DNA aptamer selection for SARS-CoV-2 spike glycoprotein detection.

The rapid spread of SARS-CoV-2 infection throughout the world led to a global public health and economic crisis triggering an urgent need for the development of low-cost vaccines, therapies and high-throughput detection assays. In this work, we used a combination of Ideal-Filter Capillary Electrophoresis SELEX (IFCE-SELEX), Next Generation Sequencing (NGS) and binding assays to isolate and validate single-stranded DNA aptamers that can specifically recognize the SARS-CoV-2 Spike glycoprotein. Two selected non-competing DNA aptamers, C7 and C9 were successfully used as sensitive and specific biological recognition elements for the development of electrochemical and fluorescent aptasensors for the SARS-CoV-2 Spike glycoprotein with detection limits of 0.07 fM and 41.87 nM, respectively.

Ratiometric electrochemical aptasensor with strand displacement for insulin detection in blood samples.

BACKGROUND: Diabetes patients suffer either from insulin deficiency or resistance with a high risk of severe long-term complications, thus the quantitative assessment of insulin level is highly desired for diabetes surveillance and management. Utilizing insulin-capturing aptamers may facilitate the development of affordable biosensors however, their rigid G-quadruplex structures impair conformational changes of the aptamers and diminish the sensor signals. RESULTS: Here we report on a ratiometric, electrochemical insulin aptasensor which is achieved by hybridization of an insulin-capturing aptamer and a partially complementary ssDNA to break the rigid G-quadruplex structures. To improve the durability of the aptasensor, the capturing aptamer was immobilized on gold electrodes via two dithiol-phosphoramidite functional groups while methoxy-polyethylene glycol thiol was used as a blocking molecule. The exposure of the sensor to insulin-containing solutions induced the dissociation of the hybridized DNA accompanied by a conformational rearrangement of the capturing aptamer back into a G-quadruplex structure. The reliability of sensor readout was improved by the adoption of an AND logic gate utilizing anthraquinone and methylene blue redox probes associated to the aptamer and complementary strand, respectively. Our aptasensor possessed an improved detection limit of 0.15 nM in comparison to aptasensors without strand displacement. SIGNIFICANCE: The sensor was adapted for detection in real blood and is ready for future PoC diagnostics. The capability of monitoring the insulin level in an affordably manner can improve the treatment for an increasing number of patients in developed and developing nations. The utilization of low-cost and versatile aptamer receptors together with the engineering of ratiometric electrochemical signal recording has the potential to considerably advance the current insulin detection technology toward multi-analyte diabetes sensors.

Highly sensitive detection of malaria biomarker through matching channel and gate capacitance of integrated organic electrochemical transistors.

Organic electrochemical transistors (OECTs) possess versatile advantages for biochemical and electrophysiological applications due to electrochemical gating and ion-to-electron conversion capability. Although OECTs have been successfully applied for biochemical sensing, the effect of relative capacitance for specific sensing events is still unclear. In the present work, we design integrated interdigitated OECTs (iOECTs) with on-plane gold gate and different channel geometries for point-of-care diagnosis of malaria using aptamer as receptor. The transconductance of the iOECTs gated with micro-size gold electrodes decreased with increasing the channel thicknesses, especially for devices with large channel areas, which is inconsistent with devices gated by typical Ag/AgCl electrodes, attributing to the limited gating efficiency of the micro-size gate electrode. The capacitance of gate electrode was heavily suppressed by receptors but increased with the incubation of targets. In addition, the integrated iOECTs with thin channels exhibited superior sensitivity for malaria detection with the detection limit as low as 3.2 aM, much lower than their thick channel counterpart and other state-of-the-art biosensors. These deviations could be caused by the high relative capacitances, with respect to the gate and channel capacitance (Cg/Cch), resulting in a high gate potential drop over the organic channel and thus entirely gating on the thin channel device. These findings provide guidance to optimize the geometry of OECT devices with on-chip integrated gates and the fabrication of miniaturized OECTs for biosensing applications.

Truncated Electrochemical Aptasensor with Enhanced Antifouling Capability for Highly Sensitive Serotonin Detection.

Accurate determination of serotonin (ST) provides insight into neurological processes and enables applications in clinical diagnostics of brain diseases. Herein, we present an electrochemical aptasensor based on truncated DNA aptamers and a polyethylene glycol (PEG) molecule-functionalized sensing interface for highly sensitive and selective ST detection. The truncated aptamers have a small size and adopt a stable stem-loop configuration, which improves the accessibility of the aptamer for the analyte and enhances the sensitivity of the aptasensor. Upon target binding, these aptamers perform a conformational change, leading to a variation in the Faraday current of the redox tag, which was recorded by square wave voltammetry (SWV). Using PEG as blocking molecules minimizes nonspecific adsorption of other interfering molecules and thus endows an enhanced antifouling ability. The proposed electrochemical aptamer sensor showed a wide range of detection lasting from 0.1 nM to 1000 nM with a low limit of detection of 0.14 nM. Owing to the unique properties of aptamer receptors, the aptasensor also exhibits high selectivity and stability. Furthermore, with the reduced unspecific adsorption, assaying of ST in human serum and artificial cerebrospinal fluid (aCSF) showed excellent performance. The reported strategy of utilizing antifouling PEG describes a novel approach to building antifouling aptasensors and holds great potential for neurochemical investigations and clinical diagnosis.

