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Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary 'antisense' strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a 'fit-for-purpose' model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand. [ABSTRACT FROM AUTHOR]

Abstract Aptamers have emerged as prominent ligands in clinical diagnostics because they provide various advantages over antibodies, such as quicker generation time, reduced manufacturing costs, minimal batch-to-batch variability, greater modifiability, and improved thermal stability. In the present study, we isolated and characterized DNA aptamers that can specifically bind to the hemolysin E (HlyE) antigen of Salmonella Typhi for future development of typhoid diagnostic tests. The DNA aptamers against Salmonella Typhi HlyE were isolated using systematic evolution of ligands by exponential enrichment (SELEX), and their binding affinity and specificity were assessed utilizing enzyme-linked oligonucleotide assay (ELONA). A total of 11 distinct aptamers were identified, and the binding affinities and species selectivities of the three most probable aptamers were determined. Kd values were obtained in the nanomolar range, with the highest affinity of 83.6 nM determined for AptHlyE97. In addition, AptHlyE11, AptHlyE45 and AptHlyE97 clearly distinguished S. Typhi HlyE from other tested bacteria, such as Salmonella Paratyphi A, Salmonella Paratyphi B, Shigella flexneri, Klebsiella pneumonia and Escherichia coli, therefore displaying desirable specificity. These novel aptamers could be used as diagnostic ligands for the future development of inexpensive and effective point-of-care tests for typhoid surveillance, especially in developing countries of the tropics and subtropics.

Abstract Background Spores produced by the filamentous fungus Aspergillus niger are abundant in a variety of environments. The proliferation of this fungus in indoor environments has been associated to health risks and its conidia can cause allergic reaction and severe invasive disease in animals and humans. Therefore, the detection and monitoring of Aspergillus conidia is of utmost importance to prevent serious fungal infections and contaminations. Among others, aptamers could serve as biosensors for the specific detection of fungal spores. Results In this study, DNA aptamers specific to conidia of A. niger were developed by optimizing a whole-cell SELEX approach. Three whole-cells SELEX experiments were performed in parallel with similar conditions. Quantification of recovered ssDNA and melting curve analyses were applied to monitor the ongoing SELEX process. Next-generation sequencing was performed on selected recovered ssDNA pools, allowing the identification of DNA aptamers which bind with high affinity to the target cells. The developed aptamers were shown to be species-specific, being able to bind to A. niger but not to A. tubingensis or to A. nidulans. The binding affinity of two aptamers (AN01-R9-006 and AN02-R9-185) was measured to be 58.97 nM and 138.71 nM, respectively, which is in the range of previously developed aptamers. Conclusions This study demonstrates that species-specific aptamers can be successfully developed via whole-cell SELEX to distinguish different Aspergillus species and opens up new opportunities in the field of diagnostics of fungal infections.

Alzheimer's disease (AD) is considered the most common and prevalent form of dementia of adult-onset with characteristic progressive impairment in cognition and memory. The cure for AD has not been found yet and the treatments available until recently were only symptomatic. Regardless of multidisciplinary approaches and efforts made by pharmaceutical companies, it was only in the past two years that new drugs were approved for the treatment of the disease. Amyloid beta (Aβ) immunotherapy is at the core of this therapy, which is one of the most innovative approaches looking to change the course of AD. This technology is based on synthetic peptides or monoclonal antibodies (mAb) to reduce Aβ levels in the brain and slow down the advance of neurodegeneration. Hence, this article reviews the state of the art about AD neuropathogenesis, the traditional pharmacologic treatment, as well as the modern active and passive immunization describing approved drugs, and drug prototypes currently under investigation in different clinical trials. In addition, future perspectives on immunotherapeutic strategies for AD and the rise of the aptamer technology as a non-immunogenic alternative to curb the disease progression are discussed.

This study presents a cancer diagnostics method through the development of a graphitic carbon nitride (g-C3N4)-based electrochemical sensor for detecting circulating tumor cells (CTCs) in blood. Utilizing the unique properties of g-C3N4 nanosheets combined with gold nanoparticles functionalized with DNA aptamers, the sensor exhibits remarkable sensitivity and selectivity in CTC detection. The aptamers specifically target cell surface biomarkers, enabling the precise capture and quantification of CTCs. The sensor's detection limit is an impressive 5 cells/mL, with a broad detection range from 10 to 105 cells/mL. This high-performance sensing platform not only achieves rapid and accurate quantification of CTCs but also demonstrates robustness against interference from other cell types, making it highly suitable for clinical applications. The optimized conditions for aptamer-functionalized nanoparticle concentration and incubation time ensure the sensor's efficacy, as validated in spiked blood samples. This work provides a practical and efficient approach for non-invasive cancer diagnostics, offering significant improvements over existing methods in terms of simplicity, sensitivity, and specificity for early detection and monitoring of cancer progression.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy, characterized by progressive and asymmetric muscle atrophy, primarily affecting muscles of the face, shoulder girdle, and upper arms before affecting muscles of the lower extremities with age and greater disease severity. FSHD is a disabling condition, and patients may also present with various extramuscular symptoms. FSHD is caused by the aberrant expression of double homeobox 4 (DUX4) in skeletal muscle, arising from compromised epigenetic repression of the D4Z4 array. DUX4 encodes the DUX4 protein, a transcription factor that activates myotoxic gene programs to produce the FSHD pathology. Therefore, sequence-specific oligonucleotides aimed at reducing DUX4 levels in patients is a compelling therapeutic approach, and one that has received considerable research interest over the last decade. This review aims to describe the current preclinical landscape of oligonucleotide therapies for FSHD. This includes outlining the mechanism of action of each therapy and summarizing the preclinical results obtained regarding their efficacy in cellular and/or murine disease models. The scope of this review is limited to oligonucleotide-based therapies that inhibit the DUX4 gene, mRNA, or protein in a way that does not involve gene editing. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) is a prevalent gastrointestinal cancer characterized by high mortality and an unfavorable prognosis. While combination therapies involving surgery, chemotherapy, and radiation therapy are advancing, targeted therapy for ESCC remains underdeveloped. As a result, the overall five‐year survival rate for ESCC is still below 20%. Herein, ESCC‐specific DNA aptamers and an innovative aptamer‐modified nano‐system is introduced for targeted drug and gene delivery to effectively inhibit ESCC. The EA1 ssDNA aptamer, which binds robustly to ESCC cells with high specificity and affinity, is identified using cell‐based systematic evolution of ligands by exponential enrichment (cell‐SELEX). An EA1‐modified nano‐system is developed using a natural egg yolk lipid nanovector (EA1‐EYLNs‐PTX/siEFNA1) that concurrently loads paclitaxel (PTX) and a small interfering RNA of Ephrin A1 (EFNA1). This combination counters ESCC's proliferation, migration, invasion, and lung metastasis. Notably, EFNA1 is overexpressed in ESCC tumors with lung metastasis and has an inverse correlation with ESCC patient prognosis. The EA1‐EYLNs‐PTX/siEFNA1 nano‐system offers effective drug delivery and tumor targeting, resulting in significantly improved therapeutic efficacy against ESCC tumors. These insights suggest that aptamer‐modified nano‐systems can deliver drugs and genes with superior tumor‐targeting, potentially revolutionizing targeted therapy in ESCC. [ABSTRACT FROM AUTHOR]

