Redox Tag Research Articles

Electrocatalytic Radical Cation Diels-Alder Reactions Using Enol Ethers As Dienophiles

Single-electron oxidation enables the conversion of electron-rich alkenes such as styrene derivatives to radical cation species. We have been developing various cycloaddition reactions of radical cations produced by single-electron oxidation using electrochemical and TiO2 photochemical methods. Introducing an electron-rich aryl group which we call a redox tag is a critical part of the process to control the reactivity of the radical cations. During carbon-carbon bonds formation the motif functions as both electron donor and electron acceptor. In addition to styrene derivatives, enol ethers can also be readily oxidized by single electron transfer (SET) electrochemically or photochemically to generate the radical cations. For instance, enol ethers tethered redox tag serves efficient radical cation precursors for [2+2] cycloadditions.[1] The cycloadditions are net redox-neutral reactions and are initiated by the formation of a radical cation by single-electron oxidation and completed by reduction after ring formation.Herein, we present [4+2] cycloadditions using enol ethers as dienophiles by electrochemical oxidation in lithium perchlorate (LiClO4)/nitromethane (MeNO2) system. In the case of disubstituted enol ethers prepared from aldehydes, the approach involving redox tag was successful in obtaining the corresponding cycloadducts, while the desired products were not obtained when the substrates without redox tag were subjected.[2] This result suggests that intramolecular electron transfer from the redox tag is crucial for the formation of the cyclohexene ring systems. In contrast, the trisubstituted enol ethers prepared from ketones allowed the cycloaddition reaction to proceed without the involvement of redox tag. It is expected that the methyl group significantly stabilizes the cyclohexene radical cations to have enough lifetime to be reduced by intermolecular SET. Furthermore, since the cycloaddition reaction of enol ether was completed with a catalytic amount of electricity (0.4 F/mol), a radical cation chain mechanism is proposed in which electron acts as a catalyst.

Single-Response Duplexing of Electrochemical Label-Free Biosensor from the Same Tag.

Multiplexing is a valuable strategy to boost throughput and improve clinical accuracy. Exploiting the vertical, meshed design of reproducible and low-cost ultra-dense electrochemical chips, the unprecedented single-response multiplexing of typical label-free biosensors is reported. Using a cheap, handheld one-channel workstation and a single redox probe, that is, ferro/ferricyanide, the recognition events taking place on two spatially resolved locations of the same working electrode can be tracked along a single voltammetry scan by collecting the electrochemical signatures of the probe in relation to different quasi-reference electrodes, Au (0V) and Ag/AgCl ink (+0.2V). This spatial isolation prevents crosstalk between the redox tags and interferences over functionalization and binding steps, representing an advantage over the existing non-spatially resolved single-response multiplex strategies. As proof of concept, peptide-tethered immunosensors are demonstrated to provide the duplex detection of COVID-19 antibodies, thereby doubling the throughput while achieving 100% accuracy in serum samples. The approach is envisioned to enable broad applications in high-throughput and multi-analyte platforms, as it can be tailored to other biosensing devices and formats.

Development of an Electrochemical, Aptamer-Based Sensor for Dynamic Detection of Neuropeptide Y.

The ability to monitor dynamic changes in neuropeptide Y (NPY) levels in complex environments can have an impact on many fields, including neuroscience and immunology. Here, we describe the development of an electrochemical, aptamer-based (E-AB) sensor for the dynamic (reversible) measurement of physiologically relevant (nanomolar) concentrations of neuropeptide Y. The E-AB sensors are fabricated using a previously described 80 nucleotide aptamer1 reported to specifically bind NPY with a binding affinity Kd = 0.3 ± 0.2 uM. We investigated two redox tag placement locations on the aptamer sequence (terminal vs internal) and various sensor fabrication and interrogation parameters to tune the performance of the resulting sensor. The best-performing sensor architecture displayed a physiologically relevant dynamic range (nM) and low limit of detection and is selective among competitors and similar molecules. The development of this sensor accomplishes two breakthroughs: first, the development of a nonmicrofluidic aptamer-based electrochemical sensor that can detect NPY on a physiologically relevant (seconds to minutes) time scale and across a relevant concentration range; second, the expansion of the range of molecules for which an electrochemical, aptamer-based sensor can be used.

