Practical biophysics: Sensors for rapid detection of biological targets utilizing target-induced oligonucleotide annealing

Detection and quantitation of biomolecules is one of the most commonly performed measurements in biomedical research and clinical diagnostics. There is high demand for convenient, rapid and sensitive biomolecule detection methodologies. In this review we discuss a family of sensors that have been developed in our laboratory that share a common simple biophysical mechanism of action and that are capable of rapid detection of a diverse range of biological targets. The sensors generate fluorescence signal in the presence of the target molecule through target-induced association of short fluorochrome-labeled complementary oligonucleotides that are attached to target recognition elements of the sensors (antibodies, aptamers, etc.) via nanometer scale flexible linkers. This sensor design can be used for detecting proteins, antibodies, nucleic acids and whole cells. The assays using these sensors require only adding a sample to the sensor mix followed by simple fluorescence intensity readout. The simplicity, the speed of detection and the potential for miniaturization are the main assets of these sensors.     

A flow-through amperometric sensor based on dialysis tubing and free enzyme reactors.

A generic flow-through amperometric microenzyme sensor is described, which is based on semi-permeable dialysis tubing carrying the sample to be analyzed. This tubing (300 microm OD) is led through a small cavity, containing the working and reference electrode. By filling this cavity with a few microl of an appropriate enzyme solution, an amperometric enzyme sensor results. As the dialysis tubing is impermeable for large molecular species such as enzymes, this approach does not require any immobilization chemistry, and as a consequence the enzyme is present in its natural free form. Based on this principle, amperometric sensors for lactate, glucose, and glutamate were formed by filling cavities, precision machined in Perspex, with buffered solutions containing respectively, lactate-, glucose-, and glutamate-oxidase. All sensors showed a large linear range (0-35 mM for glucose, 0-3 mM for lactate, and 0-5 mM for glutamate) covering the complete physiological range. The lower detection limit was in the order of 15-50 microM. Applicability in flow injection analysis systems is demonstrated.     

Non-invasive self-referencing electrochemical sensors for quantifying real-time biofilm analyte flux

Current techniques for characterizing biofilm physiology lack the signal filtering capability required for quantifying signals associated with real time biologically active transport. Though a great deal was learned from previous investigations, no results have been reported on the characterization of in vivo, real time biofilm flux using non-invasive (non-destructive) techniques. This article introduces the self-referencing technique for applications in biofilm physiology. Self-referencing is a non-invasive sensing modality which is capable of sensing changes in biologically active analyte flux as small as 10 fmol cm(-2) s(-1). Studies directly characterizing flux, as opposed to concentration, have the advantage of quantifying real time changes in biologically active transport which are otherwise lost to background noise. The use of this modality for characterizing biofilm physiology is validated with a reversible enzyme inhibition study. The experiment used self-referencing potentiometric sensors for quantifying real time ammonium and nitrite flux. Amperometric and optical sensing methods, though not presented herein, are also powerful sensing tools which benefit from operation in self-referencing mode. Reversible ammonia monooxygenase inhibition by a copper chelator (thiourea), and subsequent relief by excess copper addition was successfully demonstrated using self-referencing ion-selective microelectrodes for a mature Nitrosomonas europaea biofilm.     

Bimetallic PtM (M=Pd, Ir) nanoparticle decorated multi-walled carbon nanotube enzyme-free, mediator-less amperometric sensor for H₂O₂

A new highly catalytic and intensely sensitive amperometric sensor based on PtM (where M=Pd, Ir) bimetallic nanoparticles (NPs) for the rapid and accurate estimation of hydrogen peroxide (H(2)O(2)) by electrooxidation in physiological conditions is reported. PtPd and PtIr NPs-decorated multiwalled carbon nanotube nanocatalysts (PtM/MWCNTs) were prepared by a modified Watanabe method, and were characterized by XRD, TEM, ICP, and XAS. The sensors were constructed by immobilizing PtM/MWCNTs nanocatalysts in a Nafion film on a glassy carbon electrode. Both PtPd/MWCNTs and PtIr/MWCNTs assemblies catalyzed the electrochemical oxidation of H(2)O(2). Cyclic voltammetry characterization measurements revealed that both the PtM (M=Pd, Ir)/MWCNTs/GCE possessed similar electrochemical surface areas (～0.55 cm(2)), and electron transfer rate constants (～1.23 × 10(-3)cms(-1)); however, the PtPd sensor showed a better performance in H(2)O(2) sensing than did the PtIr counterpart. Explanations were sought from XAS measurements to explain the reasons for differences in sensor activity. When applied to the electrochemical detection of H(2)O(2), the PtPd/MWCNTs/GC electrode exhibited a low detection limit of 1.2 μM with a wide linear range of 2.5-125 μM (R(2)=0.9996). A low working potential (0V (SCE)), fast amperometric response (<5s), and high sensitivity (414.8 μA mM(-1)cm(-2)) were achieved at the PtPd/MWCNTs/GC electrode. In addition, the PtPd/MWCNTs nanocatalyst sensor electrode also exhibited excellent reproducibility and stability. Along with these attractive features, the sensor electrode also displayed very high specificity to H(2)O(2) with complete elimination of interference from UA, AA, AAP and glucose.     

