Molecular Recognition Elements Research Articles

Functional Nucleic Acids as Bacterial Biosensors: A Focus on Clostridioides difficile Infection

Clostridioides difficile (CD) is a Gram‐positive, anaerobic, and spore‐forming bacillus that colonizes the human gut and causes a range of diseases, such as pseudomembranous colitis and antibiotic‐associated diarrhea, that are generally known as CD infection (CDI). Rapid and accurate detection of CDI with high sensitivity and specificity is crucial for patient treatment, infection control, and epidemiological monitoring. Current diagnostic methods for CDI have several limitations, such as high cost, long turnaround time, suboptimal sensitivity, and the need for specialized equipment. Hence, novel detection methods that can overcome these limitations are needed. Functional nucleic acids (FNAs) are a promising class of molecular recognition element (MRE) that can be incorporated into biosensors for detecting infectious pathogens. Several FNAs have been developed for detecting CD. In this review, an overview of CD, CDI, and current diagnostic methods for CDI and their drawbacks are provided. Furthermore, the design principles and working mechanisms of FNAs as well as their applications for the detection of pathogenic bacteria, including CD, are discussed. The potential for developing point‐of‐care paper sensors using currently available CD‐selective FNAs is also highlighted.

Ligand-Induced Folding in a Dopamine-Binding DNA Aptamer.

Aptamers are often employed as molecular recognition elements in the development of different types of biosensors. Many of these biosensors take advantage of the aptamer having a ligand-induced structure-formation binding mechanism. However, this binding mechanism is poorly understood. Here we use isothermal titration calorimetry, circular dichroism spectroscopy and NMR spectroscopy to study the binding and ligand-induced structural change exhibited by a dopamine-binding DNA aptamer. We analysed a series of aptamers where we shorten the terminal stem that contains the 5´and 3´termini of the aptamer sequence. All aptamers bind dopamine in an enthalpically driven process coupled with an unfavorable entropy. A general trend of the aptamer having a weaker binding affinity is observed as the terminal stem is shortened. For all aptamers studied, numerous signals appear in the imino region of the1H NMR spectrum indicating that new structure forms with ligand binding. However, it is only when this region of structure formation in the aptamer is brought close to the sensor surface that we obtain a functional electrochemical aptamer-based biosensor.

Low-tech innovation: biomimetic solid-contact potentiometric sensor for nanomolar-level atrazine detection.

Despite the fact that there are already a number of solid-contact-based ion-selective electrodes designed for atrazine detection, our ground-breaking contribution lies in introducing the first-ever atrazine potentioselectrode, enabling the ultra-sensitive detection of atrazine at nanomolar levels. Solid-contact ion-selective electrodes can offer advantages, such as improved stability, reproducibility, sensitivity, and selectivity compared to their liquid-contact counterparts. Here, a biomimetic potentiometric sensor for Atrazine was developed using economic, light weight, and flexible carbon cloth as solid-contact material. Our methodology entails the synthesis of a molecularly imprinted polymer (MIP) through straightforward precipitation polymerization, showcasing a streamlined and efficient method for creating highly specific molecular recognition elements. The validation of template removal is confirmed via meticulous analysis employing EDX and FTIR techniques, ensuring the efficacy of our methodology. The resulting sensing membrane are casted by dispersing the MIP in 2-nitrophenyl octyl ether plasticizer and embedding it within a PVC matrix containing sodium tetraphenyl borate as a lipophilic additive. The developed sensor responds to atrazine in the pH range of 2.8-3.3 over a wide concentration range of 1 × 10-8M to 1 × 10-5M & 1 × 10-5M to 1 × 10-1M with respective slopes of 29.2 mv & 58.7mV and a limit of detection of 1 × 10-9M. An impressive feature of this sensor lies in its swift response time, registering a rapid reaction within a mere 10s. Emphasize the sensor's commendable attributes of reproducibility, selectivity, and sensitivity underscoring its successful application in field monitoring.

