Molecular Recognition Elements Research Articles

DNAzyme biosensors for the detection of pathogenic bacteria

• DNAzymes can be used for the detection and diagnosis of pathogenic bacteria. • DNAzymes can be used to detect pathogenic bacteria in food and environments. • DNAzymes have potential applications in molecules detection. • RNA-cleaving DNAzymes as a molecular beacon have great advantages in fabrication of sensors. Pathogenic bacteria contamination can cause many problems, such as infections in humans and animals and environmental pollution. Early prevention and detection of pathogenic bacteria outbreaks is crucial to managing these problems. Some traditional detection methods such as cell culturing and polymerase chain reaction (PCR) are time-consuming, labor-intensive, insensitive, and inconvenient for detection. Therefore, simple and efficient pathogenic bacteria detection methods involving DNAzymes signaling mechanisms have been reported, including fluorescence and color detection. DNAzymes are divided into cases as a molecular recognition element (RNA-cleaving DNAzyme) and as a reporter element (peroxidase mimicking DNAzyme), which have proven compatible with isothermal amplification technology for pathogenic bacteria detection. In this review, we summarize the current methods of detecting pathogenic bacteria based on various DNAzyme sensors. We include the design principles, working mechanisms, and detection applications of these sensors and discuss the experimental and theoretical bases for their application in the diagnosis and prevention of diseases caused by pathogenic bacteria.

Highly toxic type Ⅱ ribosome-inactivating proteins ricin and abrin and their detection methods: a review

Ⅱ型核糖体失活蛋白(RIPs)是一类重要的蛋白毒素,该类毒素大都具有一对二硫键连接的A-B链结构特征,B链具有半乳糖结合特性,能够与真核细胞膜表面受体特异性结合,将具有N-糖苷酶活性的A链导入细胞,与核糖体特定位点发生脱嘌呤作用使核糖体失活,最终通过抑制蛋白质合成而展现出细胞毒性。Ⅱ型RIPs毒素毒性极强,来源于植物的蓖麻毒素(ricin)和相思子毒素(abrin)的毒性分别是神经性毒剂维埃克斯(Vx)的385倍和2885倍。同时,该类毒素来源广泛、易于制备、稳定性好,成为一类潜在化生恐怖战剂,受到国内外广泛关注,其中蓖麻毒素作为唯一的蛋白毒素被收录于禁止化学武器公约禁控清单。近年来发生的多次蓖麻毒素邮件恐怖事件,进一步促进了有关Ⅱ型RIPs毒素的准确、灵敏、快速的检测鉴定技术的发展。剧毒性Ⅱ型RIPs毒素的检测鉴定方法主要涉及免疫分析法为代表的特异性识别和生物质谱分析为主的定性定量检测方法,以及基于脱嘌呤反应活性和细胞毒性的毒素活性检测方法。基于抗原-抗体作用的免疫检测法及基于寡核苷酸适配体的特异性识别检测法具有速度快、灵敏度高的优势,但对于复杂样品中高度同源蛋白的检测,易产生假阳性结果。随着生物质谱技术的快速发展,电喷雾离化(ESI)或基质辅助激光解吸离化(MALDI)等技术广泛应用于蛋白质的准确鉴定,不仅能够提供蛋白毒素的准确分子量和结构序列信息,而且能够实现准确定量。酶解质谱法是应用最为广泛的检测鉴定方法,通过酶解肽指纹谱分析,实现蛋白毒素的准确鉴定;对于复杂样品中蛋白毒素的分析,通过多种蛋白酶酶解策略获得丰富的特异性肽段标志物,然后进行肽段标志物的靶向质谱分析从而获得准确的定性及定量信息,方法有效提升了Ⅱ型RIPs毒素鉴定的准确度和灵敏度。免疫分析法和生物质谱法能够准确鉴定Ⅱ型RIPs毒素,但无法识别毒素是否还保持毒性。对于Ⅱ型RIPs毒素的活性分析,主要包括基于N-糖苷酶活性的脱嘌呤反应测定法和细胞毒性测定法,两种方法均可实现毒素毒性的简便、快速、灵敏的分析检测,是Ⅱ型RIPs毒素检测方法的有效补充。由于该类毒素的高度敏感性,国际禁止化学武器组织(OPCW)对相关样品中Ⅱ型RIPs毒素的分析提出了唯一性鉴定的技术要求。该文引用了Ⅱ型RIPs毒素及其检测方法相关的70篇文献,综述了以上Ⅱ型RIPs毒素的结构性质、中毒机理及典型剧毒性Ⅱ型RIPs毒素检测方法的研究进展,对不同检测方法的特点和应用潜力进行了总结,并结合OPCW对Ⅱ型RIPs毒素唯一性鉴定的技术需求,展望了未来Ⅱ型RIPs毒素检测技术研究的发展趋势。

