Application In Cell Imaging Research Articles

Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells.

Protein-protein-interactions play an important role in many cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced "blinking" and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N&B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for protein-protein-interactions quantification via fluorescence fluctuation-based methods.

Fluorescent Coumarin-based Probe for Detection of Biological Thiols

Abstract: Nature-derived thiols such as cysteine, homocysteine, and reduced glutathione play diverse and important roles in both plant and animal cells. In plants, they maintain redox homeostasis and affect photosynthesis and signal transduction processes. In animals, changes in the levels of biothiols have been linked to cardiovascular disease, Alzheimer's disease, neuropsychiatric disorders, or stroke. The detection of biothiols in biological samples is, therefore, very important. For this purpose, rapid, sensitive, and non-invasive methods for their sensing in living cells are highly desirable. : This perspective reviews the past five years of advances in coumarin fluorescent probes for biothiol detection. The design of the probes, the mechanisms of thiol detection, and the applications in live cell imaging are presented. The classification of probes based on the coumarin backbone was made on the basis of the mode of their interactions with the analyte.

Red emission upconversion of NaYF4: Yb3+/Er3+ nanoparticles: Rapid preparation via single-mode focused microwave method and their application in cell imaging

The red emission of upconversion nanoparticles (UCNPs) is located in an “optical window” range of the biological tissue, which has a promising prospect in biology application. In this work, novel red-emitting NaYF4:Yb3+/Er3+ UCNPs with CH3COO− modifications were synthesized by single-mode focused microwave method. The morphology, structure, and luminescence properties of as-prepared UCNPs were characterized. The results showed that single-mode focused microwave method was an effective approach to prepare hydrophilic and strongly red emission UCNPs within a drastically reduced reaction time (only 10 min) compared to the traditional methods. Importantly, we also demonstrated that the CH3COO− modified NaYF4:Yb3+/Er3+ UCNPs could be successfully applied for in vitro imaging of HeLa cells. This work not only provides a rapidly synthetic methodology for the preparation of red-emitting UCNPs but also offers an effective material for cancer cell imaging.

Base mismatching-fueled highly efficient catalytic hairpin assembly to regulate Ag nanoclusters brightness for amplifiable fluorescence biosensor

Exploring base mismatching-fueled catalytic hairpin assembly (mfCHA) is intriguing to regulate the fluorescence of DNA-scaffolded red Ag nanoclusters (rAgNC) for rapid and amplifiable biosensing of miRNA-155 (miR-155). Herein, we report the first design of a miR-155-responsive mfCHA route with rapid reaction kinetics and high conversion efficiency to light-up the fluorescence of rAgNC. The mfCHA was operated by a signaling hairpin (SH) programmed with the rAgNC template sequence and miR-155-discernible segment, and a G-rich (G12) enhancing hairpin (EH*). To implement faster and more efficient mfCHA amplification as a big challenge in traditional CHA, the locked stem of SH and the loop of EH* were encoded with single mismatched base in different sites, endowing mfCHA with more negative reaction standard free energy as driving force. Note that the originally synthesized rAgNC in SH was non-emissive. However, the presence of miR-155 initiated hairpin assembly between SH and EH* , leading to the displacement of miR-155 for repeated recycling and progressive mfCHA. Rationally, the red fluorescence of rAgNC was remarkably lighted-up due to the proximity to G12 enhancer in the shared dsDNA duplex products. Thus, this mfCHA-based assay platform displayed good simplicity, cost-effectiveness, rapidness, high sensitivity, and potential application for cell imaging.

A terpyridyl-rhodamine hybrid fluorescent probe for discriminative sensing of Hg (II) and Cu (II) in water and applications for molecular logic gate and cell imaging