Redox-Tag-Guided Radical Cation Diels–Alder Reactions: Use of Enol Ethers as Dienophiles

AbstractAlthough radical cation Diels–Alder reactions enable the formation of cyclohexene ring systems between electronically mismatched (both electron-rich) dienes and dienophiles, which is otherwise difficult or impossible to achieve under thermal conditions, the substrate scope has been limited. Herein, we disclose that a radical cation Diels–Alder reaction using an enol ether as an electron-rich (and therefore oxidizable) dienophile is possible through a rationally designed redox tag strategy. Electrochemical and TiO2 photochemical approaches are effective in driving the reaction, where both intermolecular and intramolecular electron transfers are the key.

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.

Amplification-Free Ratiometric Electrochemical Aptasensor for Point-of-Care Detection of Therapeutic Monoclonal Antibodies.

The rapid quantification of therapeutic monoclonal antibodies (mAbs) is of great significance to their pharmacokinetics/pharmacodynamics (PK/PD) research and the personalized medication for disease treatment. Taking advantage of the direct decoration of tens of redox tags to the target of interest, we illustrate herein an amplification-free ratiometric electrochemical aptasensor for the point-of-care (POC) detection of trace amounts of therapeutic mAbs. The POC detection of therapeutic mAbs involved the use of the methylene blue (MB)-conjugated aptamer as the affinity element and the decoration of therapeutic mAbs with ferrocene (Fc) tags via the boronate crosslinking, in which the MB-derived peak current was used as the reference signal, and the peak current of the Fc tag was used as the output signal. As each therapeutic mAb carries tens of diol sites for the site-specific decoration of the Fc output tags, the boronate crosslinking enabled the amplification-free detection, which is cost-effective and quite simple in operation. In the presence of bevacizumab (BevMab) as the target, the resulting ratiometric signal (i.e., the IFc/IMB value) exhibited a good linear response over the range of 0.025-2.5 μg/mL, and the limit of detection (LOD) of the electrochemical aptasensor was 6.5 ng/mL. Results indicated that the aptamer-based affinity recognition endowed the detection of therapeutic mAbs with high selectivity, while the ratiometric readout exhibited satisfactory reproducibility and robustness. Moreover, the ratiometric electrochemical aptasensor is applicable to the detection of therapeutic mAbs in serum samples. Taking together, the amplification-free ratiometric electrochemical aptasensor holds great promise in the POC detection of therapeutic mAbs.

Ni-catalyzed mild hydrogenolysis and oxidations of C–O bonds via carbonate redox tags

Oxygenated molecules are omnipresent in natural as well as artificial settings making the redox transformation of the present C–O bonds a central tool for their processing. However, the required (super)stoichiometric redox agents which traditionally include highly reactive and hazardous reagents pose multiple practical challenges including process safety hazards or special waste management requirements. Here, we report a mild Ni-catalyzed fragmentation strategy based on carbonate redox tags for redox transformations of oxygenated hydrocarbons in the absence of any external redox equivalents or other additives. The purely catalytic process enables the hydrogenolysis of strong C(sp2)–O bonds including that of enol carbonates as well as the catalytic oxidation of C–O bonds under mild conditions down to room temperature. Additionally, we investigated the underlying mechanism and showcased the benefits of carbonate redox tags in multiple applications. More broadly, the work herein demonstrates the potential of redox tags for organic synthesis.

Dual-label vertical flow-based electrochemical immunosensor for rapid and simultaneous detection of hepatitis B surface and e virus antigens

The detection of hepatitis B virus (HBV)-associated biomarkers, especially hepatitis B surface (HBsAg) and hepatitis B e (HBeAg) antigens, is of critical significance for the early diagnosis and monitoring of disease progression. However, the detection of these biomarkers has seldom been reported. Herein, we first present a dual-label electrochemical vertical flow-based immunosensor (DL-eVFIS) for the simultaneous detection of HBsAg and HBeAg. DL-eVFIS was engineered to integrate multiple time-sequenced detection procedures into a single device, thus considerably shortening the overall assaying time to 6 min. To enable simultaneous determination, thionine (Th) and ferrocenecarboxylic acid (Fc) were employed as signal tags, as they demonstrate nonoverlapping specific reduction peaks. The appearance of target antigens resulted in a substantial increase in the electrochemical signal response of the redox tags. While requiring only a miniscule sample volume (5 µL), superior performance was achieved in terms of their limits of detection (LODs) at 1.66 pg/mL for HBsAg and 5.32 pg/mL for HBeAg. DL-eVFIS further demonstrates excellent specificity and selectivity in clinical sample analysis compared with the conventional method. This study contributes to next-generation point-of-care (POC) testing devices for HBV diagnosis and a wide range of biosensing 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.

