OBJECTIVE: Staphylococcus aureus is the most common and impactful multi-drug resistant pathogen implicated in (periprosthetic) joint infections (PJI) and fracture-related infections (FRI). Therefore, the present proof-of-principle study was aimed at the rapid detection of S. aureus in synovial fluids and biofilms on extracted osteosynthesis materials through bacteria-targeted fluorescence imaging with the \'smart-activatable\' DNA-based AttoPolyT probe. This fluorogenic oligonucleotide probe yields large fluorescence increases upon cleavage by micrococcal nuclease, an enzyme secreted by S. aureus.
METHODS: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria.
RESULTS: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min.
CONCLUSIONS: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials.

Deep tissue noninvasive high-resolution imaging with light is challenging due to the high degree of light absorption and scattering in biological tissue. Photoacoustic imaging (PAI) can overcome some of the challenges of pure optical or ultrasound imaging to provide high-resolution deep tissue imaging. However, label-free PAI signals from light absorbing chromophores within the tissue are nonspecific. The use of exogeneous contrast agents (probes) not only enhances the imaging contrast (and imaging depth) but also increases the specificity of PAI by binding only to targeted molecules and often providing signals distinct from the background.
We aim to review the current development and future progression of photoacoustic molecular probes/contrast agents.
First, PAI and the need for using contrast agents are briefly introduced. Then, the recent development of contrast agents in terms of materials used to construct them is discussed. Then, various probes are discussed based on targeting mechanisms, in vivo molecular imaging applications, multimodal uses, and use in theranostic applications.
Material combinations are being used to develop highly specific contrast agents. In addition to passive accumulation, probes utilizing activation mechanisms show promise for greater controllability. Several probes also enable concurrent multimodal use with fluorescence, ultrasound, Raman, magnetic resonance imaging, and computed tomography. Finally, targeted probes are also shown to aid localized and molecularly specific photo-induced therapy.
The development of contrast agents provides a promising prospect for increased contrast, higher imaging depth, and molecularly specific information. Of note are agents that allow for controlled activation, explore other optical windows, and enable multimodal use to overcome some of the shortcomings of label-free PAI.

Precise and rapid determination of free bilirubin (BR), a key biomarker of pathological conditions of the liver, is important clinical issue. The present study demonstrates that the combination of the strong specific affinic properties of protein, bovine serum albumin (BSA), toward bilirubin and luminescence of well-characterized semiconductor quantum dots (QDs) can offer a simple, fast, and sensitive technique for the determination of free bilirubin through quenching analysis. Here, BSA molecule not only stabilizes the quantum dots in an aqueous environment but also helps bring BR closer to QDs during the interactions of CdSe-BSA QDs with BR. Further, it is revealed through photophysical investigation that the conformation of protein molecule may play an important role in biomolecular sensing considering bilirubin as a model target molecule. The luminescence of CdSe-BSA QDs was highly responsive toward bilirubin, where nearly 90% of emission intensity was quenched on adding only 40 μM bilirubin, suggesting strong interactions involved between synthesized QDs and bilirubin. Solvent polarity dependence on luminescence changes confirms strong electrostatic interaction between the QDs and BR. The applicability of the synthesized quantum dots in sensing bilirubin has been checked in the presence of different possible interfering agents and also with plasma isolated from real blood samples of both normal and hepatitis patients. It was observed that bilirubin as control sample as well as in human serum sample can be optimally measured at pH 7.5, 25 °C. Thus, the proposed strategy being able to measure free BR even at least two orders of magnitude lower than bilirubin level in normal blood may provide a reasonable protocol to determine BR in the pathophysiology of many critical human diseases, like hepatitis and Gilbert\'s syndrome in the near future.

UNASSIGNED: The utilization of fluorescein-guided biopsies and resection has been recently discussed as a suitable strategy to improve and expedite operative techniques for the resection of central nervous system (CNS) tumors. However, little is known about the optical properties of sodium fluorescein (NaFl) in human tumor tissue and their potential impact on ex vivo analyses involving fluorescence-based methods.
UNASSIGNED: Tumor tissue was obtained from a study cohort of an observational study on the utilization of fluorescein-guided biopsy and resection (n=5). The optical properties of fluorescein-stained tissue were compared to the optical features of the dye in vitro and in control samples consisting of tumor tissue of high-grade glioma patients (n=3) without intravenous (i.v.) application of NaFl. The dye-exposed tumor tissues were used for optical measurements to confirm the detectability of NaFl emission ex vivo. The tissue samples were fixed in 4%PFA, immersed in 30% sucrose, embedded in Tissue-Tek OCT compound, and cut to 10 μm cryosections. Spatially resolved emission spectra from tumor samples were recorded on representative slides with a Confocal Laser Scanning Microscope FV1000 (Olympus GmbH, Hamburg, Germany) upon excitation with λexc = 488 nm.
UNASSIGNED: Optical measurements of fluorescein in 0.9% sodium chloride (NaCl) under in vitro conditions showed an absorption maximum of λmax abs = 479 nm as detected with spectrophotometer Specord 200 and an emission peak at λmax em = 538 nm recorded with the emCCD detection system of a custom-made microscope-based single particle setup using a 500 nm long-pass filter. Further measurements revealed pH- and concentration-dependent emission spectra of NaFl. Under ex vivo conditions, confocal laser scanning microscopy of fluorescein tumor samples revealed a slight bathochromic shift and a broadening of the emission band.
UNASSIGNED: Tumor uptake of NaFl leads to changes in the optical properties - a bathochromic shift and broadening of the emission band - possibly caused by the dye\'s high pH sensitivity and concentration-dependent reabsorption acting as an inner filter of the dye\'s emission, particularly in the short wavelength region of the emission spectrum where absorption and fluorescence overlap. Understanding the ex vivo optical properties of fluorescein is crucial for testing and validating its further applicability as an optical probe for intravital microscopy, immunofluorescence localization studies, and flow cytometry analysis.

