Plate Reader Research Articles

Evaluating various properties of nanohydroxyapatite synthesized from eggshells and dual-doped with Si4+ and Zn2+: An in vitro study

BackgroundThis study aimed to evaluate morphological, chemical and biocompatible properties of nanohydroxyapatite (N-HA) synthesized from eggshells and dual-doped with Si4+ and Zn2+. MethodsIn the current study, N-HA was synthesized from chicken eggshells using the wet chemical precipitation method and doped with Si4+ and Zn2+. The physical assessment was carried out using field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDX) analysis, and X-ray diffraction (XRD) analysis. Crystal size was calculated using the Scherrer equation. Cytotoxicity was studied in vitro using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) cytotoxicity assay. The optical density (OD) of each well was obtained and recorded at 570 nm for 24 h (t1), 48 h (t2), 72 h (t3), and 5 days (t4) using a microplate reader. ResultsThe results of Si–Zn-doped HA showed a high specific surface area with an irregular nano-sized spherical particle structure. The atomic percentage provided the ratio of calcium to phosphate; for non-doped HA, the atomic Ca/P ratio was 1.6, but for Si–Zn-doped HA, where Zn+2 Ca and Si + replaced 4 substituted P, the atomic ratio (Ca + Zn)/(P + Si) was 1.76. The average crystal size of Si–Zn-doped HA was 46 nm, while for non-doped HA it was 61 nm. both samples were non-toxic and statistically significantly less viable than the control group After 5 days, the mean cell viability of Si–Zn-doped HA (79.17 ± 2.18) was higher than that of non-doped HA (76.26 ± 1.71) (P = 0.091). ConclusionsThe MTT assay results showed that Si–Zn-doped HA is biocompatible. In addition, it showed characteristic physiochemical properties of a large surface area with interconnected porosity.

Comparative Assessment of Lipid Peroxidase in Oral Cancer and Oral Potentially Malignant Disorders.

Introduction Oral potentially malignant disorders (OPMD) are abnormally altered tissues that could potentially develop into oral cancer. From the literature, it is understandable that not all OPMDs develop into oral cancer. Hence, it is essential to identify the high-risk lesions that are more likely to develop into oral cancer. Lipid peroxidase (LPO) is a byproduct of phospholipidmetabolism, and its levels are an oxidative stress marker that can probably help us predict the onset of cancer in OPMDS. This study aimed to assess the levels of LPO in OPMD, oral cancer, and normal patients. Materials and methods The sample size estimated was 15 per group. There were four groups in total. The estimation was done with the Abbkine LPO enzyme-linked immunosorption assay (ELISA) kit (Atlanta, Georgia, USA). An enzyme-substrate reaction was carried out, and the degree of the color change was measured using a microplate reader. The values were tabulated, and statistics were carried out using Statistical Package for Social Sciences(SPSS) version 26.0 (IBM Corp., Armonk, NY, USA). Both descriptive and inferential statistics were carried out. Results LPO levels (nmol/L) in each of the four groups were as follows: Group 1 (oral cancer): 171.86±78.86, Group 2 (controls): 71.66±28.36, Group 3: (oral leukoplakia): 127.50±103.53, and Group 4 (oral submucous fibrosis and oral lichen planus): 100.39±41.06.The results, when compared, were statistically significant (P< 0.05). Discussion From the above results, it is understandable that oral cancer patients experience increased oxidative stress compared to the OPMD group. The current study concluded that the obtained results showed differences in LPO levels, suggesting LPO could be used as a marker and screening tool to assess the rate and severity of cellular damage in patients with oral potentially malignant disorders.

Inhibition of nitric oxide production in RAW 264.7 cells and cytokines IL-1β in osteoarthritis rat models of 70 % ethanol extract of Arcangelisia flava (L.) merr stems

