A novel cascade signal amplification strategy integrating CRISPR/Cas13a and branched hybridization chain reaction for ultra-sensitive and specific SERS detection of disease-related nucleic acids.

The molecular diagnosis of disease by high-sensitively and specifically detecting extremely trace amounts of nucleic acid biomarkers in biological samples is still a great challenge, and the powerful sensing strategy has become an urgent need for basic researches and clinical applications. Herein, a novel one-pot cascade signal amplification strategy (Cas13a-bHCR) integrating CRISPR/Cas13a system (Cas13a) and branched hybridization chain reaction (bHCR) was proposed for ultra-highly sensitive and specific SERS assay of disease-related nucleic acids on SERS-active silver nanorods sensing chips. The Cas13a-bHCR based SERS assay of gastric cancer-related miRNA-106a (miR-106a) can be achieved within 60 min and output significantly enhanced SERS signal due to the multiple signal amplification, which possesses a good linear calibration curve from 10 aM to 1 nM with the limit of detection (LOD) low to 8.55 aM for detecting gastric cancer-related miR-106a in human serum. The Cas13a-bHCR based SERS sensing also shows good specificity, uniformity, repeatability and reliability, and has good practicability for detection of miR-106a in clinical samples, which can provide a potential powerful tool for SERS detection of disease-related nucleic acids and promise brighter prospects in the field of clinical diagnosis of early disease.

Novel Fluorescent Probe Based on Rare-Earth Doped Upconversion Nanomaterials and Its Applications in Early Cancer Detection.

In this paper, a novel rare-earth-doped upconverted nanomaterial NaYF4:Yb,Tm fluorescent probe is reported, which can detect cancer-related specific miRNAs in low abundance. The detection is based on an upconversion of nanomaterials NaYF4:Yb,Tm, with emissions at 345, 362, 450, 477, 646, and 802 nm, upon excitation at 980 nm. The optimal Yb3+:Tm3+ doping ratio is 40:1, in which the NaYF4:Yb,Tm nanomaterials have the strongest fluorescence. The NaYF4:Yb, Tm nanoparticles were coated with carboxylation or carboxylated protein, in order to improve their water solubility and biocompatibility. The two commonly expressed proteins, miRNA-155 and miRNA-150, were detected by the designed fluorescent probe. The results showed that the probes can distinguish miRNA-155 well from partial and complete base mismatch miRNA-155, and can effectively distinguish miRNA-155 and miRNA-150. The preliminary results indicate that these upconverted nanomaterials have good potential for protein detection in disease diagnosis, including early cancer detection.

The exploration of quantum dot-molecular beacon based MoS2 fluorescence probing for myeloma-related Mirnas detection.

Highly sensitive and reliable detection of multiple myeloma remains a major challenge in liquid biopsy. Herein, for the first time, quantum dot-molecular beacon (QD-MB) functionalized MoS2 (QD-MB @MoS2) fluorescent probes were designed for the dual detection of multiple myeloma (MM)-related miRNA-155 and miRNA-150. The results indicate that the two probes can effectively detect miRNA-155 and miRNA-150 simultaneously with satisfactory recovery rates, and the limit of detections (LODs) of miRNA-155 and miRNA-150 in human serum are low to 7.19 fM and 5.84 fM, respectively. These results indicate that our method is the most sensitive detection so far reported and that the designed fluorescent probes with signal amplification strategies can achieve highly sensitive detection of MM-related miRNAs for MM diagnosis.

A disposable paper-based hydrophobic substrate for highly sensitive surface-enhanced Raman scattering detection.

