Multi-level Biomarker Research Articles

Integrated assessment of trace elements in a marine ranching area based on multi-species and multi-level biomarkers: a case study in China’s national-level marine ranching demonstration area

The goal of this study was to evaluate the trace element pollution in a marine ranching area in China based on molecular responses (expression of metallothionein and antioxidant enzyme genes), and biochemical biomarkers (metallothionein content, antioxidant enzyme activities, and malonaldehyde level) in four indicator species. We collected samples of two fish (Lateolabrax maculatus and Sebastes schlegelii), one crustacean (Charybdis japonica), and one gastropod (Rapana venosa) from the western Furong Island marine ranching area and from an adjacent area in March 2022 and measured the trace element content in these indicator species as well as in the seawater and sediment. We found that the bioaccumulation characteristics of trace elements and the response patterns of biomarkers were species specific. Moreover, not every biomarker was significantly correlated with environmental trace element content. We then established two biomarker combinations indicative of trace element pollution in seawater and sediment, respectively, based on the correlation between biomarkers and trace element contents. The selected biomarkers were integrated using integrated biomarker response version 2 (IBRv2). IBRv2 values in the studied marine ranching area were lower than those in the adjacent area. Additionally, these values were consistent with the bioaccumulation of trace elements in the indicator species, the integrated trace element pollution index for seawater, and the potential risk index for sediment. These results show that this multi-biomarker and multi-species IBRv2 approach provided a comprehensive diagnosis of trace element pollution in the marine ranching area. Therefore, its application may be beneficial for marine environmental monitoring and management in view of the ecotoxicological impact of pollutants on organisms.

Multi-Level Biomarkers for Early Diagnosis of Ischaemic Stroke: A Systematic Review and Meta-Analysis.

Blood biomarkers hold potential for the early diagnosis of ischaemic stroke (IS). We aimed to evaluate the current weight of evidence and identify potential biomarkers and biological pathways for further investigation. We searched PubMed, EMBASE, the Cochrane Library and Web of Science, used R package meta4diag for diagnostic meta-analysis and applied Gene Ontology (GO) analysis to identify vital biological processes (BPs). Among 8544 studies, we included 182 articles with a total of 30,446 participants: 15675 IS, 2317 haemorrhagic stroke (HS), 1798 stroke mimics, 846 transient ischaemic attack and 9810 control subjects. There were 518 pooled biomarkers including 203 proteins, 114 genes, 108 metabolites and 88 transcripts. Our study generated two shortlists of biomarkers for future research: one with optimal diagnostic performance and another with low selection bias. Glial fibrillary acidic protein was eligible for diagnostic meta-analysis, with summary sensitivities and specificities for differentiating HS from IS between 3 h and 24 h after stroke onset ranging from 73% to 80% and 77% to 97%, respectively. GO analysis revealed the top five BPs associated with IS. This study provides a holistic view of early diagnostic biomarkers in IS. Two shortlists of biomarkers and five BPs warrant future investigation.

Screening potential toxicity of currently used herbicides in the freshwater amphipod Gammarus fossarum based on multi-level biomarker responses to field-realistic exposures

