CRISPR/Cas14 and G-Quadruplex DNAzyme-Driven Biosensor for Paper-Based Colorimetric Detection of African Swine Fever Virus.

The highly contagious nature and 100% fatality rate contribute to the ongoing and expanding impact of the African swine fever virus (ASFV), causing significant economic losses worldwide. Herein, we developed a cascaded colorimetric detection using the combination of a CRISPR/Cas14a system, G-quadruplex DNAzyme, and microfluidic paper-based analytical device. This CRISPR/Cas14a-G4 biosensor could detect ASFV as low as 5 copies/µL and differentiate the wild-type and mutated ASFV DNA with 2-nt difference. Moreover, this approach was employed to detect ASFV in porcine plasma. A broad linear detection range was observed, and the limit of detection in spiked porcine plasma was calculated to be as low as 42-85 copies/µL. Our results indicate that the developed paper platform exhibits the advantages of high sensitivity, excellent specificity, and low cost, making it promising for clinical applications in the field of DNA disease detection and suitable for popularization in low-resourced areas.

Novel Anti-CRISPR-Assisted CRISPR Biosensor for Exclusive Detection of Single-Stranded DNA (ssDNA).

Nucleic acid analysis plays an important role in disease diagnosis and treatment. The discovery of CRISPR technology has provided novel and versatile approaches to the detection of nucleic acids. However, the most widely used CRISPR-Cas12a detection platforms lack the capability to distinguish single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). To overcome this limitation, we first employed an anti-CRISPR protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA (crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system. Only ssDNA has the ability to recruit the cleaved crRNA fragment to recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA cannot accomplish this. By measuring the recovered cleavage activity of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR biosensor is capable of distinguishing ssDNA from dsDNA, providing a simple and reliable method for the detection of ssDNA. Furthermore, we demonstrated our developed AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of helicase and screen its inhibitors.

On-Site Fluorescent Detection of Sepsis-Inducing Bacteria using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System.

Sepsis is an extremely dangerous medical condition that emanates from the body's response to a pre-existing infection. Early detection of sepsis-inducing bacterial infections can greatly enhance the treatment process and potentially prevent the onset of sepsis. However, current point-of-care (POC) sensors are often complex and costly or lack the ideal sensitivity for effective bacterial detection. Therefore, it is crucial to develop rapid and sensitive biosensors for the on-site detection of sepsis-inducing bacteria. Herein, we developed a graphene oxide CRISPR-Cas12a (GO-CRISPR) biosensor for the detection of sepsis-inducing bacteria in human serum. In this strategy, single-stranded (ssDNA) FAM probes were quenched with single-layer graphene oxide (GO). Target-activated Cas12a trans-cleavage was utilized for the degradation of the ssDNA probes, detaching the short ssDNA probes from GO and recovering the fluorescent signals. Under optimal conditions, we employed our GO-CRISPR system for the detection of Salmonella Typhimurium (S. Typhimurium) with a detection sensitivity of as low as 3 × 103 CFU/mL in human serum, as well as a good detection specificity toward other competing bacteria. In addition, the GO-CRISPR biosensor exhibited excellent sensitivity to the detection of S. Typhimurium in spiked human serum. The GO-CRISPR system offers superior rapidity for the detection of sepsis-inducing bacteria and has the potential to enhance the early detection of bacterial infections in resource-limited settings, expediting the response for patients at risk of sepsis.

Fe-Doped Carbon Dots as NIR-II Fluorescence Probe for In Vivo Gastric Imaging and pH Detection.

