Electrochemical activation of engineered protein switches.

Engineered protein switches have a large dynamic range, high specificity for the activating ligand, and a modular architecture, and have been explored for a wide range of applications including biosensors and therapeutics. The ability to externally control switch function is important in extending applications for protein switches. We recently demonstrated that the on/off state could be controlled by the redox state of disulfide bonds introduced into the switches at select locations. Here, we demonstrate that an electrochemical signal can be used as an exogenous input to control switch function via reduction of the engineered disulfide bonds. This study suggests that disulfide-containing protein switch is a potentially useful platform for bioelectronic sensors with remote control of the sensing ability.

Ultra-sensitive and rapid detection of nucleic acids and microorganisms in body fluids using single-molecule tethering.

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL-1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT's clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.

Semiconductor quantum dots for bioanalysis.

Semiconductor nanoparticles, or quantum dots (QDs), have unique photophysical properties, such as size-controlled fluorescence, have high fluorescence quantum yields, and stability against photobleaching. These properties enable the use of QDs as optical labels for the multiplexed analysis of immunocomplexes or DNA hybridization processes. Semiconductor QDs are also used to probe biocatalytic transformations. The time-dependent replication or telomerization of nucleic acids, the oxidation of phenol derivatives by tyrosinase, or the hydrolytic cleavage of peptides by proteases are probed by using fluorescence resonance energy transfer or photoinduced electron transfer. The photoexcitation of QD-biomolecule hybrids associated with electrodes enables the photoelectrochemical transduction of biorecognition events or biocatalytic transformations. Examples are the generation of photocurrents by duplex DNA assemblies bridging CdS NPs to electrodes, and by the formation of photocurrents as a result of biocatalyzed transformations. Semiconductor nanoparticles are also used as labels for the electrochemical detection of DNA or proteins: Semiconductor NPs functionalized with nucleic acids or proteins bind to biorecognition complexes, and the subsequent dissolution of the NPs allows the voltammetric detection of the related ions, and the tracing of the recognition events.

Electronic aptamer-based sensors.

The selection of aptamers-nucleic acids that specifically bind low-molecular-weight substrates or proteins-by the SELEX (systematic evolution of ligands by exponential enrichment) procedure has attracted recent efforts directed to the development of new specific recognition units. In particular, extensive activities have been directed to the application of aptamers as versatile materials for the design of biosensors. The Minireview summarizes the recent accomplishments in developing electronic aptamer-based sensors (aptasensors), which include electrochemical, field-effect transistor, and microgravimetric quartz crystal microbalance sensors, and describes methods to develop amplified aptasensor devices and label-free aptasensors.

Controlling the direction of photocurrents by means of CdS nanoparticles and cytochrome c-mediated biocatalytic cascades.

Cathodic or anodic photocurrents are generated by a monolayer of CdS nanoparticles in the presence of the oxidized or reduced states of cytochrome c, respectively, and the photocurrents are amplified by enzyme-generated biocatalytic cascades mediated by cytochrome c.

Protein imprinting in polyacrylamide-based gels.

Protein imprinting in hydrogels is a method to produce materials capable of selective recognition and capture of a target protein. Here we report on the imprinting of fluorescently-labeled maltose binding protein (MBP) in acrylamide (AAm)/N-isopropylacrylamide (NIPAm) hydrogels. The targeting efficiency and selectivity of protein recognition is usually characterized by the imprinting factor, which in the simplest case is the ratio of protein uptake in an imprinted film divided by the uptake by the corresponding non-imprinted film. Our objective in this work is to study the dynamics of protein binding and elution in imprinted and non-imprinted films to elucidate the processes that control protein recognition. Protein elution from imprinted and non-imprinted films suggests that imprinting results in sites with a distribution of binding energies, and that only a relatively small fraction of these sites exhibit strong binding.

Influence of basement membrane proteins and endothelial cell-derived factors on the morphology of human fetal-derived astrocytes in 2D.

