Low-cost, immediate, general-purpose, and high-throughput (LIGHt) smartphone colorimetric screening assay for water-soluble protein.

An efficient and rapid method for the detection of total soluble protein in tobacco leaves, utilizing a smartphone-based colorimetric approach has been developed. The proposed low-cost, immediate, general-purpose, and high-throughput (LIGHt) smartphone colorimetric screening assay integrates commercially available microplates, enabling on-site, high-throughput screening of tobacco leaf quality. The study involves preparing protein standard solutions and constructing standard curves using both spectrophotometric and smartphone-based methods. The LIGHt smartphone colorimetry yielded an average relative standard deviation of 10.6 %, a limit of detection of 2 µg/mL, and an average recovery of 93 %. The results demonstrated a comparable performance between intensities from the blue channel and the absorbance values in reflecting protein concentrations, validating the feasibility of utilizing smartphone colorimetry for protein concentration determination. Our approach demonstrates the potential for practical implementation in the field, providing a cost-effective and user-friendly solution for rapid quality assessment in the tobacco industry. The LIGHt smartphone colorimetry enhances quality control practices in the tobacco sector and offers a promising tool for on-site production quality testing in various industries, such as fruits and vegetables.

Differences in bile acid profiles between cholestatic diseases - Development of a high throughput assay for dried bloodspots.

BACKGROUND: Cholestasis causes accumulation of bile acids (BAs) and changes the circulating bile acid profile. Quantification of circulating BAs in dried bloodspots (DBS) may demonstrate obstruction of bile flow and altered bile acid metabolism in the liver. High sample throughput enables rapid screening of cholestatic diseases. MATERIALS AND METHODS: Ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used for optimizing separation and detection of the primary unconjugated BAs cholic acid (CA) and chenodeoxycholic acid (CDCA); the secondary unconjugated BAs ursodeoxycholic acid (UDCA), hyodeoxycholic acid (HDCA) and deoxycholic acid (DCA), as well as the glycine- and taurine-conjugated variants of CA, CDCA, DCA and UDCA. Donor blood was obtained to prepare DBS calibrators and quality controls for method development and validation. RESULTS: We developed a quantitative bile acid assay with a run-time of two minutes, and one-step sample preparation of 3.2 mm DBS discs. Validation results demonstrated overall good performance and was considered fit for purpose. Children with Alagille syndrome, Aagenaes syndrome and alpha-1 antitrypsin deficiency had increased BAs in DBS from newborn screening samples compared with age matched controls, and had different bile acids profiles. CONCLUSION: We propose that our high throughput assay allows bile acid profiling in DBS that can be a valuable assessment tool for early screening of cholestasis in children. Assaying BAs in dried bloodspots is key for early detection of cholestasis, and provides transferability to a newborn screening setting.

Simultaneous Plate-Reader Characterization of Promoter Activity and Cell Growth in Engineered Mammalian Cells.

Automated high-throughput methods that support tracking of mammalian cell growth are currently needed to advance cell line characterization and identification of desired genetic components required for cell engineering. Here, we describe a high-throughput noninvasive assay based on plate reader measurements. The assay relies on the change in absorbance of the pH indicator phenol red. We show that its basic and acidic absorbance profiles can be converted into a cell growth index consistent with cell count profiles, and that, by adopting a computational pipeline and calibration measurements, it is possible to identify a conversion that enables prediction of cell numbers from plate measurements alone. The assay is suitable for growth characterization of both suspension and adherent cell lines when these are grown under different environmental conditions and treated with chemotherapeutic drugs. The method also supports characterization of stably engineered cell lines and identification of desired promoters based on fluorescence output.

Efficient arabinoxylan assay for wheat: Exploring variability and molecular marker associations in Wholemeal and refined flour.

In this study, we present a modified high throughput phloroglucinol colorimetric assay for the quantification of arabinoxylans (AX) in wheat named PentoQuant. The method was downscaled from a 10 ml glass tube to 2 ml microcentrifuge tube format, resulting in a fivefold increase in throughput while concurrently reducing the overall cost and manual labor required for the analysis. Comparison with established colorimetric assays and gas chromatography validates the modified protocol, demonstrating its superior repeatability, rapidity, and simplicity. The effectiveness of the protocol was tested on 606 unique whole meal (WM) and refined flour (RF) bread wheat samples which revealed the presence of more than a twofold variation in both the soluble (WE-AX) and total (TOT-AX) AX fractions in WM (TOT-AX = 31.9-76.1 mg/g; WE-AX = 4.4-12.6 mg/g) and RF (TOT-AX = 7.7-22.4 mg/g; WE-AX = 3.9-11.4 mg/g). Results obtained from the AX quantification were used to test the effectiveness of four molecular markers associated with AX variation and targeting two major genomic regions on the 1BL and 6BS chromosomes. These markers appeared to be particularly relevant for the WE-AX fraction, providing insights to enable marker-assisted breeding.

