Real-time Fluorescent Analysis Research Articles

Atom-Modified gDNA Enhances Cleavage Activity of TtAgo Enabling Ultra-Sensitive Nucleic Acid Testing.

The DNA-guided (gDNA) Argonaute from Thermus thermophilus (TtAgo) has little potential for nucleic acid detection and gene editing due to its poor dsDNA cleavage activity at relatively low temperature. Herein, the dsDNA cleavage activity of TtAgo is enhanced by using 2'-fluorine (2'F)-modified gDNA and developes a novel nucleic acid testing strategy. This study finds that the gDNA with 2'F-nucleotides at the 3'-end (2'F-gDNA) can promote the assembly of the TtAgo-guide-target ternary complex significantly by increasing its intermolecular force to target DNA and TtAgo, thereby providing ≈40-fold activity enhancement and decreasing minimum reaction temperature from 65 to 60°C. Based on this outstanding advance, a novel nucleic acid testing strategy is proposed, termed FAST, which is performed by using the 2'F-gDNA/TtAgo for target recognition and combining it with Bst DNA polymerase for nucleic acid amplification. By integrating G-quadruplex and Thioflavin T, the FAST assay achieves one-pot real-time fluorescence analysis with ultra-sensitivity, providing a limit of detection up to 5 copies (20µL reaction mixture) for miR-21 detection. In summary, an atom-modification-based strategy has been developed for enhancing the cleavage activity of TtAgo efficiently, thereby improving its practicability and establishing a TtAgo-based nucleic acid testing technology with ultra-sensitivity and high-specificity.

Highly sensitive and rapid determination of Mycobacterium leprae based on real-time multiple cross displacement amplification

BackgroundMycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly induces leprosy by causing permanent damage to the skin, nerves, limbs and eyes as well as deformities and disabilities. Moreover, ML grows slowly and is nonculturable in vitro. Given the prevalence of leprosy, a highly sensitive and rapid method for the early diagnosis of leprosy is urgently needed.ResultsIn this study, we devised a novel tool for the diagnosis of leprosy by combining restriction endonuclease, real-time fluorescence analysis and multiple cross displacement amplification (E-RT-MCDA). To establish the system, primers for the target gene RLEP were designed, and the optimal conditions for E-RT-MCDA at 67 °C for 36 min were determined. Genomic DNA from ML, various pathogens and clinical samples was used to evaluate and optimize the E-RT-MCDA assay. The limit of detection (LoD) was 48.6 fg per vessel for pure ML genomic DNA, and the specificity of detection was as high as 100%. In addition, the detection process could be completed in 36 min by using a real-time monitor.ConclusionThe E-RT-MCDA method devised in the current study is a reliable, sensitive and rapid technique for leprosy diagnosis and could be used as a potential tool in clinical settings.

One-tube, two-step isothermal amplification of histatin 3 mRNA for saliva screening

Saliva samples are frequently collected at crime scenes. Salivary mRNA profiling, such as that of histatin 3 (HTN3), is a highly specific approach that overcomes the limitation of traditional amylase tests. However, typical mRNA detection methods based on reverse transcription PCR (RT-PCR) are time-consuming and labor-intensive. Here, we report a one-tube, two-step isothermal amplification assay for HTN3 mRNA, which enables rapid, simple, and sensitive screening of saliva. The first step is an RT-recombinase polymerase amplification (RT-RPA) assay at 42 °C for 20 min; the second step is a loop-mediated isothermal amplification (LAMP) assay at 65 °C for 30 min. The reactions can be performed in a closed tube, and the products are detected using real-time fluorescence analysis. The assay sensitivity was 0.5 µL of saliva samples. It also detected HTN3 mRNA in mixed and mock samples, demonstrating its applicability to actual forensic samples. These findings suggest that our strategy is promising for screening of saliva from forensic samples.

Rapid, sensitive, and highly specific detection of monkeypox virus by CRISPR-based diagnostic platform.

