Noncompetitive Determination of Small Analytes by Sandwich-Type Lateral Flow Assay Based on an Aptamer Kissing Complex.

Here, we present the proof-of-concept of a lateral flow assay (LFA) that is capable of detecting small-molecule targets in a noncompetitive manner by deploying a sandwich-type format based on the aptamer kissing complex (AKC) strategy. A fluorescently labeled hairpin aptamer served as the signaling agent, while a specific RNA hairpin grafted onto the strip served as the capture element. The hairpin aptamer switched from an unfolded to a folded form in the presence of the target, resulting in kissing interactions between the loops of the reporter and the capture agents. This design triggered a target-dependent fluorescent signal at the test line. The AKC-based LFA was developed for the detection of adenosine, achieving a detection limit in the micromolar range. The assay revealed the presence of the same analyte in urine. The method also proved effective with another small molecule (theophylline). We believe that the AKC-based LFA approach could overcome many of the shortcomings associated with conventional signal-off methods and competitive processes.

Non-invasive Monitoring of α-Synuclein in Saliva for Parkinson's Disease Using Organic Electrolyte-Gated FET Aptasensor.

Parkinson's disease (PD) currently affects more than 1 million people in the US alone, with nearly 8.5 million suffering from the disease worldwide, as per the World Health Organization. However, there remains no fast, pain-free, and effective method of screening for the disease in the ageing population, which also happens to be the most susceptible to this neurodegenerative disease. αSynuclein (αSyn) is a promising PD biomarker, demonstrating clear delineations between levels of the αSyn monomer and the extent of αSyn aggregation in the saliva of PD patients and healthy controls. In this work, we have demonstrated a laboratory prototype of a soft fluidics integrated organic electrolyte-gated field-effect transistor (OEGFET) aptasensor platform capable of quantifying levels of αSyn aggregation in saliva. The aptasensor relies on a recently reported synthetic aptamer which selectively binds to αSyn monomer as the bio-recognition molecule within the integrated fluidic channel of the biosensor. The produced saliva sensor is label-free, fast, and reusable, demonstrating good selectivity only to the target molecule in its monomer form. The novelty of these devices is the fully isolated organic semiconductor, which extends the shelf life, and the novel fully integrated soft microfluidic channels, which simplify saliva loading and testing. The OEGFET aptasensor has a limit of detection of 10 fg/L for the αSyn monomer in spiked saliva supernatant solutions, with a linear range of 100 fg/L to 10 µg/L. The linear range covers the physiological range of the αSyn monomer in the saliva of PD patients. Our biosensors demonstrate a desirably low limit of detection, an extended linear range, and fully integrated microchannels for saliva sample handling, making them a promising platform for non-invasive point-of-care testing of PD.

Aptamer-based colorimetric and lateral flow assay approaches for the detection of toxic metal ions, thallium(i) and lead(ii).

Thallium(i) and lead(ii) ions are heavy metals and extremely toxic. These metals are environmental pollutants, posing a severe risk to the environment and human health. In this study, two approaches were examined using aptamer and nanomaterial-based conjugates for thallium and lead detection. The first approach utilized an in-solution adsorption-desorption approach to develop colorimetric aptasensors for the detection of thallium(i) and lead(ii) using gold or silver nanoparticles. The second approach was the development of lateral flow assays, and their performance was tested with thallium (limit of detection is 7.4 µM) and lead ion (limit of detection is 6.6 nM) spiked into real samples. The approaches assessed are rapid, inexpensive, and time efficient with the potential to become the basis for future biosensor devices.

Electrochemical DNAzyme-based biosensors for disease diagnosis.

DNAzyme-based electrochemical biosensors provide exceptional analytical sensitivity and high target recognition specificity for disease diagnosis. This review provides a critical perspective on the fundamental and applied impact of incorporating DNAzymes in the field of electrochemical biosensing. Specifically, we highlight recent advances in creating DNAzyme-based electrochemical biosensors for diagnosing infectious diseases, cancer and regulatory diseases. We also develop an understanding of challenges around translating the research in the field of DNAzyme-based electrochemical biosensors from labs to clinics, followed by a discussion on different strategies that can be applied to enhance the performance of the currently existing technologies to create truly point-of-care electrochemical DNAzyme biosensors.

