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RSC Advances May 2024Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene...
Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene (PS) microwell plates form a part of this waste. These plates are the backbone of high throughput colorimetric measurements in academic, research, and healthcare settings for detection/quantification of wide-ranging analytes including proteins, carbohydrates, nucleic acids, and enzyme activity. Polystyrene (PS) microwell plates serve as a platform for holding samples and reagents, where mixing initiates chemical reaction(s), and the ensuing color changes are quantified using a microplate reader. However, these plates are rarely reused or recycled, contributing to the staggering amounts of plastic waste generated in scientific laboratories. Here, we are reporting the fabrication of cellulose acetate (CA) microwell plates as a greener alternative to non-biodegradable PS plates and we demonstrate their application in colorimetric assays. These easy to fabricate, lighter weight, customizable, and environmentally friendly plates were fabricated in 96- and 384-well formats and made water impermeable through chemical treatment. The plates were tested in three different colorimetric analyses: (i) bicinchoninic acid assay (BCA) for protein quantification; (ii) chymotrypsin (CT) activity assay; and (iii) alkaline phosphatase (AP) activity assay. Color intensities were quantified using a freely available smartphone application, Spotxel® Reader (Sicasys Software GmbH). To benchmark the performance of this platform, the same assays were performed in commercial PS plates too and quantified using a UV/Vis microplate reader. The two systems yielded comparable linear correlation coefficients, LOD and LOQ values, thereby validating the CA plate-cell phone based analytical method. The CA microwell plates, coupled with smart phone optical data capture, provide greener, accessible, and scalable tools for all laboratory settings and are particularly well-suited for resource- and infrastructure-limited environments.
PubMed: 38741966
DOI: 10.1039/d4ra01317d -
Sensors (Basel, Switzerland) Oct 2018Enzyme-linked immunosorbent assays (ELISA) show extensive application in immunoassays, to detect and monitor protein biomarkers in clinical diagnosis. Nevertheless, the...
Enzyme-linked immunosorbent assays (ELISA) show extensive application in immunoassays, to detect and monitor protein biomarkers in clinical diagnosis. Nevertheless, the time required and its multiple steps limit its application. We take advantage of a polyethyleneimine (PEI) gold nanoparticle (GNP) coated microwell plate to perform blocking-free ELISA, in which no nonspecific protein adsorption appears on the GNP layer. If the PEI-GNP coated microwell plate and immobilization of captured antibodies on the plate are prepared in advance, such as using an ELISA kit, the whole ELISA process can be finished in less than 2 h. Meanwhile, we have ensured that the GNP layer can preserve the precision and good linearity of ELISA without causing negative effects on the plate.
Topics: Adsorption; Enzyme-Linked Immunosorbent Assay; Gold; Humans; Immunoassay; Metal Nanoparticles; Polyethyleneimine; Proteins
PubMed: 30347684
DOI: 10.3390/s18103537 -
Journal of Personalized Medicine Apr 2021Altered miRNA expression and DNA methylation have highly active and diverse roles in carcinogenesis. Simultaneous detection of the molecular aberrations may have a...
Altered miRNA expression and DNA methylation have highly active and diverse roles in carcinogenesis. Simultaneous detection of the molecular aberrations may have a synergistic effect on the diagnosis of malignancies. Herein, we develop a high-throughput assay for detecting multiple miRNAs and DNA methylation using droplet digital PCR (ddPCR) coupled with a 96-microwell plate. The microplate-based ddPCR could absolutely and reproducibly quantify 15 miRNAs and 14 DNA methylation sites with a high sensitivity (one copy/µL and 0.1%, respectively). Analyzing sputum and plasma of 40 lung cancer patients and 36 cancer-free smokers by this approach identified an integrated biomarker panel consisting of two sputum miRNAs (miRs-31-5p and 210-3p), one sputum DNA methylation (RASSF1A), and two plasma miRNAs (miR-21-5p and 126) for the diagnosis of lung cancer with higher sensitivity and specificity compared with a single type of biomarker. The diagnostic value of the integrated biomarker panel for the early detection of lung cancer was confirmed in a different cohort of 36 lung cancer patients and 39 cancer-free smokers. The high-throughput assay for quantification of multiple molecular aberrations across sputum and plasma could improve the early detection of lung cancer.
