Low Background Signal Research Articles

A new AgNC fluorescence regulation mechanism caused by coiled DNA and its applications in constructing molecular beacons with low background and large signal enhancement.

Herein, we report a new fluorescence regulation mechanism of DNA-templated AgNCs caused by coiled DNA. Based on these phenomena, a novel dual fluorescent AgNC-MB with exponential signal enhancement and remarkably low background was developed. The AgNC-MB could effectively facilitate template design and avoid the disturbance caused by undesirable hybridization.

A Turn-On Fluorescent Sensor for Selective and Sensitive Detection of Alkaline Phosphatase Activity with Gold Nanoclusters Based on Inner Filter Effect.

In this work, a novel approach for simple and sensitive determination of alkaline phosphatase (ALP) is developed on the basis of an inner filter effect of p-nitrophenylphosphate (PNPP) on the fluorescence of gold nanoclusters (AuNCs). AuNCs with a high quantum yield of 12% were synthesized by one-pot strategy and were directly applied as fluorescent substance. When AuNCs were mixed with PNPP, the fluorescence of the AuNCs was remarkably quenched or was decreased via the inner filter effect since the absorption spectrum of PNPP overlaps well with the excitation spectrum of the AuNCs. While in the presence of ALP, PNPP was catalytically hydrolyzed into p-nitrophenol, which has different absorption characteristics from those of PNPP, resulting in the recovery of the AuNCs fluorescence. Thus, a novel "turn-on" fluorescent sensor for detecting ALP was established with a detection limit as low as 0.002 U/L (signal-to-noise ratio of 3). The turn-on fluorescent sensor exhibits many merits such as high sensitivity, excellent selectivity, and high signal output because of the low background signals. In addition, the developed sensing method was successfully applied to investigate ALP inhibitors and ALP determination in serum samples. A good linear relationship was obtained in the range from 0.02 to 50 U/L, and satisfactory recoveries at four spiking levels of ALP ranged from 95% to 106% with precision below 5%. The very simple sensing approach proposed here should promote the development of fluorescence turn-on chemosensors for chemo/biodetection.

A sensitive aptasensor based on molybdenum carbide nanotubes and label-free aptamer for detection of bisphenol A.

To specifically and sensitively identify bisphenol A (BPA) with a simple and rapid method is very important for food safety. Using an anti-BPA aptamer and Mo2C nanotubes, we developed a label-free and low-background signal biosensor for BPA detection. The anti-BPA aptamer drastically increased the fluorescence signal of N-methylmesoporphyrin IX under an assistance of Help-DNA. Additionally, BPA can interact with the anti-BPA aptamer and switch its conformation to prevent the formation of a G-quadruplex, resulting in fluorescence quenching. Simultaneously, Mo2C nanotubes can reduce the background signals due to the adsorption of Help-DNA on their surface. This method shows a linear range of 2-20nM with a detection limit of 2nM for detecting BPA. This label-free BPA aptasensor with low background signal is inexpensive, easy to use, and can be applied to determine BPA in real water samples. Graphical Abstract A low-background and label-free biosensor was designed based on Mo2C nanotubes and aptamer for BPA detection.

A graphene oxide-based strand displacement amplification platform for ricin detection using aptamer as recognition element

The toxic plant protein ricin is a potential agent for criminal or bioterrorist attacks due to the wide availability and relative ease of preparation. Herein, we developed a novel strategy for the detection of ricin B-chain (RTB) based on isothermal strand-displacement polymerase reaction (ISDPR) by using aptamer as a recognition element and graphene oxide (GO) as a low background platform. In this method, ricin-binding aptamer (RBA) hybridized with a short blocker firstly, and then was immobilized on the surface of streptavidin-coated magnetic beads (MBs). The addition of RTB could release the blocker, which could hybridize with the dye-modified hairpin probe and trigger the ISDPR, resulting in high fluorescence intensity. In the absence of RTB, however, the fluorescence of the dye could be quenched strongly by GO, resulting in the extremely low background signal. Thus, RTB could be sensitively detected by the significantly increased fluorescence signal. The linear range of the current analytical system was from 0.75μg/mL to 100μg/mL and the limit of detection (3σ) was 0.6μg/mL. This method has been successfully utilized for the detection of both the RTB and the entire ricin toxin in real samples, and it could be generalized to any kind of target detection based on an appropriate aptamer.