This study presents a cancer diagnostics method through the development of a graphitic carbon nitride (g-C 3 N 4)-based electrochemical sensor for detecting circulating tumor cells (CTCs) in blood. Utilizing the unique properties of g-C 3 N 4 nanosheets combined with gold nanoparticles functionalized with DNA aptamers, the sensor exhibits remarkable sensitivity and selectivity in CTC detection. The aptamers specifically target cell surface biomarkers, enabling the precise capture and quantification of CTCs. The sensor's detection limit is an impressive 5 cells/mL, with a broad detection range from 10 to 105 cells/mL. This high-performance sensing platform not only achieves rapid and accurate quantification of CTCs but also demonstrates robustness against interference from other cell types, making it highly suitable for clinical applications. The optimized conditions for aptamer-functionalized nanoparticle concentration and incubation time ensure the sensor's efficacy, as validated in spiked blood samples. This work provides a practical and efficient approach for non-invasive cancer diagnostics, offering significant improvements over existing methods in terms of simplicity, sensitivity, and specificity for early detection and monitoring of cancer progression. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is considered the most common and prevalent form of dementia of adult-onset with characteristic progressive impairment in cognition and memory. The cure for AD has not been found yet and the treatments available until recently were only symptomatic. Regardless of multidisciplinary approaches and efforts made by pharmaceutical companies, it was only in the past two years that new drugs were approved for the treatment of the disease. Amyloid beta (A β) immunotherapy is at the core of this therapy, which is one of the most innovative approaches looking to change the course of AD. This technology is based on synthetic peptides or monoclonal antibodies (mAb) to reduce A β levels in the brain and slow down the advance of neurodegeneration. Hence, this article reviews the state of the art about AD neuropathogenesis, the traditional pharmacologic treatment, as well as the modern active and passive immunization describing approved drugs, and drug prototypes currently under investigation in different clinical trials. In addition, future perspectives on immunotherapeutic strategies for AD and the rise of the aptamer technology as a non-immunogenic alternative to curb the disease progression are discussed. Processing of APP to produce A β peptides which are the target of sequence-derived monoclonal antibodies currently approved for the treatment of AD. [Display omitted] [ABSTRACT FROM AUTHOR]

There is a significant need to develop new environmentally friendly, extraction-free sample collection mediums that can effectively preserve and protect genetic material for point-of-care and/or self-collection, home-collection, and mail-back testing. Systematic evolution of ligands by exponential enrichment (SELEX) was used to create anti-ribonuclease (RNase) deoxyribonucleic acid (DNA) aptamers against purified RNase A conjugated to paramagnetic carboxylated beads. Following eight rounds of SELEX carried out under various stringency conditions, e.g., selection using Xtract-Free™ (XF) specimen collection medium and elevated ambient temperature of 28 °C, a panel of five aptamers was chosen following bioinformatic analysis using next-generation sequencing. The efficacy of aptamer inactivation of RNase was assessed by monitoring ribonucleic acid (RNA) integrity via fluorometric and real-time RT-PCR analysis. Inclusion of aptamers in reaction incubations resulted in an 8800- to 11,200-fold reduction in RNase activity, i.e., digestion of viral RNA compared to control. Thus, anti-RNase aptamers integrated into XF collection medium as well as other commercial reagents and kits have great potential for ensuring quality intact RNA for subsequent genomic analyses. [ABSTRACT FROM AUTHOR]

Abstract Esophageal squamous cell carcinoma (ESCC) is a prevalent gastrointestinal cancer characterized by high mortality and an unfavorable prognosis. While combination therapies involving surgery, chemotherapy, and radiation therapy are advancing, targeted therapy for ESCC remains underdeveloped. As a result, the overall five‐year survival rate for ESCC is still below 20%. Herein, ESCC‐specific DNA aptamers and an innovative aptamer‐modified nano‐system is introduced for targeted drug and gene delivery to effectively inhibit ESCC. The EA1 ssDNA aptamer, which binds robustly to ESCC cells with high specificity and affinity, is identified using cell‐based systematic evolution of ligands by exponential enrichment (cell‐SELEX). An EA1‐modified nano‐system is developed using a natural egg yolk lipid nanovector (EA1‐EYLNs‐PTX/siEFNA1) that concurrently loads paclitaxel (PTX) and a small interfering RNA of Ephrin A1 (EFNA1). This combination counters ESCC's proliferation, migration, invasion, and lung metastasis. Notably, EFNA1 is overexpressed in ESCC tumors with lung metastasis and has an inverse correlation with ESCC patient prognosis. The EA1‐EYLNs‐PTX/siEFNA1 nano‐system offers effective drug delivery and tumor targeting, resulting in significantly improved therapeutic efficacy against ESCC tumors. These insights suggest that aptamer‐modified nano‐systems can deliver drugs and genes with superior tumor‐targeting, potentially revolutionizing targeted therapy in ESCC.

Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary ‘antisense’ strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a ‘fit-for-purpose’ model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand.

