Electrochemical aptamer-based (E-AB) sensors using conformational change-induced electron transfer kinetics are sensitive, reagent-less, and cost-effective tools for molecular sensing. Current advances in this technology can allow continuous drug pharmacokinetic monitoring in living animals (Dauphin-Ducharme et al., ACS Sens 4(10):2832-2837, 2019; Idili et al., Chem Sci 10(35):8164-8170, 2019), as well as automated analysis of hormone pulsatility (Liang et al., Nat Commun 10(1):852, 2019). In this chapter, we provide the methodology for an automated E-AB conformational change-based robotic sensing platform. By using an open-source programmable robotic system, this method can be adapted to a wide range of experimental scenarios.

Malaria is an infectious disease caused by parasitic protozoans from the genus Plasmodium, with the species P. falciparum causing the highest number of deaths worldwide. Rapid diagnostic tests (RDTs) have become critical in the management of malaria, but current RDTs that detect P. falciparum are primarily antibody-based, which can have drawbacks in cost and robustness. Here, we report the development of an electrochemical aptamer-based (E-AB) biosensing alternative. Through selective evolution of ligands by exponential enrichment, we identify DNA aptamers that bind specifically to P. falciparum histidine-rich protein II (PfHRP2). The aptamer is modified with a methylene blue reporter and attached to a gold sensor surface for square-wave voltammetry interrogation. Through this method we are able to quantify PfHRP2 in human serum with an LOD of 3.73 nM. We further demonstrate the biosensor is stable in serum buffers and reusable for multiple detection rounds. These findings provide a promising alternative to conventional PfHRP2 detection for malaria diagnosis, while also expanding the capabilities of E-AB biosensors.

Normal reproductive functioning is critically dependent on pulsatile secretion of luteinising hormone (LH). Assessment of LH pulsatility is important for the clinical diagnosis of reproductive disorders, but current methods are hampered by frequent blood sampling coupled to expensive serial immunochemical analysis. Here, we report the development and application of a Robotic APTamer-enabled Electrochemical Reader (RAPTER) electrochemical analysis system to determine LH pulsatility. Through selective evolution of ligands by exponential enrichment (SELEX), we identify DNA aptamers that bind specifically to LH and not to related hormones. The aptamers are integrated into electrochemical aptamer-based (E-AB) sensors on a robotic platform. E-AB enables rapid, sensitive and repeatable determination of LH concentration profiles. Bayesian Spectrum Analysis is applied to determine LH pulsatility in three distinct patient cohorts. This technology has the potential to transform the clinical care of patients with reproductive disorders and could be developed to allow real-time in vivo hormone monitoring.

