Signal Amplification Mechanism Research Articles

A molecular machine biosensor: Construction, predictive models and experimental studies

This paper describes the construction, operation and predictive modeling of a molecular machine, functioning as a high sensitivity biosensor. Embedded gramicidin A (gA) ionchannels in a self-assembled tethered lipid bilayer act as biological switches in response to target molecules and provide a signal amplification mechanism that results in high sensitivity molecular detection. The biosensor can be used as a rapid and sensitive point of care diagnostic device in different media such as human serum, plasma and whole blood without the need for pre and post processing steps required in an enzyme-linked immunosorbent assay. The electrical reader of the device provides the added advantage of objective measurement. Novel ideas in the construction of the molecular machine, including fabrication of biochip arrays, and experimental studies of its ability to detect analyte molecules over a wide range of concentrations are presented. Remarkably, despite the complexity of the device, it is shown that the response can be predicted by modeling the analyte fluid flow and surface chemical reactions. The derived predictive models for the sensing dynamics also facilitate determining important variables in the design of a molecular machine such as the ion channel lifetime and diffusion dynamics within the bilayer lipid membrane as well as the bio-molecular interaction rate constants.

STED Nanoscopy of Cardiac RyR2 Clusters and Sub-Structure Analysis After Myocardial Infarction

Cardiac ryanodine receptor (RyR2) Ca2+ release channels occur as multi-channel clusters at subcellular Ca2+ release sites in cardiomyocytes. RyR2 clusters are thought to underlie important signal amplification mechanisms including coupled gating of physically associated Ca2+ release channels. The nature of RyR2 channel organization in intact intracellular channel clusters is not known.We have investigated the nanostructure of RyR2 clusters using Stimulated Emission Depletion (STED) nanoscopy with ∼60 nm lateral resolution and indirect immunofluorescence in cardiomyocytes. We compared RyR2 clusters in adult cardiomyocytes from sham hearts with hearts 8 weeks after myocardial infarction (post-MI).Peripheral RyR2 clusters were detected at a density of 1.3 ± 0.2 clusters per μm2 in control cells (2237 clusters) and 1.6 ± 0.1 in diseased cells (1752 clusters). Individual clusters were analyzed by incrementally detecting signal edges which defined intra-cluster substructures and intensity peaks. Within individual clusters, nearest-neighbour distances between intensity peaks showed an asymmetric distribution with a maximum at 100 ± 1 nm in healthy cardiomyocytes. In post-MI cells this distribution showed a left shift with a maximum at 85 ± 3 nm (p < 0.05 Mann-Whitney test; mean distance sham 150.6 ±3.2 nm vs post-MI 129.5 ± 2.7 nm).In conclusion, immuno-labeled cardiac RyR2 clusters showed distinct non-randomly organized sub-structures as evidenced by characteristic intra-cluster peak-to-peak distances. Our data suggest that continuous RyR2 clusters contain substructures which occur repeatedly as densely organized groups of RyR2 channels. In addition, we showed altered RyR2 clusters after myocardial infarction with smaller intra-cluster peak-to-peak distances which implies tighter channel packing. We are preparing a working mathematical model of normal RyR2 cluster function and the role of potential substructures and their changes in heart disease.

Self-inducible secretion of glucagon-like peptide-1 (GLP-1) that allows MIN6 cells to maintain insulin secretion and insure cell survival

Based on the hypothesis that MIN6 cells could produce glucagon-like peptide-1 (GLP-1) to maintain cell survival, we analyzed the effects of GLP-1 receptor agonist, exendin-4 (Ex4), and antagonist, exendin-(9–39) (Ex9) on cell function and cell differentiation.MIN6 cells expressed proglucagon mRNAs and produced GLP-1, which was accelerated by Ex4 and suppressed by Ex9. Moreover, Ex4 further enhanced glucose-stimulated GLP-1 secretion, suggesting autocrine loop-contributed amplification of the GLP-1 signal. Ex4 up-regulated cell differentiation- and cell function-related CREBBP, Pdx-1, Pax6, proglucagon, and PC1/3 gene expressions. The confocal laser scanning images revealed that GLP-1 positive cells were dominant in the early stage of cells, but positive for insulin were more prominent in the mature stage of cells. Ex4 accelerated cell viability, while Ex9 and anti-GLP-1 receptor antibody enhanced cell apoptosis.MIN6 cells possess a mechanism of GLP-1 signal amplification in an autocrine fashion, by which the cells maintained insulin production and cell survival.

