Signal Generation Mechanism Research Articles

A lateral flow channel immunoassay combining a particle binding zone geometry with nanoparticle labelling amplification

A lateral flow channel immunoassay is described which combines two strategies for signal enhancement. A packed silicagel microparticle matrix, covalently labelled with capture antibodies, was incorporated into a microchannel, providing a detection surface to sample volume ratio at least 20 times the standard wall modified microfluidic channel format. Strong signal amplification was achieved with non-direct fluorescence labelling. Fluorescein diacetate nanoparticles, synthesized by a simple top-down method, increase the number of fluorophores per antibody binding event, compared with a classical molecular fluorescein label. The fluorescent signal generation mechanism, triggered by the addition of a solution of NaOH:DMSO, was optimised to produce highly fluorescent uranin. The packed channel immunoassay showed a linear relationship between fluorescence intensity and antibody concentration for detection of 0.4–1.67 nM in a model system of rabbit polyclonal antibodies (RbIgG) and goat anti-rabbit polyclonal antibodies (Gt-α RbIgG), potentially enabling this format to achieve semi-quantitative or quantitative measurement of multiple target analytes, where packed silica zones modified with different capture antibodies are incorporated into the same microchannel system.

The net water uptake by excitable cells is a primary mechanism for pain signal generation

An abnormal (hyper) excitation of neuronal and muscle membranes, which is transmitted into central nervous system (CNS) and generates pain sensation. Hence, the bioequivalence of the water by the cells in the body determines the phenomenon. Since pain can be generated by different phenomena, starting from mechanical damage to the breakdown of different metabolic pathways, there must be a common cellular mechanism through which various physical, chemical and metabolic factors generate abnormal excitation of cell membrane. It is known that pain sensation can be changed upon the effect of extremely weak chemical and physical signals, having intensity even less than thermal threshold and non-linear dose-dependent character.

Structural Disruption of an Adenosine-Binding DNA Aptamer on Graphene: Implications for Aptasensor Design.

We report on the predicted structural disruption of an adenosine-binding DNA aptamer adsorbed via noncovalent interactions on aqueous graphene. The use of surface-adsorbed biorecognition elements on device substrates is needed for integration in nanofluidic sensing platforms. Upon analyte binding, the conformational change in the adsorbed aptamer may perturb the surface properties, which is essential for the signal generation mechanism in the sensor. However, at present, these graphene-adsorbed aptamer structure(s) are unknown, and are challenging to experimentally elucidate. Here we use molecular dynamics simulations to investigate the structure and analyte-binding properties of this aptamer, in the presence and absence of adenosine, both free in solution and adsorbed at the aqueous graphene interface. We predict this aptamer to support a variety of stable binding modes, with direct base-graphene contact arising from regions located in the terminal bases, the centrally located binding pockets, and the distal loop region. Considerable retention of the in-solution aptamer structure in the adsorbed state indicates that strong intra-aptamer interactions compete with the graphene-aptamer interactions. However, in some adsorbed configurations the analyte adenosines detach from the binding pockets, facilitated by strong adenosine-graphene interactions.

Two-Dimensional Resonance Raman Signatures of Vibronic Coherence Transfer in Chemical Reactions.

Two-dimensional resonance Raman (2DRR) spectroscopy has been developed for studies of photochemical reaction mechanisms and structural heterogeneity in condensed phase systems. 2DRR spectroscopy is motivated by knowledge of non-equilibrium effects that cannot be detected with traditional resonance Raman spectroscopy. For example, 2DRR spectra may reveal correlated distributions of reactant and product geometries in systems that undergo chemical reactions on the femtosecond time scale. Structural heterogeneity in an ensemble may also be reflected in the 2D spectroscopic line shapes of both reactive and non-reactive systems. In this chapter, these capabilities of 2DRR spectroscopy are discussed in the context of recent applications to the photodissociation reactions of triiodide. We show that signatures of "vibronic coherence transfer" in the photodissociation process can be targeted with particular 2DRR pulse sequences. Key differences between the signal generation mechanisms for 2DRR and off-resonant 2D Raman spectroscopy techniques are also addressed. Overall, recent experimental developments and applications of the 2DRR method suggest that it will be a valuable tool for elucidating ultrafast chemical reaction mechanisms.

Cancer Immunotherapy Getting Brainy: Visualizing the Distinctive CNS Metastatic Niche to Illuminate Therapeutic Resistance.

