Innovative Biosensor Research Articles

Illuminating Cellular Biochemistry: Fluorogenic Chemogenetic Biosensors for Biological Imaging

Cellular activity is defined by the precise spatiotemporal regulation of various components, such as ions, small molecules, or proteins. Studying cell physiology consequently requires the optical recording of these processes, notably by using fluorescent biosensors. The recent development of various fluorogenic systems greatly expanded the palette of reporters to be included in these sensors design. Fluorogenic reporters consist of a protein or RNA tag that can complex either an endogenous or a synthetic fluorogenic dye (so-called fluorogen). The intrinsic nature of these tags, along with the high tunability of their cognate chromophore provide interesting features such as far-red to near-infrared emission, oxygen independence, or unprecedented color versatility. These engineered photoreceptors, self-labelling proteins, or noncovalent aptamers and protein tags were rapidly identified as promising reporters to observe biological events. This Minireview focuses on the new perspectives they offer to design unique and innovative biosensors, thus pushing the boundaries of cellular imaging.

Novel Plasmonic Nanocavities for Optical Trapping‐Assisted Biosensing Applications

AbstractPlasmonic nanocavities have proved to confine electromagnetic fields into deep subwavelength volumes, implying their potentials for enhanced optical trapping and sensing of nanoparticles. In this review, the fundamentals and performances of various plasmonic nanocavity geometries are explored with specific emphasis on trapping and detection of small molecules and single nanoparticles. These applications capitalize on the local field intensity, which in turn depends on the size of plasmonic nanocavities. Indeed, properly designed structures provide significant local field intensity and deep trapping potential, leading to manipulation of nano‐objects with low laser power. The relationship between optical trapping‐induced resonance shift and potential energy of plasmonic nanocavity can be analytically expressed in terms of the intercavity field intensity. Within this framework, recent experimental works on trapping and sensing of single nanoparticles and small molecules with plasmonic nanotweezers are discussed. Furthermore, significant consideration is given to conjugation of optical tweezers with Raman spectroscopy, with the aim of developing innovative biosensors. These devices, which take the advantages of plasmonic nanocavities, will be capable of trapping and detecting nanoparticles at the single molecule level.

Detection of xanthine in food samples with an electrochemical biosensor based on PEDOT:PSS and functionalized gold nanoparticles.

An innovative biosensor assembly relying on glassy carbon electrodes modified with nanocomposites consisting of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) as a host matrix with functionalized gold nanoparticles (GCE/PEDOT:PSS-AuNPs) is presented for the selective and sensitive detection of xanthine (XA). The developed sensor was successfully applied for the quantification of XA in the presence of significant interferents like hypoxanthine (HXA) and uric acid (UA). Different spectroscopy and electron microscopy analyses were done to characterize the as-prepared nanocomposite. Calibration responses for the quantification of XA was linear from 5.0 × 10−8 to 1.0 × 10−5 M (R2 = 0.994), with a detection limit as low as 3.0 × 10−8 (S/N = 3). Finally, the proposed sensor was applied for the analyses of XA content in commercial fish and meat samples and satisfactory recovery percentage was obtained.

Controlling the Bioreceptor Spatial Distribution at the Nanoscale for Single Molecule Counting in Microwell Arrays.

The ability to detect low concentrations of protein biomarkers is crucial for the early-stage detection of many diseases and therefore indispensable for improving diagnostic devices for healthcare. Here, we demonstrate that by integrating DNA nanotechnologies like DNA origami and aptamers, we can design innovative biosensing concepts for reproducible and sensitive detection of specific targets. DNA origami structures decorated with aptamers were studied as a novel tool to structure the biosensor surface with nanoscale precision in a digital detection bioassay, enabling control of the density, orientation, and accessibility of the bioreceptor to optimize the interaction between target and aptamer. DNA origami was used to control the spatial distribution of an in-house-generated aptamer on superparamagnetic microparticles, resulting in an origami-linked digital aptamer bioassay to detect the main peanut antigen Ara h1 with 2-fold improved signal-to-noise ratio and 15-fold improved limit of detection compared to a digital bioassay without DNA origami. Moreover, the sensitivity achieved was 4 orders of magnitude higher than commercially available and literature-reported enzyme-linked immunosorbent assay techniques. In conclusion, this novel and innovative approach to engineer biosensing interfaces will be of major interest to scientists and clinicians looking for new molecular insights and ultrasensitive detection of a broad range of targets, and, for the next generation of diagnostics.

