High Background Signal Research Articles

Amplification of aptamer sensor signals by four orders of magnitude via interdigitated organic electrochemical transistors

Electrochemical aptamer receptor/transducer systems are key elements of emerging E-AB sensors (aptasensor) used for the detection of various kinds of targets. However, the performance of these amperometric sensors is often limited by the low density of receptors attached to the sensor surface and high background signals. In the present work, interdigitated organic electrochemical transistors (iOECT) were used as a transducer to enhance the sensitivity and dynamic detection range of aptasensors. Therefore, the electrode of an amperometric sensor was utilized as gate electrode to operate the iOECT. This device was used to detect the low weight target molecule adenosine triphosphate (ATP), a common biomarker, which plays an important role for cardiovascular, neurodegenerative, and immune deficiency diseases. The novel aptasensor can selectively detect ATP with ultrahigh sensitivity down to the concentration of 10 pM, which is four orders of magnitude lower than the detection limit of the same aptasensor using an amperometric transducer principle (limit-of-detection of 106 nM) and most other previously reported electrochemical sensors. Furthermore, sensor regeneration was demonstrated, which facilitates reusability of OECT aptasensors. The small device size in combination with high transconductances paves the way for the development of highly sensitive integrated micro-biosensors for point-of-care applications.

Homogeneous Differential Magnetic Assay.

Assays are widely used for detection of various targets, including pathogens, drugs, and toxins. Homogeneous assays are promising for the realization of point-of-care diagnostics as they do not require separation, immobilization, or washing steps. For low concentrations of target molecules, the speed and sensitivity of homogeneous assays have hitherto been limited by slow binding kinetics, time-consuming amplification steps, and the presence of a high background signal. Here, we present a homogeneous differential magnetic assay that utilizes a differential magnetic readout that eliminates previous limitations of homogeneous assays. The assay uses a gradiometer sensor configuration combined with precise microfluidic sample handling. This enables simultaneous differential measurement of a positive test sample containing a synthesized Vibrio cholerae target and a negative control sample, which reduces the background signal and increases the readout speed. Very low concentrations of targets down to femtomolar levels are thus detectable without any additional amplification of the number of targets. Our homogeneous differential magnetic assay method opens new possibilities for rapid and highly sensitive diagnostics at the point of care.

Michael Addition/S,N-Intramolecular Rearrangement Sequence Enables Selective Fluorescence Detection of Cysteine and Homocysteine.

Acrylate has been widely used as the recognition unit for Cys fluorescent probes. Despite this widespread use, a potential drawback of this probe type is that the ester linkage between the fluorophore and acryloyl recognition unit is liable to be hydrolyzed by abundant esterase in the cytosol, thus affording a high background signal. To solve this problem, we herein put forward a new strategy to construct a selective fluorescent probe for cysteine (Cys)/homocysteine (Hcy) with propynamide as the recognition moiety. The free probe CPA displays weakly fluorescent emission in aqueous media because of the donor-excited photoinduced electron transfer (d-PET) process within the molecule. The Michael addition of Cys (or Hcy) thiols to the conjugated alkyne of CPA gives the expected β-sulfido-α,β-unsaturated amides (1a/1b), which subsequently undergo an intramolecular S,N rearrangement, yielding β-amino-α,β-unsaturated amides (2a/2b) as the final products. The above cascade reaction results in the blockage of d-PET within CPA, thus affording a dramatic fluorescence enhancement at 495 nm. The involvement of the sulfhydryl and the adjacent amino groups in the sensing process renders CPA high selectivity for Cys/Hcy over glutathione as well as other amino acids. The probe has been successfully applied to image Cys in different cell lines. Further, CPA shows two-photon fluorescence properties, and its ability to monitor Cys in deep tissues has been demonstrated by using two-photon microscopy.

Electrocatalytic Efficiency Regulation between Target-Induced HRP-Mimicking DNAzyme and GOx with Low Background for Ultrasensitive Detection of Thrombin

Herein, an efficient target-activated enzyme cascade electrocatalysis with low background signal was employed to establish electrochemical biosensor for ultrasensitive detection of thrombin via regulating electrocatalytic efficiency between target-induced hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) and glucose oxidase (GOx).Impressively, only when the target thrombin was introduced, the HRP-mimicking DNAzyme acting simultaneously as electrochemical signal probe would be formed to activate high-efficiency enzyme cascade electrocatalysis for reducing background signal significantly, which could overcome the defect of inevitable high background signal during the detection of target in the traditional cascade electrocatalysis of two existing bioenzymes. In addition, the detection sensitivity could be further improved by regulating the side length of rigid DNA tetrahedron (TDN) scaffold anchored HRP-mimicking DNAzyme and GOx at adjacent vertices for high enzyme cascade electrocatalytic efficiency. Consequently, the proposed biosensor demonstrated a low detection limit down to 0.3 fM for target thrombin, which provided a promising method for ultrasensitive monitoring of biomolecules in sensing analysis and disease diagnosis.

