Method For Rapid Quantification Research Articles

A simple and efficient method for betalain quantification in RUBY-expressing plant samples

The RUBY reporter system has demonstrated great potential as a visible marker to monitor gene expression in both transiently and stably transformed plant tissues. Ectopic expression of the RUBY reporter leads to bright red pigmentation in plant tissues that do not naturally accumulate betalain. Unlike traditional visual markers such as β-glucuronidase (GUS), luciferase (LUC), and various fluorescent proteins, the RUBY reporter system does not require sample sacrifice or special equipment for visualizing the gene expression. However, a robust quantitative analysis method for betalain content has been lacking, limiting accurate comparative analyses. In this work, we present a simple and rapid protocol for quantitative evaluation of RUBY expression in transgenic plant tissues. Using this method, we demonstrate that differential RUBY expression can be quantified in transiently transformed leaf tissues, such as agroinfiltrated Nicotiana benthamiana leaves, and in stable transgenic maize tissues, including seeds, leaves, and roots. We found that grinding fresh tissues with a hand grinder and plastic pestle, without the use of liquid nitrogen, is an effective method for rapid betalain extraction. Betalain contents estimated by spectrophotometric and High-Performance Liquid Chromatography (HPLC) analyses were highly consistent, validating that our rapid betalain extraction and quantification method is suitable for comparative analysis. In addition, betalain content was strongly correlated with RUBY expression level in agroinfiltrated N. benthamiana leaves, suggesting that our method can be useful for monitoring transient transformation efficiency in plants. Using our rapid protocol, we quantified varying levels of betalain pigment in N. benthamiana leaves, ranging from 110 to 1066 mg/kg of tissue, and in maize samples, ranging from 15.3 to 1028.7 mg/kg of tissue. This method is expected to streamline comparative studies in plants, providing valuable insights into the effectiveness of various promoters, enhancers, or other regulatory elements used in transgenic constructs.

Rapid Analytical Method for Quantification of Gamma-Hydroxybutyrate (GHB) in Hair by UPLC-MS/MS.

Gamma-hydroxybutyrate (GHB), an endogenous compound related to the neurotransmitter gamma-aminobutyric acid (GABA), is used as a therapeutic and recreational drug and as a "weapon" in drug-facilitated crimes. The very short window of detection of GHB in conventional matrices (blood and urine) makes necessary the use of alternative matrices like hair. Hair has a long window of detection and the possibility to perform segmental analysis, which makes it very useful for proving GHB intake. In the present work, a method for quantification of GHB in hair was developed and validated. Hair (10 mg) was washed twice with dichloromethane and then incubated at room temperature with Milli-Q water in an ultrasound bath for 30 min. Analysis was performed by UPLC-MS/MS using a CORTECS UPLC HILIC (1.6 μm), 2.1 × 100-mm column, and a gradient with acetonitrile and ammonium acetate (10 mM) at pH 6.0, with a total run-time of 10 min. For detection, a triple quadrupole mass spectrometer in ESI negative mode was used. The method was validated, following the criteria established in the "AAFS Standard Practices for Method Validation in Forensic Toxicology" guideline, obtaining satisfactory results for linearity (0.5-50 ng/mg), accuracy (95.0%-103.2%), imprecision (< 10.2%), limit of detection (0.1 ng/mg) and quantification (0.5 ng/mg), exogenous selectivity (no interferences), matrix effect (less than -44.2%), extraction efficiency (> 86.4%), process efficiency (> 46.1%), and autosampler stability (< 4.3%). The method was used for the analysis of 26 authentic hair samples, 25 from non-drug users, obtaining values between < LOQ and 6.25 ng/mg of endogenous GHB and 1 from a former GHB chronic user to prove abstinence.

