Non-target Molecules Research Articles

A selective supramolecular photochemical sensor for dopamine

Dopamine (DA) is an important biomarker for diseases and biological disorders. Existing techniques for DA detection suffer from drawbacks including low sensitivity and selectivity as well as interfering signals from non-target molecules. A simple and selective photochemical sensor for the determination of DA in a supramolecular manner is presented. This approach utilises the complexation properties of a highly fluorescent water-soluble complex of perylene bis(diimide) dye with the macrocyclic host cucurbit[8]uril. The method can be used for the determination of DA in aqueous media, with detection limits below 2 × 10− 5 M, even in the presence of known interferents including ascorbic acid and the catecholamines epinephrine and norepinepherine.

Molecular Nanoshearing: An Innovative Approach to Shear off Molecules with AC-Induced Nanoscopic Fluid Flow

Early diagnosis of disease requires highly specific measurement of molecular biomarkers from femto to pico-molar concentrations in complex biological (e.g., serum, blood, etc.) samples to provide clinically useful information. While reaching this detection limit is challenging in itself, these samples contain numerous other non-target molecules, most of which have a tendency to adhere to solid surfaces via nonspecific interactions. Herein, we present an entirely new methodology to physically displace nonspecifically bound molecules from solid surfaces by utilizing a newly discovered “tuneable force”, induced by an applied alternating electric field, which occurs within few nanometers of an electrode surface. This methodology thus offers a unique ability to shear-off loosely bound molecules from the solid/liquid interface. Via this approach, we achieved a 5-fold reduction in nonspecific adsorption of non-target protein molecules and a 1000-fold enhancement for the specific capture of HER2 protein in human serum.

Benzimidazole-biologically attractive scaffold for protein kinase inhibitors

Great advances in elucidating molecular structures allow the precise determination of the interactions between a protein and a therapeutic agent. Enzyme inhibitors are used as a therapeutic agent with organic molecules, that interact with their targets through the weak linkages of hydrogen bonding and van der Waals interactions. These reduce the undesirable side effects and allow more non-specific interactions with non-target molecules. Benzimidazole acts as an enzyme inhibitor that may interact with different proteins and enzymes and has inspired chemists to carry out various structural variations of it. This review discusses the development of distinct benzimidazoles with an array of enzyme inhibitors viz., aurora kinase inhibitors, cyclin-dependent kinase inhibitors, mitogen activated protein kinase inhibitors, polo like kinase inhibitors, Tie kinase inhibitors, lymphocyte specific kinase inhibitors etc., also highlighting the molecular interaction with enzyme inhibitors. Various derivatives of benzimidazole, with different inhibitory activities, have been described on the basis of substitution around the central moiety, with an aim to help medicinal chemists to develop structure–activity relationships. The reviews in the literature till now are focused only on the biological activities of benzimidazole viz., antiviral, anticancer and antifungal, but the present review focuses on the latest work, describing the inhibitor aspects and the potential of the benzimidazole ring. This discussion will further help in the development of novel benzimidazole compounds.

Tamavidin 2-HOT, a highly thermostable biotin-binding protein

Tamavidin 2 is a fungal tetrameric protein that binds with high affinity to biotin, like avidin and streptavidin. We replaced asparagine-115, which lies in a subunit–subunit interface of tamavidin 2, with cysteine to generate the novel, highly thermostable protein tamavidin 2-HOT. Tamavidin 2-HOT retained more than 80% of its biotin-binding activity even after incubation at 99.9°C for 60min and was fully active in 70% dimethylsulfoxide for 30min, whereas in these harsh conditions, avidin, streptavidin, and tamavidin 2 lost their activities (less than 20% of their biotin-binding activities). The Tm in which the biotin-binding activity becomes half of tamavidin 2-HOT was 105°C, at least 20°C higher than those of avidin, streptavidin, and tamavidin 2. Because a reducing agent removed the thermal stability of tamavidin 2-HOT, the N115C mutation likely created disulfide bridges that stabilized inter-subunit associations. Tamavidin 2-HOT is efficiently produced in the soluble form by Escherichia coli for practical use. The isoelectric point of tamavidin 2-HOT (7.4) is sufficiently low to reduce the chance for non-specific binding of non-target molecules due to high positive charges. Therefore, tamavidin 2-HOT may be useful in diverse novel applications that take advantage of its high biotin-binding capability that can withstand harsh conditions.