Monkeypox (MPX), caused by the Monkeypox virus (MPXV), has incurred global attention since it broke out in many countries in recent times, which highlights the need for rapid and reliable diagnosis of MPXV infection. We combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based detection to devise a diagnostic test for detection of MPXV and differentiation of its two clades [Central Africa clade (MPXV-CA) and West Africa clade (MPXV-WA)], and called it MPXV-RCC. The sensitivity, specificity and practicability of this method have been analyzed. The optimal conditions of MPXV-RCC assay include two RPA reactions at 38°C for 25 min and a CRISPR/Cas12a-gRNA detection at 37°C for 10 min. The results of MPXV-RCC assay were indicated by a real-time fluorescence analysis software. Thus, the whole detection process, including rapid template preparation (20 min), RPA reaction (25 min) and CRISPR-based detection (10 min), could be finished within 1 hour. The sensitivity of MPXV-RCC for MPXV-CA and MPXV-WA detection was down to 5~10 copies of recombination plasmids and pseudovirus per reaction. Particularly, MPXV-RCC assay could clearly differentiate MPXV-CA from MPXV-WA, and had no cross-reactivity with other pathogens. In addition, the feasibility of MPXV-RCC assay was further validated by using spiked clinical samples. The MPXV-RCC assay developed here is a promising tool for quick and reliable diagnosis of MPXV infection.

Development of CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Technique for the Detection of Mycobacterium tuberculosis Complex in Clinical Settings.

Tuberculosis (TB) is a chronic infectious disease with high mortality caused by the Mycobacterium tuberculosis complex (MTC). Its clinical symptoms include a prolonged cough with mucus, pleuritic chest pain, hemoptysis, etc., and predominant complications such as tuberculous meningitis and pleural effusion. Thus, developing rapid, ultrasensitive, and highly specific detection techniques plays an important role in controlling TB. Here, we devised CRISPR/CRISPR-associated 12b nuclease (CRISPR/Cas12b)-based multiple cross displacement amplification technique (CRISPR-MCDA) targeting the IS6110 sequence and used it to detect MTC pathogens. A newly engineered protospacer adjacent motif (PAM) site (TTTC) was modified in the linker region of the CP1 primer. In the CRISPR-MCDA system, the exponentially amplified MCDA amplicons with the PAM sites can guide the Cas12b/gRNA complex to quickly and accurately recognize its target regions, which successfully activates the CRISPR/Cas12b effector and enables ultrafast trans-cleavage of single-stranded DNA reporter molecules. The limit of detection of the CRISPR-MCDA assay was 5 fg/μL of genomic DNA extracted from the MTB reference strain H37Rv. The CRISPR-MCDA assay successfully detected all examined MTC strains and there was no cross-reaction with non-MTC pathogens, confirming that its specificity is 100%. The entire detection process can be completed within 70 min using real-time fluorescence analysis. Moreover, visualization detection (under UV light) was also designed to verify the results, eliminating the use of specialized instruments. In conclusion, the CRISPR-MCDA assay established in this report can be used as a valuable detection technique for MTC infection. IMPORTANCE The Mycobacterium tuberculosis complex pathogen is a crucial infectious agent of tuberculosis. Hence, improving the capability of MTC detection is one of the most urgently required strategies for preventing and controlling TB. In this report, we successfully developed and implemented CRISPR/Cas12b-based multiple cross displacement amplification targeting the IS6110 sequence to detect MTC pathogens. These results demonstrated that the CRISPR-MCDA assay developed in this study was a rapid, ultrasensitive, highly specific, and readily available method which can be used as a valuable diagnostic tool for MTC infection in clinical settings.

A novel, ultrafast, ultrasensitive diagnosis platform for the detection of SARS-COV-2 using restriction endonuclease-mediated reverse transcription multiple cross displacement amplification.

