Biological Detection Systems Research Articles

Carbon Nanotube Sensor Platforms for the Detection of Cellular Nitric Oxide Efflux Gradients

Cell communication is the ability to receive, process, and transmit signals from the environment and within cells to ensure functional coordination during physiological events. Cells communicate between themselves using waves of chemical concentration that change in both direction and time. Nitric oxide (NO) is a gaseous, ubiquitous, intra - and inter-cellular signaling molecule. NO’s small size and lipophilic nature allows it to diffuse through cell membranes and reach intracellular compartments of nearby cells. However, NO detection in biological systems represents a challenge due to NO’s short half-life (in the range of seconds) before breaking down into different subproducts. The lack of spatial NO detection, not an upstream or downstream indicator of NO, has hindered understanding of the extracellular NO dynamic signaling during both physiological and pathological events. However, single wall carbon nanotube (SWNT)-based fluorescent sensors allow for the detection of NO, and when immobilized onto a glass substrate, they can be used to quantify extracellular NO in a spatial-temporal manner. We detected changes in extracellular NO efflux from different macrophage cell lines known for their NO production under inflammatory responses, specifically human THP-1 monocytes and murine macrophage RAW 264.7. The cells were seeded on the SWNT platform and treated with Liposaccharides (LPS) 24 h to increase their NO release. The SWNT platform’s sensing response to NO was detected by changes in fluorescence intensity. Fluorescence data was collected on a custom-built hyperspectral microscope to give us near infrared (nIR) signal quantification with spatial resolution. Images were collected before and after the respective NO induction method. To analyze the extracellular NO mappings, bright field images of the cells were used to extract the cell coordinates and translate them to fluorescent images. Hyperspectral image analysis was performed with MATLAB to determine SWNT fluorescence below each cell and its surrounding area. To validate that the change in the fluorescence response was attributed to NO and not from cellular interaction with the SWNT sensor, we added 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a NO scavenger, after visualizing the SWNT quenching to recover the initial fluorescence signal, showing that the cells didn’t affect SWNT functionality. Through fluorescence intensity changes with pixel specificity on the image acquisition, we were able to spatially quantify NO released by cells with dynamic diffusion gradients. The newly developed platform provides the means to quantify extracellular NO. In this study, only two cell lines were studied, but the platforms will allow for the quantification of NO for other cell types as well. By comparing extracellular NO concentrations for different types of cells, we will be able to better understand physiological and pathological patterns of NO release, leading to a better understanding of cellular signaling.

An Innovative Aggregation-Induced Emission-Based NIR Fluorescent Probe for Visualizing Carboxylesterases in Living Cells, Zebrafish, and Tumor-Bearing Mice.

In the human body, carboxylesterases (CEs) play crucial roles in xenobiotic metabolism and lipid homeostasis. But abnormal expression of CEs is highly associated with some diseases, such as hyperlipidemia, diabetes, and liver cancer. Therefore, it is of great importance to develop an efficient tool for the accurate detection of CEs in living organisms. Herein, an innovative near-infrared (NIR) fluorescent probe, TTAP-AB, was designed for CE detection based on the aggregation-induced emission (AIE) mechanism. This probe exhibits rapid response (2 min), excellent sensitivity (limit of detection = 8.14 × 10-6 U/mL), and high selectivity to CEs. Additionally, owing to its good biocompatibility, the TTAP-AB probe enables the monitoring of dynamic changes in CE levels under drug-induced modulation in living cells and zebrafish. More importantly, the TTAP-AB probe was successfully employed to image liver tumors and assist in tumor resection through the real-time monitoring of CEs, indicating that TTAP-AB is promising to guide liver cancer surgery. Therefore, the TTAP-AB probe can not only enrich the strategies for CE detection in biological systems but also has great potential for some clinical imaging applications, including medical diagnosis, preclinical research, and imaging-guided surgery.

