SERS-based immunocapture and detection of pathogenic bacteria using a boronic acid-functionalized polydopamine-coated Au@Ag nanoprobe.

A surface-enhanced Raman scattering (SERS)-based immunocapture nanoprobe is described for the detection of pathogenic bacteria. The probe uses boronic acid-functionalized polydopamine-coated Au@Ag nanoparticles as an advanced SERS nanotag. Modified magnetic IgG@Fe3O4 nanoparticles are used for magnetic separation. Au@Ag@PDA nanoparticles, where PDA stands for polydopamine, were functionalized with boronic acid to bind to pathogenic bacteria and induce signal amplification. The Raman signal is amplified 108 times when the SERS tag binds the surface of bacteria. The SERS spectra exhibit fingerprint-like patterns that enable bacterial classification. The results of principal component analysis (PCA) and hierarchical cluster analysis (HCA) of the spectral regions were compared. The bacterial surface protein and glycan signals (1300-1450 cm-1) were the best regions for bacterial classification. Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Pseudomonas aeruginosa, and Klebsiella pneumonia were successfully classified by this method. The lowest detection limit was 10 colonies/mL (CFU·mL). The assay can be completed within 30 min. Conceivably, this method may be extended to the quantitative detection or classification of bacteria under various other conditions. Graphical abstract Schematic representation of immunocapture and detection of pathogenic bacteria using boronic acid-functionalized polydopamine-coated Au@Ag nanoprobe through the bacterial surface protein and glycan signals. Green arrow: laser; black arrow: SERS; red ball: bacteria; grey ball: IgG@Fe3O4; golden ball: boronic acid-functionalized Au@Ag@PDA.

Solid-film sampling method for the determination of protein secondary structure by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy is one of the widely used vibrational spectroscopic methods in protein structural analysis. The protein solution sample loaded in demountable CaF2 liquid cell presents a challenge and is limited to high concentrations. Some researchers attempted the simpler solid-film sampling method for the collection of protein FTIR spectra. In this study, the solid-film sampling FTIR method was studied in detail. The secondary structure components of some globular proteins were determined by this sampling method, and the results were consistent with those data determined by the traditional solution sampling FTIR method and X-ray crystallography, indicating that this sampling method is feasible and efficient for the structural characterization of proteins. Furthermore, much lower protein concentrations (~0.5 mg/mL) were needed to obtain high-quality FTIR spectra, which expands the application of FTIR spectroscopy to almost the same concentration range used for circular dichroism and fluorescence spectroscopy, making comparisons among three commonly used techniques possible in protein studies. Graphical Abstract ᅟ.

CaCO3-Encapsulated Au Nanoparticles Modulate Macrophages toward M1-like Phenotype.

Macrophage cells are plastic and can be polarized into opposing phenotypes, pro-inflammatory (M1-like cells) or anti-inflammatory (M2-like cells). Reprograming of M2-like cells into M1 phenotype will contribute significantly to combatting cancer. Gold nanoparticles (AuNPs) are intensively studied in various fields for their distinctive photo-chemical properties. However, the immune response of AuNPs is still unclear. In this study, AuNPs and CaCO3-encapsulated Au nanoparticles (Au@CaCO3 NPs) were synthesized as stimuli for macrophage modulation. Co-incubation of AuNPs and macrophages leads to a dramatically elongated macrophage cell morphology. Moreover, increased expression of M2 biomarker and M2-inducing cytokines suggests that AuNPs induce macrophage polarization toward M2 phenotype. More interestingly, the co-incubation of Au@CaCO3 NPs and macrophage cells resulted in a round cellular morphology and induced the secretion of M1 biomarker and inflammatory cytokines. Our studies demonstrate that the strategy of CaCO3-encapsulated Au nanoparticles can be used in modulating the polarization of M1 macrophages. Our strategy provides an efficient method for activating inflammation in macrophages, which will be useful for the application of nanoparticles in cancer therapy.

