Redox Label Research Articles

Antimicrobial Peptide-Based Sensor for the Electrochemical Detection of Methicillin-Resistant Staphylococcus Aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a gram-positive bacterium that colonizes on the skin and nasal track of humans, which can lead to serious illnesses such as skin and soft tissue infections, pneumonia, and septicemia.MRSA is resistant to an entire class of antibiotics called beta-lactams, including penicillin, amoxicillin, and oxacillin, making it more difficult to treat. Culture based methods and molecular based methods like PCR are currently being used for the detection of MRSA, but these methods possess the drawbacks of being slow and expensive, respectively. Thus, we focused on developing a reagentless, cost-effective, near real-time detection method for intact MRSA cells.Antimicrobial peptides (AMPs) possess the ability to interact with bacterial cells by inhibiting their growth or by lysing them.However, this initial interaction between the cationic AMPs and the negatively charged bacterial cells can be used for the detection of pathogens like MRSA. Here, we used a MRSA-specific antimicrobial peptide, DP7, to fabricate an electrochemical peptide-based (E-PB) sensor. The DP7 probe was further modified with a methylene blue redox label and an alkanethiol chain which allows for the formation of a thiol-gold self-assembled monolayer. The sensor surface was passivated with both 6-mercapto-1-hexanol and a thiolated tetra-thymine sequence to prevent surface fouling. Electrochemical detection of MRSA was accomplished using alternating current voltammetry. Control experiments were performed using two gram-positive bacteria, Bacillus subtilis and Staphylococcus epidermidis, and two gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa. The DP7 sensor responded to MRSA in a concentration-dependent manner and was proven to be selective for MRSA over the other four microbes. Specifically, the % signal suppression obtained for MRSA was much larger when compared to those obtained for the other microbes. The same behavior was observed in pure buffer and 5% synthetic wound fluid. In addition, the detection time was relatively short, with signal saturation in less than 60 min. Overall, the DP7 sensor has demonstrated to possess attributes similar to other E-PB sensors. It has the potential to be developed as a rapid, reliable, and cost-effective diagnostic tool for MRSA, which will be useful in preventing the spread of the pathogen in the community. Furthermore, this sensing platform is versatile and can be redesigned for detection of other pathogenic bacteria.

Utilization of Spontaneous Alkyne-Gold Self-Assembly Chemistry as an Alternative Method for Fabricating Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (E-AB) sensors are a promising class of biosensors which use structure-switching redox-labeled oligonucleotides (aptamers) codeposited with passivating alkanethiol monolayers on electrode surfaces to specifically bind and detect target analytes. Signaling in E-AB sensors is an outcome of aptamer conformational changes upon target binding, with the sequence of the aptamer imparting specificity toward the analyte of interest. The change in conformation translates to a change in electron transfer between the redox label attached to the aptamer and the underlying electrode and is related to analyte concentration, allowing specific electrochemical detection of nonelectroactive analytes. E-AB sensor measurements are reagentless with time resolutions of seconds or less and may be miniaturized into the submicron range. Traditionally these sensors are fabricated using thiol-on-gold chemistry. Here we present an alternate immobilization chemistry, gold-alkyne binding, which results in an increase in sensor lifetimes under ideal conditions by up to ∼100%. We find that gold-alkyne binding is spontaneous and supports efficient E-AB sensor signaling with analytical performance characteristics similar to those of thiol generated monolayers. The surface modification differs from gold-thiol binding only in the time and aptamer concentration required to achieve similar aptamer surface coverages. In addition, alkynated aptamers differ from their thiolated analogues only by their chemical handle for surface attachment, so any existing aptamers can be easily adapted to utilize this attachment strategy.

Development of a Neuropeptide Y-Sensitive Implantable Microelectrode for Continuous Measurements.

In this work, we present the development of the first implantable aptamer-based platinum microelectrode for continuous measurement of a nonelectroactive molecule, neuropeptide Y (NPY). The aptamer immobilization was performed via conjugation chemistry and characterized using cyclic voltammetry before and after the surface modification. The redox label, methylene blue (MB), was attached at the end of the aptamer sequence and characterized using square wave voltammetry (SWV). NPY standard solutions in a three-electrode cell were used to test three aptamers in steady-state measurement using SWV for optimization. The aptamer with the best performance in the steady-state measurements was chosen, and continuous measurements were performed in a flow cell system using intermittent pulse amperometry. Dynamic measurements were compared against confounding and similar peptides such as pancreatic polypeptide and peptide YY, as well as somatostatin to determine the selectivity in the same modified microelectrode. Our Pt-microelectrode aptamer-based NPY biosensor provides signals 10 times higher for NPY compared to the confounding molecules. This proof-of-concept shows the first potential implantable microelectrode that is selectively sensitive to NPY concentration changes.