Fluorescent detection of singlet oxygen: Amplifying signal transduction and improving sensitivity based on intramolecular FRET of anthryl appended porphyrins

Two new anthryl-appended porphyrin dyads have been synthesized and used as highly selective and sensitive fluorescence probes for singlet oxygen (1O2). The design strategy for the probes is directed by the idea of intramolecular fluorescence resonance energy transfer (FRET) interactions and carried out by incorporation of an electron-rich fluorophore (porphyrin) with a reactive anthracene for 1O2. The molecular recognition is based on the specific interaction of 1O2 with the inner anthracene moiety, and the signal reporter for the recognition process is the porphyrin fluorescence. As a result of overlap of the emission band of the anthracene with the absorbance band of the porphyrin, intramolecular FRET occurs between the anthracene (donor) and the porphyrin (acceptor). The effective light absorbed by the porphyrin and, concomitantly, the emitted light intensity are thus modulated by the emission intensity of the anthracene. Upon reaction with reactive oxygen species such as hydrogen peroxide, hypochlorite, superoxide, hydroxyl radicals, and 1O2, the probes exhibit a selective response toward 1O2. In addition, significant amplification of the signal transducer is observed. The feasibility of the design was demonstrated by monitoring the 1O2 generated from a MoO42−/H2O2 system. The results clearly demonstrate that the synthesized probes exhibit both high selectivity and high sensitivity for 1O2. The fluorescence reaction and signal amplification mechanism of the system were both discussed, clearly confirming that the introduction of electron-rich porphyrin units into the 9,10-positions of anthracene can improve the response sensitivity and activate the probe’s reactivity toward 1O2.

Beneficial effect of chromium supplementation on glucose, HbA 1C and lipid variables in individuals with newly onset type-2 diabetes

Project Chromium is an essential nutrient involved in normal carbohydrate and lipid metabolism. It influences glucose metabolism by potentiating the action as taking part in insulin signal amplification mechanism. A placebo-controlled single blind, prospective study was carried out to investigate the effect of chromium supplementation on blood glucose, HbA 1C and lipid profile in newly onset patients with type-2 diabetes. Procedure Total 40 newly onset type-2 diabetics were selected and after 1 month stabilization further randomly divided into two groups viz. study group and placebo group. The study group received 9 g brewer's yeast (42 μg Cr) daily and the other placebo group received yeast devoid of chromium for 3 months. Subjects were instructed not to change their normal eating and living habits. Fasting blood glucose, HbA 1C and lipid profile were analyzed at beginning and completion of the study. Results Results revealed that fasting blood glucose level significantly reduced in the subjects consuming yeast supplemented with chromium (197.65 ± 6.68 to 103.68 ± 6.64 mg/dL; p < 0.001). HbA 1C values improved significantly from 9.51 ± 0.26% to 6.86 ± 0.28%; p < 0.001 indicating better glycaemic control. In experimental group total cholesterol, TG and LDL levels were also significantly reduced from 199.66 ± 3.11 to 189.26 ± 3.01 mg/dL; p < 0.02, 144.94 ± 8.31 to 126.01 ± 8.26; p < 0.05 and 119.19 ± 1.71 to 99.58 ± 1.10; p < 0.001 respectively. Conclusions These data demonstrate beneficial effect of chromium supplementation on glycaemic control and lipid variables in subjects with newly onset type-2 diabetes.

Two-Component Dendritic Chain Reactions: Experiment and Theory

New analytical diagnostic techniques that are based on signal-amplification mechanisms could significantly improve the sensitivity of detection of various analytes. We have developed a new approach to achieving exponential amplification of a diagnostic signal through a two-component dendritic chain reaction. The chain reaction generated the analyte of interest and thereby initiated additional diagnostic cycles. The system was designed for the detection of hydrogen peroxide and produced significantly larger intensity of diagnostic signal than a classic probe. In addition, a mathematical model that simulates the disassembly kinetics of one-component and two-component reactions was developed and shown to correlate well with the observed experimental data. The modularity and flexibility of a two-component detection system should allow extension to the detection of other analytes.