Designs,applications, and limitations of genetically encoded fluorescent sensors to explore plant biology

The understanding of signaling and metabolic processes in multicellular organisms requires knowledge of the spatial dynamics of small molecules and the activities of enzymes, transporters, and other proteins in vivo, as well as biophysical parameters inside cells and across tissues. The cellular distribution of receptors, ligands, and activation state must be integrated with information about the cellular distribution of metabolites in relation to metabolic fluxes and signaling dynamics in order to achieve the promise of in vivo biochemistry. Genetically encoded sensors are engineered fluorescent proteins that have been developed for a wide range of small molecules, such as ions and metabolites, or to report biophysical processes, such as transmembrane voltage or tension. First steps have been taken to monitor the activity of transporters in vivo. Advancements in imaging technologies and specimen handling and stimulation have enabled researchers in plant sciences to implement sensor technologies in intact plants. Here, we provide a brief history of the development of genetically encoded sensors and an overview of the types of sensors available for quantifying and visualizing ion and metabolite distribution and dynamics. We further discuss the pros and cons of specific sensor designs, imaging systems, and sample manipulations, provide advice on the choice of technology, and give an outlook into future developments.

Different types of genetically encoded sensors in plants can be used to quantify and visualize ion and metabolite distributions and dynamics.     

Combined physico-chemical and biological sensing in environmental monitoring

Single-use conductivity and microbial sensors were used to investigate the effect of both species (chloride, nitrate, and sulphate) and concentration/osmolarity of anions on the metabolic activity of Escherichia coli. A new disposable, single-use conductivity sensor is described which is compatible with the CellSense mediated amperometric biosensor system. The effect of changing salinity and nitrate concentration on the response of E. coli to 3,5-dichlorophenol and mercuric chloride was determined. The implications for toxicity assessment of a hybrid sensing system, allowing the simultaneous monitoring of physico-chemical and biological data, are discussed.     

An electrochemical sensor array system for the direct, simultaneous in vitro monitoring of nitric oxide and superoxide production by cultured cells

A new approach for an amperometric array sensor platform employing arrays of sensors in a 24-well cell culture plate format has been developed for simultaneous in vitro determination of nitric oxide (NO) and superoxide free radicals (O(2)(-)) produced by stimulated cells. The work reported focuses on the direct, real-time monitoring of extracellular production of these two analytes, as well as the effects of their interaction. The sensor platform was manufactured by a combination of sputtering gold electrodes and screen-printing carbon electrodes. The O(2)(-) sensor uses covalent immobilization of cytochrome c via a binder, DTSSP (3,3'-dithio-bis(sulphosuccinimidylpropionate) onto the surface of the Au electrodes, whereas the NO sensor system involves an NiTSPc (nickel tetrasulfonated phthalocyanine) film electrodeposited onto the surface of the carbon electrodes and subsequently covered with an external layer of Nafion. For in vitro demonstration of the platforms as a potential drug-screening system, A172 glioblastoma cells were cultured and transferred into the 24-well arrays. Simultaneous and direct monitoring of NO and O(2)(-) production as a response to chemicals of biomedical relevance was carried out. The results obtained demonstrated that it would be possible to envisage a drug screening platform for compounds designed to be inhibitors of nitric oxide synthase or to have an inhibitory effect on superoxide free radical production. By suitable modification of the electrodes employed it would also be possible to extend the platform to measure alternative species.     