Measurement of neuropeptide Y in aptamer-modified planar electrodes

Electrochemical impedance spectroscopy (EIS) is a powerful technique for studying the interaction at electrode/solution interfaces. The adoption of EIS for obtaining analytical signals in biosensors based on aptamers is gaining popularity because of its advantageous characteristics for molecular recognition. Neuropeptide Y (NPY), the most abundant neuropeptide in the body, plays a crucial role with its stress-relieving properties. Quantitative measurement of NPY is imperative for understanding its role in these and other biological processes. Although aptamer-modified electrodes for NPY detection using EIS present a promising alternative, the correlation between the data obtained and the adsorption process on the electrodes is not fully understood. Various studies utilize the change in charge transfer resistance when employing an active redox label. In contrast, label-free measurement relies on changes in capacitance. To address these challenges, we focused on the interaction between aptamer-modified planar electrodes and their target, NPY. We proposed utilizing −ω*Zimag as the analytical signal, which facilitated the analysis of the adsorption process using an analogous Langmuir isotherm equation. This approach differs from implantable microelectrodes, which adhere to the Freundlich adsorption isotherm. Notably, our method obviates the need for a redox label and enables the detection of NPY at concentrations as low as 20 pg/mL. This methodology demonstrated exceptional selectivity, exhibiting a signal difference of over 20-to-1 against potential interfering molecules.

Electrochemical biosensor using direct electron transfer and an antibody–aptamer hybrid sandwich for target detection in complex biological samples

Direct electron transfer (DET) between an electrode and redox labels is feasible in electrochemical biosensors using small aptamer–aptamer sandwiches; however, its application is limited in biosensors that rely on larger antibody–antibody sandwiches. The development of sandwich-type biosensors utilizing DET is challenged by the scarcity of aptamer–aptamer sandwich pairs with high affinity in complex biological samples. Here, we introduce an electrochemical biosensor using an antibody–aptamer hybrid sandwich for detecting thrombin in human serum. The biosensor enables rapid DET through an antibody–aptamer hybrid configuration comprising (i) an antibody capture probe that provides high and specific affinity to the target in human serum, (ii) the target thrombin, and (iii) an aptamer detection probe that facilitates convenient terminal conjugation with long flexible spacer DNA and polylinker peptide containing multiple amine-reactive phenazine ethosulfate (arPES) redox labels, allowing the conjugated labels to easily approach the electrode. Rapid repeated DET using arPES-catalyzed NADH oxidation strongly enhanced the electrochemical signals. Properly sized spacer and polylinker provided low nonspecific adsorption of the aptamer probe conjugated with multiple arPESs and low interference with the binding of the aptamer probe. Methods for immobilizing thiol-terminated antibodies on Au electrodes were compared and optimized. The developed biosensor using the antibody–aptamer hybrid sandwich exhibited high sensitivity and selectivity in detecting thrombin, surpassing the limitations of an aptamer–aptamer sandwich owing to the low affinity of thrombin aptamers in human serum. The calculated detection limit of the biosensor was ∼1.5 pM in buffer and ∼2.7 nM in human serum.

Introducing common oxazine fluorophores as new redox labels for electrochemical DNA sensors

The electrochemical properties of three oxazine fluorophores, ATTO655, ATTO680 and ATTO700 have been investigated at gold electrodes. They display a reversible or quasi-reversible voltammetric behaviour involving either a 2e−, 2H+ or a 2e−, 1H+ redox process depending on the pH, at a formal potential located in the stability range of thiolate self-assembled monolayers (E°' ≈ −0.33 V vs. Ag|AgCl|3M KCl at pH 7.2). The performance of ATTO655 as redox label for electrochemical nucleic acid sensing was evaluated in a typical E-DNA configuration. The redox label has no detrimental impact on the folding of DNA, as shown with the i-motif forming sequence investigated here. An electron transfer rate constant around 40 s−1 was determined, which is comparable to the values reported for the popular methylene blue label. Hybridisation experiments show a significant signal variation between ssDNA and dsDNA, though it is emphasised that the sign and amplitude of the variation are highly dependent on the electrochemical parameters such as the frequency in square wave voltammetry.