A novel “turn-on” NIR fluorescent probe based on benzothianone for the detection of palladium species and its application in cell and zebrafish

The development of an efficient palladium probe holds significant application value, considering the detrimental impact of palladium contaminants on human health. Thus, it is critical to create a sensitive detection method. To this end, a fluorescent probe TM-TPA-Pd based on benzothianone structure was designed, using allyl carbonate as the Pd0 recognition unit. TM-TPA-Pd exhibited high sensitivity (1.4 eq), selectivity, near-infrared (NIR) fluorescence (798 nm), and low detection limit (0.46 μM) for Pd0 with a rapid “turn-on” fluorescence signal (5 min). Furthermore, TM-TPA-Pd has extremely low cytotoxicity and has been successfully applied to detecting cells and zebrafish, which has great potential for palladium detection in biological systems.

Possible detection of atmospheric bioaerosol via LiDAR: a wavelength-based simulation study

This study explores potential of LiDAR technology to rapidly detect aerosolized biological terror agents in the atmosphere. It assesses the application by simulating extinction coefficients and the Ångström exponent at various wavelengths (266, 1064, 1571, and 2000 nm), focusing on differentiating bioaerosols from typical atmospheric particles. The simulation analysis evaluates changes in aerosol distributions and related extinction coefficient and Ångström exponent shifts under clean, normal, and bad atmospheric conditions. The findings indicate that the 1064 nm wavelength effectively detects bioaerosol presence, with a combination of 1064 nm and 1571 nm providing optimal Ångström exponent use for particle size differentiation. This dual-wavelength approach is highlighted as a practical method for bioaerosol detection, showcasing a significant sensitivity to variations in particle quantity and size, which are critical in biological threat scenarios. In conclusion, the study offers guidance for selecting LiDAR wavelengths for biological agent detection systems. While providing a theoretical framework for practical applications, it also underlines the need for further experimental work to confirm findings and fine-tune technology for real-world monitoring and threat management. This research contributes to the development of effective monitoring strategies against the backdrop of biological terror threats.Graphical

Highly focused beam generated with a height tuned micro-optical structure for high contrast microscopic imaging

Light sheet illumination technology improves the signal-to-noise ratio, resolution, and reduces scattered backgrounds for biological microscopic detection system. Here, we developed a novel micro-optical structure to produce a focused and uniform beam for the enhancement of imaging contrast. The beam intensity and working distance can be modified by adjusting the height and period of the structure. Our experiments successfully recorded structured light illumination, demonstrating the ability of the structure to capture high-contrast imaging data. We compared the light fields generated with and without the structure to assess the imaging quality, revealing a maximum 4.78-fold improvement in the signal-to-noise ratio. This work provides a potential method for high-resolution and high-contrast light sheet fluorescence microscopic detection.

Ferrocene probe-assisted fluorescence quenching of PEI-carbon dots for NO detection and the logic gates based sensing of NO enabled by trimodal detection

Our research demonstrates the effectiveness of fluorescence quenching between polyethyleneimine functionalised carbon dots (PEI-CDs) and cyclodextrin encapsulated ferrocene for fluorogenic detection of nitric oxide (NO). We confirmed that ferrocene can be used as a NO probe by observing its ability to quench the fluorescence emitted from PEI-CDs, with NO concentrations ranging from 1 × 10–6 M to 5 × 10–4 M. The photoluminescence intensity (PL) of PEI-CDs decreased linearly, with a detection limit of 500 nM. Previous studies have shown that ferrocene is a selective probe for NO detection in biological systems by electrochemical and colorimetric methods. The addition of fluorogenic NO detection using ferrocene as a probe enables the development of a three-way sensor probe for NO. Furthermore, the triple mode NO detection (electrochemical, colorimetric, and fluorogenic) with ferrocene aids in processing sensing data in a controlled manner similar to Boolean logic operations. This work presents key findings on the mechanism of fluorescence quenching between ferrocene hyponitrite intermediate and PEI-CDs, the potential of using ferrocene for triple channel NO detection as a single molecular entity, and the application of logic gates for NO sensing.

Singlet Oxygen Detection by Chemiluminescence Probes in Living Cells.