Coronavirus disease 2019 (COVID-19) is a highly infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). Though many methods have been used for detecting SARS-COV-2, development of an ultrafast and highly sensitive detection strategy to screen and/or diagnose suspected cases in the population, especially early-stage patients with low viral load, is significant for the prevention and treatment of COVID-19. In this study, a novel restriction endonuclease-mediated reverse transcription multiple cross displacement amplification (MCDA) combined with real-time fluorescence analysis (rRT-MCDA) was successfully established and performed to diagnose COVID-19 infection (COVID-19 rRT-MCDA). Two sets of specific SARS-COV-2 rRT-MCDA primers targeting opening reading frame 1a/b (ORF1ab) and nucleoprotein (NP) genes were designed and modified according to the reaction mechanism. The SARS-COV-2 rRT-MCDA test was optimized and evaluated using various pathogens and clinical samples. The optimal reaction condition of SARS-COV-2 rRT-MCDA assay was 65°C for 36 min. The SARS-COV-2 rRT-MCDA limit of detection (LoD) was 6.8 copies per reaction. Meanwhile, the specificity of SARS-COV-2 rRT-MCDA assay was 100%, and there was no cross-reaction with nucleic acids of other pathogens. In addition, the whole detection process of SARS-COV-2 rRT-MCDA, containing the RNA template processing (15 min) and real-time amplification (36 min), can be accomplished within 1 h. The SARS-COV-2 rRT-MCDA test established in the current report is a novel, ultrafast, ultrasensitive, and highly specific detection method, which can be performed as a valuable screening and/or diagnostic tool for COVID-19 in clinical application.

Ectopic expression of NAC transcription factor HaNAC3 from Haloxylon ammodendron increased abiotic stress resistance in tobacco.

HaNAC3 is a transcriptional activator located in the nucleus that may be involved in the response to high temperature, high salt and drought stresses as well as phytohormone IAA and ABA treatments. Our study demonstrated that HaNAC3 increased the tolerance of transgenic tobacco to abiotic stress and was involved in the regulation of a range of downstream genes and metabolic pathways. This also indicates the potential application of HaNAC3 as a plant tolerance gene. NAC transcription factors play a key role in plant growth and development and plant responses to biotic and abiotic stresses. However, the biological functions of NAC transcription factors in the desert plant Haloxylon ammodendron are still poorly understood. In this study, the NAC transcription factor HaNAC3 was isolated and cloned from a typical desert plant H. ammodendron, and its possible biological functions were investigated. Bioinformatics analysis showed that HaNAC3 has the unique N-terminal NAC structural domain of NAC transcription factor. Quantitative real-time fluorescence analysis showed that HaNAC3 was able to participate in the response to simulated drought, high temperature, high salt, and phytohormone IAA and ABA treatments, and was very sensitive to simulated high temperature and phytohormone ABA treatments. Subcellular localization analysis showed that the GFP-HaNAC3 fusion protein was localized in the nucleus of tobacco epidermal cells. The transcriptional self-activation assay showed that HaNAC3 had transcriptional self-activation activity, and the truncation assay confirmed that the transcriptional activation activity was located at the C-terminus. HaNAC3 gene was expressed exogenously in wild-type Nicotiana benthamiana, and the physiological function of HaNAC3 was verified by simulating drought and other abiotic stresses. The results indicated that transgenic tobacco had better resistance to abiotic stresses than wild-type B. fuminata. Further transcriptome analysis showed that HaNAC3 was involved in the regulation of a range of downstream resistance genes, wax biosynthesis and other metabolic pathways. These results suggest that HaNAC3 may have a stress resistance role in H. ammodendron and has potential applications in plant molecular breeding.

A New Optical Method for Quantitative Detection of Microplastics in Water Based on Real-Time Fluorescence Analysis

Microplastics (MPs) have recently emerged as a new major and ubiquitous environmental pollutant with still undefined, yet potentially high risks for human health and ecosystems. This has prompted growing public concern along with an increasing number of scientific studies. In particular, recent research has highlighted the need for a standardized methodology to monitor microplastics in different media, particularly in water. This study reports on the use of a new particle counter for the quantitative detection of MPs in water samples based on real-time analysis of fluorescence emissions. The instrument was calibrated using two types of plastic particles, i.e., polyvinyl chloride and high-density polyethylene, selected as examples of high- and low-density plastics, respectively. Specific solvents were used to match the different plastic densities. Measurements were also carried out on particles obtained from wastewater samples collected at the inlet and outlet of specific units of a municipal wastewater treatment plant after sieving, filtering, digestion with hydrogen peroxide to remove degradable organic matter and resuspension in the solvents employed during the calibration step. A Fourier transform infrared spectroscopy analysis performed on the same wastewater samples confirmed the presence of MPs, and, in particular, of polyethylene, in some of the samples in which the highest concentrations were measured applying the proposed method. Therefore, the novel particle counter described in this paper could represent a promising method to quantitatively measure MP concentrations in water samples.

