An Enhanced Strip Biosensor for Rapid and Sensitive Detection of Histone Methylation

Histone methylation is a crucial epigenetic modification of chromosomes. In this work, we describe an enhanced strip biosensor using oligonucleotide-functionalized gold nanoparticles as an enhancer probe (AuNP-DNA) for rapid and sensitive detection of histone methylation. In conventional strip biosensor, methylated histone is captured on the test zone through the formation of antibody/methylated histone/antibody-labeled AuNP sandwich structures. Whereas, in the enhanced strip biosensor, the AuNPs in the sandwich structures are dual labeled with an antibody and another oligonucleotide (c-DNA). The sequence of the c-DNA is complementary to the oligonucleotide on the enhancer probe. The enhancer probe, AuNP-DNA, hybridizes with the c-DNA on the dual labeled AuNPs, and the color intensity of the red band on the test zone is then enhanced dramatically. The enhanced strip biosensor has been used for the visual detection of trimethylated lysine 9 of histone H3 (H3K9me3) in 20 ng of histone extract from HeLa cells within 15 min. The detection limit is 10-fold and 15-fold lower than the conventional strip biosensor and Western blot, respectively

A simple <i>in vitro</i> tumor chemosensitivity assay based on cell penetrating peptide tagged luciferase

<div><p>The analysis of intracellular ATP can reveal the response of cells to different treatments and is important for individualized medicine. In the present study, we developed a cell penetrating peptides (CPPs) tagged luciferase (TAT-LUC) for tumor chemosensitivity assay. The activity of recombinant TAT-LUC was evaluated using ATP standard solution and tumor cells. This recombinant TAT-LUC was then used for the analysis of sensitivity index (SI) of four strains of tumor cells. The results showed that TAT-LUC could detect less than 10 nM extracellular ATP with a strong correlation between the luminescence intensity and the ATP content (R² = 0.994). Without cell lysis, the detection limit for intracellular ATP analysis was 40 tumor cells. Furthermore, chemosensitivity of four strains of tumor cells (Skov-3/DDP, A549/DDP, MDA-MB-231, Huh-7) was determined by this assay successfully. The cell penetration ability of TAT-LUC enables the assay not only to reflect drug resistance of tumor cells real-timely but also to minimize the test time, which can be a valuable aid for personalized cancer chemotherapy.</p></div

Drugs used in the study and their 100% PPC.

<p>Drugs used in the study and their 100% PPC.</p

Identification of TAT-LUC.

<p>(A) Lane 1: PCR amplification products; Lane M: 1 kb DNA marker. (B) Double digestion of recombinant plasmids pET-28a-TAT-LUC with <i>Xho</i> I and <i>Bam</i>H I. Lane M: 1 kb DNA marker; lane 1: TAT-LUC plasmid digested products.</p

SDS-PAGE of TAT-LUC protein.

<p>(A) The protein was induced with IPTG (0.5 mM) at 22°C for 16 h in E. coli BL21(DE3) cells, and analyzed by 10% polyacrylamide SDS-PAGE. Lane 1, uninduced whole bacterial proteins; lane 2, supernatant of uninduced cell lysate; lane 3, pellet of uninduced cell lysate; lane 4, induced whole bacterial proteins; lane 5, supernatant of induced cell lysate; lane 6, pellet of induced cell lysate; Lane M, molecular weight marker. (B) Isolation of TAT-LUC. Lane 1, supernatant of cell lysate; Lane 2, purified TAT-LUC; lane M, molecular weight marker.</p

Chemotherapy dose-inhibition curve in the TAT-LUC based assay.

<p>The X axis is the test drug concentration percentage, and the Y axis is the inhibition rate of tumor cells proliferation. (A) Chemosensitivity of Skov-3/DDP. (B) Chemosensitivity of A549/DDP. (C) Chemosensitivity of MDA-MB-231. (D) Chemosensitivity of Huh-7. PTX, paclitaxel; DDP, cisplatin; CBP, carboplatin; GEM, gemcitabine; NVB, vinorelbine; DOX, doxorubicin; 5-FU, 5-fluorouracil; MMC, mitomycin; VCR, vincristine.</p

Schematic illustration of the TAT-LUC for intracellular ATP detection.

<p>(A) Bioluminescent method based on the luciferin-luciferase reaction (emission wavelength 562 nm). (B) TAT-LUC penetrates cell membrane and ATP powers the luciferase-mediated luminescence production. (C) Construction of the expression plasmid pET-28a-TAT-LUC. TAT-LUC was cloned into the multiple cloning site of the pET-28a vector using <i>Xho</i> I and <i>Bam</i>H I as indicated. Lac I, the lac repressor; Kan, kanamycin resistance gene; ori, origin of replication.</p

Molecular changes related to migration.

<p>Heatmap for genes differentially expressed after MPA treatment for 0h, 12h, 24h, 48h and 72h. Each sample is represented in a column and each gene is represented in a row. Increased expression is indicated as red and decreased expression is indicated as green. Representative genes are shown on the right panel and gene clusters are indicated on the left. </p

Colorimetric Detection of Copper(II) Ion Using Click Chemistry and Hemin/G-Quadruplex Horseradish Peroxidase-Mimicking DNAzyme

G-quadruplex-forming sequence can be formed through a copper­(I) ion (Cu⁺)-catalyzed click chemistry between azide- and alkyne-modified short G-rich sequences in aqueous solution, eliminating immobilization and washing steps of conventional assays. The source for Cu⁺ was generated from the reduction of Cu²⁺ with the reductant of sodium ascorbate. In the presence of hemin and K⁺, the self-assembly of hemin/G-quadruplex structure has the activity of horseradish peroxidase (HRP), which can catalyze its colorless substrate tetrazmethyl benzidine (TMB) into a colored product. Hence, the concentration of Cu²⁺ can be evaluated visually for qualitative analysis according to the color change of the solution, and the optical density (OD) value of the resulting solution at 450 nm was also recorded using a microplate reader for quantitative analysis

A colorimetric assay for vanillin detection by determination of the luminescence of o-toluidine condensates

<div><p>Vanillin (4-hydroxy-3-methoxybenzaldehyde), a food additive with rich milk flavor, is commonly used in the food, beverage and cosmetic industries. However, excessive consumption of vanillin may cause liver and kidney damage. Therefore, methods for detecting and controlling the level of vanillin in food, especially in infant powder, have important practical significance. In this study, we established a colorimetric assay for vanillin detection. The detection was performed under high-temperature and acidic conditions, which can induce the reaction of the aldehyde group of vanillin with the amino group of o-toluidine. The resulting product had a maximum absorption at 363 nm, which was quantified by a UV spectrophotometer. This assay had a limit of detection (<i>LOD</i>) of 1 pg mL⁻¹ and a linear range between 1 μg mL⁻¹ and 100 μg mL⁻¹. The average recoveries at three spiked levels were in the range from 91.1% to 101.6% with a relative standard deviation (<i>RSD</i>) of 4.62% ~ 7.27%.</p></div