Liquid-Phase Electron Microscopy with Controllable Liquid Thickness

Liquid-phase electron microscopy (LPEM) is capable of imaging nanostructures and processes in a liquid environment. The spatial resolution achieved with LPEM critically depends on the thickness of the liquid layer surrounding the object of interest. An excessively thick liquid results in broadening of the electron beam and a high background signal that decreases the resolution and contrast of the object in an image. The liquid thickness in a standard liquid cell, consisting of two liquid enclosing membranes separated by spacers, is mainly defined by the deformation of the SiN membrane windows toward the vacuum side, and the effective thickness may differ from the spacer height. Here, we present a method involving a pressure controller setup to balance the pressure difference over the membrane windows, thus manipulating the shape profiles of the used silicon nitride membrane windows. Electron energy loss spectroscopy (EELS) measurements to determine the liquid thickness showed that it is possible to control the thickness precisely during an LPEM experiment by regulating the interior pressure of the liquid cell. We demonstrated atomic resolution on gold nanoparticles and the phase contrast using silica nanoparticles in liquid with controlled thickness.

Target-activated and ratiometric photochromic probe for “double-check” detection of toxic thiols in live cells

Photochromic molecules can achieve reversible isomerization upon alternate light irradiations, which offers a great opportunity to improve the precision of analytes detection and imaging in complicated biological environments. Previous reported photochromic probe exhibited only mono-color switching and an initially fluorescence-ON state that may cause high background signal and impose an adverse impact on the desired sensing precision. To overcome this set-back, we developed a novel photochromic probe with an analyte-activation mode for ratiometric sensing of toxic thiols in both real water samples and live cells. The dynamic dual-fluorescence signal is released only after the fast and selective cleavage of the 2,4-dinitrophenyl sulfonate by the targeted thiophenol derivatives. Consequently, a "double-check" with synchronized dual-fluorescence blinking for analyte detection is successfully employed upon alternate light triggers with rapid response (k=7.2x10(-2) s(-1)), high sensitivity (LOD=6.1 nM) as well as selectivity of thiophenol derivatives over other common thiol species (e.g., GSH, Cys and Hcy). The photochromic probe was successfully introduced to the fast and on-site detection of highly toxic thiophenols in real waste water samples. Moreover, by using confocal laser-scanning microscopy (CLSM) and flow cytometric analysis, the potential applications of this ratiometric photochromic probe for trace toxic thiol sensing in live cells are examined.

Improvement of surface-enhanced Raman scattering detection and imaging by multivariate curve resolution methods

Surface-enhanced Raman scattering (SERS) tags have been intensively applied in biological detection and imaging in recent years. However, both applications often suffer from high Raman background signals from containers such as 96-well plates or autofluorescence signals from biological tissues. Here, we greatly mitigate the influence of these high background Raman or fluorescent signals in both biological detection and imaging using two multivariate curve resolution (MCR) methods including negative matrix factorization and classical least squares. The limit of detection is lowered by one order of magnitude after applying MCR methods to detect target SERS tags in a 96-well plate. Additionally, in a multiplexed cell imaging assay, both false-negative and false-positive results were eliminated with the aid of MCR methods. Accordingly, we suggest a wider application of MCR methods during both biological detection and imaging of SERS tags with high background signals.

Electrochemiluminescence Imaging for Bioanalysis.

Electrochemiluminescence (ECL) is a widely used analytical technique with the advantages of high sensitivity and low background signal. The recent and rapid development of electrochemical materials, luminophores, and optical elements significantly increases the ECL signals and, thus, ECL imaging with enhanced spatial and temporal resolutions is realized. Currently, ECL imaging is successfully applied to high-throughput bioanalysis and to visualize the distribution of molecules at single cells. Compared with other optical bioassays, no optical excitation is involved in imaging, so the approach avoids a background signal from illumination and increases the detection sensitivity. This review highlights some of the most exciting developments in this field, including the mechanisms, electrode designs, and the applications of ECL imaging in bioanalysis and at single cells and particles.

Development of a "Dual Gates" Locked, Target-Triggered Nanodevice for Point-of-Care Testing with a Glucometer Readout.