The advent of cancer immunotherapy (CIT) and its success in treating primary and metastatic cancer may offer substantially improved outcomes for patients. Despite recent advancements, many malignancies remain resistant to CIT, among which are brain metastases, a particularly virulent disease with no apparent cure. The immunologically unique niche of the brain has prompted compelling new questions in immuno-oncology such as the effects of tissue-specific differences in immune response, heterogeneity between primary tumors and distant metastases, and the role of spatiotemporal dynamics in shaping an effective anti-tumor immune response. Current methods to examine the immunobiology of metastases in the brain are constrained by tissue processing methods that limit spatial data collection, omit dynamic information, and cannot recapitulate the heterogeneity of the tumor microenvironment. In the current review, we describe how high-resolution, live imaging tools, particularly intravital microscopy (IVM), are instrumental in answering these questions. IVM of pre-clinical cancer models enables short- and long-term observations of critical immunobiology and metastatic growth phenomena to potentially generate revolutionary insights into the spatiotemporal dynamics of brain metastasis, interactions of CIT with immune elements therein, and influence of chemo- and radiotherapy. We describe the utility of IVM to study brain metastasis in mice by tracking the migration and growth of fluorescently-labeled cells, including cancer cells and immune subsets, while monitoring the physical environment within optical windows using imaging dyes and other signal generation mechanisms to illuminate angiogenesis, hypoxia, and/or CIT drug expression within the metastatic niche. Our review summarizes the current knowledge regarding brain metastases and the immune milieu, presents the current status of CIT and its prospects in targeting brain metastases to circumvent therapeutic resistance, and proposes avenues to utilize IVM to study CIT drug delivery and therapeutic efficacy in preclinical models that will ultimately facilitate novel drug discovery and innovative combination therapies.

Plasmon-Based Colorimetric Nanosensors for Ultrasensitive Molecular Diagnostics.

Colorimetric detection of target analytes with high specificity and sensitivity is of fundamental importance to clinical and personalized point-of-care diagnostics. Because of their extraordinary optical properties, plasmonic nanomaterials have been introduced into colorimetric sensing systems, which provide significantly improved sensitivity in various biosensing applications. Here we review the recent progress on these plasmonic nanoparticles-based colorimetric nanosensors for ultrasensitive molecular diagnostics. According to their different colorimetric signal generation mechanisms, these plasmonic nanosensors are classified into two categories: (1) interparticle distance-dependent colorimetric assay based on target-induced forming cross-linking assembly/aggregate of plasmonic nanoparticles; and (2) size/morphology-dependent colorimetric assay by target-controlled growth/etching of the plasmonic nanoparticles. The sensing fundamentals and cutting-edge applications will be provided for each of them, particularly focusing on signal generation and/or amplification mechanisms that realize ultrasensitive molecular detection. Finally, we also discuss the challenge and give our future perspective in this emerging field.

Recent developments in protease activity assays and sensors

Proteases play a pivotal role in regulating important physiological processes from food digestion to blood clotting. They are also important biomarkers for many diseases such as cancers. The importance of proteases has led to extensive efforts in the screening of proteases and their inhibitors as potential drug molecules. For example, human immunodeficiency virus (HIV) patients have been treated with HIV-1 protease inhibitors to prolong the life expectancy of patients. Such a close relationship between diseases and proteases provides a strong motivation for developing sensitive, selective, and robust protease assays and sensors, which can be exploited to discover new proteases and inhibitors. In this aspect, protease assays based on levels of proteolytic activities are more relevant than protease affinity assays such as immunoassays. In this review, recent developments of protease activity assays based on different detection principles are discussed and compared. For homogenous assays, fluorescence-based techniques are the most popular due to their high sensitivity and quantitative results. However, homogeneous assays have limited multiplex sensing capabilities. In contrast, heterogeneous assays can be employed to detect multiple proteases simultaneously, given the microarray technology that is already available. Among them, electrochemical methods, surface spectroscopy techniques, and enzyme-linked peptide protease assays are commonly used. Finally, recent developments in liquid crystal (LC)-based protease assays and their applications for detecting proteases and their inhibitors are discussed.

Mathematical Models for Interrelation of Characteristics of the Developing Defects with the Parameters of Acoustic Emission Signals

Mathematical models and mechanisms of acoustic emission signal generation are presented. It is shown that the reasons of acoustic emission signal origin are related to the local alterations of microstructure of materials and processes of movement of distribution at formation of tensions in solids. It is proved that the origination of signals is based on the analysis of acoustic wave energy released per load cycle and the work of the external forces at elastic deflection.