Solid-Phase Colorimetric Sensor Based on Gold Nanoparticle-Loaded Polymer Brushes: Lead Detection as a Case Study

We introduce a novel solid-phase colorimetric sensor facilely fabricated by loading unmodified gold nanoparticles into poly(oligo(ethylene glycol)methacrylate) (POEGMA) brushes grown on glass. Our work reports the first synergistic combination of metallic nanoparticles acting as a colorimetric sensing module with a nonfouling polymer matrix acting both as a nonrigid scaffold and a screen to reduce interference from nontarget molecules. In addition, as the nanocomposite is formed on a transparent substrate, solid-phase detection can be performed in the same manner as in the solution-phase. We demonstrate the use of this unique platform for label-free lead detection based on the release of gold nanoparticles from the polymer brush upon exposure to lead ions. An ultralow limit-of-detection of 25 pM (S/N = 3) and a dynamic range of 100 pM to 100 nM (R(2) = 0.987) are achieved. Furthermore, the detection is up to 1000-fold more selective to lead over other common heavy metal ions.

Liquid Chromatography-Mass Spectrometric Multiple Reaction Monitoring-based Strategies for Expanding Targeted Profiling towards Quantitative Metabolomics

Recent advances in analytical methodologies have made it possible to bring metabolomic profiling into quantitative metabolomics that permits precise measurements of comprehensive small-molecule profiles. Modern liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) mode serves as the foundation for accurate simultaneous multi-analyte quantitation across large sample sets to provide high-quality information on target molecular profiles in complex systems. Despite the intrinsic multiplexing potential of the LC-MRM-MS technique, the key bottleneck in current LC-MRM-based assays is generally the limited analyte coverage and throughput capacity. Nowadays, the MRM-based approach has emerged as an attractive strategy for quantitative proteomic analysis and high-throughput biomarker discovery. So far, the full potential of the contemporary LCMRM methodology unleashed for quantitative metabolite profiling and metabolomic measurements of non-peptidic small molecules is rarely discussed. In this review we attempt to provide an overview on the major recent developments in LC-MRM-based strategies for quantitative profiling of multi- and non-target small molecules in biological samples. This article highlights the utility and power of the LC-MRM-based targeted approaches as valuable bioanalytical tools for low-cost, multiplexed quantitation on a large scale, with special emphasis on the promise of combining various strategies for expanding coverage and throughput of the LC-MRM-based assays to cover the gap between a widely targeted profiling and large-scale unknown screening towards comparative or quantitative metabolomics. General issues raised in metabolite profiling, such as basic aspects of bioanalysis, methodological dilemmas and challenges in quantitative metabolomics are addressed, and different strategies to circumvent the existing bottleneck and potential pitfalls of the current LC-MRM-MS techniques are outlined. In addition, the rudiments of LC-MRM-MS and its recent applications in combination with such strategies for biomarker quantitation and verification is also described.

Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics.

Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu.

Selective decomposition of nucleic acids by laser irradiation on probe-tethered gold nanoparticles in solution

We have developed a new method for selective decomposition of nucleic acids. The method utilizes a high temperature and pressure region (HTP region, hereafter) around a gold nanoparticle, which was generated when the gold nanoparticle was irradiated with a pulsed laser in aqueous solution. A probe DNA molecule whose sequence was complementary to a part of a target DNA molecule was bound to the gold nanoparticle surface. In a solution containing both the target and non-target DNA molecules, the gold nanoparticle selectively attached to the target DNA through hybridization of the probe DNA. When the gold nanoparticle was excited by a pulsed laser, the HTP region was generated in the close vicinity of the gold nanoparticle and then the target DNA molecules inside of this region were decomposed. The non-target DNA molecules having no part complementary to the probe DNA were scarcely decomposed by laser irradiation. When the gold nanoparticle was excited by an intense laser, the non-target DNA molecules were also decomposed, because some of them were located inside the inflated HTP region. We discussed the mechanism of the decomposition of the DNA molecules by the HTP region.

Nanofluidic Concentration of Selectively Extracted Biomolecule Analytes by Microtubules

We present a novel device for the selective extraction and high concentration of biomolecule analytes by integrating microtubules, one of cytoskeletal filaments, with nanofluidic technologies. Microtubules can be functionalized to provide numerous, nanoscopic binding sites for specific target biomolecules. The functionalized microtubules, with the target biomolecules bound to their surface, can be transported in the opposite direction to nontarget molecules using electrokinetic separation. Subsequently, the target molecule-bound microtubules are concentrated by a flat nanochannel structure, which filters the microtubules but allows ionic current and flow to pass through it. This device makes it possible to selectively extract target molecules such as streptavidin and bovine serum albumin and then highly concentrate them up to higher than 5 orders of magnitude from a complex mixture of analytes ranging from 1 nM to 10 fM. In addition, the device performs both extraction (separation) and concentration process simultaneously, which are typically performed in order in other devices, so that we significantly reduce analysis time and labor and even enable preconcentrated, identified target molecules to be available for postanalysis. Thus, we believe that the use of functionalized microtubules with nanofluidics will be a useful means to facilitate biochemical analysis systems.

