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.

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. 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. 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.

In this work, we combined plasmon-enhanced fluorescence and electrochemical (PEF-EC) transduction mechanisms to realize a highly sensitive dual-transducer aptasensor. To implement two traducers in one biosensor, a novel large-scale nanoimprint lithography process was introduced to fabricate gold nanopit arrays (AuNpA) with unique fringe structures. Light transmitting through the AuNpA samples exhibited a surface plasmon polariton peak overlapping with the excitation peak of the C7 aptamer-associated fluorophore methylene blue (MB). We observed a five and seven-times higher average fluorescence intensity over the AuNpA and fringe structure, respectively, in comparison to a plane Au film. Furthermore, the MB fluorophore was simultaneously utilized as a redox probe for electrochemical investigations and is described here as a dual transduction label for the first time. The novel dual transducer system was deployed for the detection of SARS-CoV-2 Spike protein via a C7 aptamer in combination with a strand displacement protocol. The PEF transducer exhibited a detection range from 1 fg/mL to 10 ng/mL with a detection limit of 0.07 fg/mL, while the EC traducer showed an extended dynamic range from 1 fg/mL to 100 ng/mL with a detection limit of 0.15 fg/mL. This work provides insights into an easy-to-perform, large-scale fabrication process for nanostructures enabling plasmon-enhanced fluorescence, and the development of an advanced but universal aptasensor platform.

Accurate and simultaneous determination of multiple neurotransmitters (NTs) is crucial for a thorough understanding of brain functions and for diagnosing neurological disorders. We have developed an electrochemical biosensor utilizing aptamer recognition for the highly sensitive and simultaneous detection of multiple neurotransmitters, including glutamate quantification for the first time in multiplex detection. Microelectrode arrays (MEAs) served as biosensing platforms to facilitate the recording of signals from multiple channels, rapid mass transfer rates, and high spatial resolution, fundamental for studying NTs release in nervous tissue. To enhance aptamer receptor loading, gold nanostructures were electrodeposited onto the microelectrodes, enhancing the active surface area and electrode morphology. A potential-pulse-assisted method was employed to achieve site-selective immobilization of three different aptamers on a single MEA chip within 30 min, enabling fast and reproducible sensor fabrication. This unique strategy ensured aptamer-specific immobilization, minimized cross-talk, and allowed for multiplex detection of serotonin, glutamate, and dopamine with high sensitivity and specificity. Additionally, using polyethylene glycol (PEG) as a blocking molecule, the aptamer-functionalized MEAs showed enhanced antifouling properties and maintained detection capabilities in complex environments. This multiplexed detection strategy enables a high-performance and robust biosensor platform with strong clinical relevance, offering substantial potential for in vivo monitoring of neurotransmitter release and the diagnosis of neurological disorders.

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 $\mathsf{\mu }$M, while the optical transducer covered high concentrations from 100 $\mathsf{\mu }$M to 600 $\mathsf{\mu }$M. 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.