Perspective: Two-dimensional resonance Raman spectroscopy.

Two-dimensional resonance Raman (2DRR) spectroscopy has been developed for studies of photochemical reaction mechanisms and structural heterogeneity in complex systems. The 2DRR method can leverage electronic resonance enhancement to selectively probe chromophores embedded in complex environments (e.g., a cofactor in a protein). In addition, correlations between the two dimensions of the 2DRR spectrum reveal information that is not available in traditional Raman techniques. For example, distributions of reactant and product geometries can be correlated in systems that undergo chemical reactions on the femtosecond time scale. Structural heterogeneity in an ensemble may also be reflected in the 2D spectroscopic line shapes of both reactive and non-reactive systems. In this perspective article, these capabilities of 2DRR spectroscopy are discussed in the context of recent applications to the photodissociation reactions of triiodide and myoglobin. We also address key differences between the signal generation mechanisms for 2DRR and off-resonant 2D Raman spectroscopies. Most notably, it has been shown that these two techniques are subject to a tradeoff between sensitivity to anharmonicity and susceptibility to artifacts. Overall, recent experimental developments and applications of the 2DRR method suggest great potential for the future of the technique.

On the link between the speckle free nature of optoacoustics and visibility of structures in limited-view tomography

Similar to pulse-echo ultrasound, optoacoustic imaging encodes the location of optical absorbers by the time-of-flight of ultrasound waves. Yet, signal generation mechanisms are fundamentally different for the two modalities, leading to significant distinction between the optimum image formation strategies. While interference of back-scattered ultrasound waves with random phases causes speckle noise in ultrasound images, speckle formation is hindered by the strong correlation between the optoacoustic responses corresponding to individual sources. However, visibility of structures is severely hampered when attempting to acquire optoacoustic images under limited-view tomographic geometries. In this tutorial article, we systematically describe the basic principles of optoacoustic signal generation and image formation for objects ranging from individual sub-resolution absorbers to a continuous absorption distribution. The results are of relevance for the proper interpretation of optoacoustic images acquired under limited-view scenarios and may also serve as a basis for optimal design of tomographic acquisition geometries and image formation strategies.

Fluorescence Immunoassay System via Enzyme-Enabled in Situ Synthesis of Fluorescent Silicon Nanoparticles.

The emergence of fluorescent nanomaterials with excellent performances has triggered the development of fluorescence analysis technique, which possesses several advantages in the research and clinical applications. However, current strategies for fluorescence immunoassay usually involve the routine fluorophore-labeled antibody and/or awkward signal generation procedure that may not be available in conventional enzyme-linked immunosorbent assay (ELISA) systems. Herein, we circumvent this problem by imparting an exquisite signal generation mechanism to commercially available alkaline phosphatase (ALP)-based ELISA platform and putting forward a conceptual fluorescent ELISA system based on an original ALP-enabled in situ synthesis of fluorescent nanomaterials. After adding target antigen, the presence of ALP labeled on antibody catalyzes the transformation of the substrate ascorbic acid 2-phosphate into ascorbic acid. Then the resultant ascorbic acid (i.e., ascorbate) interacts with amine-containing silane molecules (no fluorescence) to produce intense cyan fluorescent silicon nanoparticles. For the proof-of-concept, alpha-fetoprotein and human immunoglobulin G are chosen as the model antigen targets, and our proposed immunoassay (designated as the nanoparticles generation-based fluorescent ELISA) enables the detection with either fluorescence spectroscopy or naked-eye readout under the ultraviolet lamp. The convincing recognition mechanism and assay performance ensure fluorescent ELISA to quantitatively evaluate the alpha-fetoprotein level in serologic test and potentially apply in the clinic diagnosis of hepatocellular carcinoma.