Aptamers in biomedicine: Selection strategies and recent advances

Aptamers have come in the spotlight as bio-mimetic molecular recognition elements in the field of biomedicine due to various applications in diagnostics, drug delivery, therapeutics, and pharmaceutical analysis. Aptamers are composed of nucleic acid strands (DNA or RNA) that can specifically interact in a three-dimensional tailored design with the target molecule.The basic method to generate aptamers is Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Recent technological advances in aptamer selection allow for faster and cheaper production of a new generation of high-affinity aptamers compared to the traditional SELEX, which can last up to several months. Rigorous characterization performed by multiple research groups endorsed several well-defined aptamer sequences. Binding affinity, nature of the biomolecular interactions and structural characterization are of paramount importance for aptamer screening and development of applications.However, remarkable challenges still need to be dealt with before the aptamers can make great contributions to the biomedical field. Poor specificity and sensitivity, questionable clinical use, low drug loading, in vivo stability and toxicity are only some of the identified challenges.This review accounts for the 30th celebration of the SELEX technology underlining the most important aptamers’ achievements in the biomedical field within mostly the past five years. Aptamers’ advantages over antibodies are discussed. Because of potential clinical translational utility, insights of remarkable developments in aptamer-based methods for diagnosis and monitoring of disease biomarkers and pharmaceuticals are discussed focusing on the recent studies (2015–2020). The current challenges and promising opportunities for aptamers for therapeutic and theragnostic purposes are also presented.

Aptamer Conformational Change Enables Serotonin Biosensing with Nanopipettes.

We report artificial nanopores in the form of quartz nanopipettes with ca. 10 nm orifices functionalized with molecular recognition elements termed aptamers that reversibly recognize serotonin with high specificity and selectivity. Nanoscale confinement of ion fluxes, analyte-specific aptamer conformational changes, and related surface charge variations enable serotonin sensing. We demonstrate detection of physiologically relevant serotonin amounts in complex environments such as neurobasal media, in which neurons are cultured in vitro. In addition to sensing in physiologically relevant matrices with high sensitivity (picomolar detection limits), we interrogate the detection mechanism via complementary techniques such as quartz crystal microbalance with dissipation monitoring and electrochemical impedance spectroscopy. Moreover, we provide a novel theoretical model for structure-switching aptamer-modified nanopipette systems that supports experimental findings. Validation of specific and selective small-molecule detection, in parallel with mechanistic investigations, demonstrates the potential of conformationally changing aptamer-modified nanopipettes as rapid, label-free, and translatable nanotools for diverse biological systems.

DNA Electrochemical Biosensors for In Situ Probing of Pharmaceutical Drug Oxidative DNA Damage.