Consensus Guidelines for the Design and In Vitro Preclinical Efficacy Testing N-of-1 Exon Skipping Antisense Oligonucleotides.

Antisense oligonucleotides (ASOs) can modulate pre-mRNA splicing. This offers therapeutic opportunities for numerous genetic diseases, often in a mutation-specific and sometimes even individual-specific manner. Developing therapeutic ASOs for as few as even a single patient has been shown feasible with the development of Milasen for an individual with Batten disease. Efforts to develop individualized ASOs for patients with different genetic diseases are ongoing globally. The N = 1 Collaborative (N1C) is an umbrella organization dedicated to supporting the nascent field of individualized medicine. N1C recently organized a workshop to discuss and advance standards for the rigorous design and testing of splice-switching ASOs. In this study, we present guidelines resulting from that meeting and the key recommendations: (1) dissemination of standardized experimental designs, (2) use of standardized reference ASOs, and (3) a commitment to data sharing and exchange.

Digital immunoassay for biomarker concentration quantification using solid-state nanopores.

Single-molecule counting is the most accurate and precise method for determining the concentration of a biomarker in solution and is leading to the emergence of digital diagnostic platforms enabling precision medicine. In principle, solid-state nanopores-fully electronic sensors with single-molecule sensitivity-are well suited to the task. Here we present a digital immunoassay scheme capable of reliably quantifying the concentration of a target protein in complex biofluids that overcomes specificity, sensitivity, and consistency challenges associated with the use of solid-state nanopores for protein sensing. This is achieved by employing easily-identifiable DNA nanostructures as proxies for the presence ("1") or absence ("0") of the target protein captured via a magnetic bead-based sandwich immunoassay. As a proof-of-concept, we demonstrate quantification of the concentration of thyroid-stimulating hormone from human serum samples down to the high femtomolar range. Further optimization to the method will push sensitivity and dynamic range, allowing for development of precision diagnostic tools compatible with point-of-care format.

Development and characterization of a DNA aptamer for MLL-AF9 expressing acute myeloid leukemia cells using whole cell-SELEX.

Current classes of cancer therapeutics have negative side effects stemming from off-target cytotoxicity. One way to avoid this would be to use a drug delivery system decorated with targeting moieties, such as an aptamer, if a targeted aptamer is available. In this study, aptamers were selected against acute myeloid leukemia (AML) cells expressing the MLL-AF9 oncogene through systematic evolution of ligands by exponential enrichment (SELEX). Twelve rounds of SELEX, including two counter selections against fibroblast cells, were completed. Aptamer pools were sequenced, and three candidate sequences were identified. These sequences consisted of two 23-base primer regions flanking a 30-base central domain. Binding studies were performed using flow cytometry, and the lead sequence had a binding constant of 37.5 + / - 2.5 nM to AML cells, while displaying no binding to fibroblast or umbilical cord blood cells at 200 nM. A truncation study of the lead sequence was done using nine shortened sequences, and showed the 5' primer was not important for binding. The lead sequence was tested against seven AML patient cultures, and five cultures showed binding at 200 nM. In summary, a DNA aptamer specific to AML cells was developed and characterized for future drug-aptamer conjugates.

Biosensing with DNAzymes.

This article provides a comprehensive review of biosensing with DNAzymes, providing an overview of different sensing applications while highlighting major progress and seminal contributions to the field of portable biosensor devices and point-of-care diagnostics. Specifically, the field of functional nucleic acids is introduced, with a specific focus on DNAzymes. The incorporation of DNAzymes into bioassays is then described, followed by a detailed overview of recent advances in the development of in vivo sensing platforms and portable sensors incorporating DNAzymes for molecular recognition. Finally, a critical perspective on the field, and a summary of where DNAzyme-based devices may make the biggest impact are provided.

Exploring the Unique Contrast Properties of Aptamer-Gadolinium Conjugates in Magnetic Resonance Imaging for Targeted Imaging of Thrombi.