Optimization of a deep mutational scanning workflow to improve quantification of mutation effects on protein-protein interactions.

Deep Mutational Scanning (DMS) assays are powerful tools to study sequence-function relationships by measuring the effects of thousands of sequence variants on protein function. During a DMS experiment, several technical artefacts might distort non-linearly the functional score obtained, potentially biasing the interpretation of the results. We therefore tested several technical parameters in the deepPCA workflow, a DMS assay for protein-protein interactions, in order to identify technical sources of non-linearities. We found that parameters common to many DMS assays such as amount of transformed DNA, timepoint of harvest and library composition can cause non-linearities in the data. Designing experiments in a way to minimize these non-linear effects will improve the quantification and interpretation of mutation effects.

High-throughput and green optical sensing of thiocyanate in human saliva based on microplates and an overhead book scanner as detector.

An instrumental-free, high-throughput assay has been developed for the quantification of thiocyanate in human saliva. The proposed green method is based on the rapid reaction of the analyte with Fe(III) under acidic pH in a microplates format to form a colored complex that is captured as an image by an overhead book scanner. Optimization included the effects of the amount concentration of Fe(III), acidity and reaction time / complex stability using a total volume of 300 µL per well. Validation towards the matrix effect was focused on the specific application and was performed using both artificial and human saliva. The linearity of the developed assay was up to 500 µM thiocyanate offering a lower limit of quantification (LLOQ) of 30 µM. The green potentials were evaluated by both the Green Analytical Procedure (GAPI) and Blue Applicability Grade (BAGI) indexes. The thiocyanate content in the saliva of non-smoking volunteers ranged between 750 and 1350 µΜ, while elevated concentrations were verified in smoking individuals (1860-3080 µΜ). Statistical agreement with a corroborative method was assessed using the Bland-Altman plot.

Arabidopsis transcriptome responses to low water potential using high-throughput plate assays.

Soil-free assays that induce water stress are routinely used to investigate drought responses in the plant Arabidopsis thaliana. Due to their ease of use, the research community often relies on polyethylene glycol (PEG), mannitol, and salt (NaCl) treatments to reduce the water potential of agar media, and thus induce drought conditions in the laboratory. However, while these types of stress can create phenotypes that resemble those of water deficit experienced by soil-grown plants, it remains unclear how these treatments compare at the transcriptional level. Here, we demonstrate that these different methods of lowering water potential elicit both shared and distinct transcriptional responses in Arabidopsis shoot and root tissue. When we compared these transcriptional responses to those found in Arabidopsis roots subject to vermiculite drying, we discovered many genes induced by vermiculite drying were repressed by low water potential treatments on agar plates (and vice versa). Additionally, we also tested another method for lowering water potential of agar media. By increasing the nutrient content and tensile strength of agar, we show the 'hard agar' (HA) treatment can be leveraged as a high-throughput assay to investigate natural variation in Arabidopsis growth responses to low water potential.

A high-throughput lysosome trafficking assay guides ligand selection and elucidates differences in CD22-targeted nanodelivery.

Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand.

The ability of this pH-sensitive reporter platform to rapidly screen ligands in nanoparticle format will enable identification and production of targeted NPs with desired lysosome trafficking properties.

Polysaccharide sulfotransferases: the identification of putative sequences and respective functional characterisation.

