Fluorescence Detection System Research Articles

A portable LED-induced fluorescence system for quantitative detection of different kinds of vegetable oil adulteration

This study developed a portable LED-induced fluorescence detection system for quantitative detection of various vegetable oil adulteration. Eight common vegetable oils and 14 different adulterated samples with adulteration concentrations ranging from 0 to 50% were prepared. Before quantitative analysis, different classification models were established for the determination of types of adulteration oil. The overall recognition accuracy was greater than 98%. Furthermore, with a simple calculation, the proposed normalized spectral ratio (NSR) preprocessing method was used to eliminate the light scattering effects in the raw fluorescence spectra. In addition, the competitive adaptive reweighted sampling (CARS) method was used to select characteristic wavelengths. The final oil adulteration quantitative analysis model was NSR_CARS+PLS. The range of correlation coefficient (Rp) and root-mean-square-error (RMSEP) for the prediction datasets were 0.9548-0.9974 and 1.0265%-5.0236%, respectively.

Fluorescence Immunoassay of Prostate-Specific Antigen Using 3D Paddle Screw-Type Devices and Their Rotating System.

In this paper, we present a sensitive and highly reproducible fluorescence immunosensor for detecting PSA in human serum. A unique feature of this study is that it uses creatively designed paddle screw-type devices and their custom-made rotating system for PSA immunoassay. The paddle screw devices were designed to maximize the surface-to-volume ratio over which the immunoassay reaction could occur to improve detection sensitivity. This paddle screw-based immunoassay offers an accessible and efficient method with a short analysis time of less than 30 min. Active rotation of the paddle screw plays a crucial role in fast and accurate analysis of PSA. Additionally, a paddle screw-based immunoassay and subsequent fluorescence detection using a custom prototype fluorescence detection system were compared to a typical well plate-based immunoassay system. Results of PSA detection in human serum showed that the detection sensitivity through the paddle screw-based analysis improved about five times compared to that with a well plate-based analysis.

An intelligent and portable fiber optic real-time fluorescence detection system for pathogenic microorganisms detection

The detection of pathogenic microorganisms poses an increasingly serious challenge to food safety. This paper proposes an intelligent fiber optic fluorescence sensing system for detection of pathogenic microorganisms. The system integrates light-emitting diodes and fiber optic sensing arrays. Compact and multi-channel detection is achieved by this kind of design. Environmental parameters such as temperature, humidity, and location are intelligently analyzed together with sampled fluorescence signal, which successfully achieved a high accuracy detection result without affected or interfered by environmental parameters. A Radial Basis Function Neural Network (RBFNN) algorithm is utilized for intelligent analysis and prediction of pathogenic microorganisms’ concentration with environmental parameters. Under different environmental parameters, the system has successfully detected African swine fever virus or salmonella in real-time with high accuracy and sensitivity. Since location of the sensing system can be determined by GPS module integrated in the system, the change of pathogenic microorganisms with different locations can be determined even during transportation. This innovative solution provides a convenient and accurate method for high accurate on-site real time sensing of pathogenic microorganisms.

Compact Laser-Induced Fluorescence Detector with Adjustable Laser Focal Spot for Multiple Purposes.

In many research fields, the demand for miniaturized laser-induced fluorescence (LIF) detection systems has been increasing. This work has developed a compact LIF detector, employing a laser diode as the excitation source and a photodiode as the photodetector with an adjustable laser focal spot, to meet the diverse requirements of various observation targets, such as capillaries, PCR tubes, and microfluidic chips. It features the functionalities of background fluorescence correction, the adaptive adjustment of the dynamic range, and constant power control for the laser. The influence of the excitation power on the detection limit was studied through experiments, and the configuration results for LED/LD as light sources and 487/450 nm wavelengths were compared and optimized. A fully integrated, compact, modular epifluorescence LIF detector was subsequently constructed, measuring 40 × 22 × 38 mm3 in total size, with a cost of USD 320, and achieving a detection limit of 0.4 nM for fluorescein sodium. Finally, the detector was integrated into a nucleic acid detection system with a microfluidic chip on the Chinese Space Station (CSS) and was also tested with PCR tubes and capillaries, proving its broad practicality and adaptability to various analytical systems.

