Visual Assay Research Articles

Selective and visual detection of triphosgene by a red turn-on fluorescent probe

Triphosgene has been successfully and widely applied in many fields such as medicine, pesticides, spices, dyes and polymer materials. Though triphosgene itself is low toxic, the potential presence of nucleophiles would lead to the release of cracked gas phosgene which is highly toxic and irritant if triphosgene was inhaled into the lungs, resulting in more serious and sustained respiratory distress. The risk of inhalation poisoning is especially acute, because phosgene content can be mortal prior to its smell being recognizable. Hence, triphosgene may pose a threat to public health and security due to its intentional use in terrorist activities and accidental leakage in industrial accidents. There have been few special fluorescent probe developed specifically for triphosgene. Therefore, it is urgent to develop an efficient and reliable method to detect and identify triphosgene. The macroscopical red color has the function of stimulating people's senses and emotions. A visual red colour and fluorescent assay method based on a rhodamine dye for triphosgene was developed with high selectivity and sensitivity. Furthermore, the probe RDM-670 loaded test strips are prepared and display practical utility in sensitive fluorescent detection of triphosgene.

Photocatalysis engineered hydrophilic reactors on hydrophobic paper for the visual and colorimetric assay of alkaline phosphatase activity

Taking advantage of the intrinsic photocatalysis of TiO2, hydrophilic reactor arrays were lithographically patterned on a hydrophobic paper via a simple UV irradiation. As a proof-of-concept, alkaline phosphatase (ALP) was used as the model analyte for colorimetric analysis. As ALP can induce hydrolysis of pyrophosphate-Zn(II) framework, the released Zn2+ ions are subsequently coordinated with red-colored zincon to form blue-colored zincon-Zn(II) chelate complex, and these color differences were applied for further colorimetric assay. The sensing platform showed response to ALP ranging from 20 ~ 800 U L-1 with a detection limit of 3 U L-1, and the recoveries of ALP in serum samples were in the range 95.7 ~ 104.5% with relative standard deviations from 2.10 to 3.84%. Additionally, the distinct wettability features of the proposed sensing platform effectively prevent lateral fluid spread out of hydrophilic reactors, thus allowing not only the use ofminimum amount of analyte but it hasalso a high potential for simultaneous quantification of multiple samples.

Highly selective and multicolor ultrasensitive assay of dipicolinic acid: The integration of terbium(III) and gold nanocluster

A novel multicolor fluorescent nano-probe based on the hybridization of Tb3+ ion with gold nanoclusters (Au NCs) was synthesized to monitor and on-site visual assay of 2,6-pyridinedicarboxylic acid (DPA), a biomarker of bacterial spores. DPA can replace the water molecule in the center of Tb3+ and strongly coordinate with Tb3+ based on the analyte-triggered antenna effect. Simultaneously, the red fluorescence of Au NCs is not influenced after addition of DPA and can be used as steady inside fluorescence reference channel to measure background noise. On this basis, the multicolor fluorescence nano-probe based on Tb3+-doped Au NCs for fast analysis of DPA was fabricated. The linear range of this method is 0 to 12.5 μM and the limit of detection is 3.4 nM, which is well below the quantity of DPA concentration of 60 μM released by the spore transmission dose of anthrax infection. The proposed multicolor fluorescence nano-probe was successfully detecting DPA in actual sample with good sensitivity and specificity. In addition, the visual paper-based nano-probe is designed to detect DPA by using the color scanning application of smart phone. This developed platform possesses abroad application prospects with advantages of effective, convenient carrying, simple operation, good selectivity and repeatability.

Rapid and Visual RPA-Cas12a Fluorescence Assay for Accurate Detection of Dermatophytes in Cats and Dogs.