A self referencing platinum nanoparticle decorated enzyme-based microbiosensor for real time measurement of physiological glucose transport

Glucose is the central molecule in many biochemical pathways, and numerous approaches have been developed for fabricating micro biosensors designed to measure glucose concentration in/near cells and/or tissues. An inherent problem for microsensors used in physiological studies is a low signal-to-noise ratio, which is further complicated by concentration drift due to the metabolic activity of cells. A microsensor technique designed to filter extraneous electrical noise and provide direct quantification of active membrane transport is known as self-referencing. Self-referencing involves oscillation of a single microsensor via computer-controlled stepper motors within a stable gradient formed near cells/tissues (i.e., within the concentration boundary layer). The non-invasive technique provides direct measurement of trans-membrane (or trans-tissue) analyte flux. A glucose micro biosensor was fabricated using deposition of nanomaterials (platinum black, multiwalled carbon nanotubes, Nafion) and glucose oxidase on a platinum/iridium microelectrode. The highly sensitive/selective biosensor was used in the self-referencing modality for cell/tissue physiological transport studies. Detailed analysis of signal drift/noise filtering via phase sensitive detection (including a post-measurement analytical technique) are provided. Using this highly sensitive technique, physiological glucose uptake is demonstrated in a wide range of metabolic and pharmacological studies. Use of this technique is demonstrated for cancer cell physiology, bioenergetics, diabetes, and microbial biofilm physiology. This robust and versatile biosensor technique will provide much insight into biological transport in biomedical, environmental, and agricultural research applications.     

Microscopy-based multicolor tissue cytometry at the single-cell level.

Cytomics is a novel perspective from which to look at life. As with genomics and proteomics before, this discipline requires novel and innovative techniques and technologies to focus on its substrate of research--the cytome. With cytomics being the discipline that analyzes cellular systems and their interdependencies, advanced microscopy represents a key technology in cytomics research. Yet, conventional microscopy-based investigations, i.e., "look and conclude" analyses, do not meet the major cytomics criteria of 1) relating multiple parameters to each other, 2) within large populations of cells, 3) on a single-cell basis, and 4) in a quantitative and observer-independent manner. However, emerging improvements in the fields of fluorophore technology, sensitive fluorescence detection devices, and sophisticated image analysis procedures, are important and necessary steps into the cytomics era. Tissue represents an important class of cytomes, hence tissue cytometry--on the single cell level--can be expected to become an important cytomics technology. In this report, the techniques and technologies of microscopy-based multicolor tissue cytometry (MMTC) are outlined and applications are discussed, including the phenotypic characterization of tissue infiltrating leukocytes, in situ quantification of proliferation markers and tumor suppressors, and in situ quantification of apoptosis.     

Low-potential sensitive H2O2 detection based on composite micro tubular Te adsorbed on platinum electrode

In this work a new original amperometric sensor for H(2)O(2) detection based on a Pt electrode modified with Te-microtubes was developed. Te-microtubes, synthesized by the simple thermal evaporation of Te powder, have a tubular structure with a hexagonal cross-section and are open ended. Modified electrode was prepared by direct drop casting of the mixture of Te-microtubes dispersed in ethanol on Pt surface. The spectroscopic characterization of synthesized Te-microtubes and Pt/Te-microtubes modified electrodes was performed by scanning electron microscopy (SEM), energy-dispersive X-rays microanalysis (EDX), X-ray diffraction analysis (XRD) and X-ray photoelectron spectroscopy (XPS). Moreover a complete electrochemical characterization of the new composite material Pt/Te-microtubes was performed by cyclic voltammetry (CV) and cronoamperometry (CA) in phosphate buffer solution (PBS) at pH 7. Electrochemical experiments showed that the presence of Te-microtubes on modified electrode was responsible for an increment of both cathodic and anodic currents in presence of H(2)O(2) with respect to bare Pt. Specifically, data collected from amperometric experiments at -150 mV vs. SCE in batch and -200 mV vs. SCE in flow injection analysis (FIA) experiments show a remarkable increment of the cathodic current. The electrochemical performances of tested sensors make them suitable for the quantitative determination of H(2)O(2) substrate both in batch and in FIA.     

