Indirect Detection Strategies Research Articles

A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine.

More than 170 posttranscriptional RNA modifications are so far known on both coding and noncoding RNA species. Within this group, pseudouridine (Ψ) and queuosine (Q) represent conserved RNA modifications with fundamental functional roles in regulating translation. Current detection methods of these modifications, which both are reverse transcription (RT)-silent, are mostly based on the chemical treatment of RNA prior to analysis. To overcome the drawbacks associated with indirect detection strategies, we have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT signatures specific for Ψ or Q without prior chemical treatment of the RNA samples. Combining this polymerase with next-generation sequencing techniques allows the direct identification of Ψ and Q sites of untreated RNA samples using a single enzymatic tool.

An indirect detection strategy-assisted self-cleaning electrochemical platform for in-situ and pretreatment-free detection of endogenous H2S from sulfate-reducing bacteria (SRB)

The endogenous hydrogen sulfide (H2S) can be adopted as an indicator for the indirect detection of sulphate-reducing bacteria (SRB), which considered to be closely related to pipeline corrosion and human intestinal health. Unfortunately, the in-situ detection of endogenous H2S from SRB in the complex culture medium still faces huge challenges. Besides nonspecific adsorption from the culture medium of SRB, the problem of electrode passivation by produced elemental sulfur during electrochemical detection processes of H2S cannot be ignored. To address these challenges, herein a synergistic sensing platform based on self-cleaning electrode interface and indirect detection strategy (specific H2S-induced chemical reaction) is developed. This indirect sensing strategy-assisted self-cleaning electrochemical platform showed a relatively good linear response toward H2S in the range of 0.5 − 5 μM, and the corresponding limit of detection (LOD) was calculated to be 5.09 nM. More importantly, the satisfactory self-cleaning electrode interface in indirect detection system (with only a 4.10% decrease in signal over 50 electrochemical repeated cycles) showed the electrode surface not being disturbed by elemental sulfur. Furthermore, this good selectivity of the indirect detection strategy in combination with the reproducibility, stability, and antifouling activity of the self-cleaning interface, enabled a synergistic sensing platform to detect H2S directly in the complex culture medium of SRB without time-consuming sample pretreatments. Moreover, this proposed construction strategy of synergetic sensing platform could be explored to other endogenous molecules in complex environment based on different antifouling materials and specific reactions.

Rapid urinary albumin detection using a simple redox cycling process coupled with a paper-based device

• An electrochemical-chemical (EC) redox cycling process using a mixture of potassium ferricyanide and methylene blue was proposed for sensitive and rapid detection of urinary albumin in 1 min. • A disposable electrochemical paper-based analytical device (ePAD) was proposed as a new detection platform and easily get rid of waste. • The highly sensitive and selective albumin detection was obtained without creatinine correction. • The main features are the use of low-cost devices, rapid detection, and simple procedures. A novel electrochemical-chemical (EC) redox cycling process was proposed for the sensitive, simple, and rapid detection of urinary albumin. This system employs a unique detection scheme based on the combination of ferricyanide ([Fe(CN) 6 ] 3− ) and methylene blue (MB) for signal amplification without the need for any electrode surface modifications. Measurements of bovine serum albumin (BSA) were performed as proof of concept using disposable electrochemical paper-based analytical devices (ePADs) as detection platforms. The quantification of albumin is accomplished by using an indirect detection strategy. In the absence of BSA, electron transfer via EC redox cycling process occurs readily between the redox species and the electrode surface, resulting in a large electrochemical signal. The redox species solution containing BSA, on the other hand, provides a low signal current due to the hindrance of BSA on the electrode surface, which impedes the electron transfer of the proposed redox probe. Under the optimized conditions, the linear range was 1–500 mg dL −1 with a detection limit of 0.072 mg dL −1 . This finding leads to the development of a powerful method for the direct detection of albumin at a low concentration and no requirement for correction by creatinine. The proposed devices were successfully used to detect albumin in real urine samples with satisfactory results, demonstrating good agreement with the standard method and the reliability. Aside from the ease and rapidity, the proposed method is inexpensive, sensitive, and a promising tool for clinical diagnosis of chronic kidney disease and suitable for point-of-care testing in developing countries.

Exploration of a magnetite deposit buried under thick and conductive sediments with multiple-source CSEM

The North China Craton (NCC) hosts abundant mineral resources, making it economically an important Precambrian block in China. The thick conductive loess strata in NCC presents a great challenge for successful application of controlled-source electromagnetic method (CSEM) by masking the deeper targets due to thick loess strata of conductive nature. In this work, we proposed a multiple-source CSEM method that addresses these weaknesses by using multiple-sources with inline and broadside configurations. The short offset was adopted to increase the S/N. Joint inversion of inline and broadside sources datasets enhance the detection accuracy. Based on an indirect detection strategy and the known ore-forming information, a deeply seated ore body at 1157 m was delineated.

