Recent advances in bioprobes and biolabels based on cyanine dyes.

As a functional dye, cyanine dye promotes the widespread application of bioprobes in the fields of medicine, genetics and environment, owing to its advantages of good photophysical properties, excellent biocompatibility and low toxicity to biological systems. Nowadays, it is mainly used in the fields of life sciences such as fluorescent labeling of biological macromolecules, disease diagnosis, immunoassay and DNA detection, all of which lie at the core of this review. First, we briefly introduced the characteristics and principles of the cyanine dye bioprobe. Afterward, we paid attention to the recent progress of cyanine dye bioprobes widely used in the 10 years from 2010 to 2020. The application of cyanine dyes as bioprobes with different identification elements, including enzymes, organelles, immunity and DNAs, was mainly summarized. Finally, this review gave an outlook on the future development trend of cyanine dye bioprobes. This facilitates the construction of a new type of multifunctional fluorescent probe and promotes its clinical application.

Carbon dots for multicolor cell imaging and ultra-sensitive detection of multiple ions in living cells: One Stone for multiple Birds.

Given the significant impact of ions on environment pollution and human health, it is urgently needed to establish effective and convenient ion detection approaches, particularly in living cells. In this paper, we constructed multicolor N-doped-carbon dots (mPD-CDs) by facile one-step hydrothermal carbonization of m-phenylenediamine (mPD). mPD-CDs were successfully deployed for multicolor cellular imaging for animal cells, fungi, and bacteria in a wash-free way with high photostability and satisfactory biocompability. Moreover, mPD-CDs can be used as a fluorescent sensing probe for ultrasensitive detection of both iodide ion (I-) and typical heavy metals such as cadmium (Cd2+), copper (Cu2+), mercury (Hg2+), gadolinium (Gd3+), ferrous ion (Fe2+), Zinc (Zn2+), and ferric ion (Fe3+). This is the first report using CDs as optical sensing probe for the detection of Gd3+, and for detection of Fe3+ with fluorescence "turn on". More significantly, with these versatile and fascinating properties, we applied mPD-CDs for intracellular ion detection in living cells like Hep G2 and S. cerevisiae, and zebra fish. Altogether, mPD-CDs displayed great potential for multicolor cell imaging and the multiple ion detection in vitro and in vivo, presenting a promising strategy for in-situ ultrasensitive sensing of multiple metal ions in the environment and the biological systems.

New 1,3-Disubstituted Benzo[h]Isoquinoline Cyclen-Based Ligand Platform: Synthesis, Eu3+ Multiphoton Sensitization and Imaging Applications.

The development of lanthanide-based luminescent probes with a long emission lifetime has the potential to revolutionize imaging-based diagnostic techniques. By a rational design strategy taking advantage of computational predictions, a novel, water-soluble Eu3+ complex from a cyclen-based ligand bearing 1,3-disubstituted benzo[h]isoquinoline arms was realized. The ligand has been obtained overcoming the lack of reactivity of position 3 of the isoquinoline moiety. Notably, steric hindrance of the heteroaromatic chromophore allowed selective and stoichiometry-controlled insertion of two or three antennas on the cyclen platform without any protection strategy. The complex bears a fourth heptanoic arm for easy conjugation to biomolecules. This new chromophore allowed the sensitization of the metal center either with one or two photons excitation. The suitability as a luminescent bioprobe was validated by imaging BMI1 oncomarker in lung carcinoma cells following an established immunofluorescence approach. The use of a conventional epifluorescence microscope equipped with a linear structured illumination module disclosed a simple and inexpensive way to image confocally Ln-bioprobes by single photon excitation in the 350-400 nm window, where ordinary confocal systems have no excitation sources.

Immobilization of tyrosinase on Fe3o4@Au core-shell nanoparticles as bio-probe for detection of dopamine, phenol and catechol.

