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Glucose monitoring has become a crucial part of diabetes care, and is also of importance in the food industry. However, the measurements are subject to certain limitations and errors. These may result from many factors, including the strip manufacturing process, inappropriate storage, temperature, coding, aging, etc. Here, we discuss the measurement of glucose in real samples using techniques such as photometric assay, glucometers, and using a proposed biosensor. Biosensor platforms based on the multicomponent material magnetite/lignin/polydopamine-glucose oxidase with the addition of ferrocene and a dedicated carbon paste electrode (CPE/Fe3O4/Lig/PDA/GOx/Fc) were fabricated for glucose detection in real, commercially available glucose-based samples. To determine the morphological features of the materials, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), atomic force microscopy (AFM) and zeta potential measurements were carried out. Moreover, to determine the effectiveness of glucose oxidase immobilization the Bradford assays was applied. Analysis of glucose-based products was carried out based on photometric measurements using an AU480 Chemical Analyzer from Beckam Coulter, with the use of three glucometers (Wellion Calla light® WELL 900LB, CERA-CHEK 1Code®Model G400, iXell®Rev.04/10-PL), and with the use of our proposed CPE/Fe3O4/Lig/PDA/GOx/Fc biosensor. Tests on real samples demonstrated the repeatability of the results. The results showed that this biosensor has excellent potential for application in the determination of glucose in various commercial products.
Lipopolysaccharides (LPS) are endotoxins, hazardous and toxic inflammatory stimulators released from the outer membrane of Gram-negative bacteria, and are the major cause of septic shock giving rise to millions of fatal illnesses worldwide. There is an urgent need to identify and detect these molecules selectively and rapidly. Pathogen detection has been done by traditional as well as biosensor-based methods. Nanomaterial based biosensors can assist in achieving these goals and have tremendous potential. The biosensing techniques developed are low-cost, easy to operate, and give a fast response. Due to extremely small size, large surface area, and scope for surface modification, nanomaterials have been used to target various biomolecules, including LPS. The sensing mechanism can be quite complex and involves the transformation of chemical interactions into amplified physical signals. Many different sorts of nanomaterials such as metal nanomaterials, magnetic nanomaterials, quantum dots, and others have been used for biosensing of LPS and have shown attractive results. This review considers the recent developments in the application of nanomaterials in sensing of LPS with emphasis given mainly to electrochemical and optical sensing.
Future biosensing applications will require high performance, including real-time monitoring of physiological events, incorporation of biosensors into feedback-based devices, detection of toxins, and advanced diagnostics. Such functionality will necessitate biosensors with increased sensitivity, specificity, and throughput, as well as the ability to simultaneously detect multiple analytes. While these demands have yet to be fully realized, recent advances in biofabrication may allow sensors to achieve the high spatial sensitivity required, and bring us closer to achieving devices with these capabilities. To this end, we review recent advances in biofabrication techniques that may enable cutting-edge biosensors. In particular, we focus on bioprinting techniques (e.g., microcontact printing, inkjet printing, and laser direct-write) that may prove pivotal to biosensor fabrication and scaling. Recent biosensors have employed these fabrication techniques with success, and further development may enable higher performance, including multiplexing multiple analytes or cell types within a single biosensor. We also review recent advances in 3D bioprinting, and explore their potential to create biosensors with live cells encapsulated in 3D microenvironments. Such advances in biofabrication will expand biosensor utility and availability, with impact realized in many interdisciplinary fields, as well as in the clinic.
Single-molecule techniques have become impactful in bioanalytical sciences, though the advantages for continuous biosensing are yet to be discovered. Here we present a multiplexed, continuous biosensing method, enabled by an analyte-sensitive, single-molecular nanoswitch with a particle as a reporter. The nanoswitch opens and closes under the influence of single target molecules. This reversible switching yields binary transitions between two highly reproducible states, enabling reliable quantification of the single-molecule kinetics. The multiplexing functionality is encoded per particle via the dissociation characteristics of the nanoswitch, while the target concentration is revealed by the association characteristics. We demonstrate by experiments and simulations the multiplexed, continuous monitoring of oligonucleotide targets, at picomolar concentrations in buffer and in filtered human blood plasma.