Dermatophytosis, an infectious disease caused by several fungi, can affect the hair, nails, and/or superficial layers of the skin and is of global significance. The most common dermatophytes in cats and dogs are Microsporum canis and Trichophyton mentagrophytes. Wood’s lamp examination, microscopic identification, and fungal culture are the conventional clinical diagnostic methods, while PCR (Polymerase Chain Reaction) and qPCR (Quantitative PCR) are playing an increasingly important role in the identification of dermatophytes. However, none of these methods could be applied to point-of-care testing (POCT). The recent development of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic platform promises a rapid, accurate, and portable diagnostic tool. In this paper, we present a Cas12a-fluorescence assay to detect and differentiate the main dermatophytes in clinical samples with high specificity and sensitivity. The Cas12a-based assay was performed with a combination of recombinase polymerase amplification (RPA). The results could be directly visualized by naked eyes under blue light, and all tested samples were consistent with fungal culture and sequencing results. Compared with traditional methods, the RPA-Cas12a-fluorescence assay requires less time (about 30 min) and less complicated equipment, and the visual changes can be clearly observed with naked eyes, which is suitable for on-site clinical diagnosis.

Ultrasensitive visual detection of miRNA-143 using a CRISPR/Cas12a-based platform coupled with hyperbranched rolling circle amplification

MicroRNAs are proposed novel biomarker for noninvasive diagnosis of cancer. miRNA-143 is reported to be associated with the development of prostate cancer. However, detection of miRNAs is still challenging due to their unique characteristics, such as small size and high sequence homology among family members. We here developed a gold nanoparticle (AuNP)-based visual assay that combines with CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (HRCA), which is called HRCA enhanced CRISPR/Cas12a-based assay (HECA) for sensitive detection of miRNA-143. The sequence-specific recognition character of CRISPR/Cas12a and HRCA signal amplification strategy enables the HECA outstanding specificity and sensitivity. In optimal condition, 1 fM miRNA-143 could be detected by naked eyes, and down to aM level with the aid of UV–Vis instrument. The diagnostic performance of the HECA for clinical samples was also evaluated based on the receiving operating characteristic algorithm (ROC), and our results suggest the miR-143 is a promising biomarker for noninvasive diagnosis of prostate cancer. This method is simple in operation and requires minimum instrument. We expect it to be widely applied in clinical diagnostics, especially in low-resource settings.

High Visual Colorimetric Determination of F‐ Ions by Exploiting the Inhibition of Oxidase Mimicking Activity of FeMnO4@GQD Nanocomposite

AbstractHuman health is significantly impacted by fluorides in water.Too much fluoride consumption through drinking water causes chronic dental and bones diseases. A trace number of fluorides in drinking water can be determined, which is immensely vital for human health. In the current study, we have designed a visual colorimetric chemosensor assay using the brilliant oxidase mimicking activity of biosynthesized FeMnO4@GQD nanocomposite. FeMnO4@GQD nanocomposites showed excellent mimicking activity in mild acid medium pH‐5, and obtained a Vmax value of 1.71603×10−6 and an important Km value of 0.0684 mM based on Hanes‐Woolf kinetics. The robust nanocomposites were shown to be significant stability for detecting oxidases under conditions. As a consequence, oxidase‐like activity was inhibited in the presence of fluoride ions. A linear correlation was established by the development of blue colour with fluoride ions concentration and appeared at a low limit of detection of 1.2 μg/ml without interfering with other common ions.

Colorimetric nano-beacon and magnetic separation-based rapid and visual assay for gram-negative bacteria

