Direct Fluorescence Research Articles

Direct frequency domain fluorescence lifetime imaging using simultaneous ultraviolet and visible excitation.

Due to the complexity, limited practicality, and cost of conventional fluorescence lifetime imaging/microscopy (FLIM) instrumentation, FLIM adoption has been mostly limited to academic settings. We present a novel point scanning frequency-domain (FD) FLIM instrumentation design capable of simultaneous multi-wavelength excitation, simultaneous multispectral detection, and sub-nanosecond to nanosecond fluorescence lifetime estimation. Fluorescence excitation is implemented using intensity-modulated CW diode lasers that are available in a selection of wavelengths spanning the UV-VI-NIR range (375-1064 nm). Digital laser intensity modulation was adopted to enable simultaneous frequency interrogation at the fundamental frequency and corresponding harmonics. Time-resolved fluorescence detection is implemented using low-cost, fixed-gain, narrow bandwidth (100 MHz) avalanche photodiodes, thus, enabling cost-effective fluorescence lifetime measurements at multiple emission spectral bands simultaneously. Synchronized laser modulation and fluorescence signal digitization (250 MHz) is implemented using a common field-programmable gate array (FPGA). This synchronization reduces temporal jitter, which simplifies instrumentation, system calibration, and data processing. The FPGA also allows for the implementation of the real-time processing of the fluorescence emission phase and modulation at up to 13 modulation frequencies (processing rate matching the sampling rate of 250 MHz). Rigorous validation experiments have demonstrated the capabilities of this novel FD-FLIM implementation to accurately measure fluorescence lifetimes in the range of 0.5-12 ns. In vivo endogenous, dual-excitation (375nm/445nm), multispectral (four bands) FD-FLIM imaging of human skin and oral mucosa at 12.5 kHz pixel rate and room-light conditions was also successfully demonstrated. This versatile, simple, compact, and cost-effective FD-FLIM implementation will facilitate the clinical translation of FLIM imaging and microscopy.

Ultrasensitive Hybridization Chain Reaction-Assisted Multisite Exonuclease III Amplification Strategy Combined with a Direct Quantitative Fluorescence Lateral Flow Technique for Multiple Bacterial 16S rRNA Detection.

Accurate and in-time detection of bacteria conduces to preventing their rapid spread around the environment, while a nucleic acid test (NAT) is a powerful tool for early diagnosis of pathogens. Herein, we propose a hybridization chain reaction (HCR)-mediated multisite exonuclease III (Exo-III) amplification strategy (HCR/Exo-III amplifier) to achieve the one-pot and ultrasensitive isothermal amplification of bacterial 16S rRNA and a portable fluorescence detection device (PFD) to directly read signals in a lateral flow assay (LFA). In detail, the target-initiated HCR products present multiple binding sites for triggering the Exo-III amplifier that produces numerous target amplicons. Following that, the target amplicons travel up on the strip and bridge between the DNA-CdTe/CdS probes and the capture DNA to form a positive fluorescence line. After that, the strip is inserted into the PFD to accomplish the fluorescence signal reading. The constructed HCR/Exo-III amplifier-based PFD-LFA implemented the simultaneous and specific detection of three bacteria with a detection limit of a few tenths of fM for synthetic 16S rRNA fragments and dozens of CFU/mL for Staphylococcus aureus, Listeria monocytogenes, and Salmonella typhimurium in pure cultures. The sensing platform features isothermal amplification, convenient operation, and good economy, displaying great potential for on-site testing toward multiple nucleic acid analytes.

Evaluation of Reliability and Validity of Occlusal Caries Detection by Direct Visual, Indirect Visual and Fluorescence Camera Using ICDAS II (Codes 0, 1, and 2): An In Vivo Study.

