Conventional Bioassays Research Articles

Deep Learning Bridged Bioactivity, Structure, and GC-HRMS-Readable Evidence to Decipher Nontarget Toxicants in Sediments.

Identifying causative toxicants in mixtures is critical, but this task is challenging when mixtures contain multiple chemical classes. Effect-based methods are used to complement chemical analyses to identify toxicants, yet conventional bioassays typically rely on an apical and/or single endpoint, providing limited diagnostic potential to guide chemical prioritization. We proposed an event-driven taxonomy framework for mixture risk assessment that relied on high-throughput screening bioassays and toxicant identification integrated by deep learning. In this work, the framework was evaluated using chemical mixtures in sediments eliciting aryl-hydrocarbon receptor activation and oxidative stress response. Mixture prediction using target analysis explained <10% of observed sediment bioactivity. To identify additional contaminants, two deep learning models were developed to predict fingerprints of a pool of bioactive substances (event driver fingerprint, EDFP) and convert these candidates to MS-readable information (event driver ion, EDION) for nontarget analysis. Two libraries with 121 and 118 fingerprints were established, and 247 bioactive compounds were identified at confidence level 2 or 3 in sediment extract using GC-qToF-MS. Among them, 12 toxicants were analytically confirmed using reference standards. Collectively, we present a "bioactivity-signature-toxicant" strategy to deconvolute mixtures and to connect patchy data sets and guide nontarget analysis for diverse chemicals that elicit the same bioactivity.

Synergistic effect of Erythrina variegata L. and tamoxifen against the growth of breast cancer cells

Breast cancer is the most common malignancy and the second leading cause of death in women. While tamoxifen (TAM) remains to be a first-line agent for prevention and treatment of estrogen-receptor (ER) positive breast tumors, TAM resistance represents one of the biggest clinical challenges in breast cancer therapy. Therefore, there has been growing interest in the identification of novel agents including those of plant origin that enhance the TAM sensitivity of breast cancer cells. In the present study, we aimed to evaluate the synergistic effect of Erythrina variegata extract (EVE) and TAM on the suppression of breast cancer cell growth using MCF-7 cell model and conventional bioassays such as MTT and colony formation assay, cell cycle analysis, and immunocytochemistry. Interestingly, MTT assay in combination with Chou-Talalay analysis demonstrated the potent synergism between EVE and tamoxifen against MCF-7 cell survival. Additionally, co-treatment of MCF-7 cells with EVE and TAM more significantly suppressed colony formation, cell cycle progression, and the nuclear expression of proliferative marker Ki67 compared to TAM alone. These findings imply that EVE potentiates the anti-proliferative effect of TAM in MCF-7 breast cancer cells and may become a promising agent in the treatment of ER positive breast cancer.

Plasmonic biochips with enhanced stability in harsh environments for the sensitive detection of prostate-specific antigen.

Hollow and porous plasmonic nanomaterials have been demonstrated for highly sensitive biosensing applications due to their distinctive optical properties. Immunosensors, which rely on antibody-antigen interactions, are essential constituents of diverse biosensing platforms owing to their exceptional binding affinity and selectivity. The majority of immunosensors and conventional bioassays needs special storage conditions and cold chain systems for transportation. Prostate-specific antigen (PSA), a serine protease, is widely employed in the diagnosis of prostate cancer. In this study, we present the successful utilization of a biopolymer-preserved plasmonic biosensor with improved environmental stability for the sensitive detection of PSA. The preserved plasmonic biosensors exhibited sustained sensitivity in the detection of PSA, achieving a limit of detection of 10 pg mL-1. Furthermore, these biosensors exhibited remarkable stability at elevated temperatures for one week.

Can Electrochemical Aptasensors Achieve the Commercial Success of Glucose Biosensors?