Objective: An important clinical question in the determination of the extent of thrombosis-related vascular conditions is the identification of blood clot location. Fibrin is a major molecular constituent of blood clots and can, therefore, be utilized in molecular imaging. In this proof-of-concept study, we sought to prepare a fibrin-targeting magnetic resonance imaging contrast agent, using a Gd(III)-loaded fibrinogen aptamer (FA) chelate conjugate (Gd(III)-NOTA-FA) (NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid), to endow the ability to specifically accumulate at the location of blood clots, thereby enhancing contrast capabilities. Methods: The binding affinity of FA for fibrin was confirmed by fluorescence microscopy and microscale thermophoresis. The preparation and effective loading of the chelate-aptamer conjugates were confirmed by mass spectrometry and a xylenol orange colorimetric test. Longitudinal and transverse relaxivities and the effects of target binding were assessed using T1- and T2-map sequences at 7 T. T1- and T2-weighted images were acquired after blood clots were treated with Gd(III)-NOTA-FA. Collagen was used as the protein control, while an unrelated aptamer sequence, FB139, was used as the aptamer control. Results: FA demonstrated a high affinity and selectivity toward the polymeric protein, with a Kd of 16.6 nM, confirming an avidity over fibrinogen. The longitudinal (r1) and transverse (r2) relaxivities of Gd(III)-NOTA-FA demonstrated that conjugation to the long aptamer strand shortened T1 relaxation times and increased T2 relaxation times (3.04 and 38.7 mM-1 s-1, respectively). These effects were amplified by binding to the fibrin target (1.73 and 46.5 mM-1 s-1, respectively). In vitro studies with thrombin-polymerized human blood (clots) in whole blood showed an unexpected enhancement of signal intensity (hyperintense) produced exclusively at the location of the clot during the T2-weighted scan, while the presence of fibrinogen within a whole blood pool resulted in T1 signal intensity enhancement throughout the pool. This is advantageous, as simply reversing the type of a scan from a typical T1-weighted to a T2-weighted would allow to selectively highlight the location of blood clots. Conclusions: Gd(III)-NOTA-FA can be used for molecular imaging of thrombi, through fibrin-targeted delivery of contrast to the location of blood clots in T2-weighted scans.

DNAzymes as key components of biosensing systems for the detection of biological targets.

DNAzymes are synthetic functional nucleic acids that have found widespread use in biosensing applications both for molecular recognition and signal generation. Two classes of DNAzymes have proved particularly effective for use in proof-of-concept biosensing systems, namely RNA-cleaving DNAzymes (RCDs) and peroxidase mimicking DNAzymes (PMDs). RCDs catalyze the site-specific cleavage reaction of an RNA dinucleotide junction, generating two cleavage fragments. PMDs are capable of catalyzing peroxidation reactions of chromophores, thereby generating a measurable signal. Herein, we review the use of these DNAzymes in biomedical assays and diagnostics, and show that this emerging field should have great promise for biosensor development over the next few decades.

Selection and Characterization of an RNA-Cleaving DNAzyme Activated by Legionella pneumophila.

Legionella pneumophila is a deadly bacterial pathogen that has caused numerous Legionnaires' disease outbreaks, where cooling towers were the most common source of exposure. Bacterial culturing is used for L. pneumophila detection, but this method takes approximately 10 days to complete. In this work, an RNA-cleaving fluorogenic DNAzyme, named LP1, was isolated. Extensive characterization revealed that LP1 is reactive with multiple infectious isolates of L. pneumophila but inactive with 25 other common bacterial species. LP1 is likely activated by a protein target, capable of generating a detectable signal in the presence of as few as 10 colony-forming units of L. pneumophila, and able to maintain its activity in cooling tower water from diverse sources. Given that similar DNAzymes have been incorporated into many sensitive assays for bacterial detection, LP1 holds the potential for the development of biosensors for monitoring the contamination of L. pneumophila in exposure sources.

Aptamer-Based Biosensors for Environmental Monitoring.