Layer-by-layer (LbL) immobilization of DNA aptamers in the realm of electrochemical detection of heavy metal ions (HMIs) offers an enhancement in specificity, sensitivity, and low detection limits by leveraging the cross-reactivity obtained from multiple interactions between immobilized aptamers and developed material surfaces. In this research, we present a LbL approach for the immobilization of thiol- and amino-modified DNA aptamers on a Ag-incorporated cobalt-succinate metal–organic framework (MOF) (Ag@Co-Succinate) to achieve a cross-reactive effect on the electrochemical behavior of the sensor. The solvothermal method was utilized to synthesize Ag@Co-Succinate, which was also characterized through various techniques to elucidate its structure, morphology, and presence of functional groups, confirming its suitability as a host matrix for immobilizing both aptamers. The Ag@Co-Succinate aptasensor exhibited extraordinary sensitivity and selectivity towards Hg(II) ions in electrochemical detection, attributed to the unique binding properties of the immobilized aptamers. The exceptional limit of detection of 0.3 nM ensures the sensor's suitability for trace-level Hg(II) detection in various environmental and analytical applications. Furthermore, the developed sensor demonstrated outstanding repeatability, highlighting its potential for long-term and reliable monitoring of Hg(II). [ABSTRACT FROM AUTHOR]

Due to their unique three-dimensional structure, DNA or RNA oligonucleotide aptamers bind to various molecules with high affinity and specificity. Aptamers, alone or in combination with antibodies, can be used to sensitively quantify target molecules by quantitative real-time polymerase chain reaction (qPCR). However, the assays are often complicated and unreliable. In this study, we explored the feasibility of performing the entire assay on wells of routinely used polypropylene PCR plates. We found that polypropylene wells efficiently bind proteins. This allows the entire assay to be run in a single well. To minimize nonspecific binding of the assay components to the polypropylene wells, we tested various blocking agents and identified methylcellulose as an effective alternative to the commonly used BSA. Methylcellulose not only demonstrates comparable or superior blocking capabilities but also offers the advantage of a well-defined composition and non-animal origin. Our findings support the utilization of aptamers, either alone or in combination with antibodies, for sensitive quantification of selected molecules immobilized in polypropylene PCR wells in a streamlined one-well qPCR assay under well-defined conditions. [ABSTRACT FROM AUTHOR]

Silver ions are regarded as one of the most hazardous metal contaminants, endangering both the ecological environment and human health. Traditional silver ions detection technologies are hampered by their high cost, time-consuming nature, and labor-intensive operation. DNA aptamers play an essential part in the field of biosensors due to their ease of synthesis, ease of modification, and low cost. This paper focuses on reviewing the research progress of DNA aptamer-based biosensors in silver ions detection. According to the types of signal transduction, they are classified into four forms of signal transduction: fluorescent biosensors, colorimetric biosensors, electrochemical biosensors, and surface-enhanced Raman spectroscopy biosensors. In addition, this paper gives a perspective on the application prospects outlook and development directions of DNA aptamer-based biosensors, in order to provide theoretical ideas for the future development of more sensitive DNA aptamer biosensors for rapid detection of silver ions.

Bacterial lipopolysaccharides (LPSs) are important indicators of a bacteria presence in any samples. They can therefore be used for the detection of microbiological contamination in food and dairy products. We performed a comparative analysis of different bacterial models by the application of liposomes containing LPS from Salmonella enterica serotype typhimurium on the surface of an 11-mercaptoundecanoic acid (MUA) monolayer chemisorbed on the gold surface of quartz crystal. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we were able to monitor the formation of the lectin, concanavalin A (ConA), layer on the MUA surface. We determined the optimal concentration of the ConA for the layer formation. ConA of 0.3 mg/mL was selected as the most suitable adsorption of liposomes containing LPS. Using the Sauerbrey equation, we calculated that approximately 1.13 × 1012 ConA molecules per cm2 was adsorbed on the MUA surface, which closely corresponds to the 1.19 × 1012 molecules per cm2 by theoretical models. Later, mixed LPS liposomes containing dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl ethanolamine (DPPE) and cholesterol successfully interacted with the ConA layer, which resulted in a decrease in the resonant frequency and an increase in dissipation. We compared the adsorption of liposomes with different fractions of LPS and containing LPS from different bacteria. Lack of any LPS in liposomes caused weaker adsorption on the ConA layer. Liposomes containing 50% LPS caused the most prominent adsorption and were suitable for interaction with DNA aptamers specific to certain LPS. The addition of the aptamers to the surface of ConA covered by LPS-containing liposomes resulted in a decrease in resonant frequency and an increase in the dissipation. Using the Kelvin–Voigt viscoelastic model and multiharmonic response of acoustic sensors, we also determined changes in viscoelastic values of the molecular films during interaction with liposomes and the ConA layer.

Massive use of antibiotics in veterinary medicine has led to their accumulation in meat and dairy products. Consumption of antibiotic-contaminated food can trigger the development of antibiotic-resistant bacteria, endangering human lives. Among antibiotics, the oxytetracycline (OTC) family of antibiotics is most widely used in veterinary medicine. Strict control of the antibiotics in food necessitates the development of fast and effective methods for OTC detection, for instance, in milk. One of the most promising approaches to OTC detection is based on the use of specially designed DNA aptamers. These DNA aptamers are relatively short, 15–60 bases, nucleotides folded in the solution in a 3D structure, forming a binding site for the target antibiotic. Aptamers can be chemically modified for attachment to sensor electrodes. In this work, we investigated the electrochemical detection of OTC using DNA aptamers specific to OTC that have been covalently immobilized onto the nanocomposite surface of a glassy carbon electrode with electrodeposited reduced graphene oxide and multiwalled carbon nanotubes. Differential pulse voltammetry, DPV, in the presentence of a ferri/ferrocyanide redox couple was used as an internal standard and to monitor the redox current. In the presence of OTC, the amplitude of DPV decreased, evidencing the blocking of charge transfer. After system optimization, we reached the limit of detection of 0.45 ng OTC/mL, which is 200 times lower than the maximum residue limit established by the European Commission of 100 ng OTC/mg.