Normalized Multipotential Redox Coding of DNA Bases for Determination of Total Nucleotide Composition.

The previously reported approach of orthogonal multipotential redox coding of all four DNA bases allowed only analysis of the relative nucleotide composition of short DNA stretches. Here, we present two methods for normalization of the electrochemical readout to facilitate the determination of the total nucleotide composition. The first method is based on the presence or absence of an internal standard of 7-deaza-2'-deoxyguanosine in a DNA primer. The exact composition of the DNA was elucidated upon two parallel analyses and the subtraction of the electrochemical signal intensities. The second approach took advantage of a 5'-viologen modified primer, with this fifth orthogonal redox label acting as a reference for signal normalization, thus allowing accurate electrochemical sequence analysis in a single read. Both approaches were tested using various sequences, and the voltammetric signals obtained were normalized using either the internal standard or the reference label and demonstrated to be in perfect agreement with the actual nucleotide composition, highlighting the potential for targeted DNA sequence analysis.

An Ultrasensitive Voltammetric Genosensor for the Detection of Bacteria Vibrio cholerae in Vegetable and Environmental Water Samples.

In view of the presence of pathogenic Vibrio cholerae (V. cholerae) bacteria in environmental waters, including drinking water, which may pose a potential health risk to humans, an ultrasensitive electrochemical DNA biosensor for rapid detection of V. cholerae DNA in the environmental sample was developed. Silica nanospheres were functionalized with 3-aminopropyltriethoxysilane (APTS) for effective immobilization of the capture probe, and gold nanoparticles were used for acceleration of electron transfer to the electrode surface. The aminated capture probe was immobilized onto the Si-Au nanocomposite-modified carbon screen printed electrode (Si-Au-SPE) via an imine covalent bond with glutaraldehyde (GA), which served as the bifunctional cross-linking agent. The targeted DNA sequence of V. cholerae was monitored via a sandwich DNA hybridization strategy with a pair of DNA probes, which included the capture probe and reporter probe that flanked the complementary DNA (cDNA), and evaluated by differential pulse voltammetry (DPV) in the presence of an anthraquninone redox label. Under optimum sandwich hybridization conditions, the voltammetric genosensor could detect the targeted V. cholerae gene from 1.0 × 10-17-1.0 × 10-7 M cDNA with a limit of detection (LOD) of 1.25 × 10-18 M (i.e., 1.1513 × 10-13 µg/µL) and long-term stability of the DNA biosensor up to 55 days. The electrochemical DNA biosensor was capable of giving a reproducible DPV signal with a relative standard deviation (RSD) of <5.0% (n = 5). Satisfactory recoveries of V. cholerae cDNA concentration from different bacterial strains, river water, and cabbage samples were obtained between 96.5% and 101.6% with the proposed DNA sandwich biosensing procedure. The V. cholerae DNA concentrations determined by the sandwich-type electrochemical genosensor in the environmental samples were correlated to the number of bacterial colonies obtained from standard microbiological procedures (bacterial colony count reference method).

Towards selective tetracycline recognition in wastewater based on gold nanovoids@aptamer sensing

In recent years, tetracycline antibiotic residues in food and water have been of great concern to regulators and consumers, with adverse effects on both ecosystems and human health. In this work, we developed a novel 3D honeycombed goldnanovoids@aptamer nanostructured platform with biorecognition and signal enhancement properties for the detection of tetracycline. Direct electrochemical detection was enabled by differential pulse voltammetry through the Ferrocene redox label at the aptamer strands which was effectively influenced by the tetracycline-aptamer specific interaction. The aptasensor can detect tetracycline at a concentration from 1.25∙10−8 M to 1.00∙10−6 M, and the limit of detection and limit of quantification were calculated as 1.20∙10−9 M (0.53 ng/mL) and 4.23∙10−9 M (1.87 ng/mL), respectively. Furthermore, the aptasensor presented excellent selectivity in detecting tetracycline in the presence of various interference analytes. In addition, the proposed sensor proved its practical feasibility to quickly and accurately quantify tetracycline residues in wastewater samples with satisfactory recoveries (from 93.18 to 112.64 %, RSD 1.12 % – 6.13 %). We have designed and synthesized a 3D honeycombed gold nanostructured platform for the highly tailored aptamer functionalization for the specific detection of tetracycline, expanding the range of electrochemical detection aptasensors and offering great promise for in-situ water monitoring.