A simple ultraperformance liquid chromatography mass spectrometry method for the determination of cortisol level in sea bass plasma

A simple, precise, and rapid ultra-performance liquid chromatography method for cortisol quantification in sea bass plasma, based on LC-ESI-UHR-QqTOF-MS, was optimised using tolperisone as internal standard. Sea bass plasma samples containing cortisol were spiked with tolperisone as internal standard and extracted with acetonitrile. Samples were vortexed and centrifuged at 15,000g for 10 min at 4 °C. The final extract, diluted in ammonium formate (1:1), was injected in the LC-ESI-UHR-QqTOF-MS system for analysis. Mass spectrometry acquisition parameters were set using positive ionisation mode over a m/z range from 150 to 2200. Cortisol and tolperisone were detected and quantified at m/z 363.21 and 246.18, respectively, in an Auto MS (MS/MS) mode. The detection and quantification limits of cortisol in sea bass plasma were 0.01 and 0.02 μg/mL, respectively. The mean extraction recovery of cortisol was of 99.1 ± 4.0 %. The within- and between-day precision presents a relative standard deviation (RSD) below 2.9 % and 5.3, respectively. Furthermore, the effect of four different diets (fish feed 1–4) in the basal and stress-induced plasma cortisol levels of sea bass was also assessed.

Development and validation of a UPLC-MS/MS method for rapid and simultaneous quantification of BPI-460372 and its metabolites BPI-460444 and BPI-460456 in human plasma

In cancer development and progression, the Hippo signaling pathway functions. The transcriptional enhanced associate domain (TEAD) stands out as a pivotal transcription factor within this pathway, and the suppression of TEAD represents a promising approach for cancer treatment. The primary aim of the study was to establish an analytical method for the concurrent quantification of a novel TEAD target inhibitor, BPI-460372, and its principal metabolites, BPI-460444 and BPI-460456, in human plasma. The chromatographic separation utilized a XSelect™ HSS C18 column (2.1 × 100 mm, 2.5 µm), while quantification was conducted on a SCIEX API 4000 mass spectrometer. 22 plasma samples were tested via the developed method. The calibration curve for BPI-460372 exhibited linearity from 2 to 2000 ng/mL, while its metabolites BPI-460444 and BPI-460456 had linearity between 1 and 1000 ng/mL (r > 0.99). The precision (RSD) was ≤ 17.1 %, and the accuracy (RE) fell within the range of −17.7 % to 15.0 %, all meeting acceptance criteria. The matrix effect was from 101.0 % to 105.8 %. The extraction recovery of analytes fell within the range of 96.8 % to 104.1 % with an RSD of less than 7.4 %. The developed method was effectively utilized in an advanced solid tumor patient, and the concentration trends of the three analytes in plasma were found to be largely consistent. The established analytical method showed great sensitivity, simplicity, accuracy, and reliability for the rapid and simultaneous analysis of the TEAD target inhibitor BPI-460372, alongside its major metabolites BPI-460444 and BPI-460456 in human plasma. This analytical method provided essential support for future clinical investigations and pharmacokinetic analysis.

Determination of Some Element’s Migrants in Aqueous Simulant from Plastic Food Contact Products by Inductively Coupled Plasma Mass Spectrometer

AbstractVarious chemicals present at different stages in the food supply chain can lead to the leaching of heavy metals. These metals can accumulate in the human body through the consumption of contaminated food. Consequently, it is necessary to validate an analytical technique for the quantification chemical that could contaminate food. This study presents a rapid, straightforward, and efficient analytical method for the direct quantification of some potentially toxic elements in aqueous simulants from plastic food contact products using an inductively coupled mass spectrometer (ICP-MS). The method’s validation encompassed the study of the estimated detection limits, practical quantification limits, linearity, accuracy, and measurement uncertainty of aluminium (Al), antimony (Sb), arsenic (As), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), nickel (Ni), and zinc (Zn) under optimized ICP-MS conditions. The estimated detection limits ranged from 7.5 × 10−4 to 0.074 mg/kg, while practical quantification limits spanned from 0.02 to 0.8 mg/kg. The average recoveries ± standard deviations at different spiking levels were varied between 85.7 ± 1.51 and 115.6 ± 0.88% with coefficients of variation between 0.42 and 5.85%. The method trueness was verified by using references materials (test material in aqueous acetic acid) purchased from Food Chemistry Proficiency Testing and Analysis (FAPAS) yielding satisfactory results within acceptable recovery and Z-score values. The method precision, in terms of relative standard deviation (RSD), was being below 4.22%. The method uncertainty expressed as expanded uncertainty of all validated elements was found to be ≤ 21.9%. Validated method was employed to determine specific elements in aqueous simulants of thirty commercial plastic food packaging samples, representing three distinct types of plastic polymers. The results showed that the mean concentrations, in mg/kg, were as follows: 2.04 (Al), 0.02 (As), 0.02 (Cd), 0.02 (Co), 0.06 (Cr), 0.41 (Cu), 1.55 (Fe), 0.09 (Mn), 0.15 (Ni), 0.07 (Pb), 0.05 (Sb), and 0.81 (Zn). Furthermore, 30% of analyzed samples exceeding the maximum permissible limits of Al for plastic materials and articles intended to come into contact with food.

