Magnetic transferrin nanoparticles (MTNs) assay as a novel isolation approach for exosomal biomarkers in neurological diseases.

BACKGROUND: Brain-derived exosomes released into the blood are considered a liquid biopsy to investigate the pathophysiological state, reflecting the aberrant heterogeneous pathways of pathological progression of the brain in neurological diseases. Brain-derived blood exosomes provide promising prospects for the diagnosis of neurological diseases, with exciting possibilities for the early and sensitive diagnosis of such diseases. However, the capability of traditional exosome isolation assays to specifically isolate blood exosomes and to characterize the brain-derived blood exosomal proteins by high-throughput proteomics for clinical specimens from patients with neurological diseases cannot be assured. We report a magnetic transferrin nanoparticles (MTNs) assay, which combined transferrin and magnetic nanoparticles to isolate brain-derived blood exosomes from clinical samples. METHODS: The principle of the MTNs assay is a ligand-receptor interaction through transferrin on MTNs and transferrin receptor on exosomes, and electrostatic interaction via positively charged MTNs and negatively charged exosomes to isolate brain-derived blood exosomes. In addition, the MTNs assay is simple and rapid (< 35 min) and does not require any large instrument. We confirmed that the MTNs assay accurately and efficiently isolated exosomes from serum samples of humans with neurodegenerative diseases, such as dementia, Parkinson's disease (PD), and multiple sclerosis (MS). Moreover, we isolated exosomes from serum samples of 30 patients with three distinct neurodegenerative diseases and performed unbiased proteomic analysis to explore the pilot value of brain-derived blood protein profiles as biomarkers. RESULTS: Using comparative statistical analysis, we found 21 candidate protein biomarkers that were significantly different among three groups of neurodegenerative diseases. CONCLUSION: The MTNs assay is a convenient approach for the specific and affordable isolation of extracellular vesicles from body fluids for minimally-invasive diagnosis of neurological diseases.

Hot-Spot-Specific Probe (HSSP) for Rapid and Accurate Detection of KRAS Mutations in Colorectal Cancer.

Detection of oncogene mutations has significance for early diagnosis, customized treatment, treatment progression, and drug resistance monitoring. Here, we introduce a rapid, sensitive, and specific mutation detection assay based on the hot-spot-specific probe (HSSP), with improved clinical utility compared to conventional technologies. We designed HSSP to recognize KRAS mutations in the DNA of colorectal cancer tissues (HSSP-G12D (GGT→GAT) and HSSP-G13D (GGC→GAC)) by integration with real-time PCR. During the PCR analysis, HSSP attaches to the target mutation sequence for interference with the amplification. Then, we determine the mutation detection efficiency by calculating the difference in the cycle threshold (Ct) values between HSSP-G12D and HSSP-G13D. The limit of detection to detect KRAS mutations (G12D and G13D) was 5-10% of the mutant allele in wild-type populations. This is superior to the conventional methods (≥30% mutant allele). In addition, this technology takes a short time (less than 1.5 h), and the cost of one sample is as low as USD 2. We verified clinical utility using 69 tissue samples from colorectal cancer patients. The clinical sensitivity and specificity of the HSSP assay were higher (84% for G12D and 92% for G13D) compared to the direct sequencing assay (80%). Therefore, HSSP, in combination with real-time PCR, provides a rapid, highly sensitive, specific, and low-cost assay for detecting cancer-related mutations. Compared to the gold standard methods such as NGS, this technique shows the possibility of the field application of rapid mutation detection and may be useful in a variety of applications, such as customized treatment and cancer monitoring.

Chimeric nanocomposites for the rapid and simple isolation of urinary extracellular vesicles.

Cancer cell-derived extracellular vesicles (EVs) are promising biomarkers for cancer diagnosis and prognosis. However, the lack of rapid and sensitive isolation techniques to obtain EVs from clinical samples at a sufficiently high yield limits their practicability. Chimeric nanocomposites of lactoferrin conjugated 2,2-bis(methylol)propionic acid dendrimer-modified magnetic nanoparticles (LF-bis-MPA-MNPs) are fabricated and used for simple and sensitive EV isolation from various biological samples via a combination of electrostatic interaction, physically absorption, and biorecognition between the surfaces of the EVs and the LF-bis-MPA-MNPs. The speed, efficiency, recovery rate, and purity of EV isolation by the LF-bis-MPA-MNPs are superior to those obtained by using established methods. The relative expressions of exosomal microRNAs (miRNAs) from isolated EVs in cancerous cell-derived exosomes are verified as significantly higher than those from noncancerous ones. Finally, the chimeric nanocomposites are used to assess urinary exosomal miRNAs from urine specimens from 20 prostate cancer (PCa), 10 benign prostatic hyperplasia (BPH), patients and 10 healthy controls. Significant up-regulation of miR-21 and miR-346 and down-regulation of miR-23a and miR-122-5p occurs in both groups compared to healthy controls. LF-bis-MPA-MNPs provide a rapid, simple, and high yield method for human excreta analysis in clinical applications.

A microfluidic enrichment platform with a recombinase polymerase amplification sensor for pathogen diagnosis.

Rapid and sensitive detection of low amounts of pathogen in large samples is needed for early diagnosis and treatment of patients and surveillance of pathogen. In this study, we report a microfluidic platform for detection of low pathogen levels in a large sample volume that couples an Magainin 1 based microfluidic platform for pathogen enrichment and a recombinase polymerase amplification (RPA) sensor for simultaneous pathogenic DNA amplification and detection in a label-free and real-time manner. Magainin 1 is used as a pathogen enrichment agent with a herringbone microfluidic chip. Using this enrichment platform, the detection limit was found to be 20 times more sensitive in 10 ml urine with Salmonella and 10 times more sensitive in 10 ml urine with Brucella than that of real-time PCR without the enrichment process. Furthermore, the combination system of the enrichment platform and an RPA sensor that based on an isothermal DNA amplification method with rapidity and sensitivity for detection can detect a pathogen at down to 50 CFU in 10 ml urine for Salmonella and 102 CFU in 10 ml urine for Brucella within 60 min. This system will be useful as it has the potential for better diagnosis of pathogens by increasing the capture efficiency of the pathogen in large samples, subsequently enhancing the detection limit of pathogenic DNA.