Herbicides are widely used to control weeds and maximize crop growth. Because of agricultural runoff, these chemicals are potentially hazardous to aquatic wildlife. However, their ecotoxicity and resulting disturbance in individual performance remain scarcely documented in freshwater crustaceans. This study aimed to screen the potential toxicity of currently used herbicides in the ecosystem engineer Gammarus fossarum using multi-level biomarkers. In microcosms, gammarids were exposed for 72 h to 12 herbicides individually (quinmerac, mesotrione, bentazone, isoproturon, chlortoluron, metazachlor, chloridazone, diflufenican, flufenacet, aclonifen, prosulfocarb and metolachlor) at a field-realistic concentration (i.e. 10 μg/L). The sublethal effects were assessed by monitoring several biochemical, physiological and behavioural traits. In exposed gammarids, alterations in behavioural activities were observed, i.e. increased locomotion and respiration as a general trend. Moreover, biochemical biomarkers suggested herbicide-dependent disruptions in moulting, antioxidant responses and cell integrity. Integrating multi-metric variations through statistical analyses allowed us to identify herbicide clusters likely to trigger common sets of biological responses. Depressed antioxidant defence at the cell level and impaired respiration at the individual level were the predominant toxic effects of herbicides, related to their hydrophobic feature. Furthermore, establishing relationships between sublethal alterations in gammarids and acute lethality or chronic toxicity values defined for regulatory purposes supports the relevance of these alterations as early warnings of toxicity. Our findings demonstrate that currently used herbicides have unexpected toxicological effects in a non-target wild animal, with possible long-term alterations in population dynamics and associated ecological functions, which constitute promising diagnostic tools for risk assessment in agricultural areas.

MXene-MoS2 carbon-fiber-based flexible electrochemical interface for multiple bioanalysis in biofluids

Flexible sensing technology has received widespread attention in the monitoring of human physiological indicators. Thus, it is of great significance to develop a universal flexible sensing interface for early diagnosis of multi-level biomarkers. Herein, we reported a multifunctional 3D carbon fiber paper (CFP)-MXene-MoS2 (CMM) sensing interface, which provided a flexible electrochemical biosensing platform for the ultrasensitive detection of multiple bioanalysis in biofluids. Notably, the CMM electrochemical interface was comprised of staggered carbon fibers on which the organ-like MXene-MoS2 heterostructure covered with MoS2 nanosheet arrays was uniformly allocated, permitting local enrichment of biomolecules for enhanced biomolecular capture efficiency, laying a solid foundation for the ultrasensitive detection of multiple biomolecules. In proof-of-concept experiments, we validated that the flexible CMM interface could be applied for ultrasensitive analyzing of quite a few biomolecules like ascorbic acid (AA), uric acid (UA), dopamine (DA) and microRNA (miRNA), with charming sensitivity and reproducibility. It was worth mentioned that an ultra-high sensitivity of 3.16 aM was acquired towards detecting microRNA. Under optimal parameters, the designed electrochemical interface worked well in complex biofluids such as human serum and urine samples. Overall, this flexible and disposable CMM interface with ultrahigh sensitivity provided insights for a more portable, accessible and valuable approach for monitoring biomolecules in clinical diagnosis.

Screening Potential Toxicity of Currently Used Herbicides in the Freshwater Amphipod Gammarus Fossarum Based on Multi-Level Biomarker Responses to Field-Realistic Exposures

Screening Potential Toxicity of Currently Used Herbicides in the Freshwater Amphipod Gammarus Fossarum Based on Multi-Level Biomarker Responses to Field-Realistic Exposures

A preliminary study on multi-level biomarkers response of the tropical oyster Crassostrea brasiliana to exposure to the antifouling biocide DCOIT

This study investigated the sublethal effects of environmentally relevant concentrations of DCOIT on the neotropical oyster Crassostrea brasiliana. Gills and digestive glands of animals exposed to increasing concentrations of DCOIT were analyzed for biochemical, cellular, and histopathological responses. Exposure to DCOIT (0.2 to 151 μg L−1) for 120 h triggered oxidative stress in both tissues (through the modulation of GPX, GST, GSH and GR), which led to damage of membrane lipids (increase of LPO and reduction of the NRRT). DCOIT increased histopathological pathologies in gills, such as necrosis, lymphocyte infiltration and epithelial desquamation. This study showed that short term exposure to environmental concentrations of DCOIT causes negative effects on C. brasiliana at biochemical, physiological, and histological levels. Therefore, the use of DCOIT as a booster biocide in antifouling paints should be further assessed, as it may cause environmental hazards to marine organisms.