AbstractEnzymes and antibodies are widely available biorecognition elements in bioanalytical tools such as personal glucose monitoring (PGM) devices and lateral flow assays (LFA). Meanwhile, electrochemical aptamer‐based (EAB) sensors are promising affinity‐based bioanalytical tools with potential advantages over such conventional bioassays. However, several critical factors affect the stability of EAB sensors, pivotal for their commercialization including 1) electrode defects due to surface treatment methods, 2) hampering effects of redox molecules, 3) electrical potential‐induced aptamer detachment, 4) thermal‐induced monolayer solubilization, 5) biochemical/enzymatic degradation, 6) biofouling, and 7) inadequate statistical design and analysis in EAB sensor fabrication. Herein, antidotes for the obstacles are proposed by applying novel surface treatment methods, adapting redox molecule, tuning electrochemical tests, tweaking backfilling agents, and anti‐bio‐fouling coatings. Nonetheless, the obstacles are a driving force to clear pathways toward bringing EAB sensors to the market for therapeutic drug and metabolite monitoring, point of care sensors, macromolecule detection, and pathogen diagnostics.

Spherical Nucleic Acid‐Amplified Digital Flow Cytometric Bead Assay for the Ultrasensitive Detection of Protein and Exosome†

Comprehensive SummaryMost conventional digital bioassays rely on the use of fully‐sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction, which require specialized equipment or proprietary reagents/consumables. Herein, we report a microchamber‐free and spherical nucleic acid (SNA)‐amplified digital flow cytometric bead assay (dFBA) for ultrasensitive protein and exosome analysis with simple workflows, easily accessible instruments/reagents, and high discriminating ability towards the fluorescence‐positive and fluorescence‐negative beads. In this dFBA, microbeads are employed as independent carriers to anchor the single target molecule‐initiated signal amplification reaction, avoiding the use of sealed droplets or microwell microchambers. Meanwhile, antibody‐functionalized SNAs (FSNAs) with a high density of DNA probes act as a bridge for efficiently amplified target‐to‐DNA signal conversion, which allows the use of DNA‐based rolling circle amplification (RCA) as the fluorescence signal amplification technique to quantify non‐nucleic acid targets. Even a single target‐induced on‐bead RCA and fluorescence enriching are sufficient to make the target‐loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer (FCM). This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers, enlarging the toolbox of digital bioassays for clinical applications.

Disparate properties of Burkholderia multivorans and Pseudomonas aeruginosa regarding outer membrane chemical permeabilization to the hydrophobic substances novobiocin and triclosan.

Burkholderia multivorans causes opportunistic pulmonary infections and is intrinsically resistant to many antibacterial compounds including the hydrophobic biocide triclosan. Chemical permeabilization of the Pseudomonas aeruginosa outer membrane affects sensitization to hydrophobic substances. The purpose of the present study was to determine if B. multivorans is similarly susceptive suggesting that outer membrane impermeability properties underlie triclosan resistance. Antibiograms and conventional macrobroth dilution bioassays were employed to establish baseline susceptibility levels to hydrophobic antibacterial compounds. Outer membrane permeabilizers compound 48/80, polymyxin B, polymyxin B-nonapeptide, and ethylenediaminetetraacetic acid were used in attempts to sensitize disparate B. multivorans isolates to the hydrophobic agents novobiocin and triclosan, and to potentiate partitioning of the hydrophobic fluorescent probe 1-N-phenylnapthylamine (NPN). The lipophilic agent resistance profiles for all B. multivorans strains were essentially the same as that of P. aeruginosa except that they were resistant to polymyxin B. Moreover, they resisted sensitization to hydrophobic compounds and remained inaccessible to NPN when treated with outer membrane permeabilizers. These data support the notion that while both phylogenetically-related organisms exhibit general intrinsic resistance properties to hydrophobic substances, the outer membrane of B. multivorans either resists permeabilization by chemical modification or sensitization is mitigated by a supplemental mechanism not present in P. aeruginosa.

Morphological alterations in C57BL/6 mouse intestinal organoids as a tool for predicting chemical-induced toxicity.