Identification of horse ingredients based on recombinase polymerase amplification, high-throughput DNA isolation and magnetic separation

Adulteration of meat products remains a significant issue worldwide. In this study, we developed a high-throughput, rapid, sensitive, and selective method for qualitative and quantitative detection of horse meat adulteration in meat products. This method, which is not only innovative but also highly practical, involves coupling a 96-well high-throughput DNA isolation process with recombinant enzyme polymerase amplification (RPA), magnetic separation (MS), and high-throughput fluorescence detection (FD). We designed a set of primers targeting the mitochondrial ATP6-8 gene of horses. The upper and lower primers were labeled with 6-FAM fluorescent moiety and biotin, respectively. Following RPA amplification, the amplicons were separated and purified using streptavidin-modified magnetic beads (MB@SA). Subsequently, the amplicons were qualified and quantified using a high-throughput fluorescence detection system. We optimized the conditions for primer addition, incubation time, and the ratio of amplicon to MB@SA. Our results showed that a fluorescence threshold of 834 indicated positive detection. The method exhibited high specificity, with no positive amplification observed in DNA from the other 11 types of meat. It demonstrated high sensitivity and was capable of detecting as little as 0.1% (wt%) of horse meat in adulterated samples. Combined with our developed 96-well high-throughput DNA extraction system, the RPA-MS-FD assay process could analyze 96 samples in 31.5 min, including 11.5 min for rapid DNA extraction, 15 min for RPA amplification, and 5 min for fluorescence detection. Finally, we successfully applied this assay to analyze 21 commercial meat products, proving its practicality and effectiveness.

Copper-doped carbon dots nanozymes for multi-signal specific hydrogen sulfide assay and broad-spectrum antibacterial

The emergence of carbon dots-based nanozymes (CDNEs) has opened a broad horizon for multi-mode analysis and safety antibacterial application. However, the practical applications of CDNEs are currently hindered by unsatisfactory performance and complicated synthetic methodology. In this study, we developed a novel copper-doped CDNEs (Cu-CDNEs) with both excellent fluorescence property and peroxidase-like activity. The Cu-CDNEs can be synthesized easily by pyrolyzing a solid mixture of copper glutamate and ethylenediamine hydrochloride for 5 min. By investigating the inhibition of H2S on the peroxidase-like activity of Cu-CDNEs and the competitive interaction between H2S and 3,3′,5,5′-tetramethylbenzidine with reactive oxygen species, we established a dual-mode analysis system for colorimetric and fluorescent detection of H2S with the limit of detection of 20 nM and 25 nM, respectively. Furthermore, a convenient scanometric analysis mode also was developed and successfully applied for the accurate determination of H2S in actual water samples with recoveries of 96.8 %–107.7 %. Moreover, the designed Cu-CDNEs show good cytocompatibility from MTT results, and demonstrated broad-spectrum antibacterial capacity against Gram-positive/negative bacteria due to the surface positive charge and peroxidase-like activity. This study may provide valuable insights for rational design and broad applications in environmental and biomedical fields using functional nanozymes.

Engineering the apparent quantum yield and emission rate of fluorophore molecules by coupling fluorophore dipoles with plasmon modes of gold using low frequency electric fields

Abstract Localized surface plasmons produced by gold and silver nanostructures have been utilized to enhance the intensity of fluorophore molecules. The issue with using nanostructure plasmons for fluorescence enhancement is their short-range nature (5–50 nm from the nanostructures), which limits accessibility to a few molecules. In addition, fluorophore dipoles needed to be aligned with the plasmon electric fields to maximize the fluorescence enhancement. To address these issues, we used low-frequency electric fields (<5 MHz) and commercially available nanorod and nanosphere samples and studied their effectiveness in enhancing the fluorescence of fluorophore-labeled short single-stranded DNA molecules (22 bases). We demonstrated that DNA molecules and nanorod particles can effectively be manipulated around the charging frequency of DNA molecules (∼3 MHz). Nanorod particles enhanced the fluorescence emission rate by ∼50-fold. When the 3 MHz electric field was introduced, the emission rate increased to over 700-fold. We also found that the introduction of a 3 MHz electric field aided the enhancement of the intrinsic quantum yield fluorophore molecules, which resulted in over a 1000-fold fluorescence enhancement. This enhancement was due to the very high electric produced by polarized DNA dipoles at 3 MHz, which resulted in a torque on fluorophore dipoles and subsequently aligning the fluorophore dipole axis with the plasmon electric field. At a fundamental level, our results demonstrate the role of the low-frequency electric field in the fluorophore–plasmon coupling. These findings can directly be applied to many fluorescence detection systems, including the development of biosensors.