Calcium‐activated chloride currents in olfactory sensory neurons from mice lacking bestrophin‐2

Olfactory sensory neurons use a chloride-based signal amplification mechanism to detect odorants. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca(2+) into the cilia. Ca(2+) activates a Cl(-) current that produces an efflux of Cl(-) ions and amplifies the depolarization. The molecular identity of Ca(2+)-activated Cl(-) channels is still elusive, although some bestrophins have been shown to function as Ca(2+)-activated Cl(-) channels when expressed in heterologous systems. In the olfactory epithelium, bestrophin-2 (Best2) has been indicated as a candidate for being a molecular component of the olfactory Ca(2+)-activated Cl(-) channel. In this study, we have analysed mice lacking Best2. We compared the electrophysiological responses of the olfactory epithelium to odorant stimulation, as well as the properties of Ca(2+)-activated Cl(-) currents in wild-type (WT) and knockout (KO) mice for Best2. Our results confirm that Best2 is expressed in the cilia of olfactory sensory neurons, while odorant responses and Ca(2+)-activated Cl(-) currents were not significantly different between WT and KO mice. Thus, Best2 does not appear to be the main molecular component of the olfactory channel. Further studies are required to determine the function of Best2 in the cilia of olfactory sensory neurons.

Recruitment of the cytoplasmic adaptor Grb2 to surface IgG and IgE provides antigen receptor–intrinsic costimulation to class-switched B cells

The improved antibody responses of class-switched memory B cells depend on enhanced signaling from their B cell antigen receptors (BCRs). However, BCRs on both naive and antigen-experienced B cells use the canonical immunoglobulin-associated alpha and beta-protein signaling subunits. Here we identified a BCR isotype-specific signal-amplification mechanism. Whereas immunoglobulin M (IgM)-containing BCRs initiated intracellular signals exclusively through immunoglobulin-associated alpha- and beta-proteins, IgG- and IgE-containing BCRs also used a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. When phosphorylated, this tyrosine recruited the adaptor Grb2, resulting in sustained protein kinase activation and prolonged generation of second messengers, which together culminated in enhanced B cell proliferation. Hence, membrane-bound IgG and IgE exert antigen recognition as well as costimulatory functions, thereby rendering memory B cells less dependent on T cell help.

A sensitive method to detect Escherichia coli based on immunomagnetic separation and real-time PCR amplification of aptamers

Aptamers, single-stranded nucleic acids, provide a unique opportunity as amplifiable molecules using polymerase chain reaction (PCR) as well as recognition molecules like antibodies. We report a highly sensitive detection of Escherichia coli by taking advantage of the aptamer amplification as well as the specific binding of aptamers onto E. coli. This unique approach consists of three steps. First, the target E. coli was captured by antibody-conjugated magnetic beads. Second, the RNA aptamers were bound onto the surface of captured E. coli in a sandwich way. Finally, the heat-released aptamers were amplified by using real-time reverse-transcriptase-PCR (RT-PCR). The aptamer amplification in this approach has enabled a sensitive detection of microorganisms, such as the detection of 10 E. coli in 1 ml sample. When compared to the amplification of nucleic acids extracted from the target microorganisms, this approach can not only prevent the loss of target nucleic acids during the sample preparation by obviating the necessity of cell lysis, but also provide an additional mechanism of signal amplification due to the binding of many aptamers to the surface of each E. coli. Detection of E. coli in this approach showed a wide dynamic range from 10 1 to 10 7 E. coli per ml, which can be explained by the exponential amplification of aptamers. This report has demonstrated, for the first time, the effective use of aptamer amplification in the development of sensitive microorganism detection. It is anticipated that the present approach will be easily expanded and employed in various types of microorganism detection.

Signal amplification in biological and electrical engineering systems: Universal role of cascades

In this paper we compare the cascade mechanisms of signal amplification in biological and electrical engineering systems, and show that they share the capacity to considerably amplify signals, and respond to signal changes both quickly and completely, which effectively preserves the form of the input signal. For biological systems, these characteristics are crucial for efficient and reliable cellular signaling. We show that this highly-efficient biological mechanism of signal amplification that has naturally evolved is mathematically fully equivalent with some man-developed amplifiers, which indicates parallels between biological evolution and successful technology development.