A Combined Plasmonic and Electrochemical Aptasensor Based on Gold Nanopit Arrays for the Detection of Human Serum Albumin.

Electrochemical and optical platforms are commonly employed in designing biosensors. However, one signal readout can easily lead to inaccuracies due to the effect of nonstandard test procedures, different operators, and experimental environments. We have developed a dual-signal protocol that combined two transducer principles in one aptamer-based biosensor by simultaneously performing electrochemical- and extraordinary optical transmission (EOT)-based plasmonic detection using gold nanopit arrays (AuNpA). Compared with full hole structures, we found that nanopits, that did not fully penetrate the gold film, not only exhibited a better plasmonic bandwidth and refractive index sensitivity both in the finite-difference time-domain simulation and in experiments by shielding the gold/quartz mode but also enlarged the electrochemical active surface area. Therefore, the periodic non-fully penetrating AuNpA were modified with ferrocene-labeled human serum albumin aptamer receptors. The formation of the receptor layer and human serum albumin binding complex induced a conformational change, which resulted in variation in the electron transfer between the electro-active ferrocene units and the AuNpA surface. Simultaneously, the binding event caused a surface plasmon polaritons wavelength shift corresponding to a change in the surface refractive index. Interestingly, although both transducers recorded the same binding process, they led to different limits of detection, dynamic ranges, and sensitivities. The electrochemical transducer showed a dynamic detection range from 1 nM to 600 µM, while the optical transducer covered high concentrations from 100 µM to 600 µM. This study not only provides new insights into the design of plasmonic nanostructures but also potentially opens an exciting avenue for dual-signal disease diagnosis and point-of-care testing applications.

Electrochemical Immunosensor Using Electroactive Carbon Nanohorns for Signal Amplification for the Rapid Detection of Carcinoembryonic Antigen.

In this work, a novel sandwich-type electrochemical immunosensor was developed for the quantitative detection of the carcinoembryonic antigen, an important tumor marker in clinical tests. The capture antibodies were immobilized on the surface of a gold disk electrode, while detection antibodies were attached to redox-tagged single-walled carbon nanohorns/thionine/AuNPs. Both types of antibody immobilization were carried out through Au-S bonds using the novel photochemical immobilization technique that ensures control over the orientation of the antibodies. The electroactive SWCNH/Thi/AuNPs nanocomposite worked as a signal tag to carry out both the detection of carcinoembryonic antigen and the amplification of the detection signal. The current response was monitored by differential pulse voltammetry. A clear dependence of the thionine redox peak was observed as a function of the carcinoembryonic antigen concentration. A linear detection range from 0.001-200 ng/mL and a low detection limit of 0.1385 pg/mL were obtained for this immunoassay. The results showed that carbon nanohorns represent a promising matrix for signal amplification in sandwich-type electrochemical immune assays working as a conductive and binding matrix with easy and versatile modification routes to antibody and redox tag immobilization, which possesses great potential for clinical diagnostics of CEA and other biomarkers.

Delineating charge and capacitance transduction in system-integrated graphene-based BioFETs used as aptasensors for malaria detection.

Despite significant eradication efforts, malaria remains a persistent infectious disease with high mortality due to the lack of efficient point-of-care (PoC) screening solutions required to manage low-density asymptomatic parasitemia. In response, we demonstrate a quantitative electrical biosensor based on system-integrated two-dimensional field-effect transistors (2DBioFETs) of reduced graphene oxide (rGO) as transducer for high sensitivity screening of the main malaria biomarker, Plasmodium falciparum lactate dehydrogenase (PfLDH). The 2DBioFETs were biofunctionalized with pyrene-modified 2008s aptamers as specific PfLDH receptors. While we systematically optimize biosensor interface for optimal performance, aptamer-protein transduction at 2DBioFETs is elucidated based on delineation of charge and capacitance in an updated analytical model for two-dimensional rGO/biofunctional layer/electrolyte (2DiBLE) interfaces. Our 2DBioFET-aptasensors display a limit-of-detection down to 0.78 fM (0.11 pg/mL), dynamic ranges over 9 orders of magnitude (subfemto to submicromolar), high sensitivity, and selectivity in human serum validating their diagnostic potential as rapid PoC tests for malarial management.

An electrochemical aptamer-based biosensor targeting Plasmodium falciparum histidine-rich protein II for malaria diagnosis.

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.

Electrochemical dual-aptamer biosensors based on nanostructured multielectrode arrays for the detection of neuronal biomarkers.