Green synthesis of soybean residue-based nitrogen-chlorine co-doped carbon dots based on deep eutectic solvents: Construction of a PNP fluorescence detection system under the IFE mechanism

How to manage the potential environmental pollution of waste biomass while realizing the resource utilization of the waste is a topic worthy of in-depth investigation. In this paper, after successfully preparing a deep eutectic solvents, we constructed N, Cl-CDs fluorescent probes with high fluorescence yield and biocompatibility by utilizing waste soybean residue as a cheap nitrogen-rich carbon-sourced biomass precursor. The results showed that the fluorescence quenching of N, Cl-CDs has a good linear relationship with the concentration of p-nitrophenol in the range of 0.1–60 μM, with the detection limit of 30 nM, and the fluorescence quenching mechanism was the inner filter effect. This paper demonstrates that the carbon dots fluorescence system has a superior prospect for the highly selective detection of p-nitrophenol, and provides a reliable basis for the future exploration of efficient and environmentally friendly carbon dots synthetic method as well as the economic recycling of waste.

Quantitative Detection of the CP4-EPSPS Protein in Genetically Modified Soybeans Using a Novel Fluorescence Immunoassay Assay.

Protein-based detection methods, enzyme-linked immunosorbent assays (ELISAs) and lateral flow strips, have been widely used for rapid, specific, and sensitive detection of genetically modified organisms (GMOs). However, the traditional ELISA method for the quantitative detection of GMOs has certain limitations. Herein, a quantum dot (QD)-based fluorescence-linked immunosorbent assay was developed using QDs as fluorescent markers for the detection of glyphosate-resistant protein (CP4-EPSPS) in the MON89788 soybean. The end-point fluorescent detection system was carried out using QDs conjugated with a goat anti-rabbit secondary antibody. Compared with the conventional sandwich ELISA method, the newly developed fluorescence-linked immunosorbent assay was highly sensitive and accurate for detecting the CP4-EPSPS protein. The quantified linearity was achieved in the range of 0.05-5% (w/w) for the MON89788 soybean sample. The recovery of protein extracted from mixed MON89788 soybean samples ranged from 87.67% to 116.83%. The limits of detection and limits of quantification were 0.7101 and 2.152 pg/mL, respectively. All of the results indicated that the QD-based fluorescence-linked immunosorbent assay was a highly specific and sensitive method for monitoring the CP4-EPSPS protein in GMOs.

Portable on–off visual-mode detection using intrinsic fluorescent zinc-based metal–organic framework for detection of diclofenac in pharmaceutical tablets

On-site, robust, and quantitative detection of diclofenac (DCF) is highly significant in bioanalysis and quality control. Fluorescence-based metal–organic frameworks (MOFs) play a pivotal role in biochemical sensing, offering a versatile platform for detecting various biomolecules. However, conventional fluorescent MOF sensors often rely on lanthanide metals, which can pose challenges in terms of cost, accessibility, and environmental impact. Herein, an intrinsic blue fluorescent zinc-based metal–organic framework (FMOF-5) was prepared free from lanthanide metals. Coordination-induced emission as an effective strategy was followed wherein a non-fluorescent ligand is converted to a fluorescent one after insertion in a framework. Conventional fluorometry and smartphone-assisted visual methods were employed for the detection of DCF. The fluorescence emission of the FMOF-5 was effectively quenched upon the addition of the DCF, endowing it an “off” condition, which permits the construction of a calibration curve with a wide linear range of 30–670 µM and a detection limit of about 4.1 µM. Other analytical figures of merit, such as linearity, sensitivity, selectivity, accuracy, and precision were studied and calculated. Furthermore, the proposed sensor was successfully applied to quantify DCF in pharmaceutical tablets with reliable recovery and precision. Importantly, the elimination of lanthanide metals from the fluorescence detection system enhances its practicality and sustainability, making it a promising alternative for DCF detection in pharmaceutical analysis applications.

