ABSTRACT
In this study, a nanocomposite was synthesized by incorporating graphitic carbon nanosheets, carboxyl-functionalized multiwalled carbon nanotubes, and zirconium oxide nanoparticles. The resulting nanocomposite was utilized for the modification of a glassy carbon electrode. Subsequently, matrix metalloproteinase aptamer (AptMMP-9) was immobilized onto the electrode surface through the application of ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride-N-hydroxysuccinimide (EDC-NHS) chemistry. Morphological characterization of the nanomaterials and the nanocomposite was performed using field-emission scanning electron microscopy (FESEM). The nanocomposite substantially increased the electroactive surface area by 205%, facilitating enhanced immobilization of AptMMP-9. The efficacy of the biosensor was evaluated using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Under optimal conditions, the fabricated sensor demonstrated a broad range of detection from 50 to 1250 pg/mL with an impressive lower limit of detection of 10.51 pg/mL. In addition, the aptasensor exhibited remarkable sensitivity, stability, excellent selectivity, reproducibility, and real-world applicability when tested with human serum and saliva samples. In summary, our developed aptasensor exhibits significant potential as an advanced biosensing tool for the point-of-care quantification of MMP-9, promising advancements in biomarker detection for practical applications.
Subject(s)
Aptamers, Nucleotide , Graphite , Nanocomposites , Nanotubes, Carbon , Nitrogen Compounds , Zirconium , Humans , Nanotubes, Carbon/chemistry , Matrix Metalloproteinase 9 , Electrochemical Techniques/methods , Saliva , Reproducibility of Results , Limit of Detection , Aptamers, Nucleotide/chemistry , Nanocomposites/chemistryABSTRACT
A versatile and highly sensitive sensing platform based on nanocellulose/MXene/ZrO2 nano framework has been developed at the surface of a glassy carbon electrode (GCE) for detecting ovalbumin (Ova). To create this innovative nano framework, dialdehyde groups were introduced onto the surface of cellulose nanofibers (CNFs), which were then decorated with MXene nanosheets and nanostructured zirconia. Nanocellulose/MXene/ZrO2 nano framework was used as electrochemical mediator and immobilization environment that provided the large surface area and 197 % increment in the electrochemical signal which allowed the Ova detection in the femtomolar range. Ovalbumin antibody was immobilized on the surface of dialdehyde cellulose nanofiber through covalent bonding between amino groups of Ova and dialdehyde groups of CNFs. The assembly process of nano framework, anti-Ova, and Ova antigen were characterized using electrochemical approaches (CV and DPV). The fabricated immunosensor is further applied to DPV detection of Ova and it demonstrated a linear response to Ova antigen in the linear range of 0.01-1000 pg/mL. With optimal experimental conditions, the detection limit, quantification limit and sensitivity of Ova were found to be 1.1 fg/mL, 0.01 pg/mL and 0.1497 µA pg/mL cm-2, respectively. The fabricated immunosensor exhibited high selectivity, reproducibility, and interference resistance and achieved excellent recoveries in real food samples spiked with Ova, indicating its potential applicability in food safety monitoring.
Subject(s)
Biosensing Techniques , Nitrites , Transition Elements , Ovalbumin , Immunoassay , Reproducibility of Results , Titanium , Electrochemical Techniques , Limit of Detection , Gold/chemistryABSTRACT
We propose a CRISPR/Cas12a-mediated recombinase polymerase amplification (RPA) detection method that combines RPA with Cas12a cleavage for the detection of halal food adulteration, which is of global concern, particularly for Muslim consumers. We optimized the reagent concentrations for the Cas12a cleavage steps and designed and screened gRNA targeting a conserved area of the mitochondrial cytochrome C oxidase subunit I (COX1) gene. This procedure successfully detected the presence of porcine components as low as 5 pg/µL in the linear range of 5-1000 pg/µL. The assay's detection limit was 500 times lower than CRISPR-based approaches that exclude a preamplification step, allowing the detection of trace porcine DNA in food samples. The assay additionally showed no cross-reaction with nontarget species. Therefore, this detection platform shows tremendous potential as a method for the quick, sensitive, and specific detection of porcine-derived components.