Singlet oxygen is a reactive oxygen species that causes oxidative damage to plant cells, but intriguingly it can also act as a signalling molecule to reprogram gene expression required to induce plant physiological/cellular responses. Singlet oxygen photosensitization in plants mainly occurs in chloroplasts after the molecular collision of ground-state molecular oxygen with triplet-excited-state chlorophyll. Singlet oxygen direct detection through phosphorescence emission in chloroplasts is a herculean task due to its extremely low luminescence quantum yield. Because of this, indirect alternative methods have been developed for its detection in biological systems, for example, by measuring the changes in the EPR signal or fluorescence intensity of singlet oxygen reaction-based probes. The singlet oxygen chemiluminescence (SOCL) is a chemiluminescence probe with high sensitivity and selectivity towards singlet oxygen and promising use to detect it in living cells without the inconvenience of low stability of the EPR signal of spin probes in the presence of redox compounds, spurious light scattering coming from the light source required for the excitation of fluorescence probes or the light emission of endogenous fluorescent molecules like chlorophyll in chloroplasts. The protocol presented in this chapter describes the first steps to characterizing singlet oxygen production within the biological system under study; this is accomplished through monitoring molecular oxygen consumption by SOCL using a Clark-type oxygen electrode and measuring the chemiluminescence generated by SOCL 1,2-dioxetane using a spectrofluorometer. For singlet oxygen detection within living cells, a version of SOCL with increased membrane permeability (SOCL-CPP) is described.

Umbelliferone and Its Synthetic Derivatives as Suitable Molecules for the Development of Agents with Biological Activities: A Review of Their Pharmacological and Therapeutic Potential.

Umbelliferone (UMB), known as 7-hydroxycoumarin, hydrangine, or skimmetine, is a naturally occurring coumarin in the plant kingdom, mainly from the Umbelliferae family that possesses a wide variety of pharmacological properties. In addition, the use of nanoparticles containing umbelliferone may improve anti-inflammatory or anticancer therapy. Also, its derivatives are endowed with great potential for therapeutic applications due to their broad spectrum of biological activities such as anti-inflammatory, antioxidant, neuroprotective, antipsychotic, antiepileptic, antidiabetic, antimicrobial, antiviral, and antiproliferative effects. Moreover, 7-hydroxycoumarin ligands have been implemented to develop 7-hydroxycoumarin-based metal complexes with improved pharmacological activity. Besides therapeutic applications, umbelliferone analogues have been designed as fluorescent probes for the detection of biologically important species, such as enzymes, lysosomes, and endosomes, or for monitoring cell processes and protein functions as well various diseases caused by an excess of hydrogen peroxide. Furthermore, 7-hydroxy-based chemosensors may serve as a highly selective tool for Al3+ and Hg2+ detection in biological systems. This review is devoted to a summary of the research on umbelliferone and its synthetic derivatives in terms of biological and pharmaceutical properties, especially those reported in the literature during the period of 2017-2023. Future potential applications of umbelliferone and its synthetic derivatives are presented.

Novel NIR fluorescent probe for hypochlorite ion detection in biological systems

This study presents the synthesis and application of a novel fluorescent probe, NR-ClO, for the detection of hypochlorite ion (ClO–) in biological systems. The probe was synthesized through a nucleophilic substitution reaction between Nile red and dimethylcarbamothioic chloride. The synthesized probe had high sensitivity and selectivity towards ClO–, with a detection limit of 75 nM and a linear range of 0.1–200 μM. The probe's efficacy was validated through in vitro studies using HepG2 cells and in vivo experiments using a mouse model of rheumatoid arthritis. The findings demonstrate that the NR-ClO probe is a promisingly reliable tool for real-time monitoring of ClO– in complex biological environments.

In-situ determination of ferric ions in human blood serum using highly fluorescence nitrogen and boron co-doped carbon dots nanoprobe without serum preparation step: A direct and solvent-free method

Different methods have been reported for Fe3+ ions determination as an essential element in many physiological processes, but most of these methods suffer from the consumption of solvent and time in the blood serum sample preparation stage. In this study, we report fabrication of a selective and sensitive high fluorescent nanoprobe based on the boron and nitrogen co-doped carbon dots for Fe3+ determination in aqueous solution and human blood serum as an important biological sample. Interestingly, under mildly acidic conditions, adjusted ionic strength, and the existence of EDTA, Fe3+ ions can be released from human serum transferrin, with no need for serum protein removal process as a separate step in sample preparation. Indeed, based on the innovative approach, both steps (consisting of Fe3+ ion release of protein and its detection) are in-situ performed. Notably, limit of detection of Fe3+ was improved from 4.72 μM to 0.61 μM and linear range from 40–250 μM to 1–500 μM, after optimization of conditions. Evaluation of obtained results by proposed method, was done using atomic absorption spectroscopy. The excellent precision and accuracy of achievement results, appear that designed nanoprobe is a promising novel method for ferric ion detection in biological systems.

