An Enzyme-Free DNA Circuit-Assisted Graphene Oxide Enhanced Fluorescence Anisotropy Assay for MicroRNA Detection with Improved Sensitivity and Selectivity

Graphene oxide (GO) has been proven as an outstanding fluorescence anisotropy (FA) amplifier. Yet the traditional GO amplified FA assays lack high sensitivity because of the 1:1 binding ratio between target and dye-modified probe. Herein, we report a new target-catalyzed hairpin assembly (CHA), an enzyme-free DNA circuit, assisted GO amplified FA strategy for microRNA-21 (miRNA-21) detection. In the presence of miRNA-21, the CHA was initiated and plenty of H1–H2 duplexes were produced continuously. The obtained H1–H2 duplex could induce the formation of a H1–H2–probe DNA (pDNA) complex by the toehold-mediated strand exchange reaction, which led the dye-modified pDNA to leave away from the GO surface, resulting in a decreased FA of the system. By monitoring the decrease of FA, miRNA-21 could be detected in the range of 0–16 nM. The limit of detection (LOD, 3σ) was 47 pM, which was 194 times lower than that without CHA. In addition, the selectivity of this method has also been enhanced greatly as compared to that without CHA. Our method has great potential to be applied for detecting different types of targets and monitoring diverse molecular interactions by adapting the corresponding nucleotide sequence

Effect of vitamin K₁ (VK₁) on apoptosis of RBL-2H3 cells by fluorescence staining.

<p>AnnexinV-FITC-stained cells appear green at the early stage of apoptosis. PI caused the cells to appear red during the middle to late stages of apoptosis. The magnification is 400×. The merged green and red images represent total apoptosis. VK₁-FE: vitamin K₁-fat emulsion.</p

The scores of the symptoms shown by the dogs after three sensitizations with vitamin K₁ (VK₁) injection and vitamin K₁-fat emulsion (VK₁-FE).

<p>The dogs were sensitized by intravenous drugs every other day for a total of three administrations via a micro-injection pump at 0.4 ml/min; symptoms were then observed and recorded for 30 min. The sum of the scores was determined for each symptom. The values are shown as the mean ± SE, n = 6.</p><p>**<i>P</i><0.01 vs. control.</p

The change in plasma histamine concentrations after the first intravenous administration.

<p>Blood samples were taken before and 10 min after drug administration. The plasma histamine concentrations were quantified by ELISA. The values are shown as the mean ± SE, n = 6.</p><p>*<i>P</i><0.05 vs. control.</p><p>VK₁: vitamin K₁; VK₁-FE: vitamin K₁-fat emulsion.</p

Effect of vitamin K₁ (VK₁) injection on histamine release in RBL-2H3 cells.

<p>RBL-2H3 cells were treated with the different drugs for 30 min. The supernatants were collected and measured by spectrofluorometry. PBS was used as a blank, and the supernatant from cells stimulated with Triton was considered to represent the total histamine release. The histamine release rates are shown as the mean ± SE, n = 6. ^*<i>P</i><0.05, ^**<i>P</i><0.01 vs. control (conl). VK₁-FE: vitamin K₁-fat emulsion.</p

The grades and scores of symptoms in dogs after the challenge with vitamin K₁ (VK₁) injection and vitamin K₁-fat emulsion (VK₁-FE).

<p>The dogs were intravenously stimulated with double doses of drug via a micro-injection pump at 0.4 ml/min, and symptoms were then observed and recorded for 30 min. The sum of the scores was determined for each symptom. The values are shown as the mean ± SE, n = 6.</p><p>**<i>P</i><0.01 vs. control.</p

Effect of vitamin K₁ (VK₁) injection on the apoptosis of RBL-2H3 cells by flow cytometry.

<p>Apoptotic cells were identified by Annexin V-FITC and PI staining. The concentrations of Tween-80 are 1000, 100, and 10 µg/mL, from top to bottom. The values are shown as the mean ± SE, n = 6. ^*<i>P</i><0.05, ^**<i>P</i><0.01 vs. control (conl). VK₁-FE: vitamin K₁-fat emulsion.</p

Effect of vitamin K₁ (VK₁) injection on systolic blood pressure (A) and diastolic blood pressure (B) in Beagle dogs.

<p>The change in blood pressure is presented as the decrease in blood pressure from before to after drug administration. The values are shown as the mean ± SE, n = 6. ^*<i>P</i><0.05, ^**<i>P</i><0.01 vs. control. VK₁-FE: vitamin K₁-fat emulsion.</p

Grades and scores for the symptoms presented by the dogs in a cross-challenge experiment.

<p>Dogs sensitized with 0.25 mg/kg vitamin K₁ (VK₁) injection were challenged with 0.5 mg/kg vitamin K1-fat emulsion (VK₁-FE), and dogs sensitized with 0.25 mg/kg VK₁-FE were challenged with 0.25 mg/kg VK₁ injection. Symptoms were observed and recorded for 30 min. The sum of the scores was determined for each symptom. The values are shown as the mean ± SE, n = 6.</p><p>**<i>P</i><0.01 vs. control.</p

An assessment norm for the anaphylactoid reactions and anaphylaxis in dogs.

<p>Each symptom in the dogs received a score corresponding to that symptom.</p