The vast structural diversity of sulfated polysaccharides demands an equally diverse array of enzymes known as polysaccharide sulfotransferases (PSTs). PSTs are present across all kingdoms of life, including algae, fungi and archaea, and their sulfation pathways are relatively unexplored. Sulfated polysaccharides possess anti-inflammatory, anticoagulant and anti-cancer properties and have great therapeutic potential. Current identification of PSTs using Pfam has been predominantly focused on the identification of glycosaminoglycan (GAG) sulfotransferases because of their pivotal roles in cell communication, extracellular matrix formation and coagulation. As a result, our knowledge of non-GAG PSTs structure and function remains limited. The major sulfotransferase families, Sulfotransfer_1 and Sulfotransfer_2, display broad homology and should enable the capture of a wide assortment of sulfotransferases but are limited in non-GAG PST sequence annotation. In addition, sequence annotation is further restricted by the paucity of biochemical analyses of PSTs. There are now high-throughput and robust assays for sulfotransferases such as colorimetric PAPS (3'-phosphoadenosine 5'-phosphosulfate) coupled assays, Europium-based fluorescent probes for ratiometric PAP (3'-phosphoadenosine-5'-phosphate) detection, and NMR methods for activity and product analysis. These techniques provide real-time and direct measurements to enhance the functional annotation and subsequent analysis of sulfated polysaccharides across the tree of life to improve putative PST identification and characterisation of function. Improved annotation and biochemical analysis of PST sequences will enhance the utility of PSTs across biomedical and biotechnological sectors.

High-throughput assay for regulated secretion of neuropeptides in mouse and human neurons.

Neuropeptides are the largest group of chemical signals in the brain. More than 100 different neuropeptides modulate various brain functions and their dysregulation has been associated with neurological disorders. Neuropeptides are packed into dense core vesicles (DCVs), which fuse with the plasma membrane in a calcium-dependent manner. Here, we describe a novel high-throughput assay for DCV exocytosis using a chimera of Nanoluc luciferase and the DCV-cargo neuropeptide Y (NPY). The NPY-Nanoluc reporter colocalized with endogenous DCV markers in all neurons with little mislocalization to other cellular compartments. NPY-Nanoluc reported DCV exocytosis in both rodent and induced pluripotent stem cell-derived human neurons, with similar depolarization, Ca2+, RAB3, and STXBP1/MUNC18 dependence as low-throughput assays. Moreover, NPY-Nanoluc accurately reported modulation of DCV exocytosis by known modulators diacylglycerol analog and Ca2+ channel blocker and showed a higher assay sensitivity than a widely used single-cell low-throughput assay. Lastly, we showed that Nanoluc coupled to other secretory markers reports on constitutive secretion. In conclusion, the NPY-Nanoluc is a sensitive reporter of DCV exocytosis in mammalian neurons, suitable for pharmacological and genomic screening for DCV exocytosis genes and for mechanism-based treatments for central nervous system disorders.

Use of Lentivirus-Based Method for Establishing TK6 Human Cell Lines Expressing Cytochrome P450 and its Applications in Genotoxicity Testing.

The human lymphoblastoid cell line TK6 stands out as the most widely employed human cell line in genotoxicity testing, as recommended by various testing guidelines for in vitro assessments. Nevertheless, like many testing cell lines, TK6 lacks functional phase I drug-metabolizing enzymes crucial for chemical genotoxicity evaluations. This protocol introduces a lentivirus-based methodology for establishing a panel of TK6-derived cell lines, each expressing one of 14 cytochrome P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7). The utilization of a lentiviral expression system ensures stable transduction, offering notable advantages such as sustained transgene expression, high transduction efficiency, positive selection feasibility, and user-friendly application. Additionally, we present a detailed procedure for validating the enhanced expression of each CYP in the established cell lines through real-time PCR, western blotting, and mass spectrometry analysis. Lastly, we exemplify the application of these CYP-expressing TK6 cell lines in genotoxicity testing, employing a flow-cytometry-based in vitro micronucleus test. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Lentivirus production and transduction for TK6 cells Support Protocol: Selecting a single clone of CYP-expressing TK6 cells Basic Protocol 2: Validation of CYP expression in TK6 cell lines Basic Protocol 3: Application of transduced cell lines in flow-cytometry-based micronucleus assay.

Multiplex digital microfluidics using serial controls and its applications in glucose sensing.

Digital microfluidics (DMF) has found great applications in vitro diagnostics (IVD). Compared to the microfabrication-based DMF, printed circuit board (PCB)-based DMF is more economical and compatible with existing IVD instruments. Despite that, current PCB-based DMF is oftentimes limited by the available droplets that can be controlled simultaneously, compromising their throughput and applications as point-of-care tools. In this work, a platform that simultaneously controls multiple PCB-based DMF plates was constructed. The software and hardware were first developed, followed by the reliability tests. Colorimetric analysis of glucose was applied to the PCB-based DMF, demonstrating the capability of this platform. With the high throughput enabled by simultaneous operations of multiple plates, this PCB-based DMF can potentially allow point-of-care testing with low cost for resource-limited settings.

Nanoscale Bilirubin Analysis in Translational Research and Precision Medicine by the Recombinant Protein HUG.

