Nanomaterials that can cir culate in the body hold great potential to diagnose and treat disease 1‐4 . For such applications, it is important that the nanomaterials be harmlessly eliminated from the body in a reasonable period of time after they carry out their diagnostic or therapeutic function. Despite efforts to improve their targeting efficiency, significant quantities of systemically administered nanomaterials are cleared by the mononuclear phagocytic system before finding their targets, increasing the likelihood of unintended acute or chronic toxicity. However, there has been little effort to engineer the self-destruction of errant nanoparticles into non-toxic, systemically eliminated products. Here, we present luminescent porous silicon nanoparticles (LPSiNPs) that can carry a drug payload and of which the intrinsic near-infrared photoluminescence enables monitoring of both accumulation and degradation in vivo. Furthermore, in contrast to most optically active nanomaterials (carbon nanotubes, gold nanoparticles and quantum dots), LPSiNPs self-destruct in a mouse model into renally cleared components in a relatively short period of time with no evidence of toxicity. As a preliminary in vivo application, we demonstrate tumour imaging using dextran-coated LPSiNPs (D-LPSiNPs). These results demonstrate a new type of multifunctional nanostructure with a low-toxicity degradation pathway forinvivo applications. The in vivo use of nanomaterials as therapeutic and diagnostic agents is of intense interest owing to their unique properties such as large specific capacity for drug loading 2 , strong superparamagnetism 3 ,efficientphotoluminescence 1,5 ordistinctive Raman signatures 4 , among others. Materials with sizes in the range of 20200nm can avoid renal filtration, leading to prolonged resi

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Biodegradable luminescent porous silicon nanoparticles for in vivo applications.

The use of Forster or fluorescence resonance energy transfer (FRET) as a spectroscopic technique has been in practice for over 50 years. A search of ISI Web of Science with just the acronym "FRET" returns more than 2300 citations from various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterials. The advent of new classes of fluorophores including nanocrystals, nanoparticles, polymers, and genetically encoded proteins, in conjunction with ever more sophisticated equipment, has been vital in this development. This review gives a critical overview of the major classes of fluorophore materials that may act as donor, acceptor, or both in a FRET configuration. We focus in particular on the benefits and limitations of these materials and their combinations, as well as the available methods of bioconjugation.

Materials for Fluorescence Resonance Energy Transfer Analysis: Beyond Traditional Donor-Acceptor Combinations

Fluorescent imaging of biological systems in the second near-infrared window (NIR-II) can probe tissue at centimetre depths and achieve micrometre-scale resolution at depths of millimetres. Unfortunately, all current NIR-II fluorophores are excreted slowly and are largely retained within the reticuloendothelial system, making clinical translation nearly impossible. Here, we report a rapidly excreted NIR-II fluorophore (∼90% excreted through the kidneys within 24 h) based on a synthetic 970-Da organic molecule (CH1055). The fluorophore outperformed indocyanine green (ICG)—a clinically approved NIR-I dye—in resolving mouse lymphatic vasculature and sentinel lymphatic mapping near a tumour. High levels of uptake of PEGylated-CH1055 dye were observed in brain tumours in mice, suggesting that the dye was detected at a depth of ∼4 mm. The CH1055 dye also allowed targeted molecular imaging of tumours in vivo when conjugated with anti-EGFR Affibody. Moreover, a superior tumour-to-background signal ratio allowed precise image-guided tumour-removal surgery. A renally cleared, water-soluble dye emitting in the near-infrared-imaging (NIR)-II window outperforms a clinically approved NIR-I dye in the in vivo imaging of tumours and their nearby blood and lymphatic vasculatures.

A small-molecule dye for NIR-II imaging.

Small-molecule fluorescent probes embody an essential facet of chemical biology. Although numerous compounds are known, the ensemble of fluorescent probes is based on a modest collection of modular “core” dyes. The elaboration of these dyes with diverse chemical moieties is enabling the precise interrogation of biochemical and biological systems. The importance of fluorescence-based technologies in chemical biology elicits a necessity to understand the major classes of small-molecule fluorophores. Here, we examine the chemical and photophysical properties of oft-used fluorophores and highlight classic and contemporary examples in which utility has been built upon these scaffolds.

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Bright Ideas for Chemical Biology

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.

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Extrinsic fluorescent dyes as tools for protein characterization.

Relative fluorescence quantum yields are determined using a computer-controlled luminescence spectrometer. The relative absorbances of the standards and unknowns are measured using the same instrument as for the fluorescence measurements. Relative quantum yields are presented for a wide range of compounds at room temperature.

Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer

Recent developments in photoluminescence and chemiluminescence spectroscopy are summarised, with particular reference to methods for improving the selectivity of luminescence analyses. The acquisition and manipulation of contour spectra and the problems and benefits of generally available corrected fluorescence spectra are discussed. Fluorescence and chemiluminescence immunoassays are evaluated and some probable future developments in these areas are indicated.

Recent developments in fluorescence and chemiluminescence analysis. Plenary lecture

Energy transfer phenomena, in which excited fluorophores transfer energy to neighbouring chromophores, are well characterised in photochemistry and have found a wide range of applications in analytical biochemistry. The transfer of energy from a donor to an acceptor group is only significant over distances of a few nm, so it can be used as a spectroscopic ruler and as a means of detecting molecular interactions and conformational changes. Such methods usually retain the great sensitivity and sample handling flexibility of conventional fluorescence techniques. As a result many assays involving enzymes, antibodies and nucleotides utilise energy transfer measurement principles. This article outlines these principles for the main types of energy transfer, and summarises some of their most important areas of application.

James N. Miller

Papers

Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer

Recent developments in fluorescence and chemiluminescence analysis. Plenary lecture

Fluorescence energy transfer methods in bioanalysis

Automation of an energy-transfer immunoassay by using stopped-flow injection analysis with merging zones

Determination of pH with acid—base indicators: Implications for optical fibre probes