IntroductionOne of the most frequent types of arthritis is osteoarthritis, also referred to as a degenerative joint disease. Interleukin-1β (IL-1β) and nitric oxide (NO) are essential factors in the pain response; IL-1β and NO are responsible for increasing the production of matrix metalloproteinases (MMP) and a disintegrin-like and metalloproteinases with thrombospondin motifs (ADAMS) in chondrocytes. Arcangelisia flava (L.) Merr. Has been traditionally used to treat jaundice, liver disease, diarrhea, fever, and inflammation. MethodsThis study used in vitro and in vivo models to determine the effect of a 70 % ethanol extract of Arcangelisia flava (L.) Merr. stems on the inhibition of NO production in RAW 264.7 cells induced with lipopolysaccharide (LPS) and IL-1β in osteoarthritis rats induced with monosodium iodoacetate (MIA). The NO inhibition test was determined by the NO colorimetric assay using Griess reagent and measured by the ELISA plate reader. The measurement of joint diameter and hyperalgesia in osteoarthritis rats was carried out once a week for 7 weeks, and then the IL-1β levels were measured at weeks 3 and 7. ResultThe viability of cell line this extract was greater than 80 %, and the extract at 25, 50, and 100 μg/mL significantly inhibited NO production (p < 0.0001) in RAW 264.7 cells induced with LPS. Meanwhile, this extract at 10, 30, and 90 mg/200g BW increased latency time, reduced joint swelling, and reduced IL-1β levels in the serum in the osteoarthritis rat model. Conclusion70 % ethanol extract of Arcangelisia flava (L.) Merr. Has the potential to be an anti-osteoarthritis drug.

Aspergillus Cell Surface Structural Analysis and Its Applications to Industrial and Medical Use

The hyphal surface of cells of filamentous fungi is covered with cell wall, which is mainly composed of polysaccharides. Since the cell wall is the first structure to come in contact with the infection host, the environment, and the fungus itself, the elucidation of the cell wall structure and biogenesis is essential for understanding fungal ecology. Among filamentous fungi, the genus Aspergillus is an important group in the industrial, food, and medical fields. It is known that Aspergillus species form hyphal pellets in shake liquid culture. The authors previously found the role of α-1,3-glucan in hyphal aggregation in Aspergillus species. In addition, extracellular polysaccharide galactosaminogalactan contributed to hyphal aggregation as well, and dual disruption of biosynthesis genes of α-1,3-glucan and galactosaminogalactan resulted in complete hyphal dispersion in shake liquid culture. The characteristic of mycelia to form pellets under liquid culture conditions was the main reason why the growth measurement methods used for unicellular organisms could not be applied. We reported that hyphal growth of the dual disruption mutant could be measured by optical density. A real-time plate reader could be used to determine the growth curve of the mycelial growth of the dual disruption mutant. This measurement approach not only provides basic microbiological insights in filamentous fungi, but also has the potential to be applied to high-throughput screening of anti-Aspergillus drugs.

Development of a Polymersome Blood Ammonia Assay Coupled with a Portable Near-Infrared Fluorometer.

Ammonia is a key biomarker in inborn and acquired liver disease. As clinical point-of-care blood ammonia assays are lacking, we developed a polymersome formulation for point-of-care blood ammonia sensing combined with a portable fluorometer. A pH-sensitive near-infrared (NIR) fluorescent dye was identified, which showed a strong fluorescence increase at acidic pH values. Building on reports on ammonia-selective PS-b-PEG polymersomes, these polymersomes were loaded with the NIR dye. These NIR fluorescent polymersomes sensed ammonia in a clinically relevant range in ammonia-spiked fresh whole blood with high linearity (R 2 = 0.9948) after 5 min using a conventional tabletop plate reader. Subsequently, the assay was tested with a portable fluorometer. An ammonia-dependent fluorescence increase was detected in ammonia-spiked fresh mouse blood after 5 min using the portable fluorometer. The NIR dye-loaded PS-b-PEG polymersomes rapidly sensed ammonia with high linearity in whole blood. This assay was successfully combined with a portable fluorometer and only required 3 μL of blood. These findings motivate a further development and clinical translation of this point-of-care blood ammonia assay.

A sensitive fluorescence assay of serum dipeptidyl peptidase IV activity to predict the suitability of its inhibitors in patients with type 2 diabetes mellitus

