Daptomycin-modified magnetic beads integrated with lysostaphin for selective analysis of Staphylococcus.

An antibiotic-affinity method was developed for analyzing Staphylococcus on the basis of the strong binding capability of daptomycin towards Gram-positive bacteria cellular membrane, as well as the selective lytic action of lysostaphin towards Staphylococcus. Daptomycin-modified magnetic beads were adopted to enrich Staphylococcus from sample matrix. Afterwards lysostaphin was adopted to lyse Staphylococcus, which can hydrolyze pentaglycine cross-linkers of peptidoglycan composing the cellular wall of Staphylococcus. The concentration of Staphylococcus was quantified by collecting the bioluminescent signal of the released intracellular adenosine triphosphate of the enriched Staphylococcus. Staphylococcus aureus (S. aureus) was analyzed as a model bacterium to study the feasibility of the proof-of-principle work. For bioluminescent analysis of S. aureus with the developed method, the linear range was 5.0 × 102-5.0 × 106 colony forming units mL-1, and the limit of detection was 3.8 × 102 colony forming units mL-1. The analytical procedure consisting of bacterial enrichment, cell lysis and signal collection can be accomplished within 20 min. Some common Gram-positive bacteria and Gram-negative bacteria all indicated very low interference to the analysis of the target bacterium. It has been successfully used to analyze S. aureus in milk as well as physiological saline injection, indicating its application potential for real samples.

Label-free electrochemiluminescent biosensor for rapid and sensitive detection of pseudomonas aeruginosa using phage as highly specific recognition agent.

A virulent phage named as PaP1 was isolated from hospital sewage based on a lambda phage isolation protocol. This phage showed a strong and highly specific binding ability to Pseudomonas aeruginosa (P. aeruginosa). Using this isolated phage as a recognition agent, a novel electrochemiluminescent (ECL) biosensor was developed for label-free detection of P. aeruginosa. The biosensor was fabricated through depositing phage-conjugated carboxyl graphene onto the surface of a glass carbon electrode. After specific binding of the host bacteria through the adsorption of P. aeruginosa cell wall by phage tail fibers and baseplate, the ECL signal of luminol suffered a decrease since the formed non-conductive biocomplex obstructed the interfacial electron transfer and blocked the diffusion of the ECL active molecules. The ECL emission declined linearly with P. aeruginosa concentration in the range of 1.4×102 -1.4×106CFUmL-1, with a very low detection limit of 56CFUmL-1. The whole detection process could be completed within 30min as a ready-for-use biosensor was adopted. This biosensor was successfully applied to quantitate P. aeruginosa in milk, glucose injection and human urine with acceptable recovery values ranging from 78.6% to 114.3%.

Highly Specific Bacteriophage-Affinity Strategy for Rapid Separation and Sensitive Detection of Viable Pseudomonas aeruginosa.

A virulent bacteriophage highly specific to Pseudomonas aeruginosa (P. aeruginosa) was isolated from hospital sewage using a lambda bacteriophage isolation protocol. The bacteriophage, named as PAP1, was used to functionalize tosyl-activated magnetic beads to establish a bacteriophage-affinity strategy for separation and detection of viable P. aeruginosa. Recognition of the target bacteria by tail fibers and baseplate of the bacteriophage led to capture of P. aeruginosa onto the magnetic beads. After a replication cycle of about 100 min, the progenies lysed the target bacteria and released the intracellular adenosine triphosphate. Subsequently, firefly luciferase-adenosine triphosphate bioluminescence system was used to quantitate the amount of P. aeruginosa. This bacteriophage-affinity strategy for viable P. aeruginosa detection showed a linear range of 6.0 × 102 to 3.0 × 105 CFU mL-1, with a detection limit of 2.0 × 102 CFU mL-1. The whole process for separation and detection could be completed after bacteria capture, bacteriophage replication, and bacteria lysis within 2 h. Since the isolated bacteriophage recognized the target bacteria with very high specificity, the proposed strategy did not show any signal response to all of the tested interfering bacteria. Furthermore, it excluded the interference from inactivated P. aeruginosa because the bacteriophage could replicate only in viable cells. The proposed strategy had been applied for detection of P. aeruginosa in glucose injection, human urine, and rat plasma. In the further work, this facile bacteriophage-affinity strategy could be extended for detection of other pathogens by utilizing virulent bacteriophage specific to other targets.