Bioanalytical Applications Research Articles

High efficiency luminescent polystrene beads with a novel europium phenanthrene diketonate

Fluorescent or luminescent polymer particles (beads) have become an important tool in bioanalytical and other applications, the size regime of interest ranging from some ten to a few hundred nanometers. Bead-imbedded rare earth complexes are of particular interest, which is owed to their narrow line emissions, large separation between excitation and emissions (apparent Stokes shifts) and extrordinarily longs decay times of the emission. The latter is advantageous for the discrimination against undesired background (e.g. autofluorescence) via time gated analysis; high quantum yields and high absorptions of the incident radiation at the same time are dominant optical criteria. We here describe and characterize a new Eu3+ diketonate comprising tris(3-phenanthryl-3,3,3-trifluoroacetone)Eu(III)-di(tri-n-octyl phosphine oxide), Eu(pta)3(TOPO)2, in polystyrene with high quantum efficiencies (up to 90%) and long decay times (up to 680 μs).

Nanomaterials with Glucose Oxidase-Mimicking Activity for Biomedical Applications.

Glucose oxidase (GOD) is an oxidoreductase that catalyzes the aerobic oxidation of glucose into hydrogen peroxide (H2O2) and gluconic acid, which has been widely used in industrial raw materials production, biosensors and cancer treatment. However, natural GOD bears intrinsic disadvantages, such as poor stability and a complex purification process, which undoubtedly restricts its biomedical applications. Fortunately, several artificial nanomaterials have been recently discovered with a GOD-like activity and their catalytic efficiency toward glucose oxidation can be finely optimized for diverse biomedical applications in biosensing and disease treatments. In view of the notable progress of GOD-mimicking nanozymes, this review systematically summarizes the representative GOD-mimicking nanomaterials for the first time and depicts their proposed catalytic mechanisms. We then introduce the efficient modulation strategy to improve the catalytic activity of existing GOD-mimicking nanomaterials. Finally, the potential biomedical applications in glucose detection, DNA bioanalysis and cancer treatment are highlighted. We believe that the development of nanomaterials with a GOD-like activity will expand the application range of GOD-based systems and lead to new opportunities of GOD-mimicking nanomaterials for various biomedical applications.

Iodide-Enhanced Perovskite Nanozyme-Based Colorimetric Platform for Detection of Urinary Nuclear Matrix Protein 22.

In the last decade, perovskite nanocrystals (PNCs) have brought extensive thinking owing to their excellent optical properties. Recently, we have uncovered the peroxidase-like activity of PNCs and used this for detecting many small molecules; however, the low enzymatic activity makes them unsuitable for fluorescence analysis, which is easily disturbed by the autofluorescence of biological media. This greatly limits their application in bioanalysis. Thus, the development of a method to facilely modulate the activity of PNCs for the instrument-free colorimetric detection is highly desirable. Herein, we demonstrated an iodide-enhanced perovskite nanozyme-based colorimetric platform for the visual assay of urinary nuclear matrix protein 22 (NMP22), a typical biomarker for the diagnosis of bladder cancer. We discovered that halogen could regulate the activity of perovskite nanozymes through a simple anion replacement reaction. Experimental analysis suggested that CsPbI3 nanocrystals (NCs) displayed 24-fold higher catalytic efficiency than classical CsPbBr3 NCs. As a proof-of-concept assay, the CsPbI3 NCs could be explored into an immunoassay for the detection of NMP22 in clinical urine specimens, resulting in a low detection limit of 0.03 U/mL. This iodide-enhanced immunoassay deepens our understanding of perovskite nanozymes and also provides great potential for bioanalysis.

Enhancement Effects and Mechanism Studies of Two Bismuth-Based Materials Assisted by DMSO and Glycerol in GC-Rich PCR.