Supramolecular Microgels with Molecular Beacons at the Interface for Ultrasensitive, Amplification-Free, and SNP-Selective miRNA Fluorescence Detection.

In this study, a supramolecular structure with femtomolar biorecognition properties is proposed for use in analytical devices. It is obtained by an innovative interface between synthetic hydrogel polymers and molecular beacon (mb) probes. Supramolecularly structured microgels are synthetized with a core-shell architecture with specific dyes polymerized in a desired compartment. Mb probes are opportunely conjugated at the microgel interface so that their recognition mechanism is preserved and their spatial distribution is optimized to avoid crowding effects. The miR-21, a microRNA involved in various biological processes and usually used as a biomarker in early cancer diagnosis, has been selected as the target. The results demonstrate that by tuning the spatial distribution of molecular probes immobilized on the microgel and/or the amount of microgels, the assay shows scalable sensitivity reaching a limit of detection down to about 10 fM, without amplification steps and with detection time as short as 1 h. The assay results specific toward single mutated targets, and it is stable in the presence of high-interfering oligonucleotides concentrations. The miRNA target is also detected in human serum with performances similar to those observed in PBS buffer because of microgel antifouling properties without the need of any surface treatment. All tests were performed in a low sample volume (20 μL). As a result, mb-microgel represents an innovative biosensor to precisely quantify microRNAs in a direct (mix&read), scalable, and selective way. Such an approach paves the way for creating innovative biosensing interfaces with other probes, such as hairpins, aptamers, and PNA.

An optical H2S biosensor based on the chemoselective Hb-I protein tethered to a transparent, high surface area nanocolumnar electrode

Sensitive and selective detection of analytes in complex biological fluids can be an extremely challenging issue. The constructive association of biomolecules and transparent mesoporous electrodes is of interest in this area, as it can lead to innovative biosensors combining optical and electrochemical detection modes. This concept, however, requires the development of appropriate surface functionalization methodologies that are robust enough for long-term operation in physiological environments. In the present work, the high-surface area of 3D transparent mesoporous indium-tin oxide (ITO) electrodes (prepared by glancing angle deposition or GLAD) has been chemically functionalized with recombinant hemoglobin I from Lucina pectinata according to a versatile 2-step process. First, 4-diazoniumbenzoic acid salt is covalently electrografted onto the ITO surface, followed by amide coupling of the protein. The resulting electrodes were quantitatively characterized by cyclic voltammetry and UV–vis absorption spectroscopy, demonstrating high surface coverages (up to 45% of a closed-packed monolayer for Hemoglobin-I) and homogeneous distribution across the entire thickness of the GLAD mesoporous structure. Good stability is also observed when the modified electrodes are immersed for prolonged times in a high ionic strength saline buffer. We also show that the hemoglobin I-modified electrode can be used as an optical biosensor for the selective, reversible, and fast detection of H2S in aqueous solutions over a ∼two-decade concentration range (i.e. up to 10 μM) and with a limit of detection of 0.35 μM. Good analytical performance was also achieved in human plasma without significant interference from the biological matrix.

The Potential of Bioelectrochemical Sensor for Monitoring of Acetate During Anaerobic Digestion: Focusing on Novel Reactor Design.