Sensitive and discriminative sensing of more than one analyte with a single fluorescent probe is significant and challenging. Herein a new terpyridyl-rhodamine hybrid, namely TRH, has been rationally designed and prepared with two responsive groups in the molecular structure, which facilitate the discriminative detection of Hg2+ and Cu2+ ions in water with detection limits of 4.9 and 53.3 nM by ratiometric fluorescence change (F595/F485) and fluorescence quenching, respectively. Investigations show that the selectivity to Hg2+ ions can be attributed to Hg2+-promoted spirolactam ring opening and further hydrolysis, followed by a through-bond energy transfer (TBET) process. The selective fluorescence quenching to Cu2+ ions probably can be ascribed to the binding Cu2+ to terpyridyl that triggers a ligand-to-metal charge transfer (LMCT) process, which can also efficiently inhibit the TBET process induced by Hg2+ ions and promotes the discriminative sensing of Cu (II) and Hg (II). In addition, the fluorescent responses to Hg2+ and Cu2+ ions cover a wide pH range. Moreover, a combinatorial logic gate with the functions of NOR and INHIBIT has been fabricated by using Hg2+ and Cu2+ ions as chemical input signals, and fluorescence at 485 and 595 nm as output signals. Besides, TRH also exhibits sensitive and discriminative sensing ability to Hg2+ and Cu2+ ions by the fluorescence of rhodamine fluorophore. Significantly, based on the fluorescence signal output of rhodamine moiety, TRH can be used as a tracer for the discriminative sensing of Hg2+ and Cu2+ ions by using living cells.

Easily synthesizable molecular probe for the nanomolar level detection of Cd2+ in near aqueous media: Theoretical investigations and live cell imaging

The present investigation highlights a quinoline-based small molecule probe (DEQ) for the detection of Cd2+ among other metal ions in near-aqueous media. The probe DEQ and its Cd2+ complex (DEQ-Cd) have been synthesized and characterized by all possible spectroscopic methods. The weakly emissive DEQ showed its strong emission in the presence of Cd2+, which is attributed to the photoinduced electron transfer (PET) along with the chelation-enhanced fluorescence (CHEF) mechanism. The 1:1 binding mode between ligand and Cd2+ is confirmed by single crystal XRD analysis, which is further supported by Job’s plot and HRMS. The detection limit of the probe to recognize Cd2+ was found to be as low as 89 nM. Furthermore, DEQ can act as a reversible fluorescence probe with the off-on-off mechanism by the alternative addition of Cd2+ and EDTA. DFT and TD-DFT studies exposed the proposed mechanism after Cd2+ insertion and the obtained results for electronic spectra are in line with the experimental results. The response towards pH was quite interesting and allowed us to study its application in live cell imaging. With all the positive results, the proposed ligand DEQ can be used as a potential probe for the detection of Cd2+ in real-life applications.

Design of carbon dots for bioimaging and behavior regulation of stem cells.

Carbon dots (CDs) have been widely used in bioimaging, biosensing and biotherapy because of their good biocompatibility, optical properties and stability. In this review, we comprehensively summarize the research on CDs in terms of synthesis methods, optical propertiesand biotoxicity. We describe and envisage the directions for CDs application in stem cell imaging and differentiation, with the aim of stimulating the design of future related CDs. We used 'carbon dots', 'stem cells', 'cell imaging', 'cell differentiation' and 'fate control' as keywords to search for important articles. The Web of Science database was used to extract vital information from a total of 357 papers, 126 review articlesand 231 article proceedings within 12years (2011-2022).

Monoclinic yttrium oxide quantum dots surface modified by biotin for bioimaging applications

Semiconductor nanoparticles or quantum dots are one of the central topics of interest in material science research due to its size dependent physical and chemical characteristics and applications in different areas from display to biomedical fields. This work reports the first-time synthesis of biotin functionalized monoclinic yttrium oxide quantum dots at low temperature and its application in live cell imaging. The presence of biotin was confirmed through FTIR measurements. HRTEM analysis confirmed the formation of small spherical quantum dots with an average size of 3.8 nm. Zeta potential measurements gave a negative surface charge of -26.9 mV confirming colloidal stability. The quantum dots show sharp absorption in the short UV region and PL experiments delivered defect related intense blue fluorescence emission. The material is nontoxic to both normal (L929) and cancer (MCF 7) cells and the live cell imaging experiments performed on MCF 7 cells show specific binding to the cells and presented bright fluorescence emission from the cells thus confirming the applicability of the material in both display industry and cancer cell imaging.