Food-borne diseases caused by pathogenic bacteria are one of the serious factors affecting human health. However, the most commonly used detection methods for pathogenic bacteria not only require expensive instruments, but also take a long time due to the complicated and cumbersome detection process. Therefore, the development of a fast, simple, and low-cost detection method for pathogenic bacteria is crucial for food safety and human health. In this work, based on the high binding ability of antimicrobial peptide (AMP) and polymyxin B (PMB) to bacteria, combined with magnetic separation technology, a new enzyme-free colorimetric strategy was constructed to achieve visual detection of Gram-negative bacteria in complex samples. The sensor system was divided into the following two parts: a colorimetric signal amplification nanoprobe, which was modified with AMP to enable effective binding of the colorimetric probe to the surface of bacteria, and a PMB-modified magnetic nanobead (MNB), which was used as the capture and enrichment unit of Gram-negative bacteria, as a result of which PMB could effectively distinguish Gram-negative bacteria from Gram-positive bacteria. Under optimized conditions, the detection limit of the method for Gram-negative bacteria (e.g. E. coli (G−)) was as low as 10 CFU/mL, and it was successfully applied to complex real samples. In addition, the developed colorimetric sensor offered advantages, such as fast response, less time consumption, high sensitivity, and low cost. It can be expected to become a new diagnostic tool for on-site detection of pathogenic bacteria in remote areas.

Dual-Signaling Photoelectrochemical Biosensor Based on Biocatalysis-Induced Vulcanization of Bi2MoO6 Nanosheets.

A magnetic-assisted photoelectrochemical (PEC) and colorimetric (CL) dual-modal biosensing platform with high precision was established to monitor prostate-specific antigen (PSA) based on Bi2MoO6 nanosheets (BMO) by coupling the aptamer-guided hybridization chain reaction (HCR) with the hydrolysate-induced vulcanization reaction of Bi2MoO6 nanosheets. Upon addition of PSA, trigger DNA (tDNA) was released by the interaction between the target analyte and the aptamer and then further hybridized with anchor DNA (aDNA) conjugated on magnetic beads (MBs). The as-released tDNA initiated the target-assisted HCR in the presence of two alternating hairpin sequences (Bio-H1 and Bio-H2) to produce nicked long double-stranded DNA on the surface of MBs, where numerous alkaline phosphatase (ALP) enzymes could assemble with MBs through the biotin-avidin reaction, resulting in the hydrolysis of sodium thiophosphate (TP) to H2S. The as-produced H2S reacted with BMO to form vulcanized BMO (BMO-S), thus leading to obvious enhanced PEC performance under visible light with the color change from light yellow to brown. Having optimized the test conditions, the magnetic-assisted biosensing system holds a good quantitative diagnosis sensitivity area in a range of 5.0 pg mL-1-100 ng mL-1 with a calculated detection limit down to 3.5 pg mL-1. Meanwhile, a visual colorimetric assay on basis of the change in the color of the materials was also realized. Given the exceptional performance of the constructed biosensor, it may possess great promise as an advanced bioanalytical tool for practical applications.

Visual detection of Shigella in milk by competitive annealing‐mediated isothermal amplification with paper‐based DNA extraction method

SummaryShigella is the main cause of endemic diarrhoea in low‐income countries. Fast and accurate detection of this pathogen can effectively prevent the consumption of contaminated food and reduce the risk of diarrhoea outbreaks. Recently, a competitive annealing‐mediated isothermal amplification (CAMP) assay was proposed as a novel nucleic acid detection technology. In this study, we developed a visual CAMP assay based on the invasive plasmid antigen H (ipaH) gene for rapid detection of Shigella in milk. Without enrichment culture, the visual CAMP assay could reliably detect Shigella at 2.6 × 102 CFU mL−1 or higher contamination levels. With enrichment culture, even if the spiked level is 1 CFU mL−1, the visual CAMP assay could reliably detect Shigella from the milk sample. Furthermore, a simple paper‐based DNA extraction method was established. The efficiency of the paper‐based method was comparable to that of the commercial kit method, and the entire extraction procedure can be completed in 15 min. The visual CAMP assay combined with the paper‐based DNA extraction method showed higher sensitivity and specificity for Shigella on‐site detection in milk samples.