Intestinal organoid may serve as an alternative model for toxicity testing. However, the linkage between specific morphological alterations in organoids and chemical-induced toxicity has yet to be defined. Here, we generated C57BL/6 mouse intestinal organoids and conducted a morphology-based analysis on chemical-induced toxicity. Alterations in morphology were characterized by large spheroids, hyperplastic organoids, small spheroids, and protrusion-loss organoids, which responded in a concentration-dependent manner to the treatment of four metal(loid)s including cadmium (Cd), lead (Pb), hexavalent chromium (Cr-VI), and inorganic trivalent arsenic (iAs-III). Notably, alterations in organoid morphology characterized by abnormal morphology rate were correlated with specific intestinal toxic effects, including reduction in cell viability and differentiation, induction of apoptosis, dysfunction of mucus production, and damage to epithelial barrier upon repeated administration. The benchmark dose (BMDL10) values of morphological alterations (0.007-0.195μM) were lower than those of conventional bioassays (0.010-0.907μM). We also established that the morphologic features of organoids upon Cd, Pb, Cr-VI, or iAs-III treatment were metal specific, and mediated by Wnt, bone morphogenetic protein, apoptosis induction, and Notch signaling pathways, respectively. Collectively, these findings provide novel insights into the relevance of morphological alterations in organoids to specific toxic endpoints and identify specific morphological alterations as potential indicators of enterotoxicity.

Quantitative Single‐Molecule Electrochemiluminescence Bioassay

AbstractA single‐molecule electrochemiluminescence bioassay is developed here which allows imaging and direct quantification of single biomolecules. Imaging single biomolecules is realized by localizing the electrochemiluminescence events of the labeled molecules. Such an imaging system allows mapping the spatial distribution of biomolecules with electrochemiluminescence and contains quantitative single‐molecule insights. We further quantify biomolecules by spatiotemporally merging the repeated reactions at one molecule site and then counting the clustered molecules. The proposed single‐molecule electrochemiluminescence bioassay is used to detect carcinoembryonic antigen, showing a limit of detection of 67 attomole concentration which is 10 000 times better than conventional electrochemiluminescence bioassays. This spatial resolution and sensitivity enable single‐molecule electrochemiluminescence bioassay a new toolbox for both specific bioimaging and ultrasensitive quantitative analysis.

Quantitative Single-Molecule Electrochemiluminescence Bioassay.

A single-molecule electrochemiluminescence bioassay is developed here which allows imaging and direct quantification of single biomolecules. Imaging single biomolecules is realized by localizing the electrochemiluminescence events of the labeled molecules. Such an imaging system allows mapping the spatial distribution of biomolecules with electrochemiluminescence and contains quantitative single-molecule insights. We further quantify biomolecules by spatiotemporally merging the repeated reactions at one molecule site and then counting the clustered molecules. The proposed single-molecule electrochemiluminescence bioassay is used to detect carcinoembryonic antigen, showing a limit of detection of 67 attomole concentration which is 10 000 times better than conventional electrochemiluminescence bioassays. This spatial resolution and sensitivity enable single-molecule electrochemiluminescence bioassay a new toolbox for both specific bioimaging and ultrasensitive quantitative analysis.

On-Chip Enrichment System for Digital Bioassay Based on Aqueous Two-Phase System

We developed an on-chip enrichment method based on an aqueous two-phase system of dextran/polyethylene glycol mix, DEX/PEG ATPS, for digital bioassay. Accordingly, we prepared an array device of femtoliter reactors that displays millions of uniformly shaped DEX-rich droplets under a PEG-rich medium. The DEX-rich droplets effectively enriched DNA molecules from the PEG-rich medium. To quantify the enrichment power of the system, we performed a digital bioassay of alkaline phosphatase (ALP). Upon genetically tagging ALP molecules with the DEX-binding domain (DBD) derived from dextransucrase, the ALP molecules were enriched 59-fold in the DEX droplets in comparison to that in a conventional digital bioassay. Subsequently, we performed a Cas13-based digital SARS-CoV-2 RNA detection assay to evaluate the performance of this system for a more practical assay. In this assay, the target RNA molecules bound to the DBD-tagged Cas13 molecules were effectively enriched in the DEX droplets. Consequently, an enrichment factor of 31 was achieved. Enrichment experiments for nonlabeled proteins were also performed to test the expandability of this technique. The model protein, nontagged β-galactosidase, was enriched in DEX droplets containing DBD-tagged antibody, with an enrichment factor of over 100. Thus, this system enabled effective on-chip enrichment of target molecules to enhance the detection sensitivity of digital bioassays without using external instruments or an external power source, which would be applicable for on-site bioassays or portable diagnostic tests.