Due to their relative synthetic and chemical simplicity compared to antibodies, aptamers afford enhanced stability and functionality for the detection of environmental contaminants and for use in environmental monitoring. Furthermore, nucleic acid aptamers can be selected for toxic targets which may prove difficult for antibody development. Of particular relevance, aptamers have been selected and used to develop biosensors for environmental contaminants such as heavy metals, small-molecule agricultural toxins, and water-borne bacterial pathogens. This review will focus on recent aptamer-based developments for the detection of diverse environmental contaminants. Within this domain, aptamers have been combined with other technologies to develop biosensors with various signal outputs. The goal of much of this work is to develop cost-effective, user-friendly detection methods that can complement or replace traditional environmental monitoring strategies. This review will highlight recent examples in this area. Additionally, with innovative developments such as wearable devices, sentinel materials, and lab-on-a-chip designs, there exists significant potential for the development of multifunctional aptamer-based biosensors for environmental monitoring. Examples of these technologies will also be highlighted. Finally, a critical perspective on the field, and thoughts on future research directions will be offered.

In Vitro Selection of New DNA Aptamers for Human Vascular Endothelial Growth Factor 165.

Two DNA aptamers that bind the heparin-binding domain (HBD) of the human vascular endothelial growth factor 165 (VEGF-165) have been previously reported. Although VEGF-165 is a homodimeric protein and the two aptamers have different sequences and secondary structures, the aptamers appear to occupy the same binding site and cannot form a 2 : 1 aptamer/protein complex, thus making them unsuitable for creating a higher-affinity dimeric DNA aptamer. This has motivated us to conduct a new in vitro selection experiment to search for new VEGF-165-binding DNA aptamers with different properties. We undertook a multistream selection strategy in which the concentration of VEGF-165 was varied significantly. We carried out 11 rounds of selection, and next-generation sequencing was conducted for every round in each stream. From comprehensive sequence analysis, we identified four classes of DNA aptamers, of which two were reported before, but two are new DNA aptamers. One of the new aptamers exhibits a unique property that has never been observed before: it is capable of forming the 2 : 1 aptamer/protein complex with VEGF-165. This work has expanded the repertoire of VEGF-165-binding DNA aptamers and creates a possibility to engineer a higher affinity homodimeric aptamer for VEGF-165.

Advances in functional nucleic acid based paper sensors.

Recently, portable sensing devices with point of care testing (POCT) capability have attracted great attention due to their inherent affordability and accessibility in low resource areas. Paper sensors possess excellent potential as POCT platforms because of low cost, ease of operation, disposability and high-volume manufacturing. Paper sensors that incorporate functional nucleic acids (FNAs) as recognition elements are particularly attractive given that FNAs can be isolated from random-sequence nucleic acid pools to recognize, or respond to, virtually any target of interest. In this review, the advantages of FNAs, particularly DNA aptamers and DNAzymes, as recognition elements for the design of paper sensors are first discussed. This is followed by reviewing three specific types of FNA based paper sensors: dot blots, lateral flow assays, and microfluidic paper-based analytical devices. Furthermore, advances in the signal reporting methods used by FNA based paper sensors are summarized. Finally, limitations of current FNA based paper sensors are discussed along with considerations of future research directions.

Highly Sensitive RNA-Cleaving DNAzyme Sensors from Surface-to-Surface Product Enrichment.

The engineering of easy-to-use biosensors with ultra-low detection sensitivity remains a major challenge. Herein, we report a simple approach for creating such sensors through the use of an RNA-cleaving DNAzyme (RcD) and a strategy designed to concentrate its cleavage product significantly. The assay uses micron-sized beads loaded with a target-responsive RcD and a paper strip containing a microzone covered with a DNA oligonucleotide capable of capturing the cleavage product of the RcD through Watson-Crick hybridization. Placing the beads and the paper strip in a target-containing test sample allows the bead-bound RcD molecules to undergo target-induced RNA cleavage, releasing a DNA fragment that is captured by the paper strip. This strategy, though simple, is very effective in achieving high levels of detection sensitivity, being able to enrich the concentration of the cleavage product by three orders of magnitude. It is also compatible with both fluorescence-based and colorimetric reporting mechanisms. This work provides a simple platform for developing ultrasensitive biosensors that take advantage of the widely available RcDs as molecular recognition elements.

Unraveling Determinants of Affinity Enhancement in Dimeric Aptamers for a Dimeric Protein.