Template-free synthesis of SERS-active gold nanopopcorn for rapid detection of chlorpyrifos residues

Chlorpyrifos, one of the broad-spectrum organophosphorus insecticides, is utilized extensively in agriculture. However, chlorpyrifos residues in food and water pose potential risks to human health. In this work, popcorn-like gold nanoparticles were synthesized under mild condition without any surfactant as template and serviced as SERS-active substrate for sensing chlorpyrifos by using a portable Raman spectrometer. The obtained substrate had extremely low background signal and remarkably enhanced SERS signal for detecting trace chlorpyrifos residues in fruit samples due to the “hot spots” formed between tips of gold nanopopcorns. As a result, the portable SERS-sensor system had a limit of detection at 1μM of chlorpyrifos, which met the requirement of national standard.

Targeting 24 bp within Telomere Repeat Sequences with Tandem Tetramer Pyrrole-Imidazole Polyamide Probes.

Synthetic molecules that bind sequence-specifically to DNA have been developed for varied biological applications, including anticancer activity, regulation of gene expression, and visualization of specific genomic regions. Increasing the number of base pairs targeted by synthetic molecules strengthens their sequence specificity. Our group has been working on the development of pyrrole-imidazole polyamides that bind to the minor groove of DNA in a sequence-specific manner without causing denaturation. Recently, we reported a simple synthetic method of fluorescent tandem dimer polyamide probes composed of two hairpin moieties with a linking hinge, which bound to 12 bp in human telomeric repeats (5'-(TTAGGG)n-3') and could be used to specifically visualize telomeres in chemically fixed cells under mild conditions. We also performed structural optimization and extension of the target base pairs to allow more specific staining of telomeres. In the present study, we synthesized tandem tetramer polyamides composed of four hairpin moieties, targeting 24 bp in telomeric repeats, the longest reported binding site for synthetic, non-nucleic-acid-based, sequence-specific DNA-binding molecules. The novel tandem tetramers bound with a nanomolar dissociation constant to 24 bp sequences made up of four telomeric repeats. Fluorescently labeled tandem tetramer polyamide probes could visualize human telomeres in chemically fixed cells with lower background signals than polyamide probes reported previously, suggesting that they had higher specificity for telomeres. Furthermore, high-throughput sequencing of human genomic DNA pulled down by the biotin-labeled tandem tetramer polyamide probe confirmed its effective binding to telomeric repeats in the complex chromatinized genome.

Probe-Specific Procedure to Estimate Sensitivity and Detection Limits for 19F Magnetic Resonance Imaging.

Due to low fluorine background signal in vivo, 19F is a good marker to study the fate of exogenous molecules by magnetic resonance imaging (MRI) using equilibrium nuclear spin polarization schemes. Since 19F MRI applications require high sensitivity, it can be important to assess experimental feasibility during the design stage already by estimating the minimum detectable fluorine concentration. Here we propose a simple method for the calibration of MRI hardware, providing sensitivity estimates for a given scanner and coil configuration. An experimental “calibration factor” to account for variations in coil configuration and hardware set-up is specified. Once it has been determined in a calibration experiment, the sensitivity of an experiment or, alternatively, the minimum number of required spins or the minimum marker concentration can be estimated without the need for a pilot experiment. The definition of this calibration factor is derived based on standard equations for the sensitivity in magnetic resonance, yet the method is not restricted by the limited validity of these equations, since additional instrument-dependent factors are implicitly included during calibration. The method is demonstrated using MR spectroscopy and imaging experiments with different 19F samples, both paramagnetically and susceptibility broadened, to approximate a range of realistic environments.

Glucose detection using 4-mercaptophenyl boronic acid-incorporated silver nanoparticles-embedded silica-coated graphene oxide as a SERS substrate

In this work, 4-mercaptophenyl boronic acid (4-MPBA) was self-assembled on the surface of silver nanoparticle-embedded silica-coated graphene oxide (GO@SiO2@Ag NPs@MPBA) to detect glucose by surface enhanced Raman scattering (SERS). The SERS intensity of 4-MPBA on the GO@SiO2@Ag NPs was 2.2-fold greater than that of GO@Ag NPs. Moreover, silica-coated GO exhibited lower background signals compared to GO. The SERS intensity of GO@SiO2@Ag NPs@MPBA peaked at 1 mM 4-MPBA. pH-dependent behavior of 4-MPBA on the GO@SiO2 @Ag NPs was investigated; the highest SERS signal intensity was detected at pH 3. The binding of glucose to 4-MPBA-incorporated GO@SiO2@Ag NPs increased the SERS signals at both 1,072 and 1,588 cm-1. The linear range was estimated from 2 to 20 mM glucose. These results provide insight into detection of glucose and the development of SERS-based biosensors using graphene oxide.