Research into extracellular vesicles (EVs) has grown significantly over the last few decades with EVs being widely regarded as a source of biomarkers for human health and disease with massive clinical potential. Secreted by every cell type in the body, EVs report on the internal cellular conditions across all tissue types. Their presence in readily accessible biofluids makes the potential of EV biosensing highly attractive as a noninvasive diagnostic platform via liquid biopsies. However, their small size (50-250 nm), inherent heterogeneity, and the complexity of the native biofluids introduce challenges for effective characterization, thus, limiting their clinical utility. This has led to a surge in the development of various novel EV biosensing techniques, with capabilities beyond those of conventional methods that have been directly transferred from cell biology. In this review, key detection principles used for EV biosensing are summarized, with a focus on some of the most recent and fundamental developments in the field over the last 5 years. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > In Vitro Nanoparticle-Based Sensing.
While chemical vapor deposition of diamond films is currently cost prohibitive for biosensor construction, in this paper, we show that sonication-assisted nanostructuring of biosensing electrodes with nanodiamonds (NDs) allows harnessing the hydrolytic stability of the diamond biofunctionalization chemistry for real-time continuous sensing, while improving the detector sensitivity and stability. We find that the higher surface coverages were important for improved bacterial capture and can be achieved through proper choice of solvent, ND concentration, and seeding time. A mixture of methanol and dimethyl sulfoxide provides the highest surface coverage (33.6 ± 3.4%) for the NDs with positive zeta-potential, compared to dilutions of dimethyl sulfoxide with acetone, ethanol, isopropyl alcohol, or water. Through impedance spectroscopy of ND-seeded interdigitated electrodes (IDEs), we found that the ND seeds serve as electrically conductive islands only a few nanometers apart. Also we show that the seeded NDs are amply hydrogenated to be decorated with antibodies using the UV-alkene chemistry, and higher bacterial captures can be obtained compared to our previously reported work with diamond films. When sensing bacteria from 10(6) cfu/mL E. coli O157:H7, the resistance to charge transfer at the IDEs decreased by ∼ 38.8%, which is nearly 1.5 times better than that reported previously using redox probes. Further in the case of 10(8) cfu/mL E. coli O157:H7, the charge transfer resistance changed by ∼ 46%, which is similar to the magnitude of improvement reported using magnetic nanoparticle-based sample enrichment prior to impedance detection. Thus ND seeding allows impedance biosensing in low conductivity solutions with competitive sensitivity.
From the bench-mark work on microfluidics from the Whitesides's group in 2007, paper technology has experienced significant growth, particularly regarding applications in biomedical research and clinical diagnostics. Besides the structural properties supporting microfluidics, other advantageous features of paper materials, including their versatility, disposability and low cost, show off the great potential for the development of advanced and eco-friendly analytical tools. Consequently, paper was quickly employed in the field of electrochemical sensors, being an ideal material for producing custom, tailored and miniaturized devices. Stencil-, inkjet-, or screen-printing are the preferential techniques for electrode manufacturing. Not surprisingly, we witnessed a rapid increase in the number of publications on paper based screen-printed sensors at the turn of the past decade. Among the sensing strategies, various biosensors, coupling electrochemical detectors with biomolecules, have been proposed. This work provides a critical review and a discussion on the future progress of paper technology in the context of miniaturized printed electrochemical biosensors.
Phytopathogenic bacteria cause severe economic losses in agricultural production worldwide. The spread rates, severity, and emerging plant bacterial diseases have become serious threat to the sustainability of food sources and the fruit industry. Detection and diagnosis of plant diseases are imperative in order to manage plant diseases in field conditions, greenhouses, and food storage conditions as well as to maximize agricultural productivity and sustainability. To date, various techniques including, serological, observation-based, and molecular methods have been employed for plant disease detection. These methods are sensitive and specific for genetic identification of bacteria. However, these methods are specific for genetic identification of bacteria. Currently, the innovative biosensor-based disease detection technique is an attractive and promising alternative. A biosensor system involves biological recognition and transducer active receptors based on sensors used in plant-bacteria diagnosis. This system has been broadly used for the rapid diagnosis of plant bacterial pathogens. In the present review, we have discussed the conventional methods of bacterial-disease detection, however, the present review mainly focuses on the applications of different biosensor-based techniques along with point-of-care (POC), robotics, and cell phone-based systems. In addition, we have also discussed the challenges and limitations of these techniques.