Polymerase chain reaction (PCR) has extensive bioanalytical applications in molecular diagnostics and genomic research studies for rapid detection and precise genomic amplification. Routine integrations for analytical workflow indicate certain limitations, including low specificity, efficiency, and sensitivity in conventional PCR, particularly towards amplifying high guanine-cytosine (GC) content. Further, there are many ways to enhance the reaction, for example, using different PCR strategies such as hot-start/touchdown PCR or adding some special modifications or additives such as organic solvents or compatible solutes, which can improve PCR yield. Due to the widespread use of bismuth-based materials in biomedicine, which have not yet been used for PCR optimization, this attracts our attention. In this study, two bismuth-based materials that are inexpensive and readily available were used to optimize GC-rich PCR. The results demonstrated that ammonium bismuth citrate and bismuth subcarbonate effectively enhanced PCR amplification of the GNAS1 promoter region (∼84% GC) and APOE (75.5% GC) gene of Homo sapiens mediated by Ex Taq DNA polymerase within the appropriate concentration range. Combining DMSO and glycerol additives was critical in obtaining the target amplicons. Thus, the solvents mixed with 3% DMSO and 5% glycerol were used in bismuth-based materials. That allowed for better dispersion of bismuth subcarbonate. As for the enhanced mechanisms, the surface interaction of PCR components, including Taq polymerase, primer, and products with bismuth-based materials, was maybe the main reason. The addition of materials can reduce the melting temperature (Tm), adsorb polymerase and modulate the amount of active polymerase in PCR, facilize the dissociation of DNA products, and enhance the specificity and efficiency of PCR. This work provided a class of candidate enhancers for PCR, deepened our understanding of the enhancement mechanisms of PCR, and also explored a new application field for bismuth-based materials.

Artificial intelligence-assisted smartphone-based sensing for bioanalytical applications: A review

Artificial intelligence (AI) has received great attention since the concept was proposed, and it has developed rapidly in recent years with applications in many fields. Meanwhile, newer iterations of smartphone hardware technologies which have excellent data processing capabilities have leveraged on AI capabilities. Based on the desirability for portable detection, researchers have been investigating intelligent analysis by combining smartphones with AI algorithms. Various examples of the application of AI algorithm-based smartphone detection and analysis have been developed. In this review, we give an overview of this field, with a particular focus on bioanalytical detection applications. The applications are presented in terms of hardware design, software algorithms, and specific application areas. We also discuss the existing limitations of AI-based smartphone detection and analytical approaches, and their future prospects. The take-home message of our review is that the application of AI in the field of detection analysis is restricted by the limitations of the smartphone's hardware as well as the model building of AI for detection targets with insufficient data. Nevertheless, at this juncture, while bioanalytical diagnostics and health monitoring have set the pace for AI-based smartphone applicability, the future should see the technology making greater inroads into other fields. In relation to the latter, it is likely that the ordinary or average person will play a greater participatory role.

Cascaded, Feedback-Driven, and Spatially Localized Emergence of Constitutional Dynamic Networks Driven by Enzyme-Free Catalytic DNA Circuits.

The enzyme-free catalytic hairpin assembly (CHA) process is introduced as a functional reaction module for guided, high-throughput, emergence, and evolution of constitutional dynamic networks, CDNs, from a set of nucleic acids. The process is applied to assemble networks of variable complexities, functionalities, and spatial confinement, and the systems provide possible mechanistic pathways for the evolution of dynamic networks under prebiotic conditions. Subjecting a set of four or six structurally engineered hairpins to a promoter P1 leads to the CHA-guided emergence of a [2 × 2] CDN or the evolution of a [3 × 3] CDN, respectively. Reacting of a set of branched three-arm DNA-hairpin-functionalized junctions to the promoter strand activates the CHA-induced emergence of a three-dimensional (3D) CDN framework emulating native gene regulatory networks. In addition, activation of a two-layer CHA cascade circuit or a cross-catalytic CHA circuit and cascaded driving feedback-driven evolution of CDNs are demonstrated. Also, subjecting a four-hairpin-modified DNA tetrahedron nanostructure to an auxiliary promoter strand simulates the evolution of a dynamically equilibrated DNA tetrahedron-based CDN that undergoes secondary fueled dynamic reconfiguration. Finally, the effective permeation of DNA tetrahedron structures into cells is utilized to integrate the four-hairpin-functionalized tetrahedron reaction module into cells. The spatially localized miRNA-triggered CHA evolution and reconfiguration of CDNs allowed the logic-gated imaging of intracellular RNAs. Beyond the bioanalytical applications of the systems, the study introduces possible mechanistic pathways for the evolution of functional networks under prebiotic conditions.

Tetrameric Antibody Complexes and Affinity Tag Peptides for the Selective Immobilization and Imaging of Single Quantum Dots.