Noninvasive measurement of hydrogen and potassium ion flux from single cells and epithelial structures

This review introduces newdevelopments in a technique for measuring the movement of ions acrossthe plasma membrane. With the use of a self-referencing ion-selective(Seris) probe, transport mechanisms can be studied on a variety ofpreparations ranging from tissues to single cells. In this paper weillustrate this versatility with examples from the vas deferens andinner ear epithelium to large and small single cells represented bymouse single-cell embryos and rat microglia. Potassium and hydrogen ionfluxes are studied and pharmacological manipulation of the signals arereported. The strengths of the self-referencing technique are reviewedwith regard to biological applications, and the expansion ofself-referencing probes to include electrochemical and enzyme-based sensors is discussed.

     

Intravascular glucose/lactate sensors prepared with nitric oxide releasing poly(lactide-co-glycolide)-based coatings for enhanced biocompatibility

Intravenous amperometric needle-type enzymatic glucose/lactate sensors intended for continuous monitoring are prepared with a novel nitric oxide (NO) releasing layer to improve device hemocompatibility. To create an underlying NO release coating, the sensors with immobilized enzymes (either glucose oxidase or lactate oxidase) are prepared with a thin layer of poly(lactide-co-glycolide) (PLGA) loaded with lipophilic diazeniumdiolate species that slowly release NO via a proton driven reaction. An outer thin layer (ca. 30 μm) of PurSil (polyurethane/dimethylsiloxane copolymer) limits the flux of glucose and lactate to the inner layer of enzyme, to provide the desired linear amperometric response. A 30 μm coating of PLGA containing 33 wt% of the appropriate NO donor (N-diazeniumdiolated dibutylhexanediamine, DBHD/N?O?) can release NO at a physiologically relevant rate > 1 × 10?1?mol min?1 cm?2 for at least 7 days without influencing the analytical performance of the glucose/lactate sensors. In vitro, the sensors exhibit relatively stable amperometric response over a one-week period with high selectivity over interferences (e.g., ascorbic acid) required for blood monitoring applications. Glucose sensors implanted in the veins of rabbits for 8h exhibit significantly enhanced hemocompatibility for the NO release sensors vs. corresponding controls (without NO release in same animals), with greatly reduced thrombus formation on their surfaces. Further, the analytical performance of the NO release glucose sensors are superior to controls placed in the veins of the same animals, with a greater accuracy in measuring blood glucose levels as evaluated using a Clarke error grid type analysis.     

The role of H2O2 outer diffusion on the performance of implantable glucose sensors

The performance of an implantable glucose sensor is strongly dependent on the ability of their outer membrane to govern the diffusion of the various participating species. In this contribution, using a series of layer-by-layer (LBL) assembled outer membranes, the role of outwards of H(2)O(2) diffusion through the outer membrane of glucose sensors has been correlated to sensor sensitivity. Glucose sensors with highly permeable humic acids/ferric cations (HAs/Fe(3+)) outer membranes displayed a combination of lower sensitivities and better linearities when compared with sensors coated with lesser permeable outer membranes (namely HAs/poly(diallyldimethylammonium chloride) (PDDA) and poly(styrene sulfonate) (PSS)/PDDA). On the basis of a comprehensive evaluation of the oxygen dependence of these sensors in conjunction with the permeability of H(2)O(2) through these membranes, it was concluded that the outer diffusion of H(2)O(2) is crucial to attain optimized sensor performance. This finding has important implications to the design of various bio-sensing elements employing perm-selective membranes.     

A three-enzyme microelectrode sensor for detecting purine release from central nervous system

As the purines, in particular adenosine, are important signaling agents in the nervous system we have devised a new biosensor for directly measuring their production in real time during physiological activity. Our amperometric adenosine biosensor is made by entrapping 3 enzymes (xanthine oxidase, purine nucleoside phosphorylase and adenosine deaminase) in a composite lactobionamide and amphiphillic polypyrrole matrix around a Pt microelectrode. The resulting sensors are small (25-100 microm diameter), fast responding (10-90% rise time, 2+/-0.23 s), sensitive (100-222 mA M(-1) cm(-2)) and stable (100% activity after 5 days). The sensor was used in vivo to demonstrate the spatial localization of release of adenosine from Xenopus embryo spinal cord during fictive swimming.     