Transient exposure to sublethal concentrations of a pesticide mixture (chlorpyrifos–difenoconazole) caused different responses in fish species from different trophic levels of the same community

The assessment of early effects caused in biota by sublethal exposure to pesticide mixtures should enhance the realism in the ecological risk assessment for agricultural landscapes. This study aimed to evaluate sub-individual responses in fish, which can be linked with outcomes at higher levels of biological organization and affect their trophic relationships. A multilevel biomarker approach was applied to assess the effects of a 48 h exposure of two freshwater mesoamerican fish species (Parachromis dovii and Poecilia gillii) to a mixture of sublethal concentrations of chlorpyrifos (5 μg/L) and difenoconazole (325 μg/L). Transcriptomic induction of cyp1A and the activities of 7-ethoxy-resorufin-O-distillase (EROD) and glutathione S-transferase (GST) were measured as biotransformation-related biomarkers; cholinesterase activity (ChE) was assessed as a neurotoxicity biomarker; resting metabolic rate (RMR) was measured as a physiological biomarker; and the movement of fish in a dark–light environment as a behavior biomarker. The exposure to the mixture had evident effects on P. gillii, with significant induction of cyp1A transcription, increased EROD activity, ChE inhibition in muscle, and increased permanence in the light side of the dark–light environment. Meanwhile, P. dovii only showed significant induction of cyp1A, without evidence of neurotoxicity or changes in behavior. This study demonstrates that the severity of the effects caused by the exposure to a mixture of pesticides can differ among species from the same trophic chain. The potential impairment of predator-prey relationships is a relevant effect that pesticide pollution can cause and it should be considered for the risk assessment of such contaminants.

Adverse effects of dietary virgin (nano)microplastics on growth performance, immune response, and resistance to ammonia stress and pathogen challenge in juvenile sea cucumber Apostichopus japonicus (Selenka)

It has been well documented that micro- and nanoplastics are emerging pollutants in aquatic environments, and their potential toxic effects has attracted widespread concerns. Here, we evaluated the adverse effects of dietary polystyrene nanoplastics and microplastics (PS-N/MPs) on growth performance, oxidative stress induction, immune response, ammonia detoxification, and bacterial pathogen resistance of sea cucumber Apostichopus japonicus. After collection and acclimation, sea cucumbers were randomized into 3 groups (i.e., control, 100 nm PS-NPs and 20 µm PS-MPs at 100 mg kg−1 diet) for 60-day feeding experiment. Every group contained 360 sea cucumbers which were equally divided into 3 aquaria as biological triplicates. The results showed that the specific growth rate and final weight of the sea cucumbers fed with diets containing PS-N/MPs were significantly lower than those of control group. Dietary virgin PS-N/MPs significantly increased the reactive oxygen species production and malondialdehyde content in coelomic fluid, causing oxidative stress and damage to the growth and development of A. japonicus. During the experiment, 100 nm PS-NPs significantly induced the depletion in cellular and humoral immune parameters. The calculated IBR values based on multi-level biomarkers revealed the size-dependent toxic differences of PS-NPs > PS-MPs. The relative expression levels of GDH and GS mRNA showed first rise and then fall trends after exposure to ammonia, and 100 nm PS-NPs had a more profound impact on suppressing ammonia detoxification compared with 20 µm PS-MPs. Moreover, the expression of Hsp90, Hsp70, CL, TLR, and CASP2 genes were all down-regulated by ammonia exposure. Taken together of IBR results, ammonia stress test and pathogen challenge, we deduced that dietary 100 nm PS-NPs are more potentially hazardous than 20 µm PS-MPs. These findings provide valuable information for understanding the size-dependent toxic effects of PS-N/MPs and early risk warning on marine invertebrates.

Risk factors for Epstein-Barr virus reactivation after renal transplantation: Results of a large, multi-centre study.