High-affinity aptamers can be derived de novo by using stringent conditions in SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments or can be engineered post SELEX via dimerization of selected aptamers. Using electrophoretic mobility shift assays, we studied a series of heterodimeric and homodimeric aptamers, constructed from two DNA aptamers with distinct primary sequences and secondary structures, previously isolated for VEGF-165, a homodimeric protein. We investigated four factors envisaged to impact the affinity of a dimeric aptamer to a dimeric protein: (1) length of the linker between two aptamer domains, (2) linking orientation, (3) binding-site compatibility of two component aptamers in a heterodimeric aptamer, and (4) steric acceptability of the two identical aptamers in a homodimeric aptamer. All heterodimeric aptamers for VEGF-165 were found to exhibit monomeric aptamer-like affinity and the lack of affinity enhancement was attributed to binding-site overlap by the constituent aptamers. The best homodimeric aptamer showed 2.8-fold better affinity than its monomeric unit (Kd = 13.6 ± 2.7 nM compared to 37.9 ± 14 nM), however the barrier to further affinity enhancement was ascribed to steric interference of the constituent aptamers. Our findings point to the need to consider the issues of binding-site compatibility and spatial requirement of aptamers for the development of dimeric aptamers capable of bivalent recognition. Thus, determinants highlighted herein should be assessed in future multimerization efforts.

In Vivo Use of a Multi-DNA Aptamer-Based Payload/Targeting System To Study Dopamine Dysregulation in the Central Nervous System.

The delivery of therapeutics across the blood-brain barrier remains a considerable challenge in investigating central nervous system related processes. In this work, a liposome vehicle was surface-modified with an aptamer that binds to the transferrin receptor and was loaded with two different dopamine-binding aptamer payloads. This system was effectively used to promote the delivery of the aptamer cargo from the peripheral injection site into the brain. The effect of these delivered aptamers on behavior was investigated in vivo in a locomotor task. The first dopamine binding aptamer assessed was a DNA aptamer, the binding of which had been previously validated through the aptamer-based biosensor development reported by several independent research groups. The second aptamer investigated was the result of a novel in vitro selection experiment described herein. Our data suggest that systemic administration of the modified liposomes led to delivery of the dopamine aptamers into the brain. Fluorescence microscopy revealed differential distribution of fluorescence based on the presence or absence of the transferrin receptor aptamer on the surface of fluorescently modified liposomes. In a behavioral experiment using cocaine administration to induce elevated concentrations of neural dopamine, systemic pretreatment with the dopamine aptamer-loaded liposomes reduced cocaine-induced hyperlocomotion. Multiple controls including a transferrin-negative liposome control and transferrin-positive liposomes loaded with either a nonbinding, base-substituted dopamine aptamer or a random oligonucleotide were investigated. None of these controls altered cocaine-induced hyperlocomotion. Chronic systemic administration of the modified liposomes produced no deleterious neurobehavioral or neural degenerative effects. Importantly, this work is one example of an application for this versatile multiaptamer payload/targeting system. Its general application is limited only by the availability of aptamers for specific neural targets.

Colorimetric Detection of Uranyl Using a Litmus Test.

Ingestion of water containing toxic contaminants above levels deemed safe for human consumption can occur unknowingly since numerous common contaminants in drinking water are colorless and odorless. Uranyl is particularly problematic as it has been found at dangerous levels in sources of drinking water. Detection of this heavy metal-ion species in drinking water currently requires sending a sample to a laboratory where trained personnel use equipment to perform the analysis and turn-around times can be long. A pH-responsive colorimetric biosensor was developed to enable detection of uranyl in water which coupled the uranyl-specific 39E DNAzyme as a recognition element, and an enzyme capable of producing a pH change as the reporter element. The rapid colorimetric assay presented herein can detect uranyl in lake and well water at concentrations relevant for environmental monitoring, as demonstrated by the detection of uranyl at levels below the limits set for drinking water by major regulatory agencies including the World Health Organization (30 µg/L). This simple and inexpensive DNAzyme-based assay enabled equipment-free visual detection of 15 µg/L uranyl, using both solution-based and paper-based pH-dependent visualization strategies.