Astrocytes are the most prevalent type of glial cell in the brain, participating in a variety of diverse functions from regulating cerebral blood flow to controlling synapse formation. Astrocytes and astrocyte-conditioned media are widely used in models of the blood-brain barrier (BBB), however, very little is known about astrocyte culture in 2D. To test the hypothesis that surface coating and soluble factors influence astrocyte morphology in 2D, we quantitatively analyzed the morphology of human fetal derived astrocytes on glass, matrigel, fibronectin, collagen IV, and collagen I, and after the addition soluble factors including platelet-derived growth factor (PDGF), laminin, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF). Matrigel surface coatings, as well as addition of leukemia inhibitory factor (LIF) to the media, were found to have the strongest effects on 2D astrocyte morphology, and may be important in improving existing BBB models. In addition, the novel set of quantitative parameters proposed in this paper provide a test for determining the influence of compounds on astrocyte morphology, both to screen for new endothelial cell-secreted factors that influence astrocytes, and to determine in a high-throughput way which factors are important for translation to more complex, 3D BBB models.

Surface-tethered protein switches.

Protein switches are engineered fusion proteins with an input domain that recognizes and responds to an input signal and an output domain whose function is regulated by the state of the input domain. Here we demonstrate a fully functional surface tethered protein switch that offers a potential route to a universal biosensing platform.

Photoelectrochemical and optical applications of semiconductor quantum dots for bioanalysis.

Semiconductor nanoparticles (NPs) or quantum dots (QDs) exhibit unique photophysical properties reflected by size-controlled fluorescence, high fluorescence quantum yields, and stability against photobleaching. These properties are utilized by applying the QDs as optical labels for the multiplexed analysis of immunocomplexes and DNA hybridization. Also, semiconductor QDs are used to probe biocatalytic transformations. The time-dependent replication or telomerization of nucleic acids, the oxidation of phenol derivatives by tyrosinase, and the hydrolytic cleavage of peptides by proteases are probed by using fluorescence resonance energy transfer or photoinduced electron transfer. The photoexcitation of semiconductor NP-biomolecule hybrids associated with electrodes enables the photoelectrochemical transduction of biorecognition events or biocatalytic transformations. This is exemplified with the generation of photocurrents by duplex DNA assemblies bridging CdS NPs to electrodes, and by the formation of photocurrents as a result of biocatalyzed transformations, or redox protein-mediated electron transfer in the presence of the NPs.

DNAzymes for sensing, nanobiotechnology and logic gate applications.

Catalytic nucleic acids (DNAzymes or ribozymes) are selected by the systematic evolution of ligands by exponential enrichment process (SELEX). The catalytic functions of DNAzymes or ribozymes allow their use as amplifying labels for the development of optical or electronic sensors. The use of catalytic nucleic acids for amplified biosensing was accomplished by designing aptamer-DNAzyme conjugates that combine recognition units and amplifying readout units as in integrated biosensing materials. Alternatively, "DNA machines" that activate enzyme cascades and yield DNAzymes were tailored, and the systems led to the ultrasensitive detection of DNA. DNAzymes are also used as active components for constructing nanostructures such as aggregated nanoparticles and for the activation of logic gate operations that perform computing.

Analysis of dopamine and tyrosinase activity on ion-sensitive field-effect transistor (ISFET) devices.

Dopamine (1) and tyrosinase (TR) activities were analyzed by using chemically modified ion-sensitive field-effect transistor (ISFET) devices. In one configuration, a phenylboronic acid functionalized ISFET was used to analyze 1 or TR. The formation of the boronate-1 complex on the surface of the gate altered the electrical potential associated with the gate, and thus enabled 1 to be analyzed with a detection limit of 7x10(-5) M. Similarly, the TR-induced formation of 1, and its association with the boronic acid ligand allowed a quantitative assay of TR to be performed. In another configuration, the surface of the ISFET gate was modified with tyramine or 1 to form functional surfaces for analyzing TR activities. The TR-induced oxidation of the tyramine- or 1-functionalized ISFETs resulted in the formation of the redox-active dopaquinone units. The control of the gate potential by the redox-active dopaquinone units allowed a quantitative assay of TR to be performed. The dopaquinone-functionalized ISFETs could be regenerated to give the 1-modified sensing devices by treatment with ascorbic acid.