Epstein-Barr virus (EBV) reactivation is a very common and potentially lethal complication of renal transplantation. However, its risk factors and effects on transplant outcome are not well known. Here, we have analysed a large, multi-centre cohort (N=512) in which 18.4% of the patients experienced EBV reactivation during the first post-transplant year. The patients were characterized pre-transplant and two weeks post-transplant by a multi-level biomarker panel. EBV reactivation was episodic for most patients, only 12 patients showed prolonged viraemia for over four months. Pre-transplant EBV shedding and male sex were associated with significantly increased incidence of post-transplant EBV reactivation. Importantly, we also identified a significant association of post-transplant EBV with acute rejection and with decreased haemoglobin levels. No further severe complications associated with EBV, either episodic or chronic, could be detected. Our data suggest that despite relatively frequent EBV reactivation, it had no association with serious complications during the first post-transplantation year. EBV shedding prior to transplantation could be employed as biomarkers for personalized immunosuppressive therapy. In summary, our results support the employed immunosuppressive regimes as relatively safe with regard to EBV. However, long-term studies are paramount to support these conclusions.

Evaluation of the combined toxicity of multi-walled carbon nanotubes and cadmium on earthworms in soil using multi-level biomarkers

The coexistence of multi-walled carbon nanotubes (MWCNTs) with cadmium (Cd) in soil may cause the combined biological effects, but few study reported about their joint toxic effects on earthworms. Therefore, this study investigated the effects of sub-lethal levels of MWCNTs (10, 50, 100 mg/kg) and Cd (2.0, 10 mg/kg) on earthworms Eisenia fetida for 14 days. The changes in multi-level biomarkers of growth inhibition rate, cytochrome P450 isoenzymes (CYP1A2, 2C9 and 3A4), and small molecular metabolites (metabolomics) were determined. The toxic interaction between MWCNTs and Cd was characterized by the combination of the biomarker integration index (BRI), joint effect index concentration addition index (CAI), and the effect concentration addition index (EAI). The results showed that the single MWCNTs exposure caused insignificant change in most biomarkers, while the combined exposure of MWCNTs (50–100 mg/kg) and 10 mg/kg Cd led to significant changes in ten most important metabolites identified by metabolomics and activities of CYP1A2, 2C9, and 3A4. Compared with the toxicity of Cd alone, the combined toxicity of the mixture was significantly reduced. According to the integration of BRI and CAI/EAI, a clearly antagonistic interaction at relatively low effects was observed between MWCNTs and Cd. The responses of multiple biomarkers suggest the toxic action mode of the mixture on earthworms was related to the oxidative injury, and the disruption of amino acid, purine, and pyrimidine metabolism, and the urea cycle.

Behavioural and biochemical alterations in gammarids as induced by chronic metallic exposures (Cd, Cu and Pb): Implications for freshwater biomonitoring

In freshwater species, metal toxicity is usually assessed through short-term exposures, hence limiting the practical usefulness of biomarkers for monitoring long-term impacts on wildlife populations. This study investigates the biological alterations elicited by chronic metallic exposures in Gammarus fossarum using multi-level biomarkers. In aquaria, gammarids were exposed for 10 weeks to field-realistic concentrations of Cd, Cu or Pb (0.25, 1.5 or 5.0 μg/L). At the individual level, behavioural traits (respiration, locomotion and feeding) were compared between naive and chronically-exposed gammarids. At the cellular level, enzymatic activities involved in digestion, moult and cell stress were monitored after 2, 6 and 10 weeks of exposure in males and females to consider the temporal feature of their responses. Results showed that the inhibitory effects of Cd and Pb on respiration and locomotion disappeared in chronically-exposed gammarids, reflecting acclimation to maintain these processes, unlike Cu. Chronic Cu- and Pb-elicited feeding inhibition was associated with the inhibitions of digestion enzymes. Chitobiase was inhibited by Cu in males and, by Cd and Pb in females, suggesting gender-dependent disturbances in moulting. In both genders, Cd generated cellular stress by stimulating acidic phosphatase and peroxidase activities. To conclude, such cellular impairments and alterations in individual performances are likely to disturb individual growth, population dynamics and litter decomposition in the long-term. Besides, obtaining biological responses, common to metals or specific to a metal or a gender, supports the development of biomarkers highlighting long-term impacts of metals on the health of organisms and their associated ecological functions in natural environments.