Spectral reflectometry is a spectroscopic measurement technique based on thin-film interference, which has been widely applied in industries to measure thicknesses of thin dielectric layers at the nanoscale. Recent advances in the understanding of biological nanostructures have opened a new field of spectral reflectometry in biomedicine from molecular level sensing to biomedical imaging. This chapter comprehensively covers the relevant topics on spectral reflectometry in biomedicine from its principle to applications.

Near-infrared quantum dots (NIR QDs) are promising candidate for the fluorescent probes due to their better penetration depth, long-lived luminescence with size-tunable photoluminescence wavelengths. Glutathione-coated silver sulfide quantum dots (GSH-Ag2S QDs) were synthesized using AgNO3 and Na2S in the aqueous media and they can give reaction with glutathione reductase (GR) and glutathione-s transferase (GST) enzymes as acting substrate analogue in vitro. Investigation of the toxicity of the nanomaterials are necessary to use them in the medical field and biomedical applications. Thus, in this study we investigated biocompatibility of the GSH-Ag2S QDs in vitro using 293 T and CFPAC-1 cell lines. Cell viability by MTT assay, light microscopy, fluorescence microscopy, oxidative stress enzyme activities and ICP-MS analysis were performed to evaluate the cytotoxicity and internalization of the GSH-Ag2S QDs. GSH-Ag2S QDs showed great biocompatibility with both cell lines and did not cause imbalance in the oxidative stress metabolism. The ultralow solubility product constant of Ag2S QDs (Ksp = 6.3 × 10-50) prevents release of Ag ions into the biological systems that is in agreement with data obtained by ICP-MS. In conclusion, this data prove potential of GSH-Ag2S QDs as a biocompatible optical probe to be used for the detection and/or targeting of GSH impaired diseases including cancer.

A simple, colorimetric and visual method is described for the determination of cysteamine (CA) using polyvinylpyrrolidone-stabilized silver nanoparticles (PVP-AgNPs) as a colorimetric probe. The sensing method was based on the aggregation of PVP-AgNPs that led to the changes in the color and absorption profile of the probe. The aggregation of PVP-AgNPs in the presence of CA was evidenced by using transmission electron microscopy (TEM), zeta and dynamic light scattering (DLS) measurements. A distinct color transition could be observed with the naked eye from pale yellow color of PVP-AgNPs to purple. PVP-AgNPs probe showed an excellent selectivity towards CA versus other interfering biomolecules, cations and anions. Furthermore, the colorimetric probe had a linear response for CA from 0.1 to 1.0 μM concentration range with the limit of detection (LOD) of 4.9 nM. The prepared probe was successfully utilized for the determination of CA in blood serum as biological samples.

Silver nanoparticles (AgNPs) coated with whey peptides are shown to be a useful optical nanoprobe for the highly sensitive determination of Pd(II). The peptidic surface of the AgNPs works as a molecular receptor for the rapid detection of Pd(II) via a color change from dark yellow to orange/red along with a spectral red-shift with a gap about 120 nm. The effect is caused by the formation of a coordination complex between Pd(II) and the peptide ligands. This results in the aggregation of AgNPs and an absorbance spectral shift from 410 to 530 nm. The absorbance response is linear in the range 0.1 to 1.3 μM Pd(II) with a low detection limit of 115 nM. The nanoprobe responds within a few minutes and is not interfered by other metal ions except for Mg(II). The probe potentially can be applied to the determination of Pd(II) contamination in the products of Pd(II)-catalyzed organic reactions and in pharmaceutical settings. Graphical abstractSchematic representation of the nanoprobe for Pd(II). (a) Synthesis of whey peptide-coated silver nanoparticles (AgNPs), (b) the nanoprobe design for Pd(II) detection, (c) HR-TEM imaging and elemental mapping, (d) quantitative determination of Pd(II) (Inset shows colorimetric results).

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Silicon-doped carbon quantum dots (Si-CQDs) were employed to fabricate a ratiometric fluorometric probe that shows high selectivity for hydroquinone (HQ). The Si-CQDs were prepared through hydrothermal treatment of N-[3-(trimethoxysilyl)propyl]-ethylenediamine. If HQ is oxidized in a solution of the Si-CQDs, 1,4-benzoquinone will be formed which quenches the blue fluorescence (with excitation/emission peaks at 360/435 nm) of the Si-CQDs. Simultaneously, intense green fluorescence (with a emission peak at 513 nm) appears, probably due to the formation of n-π clathrates or of a quinone imine between 1,4-benzoquinone and amino groups on the surface of the Si-CQDs. The ratio of the green and blue fluorescence can be applied to the determination of HQ with a 0.077 μM detection limit. The analytical range extends from 1 to 40 μM. Graphical abstract Schematic of a silicon-doped carbon quantum dot-based ratiometric fluorescence probe with blue and green emission for the visual and fluorometric determination of hydroquinone.