A low-voltage alternant direct current electroporation chip for ultrafast releasing the genome DNA of Helicobacter pylori bacterium.

Nucleic acid detection, as an important molecular diagnostic method, is widely used in bacterial identification, disease diagnosis. For detecting the nucleic acid of bacteria, the prerequisite is to release nucleic acids inside the bacteria. The common means to release nucleic acids is the chemical method, which involves complex processes, is time-consuming, and remains chemical inhibitors. Compared with chemical methods, electroporation as a physical method has the advantages of easy operation, short-time consumption, and chemical reagents free. However, the current works using electroporation often necessitates high-frequency or high-voltage conditions, entailing bulky power devices. Herein, we propose a low-voltage alternant direct current (LADC) electroporation chip and the corresponding miniature device for ultrafast releasing the genome DNA from Helicobacter pylori (H. pylori) for detection. We connected a micrometer-interdigital electrode in the chip with a 20 V portable battery to make the miniature device. Using this low-voltage device, our chip released genome DNA of H. pylori within only 5 ms, achieving a cell lysis rate of 99.5%. We further combined this chip with a colorimetric loop-mediated isothermal amplification assay to visually detect H. pylori within ~ 25 min at 10 CFU/µL. We detected 11 clinical samples using the chip, and the detection results were consistent with those of the clinical standard. The results indicate that the LADC electroporation chip is useful for ultrafast release of genome DNA from bacteria and is expected to promote the development of nucleic acid detection in POCT and other scenarios.

Oligo(ethylene glycol)-Functionalized Ratiometric Fluorescent Probe for the Detection of Hydrazine in Vitro and in Vivo.

Hydrazine induced toxicity causes serious harm to the health of humans. The detection of N2H4 in vitro and in vivo has attracted a great deal of attention, especially in the context of fluorescent probes. Although some fluorescent N2H4 probes have been reported, only a few operate in purely aqueous media and, as a result, require the use of organic cosolvents which hinders their use in analysis of real samples. In addition, most of the current N2H4 probes are either "off-on" or "on-off" types, in which it is difficult to eliminate interference from background fluorescence commonly occurring in in vitro and in vivo systems. Furthermore, some probes are unable to differentiate hydrazine from other organic amines. To address the above problems, we developed a novel oligo(ethylene glycol)-functionalized fluorescent probe for the detection of N2H4. The probe, which has a donor-π-acceptor (D-π-A)-type structure, is water-soluble, and it can be utilized to selectively detect N2H4 in both colorimetric and ratiometric mode. Furthermore, the probe is able to differentiate hydrazine from other organic amines and can be used to detect hydrazine vapor and for imaging A549 cells and zebrafish.

Ultrasensitive Detection of miRNA via CRISPR/Cas12a Coupled with Strand Displacement Amplification Reaction.

MicroRNA (miRNA) is a promising biomarker for the diagnosis, monitoring, and prognostic evaluation of diseases, especially cancer. The existing miRNA detection methods usually need external instruments for quantitative signal output, limiting their practical applications in point-of-care (POC) settings. Here, we propose a distance-based biosensor through a responsive hydrogel, in combination with a CRISPR/Cas12a system and target-triggered strand displacement amplification (SDA) reaction for visual quantitative and sensitive measurement of miRNA. The target miRNA is first converted into plenty of double-stranded DNA (dsDNA) via target-triggered SDA reaction. Then, the dsDNA products trigger the collateral cleavage activity of CRISPR/Cas12a, leading to the release of trypsin from magnetic beads (MBs). The released trypsin can hydrolyze gelatin, and hence the permeability of gelatin-treated filter paper is increased, resulting in a visible distance signal on a cotton thread. Using this system, the concentration of the target miRNA can be quantified visually without any assistance of instruments, and a detection limit of 6.28 pM is obtained. In addition, the target miRNA in human serum samples and cell lysates can also be detected accurately. Owing to the characteristics of simplicity, sensitivity, specificity, and portability, the proposed biosensor provides a new tool for miRNA detection and holds great promise in POC applications.

Cotton thread-based multi-channel photothermal biosensor for simultaneous detection of multiple microRNAs.

The abnormal expression of microRNAs (miRNAs) is associated with various diseases. Developing simple and portable methods for sensitive, rapid and simultaneous detection of multiple miRNAs is critical to achieve accurate and timely diagnosis. Herein, a cotton thread-based multi-channel photothermal biosensor was proposed for simultaneous detection of three breast cancer-related miRNAs including miRNA-10b, miRNA-27a and miRNA-let-7a. Three cotton thread-based channels with one input were designed and the capture probes for detecting different miRNAs were immobilized on the test zones of the corresponding channels. Cu2-xS nanostrings prepared on the basis of hybridization chain reaction (HCR) were taken as the photothermal agents for signal transduction and amplification. The formation of a sandwich structure among the capture probe, target miRNA, and Cu2-xS nanostrings led to the accumulation of the Cu2-xS nanostrings on the test zones and transformed the concentration of miRNA into temperature signal under 808 nm laser irradiation. The temperature changes were quantified by a portable thermal camera and directly reflected the concentration of miRNAs. Under the optimal conditions, the developed multi-channel photothermal biosensor showed excellent specificity and sensitivity with the detection limits of 37 pM, 38 pM and 38 pM for miRNA-10b, miRNA-27a and miRNA-let-7a, respectively. Furthermore, a simultaneous detection of the three miRNAs in cell lysates were achieved and the results were in accordance with that obtained by the quantitative reverse transcription polymerase chain reaction (qRT-PCR), indicating its excellent capacity for practical applications. The developed biosensor provided an important tool for analysis of multiple targets and showed great potential in clinical diagnosis.