Analytical Chemistry最新文献

Specific and sensitive detection of microRNA (miRNAs) is significant for the early diagnosis of cancer. Herein, we developed a cascade amplification strategy that couples DNAzyme with rolling circle amplification (RCA). The amplified DNA products serve as templates for synthesizing fluorescent copper nanoclusters (CuNCs), thereby enabling the label-free and sensitive detection of miRNA-21. First, miRNA-21 is amplified into abundant single-stranded DNA (ssDNA) fragments through a DNAzyme-assisted primary amplification module. Subsequently, the output DNA fragments trigger RCA, generating repetitive hairpin DNA (HP-DNA) structures with polythymine (T) sequences in the loop region. Finally, the amplified products act as templates for synthesizing fluorescent CuNCs, owing to the crowded microenvironment provided by the HP-DNA. The synthesized CuNCs exhibited significantly enhanced photoluminescence (PL) intensity compared to those templated by conventional poly-T single-stranded DNA. Concurrently, the cascade DNAzyme-RCA amplification is significantly more sensitive than traditional RCA, achieving a 4.1 pM detection limit. In addition, the biosensor was used to analyze the expression level of miRNA-21 in human serum and various cell lysates. The proposed detection method features simple design, low cost, and high sensitivity and eliminates the need for fluorophore labeling, holding great potential for clinical diagnostics.

Mass spectrometry imaging (MSI) can directly detect surface analytes, enabling spatial mapping of metabolite synthesis and migration within tissues. However, MSI often lacks the sensitivity for routine analysis of low exposure, low molecular weight endogenous metabolites. Amine-containing metabolites, such as methylamine, dimethylamine, trimethylamine, lysine, dopamine, and 5-hydroxytryptamine, are widely used as medical biomarkers due to their potential biological activity or toxicity. Herein, we innovatively developed a universal in situ derivatization workflow combined with desorption electrospray ionization MS/MS (DESI-MS/MS) for mapping endogenous amine-containing metabolites, with trimethylamine (TMA) selected as a case study. For the first time, tert-butyl bromoacetate (TBBA) was used as an in situ derivatization reagent, which improved sensitivity and reproducibility by overcoming the strong matrix effect and signal instability of low molecular weight compounds in MS analysis. Our data suggested that the derivatization efficiency was affected by the alkalinity of the tissue surface, while spray/extraction solvent significantly affected the sensitivity for TMA-TBBA. Notably, DESI-MS/MS exhibited good linearity, reproducibility, and stability in the analysis of amine-containing metabolites derived from TBBA. The optimized workflow was utilized for the visual analysis of endogenous TMA distribution in the brain and serum of mice, revealing the enrichment characteristics of TMA in the cortex and hippocampus, as well as the severe accumulation of TMA in aged animals. Therefore, this targeted DESI-MS/MS-MSI approach, combined with in situ derivatization, offers novel avenues for the routine quantitation of intratissue amine-containing metabolites. In principle, this assay can be extended to a wide variety of metabolites in different biological samples.

The rapid diagnosis of bloodstream infections is essential for guiding targeted treatment, reducing antibiotic resistance, and improving patient outcomes. We report an integrated centrifugal microfluidic point-of-care low-cost chip for simultaneous detection of six types of pathogen nucleic acid in 50 min. All of the reagents were prestored in dry powder form inside the microfluidic chip for real-time fluorescence loop-mediated isothermal amplification. Through a simple lysis and dilution process, clinically positive blood culture samples from patients can be loaded into the microfluidic chip for pathogen nucleic acid identification. The developed method demonstrated high consistency with the standard method used by the hospital in the testing of 120 clinical patient samples. It is envisioned that this work may provide a useful platform for rapid diagnosis of bloodstream infections by identification of pathogen nucleic acid.

DNA damage refers to chemical alterations in genomic DNA arising from endogenous or exogenous factors, and delineating the types and levels of these lesions is essential for elucidating the molecular mechanisms and regulatory networks underlying DNA repair. However, current methods are limited to one specific type of damage and often rely on DNA extraction and in vitro processing, which fails to capture the spatial distribution of diverse DNA damages within cellular context. Here we present intracellular strand break end extension-encoded amplification (ISBEA), an in situ imaging strategy that enables simultaneous visualization of multiple DNA lesion types. ISBEA uses a sequential enzymatic workflow in which glycosylases, endonucleases and exonucleases selectively recognize oxidized purines, AP sites and strand breaks, converting them stepwise into uniform 3'-hydroxyl termini. These activated termini undergo programmable extension and serve as primers to initiate DNA-encoded amplification, ultimately generating fluorescence signals present to each damage type. With spatially resolved visualization of diverse DNA damages within cells, ISBEA provides a robust platform for investigating the origins, repair pathways and biological consequences of DNA damage, offering valuable methodological support for advancing the understanding of genome stability maintenance and the development of related diseases.