Use Of DNA Aptamers Research Articles

Synthetic Chemistry and Microelectrode Arrays for Point of Care Diagnostics

Molecular diagnostics typically require a molecular interaction that identifies a specific target, a device that detects, quantifies, and records that interaction, and an interface between those two elements. Each feature is critical for the success of the device. Yet while the first two are matters of intense interest an research, the surface itself is often ignored. This is problematic because it provides the platform for the whole "experiment". For a "point-of-care" diagnostic, the surface on the device must be stable both in terms of chemistry and time while still allowing the signal from the molecular interaction to be sensed and recorded.With this in mind, we have been focusing on the use of diblock copolymers as molecular surfaces for the construction of "point-of-care" diagnostics. We have developed a toolbox of chemical reactions that can be used site-selectively on such surfaces, and we have recently demonstrated that the approach taken is compatible with the use of DNA-aptamers to do multiplex sensing on a high density microelectrode array using a surface that is stable for a year. We have also illustrated how the quality of the signaling study conducted on an array is dependent on optimization of the chemical reaction.But exactly how good are the chemical reactions employed on the arrays to build a surface in a site-selective manner? One can tell with the use of fluorescence based studies that reactions happen where you want then to happen, but are there background reactions that can be problematic for a multiplex sensing experiment, do the reactions happen in high yield, and how much of an expensive, difficult to obtain biological reagent might be lost due to the confinement strategy? Are some reactions better than others in this regard? In short, if we are to optimize the surfaces constructed on a diagnostic device, then we need not only have tools in place for conducting synthetic chemistry on the device, but also the tools in place necessary to analyze those reactions. Understanding the chemistry that can and/or does occur at any electrode in a high density array requires more than looking at pretty pictures of fluorescent spots. In the talk to be given, we will utilize a combination of a "Kenner-safety-catch" linker strategy and molecular biology techniques to answer key questions about the synthetic chemistry used to construct the DNA-aptamer based multiplex sensor mentioned above.

A Path towards Timely VAP Diagnosis: Proof-of-Concept Study on Pyocyanin Sensing with Cu-Mg Doped Graphene Oxide.

In response to the urgent requirement for rapid, precise, and cost-effective detection in intensive care units (ICUs) for ventilated patients, as well as the need to overcome the limitations of traditional detection methods, researchers have turned their attention towards advancing novel technologies. Among these, biosensors have emerged as a reliable platform for achieving accurate and early diagnoses. In this study, we explore the possibility of using Pyocyanin analysis for early detection of pathogens in ventilator-associated pneumonia (VAP) and lower respiratory tract infections in ventilated patients. To achieve this, we developed an electrochemical sensor utilizing a graphene oxide-copper oxide-doped MgO (GO - Cu - Mgo) (GCM) catalyst for Pyocyanin detection. Pyocyanin is a virulence factor in the phenazine group that is produced by Pseudomonas aeruginosa strains, leading to infections such as pneumonia, urinary tract infections, and cystic fibrosis. We additionally investigated the use of DNA aptamers for detecting Pyocyanin as a biomarker of Pseudomonas aeruginosa, a common causative agent of VAP. The results of this study indicated that electrochemical detection of Pyocyanin using a GCM catalyst shows promising potential for various applications, including clinical diagnostics and drug discovery.

Sensitive Detection of Tumor Cells Using Protein Nanoparticles with Multiple Displays of DNA Aptamers and Bioluminescent Reporters.