Electrochemical biosensor for trypsin activity assay based on cleavage of immobilized tyrosine-containing peptide

In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label. The peptides were immobilized on SPE by formation of chemical bonds between mercapto groups of the N-terminal cysteine residues and AuNPs. After the incubation with trypsin, time-dependent cleavage of the immobilized peptides was observed by decline in tyrosine electrochemical oxidation signal. The kinetic parameters of trypsin, such as the catalytic constant (kcat), the Michaelis constant (KM) and the catalytic efficiency (kcat/KM), toward the CGGGRYR peptide were determined as 0.33 ± 0.01 min−1, 198 ± 24 nM and 0.0016 min−1 nM−1, respectively. Using the developed biosensor, we demonstrated the possibility of analysis of trypsin specificity toward the peptides with amino acid residues disrupting proteolysis. Further, we designed the peptides with proline or glutamic acid residues after the cleavage site (CGGRPYR and CGGREYR), and trypsin had reduced activity toward both of them according to the existing knowledge of the enzyme specificity. The developed biosensor system allows one to perform a comparative analysis of the protease steady-state kinetic parameters and specificity toward model peptides with different amino acid sequences.

Coordination Chemistry of Uranyl Ions with Surface-Immobilized Peptides: An XPS Study.

The coordination chemistry of uranyl ions with surface immobilized peptides was studied using X-ray photoemission spectroscopy (XPS). All the peptides in the study were modified using a six-carbon alkanethiol as a linker on a gold substrate with methylene blue as the redox label. The X-ray photoemission spectra reveal that each modified peptide interacts differently with the uranyl ion. For all the modified peptides, the XPS spectra were taken in both the absence and presence of the uranium, and their comparison reveals that the interaction depends on the chemical group present in the peptides. The XPS results show that, among all the modified peptides in the current study, the (arginine)9 (R9) modified peptide showed the largest response to uranium. In the order of response to uranium, the second largest response was shown by the modified (arginine)6 (R6) peptide followed by the modified (lysine)6 (K6) peptide. Other modified peptides, (alanine)6 (A6), (glutamic acid)6 (E6) and (serine)6 (S6), did not show any response to uranium.

Supramolecular Enzymatic Labeling for Aptamer Switch-Based Electrochemical Biosensor.

Here we report a novel labeling strategy for electrochemical aptasensors based on enzymatic marking via supramolecular host–guest interactions. This approach relies on the use of an adamantane-modified target-responsive hairpin DNA aptamer as an affinity bioreceptor, and a neoglycoconjugate of β-cyclodextin (CD) covalently attached to a redox enzyme as a labeling element. As a proof of concept, an amperometric aptasensor for a carcinoembryonic antigen was assembled on screen-printed carbon electrodes modified with electrodeposited fern-like gold nanoparticles/graphene oxide and, by using a horseradish peroxidase-CD neoglycoenzyme as a biocatalytic redox label. This aptasensor was able to detect the biomarker in the concentration range from 10 pg/mL to 1 ng/mL with a high selectivity and a low detection limit of 3.1 pg/mL in human serum samples.

(Invited) Designed DNA Nano-Switches As Sensitive Electrochemical Biosensors

Functional nucleic acid receptors (aptamers) have emerged as effective and robust recognition elements for state-of-the-art biosensors. Analytical readouts from aptamer-based biosensors (whether optical, electrochemical, or otherwise) derive primarily from global-scale conformational changes induced in the aptamer domain by analyte binding. Herein, we describe a unique biosensor design principle that represents a distinct alternative to this paradigm; particularly we demonstrate the ready applicability of this design principle in the de novo creation of electrochemical sensors for a clinical analyte of current interest. The function of the class of biosensors we describe, termed “DNA nano-switches”, is designed to depend on the integrity of duplex DNA-mediated charge transfer between an electrode and a redox label.[1] Thomas, J. M.; Chakraborty, B.; Sen, D.; Yu, H.-Z. J. Am. Chem. Soc. 2012, 134, 13823–13833.[2] Tang, Y.; Ge, B.; Sen, D.; Yu, H.-Z. Chem. Soc. Rev. 2014, 43, 518–529[3] Ma, F.; Qi, L.; Einarson, O.; Sen, D.; Yu, H.-Z. Anal. Chem. 2019, 91, 8244−8251. Figure 1