Advanced X-ray absorption spectroscopy under high pressures at the ESRF beamline ID12

ABSTRACT This paper reports on advanced instrumentation for high pressure research developed for the ESRF beamline ID12 which is dedicated to polarization-dependent X-ray absorption spectroscopy at photon energies from 2 to 15 keV. Emphasis is laid on X-ray optics, the design of diamond anvil cell adapted for tender X-ray range and compatible with low temperatures and with high magnetic field as well as a highly efficient X-ray fluorescence detection system. Several examples have been selected to illustrate the performance of the beamline and show what type of advanced spectroscopic techniques can be used to study the local electronic and magnetic properties of materials under pressure.

Development of a novel testis-on-a-chip that demonstrates reciprocal crosstalk between Sertoli and Leydig cells in testicular tissue

The reciprocal crosstalk between testicular Sertoli and Leydig cells plays a vital role in supporting germ cell development and maintaining testicular characteristics and spermatogenesis. Conventional 2D and the recent 3D assay systems fail to accurately replicate the dynamic interactions between these essential endocrine cells. Furthermore, most in vitro testicular tissue models lack the ability to capture the complex multicellular nature of the testis. To address these limitations, we developed a 3D multicellular testis-on-a-chip platform that effectively demonstrates the reciprocal crosstalk between Sertoli cells and the adjacent Leydig cells while incorporating various human testicular tissue constituent cells and various natural polymers infused with blood coagulation factors. Additionally, we identified SERPINB2 as a biomarker of male reproductive toxicity that is activated in both Sertoli and Leydig cells upon exposure to various toxicants. Leveraging this finding, we designed a fluorescent reporter-conjugated toxic biomarker detection system that enables both an intuitive and quantitative assessment of material toxicity by measuring the converted fluorescence intensity. By integrating this fluorescent reporter system into the Sertoli and Leydig cells within our 3D multicellular chip platform, we successfully developed a testis-on-chip model that can be utilized to evaluate the male reproductive toxicity of potential drug candidates. This innovative approach holds promise for advancing toxicity screening and reproductive research.

Self-designed portable dual-mode fluorescence device with custom python-based analysis software for rapid detection via dual-color FRET aptasensor with IoT capabilities

An innovative aptasensor incorporating MoS2-modified bicolor quantum dots and a portable spectrometer, designed for the simultaneous detection of ochratoxin A (OTA) and aflatoxin B1 (AFB1) in corn was developed. Carbon dots and CdZnTe quantum dots were as nano-donors to label OTA and AFB1 aptamers, respectively. These labeled aptamers were subsequently attached to MoS2 receptors, enabling fluorescence resonance energy transfer (FRET). With targets, the labeled aptamers detached from the nano-donors, thereby disrupting the FRET process and resulting in fluorescence recovery. Furthermore, a portable dual-mode fluorescence detection system, complemented with customized python-based analysis software, was developed to facilitate rapid and convenient detection using this dual-color FRET aptasensor. The developed host program is connected to the spectrometer and transmits data to the cloud, enabling the device to have Internet of Things (IoT) characteristics. Connected to the cloud, this IoT-enabled device offers convenient and reliable fungal toxin detection for food safety.

Split T7 switch-mediated cell-free protein synthesis system for detecting target nucleic acids

Cell-free protein synthesis (CFPS) reactions can be used to detect nucleic acids. However, most CFPS systems rely on a toehold switch and exhibit the following critical limitations: (i) off-target signals due to leaky translation in the absence of target nucleic acids, (ii) a suboptimal detection limit of approximately 30 nM without pre-amplification, and (iii) labor-intensive screening processes due to sequence constraints for the target nucleic acids. To overcome these shortcomings, we developed a new split T7 switch-mediated CFPS system in which the split T7 promoter was applied to a three-way junction structure to selectively initiate transcription–translation only in the presence of target nucleic acids. Both fluorescence and colorimetric detection systems were constructed by employing different reporter proteins. Notably, we introduced the self-complementation of split fluorescent proteins to streamline preparation of the proposed system, enabling versatile applications. Operation of this one-pot approach under isothermal conditions enabled the detection of target nucleic acids at concentrations as low as 10 pM, representing more than a thousand times improvement over previous toehold switch-based approaches. Furthermore, the proposed system demonstrated high specificity in detecting target nucleic acids and compatibility with various reporter proteins encoded in the expression region. By eliminating issues associated with the previous toehold switch system, our split T7 switch-mediated CFPS system could become a core platform for detecting various target nucleic acids.