Deoxyribonucleic acid (DNA) electrochemical biosensors are devices that incorporate immobilized DNA as a molecular recognition element on the electrode surface, and enable probing in situ the oxidative DNA damage. A wide range of DNA electrochemical biosensor analytical and biotechnological applications in pharmacology are foreseen, due to their ability to determine in situ and in real-time the DNA interaction mechanisms with pharmaceutical drugs, as well as with their degradation products, redox reaction products, and metabolites, and due to their capacity to achieve quantitative electroanalytical evaluation of the drugs, with high sensitivity, short time of analysis, and low cost. This review presents the design and applications of label-free DNA electrochemical biosensors that use DNA direct electrochemical oxidation to detect oxidative DNA damage. The DNA electrochemical biosensor development, from the viewpoint of electrochemical and atomic force microscopy (AFM) characterization, and the bottom-up immobilization of DNA nanostructures at the electrode surface, are described. Applications of DNA electrochemical biosensors that enable the label-free detection of DNA interactions with pharmaceutical compounds, such as acridine derivatives, alkaloids, alkylating agents, alkylphosphocholines, antibiotics, antimetabolites, kinase inhibitors, immunomodulatory agents, metal complexes, nucleoside analogs, and phenolic compounds, which can be used in drug analysis and drug discovery, and may lead to future screening systems, are reviewed.

Sarcomeric Protein Fatz Forms a Tight Fuzzy Complex with α-Actinin and Phase-Separates in Vitro

In sarcomeres, α-actinin crosslinks actin filaments and anchors them to the Z-disk. FATZ proteins (filamin, α-actinin and telethonin binding proteins of the Z-disk) interact with α-actinin and five other core Z-disk proteins, contributing to myofibril assembly and maintenance as a protein interaction hub. However, the molecular mechanism behind the FATZ scaffolding function is unknown. Here, we investigated the interaction of FATZ-1 with α-actinin-2 using an integrative structural biology approach. We provide the first structure of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which alone is best described as a dynamic ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2, and propose a molecular mechanism for interaction via molecular recognition elements and additional transient binding sites, leading to differential affinities of two FATZ-1 molecules for the α-actinin-2 dimer and a polar architecture of the assembly. in combination with FATZ-1 multivalent scaffolding function, this assembly is likely to organise interaction partners and stabilise the preferential orientation of α-actinin-2 in Z-disk.Finally, we uncover the ability of FATZ-1 to phase-separate and form biomolecular condensates with α-actinin-2, suggesting an intriguing hypothesis of FATZ proteins as an interaction hub for Z-disk partners through membrane-less compartmentalization at initial stages of myofibrillogenesis.

Fabrication of polydopamine/hemin-cyclodextrin supramolecular assemblies for mimicking natural peroxidases and their sensitive detection of cholesterol

The development of peroxidase mimics can overcome shortcomings of natural peroxidases and expand their biological applications. Herein, inspired by Hemeproteins family such as peroxidase and cytochromes, polydopamine/hemin-β-cyclodextrin (PDA/Hemin-CD) supramolecular assemblies were prepared to mimick natural peroxidases using hemin as the active center. Polydopamine acts as skeleton material which could encapsulate hemin molecules during the self-assembly procedure. Mono-(6-Mercapto-6-deoxy)-β-Cyclodextrin (β-CD-SH) were decorated on the surface of PDA/Hemin serving as the molecular recognition elements. Interestingly, the addition of cholesterol could “turn off” the peroxidase activity of PDA/Hemin-CD due to the specific recognition of β-CD to cholesterol. The excellent peroxidase activity as well as molecular recognition nature of PDA/Hemin-CD make it promising candidate to construct biosensors. Based on this, a colorimetric assay for the detection of cholesterol was developed.

A Highly Specific DNA Aptamer for RNase H2 from Clostridium difficile.

Molecular recognition elements with high specificity are of great importance for the study of molecular interactions, accurate diagnostics, drug design, and personalized medicine. Herein, a highly specific DNA aptamer for RNase H2 from Clostridium difficile (C. difficile) was generated by SELEX and minimized to 40 nucleotides. The aptamer exhibits a dissociation constant (Kd) of 1.8 ± 0.5 nM and an inhibition constant (IC50) of 7.1 ± 0.6 nM for C. difficile RNase H2, both of which are 2 orders of magnitude better for the same enzyme from other control bacteria. The fluorescent version of the aptamer can distinguish C. difficile from several other control bacteria in a cell lysate assay. This work demonstrates that a ubiquitous protein like RNase H2 can still be used as the target for the development of highly specific aptamers and the combination of the protein and the aptamer can achieve the recognition specificity needed for a diagnostic test and drug development.