Electrochemistry of Cobalta Bis(dicarbollide) Ions Substituted at Carbon Atoms with Hydrophilic Alkylhydroxy and Carboxy Groups

In this study we explore the effect on the electrochemical signals in aqueous buffers of the presence of hydrophilic alkylhydroxy and carboxy groups on the carbon atoms of cobalta bis(dicarbollide) ions. The oxygen-containing exo-skeletal substituents of cobalta bis(dicarbollide) ions belong to the perspective building blocks that are considered for bioconjugation. Carbon substitution provides wider versatility and applicability in terms of the flexibility of possible chemical pathways. However, until recently, the electrochemistry of compounds substituted only on boron atoms could be studied, due to the unavailability of carbon-substituted congeners. In the present study, electrochemistry in aqueous phosphate buffers is considered along with the dependence of electrochemical response on pH and concentration. The compounds used show electrochemical signals around −1.3 and +1.1 V of similar or slightly higher intensities than in the parent cobalta bis(dicarbollide) ion. The signals at positive electrochemical potential correspond to irreversible oxidation of the boron cage (the C2B9 building block) and at negative potential correspond to the reversible redox process of (CoIII/CoII) at the central atom. Although the first signal is typically sharp and its potential can be altered by a number of substituents, the second signal is complex and is composed of three overlapping peaks. This signal shows sigmoidal character at higher concentrations and may be used as a diagnostic tool for aggregation in solution. Surprisingly enough, the observed effects of the site of substitution (boron or carbon) and between individual groups on the electrochemical response were insignificant. Therefore, the substitutions would preserve promising properties of the parent cage for redox labelling, but would not allow for the further tuning of signal position in the electrochemical window.

Wash-Free, Sandwich-Type Protein Detection Using Direct Electron Transfer and Catalytic Signal Amplification of Multiple Redox Labels.

Direct electron transfer (DET) between a redox label and an electrode has been used for sensitive and selective sandwich-type detection without a wash step. However, applying DET is still highly challenging in protein detection, and a single redox label per probe is insufficient to obtain a high electrochemical signal. Here, we report a wash-free, sandwich-type detection of thrombin using DET and catalytic signal amplification of multiple redox labels. The detection scheme is based on (i) the redox label-catalyzed oxidation of a reductant, (ii) the conjugation of multiple redox labels per probe using a poly-linker, (iii) the low nonspecific adsorption of the conjugated poly-linker due to uncharged, reduced redox labels, and (iv) a facile DET using long, flexible poly-linker and spacer DNA. Amine-reactive phenazine ethosulfate and NADH were used as the redox label and reductant, respectively. N3-terminated polylysine was used as the poly-linker for the conjugation between an aptamer probe and multiple redox labels. Approximately 11 redox labels per probe and rapid catalytic NADH oxidation enable high signal amplification. Thrombin in urine could be detected without a wash step with a detection limit of ∼50 pM, which is practically promising for point-of-care testing of proteins.

Advances in Design Strategies of Multiplex Electrochemical Aptasensors.

In recent years, the need for simple, fast, and economical detection of food and environmental contaminants, and the necessity to monitor biomarkers of different diseases have considerably accelerated the development of biosensor technology. However, designing biosensors capable of simultaneous determination of two or more analytes in a single measurement, for example on a single working electrode in single solution, is still a great challenge. On the other hand, such analysis offers many advantages compared to single analyte tests, such as cost per test, labor, throughput, and convenience. Because of the high sensitivity and scalability of the electrochemical detection systems on the one hand and the specificity of aptamers on the other, the electrochemical aptasensors are considered to be highly effective devices for simultaneous detection of multiple-target analytes. In this review, we describe and evaluate multi-label approaches based on (1) metal quantum dots and metal ions, (2) redox labels, and (3) enzyme labels. We focus on recently developed strategies for multiplex sensing using electrochemical aptasensors. Furthermore, we emphasize the use of different nanomaterials in the construction of these aptasensors. Based on examples from the existing literature, we highlight recent applications of multiplexed detection platforms in clinical diagnostics, food control, and environmental monitoring. Finally, we discuss the advantages and disadvantages of the aptasensors developed so far, and debate possible challenges and prospects.