Early detection of carious lesions paves the way for the preservation of tooth structures by remineralization strategies. Visual examination using international caries detection and assessment system (ICDAS) II has been validated with the gold standard of histological classification. A fluorescence camera is yet another diagnostic aid for the early detection of carious lesions based on the autofluorescence of the enamel and dentin. This study was done to evaluate the validity of the fluorescence camera (Soprocare and Acteone) in detecting early carious lesions. A total of 690 occlusal surfaces of non-cavitated premolars and molars were examined by examiners 1 and 2, trained in the ICDAS II grading system. The photographs of the occlusal surface and the fluorescence images, which were taken using a Soprocare (Acetone) intraoral camera, were examined by examiners 3 and 4. The scoring was tabulated and correlated. The κ values for interexaminer reproducibility of indirect visual was 0.841 (good), and his fluorescence camera was 1.00, which is very good. The correlation analysis revealed that there was a higher correlation between direct visual and indirect visual for both examiners when compared to direct and fluorescence cameras. There was a positive relationship between indirect visual and fluorescence cameras for both the experimenter). The indirect visual method for the detection of carries has high sensitivity and specificity irrespective of the examiner. Examination by fluorescence camera has a low sensitivity and high specificity. The specificity of the caries detection method by indirect visual examination based on ICDAS II coding that of the Soprocare fluorescence camera was consistent and reliable, whereas indirect visual examination had a high sensitivity for detecting ICDAS codes 1 and 2. Soprocare showed a very low sensitivity in detecting ICDAS code 1 and 2 lesions. Dhanavel C, Sai CK, Neelamurthy PS, et al. Evaluation of Reliability and Validity of Occlusal Caries Detection by Direct Visual, Indirect Visual and Fluorescence Camera Using ICDAS II (Codes 0, 1, and 2): An In Vivo Study. Int J Clin Pediatr Dent 2023;16(1):74-78.

Rapid and sensitive fluorescence determination of oxytocin using nitrogen-doped carbon dots as fluorophores

In this work, a novel nitrogen (N)-doped carbon dots (N-CDs) was prepared with quercetin as the carbon source and o-phenylenediamine as the nitrogen source by hydrothermal synthesis, and their application as fluorophores for selective and sensitive determination of oxytocin were reported. The fluorescence quantum yield of the as-prepared N-CDs, which exhibited good water solubility and photostability, was about 6.45 % using rhodamine 6 G as reference substance, and the maximum excitation (Ex) and emission (Em) wavelength were 460 nm and 542 nm, respectively. The results illustrated that the direct fluorescence quenching of N-CDs fluorophore for the detection of oxytocin achieved good linearity in the range of 0.2–5.0 IU/mL and 5.0–10.0 IU/mL, the correlation coefficients were 0.9954 and 0.9909, respectively, and the detection limit was 0.0196 IU/mL (S/N = 3). The recovery rates were 98.8∼103.8 % with RSD= 0.93 %. The interference experiments showed that common metal ions, possible impurities introduced in production and coexisting excipients in the preparation had little adverse influence on selective detection of oxytocin by the developed N-CDs based fluorescent detection method. The mechanism study on the fluorescence quenching of N-CDs by oxytocin concentrations under the given experimental conditions demonstrated that there were internal filtration effect and static quenching in the system. The developed fluorescence analysis platform for the detection of oxytocin had been proved to be rapid, sensitive, specific and accurate, and to be used for the quality inspection of oxytocin.

High-throughput, modular and logic analysis platform based on binary photonic crystals array and entropy-driven amplifier for multiplex biomarkers detection