Acetate as the dominant fraction of volatile fatty acids (VFAs) is an important intermediate in metabolic pathways of methanogenesis, which could reflect the stability status of anaerobic digestion (AD) process. Bioelectrochemical sensors for environmental or bioprocess monitoring have become increasingly attractive in recent years. Although it was more favorable, several challenges still need to be addressed for acetate detection, including large electrode spacing, low stability, biofouling at the cathode and low detection range. In this study, an innovative biosensor on the basis of a three-chamber microbial electrochemical system was proposed to monitor the acetate during the AD process. In such a system, acetate was first transferred from sample chamber through the anion exchange membrane (AEM) to anode due to the driven force of concentration difference and then oxidized by anodic biofilm as a substrate for the current generation. With such design, the influence of waste properties fluctuation in the cathodic reaction could be avoided. The response of current density to different acetate concentrations was investigated. The selectivity, the influence of the sample temperature and the external resistance were also evaluated. The correlation (R2 > 0.99) between the current densities and acetate concentrations (up to 160 mM) was established at specific reaction time (from 2 to 5 h). Current densities after 5 h reaction were improving about 20% when the sample temperature was high (e.g., 37 and 55°C). The detection range increased along with the decrease of external resistance. The acetate concentrations of AD effluents as determined by the biosensor where within 24.2% of the ones determined by gas chromatography. Nevertheless, the application of the biosensor for monitoring acetate in environmental samples could still be promising.

A Bioelectronic System to Measure the Glycolytic Metabolism of Activated CD4+ T Cells.

The evaluation of glucose metabolic activity in immune cells is becoming an increasingly standard task in immunological research. In this study, we described a sensitive, inexpensive, and non-radioactive assay for the direct and rapid measurement of the metabolic activity of CD4+ T cells in culture. A portable, custom-built Cell Culture Metabolite Biosensor device was designed to measure the levels of acidification (a proxy for glycolysis) in cell-free CD4+ T cell culture media. In this assay, ex vivo activated CD4+ T cells were incubated in culture medium and mini electrodes were placed inside the cell free culture filtrates in 96-well plates. Using this technique, the inhibitors of glycolysis were shown to suppress acidification of the cell culture media, a response similar to that observed using a gold standard lactate assay kit. Our findings show that this innovative biosensor technology has potential for applications in metabolic research, where acquisition of sufficient cellular material for ex vivo analyses presents a substantial challenge.

Determination of proline in human plasma samples using the encapsulation of proline dehydrogenase enzyme in dendritic silica: a new platform for the enzymatic biosensing of amino acids

An innovative electrochemical biosensor for the sensitive and specific detection of l-proline (l-Pro) in human plasma samples was prepared by the encapsulation of proline dehydrogenase on amine functionalized dendritic fibrous nano-silica KCC-1.

Graphene-Based Raman Spectroscopy for pH Sensing of X-rays Exposed and Unexposed Culture Media and Cells.

Graphene provides a unique way of sensing the local pH level of substances on the micrometric scale, with important implications for the monitoring of cellular metabolic activities where proton excretion could occur. Accordingly, an innovative biosensing approach for the quantification of the pH value of biological fluids, to be used also with small amounts of fluids, was realized and tested. It is based on the use of micro-Raman spectroscopy to detect the modifications of the graphene doping level induced by the contact of the graphene with the selected fluids. The approach was preliminarily tested on aqueous solutions of known pH values. It was then used to quantify the pH values of cell culture media directly exposed to different doses of X-ray radiation and to media exposed to X-ray-irradiated cells. The Raman response of cells placed on graphene layers was also examined.

Mercury Selective GaN HEMT Sensor for Dynamic Water Quality Monitoring

Mercury is an extremely toxic metal that can cause irreversible damage to human, animal and plant life. Even a very low level exposure to mercury over a long period of time is detrimental. Monitoring mercury levels in drinking water, water bodies and other drinks are thus necessary. A method which can detect mercury level instantly from the sample would be ideal, but traditional methods are really complex and time consuming, they also require bulky and costly apparatus and in some cases requires really high temperature up to 10000K. Recently ion-selective electrodes have been quite popular due to their portability and selectivity also they are economical than the traditional methods. In our research, we utilize an AlGaN/GaN high electron mobility transistor by functionalizing the gate area with a polyvinyl chloride (PVC) based ion selective membrane. The functionalized HEMT device is applied with pulsed gate bias at reference gate electrode positioned at fixed distance from the membrane functionalized channel region. The gate bias drops across the test solution, influencing the membrane on the channel where specific ionophore captures the target heavy metal ion. Thus the liquid gated HEMT’s drain current response is a function of the concentration of the target heavy metal ion. The unique sensing structure design enables direct detection of target ions in a background matrix of high ionic concentration solution. Ionophores are added to the membrane which increases the affinity to the particular ion and promises high specificity and low affinity for other ions. The sensing technique enables direct detection of target ions in the medium using GaN HEMT and innovative biosensor packaging methodology, a portable biosensor system has been designed and fabricated which can provide quantitative detection in 10 minutes with a 10 µl sample. The sensor showed a rapid and stable response when introduced to solutions of varying Hg2+ concentrations. An interesting thing about the technique is that that the pH of the sample is between 6-7, which helps in real time testing and have a fast response time. ISFET sensors are portable and robust, they also show better selectivity and sensitivity when compared to traditional electrochemical ion selective electrodes and for AlGaN/GaN devices with other forms of functionalization. These devices have much greater possibilities for development and integration as they don't require a reference electrode which reduces the manufacturing costs of ISFET. The development of this sensor will help in providing real time monitoring of water quality in rivers or water bodies, tap water and even beverages which will be a great boon to humankind. Figure 1

