Visual Assay Research Articles

Application of surface selective site-directed crystallization in a visual assay of DNA

Single crystals were used as digital signals for a DNA visual quantification assay using a smartphone.

Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

Development of a novel visual assay for ultrasensitive detection of Listeria monocytogenes in milk and chicken meat harnessing helix loop-mediated isothermal amplification (HAMP)

Development of a novel visual assay for ultrasensitive detection of Listeria monocytogenes in milk and chicken meat harnessing helix loop-mediated isothermal amplification (HAMP)

Visualized sensing of erythritol using a simple enzyme-free catechol-based hydrogel film.

Based on the versatile properties of bio-derived materials, non-enzymatic assays in combination with electronic devices have attracted increasing interest. Here, we report a novel enzyme-free visualization approach for the detection of erythritol, which is a zero-calorie natural sweetener and serves as an ideal sucrose substitute for diabetics or overweight people who need sugar control. The recognition element of the electrochemical biosensor was constructed by catechol modification on a chitosan-based hydrogel film. The signal transduction was achieved by the competitive binding assay of sweeteners. The results show that 2-fluorophenylboronic acid (FPBA) can form a cyclic boronate ester with the ortho-hydroxyls of both reduced catechol and oxidized quinone, impeding the electron transfer and leading to redox signal attenuation. The addition of sweeteners caused a competitive reaction resulting in bonding between the 1,2-diols and FPBA moieties, and in the recovery of the redox signals. Importantly, the pattern of redox signal changes of catechol can be detected optically, as the oxidized quinone state is darker in color than the reduced catechol state. Using a simple cell phone imaging application, we demonstrate that erythritol can be distinguished from other sweeteners in real samples using the oxidized catechol-Chit0/agarose hydrogel film. Thus, we envision that this method could allow diabetics and people who need to control their sugar intake to detect whether the product contains only erythritol in the field or at home. In addition, this work further illustrates the potential of bio-derived materials for performing redox-based functions and enzyme-free visualization assays.

Portable visual assay for anthrax biomarker based on lanthanide coordination polymer nanoparticles and smartphone-integrated mini-device

A novel portable sensor based on LML-Tb3+-AMP NPs and a custom-built device was fabricated for real-time and visual detection of anthracis biomarker.

Visual detection of water content in liquor with near-infrared fluorescence sensor assisted by smartphone

Water content was an essential indicator in organic solvents, and it was necessary to develop a facile, cheap and readily available tool for the real-time, specifical and sensitive detection of water content. In this work, two novel D-π-A type near-infrared fluorescence sensors (DCM-1 and DCM-2) were designed and synthesized for the detection of trace water in organic solvents. DCM-1 and DCM-2 with solvent-dependent effects and large Stokes shift (>120 nm) showed good linear “intensity-to-content” relationships in four commonly-used organic solvents, and accomplished the ultra-fast and high-accuracy detection of the trace water in organic solvents. More importantly, a portable, fast, and accurate smartphone-assisted visual assay was designed for visual quantitative detection of the water content in organic solvents with a detection limit as low as 1.028 % v/v (e.g. in ethanol) and a wide detection range (0–60 % v/v). The smartphone-based visual assay was further applied to estimate the water content in disinfection alcohol and commercial liquor, which furnished a new strategy and broad prospects to achieve the accurate onsite detection of water content.

Fully Integrated Ratiometric Fluorescence Enrichment Platform for High-Sensitivity POC Testing of Salivary Cancer Biomarkers.

The point-of-care (POC) testing of cancer biomarkers in saliva with both high sensitivity and accuracy remains a serious challenge in modern clinical medicine. Herein, we develop a new fully integrated ratiometric fluorescence enrichment platform that utilizes acoustic radiation forces to enrich dual-emission sandwich immune complexes for a POC visual assay. As a result, the color signals from red and green fluorescence (capture probe and report probe, respectively) are enhanced by nearly 10 times, and colorimetric sensitivity is effectively improved. When illuminated using a portable UV lamp, the fluorescence color changing from red to green can be clearly seen with the naked eye, which allows a semiqualitative assessment of the carcinoembryonic antigen (CEA) level. In combination with a homemade smartphone-based portable device, cancer biomarkers like CEA are quantified, achieving a limit of detection as low as 0.012 ng/mL. We also directly quantify CEA in human saliva samples to investigate the reliability of this fully integrated platform, thus validating the usefulness of the proposed strategy for clinical diagnosis and home monitoring of physical conditions.

