Bioimaging Probes Research Articles

Harnessing aggregation-induced emission property of indolizine derivative as a fluorogenic bioprobe for endoplasmic reticulum

For fluorogenic bioimaging of the endoplasmic reticulum, a new aggregation-induced emission luminogen (AIEgen) core skeleton was conjugated with glibenclamide. Click chemistry was successfully employed for the efficient conjugation between azide-modified glibenclamide and alkyne-containing indolizine core skeleton to generate Aru68. When the volume fraction of water is increases in the solution of Aru68 in water and DMF induced aggregates formation and simultaneous increase of fluorescence intensity up to 100-fold was observed. Solvatochromism and restriction of molecular motion study by perturbation of solvent polarity index and volume fraction of glycerin in glycerin/methanol mixture provided a mechanistic insight into the turn-on phenomenon of Aru68 as AIEgen. The final bio-imaging demonstration of Aru68 in live cell conditions revealed great potential not only of the probe for wash-free bioimaging of the endoplasmic reticulum but also of the indolizine core skeleton for efficient fluorogenic bioprobe development based on aggregation-induced emission processes.

The Multifarious Applications of Copper Nanoclusters in Biosensing and Bioimaging and Their Translational Role in Early Disease Detection.

Nanoclusters possess an ultrasmall size, amongst other favorable attributes, such as a high fluorescence and long-term colloidal stability, and consequently, they carry several advantages when applied in biological systems for use in diagnosis and therapy. Particularly, the early diagnosis of diseases may be facilitated by the right combination of bioimaging modalities and suitable probes. Amongst several metallic nanoclusters, copper nanoclusters (Cu NCs) present advantages over gold or silver NCs, owing to their several advantages, such as high yield, raw abundance, low cost, and presence as an important trace element in biological systems. Additionally, their usage in diagnostics and therapeutic modalities is emerging. As a result, the fluorescent properties of Cu NCs are exploited for use in optical imaging technology, which is the most commonly used research tool in the field of biomedicine. Optical imaging technology presents a myriad of advantages over other bioimaging technologies, which are discussed in this review, and has a promising future, particularly in early cancer diagnosis and imaging-guided treatment. Furthermore, we have consolidated, to the best of our knowledge, the recent trends and applications of copper nanoclusters (Cu NCs), a class of metal nanoclusters that have been gaining much traction as ideal bioimaging probes, in this review. The potential modes in which the Cu NCs are used for bioimaging purposes (e.g., as a fluorescence, magnetic resonance imaging (MRI), two-photon imaging probe) are firstly delineated, followed by their applications as biosensors and bioimaging probes, with a focus on disease detection.

Photoluminescent nanocluster-based probes for bioimaging applications.

In the continuous search for versatile and better performing probes for optical bioimaging and biosensing applications, many research efforts have focused on the design and optimization of photoluminescent metal nanoclusters. They consist of a metal core composed by a small number of atoms (diameter < 2-3nm), usually coated by a shell of stabilizing ligands of different nature, and are characterized by molecule-like quantization of electronic states, resulting in discrete and tunable optical transitions in the UV-Vis and NIR spectral regions. Recent advances in their size-selective synthesis and tailored surface functionalization have allowed the effective combination of nanoclusters and biologically relevant molecules into hybrid platforms, that hold a large potential for bioimaging purposes, as well as for the detection and tracking of specific markers of biological processes or diseases. Here, we will present an overview of the latest combined imaging or sensing nanocluster-based systems reported in the literature, classified according to the different families of coating ligands (namely, peptides, proteins, nucleic acids, and biocompatible polymers), highlighting for each of them the possible applications in the biomedical field.

Small Molecule Fluorescent Probes for Sensing and Bioimaging of Nitroreductase

AbstractHypoxia is a condition caused by a decrease in oxygen and is often seen in cases of cardiac ischemia, solid tumors, inflammation, and other diseases. Tumor hypoxia can result in cancer invasion, metastasis, decreased apoptosis, and resistance to various therapies (chemotherapy, immunotherapy, and radiotherapy), ultimately reducing overall survival rates. These effects of hypoxia underline the need to monitor hypoxia‐associated specific markers for more effective therapeutic intervention. Nitroreductase (NTR) is a common biomarker of hypoxia, via its reduction activity with nitroaromatic compounds to corresponding amino derivatives using NADH and NADPH as cofactor. The NTR expression level can be correlated to cellular hypoxia. Therefore, developing a diagnostic tool for measuring NTR activity is of immense importance for improving treatment efficacies. To leverage NTR activity as a proxy of hypoxia, small molecule based fluorescent probes have been developed as quantifiable probes. To date, several NTR‐sensitive fluorescent probes have been reported. This review is focuses on the advances in these fluorescent NTR probes over the last five years.