DPP-IV inhibitors, which are close to the natural hypoglycemic pathway of human physiology and have few side effects, have been extensively employed in the management of type 2 diabetes mellitus (T2DM). However, there are currently no specific blood indicators that can indicate or predict a patient's suitability for DPP-IV inhibitors. In this study, based on the self-developed high-specificity fluorescent substrate glycyl-prolyl-N-butyl-4-amino-1, 8-naphthimide (GP-BAN), a detection method of human serum DPP-IV activity was established and optimized. The method demonstrates a favorable lower limit of detection (LOD) at 0.32 ng/mL and a satisfactory lower limit of quantification (LOQ) of 1.12 ng/mL, and can be used for the detection of DPP-IV activity in trace serum (2 μL). In addition, Vitalliptin and Sitagliptin showed similar IC50 values when human recombinant DPP-IV and human serum were used as enzyme sources, and the intra-day and inter-day precision obtained by the microplate analyzer were less than 15 %. These results indicate that the microplate reader based detection technique has good accuracy, repeatability and reproducibility. A total of 700 volunteers were recruited, and 646 serum samples were tested for DPP-IV activity. The results showed that serum DPP-IV activity was higher in patients with T2DM than in controls (P < 0.01). However, the statistical data of family history of diabetes, gender and age of diabetic patients showed no statistical significance, and there was no contrast difference. The DPP-IV activity of serum in T2DM patients ranged from 2.4 μmol/min/L to 78.6 μmol/min/L, with a huge difference of up to 32-fold. These results suggest that it is necessary to test DPP-IV activity in patients with T2DM when taking DPP-IV inhibitors to determine the applicability of DPP-IV inhibitors in T2DM patients. These results suggest that it is necessary to detect the activity of DPP-IV in blood before taking DPP-IV inhibitors in patients with T2DM to judge the applicability of DPP-IV inhibitors in patients with T2DM.

Dihydroisotanshinone I regulates ferroptosis via PI3K/AKT pathway to enhance cisplatin sensitivity in lung adenocarcinoma.

Dihydroisotanshinone I (DT) is a kind of diterpenoid compound extracted from the dried roots of Salvia miltiorrhiza Bunge, and exhibits multiple biological activities including anti-tumor activity. Cisplatin is one of the first-line drugs for the treatment of lung adenocarcinoma (LAUD), but the drug resistance and toxicity limit its efficacy. DT is known to induce apoptosis and ferroptosis, but it is unclear whether DT can inhibit the cisplatin-resistant LAUD cells and reverse the drug resistance in LAUD. Therefore, our study intends to establish the cisplatin-resistant human LAUD cells (A549/DDP), and figure out the influence and related mechanisms of DT reversing cisplatin resistance in A549/DDP cells, so as to provide a theoretical basis for the DT as a new natural candidate for the treatment of LAUD. The establishment of A549/DDP was the continuous stimulation by exposing A549 to gradient concentrations of Cisplatin. The cell viability of A549 and A549/DDP was detected by CCK-8 kit, and the IC50 value was calculated. The morphological changes of A549 and A549/DDP cells were observed by an inverted microscope. The contents of malondialdehyde (MDA) and glutathione (GSH) in A549/DDP cells after drug treatment were detected by related kits. The levels of Fe2+, cytosolic reactive oxygen species (ROS), and lipid reactive oxygen species (lipid ROS) were detected by a fluorescence microplate reader or fluorescence cell imager according to the related fluorescent probe kit instructions. Western blot was used to detect the expressions of PI3K, phospho-PI3K, AKT, phospho-AKT, MDM2, p53, GPX4, and SLC7A11 in A549/DDP after different drug treatments. Our study demonstrated that the inhibitory effect of DT on A549 and A549/DDP cells was time-dependent and concentration-dependent, and DT and DDP had a synergistic effect on inhibiting the proliferation of A549/DDP cells. Furthermore, DT mainly induced ferroptosis in A549/DDP cells and synergized with cisplatin to promote ferroptosis in A549/DDP cells. The result of KEGG pathway analysis, molecular docking and western blot showed that DT could enhance the cisplatin sensitivity of A549/DDP by inhibiting PI3K/MDM2/P53 signaling pathway. Consequently, we concluded that DT promotes ferroptosis in cisplatin-resistant LAUD A549/DDP cells. Additionally, DT reverses cisplatin resistance by promoting ferroptosis via PI3K/MDM2/P53 pathway in A549/DDP cells.

In Situ Measurement of Nanoparticle-Blood Protein Adsorption and Its Heterogeneity with Single-Nanoparticle Resolution via Dual Fluorescence Quantification.

The formation of a protein corona gives nanomedicines a distinct biological identity, profoundly influencing their fate in the body. Nonspecific nanoparticle-protein interactions are typically highly heterogeneous, which can lead to unique biological behaviors and in vivo fates for individual nanoparticles that remain underexplored. To address this, we have established an in situ approach that allows quantitative examination of nanoparticle-protein adsorption at the individual nanoparticle level. This method integrates dual fluorescence quantification techniques, wherein the nanoparticles are first individually analyzed via nanoflow cytometry to detect fluorescent signals from adsorbed proteins. The obtained fluorescence intensity is then translated into protein quantities through calibration with microplate reader quantification. Consequently, this approach enables analysis of interparticle heterogeneity of nano-protein interactions, as well as in situ monitoring of protein adsorption kinetics and nanoparticle aggregation status in blood serum, preconditioning for a comprehensive understanding of nano-bio interactions, and predicting in vivo fate of nanomedicines.