Faradaic electrochemical impedance spectroscopy for enhanced analyte detection in diagnostics

Electrochemical impedance spectroscopy (EIS) is a widely implementable technique that can be applied to many fields, ranging from disease detection to environmental monitoring. EIS as a biosensing tool allows detection of a broad range of target analytes in point-of-care (POC) and continuous applications. The technique is highly suitable for multimarker detection due to its ability to produce specific frequency responses depending on the target analyte and molecular recognition element (MRE) combination. EIS biosensor development has shown promising results for the medical industry in terms of diagnosis and prognosis for various biomarkers. EIS sensors offer a cost-efficient system and rapid detection times using minimal amounts of sample volumes, while simultaneously not disturbing the sample being studied due to low amplitude perturbations. These properties make the technique highly sensitive and specific. This paper presents a review of EIS biosensing advancements and introduces different detection techniques and MREs. Additionally, EIS's underlying theory and potential surface modification techniques are presented to further demonstrate the technique's ability to produce stable, specific, and sensitive biosensors.

Autocatalytic MNAzyme-integrated surface plasmon resonance biosensor for simultaneous detection of bacteria from nosocomial bloodstream infection specimens

Early accurate profiling of pathogenic bacteria in patients with nosocomial bloodstream infections (nBSIs) is crucial to optimize antibiotic therapy and patient survival. We present a novel autocatalytic Multi-component DNAzyme (MNAzyme)-integrated surface plasmon resonance (SPR) biosensor assay that rapidly and sensitively detects the DNA of three nBSI pathogenic bacteria. This strategy combines autocatalytic MNAzyme cleavage on magnetic beads, as the molecular recognition and signal amplification element, with hybridization chain reaction (HCR)-mediated nanowire assembly on SPR sensing film, as the signal readout and further amplification. Once the MNAzyme complex was activated as a biocatalyst by co-recognizing the bacterial DNA and initial probes, the MNAzyme substrate was catalytically cleaved into two DNA segments. One hybridized with immobilized probes triggering folded hairpin probes to self-assemble into massive nanowire structures via HCR on the sensing interface, and the other co-recognized the initial probes trigging another cycle of multi-turnover cleavage. As a result, the amount of cleaved DNA segments was increasing and greatly enhancing the SPR signal from the analyte. This assay system can provide the fast and quantitative analysis at low concentration (67 cfu/mL of S. aureus, 57 cfu/mL of K. pneumonia and 61 cfu/mL of E. coli, respectively) in a nonenzymatic manner. More importantly, the biosensor was successfully applied to DNA extracted from clinical blood specimens of patients with nBSIs, showing good consistence and practicability. The developed autocatalytic MNAzyme-integrated SPR biosensor provides a sensitive, rapid, reliable and culture-independent approach to efficiently determine multiple pathogenic bacteria for the early diagnosis of nBSIs.

Development of Insulin Sensors Based on Faradaic and Non-Faradaic Electrochemical Impedance Spectroscopy Detection Principle Using Single Chain Antibody (scFv)

