Peptidoglycan-Binding Protein Metamaterials Mediated Enhanced and Selective Capturing of Gram-Positive Bacteria and Their Specific, Ultra-Sensitive, and Reproducible Detection via Surface-Enhanced Raman Scattering

Abstract

Biological metamaterials with a specific size and spacing are necessary for developing highly sensitive and selective sensing systems to detect hazardous bacteria in complex solutions. Herein, the construction of peptidoglycan-binding protein (PGBP)-based metamaterials to selectively capture Gram-positive cells with high efficacy is reported. Nanoimprint lithography was used to generate a nanohole pattern as a template, the inside of which was modified with nickel(II)-nitrilotriacetic acid (Ni-NTA). Then, PGBP metamaterials were fabricated by immobilizing PGBP via chelation between Ni-NTA and six histidines on PGBP. Compared to the flat and spread PGBP-covered bare substrates, the PGBP-based metamaterials enabled selective capturing of Grampositive bacteria with high efficacy, owing to enhanced interactions between the metamaterials and bacterial surface not shown in bulk materials. Thereafter, the specific strain and quantitative information of the captured bacteria was obtained by surface-enhanced Raman scattering mapping analysis in the 1 to 1 × 10 6 cfu/mL range within 30 min. It should be noted that no additional signal amplification process was required for lowly abundant bacteria, even at the single-bacterium level. The PGBP-based metamaterials could be regenerated multiple times with preserved sensing efficiency. Finally, this assay can detect specific Gram-positive bacteria, such as Staphylococcus aureus, in human plasma.

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“…These small nanogaps contributed to the boosting of the signal in the Raman spectra after the rhodamine 6G (R6G) target molecules were loaded (Figure b). X-ray diffraction (XRD) studies were also performed to confirm the highly crystalline structure of the fabricated SERS substrates (Figure c). − Furthermore, X-ray photoelectron spectra (XPS) demonstrated successful silver deposition after the electrodeposition process. Figure d,e represents the Au 4f and Ag 3d orbitals at a high resolution.…”

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“…Bacteria are everywhere in nature, and many bacterial strains are pathogenic. [ 172 ] For example, Escherichia coli can cause diarrhoea, infections and its presence is also an indicator for environmental monitoring. [ 32 ] The conventional microbial detection methods like polymerase chain reaction (PCR), flow cytometry, fluorescence and electrochemical methods are complex, time‐consuming and expensive with poor portability.…”

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“…78 The quantitative analysis of the captured bacteria was accomplished by SERS within 30 min and detected Staphylococcus aureus associated with sepsis at a low concentration of 10 colony forming units (cfu)/mL in human plasma. 78 In another study, De Plano et al chose phage clones that explicitly bind the surface of Staphyloccocus aureus, Pseudomonas aeruginosa, and Escherichia coli from M13 phage display libraries and functionalized them with commercial magnetic beads. 79 These were used to capture and concentrate the bacteria involved in sepsis from the blood, which was detected using spontaneous Raman microspectroscopy with a detection limit of 10 cfu in 7 mL of blood.…”

“…Lee et al constructed peptidoglycan-binding protein (PBPMs) and used it to capture Gram-positive bacteria with high efficiency . The quantitative analysis of the captured bacteria was accomplished by SERS within 30 min and detected Staphylococcus aureus associated with sepsis at a low concentration of 10 colony forming units (cfu)/mL in human plasma .…”

“…Lee et al constructed peptidoglycan-binding protein (PBPMs) and used it to capture Gram-positive bacteria with high efficiency . The quantitative analysis of the captured bacteria was accomplished by SERS within 30 min and detected Staphylococcus aureus associated with sepsis at a low concentration of 10 colony forming units (cfu)/mL in human plasma . In another study, De Plano et al chose phage clones that explicitly bind the surface of Staphyloccocus aureus , Pseudomonas aeruginosa , and Escherichia coli from M13 phage display libraries and functionalized them with commercial magnetic beads .…”