An optical bio-probe based on the immobilized tyrosinase on the surface of Fe3O4@Au was described for the detection of dopamine, phenol and catechol. The prepared bio-probe (Fe3O4@Au@tyrosinase) was characterized by means such as TEM, SEM, VSM, DLS and TGA. In the presence of the bio-probe, the phenol, catechol and dopamine were converted to benzoquinone, o-quinone and dopaquinone, and the fluorescence spectra appeared at 308 nm, 329 nm and 336 nm with ex = 270 nm, respectively. However, by increasing the concentration of phenolic compounds in the bio-probe, the amount of products (benzoquinone, o-quinone and dopaquinone) was increased which was the reason for the increase in fluorescence intensity. Using this mechanism, a bio-probe was designed such that the intensity of the fluorescence spectra increased proportionally with the increase of the substrate concentrations after different time periods. The 0.003 mg/mL of tyrosinase was loaded on 1.65 mg/mL of the Fe3O4@Au. The highest performance for a bio-probe was demonstrated at room temperature and pH 6.8. By investigating the characteristics of the response of the bio-probe to different phenolic compounds, it was found that the bio-probe had a linear response in the concentration range 5.0-75.0 µM, 10.0-100.0 µM for phenol and dopamine and 50.0-500.0 M for catechol. The Michaelis-Menten constant (Km) of the bio-probe was calculated as 0.6 µM. Finally, the bio-probe seems to be stable and efficient even after about 2 months. A novel and easy method for the detection of dopamine, phenol and catechol by florescence that uses oxide capability to identify the phenolic compounds was introduced.

Plasma membrane imaging with a fluorescent benzothiadiazole derivative.

This work describes a novel fluorescent 2,1,3-benzothiadiazole derivative designed to act as a water-soluble and selective bioprobe for plasma membrane imaging. The new compound was efficiently synthesized in a two-step procedure with good yields. The photophysical properties were evaluated and the dye proved to have an excellent photostability in several solvents. DFT calculations were found in agreement with the experimental data and helped to understand the stabilizing intramolecular charge-transfer process from the first excited state. The new fluorescent derivative could be applied as selective bioprobe in several cell lines and displayed plasma-membrane affinity during the imaging experiments for all tested models.

Fluorescent 6-amino-6-deoxyglycoconjugates for glucose transporter mediated bioimaging.

Two novel fluorescent bioprobes, namely, 6N-Gly-Cy3 and 6N-Gly-Cy5, were designed and synthesized for real-time glucose transport imaging as well as potentially useful tracer for galactokinase metabolism. The structure of the bioprobes was fully characterized by 1H NMR, 13C NMR, IR, and HRMS. The fluorescence properties, glucose transporter (GLUT) specificity, and the quenching and safety profiles were studied. The cellular uptake of both bioprobes was competitively diminished by d-glucose, 2-deoxy-d-glucose and GLUT specific inhibitor in a dose-dependent manner in human colon cancer cells (HT29). Comparison study results revealed that the 6N-derived bioprobes are more useful for real-time imaging of cell-based glucose uptake than the structurally similar fluorescent tracer 6-NBDG which was not applicable under physiological conditions. The up to 96 h long-lasting quenching property of 6N-Gly-Cy5 in HT29 suggested the potential applcability of the probe for cell labeling in xenograft transplantation as well as in vivo animal imaging studies.

Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA).

A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C6 linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λex 295nm and it was found to be very high Ka∼1.39×10(7) M(-1) in case of ABG complex as compared to GlcNAc only Ka∼1.01×10(4) M(-1) with the phenomenon proven to be due to glycocluster effect.

Spectral surface plasmon resonance imaging for the detection of clenbuterol via three-dimensional immobilization of bioprobes.

A method of immobilizing clenbuterol (CLEN) on the sensor chip for spectral surface plasmon resonance imaging (SPRi) was experimentally investigated. The bioprobes on the sensor chip were prepared by immobilizing bovine serum albumin (BSA) protein and conjugating CLEN molecules to BSA, which provides more active points and free orientations for specific binding. The calibration curve showed that the wavelength resonance shift decreased as the concentration of CLEN analyte increased, consistent with the inhibition principle. The limit of detection (LOD) was estimated to be 6.32 µg/ml. This method proved to be highly specific, high throughput, label free, and operationally convenient.