IntroductionIn this paper, we report novel insulin sensors using insulin single-chain antibody (scFv) based on two electrochemical impedance spectroscopy (EIS) methods; faradaic EIS and non-faradaic EIS. For faradaic EIS measurement, varieties of molecular recognition elements (MREs) can be utilized, such as IgG and binding proteins, without consideration of their molecular size. However, a solubilized redox probe is necessary to aid the transfer of electrons and detection is based on the flux of electrons and the obstruction that is present on the electrode surface. Non-faradaic EIS does not require a solubilized redox probe and is based around changes within the electrical double layer. However, there are the limitation of the size for MREs, since the ideal size of MREs should be smaller than the size of electrical double layer (EDL). In this paper, thanks to the small size of scFv as MRE for EIS, we construct two different types of EIS based insulin sensors, and compare the suitability in the use for insulin monitoring, being dedicated for the management of diabetic patients.MethodsDevelopment of this sensor requires immobilization of a molecular recognition element (MRE) that binds with insulin. For this purpose, we have cultivated single-chain insulin antibody by transforming an Escherichia coli strain, harvesting and filtering the protein culture, and purifying the protein culture. Through this cultivation procedure, we obtained insulin scFv that can be immobilized on a gold surface electrode. For faradaic EIS measurement, we employed potassium ferricyanide as the solubilized redox probe to conduct insulin measurement. Binding of insulin concentrations with insulin scFv caused a change in electron flux, which thereby caused changes in charge-transfer resistance (Rct) or impedance. In non-faradaic EIS measurement, ionic liquid is typically used as the buffer to redistribute the dielectric bilayer. Thanks to the small size of insulin scFv, capacitance changes can be directly measured within EDL. Both detection principles were compared by selecting an optimized frequency which best reflects binding between insulin and the MRE.ResultsWe were able to identify an optimized frequency for both detection principles when measuring insulin, establishing a unique change in impedance. A change in Rct was noticed through faradaic EIS when measuring insulin against the insulin scFv. Additionally, at the optimized frequency the sensor had a slope of 701 Ohms/Ln(nM). With the non-Faradaic EIS detection principle, the sensor displayed changes within EDL.DiscussionWe were able to determine that faradaic and non-faradaic EIS could be used to measure changing insulin concentrations when using insulin scFv as the MRE. Differences between both detection principles existed due to the use of a solubilized redox probe for faradaic EIS versus the absence of it when conducing non-Faradaic EIS. Future work will include comparing the sensitivity and selectivity of both detection methods when measuring insulin.

The Abtamer: A Novel Antibody-Aptamer Conjugate for Electrochemical Biosensing Applications

Introduction: We propose an innovated semisynthetic biosensing molecule, the Abtamer, an antibody-aptamer one-to-one complex. An important consideration in the design of electrochemical biosensors is the molecular recognition element (MRE) from which the signal to be transduced is generated. Among MREs utilized for affinity principle based electrochemical biosensors, antibodies are commonly used due to their high sensitivity and selectivity towards specific targets. However, antibodies’ structural rigidity proves limiting in biosensing applications.In contrast, DNA/RNA aptamers are easily-synthesized, versatile biomolecules, which are able to exhibit a high affinity and sensitivity towards their selected target [1]. Owing to their structure, aptamers are inherently flexible which allows for pronounced conformational changes upon binding. Furthermore, their small size allows for the immobilization of a larger density of molecules on the sensor surface, aiding in their sensitivity [2]. However, these same characteristics put aptamers at a disadvantage in terms of their selectivity towards targets. Without various post-synthesis modifications, DNA/RNA oligonucleotides can exhibit nonspecific electrostatic interactions with other analytes in solution.To this end, we propose a new biosensing molecule for biomedical sensor applications based on antibody-aptamer conjugates, designated as “Abtamers,” which includes advantages of both antibodies and aptamers; e.g. high sensitivity, selectivity with molecular dynamics upon target binding, facilitating signals for sensing. We propose to use insulin as a model target for an electrochemical sensor employing this novel biorecognition element. The electrochemical detection of insulin levels in persons with diabetes would provide another useful metric in diabetes technology, as insulin measurements are typically done via ELISA. Methods: The antibody-aptamer conjugate constructed utilized an insulin single chain variable fragment (scFv) and an insulin aptamer. To circumvent the non-optimal binding of polypeptides to oligonucleotides, we utilized the SpyCatcher/SpyTag complex as the bioconjugation system to couple the scFv to the aptamer in a one-to-one manner [3-4]. We created a recombinant fusion molecule of the insulin scFv and SpyCatcher, and created a SpyTag-insulin aptamer fusion molecule. The combination of these two fusion molecules results in an insulin antibody-insulin aptamer conjugate. We aim to employ this new MRE to detect insulin via square wave voltammetry. Results and Discussion: Through the binding of the two fusion molecules, we have created an irreversibly bound conjugate, conserving the structures of both the antibody and aptamer. The modification of a redox probe to the insulin aptamer domain in the Abtamer conjugate allows for a simple and facile method to create an electroactive biomolecule which can be immobilized to an electrode surface. We expect the binding affinity of this molecule to be higher towards specified targets due to both an antibody and aptamer binding event. Furthermore, the flexibility of the aptamer allows for conformational changes upon binding which can be exploited in electrochemical sensing applications. The developed insulin antibody-insulin aptamer conjugate can be applied as a biorecognition element for an electrochemical sensing platform, thereby realizing a unique biosensing principle, the “Abta-sensor”