PubMed: 33946992
DOI: 10.3390/jpm11050359 -
Sensors (Basel, Switzerland) Dec 2017Cardiovascular disease (CVD) and cancer are two leading causes of death worldwide. CVD and cancer share risk factors such as obesity and diabetes mellitus and have... (Review)
Review
Cardiovascular disease (CVD) and cancer are two leading causes of death worldwide. CVD and cancer share risk factors such as obesity and diabetes mellitus and have common diagnostic biomarkers such as interleukin-6 and C-reactive protein. Thus, timely and accurate diagnosis of these two correlated diseases is of high interest to both the research and healthcare communities. Most conventional methods for CVD and cancer biomarker detection such as microwell plate-based immunoassay and polymerase chain reaction often suffer from high costs, low test speeds, and complicated procedures. Recently, lab-on-a-chip (LoC)-based platforms have been increasingly developed for CVD and cancer biomarker sensing and analysis using various molecular and cell-based diagnostic biomarkers. These new platforms not only enable better sample preparation, chemical manipulation and reaction, high-throughput and portability, but also provide attractive features such as label-free detection and improved sensitivity due to the integration of various novel detection techniques. These features effectively improve the diagnostic test speed and simplify the detection procedure. In addition, microfluidic cell assays and organ-on-chip models offer new potential approaches for CVD and cancer diagnosis. Here we provide a mini-review focusing on recent development of LoC-based methods for CVD and cancer diagnostic biomarker measurements, and our perspectives of the challenges, opportunities and future directions.
Topics: Biomarkers; Cardiovascular Diseases; Humans; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Microfluidics; Neoplasms; Point-of-Care Systems
PubMed: 29258216
DOI: 10.3390/s17122934 -
Pharmaceutics Jan 2021High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing...
High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing multi-well plate formats. High-throughput plate readers are helpful tools to assess the aggregation tendency and colloidal stability of biological drug candidates based on the diffusion self-interaction parameter (). However, plate readers evoke issues about the precision and variability of determined data. In this article, we report about the statistical evaluation of intra- and inter-plate variability (384-well plates) for the analysis of protein and peptide solutions. ANOVA revealed no significant differences between the runs. In conclusion, the reliability and precision of was dependent on the plate position of the sample replicates and value. Positive values (57.0 mL/g, coefficients of variation () 8.9%) showed a lower variability compared to negative values (-14.8 mL/g, 13.4%). The variability of was not reduced using more data points (120 vs. 30). A analysis exclusively based on center wells showed a lower (<2%) compared to edge wells (5-12%) or a combination of edge and center wells (2-5%). We present plate designs for analysis within the early formulation development, screening up to 20 formulations consuming less than 50 mg of active pharmaceutical ingredient (API).
PubMed: 33514069
DOI: 10.3390/pharmaceutics13020172 -
Journal of Visualized Experiments : JoVE Aug 2023Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the...
Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems. Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro's components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.
Topics: Saccharomyces cerevisiae; Optogenetics; Saccharomycetales; Automation; High-Throughput Screening Assays
PubMed: 37590537
DOI: 10.3791/65686 -
Microorganisms Nov 2022induces heavy mortality in aquaculture. The detection of is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously...
induces heavy mortality in aquaculture. The detection of is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously published real-time quantitative PCR (qPCR) assay to confirm the presence of in fish and in water using environmental DNA (eDNA) quantification. Analytical sensitivity and specificity of the assay was assessed in silico, in vitro and the qPCR assay was compared with microbiological cultivation methods to detect and quantify in water samples from a controlled fish exposure experiment and from fish farms. Furthermore, we compared the use of an agar cultivation method and the qPCR assay to detect directly from mucus samples taken from the fish surface. The analytical sensitivity and specificity of the qPCR assay were high. The qPCR assay detected 100% of -positive water samples. In a field study, the qPCR assay and a microwell plate (MWP) enumeration method correlated significantly. Furthermore, the qPCR assay could be used to confirm the presence of in skin mucus. Thus, the qPCR assay could complement diagnostic methods in specifically detecting saprolegniosis in fish and used as a surveillance method for pathogen in aquaculture environments.
PubMed: 36363778
DOI: 10.3390/microorganisms10112186 -
Scientific Reports Aug 2018Microwell platforms show great promise in single-cell studies and protein measurements because of their low volume sampling, rapid analysis and high throughput screening...