Optical protein sensor for detecting cancer markers in saliva

A surface immobilized optical protein sensor has been utilized to detect Interleukin-8 (IL-8) protein, an oral cancer marker, and can reach limit of detection (LOD) at 1.1pM in buffer without using enzymatic amplification. Only after applying enzymatic amplification to increase the signal level by a few orders of magnitude, ELISA can reach the LOD of 1pM level. We then develop the confocal optics based sensor for further reducing the optical noise and can extend the LOD of the surface immobilized optical protein sensor two orders in magnitude. These improvements have allowed us to detect IL-8 protein at 4.0fM in buffer. In addition, these sensitive LODs were achieved without the use of enzymatic signal amplification, such that the simplified protocol can further facilitate the development of point-of-care devices.The ultra sensitive optical protein sensor presented in this paper has a wide number of applications in disease diagnoses. Measurements for detecting biomarkers in clinical sample are much more challenging than the measurements in buffer, due to high background noise contributed by large collections of non-target molecules. We used clinical saliva samples to validate the functionality of the optical protein sensor. Clinical detection of disease-specific biomarkers in saliva offers a non-invasive, alternative approach to using blood or urine. Currently, the main challenge of using saliva as a diagnostic fluid is its inherently low concentration of biomarkers. We compare the measurements of 40 saliva samples; half from oral cancer patients and half from a control group. The data measured by the optical protein sensor is compared with the traditional Enzyme-Linked Immunosorbant Assay (ELISA) values to validate the accuracy of our system. These positive results enable us to proceed to using confocal optical protein sensor to detect other biomarkers, which have much lower concentrations.

Enhanced Photocatalytic Degradation and Selective Removal of Nitrophenols by Using Surface Molecular Imprinted Titania

Poor selectivity of titania (TiO2) photocatalysis is unfavorable to photocatalytic removal of highlytoxic low-level organic pollutants in polluted waters in the presence of other less toxic high-level pollutants. A new strategy of increasing this selectivity is the surface modification of TiO2 via coating a thin layer of molecular imprinted polymer (MIP), which provides molecular recognition ability toward the template molecules. By using 2-nitrophenol and 4-nitrophenol as target pollutants, MIP-coated TiO2 photocatalysts were prepared via surface molecular imprinting and were observed to have high activity and selectivity toward the photodegradation of the targets. In the presence of bisphenol A (50 mg L(-1)) as a nontarget pollutant, the apparent rate constant for the photodegradation of the target 2-nitrophenol and 4-nitrophenol (1.8 mg L(-1)) over the corresponding MIP-coated TiO2 was 10.73 x 10(-3) and 7.06 x 10(-3) min(-1), being 2.46 and 4.61 times of that (4.36 x 10(-3) and 1.53 x 10(-3) min(-1)) over neat TiO2, respectively. The enhanced photocatalytic selectivity was increased when the concentration of the target was decreased and/or when the difference in both the chemical structure and molecule size between the target and nontarget molecules was increased. The increased selectivity was mainly attributed to the special interaction between the target molecules and the footprints polymer via the functional groups (-OH and -NO2).

SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem–loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.

Spinning-disk self-referencing interferometry of antigen-antibody recognition.

A gold ridge microstructure fabricated to a height of lambda/8 on a high-reflectivity substrate behaves as a wave-front-splitting self-referencing interferometer in phase quadrature when illuminated by a Gaussian laser beam and observed in the far field along the optic axis. When immuno-gammaglobulin (IgG) antibodies are selectively immobilized on the gold microstructure, they recognize and bind to a specific antigen, which shifts the relative optical phase of the interferometer and modifies the far-field diffracted intensity. We detect bound antigen interferometrically on spinning disks at a sampling rate of 100 kHz and verify the interferometric nature of the signal by using two quadratures of opposite sign to rule out effects of dynamic light scattering. Strong molecular recognition is demonstrated by the absence of binding to nontarget molecules but strong signal change in response to a specific antigen. This BioCD has the potential to be applied as a spinning-disk interferometric immunoassay and biosensor.

Specifically associated PCR products probed by coincident detection of two-color cross-correlated fluorescence intensities in human gene polymorphisms of methylene tetrahydrofolate reductase at site C677T: a novel measurement approach without follow-up mathematical analysis

Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born.