Colorimetric determination of staphylococcal enterotoxin B via DNAzyme-guided growth of gold nanoparticles

The authors describe a colorimetric method for the determination of the staphylococcal enterotoxin B (SEB) that also allows for visual readout. The assay is based on the growth of gold nanoparticles (AuNPs) mediated by a hemin/G-quadruplex DNAzyme which generates a color change from red to blue in the presence of SEB. The method is enzyme-free and does not require a label. The kinetics of the formation of the AuNPs is controlled by the hemin/G-quadruplex DNAzyme and this is key to the signal generation mechanism. In the presence of SEB, the reactions between aptamer and target modulated the amount of single probe G strands that form DNAzyme capable of consuming hydrogen peroxide. The growth process of AuNPs is influenced by the resulting concentration of H2O2 and leads to the color change. Under optimal conditions, a linear relationship exists between absorbance and SEB concentration in the range from 0.1 to 500 pg·mL‾1 which covers the clinically relevant range. In case of visual detection, the lower limit of detection is 1 pg·mL−1. The assay described here is sensitive, comparably inexpensive and can detect SEB rapidly without the need for sophisticated equipment. In our perception, the method has a wide scope in that it may be adapted to various nucleic acids, proteins and other biomolecules if respective aptamers are available.

Feasibility Study on a Microwave-Based Sensor for Measuring Hydration Level Using Human Skin Models.

Tissue dehydration results in three major types of exsiccosis—hyper-, hypo-, or isonatraemia. All three types entail alterations of salt concentrations leading to impaired biochemical processes, and can finally cause severe morbidity. The aim of our study was to demonstrate the feasibility of a microwave-based sensor technology for the non-invasive measurement of the hydration status. Electromagnetic waves at high frequencies interact with molecules, especially water. Hence, if a sample contains free water molecules, this can be detected in a reflected microwave signal. To develop the sensor system, human three-dimensional skin equivalents were instituted as a standardized test platform mimicking reproducible exsiccosis scenarios. Therefore, skin equivalents with a specific hydration and density of matrix components were generated and microwave measurements were performed. Hydration-specific spectra allowed deriving the hydration state of the skin models. A further advantage of the skin equivalents was the characterization of the impact of distinct skin components on the measured signals to investigate mechanisms of signal generation. The results demonstrate the feasibility of a non-invasive microwave-based hydration sensor technology. The sensor bears potential to be integrated in a wearable medical device for personal health monitoring.

Application of angular–temporal spectrum to exploratory analysis of generalized angular–temporal deterministic signals

Vibration-based condition monitoring of rotary machinery develops toward application for highly non-stationary operational conditions, mainly rotational speed and load. If speed and load vary during the acquisition of vibration signal, both, instantaneous amplitudes and instantaneous frequencies of signal components vary accordingly. This scenario causes a strong demand for novel signal processing approach suitable for analysis of such processes. In this paper, angular–temporal spectrum is introduced as a novel and practical solution for analysis of such signals. The tool is based on well-established principles of angular–temporal determinism, and allows for representation of energy of analyzed signal on bi-frequency plane related to angular and temporal properties of the signal. Additionally, proposed approach includes amplitude normalization technique in order to give more interpretable measures of amplitude related to varying speed and load. The paper gives overall description of the proposed tool as well as its intuitive explanation from the perspective of signal generation mechanism. The outcome of the angular–temporal spectrum is presented using generated signal, vibration signal measured on lab test-rig operating with damaged bearing during run-up process, and finally on industrial data from a wind turbine.

Measurement of the thermal expansion coefficient of Guadua angustifolia-Kunth using the photoacoustic technique

In this paper, the Linear Thermal Expansion Coefficient of Guadua angustifolia- Kunth samples was measured using the Photoacoustic (PA) technique in a heat transmission configuration and considering the thermoelastic bending as a PA signal generation mechanism in addition to the thermodiffusion ones. The obtained value of (27±7)x10-6K-1 is a reasonable value compared with that reported for similar materials such as wood.

Emerging techniques for ultrasensitive protein analysis.

Many important biomarkers for devastating diseases and biochemical processes are proteins present at ultralow levels. Traditional techniques, such as enzyme-linked immunosorbent assays (ELISA), mass spectrometry, and protein microarrays, are often not sensitive enough to detect proteins with concentrations below the picomolar level, thus requiring the development of analytical techniques with ultrahigh sensitivities. In this review, we highlight the recent advances in developing novel techniques, sensors, and assays for ultrasensitive protein analysis. Particular attention will be focused on three classes of signal generation and/or amplification mechanisms, including the uses of nanomaterials, nucleic acids, and digital platforms.

Oriented assembly of invisible probes: towards single mRNA imaging in living cells.

