphene oxide (GO) fluorescence-based sensor. Specific lncRNA primers with the T7 promoter overhang were designed and further had with the efficient amplification ability of T7 RNA polymerase. This detection platform distinguished the target lncRNA-ATB from other lncRNAs. In addition, the super fluorescence quenching ability of GO resulted in the development of a switch on/off fluorescence sensor. The resulting platform was able to detect target lncRNAs from samples of cervical cancer cell lines (HeLa) and human sera with high selectivity and a low detection limit of 1.96 pg. Therefore, the assay developed in this study demonstrated a high potential as an alternative tool for lncRNA quantification in clinical diagnosis.

Metabolomics approach using HR-MAS NMR spectroscopy for the assessment of metabolic profiles of uterine fibroids

The aim of this study is to determine dysregulated metabolites and metabolic pathways in uterine fibroids and in the myometrial tissue from which uterine fibroids are derived. Fifteen (15) patients underwent hysterectomy because of uterine fibroids and 14 controls were included in this study.

Single-tube four-target lateral flow assay detects human papillomavirus types associated with majority of cervical cancers

Isothermal nucleic acid amplification methods have many advantages for use at the point of care. However, there is a lack of multiplexed isothermal amplification tests to detect multiple targets in a single reaction, which would be valuable for many diseases, such as infection with high-risk human papillomavirus (hrHPV). In this study, we developed a multiplexed loop-mediated isothermal amplification (LAMP) reaction to detect the three most common hrHPV types that cause cervical cancer (HPV16, HPV18, and HPV45) and a cellular control for sample adequacy. First, we characterized the assay limit of detection (LOD) in a real-time reaction with fluorescence readout; after 30 min of amplification the LOD was 100, 10, and 10 copies/reaction of HPV16, HPV18, and HPV45, respectively, and 0.1 ng/reaction of human genomic DNA (gDNA). Next, we implemented the assay on lateral flow strips, and the LOD was maintained for HPV16 and HPV18, but increased to 100 copies/reaction for HPV45 and to 1 ng/reaction for gDNA. Lastly, we used the LAMP test to evaluate total nucleic acid extracted from 38 clinical samples; compared to qPCR, the LAMP test had 89% sensitivity and 95% specificity. When integrated with sample preparation, this multiplexed LAMP assay could be useful for point-of-care testing.

Optical-based biosensor for detection of oncomarker CA 125, recent progress and current status

Ovarian cancer (OV) is the second most mortal gynecological malignancy. The oncomarker CA125 has been used as the main ovarian cancer marker for diagnosing and screening ovarian cancer in stages I and II. Therefore, sensitive and real-time detection of CA 125 is critical in ovarian cancer monitoring. Various tests are used to diagnose the CA 125. In recent years, modern methods such as biosensor technology have replaced the old tests for rapid, sensitive and specific detection of CA 125. Various types of biosensors are being developed, among which Surface Plasmon resonance (SPR) biosensors are one of the most important and remarkable types. Considering the importance of SPR biosensors in the diagnosis of enocomarker CA 125, the main focus of the present study is to consolidate the research work from the past two decade to the present. Also, the advantages and challenges in SPR biosensors development have been considered in the detection of CA 125 oncomarker.

Aptamer-based carbohydrate antigen 125 sensor with molybdenum disulfide functional hybrid materials

Epithelial ovarian cancer is a malignant tumor of the female reproductive system with insidious symptoms, aggressiveness, risk of metastasis, and high mortality. Carbohydrate antigen 125 (CA125), a standard biomarker for screening epithelial ovarian cancer, can be applied to track cancer progression and treatment response. Here, we constructed an aptamer-based electrochemical biosensor to achieve sensitive detection of CA125. Molybdenum disulfide (MoS

Development of a kind of RG108-Fluorescein conjugates for detection of DNA methyltransferase 1 (DNMT1) in living cells

DNA methyltransferase 1 (DNMT1) is one of the most essential proteins in propagating DNA methylation patterns during replication. Developing methods to assess the expression level of DNMT1 will enable study of gene methylation abnormalities. Thus, a series of fluorescein-conjugated RG108 derivatives were designed and synthesized in the current study. The affinity of the derivatives with DNMT1 was evaluated using surface plasmon resonance. Permeability of the derivatives through the cytomembrane and nuclear envelope was evaluated via confocal imaging. Probe 8a was found to compete with RG108 binding to DNMT1 in the nucleus of HeLa cells, suggesting that probe 8a and RG108 share the same binding site. A HeLa cell model with 4.05-fold overexpression of DNMT1 was constructed and used to evaluate probe 8a. Probe 8a was found to be significantly increased in the nucleus of DNMT1 overexpressing cells. These results indicate that fluorescent probes derived from RG108 have the potential to be used for evaluating the expression level of DNMT1 in living cells.

Sensitive and reliable analysis of endometrial cancer related microRNA using ternary hybridization hairpin probe

MicroRNA (miRNA), as a kind of small non-coding ribonucleic acid (RNA) that plays a crucial role in regulating transcriptional activities, is a potential biomarker for EC diagnosis. However, reliable detection of miRNA remains a huge challenge, especially for these methods that require multiple probes for signal amplifications, due to the detective deviation caused by variation of probe concentrations. Herein, we present a novel approach for miRNA-205 identification and quantification by employing simply a ternary hairpin probe (TH probe). The ternary hybridization of three sequences results in the construction of the TH probe, which combines high-efficient signal amplification and specific target recognition. A significant number of G-rich sequences have been produced as a result of the enzymes assisted signal amplification process. The G-rich sequences can fold into G-quadruplexes, which can then be detected in a label-free manner by a common fluorescent dye (thioflavin T). Eventually, the approach exhibits a low limit of detection of 278 aM with a wide detection range of 7 orders of magnitude. In summary, the proposed approach possesses a great potential for both clinical diagnosis of EC and fundamental biomedical researches.