Working principle of real-time Bi-PAP.

<p>The working principle is schematically illustrated for the detection of a mutant template (shown as GC-containing, left panel) using a pair of Bi-PAP primers and a molecular beacon. The four steps: primer annealing, pyrophosphorolysis, primer extension/probe hybridization, and fluorescence detection, are shown from the top to bottom. As a result, fluorescence generated from the probe hybridization is detected in the form of amplification profile. In case of a wild-type template (shown as TA-containing, right panel), two Bi-PAP primers cannot be activated and extended owing to 3′-terminal mismatch, leading to no probe hybridization and thus no fluorescence signal.</p

Quantitative performance of the three-color triplex real-time Bi-PAP.

<p>Amplification curves of 50%, 10%, 1%, 0.1%, 0.01%, and 0% mutant (from left to right) are shown for L858R (A) and T790M (B) in the upper panel. The linear relationship of the Cq difference between the mutation and the internal control (ΔCq =  Cq – Cq^IC) with respect to the logarithmic mutation percentages are shown in the lower panel. Amplification curves of the internal control (C) with different mutant percentages are shown in the upper panel. The linear relationship between the Cq values of the internal control and the logarithmic mutant percentages is given in the lower panel.</p

Study on the sensitivity and specificity of the singleplex real-time Bi-PAP.

<p>(A) Amplification curves of 5000, 500, 50, 5 copies per reaction (from the left to right) of G12R mutant plasmids. (B) Amplification curves of 500, 100, 10, 1 ng wild-type genomic DNA per reaction. Water was used as non-template control (NTC).</p

Real-Time Bidirectional Pyrophosphorolysis-Activated Polymerization for Quantitative Detection of Somatic Mutations

<div><p>Detection of somatic mutations for targeted therapy is increasingly used in clinical settings. However, due to the difficulties of detecting rare mutations in excess of wild-type DNA, current methods often lack high sensitivity, require multiple procedural steps, or fail to be quantitative. We developed real-time bidirectional pyrophosphorolysis-activated polymerization (real-time Bi-PAP) that allows quantitative detection of somatic mutations. We applied the method to quantify seven mutations at codons 12 and 13 in <i>KRAS</i>, and 2 mutations (L858R, and T790M) in <i>EGFR</i> in clinical samples. The real-time Bi-PAP could detect 0.01% mutation in the presence of 100 ng template DNA. Of the 34 samples from the colon cancer patients, real-time Bi-PAP detected 14 <i>KRAS</i> mutant samples whereas the traditional real-time allele-specific PCR missed two samples with mutation abundance <1% and DNA sequencing missed nine samples with mutation abundance <10%. The detection results of the two <i>EGFR</i> mutations in 45 non-small cell lung cancer samples further supported the applicability of the real-time Bi-PAP. The real-time Bi-PAP also proved to be more efficient than the real-time allele-specific PCR in the detection of templates prepared from formalin-fixed paraffin-embedded samples. Thus, real-time Bi-PAP can be used for rapid and accurate quantification of somatic mutations. This flexible approach could be widely used for somatic mutation detection in clinical settings.</p></div

Quantitative performance of the two-color duplex real-time Bi-PAP.

<p>(A) Amplification curves of 100%, 10%, 1%, 0,1%, 0.01%, and 0% G12R mutant (from left to right) in the presence of 100 ng wild-type genomic DNA (upper panel). The linear relationship of the Cq difference between the mutation and the internal control (ΔCq =  Cq – Cq^IC) with respect to the logarithmic mutation percentages (lower panel). (B) Amplification curves of the internal control with different mutant percentages (upper panel). The linear relationship between the Cq values of the internal control and the logarithmic mutant percentages (lower panel).</p

The limit of detection of the two-color duplex real-time Bi-PAP for each KRAS mutation in the presence of 100 ng wild-type genomic DNA.

<p>For each mutation, the ΔCq values for wild-type (red), 0.01% mutant (blue), and 0.1% (black) were detected in 10 replicates and calculated. The line within the box denotes the median, the square within the box denotes the mean, the horizontal borders of each box denote the SD, and the whiskers denote the minimum and maximum.</p

Performance comparison of real-time Bi-PAP and real-time ARMS PCR in the detection of template DNA derived from frozen tissue and FFPE tissue samples.