Simultaneous determination of three tyrosine kinase inhibitors and three triazoles in human plasma by LC-MS/MS: applications to therapeutic drug monitoring and drug-drug interaction studies

Tyrosine kinase inhibitors (TKIs) and triazole antifungals are the first-line drugs for treating chronic myeloid leukemia (CML) and fungal infections, respectively, but both suffer from large exposure differences and narrow therapeutic windows. Moreover, these two types of drugs are commonly used together in CML patients with fungal infections. Multiple studies and guidelines have suggested the importance of therapeutic drug monitoring (TDM) of TKIs and triazoles. Currently, methods for the simultaneous determination of both types of drugs are limited. We developed a simple, rapid, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of three commonly used TKIs (imatinib, dasatinib, and nilotinib) and three commonly used triazoles (voriconazole, itraconazole, and posaconazole) in human plasma. The analytes were eluted on a Welch XB-C18 analytical column (50 × 2.1 mm, 5 µm) at 0.7 mL/min, using a gradient elution of 10 mM ammonium formate (A) and methanol–acetonitrile-isopropanol (80:10:10, v/v/v) containing 0.2 % formic acid (B) with a total analysis time of 3.5 min. The calibration curves were linear over the range from 20 to 4000 ng/mL for imatinib and nilotinib, from 2 to 400 ng/mL for dasatinib, and from 50 to 10,000 ng/mL for voriconazole, itraconazole, and posaconazole. Selectivity, accuracy, precision, recovery, matrix effect, and stability all met the validation requirements. The method was successfully used for TDM in CML patients who co-treated with both TKIs and triazoles. Drug-drug interaction analysis between TKIs and triazoles showed that a significant positive correlation was observed between imatinib and voriconazole, as well as dasatinib and voriconazole. Therefore, this method can be well applied in clinical TDM for patients receiving TKIs, triazoles, or both simultaneously.

Towards Digital Quantification of Ploidy from Pan-Cancer Digital Pathology Slides using Deep Learning.

Abnormal DNA ploidy, found in numerous cancers, is increasingly being recognized as a contributor in driving chromosomal instability, genome evolution, and the heterogeneity that fuels cancer cell progression. Furthermore, it has been linked with poor prognosis of cancer patients. While next-generation sequencing can be used to approximate tumor ploidy, it has a high error rate for near-euploid states, a high cost and is time consuming, motivating alternative rapid quantification methods. We introduce PloiViT, a transformer-based model for tumor ploidy quantification that outperforms traditional machine learning models, enabling rapid and cost-effective quantification directly from pathology slides. We trained PloiViT on a dataset of fifteen cancer types from The Cancer Genome Atlas and validated its performance in multiple independent cohorts. Additionally, we explored the impact of self-supervised feature extraction on performance. PloiViT, using self-supervised features, achieved the lowest prediction error in multiple independent cohorts, exhibiting better generalization capabilities. Our findings demonstrate that PloiViT predicts higher ploidy values in aggressive cancer groups and patients with specific mutations, validating PloiViT potential as complementary for ploidy assessment to next-generation sequencing data. To further promote its use, we release our models as a user-friendly inference application and a Python package for easy adoption and use.