A one-tube rapid visual CRISPR assay for the field detection of Japanese encephalitis virus

Early and rapid detection of Japanese encephalitis virus (JEV) is necessary for timely preventive and control measures. However, JEV RNA detection remains challenging due to the low level of viremia. In this study, a RApid VIsual CRISPR (RAVI-CRISPR) assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and CRISPR/Cas12a targeting was developed for easy detection of JEV in the field. We showed successful detection of 8.97 or more copies of the C gene sequence of JEV RNA within approximately 60 min. This assay also displayed no cross-reactivity with other porcine pathogens. We applied our one-tube RAVI-CRISPR assay to 18 brain tissue sample for JE diagnosis. The results from both fluorescence intensity measurements and directly naked-eye visualization were consistent with those from real-time PCR analysis. Taken together, our results showed that one-tube RAVI-CRISPR assay is robust, convenient, sensitive, specific, affordable, and potentially adaptable to on-site detection or surveillance of JEV in clinical and vector samples.

Assessment of the Quality of Injectable Antibiotics in Benin.

Substandard and falsified medicines are an enormous threat to global health. Poor quality antibiotic preparations contribute to the development of antimicrobial resistance. In surgery, where the occurrence of healthcare-associated infections is high, healthcare teams need to rely on the quality of antibiotic prophylaxis to prevent infections. We assessed the quality of antibiotics used for surgical infection prophylaxis in Benin. Thirty-three samples were collected from six hospitals located in various departments in Benin. The antibiotics (powders for injection: amoxicillin + clavulanic acid, ampicillin, ceftriaxone; solutions for injection: ciprofloxacin, gentamicin, metronidazole) were assessed using visual inspection, pharmacotechnical tests (including uniformity of mass, pH measure, sterility test, and active pharmaceutical ingredient identification), and assay tests (including a simple analytical method thin layer chromatography) and complex analytical techniques (ultraviolet-visible spectrophotometry, high-performance liquid chromatography-diode-array detection, conductometry). Because the material needed for the methods recommended by the pharmacopeias to assess the dosage of gentamicin was not available, we developed and validated a conductometry method. Results showed that 97% (n = 32) of the samples passed visual inspection; 100% (n = 33) of the samples passed the pharmacotechnical tests, identification of active ingredients, and sterility test; 88% (n = 29) passed the test for percentage of active pharmaceutical ingredients. Overall, 15% of the samples did not pass the quality test (3% on visual inspection and 12% for excess active ingredients). Although most of the samples passed the quality tests, it appears important to perform routine quality control for intravenous medicines.

A Visual Assay of a Loop-Mediated Isothermal Amplification Based Vertical Immunoassay for SARS-CoV-2 RNA Detection.

SARS-CoV-2 is a novel coronavirus that has caused a global pandemic. To date, 504,907,616 people have been infected and developed coronavirus disease 2019 (COVID-19). A rapid and simple diagnostic method is needed to control this pandemic. In this study, a visual nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification and a vertical flow visualization strip (RT-LAMP-VF) was successfully established and could detect 20 copies/μl of SARS-CoV-2 RNA transcript within 50 min at 61°C. This assay had no cross-reactivity with a variety of coronaviruses, including human coronavirus OC43, 229E, HKU1, NL63, severe acute respiratory syndrome-related coronavirus (SARSr-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and bat coronavirus HKU4, exhibiting very high levels of diagnostic sensitivity and specificity. Most strikingly, this method can be used for detecting multiple SARS-CoV-2 variants, including the Wuhan-Hu-1 strain, Delta, and Omicron variants. Compared with the RT-qPCR method recommended by the World Health Organization (WHO), RT-LAMP-VF does not require special equipment and is easy to perform. As a result, it is more suitable for rapid screening of suspected SARS-CoV-2 samples in the field and local laboratories.

CRISPR-Cas13a Based Visual Detection Assays for Feline Calicivirus Circulating in Southwest China.