The development of high-throughput and modular assay of multiplex biomarkers has great significance for disease diagnosis but it still faces some difficulty. Herein, a high-throughput, modular and logic analysis platform combining binary photonic crystals (PCs) array and entropy-driven DNA amplifier (EDA) was developed to achieve simple, high-sensitive, and nonenzymic detection of multiplex biomarkers. In this platform, three modules including logic-recognition, EDA and binary PCs array were integrated, and each module had independent and universal design but connected functions. The binary PCs array was fabricated conveniently through stacking self-assembly of two kinds of colloidal nanospheres, and could specifically enhance two kinds of fluorescence based on the match between emission peak and photonic band gap. Functionally, the input of two mRNA biomarkers initiated the logical operation of logic-recognition module, and the released DNA strand triggered corresponding EDA module for amplified fluorescence signal output (FAM or Cy5). The whole DNA circuit was performed in one step. Then, binary PCs array module further enhanced both two fluorescence signals, and got direct fluorescence signal readout by microplate reader (scanning rate of 96 samples/ 7 min). It is the first time that the binary PCs array-based high-throughput platform has achieved logical and sensitive detection of multi-biomarkers by employing logic gate and EDA. Therefore, the proposed platform has merits of logical analysis, high sensitivity, programmable design, high throughput and multiplex detection, showing potential application in bioanalysis and clinical diagnostics.

Survey on Dermatophytes Isolated from Animals in Switzerland in the Context of the Prevention of Zoonotic Dermatophytosis

Most inflammatory dermatophytoses in humans are caused by zoophilic and geophilic dermatophytes. Knowledge of the epidemiology of these fungi in animals facilitates the prevention of dermatophytosis of animal origin in humans. We studied the prevalence of dermatophyte species in domestic animals in Switzerland and examined the effectiveness of direct mycological examination (DME) for their detection compared to mycological cultures. In total, 3515 hair and skin samples, collected between 2008 and 2022 by practicing veterinarians, were subjected to direct fluorescence microscopy and fungal culture. Overall, 611 dermatophytes were isolated, of which 547 (89.5%) were from DME-positive samples. Cats and dogs were the main reservoirs of Trichophyton mentagrophytes and Microsporum canis, whereas Trichophyton benhamiae was predominantly found in guinea pigs. Cultures with M. canis significantly (p < 0.001) outnumbered those with T. mentagrophytes in DME-negative samples (19.3% versus 6.8%), possibly because M. canis can be asymptomatic in cats and dogs, unlike T. mentagrophytes, which is always infectious. Our data confirm DME as a reliable, quick, and easy method to identify the presence of dermatophytes in animals. A positive DME in an animal hair or skin sample should alert people in contact with the animal to the risk of contracting dermatophytosis.

The Distinction between Dematiaceous Molds and Non-Dematiaceous Fungi in Clinical and Spiked Samples Treated with Hydrogen Peroxide Using Direct Fluorescence Microscopy

Dematiaceous fungi are pigmented molds with a high content of melanin in their cell walls that can cause fatal infections in immunocompromised hosts. Direct microscopy is the main method for the rapid diagnosis of dematiaceous fungi in clinical specimens. However, it is often difficult to distinguish their hyphae from non-dematiaceous hyphae and yeast pseudohyphae. Our aim was to develop a fluorescence staining method that targets melanin for the detection of dematiaceous molds in clinical specimens. Glass slide smears of clinical samples and sterile bronchoalveolar lavage spiked with dematiaceous and non-dematiaceous fungi were treated with hydrogen peroxide, and digital images were recorded using direct microscopy with different fluorescent filters. The images of fungi were compared for their fluorescence intensity using the NIS-Elements software. The fluorescent signal between dematiaceous and non-dematiaceous fungi demonstrated a markedly increased mean intensity for dematiaceous molds following hydrogen peroxide treatment (7510.3 ± 10,427.6 vs. 0.3 ± 3.1, respectively, p < 0.0001). No fluorescent signal was detected in the absence of hydrogen peroxide. "Staining" fungal clinical specimens with hydrogen peroxide, followed by fluorescence microscopy examination, can differentiate between dematiaceous and non-dematiaceous fungi. This finding can be used for the detection of dematiaceous molds in clinical specimens and enables the early and appropriate treatment of infections.

Room-Temperature Photochromism of Silicon Vacancy Centers in CVD Diamond.