A Disposable Vancomycin Sensor Using Molecularly Imprinted Carbon Paste on a Ceramic Chip

Introduction Vancomycin (VCM) is the first-choice drug for the therapy of MRSA infection, which is the most typical nosocomial infection. However, VCM resistant bacteria is a severe worldwide problem [1]. Therapeutic drug monitoring (TDM) of VCM in the blood is useful to prevent the creation of resistant bacteria. Nevertheless, conventional TDM using immunoassay requires economical and labor burdens to hospitals [2]. Thus, an inexpensive disposable sensor that can determine the VCM concentration in blood with simple operation as that of blood sugar-sensor is required. In this study, we developed a VCM sensor using a carbon paste electrode grafted with a molecularly imprinted polymer (MIP) and a ceramic platform. MIPs are a molecular recognition element prepared by simple radical copolymerization in the presence of the target as a template. We have discovered that redox current at a MIP-grafted carbon paste electrode is sensitive to the target [3]. Method Figure 1(a) shows the scheme of the ceramic chip composed of aluminum oxide ceramic body and platinum wiring. It contains three holes for electrodes with 2.0 mm diameter and 0.4 mm depth, connected with platinum wiring at the bottom. The chip also comprises a square sample-reservoir of side 10 mm. VCM imprinted poly(methylene bisacrylamide-co-methacrylic acid-co-Allylaminocarboxypropyonic-3-ferrocene) was grafted on graphite particle surface by a procedure similar to our previous work [3], This grafted graphite was mixed with silicone oil to make a paste to use as the sensing material. A non-treated polymer was also synthesized in a similar way as that of VCM MIP, but without the template.Two of the holes of 2.0 mm diameter were each packed with grafted graphite and Ag/AgCl paste to act as the working electrode a reference electrode, respectively. The Pt wire of the third hole was used as the counter electrode. We performed differential pulse voltammetry with a VCM sample solution (0-60 mgmL-1, both in phosphate buffer and in bovine blood) filled in the sample-reservoir. Results and Conclusions The execution time of each voltammetry was 10 min only. Figure 1(b) shows the influence of the VCM concentration on the redox current at 0.8 V at both non-treated and the MIP-carbon paste electrode. It is quite evident from the figure that only MIP responds to VCM. The dynamic range at the MIP electrode versus the concentration of VCM is seen in the range of 0-60 mgmL-1, which corresponds covers the therapeutically effective vancomycin concentration level in plasma varying from 20 to 40 mgL-1 [4], [5]. Additionally, the R2 value of 0.9963 indicates a linear behavior of the sensor with change in drug concentration. From these results, it can be concluded that the proposed method is useful for quick and straightforward TDM to prevent both of the toxic side effects by overdose and the creation of antibacterials by underdose.

Electrogenerated chemiluminescence aptamer-based biosensing for the determination of mucin and specific cells based on bio-bar-code amplification