Feline calicivirus (FCV) is a well-known causative pathogen for upper respiratory infection in cats. Its high genetic variability challenges existing molecular diagnostic methods in clinical settings. Thus, we developed two sensitive and visual assays for FCV nucleic acid detection based on RPA reaction and CRISPR-Cas13a trans-cleavage activity. Recombinant plasmid DNA, crRNAs, and RPA primers were designed and prepared, respectively, targeting to FCV ORF1 gene. Besides, purified LwCas13a protein was produced by E.coli prokaryotic expression system. To confirm the validity of FCV-Cas13a assays, seven reaction systems (RSs) with different components were tested, and visual readouts were displayed by lateral flow dipstick (FCV-Cas13a-LFD) and fluorescence detector (FCV-Cas13a-FLUOR), respectively. The established FCV-Cas13a assays were capable of detecting FCV nucleic acid in presetting RSs without cross-reaction with other feline-associated pathogens, and the detection limit was as low as 5.5 copies/μl for both visual methods. Moreover, the positive rate of 56 clinical specimens detected by FCV-Cas13a assays (67.9%, 38/56) was notably higher than that of RT-qPCR (44.6%, 25/56) (p < 0.001), including 13 presumptive positive specimens. Taken together, FCV-Cas13a assays provided reliable and visual diagnostic alternatives for FCV field detection.

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 µm and 300 µm diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Benzotrithiophene-based covalent organic frameworks for real-time visual onsite assays of enrofloxacin

Enrofloxacin (ENR) is a fluoroquinolone antibiotic that has been used to treat bacterial diseases in aquaculture extensively. ENR might accumulate in the body and cause severe liver damage. Therefore, it is necessary to develop a method for rapid detection of ENR. Herein, amine monomers with different numbers of amino groups, charge and length (1,3-diaminoguanidine monohydrochloride: DMGH, triaminoguanidine hydrochloride: TAGH, hydrazine: H) were used to regulate the fluorescence of benzotrithiophene tricar-baldehyde (BTT)-based covalent organic frameworks (COFs) for real-time visual onsite assays of ENR for the first time. The C3 symmetric COFBTT-TAGH only had one emission peak at 540 nm, while C6 symmetric COFBTT-DMGH and COFBTT-H had two emission peaks. COFBTT-TAGH could achieve the color transition from green to blue with increasing ENR. Thus test paper and gel were designed as real-time detection tools, combined with a smartphone APP. The detection limits obtained by RGB analysis were 106.2 nM for test paper and 26.00 nM for test gel, respectively. This method was applied to detect ENR in fish and clam metabolite successfully. This work also provides important reference to regulate fluorescence of COFs according to actual application.

Closed-tube saltatory rolling circle amplification with hydroxynaphthol blue for visual on-site detection of peanut as an allergenic food

Peanut allergy as one of the major causes of food-induced anaphylaxis is usually life-threatening for specific individuals. Nowadays, the effective remedy is avoidance of consuming allergen-containing food, urging monitoring the presence of peanut in foods. Herein, we proposed a closed-tube saltatory rolling circle amplification (SRCA) assay with hydroxynaphthol blue (HNB) for visual on-site detection of peanut allergen, displaying highly specificity for Ara h 1-encoding gene. The results can be discriminated by naked eye without opening the tube, averting costly sophisticated apparatus and reducing the risk of cross-contamination. The assay was 1000-, 100- and 10-fold more sensitive than PCR, LAMP and SRCA, respectively. The relative limit of detection is 0.01% in a binary mixture. The relative sensitivity, specificity and accuracy of this method were 100%, 97.50% and 98.67%, respectively. Thus, the visual closed-tube HNB-SRCA assay with superiorities of easy-to-perform, cost-effective and user-friendly has excellent potential for on-site application of food allergy.