The silicon vacancy (SiV) center in diamond is typically found in three stable charge states, SiV0, SiV-, and SiV2-, but studying the processes leading to their formation is challenging, especially at room temperature, due to their starkly different photoluminescence rates. Here, we use confocal fluorescence microscopy to activate and probe charge interconversion between all three charge states under ambient conditions. In particular, we witness the formation of SiV0 via the two-step capture of diffusing, photogenerated holes, a process we expose both through direct SiV0 fluorescence measurements at low temperatures and confocal microscopy observations in the presence of externally applied electric fields. In addition, we show that continuous red illumination induces the converse process, first transforming SiV0 into SiV- and then into SiV2-. Our results shed light on the charge dynamics of SiV and promise opportunities for nanoscale sensing and quantum information processing.

Recent Advances in N-Heterocyclic Small Molecules for Synthesis and Application in Direct Fluorescence Cell Imaging.

Nitrogen-containing heterocycles are ubiquitous in natural products and drugs. Various organic small molecules with nitrogen-containing heterocycles, such as nitrogen-containing boron compounds, cyanine, pyridine derivatives, indole derivatives, quinoline derivatives, maleimide derivatives, etc., have unique biological features, which could be applied in various biological fields, including biological imaging. Fluorescence cell imaging is a significant and effective imaging modality in biological imaging. This review focuses on the synthesis and applications in direct fluorescence cell imaging of N-heterocyclic organic small molecules in the last five years, to provide useful information and enlightenment for researchers in this field.

UHECR anisotropy and extragalactic magnetic fields with the Telescope Array

We study the energy-dependent distribution of ultra-high energy cosmic ray arrival directions with respect to luminous matter in the local Universe. We use a specially designed test statistic (TS) that is robust to uncertainties of the galactic magnetic field. We generate realistic mock UHECR sets assuming various injected compositions, and different strengths of the extragalactic magnetic field (EGMF). Applying the TS to both mock sets and the Telescope Array Surface Detector data we constrain, for a given EGMF strength, the UHECR injected mass composition at energies above 10 EeV. At highest energies, higher than 100 EeV, only heavy composition — iron or at least silicon — is compatible with the data, irrespective of the EGMF strength. We then compare the obtained results with the direct Telescope Array fluorescence measurements of the UHECR mass composition. Requiring that the TA composition measurements are compatible with the arrival direction distribution allows us to constrain the parameters of the EGMF. It appears that light composition, measured by TA at around 10 EeV, is compatible with the arrival directions if the EGMF has strength of order 1 nG.

Direct fluorescence labelling of NO inside plant cells.

Nitric oxide (NO) plays a key role in regulating plant growth, enhances nutrient uptake, and activates disease and stress tolerance mechanisms in most plants. NO is marked as a potential tool for improving the yield and quality of horticultural crop species. Research on NO in plant species can provide an abundance of valuable information regarding this. Hence, we have prepared a simple chemosensor (NPO) for the detection of endogenous NO in chickpea saplings. NPO selectively interacts with NO as determined through a chemodosimetric method to clearly show both the colorimetric and fluorometric changes. After the interaction with NO, the colorless NPO turns yellow as observed by the naked eye and shows bright cyan-blue fluorescence under a UV lamp. The 1 : 1 stoichiometric ratio between NPO and NO is determined from Job's plot resulting in a stable diazeniumdiolate product. The interaction mechanism is well established by absorption, fluorescence titration, NMR titration, HRMS, and DFT calculations. This method has successfully been employed in the plant's root and stem systems to label NO. Confocal microscopy images might help us to understand the endogenous NO generation and the mechanism that happens inside plant tissues.

Hematological and serum biochemical profiles of a natural African swine fever virus infection in pigs.