Rational development of a peptide-based probe for fluorescence and colorimetric dual-mode detection of Cu2+ and S2− ions: Real application in cell imaging and test strips

A new dual-mode probe FAM-SSH with fluorescence and colorimetric properties was developed by solid-phase peptide synthesis, comprising 5-carboxy fluorescein (5-FAM) as a fluorophore, and tripeptide (Ser-Ser-His) as a recognition group. FAM-SSH not only displayed highly selective detection of Cu2+ based on fluorescence quenching mode, but also achieved colorimetric recognition of Cu2+ in solution, wherein a color change was observable to the naked eye. Additionally, the FAM-SSH-Cu2+ ensemble was highly selective for S2− over a wide pH range (7.0–12.0), characterized by a fluorescence enhanced response and colorimetric recognition, which was caused by the release of FAM-SSH and the precipitation of CuS. Moreover, the limit of detection (LOD) values for Cu2+ and S2− were 55.5 nM and 31.1 nM, respectively. Results of sample analyses and cell imaging experiments indicated that FAM-SSH has exciting field practicability and good cell permeability, and would be further useful for detection and imaging in environmental systems and living cells. Finally, test strips were produced by immersing them in FAM-SSH solution, thereby creating a method for portable visual detection. More importantly, a smartphone-assisted visual sensing platform was also developed for semi-quantitative Cu2+ and S2− detection with LOD values of 0.48 μM and 1.22 μM, respectively.

Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array.

Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples. We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM. A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells. An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a to acquisition speed advantage for the light sheet compared to the laser scanning geometry. FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

Functionalized β-NaGdF4:YbIII luminescent nanothermometer based on the direct population of triplet states and NIR emission

Lanthanide (LnIII)-doped NaGdF4 nanoparticles functionalized with organic ligands have been investigated for cell imaging and luminescence thermometry for instance due to their versatility regarding radiation conversion, e.g, near-infrared (NIR)-to-NIR, NIR-to-visible, or UV-to-NIR. Yet, in the later case, the high energy of radiation into the human tissue is an issue, aiming cell imaging application. This drawback could be surpassed by shifting the absorption bands of the organic luminescent sensitizers attached on the particle to longer wavelengths, which can be achieved by singlet (S0) → triplet (Tn) transitions instead of the classic S0 → Sn processes. Herein, this possibility is discussed for hybrid systems based on spheroidal β-NaGdF4:YbIIII nanoparticles (average diameter of 7.8 nm) functionalized on the surface with 3,5-dibromobenzoate (3,5-bbza-) coordinated to LnIII ions. The Tn states of 3,5-bbza- are directly populated upon excitation at 368 nm or 430 nm, followed by energy transfer to YbIII that emits within the NIR spectral region. On the other hand, the ligand singlet states are populated at 300 nm. Taking advantage of the intensity ratio of the emission spectrum collected upon S0 → Tn (368 nm or 430 nm) or S0 → Sn (300 nm) excitation, luminescence thermometry was performed for the hybrid system, which displays maximum relative thermal sensitivity of 0.58% K−1 at 77 K. Therefore, the S0 → Tn transitions observed in the NaGdF4:YbIIII nanoparticles functionalized with 3,5-bbza- enable visible-shifted excitation bands and luminescence thermometry, which are interesting features aiming application in cell imaging and temperature probing, respectively.

Circularly Polarized Luminescence Switching, Chirality Self‐Sorting, and Cell Imaging of Chiral Rhodamine Dyes

AbstractRhodamine dyes, as star molecules of fluorescent dyes, are critical tools in modern analytical and biochemical research. However, switching their chiroptical properties still remains a big challenge because of the absence of a suitable synthesis method to incorporate through conjugation a chiral fragment into the chromophore core of rhodamine. Herein, a simple and straightforward way is utilized to link two rhodamine chromophores by different chiral bridges. The conjugated RRR/SSS birhodamine dyes with one axial chirality, which is constructed directly by two rhodamine chromophores, and two spirocyclic chiralities show the interesting circular dichroism and circularly polarized luminescence switching and lysosome‐targetable properties. On the other hand, the unconjugated RR/SS birhodamine dyes with two chiral carbons do not have such chiroptical switching properties but do have different amazing cellular staining properties. The RR birhodamine dye can stain both lysosomes and mitochondria, but SS birhodamine dye can stain lysosomes only. Moreover, the mechanisms of chiroptical switching and diastereoselectivity are investigated as well, based on single‐crystal structures and density functional theory calculations. Therefore, a new platform is afforded here in designing chiral dyes for chiroptical switching and cell imaging applications.

Highly sensitive fluorescent probes for selective detection of hypochlorite: Applications in blood serum and cell imaging.