Development of a rapid dot-blot assay for ochratoxin A (OTA) detection using peptide conjugated gold nanoparticles for bio-recognition and detection

Ochratoxin A is a secondary metabolite produced by fungi and a major mycotoxin contaminating cereal grains and cereal-based products. Although, ochratoxin A is hepatotoxic, nephrotoxic and carcinogenic, its detection is limited due to low awareness, time-consuming conventional methods and false positive results. In this study, a rapid, user-friendly dot-blot assay was developed using peptide conjugated gold nanoparticles (AuNPs) as a detection agent. The peptide binding to ochratoxin A was evaluated by the apparent dissociation constant ( K d app ) measurement using indirect enzyme linked immunoabsorbent assay. A low K d value (1.046 μM) suggested high binding of peptide with ochratoxin A. The potential of peptide-based ochratoxin A recognition was translated to a portable platform using streptavidin functionalized AuNPs conjugated to a biotinylated 11-mer peptide. Dot-blot assay was developed for detection of ochratoxin A with a limit of detection of 0.49 μg/kg. Negligible cross-reactivity with low spot intensities were observed for high concentrations of other mycotoxins like aflatoxin B1 and citrinin, in comparison to control. Further, percent recovery of ochratoxin A from spiked wheat samples by dot-blot was comparable to the conventional high performance liquid chromatography (HPLC). Moreover, detection of ochratoxin A by dot-blot was validated by HPLC in 65 whole wheat, crushed wheat coarse, crushed wheat fine and wheat flour samples with a high correlation with R 2 = 0.93. HPLC and dot-blot confirmed the presence of ochratoxin A ˂ 5 μg/kg in the samples. Therefore, a portable, easy-to-use dot-blot assay for ochratoxin A detection with potential of application at each stage of harvesting, storage, production and distribution can contribute to safe and healthy food and feed. • A rapid, on-site, sensitive visual Dot-blot assay was developed for ochratoxin detection. • Peptide conjugated gold nanoparticles were employed as a recognition label to replace expensive antibodies. • The developed Dot-blot assay had a limit of detection of 0.49 μg/kg for ochratoxin. • High sensitivity and specificity for ochratoxin detection.

Rapid on-site identification of Lepiota brunneoincarnata-induced mushroom poisoning by simple DNA extraction and loop-mediated isothermal amplification strategy

Lepiota brunneoincarnata-induced mushroom poisoning has occurred widely in some areas of the world in recent years, resulting in high mortality and posing potential health risks to humans and pets. However, a rapid and sensitive method for the identification of L. brunneoincarnata is scarce. Loop-mediated isothermal amplification (LAMP) combined with visual dye reaction or lateral flow dipstick (LFD) was established for rapid on-site detection of L. brunneoincarnata. Simple DNA extraction was completed by the heating lysis method within 15 min. In addition, a specific LAMP primer set was designed targeting the internal transcribed spacer (ITS) sequence of L. brunneoincarnata, with no cross-reactivity against 22 other mushroom species. Sensitivity evaluation showed that the detection limits of visual LAMP and LAMP-LFD assays were 10 and 0.1 pg/μL genomic DNA of L. brunneoincarnata, respectively. Moreover, the developed method was successfully applied to cooked mushroom and simulated vomitus, with detection limits of 1% admixture for the visual LAMP assay and 0.1% admixture for the LAMP-LFD assay. The whole detection protocol can be completed in 60 min without expensive instruments, which is suitable for the rapid identification of Lepiota brunneoincarnata-induced mushroom poisoning in the field.

Engineering of 2D artificial nanozyme-based blocking effect-triggered colorimetric sensor for onsite visual assay of residual tetracycline in milk.