Redox-Controlled Energy Transfer Quenching of Fluorophore-Labeled DNA SAMs Enables In Situ Study of These Complex Electrochemical Interfaces.

Interfaces modified by a molecular monolayer can be challenging to study, particularly in situ, requiring novel approaches. Coupling electrochemical and optical approaches can be useful when signals are correlated. Here we detail a methodology that uses redox electrochemistry to control surface-based fluorescence intensity for detecting DNA hybridization and studying the uniformity of the surface response. A mixed composition single-strand DNA SAM was prepared using potential-assisted thiol exchange with two alkylthiol-modified ssDNAs that were either labeled with a fluorophore (AlexaFluor488) or a methylene blue (MB) redox tag. A significant change in fluorescence was observed when reducing MB to colorless leuco-MB. In situ fluorescence microscopy on a single-crystal gold bead electrode showed that fluorescence intensity depended on (1) the potential controlling the oxidation state of MB, (2) the surface density of DNA, (3) the MB:AlexFluor488 ratio in the DNA SAM, and (4) the local environment around the DNA SAM. MB efficiently quenched AlexaFluor488 fluorescence. Reduction of MB showed a significant increase in fluorescence resulting from a decrease in quenching or energy transfer efficiency. Hybridization of DNA SAMs with its unlabeled complement showed a large increase in fluorescence due to MB reduction for surfaces with sufficient DNA coverage. Comparing electrochemical-fluorescence measurements to electrochemical (SWV) measurements showed an improvement in detection of a small fraction of hybridized DNA SAM for surfaces with optimal DNA SAM composition and coverage. Additionally, this coupled electrochemical redox-fluorescence microscopy method can measure the spatial heterogeneity of electron-transfer kinetics and the influence of the local interfacial environment.

The Characterization of Binding between Aptamer and Bisphenol A and Developing Electrochemical Aptasensors for Bisphenol A with Rationally Engineered Aptamers.

Bisphenol A (BPA) is widely used in the manufacture of polycarbonate and epoxy-resin-based products, and BPA contamination often happens in a variety of types of environment and food stuffs. BPA can cause many harmful effects to health due to its high toxicity. The rapid detection of BPA is of great significance in environmental monitoring and food safety. Nucleic acid aptamers show advantages in biosensors due to good chemical stability, the ease of labeling functional groups, and target binding that induces conformation change. Here, we performed a thorough characterization of the binding performance of one 60-nt anti-BPA DNA aptamer with isothermal titration calorimetry (ITC). We found the crucial region of the aptamer sequence for affinity binding with BPA, and the aptamer was able to be truncated to 29-nt DNA without losing affinity. We then developed a simple reagent-less electrochemical aptamer-based sensor for rapid BPA detection with this engineered aptamer. The truncated aptamer with a redox tag methylene blue (MB) was immobilized on a gold electrode. BPA-binding induced the conformation change of the MB-labeled aptamer, moving the MB close to the electrode surface and causing a significant current increase in MB in square wave voltammetry (SWV). Under optimized conditions, we achieved the quantitative detection of BPA with a detection limit of BPA at 0.1 μM. This sensor showed quick response to BPA and could be regenerated by washing with deionized water. This sensor was selective, and it allowed detecting BPA in complex samples, showing its potential in practice. This study will help in further applications of the aptamers of BPA.

(Invited, Digital Presentation) Development of Multiplex Electrode Array Sensors for Proteases Activity Profiling Toward Cancer Diagnosis