Rational Design and Synthesis of Large Stokes Shift 2,6-Sulphur-Disubstituted BODIPYs for Cell Imaging

Five new disubstituted 2,6-thioaryl-BODIPY dyes were synthesized via selective aromatic electrophilic substitution from commercially available thiophenols. The analysis of the photophysical properties via absorption and emission spectroscopy showed unusually large Stokes shifts for BODIPY fluorophores (70–100 nm), which makes them suitable probes for bioimaging. Selected compounds were evaluated for labelling primary immune cells as well as different cancer cell lines using confocal fluorescence microscopy.

Polyphenol derived bioactive carbon quantum dot-incorporated multifunctional hydrogels as an oxidative stress attenuator for antiaging and in vivo wound-healing applications.

Upregulation of certain enzymes, such as collagenase, tyrosinase, and elastase, is triggered by several extrinsic environmental factors, such as temperature, UV radiation, humidity, and stress, and leads to elasticity loss and skin pigmentation. Herein, dual-emissive polyaromatic carbon quantum dots (CQDs) with abundant phenolic moieties, that is green and yellow CQDs (G-CQDs and Y-CQDs, respectively), were prepared using a three-fold symmetric molecule, 1,3,5-trihydroxybenzene. The significant inhibition efficacy of the fabricated CQDs against collagenase, elastase, and tyrosinase, which play important roles in skin aging, revealed their excellent antiaging potential. Y-CQDs with large polyphenolic-polyaromatic domains and abundant -OH groups exhibited high enzyme inhibitory efficacy against skin aging, and their collagenase, elastase, and tyrosinase inhibitory efficacies were ∼75 ± 4.2%, ∼52 ± 3.1%, and ∼35.3 ± 4.2%, respectively, at a concentration of 100 μg mL-1. The most critical factor that delays wound healing is oxidative stress, which is caused by the overproduction of free radicals around inflamed tissue. CQDs were effective in suppressing UV-induced reactive oxygen species at the cellular level and improved the cell viability. Subsequently, CQD-incorporated dual-emissive biocompatible gelatin-methacryloyl hydrogels were constructed as wound dressing materials to promote wound healing via inducing the proliferation of fibroblasts, enhancing cell migration and alleviating inflammation and to provide antiaging benefits. Our results demonstrated that the fabricated CQDs with remarkable optical features, low cytotoxicity, and excellent antioxidant and antiaging properties can be used as bio-imaging probes, antiaging agents, and wound dressing materials for oxidative stress-related diseases in the nanomedicine and cosmetics industries.

Conjugated polymer-based luminescent probes for ratiometric detection of biomolecules.

Accurate monitoring of the biomolecular changes in biological and physiological environments is of great significance for the pathogenesis, development, diagnosis and treatment of diseases. Compared with traditional luminescent probes on the basis of an intensity-dependent single-channel readout, ratiometric fluorescence detection is a more reliable sensing or imaging method which can monitor different emission signals in two or more channels with a built-in self-calibration functionality, attracting growing attention in biomolecule detection. As a kind of luminescent material with many prospects, conjugated polymers with an easily functionalized organic molecular structure, high brightness, superior stability, tunable emission, and superior biocompatibility have been widely adopted as ratiometric fluorescent probes in biosensing and bioimaging. This review first summarizes the design principles of luminescent conjugated polymers that have been developed as methods for the ratiometric measurement of biomolecules. Additionally, their potential in accurate biodetection of living biosystems was investigated. This paper aims to provide a comprehensive review of the existing challenges and latest advancements in ratiometric detection of various biomolecules with high selectivity and sensitivity. We sincerely expect that the information presented in this review could inspire broader interests across various disciplines and stimulate more exciting achievements in biodetection for the benefit of biomedical research.