Carbon dots (CDs) with excellent cytocompatibility, tunable optical properties, and simple synthesis routes are highly desirable for use in optical bioimaging. However, the majority of existing CDs are triggered by ultraviolet/blue light, presenting emissions in the visible/first near-infrared (NIR-I) regions, which do not allow deep tissue penetration. Emerging research into CDs with NIR-II emission in the red region has generated limited designs with poor quantum yield, restricting their in vivo imaging applications due to low penetration depth. Developing novel CDs with NIR-II emissions and high quantum yield has significant and far-reaching applications in bioimaging and photodynamic therapy. Here, it is developed for the first time Fe-doped CDs (Fe-CDs) exhibiting the excellent linear relationship between 900-1200 nm fluorescence-emission and pH values, and high quantum yield (QY-1.27%), which can be used as effective probes for in vivo NIR-II bioimaging. These findings demonstrate reliable imaging accuracy in tissue as deep as 4 mm, reflecting real-time pH changes comparable to a standard pH electrode. As an important example application, the Fe-CDs probe can non-invasively monitor in vivo gastric pH changes during the digestion process in mice, illustrating its potential applications in aiding imaging-guided diagnosis of gastric diseases or therapeutic delivery.

Mitochondria-targeted polydopamine nanoprobes for visualizing endogenous sulfur dioxide derivatives in a rat epilepsy model.

Epilepsy is the fourth most common neurological disorder, and aberrantly elevated sulfur dioxide derivatives (SO32-/HSO3-) are thought to underlie the hippocampal neuronal apoptosis in epilepsy. We have designed and synthesized a mitochondria-targeted polydopamine nanoprobe for visualizing endogenous SO32-/HSO3- by the nucleophilic addition reaction. The nanoprobe was used for imaging SO2 derivatives both in the mitochondria of cultured cells and zebrafish, and successfully applied in the hippocampus of a rat model of epilepsy. The PDAD nanoprobe could be of great value for the elucidation of mechanisms of abnormal SO32-/HSO3- involved in diseases such as epilepsy.

Polydopamine Dots-Based Fluorescent Nanoswitch Assay for Reversible Recognition of Glutamic Acid and Al3+ in Human Serum and Living Cell.

We developed a facile and feasible fluorescent nanoswitch assay for reversible recognition of glutamate (Glu) and Al3+ in human serum and living cell. The proposed nanoswitch assay is based on our recently developed method for controlled synthesis of fluorescent polydopamine dots (PDADs) at room temperature with dopamine as the sole precursor. The fluorescence of nanoswitch assay could be quickly and efficiently quenched by Glu (turn-Off), and the addition of Al3+ could recover the fluorescence of the PDADs-Glu system (turn-On). Meanwhile, the reversible recognition of Glu and Al3+ in this nanoswitch system was stable after three cycles. Additionally, the system displayed excellent performance for Glu and Al3+ determination with a low detection limit of 0.12 and 0.2 µM, respectively. Moreover, PDADs are successfully applied to determine Glu and monitor Al3+ in human serum. Noteworthy, the nanoswitch assay is transported into HepG2 cells and realized "Off" detection of Glu and "On" sensing Al3+ in the living cells. Therefore, this PDADs-based nanoswitch assay provides a strategy to develop reversible recognition biosensors for intracellular and external molecular analysis.

Mitochondrial Specific H2S n Fluorogenic Probe for Live Cell Imaging by Rational Utilization of a Dual-Functional-Photocage Group.

Reactive sulfur species play a very important role in modulating neural signal transmission. Hydrogen polysulfides (H2S n, n > 1) are recently suggested to be the actual signaling molecules. There are still few spatiotemporal controllable-based probes to detect H2S n. In this work, for the first time, we proposed the photocleavage product of the common photoremovable protecting group (2-nitrophenyl moiety) capable of trapping H2S n. Taking advantage of this, we constructed the probe H1 containing a photocontrollable group, a mitochondrial directing unit and a signal reporter fluorescein dye. H1 exhibited excellent fluorescence enhancement (50-fold) in response to H2S n under the aqueous buffer only after UV irradiation. H1 also showed high selectivity and sensitivity for H2S n over other reactive sulfur species, reactive oxygen species, and other analytes, especially biothoils including hydrogen sulfide, cysteine, homocysteine, and glutathione. We showed the utility of H1 to image H2S n in living cells with high spatiotemporal resolution.