Proteases are a large family of enzymes involved in many important biological processes. Quantitative detection of the activity profile of specific target proteases is in high demand for the diagnosis and treatment monitoring of diseases such as cancers. The overexpression and enhanced activity of proteases were found to correlate well with cancer development. Protease inhibitors are also a class of major anti-cancer drug candidates. However, due to the presence of a large number (over 600) of proteases in human body, their complex interactions with each other, and large influence by the surrounding factors (such as buffer composition, pH value and temperature), it is challenging to accurately detect the activity of specific proteases in complex media. It is even more difficult to measure the activity profiles of a large set of proteases that are relevant to cancers. Here we summarize our studies on the development of a multiplex electrode array biosensor platform toward cancer diagnosis based on rapid profiling of multiple protease activities. Both nano- and micro-electrode arrays have been demonstrated for electrochemical detection of protease activities. The individually addressed 3x3 gold thin-film microelectrode array is particularly attractive for rapid profiling of multiple protease activities.The general sensor scheme involves attaching short peptides with specific sequences of 4 to 8 amino acids on the electrode surface of the electrochemical sensor chip, which serve as the substrates for selective proteolysis by the cognate proteases. The far end is covalently attached with a ferrocene (Fc) group as the redox tag for electrochemical measurements. The quantity of the intact peptides on the electrode surface is measured with an AC voltammetry (ACV) method, which is proportional to the peak current at the specific electrode potential when Fc oxidation is oxidized into ferrocenium (Fc+). As the peptide is cleaved by the cognate protease, the peak current decreases continuously in the continuously repeated ACV curves. The recorded kinetic proteolytic curve can be quantitatively described by the heterogeneous Michaelis-Menten model following an exponential decay. The inverse of exponential decay time constant, i.e., 1/τ, is found to represent the protease activity which equals to [E](kcat /KM), where [E] is the protease concentration and kcat /KM is the specificity constant defined by the kinetic proteolytic reaction constant kcat and the Michaelis equilibrium constant KM. The kcat /KM values have been investigated versus the substrate peptide sequence and length, temperature, and buffer composition to optimize the detection for activity of cathepsin B, a potential cancer biomarker. The limit of detection (LOD) for activated cathepsin B can reach down to 0.57 pM, sufficient to directly detect cathepsin B in diluted human serum.This electrochemical method has been compared and validated with the commonly used affinity assay, i.e., enzyme-linked immunosorbent assay (ELISA). The ELISA measurements were found to be more sensitive to the concentration of proenzyme (zymogen), while the electrochemical method detects the activity of the proteases which reflects the intrinsic biological function. This provides more critical information than the concentration derived from ELISA.Currently, two sets of peptides have been synthesized which are selective for detection of cathepsin B and ADAM-17, respectively. They are functionalized to the 3x3 gold thin-film microelectrode array and used to simultaneously detect these two types of proteases in serum samples from breast cancer patients at different stages. The activity profiles will be correlated to the cancer development. Figure 1

A Programmable Electrochemical Y‐Shaped DNA Scaffold Sensor for the Single‐Step Detection of Antibodies and Proteins in Untreated Biological Fluids

AbstractProteins and antibodies are key biomarkers for diagnosing and monitoring specific medical conditions. Currently, gold standard techniques used for their quantification require laborious multi‐step procedures, involving high costs and slow response times. It is possible to overcome these limitations by exploiting the chemistry and programmability of DNA to design a reagentless electrochemical sensing platform. Specifically, three DNA single strands are engineered that can self‐assemble into a Y‐shaped DNA nanostructure that resembles one of the IgGs. In order to convert this DNA nanostructure into a responsive DNA‐scaffold bioreceptor, it is modified including two recognition elements, two redox tag molecules, and a thiol group. In the absence of the target, the scaffold receptor can efficiently collide with the electrode surface and generate a strong electrochemical signal. The presence of the target induces its bivalent binding, which produces steric hindrance interactions that limit the receptor's collisional activity. In its bound state, the redox tags can therefore approach the surface at a slower rate, leading to a signal decrease that is quantitatively related to the target concentration. The Y‐shape DNA scaffold sensor can detect nanomolar concentrations of antibodies and proteins in <15 min with a single‐step procedure directly in untreated biological fluids.

Hemoglobin-catalyzed atom transfer radical polymerization for ultrasensitive electrochemical DNA detection

The use of hemoglobin (Hb) to drive atom transfer radical polymerization (ATRP) process (Hb-ATRP) for detection of lung cancer related nucleic acid is firstly reported. Hb does not need to be treated prior to using indicating the potential for synthetic engineering in complex biological microenvironments without the need for in vitro techniques. Here, we report a new signal amplification strategy using Hb-mediated graft of nitronyl niroxide monoradical polymers as a signal-on electrochemical biosensor for ultralow level DNA highly selective detection. Building DNA biosensors includes: (i) the fixation of peptide nucleic acid (PNA) probe (no phosphate group) via the 5′ terminus-SH; (ii) the modification of transition metal; (iii) Site-specific markers of Hb-ATRP promoter, and (iv) the grafting of polymers with electrochemical signal by Hb-ATRP process. Through the Hb-ATRP process of nitronyl nitroxide monoradical (TEMPO), the presence of a small amount of DNA can eventually result in calling a certain number of TEMPO redox tags. Obviously, the Hb-ATRP is a method of easy source of raw materials, simple operation and no need for complex equipment. The constructed biosensor, as expected, is highly selective and sensitive to target DNA. The detection limit can be calculated as 15.96 fM under optimal conditions. The excellent performance also shows that the constructed DNA biosensor is suitable for DNA screening and DNA concentration determination in complex sample matrix.