Fluorescent polymer dots and graphene oxide based nanocomplexes for "off-on" detection of metalloproteinase-9.

Numerous studies have demonstrated that cancer-related matrix metalloproteinase-9 (MMP-9) is an ideal biomarker for cancer diagnosis. However, most MMP-9 detection methods are expensive and time-consuming, and more convenient and specific MMP-9 detection methods are needed both clinically and in research. In the present study, peptide-linked polymer dots were assembled onto a graphene oxide surface to construct a graphene oxide-peptide-polymer dot (GO-Pep-Pdot) nanocomplex for sensitive, rapid, and accurate detection of MMP-9. In the absence of MMP-9, the nanocomplex was in an "off" state, whereas in the presence of MMP-9, the nanocomplex was turned "on", resulting in the emission of a fluorescence signal that is linearly correlated with the MMP-9 concentration. The limit of detection of the nanocomplex was 3.75 ng mL-1, lower than most methods. This method was successfully verified by detecting MMP-9 in clinical serum samples of prostate cancer. The results suggest that this protease nanocomplex is generic and can be adopted to respond to other proteases by selecting specific peptides with suitable cleavage sites in clinics.

Novel superparamagnetic core-shell molecular imprinting microspheres towards high selective sensing.

Novel superparamagnetic core-shell imprinting microspheres (MCSIMs) were synthesized using magnetite microspheres with 350 nm diameter and 70 nm thickness silica gel to form core-shell Fe(3)O(4)/SiO(2) composite for template phenylephrine (Phen) recognition and high efficiency separation. Compared to the previous imprinting recognition, the main advantage of this strategy lies in two aspects: one is the high stability and monodispersity of the MCSIMs structure, the other is the use of superparamagnetic Fe(3)O(4)/SiO(2) microspheres as an immobilization matrix and separation tool, thus greatly simplifying time-consuming washing steps. The affinity and selectivity of the MCSIMs were monitored by QCM and electrochemistry measurements. Imprinting microspheres have a remarkable affinity to Phen over that of structurally related molecules, including DA, EP, Phe and Tyr. The relative binding selectivity for different analytes estimated from amperometric signals was Phen : DA : EP = 40 : 5 : 1. The MCSIMs sensor showed a high sensitivity (400 microA mM(-1)), short response time (reaching 98% within 10 s), and broad linear response range from 1 microM to 0.1 mM and low detection limit (0.1 microM). Additionally, the results of control experiments showed that only negligible signal was obtained for non-imprinting microspheres. This could be reasonably attributed to the unique surface pores, charges and especially the nature of the functional groups inside MCSIMs cavities.

Site-specific protein immobilization in a microfluidic chip channel via an IEF-gelation process.

A novel strategy for site-specific protein immobilization via combining chip IEF with low-temperature sol-gel technology, called IEF-GEL here, in the channel of a modified poly(methyl methacrylate) (PMMA) microfluidic chip is proposed in this work. The IEF-GEL process involves firstly IEF for homogeneously dissolved protein in PBS containing alumina sol and carrier ampholyte with prearranged pH gradient, and then gelation locally for protein encapsulation. The process and feasibility of proposed IEF-GEL were investigated by EOF measurements, fluorescence microscopic photography, Raman spectrum and further demonstrated by glucose oxidase (GOx) reactors integrated with end-column electrochemical detection. Site-controllable immobilization of protein was realized in a 30 mm long microfluidic chip channel by the strategy to create a approximately 1.7 mm concentrated FITC-BSA band, which leads to great improvement of the elute peak shape, accomplished with remarkably increased sensitivity, approximately 20 times higher than that without IEF-GEL treatment to GOx reactors. The kinetic response of GOx after IEF-GEL treatment was also investigated. The proposed system holds the advantages of IEF and low-temperature sol-gel technologies, i.e. concentrating the protein to be focused and retaining the biological activity for the gel-embedded protein, thus realizes site-specific immobilization of low-concentration protein at nL volume level.