Trastuzumab (T) detection in serum using signal amplification strategy for non-faradaic impedimetric sensing quartz crystal microbalance (QCM) based on peptide piezo-immunosensors and its role in breast cancer treatment.

e12502 Background: Her2Neu (H) antigen, expressed on 20% of Breast cancers, is an established target for antibody therapy with T. Immunohistochemistry is still the most widely used technique to detect h level which is time consuming and does not reveal any details of interaction between the molecules. We have developed a new innovative biosensor based novel technique to study real time interaction of h antigens with T using QCM Piezo-immunosensor. This quantitative label free peptide based assay can be used to characterize cell surface antigen, to study antigen- antibody interactions and obtain understanding of mechanisms of resistance. Methods: A label free and reagent free peptide mimotope capacitive biosensor is developed for T quantification based on non-Faradaic readout. The low sensitivity issue of capacitive biosensor was overcome with two innovations: peptide mimotope mixed SAM biointerface and dilution of the testing buffer. Signal amplification was achieved through dilution of the PBS buffer to tune Cdl to dominate the overall capacitance change upon target binding. After 1000 times dilution, limit of detection is lowered 500 times (0.22 µg/mL) and the sensitivity increased 20 times (0.04192 (µg/mL)-1). Results: Binding was very specific. Signal amplification strategy is practical. Further applied to planar electrode for optimizing sensing, response time in less than 1 minute. Conclusions: This is the first report of T detection using electrochemical method based on non-Faradaic impedance. h antigen density and interactions of antigens will help physicians to determine the clinical efficacy and resistance mechanisms to targeted antibodies like T and ado-Trastuzumab.For the first time, we have established a low cost, highly sensitive, fast, synthetic, QCM assay which could be used as a basis for developing a new generation of affinity-based Immunosensor assays. This real time capability and its simplicity of operation are highly suitable for multipurpose studies on living cells including cell immobilization, cytotoxicity of drugs, and the cell action mechanisms

Blood Based Biomarker Detection Using FET Biosensor: Towards Self-Health Management