Aptamers are synthetic single-stranded oligonucleotides that exhibit selective binding properties to specific targets, thereby providing a powerful basis for the development of selective and sensitive (bio)chemical assays. Electrochemical biosensors utilizing aptamers as biological recognition elements, namely aptasensors, are at the forefront of current research. They exploit the combination of the unique properties of aptamers with the advantages of electrochemical detection with the view to fabricate inexpensive and portable analytical platforms for rapid detection in point-of-care (POC) applications or for on-site monitoring. The immobilization of aptamers on suitable substrates is of paramount importance in order to preserve their functionality and optimize the sensors' sensitivity. This work describes different immobilization strategies for aptamers on the surface of semiconductor-based working electrodes, including metal oxides, conductive polymers, and carbon allotropes. These are presented as platforms with tunable band gaps and various surface morphologies for the preparation of low cost, highly versatile aptasensor devices in analytical chemistry. A survey of the current literature is provided, discussing each analytical method. Future trends are outlined which envisage aptamer-based biosensing using semiconductors. [ABSTRACT FROM AUTHOR]

Cytochrome c (cyt c) is an important indicator of cell apoptosis and can, therefore, be used for the diagnosis of cancer. We performed a comparative analysis of cyt c detection on the surface of lipid films or a monolayer of 11-mercaptoundecanoic acid (MUA) with immobilized specific or nonspecific DNA aptamers. A quartz crystal microbalance with dissipation monitoring (QCM-D) in a multiharmonic mode was used to study the interaction of cyt c with various surfaces. For this purpose, changes in the resonant frequency, Δf, and dissipation, ΔD, were determined. The strongest interaction of cyt c was observed with sensors based on specific DNA aptamers that were accompanied by a decrease in frequency and an increase in dissipation. The limit of detection (LOD) for this aptasensor was established as 2.89 ± 0.12 nM. The interaction of cyt c with supported lipid films also resulted in a decrease in resonant frequency, but significant changes occurred only in the µM concentration range of cyt c. Changes in dissipation were much lower in comparison with aptamer-based surfaces, which suggests a weaker contribution of cyt c adsorption to the viscosity. [ABSTRACT FROM AUTHOR]

The functionality of a semisynthetic DNA in the biological environment will depend on the base pair nature of its complementary base pairs. To understand this, base pair interactions between complementary bases of recently proposed eight second-generation artificial nucleobases are studied herein by considering their rare tautomeric conformations and a dispersion-corrected density functional theoretic method. It is found that the binding energies of two hydrogen-bonded complementary base pairs are more negative than those of the three hydrogen-bonded base pairs. However, as the former base pairs are endothermic, the semisynthetic duplex DNA would involve the latter base pairs. [ABSTRACT FROM AUTHOR]

One of the promising novel methods for radical tumor resection at a single-cell level is magneto-mechanical microsurgery (MMM) with magnetic nano- or microdisks modified with cancer-recognizing molecules. A low-frequency alternating magnetic field (AMF) remotely drives and controls the procedure. Here, we present characterization and application of magnetic nanodisks (MNDs) as a surgical instrument ("smart nanoscalpel") at a single-cell level. MNDs with a quasi-dipole three-layer structure (Au/Ni/Au) and DNA aptamer AS42 (AS42-MNDs) on the surface converted magnetic moment into mechanical and destroyed tumor cells. The effectiveness of MMM was analyzed on Ehrlich ascites carcinoma (EAC) cells in vitro and in vivo using sine and square-shaped AMF with frequencies from 1 to 50 Hz with 0.1 to 1 duty-cycle parameters. MMM with the "Nanoscalpel" in a sine-shaped 20 Hz AMF, a rectangular-shaped 10 Hz AMF, and a 0.5 duty cycle was the most effective. A sine-shaped field caused apoptosis, whereas a rectangular-shaped field caused necrosis. Four sessions of MMM with AS42-MNDs significantly reduced the number of cells in the tumor. In contrast, ascites tumors continued to grow in groups of mice and mice treated with MNDs with nonspecific oligonucleotide NO-MND. Thus, applying a "smart nanoscalpel" is practical for the microsurgery of malignant neoplasms. [ABSTRACT FROM AUTHOR]

Layer-by-layer (LbL) immobilization of DNA aptamers in the realm of electrochemical detection of heavy metal ions (HMIs) offers an enhancement in specificity, sensitivity, and low detection limits by leveraging the cross-reactivity obtained from multiple interactions between immobilized aptamers and developed material surfaces. In this research, we present a LbL approach for the immobilization of thiol- and amino-modified DNA aptamers on a Ag-incorporated cobalt-succinate metal–organic framework (MOF) (Ag@Co-Succinate) to achieve a cross-reactive effect on the electrochemical behavior of the sensor. The solvothermal method was utilized to synthesize Ag@Co-Succinate, which was also characterized through various techniques to elucidate its structure, morphology, and presence of functional groups, confirming its suitability as a host matrix for immobilizing both aptamers. The Ag@Co-Succinate aptasensor exhibited extraordinary sensitivity and selectivity towards Hg(II) ions in electrochemical detection, attributed to the unique binding properties of the immobilized aptamers. The exceptional limit of detection of 0.3 nM ensures the sensor’s suitability for trace-level Hg(II) detection in various environmental and analytical applications. Furthermore, the developed sensor demonstrated outstanding repeatability, highlighting its potential for long-term and reliable monitoring of Hg(II).

Massive use of antibiotics in veterinary medicine has led to their accumulation in meat and dairy products. Consumption of antibiotic-contaminated food can trigger the development of antibiotic-resistant bacteria, endangering human lives. Among antibiotics, the oxytetracycline (OTC) family of antibiotics is most widely used in veterinary medicine. Strict control of the antibiotics in food necessitates the development of fast and effective methods for OTC detection, for instance, in milk. One of the most promising approaches to OTC detection is based on the use of specially designed DNA aptamers. These DNA aptamers are relatively short, 15–60 bases, nucleotides folded in the solution in a 3D structure, forming a binding site for the target antibiotic. Aptamers can be chemically modified for attachment to sensor electrodes. In this work, we investigated the electrochemical detection of OTC using DNA aptamers specific to OTC that have been covalently immobilized onto the nanocomposite surface of a glassy carbon electrode with electrodeposited reduced graphene oxide and multiwalled carbon nanotubes. Differential pulse voltammetry, DPV, in the presentence of a ferri/ferrocyanide redox couple was used as an internal standard and to monitor the redox current. In the presence of OTC, the amplitude of DPV decreased, evidencing the blocking of charge transfer. After system optimization, we reached the limit of detection of 0.45 ng OTC/mL, which is 200 times lower than the maximum residue limit established by the European Commission of 100 ng OTC/mg. [ABSTRACT FROM AUTHOR]

