No abstract

Thrombin is an important biomarker, and its detection is of great significance for diagnosis and prevention of related diseases. Conventional methods such as enzyme-linked immunosorbent assay are sensitive but require multiple steps. In this work, a label-free biosensor that only required one step of incubation was developed based on target-triggered release of the cargo molecules from gold nanocages, which achieved highly sensitive and specific detection of thrombin. The proposed biosensor consists of an array of gold nanocages loaded with molecules in their interiors and the DNA probes immobilized on their surface for hybridization with thrombin-specific aptamers to seal their pores. Upon interaction with thrombin, the surface aptamers were lifted off the gold nanocages, resulting in release of the cargo molecules. The loss of cargo molecules was detected by quartz crystal microbalance (QCM). The signal could be amplified by the choice of cargo molecules. The use of polyamidoamine as cargo molecules allowed us to achieve a label-free biosensor with a linear detection range of 0.0086−86 nM and a limit of detection of 7.7 pM. The biosensor was also tested with spiked human serum samples with a limit of detection of 1.2 nM. The detection could be carried out within 1.5 h with only one incubation step of 45 min. The specificity of the biosensor was confirmed by testing against bovine serum albumin and lysozyme at 1 μM.

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Background: Foodborne pathogenic bacteria threaten worldwide public health, and simple bacterial detection methods are in urgent need. Here, we established a lab-on-a-tube biosensor for simple, rapid, sensitive, and specific detection of foodborne bacteria. Methods: A rotatable Halbach cylinder magnet and an iron wire netting with magnetic silica beads (MSBs) were used for simple and effective extraction and purification of DNA from the target bacteria, and recombinase-aided amplification (RAA) was combined with clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins12a(CRISPR-Cas12a) to amplify DNA and generate fluorescent signal. First, 15 mL of the bacterial sample was centrifuged, and the bacterial pellet was lysed by protease to release target DNA. Then, DNA-MSB complexes were formed as the tube was intermittently rotated and distributed uniformly onto the iron wire netting inside the Halbach cylinder magnet. Finally, the purified DNA was amplified using RAA and quantitatively detected by the CRISPR-Cas12a assay. Results: This biosensor could quantitatively detect Salmonella in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 102 CFU/mL Salmonella Typhimurium was over 2000 RFU, while 104 CFU/mL Listeria monocytogenes, Bacillus cereus, and E. coli O157:H7 were selected as non-target bacteria and had signals less than 500 RFU (same as the negative control). Conclusions: This lab-on-a-tube biosensor integrates cell lysis, DNA extraction, and RAA amplification in one 15 mL tube to simplify the operation and avoid contamination, making it suitable for low-concentration Salmonella detection.