Diseases that are the leading causes of mortality worldwide such as cardiovascular diseases (CVD), cancer and infectious diseases can be better managed and cured with early and prompt diagnosis and prognosis. Molecular biomarkers in whole blood (blood serum proteins) have predictive, diagnostic and prognostic capacity and semiconductor based electronic biosensors can be used to deliver reliable and affordable point-of-care disease monitoring. GaN High Electron Mobility Transistor (HEMT) based sensors have previously been used in a variety of applications such as ion, gas and biomolecule sensing. However, the limitations of FET biosensors set by the charge screening effect in high ionic strength solutions required additional sample processing steps, making them practically unusable in terms of point-of-care testing. With a novel biosensing technique enabling direct detection of target proteins in high salt concentration medium using GaN HEMT and innovative biosensor packaging methodology, a portable biosensor system has been designed and fabricated which can provide quantitative blood biomarker analysis in 5 minutes with less than 5 µl sample. Antibody or aptamer functionalized reference gate electrode and transistor channel form an electrical double layer (EDL) FET structure that can detect binding of target proteins with very high sensitivity. The sensing and packaging techniques have been specifically optimized to meet the requirements and convenience of a mobile diagnostic system that can be used by anyone, at all times. HEMT chip is fabricated in a 4-5 mask process: mesa isolation, ohmic metal deposition and annealing, interconnect metal deposition and device passivation. A simple and robust two step epoxy based planar HEMT chip packaging allows for easy integration with commercially available reader sockets, much like the micro-SD card. A single step surface functionalization strategy is followed for immobilizing receptors which allows for high sensitivity with a wide dynamic range of detection. Pulsed gate voltage modulates the channel conductivity which behaves as a function of the charge distribution within the EDL structure. Thus, without complex sample pre-treatments and extremely low sampling requirements, quantitative detection of protein biomarkers can be achieved. The structural design of EDL FET brings the potential to investigate the binding events that were previously undetectable in traditional FET based biosensors. Results from testing purified proteins and clinical serum samples elucidate the potential of the sensing technique to overcome the charge screening effect and offer sensing beyond Debye length in physiological salt environment, with high sensitivity and ultra-low detection limit. This diagnostic technique can be applied to all types of FET based sensors that uses immunological reactions to detect the target. Owing to standard semiconductor fabrication processes and minimal or no sampling requirements, the portable sensing system can be affordable and accessible to all. Enabled with cloud management, this diagnostic system houses the potential to be a pioneering IoT biosensor. Figure 1

Bio-electrolytic sensor for rapid monitoring of volatile fatty acids in anaerobic digestion process

This study presents an innovative biosensor that was developed on the basis of a microbial electrolysis cell for fast and reliable measurement of volatile fatty acids (VFA) during anaerobic digestion (AD) process. The bio-electrolytic sensor was first tested with synthetic wastewater containing varying concentrations of VFA. A linear correlation (R2 = 0.99) between current densities (0.03 ± 0.01 to 2.43 ± 0.12 A/m2) and VFA concentrations (5–100 mM) was found. The sensor performance was then investigated under different affecting parameters such as the external voltage, VFA composition ratio, and ionic strength. Linear relationship between the current density and VFA concentrations was always observed. Furthermore, the bio-electrolytic sensor proved ability to handle interruptions such as the presence of complex organic matter, anode exposure to oxygen and low pH. Finally, the sensor was applied to monitor VFA concentrations in a lab-scale AD reactor for a month. The VFA measurements from the sensor correlated well with those from GC analysis which proved the accuracy of the system. Since hydrogen was produced in the cathode as byproduct during monitoring, the system could be energy self-sufficient. Considering the high accuracy, short response time, long-term stability and additional benefit of H2 production, this bio-electrolytic sensor could be a simple and cost-effective method for VFA monitoring during AD and other anaerobic processes.

Use of Piezo-Immunosensors in Detecting Antigens and Antibodies Using Quartz Crystal Microbalance (QCM): a Novel Technique for Characterization, Understanding Interactions and Mechanisms of Action and Resistance of Monoclonal Antibodies