Research Progress on Chiral Supramolecular Sensors for Enantiomer Detection

Chiral substances occur naturally in abiotic and living systems. The recognition and detection of chiral substances in the natural environment or their analysis and detection in biological systems are crucial. Chiral recognition is a research hotspot in clinical medicine, pharmacology, biochemistry, and other fields. Indeed, many researchers have developed various sensors with different functionalized materials for detecting and analyzing enantiomers. Supramolecular systems have important applications in the development of molecular recognition technologies, and the development of supramolecular chemistry is closely related to research on molecular devices. Therefore, this review summarizes the principle of chiral supramolecular sensors for the detection of enantiomers from the perspective of various sensor types, including optical, electrochemical, electrochemical luminescence, photoelectric, and supramolecular chemical sensors. This review also summarizes the relevant reports on chiral supramolecular sensors in the last five years. Finally, we highlight the prospects of supramolecular chiral sensors in future research.

Genetically encoded protein sensors for metal ion detection in biological systems: a review and bibliometric analysis.

Metal ions are indispensable elements in living organisms and are associated with regulating various biological processes. An imbalance in metal ion content can lead to disorders in normal physiological functions of the human body and cause various diseases. Genetically encoded fluorescent protein sensors have the advantages of low biotoxicity, high specificity, and a long imaging time in vivo and have become a powerful tool to visualize or quantify the concentration level of biomolecules in vivo and in vitro, temporal and spatial distribution, and life activity process. This review analyzes the development status and current research hotspots in the field of genetically encoded fluorescent protein sensors by bibliometric analysis. Based on the results of bibliometric analysis, the research progress of genetically encoded fluorescent protein sensors for metal ion detection is reviewed, and the construction strategies, physicochemical properties, and applications of such sensors in biological imaging are summarized.

Fluorescent Probes for HOCl Detection in Living Cells

Hypochlorous acid (HOCl) plays an important role in the immune system not only protecting the organism from pathogens, but also, due to its high reactivity, provoking the development and complication of many diseases. Therefore, the development of highly sensitive and selective HOCl biosensors, including fluorescent probes, to better understand the roles of HOCl in living systems, is of great importance. Nevertheless, there are many difficulties associated with HOCl registration in biological probes (chemo- and photostability, cytotoxicity, fluorescence and absorption characteristics, selectivity, sensitivity, etc.). Thus, the development of new chemosensors has been relevant for many years. In this review, we describe classification of small-molecule probes for HOCl detection in biological systems, as well as summarize the results of the development of chemosensors, their photophysical properties, and biological applications. Particular attention is paid to the achievements in this area over the years 2016–2021. A number of problems related to the design and application of small-molecule probes are formulated. Due to the absence of a “gold standard” among the HOCl chemosensors, which is associated with the commercial unavailability or complex methods of synthesis of new sensors as well, some recommendations are given regarding the field of the chemosensor application. Trends in the development of fluorescent chemosensors for HOCl visualization in living cells are outlined.

Label-free biological sample detection and non-contact separation system based on microfluidic chip.

The detection and separation of biological samples are of great significance for achieving accurate diagnoses and state assessments. Currently, the detection and separation of cells mostly adopt labeling methods, which will undoubtedly affect the original physiological state and functions of cells. Therefore, in this study, a label-free cell detection method based on microfluidic chips is proposed. By measuring the scattering of cells to identify cells and then using optical tweezers to separate the target cells, the whole process without any labeling and physical contact could realize automatic cell identification and separation. Different concentrations of 15 µm polystyrene microspheres and yeast mixed solution are used as samples for detection and separation. The detection accuracy is over 90%, and the separation accuracy is over 73%.