Determination of Cannabinoids in Meat Products and Animal Feeds in Singapore Using Liquid Chromatography-Tandem Mass Spectrometry.

There has been a growing interest in the use of hemp as an animal feed ingredient considering its economic value and nutritional properties. However, there is a paucity of research regarding the safety of hemp-based animal feed currently. Thus, this raises safety concerns on the potential transfer of cannabinoids from hemp-based animal feed to animal products intended for human consumption and its health effects. As such, the detection and quantification of cannabinoids in meat and animal feeds would be desirable for monitoring purposes. In this study, a simple, rapid and sensitive method for the simultaneous quantification of four major cannabinoids (delta-9-tetrahydrocannabinol, cannabidiol, cannabinol and tetrahydrocannabinolic acid) in meat and animal feeds by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was successfully developed and validated. The method was selective and sensitive, achieving limits of detection and quantification for the four cannabinoids from 5 to 7 µg/kg and 15 to 21 µg/kg, respectively. The overall recovery with matrix-matched calibration curves for the cannabinoids ranged from 87-115%. The coefficients of variation were between 2.17-13.38% for intraday precision and 3.67-12.14% for inter-day precision. The method was subsequently applied to monitor cannabinoids in 120 meat and 24 animal feed samples. No cannabinoid was detected, suggesting no imminent food safety concerns arising from the potential incorporation of hemp and by-products in animal feed and nutrition under the promotion of sustainable agricultural practices.

Assessment of 5-Hydroxymethylfurfural in Food Matrix by an Innovative Spectrophotometric Assay.

Foods contaminants pose a challenge for food producers and consumers. Due to its spontaneous formation during heating and storage, hydroxymethylfurfural (HMF) is a prevalent contaminant in foods rich in carbohydrates and proteins. Colorimetric assays, such as the Seliwanoff test, offer a rapid and cost-effective method for HMF quantification but require careful optimization to ensure accuracy. We addressed potential interference in the Seliwanoff assay by systematically evaluating parameters like incubation time, temperature, and resorcinol or hydrochloric acid concentration, as well as the presence of interfering carbohydrates. Samples were analyzed using a UV-Vis spectrophotometer in scan mode, and data obtained were validated using HPLC, which also enabled quantification of unreacted HMF for assessing the protocol's accuracy. Incubation time and hydrochloric acid percentage positively influenced the colorimetric assay, while the opposite effect was observed with the increase in resorcinol concentration. Interference from carbohydrates was eliminated by reducing the acid content in the working reagent. HPLC analyses corroborated the spectrophotometer data and confirmed the efficacy of the proposed method. The average HMF content in balsamic vinegar samples was 1.97 ± 0.94 mg/mL. Spectrophotometric approaches demonstrated to efficiently determine HMF in complex food matrices. The HMF levels detected in balsamic vinegars significantly exceeded the maximum limits established for honey. This finding underscores the urgent need for regulations that restrict contaminant levels in various food products.

Rapid and Near-Analytical Planning Method for Entry Trajectory under Time and Full-State Constraints

A rapid trajectory-planning method based on an analytical predictor–corrector design of drag acceleration profile and a bank-reversal logic based on double-stage adaptive adjustment is proposed to solve the entry issue under time and full-state constraints. First, an analytical predictor–corrector algorithm is used to design the profile parameters to satisfy the terminal of altitude, velocity, range, time, and flight-path angle constraints. Subsequently, an adaptive lateral planning algorithm based on heading adjustment and maintenance is proposed to achieve the flight stage adaptive division and determination of the bank-reversal point, thereby satisfying the terminal position and heading angle constraints. Concurrently, a rapid quantification method is proposed for the adjustable capacity boundary of the terminal heading angle. On this basis, a range-and-time correction strategy is designed to achieve high precision and the rapid generation of a three-degree-of-freedom entry trajectory under large-scale lateral maneuvering. The simulation results demonstrated that compared with the existing methods, the proposed method can adaptively divide flight stages, ensuring better multitask applicability and higher computational efficiency.