Screening and Specificity Analysis of Aptamer Based on MCF‐7 Breast Cancer Cell Culture Media

AbstractBreast cancer has been the most common cancer and the most prevalent tumor in women worldwide. However, a rapid, sensitive, and painless test for breast cancer has not yet been discovered. Aptamers, as sensitive molecular recognition elements, which can specifically recognize target molecules and be used in the early diagnosis of cancer. In this study, aptamers of proteins secreted by MCF‐7 breast cancer cells were screened using Med‐SELEX technology. According to the enrichment degree and homology of aptamers, the aptamer family tree was constructed. The structure of the aptamers and the docking results with the targets were then predicted. Aptamer affinity and specificity were determined by qPCR, and the optimal aptamer AY 20 was obtained. Colloidal gold colorimetry confirmed that AY20 had high specificity for breast cancer cell culture media. Therefore, the aptamer AY20 can be used as an early diagnostic tool for breast cancer. The work provides a new idea for the rapid detection of breast cancer.

Transient Non-local Interactions Dominate the Dynamics of Measles Virus NTAIL.

The RNA genome of measles virus is encapsidated by the nucleoprotein within a helical nucleocapsid that serves as template for both transcription and replication. The intrinsically disordered domain of the nucleoprotein (NTAIL), partly protruding outward from the nucleocapsid, is essential for binding the polymerase complex responsible for viral transcription and replication. As for many IDPs, binding of NTAIL occurs through a short molecular recognition element (MoRE) that folds upon binding, with the majority of NTAIL remaining disordered. Though NTAIL regions far from the MoRE influence the binding affinity, interactions between them and the MoRE have not been investigated in depth. Using an integrated approach, relying on photo-induced electron transfer (PET) experiments between tryptophan and cysteine pairs placed at different positions in the protein under varying salt and pH conditions, combined with simulations and analytical models, we identified transient interactions between two disordered regions distant in sequence, which dominate NTAIL dynamics, and regulate the conformational preferences of both the MoRE and the entire NTAIL domain. Co-evolutionary analysis corroborates our findings, and suggests an important functional role for the same intramolecular interactions. We propose mechanisms by which these non-local interactions may regulate binding to the phosphoprotein, polymerase recruitment, and ultimately viral transcription and replication. Our findings may be extended to other IDPs, where non-local intra-protein interactions affect the conformational preferences of intermolecular binding sites.

Empowering Exosomes with Aptamers for Precision Theranostics.

As information messengers for cell-to-cell communication, exosomes, typically small membrane vesicles (30-150nm), play an imperative role in the physiological and pathological processes of living systems. Accumulating studies have demonstrated that exosomes are potential biological candidates for theranostics, including liquid biopsy-based diagnosis and drug delivery. However, their clinical applications are hindered by several issues, especially their unspecific detection and insufficient targeting ability. How to upgrade the accuracy of exosome-based theranostics is being widely explored. Aptamers, benefitting from their admirable characteristics, are used as excellent molecular recognition elements to empower exosomes for precision theranostics. With high affinity against targets and easy site-specific modification, aptamers can be incorporated with platforms for the specific detection of exosomes, thus providing opportunities for advancing disease diagnostics. Furthermore, aptamers can be tailored and functionalized on exosomes to enable targeted therapeutics. Herein, this review emphasizes the empowering of exosomes by aptamers for precision theranostics. A brief introduction of exosomes and aptamers is provided, followed by a discussion of recent progress in aptamer-based exosome detection for disease diagnosis, and the emerging applications of aptamer-functionalized exosomes for targeted therapeutics. Finally, current challenges and opportunities in this research field are presented.

The diagnostic potentiality of the RNA aptamer against progesterone receptor isolated by crush and soak (CRUSOAK)-SELEX.

Aptamers are a class of molecular recognition elements that exhibit high binding affinity and specificity against their respective targets. In view of the many advantages aptamers harbor over their counterpart antibodies, we were impelled to isolate an RNA aptamer against progesterone receptor, particularly its DNA binding domain. A total of eight SELEX cycles were executed against the recombinant Progesterone Receptor DNA-binding domain (PR DBD). The RNA-protein complex in the gel shift assay was subjected to crush and soak method to elute the binders prior to conventional sequencing, the step of which was based upon to coin the term CRUSOAK-SELEX. The sequencing revealed three different classes of sequences, with one class termed, PRapt-3, showing the strongest binding against PR DBD. The dissociation constant of PRapt-3 RNA aptamer was estimated at 380nM ± 35nM. PRapt-3 was successfully used to develop aptamer-based diagnostic assays such as ELASA, aptamer-based dot blot, and aptamer-based western blot. The prominent highlight is the performance of the aptamer in aptacytostaining, which was unachievable with antibodies. Compared to its counterpart antibodies, PRapt-3 has a better penetration capacity in aptahistostaining using the formalin-fixed paraffin-embedded (FFPE) breast cancer cells and tissue blocks. This study represents the first ever demonstration of an aptamer against progesterone receptor and its diagnostic capacity.

