Chemiluminescent Detection Of Gram-positive Bacteria Utilizing The Molecular Recognition Of Phe¹¹-protonectin

Pathogenic bacteria are a class of microorganisms that can cause diseases via infecting organisms.They can cause serious threat to human health and global public security because they are widespread,and can grow rapidly.Food-borne infections are common in developing countries,while hospital-acquired infections and coinfections are serious problems in developed countries.As one of the common pathogens,Gram-positive bacteria are believed to be the main causes of many infectious diseases.Therefore,the development of simple,rapid,sensitive and effective methods for the detection of Gram-positive bacteria is still an important mission for the prevention and clinical diagnosis of infectious disease.In our works,utilizing the affinity between the antimicrobial peptide Phe¹¹-protonectin and the cell membrane of Gram-positive bacteria,two facile methods for the detection of Gram-positive bacteria were developed based on bioluminescence and chemiluminescence.（1）A method for the detection of viable Gram-positive bacteria utilizing Phe¹¹-protonectin-functionalized magnetic beadsA rapid and facile bioluminescence method utilizing the affinity of the Phe¹¹-protonectin to Gram-positive bacteria was developed for rapid detection of viable Gram-positive bacteria.Phe¹¹-protonectin was designed and synthesized by replacing the eleventh amino acid of protonectin（a natural cationic broad-spectrum antimicrobial peptide）with phenylalanine.Compared with broad-spectrum protonectin,Phe¹¹-protonectin showed greatly improved selectivity against Gram-positive bacteria.Therefore,Phe¹¹-protonectin-functionalized magnetic beads were chosen as the recognition reagents to efficiently enrich and separate Gram-positive bacteria from various sample matrices.It is known that the amount of adenosine triphosphate in living bacteria is approximate 10^-18 mol.However,dead bacteria contain almost no adenosine triphosphate because of the enzymatic degradation reaction.Besides,adenosine triphosphate is able to emit bioluminescence via the reaction with luciferin and luciferase.Therefore,the amount of viable Gram-positive bacteria can be determined by the quantitative detection of adenosine triphosphate released from bacteria.Staphylococcus aureus（S.aureus）,Streptococcus mutans and Bacillus cereus were detected as model bacteria to explore the feasibility of this method for the detection of Gram-positive bacteria.The results showed that the linear range for Gram-positive bacteria was 2.3×10³-1.2×10⁷ CFU mL^-1,and the limit of detection was 2.2×10² CFU mL^-1.The whole process can be completed within 33 minutes.The method was specific for viable Gram-positive bacteria detection.Gram-negative bacteria and Gram-positive dead bacteria showed minor interference.The method was successfully applied to detect Gram-positive bacteria in peach juice,glucose injection,human urine and lake water,indicating its good application prospect in food,medicine,biological samples and environmental samples analysis.（2）A dual-site recognition chemiluminescence method for the detection of S.aureus using antimicrobial peptide Phe¹¹-protonectinA chemiluminescent method for the detection of S.aureus was developed utilizing the strong binding between Phe¹¹-protonectin and cellular membrane,as well as the recognition of immunoglobulin G to protein A on the cell wall.As a designed cationic antimicrobial peptide with the ability to bind Gram-positive bacteria,Phe¹¹-protonectin can be used as a novel recognition element for the detection of Gram-positive bacteria,such as S.aureus.At the same time,immunoglobulin G was selected as the capture agent for S.aureus to improve the selectivity of the method,since protein A on the cell wall of S.aureus can specifically bind to the Fc fragment of immunoglobulin G.Alkaline phosphatase-labeled Phe¹¹-protonectin was used as a tracer.Thus,a sandwich chemiluminescent protocol for the detection of S.aureus based on two-site recognition was established.The results showed that the linear range for S.aureus detection was1.0×10³-1.0×10⁷ CFU mL^-1,and the limit of detection was 2.9×10² CFU mL^-1.The whole process can be completed within 130 minutes.Gram-negative bacteria and other Gram-positive bacteria showed minor interference.Thus,this method indicated good specificity for the detection of S.aureus.The method was applied to the detection of S.aureus in physiological saline injection,milk and human urine,with recovery values from 83.0%to 110.0%,which indicated its good application prospects in clinical diagnosis,drug and food safety monitoring.