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Abstract: Herein we describe a novel “turn-off” biosensing strategy for thrombin detection based on Surface Enhanced Raman scattering (SERS) and the mediation of spacing between 4-mercaptobenzoic acid (4-MBA) labeled gold nanoparticles (AuNPs). The multiple arginine peptides that initiated gold nano-aggregates incorporating Raman reporter molecules due to the formation of “Hot Spots”, are induced to disband by the addition of thrombin in which the peptides are catalytically cleaved into fragments and thus SERS signals of 4-MBA are sharply declined because of the weakened ability of fragments to induce aggregation. Through this strategy, a novel “turn-off” SERS biosensor for thrombin based on enzymatic amplification is established with sensitivity, selectivity and simplicity as AuNPs and peptides are easily accessible. Compared with non-enzymatic amplification based methods, this newly proposed method has improved sensitivity. The limit of detection was 160fM (at the ratio of signal to noise, S/N=3:1). Further, controlled experiments showed that the method exhibited good selectivity over other proteases. The method demonstrated the capability and the potential for application in complex matrix and future biomarker development. The results also presented the potential and superiority of SERS biosensor based on signal amplification. [Copyright &y& Elsevier]

We describe herein a novel' “turn-on” SERS-based strategy for protease detection based on surface enhanced Raman scattering (SERS) and the mediation of spacing between 4-mercaptobenzoic acid (4-MBA) labeled gold nanoparticles (AuNPs) through enzyme assays. The method employed non-cross-linking aggregation of 4-MBA-modified AuNPs by peptides after treatment with target protease. Thus, SERS signals of 4-MBA are sharply increased because of the decreased electrostatic stability of AuNPs that initiated gold nanoaggregates incorporating Raman reporter molecules due to the formation of “Hot Spots”. Through this strategy, a novel and facile “turn-on” SERS biosensor for trypsin and thrombin based on enzymatic cleavage activity is established with sensitivity, selectivity and simplicity as AuNPs and peptides are easily accessible. Compared with other methods, this newly proposed method has improved sensitivity. The limit of detection was 85 fM (at the ratio of signal to noise, S/N=3:1) for trypsin. Controlled experiments showed that the method exhibited good selectivity over other proteases. We also proved that this principle could be easily adapted to detection of other proteases such as thrombin. The method demonstrated the capability for application in complex matrix samples. The results also presented the potential and superiority of SERS biosensor as a general approach for proteases based on enzyme activity. [ABSTRACT FROM AUTHOR]

Catalytic hairpin assembly (CHA) is a promising enzyme-free, isothermal signal amplification strategy, but the relatively time-consuming strand replacement limits its application scenarios. Here, we developed an ultrasound-enhanced catalytic hairpin assembly (UECHA) biosensing platform for early screening of Alzheimer's disease by introducing a portable acoustic-drive platform with functionalized microspheres for effective biomarkers enrichment and fluorescence enhancement. By constructing a gradient ultrasonic field in a microcavity, the platform concentrates the functionalized microspheres in a central position, accompanied by an enhanced fluorescence signal with a specific release. In addition, the programmable frequency modulation can also modify the acoustic potential well and effectively promote non-equilibrium chemical reactions such as CHA (25 min). Compared with the conventional catalytic hairpin assembly (CHA), UECHA allows for direct and quantitative measurement of AD miRNAs down to 3.55 × 10−15 M in 1 μL samples. This visual analysis of ultra-trace biomarkers based on acoustic enrichment and promotion provides a new perspective for the rapid and highly sensitive clinical detection of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Mitochondrial genetic diseases are often characterized by heteroplasmic single nucleotide polymorphisms (SNPs) where both wild-type (WT) and mutant-type (MT) coexist, making detection of accurate SNP abundance critical for diagnosis. Here, we present RatioCRISPR, an automated ratiometric biochip sensor based on the CRISPR/Cas12a system for detecting multiple heteroplasmic SNPs in mitochondrial DNA (mtDNA). The ratiometric sensor output is only influenced by the relative abundance of WT and MT, with minimal impact from sample concentration. Biochips allow the simultaneous detection of multiple SNP sites for more accurate disease diagnosis. RatioCRISPR can accurately detect 8 samples simultaneously within 25 min with a limit of detection (LOD) of 15.7 aM. We successfully detected 13 simulated samples of three mtDNA point mutations (m.3460G>A, m.11778G>A, and m.14484T>C), which lead to Leber's hereditary optic neuropathy (LHON) and set a threshold (60%) of heteroplasmy to evaluate disease risk. This automated and accurate biosensor has broad applications in diagnosing multiple SNPs, especially those with heteroplasmic variations, making it an advanced and convenient tool for mtDNA disease diagnosis. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas system is known as one of the directions of the next generation of mainstream molecular diagnostic technology. However, most current CRISPR/Cas molecular diagnostics still rely on the pre-amplification of nucleic acid due to the limited sensitivity of CRISPR/Cas alone, which has no significant advantage over commercial Taqman-PCR and TwistAmp® Exo kits. Herein, we report an aM-level sensitive cascade CRISPR-Dx system (ASCas) that eliminates nucleic acid pre-amplification, thus avoiding aerosol contamination and greatly reducing the testing environment and personnel skill requirements for molecular diagnostics. Most importantly, the Cas13a nucleases with high sensitivity and trans -cleavage efficiency can rapidly cleaved RNA bubbles on the hybridized cascade probe at low concentration target RNA detection, which results in the destruction of the cascade probe and releases a large amount of trigger DNA for further signal amplification of secondary Cas12a reactions. Therefore, the ASCas system achieves amplification-free, ultra-sensitivity (1 aM), and ultra-fast (20 min) RNA detection. In addition, the ASCas system replaces the complicated screening process of primers and probes with the programmed Cas13a-crRNA design so that a suitable detection system can be constructed more quickly and straightforwardly for the mutation-prone SARS-CoV-2 virus. • A novel cascade CRISPR-Dx system achieves aM-level sensitivity and detects SARS-CoV-2 standard within 20 min. • The cascade probe superimposes the Cas12a and Cas13a reactions, enabling detection without nucleic acid amplification. • This system reduces specialized equipment requirements and is suitable for developing countries. • The ASCas system is suitable for detecting other RNA targets and has potential applications in cancer diagnosis. [ABSTRACT FROM AUTHOR]