Covalent Attachment of Horseradish Peroxidase to Single‐Walled Carbon Nanotubes for Hydrogen Peroxide Detection

AbstractSingle‐walled carbon nanotubes (SWCNTs) are desirable nanoparticles for sensing biological analytes due to their photostability and intrinsic near‐infrared fluorescence. Previous strategies for generating SWCNT nanosensors have leveraged nonspecific adsorption of sensing modalities to the hydrophobic SWCNT surface that often require engineering new molecular recognition elements. An attractive alternate strategy is to leverage pre‐existing molecular recognition of proteins for analyte specificity, yet attaching proteins to SWCNT for nanosensor generation remains challenging. Toward this end, a generalizable platform is introduced to generate protein‐SWCNT‐based optical sensors and use this strategy to synthesize a hydrogen peroxide (H2O2) nanosensor by covalently attaching horseradish peroxidase (HRP) to the SWCNT surface. A concentration‐dependent response is demonstrated to H2O2, confirming the nanosensor can image H2O2 in real‐time, and assess the nanosensor's selectivity for H2O2 against a panel of biologically relevant analytes. Taken together, these results demonstrate successful covalent attachment of enzymes to SWCNTs while preserving both intrinsic SWCNT fluorescence and enzyme function. It is anticipated this platform can be adapted to covalently attach other proteins of interest including other enzymes for sensing or antibodies for targeted imaging and cargo delivery.

Solubilization and Photostabilization in a Sodium Deoxycholate Hydrogel of a Neutral Conjugated Thiophene Oligomer and Polymer.

Oligo(3-hexylthiophene-co-1,4-phenylene) and poly(3-hexylthiophene) were solubilized in sodium deoxycholate self-assemblies in water solutions and hydrogels, with the goal of solubilizing sufficient material in a hydrogel for fluorescence applications. The neutral conjugated oligomer and polymer were incorporated as monomers into the self-assemblies with sodium deoxycholate aggregates, leading to the photoprotection of these neutral conjugated and water-insoluble molecules. Dynamic light scattering, rheology, and fluorescence experiments established that the deoxycholate aggregation and gel formation properties were not altered with the incorporation of the oligomer or polymer into the deoxycholate self-assemblies, showing that this adaptable host system with some molecular recognition elements is a viable strategy to incorporate into hydrogels neutral conjugated molecules as isolated monomers. This strategy has the potential to be used when conjugated molecules are used for fluorescence applications in hydrogels.

Design of a highly sensitive and precise long-range surface plasmon resonance sensor for early detection of malaria

This manuscript presents an innovative Kretschmann configured for Long Range Surface Plasmon Resonance (LRSPR) sensor for malaria detection. It utilizes tungsten diselenide (WSe2) as a bio+molecular recognition element (BRE) layer to attach target analyte. The optimized sensor design demonstrates exceptional performance in detecting different malaria stages (Schizont with refractive index (RI) = 1.371, Trophozoite with RI = 1.381, and Ring with RI = 1.396) at a 633 nm wavelength. In comparison to conventional surface plasmon resonance (CSPR), the LRSPR biosensor exhibits a substantial improvement, a 6.67 to 10 times increase in detection accuracy (DA), an impressive 40 to 86.55 times enhancement in imaging figure of merit (IFOM), and a 6.0 to 8.66 times boost in imaging sensitivity (Simg.) for the sequential detection of different stages of malaria. COMSOL Multiphysics simulations highlight a deeper penetration depth (PD) of 258.71 nm, 276.85 nm, and 373.04 nm for the detection of Schizont, Trophozoite, and Ring stages, respectively.