Magnetic porous carbon nanocomposites derived from metal-organic frameworks as a sensing platform for DNA fluorescent detection

Metal-organic frameworks (MOFs) have emerged as very fascinating functional materials due to their tunable nature and diverse applications. In this work, we prepared a magnetic porous carbon (MPC) nanocomposite by employing iron-containing MOFs (MIL-88A) as precursors through a one-pot thermolysis method. It was found that the MPC can absorb selectively single-stranded DNA (ssDNA) probe to form MPC/ssDNA complex and subsequently quench the labelled fluorescent dye of the ssDNA probe, which is resulted from the synergetic effect of magnetic nanoparticles and carbon matrix. Upon the addition of complementary target DNA, however, the absorbed ssDNA probe could be released from MPC surface by forming double-stranded DNA with target DNA, and accompanied by the recovery of the fluorescence of ssDNA probe. Based on these findings, a sensing platform with low background signal for DNA fluorescent detection was developed. The proposed sensing platform exhibits high sensitivity with detection limit of 1 nM and excellent selectivity to specific target DNA, even single-base mismatched nucleotide can be distinguished. We envision that the presented study would provide a new perspective on the potential applications of MOF-derived nanocomposites in biomedical fields.

A dual-probe surface-co-immobilization-based electrochemical sensor for highly sensitive and tunable specific detection of a designated DNA

A “signal on” electrochemical sensing method was established for the detection of a designated 24-base DNA. This sensor system shows a simple structure with only composing of three probes. One capturing probe (CP) and one assistant probe (AP) are co-immobilized onto the Au electrode surface in a short CP/AP duplex state, and one methylene blue (MB) modified signaling probe (SP) is free in the detection solution. Before adding the target, a low background signal helpful for improving the sensitivity is obtained with low efficient electron transfer because of the MB labels far from the electrode surface; after adding the target, the longer and more stable CP/target duplex formed between CP and the target undoes the former CP/AP duplex to release the AP, which induces the formation of another AP/SP duplex. This structure can drive the MB labels close to the electrode surface with much higher efficient electron transfer, thereby producing a large detection signal. The proposed sensor features a high sensitivity with the detection limit (DL) of ∼10 fM for the target. When used for discriminating the mismatched target, the sensor also exhibits a good ability and its high adjustability.

Screening of nerve agent markers with hollow fiber-chemosorption of phosphonic acids.

This report describes a method developed for extracting nerve gas markers such as phosphonic acids from urine and other aqueous samples. It involves single-step microextraction with chemosorption to hollow fibers that have been pre-soaked in a solution containing a derivatization reagent (3,5 triflouro methyl benzene diazomethane). The derivatives it forms with phosphonic acids can be sensitively detected by mass spectrometric detectors operating in negative chemical ionization (NCI) mode. Limits of quantification obtained in analyses of water and urine extracts by GC/MS in negative chemical ionization and selected ion monitoring mode were 0.1-10 and 0.5-10ng/mL, respectively. Pentaflourophenyl diazomethane can also be used as a derivatization reagent, and the micro-extracts (which generate low background signals) can be sensitively analyzed by GC-MS/MS in NCI selected reaction monitoring (SRM) mode, using two specific transitions for both reagents. Thus, this sensitive approach can be flexibly modified to obtain confirmatory information, or address potential problems caused by interferences in some samples.

Mirrored stainless steel substrate provides improved signal for Raman spectroscopy of tissue and cells

Raman spectroscopy (RS) is a powerful technique that permits the non‐destructive chemical analysis of cells and tissues without the need for expensive and complex sample preparation. To date, samples have been routinely mounted onto calcium fluoride (CaF2) as this material possesses the desired mechanical and optical properties for analysis, but CaF2 is both expensive and brittle and this prevents the technique from being routinely adopted. Furthermore, Raman scattering is a weak phenomenon and CaF2 provides no means of increasing signal. For RS to be widely adopted, particularly in the clinical field, it is crucial that spectroscopists identify an alternative, low‐cost substrate capable of providing high spectral signal to noise ratios with good spatial resolution. Results show that these desired properties are attainable when using mirrored stainless steel as a Raman substrate. When compared with CaF2, data show that stainless steel has a low background signal and provides an average signal increase of 1.43 times during tissue analysis and 1.64 times when analyzing cells. This result is attributed to a double‐pass of the laser beam through the sample where the photons from the source laser and the forward scattered Raman signal are backreflected and retroreflected from the mirrored steel surface and focused towards collection optics. The spatial resolution on stainless steel is at least comparable to that on CaF2 and it is not compromised by the reflection of the laser. Steel is a fraction of the cost of CaF2 and the reflection and focusing of photons improve signal to noise ratios permitting more rapid mapping. The low cost of steel coupled with its Raman signal increasing properties and robust durability indicates that steel is an ideal substrate for biological and clinical RS as it possesses key advantages over routinely used CaF2. © 2016 The Authors. Journal of Raman Spectroscopy Published by John Wiley & Sons Ltd.