β-Cyclodextrin-Stabilized Au Nanoparticles for the Detection of Butyl Benzyl Phthalate

Currently, developing new types of simple and effective detection methods of phthalate plasticizers (PAEs) remains challenging, whereas the detection process of the traditional instruments is tedious and laborious. With regard to PAEs, their intrinsically poor Raman activity and very low affinity onto metallic surfaces seriously obstruct the realization of directly effective surface-enhanced Raman spectroscopy (SERS) detection. Here, a dual-mode sensing system with the effective and specific detection of trace butyl benzyl phthalate (BBP, one of PAEs) was demonstrated by constructing β-cyclodextrin (β-CD) stabilized AuNPs (AuNPs@β-CD) colloid. The AuNPs@β-CD assemblies could achieve the enrichment of trace BBP by the hydrophobic cavities of β-CD units, which significantly improved the detection sensitivity. Serving as macromolecular binder and switchable Raman reporters, BBP could assemble AuNPs@β-CD into photonic clusters with different shapes. The obtained BBP-triggered AuNPs@β-CD clusters are able to amplify the corresponding SERS signals due to the abundant hot spots with a lowest detection limit of 0.01 μM. In the colorimetric analysis of detecting BBP, the colorimetric distinguishable response can be accurately quantified via UV–vis spectroscopy with AuNPs@β-CD clusters acting as naked-eye indicators and a detection limit as low as 14.9 nM. Particularly, the proposed colorimetric/SERS sensor was applied to evaluate BBP in liquor and rice wine and the recovery of BBP added to liquor and rice wine samples was 90–108% for UV–vis spectroscopy and 87–109% for SERS assay. The corresponding vibrational bands in supramolecular complexes composed of BBP and β-CD was recognized by the density functional theoretical simulation. Our proposed indirect detection strategy of BBP opens a new avenue to design novel and effective solvatochromic and/SERS-based sensing systems in the field of trial applications.

Challenges in the electrochemical (bio)sensing of nonelectroactive food and environmental contaminants

Abstract Electrochemical detection of nonelectroactive contaminants can be successfully faced via the use of indirect detection strategies. These strategies can provide sensitive and selective responses often coupled with portable and user-friendly analytical tools. Indirect detection strategies are usually based on the change in the signal of an electroactive probe, induced by the presence of the target molecule at a modified electrode. This critical review aims at addressing the developments in indirect electrosensing strategies for nonelectroactive contaminants in food and environmental analysis in the last few years. Emphasis is given to the strategy design, the electrode modifiers used and the feasibility of technological transfer.

An ultra-sensitive aptasensor on optical fibre for the direct detection of bisphenol A

We present a plasmonic biosensor capable of detecting the presence of bisphenol A in ultra-low concentrations, yielding a wavelength shift of 0.15 ± 0.01 nm in response to a solution of 1 fM concentration with limit of detection of 330 ± 70 aM The biosensing device consists of an array of gold nano-antennae with a total length of 2.3 cm that generate coupled localised surface plasmons (cLSPs) and is covalently modified with an aptamer specific for bisphenol A recognition. The array of nano-antennae is fabricated on a lapped section of standard telecommunication optical fibre, allowing for potential multiplexing and its use in remote sensing applications. These results have been achieved without the use of enhancement techniques and therefore the approach allows the direct detection of bisphenol A, a low molecular weight (228 Da) target usually detectable only by indirect detection strategies. Its detection at such levels is a significant step forward in measuring small molecules at ultra-low concentrations. Furthermore, this new sensing platform paves the way for the development of portable systems for in-situ agricultural measurements capable of retrieving data on a substance of very high concern at ultra-low concentrations.

Achievements and Challenges in Anti-Doping Research.