Label-free and reagentless aptamer-based sensors for small molecules.

A label free, reagentless aptasensor for adenosine is developed on an ISFET device. The separation of an aptamer/nucleic acid duplex by adenosine leads to the aptamer/adenosine complex that alters the gate potential of the ISFET. The sensitivity limit of the device is 5 x 10-5 M. Also, the immobilization of the aptamer/nucleic acid duplex on an Au-electrode and the separation of the duplex by adenosine mono-phosphate (AMP) enable the electrochemical detection of adenosine by faradaic impedance spectroscopy. The separation of the aptamer/nucleic acid duplex by adenosine and the formation of the aptamer/adenosine complex results in a decrease in the interfacial electron-transfer resistance in the presence of [Fe(CN)6]3-/4- as redox active substrate.

Dopamine-, L-DOPA-, adrenaline-, and noradrenaline-induced growth of Au nanoparticles: assays for the detection of neurotransmitters and of tyrosinase activity.

The neurotransmitters dopamine (1), L-DOPA (2), adrenaline (3), and noradrenaline (4) mediate the generation and growth of Au nanoparticles (Au-NPs). The plasmon absorbance of the Au-NPs allows the quantitative colorimetric detection of the neurotransmitters. Neurotransmitters 1, 2, and 4 are sensed with a detection limit of 2.5 x 10(-6) M, whereas the detection limit for analyzing 3 corresponds to 2 x 10(-5) M. The neurotransmitter-mediated growth of the Au-NPs is also used to probe the activity of tyrosinase. The later biocatalyst oxidizes tyrosine to L-DOPA that mediates the growth of the Au-NPs. The analysis of tyrosinase activity is important for detecting melanoma cells and Parkinson disease.

Biocatalytic growth of Au nanoparticles: from mechanistic aspects to biosensors design.

The H(2)O(2)-mediated enlargement of Au nanoparticles (NPs) and the growth mechanism are described. In addition to the deposition of gold on the NP faces, the formation of nanocrystalline clusters at the intersection of the faces is observed. The detachment of the latter nanoclusters provides additional seeds for the deposition of gold. The biocatalyzed generation of H(2)O(2) in the presence of O(2)/glucose and glucose oxidase enabled the development of an optical biosensor for glucose.

Reconstitution of apo-glucose dehydrogenase on pyrroloquinoline quinone-functionalized au nanoparticles yields an electrically contacted biocatalyst.

An electrically contacted glucose dehydrogenase (GDH) enzyme electrode is fabricated by the reconstitution of the apo-GDH on pyrroloquinoline quinone (PQQ)-functionalized Au nanoparticles (Au-NPs), 1.4 nm, associated with a Au electrode. The Au-NPs functionalized with a single amine group were attached to the Au surface by 1,4-benzenedithiol bridges, and PQQ was covalently linked to the Au-NPs. The apo-GDH was then reconstituted on the PQQ cofactor sites. The surface coverage of GDH corresponded to 1.4 x 10(-12) mol cm(-2). The reconstituted enzyme revealed direct electrical contact with the electrode surface, and the bioelectrocatalytic oxidation of glucose occurred with a turnover number of 11,800 s(-1). In contrast, a system that included the covalent attachment of GDH to the PQQ-Au-NPs monolayer in a random, nonaligned, configuration revealed lack of electrical communication between the enzyme and the electrode, albeit the enzyme existed in a bioactive structure. The bioelectrocatalytic function of the later system was, however, activated by the diffusional electron mediator 2,6-dichlorophenol-indophenol. The results imply that the alignment of GDH on a Au-NP through the reconstitution process leads to an electrically contacted enzyme-electrode, where the Au-NP acts as a charge-transfer mediator.

Electrical contacting of flavoenzymes and NAD(P)+-dependent enzymes by reconstitution and affinity interactions on phenylboronic acid monolayers associated with Au-electrodes.