Marine Bacteria under Low-Intensity Radioactive Exposure: Model Experiments

Radioactive contaminants create problems all over world, involving marine ecosystems, with their ecological importance increasing in the future. The review focuses on bioeffects of a series of alpha and beta emitting radioisotopes (americium-241, uranium-(235 + 238), thorium-232, and tritium) and gamma radiation. Low-intensity exposures are under special consideration. Great attention has been paid to luminous marine bacteria as representatives of marine microorganisms and a conventional bioassay system. This bioassay uses bacterial bioluminescence intensity as the main testing physiological parameter; currently, it is widely applied due to its simplicity and sensitivity. Dependences of the bacterial luminescence response on the exposure time and irradiation intensity were reviewed, and applicability of hormetic or threshold models was discussed. A number of aspects of molecular intracellular processes under exposure to low-intensity radiation were analyzed: (a) changes in the rates of enzymatic processes in bacteria with the bioluminescent system of coupled enzymatic reactions of NADH:FMN-oxidoreductase and bacterial luciferase taken as an example; (b) consumption of an intracellular reducer, NADH; (c) active role of reactive oxygen species; (d) repairing of the DNA damage. The results presented confirm the function of humic substances as natural radioprotectors.

Larvicidal Activities of Merremia dissecta (Jacquin) Hallier f. and Peltrophorum pterocarpum (DC.) Backer ex k. Heyne Leaves against Aedes aegypti (Diptera: Culicidae)

Aim: Aedes aegypti is the principal vector of Dengue, Chikungunya, Zika. The burden of these diseases is typically highest in tropical and subtropical areas, affecting the most impoverished populations. This laboratory study accessed the larvicidal and pupicidal activities of ethanol, methanol, acetone leaf extracts of Merremia dissecta and Peltrophorum pterocarpum against the Ae. aegypti.&#x0D; Study Design: Conventional bioassay.&#x0D; Place and Duration of Study: Department of Zoology, Government Arts College, Coimbatore – 641018, Tamil Nadu, India, between July 2021 July 2022.&#x0D; Methodology: Larvae and pupae were exposed to various concentrations of plant leaf extracts for 24 hrs. at the laboratory. The probit analyses were used to analyze the data.&#x0D; Results: Results revealed that the plant extracts recorded the highest larval mortality at 0.421% and 0.634% ethanol, acetone leaf extracts of M. dissecta; 0.713%. and 0.621% methanol and ethanol leaf extract of P. pterocarpum against fourth instar of Ae. aegypti after 24 hrs.&#x0D; Conclusion: The results suggest that this plant extracts can be used as an good larvicidal and pupicide against the dengue and zika virus vector Ae. aegypti.

C-Series Coelenterazine-Driven Bioluminescence Signature Imaging.

The present study introduces a unique BL signature imaging system with novel CTZ analogues named "C-series." Nine kinds of C-series CTZ analogues were first synthesized, and BL intensity patterns and spectra were then examined according to the marine luciferases. The results show that the four CTZ analogues named C3, C4, C6, and C7, individually or collectively luminesce with completely distinctive BL spectral signatures and intensity patterns according to the luciferases: Renilla luciferase (RLuc), NanoLuc, and artificial luciferase (ALuc). The signatural reporters were multiplexed into a multi-reporter system comprising RLuc8.6-535SG and ALuc16. The usefulness of the signatural reporters was further determined with a multi-probe system that consists of two single-chain probes embedding RLuc8 and ALuc23. This study is a great addition to the study of conventional bioassays with a unique methodology, and for the specification of each signal in a single- or multi-reporter system using unique BL signatures and patterns of reporter luciferases.

Planar chromatography-bioassays for the parallel and sensitive detection of androgenicity, anti-androgenicity and cytotoxicity