ABSTRACT
This study introduces CRISPR/Cas-based aptasensor for the highly sensitive and specific detection of the antibiotic, ampicillin. Ampicillin (AMPI) is a commonly used antibiotic for treating pathogenic bacteria and is additionally added to livestock feed in agriculture. This study can enable early detection of antibiotic residues, prevent their accumulation in the environment, and ensure compliance with food safety regulations. Herein, the aptasensor was developed with the CRISPR/Cas system by utilizing three different ampicillin-specific aptamers, each conjugated with a biotin at the 5'-end. The ssDNA activator was bound to the aptamers through complementary base pairings. The attraction of the aptamers to the ampicillin target released the bound ssDNA, causing the activation of the CRISPR/Cas system. The DNA reporter probe, labelled with Cy3 and a quencher, turns on the fluorescence signal when cleaved by the activated Cas12a through trans-cleavage measured using a fluorescence spectrophotometer at 590 nm. The fluorescence signal was linearly proportional to the ampicillin target concentration with a 0.01 nM limit of detection and a read-out time of 30 min. This aptasensor showed high sensitivity towards ampicillin even in the presence of other antibiotics. The method was also successfully implemented for ampicillin detection in spiked food samples.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , CRISPR-Cas Systems/genetics , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Ampicillin , Anti-Bacterial Agents/pharmacology , DNA, Single-StrandedABSTRACT
For early detection and diagnosis of cancer, it is essential to develop an electrochemical biosensor that is quick, accurate, and sensitive. Here, we use gold nanorod (AuNR) and gold nanowire (AuNW) nanocomposites (AuNR/AuNW/CS) as electrode modifiers on a glassy carbon electrode (GCE) to construct a sensitive label-free electrochemical aptasensor to detect ApoA1. The thiolated ApoA1-specific aptamers were immobilized onto the modified electrode surface through self-assembled monolayers. Electrochemical techniques, such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV), were used to analyze the fabrication steps. The concentration of ApoA1 was measured with DPV on the aptasensor, with a linear range of 0.1 to 1000 pg mL-1 and a detection limit of 0.04 pg mL-1. When compared to results from ELISA tests (which have a detection limit of 80 pg mL-1), the results achieved here were over 2000 times better. The aptasensor's performance was successfully evaluated using human serum spiked with ApoA1, suggesting that it has great potential for practical application. The electrochemical apatsensor additionally demonstrated outstanding selectivity responses and strong stability toward the target analyte.
ABSTRACT
A molybdenum sulfide/zirconium oxide/Nafion (MoS2/ZrO2/Naf) based electrochemiluminescence (ECL) aptasensor for the selective and ultrasensitive detection of ApoA1 is proposed, with Ru(bpy)3 2+ as the luminophore. The chitosan (CS) modification on the nanocomposite layer allowed glutaraldehyde (GLUT) cross-linking, resulting in the immobilization of ApoA1 aptamers. Scanning electron microscopy, tunneling electron microscopy, and energy dispersive X-ray spectroscopy were used to characterize the nanocomposite, while electrochemiluminescence (ECL), cyclic voltammetry, and electrochemical impedance spectroscopy were used to analyze the aptasensor assembly. The nanocomposite was used as an electrode modifier, which increased the intensity of the ECL signal. Due to the anionic environment produced on the sensor surface following the specific interaction of the ApoA1 biomarker with the sensor, more Ru(bpy)3 2+ were able to be electrostatically attached to the aptamer-ApoA1 complex, resulting in enhanced ECL signal. The ECL aptasensor demonstrated outstanding sensitivity for ApoA1 under optimal experimental conditions, with a detection limit of 53 fg/mL and a wide linear dynamic range of 0.1-1000 pg/mL. The potential practical applicability of this aptasensor was validated by analyzing ApoA1 in human serum samples, with recovery rates of 94-108% (n = 3). The proposed assay was found to be substantially better compared to the commercially available enzyme-linked immunosorbent assay method, as reflected from over 1500 times improvement in the detection limit for ApoA1.
ABSTRACT
Nanozymes have piqued the curiosity of scientists in recent years because of their ability to demonstrate enzyme-like activity combined with advantages such as high stability, inexpensive availability, robust activity, and tunable properties. These attributes have allowed the successful application of nanozymes in sensing to detect various chemical and biological target analytes, overcoming the shortcomings of conventional detection techniques. In this review, we discuss recent developments of nanozyme-based sensors to detect biomarkers associated with food quality and safety. First, we present a brief introduction to this topic, followed by discussing the different types of sensors used in food biomarker detection. We then highlight recent studies on nanozyme-based sensors to detect food markers such as toxins, pathogens, antibiotics, growth hormones, metal ions, additives, small molecules, and drug residues. In the subsequent section, we discuss the challenges and possible solutions towards the development of nanozyme-based sensors for application in the food industry. Finally, we conclude the review by discussing future perspectives of this field towards successful detection and monitoring of food analytes.
