Yong Li

For the precise molecular detection of bloodstream infection, sample processing is critical, as it involves purifying pathogenic DNA to circumvent the inhibitory effect of complex blood components. To integrate the cumbersome workflow encompassing the entire process to realize automated operation, passive methods such as gravity-driven microfluidics eliminate additional driving and controlling modules to simplify device setup and operations. Nevertheless, gravity-driven microfluidics encounters challenges in handling complex flow control and necessitates a relatively large volume of fluid to initiate the flow. In this study, to harness the simplicity of gravity for manipulating the cumbersome processing and achieving the automated detection of bacterial pathogens from whole blood, a novel rotation-programmed microfluidic platform was developed. Through simple procedural rotation of the chip, DNA extraction, LAMP reaction, and detection were integrated into the gravity-driven microfluidic platform. Complex handling procedures, such as sample processing including adsorption, washing, and elution, can be readily automated by antibiotic-modified magnetic nanoparticles on the chip. The sample-to-answer process was completed within 1.5 h, and the limit of detection for S. aureus from whole blood was 10² CFU mL^–1. Machine learning-based image processing enabled rapid and accurate result readout through colorimetric detection. Beyond the diagnosis of bloodstream infections, this platform can be potentially extended to other complex and multistep processing applications, such as protein assay, by adjusting the design and programming.

Although polymerase chain reaction (PCR)-based molecular sensing methods offer faster and more accurate identification of bloodstream infection (BSI) pathogens compared to blood culture methods, conventional PCR platforms still face significant limitations, including bulky instrumentation, high energy consumption, and relatively long assay duration. Photothermal PCR enables accelerated thermal cycling and lower energy input; however, it often suffers from nonuniform heat distribution during ultrashort heating intervals, fluorescence quenching induced by nanomaterials, nonspecific adsorption of biomolecules, and complex device fabrication. To address these challenges, this study presents a novel microfluidic PCR system based on a patterned reduced graphene oxide/polydimethylsiloxane (rGO/PDMS) composite that enables three-dimensional photothermal heating. Beyond its simplified fabrication process, the platform demonstrates uniform heat transfer and eliminates nonspecific adsorption during amplification. Gram-negative bacteria constitute a substantial proportion of pathogens responsible for BSI. To further streamline bacterial detection from whole blood, a novel Gram-negative bacterial DNA extraction method employing antibiotic-modified magnetic beads was integrated into the workflow. The entire detection process, from sample preparation to result readout, can be completed within 40 min, with 40 PCR cycles accomplished in just 5 min. The system achieves a limit of detection for bacteria as low as 10² CFU mL^-1 in whole blood.

DNA methylation serves as a crucial biomarker for early cancer detection, holding great promise in liquid biopsy. However, its low abundance in plasma and interference from complex matrices pose significant challenges to clinical detection sensitivity. To this end, this study developed a novel direct enrichment platform based on methyl-binding domain (MBD) protein-functionalized magnetic nanoparticles (MBD@MNPs). Through systematic optimization, the methylated DNA (mDNA) capture efficiency of MBD@MNPs reached 86.6% with an elution efficiency of 44.9% in simulated systems, while bypassing the cumbersome step of total cell-free DNA (cfDNA) extraction and overcoming plasma matrix interference. In comparison, this strategy enhanced the sensitivity of methylation-specific PCR by 25-fold, with a detection limit as low as 0.04 ng/mL. Applied to plasma samples from patients with various tumors (including colorectal, lung, breast, cervical, liver, and gastric cancers), the technology significantly improved the detection rates up to 100% of tumor-specific methylation biomarkers, such as SDC2, SHOX2, RASSF1A, ZNF671, Septin9, and BMP3. Therefore, this study provides an efficient, universal and user-friendly enrichment and accurate detection of mDNA, laying a methodological foundation for early cancer screening and prognosis assessment.