Simple and effective detection methods for circulating tumor cells are essential for early detection and progression monitoring of tumors. The use of DNA aptamer and bioluminescence is expected to be a key tool for the simple, effective, and sensitive detection of tumor cells. Herein, we designed multifunctional protein nanoparticles for the detection of tumor cells using DNA aptamer and bioluminescence. Fusion proteins (ELP-poly(d)-POIs), composed of elastin-like polypeptide (ELP) fused with protein of interests (POIs) via poly(aspartic acid) (poly(d)), formed the protein nanoparticles based on the temperature responsivity of ELP sequences, leading to multiply displayed POIs on the protein nanoparticles. In the present study, we focused on porcine circovirus type 2 replication initiation protein (Rep), which covalently conjugated with DNA aptamers, and NanoLuc luciferase (Nluc), which emitted a strong bioluminescence, as POIs. ELP-poly(d)-Rep and ELP-poly(d)-Nluc were constructed and formed the protein nanoparticles with multiply displayed Nluc and Rep (DNA aptamer) that amplified the bioluminescence signal and tumor recognition ability. Mucin-1 (MUC1)-overexpressing human breast tumor MCF7 cells and MUC1-recognizing aptamer (MUC1 aptamer) were selected as models. The MUC1 aptamer-conjugated protein nanoparticles exhibited a 13.7-fold higher bioluminescence signal to MCF-7 cells than to human embryonic kidney 293 (HEK293) cells, which express low levels of MUC1. Furthermore, the protein nanoparticles could detect up to 70.7 cells/mL of MCF-7 cells from a cell suspension containing HEK-293. The protein nanoparticles with multiple Rep and Nluc show a great potential as a material for detecting CTCs.

Electrochemical aptasensors for diagnostics and analysis of biological samples. Important aspects

The transition to personalized medicine requires a continuous improvement in the quality of diagnostics, and, consequently, improvement and effi ciency of the use of diagnostic tools. Biosensors have high potential in the fi eld of clinical diagnosis, especially in the early stages of diseases. Th e use of postgenomic technologies, in particular the use of DNA-aptamers, which are recognizing biomolecules, as part of biosensors, in combination with highly sensitive methods of analysis, such as electrochemical ones, can further increase the potential of using biosensor systems. Because it becomes possible to determine the presence of target objects (for example, tumor markers or markers of other diseases) with high sensitivity and selectivity of analysis. However, despite the rapid growth in the number of scientifi c research and publications in the fi eld of the development of electrochemical biosensors based on aptamers (aptasensors), at the moment the commercialization of the products obtained occurs slowly and to a low level. Th e market of biosensors is still dominated by enzymes and antibodies-based sensors that are standardized in the industry. Th is is probably due to the fact that the business originated from enzymes/antibodies utilization at fi rst. But also the general lack of knowledge on the performance of aptasensors in research and in practical use plays an important role [1]. Th e latter, inter alia, is a consequence of the lack of a proven theoretical base to date, of confi rmed key mechanisms and patterns governing the processes of obtaining the response of an electrochemical aptasensor. However, it is obvious that the confi rmed knowledge of the key patterns and mechanisms of the biosensor response formation in the presence of the target object will allow a suffi ciently high degree of control of this process to optimize the analysis procedure and increase its effi ciency and stability of the aptasensor making it suitable for the practical use. A large variety of approaches are used for new electrochemical aptasensors development. Among them there are diff erent signal amplifi cation strategies, nanoparticles and other biomolecules addition to the aptasensor’s construction, more complicated signal formation schemes, and so on [2]. So, the fi rst important aspect is the fact that every new-developed approach requires theoretical consideration and deep study of the sensing mechanism. Before developing an aptasensor for the commercial purposes, one should consider the target task and the specifi c requirements, namely sampling and samples’ preparation, analyte’s type and the targeted concentration range, the possible composition of the matrix, noise, the fi nal cost of the device, etc. [2-5]. Th us, two next important aspects are the necessity to take into account the real samples’ specifi city and the request for the minimal possible price. Th e last important point that needs to be highlighted is the analytical signal of the electrochemical aptasensor itself. Normally, for the entire electrochemical response recorded in the form of dependences of one or more parameters on the programmed eff ect (current, potential, frequency), one feature is selected (called an analytical signal), by which all the data is analyzed. More frequently, to detect the presence of the target analyte, the magnitude of the selected feature is compared for the response of the biosensor before and aft er contact with the test sample (for example, [6]). However, the responses are complex and are composed of many features. Th e signal can be either a nontrivial feature, or it might be their pair or a triple combination. Identifi cation of the informative part of the response is the work of obtaining/generating an aptasensor signal. So, in the present work, four important aspects that need to be considered during electrochemical aptasensor development were pointed out. Not all of them are widely presented in the literature, but still, they require the attention of the researchers.