Microwell platforms show great promise in single-cell studies and protein measurements because of their low volume sampling, rapid analysis and high throughput screening ability. However, the existing actuation mechanisms to manipulate the target samples and fabrication procedures involved in the microwell-based microfluidic devices are complex, resource-intensive and require an external power source. In this work, we present proof of concept of a simple, power-free and low-cost closed magnet digital microfluidics device for isolating biological entities in femtoliter-sized microwells. The target biological entities were encapsulated in magnetic liquid marbles and shuttled back and forth between micropatterned top and bottom plates in the microdevice to obtain high loading efficiency and short processing time. The microdevice performance was studied through fluorescent detection of three different entities: microbeads, bovine serum albumin (BSA) and Escherichia coli, captured in the microwell array. Almost 80% of the microwells were loaded with single microbeads in five shuttling cycles, in less than a minute. Further, a low volume of BSA was compartmentalized in the microwell array over a two order range of concentration. The microdevice exhibits two unique features: lotus leaf stamps were used to fabricate micropatterns (microwells and micropillars) on top and bottom plates to impart functionality and cost-effectiveness, and the target samples were actuated by a permanent magnet to make the microdevice power-free and simple in operation. The developed biomimetic microdevice is therefore capable of capturing a multitude of biological entities in low-resource settings.
Topics: Animals; Biomimetic Materials; Biomimetics; Cattle; Escherichia coli; Fluorescein-5-isothiocyanate; Fluorescence; Hydrophobic and Hydrophilic Interactions; Magnets; Microfluidics; Nanoparticles; Serum Albumin, Bovine; Signal Processing, Computer-Assisted; Wettability
PubMed: 30143719
DOI: 10.1038/s41598-018-31274-z -
Journal of Colloid and Interface Science Oct 2022Microscopic self-propelled motors (SPMs) are an area of active research, but very little investigation has been conducted on millimetre-scale or macroscopic SPMs and...
HYPOTHESIS
Microscopic self-propelled motors (SPMs) are an area of active research, but very little investigation has been conducted on millimetre-scale or macroscopic SPMs and exploring their potential in biomedical research. In this study, we tested if 3D reactive inkjet (RIJ) printing could be used for precise fabrication of millimetre-scale self-propelled motors (SPMs) with well-defined shapes from regenerated silk fibroin (RSF) by converting water soluble RSF (silk I) to insoluble silk fibroin (silk II). Secondly, we compared the different propulsion behaviour of the SPMs to put forward the best geometry and propulsion mechanism for potential applications in enhancing the sensitivity of diffusion-rate limited biomedical assays by inducing fluid flow.
EXPERIMENTS
SPMs with four different geometric shapes and propelled by two different mechanisms (catalysis and surface tension gradient) were fabricated by 3D RIJ printing and compared. For bubble propulsion, the structures were selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment in hydrogen peroxide solutions. For surface tension propulsion, PEG-doped structures were propelled through surface tension gradients caused by leaching of PEG surfactant in deionized water.
FINDINGS
The results demonstrated the ability of 3D inkjet printing to fabricate SPMs with desired propulsion mechanism and fine-tune the propulsion by precisely fabricating the different geometric shapes. The resulting 3D structures were capable of generating motion without external actuation, thereby enabling applications in biomedicine such as micro-stirring small fluid volumes to enhance biological assay sensitivity. The surface tension gradient caused by the leaching of surfactant led to faster propulsion velocities with smooth deceleration, whereas, in comparison, catalysis-induced bubble propulsion tended to be jerky and uneven in deceleration, and therefore less suitable for aforementioned applications. Computational fluid dynamic simulations were used to compare the various experimental SPMs ability to enhance mixing when deployed within 96-well plate microwells, to reveal the effect of both SPM shape and motion character on performance, and show viability for small scale mixing applications.
Topics: Fibroins; Printing, Three-Dimensional; Silk; Surface-Active Agents; Water
PubMed: 35576653
DOI: 10.1016/j.jcis.2022.05.011 -
Scientific Reports Mar 2022Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 10-10 islet-like clusters per patient. Although thousands of...
Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 10-10 islet-like clusters per patient. Although thousands of uniform cell clusters can be produced using a conventional microwell plate, numerous obstacles need to be overcome for its clinical use. In this study, we aimed to develop a novel bag culture method for the production of uniform cell clusters on a large scale (10-10 clusters). We prepared small-scale culture bags (< 10 clusters) with microwells at the bottom and optimized the conditions for producing uniform-sized clusters in the bag using undifferentiated induced pluripotent stem cells (iPSCs). Subsequently, we verified the suitability of the bag culture method using iPSC-derived pancreatic islet cells (iPICs) and successfully demonstrate the production of 6.5 × 10 uniform iPIC clusters using a large-scale bag. In addition, we simplified the pre- and post-process of the culture-a degassing process before cell seeding and a cluster harvesting process. In conclusion, compared with conventional methods, the cluster production method using bags exhibits improved scalability, sterility, and operability for both clinical and research use.
Topics: Cell Differentiation; Diabetes Mellitus, Type 1; Humans; Induced Pluripotent Stem Cells; Pluripotent Stem Cells
PubMed: 35338209
DOI: 10.1038/s41598-022-09124-w