Anti-androgens entering the aquatic environment, e.g., by effluents from wastewater treatment plants or agricultural settings are contributing to endocrine disruption in wildlife and humans. Due to the simultaneous presence of agonistic compounds, common in vitro bioassays can underestimate the risk posed by androgen antagonists. On the other hand, cytotoxic effects might lead to false positive assessments of anti-androgenic effects in conventional bioassays. In the present study, a combination of normal phase high-performance thin-layer chromatography (NP-HPTLC) with a yeast-based reporter gene assay is established for the detection of anti-androgenicity as a promising tool to reduce interferences of androgenic and anti-androgenic compounds present in the same sample. To avoid a misinterpretation of anti-androgenicity with cytotoxic effects, cell viability was assessed in parallel on the same plate using a resazurin viability assay adapted to HPTLC plates. The method was characterized by establishing dose-response curves for the model compounds flutamide and bisphenol A. Calculated effective doses at 10% (ED10) were 27.9 ± 1.3 ng zone−1 for flutamide and 20.1 ± 5.1 ng zone−1 for bisphenol A. Successful distinction between anti-androgenicity and cytotoxicity was exemplarily demonstrated with 4-nitroquinoline 1-oxide. As a proof of concept, the detection and quantification of anti-androgenicity in an extract of a landfill leachate is demonstrated. This study shows that the hyphenation of HPTLC with the yeast anti-androgen screen is a matrix-robust, cost-efficient and fast screening tool for the sensitive and simultaneous detection of anti-androgenic and cytotoxic effects in environmental samples. The method offers a wide range of possible applications in environmental monitoring and contributes to the identification of anti-androgenicity drivers in the course of an effect-directed analysis.

Special Issue on the Development of Microfluidic Devices for Medical Applications

Microfluidics refers to the science of handling fluids in microstructures and has been widely used in the medical field to miniaturize conventional drug delivery systems, bioassays, and diagnostics [...]

Ovicidal and Larvicidal Activities of n-hexane Extract of Murraya paniculata (L.) Leaves against Aedes aegypti

Aims: Aedes aegypti is a widely distributed mosquito and the primary vector of several arboviral diseases attracting major global health concerns. This laboratory study accessed the ovicidal and larvicidal activities of n-hexane extract of Murraya paniculata (L.) Jack leaves on eggs and first instar larvae of Aedes aegypti.&#x0D; Study Design: Conventional bioassay,&#x0D; Place and Duration of Study: Department of Zoology, ETF Laboratory and Central research laboratory, College of Medicine, University of Lagos, Idi-araba, between February and July 2021.&#x0D; Methodology: Eggs and larvae were exposed to various quantities of test plant extract ranging from 20ppm to 80ppm for 72 hours at ambient laboratory temperature(30\(\pm\)0.570c). Probit analyses and analysis of variance were used to analyze the data.&#x0D; Results: Results revealed that the plant extract recorded the highest ovicidal activity of 61% at 80 ppm concentration against the eggs of Aedes aegypti, after 72 hours. The LC50 values of ovicidal and larvicidal activities at 72hrs were 85.162ppm and 80.270ppm respectively, there was a significant difference between the ovicidal and larvicidal activity of each concentration while mortality increased with increasing concentration.&#x0D; Conclusion: The results suggest that n-hexane extract of M. paniculata can be used as an environmentally friendly method for the control of Aedes aegypti.

Preparation of freeze-dried bioluminescent bacteria and their application in the detection of acute toxicity of bisphenol A and heavy metals.

Current chemical analysis approaches for contaminants have failed to reveal their biotoxicity. Moreover, conventional bioassays are time consuming and exhibit poor repeatability. In this study, we performed the acute toxicity detection of various contaminants (chromium (Cr), cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg), tin (Sn), nickel (Ni), and bisphenol A (BPA)) with four bioluminescent bacteria (Vibrio qinghaiensis Q67, V. fischeri, Photobacterium phosphoreum T3, and P. phosphoreum 502) using a rapid, flexible, and low‐cost bioassay. We found that the temperature affected the bacterial luminescence, and freeze‐dried cells exhibited sensitive toxic responses to contaminants. Indeed, the optimized protectants containing 12% (w/v) trehalose, 4% sucrose, and 2% sorbitol displayed better luminescence and toxic sensitivity. Furthermore, freeze‐dried powders of these strains were prepared and subjected to acute toxicity detection. The results showed that all contaminants exhibited acute toxicity toward Q67, but the other strains did not show obvious response to nickel and tin. The relative half‐maximal effective concentration (EC50) values of BPA, Cr, Cd, Pb, As, Hg, Ni, and Sn to Q67 were 0.674, 1.313, 11.137, 5.921, 4.674, 0.911, 5.941, and 54.077 mg/L, respectively. In addition, the EC50 values of contaminants toward different strains were suggested to be statistically significant. Freeze‐dried Q67 exhibited toxic responses to more contaminants than the other bioluminescent strains; therefore, Q67 was selected to be more suitable than the other strains for single and mixture toxicity detection tests. Compared with other strains, Q67 was more appropriate for the rapid screening of the mixture toxicity of contaminants in samples as a nonspecific screening sensor before the use of standard analysis approaches.