ABSTRACT
An electrochemiluminescence (ECL) aptasensor for the detection of the milk protein allergen ß-lactoglobulin (ß-LG) using nanocomposite as luminophore was fabricated. The Ru-AuNPs/GNP/Naf complex was formed by combining the Rubpy32+-AuNPs complex (Ru-AuNPs), prepared by modifying the negatively charged surface of gold nanoparticles (AuNPs) with positively charged Rubpy32+ through electrostatic interactions and the graphene nanoplatelets-Nafion (GNP/Naf) at a ratio of 2:1. The nanocomposite was coated on the surface of the screen-printed electrode (SPCE) through the film-forming properties of Nafion. A layer of chitosan (CS) was coated onto this modified electrode, and later amine-terminated ß-LG aptamers were covalently attached to the CS/Ru-AuNP/GNP/Naf via glutaraldehyde (GLUT) cross-linking. When ß-LG was incubated with the aptasensor, a subsequent decrease in ECL intensity was recorded. Under the optimal conditions, the ECL intensity of the aptasensor changed linearly with the logarithmic concentration of ß-LG, in the range 0.1 to 1000 pg/ml, and the detection limit was 0.02 pg/mL (3σ/m). The constructed aptasensor displayed simple and fast determination of ß-LG with excellent reproducibility, stability, and high specificity. Additionally, the proposed ECL aptasensor displayed high recoveries (92.5-112%) and low coefficients of variation (1.6-7.8%), when ß-LG fortified samples were analyzed. Integrating Ru-AuNPs/GNP/Naf nanocomposite in the ECL aptasensor paves the way towards a cost-effective and sensitive detection of the milk allergen ß-LG.
Subject(s)
Biosensing Techniques , Metal Nanoparticles , Nanocomposites , Electrochemical Techniques , Gold , Lactoglobulins , Luminescent Measurements , Reproducibility of ResultsABSTRACT
Ultrasensitive biosensors have become a necessity in the world of scientific research, and several signal enhancement strategies have been employed to attain exceptionally low detection limits. Nanotechnology turns out to be a strong contender for signal amplification, as they can be employed as platform modifiers, catalysts, carriers or labels. Here, we have described the most recent advancements in the utilization of nanomaterials as signal amplification components in aptamer-based electrochemical biosensors. We have briefly reviewed the methods that utilized nanomaterials, namely gold and carbon, as well as nanocomposites such as: graphene, carbon nanotubes, quantum dots, and metal-organic frameworks.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Graphite , Nanostructures , Nanotubes, Carbon , Biosensing Techniques/methods , GoldABSTRACT
Rac1 GTPase is hyperactivated in tumors and contributes to malignancy. Rac1 disruption of junctions requires its effector PAK1, but the precise mechanisms are unknown. Here, we show that E-cadherin is internalized via micropinocytosis in a PAK1-dependent manner without catenin dissociation and degradation. In addition to internalization, PAK1 regulates E-cadherin transport by fine-tuning Rab small GTPase function. PAK1 phosphorylates a core Rab regulator, RabGDIß, but not RabGDIα. Phosphorylated RabGDIß preferentially associates with Rab5 and Rab11, which is predicted to promote Rab retrieval from membranes. Consistent with this hypothesis, Rab11 is activated by Rac1, and inhibition of Rab11 function partially rescues E-cadherin destabilization. Thus, Rac1 activation reduces surface cadherin levels as a net result of higher bulk flow of membrane uptake that counteracts Rab11-dependent E-cadherin delivery to junctions (recycling and/or exocytosis). This unique small GTPase crosstalk has an impact on Rac1 and PAK1 regulation of membrane remodeling during epithelial dedifferentiation, adhesion, and motility.