Due to the complexity of biological systems and the ultralow concentration of analytes, improving the signal-to-noise ratio and lowering the limit of detection to allow highly sensitive detection is key to biomolecule analysis, especially intracellular analysis. Here, we present a method for highly sensitive imaging of mRNA in living cells by using novel invisible oriented probes to construct a turn-on signal generation mechanism from zero background. Two DNA probes (S1 and S2) are asymmetrically modified on two small gold nanoparticles (AuNPs) with a diameter of 20 nm. The hybridization of the two DNA probes with a single target mRNA leads to the formation of an AuNP dimer which shows a prominent plasmonic coupling effect. It generates a strong scattering signal from zero-background under a dark-field spectral analysis system. The unique design of the oriented assembly dimer has the ability to easily discriminate the target signal from the inherent cellular background noise in intracellular detection, thus making this approach a valuable technique for imaging single survivin mRNA and monitoring the distribution of survivin mRNA in tumor cells.

Nanoparticles functionalized with phenylboronic acid for the potentiometric detection of saccharides

Abstract We have proposed a concept for the analytical application of modified gold nanoparticles in potentiometric sensors. The main idea is to embed modified gold nanoparticles as receptor units during electropolymerization into a polymeric matrix which should serve as a transducer of the potentiometric signal. The concept was verified using a model system based on gold nanoparticles modified with mercaptophenylboronic acid (MPBA–AuNPs) as a receptor of saccharides in a pH-sensitive polymer matrix, namely polyaniline (PANI). The determination of saccharides, in particular d -glucose, is based on the determination of released protons as products of the reaction between mercaptophenylboronic acid and saccharides. Differential pulse voltammetry showed that approximately 33% of the MPBA–AuNPs present in the polymerization mixture were built into the PANI polymeric film. The absorption and reflectivity spectra confirmed the recognition process occurring between mercaptophenylboronic acid and d -glucose inside the pH-sensitive polymeric matrix. A potentiometric response of MPBA–AuNP-modified PANI electrodes towards d -glucose in the concentration range from 0.31 up to 33 mM with a sensitivity of + 47 mV/decade was verified. The MPBA–AuNP-modified PANI film can be considered to be a saccharide-sensitive receptor layer exhibiting an indirect mechanism of signal generation.

Camera‐based high frequency heterodyne lock‐in carrierographic (frequency‐domain photoluminescence) imaging of crystalline silicon wafers

A heterodyne lock‐in imaging scheme is introduced to overcome NIR camera speed limitations and poor signal‐to‐noise ratios, thereby generating high‐frequency carrierographic imaging up to 10 kHz. A theoretical model was established to investigate the heterodyne signal generation mechanism as well as to extract the major carrier transport parameters by best‐fitting the entire modulation frequency dependence. Good agreement was found between the theoretically predicted and experimentally measured heterodyne amplitude dependence on excitation intensity. A contrast inversion feature in the high‐frequency heterodyne amplitude images was observed. High‐frequency information presents a method for resolving local near‐surface carrier transport parameters from the lumped effective lifetime, thereby providing ac‐diffusion‐length‐controlled depth‐selective/resolved imaging.Heterodyne lock‐in carrierography (LIC) amplitude images of a crystalline silicon wafer at different frequencies.

Electrochemical label-free and reagentless genosensor based on an ion barrier switch-off system for DNA sequence-specific detection of the avian influenza virus.

This paper concerns the development of genosensors based on redox-active monolayers incorporating (dipyrromethene)2Cu(II) and (dipyrromethene)2Co(II) complexes formed step by step on a gold electrode surface. They were applied for electrochemical determination of oligonucleotide sequences related to avian influenza virus (AIV) type H5N1. A 20-mer probe (NH2-NC3) was covalently attached to the gold electrode surface via a reaction performed in the presence of ethyl(dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC/NHS) between the amine group present in the probe and carboxylic groups present on the surface of the redox-active layer. Each modification step has been controlled with Osteryoung square-wave voltammetry. The genosensor incorporating the (dipyrromethene)2Cu(II) complex was able to detect a fully complementary single-stranded DNA target with a detection limit of 1.39 pM. A linear dynamic range was observed from 1 to 10 pM. This genosensor displays good discrimination between three single-stranded DNA targets studied: fully complementary, partially complementary (with only six complementary bases), and totally noncomplementary to the probe. When the (dipyrromethene)2Co(II) complex was applied, a detection limit of 1.28 pM for the fully complementary target was obtained. However, this genosensor was not able to discriminate partially complementary and totally noncomplementary oligonucleotide sequences to the probe. Electrochemical measurements, using both types of genosensors in the presence of different supporting electrolytes, were performed in order to elaborate a new mechanism of analytical signal generation based on an ion barrier "switch-off" system.