<p indent=0mm>A highly sensitive electrogenerated chemiluminescence (ECL) aptamer-based biosensing based on bio-bar-code amplification was developed for the determination of cell surface proteins and specific cells. MCF-7 cells were used as target cells, mucin (MUC1) was used as target proteins, anti-MUC1 specific aptamers (rcDNA) were used as molecular recognition elements and Ru(phen)₃²⁺ was used as ECL signal molecules. A hairpin DNA (hpDNA) and a rcDNA were self-assembled on the surface of gold nanoparticles (AuNP) by self-assembly technique to obtain the bio-bar-code probe hpDNA/AuNP/rcDNA. rcDNA was modified onto the surface of gold electrodes to form a biosensing platform. A sandwich complex among rcDNA, target MUC1 (or MCF-7 cells) and hpDNA/AuNP/rcDNA was formed on the surface of electrodes. After Ru(phen)₃²⁺ was intercalated into hpDNA, strong ECL signals were generated in the presence of co-reactants at the constant potential of <sc>0.95 V</sc>. The increased ECL intensity was proportional to the logarithm of MUC1 concentration in the range of 4~800 pg/mL with a detection limit of 0.5 pg/mL. The increased ECL intensity was proportional to the logarithm concentration of MCF-7 cells in the range of 30~5×10⁴ cells/mL with a detection limit of 9 cells/mL. In addition, the developed ECL method was used to discriminate the MUC1 expression on different types of the cells and to monitor the dynamics of MUC1 on the surface of MCF-7 cells under the external drug stimulation, including apigenin and H₂O₂. The decline of MUC1 expression on the surface of MCF-7 cells was observed under apigenin treatment while inconspicuous MUC1 expression was observed under H₂O₂ treatment.

The intrinsically disordered region of GCEprotein adopts a more fixed structure by interacting with the LBD of the nuclearreceptor FTZ-F1

The Drosophila melanogaster Germcell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor -Methoprene tolerant protein (MET). Both proteins mediate JH function, preventingprecocious differentiation during D. melanogasterdevelopment. Despite that GCE and MET are often referred to as equivalent JHreceptors, their functions are not fully redundant and show tissue specificity. Bothproteins belong to the family of bHLH-PAS transcription factors. The similarity oftheir primary structure is limited to defined bHLH and PAS domains, while their longC-terminal fragments (GCEC, METC) show significant differences and are expected todetermine differences in GCE and MET protein activities. In this paper we presentthe structural characterization of GCEC as a coil-like intrinsically disorderedprotein (IDP) with highly elongated and asymmetric conformation. In comparison topreviously characterized METC, GCEC is less compacted, contains more molecularrecognition elements (MoREs) and exhibits a higher propensity for induced folding.The NMR shifts perturbation experiment and pull-down assay clearly demonstrated thatthe GCEC fragment is sufficient to form an interaction interface with the ligandbinding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1).Significantly, these interactions can force GCEC to adopt more fixed structure thatcan modulate the activity, structure and functions of the full-length receptor. Thediscussed relation of protein functionality with the structural data of inherentlydisordered GCEC fragment is a novel look at this protein and contributes to a betterunderstanding of the molecular basis of the functions of the C-terminal fragments ofthe bHLH-PAS family.2UuA8w5_5agRn7KffXMuPjVideo abstract.

Implantable Nanosensors for Human Steroid Hormone Sensing In Vivo Using a Self-Templating Corona Phase Molecular Recognition.

Dynamic measurements of steroid hormones in vivo are critical, but steroid sensing is currently limited by the availability of specific molecular recognition elements due to the chemical similarity of these hormones. In this work, a new, self-templating synthetic approach is applied using corona phase molecular recognition (CoPhMoRe) targeting the steroid family of molecules to produce near infrared fluorescent, implantable sensors. A key limitation of CoPhMoRe has been its reliance on library generation for sensor screening. This problem is addressed with a self-templating strategy of polymer design, using the examples of progesterone and cortisol sensing based on a styrene and acrylic acid copolymer library augmented with an acrylated steroid. The pendant steroid attached to the corona backbone is shown to self-template the phase, providing a unique CoPhMoRE design strategy with high efficacy. The resulting sensors exhibit excellent stability and reversibility upon repeated analyte cycling. It is shown that molecular recognition using such constructs is viable even in vivo after sensor implantation into a murine model by employing a poly (ethylene glycol) diacrylate (PEGDA) hydrogel and porous cellulose interface to limit nonspecific absorption. The results demonstrate that CoPhMoRe templating is sufficiently robust to enable a new class of continuous, in vivo biosensors.