Subject(s)
Adherens Junctions/physiology , Exocytosis , Keratinocytes/physiology , p21-Activated Kinases/metabolism , rab GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein/metabolism , Actins/metabolism , Adherens Junctions/chemistry , Cells, Cultured , Humans , Keratinocytes/cytology , Signal Transduction , p21-Activated Kinases/genetics , rab GTP-Binding Proteins/genetics , rac1 GTP-Binding Protein/geneticsABSTRACT
A synergistic nanocomposite film composed of gold nanourchins (AuNU), oxidised carbon nanohorns (CNH), and chitosan functioned as an electrode modifier in the fabrication of the sensitive lipocalin-2 (LCN-2) aptasensor. The AuNUs/CNH/CS composite increased the surface area and thereby amplified the signal transduction. The amine-terminated LCN-2 aptamer was immobilised through the amide bond formed between the carboxyl group of polyglutamic acid (PGA) and the amine group of aptamer. Interaction of LCN-2 with the aptamer caused conformational changes in the structure of the aptamer. This generated higher conductivity, resulting in increased DPV peak current. The DPV signal increased with increasing concentration of LCN-2, and the change in signal was used for quantitative detection. The proposed aptasensor was able to detect LCN-2 in the linear range of 0.1 - 100.0 pg mL-1, with a low detection limit of 10 fg mL-1. The aptasensor showed high sensitivity, selectivity, reproducibility, and was able to detect LCN-2 in serum samples.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Metal Nanoparticles , Nanotubes, Carbon , Electrochemical Techniques , Electrodes , Gold , Limit of Detection , Lipocalin-2 , Reproducibility of ResultsABSTRACT
This study focuses on developing a highly sensitive immunosensor by immobilizing oxidized carbon nanohorns/Nafion/Fe3O4@Pd nanocomposite on carbon screen-printed electrodes (SPEs) for the detection of tropomyosin (Tro-Ag). The performance of the fabricated immunosensor was investigated via electrochemiluminescence (ECL) method, resulting from the chemical reaction between tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) and tripropylamine (TPrA), and the peak intensity is recorded at 1.0 V. The nanocomposite is able to enhance the ECL intensity of [Ru(bpy)3]2 +/TPrA system and achieves high sensitivity of 28.16 fg/mL with a dynamic working range of 28.16 fg/mL to 100 ng/ml.. Furthermore, the immunosensor demonstrated a decent stability and good repeatability for Tro-Ag detection in food products. Graphical abstract The schematic representation of the modified carbon SPE with CNHs-OH/Nafion/Fe3O4@Pd and the signal produced in the (a) absence and (b) presence of Tro-Ag.
Subject(s)
Electrochemical Techniques/methods , Immunoassay/methods , Luminescent Measurements/methods , Nanocomposites/chemistry , Tropomyosin/analysis , Animals , Antibodies, Immobilized/immunology , Carbon/chemistry , Coordination Complexes/chemistry , Electrochemical Techniques/instrumentation , Electrodes , Ferrosoferric Oxide/chemistry , Fluorocarbon Polymers/chemistry , Organometallic Compounds/chemistry , Palladium/chemistry , Penaeidae/chemistry , Propylamines/chemistry , Rabbits , Tropomyosin/immunologyABSTRACT
Stable cell-cell contacts underpin tissue architecture and organization. Quantification of junctions of mammalian epithelia requires laborious manual measurements that are a major roadblock for mechanistic studies. We designed Junction Mapper as an open access, semi-automated software that defines the status of adhesiveness via the simultaneous measurement of pre-defined parameters at cell-cell contacts. It identifies contacting interfaces and corners with minimal user input and quantifies length, area and intensity of junction markers. Its ability to measure fragmented junctions is unique. Importantly, junctions that considerably deviate from the contiguous staining and straight contact phenotype seen in epithelia are also successfully quantified (i.e. cardiomyocytes or endothelia). Distinct phenotypes of junction disruption can be clearly differentiated among various oncogenes, depletion of actin regulators or stimulation with other agents. Junction Mapper is thus a powerful, unbiased and highly applicable software for profiling cell-cell adhesion phenotypes and facilitate studies on junction dynamics in health and disease.
Subject(s)
Cell Communication/physiology , Computational Biology/methods , Endothelial Cells/physiology , Intercellular Junctions/physiology , Keratinocytes/physiology , Myocytes, Cardiac/physiology , Animals , Cadherins/metabolism , Cell Adhesion/physiology , Cells, Cultured , Endothelial Cells/metabolism , Humans , Intercellular Junctions/metabolism , Keratinocytes/metabolism , Microscopy, Confocal , Myocytes, Cardiac/metabolism , Phenotype , Rats, Sprague-Dawley , SoftwareABSTRACT
Electrochemical bio-sensing using paper-based detection systems is the main focus of this review. The different existing designs of 2-dimensional and 3-dimensional sensors, and fabrication techniques are discussed. This review highlights the effect of adopting different sensor designs, distinct fabrication techniques, as well as different modification methods, in order to produce reliable and reproducible reading. The use of various nanomaterials have been demonstrated in order to modify the surface of electrodes during fabrication to further enhance the signal for subsequent analysis. The reviewed sensors were classified into categories based on their applications, such as diagnostics, environmental and food testing. One of the major advantages of using paper-based electrochemical sensors is the potential for miniaturization, which only requires relatively small amount of samples, and the low cost for the purpose of mass production. Additionally, most of the devices reviewed were made to be portable, making them well-suited for on-site detection. Finally, paper-based detection is an ideal platform to fabricate cost-effective, user-friendly and sensitive electrochemical biosensors, with large capacity for customization depending on functional needs.