Background: Antigens like CD20 antigen, are established targets for antibody therapy with monoclonal antibodies (mAb) like Rituximab. Mixed clinico-pathological responses to mAb have been reported either due to presence of antibodies, rapid clearance or low density of target receptor/antigens. This demands need for an assay to monitor serum mAb therapeutic levels to ensure appropriate dosage. Enzyme-linked immunosorbent assay (ELISA) is still the most widely used technique to detect mAb level in human serum which is expensive and time consuming. Understanding properties and interactions of antigens is quintessential for developing better targeted agents and overcoming resistance. Flow cytometry is still the most widely used technique to detect CD20 level in human serum which is expensive, time consuming and does not reveal any details of interaction between the molecules. We have developed a new innovative biosensor based novel technique to not only monitor levels but also study real time interaction of antigens with antibodies using QCM Piezo-immunosensor. This quantitative label free peptide based assay can be used to characterize cell surface antigen, to study antigen- antibody interactions and obtain understanding of mechanisms of resistance to therapy.Method:Mimotope was used as a substitute for the antigen like CD20 and HER2 receptor protein in QCM assays to detect mAb level. The validation samples were prepared from the standard T solution in 10% human serum at three concentrations (10, 20 and 40 ug/ml). The changes in frequencies (ΔF) of sera from 3 female patients were obtained by calculating the differences between frequency shifts in pre and post mAb infusion. mAb level was calculated by equation, (ΔF +1.0022) ÷ 0.9997 μg / ml. The real-time processes of attachment of Cells like Raji cells on the gold electrode and the subsequent binding of antibody like Rituximab to the cells were studied using QCM biosensor. The interaction between Antigen and Antibody led to the increased resonant frequency shifts (df0) in the studied antibody concentration range from 5 to 250 μg mL-1 . Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved very specific interaction between Rituximab and CD20 antigens on B cellsResults: Antigen and Antibody binding was very specific. This binding decreased the electrochemical activity and stability of the cells, supporting the cell lysis mechanisms of action of Rituximab. We showed that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface and the buffers used. QCM assay was capable of detecting mAb like Trastuzumab serum level as low as 0.038 nM (linear operating range of 0.038-0.859 nM). The time frame of assay was 20-30 minutes. These results were in concordance with previously published results using ELISA. We have shown a systematic approach for using QCM technique to quantify the apparent binding constant between antigen and antibody can reveal antigen density.Conclusion: We have established a low cost, highly sensitive, fast, synthetic peptide based QCM assay which could be used as a basis for developing a new generation of affinity-based Immunosensor assays to monitor mAb serum levels like Rituximab, Trastuzumab and other monoclonal antibodies, helping physicians to determine the clinical efficacy of these drugs and ensuring appropriate dosages. Moreover antigen density and interactions of antigens with respective monoclonal antibodies like CD20 with Rituximab will help physicians to determine the clinical efficacy and resistance mechanisms to targeted antibodies like Rituximab and Ofatumumab. This could be used as a basis for developing a new generation of affinity-based Immunosensor assays. Our study shows that peptide mimotopes have potential benefit in sensor applications as the peptide-peptide interactions in the peptide mimotopes could be manipulated by the addition of functional groups to the peptide to influence binding of the target protein as well as for surface immobilization. DisclosuresNo relevant conflicts of interest to declare.

8-hydroxy-2′-deoxyguanosine (8-OHdG) biomarker detection down to picoMolar level on a plastic antibody film

An innovative biosensor assembly relying on a simple and straightforward in-situ construction is presented to monitor urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) down to the pmol/L level. The sensing film of the biosensor consisted of a molecularly imprinted polymer (MIP) layer for 8-OHdG assembled on a gold electrode through electropolymerization of monomer combined with the template.The analytical features of the resulting biosensor were assessed by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). Some experimental parameters such as the initial concentration of the monomer and the ratio template-monomer were investigated and optimized in order to finely tune the performance of the MIP-based sensor. Under optimal conditions, the developed biosensor was able to rebind 8-OHdG with a linear response against EIS from 0.1 to 100pg/ml 3.5-3500 pM. The interference of coexisting species was tested, also with calibrations on urine samples, and good selectivity towards 8-OHdG was obtained.RAMAN spectroscopy, FTIR and SEM evaluations of the prepared films confirmed the formation of a polyphenol thin-film on the electrode surface. The presence and distribution of the imprinted cavities on the MIP layer was confirmed by confocal microscopy imaging of the film, after a post-treatment with Fluorescein Isothiocyanate (FITC) labeled 8-OHdG antibody.Overall, this label-free biosensor for urinary 8-OHdG detection constitutes a promising low-cost alternative to the conventional immunoassay approaches, due to its simplicity, stability, high sensitivity and selectivity for biological sample assays, opening new doors for other applications.