Highly selective fluorescent detection platform based on isoquinoline Schiff base ligand monitors glutathione in biological systems

Aberrant levels of intracellular glutathione (GSH) have been held responsible for various disease and pathological processes, and existing evidence underscores the significance of GSH detection in biological systems. The recognition, discrimination, and precise detection of GSH in the cellular matrix and living organisms can revolutionize our understanding of the physiological and pathological processes of GSH. Herein, we reported a new isoquinoline Schiff base fluorescence probe, N'-((7-(diethylamino)-2-oxo-2H-chromene)methylene) isoquinoline-3-carbohydrazide (NCMC), which can selectively recognize GSH by its ensemble complex NCMC-Cu2+ with a low nanomolar level detection limit (4.16 × 10−8 M) in the aqueous environment. Moreover, the binding behavior and “ON-OFF-ON” fluorescence probing pattern were illustrated in detail by UV–vis titration, ESI-MS spectroscopy, and density functional theory calculations. Notably, the nontoxic NCMC-Cu2+ ensemble platform showed great potential for detecting GSH distribution in cells and zebrafish larvae, and can also be applied in differentiating endogenous GSH in both cancer cells and normal cells, which may represent a promising GSH fluorescence probe in bioimaging and disease diagnosis.

Relationship between delayed luminescence emission and mitochondrial status in Saccharomyces cerevisiae

Delayed luminescence (DL) is gradually used in various detection of biological systems as a rapid detection technique, however, its biological mechanism was still not clear. In this study, a new model of DL detection system for liquid biological samples is established to investigate the DL emission of Saccharomyces cerevisiae cells cultured in different glucose concentrations. We analyzed the relationship between the DL emission and cell growth, cell vitality, mitochondrial morphology, mitochondrial DNA (mtDNA) copy number, adenosine triphosphate (ATP), oxygen consumption rate (OCR), as well as mitochondria membrane potential (MMP) in S. cerevisiae cells cultured with 0.01, 0.05, 0.15, 3, 10 and 20 g/L glucose respectively. It was found that the DL emission had strong correlation with mitochondrial morphology, OCR, and MMP. The results suggested that DL is an indicator of mitochondria status under different glucose supply conditions, and may be an effective method to detect mitochondrial metabolism related disorders.

Development of an esterase fluorescent probe based on naphthalimide-benzothiazole conjugation and its applications for qualitative detection of esterase in orlistat-treated biosamples

Esterase is a large hydrolysis family, and widely distributed in many kinds of cells. It is responsible for multiple physiological and pathological functions including metabolism, gene expression. While abnormality of esterase is associated with many pathological activities in obesity, Wolman's disease, and cancer. Thereby, it is essential to design an effective tool for esterase in situ detection in biological systems. Herein, a novel fluorescent probe Y-1 for monitoring esterase in living cells was rationally designed. Probe Y-1 was synthesized by the conjugation between an acetylation of 4-hydroxy naphthalimide and benzothiazole group. Benzothiazole moiety is a typical Excited-state intramolecular proton transfer (ESIPT) controller. Acetate group was selected as the responsive site and ESIPT initiator. As the acetate group could block the ESIPT effect, the probe emits no fluorescence under the excitation of 455 nm. When binding with esterase, Y-1 shows distinct fluorescence with the peak at 560 nm with short time when ESIPT is on. Y-1 displays high sensitivity (LOD is 0.216 × 10−3 U/mL), fast response (within 5 min), high selectivity and photostability towards esterase. Furthermore, the %RSD (relative standard deviation) of within-day and day-to-day precision was no more than 13.0% and the accuracy ranged from −6.5 to −12.3%. Kinetics performance of Y-1 indicates that esterase has high affinity and hydrolysis to Y-1. For biological applications, our probe is a time-dependent visualizing esterase in living HepG2 and CoLo205 cells within 15 min. After the treatment of orlistat (1 and 5 μM) for inhibiting the activity of esterase, the bright fluorescence has also been detected using our probe. Furthermore, it has been successful in monitoring the esterase in zebrafish, the data were consistent with cellular phenomena. Therefore, all these findings indicate that the robust probe Y-1 is a useful qualitative tool for detecting esterase in biological systems.

Porous Oxyhydroxide Derived from Metal-Organic Frameworks as Efficient Triphosphatase-like Nanozyme for Chromium(III) Ion Colorimetric Sensing.