Bacterial lipopolysaccharides (LPSs) are important indicators of a bacteria presence in any samples. They can therefore be used for the detection of microbiological contamination in food and dairy products. We performed a comparative analysis of different bacterial models by the application of liposomes containing LPS from Salmonella enterica serotype typhimurium on the surface of an 11-mercaptoundecanoic acid (MUA) monolayer chemisorbed on the gold surface of quartz crystal. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we were able to monitor the formation of the lectin, concanavalin A (ConA), layer on the MUA surface. We determined the optimal concentration of the ConA for the layer formation. ConA of 0.3 mg/mL was selected as the most suitable adsorption of liposomes containing LPS. Using the Sauerbrey equation, we calculated that approximately 1.13 × 1012 ConA molecules per cm2 was adsorbed on the MUA surface, which closely corresponds to the 1.19 × 1012 molecules per cm2 by theoretical models. Later, mixed LPS liposomes containing dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl ethanolamine (DPPE) and cholesterol successfully interacted with the ConA layer, which resulted in a decrease in the resonant frequency and an increase in dissipation. We compared the adsorption of liposomes with different fractions of LPS and containing LPS from different bacteria. Lack of any LPS in liposomes caused weaker adsorption on the ConA layer. Liposomes containing 50% LPS caused the most prominent adsorption and were suitable for interaction with DNA aptamers specific to certain LPS. The addition of the aptamers to the surface of ConA covered by LPS-containing liposomes resulted in a decrease in resonant frequency and an increase in the dissipation. Using the Kelvin–Voigt viscoelastic model and multiharmonic response of acoustic sensors, we also determined changes in viscoelastic values of the molecular films during interaction with liposomes and the ConA layer. [ABSTRACT FROM AUTHOR]

We analyzed the possibility of the detection of cytochrome c (cyt c) being physically adsorbed on lipid films or covalently bounded to 11-mercapto-1-undecanoic acid (MUA) chemisorbed on the gold layer using quartz crystal microbalance with dissipation monitoring (QCM-D). The negatively charged lipid film composed of a mixture of zwitterionic DMPC and negatively charged DMPG phospholipids at a molar ratio of 1:1 allowed the formation of a stable cyt c layer. Addition of DNA aptamers specific to cyt c, however, resulted in removal of cyt c from the surface. The interaction of cyt c with the lipid film and its removal by DNA aptamers were accompanied by changes in viscoelastic properties evaluated using the Kelvin–Voigt model. Cyt c covalently bound to MUA also provided a stable protein layer already at its relatively low concentrations (0.5 μM). A decrease in the resonant frequency following the addition of gold nanowires (AuNWs) modified by DNA aptamers was observed. The interaction of aptamers with cyt c on the surface can be a combination of specific and non-specific interactions due to electrostatic forces between negatively charged DNA aptamers and positively charged cyt c. [ABSTRACT FROM AUTHOR]

Novel rapid methodologies for the detection of bacteria have been recently investigated and applied. In hospital environments, infections by pathogens are very common and can cause serious health problems. Pseudomonas aeruginosa is one of the most common bacteria, which can grow in hospital equipment such as catheters and respirators. Even at low concentrations, it can cause severe infections as it is resistant to antibiotics and other treatments. Based on this subject's relevance, this work aimed to develop a colorimetric biosensor using aptamer-functionalized gold nanoparticles for identifying P. aeruginosa. The detection mechanism is based on the color change of gold nanoparticles (AuNPs) from red to blue-purple through NaCl induction after bacteria incubation and aptamer-target binding. First, AuNPs were synthesized and characterized. The influence of aptamer and sodium chloride concentration on the agglomeration of AuNPs was investigated. Optimization of aptamer concentration and salt addition were performed. The best condition for detection was 5 µM aptamers and 200 mM of NaCl. In this case, P. aeruginosa was detected after 5 h for concentrations from 108 to 105 CFU mL−1, being 105 and 104 CFU mL−1 the detection limit for color change by the naked eye and UV–Vis spectrometry, respectively. In addition, other bacteria such as E. coli, S. typhimurium, and Enterobacteriaceae bacterium were also detected with color changing from red to gray. Finally, it was confirmed that the salt incubation time can be 2 h, and that the ideal aptamer concentration is 5 µM. Thus, the colorimetric analysis can be a simple and fast detection method for P. aeruginosa in the range of 108 to 105 CFU mL−1 to the naked eye. Key Points: • A new method for rapid detection of Pseudomonas aeruginosa • Aptamers conjugated with gold nanoparticles allow pathogen detection by colorimetry • No need for previous surface modification of nanoparticles [ABSTRACT FROM AUTHOR]

Background and Aim: Salmonella is a major foodborne pathogen in the poultry industry, wherein the control measures may include sanitation and antibacterial and vaccines. However, there have been severe global restrictions on using anti-Salmonella antibacterial agents in livestock. This situation, along with rapidly increasing drug-resistant bacterial species, has led to the exploration of unconventional methods to control Salmonella infection in poultry. In recent years, selection techniques of promising DNA aptamers have begun to permeate several medical branches, resulting in the development of numerous anti-Salmonella DNA aptamers, most of which are used as sensing molecules for diagnostic purposes. These DNA aptamers have been demonstrated to interfere with bacterial growth, multiplication, and viability. Aptamers formed in rolling circle amplification products (RCA-p) could improve the potential action of aptamer interference with bacteria. This study aimed to test the use of single-stranded DNA aptamers in the form of RCA-p as a bacteriostatic to Salmonella in vitro. Materials and Methods: Salmonella Typhimurium and Salmonella Enteritidis isolates were subjected to the action of anti-ST and anti-SE DNA aptamers in the form of RCA-p. Each isolate was grown on MacConkey and Luria-Bertani agar media separately in different concentrations in the presence or absence of the cognate RCA-p. Results: The anti-Salmonella species DNA aptamer-based RCA-p were capable of reducing bacterial growth to significant levels in vitro. Conclusion: We describe a potential solution for the rapidly developing drug resistance of several bacterial species. Our findings suggested that the use of non-toxic, non-immunogenic, and low-cost DNA aptamers targeting Salmonella in the form of RCA-p could inhibit the bacterial growth rate. Unlike polymerase chain reaction, RCA yields tandem repeats of single-stranded DNA at isothermal conditions, which would increase the probability of receptor-ligand clustering and increase affinity. Furthermore, as our RCA template was bivalent with two DNA aptamer sequences, we could target multiple sites or antigens on a bacterial cell.