Super-stable cyanine@albumin fluorophore for enhanced NIR-II bioimaging

Near-infrared-II (NIR-II) dyes could be encapsulated by either exogenous or endogenous albumin to form stable complexes for deep tissue bioimaging. However, we still lack a complete understanding of the interaction mechanism of the dye@albumin complex. Studying this principle is essential to guide efficient dye synthesis and develop NIR-II probes with improved brightness, photostability, etc.Methods: Here, we screen and test the optical and chemical properties of dye@albumin fluorophores, and systematically investigate the binding sites and the relationship between dye structures and binding degree. Super-stable cyanine dye@albumin fluorophores are rationally obtained, and we also evaluate their pharmacokinetics and long-lasting NIR-II imaging abilities.Results: We identify several key parameters of cyanine dyes governing the supramolecular/covalent binding to albumin, including a six-membered ring with chlorine (Cl), the small size of side groups, and relatively high hydrophobicity. The tailored fluorophore (IR-780@albumin) exhibits much-improved photostability, serving as a long-lasting imaging probe for NIR-II bioimaging.Conclusion: Our study reveals that the chloride-containing cyanine dyes with the above-screened chemical structure (e.g. IR-780) could be lodged into albumin more efficiently, producing a much more stable fluorescent probe. Our finding partly solves the photobleaching issue of clinically-available cyanine dyes, enriching the probe library for NIR-II bioimaging and imaging-guided surgery.

Simple fluorescence chemosensor for the detection of calcium ions in water samples and its application in bio-imaging of cancer cells.

This article describes the design, synthesis and characterization of a sensor suitable for practical measurement of ionized calcium in water samples and cancer cells. Calcium is an important ion in living organs and works as a messenger in several cellular functions. A lack of Ca ions interrupts the immune system and can lead to several diseases. A novel magnetic-polydopamine nanoparticle (PDNP)/rhodamine B (RhB)/folic acid (FA) nanoparticle was developed for the determination of calcium ions in MCF 7 cell lysates and water samples. Furthermore, the produced nanoparticle was employed for bioimaging of folate receptor (FR)-overexpressed cancer cells. This nanoprobe displayed a bright photoluminescence emission at 576 nm under an excitation wavelength of 420 nm. In the presence of calcium ions, the fluorescence emission of the MNPs-PDNPs/RhB/FA probe was proportionally decreased from 20 ng mL-1 to 100 ng mL-1 and 0.5 μg mL-1 to 20 μg mL-1 with a lower limit of quantification (LLOQ) of about 20 ng mL-1. The developed sensor showed a low-interference manner in the presence of possible coexistence interfering ions. In addition, this nanomaterial showed excellent biocompatibility with favorable differentiation ability to attach to the FR-positive cancer cells. The MNPs-PDNPs/RhB/FA nanoparticle has been utilized for bioimaging of the MCF 7 cell with favorable differentiation ability.

Porous Silicon Nanoparticles with Rare Earth as Potential Contrast Agents for MRI and Luminescent Probes for Bioimaging

Nanoparticles of porous silicon with incorporated europium and gadolinium ions were prepared by using mechanical grinding of electrochemically grown mesoporous silicon films followed with impregnation with rare earth ions from aqueous solutions. The photoluminescence spectroscopy of europium doped porous silicon nanoparticles allowed us to reveal narrow lines associated with the 5D0→7F4 transitions in Eu3+ ions. Measurements of the proton relaxation in aqueous suspensions of nanoparticles with embedded Gd3+ ions showed an effect of the shortening of both the longitudinal and transverse relaxation times. Potential applications of rare earth doped porous silicon nanoparticles as contrast agents in MRI and fluorescent labels in bioimaging are discussed.

Development of an activatable hydrogen sulfide-specific two-photon fluorescent probe for bioimaging in an air pouch inflammation model.

Inflammation caused by traumatic, ischemic, infectious, autoimmune or toxic injury may further trigger cancer and even death. Overexpression of hydrogen sulfide (H2S) in vivo has been identified as a biomarker for various types of inflammation. Identification-responsive fluorescence imaging probes have broad application prospects for in vivo diagnosis of inflammation. However, it is a challenge to design an imaging probe that concurrently responds to the target molecules to improve the sensitivity and specificity of inflammation detection. Herein, we designed and synthesized an activatable two-photon fluorescent probe to detect H2S. Fl-H2S had high selectivity, excellent photostable signals and low detection limit for recognizing H2S. In addition, Fl-H2S showed excellent two-photon fluorescence properties in cell and liver tissue visualization experiments, with a penetration depth of up to 126 μm in liver tissue. Most importantly, the unique probe Fl-H2S was the first probe to monitor H2S levels in a mouse air pouch inflammation model by fluorescence imaging technology. We expect Fl-H2S to become an effective tool for longitudinal monitoring of inflammation, diagnosis of inflammation and prediction of underlying pathogenesis of related diseases by detecting H2S.