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia, yet its diagnosis remains challenging due to overlapping symptoms with other respiratory pathogens. In this study, an integrated microfluidic cartridge platform has been developed that combines three synergistic advancements: a chemical lysis reagent (GuHCl/EDTA) enabling direct genomic DNA release from M. pneumoniae collected by nasal swabs within 2 min without extraction steps, a novel dual-dye system (hydroxy naphthol blue/SYBR Green I) that leverages complementary fluorescence transitions from red to green for enhanced visual discrimination of loop-mediated isothermal amplification (LAMP) results, and a smartphone-based RGB quantification algorithm to standardize interpretation by analyzing colorimetric changes. The microfluidic cartridge integrates lysis, amplification, and detection into a closed workflow, achieving a sample-to-answer time of 40 min with a detection limit of 1 copy/μL. Clinical validation using 25 nasal swab samples demonstrated 95% sensitivity (19/20) and 100% specificity (5/5) compared with serological tests. This platform offers a rapid, cost-effective, and user-friendly solution for point-of-care diagnosis of M. pneumoniae, particularly in resource-limited settings. Graphical Abstract

Rapid and precise identification of the pathogens causing sepsis remains a significant diagnostic challenge. Blood culture is time-consuming and insensitive, while molecular diagnostic techniques, such as the polymerase chain reaction (PCR), are fast but greatly influenced by template quality. Here, we present a new approach to separate trace amounts of pathogen DNA from blood, which utilizes lysozyme to destroy bacteria and release DNA, followed by enrichment and purification using magnetic nanoparticles (MNPs) modified with kanamycin (Kan) or tobramycin (TM). We demonstrate that the prepared Kan@MNPs and TM@MNPs can efficiently adsorb DNA, with the mechanism involving interaction with the minor groove of DNA. Notably, the adoption of lysozyme ensures bacterial lysis while avoiding damage to blood cells, minimizing the interference from human genomic DNA background and inhibitory components, thereby obtaining relatively pure bacterial DNA. For artificially infected whole blood samples, our method shortens the sample processing time to 35 min and achieves a 10-fold improvement in PCR sensitivity compared to a commercial kit. Through clinical evaluation of blood samples collected from suspected infected patients, we identified positive samples that were 100% consistent with the clinical practice. Therefore, this method holds promising potential for clinical application in advancing rapid sepsis diagnosis and earlier interventions.

A DNA electrochemical interface biosensor based on screen-printed carbon electrodes (SPCEs) holds promise for point-of-care testing (POCT) detection of pathogens in food safety. Nevertheless, SPCE commonly has a rough surface and suffers from a relatively low electron transfer rate, disorder of DNA capture probes (CPs), and the steric hindrance effect of target nucleic acid binding. These issues lead to a low sensitivity. Herein, a simple and rapid electrochemical biosensor based on N-doped porous carbon (NPC)-modified SPCE and freezing-directed DNA combined with catalyzed hairpin assembly (CHA) was constructed for the one-pot detection of pathogens in food samples without time-consuming growth cultures. The biosensor was constructed by SPCE modified with NPC for enhanced electrochemical properties, and the DNA CP designed for CHA was stably fixed on the electrode for a high hybridization efficiency. Moreover, the signals amplified by CHA enable the selective and sensitive detection of pathogens without washing steps. This one-pot method is simple and sensitive with a wide detection linear range of 10¹ to 10⁷ CFU/mL and limit of detection of 5 CFU/mL for Escherichia coli and shows specificity against other coexisting pathogens. The whole detection of pathogens in complex samples is performed only within 60 min from sample-to-answer, which has great potential for POCT of pathogens in food safety.

Staphylococcus aureus is a common foodborne pathogen that causes food poisoning and infectious diseases in humans and animals. Rapid detection of S. aureus with high sensitivity is of great significance to prevent the spread of this pathogen. In this study, we developed a staggered strand exchange amplification (SSEA) method by refining denaturation bubble-mediated strand exchange amplification (SEA) to detect S. aureus at a constant temperature with high specificity and efficiency. This method employs a DNA polymerase and two sets of forward and reverse primers arranged in tandem that invade denaturation bubbles of double-stranded DNA. In comparison, the sensitivity of SSEA was 20 times that of SEA. Subsequently, magnetic bead (MB)-based DNA extraction was introduced into SSEA to establish an all-in-one SSEA platform that incorporated sample processing, amplification and detection in a single tube. The use of MBs further enhanced the sensitivity of SSEA by two orders of magnitude. Specificity tests showed that the all-in-one SSEA could specifically identify S. aureus and had no cross-reaction with other common foodborne pathogens. For artificially spiked meat samples, the method could detect 1.0 × 10² CFU g⁻¹S. aureus in pork and 1.0 × 10³ CFU g⁻¹ in either duck or scallop samples without a bacterial enrichment step. The entire assay can be completed sample-to-answer within 1 h. Thus, we believe that this easy-to-operate diagnostic platform enables sensitive and accurate detection of S. aureus and holds great promise for the food safety industry.