Aptamers are good affinity receptors for bio-assays, while colorimetric method is suitable for point-of-care sensing via direct visualization. But previously aptamers often need complex re-engineering for colorimetric measurement at the cost of affinity and performance. Here isoquinoline alkaloids are found to own unique light-activated oxidative capacity, which can be specifically triggered by unmodified aptamers. This feature is universal for two alkaloids to efficiently oxidize four chromogenic substrates with obvious color changes. Based on a dye-displacement process, we have developed a novel light-activated aptamer system for the colorimetric assay of estradiol. It shows a good sensitivity with a detection limit of 326 nM, and this homogeneous assay is reliable to avoid artifacts in previous heterogeneous scheme. Besides, it is proven to be a universal design to assay other two targets. Significantly, they do not employ any aptamers re-engineering but only simply use their parental aptamers. Therefore, this light-activated oxidative capacity of isoquinoline alkaloid can serve as an ideal tool for colorimetric assay of various targets based on aptamer's specific recognition. [Display omitted] • Isoquinoline alkaloids were found to own light-activated oxidative capacity. • A light-activated aptamer system was developed for universal colorimetric assay. • It did not employ any aptamers re-engineering. [ABSTRACT FROM AUTHOR]

Fibronectin (FN) is a well‐established hallmark of epithelial‐to‐mesenchymal transition, and may serve as an omnipresent cancer biomarker regardless of the origins of tumor cells. An ssDNA aptamer (ZY‐1) with highly selective binding affinity to mesenchymal stromal cells is previously developed, but the binding target of ZY‐1 on the cells and the underlying mechanism is yet to be understood. Here, the identification of FN as the target protein of aptamer ZY‐1 is reported for the first time and the mechanism of ZY‐1 binding to cFN is explored. The data indicate that ZY‐1 solely recognizes cellular fibronectin (cFN) rather than plasma fibronectin (pFN). The ZY‐1 binding to cFN is explored through computational modeling and the competition of heparin in binding cFN owing to steric hindrance is confirmed. The in vitro assay and noninvasive in vivo fluorescence imaging results validate the specificity of ZY‐1 in targeting cFN and sensitivity in detecting tumors. The ZY‐1‐mediated targeted cancer therapy using a proof‐of‐concept study with a ZY‐1‐based complex loaded with doxorubicin (Dox) is further proved. This study would facilitate more comprehensive studies of anti‐FN aptamers in the imaging and treatment of tumors and other FN‐associated diseases. [ABSTRACT FROM AUTHOR]

There is a critical need for the development of more potent inhibitors for osteoarthritis (OA) therapy given the poor life quality of arthritis patients. Aggrecanase ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) is an established drug target identified for osteoarthritis. In this study, we evolved and characterized two new DNA aptamer inhibitors of ADAMTS-5, namely apt21 and apt25. The aptamers exhibited nanomolar binding affinity and high specificity against ADAMTS-5. K D values of apt21 and apt25 were determined by the Enzyme-linked Oligonucleotide Assay (ELONA) at 1.54 ± 0.16 nM and 1.79 ± 0.08 nM, respectively. Circular Dichroism (CD) analysis demonstrated that both aptamers formed monovalent cation dependent G-quadruplex structures. Calcium ions did not affect the binding of the aptamers to ADAMTS-5. The inhibitory effects of apt21 and apt25 on ADAMTS-5 were evaluated by the Förster Resonance Energy Transfer (FRET) assay, in which IC 50 values of apt21 and apt25 were estimated at 52.76 ± 6.70 μM and 61.14 ± 9.67 μM, respectively. These two aptamers are the first DNA G-quadruplex aptamers demonstrated to inhibit ADAMTS-5 and could have value for OA therapy. [Display omitted] • DNA aptamers which bind and inhibit ADAMTS-5 were selected and characterized. • G-quadruplex structures of the two DNA aptamers were characterized. • Specific inhibitory effects of two DNA aptamers on ADAMTS-5 enzymatic activity were evaluated. [ABSTRACT FROM AUTHOR]

Identification of primary tumors and metastasis sites is an essential step in cancer diagnostics and the following treatment. Positron emission tomography-computed tomography (PET/CT) is one of the most reliable methods for scanning the whole organism for malignancies. In this work, we synthesized an 11C-labeled oligonucleotide primer and hybridized it to an anti-cancer DNA aptamer. The 11C-aptamer was applied for in vivo imaging of Ehrlich ascites carcinoma and its metastases in mice using PET/CT. The imaging experiments with the 11C-aptamer determined very small primary and secondary tumors of 3 mm2 and less. We also compared 11C imaging with the standard radiotracer, 2-deoxy-2-[fluorine-18]fluoro-D-glucose (18F-FDG), and found better selectivity of the 11C-aptamer to metastatic lesions in the metabolically active organs than 18F-FDG. 11C radionuclide with an ultra-short (20.38 min) half-life is considered safest for PET/CT imaging and does not cause false-positive results in heart imaging. Its combination with aptamers gives us high-specificity and high-contrast imaging of cancer cells and can be applied for PET/CT-guided drug delivery in cancer therapies.

Aptamers are synthetic single-stranded oligonucleotides that exhibit selective binding properties to specific targets, thereby providing a powerful basis for the development of selective and sensitive (bio)chemical assays. Electrochemical biosensors utilizing aptamers as biological recognition elements, namely aptasensors, are at the forefront of current research. They exploit the combination of the unique properties of aptamers with the advantages of electrochemical detection with the view to fabricate inexpensive and portable analytical platforms for rapid detection in point-of-care (POC) applications or for on-site monitoring. The immobilization of aptamers on suitable substrates is of paramount importance in order to preserve their functionality and optimize the sensors’ sensitivity. This work describes different immobilization strategies for aptamers on the surface of semiconductor-based working electrodes, including metal oxides, conductive polymers, and carbon allotropes. These are presented as platforms with tunable band gaps and various surface morphologies for the preparation of low cost, highly versatile aptasensor devices in analytical chemistry. A survey of the current literature is provided, discussing each analytical method. Future trends are outlined which envisage aptamer-based biosensing using semiconductors.