Bilirubin is a toxicological biomarker for hemolysis and liver diseases. The current automated diazo method used in clinical chemistry has limited applicability in rodent models and cannot be used in small animals relevant to toxicology, microphysiological systems, cell cultures, and kinetic studies. Here, we present a versatile fluorometric method for nanoscale analysis of bilirubin based on its highly specific binding to the recombinant bifunctional protein HELP-UnaG (HUG). The assay is sensitive (LoQ = 1.1 nM), accurate (4.5% relative standard error), and remarkably robust, allowing analysis at pH 7.4-9.5, T = 25-37 °C, in various buffers, and in the presence of 0.4-4 mg × L-1 serum albumin or 30% DMSO. It allows repeated measurements of bilirubinemia in murine models and small animals, fostering the 3Rs principle. The assay determines bilirubin in human plasma with a relative standard error of 6.7% at values that correlate and agree with the standard diazo method. Furthermore, it detects differences in human bilirubinemia related to sex and UGT1A1 polymorphisms, thus demonstrating its suitability for the uniform assessment of bilirubin at the nanoscale in translational and precision medicine.

Discovery and development of inhibitors of acetyltransferase Eis to combat Mycobacterium tuberculosis.

Aminoglycosides are bactericidal antibiotics with a broad spectrum of activity, used to treat infections caused mostly by Gram-negative pathogens and as a second-line therapy against tuberculosis. A common resistance mechanism to aminoglycosides is bacterial aminoglycoside acetyltransferase enzymes (AACs), which render aminoglycosides inactive by acetylating their amino groups. In Mycobacterium tuberculosis, an AAC called Eis (enhanced intracellular survival) acetylates kanamycin and amikacin. When upregulated as a result of mutations, Eis causes clinically important aminoglycoside resistance; therefore, Eis inhibitors are attractive as potential aminoglycoside adjuvants for treatment of aminoglycoside-resistant tuberculosis. For over a decade, we have studied Eis and discovered several series of Eis inhibitors. Here, we provide a detailed protocol for a colorimetric assay used for high-throughput discovery of Eis inhibitors, their characterization, and testing their selectivity. We describe protocols for in vitro cell culture assays for testing aminoglycoside adjuvant properties of the inhibitors. A procedure for obtaining crystals of Eis-inhibitor complexes and determining their structures is also presented. Finally, we discuss applicability of these methods to discovery and testing of inhibitors of other AACs.

NanoFIRE: A NanoLuciferase and Fluorescent Integrated Reporter Element for Robust and Sensitive Investigation of HIF and Other Signalling Pathways.

The Hypoxia Inducible Factor (HIF) transcription factors are imperative for cell adaption to low oxygen conditions and development; however, they also contribute to ischaemic disease and cancer. To identify novel genetic regulators which target the HIF pathway or small molecules for therapeutic use, cell-based reporter systems are commonly used. Here, we present a new, highly sensitive and versatile reporter system, NanoFIRE: a NanoLuciferase and Fluorescent Integrated Reporter Element. Under the control of a Hypoxic Response Element (HRE-NanoFIRE), this system is a robust sensor of HIF activity within cells and potently responds to both hypoxia and chemical inducers of the HIF pathway in a highly reproducible and sensitive manner, consistently achieving 20 to 150-fold induction across different cell types and a Z' score > 0.5. We demonstrate that the NanoFIRE system is adaptable via substitution of the response element controlling NanoLuciferase and show that it can report on the activity of the transcriptional regulator Factor Inhibiting HIF, and an unrelated transcription factor, the Progesterone Receptor. Furthermore, the lentivirus-mediated stable integration of NanoFIRE highlights the versatility of this system across a wide range of cell types, including primary cells. Together, these findings demonstrate that NanoFIRE is a robust reporter system for the investigation of HIF and other transcription factor-mediated signalling pathways in cells, with applications in high throughput screening for the identification of novel small molecule and genetic regulators.

Smartphone-Based High-Throughput Fluorimetric Assay for Histidine Quantification in Human Urine Using 96-Well Plates.

A high-throughput fluorimetric assay for histidine was developed, using a 96-well plates platform. The analyte reacts selectively with o-phthalaldehyde under mild alkaline conditions to form a stable derivative. Instrumental-free detection was carried out using a smartphone after illumination under UV light (365 nm). The method was proved to be linear up to 100 µM histidine, with an LLOQ (lower limit of quantification) of 10 µM. The assay was only prone to interference from glutathione and histamine that exist in the urine samples at levels that are orders of magnitude lower compared to histidine. Human urine samples were analyzed following minimum treatment and were found to contain histidine in the range of 280 to 1540 µM. The results were in good agreement with an HPLC corroborative method.