Lysophosphatidic Acid 2 Receptor-mediated Supramolecular Complex Formation Regulates Its Antiapoptotic Effect

The G protein-coupled lysophosphatidic acid 2 (LPA(2)) receptor elicits prosurvival responses to prevent and rescue cells from apoptosis. However, G protein-coupled signals are not sufficient for the full protective effect of LPA(2). LPA(2) differs from other LPA receptor subtypes in the C-terminal tail, where it contains a zinc finger-binding motif for the interactions with LIM domain-containing TRIP6 and proapoptotic Siva-1, and a PDZ-binding motif through which it complexes with the NHERF2 scaffold protein. In this report, we identify a unique CXXC motif of LPA(2) responsible for the binding to TRIP6 and Siva-1, and demonstrate that disruption of these macromolecular complexes or knockdown of TRIP6 or NHERF2 expression attenuates LPA(2)-mediated protection from chemotherapeutic agent-induced apoptosis. In contrast, knockdown of Siva-1 expression enhances this effect. Furthermore, a PDZ-mediated direct interaction between TRIP6 and NHERF2 facilitates their interaction with LPA(2). Together, these results suggest that in addition to G protein-activated signals, the cooperation embedded in the LPA(2)-TRIP6-NHERF2 ternary complex provides a novel ligand-dependent signal amplification mechanism that is required for LPA(2)-mediated full activation of antiapoptotic signaling.

Noise color and asymmetry in stochastic resonance with silicon nanomechanical resonators

Stochastic resonance with white noise has been well established as a potential signal amplification mechanism in nanomechanical two-state systems. While white noise represents the archetypal stimulus for stochastic resonance, typical operating environments for nanomechanical devices often contain different classes of noise, particularly colored noise with a 1/f spectrum. As a result, improved understanding of the effects of noise color will be helpful in maximizing device performance. Here we report measurements of stochastic resonance in a silicon nanomechanical resonator using 1/f noise and Ornstein-Uhlenbeck noise types. Power spectral densities and residence time distributions provide insight into asymmetry of the bistable amplitude states, and the data sets suggest that 1/fα noise spectra with increasing noise color (i.e. α) may lead to increasing asymmetry in the system, reducing the achievable amplification. Furthermore, we explore the effects of correlation time τ on stochastic resonance with the use of exponentially correlated noise. We find monotonic suppression of the spectral amplification as the correlation time increases.

Development of redox-labeled electrochemical immunoassay for polycyclic aromatic hydrocarbons with controlled surface modification and catalytic voltammetric detection

A redox-labeled direct competitive electrochemical immunoassay for polycyclic aromatic hydrocarbons (PAHs) was developed. A ruthenium tris(bipyridine)-pyrenebutyric acid conjugate was synthesized as the redox-labeled tracer. Its recognition by an anti-PAH monoclonal antibody was confirmed by surface plasmon resonance. In the immunoassay, the antibody was immobilized on (3-glycidoxypropyl)-trimethoxysilane (GPTMS)-modified indium tin oxide (ITO) electrodes. The assay was quantified by measuring the electro-catalytic current of the redox label in an oxalate-containing electrolyte which served as a sacrificial electron donor to amplify the current signal. Formation of GPTMS film on ITO and subsequent antibody immobilization were characterized by X-ray photoelectron spectroscopy (XPS) and electrochemistry. Using a ruthenium tris(bipyridine)-conjugated IgG (IgG-Ru) as the surface-bound redox probe, the highest electrochemical signal was obtained on GPTMS electrodes with 1 h modification. Under the optimized conditions for ITO modification, antibody immobilization and tracer concentration, competition curves for benzo[ a]pyrene and pyrenebutyric acid were obtained with a detection limit of 2.4 and 10 ng mL −1, respectively. The redox-labeled electrochemical immunoassay with signal amplification mechanism offers a potential analytical method for the simultaneous detection of multiple environmental organic pollutants on antibody biochips.

