Efficient pathogen enrichment and nucleic acid isolation are critical for accurate and sensitive diagnosis of infectious diseases, especially those with low pathogen levels. Our study introduces a ...biporous silica nanofilms-embedded sample preparation chip for pathogen and nucleic acid enrichment/isolation. This chip features unique biporous nanostructures comprising large and small pore layers. Computational simulations confirm that these nanostructures enhance the surface area and promote the formation of nanovortex, resulting in improved capture efficiency. Notably, the chip demonstrates a 100-fold lower limit of detection compared to conventional methods used for nucleic acid detection. Clinical validations using patient samples corroborate the superior sensitivity of the chip when combined with the luminescence resonance energy transfer assay. The enhanced sample preparation efficiency of the chip, along with the facile and straightforward synthesis of the biporous nanostructures, offers a promising solution for polymer chain reaction-free detection of nucleic acids.
Cell-free nucleic acids (cfNAs) in liquid biopsy samples are emerging as important biomarkers for cancer diagnosis and monitoring, and for predicting treatment outcomes. Many cfNA isolation methods ...have been developed recently. However, most of these techniques are time-consuming, complex, require large equipment, and yield low-purity cfNAs because the genetic background of normal cells is amplified during cell lysis, which limits their clinical application. Here, we report a rapid and simple cfNA sampling platform that can overcome the limitations of conventional methods. We synthesised a biocomposite by combining amine-modified diatomaceous earth (DE) and cucurbituril (CB). The biocomposite platform showed high capture efficiency (86.78-90.26%) with genomic DNA and amplified DNA products (777, 525 and 150 bp). The biocomposite platform allowed the isolation of high purity and quantity cfDNAs from the plasma of 13 cancer patients (three colorectal cancer and ten pancreatic cancer samples) without requiring a lysis step or special equipment. The biocomposite platform may be useful to isolate cfNAs for the diagnosis and treatment of cancers in clinical applications.
Herein, we describe the synthesis of highly water-dispersible and biocompatible 3D adsorbents via a rapid two-step strategy employing a mesoporous magnetic nanomulberry-shaped Fe
O
(MNM) on ...diatomaceous earth (DE) and cucurbituril (CB; MNM-DE-CB). Coating of CB on the surface of MNM-DE via hydrogen bonds not only enhanced the dispersibility of CB, but also improved the stability of MNM-DE. The ability of the adsorbent to remove dyes from water was investigated as a function of metal ions, solution pH, temperature, and concentration to determine optimum reaction conditions. Unlike MNM-DE, MNM-DE-CB exhibited highly efficient, rapid dye removal and recyclability in aqueous solution, and low cytotoxicity toward cancer cells in drug delivery tests. MNM-DE-CB is a promising green adsorbent with potential for diverse applications including water remediation, interface catalysis, bio-sample preparation, and drug delivery.
Cell‐free nucleic acids (cfNAs) are emerging diagnostic biomarkers for monitoring the treatment and recurrence of cancers. In particular, the biological role and clinical usefulness of cfNAs obtained ...from the plasma of patients with various cancers are popular and still intensely explored, yet most studies are limited by technical problems during cfNA isolation. A dimethyl dithiobispropionimidate (DTBP)‐based microchannel platform that enables spontaneous cfNA capture in 15 min with minimal cellular background and no requirements for use of bulky instruments is reported first. This platform identified KRAS and BRAF hot‐spot mutations following cfDNA isolation from the blood plasma and tissues obtained from 30 colorectal cancer patients. The correlation of mutations between the primary tissues and plasma from the patients was high using this platform with whole genome sequencing compared to the spin‐column method. This platform can also be combined with various detection approaches (biooptical sensor, Sanger sequencing, and polymerase chain reaction (PCR)) for rapid, simple, low‐cost, and sensitive circulating tumor DNA detection in blood plasma. The efficiency and versatility of this platform in isolating cfNAs from liquid biopsies has applications in cancer treatment and precision medicine.
Dimethyl dithiobispropionimidate platform enables spontaneous cell‐free nucleic acid (cfNA) capture in 15 min with less cellular background and no requirement for bulky instruments. This platform can be combined with various detection approaches for rapid, simple, low‐cost, and sensitive circulating tumor DNA detection in blood plasma. The efficiency and versatility of this platform in isolating cfNAs from liquid biopsies can find various cancer applications.
Infectious diseases, especially pathogenic infections, are a growing threat to public health worldwide. Since pathogenic bacteria usually exist in complex matrices at very low concentrations, the ...development of technology for rapid, convenient, and biocompatible sample enrichment is essential for sensitive diagnostics. In this study, a cucurbit6uril (CB) supermolecular decorated amine-functionalized diatom (DA) composite was fabricated to support efficient sample enrichment and in situ nucleic acid preparation from enriched pathogens and cells. CB was introduced to enhance the rate and effectiveness of pathogen absorption using the CB-DA composite. This novel CB-DA composite achieved a capture efficiency of approximately 90% at an
concentration of 10⁶ CFU/mL within 3 min. Real-time PCR analyses of DNA samples recovered using the CB-DA enrichment system showed a four-fold increase in the early amplification signal strength, and this effective method for capturing nucleic acid might be useful for preparing samples for diagnostic systems.
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.
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.
Using comparative statistical analysis, we found 21 candidate protein biomarkers that were significantly different among three groups of neurodegenerative diseases.
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.
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.
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.
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