Single and combined effects of insecticides on multi-level biomarkers in the non-target amphipod Gammarus fossarum exposed to environmentally realistic levels

Aquatic media are ultimate recipients of various contaminants including pesticides pervasively applied in agrosystems. Characterizing the ecotoxicity of pesticides and their mixtures to aquatic wildlife at field-realistic levels is thus crucial for environmental risk assessment. This study aims at assessing the effects of two current-use insecticides, imidacloprid and chlorpyrifos, on Gammarus fossarum using multi-level biomarkers. In microcosms, gammarids were exposed for 72 h to insecticides tested individually or in mixture at 0.01, 0.1 and 1 μg/L of each chemical. Multi-metric responses were assessed at the individual level (behavioural traits: locomotion, respiration and amplexus formation) and the cellular level (enzymes involved in growth, moulting, digestion and cell stress). The results showed insecticide-elicited behavioural and biochemical responses from the lowest concentration of 0.01 μg/L. Overall, single exposures stimulated behavioural traits and inhibited enzymatic activities, highlighting subtle impacts at different organizational levels but these were not dose related. For binary mixtures, antagonistic effects (i.e. less-than-additive) on biomarkers were mainly observed when compared with single exposures. Multi-variable analyses indicated the complementarity of behavioural and biochemical biomarkers in identifying sublethal biological alterations and dose-dependent multiple action sites of insecticides. Besides, the mortality observed only for the mixture at 1 μg/L demonstrated a high lethal potential of insecticides in a simple binary combination. To conclude, this study demonstrates disturbances in individual performances and cellular impairments occurring at environmentally realistic exposure levels in a non-target wild species. Since the sublethal effects, such as those identified with this multi-biomarker approach, could lead to long-term alterations in population dynamics of agricultural areas, they constitute promising early endpoints for risk assessment of insecticides.

Longitudinal multi-level biomarker analysis of BDNF in major depression and bipolar disorder.

Preliminary evidence suggests that BDNF (brain derived neurotrophic factor) rs6265 genetic polymorphism, BDNF gene promotor methylation and BDNF serum levels might play an important role in the pathogenesis of affective disorders. As studies testing the BDNF system across molecular levels are sparse, this study aimed at investigating the BDNF val66met genotype, BDNF DNA methylation changes and peripheral BDNF serum levels in acute and remitted phases of MDD (major depressive disorder) and BD (bipolar disorder) and healthy controls. We found a significant difference of methylation levels at CpG site 1-1-1 and 3-1-1 between MDD and healthy controls (p < 0.003) with MDD patients showing significantly higher methylation levels. CpG 5-2-1 revealed a statistically significant difference between MDD and healthy controls and MDD and BD (p = 0.00003). Similar to the results of the methylation analysis a significant difference between MDD and healthy controls was found in BDNF serum levels (p = 0.002) with significantly lower BDNF serum levels in MDD compared to healthy controls. A difference between the samples from admission and discharge from hospital of both BDNF gene methylation and serum levels could not be detected in the present study and no influence of the BDNF val66met genotype on neither methylation nor BDNF serum level.