Colloidal semiconductor quantum dots (QDs) are of widespread interest as fluorescent labels for bioanalysis and imaging applications. Single-particle measurements have proven to be a very powerful tool for better understanding the fundamental properties and behaviors of QDs and their bioconjugates; however, a recurring challenge is the immobilization of QDs in a solution-like environment that minimizes interactions with a bulk surface. Immobilization strategies for QD-peptide conjugates are particularly underdeveloped within this context. Here, we present a novel strategy for the selective immobilization of single QD-peptide conjugates using a combination of tetrameric antibody complexes (TACs) and affinity tag peptides. A glass substrate is modified with an adsorbed layer of concanavalin A (ConA) that binds a subsequent layer of dextran that minimizes nonspecific binding. A TAC with anti-dextran and anti-affinity tag antibodies binds to the dextran-coated glass surface and to the affinity tag sequence of QD-peptide conjugates. The result is spontaneous and sequence-selective immobilization of single QDs without any chemical activation or cross-linking. Controlled immobilization of multiple colors of QDs is possible using multiple affinity tag sequences. Experiments confirmed that this approach positions the QD away from the bulk surface. The method supports real-time imaging of binding and dissociation, measurements of Förster resonance energy transfer (FRET), tracking of dye photobleaching, and detection of proteolytic activity. We anticipate that this immobilization strategy will be useful for studies of QD-associated photophysics, biomolecular interactions and processes, and digital assays.

A Highly Stable Graphene‐Based Metal Oxide Nano Enzyme with the Peroxidase‐Mimetic Activity for Equipment‐Free Diagnosis of Diabetic Patients

AbstractNanozymes are gaining attention as promising alternatives to natural enzymes due to their unique characteristics. In this study, CuO nanoparticles were decorated on three‐dimensional nitrogen‐doped graphene‐based frameworks (CuO/3D(N)GFs) were prepared through a facile hydrothermal process. The prepared nanocomposite was characterized using XRD, EDX, FTIR, and SEM to determine its structure, composition, and morphology. The CuO/3D(N)GFs showed excellent peroxidase mimetic activity, followed Michaelis‐Menten kinetics, and exhibited better kinetic parameters (Km and Vmax) for H2O2 substrate than the natural enzyme, making it a superior catalyst. A rapid and facile colorimetric platform was developed to diagnose diabetes with the naked eye by coupling the activities of the nanozyme and glucose oxidase. This technique can quantitatively measure the glucose with high precision in diabetic patients, with a linear range of 1–10 mM and good sensitivity/selectivity toward glucose. The highest peroxidase‐like activity of the nanozyme was observed at 30 °C, but it still retained over 60 % of its activity between 20–65 °C and showed long‐term storage stability. Therefore, the CuO/3D(N)GFs nanocomposite holds promise for catalysis, biosensing, and bioanalysis applications.

Surface Engineering of MoS2 Quantum Dots via Cyclodextrin-Based Host-Guest Chemistry as Versatile Platforms for Enhanced Cell Imaging Application.

Transition metal chalcogenide quantum dots (QDs), especially MoS2 QDs, are an emerging class of novel optical probes for versatile bioanalytical applications owing to their distinct physicochemical properties. However, the reasonable use of these QDs for biological imaging has been largely restricted due to the challenge of controllable surface functionalization. In this work, we report a new strategy to engineer the surface of MoS2 QDs by taking advantage of cyclodextrin (CD)-based host-guest chemistry. The prepared β-CD-modified QDs (β-CD-MoS2 QDs) exhibit enhanced fluorescence properties, excellent biocompatibility, and good stability, making them promising as novel optical probes for bioimaging. Cellular imaging experiments revealed that these β-CD-MoS2 QDs can enter living cells through multiple internalization pathways, which differs significantly from pristine QDs. Particularly, we observed that the intracellular accumulation of MoS2 QDs in lipid droplets was enhanced owing to the specific binding of β-CD to cholesterol, which was then harnessed for monitoring the lipid metabolism in living cells via fluorescence imaging. Furthermore, we also demonstrated the potential use of β-CD-MoS2 QDs for targeted cell imaging and microplate-based cell recognition, which can be easily achieved via bioconjugation with functional motifs (e.g., folate acid) through host-guest chemistry. Altogether, these results illustrate the great potential of engineering the surface of MoS2 QDs and other analogous materials via CD-based host-guest chemistry for advancing their cell imaging applications.

