Fluorescent Benzothiadiazole Derivatives as Fluorescence Imaging Dyes: A Decade of New Generation Probes.

The current review describes advances in the use of fluorescent 2,1,3-benzothiadiazole (BTD) derivatives after nearly one decade since the first description of bioimaging experiments using this class of fluorogenic dyes. The review describes the use of BTD-containing fluorophores applied as, inter alia, bioprobes for imaging cell nuclei, mitochondria, lipid droplets, sensors, markers for proteins and related events, biological processes and activities, lysosomes, plasma membranes, multicellular models, and animals. A number of physicochemical and photophysical properties commonly observed for BTD fluorogenic structures are also described.

Advances in Molecularly Imprinting Technology for Bioanalytical Applications.

In recent years, along with the rapid development of relevant biological fields, there has been a tremendous motivation to combine molecular imprinting technology (MIT) with biosensing. In this situation, bioprobes and biosensors based on molecularly imprinted polymers (MIPs) have emerged as a reliable candidate for a comprehensive range of applications, from biomolecule detection to drug tracking. Unlike their precursors such as classic immunosensors based on antibody binding and natural receptor elements, MIPs create complementary cavities with stronger binding affinity, while their intrinsic artificial polymers facilitate their use in harsh environments. The major objective of this work is to review recent MIP bioprobes and biosensors, especially those used for biomolecules and drugs. In this review, MIP bioprobes and biosensors are categorized by sensing method, including optical sensing, electrochemical sensing, gravimetric sensing and magnetic sensing, respectively. The working mechanism(s) of each sensing method are thoroughly discussed. Moreover, this work aims to present the cutting-edge structures and modifiers offering higher properties and performances, and clearly point out recent efforts dedicated to introduce multi-sensing and multi-functional MIP bioprobes and biosensors applicable to interdisciplinary fields.

Polyanionic Polydentate Europium Complexes as Ultrabright One- or Two-photon Bioprobes.

A family of europium (III) complexes based on a polydentate ligand functionalized by charge-transfer antennae presents remarkable one- and two-photon photophysical proper-ties in water or buffer. A detailed analysis of their emission properties suggests that the wrapping of the ligand around the central rare-earth ion results in an overall Cs symmetry in agreement with the theoretical simulation and that about 65-70 % of the emission intensity is concentrated in the hypersensitive 5 D0 →7 F2 transition at 615 nm. Their brightness is excellent, in the range of the best lanthanide bioprobes making them very attractive for bio-imaging experiments.

Scalable Fabrication Framework of Implantable Ultrathin and Flexible Probes with Biodegradable Sacrificial Layers.

For long-term biocompatibility and performance, implanted probes need to further reduce their size and mechanical stiffness to match that of the surrounding cells, which, however, makes accurate and minimally invasive insertion operations difficult due to lack of rigidity and brings additional complications in assembling and surgery. Here, we report a scalable fabrication framework of implantable probes utilizing biodegradable sacrificial layers to address this challenge. Briefly, the integrated biodegradable sacrificial layer can dissolve in physiological fluids shortly after implantation, which allows the in situ formation of functional ultrathin film structures off of the initial small and rigid supporting backbone. We show that the dissolution of this layer does not affect the viability and excitability of neuron cells in vitro. We have demonstrated two types of probes that can be used out of the box, including (1) a compact probe that spontaneously forms three-dimensional bend-up devices only after implantation and (2) an ultraflexible probe as thin as 2 µm attached to a small silicon shaft that can be accurately delivered into the tissue and then get fully released in situ without altering its shape and position because the support is fully retracted. We have obtained a >93% yield of the bend-up structure, and its geometry and stiffness can be systematically tuned. The robustness of the ultraflexible probe has been tested in tissue-mimicking agarose gels with <1% fluctuation in the test resistance. Our work provides a general strategy to prepare ultrasmall and flexible implantable probes that allow high insertion accuracy and minimal surgical damages with the best biocompatibility.

Lanthanide circularly polarized luminescence: bases and applications.