Zika virus became a major public health problem in early 2015, when cases of Guillain–Barré syndrome and microcephaly were associated with viral infection. Currently, ZIKV is endemic in all tropical areas of the world, and the chance for future Zika epidemics remains very real and accurate diagnosis is crucial. The aim of this work was to select specific ssDNA aptamers that bind to the entire Zika virus and can be used to compose specific diagnostics, without cross-reactivity with other flaviviruses. Zika virus was cultivated in Vero cells and used as a target for aptamer selection. Aptamers specific for the ZIKV were selected using whole-virus SELEX, with counterselection for other flavivirus. Secondary and tertiary structures were evaluated and the molecular anchoring between the aptamers and target were simulated by the HDOCK server. Aptamer interaction was evaluated by ELISA/ELASA and the dissociation constant (Kd) was calculated by thermophoresis. Four ZIKV-specific aptamers were selected. The best two were further characterized and proved to be specific for ZIKV. Aptamers are capable of binding specifically to the ZIKV and differentiate from Dengue virus. The aptamers selected in this work can be used as capture agents in the composition of diagnostic tests to specifically detect ZIKV infection. [ABSTRACT FROM AUTHOR]

This paper presents a fully automated point-of-care device for protein quantification using short-DNA aptamers, where no manual sample preparation is needed. The device is based on our novel aptamer-based methodology combined with real-time polymerase chain reaction (qPCR), which we employ for very sensitive protein quantification. DNA amplification through qPCR, sensing and real-time data processing are seamlessly integrated into a point-of-care device equipped with a disposable cartridge for automated sample preparation. The system's modular nature allows for easy assembly, adjustment and expansion towards a variety of biomarkers for applications in disease diagnostics and personalised medicine. Alongside the device description, we also present a new algorithm, which we named PeakFluo, to perform automated and real-time quantification of proteins. PeakFluo achieves better linearity than proprietary software from a commercially available qPCR machine, and it allows for early detection of the amplification signal. Additionally, we propose an alternative way to use the proposed device beyond the quantitative reading, which can provide clinically relevant advice. We demonstrate how a convolutional neural network algorithm trained on qPCR images can classify samples into high/low concentration classes. This method can help classify obese patients from their leptin values to optimise weight loss therapies in clinical settings. [ABSTRACT FROM AUTHOR]

Chikungunya fever, caused by Chikungunya virus (CHIKV) exhibits clinical features that mimic that of other arbovirus infections such as dengue. CHIKV Envelope 2 (E2) protein, an antigenic epitope of CHIKV, has been identified as an ideal marker for diagnostics. The current CHIKV antigen detection tests are largely based on antibodies but are beleaguered by issues such as sensitivity to high temperature, expensive and prone to batch-to-batch variations. Aptamers are suitable alternatives to antibodies as they are cheaper and have no batch-to-batch variations compared to antibodies. In this study, DNA aptamer selection against CHIKV E2 proteins was performed using two different randomized ssDNA libraries. Chik-2 (96-mer) and Chik-3 (76-mer) were isolated from these two libraries and were identified as the potential aptamers against CHIKV E2 protein. The binding affinity of Chik-2 and Chik-3 against CHIKV E2 protein was estimated at 177.5 ± 32.69 nM and 30.01 ± 3.60 nM, respectively. A sandwich ELASA was developed, and this assay showed a detection limit of 2.17 x 103 PFU/mL. The sensitivity and specificity of the assay were 80 % and 100 %, respectively. The assay showed no cross-reactivity with dengue-positive samples, demonstrating the enormous diagnostic potential of these aptamers for the detection of CHIKV. [Display omitted] • In this study, DNA aptamer selection against CHIKV E2 proteins was performed using two different randomized ssDNA libraries. • Chik-2 (96-mer) and Chik-3 (76-mer) were successfully generated against CHIKV E2 protein. • A sandwich ELASA assay was developed, achieving sensitivity and specificity of 80 % and 100 %, respectively. [ABSTRACT FROM AUTHOR]

Surface engineering of nanoparticles has recently emerged as a promising technique for synthetic molecular recognition of biological analytes. In particular, the use of synthetic heteropolymers adsorbed onto the surface of a nanoparticle can yield selective detection of a molecular target. Synthetic molecular recognition has unique advantages in leveraging the photostability, versatility, and exceptional chemical stability of nanomaterials. In particular, single-walled carbon nanotubes (SWNT) exhibit a large Stokes shift and near infrared emission for maximum biological sample transparency. Optical biosensors with high signal transduction and molecular specificity can be synthesized with amphiphilic heteropolymers grafted to SWNT, and discovered by high-throughput screening. Herein, we describe the development and the characterization of surface-engineered nanoparticles, or synthetic antibodies, for protein detection.

Cytochrome c (cyt c) is an important indicator of cell apoptosis and can, therefore, be used for the diagnosis of cancer. We performed a comparative analysis of cyt c detection on the surface of lipid films or a monolayer of 11-mercaptoundecanoic acid (MUA) with immobilized specific or nonspecific DNA aptamers. A quartz crystal microbalance with dissipation monitoring (QCM-D) in a multiharmonic mode was used to study the interaction of cyt c with various surfaces. For this purpose, changes in the resonant frequency, Δf, and dissipation, ΔD, were determined. The strongest interaction of cyt c was observed with sensors based on specific DNA aptamers that were accompanied by a decrease in frequency and an increase in dissipation. The limit of detection (LOD) for this aptasensor was established as 2.89 ± 0.12 nM. The interaction of cyt c with supported lipid films also resulted in a decrease in resonant frequency, but significant changes occurred only in the µM concentration range of cyt c. Changes in dissipation were much lower in comparison with aptamer-based surfaces, which suggests a weaker contribution of cyt c adsorption to the viscosity.

The development of biosensors has become increasingly important to tackle the spread of potentially pandemic pathogens and for the decentralization of healthcare services. Despite progress, achieving selective detection of ultralow concentrations of biomarkers in complex fluids with point‐of‐care devices is a standing challenge. Here, an efficient material platform for the sensing of biomarkers down to attomolar concentrations with excellent selectivity and a tuneable linear response range is reported. It is demonstrated that by decreasing the Au nanoislands on an elsewhere passivated surface, it is possible to more than double their electrochemical response to low biomarker concentrations via a gate‐type mechanism. As an exemplary application, the first sensing of glycated albumin, a key biomarker for diabetes management, in clinically relevant levels by conjugation of a selective DNA aptamer to the Au nanoisland surface is showcased. With a limit of detection of 0.55 × 10−18m, the excellent selectivity of this platform against nonspecific biomolecules is validated. This platform shows exceptional sensitivity and selectivity in mouse serum, further highlighting its potential for clinical utility. The versatility of this platform to detect other biomarkers, like miRNAs, is also showcased. These findings provide a flexible material platform for the scalable and low‐cost engineering of future point‐of‐care biosensors. [ABSTRACT FROM AUTHOR]