Biomolecular sensors for advanced physiological monitoring.

Body-based biomolecular sensing systems, including wearable, implantable and consumable sensors allow comprehensive health-related monitoring. Glucose sensors have long dominated wearable bioanalysis applications owing to their robust continuous detection of glucose, which has not yet been achieved for other biomarkers. However, access to diverse biological fluids and the development of reagentless sensing approaches may enable the design of body-based sensing systems for various analytes. Importantly, enhancing the selectivity and sensitivity of biomolecular sensors is essential for biomarker detection in complex physiological conditions. In this Review, we discuss approaches for the signal amplification of biomolecular sensors, including techniques to overcome Debye and mass transport limitations, and selectivity improvement, such as the integration of artificial affinity recognition elements. We highlight reagentless sensing approaches that can enable sequential real-time measurements, for example, the implementation of thin-film transistors in wearable devices. In addition to sensor construction, careful consideration of physical, psychological and security concerns related to body-based sensor integration is required to ensure that the transition from the laboratory to the human body is as seamless aspossible.

Heterostructured Au@Pt/Nb2C-MXene nanocomposite as a colorimetric sensor with peroxidase-like activity for reactive oxygen species of H2O2

Bioinspired nanozymes based on two-dimensional (2D) transition metal carbide nanosheets are receiving attention because of their peroxidase-mimicking catalytic applications in bioanalysis. Herein, we report the Nb2C/Au@Pt nanocomposite-based heterostructured nanozymes for the detection of reactive oxygen species in a biological environment. The nanocomposites were fabricated by decorating 2D Nb2C-MXene with Au@Pt core–shell nanoparticles via interfacial electrostatic attraction between the transition metal carbide and noble metals. The nanocomposites were characterized using field emission scanning electron microscope, X-ray diffraction, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, UV–vis absorption spectroscopy, and zeta potential. The Nb2C-MXene with inherent electron and photothermal effects exhibited enhanced surface plasmon properties when adorned with Au@Pt. The Nb2C/Au@Pt nanocomposites demonstrated a synergistically enhanced peroxidase-like activity and efficiently catalyzed the 3,3′,5,5′-tetramethylbenzidine (TMB) reaction in the presence of hydroxyl radicals using H2O2. The Michaelis-Menten kinetics measurements revealed high catalytic activity of Nb2C/Au@Pt nanocomposites for TMB. Accordingly, a suitable label-free colorimetric sensing platform was established, considering an inhibitory effect on the specific detection of H2O2 in a linear range of 0.3–2.75 M. This study presents an effective strategy to design and fabricate MXene-based peroxidase-like nanozymes and demonstrates their potential in the fields of catalysis and biomedicine.

A fluorescence-electrochemiluminescence dual-mode sensor based on a "switch" system for highly selective and sensitive K-ras gene detection

Herein, an fluorescence (FL)−electrochemiluminescence (ECL) dual-mode biosensor is constructed based on the dual-signal “turn-on” strategy of functionalized metal-organic frameworks nanosheets (RuMOFNSs)-tetraferrocene for K-ras gene detection, and the mechanism of bursting through front-line orbital theory is explained for the first time. Amino-functionalized tetraferrocene-labeled probe DNA molecules are linked to RuMOFNSs by covalent amide bonds, acting as FL and ECL intensity switches. The target DNA, complementary to the probe DNA, triggers cyclic amplification of the target by nucleic acid exonuclease III (Exo III), repelling tetraferrocene reporter groups away from RuMOFNSs and inhibiting the electron transfer process and photoinduced electron transfer (PET) effect. These phenomena induce a double turn-on of FL and ECL signals with a high signal-to-noise ratio. The developed FL-ECL dual-mode sensing platform provides sensitive detection of the K-ras gene with detection limits of 0.01 fM (the detection range is 1 fM to 1 nM) and 0.003 fM (the detection range is 0.01 fM to 10 pM), respectively. In addition, the proposed dual-mode sensor can be easily extended to detect other disease-related biomarkers by changing the specific target and probe base sequences, depicting potential applications in bioanalysis and early disease diagnosis.

