Novel Method For Sensitive Detection Research Articles

A Novel Method for Sensitive Detection of Vibrio alginolyticus Based on Aptamer and Hybridization Chain Reaction in Aquaculture

In this study, we developed a novel method for the detection of Vibrio alginolyticus, utilizing the specific recognition of an aptamer for V. alginolyticus and signal amplification via hybridization chain reaction (HCR) and horseradish-peroxidase-conjugated streptavidin. The proposed HCR-based multivalent aptamer (multi-Apt) amplifier allows for sensitive detection of V. alginolyticus in a linear range from 10 to 107 CFU/mL. The linear equation is y = 747.5x + 126.2, R2 = 0.986, and the limit of detection (LOD) is 3 CFU/mL. Seawater and freshwater samples were utilized in the spike recovery experiment, yieldng a recovery rates ranging from 94.3% to 108.8%. The relative standard deviation (RSD) for all samples is below 6.73%. Taken together, the proposed method has great potential for application in monitoring of V. alginolyticus in aquaculture environments.

A novel method for sensitive detection of Escherichia coli O157:H7 based on an aptamer and hybridization chain reaction

This work developed a novel assay for the detection of Escherichia coli O157:H7 (E. coli O157:H7), which is based on the specific recognition of an aptamer (H0) for E. coli O157:H7, the signal amplification of a hybridization chain reaction (HCR) and the adsorption and quenching effect of graphene oxide (GO). The feasibility of this work was verified through control experiments. The concentration of GO, the HCR time, the concentration of H0, and the concentration of H1/H2 were optimized. A series of E. coli O157:H7 dilutions were detected under the optimal conditions, and the detection range was 2.6 × 102 to 2.6 × 108 CFU mL-1. The linear equation is y = 591.1x- 943.9, R2 = 0.995, and LOD is 5 × 101 CFU mL-1. Milk samples were used for the spike recovery experiment, and the recovery rate was 92.6-103.6%. This method shortens the detection time greatly and provides a new strategy for food safety monitoring.

Regulation of multiple energy transfer processes in a simple nano-system for sensitive telomerase detection

Telomerase, as a potential biomarker for early cancer diagnostics and therapies, has attracted considerable interests concerning its detection and monitoring. Herein, we develop a novel method for sensitive detection of telomerase activity by designing a gold nanoparticles/graphene oxide (AuNPs/GO) probe. The AuNPs were functionalized with a telomerase substrate (TS) primer and a 6-carboxy-fluorescein (FAM)-modified complementary DNA (P1). In the absence of telomerase, P1 exists in the random-coiled conformation, and the fluorescence resonance energy transfer (FRET) from FAM to AuNPs and GO results in efficient fluorescence quenching. In the presence of telomerase, the multiple hybridization between TS extension products and P1 leads to the conformation transition of P1 from single-stranded DNA to double-stranded rigid structure, and thus the FRET process can be prevented with the efficient fluorescence recovery. The metal enhanced fluorescence (MEF) effect between FAM and AuNPs can further effectively enhance the fluorescence of FAM, and thus the sensitivity and specificity of telomerase detection can be remarkably improved. It is worth mentioning that the proposed strategy does not need to design complex hairpin structure and allows the measurement of telomerase activity in three crude cell extracts equivalent to 7 HeLa cells, 8 A549 cells and 8 L929 cells in 1 h. In addition, the present sensing platform can be applied to inhibitor screening, in situ telomerase imaging, and intracellular drug delivery.

FERMI: A Novel Method for Sensitive Detection of Rare Mutations in Somatic Tissue.

With growing interest in monitoring mutational processes in normal tissues, tumor heterogeneity, and cancer evolution under therapy, the ability to accurately and economically detect ultra-rare mutations is becoming increasingly important. However, this capability has often been compromised by significant sequencing, PCR and DNA preparation error rates. Here, we describe FERMI (Fast Extremely Rare Mutation Identification) - a novel method designed to eliminate the majority of these sequencing and library-preparation errors in order to significantly improve rare somatic mutation detection. This method leverages barcoded targeting probes to capture and sequence DNA of interest with single copy resolution. The variant calls from the barcoded sequencing data are then further filtered in a position-dependent fashion against an adaptive, context-aware null model in order to distinguish true variants. As a proof of principle, we employ FERMI to probe bone marrow biopsies from leukemia patients, and show that rare mutations and clonal evolution can be tracked throughout cancer treatment, including during historically intractable periods like minimum residual disease. Importantly, FERMI is able to accurately detect nascent clonal expansions within leukemias in a manner that may facilitate the early detection and characterization of cancer relapse.

Study on the Interaction of the CpG Alternating DNA with CdTe Quantum Dots.