Fabrication of single cell microarrays on a double-layered hydrogel for mitochondrial activity monitoring

Single-cell microarrays may play an important role in understanding the heterogeneity of cell populations. A hydrogel-coated micropatterned surface was prepared to capture single cells from a mixture of cells and detect their mitochondrial activity using real-time fluorescence analysis. The double-layered hydrogel micropatterns were functionalized with RGD-containing peptides using a photomask. We were able to control the number of cells immobilized on each spot of the micropattern by controlling the size of the spots. Single cells were properly arranged on the peptide-functionalized spot with aligned squares (20 μm × 20 μm), with 68% frequency of single cells. In the single-cell micropattern, a mitochondria-targetable switch-on-type of probe was employed to discriminate between the types of cells and exhibit heterogeneous cell behavior in real-time. This single cell detection technology allows for the analysis of cell function and gene expression in various environments.

A Sample-to-Answer Compact Optical System for On-Site Detection of Candidatus Liberibacter Asiaticus

HighlightsA portable system based on real-time fluorescence analysis was developed for field detection of Candidatus Liberibacter asiaticus within 40 min from sample to answer.A smartphone-assisted device was designed for easy operation, reliable nucleic acid amplification, and highly sensitive fluorescence detection, with sensitivity comparable to that of a commercial instrument.A novel homemade 3D printed box was used for in-field reagent storage, and it could maintain low temperature (<4°C) for about 8 hours without power supply.This fully integrated system is stable, easy to use, inexpensive, and has great application prospects in resource-limited areas.Abstract. Candidatus Liberibacter asiaticus (Las) is a main causal agent of huanglongbing (HLB), a destructive disease that has greatly reduced citrus yields and quality. Instruments with high sensitivity and portability are urgently required for on-site testing. In this study, a novel sample-to-answer optical system for on-site detection of Las was developed. Three major functions, including DNA extraction, amplification, and detection, are integrated into a portable case. This system mainly consists of (1) a specially designed 3D printed box for on-site reagent storage that can maintain low temperature (below 4°C) for 7.5 h at ambient temperature (35°C); (2) a custom device, called the IF-Device, for DNA amplification and detection of HLB, with an optimized optical structure, a sensitive signal processing circuit, and a precise temperature control algorithm with an accuracy of ±0.1°C; and (3) a battery-based power supply for the whole system. In a typical test using sodium fluorescein as a standard model, the results showed that the sensitivity of this system (1.0 nM) could easily meet the requirements of fluorescence biosensors. The feasibility of this homemade system was evaluated with samples extracted from infected citrus leaves based on the loop-mediated isothermal amplification (LAMP) method, and the limit of detection (LOD) was approximately 1.0 × 10-4 ng µL-1. The whole detection process for eight samples could be simultaneously accomplished within 40 min, and the results could be displayed on a smartphone in real-time. Moreover, the portable case is anti-interference, low cost, and only 2 kg in weight. Considering its sensitivity, stability, and portability, this highly integrated system possesses promising prospects for in-field detection. Keywords: Field detection, Fluorescence biosensor, Huanglongbing, Isothermal amplification, Sample-to-answer.

Magnetic Fluorescence Molecularly Imprinted Polymer Based on FeOx/ZnS Nanocomposites for Highly Selective Sensing of Bisphenol A.