<p>(A) Real-time Bi-PAP. (B) TheraScreen <i>EGFR</i> RGQ PCR. (C) AmoyDx ARMS <i>EGFR</i>. Amplification curves shown are from 20 frozen tissue samples (black lines) and 25 FFPE tissue samples (red lines), respectively. The difference between the average amplification Cq values (indicated with a double-headed arrow) from the two types of samples are given for each assay in the detection of the internal control.</p

Quantitative Analysis of the Role Played by Poly(vinylpyrrolidone) in Seed-Mediated Growth of Ag Nanocrystals

This article presents a quantitative analysis of the role played by poly­(vinylpyrrolidone) (PVP) in seed-mediated growth of Ag nanocrystals. Starting from Ag nanocubes encased by {100} facets as the seeds, the resultant nanocrystals could take different shapes depending on the concentration of PVP in the solution. If the concentration was above a critical value, the seeds simply grew into larger cubes still enclosed by {100} facets. When the concentration fell below a critical value, the seeds would evolve into cuboctahedrons enclosed by a mix of {100} and {111} facets and eventually octahedrons completely covered by {111} facets. We derived the coverage density of PVP on Ag(100) surface by combining the results from two measurements: (i) cubic seeds were followed to grow at a fixed initial concentration of PVP to find out when {111} facets started to appear on the surface, and (ii) cubic seeds were allowed to grow at reduced initial concentrations of PVP to see at which concentration {111} facets started to appear from the very beginning. We could calculate the coverage density of PVP from the differences in PVP concentration and the total surface area of Ag nanocubes between these two samples. The coverage density was found to be 140 and 30 repeating units per nm² for PVP of 55 000 and 10 000 g/mol in molecular weight, respectively, for cubic seeds of 40 nm in edge length. These values dropped slightly to 100 and 20 repeating units per nm², respectively, when 100 nm Ag cubes were used as the seeds

Fluorescent Probe-Based Lateral Flow Assay for Multiplex Nucleic Acid Detection

Here we report a rapid, low cost, and disposable dipstick-type DNA biosensor that enables multiplex detection in a single assay. The fluorescent probes labeled with different fluorophores were introduced into the lateral flow nucleic acid testing system. In combination with multiple immobilized probes arranged in an array formant on the membrane, a dual-color fluorescent lateral flow DNA biosensor was developed using a portable fluorescence reader. Up to 13 human papillomavirus types could be detected simultaneously by a single-step operation in less than 30 min after linear-after-the-exponential (LATE)-PCR. The sensitivity was determined to be 10–10² copies plasmid DNA/μL. The specificity study showed no cross-reactivity among the 31 different common HPV types. In the clinical validation, 95.3% overall agreement showed very good potential for this method in the clinical application when compared to a commercial kit

Quantifying the Coverage Density of Poly(ethylene glycol) Chains on the Surface of Gold Nanostructures

The coverage density of poly(ethylene glycol) (PEG) is a key parameter in determining the efficiency of PEGylation, a process pivotal to <i>in vivo</i> delivery and targeting of nanomaterials. Here we report four complementary methods for quantifying the coverage density of PEG chains on various types of Au nanostructures by using a model system based on HS–PEG–NH₂ with different molecular weights. Specifically, the methods involve reactions with fluorescamine and ninhydrin, as well as labeling with fluorescein isothiocyanate (FITC) and Cu²⁺ ions. The first two methods use conventional amine assays to measure the number of unreacted HS–PEG–NH₂ molecules left behind in the solution after incubation with the Au nanostructures. The other two methods involve coupling between the terminal −NH₂ groups of adsorbed −S–PEG–NH₂ chains and FITC or a ligand for Cu²⁺ ion, and thus pertain to the “active” −NH₂ groups on the surface of a Au nanostructure. We found that the coverage density decreased as the length of PEG chains increased. A stronger binding affinity of the initial capping ligand to the Au surface tended to reduce the PEGylation efficiency by slowing down the ligand exchange process. For the Au nanostructures and capping ligands we have tested, the PEGylation efficiency decreased in the order of citrate-capped nanoparticles > PVP-capped nanocages ≈ CTAC-capped nanoparticles ≫ CTAB-capped nanorods, where PVP, CTAC, and CTAB stand for poly(vinyl pyrrolidone), cetyltrimethylammonium chloride, and cetyltrimethylammonium bromide, respectively