African swine fever (ASF) is a contagious viral disease that affects pigs of all ages, inducing hemorrhagic fever with high mortality and severe threat to pig production. This study investigated the hematological and serum biochemical abnormalities associated with a natural ASF infection in pigs. A total of 100 serum samples of pigs from piggery suspected of ASFV infection were screened for antibodies by ELISA. Thirty‑two blood samples from serologically positive pigs and 32 negative pigs were undergo to hematological and serum biochemical analyses following standard procedures. The results showed that the mean values of the red blood cell (RBC) count, total white blood cell (TWBC) count, absolute lymphocyte count, absolute monocyte count, serum total protein (TP) and globulin were significantly (p < 0.05) lower while the mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), absolute neutrophil count and serum gamma glutamyl transferase (GGT) were significantly (p < 0.05) higher in the infected than the healthy pigs. There were no significant differences (p > 0.05) in the mean values of the packed cell volume (PCV), hemoglobin concentration, absolute eosinophil count, cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities between the infected and healthy pigs. Hence, natural ASFV infection may have caused alterations in the hematological and serum biochemical parameters in the infected pigs. The generated data could complement the existing laboratory diagnostic techniques such as polymerase chain reaction, direct fluorescence antibody test, indirect fluorescent antibody test and ELISA in the diagnosis of ASF in pigs.

Direct blood fluorescence signal intensity of neutrophils (NEU-SFL): A predictive marker of death in hospitalized COVID-19 patients?

Coronavirus disease 2019 (COVID-19) is a respiratory disease triggered by immunopathological mechanisms that cause excessive inflammation and leukocyte dysfunction. Neutrophils play a critical role in the innate immunity and are able to produce neutrophil extracellular traps (NETs: NETosis process) to combat infections. Some NETs markers are increased in patients who died from COVID-19. Recently, the neutrophil fluorescence variable (NEU-SFL), available on certain automated complete blood count (CBC) analyzers, has been correlated with NET formation and may reflect NETosis in patients. Here we evaluate whether NEU-SFL measured after admission of COVID-19 patients is associated with in-hospital survival or death. 1,852 patients admitted for severe COVID-19 at Nîmes University Hospital in 2021 were retrospectively included in the study: 1,564 who survived the hospital stay and 288 who did not. The NEU-SFL was obtained on the Sysmex™ XN-10® analyzer and values for survivors and non-survivors were compared. The intra-patient NEU-SFL variations between the hospital entry and the last day of hospitalization were also analyzed (IRB 22.06.01, NCT05413824). Non-survivors presented higher NEU-SFL values. NEU-SFL values above the 4th quartile were independently associated with a 2.88-fold risk of death. Furthermore, the difference of NEU-SFL values between the first and the last available data during hospitalization revealed that a decrease in NEU-SFL was associated to survivors and vice versa. Our study reinforces the role of neutrophils and NETosis in the pathophysiology and prognosis of COVID-19. Further studies combining NEU-SFL with other NETosis markers could improve the management of COVID-19 patients.

Development of a Modular, Induced Fluorescence-based Instrument Family : The Aquafluosense Project

In the scope of our recently completed AQUAFLUOSENSE research and development project, the design and construction of a prototype of an electron excitation fluorescence-based analytical instrument family has been carried out for water quality measurement applications. The objective of the project was to develop a new water analysis system for natural and artificial waters, allowing complex, systematic and for main parameters in situ assessment and monitoring of water quality, by developing a modular instrument family that can be individually configured for target tasks at each monitoring point. Within the instrument family, created in a collaboration of working groups of a number of research and development institutions, different modules allow for the determination of key water parameters. A common optical characteristic of these modules is that they measure the target parameter on the basis of an induced (excitation) fluorescence signal generated in the test sample. The modules allow the determination of individual biological or chemical components based either on measuring their fluorescence directly (direct fluorescence) or by relying on detection of the fluorescence of a coupled dye (indirect fluorescence). Thus, the instrument modules provide experimental data on the algal density and the total organic carbon content, as well as the presence of certain organic micropollutants in the given water body studied, the latter target analytes detected by direct fluorescence measurement or by an immunofluorescence measurement modality.

Flapping motion as a fluorescent probe for assembly process involving highly viscous liquid-like cluster intermediates during evaporative crystallization.