Characterization of self-assembled monolayers (SAM) of peptide mimotopes of CD20 antigen and their binding with rituximab (R) using Quartz Crystal Microbalance (QCM) Piezo-Immunosensors: A novel technique to study CD20 antigen interaction.

e14004 Background: CD20 antigen is an established target for antibody therapy like R. Understanding interactions of CD20 antigens is important for developing better agents and overcoming resistance. We have developed a new innovative biosensor based quantitative technique to study real time interaction of CD20 antigens with R using QCM Piezo-immunosensor, which can be used to characterize cell surface antigen, study interactions and understand mechanisms of resistance. Methods: Binding of R with SAM of peptide mimotopes of CD20 antigen was systematically characterized. Four peptide mimotopes of CD 20 antigen were selected from literature and redesigned to allow their SAM immobilizations on gold electrodes through a peptide linker with cysteine. The bindings of these peptides with R and control antibodies (Trastuzumab, Bevacizumab) were characterized by Quartz Crystal Microbalance (QCM). Among the four peptide mimotopes initially selected, the peptide designated as CN-14 was the most selective and sensitive for R binding, which was further characterized by ellipsometry, atomic force microscopy and QCM. Results: We have shown a systematic approach for using QCM technique to quantify the apparent binding of CD20 and R. As the GLU residue pKa of CN-14 increased, the affinity of the SAM to Rituximab increased while the selectivity decreased. This was attributed to the weakening of the salt bridge between the CN-14 ARG-10 and GLU-13 at higher pKa values for GLU-13. Conclusions: Interaction of CD20 antigens with antibodies will help physicians to determine the clinical efficacy and resistance mechanisms to antibodies like R. QCM is low cost, highly sensitive, fast, synthetic, assay which could be used as a basis for developing a new generation of affinity-based Immunosensor assays. Our study shows that peptide mimotopes have potential benefit in sensor applications as the peptide-peptide interactions in the SAM could be manipulated by the addition of functional groups to the peptide to influence binding of the target protein as well as for surface immobilization via SAM.

Screening and Identification of DNA Aptamers to Tyramine Using in Vitro Selection and High-Throughput Sequencing.

Aptamers are synthetic single-stranded DNA or RNA sequences that can fold into tertiary structures allowing them to interact with and bind to targets with high affinity and specificity. This paper describes the first selection and identification of DNA aptamers able to recognize the biogenic amine tyramine. To successfully isolate aptamers to this challenging small molecule target, the SELEX methodology was adapted by combining a systematic strategy to increase the selection stringency and monitor enrichment success. As the benefits of applying high-throughput sequencing (HTS) in SELEX experiments is becoming more clear, this method was employed in combination with bioinformatics analysis to evaluate the utility of the selection strategy and to uncover new potential high affinity sequences. On the basis of the presence of consensus regions (sequence families) and family similarities (clusters), 15 putative aptamers to tyramine were identified. A recently described workflow approach to perform a primary screening and characterization of the aptamer candidates by microequilibrium dialysis and by microscale thermophoresis was next leveraged. These candidate aptamers exhibited dissociation constant (Kd) values in the range of 0.2-152 μM with aptamer Tyr_10 as the most promising one followed by aptamer Tyr_14. These aptamers could be used as promising molecular recognition tools for the development of inexpensive, robust and innovative biosensor platforms for the detection of tyramine in food and beverages.

Recent Advances in Biosensor Technology for Potential Applications - An Overview.

Imperative utilization of biosensors has acquired paramount importance in the field of drug discovery, biomedicine, food safety standards, defense, security, and environmental monitoring. This has led to the invention of precise and powerful analytical tools using biological sensing element as biosensor. Glucometers utilizing the strategy of electrochemical detection of oxygen or hydrogen peroxide using immobilized glucose oxidase electrode seeded the discovery of biosensors. Recent advances in biological techniques and instrumentation involving fluorescence tag to nanomaterials have increased the sensitive limit of biosensors. Use of aptamers or nucleotides, affibodies, peptide arrays, and molecule imprinted polymers provide tools to develop innovative biosensors over classical methods. Integrated approaches provided a better perspective for developing specific and sensitive biosensors with high regenerative potentials. Various biosensors ranging from nanomaterials, polymers to microbes have wider potential applications. It is quite important to integrate multifaceted approaches to design biosensors that have the potential for diverse usage. In light of this, this review provides an overview of different types of biosensors being used ranging from electrochemical, fluorescence tagged, nanomaterials, silica or quartz, and microbes for various biomedical and environmental applications with future outlook of biosensor technology.

A multipurpose biochip for food pathogen detection

We realized an innovative biosensing platform with high sensitivity, low-cost and label-free features forS. aureusandL. monocytogenesdetection from meat.