Developing a facile and sensitive sensing platform is of importance for point-of-care testing (POCT). Herein, a sensitive and portable POCT platform based on "dual gates" aminated magnetic mesoporous silica nanocomposites (AMMS) bearing polydopamine (PDA)-aptamer (Apt) two-tier shells, as a novel nanodevice, is designed for target detection through a target-triggered glucose (GO) release from AMMS with personal glucometer (PGM) readout. In the absence of target, GO can be firmly captured in pores by the designed "dual gates", which would decrease the high background signal of this system and ensure the accuracy of the detection results. Upon the introduction of the target molecules under acidic conditions (pH 5.5), the subsequent PDA self-degradation and the specific Apt-target reaction can cause the departure of "dual gates" and the opening of pores to release the loaded GO molecules, which could be quantitatively monitored by a portable PGM. It has been demonstrated that such POCT platform shows high sensitivity and excellent selectivity for aflatoxin B1 (AFB1) detection, accompanied by the well-presented reproducibility and stability. Importantly, this sensing platform was further validated by assaying contaminated samples, where the obtained results were well matched with that by HPLC. Regarding the features of portability, high sensitivity, and high throughput detection, the developed platform might find wide applications in POCT.

Three Color Single-Molecule FRET of Fast IDP Binding and Folding

Molecular interactions of intrinsically disordered proteins (IDPs) are different from those between folded molecules because IDP binding involves large convormational changes. To understand the mechanism of IDP binding, three-color Förster resonance energy transfer (FRET) will be very useful because both the interaction between two molecules and conformational changes can be probed simultaneously. In this work, we investigated binding of a disordered protein, the transactivation domain (TAD) of p53 and one of its binding partners, nuclear coactivator binding domain (NCBD) of CBP. TAD labeled with Alexa488 (donor) and Alexa594 (acceptor 1, A1) site-specifically was immobilized on a glass surface and incubated with CF680R (acceptor 2, A2)-labeled NCBD in solution. To determine all three FRET efficiencies, alternating excitation of the donor and A1 is typically used. However, in binding experiments, direct excitation of A2 attached to NCBD in solution by A1 excitation laser produces a high background signal. Therefore, we devised a method using a single donor excitation that determines all three FRET efficiencies by utilizing incomplete labeling of A2. When TAD binds to unlabled NCBD, the FRET efficiency between the donor and A1 (E1) can be determined. Using this information, it is possible to determine the other two FRET efficiencies in principle. However, due to the fast binding and dissociation kinetics, the bound and unbound states are not well separated in binned FRET efficiency trajectories (1 ms bin time), which prevents pre-determination of E1. We employed the maximum likelihood method developed by Gopich and Szabo that analyzes photon trajectories directly without binning the data. By globally analyzing the data using a kinetic model that includes binding to both labeled and unlabled NCBD, rate coefficients of the sub-millisecond kinetics and all three FRET efficiencies were determined accurately.

High-resolution plasma membrane-selective imaging by second harmonic generation

The plasma membrane is the site of intercellular communication and subsequent intracellular signal transduction. The specific visualization of the plasma membrane in living cells, however, is difficult using fluorescence-based techniques owing to the high background signals from intracellular organelles. In this study, we show that second harmonic generation (SHG) is a high-resolution plasma membrane-selective imaging technique that enables multifaceted investigations of the plasma membrane. In contrast to fluorescence imaging, SHG specifically visualizes the plasma membrane at locations that are not attached to artificial substrates and allows high-resolution imaging because of its subresolution nature. These properties were exploited to measure the distances from the plasma membrane to subcortical actin and tubulin fibers, revealing the precise cytoskeletal organization beneath the plasma membrane. Thus, SHG imaging enables the specific visualization of phenomena at the plasma membrane with unprecedented precision and versatility and should facilitate cell biology research focused on the plasma membrane.

Temporal Filtering to Improve Single Molecule Identification in High Background Samples.

Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported in the last decade, the technique could not reach its full potential due to a set of limitations immanent to the samples themselves. Particularly, high background signals impede the proper performance of most single-molecule identification and localization algorithms. One option is to exploit the characteristic blinking of single molecule signals, which differs substantially from the residual brightness fluctuations of the fluorescence background. To pronounce single molecule signals, we used a temporal high-pass filtering in Fourier space on a pixel-by-pixel basis. We evaluated the performance of temporal filtering by assessing statistical parameters such as true positive rate and false discovery rate. For this, ground truth signals were generated by simulations and overlaid onto experimentally derived movies of samples with high background signals. Compared to the nonfiltered case, we found an improvement of the sensitivity by up to a factor 3.5 while no significant change in the localization accuracy was observable.