Sensor and biosensor based on Prussian Blue modified gold and platinum screen printed electrodes

Gold (Au) and platinum (Pt) screen-printed electrodes were modified with Prussian Blue (PB) for the development of amperometric sensors selective for hydrogen peroxide detection. The sensors exhibited sensitivities towards H(2)O(2) equal to 2 A M(-1) cm(-2) for Au and 1 A M(-1) cm(-2) for Pt electrodes. The sensors were also employed as the basis for construction of glucose biosensors through further modification with crystallised glucose oxidase immobilised in a Nafion membrane. In order to improve the operational stability of the modified electrodes a buffer solution containing tetrabutylammonium toluene-4-sulfonate was used. The long-term performance of the sensors and biosensors were evaluated by continuous monitoring of hydrogen peroxide and glucose solutions (50 microM and 1 mM, respectively) in the flow-injection mode for 10 h.     

Cellular redox poise modulation; the role of coenzyme Q10, gene and metabolic regulation

In this communication, the concept is developed that coenzyme Q10 has a toti-potent role in the regulation of cellular metabolism. The redox function of coenzyme Q10 leads to a number of outcomes with major impacts on sub-cellular metabolism and gene regulation. Coenzyme Q10's regulatory activities are achieved in part, through the agency of its localization in the various sub-cellular membrane compartments. Its fluctuating redox poise within these membranes reflects the cell's metabolic micro-environments. As an integral part of this process, H2O2 is generated as a product of the normal electron transport systems to function as a mitogenic second messenger informing the nuclear and mitochondrial (chloroplast) genomes on a real-time basis of the status of the sub-cellular metabolic micro-environments and the needs of that cell. Coenzyme Q10 plays a major role both in energy conservation, and energy dissipation as a component of the uncoupler protein family. Coenzyme Q10 is both an anti-oxidant and a pro-oxidant and of the two the latter is proposed as its more important cellular function. Coenzyme Q10 has been reported, to be of therapeutic benefit in the treatment of a wide range of age related degenerative systemic diseases and mitochondrial disease. Our over-arching hypotheses on the central role played by coenzyme Q10 in redox poise changes, the generation of H2O2, consequent gene regulation and metabolic flux control may account for the wide ranging therapeutic benefits attributed to coenzyme Q10.     

Toward real-time continuous brain glucose and oxygen monitoring with a smart catheter

Oxygen and glucose biosensors have been designed, fabricated, characterized and optimized for real-time continuous monitoring on a new smart catheter for use in patients with traumatic brain injury (TBI). Oxygen sensors with three-electrode configuration were designed to achieve zero net oxygen consumption. Glucose sensors were based on the use of platinum nanoparticle-enhanced electrodes that were modified with polycation and glucose oxidase immobilized by chitosan matrix. An iridium oxide electrode was developed to work as a biocompatible reference electrode with enhanced durability and stability in the biological solutions. A study of the effect of temperature on oxygen sensor performance, and both temperature and oxygen effects on glucose sensor performance were accomplished to enhance their operative stability and provide useful information for in vivo applications. A new methodology for automatic correction of the temperature and oxygen dependence of biosensor outputs is demonstrated through programmed LabView™ software. In vitro experiments in both physiological and pathophysiological ranges (oxygen: 0–60 mmHg; glucose: 0.1–10 mM; temperature: 25–40 °C) with clinical samples of cerebrospinal fluid obtained from TBI patients have demonstrated stable measurements with enhanced accuracy, indicating the feasibility of the sensors for a real-time continuous in vivo monitoring.     

Monitoring blood coagulation with magnetoelastic sensors

The determination of blood coagulation time is an essential part of monitoring therapeutic anticoagulants. Standard methodologies for the measurement of blood clotting time require dedicated personnel and involve blood sampling procedures. A new method based on magnetoelastic sensors has been employed for the monitoring of blood coagulation. The ribbon-like magnetoelastic sensor oscillates at a fundamental frequency, which shifts linearly in response to applied mass loads or a fixed mass load of changing elasticity. The magnetoelastic sensors emit magnetic flux, which can be detected by a remotely located pick-up coil, so that no direct physical connections are required. During blood coagulation, the viscosity of blood changes due to the formation of a soft fibrin clot. In turn, this change in viscosity shifts the characteristic resonance frequency of the magnetoelastic sensor enabling real-time continuous monitoring of this biological event. By monitoring the signal output as a function of time, a distinct blood clotting profile can be seen. The relatively low cost of the magnetoelastic ribbons enables their use as disposable sensors. This, along with the reduced volume of blood required, make the magnetoelastic sensors well suited for at-home and point-of-care testing devices.