Lanthanide (III) luminescence is very characteristic: it is characterized by narrow emission bands, large Stokes shift, and a long excited state lifetime. Moreover, chiral lanthanide complexes can emit strongly circularly polarized light in a way that is almost precluded to purely organic molecules. Thanks to the sensitivity and specificity of the Ln circularly polarized luminescence (CPL) signal, CPL-active complexes are therefore employed as bioanalytical tools and other uses can be envisaged in many other fields. Here we present a brief overview of the most recently developed CPL-active lanthanide complexes and a selected few examples of their applications. We briefly discuss the main mechanisms that can rationalize the observed outstanding CPL properties of these systems, and some practical suggestions on how to measure and report data.

Paper-based DNA detection using lanthanide-doped LiYF4 upconversion nanocrystals as bioprobe.

A novel sensitive DNA bioassay using lanthanide-doped LiYF4 upconversion nanocrystals as luminescent marker and oligonucleotide hybridization as the selective reaction is developed in a paper-based platform, providing a detection limit of 3.6 fmol.

Tuning the cellular uptake properties of luminescent heterobimetallic iridium(III)-ruthenium(II) DNA imaging probes.

The synthesis of two new luminescent dinuclear Ir(III)-Ru(II) complexes containing tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2''',3'''-j]phenazine (tpphz) as the bridging ligand is reported. Unlike many other complexes incorporating cyclometalated Ir(III) moieties, these complexes display good water solubility, allowing the first cell-based study on Ir(III)-Ru(II) bioprobes to be carried out. Photophysical studies indicate that emission from each complex is from a Ru(II) excited state and both complexes display significant in vitro DNA-binding affinities. Cellular studies show that each complex is rapidly internalised by HeLa cells, in which they function as luminescent nuclear DNA-imaging agents for confocal microscopy. Furthermore, the uptake and nuclear targeting properties of the complex incorporating cyclometalating 2-(4-fluorophenyl)pyridine ligands around its Ir(III) centre is enhanced in comparison to the non-fluorinated analogue, indicating that fluorination may provide a route to promote cell uptake of transition-metal bioprobes.

Tapered Fiber Bioprobe Based on U-Shaped Fiber Transmission for Immunoassay.

In this paper, a tapered fiber bioprobe based on Mach-Zehnder interference (MZI) is proposed. To retain the highly sensitive straight-tapered fiber MZI sensing structure, we designed a U-shaped transmission fiber structure for the collection of optical sensing signals to achieve a miniature-insert-probe design. The spectrum responses from the conventional straight-tapered fiber MZI sensor and our proposed sensor were compared and analyzed, and experimental results showed that our proposed sensor not only has the same sensing capability as the straight-tapered fiber sensor, but also has the advantages of being flexible, convenient, and less liquid-consuming, which are attributed to the inserted probe design. The tapered fiber bioprobe obtained a sensitivity of 1611.27 nm/RIU in the refractive index detection range of 1.3326-1.3414. Finally, immunoassays for different concentrations of human immunoglobulin G were achieved with the tapered fiber bioprobe through surface functionalization, and the detection limit was 45 ng/mL. Our tapered fiber bioprobe has the insert-probe advantages of simpleness, convenience, and fast operation. Simultaneously, it is low-cost, highly sensitive, and has a low detection limit, which means it has potential applications in immunoassays and early medical diagnosis.

Heterocycle-Based Multicomponent Reactions in Drug Discovery: From Hit Finding to Rational Design.

In the context of the structural complexity necessary for a molecule to selectively display a therapeutical action and the requirements for suitable pharmacokinetics, a robust synthetic approach is essential. Typically, thousands of relatively similar compounds should be prepared along the drug discovery process. In this respect, heterocycle-based multicomponent reactions offer advantages over traditional stepwise sequences in terms of synthetic economy, as well as the fast access to chemsets to study the structure activity relationships, the fine tuning of properties, and the preparation of larger amounts for preclinical phases. In this account, we briefly summarize the scientific methodology backing the research line followed by the group. We comment on the main results, clustered according to the targets and, finally, in the conclusion section, we offer a general appraisal of the situation and some perspectives regarding future directions in academic and private research.

Effect of ytterbium amount on LaNbO4:Tm3+,Yb3+ nanoparticles for bio-labelling applications.