Detection and elimination of tetracycline: Constructing multi-mode carbon dots for ultra-sensitive visual assay and CDs/TiO2 for photocatalytic degradation

Ultrasensitive detection and degradation of tetracycline (TC) has gained increasing attention owing to their inevitable detriments to human and ecosystem. Many studies have focused on mono-functional detection or degradation. However, the design of multi-functional materials is still a challenge. Till now, a novel protocol applied to the consecutive detection and complete degradation of TC are rarely successfully reported. Herein, we not only developed a multi-mode optical carbon dots (CDs) for ultra-sensitive sensing, but also further manufactured CDs/TiO2 photocatalyst that can degrade TC 100 %, completely eliminating the TC for the first time. The as-prepared CDs can selectively detect TC with low detection limit of 5.2 nM and 10.7 nM within only 40 s, accompanied by wide linear interval of 1.0–50.0 μM and 50.0–250.0 μM, respectively. Therefore, the fabricated point-of-care testing platform can realize “in-situ” visual detection rapidly and efficiently. Furthermore, a highly active CDs/TiO2 photocatalyst was prepared by rationally modifying CDs on TiO2 and exhibited almost 100 % degradation behavior towards TC within 70 min. The mechanism of fluorescence quenching and photocatalysis were lucubrated. Notably, our proposed protocol can drive consecutive detection and elimination towards tetracycline, which powerfully reduces the potential harm and provides a new strategy for the eradication of pollutants.

Real-time thiol detection in iPSC-derived neuron cultures using SemKur-IM, a novel fluorescent dithio probe

Neurological disorders associated with inflammation and oxidative stress show reduced glutathione (GSH) levels in the human brain. Drug discovery efforts and pharmacological studies would benefit from tools (e.g. chemical probes) that detect changes to oxidative stress, from the perspective of physiologically-relevant reporters like cellular thiols, including GSH. To this end, we have developed a fluorescence visualization assay using iPSC-derived cortical glutamatergic neurons that were loaded with 25 μM of a novel thiol-detection fluorescent probe, SemKur-IM. This probe enables visualization of cellular thiol level changes in the neuronal somas and neurites, in response exposure to N-acetyl-cysteine (NAC). Cellular thiol redox state was observed to change, based on an increase in green fluorescence (485 nm excitation maximum; 525 nm emission maximum) due to changes in thiol levels, from 0 to 40 mM. Interestingly, prior to treatment with NAC, cells did not appear to have significant levels of reduced thiols. Our studies demonstrate the utility of SemKur-IM in the detection of thiol levels in live cells in response to chemical exposures, such as from drugs that return the cell to a healthier reduced state. An initial application to screening the effects of an Alzheimer's disease drug candidate, Posiphen, using fluorescence cell sorting is presented. Other potential applications include high throughput screening of central nervous system (CNS) drugs thought to work by affecting cellular redox state in neurons.

Scab Resistance in some Apples Genotypes from the UASVM Collection Cluj-Napoca

Apple is one of the most consumed fruits in the world and as well one of the most important crops in temperate areas. Apple scab, caused by Venturia inaequalis is damaging diseases that affects apple species and causes up to 70% yield losses depending of cultivar’s resistance. In order to control this disease, costly chemical pesticides are necessary, which increase the pollution of environment by their toxicity. This study is focused on checking the behaviour of certain apple varieties from the pomological collection at UASVM Cluj-Napoca against apple scab infection. Nine apple cultivars ('Fuji Kiku', 'Red Cap', 'Lena', 'Katja', 'Gala Mitchgla', 'Fiesta', 'Yellow', 'Pinova' and 'Reinette du Canada'), were analysed in terms of behaviour to scab attack on leaves and fruits. Visual observations were completed by molecular analyses for the potential presence of the Vf gene in some cases. Combining results from visual observation and molecular assays, the 'Lena' and 'Pinova' cultivars were noted as resistant to scab in our field trial conditions. This indicates a good opportunity to bring them into apple breeding programs for resistance to apple scab.

Differential Developmental Neurotoxicity and Tissue Uptake of the Per- and Polyfluoroalkyl Substance Alternatives, GenX and PFBS.

Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic chemicals with several applications. Multiple adverse health effects are reported for longer carbon chain (≤C8) PFAS. Shorter carbon chain PFAS, [e.g., hexafluoropropylene oxide dimer acid (HFPO-DA; GenX) and perfluorobutanesulfonic acid (PFBS)] were introduced as alternatives. Past studies indicate that longer-chain PFAS are neurotoxic targeting the dopamine pathway, but it is not known if shorter-chain PFAS act similarly. This study aimed to evaluate developmental neurotoxicity and tissue uptake of GenX and PFBS using the zebrafish (Danio rerio). First, acute toxicity was assessed by measuring LC50 at 120 h postfertilization (hpf). Body burden was determined after embryonic exposure (1-72 hpf) to sublethal concentrations of GenX or PFBS by LC-ESI-MS/MS. Locomotor activity using a visual motor response assay at 120 hpf and dopamine levels at 72 hpf was assessed after embryonic exposure. PFBS was more acutely toxic and bioaccumulative than GenX. GenX and PFBS caused hyperactivity at 120 hpf, but stronger behavioral alterations were observed for PFBS. An increase in whole organism dopamine occurred at 40 ppb of GenX, while a decrease was observed at 400 ppb of PFBS. Differences detected in dopamine for these two PFAS indicate differential mechanisms of developmental neurotoxicity.

Monitoring Salivary Sialic Acid and Sialidase Activity to Assess Oral Health Status: Results of a Single Site Double-Blind Study

Objective: Sialic acid [SA] represents a critical mucosal membrane component maintaining mucosal integrity. This investigation stratified adult subjects based on clinical parameters of periodontal health to examine salivary sialic acid [SA] as a health measure and develop a corresponding rapid visual chair-side assay. Methods: Adults [n = 90] were enrolled and clinically stratified into healthy [n = 30], gingivitis [n = 29] or periodontitis [n = 31] groups. Saliva from subjects was evaluated for SA using the Ninhydrin method. A novel rapid SA spot test was developed utilizing filter paper discs soaked in a sialidase substrate. Substrate-laden disks were incubated at room temperature with saliva produced a blue color with increasing color intensities due to higher sialidase activity. Subjects were recalled weekly for clinical and salivary assessments. Results: Average baseline salivary SA in healthy, gingivitis and periodontal disease groups were 64, 95 and 102 µg/mL, respectively with significant differences (&lt;0.05). Differences in SA concentrations among control and test groups were maintained throughout the study. Similarly, the differences in the color intensities in the rapid visual chair side spot test were also observed during the entire study period. Conclusions: Increasing levels of salivary SA were observed from healthy to periodontal disease with these differences remaining consistent over the study. These results corresponded with the chair-side visual assay, which is suitable for patient education or monitoring.

A novel functionalized CdTe@MOFs based fluorometric and colorimetric biosensor for dual-readout assay of creatinine

Creatinine (Cre) is a crucial biomarker in the clinical diagnosis and therapy of chronic kidney disease (CKD) and muscle diseases. Therefore, designing and developing highly sensitive, multi-signal assays for the detection of Cre is highly significant. Herein, by in situ encapsulating CdTe QDs and post-synthetically modifying pyridine-2-carboxaldehyde (2-PC), a novel ratiometric fluorescence and colorimetric dual-signal sensor CdTe@UiO-66-PC-Cu was constructed for the detection of Cre with low detection limits (LOD) 2.48 nM and 1.78 μM, respectively. Satisfactory recovery of 93.0–105.0% (RSD < 4.8%, n = 3) was also acquired for determination of Cre in actual samples. In addition, by integrating smartphone-based portable colorimetric sensing, a novel hydrogel-based colorimetric paper analytical device (c-PAD) platform was fabricated for the visual colorimetric assay of Cre in whole blood without any separation process. The dual-signal sensing system of ratiometric fluorescence and colorimetry can observably improve the sensitivity and accuracy of Cre detection. Meanwhile, this portable smartphone-integrated colorimetric sensing platform features low cost, operational simplicity, and broad adaptability, providing an ideal way to achieve point-of-care testing (POCT) of other small molecule biomarkers in human whole blood.

Lysosome-targeted dual-emissive carbon dots for ratiometric optical dual-readout and smartphone-assisted visual determination of Hg2+ and SO32−

Smartphone-assisted visual assay not only expands quantitative analysis but also enhance real-time on-site sensing capabilities. Herein, lysosome-targeted dual-emissive carbon dots (L-CDs) can not only recognize Hg2+ and SO32− by ratiometric fluorescence and ratiometric absorption, but also visually quantify Hg2+ and SO32− by smartphone-assisted method. With monitoring of intrinsic ratiometric fluorescent variation (I580/I468), L-CDs are developed as an effective sensing platform for ratiometric fluorescent successive identification of Hg2+ and SO32− accompanying with continuous fluorescence variation of blue, purplish pink, pink, and light yellow. With detecting of inherent ratiometric absorption change (A538/A206), L-CDs are also constructed as an efficacious sensing terrace for ratiometric absorption consecutive discrimination of Hg2+ and SO32−. More crucially, integrating continuous fluorescence color change and color recognizer APP in the smartphone, visual quantification of Hg2+ and SO32− can be accomplished on the basis of the ratio of red value and blue value (R/B) with linear ranges of 0–130 and 0–850 µM, respectively, as well as LOD of 8.4 and 12.9 nM, respectively. More interestingly, confocal fluorescent imaging of HeLa cells further verifies that L-CDs can be regarded as favorable biosensors for on-site, real-time differentiation of Hg2+ and SO32− in vitro and in vivo with ratiometric manners.

Portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification for antibiotic resistance gene ermB in wastewater

Wastewater is among the main sources of antibiotic resistance genes (ARGs) in the environment, but effective methods to quickly assess ARGs on-site in wastewater are lacking. Here, using the typical ARG ermB as the target, we report a portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification (LAMP) for the detection of ARGs. Six primers of LAMP and the crRNA of CRISPR/Cas12a were first designed to be preamplification with LAMP and lead Cas12a to recognize the ermB via base pairing. Due to the trans-cleavage activity of CRISPR/Cas12a after amplicon recognition, ssDNA probes modified with reporter molecules were used to implement a visual assay with lateral flow test strips and fluorescence. After a simple nucleic acid extraction with magnetic beads, the constructed biosensor possesses excellent sensitivity and selectivity as low as 2.75 × 103 copies/μL using fluorescence and later flow strips in wastewater. We further evaluated the community-wide prevalence of ermB in wastewater influent and found high mass loads of ermB during different months. This user-friendly and low-cost biosensor is applicable for rapid on-site ARG detection, providing a potential point-of-use method for rapid assessments of ARG abundance in wastewater from large city areas with many wastewater treatment plants and in resource-limited rural areas.

Rapid and visual detection of Toxoplasma gondii oocyst in cat feces using loop-mediated isothermal amplification (LAMP) assay

Toxoplasma gondii is an obligate parasitic protozoon that transmits to animals and humans via ingested food. Cats that act as T. gondii’s final hosts play a critical role in T. gondii transmission by shedding millions of oocysts. Timely diagnosis of infected cats is essential for preventing toxoplasmosis because oocysts are a putative T. gondii source in epidemiology. We developed a new visual LAMP assay targeting the B1 gene to analyze single oocysts in cat feces in this study. The amplification result could be visually estimated based on the color change. LAMP assay analytical sensitivity was 101 copies/µL for the B1 gene plasmid, which was tenfold better than the PCR reaction. There were no cross-reactions with other parasites. The LAMP assay can detect a single T. gondii oocyst in 200 mg of cat feces. The LAMP assay detected a single oocyst in 200 mg cat feces at a higher rate than the PCR assay (83.3% vs. 50.0%).

Establishment and Application of CRISPR-Cas12a-Based Recombinase Polymerase Amplification and a Lateral Flow Dipstick and Fluorescence for the Detection and Distinction of Deformed Wing Virus Types A and B.

Deformed wing virus (DWV) is one of the important pathogens of the honey bee (Apis mellifera), which consists of three master variants: types A, B, and C. Among them, DWV types A (DWV-A) and B (DWV-B) are the most prevalent variants in honey bee colonies and have been linked to colony decline. DWV-A and DWV-B have different virulence, but it is difficult to distinguish them via traditional methods. In this study, we established a visual detection assay for DWV-A and DWV-B using recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 12a fluorescence system (RPA-CRISPR-Cas12a-LFD). The limit of detection of this system was ~6.5 × 100 and 6.2 × 101 copies/μL for DWV-A and DWV-B, respectively. The assays were specific and non-cross-reactive against other bee viruses, and the results could be visualized within 1 h. The assays were validated by extracting cDNA from 36 clinical samples of bees that were suspected to be infected with DWV. The findings were consistent with those of traditional reverse transcription-quantitative polymerase chain reaction, and the RPA-CRISPR-Cas12a assay showed the specific, sensitive, simple, and appropriate detection of DWV-A and DWV-B. This method can facilitate the visual and qualitative detection of DWV-A and DWV-B as well as the monitoring of different subtypes, thereby providing potentially better control and preventing current and future DWV outbreaks.

Novel Bimolecular Fluorescence Complementation (BiFC) Assay for Visualization of the Protein-Protein Interactions and Cellular Protein Complex Localizations.