Aptamers and Nanobodies as New Bioprobes for SARS-CoV-2 Diagnostic and Therapeutic System Applications.

The global challenges posed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have underscored the critical importance of innovative and efficient control systems for addressing future pandemics. The most effective way to control the pandemic is to rapidly suppress the spread of the virus through early detection using a rapid, accurate, and easy-to-use diagnostic platform. In biosensors that use bioprobes, the binding affinity of molecular recognition elements (MREs) is the primary factor determining the dynamic range of the sensing platform. Furthermore, the sensitivity relies mainly on bioprobe quality with sufficient functionality. This comprehensive review investigates aptamers and nanobodies recently developed as advanced MREs for SARS-CoV-2 diagnostic and therapeutic applications. These bioprobes might be integrated into organic bioelectronic materials and devices, with promising enhanced sensitivity and specificity. This review offers valuable insights into advancing biosensing technologies for infectious disease diagnosis and treatment using aptamers and nanobodies as new bioprobes.

Rapid intracellular pH measurement based on electroporation- surface-enhanced Raman scattering.

In this study, electroporation-surface-enhanced Raman scattering (SERS) was applied to rapidly measure intracellular pH. The generation of a sensitive SERS probe for measuring pH in the range of 6.0-8.0 was accomplished through the conjugation of the pH-sensitive molecule 4-mercaptobenzoic acid (4-MBA) to the surface of gold nanoparticles (Au NPs) through its thiol functional group. This bioprobe was then rapidly introduced into nasopharyngeal carcinoma CNE-1 cells by electroporation, followed by SERS scanning and the fitting of intensity ratios of each detection point's Raman peaks at 1423 cm-1 and 1072 cm-1, to create the pH distribution map of CNE-1 cells. The electroporation-SERS assay introduces pH bioprobes into a living cell in a very short time and disperses the nanoprobe throughout the cytoplasm, ultimately enabling rapid and comprehensive pH analysis of the entire cell. Our work demonstrates the potential of electroporation-SERS for the biochemical analysis of live cells.

Regulation of probe density on upconversion nanoparticles enabling high-performance lateral flow assays.

Upconversion nanoparticles (UCNPs)-based fluorescence probes have shown great potential in point-of-care testing (POCT) applications, due to UCNPs' features of high photostability and background-free fluorescence. Ceaseless improvements of UCNPs-probes have been carried out to increase detection sensitivity and to broaden detection range of UCNPs-based POCT. In this paper, we optimized UCNPs-probes by regulating probe density. The optimization was verified by a traditional lateral flow assay (LFA) platform for C-reactive protein (CRP) detection. Further, the optimized UCNPs-LFA integrating with a home-made benchtop fluorescence analyzer holds the capability to achieve high-performance POCT. Finally, nearly a 20 times sensitivity enhancement with a limit of detection of 0.046 ng/mL and a broad detection range of 0.2-300 ng/mL for CRP detection was obtained. Moreover, the optimized UCNPs-LFA was applied to detecting CRP in clinical serum samples and the detection results were consistent with the clinical test, validating its clinical practicability. The proposed optimization method is also expected to optimize other nanoparticles-based bio-probes for wider POCT application.