Subject(s)
Biosensing Techniques/instrumentation , Paper , Electrochemistry , Equipment Design , HumansABSTRACT
The rapid and increasing use of the nanomaterials (NMs), nanostructured materials (NSMs), metal nanoclusters (MNCs) or nanocomposites (NCs) in the development of electrochemiluminescence (ECL) nanobiosensors is a significant area of study for its massive potential in the practical application of nanobiosensor fabrication. Recently, NMs or NSMs (such as AuNPs, AgNPs, Fe3O4, CdS QDs, OMCs, graphene, CNTs and fullerenes) or MNCs (such as Au, Ag, and Pt) or NCs of both metallic and non-metallic origin are being employed for various purposes in the construction of biosensors. In this review, we have selected recently published articles (from 2014-2017) on the current development and prospects of label-free or direct ECL nanobiosensors that incorporate NCs, NMs, NSMs or MNCs.
Subject(s)
Biosensing Techniques , Graphite , Luminescent Measurements , NanocompositesABSTRACT
Nucleic acid detection is of paramount importance in monitoring of microbial pathogens in food safety and infectious disease diagnostic applications. To address these challenges, a rapid, cost-effective label-free technique for nucleic acid detection with minimal instrumentations is highly desired. Here, we present paper microchip to detect and quantify nucleic acid using colorimetric sensing modality. The extracted DNA from food samples of meat as well as microbial pathogens was amplified utilizing loop-mediated isothermal amplification (LAMP). LAMP amplicon was then detected and quantified on a paper microchip fabricated in a cellulose paper and a small wax chamber utilizing crystal violet dye. The affinity of crystal violet dye toward dsDNA and positive signal were identified by changing the color from colorless to purple. Using this method, detection of Sus scrofa (porcine) and Bacillus subtilis (bacteria) DNA was possible at concentrations as low as 1 pg/µL (3.43 × 10 -1 copies/µL) and 10 pg/µL (2.2 × 103 copies/µL), respectively. This strategy can be adapted for detection of other DNA samples, with potential for development of a new breed of simple and inexpensive paper microchip at the point-of-need.
Subject(s)
Colorimetry/instrumentation , DNA/analysis , Gentian Violet/chemistry , Lab-On-A-Chip Devices , Nucleic Acid Amplification Techniques/instrumentation , Paper , Animals , Bacillus subtilis/genetics , DNA/chemistry , DNA, Bacterial/analysis , DNA, Bacterial/chemistry , Red MeatABSTRACT
The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34-/- mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein-lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking.
Subject(s)
Class III Phosphatidylinositol 3-Kinases/metabolism , Endocytosis , GTPase-Activating Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Autophagy , Biocatalysis , Endosomes/metabolism , Endosomes/ultrastructure , Fibroblasts/metabolism , HeLa Cells , Humans , Lysosomes/metabolism , Lysosomes/ultrastructure , Mice, Knockout , Phosphatidylinositol Phosphates/metabolism , Protein Transport , TOR Serine-Threonine Kinases/metabolism , Vacuoles/metabolism , Vacuoles/ultrastructure , rab7 GTP-Binding ProteinsABSTRACT
Autophagy is an evolutionarily conserved process that enables catabolic and degradative pathways. These pathways commonly depend on vesicular transport controlled by Rabs, small GTPases inactivated by TBC/RabGAPs. The Rac1 effector TBC/RabGAP Armus (TBC1D2A) is known to inhibit Rab7, a key regulator of lysosomal function. However, the precise coordination of signaling and intracellular trafficking that regulates autophagy is poorly understood. We find that overexpression of Armus induces the accumulation of enlarged autophagosomes, while Armus depletion significantly delays autophagic flux. Upon starvation-induced autophagy, Rab7 is transiently activated. This spatiotemporal regulation of Rab7 guanosine triphosphate/guanosine diphosphate cycling occurs by Armus recruitment to autophagosomes via interaction with LC3, a core autophagy regulator. Interestingly, autophagy potently inactivates Rac1. Active Rac1 competes with LC3 for interaction with Armus and thus prevents its appropriate recruitment to autophagosomes. The precise coordination between Rac1 and Rab7 activities during starvation suggests that Armus integrates autophagy with signaling and endocytic trafficking.