X-ray fluorescence detection system for trace elements in oil based on graphite curved crystal technology

Curved crystal technology is a key technology in the field of X-ray fluorescence trace detection. To detect trace sulfur, chlorine, and silicon in oil, the key parameters of Johann-type graphite curved crystal design were calculated based on geometrical optics theory and mathematical modeling, and a curved crystal optical system was designed. Subsequently, the hardware, firmware, software, and mechanical structure were designed according to specific requirements. Following the development of the relevant equipment, laboratory tests were performed on standard petroleum samples. Experimental results show that technology based on bent crystals can significantly reduce the detection limit of the instrument, and the detection limit of sulfur, chlorine, and silicon can reach below 1 ppm. In accordance with the experimental results, possible interference factors were analyzed, and suitable solutions were proposed. The performance of the proposed system was thus directly verified, showcasing its applicability in detecting trace elements.

Study on Circulating Tumor Cell Detection System Based on Microfluidic Chip

To achieve high throughput and high detection rate of circulating tumor cells (CTCs) in human peripheral blood, and to provide efficient and accurate early screening for cancer patients. A microfluidic chip with the integration of sorting, enrichment and detection was designed, and CTCs at the single cell level were detected by fluorescence detection system to obtain the number of CTCs in samples. The peripheral blood samples after lysed red blood cells were used for 6 experiments. When the injection rate reached 0.2 mL/h, CTCs could reach the best detection rate of 78.6%, and the correlation coefficient within the group was above 0.8. CTCs detection system can achieve high detection rate and has good reliability, which can provide a reliable reference for clinical research in related fields.

NIRis: A low-cost, versatile imaging system for near-infrared fluorescence detection of phototrophic cell colonies used in research and education.

A variety of costly research-grade imaging devices are available for the detection of spectroscopic features. Here we present an affordable, open-source and versatile device, suitable for a range of applications. We provide the files to print the imaging chamber with commonly available 3D printers and instructions to assemble it with easily available hardware. The imager is suitable for rapid sample screening in research, as well as for educational purposes. We provide details and results for an already proven set-up which suits the needs of a research group and students interested in UV-induced near-infrared fluorescence detection of microbial colonies grown on Petri dishes. The fluorescence signal confirms the presence of bacteriochlorophyll a in aerobic anoxygenic phototrophic bacteria (AAPB). The imager allows for the rapid detection and subsequent isolation of AAPB colonies on Petri dishes with diverse environmental samples. To this date, 15 devices have been build and more than 7000 Petri dishes have been analyzed for AAPB, leading to over 1000 new AAPB isolates. Parts can be modified depending on needs and budget. The latest version with automated switches and double band pass filters costs around 350€ in materials and resolves bacterial colonies with diameters of 0.5 mm and larger. The low cost and modular build allow for the integration in high school classes to educate students on light properties, fluorescence and microbiology. Computer-aided design of 3D-printed parts and programming of the employed Raspberry Pi computer could be incorporated in computer sciences classes. Students have been also inspired to do agar art with microbes. The device is currently used in seven different high schools in Finland. Additionally, a science education network of Finnish universities has incorporated it in its program for high school students. Video guides have been produced to facilitate easy operation and accessibility of the device.

Fiber optic-based integrated system for in vivo multiscale pharmacokinetic monitoring.