A novel sensitive method for detection of DNA methylation was developed with thioglycollic acid (TGA)-capped CdTe quantum dots (QDs) as fluorescence probes. Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. DNA methylation occurs most often at cytosine-guanine sites (CpG dinucleotides) of gene promoters. The QDs significantly interacted with hybridized unmethylated and methylated DNA. The interaction of CpG rich methylated and unmethylated DNA hybrid with quantum dots as an optical probe has been investigated by fluorescence spectroscopy and electrophoresis assay. The fluorescence intensity of QDs was highly dependent to unmethylated and methylated DNA. Specific site of CpG islands of Adenomatous polyposis coli (APC), a well-studied tumor suppressor gene, was used as the detection target. Under optimum conditions, upon the addition of unmethylated dsDNA, the fluorescence intensity increased in linear range from 1.0 × 10- 10 to 1.0 × 10- 6M with detection limit of 6.2 × 10- 11 M and on the other hand, the intensity of QDs showed no changes with addition of methylated dsDNA. We also demonstrated that the unmethylated and methylated DNA and QDs complexes showed different mobility in electrophoresis assay. This easy and reliable method could distinguish between methylated and unmethylated DNA sequences.

Chiroplasmonic assemblies of gold nanoparticles as a novel method for sensitive detection of alpha-fetoprotein

Serum alpha-fetoprotein (AFP) is present in enhanced concentrations in patients with liver cancer. Thus, sensitive detection of AFP plays an important role in the early diagnosis and treatment of cancer. In this study, a novel chiroplasmonic assay is presented for the determination of AFP. It is based on (a) the use of DNA-induced chiroplasmonic assemblies of gold nanoparticles (AuNPs) and (b) the high specificity and affinity constants between AFP and its aptamer. First, the AFP aptamer is hybridized with its complementary sequence immobilized on AuNP. As a result, the DNA-modified AuNPs are assembled to form AuNPs dimers which display strong chiroptical activity. If, however, AFP is added, it will bind to the aptamer and this results in a destruction of the aptamer-DNA hybrid and a reduction of the CD signal. Under optimized conditions, the CD intensity drops linearly with the log of the concentration of AFP in the range from 0.02 to 5 ng⋅mL−1. Compared to the established method, this assay does not require bisulfite conversion, PCR amplification or separation. Amounts as low as 11 pg⋅mL−1 (S/N = 3) AFP can be detected. The method was applied to spiked human serum samples and gave recoveries ranging from 92.0% to 106%, and the relative standard derivations ranged from 4.6% to 6.3%. To the best of our knowledge, this is the first chirality based assay for AFP.

Highly Sensitive Method for Assay of Drug-Induced Apoptosis Using Fluorescence Correlation Spectroscopy

Apoptosis plays a crucial role in many biological processes and pathogenesis of various malignancies and diseases of the immune system. In this paper, we described a novel method for sensitive detection of drug-induced apoptosis by using fluorescence correlation spectroscopy (FCS). The principle of this method is based on the assay of DNA fragmentation in the process of the drug-induced apoptosis. FCS is a single molecule method, and it can be used for sensitive and selective assay of DNA fragmentation without separation. We first developed a highly sensitive method for characterization of DNA fragments using a home-built FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin. Furthermore, FCS method established was used to directly detect the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data. The characteristic diffusion time of DNA fragments was used as an important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of lidamycin concentration, which implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gel electrophoresis. Compared to current methods, the method described here is sensitive and simple, and more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nL level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs.

Recognition and detection of ssDNA using 2-mercaptobenzothiazole self-assembled monolayer modified gold electrode

A novel method for sensitive detection of single-stranded DNA (ssDNA) using 2-mercaptobenzothiazole (MBT) self-assembled monolayer (SAM) modified gold electrode (Au-MBT) has been developed. Based on the intercalating effect of MBT into double stranded DNA (dsDNA), Au-MBT electrode possessed the ability to distinguish ssDNA and dsDNA. Cyclic voltammetry (CV) was used to preliminarily characterize the MBT self-assembled monolayer on gold electrode. In the mixture of excessive probe ssDNA and target ssDNA, current signal recorded by differential pulse voltammetry (DPV) after DNA hybridization reflected the extent of the hybridization. Definite relations were observed between the reductive peak currents and the concentration of target ssDNA. The quantitative detection of target ssDNA was achieved with a linear range from 1.59 × 10−8 to 2.4 × 10−6 mol/L. The detection limit was 2.38 × 10−9 mol/L (3σ, n = 11). The Au-MBT electrode possessed high reusability and reproducibility, no significant deterioration of peak currents occurring over 10 detection/regeneration cycles. Combined with the advantages of simplicity and low-cost, the developed electrochemical biosensor shows potential in continuous and quantitative detection of ssDNA in biological samples.