Péter Kele

Upconverting luminescent nanoparticles for use in bioconjugation and bioimaging

Upconverting luminescent nanoparticles (UCNPs) display the unique property of emitting visible light following photoexcitation with near-infrared laser light. This results in features such as virtually zero autofluorescence of (biological) matter and easy separation of the emission peaks from stray light. Other features include rather narrow emission bands, very high chemical stability, the lack of bleaching, and the absence of blinking effects. This article reviews the work performed in the past few years with UCNPs in terms of surface modifications, bioconjugation, and optical (cellular) imaging.

Dual labeling of biomolecules by using click chemistry: a sequential approach.

Click by click: Dual labeling of model compounds was carried out by using copper-free and copper-mediated click chemistry in a sequential manner. This method was used to introduce two labels onto biological targets or nanoparticles, thus quickly converting them into fluorescence resonance energy transfer systems.

Clickable fluorophores for biological labeling—with or without copper

The synthesis of a set of new clickable fluorophores that virtually cover the whole visible spectrum reaching the near infra-red regime is presented herein. Besides dyes that are capable of participating in classical copper catalyzed 1,3-dipolar cycloaddition reactions with the counterparting function we have also prepared dyes containing a cyclooctyne moiety, an alkyne derivative that enables copper free clicking to azides. The suitability of these dyes for fluorescent labeling of biomolecules is presented by examples on model frameworks representing major biopolymer building blocks. The versatility of these dyes is presented in cell labeling experiments as well as by labeling the azide modified surface glycans of CHO-cells either by copper catalyzed or copper-free click reaction. These dyes are expected to have a large variety of applications in (bio)orthogonal labeling schemes both in vivo and in vitro.

Fluorescent Silica Nanoparticles

We report on the preparation of fluorescent silica nanoparticles (NPs). They have been prepared by (a) modification of the NPs by amino groups and subsequent introduction of amino‐reactive fluorophores of various color and (b) by modification of the NPs by either azido groups or alkyne groups and subsequent conjugation to fluorophores by so‐called click chemistry, which is a novel approach toward modifying silica NPs. The new NPs were characterized in terms of size and spectral properties.

A Non-Fluorinated Monobenzocyclooctyne for Rapid Copper-Free Click Reactions

We designed and synthesised carboxymethylmonobenzocyclooctyne (COMBO) through a four-step reaction pathway. COMBO is a new, structurally simple, non-fluorinated, and directly conjugable copper-free click reagent, which shows excellent reaction kinetics, as also evidenced by theoretical calculations. Additionally, the carboxylic acid appendage allows further conjugation to biomolecules or fluorescent labels. The utility of COMBO in bioorthogonal labelling schemes was demonstrated when a COMBO-containing fluorescent label was employed in glycan imaging of HeLa cells (metabolically modified to have azidosialic acid residues on their cell-surface glycans).

Coumaryl crown ether based chemosensors: Selective detection of saxitoxin in the presence of sodium and potassium ions

Two novel fluorescent chemosensors in which an aza-crown is linked to 4-coumaryl fluorophores by a methylene spacer have been synthesized for sensing saxitoxin. Fluorescence enhancement was observed upon binding of the dicationic toxin molecule, whereas several metal ions produced no effect.

Probing the Activity of Matrix Metalloproteinase II with a Sequentially Click-Labeled Silica Nanoparticle FRET Probe

Selective double click labeling: The determination of nanomolar concentrations of matrix metalloproteinase II is made possible through the use of a nanoprobe that consists of fluorescently doped silica nanoparticles to which a FRET-based enzyme substrate has been conjugated through sequential click reactions.

New Generation of Bioorthogonally Applicable Fluorogenic Dyes with Visible Excitations and Large Stokes Shifts

Synthesis of a set of new, azide bearing, biorthogonally applicable fluorogenic dyes with large Stokes shifts is presented herein. To assess the fluorogenic performance of these new dyes we have labeled a genetically modulated, cyclooctyne-bearing protein in lysate medium. Studies showed that the labels produce specific signal with minimal background fluorescence. We also provide theoretical insights into the design of such fluorogenic labels.

Analysis of matrix metalloproteinase triple-helical peptidase activity with substrates incorporating fluorogenic L- or D-amino acids

The consequences of improper regulation of collagen turnover include diseases such as tumor cell metastasis and arthritis. Several fluorogenic triple-helical peptide (fTHP) substrates have been constructed presently to examine collagenolytic behavior. These substrates incorporate L- or D-2-amino-3-(7-methoxy-4-coumaryl)propionic acid (Amp) or L- or D-2-amino-3-(6,7-dimethoxy-4-coumaryl)propionic acid (Adp) as the fluorophore and N-2,4-dinitrophenyl (Dnp) as the quencher. The desired sequences were C6-(Gly-Pro-Hyp)5-Gly-Pro-[Amp/Adp]-Gly-Pro-Gln-Gly approximately Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg-(Gly-Pro-Hyp)5-NH2. All four fTHPs formed stable triple-helices. Matrix metalloproteinase-2 (MMP-2) rates of hydrolysis for all fTHPs were considerably more rapid than corresponding MMP-1 rates. Evaluation of individual kinetic parameters indicated that MMP-2 bound to the fTHPs more efficiently than MMP-1. Comparison to a triple-helical substrate incorporating the same sequence but with a different fluorophore [Lys((7-methoxycoumarin-4-yl)acetyl); Lys(Mca)] demonstrated that the shorter side chain of Amp or Adp was better tolerated by MMP-1 and MMP-2. Adp may well be the fluorophore of choice for fTHPs, as (a) fTHPs incorporating Adp were obtained in significantly higher yields than the Amp-containing fTHPs, (b) Adp has a larger Stokes shift than either Amp or Lys(Mca) and thus has less chance of self-quenching, (c) Adp has a relatively high quantum yield, (d) the Adp/Dnp pair is compatible with multiwell plate reader formats, and (e) MMPs better tolerate Adp than Lys(Mca).

Bioorthogonal fluorescent labels: A review on combined forces

This review ventures to summarize the latest developments in bioorthogonal fluorescent imaging labels with a special focus on bioimaging applications. We briefly summarize the most preferred means of bioorthogonal tagging schemes for the labeling of specific biomolecular structures. The review is structured by the type of the fluorescent labels that can address the problems that most commonly compromise fluorescent imaging techniques, i.e. the autofluorescence of biomolecules, the background fluorescence of unreacted reagents, and photobleaching. Thus, we present (i) far-red/near-infra-red emitting dyes, (ii) fluorogenic scaffolds, and (iii) nanoparticle-based signaling platforms.