Normal reproductive functioning is critically dependent on pulsatile secretion of LH. Assessment of LH pulsatility is important for the clinical diagnosis of patients with reproductive disorders, but this is not routinely available in the clinic because of the need for frequent blood sampling coupled with expensive serial immunochemical analysis. The emerging technology of electrochemical aptamer-based (E-AB) sensing could potentially offer a generalizable approach for LH pulsatility measurement in humans. E-AB sensors take advantage of the intrinsic properties of nucleic acid aptamers to specifically bind to a molecular target and undergo a reversible conformational change. The conformational change can be measured through the electrochemical signal response. Aptamers, unlike antibodies, can facilitate low-cost repeat analytical measurements. However, there is currently no aptamer-based LH sensing technology. Here, we report the development and application of a novel antibody-free DNA aptamer-mediated electrochemical analysis to accurately assess LH pulsatility in patients with reproductive disorders. Through selective evolution of ligands by exponential enrichment (SELEX), single-stranded DNA oligonucleotides (aptamers) were identified that bind specifically to LH (and that do not bind to the closely related FSH). The aptamers were integrated into E-AB sensors on a robotic platform (which we term RAPTER) to enable sensitive, rapid and repeatable detection of LH. We next determined if the RAPTER system could measure LH pulsatility in clinical samples from patients with reproductive disorders. We obtained 441 serum samples from 3 patient cohorts - young females with regular menstrual cycles (normal LH pulsatility), menopausal women (disordered LH pulsatility, high LH), and women with hypothalamic amenorrhoea (suppressed/ absent LH pulsatility, low LH). Blood sampling was performed every 10 minutes for 8 hours in these patients and samples were analysed using both the current gold standard LH immunochemical assay and RAPTER. We performed a Bland-Altman analysis and plotted the linear regression to compare the results obtained. The RAPTER assay showed an almost perfect correlation with the clinical immunochemical assay (R2= 0.94). Bayesian Spectrum Analysis was used to determine the effective LH pulse interval from both datasets (clinical assay and RAPTER assay). We are able to distinguish 3 patient cohorts based on the time interval ranges. Taken together, the RAPTER system provides a new approach for LH pulsatility determination by reliably and immediately calculating varying LH concentrations within distinct patient cohorts. This has the potential to transform the clinical care of patients with reproductive disorders as LH pulsatility could be measured easily in clinical practice to aid correct diagnosis. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Here, we integrate 3D-printing technology with low-cost open source electronics to develop a portable diagnostic platform suitable for a wide variety of diagnostic and sensing assays. We demonstrate two different clinical applications in the diagnosis of Clostridium difficile infection and malaria.

There is a critical need for better biosensors for the detection and diagnosis of malaria. We previously developed a DNA aptamer that recognises the Plasmodium falciparum lactate dehydrogenase (PfLDH) enzyme with high sensitivity and specificity. The aptamer was integrated into an Aptamer-Tethered Enzyme Capture (APTEC) assay as a laboratory-based diagnostic approach. However, a portable equipment-free point-of-care aptamer-mediated biosensor could have a significant impact on malaria diagnosis in endemic regions. Here, we present a new concept for a malaria biosensor whereby aptamers are coated onto magnetic microbeads for magnet-guided capture, wash and detection of the biomarker. A biosensor incorporating three separate microfluidic chambers was designed to enable such magnet-guided equipment-free colorimetric detection of PfLDH. A series of microfluidic biosensor prototypes were optimised to lower rates of inter-chamber diffusion, increase sensitivity, and provide a method for point-of-care sample testing. The biosensor showed high sensitivity and specificity when detecting PfLDH using both in vitro cultured parasite samples and using clinical samples from malaria patients. The high performance of the biosensor provides a proof-of-principle for a portable biosensor that could be adaptable for a variety of aptamer-mediated diagnostic scenarios.

DNA nanostructures can show dynamic responses to molecular triggers for a wide variety of applications. While DNA sequence signal triggers are now well-established, there is a critical need for a broader diversity of molecular triggers to drive dynamic responses in DNA nanostructures. DNA aptamers are ideal; they can both seamlessly integrate into DNA nanostructure scaffolds and transduce molecular recognition into functional responses. Here, we report construction and optimization of a DNA origami nanobox locked by a pair of DNA double strands where one strand is a DNA aptamer targeting the malaria biomarker protein Plasmodium falciparum lactate dehydrogenase. The protein acts as the key which enables box opening. We observe highly specific protein-mediated box opening by both transmission electron microscopy and fluorescence. Aptamer-enabled DNA boxes have significant potential for enabling direct responses to proteins and other biomolecules in nanoscale diagnostics, drug delivery and sensing devices.

Aptamers are short nucleic acid sequences capable of specific, high-affinity molecular binding. They are isolated via SELEX (Systematic Evolution of Ligands by Exponential Enrichment), an evolutionary process that involves iterative rounds of selection and amplification before sequencing and aptamer characterization. As aptamers are genetic in nature, bioinformatic approaches have been used to improve both aptamers and their selection. This review will discuss the advancements made in several enclaves of aptamer bioinformatics, including simulation of aptamer selection, fragment-based aptamer design, patterning of libraries, identification of lead aptamers from high-throughput sequencing (HTS) data and in silico aptamer optimization.