Development and Validation of a Robust RP-HPLC Method for the Simultaneous Analysis of Exemestane and Thymoquinone and Its Application to Lipid-Based Nanoformulations.

The present study describes the development and validation of a simple and rapid HPLC method for the simultaneous quantification of exemestane and thymoquinone. The separation of both compounds was performed on a 5 μ C-18 column utilizing phase A as water/methanol (45:5 v/v) and phase B as acetonitrile (50 v/v) (total ratio of A/B = 40:60 v/v) in isocratic elution mode as the mobile phase at a flow rate of 0.8 mL/min. Further, the Box-Behnken design was used for optimizing the analytical method. The proposed method was validated for various parameters, and all parameters were found to be within an acceptable range. The simultaneous detection of both drugs was monitored at 243 nm with a retention time of 5.73 and 6.93 min, respectively. Moreover, the forced degradation studies were conducted under various stress conditions, and the relevance of the validated RP-HPLC method was further explored for the estimation of drugs from lipid-based nanoformulation. Taken together, the study construed the development of an efficient and robust method that could be used for the quantification of these agents in various in vitro as well as in vivo models.

Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study.

Amino acids (AAs) and their metabolites are important building blocks, energy sources, and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Therefore, robust, reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification are required to identify candidate biomarkers and to improve screening methods. We developed a validated semi-automated robotic liquid extraction and processing workflow and a rapid method for absolute quantification of 20 free, underivatized AAs and six TRP metabolites using dual-column U(H)PLC-MRM-MS. The extraction and sample preparation workflow in a 96-well plate was optimized for robust, reproducible high sample throughput allowing for transfer of samples to the U(H)PLC autosampler directly without additional cleanup steps. The U(H)PLC-MRM-MS method, using a mixed-mode reversed-phase anion exchange column with formic acid and a high-strength silica reversed-phase column with difluoro-acetic acid as mobile phase additive, provided absolute quantification with nanomolar lower limits of quantification within 7.9 min. The semi-automated extraction workflow and dual-column U(H)PLC-MRM-MS method was applied to a human prostate cancer study and was shown to discriminate between treatment regimens and to identify metabolites responsible for discriminating between healthy controls and patients on active surveillance.

A method for rapid and reliable quantification of VEGF-cell binding activity

Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor that binds a broad spectrum of cell types and regulates diverse cellular processes, including angiogenesis, growth and survival. However, it is technically difficult to quantify VEGF-cell binding activity because of reversible nature of ligand-receptor interactions. Here we used T7 bacteriophage display to quantify and compare binding activity of three human VEGF-A (hVEGF) isoforms, including hVEGF111, 165 and 206. All three isoforms bound equally well to immobilized aflibercept, a decoy VEGF receptor. hVEGF111-Phage exhibited minimal binding to immobilized heparan sulfate, whereas hVEGF206-Phage and hVEGF165-Phage had the highest and intermediate binding to heparan, respectively. In vitro studies revealed that all three isoforms bound to human umbilical vein endothelial cells (HUVECs), HEK293 epithelial and SK-N-AS neuronal cells. hVEGF111-Phage has the lowest binding activity, while hVEGF206-Phage has the highest binding. hVEGF206-Phage was the most sensitive to detect VEGF-cell binding, albeit with the highest background binding to SK-N-AS cells. These results suggest that hVEGF206-Phage is the best-suited isoform to quantify VEGF-cell binding even though VEGF165 is the most biologically active. Furthermore, this study demonstrates the utility of T7 phage display as a platform for rapid and convenient ligand-cell binding quantification with pros and cons discussed.