Highly sensitive magnetic-microparticle-based aptasensor for Cryptosporidium parvum oocyst detection in river water and wastewater: Effect of truncation on aptamer affinity

Many methods have been reported to detect Cryptosporidium parvum (C. parvum) oocysts in the water environment using monoclonal antibodies. Herein, we report the use of DNA aptamers as an alternative ligand. We present the highly sensitive detection of C. parvum oocysts in wastewater samples based on aptamer-conjugated magnetic beads. A previously selected DNA aptamer (R4-6) that binds to C. parvum oocysts with high affinity and selectivity was rationally truncated into two minimer aptamers (Min_Crypto1 and Min_Crypto2), and conjugated to micro-magnetic beads. In flow cytometry tests with phosphate buffer, river water, and wastewater samples, both the minimers showed improved affinity and specificity toward C. parvum oocysts than the parent R4-6. Moreover, Min_Crypto2 showed higher affinity to its target than the parent aptamer when testing in wastewater, indicating superior binding properties in a complex matrix.Using a fluorescence microplate-based assay, and when incubated with different numbers of oocysts, Min_Crypto2 showed a limit of detection as low as 5 C. parvum oocysts in 300 μL of wastewater. Results described here indicate that Min_Crypto2 has superior specificity and sensitivity for the detection of C. parvum oocysts, and has a strong potential to be used successfully in a sensor.

Characterization and detection of cardiac Troponin-T protein by using ‘aptamer’ mediated biofunctionalization of ZnO thin-film transistor

In this work, we have demonstrated the use of DNA aptamer as a selective bio recognizable entity towards the qualitative detection of cardiac Troponin-T (cTnT), a clinically important cardiac marker protein. Prior to this, the ZnO thin film surface as an active semiconducting channel material was first biofunctionalized with DNA aptamer by using standard biotin-streptavidin chemistry. Biofunctionalization was further confirmed by analyzing the ZnO surface with atomic force microscope (AFM) and X- ray photoelectron spectroscopy (XPS) techniques. In order to understand the efficacy of biomolecular interaction between cardiac Troponin-T (cTnT) and its corresponding aptamer, Kelvin Probe Force Microscopy (KPFM) technique was performed to monitor the effective contact potential difference. Further, to realize the essential technological importance of this methodology as a sensor platform, a thin-film transistor (TFT) micro device was fabricated using ZnO as a channel material and characterized. These fabricated devices were further used to demonstrate the qualitative detection of cardiac Troponin-T protein by employing aptamer-mediated biofunctionalization of ZnO TFT.

Comparison against current standards of a DNA aptamer for the label-free quantification of tobramycin in human sera employed for therapeutic drug monitoring

The use of DNA aptamers in biosensors for the quantification of pharmaceuticals in the clinics would help to overcome the limitations of antibody-based detection for small molecules. The interest for such systems is proven by the ever-increasing number of aptamer-based solutions for analytics proposed in the literature as proof-of-concept demonstrators. Despite such diversity, these platforms often lack a comparative assessment of their performances against the current standard of practice in the clinics when using real samples. We employed an aptamer against tobramycin discovered in our laboratory to quantify through surface plasmon resonance the concentration of the antibiotic in clinical samples obtained from patients treated with tobramycin and undergoing therapeutic drug monitoring. We then compared the performances of our detection strategy against the current standard of practice. Our results show how, using adequate calibration and matrix complexity reduction, DNA aptamer-based direct assays can assess clinically relevant concentrations of small molecules in patient serum and with good correlation to current standards used in the clinics.