Being one of the vital reactive oxygen species (ROS), abnormal level of hypochlorite ion (ClO-) may pose detrimental threats to living organisms. Therefore, highly selective, and rapid monitoring of ClO- in living system is of prime importance to protect living organisms from its harmful effects. In this regard, design of synthetic fluorescent probes for ClO- has garnered considerable attention. However less fluorescence emission in aggregated state and less photostability of several existing probes for ClO- inspired us to design aggregation induced emission (AIE) active fluorescent probes SH1 and SH2. Probes were rationally designed by introducing thiourea moiety that selectively reacted through desulfurization reaction and resulted in highly selective detection of ClO-. Hypochlorite induced desulfurization reaction was validated through 1H NMR titration and DFT studies. Fine tuning of probes SH1 and SH2 prompted highly sensitive nanoscale (55nM and 77nM) and rapid (15 and 35 sec) detection of ClO-. Probe SH1 displayed less cytotoxic effect to live cells before it was successfully applied for bioimaging of ClO- in live MCF-7 cells. Moreover, probes displayed excellent sensing potential for ClO- in blood serum and real water samples. Advantageously, probe coated portable fluorescent films were fabricated for the easy and fast monitoring of ClO-. Of note, this work offers excellent design strategy for highly selective detection of ClO- that may lead to clinical trials.

A dual colorimetric fluorescent probe with large Stokes shift for F- detection in the near infrared and its application in cell imaging

In this study, we have successfully designed and synthesized a colorimetric and near-infrared fluorescent probe YC-Si for F- detection based on malononitrile isophorone derivatives which can selectively make Si-O bond breakage probes that can respond specifically to F- and have high sensitivity with a detection limit of 6.59 × 10-7 mol/L. The probe YC-Si has a large Stokes shift at 678 nm (168 nm). In cell imaging experiments it was demonstrated that YC-Si has cell permeability and low cytotoxicity and can be used to detect F- in living cells..

Precisely designed oxazolonaphthoimidazo[1,2-a]pyridine-based sensor for the detection of Fe3+ and DCP with cell imaging application

Two new turn-off fluorescent sensors (V3 and V4) presented in the article revealed the potential application for the precise detection of Fe3+ and diethylchlorophosphate (DCP). These sensors include oxazolonaphthoimidazo[1,2-a]pyridine scaffold integrated with anthracene and pyrene-based framework. This design has facilitated the twisted intramolecular charge transfer (TICT) and planarised intramolecular charge transfer (PLICT) mechanisms, which were confirmed using computational and photophysical studies. The V3 and V4 fluorescent probes were particularly sensitive and highly selective for detecting Fe3+ and DCP analytes. For Fe3+, V3 and V4 undergo turn-off mechanism with the detection limit of 14.1 and 4.5 nM, respectively. Intracellular detection of Fe3+via confocal live cell imaging was also demonstrated, showing its application under intracellular conditions. Our experimental data revealed the promises of V3 and V4 for instantaneous, accurate and on-spot monitoring of Fe3+ and DCP, even in the presence of other interfering analytes.

Room temperature synthesized metal organic frameworks of Lawsonia inermis: Potential candidates for sensing and cellular imaging

A novel environmentally benign synthesis of Zn and Fe based metal organic frameworks (MOFs) of Lawsonia inermis leaf extract prepared in-house by a simple room temperature extraction technique is presented. BET isotherm and HRTEM analyses revealed mesoporous nature of the newly synthesized MOFs [6.4 nm (Zn-NLw), 3.6 nm (Fe-NLw)]. Their potential for host-guest interaction facilitating sensing applications was demonstrated by selective binding of histidine (His) and tryptophan (Trp) recognized from fluorescence. Addition of His and Trp, induced significant changes in the emission maxima of MOFs (at 470 nm) demonstrating their sensing capacity for these molecules. Host-guest interaction was further investigated by electrochemical studies evidenced as a function of variation in oxidation-reduction peaks. Cytocompatibility of the MOFs were assessed by MTT assay, impact on cellular architecture, degree of cellular uptake while their application in cell imaging was investigated by confocal microscopy analysis. The results suggest excellent biocompatibility of Zn-NLw and Fe-NLw in vitro with significant level of uptake by cells. Fe-NLw exhibited time dependent subcellular localization with distinct luminescence characteristics at a short time period of 30 min as well as at 2 h. These results therefore validate the potential application of Zn-NLw and Fe-NLw in biosensing and fluorescence based bioimaging.