Development of Rapid, Simple and Low-Cost Methods for Quantification of Reducing Sugars in Cow Milk and Derivatives Using Smartphone Digital Images

Development of Rapid, Simple and Low-Cost Methods for Quantification of Reducing Sugars in Cow Milk and Derivatives Using Smartphone Digital Images

A developed and validated centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method for rapid quantification of unbound lenvatinib in human plasma

In clinical practice, the determination of unbound drug concentration is very important for dose adjustment and toxicity prediction because only the unbound fraction can achieve a pharmacological effect. A fast, sensitive and accurate analytical method of centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method was developed and applied to allow the quantification of unbound lenvatinib concentration. The application of linear regression analysis was used to examine the effects of centrifugal force, centrifugal time, and protein content on ultrafiltrate volume (Vu). The results indicated that the centrifugal force and centrifugal time have an influence on Vu that is significantly positive (P < 0.05). This developed method with good linearity (r2 = 0.9996), good accuracy (bias % ≤ 2.24 %), good precision (CV % ≤ 7.10 %), and good recovery (95.46 %−106.46 %) was suitable for routine clinical practice and studies. Particularly, the ultrafiltration membrane had no non-specific binding to lenvatinib. The unbound fractions can be separated in just 15 min. This method was applied to quantify clinical samples and to determine the plasma protein binding and unbound fraction of lenvatinib. This study provides a more effective and promising method for determination of unbound lenvatinib. It could be beneficial to measure the unbound concentration of lenvatinib in personalized medicine and therapeutic drug monitoring in routine clinical practice.

Preparation of surface molecularly‐imprinted magnetic kaolin for selective separation and determination of thiabendazole

AbstractIn this study, a selective sorbent was fabricated by combining a surface molecularly‐imprinted polymer and magnetic kaolin for reliable quantification of thiabendazole (TBZ) coupled with spectrofluorimetry. Several surface characterization methods were conducted to confirm the synthesis of TBZ‐imprinted magnetic kaolin with a polymer thickness of ~48 nm. The molecularly‐imprinted magnetic kaolin (MIP@MKLN) showed high adsorption capacity (8.7 mg/g), remarkable selectivity with an imprinting factor of 3.71 and excellent regeneration ability for up to 10 uses without significant change of its initial adsorption capacity for TBZ. The proposed method had linear range from 5 to 500 μg/mL TBZ concentration with a correlation coefficient of 0.995 and the limit of detection was 1.4 μg/mL. MIP@MKLN was used for selective separation and quantification of TBZ in orange and lemon samples. There were high recovery values ranging from 96.4% to 100.2% with lower relative standard deviations (less than 5%) in the spiked samples under optimized conditions. Overall, the proposed method based on MIP@MKLN as an excellent sorbent could produce a feasible, convenient, and promising method for rapid, selective, and sensitive quantification of TBZ in real samples.

Ex vivo method for rapid quantification of post traumatic brain injury lesion volumes using ultrasound

BackgroundStudies of traumatic brain injury often involve the quantification of the lesion volume as a major outcome measure. The determination of lesion volume typically employs the cutting and mounting of brain tissue, and the calculation of the cross-sectional area of the lesion within each section of brain after histological staining. This is a time consuming and laborious task often requiring many weeks to determine the lesion volume for an individual brain. MethodsIn this report we present a method for determining the lesion volume within the brain following traumatic brain injury that involves the use of ultrasound imaging. With this process the lesion volume can be determined within a time period of 90 min per brain rather than weeks and months. Moreover, we have developed a pipeline that will combine the cross-sectional ultrasound images of the brain with the Allen Mouse Brain Atlas to provide the precise anatomical structures that are affected by traumatic injury to the brain. The anatomical detail was lastly paired with behavioral data showing neurological deficits correlated with specific areas of brain injury. ResultsThe accuracy and precision of this method was shown to be highly consistent with the traditional histological approach. Additionally, the mapping process and behavioral data show that neurological recovery from 1 to 3 weeks post injury is not correlated with gross anatomical recovery of the TBI lesion in our TBI model. ConclusionTogether these approaches will enhance the pipeline for processing brain tissue in experimental conditions where the lesion volume is an important outcome parameter and provide more high resolution information about the identity of the damaged regions of the brain.