P53 Latency: C-TERMINAL DOMAIN PREVENTS BINDING OF p53 CORE TO TARGET BUT NOT TO NONSPECIFIC DNA SEQUENCES

The p53 transcription factor is either latent or activated through multi-site phosphorylation and acetylation of the negative regulatory region in its C-terminal domain (CTD). How CTD modifications activate p53 binding to target DNA sequences via its core domain is still unknown. It has been proposed that nonmodified CTD interacts either with the core domain or with DNA preventing binding of the core domain to DNA and that the fragments of the CTD regulatory region activate p53 by interfering with these interactions. We here characterized the sequence and target specificity of p53 activation by CTD fragments, interaction of activating peptides with p53 and target DNA, and interactions of "latent" p53 with DNA by a band shift assay and by fluorescence correlation spectroscopy. In addition to CTD fragments, several long basic peptides activated p53 and also transcription factor YY1. These peptides and CTD aggregated target DNA but apparently did not interact with p53. The potency to aggregate DNA correlated with the ability to activate p53, suggesting that p53 binds to target sequences upon interactions with tightly packed DNA in aggregates. Latent full-length p53 dissociated DNA aggregates via its core and CTD, and this effect was potentiated by GTP. Latent p53 also formed complexes via both its core and CTD with long nontarget DNA molecules. Such p53-DNA interactions may occur if latent p53 binding to DNA via CTD prevents the interaction of the core domain with target DNA sites but not with nonspecific DNA sequences.

DETECTION AND QUANTITATIVE MEASUREMENT OF TRANSFORMING GROWTH FACTOR-β1 (TGF-β1) GENE EXPRESSION USING A SEMI-NESTED COMPETITIVE PCR ASSAY

A polymerase chain reaction (PCR) technique was optimized for detection and quantification of very low concentrations (down to a few molecules) of transforming growth factor β1 (TGF-β1) mRNA. The strategy involved a combination of a competitive PCR assay and a semi-nested PCR. In the present study, the semi-nested PCR technique was tested in several rat organs containing different concentrations of target mRNA. A control fragment for TGF-β1 was used to correct for differences in amplification of various cDNA samples. TGF-β1 mRNA levels were also corrected according to the abundance of the “housekeeping” gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same samples. The differences of sensitivity among the standard (one-step) and semi-nested (two-step) competitive PCR assays for the detection of TGF-β1 are discussed. In conclusion, the semi-nested PCR protocol provides greatly enhanced sensitivity over standard PCR analysis. It is a reproducible and very specific method for quantification of only a few molecules of TGF-β1 mRNA in a background of non-target molecules.

Affinity capture electrophoresis for sequence-specific DNA purification

A new method, affinity capture electrophoresis (ACE), has been developed for the sequence-specific isolation of DNA. The target DNA is complexed with a biotinylated probe and electrophoresed in a gel equipped with a trap of immobilized streptavidin. This selectively captures the target molecules and its biotinylated probe, while other nontarget molecules pass through the trap. The target DNA is subsequently recovered from the trap by destroying the interaction between the target DNA and the biotinylated probe. Two variations of this technique, one using triple-helix formation and the other using hybridization with a uracil-containing DNA probe at the end of the target fragment, proved effective in model experiments. Since this technique requires no denaturation and handles DNA inside an agarose gel matrix, it is, in principle, applicable to the isolation of very large DNAs.

A target-radical hypothesis of radiation damage fixation and modification in oxygenated and hypoxic cells

Radicals in radiation target molecules (presumed to be DNA), are produced by ionizing radiation in at least two ways: Solute and solvent radicals R’ react with target molecules, or ionizations in target molecules become radicals. T’. An intracellular radiation-chemical reaction scheme is therefore proposed in which solute and solvent radicals react with non-target molecules S. (scavengers) or with target molecules (DNA) to produce target radicals. The probability of target radical production is affected by scavenger molecules, and the probability of target radical decomposition to become “uncommitted damage” that the cell may repair is affected by the concentrations of 02, thiols and electronaff,nic sensitizers. which compete with one another to form target-radical products TO:, T&, and TL. This uncommitted damage is then subject to biochemical modification by the cell. The modification results in either “fixation” into F,T02 or F,TL (molecular lesions that are permanent) or enzymatic “repair” through T T’+L-TL:andT+&-T.$, which produce uncommitted chemical damage. Experimental data on strand-break induction in glutathioneproficient and -deficient cells. the inactivation of cells and the computer simulations generally agree that RSH molecules act as scavengers of solvent and solute radicals (R’) in an Ol-independent manner and react with target radicals to reverse T in competition with O2 and/or electronaffinic sensitizers It is postulated that the two categories of uncommitted target lesions TO2 and TL are preferentially repaired by different pathways. Evidence favoring this notion is available in existing literature. Reported values of dose-modification factors when DNA strandbreakage (uncommitted damage) and ceil survival (fixed, unrepaired damage) are the end-points and cells are treated with 02, SH compounds, SH depletion. or electronafTinic sensitizers are compared with calculated values to evaluate the plausibility of the proposed reaction scheme.