In this study, magnetic fluorescence molecularly imprinted polymers were fabricated and used for the selective separation and fluorescence sensing of trace bisphenol A (BPA) in environmental water samples. The carboxyl-functionalized FeOx magnetic nanoparticles were conjugated with mercaptoethylamine-capped Mn2+ doped ZnS quantum dots to prepare magnetic FeOx and ZnS quantum dot nanoparticles (FeOx/ZnS NPs). Additionally, molecular imprinting on the FeOx/ZnS NPs was employed to synthesize core-shell molecularly imprinted polymers. The resulting nanoparticles were well characterized using transmission electron microscopy, Fourier transform infrared spectra, vibrating sample magnetometer and fluorescence spectra, and the adsorption behavior was investigated. Binding experiments showed that the molecularly imprinted FeOX/ZnS NPs (FeOx/ZnS@MIPs) exhibited rapid fluorescent and magnetic responses, and high selectivity and sensitivity for the detection of bisphenol A (BPA). The maximum adsorption capacity of FeOx/ZnS@MIPs was 50.92 mg·g−1 with an imprinting factor of 11.19. Under optimal conditions, the constructed fluorescence magnetic molecularly imprinted polymers presented good linearity from 0 to 80 ng mL−1 with a detection limit of 0.3626 ng mL−1 for BPA. Moreover, the proposed fluorescence magnetic polymers were successfully applied to on-site magnetic separation and real-time fluorescence analysis of target molecule in real samples.

Lamb wave-based molecular diagnosis using DNA hydrogel formation by rolling circle amplification (RCA) process

Recent developments in microfluidics enable the lab-on-a-chip-based molecular diagnosis. Rapid and accurate diagnosis of infectious diseases is critical for preventing the transmission of the disease. Here, we characterize a Lamb wave-based device using various parameters including the contact angle and viscosity of the sample droplet, the applied voltage, and the temperature increase. Additionally, we demonstrate the functionality of the Lamb wave-based device in clinical application. Optimal temperature for rolling circle amplification (RCA) process is 30 °C, and it was achieved by Lamb wave generation at 17 V. Gene amplification due to RCA process could be detected by viscosity increase due to DNA hydrogel formation in a sample droplet, which induced the acoustic streaming velocity of suspended particles to be decreased. In our Lamb wave-based device, isothermal amplification of target nucleic acids could be successfully detected within 30 min using 10 μL of sessile droplet, and was validated by comparing that of commercial real-time fluorescence analysis. Our device enables simple and low-cost molecular diagnosis, which can be applied to resource-limited clinical settings.

DNA nanomachine-assisted magnetic bead based target recycling and isothermal amplification for sensitive fluorescence determination of interferon-γ

The authors describe a protein-triggered self-assembling DNA nanomachine for the detection of interferon-γ (IFN-γ), an important cytokine associated with immuno response. The DNA nanoprobe exhibits good specificity and sensitivity for interferon-γ (IFN-γ) detection. In the presence of the target (IFN-γ), four functional DNA probes are assembled to form a DNA nanocomplex. It promotes the release of the target for recycling. The captured DNA probe (biotinylated-C1) is then brought into contact with streptavidin-functionalized magnetic beads (sa-MB). These cause the dissociation of the B3 primer signal probe (S1) from the reaction solution which then is available for loop-mediated isothermal amplification (LAMP) analysis. As the deficiency of the B3 primer inhibits the LAMP reaction, the resulting point of inflexion (POI) value (the starting point of LAMP) is increased. In the absence of IFN-γ, the operation of the DNA nanomachine is not triggered, and the LAMP reaction remains unaffected. In this way, the POI values indirectly report the concentration of IFN-γ. The feasibility of the protein-driven DNA nanomachine was first demonstrated by the gel electrophoresis and real-time fluorescence analysis. Then, under optimal conditions, IFN-γ in concentrations as low as 5 ng·mL−1 can be quantified, and the detection limit is 2.3 ng·mL−1. Besides, this strategy can effectively discriminate IFN-γ from other proteins and displayed acceptable recoveries even in serum samples. In our perception, this approach holds great promise as it may be adapted to detect numerous other proteins.