We selected a novel biotin-binding peptide for sensing biotin, biotinylated proteins, and nucleotides. From a 15-mer library displayed on the RNA coliphage Qβ, a 15-amino acid long peptide (HGHGWQIPVWPWGQG) hereby referred to as a nanotag was identified to selectively bind biotin. The target selection was achieved through panning with elution by infection. The selected peptide was tested as a transducer for an immunogenic epitope of the foot-and-mouth disease virus (FMDV) on Qβ phage platform separated by a linker. The biotin-tag showed no significant influence on the affinity of the epitope to its cognate antibody (SD6). The nanotag-bound biotin selectively fused either to the C- or N-terminus of the epitope. The epitope would not bind or recognize SD6 while positioned at the N-terminus of the nanotag. Additionally, the biotin competed linearly with the SD6 antibody in a competitive ELISA. Competition assays using the selected recombinant phage itself as a probe or transducer enable the operationalization of this technology as a biosensor toolkit to sense and quantify SD6 analyte. Herein, the published Strep II nanotag (DVEWLDERVPLVET) was used as a control and has similar functionalities to our proposed novel biotin-tag thereby providing a new platform for developing devices for diagnostic purposes.
"Clickable" organic electrochemical transistors (OECTs) allow the reliable and straightforward functionalization of electronic devices through the well-known click chemistry toolbox. In this work, we study various aspects of the click chemistry-based interface engineering of "clickable" OECTs. First, different channel architectures are investigated, showing that PEDOT-N3 films can properly work as a channel of the transistors. Furthermore, the Cu(I)-catalyzed click reaction of ethynyl-ferrocene is studied under different reaction conditions, endowing the spatial control of the functionalization. The strain-promoted and catalyst-free cycloaddition of a dibenzocyclooctyne-derivatized poly-l-lysine (PLL-DBCO) is also performed on the OECTs and validated by a fiber optic (FO)-SPR setup. The further immobilization of an azido-modified HD22 aptamer yields OECT-based biosensors that are employed for the recognition of thrombin. Finally, their performance is evaluated against previously reported architectures, showing higher density of the immobilized HD22 aptamer, and originating similar KD values and higher maximum signal change upon analyte recognition.
Printed circuit boards (PCBs) offer a promising platform for the development of electronics-assisted biomedical diagnostic sensors and microsystems. The long-standing industrial basis offers distinctive advantages for cost-effective, reproducible, and easily integrated sample-in-answer-out diagnostic microsystems. Nonetheless, the commercial techniques used in the fabrication of PCBs produce various contaminants potentially degrading severely their stability and repeatability in electrochemical sensing applications. Herein, we analyse for the first time such critical technological considerations, allowing the exploitation of commercial PCB platforms as reliable electrochemical sensing platforms. The presented electrochemical and physical characterisation data reveal clear evidence of both organic and inorganic sensing electrode surface contaminants, which can be removed using various pre-cleaning techniques. We demonstrate that, following such pre-treatment rules, PCB-based electrodes can be reliably fabricated for sensitive electrochemical biosensors. Herein, we demonstrate the applicability of the methodology both for labelled protein (procalcitonin) and label-free nucleic acid (E. coli-specific DNA) biomarker quantification, with observed limits of detection (LoD) of 2 pM and 110 pM, respectively. The proposed optimisation of surface pre-treatment is critical in the development of robust and sensitive PCB-based electrochemical sensors for both clinical and environmental diagnostics and monitoring applications.
Recent technological advances in nanoscience and material designing have led to the development of point-of-care devices for biomolecule sensing and cancer diagnosis. In situ and portable sensing devices for bedside, diagnosis can effectively improve the patient's clinical outcomes and reduce the mortality rate. Detection of exosomal RNAs by immuno-biochip with increased sensitivity and specificity to diagnose cancer has raised the understanding of the tumor microenvironment and many other technology-based biosensing devices hold great promise for clinical innovations to conquer the unbeatable fort of cancer metastasis. Electrochemical biosensors are the most sensitive category of biomolecule detection sensors with significantly low concentrations down to the atomic level. In this sense, this review addresses the recent advances in cancer detection and diagnosis by developing significant biological sensing devices that are believed to have better sensing potential than existing facilities.