Directly and ultrasensitivity detecting SARS-CoV-2 spike protein in pharyngeal swab solution by using SERS-based biosensor.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a great disaster to the economy and human society. Nowadays, SARS-CoV-2 is fading away from people's memory but it still exists around us. PCR plays an important role in detecting SARS-CoV-2 but it requires a long detecting time, equipped laboratory, and professional operators. In comparison with polymerase chain reaction (PCR), surface-enhanced Raman scattering (SERS) is a promising method for detecting SARS-CoV-2 due to its fast, easily operated, and high-sensitivity properties. In this study, the monolayer Ag nanoparticles (MAgNPs) covered with single-layer graphene (SLG) are applied as a SERS substrate. The angiotensin converting enzyme 2 (ACE2) is selected as a bio-probes that can specifically bind to the SARS-CoV-2 S protein. The SERS-based biosensor is formed by ACE2 functionalized SLG/MAgNPs and the LODs of detecting SARS-CoV-2 S protein in phosphate-buffered saline (PBS) and in pharyngeal swabs solution (PSS) are 0.1 fg mL-1 and 10 fg mL-1, respectively. This biosensor provides a way of directly detecting SARS-CoV-2 S protein with high sensitivity and specificity. It illustrates a practical potential in the rapid detection of the SARS-CoV-2 virus.

Visualizing sweetness: increasingly diverse applications for fluorescent-tagged glucose bioprobes and their recent structural modifications.

Glucose homeostasis is a fundamental aspect of life and its dysregulation is associated with important diseases, such as cancer and diabetes. Traditionally, glucose radioisotopes have been used to monitor glucose utilization in biological systems. Fluorescent-tagged glucose analogues were initially developed in the 1980s, but it is only in the past decade that their use as a glucose sensor has increased significantly. These analogues were developed for monitoring glucose uptake in blood cells, but their recent applications include tracking glucose uptake by tumor cells and imaging brain cell metabolism. This review outlines the development of fluorescent-tagged glucose analogues, describes their recent structural modifications and discusses their increasingly diverse biological applications.

Detection of S2- in Water by a Glucose Enhanced Water-Soluble Fluorescent Bioprobe.

That sulfide anions (S2-) in aquatic environments are produced by microorganisms through degrading sulfur-containing proteins and other organics are harmful to human health. Thus, it is of significance to develop a convenient method for the detection of S2- in water. Small molecular fluorescent probes are very popular for their advantages of visualization, real-time, high sensitivity, and convenience. However, low solubility in water limits the application of existing S2- probes. In this work, we found that our previously developed water-soluble glycosylated fluorescent bioprobe Cu[GluC] can achieve detection of S2- in water. Cu[GluC] can restore fluorescence within 20 s when it encounters S2- and shows good sensitivity towards S2- with a detection limit of 49.6 nM. Besides, Cu[GluC] derived fluorescent test strips were obtained by immersion and realized conveniently visual S2- detection in water by coupling with a UV lamp and a smartphone app. This work provides a fluorescent bioprobe with good water solubility as well as its derived fluorescent test strip for sensitive and simple detection of S2- in water, which shows good prospects in on-site water quality monitoring.

Metal-Doped Boron Quantum Dots for Versatile Detection of Lactate and Fluorescence Bioimaging.

To improve the stability and fluorescence (FL) of monoelemental boron nanomaterials, this work put forward a metal-coordination strategy to explore emerging metal-doped boron quantum dots, Co@BQDs. Through theoretical calculations, B-Co bonding as predicted can suppress the B-O reaction and protect the electronic structures of exfoliated two-dimensional (2D) boron from oxidation and decomposition upon exposure to oxygen. In experimental studies, Co2+ was added into a dispersion liquid of bulk boron and subjected to probe sonication to promote Co2+ adsorption on the surface of exfoliated 2D boron, followed by Co2+ coordination with exposed boron atoms. Solvothermal treatment of exfoliated 2D boron resulted in the generation of Co2+-doped 0D boron Co@BQDs. Experimental results confirm that Co@BQDs have higher colloidal and FL stability than BQDs as a reference. B-Co bonding formation to suppress the B-O reaction ensures the high stability of exfoliated boron structures. A dispersion liquid of Co@BQDs with stable and bright FL was used for visual FL imaging of solutions and solid substrates. Based on enzymatic and cascade oxidation-induced FL quenching of Co@BQDs, a novel FL bio-probe of lactate was explored. This bio-probe, with a broad detection range of 0.01-10 mM and a low detection limit of 3.1 µM, enables FL sensing of lactate in biosamples and shows high detection recoveries of 98.0-102.8%. Moreover, this bio-probe realized versatile FL imaging and visual detection of lactate in liquid/solid-phase systems. These results demonstrate great prospects of Co@BQDs as emerging and efficient imaging reagents for long-term tracking and bioimaging applications.