Use of DNA-Aptamers for Enrichment of Low Abundant Proteins in Cellular Extracts for Quantitative Detection by Selected Reaction Monitoring

Use of DNA-Aptamers for Enrichment of Low Abundant Proteins in Cellular Extracts for Quantitative Detection by Selected Reaction Monitoring

Use of DNA-aptamers for enrichment of low abundant proteins in cellular extracts for quntitative detection by selected reaction monitoring

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of target protein with aptamers immobilized on a solid phase was studied. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as model target protein. It has been demonstrated that the affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads results in an approximately 10-fold increase of the concentration of target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM is possible without an interference from other peptides present in a tryptic digest.

Aptamer based voltammetric determination of ampicillin using a single-stranded DNA binding protein and DNA functionalized gold nanoparticles.

An aptamer based method is described for the electrochemical determination of ampicillin. It is based on the use of DNA aptamer, DNA functionalized gold nanoparticles (DNA-AuNPs), and single-stranded DNA binding protein (ssDNA-BP). When the aptamer hybridizes with the target DNA on the AuNPs, the ssDNA-BP is captured on the electrode surface via its specific interaction with ss-DNA. This results in a decreased electrochemical signal of the redox probe Fe(CN)63- which is measured best at a voltage of 0.188mV (vs. reference electrode). In the presence of ampicillin, the formation of aptamer-ampicillin conjugate blocks the further immobilization of DNA-AuNPs and ssDNA-BP, and this leads to an increased response. The method has a linear reposne that convers the 1 pM to 5nM ampicillin concentration range, with a 0.38 pM detection limit (at an S/N ratio of 3). The assay is selective, stable and reproducible. It was applied to the determination of ampicillin in spiked milk samples where it gave recoveries ranging from 95.5 to 105.5%. Graphical abstract Schematic of a simple and sensitive electrochemical apta-biosensor for ampicillin detection. It is based on the use of gold nanoparticles (AuNPs), DNA aptamer, DNA functionalized AuNPs (DNA-AuNPs), and single-strand DNA binding protein (SSBP).

Evolving Aptamers with Unnatural Base Pairs.

A novel technology, genetic alphabet expansion, has rapidly advanced through the successful creation of unnatural base pairs that function as a third base pair in replication. Recently, genetic alphabet expansion has been applied to some practical areas. Among them, the application to DNA aptamer generation is a good example of the broad utility of this technology. A hydrophobic unnatural base pair, Ds-Px, which exhibits high fidelity in replication as a third base pair, has been applied to an evolutionary engineering method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) to generate DNA aptamers that bind to targets. A few Ds bases in DNA aptamers significantly increase the binding affinity to targets, enabling the use of DNA aptamers as an alternative to antibodies. This protocol describes the ExSELEX (genetic alphabet Expansion for SELEX) method to generate Ds-containing DNA aptamers. © 2017 by John Wiley & Sons, Inc.

Development of a Sensitive Multiplexed Open Circuit Potential System for the Detection of Prostate Cancer Biomarkers

We report the development of a sensitive label-free, cost-effective detection system with simultaneous multi-channel measurement of open circuit potential (OCP) variations for the detection of prostate specific antigen (PSA). We demonstrate a significant increase of 600 times in the sensitivity as compared to the reported literature. To accurately measure OCP variations, a complete monolithic field-effect transistor (FET)-input ultra-low input bias current instrumentation amplifier is used to form the electronic circuit to measure the variation between a working electrode and a reference electrode. This amplifier electronic system setup provides a differential voltage measurement with high input impedance and low input bias current. Since no current is applied to the electrochemical system, a true and accurate measurement of the variation can be performed. This is the first report on the use of DNA aptamers with an OCP system where we employed a DNA aptamer against PSA. An optimised ratio of anti-PSA DNA aptamer with 6-mercapto-1-hexanol (MCH) was used to fabricate the aptasensor using gold electrodes. The electrodes are hosted in a cell with an automated flow system. A wide range of concentrations of PSA (0.1 to 100 ng/mL) were injected through the system. The sensor could potentially differentiate 0.1 ng/mL PSA from blank measurement, which is well below the required clinical range (>1 ng/mL). The sensor was also challenged with 4% human serum albumin and human kallikrein2 as control proteins where the sensor demonstrated excellent selectivity. The developed system can be further generalised to various other targets using specific probes.