A Practical Insecticide Resistance Monitoring Bioassay for Orally Ingested Dinotefuran in Anopheles Malaria Vectors.

Simple SummaryWhen insecticides are used to control mosquitoes, resistance is likely to develop over time. It is important to monitor the trait so that an alternative insecticide class can be deployed if needed, to sustain the efficiency of the intervention. Most insecticides for control of adult malaria vectors are used in treated bed nets or sprayed on walls where mosquitoes rest, so that mosquitoes contact them through their tarsi (feet). To control mosquitoes which are becoming resistant to these tools, new insecticide-based tools using both different chemistry and mode of uptake have been developed. One example of these is Attractive Toxic Sugar Baits (ATSBs), from which mosquitoes feed and ingest insecticide that kills them. However, different methods may be needed to monitor for resistance against interventions that have different modes of uptake. This study employed a method for applying insecticide directly onto a mosquito and measuring mortality, and the results were related to mortality from the same insecticide when ingested. This demonstrated that the method may be suitable to detect signs of resistance developing in mosquito populations targeted with ATSBs. Application of the method in wild populations will provide further validation.Attractive Toxic Sugar Baits (ATSB) deployed outdoors are likely to be particularly effective against outdoor biting mosquitoes and, if they contain insecticides with a different mode of action, mosquitoes resistant to pyrethroids. One such ATSB based on the neonicotinoid dinotefuran is currently under evaluation in Africa. As with any insecticide-based intervention, it will be important to monitor for the possible emergence of vector resistance. While methods for detecting resistance to insecticides via tarsal contact are recommended by the World Health Organization (WHO), these may not be applicable for orally ingested insecticides. Here, a new ingestion assay, appropriate for a controlled laboratory setting, is described using fluorescein sodium salt (uranine) as a feeding marker. Conventional topical application bioassays, more appropriate for routine deployment, have also been used to apply dinotefuran to the thorax of adult Anopheles mosquitoes with an organic carrier to bypass lipid cuticle barriers. The two methods were compared by establishing lethal doses (LD) in several Anopheles strains. The similarity of the ratios of susceptibility to dinotefuran between pairs of pyrethroid susceptible and resistant strains validates topical application as a suitable, more practical and field applicable method for monitoring for the emergence of resistance to orally ingested dinotefuran. A discriminating dose is proposed, which will be further validated against field populations and used to routinely monitor for the emergence of resistance alongside ATSB trials.

A 4-Gene Signature of CDKN1, FDXR, SESN1 and PCNA Radiation Biomarkers for Prediction of Patient Radiosensitivity.

The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.

Dependence of Graphene Oxide (GO) Toxicity on Oxidation Level, Elemental Composition, and Size.

The mass production of graphene oxide (GO) unavoidably elevates the chance of human exposure, as well as the possibility of release into the environment with high stability, raising public concern as to its potential toxicological risks and the implications for humans and ecosystems. Therefore, a thorough assessment of GO toxicity, including its potential reliance on key physicochemical factors, which is lacking in the literature, is of high significance and importance. In this study, GO toxicity, and its dependence on oxidation level, elemental composition, and size, were comprehensively assessed. A newly established quantitative toxicogenomic-based toxicity testing approach, combined with conventional phenotypic bioassays, were employed. The toxicogenomic assay utilized a GFP-fused yeast reporter library covering key cellular toxicity pathways. The results reveal that, indeed, the elemental composition and size do exert impacts on GO toxicity, while the oxidation level exhibits no significant effects. The UV-treated GO, with significantly higher carbon-carbon groups and carboxyl groups, showed a higher toxicity level, especially in the protein and chemical stress categories. With the decrease in size, the toxicity level of the sonicated GOs tended to increase. It is proposed that the covering and subsequent internalization of GO sheets might be the main mode of action in yeast cells.