The most important element in achieving athlete compliance with anti-doping rules is the certainty of detection. Thus, scientific research plays a mission critical role in achieving clean competition. Many factors contribute to the advances in detection. Incremental advances in the ability to detect prohibited substances and methods, and identification of long-lived metabolites continue to lengthen detection windows. While the athlete biological passport hematological and steroidal modules hold great promise, experience shows that new research is needed to improve the sensitivity and specificity of the approach for current doping techniques. Indirect detection strategies using biomarkers or transcriptomic techniques have been increasingly investigated. The incorporation of more cost-effective sampling strategies using dried blood and plasma spots, oral fluid, and breath analysis show great promise toward increasing the number of tests while remaining within testing budget constraints. Despite the importance of research to ensuring rule compliance, a major challenge for anti-doping research is achieving and maintaining sufficient funding in the reality of the myriad of new substances introduced for disease treatment but abused for performance enhancement. In addition, obtaining metabolism and population reference range data, particularly for new drugs or designer drugs that have not obtained approval for administration to human subjects, remains a significant problem. Nevertheless, research continues to contribute important data to support anti-doping efforts.

Gamma-ray boxes from axion-mediated dark matter

We compute the gamma-ray output of axion-mediated dark matter and derive the corresponding constraints set by recent data. In such scenarios the dark matter candidate is a Dirac fermion that pair-annihilates into axions and/or scalars. Provided that the axion decays (at least partly) into photons, these models naturally give rise to a box-shaped gamma-ray spectrum that may present two distinct phenomenological behaviours: a narrow box, resembling a line at half the dark matter mass, or a wide box, spanning an extensive energy range up to the dark matter mass. Remarkably, we find that in both cases a sizable gamma-ray flux is predicted for a thermal relic without fine-tuning the model parameters nor invoking boost factors. This large output is in line with recent Fermi-LAT observations towards the galactic centre region and is on the verge of being excluded. We then make use of the Fermi-LAT and H.E.S.S. data to derive robust, model-independent upper limits on the dark matter annihilation cross section for the narrow and wide box scenarios. H.E.S.S. constraints, in particular, turn out to match the ones from Fermi-LAT at hundreds of GeV and extend to multi-TeV masses. Future Čerenkov telescopes will likely probe gamma-ray boxes from thermal dark matter relics in the whole multi-TeV range, a region hardly accessible to direct detection, collider searches and other indirect detection strategies.

PROBING ISOSPIN-VIOLATING DARK MATTER

We discuss experimental probes of isospin-violating dark matter (IVDM), including direct and indirect detection strategies. We point out the important role which IVDM plays in understanding recent data regarding low-mass dark matter, and describe strategies for finding evidence of IVDM at current and upcoming experiments.

Antibody microarray‐based profiling of complex specimens: systematic evaluation of labeling strategies

Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined in this study the suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis. N-hydroxysuccinimidyl ester (NHS)- and Universal Linkage System (ULS)-based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labeling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labeling were analyzed and optimized in this study. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labeling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Finally, particularly sensitive detection chemistry was applied to monitoring cytokine production of stimulated peripheral T cells. Microarray data were in accord with quantitative cytokine levels measured by ELISA and Luminex, demonstrating comparable reliability and femtomolar range sensitivity of the established microarray approach.

Separation of acetylated amino acids

Acetylated amino acids (Nac-AA) are separated as anions on a reversed stationary phase from a mobile phase containing a quaternary ammonium (R 4N +) salt as a mobile phase additive. If the counteranion accompanying the R 4N + or ionic strength salt is detector active than the separated NAc-AA derivatives can be detected by an indirect detection strategy. Variables influencing the separations are NAc-AA side chain structure and the mobile phase parameters such as hdyrophobicity of the alkyl groups in the R 4N + salt, R 4N + salt concentration, counteranion eluent strength, counteranion concentration, solvent composition, and pH. Indirect detection is influenced by these same mobile phase parameters as well as the properties of the detector active counteranion. The detection limit for indirect photometric detection at 287 nm using a tetrapentylammonium salt with a disodium 1,5-naphthalenedisulfonate—sodium benzoate counteranion mixture was about 70 pmol of NAc-AA depending on the amino acid injected as a 10-μ1 sample.

Liquid chromatography and postcolumn indirect detection of glyphosate

Glyphosate [N-(phosphonomethyl)glycine] and its metabolite aminomethylphosphonic acid (AMPA) were separated and detected by a postcolumn indirect detection strategy. Separation can be done on a cation-exchange column, where glyphosate elutes before AMPA, or on an anion-exchange column, where the elution order is reversed. Detection was achieved by using a fluorescent Al 3+—morin postcolumn reagent. When the postcolumn reagent combines with the column effluent in a mixing tee, the fluorescence decreases in the presence of both analytes. Variables affecting the postcolumn indirect fluorescence detection were established and optimized; the major factors were postcolumn pH and volume and temperature of the postcolumn reaction coil. Detection limits, defined as three times the background noise, for glyphosate and AMPA separated on an anion-exchange column were 14 and 40 ng, respectively.