A Folding-Based Electrochemical Aptasensor for the Single-Step Detection of the SARS-CoV-2 Spike Protein.

Efficient and timely testing has taken center stage in the management, control, and monitoring of the current COVID-19 pandemic. Simple, rapid, cost-effective diagnostics are needed that can complement current polymerase chain reaction-based methods and lateral flow immunoassays. Here, we report the development of an electrochemical sensing platform based on single-walled carbon nanotube screen-printed electrodes (SWCNT-SPEs) functionalized with a redox-tagged DNA aptamer that specifically binds to the receptor binding domain of the SARS-CoV-2 spike protein S1 subunit. Single-step, reagentless detection of the S1 protein is achieved through a binding-induced, concentration-dependent folding of the DNA aptamer that reduces the efficiency of the electron transfer process between the redox tag and the electrode surface and causes a suppression of the resulting amperometric signal. This aptasensor is specific for the target S1 protein with a dissociation constant (KD) value of 43 ± 4 nM and a limit of detection of 7 nM. We demonstrate that the target S1 protein can be detected both in a buffer solution and in an artificial viral transport medium widely used for the collection of nasopharyngeal swabs, and that no cross-reactivity is observed in the presence of different, non-target viral proteins. We expect that this SWCNT-SPE-based format of electrochemical aptasensor will prove useful for the detection of other protein targets for which nucleic acid aptamer ligands are made available.

Highly selective and sensitive detection of glutamate by an electrochemical aptasensor.

An electrochemical aptamer-based sensor was developed for glutamate, the major excitatory neurotransmitter in the central nervous system. Determining glutamic acid release and glutamic acid levels is crucial for studying signal transmission and for diagnosing pathological conditions in the brain. Glutamic acid-selective oligonucleotides were isolated from an ssDNA library using the Capture-SELEX protocol in complex medium. The selection permitted the isolation of an aptamer 1d04 with a dissociation constant of 12µM. The aptamer sequence was further used in the development of an electrochemical aptamer sensor. For this purpose, a truncated aptamer sequence named glu1 was labelled with a ferrocene redox tag at the 3'-end and immobilized on a gold electrode surface via Au-thiol bonds. Using 6-mercapto-1-hexanol as the backfill, the sensor performance was characterized by alternating current voltammetry. The glu1 aptasensor showed a limit of detection of 0.0013pM, a wide detection range between 0.01pM and 1nM, and good selectivity for glutamate in tenfold diluted human serum. With this enzyme-free aptasensor, the highly selective and sensitive detection of glutamate was demonstrated, which possesses great potential for implementation in microelectrodes and for in vitro as well as in vivo monitoring of neurotransmitter release.

Conformation-based stimuli-response sensors: Strategies for optimizing electrochemical and FRET transduction

Conformation-based stimuli-responsive sensors utilize a wide range of macromolecules (referred to as probe in this manuscript) and read-out methods. The review focuses on the variables associated with the mechanistic confirmation change centered around two transduction outputs: electrochemical and fluorescence resonance energy transfer. The electrochemical readout is limited in that the responsive material is bound between the electrode surface and an electroactive tag; interaction with analyte changes the distance between the electrode surface and the electroactive tag. Variables discussed in electrochemical stimuli-responsive sensor outputs include redox tag choice, electrode surface packing density, probe architectures, and redox tag location. Fluorescence resonance energy transfer readout uses a solution-free polymer distance where a conformational change induces a change in distance between a donor fluorophore and an acceptor fluorophore, thereby affecting the degree of nonradiative energy transfer from the donor to the acceptor. Variables discussed in fluorescence resonance energy transfer readouts include fluorophore types and sensor architecture. While there are already many examples of this in the literature, we believe this to be a fertile area for further development.