Fabrication of a low background signal glucose biosensor with 3D network materials as the electrocatalyst

Glucose oxidase (GOx) based biosensor is an effective method to determine glucose level. However, the biosensors embedded with high electroactive species suffered from high background signal levels, which leading to relative low sensitivity for glucose sensing. In this work, a novel 3D network materials based glucose biosensor with low background signal was constructed, which demonstrated high sensitivity and selectivity towards glucose assay. Here, the combination of ionic liquid modified graphene sheets (GS-IL) and Au nanorods (Au NRs) acted as high electroactive catalyst, and thiol-containing silica sol-gel served as a nonconductive matrix to self-assembly of GS-IL and Au NRs to form the three-dimensional (3D) network materials. Meanwhile, the doping amount of the sol-gel had significant influences on electrochemical performance of the 3D network materials based biosensor. As a result, optimized 3.75% doping 3D network materials were selected to construct the glucose biosensor, which exhibited low background signal and high sensitivity. This biosensor was successfully applied in monitoring the glucose levels of serum and brain microdialysate samples.

Cell organelles and fluorescence of parenchyma cells in Eucalyptus bosistoana sapwood and heartwood investigated by microscopy

BackgroundEucalyptus bosistoana is currently investigated in New Zealand for its potential to produce naturally durable timber in short-rotation plantations. Little is known of heartwood formation in young trees. The objective of this study was to identify conventional and confocal microscopy methods which allow the observation of cell organelles and the chemical composition in the E. bosistoana parenchyma cells before and after heartwood formation.ResultsNuclei, microtubules and peroxisomes in parenchyma cells of 2-year-old E. bosistoana stems were visualised by confocal microscopy combined with optimised immunolabelling protocols. Sequential staining of the tissue with toluidine blue and iodine/potassium iodide identified different cell organelles in parenchyma cells of sapwood. Iodine/potassium iodide stained starch (amyloplasts), while amido black stained proteins in sapwood. Fluorescence emission spectra confirmed the presence of chloroplasts in parenchyma of 2-year-old E. bosistoana. Fluorescence emission spectral (lambda) scans showed differences between parenchyma and fibre cells as well as sapwood and heartwood.ConclusionsPhysiological changes between sapwood and heartwood were visualised in parenchyma cells. Labelling of cell organelles was challenging due to unspecific binding and high background signals. Understanding heartwood formation is critical for the success of a plantation forest industry aiming to produce ground-durable timber, as heartwood formation is variable.

Which glue to choose? A neutron scattering study of various adhesive materials and their effect on background scattering

The background scattering from numerous frequently used sample adhesives has been investigated to determine the background contribution of these glues when used for inelastic neutron scattering measurements. Starting with a bare Cu sheet, different glues have been trialled, such as GE varnish, CYTOP solution, Teflon tape, Fomblin oil and two-component epoxy glue. Measurements were collected using the PELICAN cold-neutron time-of-flight spectrometer at ANSTO, which is capable of collecting data over a wide range of Q–ω space simultaneously. The results indicate that those glues containing hydrogen give much higher background signals, while those that do not contain hydrogen have a much smaller impact on the background signal. This was observed for both elastic and inelastic neutron scattering.

Single-layer graphene-coated gold chip for electrochemical surface plasmon resonance study.

Surface plasmon resonance (SPR) employs a gold (Au) thin film (ca. 50nm in thickness) chip to generate a surface plasmonic wave (SPW) for in situ monitoring of the interface/surface, which makes it intrinsically compatible with electrochemistry for combined electrochemical surface plasmon resonance (EC-SPR) investigations. However, conventional SPR Au chips suffers from a high background signal, narrow electrochemical window, and limited electrochemical stability. Presented in this work is a novel SPR chip composed of the Au/long-chain alkane thiol self-assembled monolayer/single-layer graphene (Au/SAM/G) sandwich architecture to address these problems. On this chip, the single-layer graphene serves as a working electrode for electrochemical measurement, and the underlying Au film serves as the SPW support for SPR monitoring; the sandwiched thiol monolayer enables the electrical separation of the graphene and Au film to protect the Au film from electrochemical polarization. Our experiment indicates that the electrochemical window of such a chip extends beyond the hydrogen/oxygen evolution reaction potential on Au with significantly improved electrochemical stability and suppressed background signal. Moreover, its intrinsic SPR sensitivity is completely reserved even compared to that of the conventional SPR Au chip. This Au/SAM/G chip may offer a valuable solution to the EC-SPR investigations in harsh conditions. Graphical abstract ᅟ.