Developmental toxicity of kresoxim-methyl during zebrafish (Danio rerio) larval development

Kresoxim-methyl (KM) is a broad spectrum strobilurin fungicide that has been used widely on crops around the world. In the present study, we aimed to investigate the toxic effects of KM using various sublethal endpoints during zebrafish (Danio rerio) larval development. Results showed that the LC50 values of KM to zebrafish at multiple life stages (embryo, larvae, juvenile and adult) were 0.340, 0.224, 0.328 and 0.436 mg/L, respectively. The transcription patterns of 45 genes involved in hypothalamic-pituitary-thyroid/gonadal (HPT/HPG) axis, oxidative stress and apoptosis revealed KM could affect zebrafish larval development at multiple pathways. The activities of aromatase, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), caspase 3 (Cas3) and caspase 9 (Cas9), and the levels of estradiol (E2), vitellogenin (VTG), thyroid hormones (T3 and T4), reactive oxygen species (ROS) and ATP after embryos exposed to KM for 3 d, 6 d and 10 d were correlated well with the transcription of the corresponding molecules involved in these pathways. In addition to providing the first description of the toxic effects induced by KM during larval development, the results of present study also provided the potential mechanisms of KM on multi-level biomarker responses in larval zebrafish.

Predicting the response to neoadjuvant therapy for early-stage breast cancer: tumor-, blood-, and imaging-related biomarkers.

Neoadjuvant therapy (NAT) has been used increasingly in patients with locally advanced or early-stage breast cancer. However, the accurate evaluation and prediction of response to NAT remain the great challenge. Biomarkers could prove useful to identify responders or nonresponders, or even to distinguish between early and delayed responses. These biomarkers could include markers from the tumor itself, such as versatile proteins, genes, and ribonucleic acids, various biological factors or peripheral blood cells, and clinical and pathological features. Possible predictive markers could also include multiple features from functional imaging, such as standard uptake values in positron emission tomography, apparent diffusion coefficient in magnetic resonance, or radiomics imaging biomarkers. In addition, cells that indirectly present the immune status of tumor cells and/or their host could also potentially be used as biomarkers, eg, tumor-infiltrating lymphocytes, tumor-associated macrophages, and myeloid-derived suppressor cells. Though numerous biomarkers have been widely investigated, only estrogen and/or progesterone receptors and human epidermal growth factor receptor have been proven to be reliable biomarkers to predict the response to NAT. They are the only biomarkers recommended in several international guidelines. The other aforementioned biomarkers warrant further validation studies. Some multigene profiling assays that are commercially available, eg, Oncotype DX and MammaPrint, should be used with caution when extrapolated to NAT settings. A panel of combined multilevel biomarkers might be able to predict the response to NAT more robustly than individual biomarkers. To establish such a panel and its prediction model, reliable methods and extensive clinical validation are warranted.

Assessment of adverse impacts of glyphosate-based herbicide, Excel Mera 71 by integrating multi-level biomarker responses in fishes

This work was aimed to investigate the adverse impacts of Excel Mera 71 on Anabas testudineus, Heteropneustes fossilis and Oreochromis niloticus under laboratory (17.2 mg/L) and field (750 g/acre) conditions based on multiple-level integrated biomarker responses. Haematological parameters were influenced significantly and showed species specificity after Excel Mera 71 exposure under both the conditions. Physiological indices (hepato-somatic index and condition factor) showed higher values; however, hepato-somatic index induced significantly under both the conditions (p H. fossilis > A. testudineus), while total nuclear abnormalities under field showed the order as: H. fossilis (0.49 ± 0.08) > A. testudineus (0.46 ± 0.10) > O. niloticus (0.27 ± 0.05). Moreover, multi-level integrated biomarker responses displayed responses as: O. niloticus (6.97 fold) > H. fossilis (4.01 fold) > A. testudineus (2.33 fold) in laboratory; however, under field condition, responses were totally different [A. testudineus–H. fossilis (3.98 fold) > O. niloticus (3.01 fold)]. Additionally, glyphosate concentration was significantly correlated with several multi-level biomarker responses (p < 0.05). Moreover, multi-level integrated biomarker responses clearly indicated the higher impacts under laboratory experiment. Present investigation documented that multi-level integrative biomarker responses should be considered as reliable tool for assessment of herbicidal intoxication.