High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism.

In a search for a reliable, inexpensive, and versatile technique for high-throughput kinetic assays of drug metabolism, we elected to rehire an old-school approach based on the determination of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. After evaluating several fluorometric techniques for FA detection, we chose the method based on the Hantzsch reaction with acetoacetanilide as the most sensitive, robust, and adaptable to high-throughput implementation. Here we provide a detailed protocol for using our new technique for automatized assays of cytochrome P450-dependent drug demethylations and discuss its applicability for high-throughput scanning of drug metabolism pathways in the human liver. To probe our method further, we applied it to re-evaluating the pathways of metabolism of ketamine, a dissociative anesthetic and potent antidepressant increasingly used in the treatment of alcohol withdrawal syndrome. Probing the kinetic parameters of ketamine demethylation by ten major cytochrome P450 (CYP) enzymes, we demonstrate that in addition to CYP2B6 and CYP3A enzymes, which were initially recognized as the primary metabolizers of ketamine, an important role is also played by CYP2C19 and CYP2D6. At the same time, the involvement of CYP2C9 suggested in the previous reports was deemed insignificant.

High-throughput microbead assay system with a portable, cost-effective Wi-Fi imaging module, and disposable multi-layered microfluidic cartridges for virus and microparticle detection, and tracking.

In recent years biomedical scientific community has been working towards the development of high-throughput devices that allow a reliable, rapid and parallel detection of several strains of virus or microparticles simultaneously. One of the complexities of this problem lies on the rapid prototyping of new devices and wireless rapid detection of small particles and virus alike. By reducing the complexity of microfluidics microfabrication and using economic materials along with makerspace tools (Kundu et al. 2018) it is possible to provide an affordable solution to both the problems of high-throughput devices and detection technologies. We present the development of a wireless, standalone device and disposable microfluidics chips that rapidly generate parallel readouts for selected, possible virus variants from a nasal or saliva sample, based on motorized and non-motorized microbeads detection, and imaging processing of the motion tracks of these beads in micrometers. Microbeads and SARS-CoV-2 COVID-19 Delta variant were tested as proof-of-concept for testing the microfluidic cartridges and wireless imaging module. The Microbead Assay (MA) system kit consists of a Wi-Fi readout module, a microfluidic chip, and a sample collection/processing sub-system. Here, we focus on the fabrication and characterization of the microfluidic chip to multiplex various micrometer-sized beads for economic, disposable, and simultaneous detection of up to six different viruses, microparticles or variants in a single test, and data collection using a commercially available, Wi-Fi-capable, and camera integrated device (Fig. 1).

Dynamic Measurement of Endosome-Lysosome Fusion in Neurons Using High-Content Imaging.

Endocytosis is a dynamic cellular process that actively transports particles into a cell. Late endosome fusion with the lysosome is a crucial step in the delivery of newly synthesized lysosomal proteins and endocytosed cargo for degradation. Disturbing this step in neurons is associated with neurological disorders. Thus, studying endosome-lysosome fusion in neurons will provide new insight into the mechanisms of these diseases and open new possibilities for therapeutic treatment. However, measuring endosome-lysosome fusion is challenging and time consuming, which limits the research in this area. Here we developed a high throughput method using pH-insensitive dye-conjugated dextrans and the Opera Phenix® High Content Screening System. By using this method, we successfully separated endosomes and lysosomes in neurons, and time-lapse images were collected to capture endosome-lysosome fusion events in hundreds of cells. Both assay set-up and analysis can be completed in an expeditious and efficient manner.

A Self-Assembly Method for Creating Vascularized Tumor Explants Using Biomaterials for 3D Culture.

Validation of potential therapeutic targets in cancer requires functional live assays that recapitulate the biology, anatomy, and physiology of human tumors. We present a methodology for maintaining mouse and patient tumor samples ex vivo for in vitro drug-screening as well as for the guidance of patient-specific chemotherapies. The harvested tumor biopsy, excised from mice or patients, is integrated into a support tissue that includes extended stroma and vasculature. The methodology is more representative than tissue culture assays, faster than patient-derived xenograft models, easy to implement, amenable to high-throughput assays and does not carry the ethical issues or expense associated with animal studies. Our physiologically relevant model can be successfully used for high-throughput drug screening.