An HPLC-SEC-based rapid quantification method for vesicular stomatitis virus particles to facilitate process development

Virus particle (VP) quantification plays a pivotal role in the development of production processes of VPs for virus-based therapies. The yield based on total VP count serves as a process performance indicator for evaluating process efficiency and consistency. Here, a label-free particle quantification method for enveloped VPs was developed, with potential applications in oncolytic virotherapy, vaccine development, and gene therapy. The method comprises size exclusion chromatography (SEC) separation based on high-performance liquid chromatography (HPLC) instruments. Ultraviolet (UV) was used for particle quantification and multi-angle light scattering (MALS) for particle characterization. Consistent recoveries of over 97% in the SEC were achieved upon mobile phase screenings and addition of bovine serum albumin (BSA) as sample stabilizer. A calibration curve was generated, and the method's performance and applicability to in-process samples were characterized. The assay’s repeatability variation was < 1% and its intermediate precision variation was < 3%. The linear range of the method spans from 7.08×108 to 1.72×1011 VP/mL, with a limit of detection (LOD) of 7.72×107 VP/mL and a lower limit of quantification (LLOQ) of 4.20×108 VP/mL. The method, characterized by its high precision, requires minimal hands-on time and provides same-day results, making it efficient for process development.

Development of a TaqMan Real-Time PCR for Early and Accurate Detection of Anthracnose Pathogen Colletotrichum siamense in Pachira glabra.

Anthracnose, caused by Colletotrichum siamense, is a destructive disease of Pachira glabra in southern China. Early and proper monitoring and quantification of C. siamense is of importance for disease control. A calmodulin (CAL) gene-based TaqMan real-time PCR assay was developed for efficient detection and quantification of C. siamense, which reliably detected as low as 5 pg of genomic DNA and 12.8 fg (5800 copies) of target DNA. This method could specifically recognize all tested C. siamense isolates, while no amplification was observed in other closely related Colletotrichum species. The assay could still detect C. siamense in plant mixes, of which only 0.01% of the tissue was infected. A dynamic change in the amount of C. siamense population was observed during infection, suggesting that this real-time PCR assay can be used to monitor the fungal growth progression in the whole disease process. Moreover, the method enabled the detection of C. siamense in naturally infected and symptomless leaves of P. glabra trees in fields. Taken together, this specific TaqMan real-time PCR provides a rapid and accurate method for detection and quantification of C. siamense colonization in P. glabra, and will be useful for prediction of the disease to reduce the epidemic risk.

A ratiometric electrochemical sensor for simultaneous detection of multiple heavy metal ions in water and herbal medicines based on methylene blue-functionalized metal–organic framework

Heavy metal contamination in water and herbal medicine poses a global threat to human health. There is a pressing need for the development of a simple and rapid method for simultaneous quantification. In this study, a ratiometric electrochemical sensor was first constructed using a hybrid material composed of methylene blue functionalized metal–organic framework (MB-UiO66-NH2) and multi-walled carbon nanotubes (MWCNTs). Then, MB-UiO66-NH2/MWCNTs harnessed the large specific surface area of UiO66-NH2 and good conductivity of MWCNTs, resulting in a sensor with superior analytical performance for Cd2+, Pb2+, and Hg2+ detection. Meanwhile, the ratiometric probe uses MB as an internal reference, with the ratio signal (Imetalion/IMB) serving as output. This ratiometric electrochemical sensor was able to enhance the reproducibility and reliability and eliminate interference, particularly in complex environments. Importantly, based on this ratiometric electrochemical platform, the low detection limit were 11.08 nM for Cd2+, 5.47 nM for Pb2+, and 6.42 nM for Hg2+, which realized the simultaneous, sensitive, and reliable detection. In addition, it has been successfully applied in real water and representative herbal medicine sample analysis. Therefore, this work provides a new approach for the risk assessment of heavy metal accumulation, which is conducive to safeguarding people's lives and health.