The progress of luminescent assay in clinical diagnosis and treatment of diabetes mellitus

Diabetes mellitus is a worldwide proliferated noninfectious disease in these years. Although it is not dangerous for life, but the complications such as kidney disease, blindness, foot disease or nerve damage will result in severe degradation of the vital quality of the diabetics. So, the monitoring of those diabetes related indexes is very important for controlling and treatment of the disease. More accurate and convenient methods for their assay are in expect. The electrochemiluminescent (ECL) and chemiluminescent (CL) analysis have received high attention because of their excellent sensitivity, low back-ground signal and other advantages. Especially combined with some biological measures, they have shown excellent specificity, and flexibility for a wide range of analytes, leading to their usefulness in clinical tests including diabetes. This paper will review the progress of this luminescent assay for diabetes mellitus in present years.

Microelectrodes Integrated into a Microfluidic Chip for the Detection of CCRF‐CEM Cells Based on the Electrochemical Oxidation of Hydrazine

AbstractWe designed one type of microfluidic chip integrated with Au microelectrodes to detect target CCRF‐CEM cells based on the electrochemical oxidation of hydrazine catalyzed by Pt nanoparticles (NPs). The microelectrode can improve the sensitivity of detection, with a low background signal and decreased nonspecific adsorption. Applying the cell aptamer as the sensing element, this method exhibits good selectivity. Owing to the strong catalytic ability of Pt NPs for the electrochemical oxidation of hydrazine, the limit of detection of this method is ten cells. By adjusting the detection area of Au microelectrodes, this chip can detect a wide range of target cells. Besides, this method successfully detected the target cells doped in blood samples, indicating its potential application in clinical diagnosis.

OMIP‐034: Comprehensive immune phenotyping of human peripheral leukocytes by mass cytometry for monitoring immunomodulatory therapies

Keywords: mass cytometry; panel design; mass-response characteristic; human blood leukocytes; immune phenotyping; immune monitoring; autoimmunity; rheumatism; systemic lupus erythematosus (SLE); biomarker and clinical trial

Conjugated Polymer-Based Hybrid Materials for Turn-On Detection of CO2 in Plant Photosynthesis.

Detection of carbon dioxide (CO2) is of fundamental importance in diverse applications ranging from environmental analysis to agricultural production. In this work, a hybrid probe based on guanidinium-pendent oligofluorene (G-OF) and water-soluble conjugated polythiophene (PTP) has been developed for the turn on detection of CO2 with low background signal, taking advantage of the efficient fluorescence quenching of the tight aggregate of G-OF/PTP. In the presence of CO2, the electrostatic repulsion between G-OF and PTP can be effectively enhanced through protonation of the side chains, leading to the disaggregation and thus the "turn-on" fluorescence. The strategy allows for the light-up visible detection of CO2 with high sensitivity. Importantly, this system is capable of sensitively monitoring the concentration changes of CO2 in the process of the photosynthesis, which represents a concept to monitor the photosynthesis based on water-soluble conjugated polymers.

Paralogous Regulators ArsR1 and ArsR2 of Pseudomonas putida KT2440 as a Basis for Arsenic Biosensor Development.