Regulation of Target-activated CRISPR/Cas12a on Surface Binding of Polymer Dots for Sensitive Electrochemiluminescence DNA Analysis.

Polymer dots (Pdots) have emerged as a type of attractive electrochemiluminescence (ECL) emitter. However, the low ECL efficiency severely limits their practicability. In this work, we develop a sensitive ECL biosensing strategy for the detection of human papilloma virus subtype (HPV-16) DNA by using target-activated CRISPR/Cas12a to regulate the binding of Pdots-DNA to biosensor and local surface plasmon resonance (LSPR) effect of electrochemically deposited Au nanoparticles (depAuNPs) to enhance the ECL emission of Pdots bound on biosensor. The biosensor is prepared by simply assembling hairpin DNA on depAuNPs modified electrode. In the presence of target DNA, the designed specific CRISPR/Cas12a can be activated to digest single-stranded assistant DNA, which decreases the amount of hairpin DNA opened by assistant DNA to bind Pdots-DNA on the biosensor surface, thus reduces the ECL emission. The integration of target DNA-triggered catalysis and the LSPR effect of depAuNPs greatly improves the sensitivity of ECL analysis. Using HPV-16 DNA as a target model, the proposed method shows a limit of detection (LOD) of 3.2 fM at a signal-to-noise ratio of 3 and a detectable concentration range of 5.0 fM to 50 pM. The high sensitivity, excellent selectivity, good testing stability, and acceptable fabrication reproducibility of the designed ECL biosensing strategy demonstrate its potential application in DNA bioanalysis.

Self-Powered DNAzyme Walker Enables Dual-Mode Biosensor Construction for Electrochemiluminescence and Electrochemical Detection of MicroRNA.

Herein, an electrochemiluminescence (ECL) and electrochemical (EC) dual-mode biosensor platform with a self-powered DNAzyme walking machine was established for accurate and sensitive detection of miRNA-21. By employing a magnesium ion (Mn2+)-dependent DNAzyme cleavage cycling reaction, the walking machine was built by assembling DNAzyme walking strands and ferrocene (Fc)-labeled substrate strands on the Au nanoparticles and graphitic carbon nitride nanosheet (g-C3N4 NS)-covered electrode. The DNAzyme walking strand was first prohibited by a blocker strand. After the addition of target miRNA-21 and Mn2+, the DNAzyme walker could be activated and produce autonomous movements along the electrode track fueled by Mn2+-dependent DNAzyme-catalyzed substrate cleavage without additional energy supply. Notably, each walking step resulted in the cleavage of a substrate strand and the release of a Fc-labeled DNA strand fragment, allowing us to acquire an extreme ECL signal recovery of g-C3N4 inhibited by Fc. Meanwhile, numerous Fc-labeled DNA fragments escaped from the surface of the electrode, directly producing an obvious decrease in the square wave voltammetry (SWV) signal from Fc on the same sensing platform. This work not only avoided difficultly assembling various signal indicators but also significantly improved the sensitivity through using self-powered DNAzyme-walker amplification. Moreover, the proposed design employed the same reaction to produce two signal output modes, which could eliminate the interference from diverse reactive pathways on the outcome to mutually improve the accuracy. Therefore, the dual-mode miRNA-21 biosensor exhibited wide detection ranges of 100 aM to 100 nM with low detection limits of 54.3 and 78.6 aM by ECL and SWV modes, respectively, which provided an efficient and universal biosensing approach with extensive applications in early disease diagnosis and bioanalysis.

Electrochemical biomimetic enzyme cascade amplification combined with target-induced DNA walker for detection of thrombin

Fe-N-doped carbon nanomaterials (Fe-N/CMs) were designed as a novel biomimetic enzyme with excellent peroxidase-like activity to achieve high-efficient enzyme cascade catalytic amplification with the aid of glucose oxidase (GOx), which was further combined with target-induced DNA walker amplification to develop a sensitive electrochemical biosensor for thrombin detection. Impressively, massive output DNA was transformed from small amounts of target thrombin by highly effective DNA walker amplification as protein-converting strategy, which could then induce the immobilization of functionalized nanozyme on the electrode surface to achieve the high-efficient electrochemical biomimetic enzyme cascade amplification. As a result, an amplified enzyme cascade catalytic signal was measured for thrombin detection ranging from 0.01pM to 1nM with a low detection limit of 3 fM. Importantly, the new biomimetic enzyme cascade reaction coupled the advantages of natural enzyme and nanozyme, which paved an avenue to construct varied artificial multienzymes amplification systems for biosensing, bioanalysis, and disease diagnosis applications.