Fluorescence probes are widely used to assess the molecular environment based on their photo-physical properties. Specifically, flexible and aromatic photo-functional system (FLAP) is unique viscosity probe owing to the excited-state planarization of anthracene wings. We have previously applied fluorescence spectroscopy to monitor the evaporative crystallization of solvents. The fluorescence color and spectral changes, which depend on the aggregation form, enable direct fluorescence visualization during evaporative crystallization. The fluorescence visualization of the liquid-like cluster intermediate proposed in the two-step nucleation model for the nucleation process has been achieved. However, the physical properties of these clusters, especially the viscosity, molecular motion, and intermolecular interactions, are still unclear. In this study, FLAPs are used as probes for local-viscosity changes and space limitations of the liquid-like cluster state during evaporative crystallization by observing the fluorescence-spectral changes and using hyperspectral-camera (HSC) imaging. Green emission originates from the monomer in the solution owing to the free-flapping motion. The fluorescence color turns blue with increasing viscosity under crowding conditions. If the survival time of the liquid-like cluster state is sufficient, crystalline phase (R-phase) formation proceeds via a 2-fold π-stacked array of the V-shaped molecules. It is difficult to form the V-shaped stacked columnar structures in the liquid-like cluster state region, resulting in the deposition of head-to-tail dimer structures, such as the yellow-emissive phase (Y-phase). In the case of the FLAP, the stacking intermediate does not form during solvent evaporation in the liquid-like cluster; rather, it is deposited in an amorphous form that exhibits blue emission (B-phase). These findings suggest that it is important to the maintenance of the survival time of the liquid-like cluster states to organize and rearrange the stacking forms. We have achieved the fluorescence probing of viscosity changes at local molecular motion with solvent depletion during solvent evaporation for the first time.

Evolution of a natural dihydropteroate synthase and development of a signal amplified fluorescence method for detection of 44 sulfonamides in milk

In this study, the recognition mechanisms and affinities of a natural dihydropteroate synthase for 44 sulfonamides were studied respectively. Then the key contact amino acid Ser222 was mutated to Arg222 by using site-directed mutagenesis method to produce a mutant. Results showed that the binding energies and affinities for the 44 drugs were generally improved. Then this mutant was used as recognition reagent to develop a direct competitive fluorescence method on 96-well microplate for determination of the 44 drugs in milk. Due to the used signal amplified fluorescent tracer, the limits of detection for the 44 drugs were in the range of 0.025–0.65 ng/mL, and the sensitivities were improved for about 6–86 folds in comparison with the conventional fluorescent tracer. After comprehensive comparison, the present method showed generally better performances than the previously reported immunoassays for sulfonamides. Therefore, this method could be used as an efficient tool for the routine screening of sulfonamides residues in large number of food samples.

Application of CD138 Immunomagnetic Bead Sorting Combined with Fluorescence in Situ Hybridization in Multiple Myeloma

To compare the effects of direct fluorescence in situ hybridization (D-FISH) detection without sorting and CD138 immunomagnetic bead sorting technology combined with FISH (MACS-FISH) on cytogenetic analysis of patients with multiple myeloma (MM). FISH test results of 229 patients with initial MM were retrospectively analyzed. The patients were divided into two groups, 140 patients were tested with D-FISH and 89 patients with MACS-FISH. The combination probe was designed as P53, D13S319, RB1, 1q21, and IgH. Cytogenetic detection results were compared between the two groups. The total detection rate of cytogenetic abnormalities in D-FISH group was 52.9%, and that in MACS-FISH group was 79.8%. There was a significant difference in the cytogenetic abnormality rate between the two groups (P=0.020). The abnormal genes with the highest detection rate in the two groups were 1q21 and IgH, respectively, while the lowest was P53. There was no significant difference in the percentage of P53 positive cells (positive rate) between the two groups, while D13S319, RB1, 1q21, and IgH showed significant difference in positive cell rate (P=0.0002, P<0.0001, P=0.0033, P=0.0032). There was no significant correlation between the proportion of plasma cells (PC) detected by bone marrow morphology and cytogenetic abnormality rate in the D-FISH group, while there was a correlation between the proportion of PC detected by flow cytometry and cytogenetic abnormality rate (r=0.364). The PC proportion detected by bone marrow morphology and flow cytometry in the MACS-FISH group had no correlation with the cytogenetic abnormality rate and positive cell rate of the 5 genes mentioned above. Additionally, the PC proportion detected by bone marrow morphology and flow cytometry showed significant difference (P<0.0001). CD138 immunomagnetic bead sorting combined with FISH technology can significantly improve the abnormality detection rate of MM cytogenetics.