Development of a Multi-Task Formaldehyde Specific Fluorescent Probe for Bioimaging in Living Systems and Decoration Materials Analysis

Development of a Multi-Task Formaldehyde Specific Fluorescent Probe for Bioimaging in Living Systems and Decoration Materials Analysis

Metal-Organic Frameworks for Bioimaging: Strategies and Challenges.

Metal-organic frameworks (MOFs), composited with metal ions and organic linkers, have become promising candidates in the biomedical field own to their unique properties, such as high surface area, pore-volume, tunable pore size, and versatile functionalities. In this review, we introduce and summarize the synthesis and characterization methods of MOFs, and their bioimaging applications, including optical bioimaging, magnetic resonance imaging (MRI), computed tomography (CT), and multi-mode. Furthermore, their bioimaging strategies, remaining challenges and future directions are discussed and proposed. This review provides valuable references for the designing of molecular bioimaging probes based on MOFs.

Ligand design and nuclearity variation towards dual emissive Pt(ii) complexes for singlet oxygen generation, dual channel bioimaging, and theranostics

Mono- and dinuclear Pt(ii) complexes of a new ditopic ligand with a thiophene cyclometalating core are reported. The mononuclear complex shows dual emission of fluorescence and phosphorescence and is demonstrated as a dual channel bioimaging probe.

Engineering colloidally stable, highly fluorescent and nontoxic Cu nanoclusters via reaction parameter optimization.

Metal nanoclusters (NCs) composed of the least number of atoms (a few to tens) have become very attractive for their emerging properties owing to their ultrasmall size. Preparing copper nanoclusters (Cu NCs) in an aqueous medium with high emission properties, strong colloidal stability, and low toxicity has been a long-standing challenge. Although Cu NCs are earth-abundant and inexpensive, they have been comparatively less explored due to their various limitations, such as ease of surface oxidation, poor colloidal stability, and high toxicity. To overcome these constraints, we established a facile synthetic route by optimizing the reaction parameters, especially altering the effective concentration of the reducing agent, to influence their optical characteristics. The improvement of the photoluminescence intensity and superior colloidal stability was modeled from a theoretical standpoint. Moreover, the as-synthesized Cu NCs showed a significant reduction of toxicity in both in vitro and in vivo models. The possibility of using such Cu NCs as a diagnostic probe toward C. elegans was explored. Also, the extension of our approach toward improving the photoluminescence intensity of the Cu NCs on other ligand systems was demonstrated.

Near-infrared-emitting upconverting BiVO4 nanoprobes for in vivo fluorescent imaging

Near-infrared (NIR) emitting BiVO4:Yb3+,Tm3+ nanoparticles are synthesized by a new solvothermal strategy using solvents of oleic acid and methanol. The obtained BiVO4:Yb3+,Tm3+ samples show an average particle size of ≈164 nm and exhibit an asymmetry monoclinic crystal structure of BiVO4. At NIR excitation of 980 nm, the BiVO4:Yb3+,Tm3+ sample exhibits a nearly single NIR emission at ≈796 nm with extremely weak blue emissions from Tm3+ ions. These high-energy visible emissions are absorbed by the semiconducting host of BiVO4 that possesses a bandgap of ≈2.2 eV. Therefore, the NIR excitation to a single intense NIR emission fluorescent BiVO4 materials could be a potential ideal probe for deep-tissue high-resolution bioimaging. To validate the ability of BiVO4 materials for bio-applications, we conduct the cytotoxicity experiments. The results show that the cytotoxicity of HeLa cells is negligible at a concentration of 0.2 mg/ml of BiVO4:Yb3+,Tm3+ , and the cell viability approaches 90% at a high dosage of 0.5 mg/ml. The Daphnia magna and Zebrafish treated with nanoparticles (0.5 mg/ml) display bright NIR emission without any background, indicating the excellent in vivo fluorescent imaging capacity of BiVO4:Yb3+,Tm3+ nanoparticles. Our findings offer an environment-friendly strategy to synthesize BiVO4 UCL nanophosphors and provide a promising new class of fluorescent probes for biological applications.