The remarkable metal resistance of many microorganisms is related to the presence of multiple metal resistance operons. Pseudomonas putida KT2440 can be considered a model for these microorganisms since its arsenic resistance is due to the action of proteins encoded by the two paralogous arsenic resistance operons ARS1 and ARS2. Both operons contain the genes encoding the transcriptional regulators ArsR1 and ArsR2 that control operon expression. We show here that purified ArsR1 and ArsR2 bind the trivalent salt of arsenic (arsenite) with similar affinities (~30 μM), whereas no binding is observed for the pentavalent salt (arsenate). Furthermore, trivalent salts of bismuth and antimony showed binding to both paralogues. The positions of cysteines, found to bind arsenic in other homologues, indicate that ArsR1 and ArsR2 employ different modes of arsenite recognition. Both paralogues are dimeric and possess significant thermal stability. Both proteins were used to construct whole-cell, lacZ-based biosensors. Whereas responses to bismuth were negligible, significant responses were observed for arsenite, arsenate, and antimony. Biosensors based on the P. putida arsB1 arsB2 arsenic efflux pump double mutant were significantly more sensitive than biosensors based on the wild-type strain. This sensitivity enhancement by pump mutation may be a convenient strategy for the construction of other biosensors. A frequent limitation found for other arsenic biosensors was their elevated background signal and interference by inorganic phosphate. The constructed biosensors show no interference by inorganic phosphate, are characterized by a very low background signal, and were found to be suitable to analyze environmental samples. Arsenic is at the top of the priority list of hazardous compounds issued by the U.S. Agency for Toxic Substances and Disease. The reason for the stunning arsenic resistance of many microorganisms is the existence of paralogous arsenic resistance operons. Pseudomonas putida KT2440 is a model organism for such bacteria, and their duplicated ars operons and in particular their ArsR transcription regulators have been studied in depth by in vivo approaches. Here we present an analysis of both purified ArsR paralogues by different biophysical techniques, and data obtained provide valuable insight into their structure and function. Particularly insightful was the comparison of ArsR effector profiles determined by in vitro and in vivo experimentation. We also report the use of both paralogues to construct robust and highly sensitive arsenic biosensors. Our finding that the deletion of both arsenic efflux pumps significantly increases biosensor sensitivity is of general relevance in the biosensor field.

Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design.

Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared – non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

Non-Invasive Imaging of Amyloid Deposits in a Mouse Model of AGel Using 99mTc-Modified Nanobodies and SPECT/CT.

Gelsolin amyloidosis (AGel), also known as familial amyloidosis, Finnish type (FAF), is an autosomal, dominant, incurable disease caused by a point mutation (G654A/T) in the gelsolin (GSN) gene. The mutation results in loss of a Ca2+-binding site in the second gelsolin domain. Subsequent incorrect folding exposes a cryptic furin cleavage site, leading to the formation of a 68-kDa C-terminal cleavage product (C68) in the trans-Golgi network. This C68 fragment is cleaved by membrane type 1-matrix metalloproteinase (MT1-MMP) during secretion into the extracellular environment, releasing 8- and 5-kDa amyloidogenic peptides. These peptides aggregate and cause disease-associated symptoms. We set out to investigate whether AGel-specific nanobodies could be used to monitor amyloidogenic gelsolin buildup. Three nanobodies (FAF Nb1-3) raised against the 8-kDa fragment were screened as AGel amyloid imaging agents in WT and AGel mice using 99mTc-based single-photon emission computed tomography (SPECT)/X-ray tomography (CT), biodistribution analysis, and immunofluorescence (IF). The quantitative characteristics were analyzed in a follow-up study with a Nb11-expressing mouse model. All three nanobodies possess the characteristics desired for a99mTc-based SPECT/CT imaging agent, high specificity and a low background signal. FAF Nb1 was identified as the most potent, based on its superior signal-to-noise ratio and signal specificity. As a proof of concept, we implemented 99mTc-FAF Nb1 in a follow-up study of the Nb11-expressing AGel mouse model. Using biodistribution analysis and immunofluorescence, we demonstrated the validity of the data acquired via 99mTc-FAF Nb1 SPECT/CT. These findings demonstrate the potential of this nanobody as a non-invasive tool to image amyloidogenic gelsolin deposition and assess the therapeutic capacity of AGel therapeutics currently under development. We propose that this approach can be extended to other amyloid diseases, thereby contributing to the development of specific therapies.

One-pot hydrothermal route to fabricate nitrogen doped graphene/Ag-TiO2: Efficient charge separation, and high-performance “on-off-on” switch system based photoelectrochemical biosensing

Charge separation is crucial for increasing the performances of semiconductor-based materials in many photoactive applications. In this paper, we designed novel nanocomposites consisting of TiO2 nanocrystals, Ag nanoparticles (NPs) and nitrogen doped graphene (NGR) via a facile one-pot hydrothermal route. The as-prepared ternary nanocomposites exhibited enhanced photoelectrochemical (PEC) performances owing to the introduction of Ag NPs and NGR, which increase the excitons’ lifetime and improve the charge transfer. In particular, it is shown by means of the transient-state surface photocurrent responses that the photocurrent intensity of the as-fabricated composites exhibited 18.2 times higher than that of pristine TiO2. Based on the robust photocurrent signal, a new kind of “on-off-on” PEC aptasensor was established with the assistance of Pb2+ aptamer, which integrates the advantages of low background signal and high sensitivity. Under optimal conditions, a wide linear response for Pb2+ detection was obtained from 1pM to 5nM as well as a detection limit down to 0.3pM. With its simplicity, selectivity, and sensitivity, this proposed strategy shows great promise for Pb2+ detection in food and environment analysis.