Novel Bis-pyrazoline Fluorescent Probe for Cu2+ and Fe3+ Detection and Application in Cell Imaging.

A fluorescent probe Y((1,1'-([1,1'-biphenyl]-4,4'-diylbis(3-(2-hydroxyphenyl)-4,5-dihydro-1H-pyrazole-5,1-diyl)) bis(ethan-1-one))) was designed and synthesized, which could be used to Cu2+ and Fe3+ sensors. Through the study of optical properties, the probe Y shows good selectivity and sensitivity to Cu2+ and Fe3+ in aqueous tetrahydrofuran solution [10.0mM HEPES, pH 7.4, THF-H2O = 9:1(v/v)] with has excellent anti-interference performance, and its detection limits were 0.931uΜ for Cu2+ and 0.401uΜ for Fe3+. The coordination mechanism of probe Y with Cu2+ and Fe3+ was speculated and verified at DFT level and HRNM. By Hela cytotoxicity and imaging tests, probe Y not only has good biocompatibility, but also can be used for sensing Cu2+ in cells.

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.

Microencapsulation of Lead-Halide Perovskites in an Oil-in-Fluorine Emulsion for Cell Imaging.

The superior optical properties of lead-halide perovskites (LHPs) inspired significant research in cell imaging applications; many encapsulating processes have improved perovskite stabilities with comparable biosafety. Herein, facile solvent evaporation encapsulation based on an oil-in-fluorine emulsion for aqueous-stable and extremely nontoxic LHP microcapsules is described. Perfluorooctane dispersed the emulsifier fluorocarbon surfactant to form a continuous fluorine phase, while LHPs and polymethylmethacrylate (PMMA) were dispersed in 1,2-dichloroethane, then emulsified in the fluorine phase to form an oil-in-fluorine emulsion. CsPbBr3 microcapsules with a dense PMMA shell that protect fragile CsPbBr3 from the external environment and inhibit lead ion release were obtained after solvent evaporation. The CsPbBr3 microcapsules not only retained 91% of fluorescence intensity after exposure to water for 30 d but also possess extremely low cytotoxicity for MCF-7 cells. After exposure to 2 mg/mL of CsPbBr3 microcapsules for 48 h, the cell viability remained >90%. The intracellular uptake of CsPbBr3 microcapsules indicates its potential use in cell imaging.

Rhodamine B-Quinoline based schiff base as fluorescent ‘turn on’ sensor of Al3+, Cr3+, HSO4− and its cytotoxicity and cell imaging application on TPC-1 and HtH-7 cell lines

A designed Rhodamine-B-quinoline based Schiff base chemosensor (RhBQ) has been synthesized and characterized using various spectroscopic techniques. It shows ‘turn on’ fluorescence emission and also colorimetric sensitivity and selectivity towards trivalent metal ions (Al3+ and Cr3+) and anion (HSO4−) present in aqueous medium. Stoichiometry of both metal (Al3+ & Cr3+) - RhBQ complexes are found to be 1:1 using Job’s plot. A notable enhancement in emission intensity for RhBQ-Al3+ (∼341 fold), RhBQ-Cr3+ (∼292 fold), and RhBQ-HSO4− (∼136 fold) complexes compare to RhBQ are observed. Sensing mechanism i.e. opening of spirolactam ring of RhBQ has been confirmed through various spectroscopic techniques such as; 1H NMR, 13C NMR, ESI-MS, TRPL, FT-IR, and DFT based computational study. The limit of detection (LOD) for Al3+, Cr3+, and HSO4− are found to be 2.2 × 10−8 M, 2.12 × 10−8 M and 8.63 × 10−7 M respectively. An advanced level 3 input OR–INHIBIT logic gate are constructed using chelating ligand EDTA. For onsite detection, TLC paper strip spot of the probe, RhBQ is prepared and the detection of Al3+ in few antacid drug are presented. In addition, cell-imaging analysis of Al3+, Cr3+, and HSO4− ions in the Human papillary thyroid carcinoma cell line (TPC-1) & Human anaplastic thyroid cancer cell lines (HtH-7) are presented and shows promising concentration and time-dependent detection results.