Highly sensitive potentiometric assay of vardenafil in pharmaceutical formulations and biological fluids

Sensitive disposable screen printed potentiometric vardenafil (VAR) sensors have been constructed based on multiwall carbon nanotubes/crown ether (MWCNTs/CE) composite. Factors affecting the sensor performance have been optimized on the basis of nature and content of the molecular recognition elements, anionic sites, membrane plasticizers and nanomaterial. The fabricated sensors showed acceptable selectivity and sensitivity within the vardenafil concentration range from 10-6 to 10-2 mol L-1 with Nernstian slope value of 62.1±0.8 mV decade-1 and detection limit 8.0 × 10-7 mol L-1. Fast response time, enhanced potential reading stability and long shelf lifetime are the promising futures of the novel sensors. The proposed method has been suggested for potentiometric assay of VAR in dosage formulation and biological fluids with average recoveries agreeable with the reported official methods.

In Vitro Selection of an ATP-Binding TNA Aptamer.

Recent advances in polymerase engineering have made it possible to isolate aptamers from libraries of synthetic genetic polymers (XNAs) with backbone structures that are distinct from those found in nature. However, nearly all of the XNA aptamers produced thus far have been generated against protein targets, raising significant questions about the ability of XNA aptamers to recognize small molecule targets. Here, we report the evolution of an ATP-binding aptamer composed entirely of α-L-threose nucleic acid (TNA). A chemically synthesized version of the best aptamer sequence shows high affinity to ATP and strong specificity against other naturally occurring ribonucleotide triphosphates. Unlike its DNA and RNA counterparts that are susceptible to nuclease digestion, the ATP-binding TNA aptamer exhibits high biological stability against hydrolytic enzymes that rapidly degrade DNA and RNA. Based on these findings, we suggest that TNA aptamers could find widespread use as molecular recognition elements in diagnostic and therapeutic applications that require high biological stability.

Analysis of pressure-driven membrane preconcentration for point-of-care assays.

Point-of-care diagnostic devices for both physicians and patients themselves are now ubiquitous, but often not sensitive enough for highly dilute analytes (e.g., pre-symptomatic viral detection). Two primary methods to address this challenge include (1) increasing the sensitivity of molecular recognition elements with greater binding affinity to the analyte or (2) increasing the concentration of the analyte being detected in the sample itself (preconcentration). The latter approach, preconcentration, is arguably more attractive if it can be made universally applicable to a wide range of analytes. In this study, pressure-driven membrane preconcentration devices were developed, and their performance was analyzed for detecting target analytes in biofluids in the form of point-of-care lateral-flow assays (LFAs). The demonstrated prototypes utilize negative or positive pressure gradients to move both water and small interferents (salt, pH) through a membrane filter, thereby concentrating the analyte of interest in the remaining sample fluid. Preconcentration up to 33× is demonstrated for influenza A nucleoprotein with a 5 kDa pore polyethersulfone membrane filter. LFA results are obtained within as short as several minutes and device operation is simple (very few user steps), suggesting that membrane preconcentration can be preferable to more complex and slow conventional preconcentration techniques used in laboratory practice.

Recent Progress in the Identification of Aptamers Against Bacterial Origins and Their Diagnostic Applications.

Aptamers have gained an increasing role as the molecular recognition element (MRE) in diagnostic assay development, since their first conception thirty years ago. The process to screen for nucleic acid-based binding elements (aptamers) was first described in 1990 by the Gold Laboratory. In the last three decades, many aptamers have been identified for a wide array of targets. In particular, the number of reports on investigating single-stranded DNA (ssDNA) aptamer applications in biosensing and diagnostic platforms have increased significantly in recent years. This review article summarizes the recent (2015 to 2020) progress of ssDNA aptamer research on bacteria, proteins, and lipids of bacterial origins that have implications for human infections. The basic process of aptamer selection, the principles of aptamer-based biosensors, and future perspectives will also be discussed.