Self-assembly Induced Enhanced Electrochemiluminescence of Copper Nanoclusters Using DNA Nanoribbon Templates.

Copper nanoclusters (CuNCs) are attractive electrochemiluminescence (ECL) emitters as Cu is comparatively inexpensive, nontoxic, and highly abundant. However, their ECL yield is relatively low. Herein, we report that orderly self-assembly of CuNCs using DNA nanoribbon as the template (DNR/CuNCs) conferred the CuNCs with improved ECL properties compared with individual CuNCs in both annihilation and co-reactant processes. The DNR/CuNCs resulted in a high ECL yield of 46.8 % in K2 S2 O8 , which was ≈68 times higher than that of individual CuNCs. This strategy was successfully extended to other ECL emitters, such as gold nanoclusters and the Ru(bpy)3 2+ /TPrA system. Furthermore, as an application of DNR/CuNCs, a DNR/CuNC-based ECL biosensor with higher sensitivity was constructed for dopamine determination (two orders of magnitude lower than that previously reported), showing that DNR/CuNCs have a potential for application in ECL bioanalysis as a new type of superior luminophore candidate.

Self‐assembly Induced Enhanced Electrochemiluminescence of Copper Nanoclusters Using DNA Nanoribbon Templates

AbstractCopper nanoclusters (CuNCs) are attractive electrochemiluminescence (ECL) emitters as Cu is comparatively inexpensive, nontoxic, and highly abundant. However, their ECL yield is relatively low. Herein, we report that orderly self‐assembly of CuNCs using DNA nanoribbon as the template (DNR/CuNCs) conferred the CuNCs with improved ECL properties compared with individual CuNCs in both annihilation and co‐reactant processes. The DNR/CuNCs resulted in a high ECL yield of 46.8 % in K2S2O8, which was ≈68 times higher than that of individual CuNCs. This strategy was successfully extended to other ECL emitters, such as gold nanoclusters and the Ru(bpy)32+/TPrA system. Furthermore, as an application of DNR/CuNCs, a DNR/CuNC‐based ECL biosensor with higher sensitivity was constructed for dopamine determination (two orders of magnitude lower than that previously reported), showing that DNR/CuNCs have a potential for application in ECL bioanalysis as a new type of superior luminophore candidate.

Tracking metal enhanced fluorescence from Eschericcia coli nano-bio-assemblies within colloidal dispersions by static and 3D spectra emissions

It was studied the interactions of single Ultraluminescent 40 nm gold Core shell Nanoparticles (d40 Au@SiO2-Fl) with Eschericcia Coli bacteria (Esc. Col). By this manner, it was tracked from initial Nano-Bio-assembly formation (Au@SiO2-Fl-Esc. Col) towards inter-Nano-Biostructure interactions. In order to do that it was determined association constants between d40 Au@SiO2-Fl and Biostructures by 3D Fluorescence, fluorescence spectroscopy measurements. The association constant was related with non-covalent Nano-Biostructure interactions of 45 ± 2 biostructure-1; accompanied with a Quantum Yield ratio between the Bio-assembly and Nano-labeller of 3.6 ± 0.1 times, with the maximal concentration of bacteria addition. This phenomenon of interaction was tracked from the bulk of the colloidal dispersion. And, from these individual interactions, it was possible to generate Bioimaging by Laser Fluorescence Microscopy in real time of the dynamic of Bio-assembly formation as well as the by inter-Nano-Biostructures. These data leaded to the correlation of 3D Fluorescence spectroscopy within different bacteria concentrations. The recorded spectra showed increased values of intensities that fitted to a linear equation as model of the experimental data records. About analytical performances, it could be noted that optimized sensitivity by 3D Fluorescence was unless 65% higher than by static Fluorescence. Moreover, it should be mentioned that two distributions of emissions were recorded that correlated with Plasmonics coupling with natural emitters within the UV and MEF Nanolabeller emissions. Thus, optimizing conditions, LODs of 0.3x10 3 bacterium*mL−1 were determined. Moreover, by In Flow Cytometry was recorded different sensitivity values depending of the Laser excitation applied. These phenomena were explained by the development of different Nano-Bio-emitters species. In addition, in order to validate the methodology, it was quantified Biostructures from fortified aqueous samples. By 3D Fluorescence it was determined recoveries of (97 ± 8)x103, and (93 ± 5)x103 from upper and lower distributions of emissions recorded in 3D Fluorescence topographies. The reproducibility in all cases was by %CV of 98–99 %; therefore the proposed Bioassays showed interesting insights in Nano-Biotechnology with impact in varied Research fields.In this way, it was showed and discussed about Bio-analytical applications for Eschericcia Coli detection and quantification within colloidal dispersion with relative simple, accessible and portable fluorescence spectroscopy set ups. Therefore, it was given insights for accessible Nano-Biodetection based on Ultra-sensitive MEF Nanoplatforms for Bio-labeling using standard spectroscopic and compared by In Flow Citometry analysis as well.