Fluorescence‐activated screening of polyester‐depolymerizing enzymes based on pseudo‐PET polythioester plastics

AbstractWhile fluorescence‐based high‐throughput screening (HTS) has been valuable for identifying enzymes and microorganisms for polyethylene terephthalate (PET) depolymerization, the traditional approach relies on cleaving ester bonds in small‐molecule probes, limiting its ability to directly detect activity towards polymeric PET substrates. This study proposes a novel fluorescence‐based screening strategy for detecting the release of sulfhydryl groups during the depolymerization of pseudo‐PET polythioesters by polyester hydrolases. The strategy successfully identifies the polyester‐depolymerizing activity of leaf‐branch compost cutinase (LCCICCG), whereas porcine liver esterase (PLE) only hydrolyses small molecular substrates. Furthermore, the strategy can be integrated with a droplet microfluidic system for high‐throughput screening of LCCICCG. This work further demonstrates the applicability of the microplate reader platform for screening polyester‐depolymerizing bacteria. This novel fluorescence‐based approach offers an efficient strategy to identify enzymes and microbial resources for depolymerizing polyester‐like plastics.

The reliability, sensitivity and specificity of fluorescent oxidation products for measuring global oxidative stress in vivo

Introduction To examine the reliability, sensitivity, and specificity of fluorescent oxidation products (FlOPs; markers of global oxidative damage) for measuring global oxidative stress. Methods To improve FlOP measurement reliability, we mixed plasma samples with the extractant at eight ratios and measured FlOPs with a fluorescent microplate reader (excitation/emission wavelengths 320/420 nm denoted FlOP_320; 360/420 nm [FlOP_360]; and 400/475 nm [FlOP_400]). In a human study, we examined the reliability of an improved FlOP measurement. In an animal study, we examined the sensitivity and specificity of FlOPs for measuring D-galactose induced global oxidative stress. Results At a 1:20 (plasma/extractant) mixture ratio, the overall inter-/intra-assay coefficients of variation (CV) for FlOP measurements among healthy and coronary heart disease participants were <3.6%/<2.7% and <4.4%/<2.0%, respectively. On day 30 of the animal experiment, the Pearson correlations (r) of FlOP_320 and FlOP_360 with D-galactose dose were 0.816 and 0.801, respectively, which were 3-12 times higher than those of malondialdehyde (0.183), 8-hydroxyguanosine (0.157), pentosidine (0.254), and nitrotyrosine (0.068) for measuring D-galactose-induced oxidative damage. FlOP_360 (r = 0.225, P = 0.045), but not FlOP_320 and FlOP_400, were significantly correlated with c-reactive protein, a marker of inflammation. Conclusions FlOP_320 are reliable, sensitive and specific markers for measuring global oxidative stress. Key policy highlights Fluorescent oxidation products (FlOPs) are a reliable marker of oxidative stress FlOPs at 320/420 nm are more sensitive than traditional markers FlOP at 320/420 nm did not reflect inflammation as quantified by c-reactive protein

Portable Near-Infrared to Near-Infrared Platform for Homogeneous Quantification of Biomarkers in Complex Biological Samples.

Förster resonance energy transfer (FRET)-based homogeneous immunoassay obviates tedious washing steps and thus is a promising approach for immunoassays. However, a conventional FRET-based homogeneous immunoassay operating in the visible region is not able to overcome the interference of complex biological samples, thus resulting in insufficient detection sensitivity and poor accuracy. Here, we develop a near-infrared (NIR)-to-NIR FRET platform (Ex = 808 nm, Em = 980 nm) that enables background-free high-throughput homogeneous quantification of various biomarkers in complex biological samples. This NIR-to-NIR FRET platform is portable and easy to operate and is mainly composed of a high-performance NIR-to-NIR FRET pair based on lanthanide-doped nanoparticles (LnNPs) and a custom-made microplate reader for readout of NIR luminescence signals. We demonstrate that this NIR-to-NIR FRET platform is versatile and robust, capable of realizing highly sensitive and accurate detection of various critical biomarkers, including small molecules (morphine and 1,25-dihydroxyvitamin D), proteins (human chorionic gonadotropin), and viral particles (adenovirus) in unprocessed complex biological samples (urine, whole blood, and feces) within 5-10 min. We expect this NIR-to-NIR FRET platform to provide low-cost healthcare for populations living in resource-limited areas and be widely used in many other fields, such as food safety and environmental monitoring.

Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays.

The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.

Unraveling the influence of CRISPR/Cas13a reaction components on enhancing trans-cleavage activity for ultrasensitive on-chip RNA detection.

The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report onthe influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To furtherimprove sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.

Direct Zeptomole Detection of RNA Biomarkers by Ultrabright Fluorescent Nanoparticles on Magnetic Beads.

Nucleic acids are important biomarkers in cancer and viral diseases. However, their ultralow concentration in biological/clinical samples makes direct target detection challenging, because it leads to slow hybridization kinetics with the probe and its insufficient signal-to-noise ratio. Therefore, RNA target detection is done by molecular (target) amplification, notably by RT-PCR, which is a tedious multistep method that includes nucleic acid extraction and reverse transcription. Here, a direct method based on ultrabright dye-loaded polymeric nanoparticles in a sandwich-like hybridization assay with magnetic beads is reported. The ultrabright DNA-functionalized nanoparticle, equivalent to ≈10 000 strongly emissive rhodamine dyes, is hybridized with the magnetic bead to the RNA target, providing the signal amplification for the detection. This concept (magneto-fluorescent sandwich) enables high-throughput detection of DNA and RNA sequences of varied lengths from 48 to 1362nt with the limit of detection down to 0.3 fm using a plate reader (15 zeptomoles), among the best reported for optical sandwich assays. Moreover, it allows semi-quantitative detection of SARS-CoV-2 viral RNA directly in clinical samples without a dedicated RNA extraction step. The developed technology, combining ultrabright nanoparticles with magnetic beads, addresses fundamental challenges in RNA detection; it is expected to accelerate molecular diagnostics of diseases.

Borneol and lactoferrin dual-modified crocetin-loaded nanoliposomes enhance neuroprotection in HT22 cells and brain targeting in mice

Crocetin (CCT), a natural bioactive compound extracted and purified from the traditional Chinese medicinal herb saffron, has been shown to play a role in neurodegenerative diseases, particularly depression. However, due to challenges with solubility, targeting, and bioavailability, formulation development and clinical use of CCT are severely limited. In this study, we used the emulsification-reverse volatilization method to prepare CCT-loaded nanoliposomes (CN). We further developed a borneol (Bor) and lactoferrin (Lf) dual-modified CCT-loaded nanoliposome (BLCN) for brain-targeted delivery of CCT. The results of transmission electron microscope (TEM) and particle size analysis indicated that the size of BLCN (∼140 nm) was suitable for transcellular transport across olfactory axons (∼200 nm), potentially paving a direct path to the brain. Studies on lipid solubility, micropolarity, and hydrophobicity showed that BLCN had a relatively high Lf grafting rate (81.11 ± 1.33 %) and CCT entrapment efficiency (83.60 ± 1.04 %) compared to other liposomes, likely due to Bor improving the lipid solubility of Lf, and the combination promoting the orderly arrangement of liposome membrane molecules. Microplate reader and fluorescence microscopy analysis showed that BLCN efficiently promoted the endocytosis of fluorescent coumarin 6 into HT22 cells with a maximal fluorescence intensity of (13.48 ± 0.80 %), which was significantly higher than that of CCT (5.73 ± 1.17 %) and CN (12.13 ± 1.01 %). BLCN also exhibited sustained function, remaining effective for more than 12 h after reaching a peak at 1 h in cells, while CN showed a significant decrease after 4 h. The uptake mechanisms of BLCN in HT22 cells mainly involve energy-dependent, caveolae-mediated, and microtubule-mediated endocytosis, as well as micropinocytosis. Furthermore, BLCN displayed a significant neuroprotective effect on HT22 cells in glutamate-, corticosterone-, and H2O2-induced models. Tissue fluorescence image analysis of mice showed that BLCN exhibited substantial retention of fluorescent DiR in the brain after nasal administration for 12 h. These findings suggest that CCT has the potential for cellular uptake, neuroprotection, and targeted delivery to the brain following intranasal administration when encapsulated in Bor and Lf dual-modified nanoliposomes.

Covalently Modified Molecular-Recognition-Capable UV-Transparent Microplate for Ultra-High-Throughput Screening of Dissolved Zn2+ and Pb2.