Aptamer-Quantum Dot Lateral Flow Test Strip Development for Rapid and Sensitive Detection of Pathogenic Escherichia coli via Intimin, O157- Specific LPS and Shiga Toxin 1 Aptamers

Background: The use of DNA aptamers to replace antibodies in lateral flow (LF) test strip assays coupled to quantum dots (QDs) has demonstrated enhanced sensitivity for the rapid and portable detection of several foodborne pathogenic bacterial species with simple ultraviolet (UV) illumination and visual assessment (Bruno, Pathogens, 2014; 3: 341-355). Objectives: The present report extends and focuses development on detection of O157- specific lipopolysaccharide (LPS), intimin protein and Shiga toxin 1 (Stx 1) using the same aptamer-QD LF assay approach to aid in rapid and sensitive detection of E. coli O157:H7 and other pathogenic serotypes of E. coli. Methods: Numerous anti-O157 LPS, intimin and anti-Stx 1 aptamer DNA sequence candidates were developed and screened by enzyme-linked aptamer assay (ELASA) followed by further screening of the best candidates in less expensive aptamer- latex particle or aptamer-colloidal gold (CG) LF formats. The highest affinity and most specific aptamer candidates were incorporated into the aptamer-QD LF format. Results: Several high affinity and specific aptamers against intimin, Stx1, and O157 LPS were developed and found to work well in various capture-reporter conjugate combinations using latex particles, CG and QDs with detection limits as low as 100 E. coli O157:H7 bacterial cells and 10 ng of Stx 1 in buffer by visual assessment. Conclusion: The seminal work in this aptamer-QD LF area has been extended to sensitive detection of E. coli O157 cells as well as detection of other pathogenic E. coli serotypes by targeting intimin and Stx 1. Keywords: Aptamer, E. coli, intimin, lateral flow, quantum dot, SELEX, Shiga toxin.

Aptamer-conjugated nanomaterials for specific cancer cell recognition and targeted cancer therapy.

Based on their unique advantages, increasing interest has been shown in the use of aptamers as target ligands for specific cancer cell recognition and targeted cancer therapy. Recently, the development of aptamer-conjugated nanomaterials has offered new therapeutic opportunities for cancer treatment with better efficacy and lower toxicity. We highlight some of the promising classes of aptamer-conjugated nanomaterials for the specific recognition of cancer cells and targeted cancer therapy. Recent developments in the use of novel strategies that enable sensitive and selective cancer cell recognition are introduced. In addition to targeted drug delivery for chemotherapy, we also review how aptamer-conjugated nanomaterials are being incorporated into emerging technologies with significant improvement in efficiency and selectivity in cancer treatment.

Thrombin-Mediated Transcriptional Regulation Using DNA Aptamers in DNA-Based Cell-Free Protein Synthesis

Realizing the potential of cell-free systems will require development of ligand-sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand-sensitive transcriptional control in cell-free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single-stranded template, results in up to a 6-fold change in gene expression in a ligand concentration-dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell-free systems.

Engineering a Biomimetic Biological Nanopore to Selectively Capture Folded Target Proteins

Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis and applications in sensing. Biological nanopores generally perform better than artificial nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analytes proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at −50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA nanopores decorated with aptamers covalently attached to the nanopore exterior. Like nuclear-pore complexes (NPC), these nanopores selectively captured and translocated cognate protein analytes into their interiors, but excluded non-cognate analytes.View Large Image | View Hi-Res Image | Download PowerPoint Slide

An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry.

Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis, and applications in sensing. Biological nanopores generally perform better than artificial nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analyte proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at -50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA nanopores decorated with covalently attached aptamers. These nanopores selectively captured and internalized cognate protein analytes but excluded noncognate analytes, in a process that resembles transport by nuclear pores.