Optical control of protein activity represents a promising strategy for precise modulation of biological processes. We report rationally designed, aptamer‐based spherical nucleic acids (SNAs) capable of noninvasive and programmable regulation of target protein activity by deep‐tissue‐penetrable near‐infrared (NIR) light. The photoresponsive SNAs are constructed by integrating activatable aptamer modules onto the surface of upconversion nanoparticles. The SNAs remain inert but can be remotely reverted by NIR light irradiation to capture the target protein and thus function as an enzyme inhibitor, while introduction of antidote DNA could further reverse their inhibition functions. Furthermore, we demonstrate the potential of the SNAs as controllable anticoagulants for the NIR light‐triggered regulation of thrombin function. Ultimately, the availability of diverse aptamers would allow the design to regulate the activities of various proteins in a programmable manner. [ABSTRACT FROM AUTHOR]

Antibiotics are often used in human and veterinary medicine for the treatment of bacterial diseases. However, extensive use of antibiotics in agriculture can result in the contamination of common food staples such as milk. Consumption of contaminated products can cause serious illness and a rise in antibiotic resistance. Conventional methods of antibiotics detection such are microbiological assays chromatographic and mass spectroscopy methods are sensitive; however, they require qualified personnel, expensive instruments, and sample pretreatment. Biosensor technology can overcome these drawbacks. This review is focused on the recent achievements in the electrochemical biosensors based on nucleic acid aptamers for antibiotic detection. A brief explanation of conventional methods of antibiotic detection is also provided. The methods of the aptamer selection are explained, together with the approach used for the improvement of aptamer affinity by post-SELEX modification and computer modeling. The substantial focus of this review is on the explanation of the principles of the electrochemical detection of antibiotics by aptasensors and on recent achievements in the development of electrochemical aptasensors. The current trends and problems in practical applications of aptasensors are also discussed. [ABSTRACT FROM AUTHOR]

Background and Aim: Salmonella is a major foodborne pathogen in the poultry industry, wherein the control measures may include sanitation and antibacterial and vaccines. However, there have been severe global restrictions on using antiSalmonella antibacterial agents in livestock. This situation, along with rapidly increasing drug-resistant bacterial species, has led to the exploration of unconventional methods to control Salmonella infection in poultry. In recent years, selection techniques of promising DNA aptamers have begun to permeate several medical branches, resulting in the development of numerous anti-Salmonella DNA aptamers, most of which are used as sensing molecules for diagnostic purposes. These DNA aptamers have been demonstrated to interfere with bacterial growth, multiplication, and viability. Aptamers formed in rolling circle amplification products (RCA-p) could improve the potential action of aptamer interference with bacteria. This study aimed to test the use of single-stranded DNA aptamers in the form of RCA-p as a bacteriostatic to Salmonella in vitro. Materials and Methods: Salmonella Typhimurium and Salmonella Enteritidis isolates were subjected to the action of anti-ST and anti-SE DNA aptamers in the form of RCA-p. Each isolate was grown on MacConkey and Luria-Bertani agar media separately in different concentrations in the presence or absence of the cognate RCA-p. Results: The anti-Salmonella species DNA aptamer-based RCA-p were capable of reducing bacterial growth to significant levels in vitro. Conclusion: We describe a potential solution for the rapidly developing drug resistance of several bacterial species. Our findings suggested that the use of non-toxic, non-immunogenic, and low-cost DNA aptamers targeting Salmonella in the form of RCA-p could inhibit the bacterial growth rate. Unlike polymerase chain reaction, RCA yields tandem repeats of single-stranded DNA at isothermal conditions, which would increase the probability of receptor-ligand clustering and increase affinity. Furthermore, as our RCA template was bivalent with two DNA aptamer sequences, we could target multiple sites or antigens on a bacterial cell. [ABSTRACT FROM AUTHOR]

Background: Biomarker detection strategies have, in recent years, been moving towards nucleic acid-based detection systems in the form of aptamers, short oligonucleotide sequences which have shown promise in pre-clinical and research settings. One such aptamer is M5-15, a DNA aptamer raised against human alpha synuclein (α-syn) the causative agent in Lewy body and Parkinson's disease (PD) associated dementia. While this aptamer has shown promise, in silicomethodologies have demonstrated a capacity to produce aptamers that have higher affinities for their targets than in vitro generated sequences. Methods: A Python script random generated library of DNA sequences were screened based on their thermodynamic stability with the use of DINAMelt server-QuickFold web server. The selected sequences were examined with MFold in order to generate secondary structure data that were used to produce 3D data with the use of RNA composer software. Further on, the structure was corrected and RNA was replaced with DNA and the virtual screening for α-syn aptamer took place with a series of molecular docking experiments with the use of CSD-Discovery-GOLD software. Results: Herein we propose an alternative in silico generated aptamer we call TMG-79 which demonstrates greater affinity for the target compared to M5-15 (M5-15 = –15.9 kcal/mol, TMG-79 = –17.77 kcal/mol) as well as better ChemPLP fitness scoring between the top poses (M5-15 = 32.33, TMG-79 = 53.32). Structural analysis suggests that while there are similarities, the greater potential flexibility of TMG-79 could be promoting greater affinity for the α-syn compared to M5-15. Conclusions: In silico methods of aptamer generation has the potential to revolutionise the field of aptamer design. We feel that further development of TMG-79 and validation in vitro will make it a viable candidate for diagnostic and research use in the future. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase-9 (MMP-9) plays major roles in extracellular matrix (ECM) remodeling and membrane protein cleavage, suggesting a high correlation with cancer cell invasion and tumor metastasis. Here, we present a contrast agent based on a DNA aptamer that can selectively target human MMP-9 in the tumor microenvironment (TME) with high affinity and sensitivity. Surface modification of plasmonic gold nanospheres with the MMP-9 aptamer and its complementary sequences allows the nanospheres to aggregate in the presence of human MMP-9 through DNA displacement and hybridization. Aggregation of gold nanospheres enhances the optical absorption in the first near-infrared window (NIR-I) due to the plasmon coupling effect, thereby allowing us to detect the aggregated gold nanospheres within the TME via ultrasound-guided photoacoustic (US/PA) imaging. Selective and sensitive detection of human MMP-9 via US/PA imaging is demonstrated in solution of nanosensors with the pre-treatment of human MMP-9, in vitro in cell culture, and in vivo in a xenograft murine model of human breast cancer.