Antibacterial and Fluorescence Staining Properties of an Innovative GTR Membrane Containing 45S5BGs and AIE Molecules In Vitro.

This study aimed to add two functional components-antibacterial 45S5BGs particles and AIE nanoparticles (TPE-NIM+) with bioprobe characteristics-to the guided tissue regeneration (GTR) membrane, to optimize the performance. The PLGA/BG/TPE-NIM+ membrane was synthesized. The static water contact angle, morphologies, and surface element analysis of the membrane were then characterized. In vitro biocompatibility was tested with MC3T3-E1 cells using CCK-8 assay, and antibacterial property was evaluated with Streptococcus mutans and Porphyromonas gingivalis by the LIVE/DEAD bacterial staining and dilution plating procedure. The fluorescence staining of bacteria was observed by Laser Scanning Confocal Microscope. The results showed that the average water contact angle was 46°. In the cytotoxicity test, except for the positive control group, there was no significant difference among the groups (p > 0.05). The antibacterial effect in the PLGA/BG/TPE-NIM+ group was significantly (p < 0.01), while the sterilization rate was 99.99%, better than that in the PLGA/BG group (98.62%) (p < 0.01). Confocal images showed that the membrane efficiently distinguished G+ bacteria from G- bacteria. This study demonstrated that the PLGA/BG/TPE-NIM+ membrane showed good biocompatibility, efficient sterilization performance, and surface mineralization ability and could be used to detect pathogens in a simple, fast, and wash-free protocol.

Synergistic integration of metal nanoclusters and biomolecules as hybrid systems for therapeutic applications.

Therapeutic nanoparticles are designed to enhance efficacy, real-time monitoring, targeting accuracy, biocompatibility, biodegradability, safety, and the synergy of diagnosis and treatment of diseases by leveraging the unique physicochemical and biological properties of well-developed bio-nanomaterials. Recently, bio-inspired metal nanoclusters (NCs) consisting of several to roughly dozens of atoms (<2 nm) have attracted increasing research interest, owing to their ultrafine size, tunable fluorescent capability, good biocompatibility, variable metallic composition, and extensive surface bio-functionalization. Hybrid core-shell nanostructures that effectively incorporate unique fluorescent inorganic moieties with various biomolecules, such as proteins (enzymes, antigens, and antibodies), DNA, and specific cells, create fluorescently visualized molecular nanoparticle. The resultant nanoparticles possess combinatorial properties and synergistic efficacy, such as simplicity, active bio-responsiveness, improved applicability, and low cost, for combination therapy, such as accurate targeting, bioimaging, and enhanced therapeutic and biocatalytic effects. In contrast to larger nanoparticles, bio-inspired metal NCs allow rapid renal clearance and better pharmacokinetics in biological systems. Notably, advances in nanoscience, interfacial chemistry, and biotechnologies have further spurred researchers to explore bio-inspired metal NCs for therapeutic purposes. The current review presents a comprehensive and timely overview of various metal NCs for various therapeutic applications, with a special emphasis on the design rationale behind the use of biomolecules/cells as the main scaffolds. In the different hybrid platform, we summarize the current challenges and emerging perspectives, which are expected to offer in-depth insight into the rational design of bio-inspired metal NCs for personalized treatment and clinical translation.

Real-Time Visualization and Monitoring of Physiological Dynamics by Aggregation-Induced Emission Luminogens (AIEgens).