A possible pathogenetic factor of sickle-cell disease based on fluorescent analysis via an optofluidic resonator

Waveguide based optofluidic resonator features high precision and high sensitivity in real-time fluorescent analysis. We present a novel optofluidic resonator following the hollow-core metal-cladding waveguide structure, which is then used to record the real-time binding process of Fe2+ and Fe3+ with protoporphyrin IX (PpIX) in PBS solution, respectively. The central fluorescent wavelength of compound with Fe2+ is in good accordance with that of the normal hemoglobin, whilst the peaks of the Fe3+ compound match the hemoglobin specimen from sickle-cell disease (SCD) patients. Similar statement holds when we monitor the real-time oxidation processes of these products by injecting oxygen into the optofluidic chip. These observations lead to the speculation that the SCD is caused by replacing the Fe2+ in hemoglobin with Fe3+, which may be insightful in the discovery of new clinical routes to cure this disease.

Real-time fluorescence analysis on molecular mechanisms for regulation of cytochrome P450scc activity upon steroidogenic stimulation in adrenocortical cells

Real-time fluorescence analysis revealed that the activity of cytochrome P450scc was related to Ca 2+ signals arising from extracellular NADPH, ACTH and ATP stimulation in adrenocortical fasciculata cells. The side-chain cleavage reaction by cytochrome P450scc was measured with 3β-hydroxy-22,23-bisnor-5-cholenyl ether (cholesterol–resorufin) by observing the distinct increase in fluorescence upon conversion of cholesterol–resorufin to resorufin and pregnenolone. Adrenocorticotropic hormone (ACTH) induced a relatively small stimulation of the P450scc activity. A significant production of resorufin was revealed after stimulation of cell cultures with 100 pM, 1 nM of ACTH for 3 h. On the other hand, extracellular NADPH was found to rapidly and greatly stimulate the resorufin production in intact cells immediately after the addition of 50–500 μM NADPH. The extracellular NADPH stimulation was prevented by the addition of thapsigargin and EGTA which abolished Ca 2+ oscillations induced by NADPH. Suramin, a specific antagonist of the P 2y type ATP receptor, also completely abolished the NADPH-induced cholesterol–resorufin conversion. These results imply that extracellular NADPH (membrane impermeable) produced Ca 2+ oscillations through its binding to ATP receptor thereby stimulating the activity of P450scc. The application of 45–500 μM extracellular ATP to cells did not, however, significantly increase the resorufin production. These three stimulators produced very different types of Ca 2+ signals. ACTH induced mainly a series of Ca 2+ spikes superimposed on a long-lasting basal Ca 2+ elevation. The Ca 2+ signals induced by NADPH showed predominantly a series of Ca 2+ spikes without elevation of the basal Ca 2+ concentration. Only long-lasting Ca 2+ elevation was induced by extracellular ATP. The stimulation of cytochrome P450scc may thus be correlated with the different patterns of Ca 2+ signals.

Solubilization and display of G protein-coupled receptors on beads for real-time fluorescence and flow cytometric analysis.

G protein-coupled receptors (GPCR) and cellular signaling elements are prime targets for drug discovery. Sensitive real-time methods that expand the analytical capabilities for these elements can play significant roles in basic research and drug discovery. Here, we describe novel approaches for the real-time fluorescence analysis of GPCRs. Using the G protein-coupled N-formyl peptide receptor (FPR) as a model system in concert with a fluorescent ligand, we showed the quantitative solubilization of his-tagged FPRs in 1% dodecyl maltoside. Solubilized receptors reconstitute in dodecyl maltoside with a mixture of bovine brain Gi/Go showing an apparent Kd of 100 nM. Solubilized receptors were also bound to Ni(2+)-silica particles and were detected in a flow cytometer by the binding of fluorescent ligand. The efficiency of receptor uptake by the particles was in excess of 80% with an apparent affinity for the bead in the nM range. The receptors had largely homogeneous dissociation characteristics, an appropriate Kd for the ligand in the low nM range and a high site number, with several million receptor molecules per particle. However, the G protein reconstitution was not detected on the beads, apparently for steric reasons. These approaches for displaying receptors could prove useful in drug discovery and in the analysis of the molecular assemblies in signal transduction.