In-vitro Clinical Diagnostics using RNA-Cleaving DNAzymes.

Over the last three decades, significant advancements have been made in the development of biosensors and bioassays that use RNA-cleaving DNAzymes (RCDs) as molecular recognition elements. While early examples of RCDs were primarily responsive to metal ions, the past decade has seen numerous RCDs reported for more clinically relevant targets such as bacteria, cancer cells, small metabolites, and protein biomarkers. Over the past 5 years several RCD-based biosensors have also been evaluated using either spiked biological matrixes or patient samples, including blood, serum, saliva, nasal mucus, sputum, urine, and faeces, which is a critical step toward regulatory approval and commercialization of such sensors. In this review, an overview of the methods used to generate RCDs and the properties of key RCDs that have been utilized for in vitro testing is first provided. Examples of RCD-based assays and sensors that have been used to test either spiked biological samples or patient samples are then presented, highlighting assay performance in different biological matrixes. A summary of current prospects and challenges for development of in vitro diagnostic tests incorporating RCDs and an overview of future directions of the field is also provided.

Electrochemical Impedimetric Platform Based on Con A@MIL-101 for Glycoprotein Detection.

An electrochemical impedimetric biosensing platform with lectin as a molecular recognition element has been established for the sensitive detection of glycoproteins, a class of important biomarkers in clinical diagnosis. One of the representative metal-organic framework materials, MIL-101(Cr)-NH2, was utilized as the supporting matrix, and its amino groups served as the anchors to immobilize the lectins of concanavalin A (Con A), constituting Con A@MIL-101(Cr)-NH2 for the determination of invertase (INV) as a model glycoprotein. The Con A concentration, immobilization time, and incubation time with INV were optimized. Under the optimal conditions, the degree of impedance increase was linearly proportional to the logarithm of INV concentration between 1.0 × 10-16 and 1.0 × 10-11 M, affording a limit of detection as low as 3.98 × 10-18 M. Good specificity, stability, reproducibility, and repeatability were demonstrated for the fabricated biosensing platform. Moreover, real mouse serum samples were spiked with different concentrations of INV. Excellent recoveries were obtained, which demonstrated the biosensing platform's capability of analyzing glycoproteins within a complex matrix.

Surface Modification of Screen-Printed Carbon Electrode through Oxygen Plasma to Enhance Biosensor Sensitivity.

The screen-printed carbon electrode (SPCE) is a useful technology that has been widely used in the practical application of biosensors oriented to point-of-care testing (POCT) due to its characteristics of cost-effectiveness, disposability, miniaturization, wide potential window, and simple electrode design. Compared with gold or platinum electrodes, surface modification is difficult because the carbon surface is chemically or physically stable. Oxygen plasma (O2) can easily produce carboxyl groups on the carbon surface, which act as scaffolds for covalent bonds. However, the effect of O2-plasma treatment on electrode performance remains to be investigated from an electrochemical perspective, and sensor performance can be improved by clarifying the surface conditions of plasma-treated biosensors. In this research, we compared antibody modification by plasma treatment and physical adsorption, using our novel immunosensor based on gold nanoparticles (AuNPs). Consequently, the O2-plasma treatment produced carboxyl groups on the electrode surface that changed the electrochemical properties owing to electrostatic interactions. In this study, we compared the following four cases of SPCE modification: O2-plasma-treated electrode/covalent-bonded antibody (a); O2-plasma-treated electrode/physical adsorbed antibody (b); bare electrode/covalent-bonded antibody (c); and bare electrode/physical absorbed antibody (d). The limits of detection (LOD) were 0.50 ng/mL (a), 9.7 ng/mL (b), 0.54 ng/mL (c), and 1.2 ng/mL (d). The slopes of the linear response range were 0.039, 0.029, 0.014, and 0.022. The LOD of (a) was 2.4 times higher than the conventional condition (d), The slope of (a) showed higher sensitivity than other cases (b~d). This is because the plasma treatment generated many carboxyl groups and increased the number of antibody adsorption sites. In summary, the O2-plasma treatment was found to modify the electrode surface conditions and improve the amount of antibody modifications. In the future, O2-plasma treatment could be used as a simple method for modifying various molecular recognition elements on printed carbon electrodes.