The preparation of integrated, electrically contacted, flavoenzyme and NAD(P)(+)-dependent enzyme-electrodes is described. The reconstitution of apo-glucose oxidase, apo-GOx, on a FAD cofactor linked to a pyrroloquinoline quinone (PQQ) phenylboronic acid monolayer yields an electrically contacted enzyme monolayer (surface coverage 2.1 x 10(-)(12) mol cm(-)(2)) exhibiting a turnover rate of 700 s(-)(1) (at 22 +/- 2 degrees C). The system is characterized by microgravimetric quartz-crystal microbalance analyses, Faradaic impedance spectroscopy, rotating disk electrode experiments, and cyclic voltammetry. The performance of the enzyme-electrode for glucose sensing is described. Similarly, the electrically contacted enzyme-electrodes of NAD(P)(+)-dependent enzymes malate dehydrogenase, MalD, and lactate dehydrogenase, LDH, are prepared by the cross-linking of affinity complexes generated between the enzymes and the NADP(+) and NAD(+) cofactors linked to a pyrroloquinoline quinone phenylboronic acid monolayer, respectively. The MalD enzyme-electrode (surface coverage 1.2 x 10(-)(12) mol cm(-)(2)) exhibits a turnover rate of 190 s(-)(1), whereas the LDH enzyme-electrode (surface coverage 7.0 x 10(-)(12) mol cm(-)(2)) reveals a turnover rate of 2.5 s(-)(1). Chronoamperometric experiments reveal that the NAD(+) cofactor is linked to the PQQ-phenylboronic acid by two different binding modes. The integration of the LDH with the two NAD(+) cofactor configurations yields enzyme assemblies differing by 1 order of magnitude in their bioelectrocatalytic activities.

Electrical contacting of glucose oxidase by surface-reconstitution of the apo-protein on a relay-boronic acid-FAD cofactor monolayer.

A new methodology for the surface-reconstitution of apo-flavoenzymes on a relay unit (pyrroloquinoline quinone, PQQ) functionalized with a boronic acid ligand as a linker to native FAD was developed. The reconstitution of apo-glucose oxidase (apo-GOx) on the PQQ-FAD monolayer yields an electrically contacted enzyme-electrode.

Probing antigen-antibody binding processes by impedance measurements on ion-sensitive field-effect transistor devices and complementary surface plasmon resonance analyses: development of cholera toxin sensors.

Impedance measurements on ISFET devices are employed to develop new immunosensors. The analysis of the transconductance curves recorded at variable frequencies, upon the formation of antigen-antibody complexes on the ISFET devices, allows determination of the biomaterial film thicknesses. Complementary surface plasmon resonance measurements of analogous biosensor systems, using Au-coated glass slides as support, reveal similar film thicknesses of the biomaterials and comparable detection limits. A dinitrophenyl antigen layer is immobilized on the ISFET gate as a sensing interface for the anti-dinitrophenyl antibody (anti-DNP-Ab). The anti-DNP-Ab is analyzed with a sensitivity that corresponds to 0.1 microg mL(-1). The assembly of the biotinylated anti-anti-DNP-Ab and avidin layers on the base anti-DNP-Ab layer is characterized by impedance measurements. The development of an ISFET-based sensor for the cholera toxin is described. The anti-cholera toxin antibody is immobilized on the ISFET device. The association of the cholera toxin (CT) to the antibody is monitored by the impedance measurements. The detection limit for analyzing CT is 1.0 x 10(-11) M.

Probing photoelectrochemical processes in Au-CdS nanoparticle arrays by surface plasmon resonance: application for the detection of acetylcholine esterase inhibitors.

The photoelectrochemical charging of Au-nanoparticles (NP) in a Au-nanoparticle/CdS-nanoparticle array assembled on a Au-coated glass surface is followed by means of surface plasmon resonance (SPR) spectroscopy upon continuous irradiation of the sample. The charging of the Au-NPs results in the enhanced coupling between the localized surface plasmon of the Au-NP and the surface plasmon of the bulk surface, leading to a shift in the plasmon angle. The charging effect of the Au-NPs is supported by concomitant electrochemical experiments in the dark. Analysis of the results indicates that ca. 4.2 electrons are associated with each Au-nanoparticle under steady-state irradiation. The photoelectrochemical charging effect of the Au-NPs in the Au-CdS NP array is employed to develop a SPR sensor for acetylcholine esterase inhibitors.