Zn2+ has a crucial role both in biology and the environment, while Pb2+ presents serious hazards in the same areas due to its toxicity, and the need for their analysis often exceeds available instrumental capacity. We report, herein, a new high-throughput optochemical screening method for Zn2+ and Pb2+ in various solutions. Moreover, we also introduced a new and generalizable three-step-microplate-modification technique, including plasma treating, linker-docking and photocatalytic copolymerization. The surface of a commercially available 96-well-cycloolefin-microplate was treated with atmospheric plasma, and then, the bottoms of the wells were covered by covalently attaching a methacrylate-containing linker-monolayer. Finally, the preactivated microplate wells were covalently functionalized by immobilizing bis(acridino)-crown ether-type sensor molecules, via photocatalytic copolymerization, to a polymethacrylate backbone. This sensing tool can be used in all microplate readers, is compatible with liquid handling platforms and provides an unprecedently fast monitoring (>1000 samples/hour, extrapolated from the time required for 96 measurements) of dissolved Zn2+ and Pb2+ among recent alternatives above the detection limits of 8.0 × 10-9 and 3.0 × 10-8 mol/L, respectively, while requiring a sample volume of only 20 µL.

HcGr76 responds to fructose and chlorogenic acid and is involved in regulation of peptide expression in the midgut of Hyphantria cunea larvae.

Sensing dietary components in the gut is important to ensure an appropriate hormonal response and metabolic regulation after food intake. The fall webworm, Hyphantria cunea, is a major invasive pest in China and has led to significant economic losses and ecosystem disruption. The larvae's broad host range and voracious appetite for leaves make H. cunea a primary cause of serious damage to both forests and crops. To date, however, the gustatory receptors (Grs) of H. cunea and their regulatory function remain largely unknown. We identified the fall webworm gustatory receptor HcGr76 as a fructose and chlorogenic acid receptor using Ca2+ imaging and determination of intracellular Ca2+ concentration by a microplate reader. Moreover, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis revealed that HcGr76 is highly expressed in the anterior and middle midgut. Knockdown of HcGr76 caused a significant reduction in the expression of neuropeptide F 1 (NPF1) and CCHamide-2, and led to a decrease in carbohydrate and lipid levels in the hemolymph. Our studies provide circumstantial evidence that HcGr76 expressed in the midgut is involved in sensing dietary components, and regulates the expression of relevant peptide hormones to alter metabolism in H. cunea larvae, thus providing a promising molecular target for the development of new insect-specific control products. © 2024 Society of Chemical Industry.

Cell-Free Translation Quantification via a Fluorescent Minihelix.

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

A 3D-Printed Do-It-Yourself ELISA Plate Reader as a Biosensor Tested on TNFα Assay.

Simple analytical devices suitable for the analysis of various biochemical and immunechemical markers are highly desirable and can provide laboratory diagnoses outside standard hospitals. This study focuses on constructing an easily reproducible do-it-yourself ELISA plate reader biosensor device, assembled from generally available and inexpensive parts. The colorimetric biosensor was based on standard 96-well microplates, 3D-printed parts, and a smartphone camera as a detector was utilized here as a tool to replace the ELISA method, and its function was illustrated in the assay of TNFα as a model immunochemical marker. The assay provided a limit of detection of 19 pg/mL when the B channel of the RGB color model was used for calibration. The assay was well correlated with the ELISA method, and no significant matrix effect was observed for standard biological samples or interference of proteins expected in a sample. The results of this study will inform the development of simple analytical devices easily reproducible by 3D printing and found on generally available electronics.

Measurement of cellular MDA content through MTBE-extraction based TBA assay by eliminating cellular interferences

Malondialdehyde (MDA) has long been served as a crucial indicator for assessing cellular oxidative stress levels. In this study, we introduce a new approach to determine cellular MDA levels based on a methyl tert-butyl ether (MTBE) extraction, aimed at eliminating interferences from cellular components during thiobarbituric acid (TBA) derivatization of MDA. By leveraging the effective MTBE extraction, we identified that the determination of the MDA-TBA adduct formed from the MTBE extraction layer can effectively eliminate the interferences from cellular proteins and metabolites. This method demonstrated acceptable linearity and precision in cellular samples and showed significant differences in H2O2 treated cellular oxidative stress models. The MTBE extraction-based MDA-TBA approach provides a reliable, cost-effective, and feasible method to determine cellular MDA levels using batch microplate reader approach for the assessment of cellular oxidative stress.