High-Resolution Plasma Membrane-Selective Imaging by Second Harmonic Generation

SummaryThe plasma membrane is the site of intercellular communication and subsequent intracellular signal transduction. The specific visualization of the plasma membrane in living cells, however, is difficult using fluorescence-based techniques owing to the high background signals from intracellular organelles. In this study, we show that second harmonic generation (SHG) is a high-resolution plasma membrane-selective imaging technique that enables multifaceted investigations of the plasma membrane. In contrast to fluorescence imaging, SHG specifically visualizes the plasma membrane at locations that are not attached to artificial substrates and allows high-resolution imaging because of its subresolution nature. These properties were exploited to measure the distances from the plasma membrane to subcortical actin and tubulin fibers, revealing the precise cytoskeletal organization beneath the plasma membrane. Thus, SHG imaging enables the specific visualization of phenomena at the plasma membrane with unprecedented precision and versatility and should facilitate cell biology research focused on the plasma membrane.

A highly sensitive electrochemiluminescence sensor for the detection of l-cysteine based on the rhombus-shaped rubrene microsheets and platinum nanoparticles

We present a highly sensitive platinum nanoparticles (PtNPs), rhombus-shaped rubrene microsheets (RubRMs) and dissolved oxygen (O2) system for the monitoring of l-cysteine by the quenched electrochemiluminescence (ECL) signals. Firstly, RubRMs has been prepared through reprecipitation method, then the metal organic composites of PtNPs-RubRMs was synthesized by a simple in situ reduction of PtNPs on RubRMs surface. The composites combined the high ECL characteristic of RubRMs, the wonderful catalytic efficiency and good electron transfer ability of PtNPs. With the synergistic effect between RubRMs and PtNPs, the proposed ECL sensors exhibited excellent responses for l-cysteine. Under the optimum conditions, the linear range was from 1.0 × 10−9 mol L-1 to 5.0 × 10-4 mol L-1 with a detection limit of 3.3 × 10-10 mol L-1 (S/N = 3). And the results shows high sensitivity, excellent stability, and low background signal. It is simple to prepare and operate for the practical application.

Magnetic ionic liquid-enhanced isothermal nucleic acid amplification and its application to rapid visual DNA analysis

Isothermal nucleic acid amplification (INAA) techniques such as loop-mediated isothermal amplification (LAMP) and isothermal multiple-self-matching-initiated amplification (IMSA) constitute simple and rapid approaches for the detection of pathogens. However, due to the employment of multiple primers, the detection of LAMP and IMSA products is easily influenced by high background signals from primer dimer-based nonspecific nucleic acid amplification (NSA) products. Moreover, time-consuming sample preparation steps are often required for the isolation of sufficiently pure nucleic acid prior to INAA. To address these drawbacks, hydrophobic magnetic ionic liquids (MILs) were used to rapidly preconcentrate DNA from complex biological samples followed by direct amplification by LAMP and IMSA. Careful control of the components within the isothermal buffer permitted direct addition of DNA-enriched MIL to the INAA reaction mixture, thereby circumventing tedious purification procedures that are ordinarily required prior to downstream DNA amplification. When added directly to INAA reactions, MIL solvents released metal ions that ultimately inhibited the primer dimer-mediated NSA, resulting in a flat or decreased baseline signal in no-template control samples and short threshold time for positive reactions. Using a MIL-based single droplet DNA extraction method, MIL-enhanced INAA reaction system, and a handheld 3D printed device for visual detection of the amplified product in customized tubes, we demonstrate the potential of the MIL-based approach for the onsite analysis of DNA from pathogens.

Maximal viral information recovery from sequence data using VirMAP

Accurate classification of the human virome is critical to a full understanding of the role viruses play in health and disease. This implies the need for sensitive, specific, and practical pipelines that return precise outputs while still enabling case-specific post hoc analysis. Viral taxonomic characterization from metagenomic data suffers from high background noise and signal crosstalk that confounds current methods. Here we develop VirMAP that overcomes these limitations using techniques that merge nucleotide and protein information to taxonomically classify viral reconstructions independent of genome coverage or read overlap. We validate VirMAP using published data sets and viral mock communities containing RNA and DNA viruses and bacteriophages. VirMAP offers opportunities to enhance metagenomic studies seeking to define virome-host interactions, improve biosurveillance capabilities, and strengthen molecular epidemiology reporting.