Accurate and low-cost onsite assay of residual antibiotics in food and agriculture-related matrixes (e.g., milk) is of significant importance for evaluating and controlling food pollution risk. Herein, we employed hybrid Cu-doped-g-C3N4 nanozyme to engineer smartphone-assisted onsite visual sensor for reliable and precise reporting the levels of tetracycline (TC) residues in milk through π-π stacking-triggered blocking effect. Benefiting from the synergetic effects of Cu2+ and g-C3N4 nanosheet, Cu-doped-g-C3N4 nanocomposite exhibited an improved peroxidase-like activity, which could effectively catalyze H2O2 to oxidate colorless TMB into steel-blue product oxTMB. Interestingly, owing to the blocking effect caused by the π-π stacking interaction between TC tetraphenyl skeleton and Cu-doped-g-C3N4 nanozyme, the affinity of Cu-doped-g-C3N4 nanocomposite toward the catalytic substrates was remarkably blocked, resulting in a TC concentration-dependent fading of solution color. Using smartphone-assisted detection a simple, low-cost, reliable, and sensitive portable colorimetric sensor-based nanozyme for onsite visual monitoring the residual TC in milk was successfully developed with a detection limit of 86.27nM. Of particular mention is that this detection limit is comparable to most other reported colorimetric methods and below most official allowable residue thresholds in milk matrixes. This work gave a novel insight to integrate two-dimensional (2D) artificial nanozymes-based π-π stacking-triggered blocking effect with smartphone-assisted detection for developing efficient and low-cost colorimetric point-of-care testing of the risk factors in food and agriculture-related matrixes.

Establishing Olfactory Based Learning and Memory Protocol in Kabuki Syndrome Mice

BackgroundKabuki Syndrome (KS) is a genetic disorder that affects multiple systems influencing learning and development. This project is focused on Type 1 KS, which is caused by a heterozygous loss‐of‐function mutation in the Lysine Methyltransferase 2D (KMT2D) gene. KMT2D encodes for kmt2d, a protein responsible for methylating histone 3 lysine 4 (H3K4) to open the chromatin and promote gene expression. Reduced kmt2d function results in reduced H3K4 methylation and gene expression capabilities. Using a mouse model of Kabuki Syndrome, our lab aims to determine effective treatments for the molecular and behavioral deficits seen in this disease. Our mouse model has a C57BL/6J genetic background and has shown to develop eye abnormalities reported in C57BL/6J mice. This has made it unreliable for us to use visual based learning assays with our mice, so we are working to establish an olfactory based learning and memory protocol with this model. We aim to answer the following questions: 1. Are these mice able to learn using an olfactory based discriminatory assay? 2. Does the light/dark cycle impact their learning capabilities? 3. Do Kmt2d+/βGeo­mice ­show a deficit in olfactory learning and memory capabilities?MethodsTraining. Mice will be trained to associate either lemon or rose scent to a saccharin reward.Dark Cycle: At the start of their dark cycle, mice will be moved to a procedure room and be allowed to sit for 2 hours. Red light lamps will be used to avoid disrupting their light cycle. Sample pots will be prepared in the hallway on a cart. The mice will then be placed into separate clean IVC cages filled with their regular bedding. The animals will receive six 10 min trials per day (3 rose and 3 lemon) in random order, with 10 min resting periods back in their home cage in between trials. Over 4 days the animals will receive 24 trials in total. Fresh pots will be used for each trial.Light Cycle: The same training procedure as the dark cycle, only without the red lamps.Testing. Discrimination tests will be performed on the day following the last training trials at approximately the same time as training, with the same light and food manipulations as in training. Prior to testing the animals will be habituated to the apparatus. Odor pots containing bedding but no odors will be placed in the middle of each end compartment of the association chamber. The mouse will be placed in the center of the apparatus to give it an opportunity to explore the apparatus. After 2 minutes, the mouse will be placed in an IVC cage for ∼5 min prior to the test phase.The rose and lemon odor pots, without saccharin, will be placed in the discrimination chamber. The mouse will be placed in the center of the chamber. The discrimination test will be recorded on video, and the time the mouse spent digging in both odor pots during a 2 min period will be recorded by a third party. At the end of each trial the test chamber will be cleaned and dried prior to testing another subject. The discrimination chamber will be rotated between subjects to counteract the influence of any room cues.SummaryThis project will establish a reliable method for learning and memory analyses in this model.