Biomarkers in basics, clinical and translational research to get the translational pipeline succeeded

The use of biomarkers in basic and clinical research as well as in translational research has become so commonplace that their presence as primary endpoints in clinical trials is now accepted almost without question. In basic research they play an important role of milestones during the verification of theory of disease pathway. In clinical practice biomarkers are used to evaluate drug efficiency and for patients' condition monitoring. And eventually translational research is hardly thinkable without a deep system of multilevel biomarkers. In this speech current progress and issues of biomarker armamentarium will be discussed.

Abstract 2249: Integrated multi-level omics-based biomarker models for ovarian cancer survival

Abstract Background: Although a number of prognostic biomarkers for ovarian cancer have been proposed, the pool of clinically useful biomarkers remains small. While the predictive power of individual biomarkers may be weak, the advent of more affordable high-throughput technologies and readily available “omics” level data point toward the possibility of high performance biomarker panels. In this study, we used tumor-based somatic mutation, mRNA and miRNA expression, and DNA methylation data from The Cancer Genome Atlas (TCGA) to construct integrated biomarker models for overall and progression-free survival. Methods: The somatic mutations, mRNA expression, and DNA methylation of 451 candidate genes, as well as the expression of the miRNAs targeting them, were individually analyzed for association with overall and progression-free survival in 488 TCGA serous cystadenocarcinoma patients of predominantly Caucasian descent. Following correction for multiple comparisons, molecular variations associated with survival were combined to construct integrated cross-validated prediction models for overall and progression-free survival. The discriminative power of these models was then evaluated for 1-, 3-, and 5-year overall and progression-free survival using ROC analysis. Results: Our cross-validated integrated molecular models were demonstrated to predict 5-year overall survival in the total patient pool with a ROC AUC of 0.801 and 5-year overall and progression-free survival in Caucasian patients with ROC AUCs of 0.866 and 0.809, respectively. Conclusions: Our findings suggest the potential utility of our multi-omics-based biomarker models in helping to inform clinical decisions following ovarian cancer diagnosis. Although extensive efforts were made to maximize the predictive potential of our models, further retrospective and prospective validation efforts are necessary before their clinical utility can be accurately assessed. Citation Format: Alan Fu, Shen-Chih Chang, Aileen Baecker, Helena R. Chang, Zuo-Feng Zhang. Integrated multi-level omics-based biomarker models for ovarian cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2249. doi:10.1158/1538-7445.AM2017-2249

Universal ratiometric electrochemical biosensing platform based on mesoporous platinum nanocomposite and nicking endonuclease assisted DNA walking strategy

The occurrence and development of many complex diseases are associated with various molecules, whose contents are rarely in the early stage of the disease. Thus a universal platform for the ultrasensitive detection of multilevel biomarkers should be developed. In this study, we introduced an electrochemical biosensing system based on nicking endonuclease (Nt.BbvCI) assisted DNA walking strategy. We successfully constructed a universal signal-off-on ratiometric electrochemical biosensor for various biomolecules, including small molecules, nucleic acids, and proteins, by progressively optimizing the schematics (schemes 1, 2, and 3). The MB-hairpin probes (MB-HPs) acted as a signal-off probe, and nanocomposites (MPNs@DOX@DNA2) acted as a conventional signal-on probe (scheme 3). With the aid of the MPNs@DOX@DNA2 and Nt.BbvCI assisted DNA walking mechanism, the designed ratiometric electrochemical biosensor showed a high sensitivity and broad detection range. In addition, the proposed method can be utilized to detect diverse targets quantitatively by changing the sequence of aptamers under optimum experimental conditions. Furthermore, it has been widely proved to realize well-accepted signal response in identifying complex samples, thereby resulting in an wide prospect for bioanalysis and clinical diagnosis.

Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform.

The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin-HRP). Hydrogen peroxide (H2O2) is then reduced by avidin-HRP in the presence of TMB (3,3',5,5'-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h.