Dextran-Functionalized Super-nanoparticle Assemblies of Quantum Dots for Enhanced Cellular Immunolabeling and Imaging

Colloidal semiconductor quantum dots (QDs) are a popular material for applications in bioanalysis and imaging. Although individual QDs are bright, some applications benefit from the use of even brighter materials. One approach to achieve higher brightness is to form super-nanoparticle (super-NP) assemblies of many QDs. Here, we present the preparation, characterization, and utility of dextran-functionalized super-NP assemblies of QDs. Amphiphilic dextran was synthesized and used to encapsulate many hydrophobic QDs via a simple emulsion-based method. The resulting super-NP assemblies or "super-QDs" had hydrodynamic diameters of ca. 90-160 nm, were characterized at the ensemble and single-particle levels, had orders-of-magnitude superior brightness compared to individual QDs, and were non-blinking. Additionally, binary mixtures of red, green, and blue (RGB) colors of QDs were used to prepare super-QDs, including colors difficult to obtain from individual QDs (e.g., magenta). Tetrameric antibody complexes (TACs) enabled simple antibody conjugation for selective cellular immunolabeling and imaging with both an epifluorescence microscope and a smartphone-based platform. The technical limitations of the latter platform were overcome by the increased per-particle brightness of the super-QDs, and the super-QDs outperformed individual QDs in both cases. Overall, the super-QDs are a very promising material for bioanalysis and imaging applications where brightness is paramount.

New High-Performance Fluorescent Dye Scaffolds: Applications for Bioimaging and Biosensing

AbstractFluorescence imaging in the shortwave infrared region (SWIR, 850–2500 nm) window has become an indispensable tool in biomedical research because it has weaker absorption, less light scattering, and less background fluorescence in this window. As we know, the design and synthesis of fluorescent dyes and fluorescent probes are the cores of fluorescence imaging and biosensing. Thus, their photophysical mechanisms exploring and bioanalytical applications are multidisciplinary and cutting-edge research topics. In this regard, we have been working on high-performance fluorescent dyes and fluorescent probes for years. In short, we have developed a series of bright, stable, aggregation-resistant, SWIR fluorescent dyes ECXs, based on a carbon-bridged spiro ring strategy. We also developed a series of high-performance SWIR fluorescent dyes CXs and Chrodols, which combine the structural advantages of cyanine and xanthenoid dyes. Based on these new SWIR scaffolds, we further constructed some activatable SWIR fluorescent probes with OFF-ON or ratiometric properties for biosensing in vivo. Therefore, the main line of our work is to gain an in-depth understanding of the photophysical mechanisms of fluorescent dyes, to create high-performance luminescent dyes, and to further develop fluorescent probes for bioimaging and biosensing.1 Introduction2 ECX Dyes Based on a Carbon-Bridged Spiro Ring Strategy3 Fluorescent Dyes and Fluorescent probes Combining the Structures of Cyanine and Xanthenoid Dyes3.1 CX Series Dyes3.2 A Functionalized Modified CX probe NRh3.3 CX-like Dyes with a Secondary Amino Py-23.4 Chrodol Series Dyes4 Conclusion and Future Prospects