Ultrasensitive prostate cancer marker PCA3 detection with impedimetric biosensor based on specific label-free aptamers

Prostate cancer (PCa) appears among the most frequently diagnosed types of malignancies in males. Because of the high demand and increasing detection rate of early PCa, alongside the specificity limitations of the gold standard clinical tools available for the diagnosis and prognosis of prostate cancer, there is an urgent need for more reliable PCa markers and highly sensitive diagnostic tools to avoid under-treatment and over-diagnosis. PCA3, or prostate cancer antigen 3, is a potential prostate cancer biomarker that is more specific and useful for preventing unnecessary repeat biopsies, particularly in men with persistently high prostate-specific antigen indices after a negative biopsy. Additionally, an electrochemically based biosensor would prove to be a powerful diagnostic tool for PCA3 detection in urine because of its simplicity, sensitivity, and cost-effectiveness, in contrast to the more traditional PCa diagnostics that depend on blood testing. This paper aimed to design a novel and simple electrochemical impedimetric biosensor based on a label-free RNA-aptamer (CG3-PCA3) as the molecular recognition element for detecting PCA3. The proposed aptasensor for the detection of PCA3 has been developed using a screen-printed carbon electrode (SPCE) modified by gold nanoparticles (AuNPs), further improving sensitivity and allowing the immobilisation of thiolate aptamers on its surface. The findings presented here demonstrated a high sensitivity to PCA3, with a detection limit of 20 fM in artificial urine and 1 fM in buffer. These results indicate that the PCA3 aptasensor could be a promising tool for routine PCa diagnosis due to its high sensitivity and cost-effectiveness.

Aptamers and Nanobodies as New Bioprobes for SARS-CoV-2 Diagnostic and Therapeutic System Applications.

The global challenges posed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have underscored the critical importance of innovative and efficient control systems for addressing future pandemics. The most effective way to control the pandemic is to rapidly suppress the spread of the virus through early detection using a rapid, accurate, and easy-to-use diagnostic platform. In biosensors that use bioprobes, the binding affinity of molecular recognition elements (MREs) is the primary factor determining the dynamic range of the sensing platform. Furthermore, the sensitivity relies mainly on bioprobe quality with sufficient functionality. This comprehensive review investigates aptamers and nanobodies recently developed as advanced MREs for SARS-CoV-2 diagnostic and therapeutic applications. These bioprobes might be integrated into organic bioelectronic materials and devices, with promising enhanced sensitivity and specificity. This review offers valuable insights into advancing biosensing technologies for infectious disease diagnosis and treatment using aptamers and nanobodies as new bioprobes.

Polydopamine functionalized dendritic fibrous silica nanoparticles as a generic platform for nucleic acid-based biosensing

Accurate and rapid detection of nucleic acid sequences is of utmost importance in various fields, including disease monitoring, clinical treatment, gene analysis and drug discovery. In this study, we developed a "turn-on" fluorescence biosensor that enables simple and highly efficient detection of nucleic acid biomarkers. Our approach involves the utilization of 6-carboxyfluorescein modified single-stranded DNA (FAM-ssDNA) as molecular recognition element, along with polydopamine-functionalized dendritic fibrous nanosilica (DFNS). FAM-ssDNA serves as both specific molecular recognition element for the target analyte and reporter capable of transducing a detectable signal through Watson–Crick base pairing. The polydopamine-functionalized DFNS (DFNS@DA) exhibits strong binding to FAM-ssDNA via polyvalent metal mediated coordination leading to effective quenching by fluorescence resonance energy transfer. In the presence of a complementary target sequence, FAM-ssDNA forms hybridized structure and detaches from DFNS@DA, which causes an increased fluorescence emission. The analytical system based on FAM-ssDNA and DFNS@DA demonstrates exceptional sensitivity, selectivity, and rapid response for the detection of nucleic acid sequences, leveraging the high adsorption and quenching properties of DFNS@DA. For the first proof of concept, we demonstrated the successful detection of microRNA (miR-21) in cancer cells using the FAM-ssDNA/DFNS@DA system. Our results highlight the promising capabilities of DFNS@DA and nucleic acid-based biosensors, offering a generic and cost-effective solution for the detection of nucleic acid-related biomarkers.Graphical abstract