The immunoglobulin tail tyrosine of membrane-bound IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched memory B cells

Improved antibody responses by class-switched memory B cells require enhanced signaling from their antigen receptor (BCR) in a coreceptor-independent manner. However, all BCR classes on newly generated and antigen-experienced B cells utilize the canonical Igα/Igβ subunit for signaling via the immunoreceptor tyrosine-based activation motif (ITAM) in their cytoplasmic domains. We have now identified the signal amplification mechanism of the activated IgG- and IgE-BCR on class-switched B cells. An evolutionary conserved tyrosine residue in the cytoplasmic segments of membrane-bound IgG and IgE heavy chains, named Immunoglobulin Tail Tyrosine (ITT), becomes phosphorylated and recruits the adaptor protein Grb2 in order to prolong activation of protein kinases and sustain the generation of second messengers. Exchange of the ITT for phenylalanine phenocopies the reduced signaling profile of the IgM-BCR expressed on naive B cells. Hence membrane-bound IgG and IgE not only recognize antigen but also exert BCR-intrinsic costimulation to render memory B cells less dependent on T cell help for activation. Moreover, our finding of a signaling competent phospho-ITT confutes the paradigm of BCR tyrosine phosphorylation being confined to ITAM-containing subunits.

The role of kinases in the Hedgehog signalling pathway

The Hedgehog (Hh) signalling pathway has a crucial role in several developmental processes and is aberrantly activated in a variety of cancers. In Drosophila, many of the canonical Hh pathway components are phosphorylated, yet the precise role of these phosphorylation events in the regulation of Hh signal transduction is unclear. Furthermore, the Hh pathway receives input from several kinases that have well-described roles in other cellular functions, some of which have both positive and negative effects on Hh signalling. Several recent studies have characterized the role of specific phosphorylation events in the Hh pathway, and have begun to shed light on how phosphorylation of Hh signalling components affects their subcellular location, stability and activity to mediate the transcriptional response to the Hh gradient.

Investigation of a Fluorescence Signal Amplification Mechanism Used for the Direct Molecular Detection of Nucleic Acids

A fluorescence signal amplification mechanism allowing detection limits for DNA in the zeptomolar range was investigated. Photophysical properties of the molecular system were studied in order to better explain the signal amplification that is observed. We show that the confinement of a fluorescent DNA hybridization transducer in aggregates improves its quantum yield and photostability. Furthermore, we show that the combination of the resonance energy transfer occurring within the aggregates with the use of a conjugated polymer as the hybridization transducer and donor allows ultrafast and efficient energy coupling to the aggregates and can lead to the excitation of a large number of acceptors by only one donor.

Mechanism of Signal Amplification in the Olfactory Sensory Cilia

Molecular mechanisms underlying olfactory signal amplification were investigated by monitoring cAMP dynamics in the intact sensory cilia. We saw that [cAMP]i increased superlinearly with time during odorant stimuli for >1 s. This time course was remarkably different from that obtained with the rapid quench method previously applied to the in vitro preparation, in which [cAMP]i change has been reported to be transient. The superlinear increase of [cAMP]i was attributable to a gradual increase of cAMP production rate that was consistent with the thermodynamical interaction model between elemental molecules, as has been revealed on the rod photoreceptor cell. It thus seems likely that the fundamental mechanism for molecular interactions between olfactory transduction elements is similar to that of the rod. In olfaction, however, cAMP production was extremely small (approximately 200,000 molecules/s/cell at the maximum), in contrast to the cGMP hydrolysis in the rod (250,000 molecules/photon). The observed numbers indicate that the olfactory receptor cell has lower amplification at the enzymatic cascade. Seemingly, such low amplification is a disadvantage for the signal transduction, but this unique mechanism would be essential to reduce the loss of ATP that is broadly used for the activities of cells. Apparently, transduction by a smaller number of second-messenger formations would be achieved by the fine ciliary structure that has a high surface-volume ratio. In addition, it is speculated that this low amplification at their enzymatic processes may be the reason why the olfactory receptor cell has acquired high amplification at the final stage of transduction channels, using Ca2+ as a third messenger.