The concept of point of darkness has received much attention for biosensing based on phase-sensitive detection and perfect absorption of light. The maximum phase change is possible at the point of darkness where the reflection is almost zero. To date, this has been experimentally realized using different material systems through the concept of topological darkness. However, complex nanopatterning techniques are required to realize topological darkness. Here, we report an approach to realize perfect absorption and extreme phase singularity using a simple metal-dielectric multilayer thin-film stack. The multilayer stack works on the principle of an asymmetric Fabry-Perot cavity and shows an abrupt phase change at the reflectionless point due to the presence of a highly absorbing ultrathin film of germanium in the stack. In the proof-of-concept phase-sensitive biosensing experiments, we functionalize the film surface with an ultrathin layer of biotin-thiol to capture streptavidin at a low concentration of 1 pM.
Staphylococcus aureus (S. aureus) is a major foodborne pathogen. Rapid and specific detection is crucial for controlling staphylococcal food poisoning. This study reported a Staphylococcus phage named LSA2302 showing great potential for applications in the rapid detection of S. aureus. Its biological characteristics were identified, including growth properties and stability under different pH and temperature conditions. The genomic analysis revealed that the phage has no genes associated with pathogenicity or drug resistance. Then, the phage-functionalized magnetic beads (pMB), serving as a biological recognition element, were integrated with ATP bioluminescence assays to establish a biosensing method for S. aureus detection. The pMB enrichment brought high specificity and a tenfold increase in analytical sensitivity during detection. The whole detection process could be completed within 30 min, with a broad linear range of 1 × 104 to 1 × 108 CFU/mL and a limit of detection (LOD) of 2.43 × 103 CFU/mL. After a 2 h pre-cultivation, this method is capable of detecting bacteria as low as 1 CFU/mL. The recoveries of S. aureus in spiked skim milk and chicken samples were 81.07% to 99.17% and 86.98% to 104.62%, respectively. Our results indicated that phage-based biosensing can contribute to the detection of target pathogens in foods.
The deposition of biomolecules on biosensing surface platforms plays a key role in achieving the required sensitivity and selectivity for biomolecular interactions analysis. Controlling the interaction between the surface and biomolecules is increasingly becoming a crucial design tool to modulate the surface properties needed to improve the performance of the assay and the detection outcome. Carboxymethyl-dextran (CMD) coating can be exploited to promote chemical grafting of proteins, providing a hydrophilic, bioinert, nonfouling surface and a high surface density of immobilized proteins. In the present work, we developed and optimized a technique to produce a cost-effective CMD-based patterned surface for the immobilization of biomolecules to be used on standard protocols optimization. They consist of silicon or glass substrates with patterned bioactive areas able to efficiently confine the sampling solution by simply exploiting hydrophilic/hydrophobic patterning of the surface. The fabrication process involves the use of low-cost instruments and techniques, compatible with large scale production. The devices were validated through a chemiluminescence assay we recently developed for the analysis of binding of DNA nanoassemblies modified with an affinity binder to target proteins immobilized on the bioactive areas. Through this assay we were able to characterize the chemical reactivity of two target proteins toward a dextran matrix on patterned surfaces and to compare it with model CMD-based surface plasmon resonance (SPR) surfaces. We found a high reproducibility and selectivity in molecular recognition, consistent with results obtained on SPR sensor surfaces. The suggested approach is straightforward, cheap, and provides the means to assess patterned functionalized surfaces for bioanalytical platforms.
Diseases and diagnoses are predominant in the human population. Early diagnosis of etiological agents plays a vital role in the treatment of bacterial infections. Existing standard diagnostic platforms are laborious, time-consuming, and require trained personnel and cost-effective procedure, though they are producing promising results. These shortcomings have led to a thirst for rapid diagnostic procedures. Fluorescence-based diagnosis is one of the efficient rapid diagnostic methods that rely on specific and sensitive bacterial detection. Emerging bio-sensing studies on conducting polymers (CPs) are gaining popularity in medical diagnostics due to their promising properties of high fluorescence efficiency, good light stability, and low cytotoxicity. Poly[2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV), is the first identified soluble polymer and model material for understanding the fundamental photophysics of conventional CPs. In this present study, MEH-PPV is used as a fluorescent dye for direct pathogen detection applications by interacting with the microbial cell surface. An optimized concentration of MEH-PPV solution used to confirm the presence of selective bacterial structures. The present study endeavours towards bacterial detection based on the emission from bacteria due to interfacial interaction between polymer and bacterial surface.