Development of Better Aptamers: Structured Library Approaches, Selection Methods, and Chemical Modifications

AbstractSystematic evolution of ligands by exponential enrichment (SELEX) has been used to discover thousands of aptamers since its development in 1990. Aptamers are short single‐stranded oligonucleotides capable of binding to targets with high specificity and selectivity through structural recognition. While aptamers offer advantages over other molecular recognition elements such as their ease of production, smaller size, extended shelf‐life, and lower immunogenicity, they have yet to show significant success in real‐world applications. By analyzing the importance of structured library designs, reviewing different SELEX methodologies, and the effects of chemical modifications, we provide a comprehensive overview on the production of aptamers for applications in drug delivery systems, therapeutics, diagnostics, and molecular imaging.

Development of Better Aptamers: Structured Library Approaches, Selection Methods, and Chemical Modifications.

Systematic evolution of ligands by exponential enrichment (SELEX) has been used to discover thousands of aptamers since its development in 1990. Aptamers are short single-stranded oligonucleotides capable of binding to targets with high specificity and selectivity through structural recognition. While aptamers offer advantages over other molecular recognition elements such as their ease of production, smaller size, extended shelf-life, and lower immunogenicity, they have yet to show significant success in real-world applications. By analyzing the importance of structured library designs, reviewing different SELEX methodologies, and the effects of chemical modifications, we provide a comprehensive overview on the production of aptamers for applications in drug delivery systems, therapeutics, diagnostics, and molecular imaging.

Production of hybrid macrolide antibiotics by exploiting the specific substrate recognition characteristics of multifunctional cytochrome P450 enzyme MycG.

Macrolide antibiotics are biosynthesized via enzymatic modifications, including glycosylation, methylation, and oxidation, after the core macro-lactone ring is generated by a polyketide synthase system. This study explored the diversification of macrolides by combining biosynthetic enzymes and reports an approach to produce unnatural hybrid macrolide antibiotics. The cytochrome (CYP) P450 monooxygenase MycG exhibits bifunctional activity, catalyzing late-stage hydroxylation at C-14 followed by epoxidation at C-12/13 during mycinamicin biosynthesis. The mycinose sugar of mycinamicin serves as a key molecular recognition element for binding to MycG. Thus, we subjected the hybrid macrolide antibiotic 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (IZI) to MycG, and confirmed that MycG catalyzed hydroxylation at C-22 and epoxidation at C-12/13 in IZI. In addition, the introduction of mycinose biosynthesis-related genes and mycG into rosamicin-producing Micromonospora rosaria enabled the fermentative production of 22-hydroxylated and 12,13-epoxidized forms of IZI. Interestingly, MycG catalyzed the sequential oxidation of hydroxylation and epoxidation in mycinamicin biosynthesis, but only single reactions in IZI. These findings highlight the potential for expanding the application of the multifunctional P450 monooxygenase MycG for the production of unnatural compounds.

Reflection-fluorescence ratios probe for the stable detection of glucose

Herein, an optical ratios probe with reflection (RE) and fluorescence (FL), named as RE-FL ratios probe, has been developed for the ratiometric detection of glucose (GLU). This probe was prepared through self-assembling monodisperse colloids into opal photonic crystal and immobilizing fluorescence imidazolium ionic liquids as molecular recognition elements. So there was a periodic three-dimensional face-centered cubic arrayed structure and emissions of strong RE and FL in the probe, which allowed dual optical detection of the target analyte. From the results, it was learned that RE and FL emissions of RE-FL ratios probe were decreased when an increase of GLU concentration, and the resulting ratiometric optical changes can be quantitatively correlated to GLU concentration from 1 to 64 mM. The detection of probe was characterized by simple operation, stability, reusability and glucose oxidase independence. Furthermore, the probe successfully used to detect GLU in the real serum sample and showed great stability compared with the only FL pattern. Different types of appropriated recognition elements are expected to be introduced into opal photonic crystals to construct variety ratiometric optical probes in the wide of applications.