The dephosphorylation that involves the removal of a phosphate group from a substrate molecule plays a significant role in living organisms. An enzyme mimic (nanozyme) with phosphatase-like catalytic activity has recently received attention in terms of its capacity for dephosphorylation. In this study, three types of highly porous oxyhydroxide with remarkable triphosphatase-like catalytic activities, ZrOOH, GdOOH, and HfOOH, have been prepared through the transformation of metal-organic frameworks (MOFs) using a simple alkaline hydrolysis method. The triphosphatase mimetic activities of ZrOOH, GdOOH, and HfOOH were then thoroughly investigated and verified. In particular, an isotopic tracing experiment revealed that abundant surface hydroxyls could serve as nucleophilic agents to directly attack the electropositive phosphorus atom, causing the cleavage of the terminal phosphoester bonds of phosphoester substrate molecules. The kinetic analysis provided calculated values of Km of 105.7, 90.5, and 46.1 μM, while the Vmax values were 3.57, 4.76, and 2.74 × 10-8 M s-1 and Ea values were estimated to be 47.52, 41.15, and 52.79 kJ/mol for ZrOOH, GdOOH, and HfOOH, respectively. The chromium(III) ions acting as "poisoning" inhibitors efficiently downregulated the triphosphatase mimetic activity of GdOOH. On the basis of this effect, a colorimetric chromium(III) ion-sensing system was explored, which provided a relevant linear response range for the detection of chromium(III) ions of 5.0-200 μM and a low detection limit of 0.84 μM. This work not only shows the great potential of ZrOOH, GdOOH, and HfOOH as triphosphatase nanozymes but also deepens our understanding of the role of surface hydroxyls on phosphatase-mimicking nanozyme catalytic dephosphorization, which could be used in the rational design of phosphatase-mimicking nanozymes. Furthermore, the developed colorimetric sensing system could be applied to chromium(III) ion detection in biological systems.

Polydopamine-mediated photothermal effect enables a new method for point-of-care testing of biothiols using a portable photothermal sensor

Abnormal levels of biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are associated with many pathological diseases. The demand for bulky and costly apparatuses represents a big impediment for point-of-care testing of biothiols. Herein, we developed a novel photothermal sensor for biothiols based on the formation of polydopamine nanoparticles (PDA NPs) using a thermometer as readout. A zeolitic imidazolate framework (ZIF-67) was first fabricated through a facile one-pot strategy. Then dopamine (DA) was immediately polymerized into PDA NPs with excellent photothermal effects (PETs) in the presence of the ZIF-67. Biothiols could disintegrate ZIF-67 by substituting ligands, causing the reaction between biothiols and cobalt ions to form complexes, and release 2-methylimidazole (2MIM) that can slowly oxidize DA, thereby "weakening" the PETs signal, which enabled us to convert biological signals into temperature signals. Therefore, the quantitative detection of Cys, Hcy and GSH can be achieved by recording temperature changes, with detection limits as low as 0.158 μM, 0.181 μM and 0.207 μM, respectively. Furthermore, the photothermal sensor has been applied in detection of biothiols in fetal bovine serum with satisfactory recovery rates, correlating well with the results by standard Ellman method, signifying its promising potential in integrated biological detection system.

A two-photon “turn-on” fluorescent probe for both exogenous and endogenous selenocysteine detection and imaging in living cells and zebrafish

Selenocysteine (Sec) is recognized as the 21st amino acid employing as an essential building block for selenoproteins (SePs), which plays a significant role in various physiological processes. Therefore, there is an urgent need to reasonably develop some reliable and rapid methods for Sec detection in biological systems. In this work, we reported a new two-photon (TP) fluorescent probe BNT-Sec for Sec detection and imaging in living cells and zebrafish with two part: (1) a D-π-A-structured naphthalene derivative as a TP fluorophore; (2) a well-know Sec responsive site with strong intromolecular charge transfer effect (ICT) to selectively detect endogenous and exogenous. In the presence of Sec, probe BNT-Sec can initiate a Se-dependent specific aromatic nucleophilic substitution reaction, which exhibited BNT-Sec had a large fluorescence intensity enhancement with ~18.9-fold at 510 nm, a high sensitivity low LOD value’ 10.6 nM, good light stability, strong specificity, pH stability and low cytotoxicity. In addition, BNT-Sec can be conveniently used to detect Sec in living cells and zebrafish for TP imaging due to the great TP measurement properties of fluorophore, exhibiting it has the potential to reveal the role of selenocysteine in physiological and pathological processes in further biological applications.