Coherent signal amplification in bistable nanomechanical oscillators by stochastic resonance

Stochastic resonance is a counterintuitive concept: the addition of noise to a noisy system induces coherent amplification of its response. First suggested as a mechanism for the cyclic recurrence of ice ages, stochastic resonance has been seen in a wide variety of macroscopic physical systems: bistable ring lasers, superconducting quantum interference devices (SQUIDs), magnetoelastic ribbons and neurophysiological systems such as the receptors in crickets and crayfish. Although fundamentally important as a mechanism of coherent signal amplification, stochastic resonance has yet to be observed in nanoscale systems. Here we report the observation of stochastic resonance in bistable nanomechanical silicon oscillators. Our nanomechanical systems consist of beams that are clamped at each end and driven into transverse oscillation with the use of a radiofrequency source. Modulation of the source induces controllable switching of the beams between two stable, distinct states. We observe that the addition of white noise causes a marked amplification of the signal strength. Stochastic resonance in nanomechanical systems could have a function in the realization of controllable high-speed nanomechanical memory cells, and paves the way for exploring macroscopic quantum coherence and tunnelling.

DNA hybridization-enhanced porous silicon corrosion: mechanistic investigations and prospect for optical interferometric biosensing

Hybridization of DNA oligonucleotides in neutral aqueous solutions with complementary sequences immobilized on highly doped p-type porous silicon matrix is shown to result in an unexpectedly large shift in the Fabry–Perot interference pattern to lower wavelengths implying a decrease in effective optical thickness of the porous matrix. We have determined that the observed optical effects are due to enhanced corrosion (oxidation–hydrolysis) of the porous silicon layer triggered by the formation of complementary DNA duplexes. Scanning force microscopy and reflectance spectroscopy were employed at various stages of the signal evolution process to monitor and establish the material changes induced by the DNA binding events. We postulate that the slow background corrosion process initiated at the exposed Si–H x groups is dramatically enhanced as a result of the change in carrier charge density of the porous silicon layer in response to the local increase in the electrostatic field generated by the nucleic acid hybridization. The proposed mechanism is consistent with the experimental observations that the characteristics of the porous silicon matrix and the charge density of the hybridized DNA complexes can both influence the corrosion process. Functionalized porous silicon matrices prepared from highly doped silicon wafers (resistivity 1 mΩ·cm) produce large corrosion rates and improved signal to noise ratios. Moreover, the enhanced decrease in the effective optical thickness could be prevented by either shielding the negative charges of the DNA duplex in the presence of Mg 2+ ions, or by using backbone charge neutral peptide nucleic acids (PNA) in the DNA hybridization experiments. The observed phenomenon is thus an example of an active sensor matrix in which the molecular recognition signal is transduced and amplified by a profound change in the chemical reactivity and physical property of the solid support itself. With the signal amplification mechanism described, binding of unlabeled complementary DNA oligonucleotides of approximately 0.1 amol/mm 2 has been detected suggesting the potential utility of this new approach in DNA sensing.

Oligonucleotide hybridization studied by a surface plasmon diffraction sensor (SPDS).

A novel label-free biosensor concept based on surface plasmon-enhanced diffraction by micro- patterned interfaces was applied to the study of hybridization reactions of target DNA oligonucleotides (15mers and 75mers) from solution to probe DNA oligonucleotides attached via streptavidin to the sensor surface. The self-referencing and quadratic signal amplification mechanism of the sensor allowed highly sensitive detection of the hybridization process. Association and dissociation processes of DNA targets could be recorded in real time and used for the quantification of their binding affinities, which differ considerably with a single base pair mismatch. An equilibrium titration approach was also applied in order to obtain the binding affinities for 15mer targets, yielding similar affinity values. The hybridization efficiencies were found to be higher for the 15mers than for the 75mers, although the latter contained the same recognition sequences. The hybridization efficiency was shown to depend on the probe density and reached nearly 100% for the 15mer fully complementary targets at a probe density of approximately 1.2 x 10(12) molecules/cm2. Using the assay as an end-point determination method, the lowest detectable coverage of a 15mer oligonucleotide was at least approximately 1.1 x 10(11) molecules/cm2. The diffraction sensing concept offers a completely novel way to integrate a reference channel in large-scale, label-free screening applications, to improve the stability and to enhance the sensitivity of microarray read-out systems.