Investigations of protein-protein interactions (PPIs) are of paramount importance for comprehending cellular processes within biological systems. The bimolecular fluorescence complementation (BiFC) assay presents a convenient methodology for visualizing PPIs within live cells. While a range of fluorescent proteins have been introduced into the BiFC system, there is a growing demand for new fluorescent proteins to accommodate the expanding requirements of researchers. This study describes the introduction of Tagged blue fluorescent protein 2 (TagBFP2) into the BiFC assay to verify the interaction between two proteins, with Enhanced yellow fluorescent protein (EYFP) employed as a positive control. Both fluorescent proteins demonstrated optimal performance in this study. Compared to EYFP, the BiFC system utilizing TagBFP2 yielded a higher signal-to-noise ratio, which facilitated differentiation of the signal of PPIs from noise and enabled employment of other fluorescent proteins within the BiFC assay. Notably, the utilization of a fluorescent secondary antibody in immunofluorescence applications or the tagging of an interest protein with a fluorescent protein occupied the green or yellow channel. Overall, the present article introduces a BiFC assay that is highly straightforward, reliable, and replicable, with the ability to be completed within 1week. This method requires neither expensive instrumentation nor technical skills of a high order.

Adverse developmental impacts in progeny of zebrafish exposed to the agricultural herbicide atrazine during embryogenesis

Atrazine (ATZ) is an herbicide commonly used on crops in the Midwestern US and other select global regions. The US Environmental Protection Agency ATZ regulatory limit is 3 parts per billion (ppb; µg/L), but this limit is often exceeded. ATZ has a long half-life, is a common contaminant of drinking water sources, and is indicated as an endocrine disrupting chemical in multiple species. The zebrafish was used to test the hypothesis that an embryonic parental ATZ exposure alters protein levels leading to modifications in morphology and behavior in developing progeny. Zebrafish embryos (F1) were collected from adults (F0) exposed to 0, 0.3, 3, or 30 ppb ATZ during embryogenesis. Differential proteomics, morphology, and behavior assays were completed with offspring aged 120 or 144 h with no additional chemical treatment. Proteomic analysis identified differential expression of proteins associated with neurological development and disease; and organ and organismal morphology, development, and injury, specifically the skeletomuscular system. Head length and ratio of head length to total length was significantly increased in the F1 of 0.3 and 30 ppb ATZ groups (p < 0.05). Based on molecular pathway alterations, further craniofacial morphology assessment found decreased distance for cartilaginous structures, decreased surface area and distance between saccular otoliths, and a more posteriorly positioned notochord (p < 0.05), indicating delayed ossification and skeletal growth. The visual motor response assay showed hyperactivity in progeny of the 30 ppb treatment group for distance moved and of the 0.3 and 30 ppb treatment groups for time spent moving (p < 0.05). Due to the changes in saccular otoliths, an acoustic startle assay was completed and showed decreased response in the 0.3 and 30 ppb treatments (p < 0.05). These findings suggest that a single embryonic parental exposure alters cellular pathways in their progeny that lead to perturbations in craniofacial development and behavior.

A luminescent MOF-based nonenzymatic probe for colorimetric/photothermal/fluorescence triple-mode assay of uric acid in body fluids

Monitoring the levels of uric acid (UA) in body fluids is of great significance in the clinical diagnosis and therapy of related diseases. Herein, a novel nanocomposite R6G@Fe-MOF based nonenzymatic probe is presented to provide a ratiometric fluorescent, colorimetric, and photothermal triple read-out signal for the visual, sensitive, and convenient assay of UA. The framework structure of the in situ encapsulated R6G@Fe-MOF is found to decompose upon the addition of UA, resulting in the reduction of Fe3+ to Fe2+. This reduction will lead to a rapid increase in fluorescence emission (FL) at 430 nm. Simultaneously, the FL at 573 nm will decrease remarkably due to the inner filter effect (IFE) between UA and R6G@Fe-MOF. Furthermore, the reaction of the generated Fe2+ with potassium ferricyanide (K3 [Fe(CN)6]) can in situ generate Prussian blue (PBNPs) with outstanding color and photothermal properties, which allow for easy colorimetric and photothermal signal readout. The detection limits (LOD) for the colorimetric, fluorometric and photothermal detection are low at 1.68 μM, 0.236 μM, and 1.32 μM respectively. Ultimately, it is successfully employed to determine UA in urine, serum, and saliva, yielding satisfactory results. The constructed R6G@Fe-MOF sensor provides a simple, sensitive, and accurate determination of UA that can be tailored to meet the needs of various applications, and also provides new perspectives for the design and development of versatile sensors for diverse uses.