PURPOSE: This work investigates how Yb3+ concentration affects the luminescent properties of LaNbO4 nanoparticles for medical imaging applications. Due to the highly transparent optical window for organic tissues in the near infrared region (650-1000 nm), upconversion fluorescence allows near infrared wavelengths to penetrate deeply into tissues, which is useful in biomedical areas such as biodetection, activated phototherapy, and screening. MATERIALS/METHOD: Upconversion nanoparticles based on LaNbO4 doped with Tm3+ and Yb3+ were prepared by the one-step industrial process called Spray Pyrolysis. Samples with different Tm3+:Yb3+ molar ratios (1:4, 1:8 and 1:16) were obtained. RESULTS: The X-ray powder diffractograms of all the samples displayed the typical peaks of a crystalline material (tetragonal phase). Emission bands emerged in the blue, red, and near infrared regions, and they corresponded to the Tm3+1G4 → 3H6 (475 nm), 1G4 → 3F4 (650 nm), 3F2,3 â†’ 3H6 (690 nm), and 3H4 → 3H6 (803 nm) transitions, which indicated a two-photon absorption process. As for bio-labelling application, the results indicated that Yb3+ concentration was directly related to signal intensity. CONCLUSIONS: The intensity of positive conversion emissions depends directly on Yb3+ concentration. The bio-labelling tests pointed to the potential application of these materials. The sample containing the highest amount of Yb3+ provided better results and was easier to detect than the standard sample.

Enzyme-Responsive Peptide-Based AIE Bioprobes.

Enzyme, which exists widely in organisms, has high specificity and high catalytic efficiency for its substrates. The absence, the reduced activity, or the overexpression of enzyme are closely related to the occurrence and development of diseases. Therefore, enzyme is often used as markers for disease detection and treatment. To detect enzyme activity and track drug release, aggregation-induced emission (AIE) bioprobes have been developed because of their excellent photostability and high signal-to-noise ratio (SNR). Among them, peptide-based AIE bioprobes with great biocompatibility and specificity are favored by an increasing number of researchers. Enzymatic hydrolysis of peptide can cause aggregation of AIE molecules and drug release. In this review, enzyme-responsive peptide-based AIE bioprobes used for biomedical application are summarized according to the three aggregation strategies triggered by various reaction between peptide and enzyme, including enzyme-triggered precipitate, enzyme-catalyzed coupling, and enzyme-instructed self-assembly. By giving some representative examples, we discuss how each aggregation strategy detects enzyme activity and treats the diseases under imaging guidance. Finally, we comment on the current problems and future prospects of enzyme-responsive peptide-based AIE bioprobes.

Dye-Loaded Quatsomes Exhibiting FRET as Nanoprobes for Bioimaging.

Fluorescent organic nanoparticles (FONs) are emerging as an attractive alternative to the well-established fluorescent inorganic nanoparticles or small organic dyes. Their proper design allows one to obtain biocompatible probes with superior brightness and high photostability, although usually affected by low colloidal stability. Herein, we present a type of FONs with outstanding photophysical and physicochemical properties in-line with the stringent requirements for biomedical applications. These FONs are based on quatsome (QS) nanovesicles containing a pair of fluorescent carbocyanine molecules that give rise to Förster resonance energy transfer (FRET). Structural homogeneity, high brightness, photostability, and high FRET efficiency make these FONs a promising class of optical bioprobes. Loaded QSs have been used for in vitro bioimaging, demonstrating the nanovesicle membrane integrity after cell internalization, and the possibility to monitor the intracellular vesicle fate. Taken together, the proposed QSs loaded with a FRET pair constitute a promising platform for bioimaging and theranostics.

Dual-emission hydrogel nanoparticles with linear and reversible luminescence-response to pH for intracellular fluorescent probes.

A type of dual-emission probe with highly recognizable luminescence-response to pH has been designed. For the prepared core-shell polymeric hydrogel nanoparticles probe (Eu(DBM)3Phen doped polystyrene (PS)-co-poly(N-isopropylacrylamide)(PNIPAM)/FITC), the red emission of inner encapsulated europium complexes Eu(DBM)3Phen is remained in the hydrophobic PS core and used as a stable reference signal. Comparingly, the green emission of outer electrostatic bonded fluorescent isothiocyanate (FITC) moieties on the hydrophilic PNIPAM shell is adjusted and acted linear and reversible luminescence-response to pH between 5.8 and 7.4 with an exact resolution of 0.1 units. As a remarkable and smart pH probe, the hydrogel nanoparticles show low biological toxicity and prolonged resistance to ions and photobleaching. Also, the probes have successfully discriminated the fluorescent imaging for cytoplasmic matrix based on different pH with minimum biologic background fluorescence. These dual-emission pH-sensitive hydrogel nanoparticles suggest potential applications in clinical medicine, such as cell imaging and disease diagnosis.