We present the adaption of an aptamer-tethered enzyme capture (APTEC) assay into point-of-care device prototypes with potential for malaria diagnosis. The assay functions by capturing the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) from samples and using its intrinsic enzymatic activity to generate a visualizable blue color in response to Plasmodium positive samples. Using three-dimensional (3D) printing rapid prototyping, a paper based syringe test and magnetic bead-based well test were developed. Both were found to successfully detect recombinant PfLDH at ng mL(-1) concentrations using low sample volumes (20 mu L) and could function using purified or spiked whole blood samples with facile sample preparation. The syringe test was found to be more analytically sensitive but required more additional preparation steps, while the well test required fewer steps and hence may be better suited for future clinical testing. Additionally, the development reagents required for the color response were fully stabilized through desiccation with sugar stabilization agents and could withstand temperatures as high as 90 degrees C. This study demonstrates how a previously reported biochemical assay can be adapted into workable point-of-care device prototypes by using 3D printing rapid prototyping. This novel technology, intended for rapid diagnostic tests (RDTs) for malaria, is distinct and carries many potential advantages relative to established lateral flow immunochromatographic approaches.

Aptamers have significant potential as affinity reagents, but better approaches are critically needed to discover higher affinity nucleic acids to widen the scope for their diagnostic, therapeutic, and proteomic application. Here, we report aptamer affinity maturation, a novel aptamer enhancement technique, which combines bioinformatic resampling of aptamer sequence data and microarray selection to navigate the combinatorial chemistry binding landscape. Aptamer affinity maturation is shown to improve aptamer affinity by an order of magnitude in a single round. The novel aptamers exhibited significant adaptation, the complexity of which precludes discovery by other microarray based methods Honing aptamer sequences using aptamer affinity maturation could help optimize a next generation of nucleic acid affinity reagents.

Nucleic acid-mediated nanomachines have significant potential in biomedical applications but new approaches that link molecular recognition of proteins to change in nucleic acid structure and function are required. Here, a split DNA aptamer is integrated into G-quadruplex tweezers, which close in the presence of the malaria biomarker protein Plasmodium falciparum lactate dehydrogenase (PfLDH). Closing of the tweezers enables G-quadruplex hemin mediated peroxidase activity, which can be observed colorimetrically. The PfLDH aptamer is split within an asymmetric internal loop and incorporated into the tweezers maintaining aptamer binding capability. Spacing between the G-quadruplex structure and split aptamer, together with extent of complementarity, is found to be critical for optimization to enhance catalytic performance. The integrated split aptamer is observed to maintain the high specificity to Plasmodium falciparum lactate dehydrogenase of the parent aptamer. Split aptamer approaches have significant potential to functionalize nucleic acid nanostructures for protein molecular recognition.

The functionalisation of microbeads with oligonucleotides has become an indispensable technique for high-throughput aptamer selection in SELEX protocols. In addition to simplifying the separation of binding and non-binding aptamer candidates, microbeads have facilitated the integration of other technologies such as emulsion PCR (ePCR) and Fluorescence Activated Cell Sorting (FACS) to high-throughput selection techniques. Within these systems, monoclonal aptamer microbeads can be individually generated and assayed to assess aptamer candidate fitness thereby helping eliminate stochastic effects which are common to classical SELEX techniques. Such techniques have given rise to aptamers with 1000 times greater binding affinities when compared to traditional SELEX. Another emerging technique is Fluorescence Activated Droplet Sorting (FADS) whereby selection does not rely on binding capture allowing evolution of a greater diversity of aptamer properties such as fluorescence or enzymatic activity. Within this review we explore examples and applications of oligonucleotide functionalised microbeads in aptamer selection and reflect upon new opportunities arising for aptamer science.