Kinetics and Energetics of Intramolecular Electron Transfer in Single-Point Labeled TUPS-Cytochrome c Derivatives.

Electron transfer within and between proteins is a fundamental biological phenomenon, in which efficiency depends on several physical parameters. We have engineered a number of horse heart cytochrome c single-point mutants with cysteine substitutions at various positions of the protein surface. To these cysteines, as well as to several native lysine side chains, the photoinduced redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) was covalently attached. The long-lived, low potential triplet excited state of TUPS, generated with high quantum efficiency, serves as an electron donor to the oxidized heme c. The rates of the forward (from the label to the heme) and the reverse (from the reduced heme back to the oxidized label) electron transfer reactions were obtained from multichannel and single wavelength flash photolysis absorption kinetic experiments. The electronic coupling term and the reorganization energy for electron transfer in this system were estimated from temperature-dependent experiments and compared with calculated parameters using the crystal and the solution NMR structure of the protein. These results together with the observation of multiexponential kinetics strongly support earlier conclusions that the flexible arm connecting TUPS to the protein allows several shortcut routes for the electron involving through space jumps between the label and the protein surface.

Real-Time Cortisol Detection Using Electrocatalytic Polymer-Based Sensor

Cortisol is a steroid hormone produced by the adrenal gland in response to stress or agitate states. Deficiency or excess cortisol level can be triggered by physical or psychological stress, which is considered a biomarker for many diseases, such as Cushing syndrome [1]. In the human body, cortisol concentrations fluctuate in a circadian rhythm, being the highest in the early morning and the lowest in the night. A reliable cortisol test cannot depend on a single or one-day average measurement. Instead, screening a patient's dynamic cortisol levels over the circadian cycle is expected. The existing clinical cortisol measurements require blood, urine, or saliva samples sent to a lab to await results. The detection methods are mainly based on immunoassay that requires labeled antigens for a competitive assay and time-consuming incubation time (15–30 min), which hinders continuous detection needed for real-time stress management. To enable accurate time course measurements, a new detection approach is required to allow real-time determination of cortisol without costly reagents, labeling processes, additional redox reagents, and cumbersome laboratory equipment.Nanomaterials that mimic the catalytic behaviors of enzymes have great potential for continuous monitoring of cortisol levels through the direct electrochemical reading from cortisol redox reaction. There are very few reports on artificial cortisol catalytic sensors which require complicated fabrication or extremely non-neutral pH conditions for sensing [2]. Copper phthalocyanine (CuPc), a macrocycle with transitional copper (Cu) ion, holds electrocatalytic behavior and can be utilized for electrochemical redox reactions of various biomolecules, including H2O2, phenol, dopamine and serotonin. However, the detection of steroid hormones using CuPc has not been demonstrated yet. Compared to similar compounds based on porphyrin, phthalocyanine has N atoms in the meso positions and rich π-electrons that permit the formation of stable metallic phthalocyanine complexes. In this work, we develop copper phthalocyanine tetrasulfonate doped polypyrrole (PPy:CuPcTS) nanocomposite onto disposable screen-printed electrodes (SPEs) and demonstrate its electrocatalytic activity for real-time cortisol sensing. The incorporation of water-soluble CuPcTS into the PPy film forms an electrochemically conducting polymer in the negative potential window and catalyzes the reduction reaction of cortisol. The PPy:CuPcTS/SPE sensor can directly determine cortisol concentrations in real-time.The PPy:CuPcTS film was formed through one-step anodic electropolymerization on SPEs. The morphology and elemental composition of the film were characterized by scanning electron microscopy (SEM) and energy dispersive X-Ray analysis (EDX), respectively. Electrochemical responses were obtained with cyclic voltammetry (CV) and chronoamperometry. Cortisol detection was performed with 1x PBS solution.During the electropolymerization of PPy with CuPcTS, parts of the pyrrole rings hold a positive charge and form a complex with the sulfonated group in the CuPcTS. The CuPcTS and the PPy rings can also bond through π-π stacking. The stacked arrangement creates more conduction pathways due to highly delocalized electrons. The chelated Cu in CuPcTS catalyzes the reduction of ketone group in cortisol to an alcohol group [3], as schematically shown in Fig. 1(a). The SEM images in Fig.1 (b) show that the polymerization of PPy in the presence of CuPcTS results in a rough surface, very different from the smooth surface morphology on an undoped PPy film. The successful doping of CuPcTS in the PPy film is further confirmed by the presence of Cu peaks in the EDX (Fig.1 (b)). The CV curves in Fig. 1(c) indicate that cortisol reduction can be achieved using PPy:CuPcTS/SPE, evidenced by the increase in cathodic current at -1 V vs. Ag/AgCl upon adding cortisol. The phenomenon was not observed with PPy/SPE. The cathodic current increases with cortisol concentrations. The proposed sensor provides a dynamic detection range from 1 nM to 500 nM (Fig. (d)). Fig. 1(e) shows that the cortisol sensor also exhibits a selective detection against common interfering molecules, including glucose, ascorbic acid, and dopamine.PPy:CuPcTS nanocomposite was successfully demonstrated for electrocatalytic cortisol sensing. The sensor was achieved by a facile fabrication method and can operate at neutral pH without redox probes and labeling steps. The new sensor possesses great potential for low-cost, real-time monitoring of stress levels.