Multielectrode arrays (MEAs) have been increasingly used for the development of biosensors due to their capability to record signals from multiple channels, fast mass transfer rates, and high spatial resolution. Alzheimer's disease (AD) is often associated with mitochondrial dysfunction, which is closely related to reduced levels of adenosine triphosphate (ATP). Therefore, simultaneous detection of ATP together with amyloid-ß oligomers (AßO), a reliable biomarker for AD, can potentially advance the early detection of Alzheimer's disease. In this work, a dual-aptamer modified MEA chip was developed that consists of microelectrodes modified with electrodeposited 3D nanostructures (3D-GMEs). Electrodeposition methods, deposition potential, and deposition time were systematically altered and the active surface areas as well as the electrode morphologies were characterized by cyclic voltammetry and scanning electron microscopy. The nanostructured microelectrodes were sequentially modified with AßO and ATP specific aptamer receptors. To achieve the modification of different aptamer receptors at different 3D-GMEs of the same MEA chip, electrochemical cleaning was applied to individual 3D-GMEs. Ferrocene labels were attached to the aptamer receptors to enable amperometric signaling after target-aptamer binding. The developed aptasensor showed a linear detection range from 1 pM to 200 nM for the detection of AßO and from 0.01 nM to 1000 nM for the detection of ATP. Finally, ATP and AßO were detected simultaneously in the same analyte solution by the same sensor chip, which could support the early detection of AD, provide comprehensive information about the health status of the patient, and be helpful for pathological studies of neurodegenerative diseases.

Polyethylene glycol-mediated blocking and monolayer morphology of an electrochemical aptasensor for malaria biomarker detection in human serum.

Better approaches are critically needed for in situ point-of-care diagnostic biosensors that enable primary care physicians, or even individual patients, to directly analyze biological fluids without complicated sample pretreatments. Additional purification steps consume time, consume reagents, often require other equipment, and can introduce false-negative results. Biosensors have been modified with blocking molecules to reduce biofouling; however, the effectiveness relies on their chemical composition and morphology. Here, we used a polyethylene glycol film to suppress unspecific binding from human serum on an electrochemical malaria aptasensor. A detailed study of the variation of the chemical and morphological composition of the aptamer/polyethylene glycol mixed monolayer as a function of incubation time was conducted. Higher resistance to matrix biofouling was found for polyethylene glycol than for hydrophobic alkanethiol films. The best sensor performance was observed for intermediate polyethylene glycol immobilization times. With prolonged incubation, phase separation of aptamer, and polyethylene glycol molecules locally increased the aptamer density and thereby diminished the analyte binding capability. Remarkably, polyethylene glycols do not affect the aptasensor sensitivity but enhance the complex matrix tolerance, the dynamic range, and the limit of detection. Careful tuning of the blocking molecule immobilization is crucial to achieving high aptasensor performance and biofouling resistance.

Amplification of aptamer sensor signals by four orders of magnitude via interdigitated organic electrochemical transistors.

Electrochemical aptamer receptor/transducer systems are key elements of emerging E-AB sensors (aptasensor) used for the detection of various kinds of targets. However, the performance of these amperometric sensors is often limited by the low density of receptors attached to the sensor surface and high background signals. In the present work, interdigitated organic electrochemical transistors (iOECT) were used as a transducer to enhance the sensitivity and dynamic detection range of aptasensors. Therefore, the electrode of an amperometric sensor was utilized as gate electrode to operate the iOECT. This device was used to detect the low weight target molecule adenosine triphosphate (ATP), a common biomarker, which plays an important role for cardiovascular, neurodegenerative, and immune deficiency diseases. The novel aptasensor can selectively detect ATP with ultrahigh sensitivity down to the concentration of 10 pM, which is four orders of magnitude lower than the detection limit of the same aptasensor using an amperometric transducer principle (limit-of-detection of 106 nM) and most other previously reported electrochemical sensors. Furthermore, sensor regeneration was demonstrated, which facilitates reusability of OECT aptasensors. The small device size in combination with high transconductances paves the way for the development of highly sensitive integrated micro-biosensors for point-of-care applications.

Amperometric Aptasensor for Amyloid-ß Oligomer Detection by Optimized Stem-Loop Structures with an Adjustable Detection Range.

Amyloid-ß oligomers (AßO) have become representative biomarkers for early diagnosis of Alzheimer's disease. Here, we report on an aptasensor based on stem-loop probes for sensitive and specific detection of AßO by an amperometric transducer principle using alternating current voltammetry (ACV). Stem-loop probes with redox-active moieties are immobilized on a gold substrate as a receptor element. The signal transduction mechanism relies on redox ferrocene (Fc) reporting via charge transfer on a molecular recognition event involving a conformational change of the molecular beacon. The stem-loop structures were optimized by considering the aptamers' stem length, spacer, and different ferrocene terminals. In addition, the sensor assembly and signal recording including aptamer concentration and ACV frequency dependence are discussed. Using the optimized stem-loop probe (B-3' Fc), the aptasensor showed a decrease of the Fc peak current induced by AßO binding within the broad concentration range spanning 6 orders of magnitude. Furthermore, the detection limit of the sensor can be further decreased by optimizing the ACV frequency, however at the cost of a narrowed detection range. In this work, a label-free electrochemical aptasensor is demonstrated, which facilitates the quantification of the concentration of AßO with high selectivity and subpicomolar sensitivity, which may be conducive to improving the diagnosis and pharmacology studies of Alzheimer's disease.