T Cells Chemotaxis Migration Studies with a Multi-Channel Microfluidic Device.

Immune surveillance is dependent on lymphocyte migration and targeted recruitment. This can involve different modes of cell motility ranging from random walk to highly directional environment-guided migration driven by chemotaxis. This study protocol describes a flow-based microfluidic device to perform quantitative multiplex cell migration assays with the potential to investigate in real time the migratory response of T cells at the population or single-cell level. The device also allows for subsequent in situ fixation and direct fluorescence analysis of the cells in the microchannel.

A Rapid Fluorescence Sensor for the Direct Quantification of Rongalite in Foodstuffs.

Rongalite was reported illegally used as a food additive for bleaching purposes and improving the tenderness of foodstuffs, which may endanger public health. At present, rongalite was mostly detected by indirect methods via derivatization or determining its decomposition products. In this study, we developed a new fluorescence sensor for the direct quantification of rongalite based on the principles: (1) dopamine reacts with resorcinol and generates strong fluorophore (azamonardine); (2) rongalite could inhibit the production of fluorophores and then result in lower fluorescence intensity. Hence, the rongalite concentration was inversely proportional to fluorescence intensity of fluorophore. Several crucial reaction conditions of fluorescence sensor were further optimized, such as dopamine and resorcinol concentration, pH values, and reaction time. Under the optimal conditions, the limit of detection of fluorescence sensor was 0.28–0.38 μg/g in vermicelli, wheat and rice powder samples, exhibiting almost 3.5-fold improvement compared to that of lateral flow immunoassay. Moreover, the detection time was substantially decreased to 20 min. The recoveries in spiked samples were 80.7–102.1% with a coefficient of variation of less than 12.6%. In summary, we developed a direct, high throughput, selective and accurate fluorescence sensor that poses a promising application for the rapid detection of rongalite in foodstuffs.

Can Direct-Immersion Aqueous–Aqueous Microextraction Be Achieved When Using a Single-Drop System?

For the analysis of biological analytes in complex matrices, it is difficult to achieve extraction of analytes and enrichment in an aqueous-aqueous single-drop microextraction system. In this study, we proposed a pH-dependent polydopamine (PDA)-coated vesicle/Fe3O4 magnetic aqueous-aqueous in a single-drop microreactor (SDMR) for the direct fluorescence detection of glutathione S-transferase (GST), a metabolic enzyme involved with crucial biological processes, in biological samples. After extracting and enriching the GST target from an aqueous-aqueous single-drop interface, the extraction process was conducted rapidly in 6 s in the SDMR system. The GST was first extracted from the sample solution via the GST-Aptamer on the polydopamine-coated vesicle/Fe3O4 nanospheres (Fe3O4@PDA@GST-Aptamer). Then, as the pH changed from weakly acidic to weakly alkaline in the SDMR system, the GST and GST-Aptamer were released from Fe3O4@PDA@GST-Aptamer nanospheres and captured by polydiacetylene vesicles via the capture probe. These changes altered the effective conjugation length and angle of the vesicle trunk, generating a highly enhanced fluorescence signal. This not only achieved the purpose of target enrichment but also reduced interferences posed by matrix effects. The approach can be used for the direct detection of GST in genuine urine and blood without any sample pretreatment. The linear range was 0.005 to 0.5 μg/mL, and the limit of detection was 0.834 ng/mL. The recoveries of GST in genuine blood samples ranged from 90.8 to 108.0% and in urine from 91.6 to 102.8%. The method has the capability of handling complex samples directly by enabling microextraction in an aqueous-aqueous single-drop system.