Physiological dynamics in living cells and tissues are crucial for maintenance and regulation of their normal activities and functionalities. Tiny fluctuations in physiological microenvironments can leverage significant influences on cell growth, metabolism, differentiation, and apoptosis as well as disease evolution. Fluorescence imaging based on aggregation-induced emission luminogens (AIEgens) exhibits superior advantages in real-time sensing and monitoring of the physiological dynamics in living systems, including its unique properties such as high sensitivity and rapid response, flexible molecular design, and versatile nano- to mesostructural fabrication. The introduction of canonic AIEgens with long-wavelength, near-infrared, or microwave emission, persistent luminescence, and diversified excitation source (e.g., chemo- or bioluminescence) offers researchers a tool to evaluate the resulting molecules with excellent performance in response to subtle fluctuations in bioactivities with broader dimensionalities and deeper hierarchies.

Fiber-optic biosensor based on the laccase immobilization on silica-functionalized fluorescent carbon dots for the detection of dopamine and multi-color imaging applications in neuroblastoma cells.

An efficient and cost-effective biosensor is of the great demand for the detection of the biologically significant neurotransmitter dopamine. In this context, enzymatic biosensors show excellent sensitivity and selectivity. In this study, we developed a laccase immobilized fiber-optic biosensor based on the fluorescence principle for the detection of dopamine. To design this biosensor, we used microwave irradiation to synthesize carbon dots (CDs) using curcumin and dimethylformamide, and the resulting CDs were called CDD-CDs. These were functionalized with a silicon precursor, 3-(aminopropyl)-triethoxysilane, and were referred to as APT-CDs. Furthermore, laccase was covalently immobilized to the APT-CDs to construct a novel bioprobe. The CDD-CDs, APT-CDs, and bioprobe showed orange (λem = 586 nm) green (λem = 533 nm), and blue-colored emissions (λem = 476 nm) at 430, 380, and 360 nm excitation wavelengths, respectively. The CDD-CDs and bioprobe showed quantum yields of 14.8% and 10.2%, respectively. The CDD-CDs displayed solvatochromism in various solvents. Bioprobe showed a significant fluorescence quenching for dopamine in the linear range of 0-30 µM with a detection limit of 41.2 nM. Bioprobe was immobilized on the tapered optical fiber using ethyl cellulose by a simple dip-coating method and investigated for multi-color imaging applications. The resulting tapered optical fiber achieved a satisfactory detection limit of 46.4 nM in the dopamine concentration range of 0-10 µM. The bioprobe demonstrated high biocompatibility, long-lasting photostability, and thermal stability, and had sufficient cytotoxicity in human neuroblastoma cells (SH-SY5Y) with excellent multi-color imaging potential. The practicality of the bioprobe was investigated in human serum and cerebrospinal fluid.

A ß-Amyloid(1-42) Biosensor Based on Molecularly Imprinted Poly-Pyrrole for Early Diagnosis of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a common form of dementia, characterized by production and deposition of ß-amyloid peptide in the brain. Thus, ß-amyloid peptide is a potentially promising biomarker used to diagnose and monitor the progression of AD. OBJECTIVE: The study aims to develop a biosensor based on a molecularly imprinted poly-pyrrole for detection of ß-amyloid. MATERIAL AND METHODS: In this experimental study, an imprinted poly-pyrrole was employed as an artificial receptor synthesized by electro-polymerization of pyrrole on screen-printed carbon electrodes in the presence of ß-amyloid. ß-amyloid acts as a molecular template within the polymer. The biosensor was evaluated by cyclic voltammetry using ferro/ferricyanide marker. The parameters influencing the biosensor performance, including electro-polymerization cycle umbers and ß-amyloid binding time were optimized to achieve the best biosensor sensitivity. RESULTS: The ß-amyloid binding affinity with the biosensor surface was evaluated by the Freundlich isotherm, and Freundlich constant and exponent were obtained as 0.22 ng mL-1 and 10.60, respectively. The biosensor demonstrated a detection limit of 1.2 pg mL-1. The biosensor was applied for ß-amyloid determination in artificial cerebrospinal fluid. CONCLUSION: The biosensor is applicable for early Alzheimer's disease detection.