Coenzyme-mediated electro-grafting for ultrasensitive electrochemical DNA biosensing

Signal amplification through the grafting of polymers has attracted increasing attention in biodetection at ultralow concentrations. Herein, an ultrasensitive peptide nucleic acid (PNA)-based electrochemical DNA biosensor is reported by using the coenzyme-mediated electro-grafting of polymers as a novel amplification method. For the capture of DNA target, PNA is used as the probe. After hybridization, reversible addition-fragmentation chain-transfer (RAFT) agents are site-specifically tethered to the deoxyribose phosphate backbone of the captured DNA targets via the carboxylate-Zr(IV)-phosphate cross-linking chemistry. Subsequently, electro-grafting of polymers through the nicotinamide adenine dinucleotide (NAD+) coenzyme-mediated electro-RAFT (NAD+-eRAFT) polymerization of ferrocenylmethyl methacrylate (FcMMA) recruits numerous ferrocene redox tags for voltammetric measurement. As the electro-grafting of polymers involves simply i) the tethering of RAFT agents and ii) the NAD+-eRAFT polymerization, it is highly efficient, easy to use, and low-cost. Under optimal conditions, the voltammetric signal correlates linearly with the logarithm of DNA concentration over the range from 0.1 fM to 0.1 nM (R2 = 0.996), with a detection limit of 0.067 fM. The PNA-based electrochemical DNA biosensor can also differentiate even single base mismatch and is applicable to DNA detection in the presence of complex serum matrices, thus showing great promise in the sequence-specific detection of DNA targets at ultralow concentrations.

Coenzyme-Mediated Electro-RAFT Polymerization for Amplified Electrochemical Interrogation of Trypsin Activity.

Trypsin is a key proteolytic enzyme in the digestive system and its abnormal levels are indicative of some pancreatic diseases. Taking advantage of the coenzyme-mediated electrografting of ferrocenyl polymers as a novel strategy for signal amplification, herein, a signal-on cleavage-based electrochemical biosensor is reported for the highly selective interrogation of trypsin activity at ultralow levels. The construction of the trypsin biosensor involves (i) the immobilization of peptide substrates (without free carboxyl groups) via the N-terminus, (ii) the tryptic cleavage of peptide substrates, (iii) the site-specific labeling of the reversible addition-fragmentation chain transfer (RAFT) agents, and (iv) the grafting of ferrocenyl polymers through the electro-RAFT (eRAFT) polymerization, which is mediated by potentiostatic reduction of nicotinamide adenine dinucleotide (NAD+) coenzymes. Through the NAD+-mediated eRAFT (NAD+-eRAFT) polymerization of ferrocenylmethyl methacrylate (FcMMA), the presence of a few tryptic cleavage events can eventually result in the recruitment of a considerable amount of ferrocene redox tags. Obviously, the NAD+-eRAFT polymerization is low-cost and easy to operate as a highly efficient strategy for signal amplification. As expected, the as-constructed biosensor is highly selective and sensitive toward the signal-on interrogation of trypsin activity. Under optimal conditions, the detection limit can be as low as 18.2 μU/mL (∼72.8 pg/mL). The results also demonstrate that the as-constructed electrochemical trypsin biosensor is applicable to inhibitor screening and the interrogation of enzyme activity in the presence of complex sample matrices. Moreover, it is low-cost, less susceptible to false-positive results, and relatively easy to fabricate, thus holding great potential in diagnostic and therapeutic applications.

Electrocatalytic Mechanism for Improving Sensitivity and Specificity of Electrochemical Nucleic Acid-Based Sensors with Covalent Redox Tags-Part I.

The design and development of advanced electrocatalysis have been extensively explored for efficient energy conversion and electrochemical biosensing. Both ferricyanide (Fe(CN)63-) and methylene blue (MB) have been widely used in the development of electrochemical biosensing strategies. However, the electrocatalytic mechanism between nucleic acid-tethered MB and Fe(CN)63- remains unexplored. In this manuscript, we aim to provide readers in our community molecular insights into the electrocatalytic mechanism. The exploration of the electrocatalytic mechanism starts with a kinetic zone diagram for a one-electron homogeneous electrocatalytic reaction. Two factors-the excess factor γ and the kinetic parameter λ-are important for a homogeneous electrocatalytic reaction; as such, we studied both. The excess factor parameter was controlled by applying Fe(CN)63- with various concentrations (50, 100, and 200 μM), and the kinetic parameter effect on the electrocatalytic process was examined by varying scan rates of cyclic voltammetry (CV) or frequencies of square-wave voltammetry (SWV). Moreover, we discovered that the probe dynamics of the nucleic acid tether is the third rate-limiting factor for the electrocatalytic reaction. As the probe dynamics switch of electrode-bound nucleic acid is often utilized as a mechanism in electrochemical nucleic acid-based sensors, we believe the electrocatalysis between nucleic acid-tethered MB and Fe(CN)63- is capable of enhancing sensitivity and specificity of electrochemical nucleic acid-based sensors with covalent redox tags.