High-performance portable graphene field-effect transistor device for detecting Gram-positive and -negative bacteria.

Current techniques for Gram-typing and for diagnosing a pathogen at the early infection stage rely on Gram stains, cultures, Enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and gene microarrays, which are labor-intensive and time-consuming approaches. In addition, a delayed or imprecise diagnosis of clinical pathogenic bacteria leads to a life-threatening emergency or overuse of antibiotics and a high-rate occurrence of antimicrobial-resistance microbes. Herein, we report high-performance antibiotics (as bioprobes) conjugated graphene micropattern field-effect transistors (ABX-GMFETs) to facilitate on-site Gram-typing and help in the detection of the presence or absence of Gram-negative and -positive bacteria in the samples. The ABX-GMFET platform, which consists of recognition probes and GM transistors conjugated with novel interfacing chemical compounds, was integrated into the microfluidics to minimize the required human intervention and facilitate automation. The mechanism of binding of ABX-GMFET was based on a charge or chemical moiety interaction between the bioprobes and target bacteria. Subsequently, ABX-GMFETs exhibited unprecedented high sensitivity with a limit of detection (LOD) of 100 CFU/mL (1-9 CFU/mL), real-time target specificity.

Laser-Activated Bioprobes with High Photothermal Conversion Efficiency for Sensitive Photoacoustic/Ultrasound Imaging and Photothermal Sensing.

Laser-activated bioprobes with high photothermal conversion efficiency (IRPDA@PFH NDs) based on biocompatible IR-780 doped polydopamine perfluorocarbon nanodroplets (NDs) were developed. When protected by gelatin microspheres, their near-spherical morphologies can be easily observed with transmission electron microscope. Doping IR-780 (3 w/w % of added dopamine hydrochloride) can significantly enhance near-infrared (NIR) absorption and photothermal conversion efficiency to 57.7%. The enhanced NIR absorption and nonradiative relaxation are preferred to stronger photoacoustic (PA) signals and higher PA imaging definition; ultrasound (US) signals also increase more than 2.5 times because of easier phase change of NDs. These bioprobes had sensitive PA/US imaging capability with highly effective substitute utilizations, in which polydopamine was used either as a PA contrast or a photothermal agent. Perfluorocarbon can be used as an US contrast agent and temperature indicator. More importantly, the gray value increments of US increase with temperature in a general range from 35 to 55 °C. Especially, an approximate linear increasing of gray value in the optimized photothermal therapy (PTT) range from 35 °C to 50 °C could be used for the temperature monitoring and control of PTT. During PTT, the heated regions and the extent of photothermal heating can be visualized by US imaging. These findings indicate their great potential for biosensing and PTT monitoring.

Enzyme-Responsive Bioprobes Based on the Mechanism of Aggregation-Induced Emission.

Enzymes play an indispensable role in maintaining normal life activities. The abnormalities of content and activity in specific enzymes are usually associated with the occurrence and the development of major diseases. Correspondingly, fluorescent bioprobes with distinctive sensing mechanisms and different functionalities have attracted growing attention as convenient tools for optical probing and monitoring the activity of enzymes. Ideally and excitedly, the recently emerged luminogens with an aggregation-induced emission (AIE) feature could perfectly overcome the aggregation-caused quenching (ACQ) effect of conventional bioprobes. Based on the fantastic characteristics of AIE luminogens (AIEgens), specific enzyme bioprobes have been designed through integration with recognition units, demonstrating many advantages including low background interference, a high signal-to-noise ratio (SNR), and superior photostability. In this review, by presenting some typical examples, we summarize the working principle and structural design of specific AIEgen-based bioprobes that are triggered by enzymes and discuss their great potential in biomedical applications, with the aim to promote the future research of fluorescent bioprobes involving enzymes.

Enhancing Antibody Serodiagnosis Using a Controlled Peptide Coimmobilization Strategy.