Poly-lysinated nanoscale carbon probe for low power two-photon bioimaging

Effective outcome from dynamic live-cell-imaging requires utilization of a probe with high emission intensity and low photobleaching. It would be preferable to achieve such properties at a low power of the applied laser to avoid any probable damage to biological cells or tissue. Most of the used small-molecule fluorophores have been reported to show significant photobleaching in a time-dependent manner and require high laser power to gain significant intensity for bioimaging. Carbon nanoparticles have recently been successfully used for cell imaging with low bleaching characteristics but require high laser power and lack optical nonlinearity at low power levels. Here, we report the preparation, characterization, and application of a Nanoscale Carbon (NC) which, on being surface decorated with crescent-shaped poly-lysine (PLNC), provides two-photon fluorescence (TPF) and low bleaching properties. PLNC was found to stain the cytoplasm of C2C12 muscle cells in the first four-hours of incubation with high TPF in the infrared range and can be useful for deep tissue imaging with further improvements.

A General Method to Develop Highly Environmentally Sensitive Fluorescent Probes and AIEgens.

Environmentally sensitive fluorescent probes (including AIEgens) play pivotal roles in numerous biological studies. Many of these functional materials are developed based on the twisted intramolecular charge transfer (TICT) mechanism. However, the TICT tendency of dialkylated amino groups in biocompatible main‐stream fluorophores (i.e., coumarins and rhodamines) is weak, limiting their sensitivities. Herein, by replacing dialkylated amino donors with an N‐methylpyrrole group to enhance TICT, a simple and general method to engineer highly environmentally sensitive fluorescent probes is reported. This method yields a platter of colorful fluorescent probes that demonstrates outstanding polarity and viscosity sensitivity with large turn‐on ratios (up to 191 times for polarity and 14 times for viscosity), as well as distinct aggregation‐induced emission (AIE) characteristics. The utilities of these probes in both wash‐free bioimaging and protein detections are also successfully demonstrated. It is expected that this molecular design strategy will inspire the creation of many environmentally sensitive probes.

Development of a human serum albumin structure-based fluorescent probe for bioimaging in living cells

Forming a stable complex is a prerequisite for intramolecular charge transfer (ICT) probe to recognize proteins. Herein, a human serum albumin (HSA) structure-based fluorescent probe DNPM was fabricated successfully with fully considering its binding to the primary sites in HSA. Molecular simulation was used to assist the probe design. Two ICT ligands DNPM and MPM were initially designed. Both DNPM and MPM had favorable HSA binding abilities, but only DNPM had a satisfactory HSA sensitivity. Electromagnetic coupling played a key role in DNPM fluorescence enhancement. Due to the electromagnetic environment difference in protein structure, DNPM only exhibited strong sensitivity to serum albumins. DNPM could bind to Sudlow site I and site II in HSA but could not be displaced from its binding sites by common site specific drugs (e.g. phenylbutazone and ibuprofen). Besides, DNPM exhibited great potential for illumining serum albumin in living cells. The results provided a beneficial approach for designing and synthesizing high sensitive and selective fluorescent probes for proteins.

Carbon nano-dot for cancer studies as dual nano-sensor for imaging intracellular temperature or pH variation

Cellular temperature and pH govern many cellular physiologies, especially of cancer cells. Besides, attaining higher cellular temperature plays key role in therapeutic efficacy of hyperthermia treatment of cancer. This requires bio-compatible, non-toxic and sensitive probe with dual sensing ability to detect temperature and pH variations. In this regard, fluorescence based nano-sensors for cancer studies play an important role. Therefore, a facile green synthesis of orange carbon nano-dots (CND) with high quantum yield of 90% was achieved and its application as dual nano-sensor for imaging intracellular temperature and pH was explored. CND was synthesized from readily available, bio-compatible citric acid and rhodamine 6G hydrazide using solvent-free and simple heating technique requiring purification by dialysis. Although the particle size of 19 nm (which is quite large for CND) was observed yet CND exhibits no surface defects leading to decrease in photoluminescence (PL). On the contrary, very high fluorescence was observed along with good photo-stability. Temperature and pH dependent fluorescence studies show linearity in fluorescence intensity which was replicated in breast cancer cells. In addition, molecular nature of PL of CND was established using pH dependent fluorescence study. Together, the current investigation showed synthesis of highly fluorescent orange CND, which acts as a sensitive bio-imaging probe: an optical nano-thermal or nano-pH sensor for cancer-related studies.