A simplified coupling of surface plasmon resonance (SPR) immuno-biosensing with ambient ionization mass spectrometry (MS) was developed. It combines two orthogonal analysis techniques: the biosensing capability of SPR and the chemical identification power of high resolution MS. As a proof-of-principle, deoxynivalenol (DON), an important mycotoxin, was captured using an SPR gold chip containing an antifouling layer and monoclonal antibodies against the toxin and, after washing, the chip could be taken out and analyzed by direct spray MS of the biosensor chip to confirm the identity of DON. Furthermore, cross-reacting conjugates of DON present in a naturally contaminated beer could be successfully identified, thus showing the potential of rapid identification of (un)expected cross-reacting molecules.
Quantum dot (Qdot) biosensors have consistently provided valuable information to researchers about cellular activity due to their unique fluorescent properties. Many of the most popularly used Qdots contain cadmium, posing the risk of toxicity that could negate their attractive optical properties. The design of a non-cytotoxic probe usually involves multiple components and a complex synthesis process. In this paper, the design and synthesis of a non-cytotoxic Qdot-chitosan nanogel composite using straight-forward cyanogen bromide (CNBr) coupling is reported. The probe was characterized by spectroscopy (UV-Vis, fluorescence), microscopy (Fluorescence, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering. This activatable ("OFF"/"ON") probe contains a core-shell Qdot (CdS:Mn/ZnS) capped with dopamine, which acts as a fluorescence quencher and a model drug. Dopamine capped "OFF" Qdots can undergo ligand exchange with intercellular glutathione, which turns the Qdots "ON" to restore fluorescence. These Qdots were then coated with chitosan (natural biocompatible polymer) functionalized with folic acid (targeting motif) and Fluorescein Isothiocyanate (FITC; fluorescent dye). To demonstrate cancer cell targetability, the interaction of the probe with cells that express different folate receptor levels was analyzed, and the cytotoxicity of the probe was evaluated on these cells and was shown to be nontoxic even at concentrations as high as 100 mg/L.
We present the development of a label-free, highly sensitive fiber-optical biosensor for online detection and quantification of biomolecules. Here, the advantages of etched fiber Bragg gratings (eFBG) were used, since they induce a narrowband Bragg wavelength peak in the reflection operation mode. The gratings were fabricated point-by-point via a nonlinear absorption process of a highly focused femtosecond-pulsed laser, without the need of prior coating removal or specific fiber doping. The sensitivity of the Bragg wavelength peak to the surrounding refractive index (SRI), as needed for biochemical sensing, was realized by fiber cladding removal using hydrofluoric acid etching. For evaluation of biosensing capabilities, eFBG fibers were biofunctionalized with a single-stranded DNA aptamer specific for binding the C-reactive protein (CRP). Thus, the CRP-sensitive eFBG fiber-optical biosensor showed a very low limit of detection of 0.82 pg/L, with a dynamic range of CRP detection from approximately 0.8 pg/L to 1.2 µg/L. The biosensor showed a high specificity to CRP even in the presence of interfering substances. These results suggest that the proposed biosensor is capable for quantification of CRP from trace amounts of clinical samples. In addition, the adaption of this eFBG fiber-optical biosensor for detection of other relevant analytes can be easily realized.
With the aim of a reliable biosensing exhibiting enhanced sensitivity and selectivity, this study demonstrates a dopamine (DA) sensor composed of conductive poly(3,4-ethylenedioxythiophene) nanotubes (PEDOT NTs) conformally coated with porphyrin-based metal-organic framework nanocrystals (MOF-525). The MOF-525 serves as an electrocatalytic surface, while the PEDOT NTs act as a charge collector to rapidly transport the electron from MOF nanocrystals. Bundles of these particles form a conductive interpenetrating network film that together: (i) improves charge transport pathways between the MOF-525 regions and (ii) increases the electrochemical active sites of the film. The electrocatalytic response is measured by cyclic voltammetry and differential pulse voltammetry techniques, where the linear concentration range of DA detection is estimated to be 2 × 10-6-270 × 10-6 m and the detection limit is estimated to be 0.04 × 10-6 m with high selectivity toward DA. Additionally, a real-time determination of DA released from living rat pheochromocytoma cells is realized. The combination of MOF5-25 and PEDOT NTs creates a new generation of porous electrodes for highly efficient electrochemical biosensing.
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