Detection of target analytes with high sensitivity and reproducibility remains a challenge for surface-enhanced Raman scattering (SERS) due to the lack of cost-effective and highly sensitive substrates. In this study, a hydrophobic SERS substrate capable of concentrating nanoparticles and analytes was prepared by spin-coating lubricating liquid onto commercial paper. The condensation effect of the paper-based hydrophobic substrate induced aggregation of gold nanoparticles (Au NPs) to generate ''hot spots'' for SERS and to drive analytes to the hot-spot areas for more sensitive detection. The obtained SERS signal intensity was 5-fold higher than that obtained using common paper, and a detection limit (LOD) of 4.3 × 10-10 M for rhodamine 6G (R6G) was achieved. Randomly selected points on the substrate and different batches of substrates all exhibited high reproducibility, and the relative standard deviation (RSD) at 1362 cm-1 is approximately 11%. A further application of the hydrophobic substrate was demonstrated by the detection of cytochrome C within a linear detection range of 3.90 × 10-8 M-1.25 × 10-6 M. In addition, the prepared substrate can obtain identifiable SERS spectra of cancer cells and non-cancer cells because a large number of AuNP or Au NPs clusters can adhere to cells, resulting in the construction of a 3D hotspot matrix. The disposable hydrophobic paper substrate eliminates the problem of solution diffusion, and also provides an effective platform for biomolecular screening detection.

Label-free discrimination of glioma brain tumors in different stages by surface enhanced Raman scattering.

According to the WHO classification criteria, the most common intracranial tumor gliomas can be divided into four grades based on their symptoms. Among them, Grade Ⅰ and Grade II are low-grade gliomas, and Grade III and Grade IV are high-grade gliomas. Because gliomas have a high lethal rate, they have received widespread attention in the medical field. Based on these circumstances, a rapid and facile surface enhanced Raman scattering (SERS) method using silver nano particle-decorated silver nanorod (AgNPs@AgNR) as substrates were developed for the discrimination of gliomas. Compared with SERS-active silver nanoparticles and silver nanorod substrates, the prepared AgNPs@AgNR substrates showed an outstanding SERS performance with an enhancement factor up to 1.37 × 109. Combined AgNPs@AgNR substrate with principal component analysis (PCA), we achieved rapid discrimination of healthy brain tissue and gliomas at different grades. The spectra obtained from the tissue illustrate prominently spectral differences which can be applied to identify whether it came from a healthy region or from a glioma. The most prominently difference between the SERS spectrum of healthy brain tissue and that of gliomas at different grades is the reduction in quotient of two characteristic peaks at 653 and 724 cm-1. Furthermore, healthy brain tissue and Grade II gliomas as low grade gliomas as well as Grade III and Grade IV as high-grade gliomas can be clearly distinguished by three-dimensional PCA. Preliminary results indicate that the SERS spectra based on AgNPs@AgNR substrates can be applied for a rapid identification owing to its simple preparation of specimen and high-speed spectral acquirement.

Ultrasensitive SERS detection of nucleic acids via simultaneous amplification of target-triggered enzyme-free recycling and multiple-reporter.

The development of ultrasensitive and specific methods for facile detection of trace nucleic acids is of great significance to human health and safety. In the present work, an ultrasensitive SERS-based strategy for detecting nucleic acids was proposed by integrating the SERS-active AgNRs array with double signal amplifications, i.e. the primary target-triggered enzyme-free amplification recycling and the secondary signal enhancement of multiple-reporter. By comparing two SERS sensing routes, i.e. solid interface recycling (Route A) and solution recycling (Route B), the superior solution recycling was determined first, and then the sensing strategy was optimized by investigating the immobilization time, surface blocking, and number of reporters utilized in the SERS sensing. The experimental results indicate that an ultrasensitive SERS strategy can be achieved via the primary amplification of target-triggered enzyme-free recycling and additional enhancement by the usage of multiple reporters. Under the optimal conditions, the SERS sensing showed good specificity and uniformity, and a linear calibration curve of DNAs in human serum solution, ranging from 1 µM to 1 fM, was obtained with LOD as low as 40.4 aM, and the following recovery rate measurements confirmed that the proposed SERS sensing had good repeatability and reliability, which shows great potential for facile detecting trace DNAs, especially disease-related nucleic acids in the liquid biopsy of early-stage cancer detection.