Antigen immunoreactivity is often determined by surface regions defined by the 3D juxtapositions of amino acids stretches that are not continuous in the linear sequence. As such, mimicking an antigen immunoreactivity by means of putative linear peptide epitopes for diagnostic purposes is not trivial. Here we present a straightforward and robust method to extend the reach of immune-diagnostic probes design by copresenting peptides belonging to the same antigenic surface. In this case study focused on a computationally predicted Zika virus NS1 protein putative antigenic region, we reached a diagnostic confidence by the oriented and spatially controlled coimmobilization of peptide sequences found adjacent within the protein fold, that cooperatively interacted to provide enhanced immunoreactivity with respect to single linear epitopes. Through our method, we were able to differentiate Zika infected individuals from healthy controls. Remarkably, our strategy fits well with the requirements to build high-throughput screening platforms of linear and mixed peptide libraries, and it could possibly facilitate the rapid identification of conformational immunoreactive regions.

Purification of quantum dot-based bioprobes via high-performance size exclusion chromatography.

Due to excellent optical properties, quantum dots (QDs) have been widely applied to sensing, labeling, and imaging. For the fabrication of QD-based bioprobes, purification is usually the crucial step. Hydrophilic octylamine grafted polyacrylic acid modified QDs (OPA-QDs) were prepared, and purified by high-performance size exclusion chromatography (HPSEC) to remove excess OPA and aggregated QDs. The percentage of suspended agglomerates of OPA-QDs in the unpurified OPA-QDs increases from 4% to 31% through a year, but the purified OPA-QDs of the same batch possess excellent colloidal stability for at least one year. Subsequently, QD-based bioprobes were fabricated by the conjugation between QDs and streptavidin (SA) or antibody (IgG), generating QD-SA and QD-IgG, respectively, which were purified via HPSEC. Finally, the resulting QD-SA and QD-IgG were adopted to detect tumour markers on slices and showed specific positive signals without nonspecific adsorption, which was contrary to the unpurified QD-IgG. Thus, the HPSEC-coupled system proposed in the current work is potent and universal for the generation of purified and monodisperse QD-based bioprobes, which is promising in the nanobiodetection field.

pH- and Temperature-Sensitive Hydrogel Nanoparticles with Dual Photoluminescence for Bioprobes.

This study demonstrates high contrast and sensitivity by designing a dual-emissive hydrogel particle system, whose two emissions respond to pH and temperature strongly and independently. It describes the photoluminescence (PL) response of poly(N-isopropylacrylamide) (PNIPAM)-based core/shell hydrogel nanoparticles with dual emission, which is obtained by emulsion polymerization with potassium persulfate, consisting of the thermo- and pH-responsive copolymers of PNIPAM and poly(acrylic acid) (PAA). A red-emission rare-earth complex and a blue-emission quaternary ammonium tetraphenylethylene derivative (d-TPE) with similar excitation wavelengths are inserted into the core and shell of the hydrogel nanoparticles, respectively. The PL intensities of the nanoparticles exhibit a linear temperature response in the range from 10 to 80 °C with a change as large as a factor of 5. In addition, the blue emission from the shell exhibits a linear pH response between pH 6.5 and 7.6 with a resolution of 0.1 unit, while the red emission from the core is pH-independent. These stimuli-responsive PL nanoparticles have potential applications in biology and chemistry, including bio- and chemosensors, biological imaging, cancer diagnosis, and externally activated release of anticancer drugs.

Ultrasensitive Luminescent In Vitro Detection for Tumor Markers Based on Inorganic Lanthanide Nano-Bioprobes.

Ultrasensitive and accurate detection of tumor markers is of vital importance for the screening or diagnosis of cancers at their early stages and for monitoring cancer relapse after surgical resection. Inorganic lanthanide (Ln3+) nanoparticles (NPs), owing to their superior physicochemical characteristics, are regarded as a new generation of luminescent nano-bioprobes in the field of cancer diagnosis and therapy. In this progress report, a focus is set on our recent efforts on the development of inorganic Ln3+-NPs as efficient luminescent nano-bioprobes for the ultrasensitive in vitro biodetection of tumor markers, with an emphasis on the dissolution-enhanced luminescent bioassay (DELBA), an emerging technique recently developed toward practical medical applications.