Iso-E-Codelock: A Rebuilding-free Electrochemical Chip with a Customizable Decoding Probe for Real-Time and Portable Pathogen Diagnostics.

In order to realize portable pathogen diagnostics with easier quantitation, digitization and integration, we develop a ready-to-use electrochemical sensing strategy (Iso-E-Codelock) for real-time detection of isothermal nucleic acid amplification. Bridged by a branched DNA as codelock, the isothermal amplicon is transduced into increased current of an electrochemical probe, holding multiple advantages of high sensitivity, high selectivity, signal-on response, "zero" background and one-pot operation. Through a self-designed portable instrument (BioAlex PHE-T), the detection can be implemented on a multichannel microchip and output real-time amplification curves just like an expensive commercial PCR machine. The microchip is a rebuilding-free and disposable component. The branch codelock probe can be customized for different targets and designs. Such high performance and flexibility have been demonstrated utilizing four virus (SARS-CoV-2, African swine fever, FluA and FluB) genes as targets, and two branch (3-way and 4-way) DNAs as codelock probes.

CRISPR/Cas12a-Responsive Hydrogels for Conjugation-Free and Universal Indicator Release in Colorimetric Detection.

Recent advances have demonstrated the significant potential and advantages to repurpose existing point-of-care reactions/devices to realize portable detection of nonoriginal targets, e.g., pathogen genes. However, pursuing this aim usually requires protein indicator-nucleic acid conjugation via a covalent bond, which may bring drawbacks such as high cost, complicated procedure, and annoying component rebuilding. Herein, we developed a conjugation-free, effective, and universal detection platform called CRIs-gel (CRISPR/Cas12a-Responsive Indicators@RCA hydrogels). Various protein indicators are pre-encapsulated into the hydrogels made of effective and high-yield rolling circle amplification (RCA). Upon a targeting sequence binding with its antisense crRNA, CRISPR/Cas12a starts its trans-cleavage activity to crush the hydrogel, which may directly release the indicator for downstream readout. Two proteins, amylase (GA) and human chorionic gonadotropin (hCG), are successfully used as model indicators to trigger the downstream amylum-I2 color change and pregnancy test strip response. After coupling with upstream isothermal nucleic acid amplification, both portable readouts may detect as few as 2 copies/µL genetic sequences of influenza A virus (FluA), human papilloma virus (HPV), SARS-CoV-2, and influenza B virus (FluB). This conjugation-free CRIs-gel platform is thus simple, sensitive, and universal and can provide innovative insights for portable point-of-care testing (POCT) development.

CLIPON: A CRISPR-Enabled Strategy that Turns Commercial Pregnancy Test Strips into General Point-of-Need Test Devices.

Desirable biosensing assays need to be sensitive, specific, cost-effective, instrument-free, and versatile. Herein we report a new strategy termed CLIPON (CRISPR and Large DNA assembly Induced Pregnancy strips for signal-ON detection) that can deliver these traits. CLIPON integrates a commercial pregnancy test strip (PTS) with four biological elements: the human chorionic gonadotropin (hCG), CRISPR-Cas12a, crRNA and cauliflower-like large-sized DNA assemblies (CLD). CLIPON uses the Cas12a/crRNA complex both to recognize a target of interest and to release CLD-bound hCG so that target presence can translate into a colorimetric signal on the PTS. We demonstrate the versatility of CLIPON through sensitive and specific detection of HPV genomic DNA, SARS-CoV-2 genomic RNA and adenosine. We also engineer a cell phone app and a hand-held microchip to achieve signal quantification. CLIPON represents an attractive option for biosensing and point-of-care diagnostics.

Label-Free and Universal CRISPR/Cas12a-Based Detection Platform for Nucleic Acid Biomarkers.

CRISPR/Cas12a has been widely used in molecular diagnostics due to its excellent trans-cleavage activity. However, conventional reporters, such as F/Q-labeled single-stranded DNA (ssDNA) reporters, enzyme-labeled reporters, and spherical nucleic acid reporters, require complex modification or labeling processes. In this study, we have developed a rapid, universal, and label-free CRISPR/Cas12a-based biomarker detection platform via designing a G-quadruplex (G4) containing a hairpin structure as the reporter. The hairpin loop design of hairpin G4 improves the cleavage efficiency of Cas12a and the signal strength of the G4 binding ligand. Meanwhile, the incorporation of a G4 binding dye (protoporphyrin IX) eliminates the need for complex modifications. The CRISPR-hairpin G4 detection platform is capable of detecting ssDNA, double-stranded DNA, genetic RNAs, and miRNAs. Moreover, this platform achieves label-free detection in clinical samples, demonstrating its practical applicability and efficiency.

Coupling nucleic acid circuitry with the CRISPR-Cas12a system for universal and signal-on detection.

We report a universal and signal-on HCR based detection platform via innovatively coupling the CRISPR-Cas12a system with HCR. By using this CRISPR-HCR pathway, we can detect different targets by only changing the crRNA. The CRISPR-HCR platform coupling with an upstream amplifier can achieve a practical sensitivity as low as ∼aM of ASFV gene in serum.

Sensitive, general and portable detection of RNAs combining duplex-specific nuclease transduction with an off-shelf signalling platform.

We report a novel RNA sensing platform by combining the RNA:DNA hybridization, duplex-specific nuclease (DSN) amplification, and personal glucose meter (PGM) readout. The so-called DSN-PGM sensing platform is sensitive, specific and general to both microRNA and long virus RNA. The detection procedure is simple and instrument-free, thus holding particular potential in the development of portable and point-of-care measurements.

[Approximate calculation of L-S coupling of aluminum 3p electronic angular momentums].

Al plasma was obtained by a pulsed laser beam, which was produced from a Nd:YAG laser set with Q-switch, ablating metal target Al in Ar. Based on the plasma, radiation of the plasma was recorded with time- and space-resolved technology, so Al characteristic radiation spectra were obtained. According to the spectra, the coupling of aluminum 3p electronic angular momentums was simplified as quasi-hydrogen particle. At the same time, the main quantum and orbit angular quantum shielding coefficients of aluminum nucleus, signed as alpha and s respectively, were carefully figured out with related theories of quantum mechanics. With our experiment, the resonance double lines of aluminum were scaled up at 396.15 and 394.40 nm respectively. As a result of our calculation, the main quantum shielding coefficient of aluminum nucleus was sigma = 10.850359, and the orbit angular quantum one was s = 7.332875. Their values were reasonable in terms of the order of magnitude.