This paper presents the development of a fiber-optic-based fluorescence detection system for multi-scale monitoring of drug distribution in living animals. The integrated system utilized dual laser sources at the wavelengths of 488 nm and 650 nm and three photomultiplier channels for multi-color fluorescence detection. The emission spectra of fluorescent substances were tracked using the time-resolved fluorescence spectroscopy module to continuously monitor their blood kinetics. The fiber bundle, consisting of 30,000 optic filaments, was designed for wide-field mesoscopic imaging of the drug's interactions within organs. The inclusion of a gradient refractive index (GRIN) lens within the setup enabled fluorescence confocal laser scanning microscopy to visualize the drug distribution at the cellular level. The system performance was verified by imaging hepatic and renal tissues in mice using cadmium telluride quantum dots (CdTe QDs) and R3. By acquiring multi-level images and real-time data, our integrated system underscores its potential as a potent tool for drug assessment, specifically within the realms of pharmacokinetic and pharmacodynamic investigations.

The homeodomain of the <i>Raphanus sativus</i> WOX4 binds to the promoter of the <i>LOG3</i> cytokinin biosynthesis gene

BACKGROUND: The WOX4 transcription factor plays a crucial role in maintaining the organisation of cambium meristem during secondary growth, but its direct targets are unknown. AIM: The aim of our work were to study the effect of WOX4 overexpression on the root development and gene expression in radish (Raphanus sativus L.), a root crop related to Arabidopsis thaliana, and to search for direct targets of the WOX4 in radish. MATERIALS AND METHODS: Radish line 19 of the St. Petersburg State University radish genetic collection was used. Plants were grown on Murashige–Skoog medium and then in soil at 23оС and 16 h of daylight. Total DNA was extracted from radish seedlings using the CTAB method. The PCR-amplified full-length RsWOX4-2 gene, gene fragments or homeobox sequence were cloned into the vectors for overexpression (pB7WG2D), RNA interference (pH7GWIWG2) and yeast one-hybrid assay (pDEST22), respectively, using the Gateway system. The vectors for overexpression and RNA interference of RsWOX4-2 were transformed into Escherichia coli DH10B and then into Agrobacteium rhizogenes Arqua chemically competent cells. Radish seedlings were transformed with A. rhizogenes containing vectors for overexpression and RNA interference of RsWOX4-2, and GUS-overexpressing A. rhizogenes was used as a control. Total RNA from transgenic radish roots was extracted with Trizol reagent. RNA reverse transcription was performed using dT-18 primers and RevertAid reverse transcriptase. qPCR was performed using the Eva Green reagent kit on a CFX96 thermocycler with fluorescence detection system. Results were processed using the 2–ΔΔCT method. Yeast transformation was performed using the competent Saccharomyces cerevisiae cells of Y2H Gold strain. For the yeast one-hybrid assay, the obtained yeast colonies transformed with plasmids containing TF homeodomain sequence and promoter regions of genes were grown on DDO and TDO selective media with different concentrations of 3-amino-1,2,4-triazole. Statistical processing based on Student’s t-test and graphing were performed using the ggplot2 package for the R programming language (v.4.0.2). RESULTS: Overexpression of the RsWOX4-2 gene affects the structure of the radish root stele and alters the number of vessels and cambium cells. Overexpression and RNA interference of the RsWOX4-2 causes changes in the expression levels of putative target genes with the WOX family transcription factor conserved binding sites in their promoters. Using the yeast one-hybrid assay, we have shown that the DNA-binding homeodomain of RsWOX4-2 interacts with the TAATCC site in the promoter of the RsLOG3 gene, which encodes the enzyme for cytokinin biosynthesis. CONCLUSIONS: We have demonstrated the effect of RsWOX4-2 overexpression on radish root stele and gene expression and identified the RsLOG3 as the putative direct target of the WOX4 transcription factor in radish.

Furan-based AIE supramolecular self-assembly system for fluorescence detection of Ag+

A novel furan-based Aggregation-Induced Emission (AIE) supramolecular self-assembling gelator (BM) was designed and synthesized, which could self-assemble into a stable supramolecular gel (OBM) in the 1,2-propanediol. OBM demonstrates “turn-off” fluorescence detection of Ag+, with excellent selectivity and a low limit of detection (LOD) of 1.168×10−8 M. The self-assembly process and ion recognition sensing mechanism of OBM were comprehensively investigated by Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray Photoelectron Spectroscopy (XPS), and rheological measurements. More importantly, single crystal were successfully obtained to validate the self-assembly process. This study provides a novel approach to constructing a multifunctional AIE self-assembly system.