Sensitive Electrochemical Detection of Microcystin-LR in Water Samples Via Target-Induced Displacement of Aptamer Associated [Ru(NH3)6]3.

In this study, we demonstrate the successful development of an electrochemical aptamer-based sensor for point-of-use detection and quantification of the highly potent microcystin-LR (MC-LR) in water. The sensor uses hexaammineruthenium(III) chloride ([Ru(NH3)6]3+) as redox mediator, because of the ability of the positively charged (3+) molecule to associate with the phosphate backbone of the nucleic acids. We quantitatively measure the target-induced displacement of aptamer associated, or surface confined, [Ru(NH3)6]3+ in the presence of MC-LR. Upon the addition of MC-LR in the water, surface-confined [Ru(NH3)6]3+ dissociates, resulting in less faradaic current from the reduction of [Ru(NH3)6]3+ to [Ru(NH3)6]2+ Sensing surfaces of highly packed immobilized aptamers were capable of recording decreasing square wave voltammetry (SWV) signals after the addition of MC-LR in buffer. As a result, SWV recorded substantial signal suppression within 15 min of target incubation. The sensor showed a calculated limit of detection (LOD) of 9.2 pM in buffer. The effects of interferents were minimal, except when high concentrations of natural organic matter (NOM) were present. Also, the sensor performed well in drinking water samples. These results indicate a sensor with potential for fast and specific quantitative determination of MC-LR in drinking water samples. A common challenge when developing electrochemical, aptamer-based sensors is the need to optimize the nucleic acid aptamer in order to achieve sensitive signaling. This is particularly important when an aptamer experiences only a small or localized conformational change that provides only a limited electrochemical signal change. This study suggests a strategy to overcome that challenge through the use of a nucleic acid-associated redox label.

Aptamer-Modified Electrode Biosensors for Npy Detection Using Methylene Blue Redox Probe

A biosensor is a device that can determine the concentration or presence of chemicals, such as neurotransmitters and neuropeptides. Certain excitatory neurotransmitters and neuropeptides can be measured by Methylene blue (MB), a synthetic dye that stains to negatively charged cell components. For the modification process in this work, aptamers are used because they possess the advantage of binding to any given target molecule and, when they bind, they undergo conformational changes, which is where MB comes into play. This research focuses on the modification of the surface of Gold (Au) and Highly Oriented Pyrolytic Graphite (HOPG) electrodes using MB as a redox label probe for the detection of neurotransmitters and neuropeptides in aptamer-modified electrodes. Electrochemical Impedance Spectroscopy (EIS) was used to understand the adsorption properties of molecules that stick to the surface of the electrode and Atomic Force Microscopy (AFM) was used to measure the topography of the electrodes before and after the aptamer modification. As results, we were able to prove that AFM cannot monitor the changes on the surface of the Au electrodes with the different modifications due to the roughness of the surface, and that, electrochemically, the capacitance of the gold electrode decreases at higher concentrations of NPY. Different NPY measurements were made, ranging from femtograms to picograms, and different molecules such as dopamine, epinephrine, and PYY were also tested to determine if the sensor system has the same affinity with different types of molecules. These results give us a better understanding of the use of these techniques for the monitoring of biomolecules.