Electrochemical ELASA: improving early cancer detection and monitoring.

The discovery of new molecular biomarkers of cancer during the last decades and the development of new diagnostic devices exploiting those have significantly contributed to the clinical analysis of cancer and to improve the outcomes. Among those, liquid biopsy sensors exploiting aptamers for the detection of cancer biomarkers in body fluids are useful and accurate tools for a fast and inexpensive non-invasive screening of population. The incorporation of aptamers in electrochemical sandwich biosensors using enzyme labels, a so-called ELASA, has demonstrated its utility to improve the detection schemes. In this review, we overview the existing ELASA assays for numerous cancer biomarkers as alternatives to the traditional ELISA and discuss their possibilities to reach the market, currently dominated by optical immunoassays.

Electron Transfer in Binary Hemin-Modified Alkanethiol Self-Assembled Monolayers on Gold: Hemin's Lateral and Interfacial Interactions.

Orientated coupling of redox enzymes to electrodes by their reconstitution onto redox cofactors, such as hemin conjugated to self-assembled monolayers (SAMs) formed on the electrodes, poses the requirements for a SAM design enabling reconstitution. We show that the kinetics of electron transfer (ET) in binary SAMs of alkanethiols on gold composed of in situ hemin-conjugated 11-amino-1-undecanethiol (AUT) and diluting OH-terminated alkanethiols with 11, 6, and 2 methylene groups (MC11OH, MC6OH, and MC2OH) depends on both the SAM composition and surface density of hemin, Γheme. In AUT/MC11OH SAMs composed of equal linker/diluent lengths, the heterogeneous ET rate constant ks decreased with the Γheme and varied between 70 and 500 s-1. For shorter diluents, the ks of 245-330 s-1 (C6) and 300-340 s-1 (C2) showed a little (if any) Γheme dependence. In AUT/MC11OH SAMs, the increasing Γheme resulted in the steric crowding of hemin species and their neighboring lateral interactions in the plane of hemin localization, affecting the potential distribution at the SAM/electrode interface and inducing local electrostatic effects interfering with hemin oxidation. In AUT/MC6OH and AUT/MC2OH SAMs, hemin discharged at the plane of the closest approach to the gold surface, equal to the diluent length and permeable to electrolyte ions, which lessened those effects. All studied binary SAMs provided steric hindrance for protein reconstitution on the hemin cofactor conjugated to the extended AUT linker. Further use of SAM-modified electrodes with the covalently attached hemin as interfaces for heme proteins' reconstitution should consider SAMs with loosely dispersed redox centers terminating more rigid molecular wires. Such wires place hemin at fixed distances from the electrode surface and thus ensure the interfacial properties required for the effective on-surface reconstitution of proteins and enzymes.

Design Strategies for Electrochemical Aptasensors for Cancer Diagnostic Devices.

Improved outcomes for many types of cancer achieved during recent years is due, among other factors, to the earlier detection of tumours and the greater availability of screening tests. With this, non-invasive, fast and accurate diagnostic devices for cancer diagnosis strongly improve the quality of healthcare by delivering screening results in the most cost-effective and safe way. Biosensors for cancer diagnostics exploiting aptamers offer several important advantages over traditional antibodies-based assays, such as the in-vitro aptamer production, their inexpensive and easy chemical synthesis and modification, and excellent thermal stability. On the other hand, electrochemical biosensing approaches allow sensitive, accurate and inexpensive way of sensing, due to the rapid detection with lower costs, smaller equipment size and lower power requirements. This review presents an up-to-date assessment of the recent design strategies and analytical performance of the electrochemical aptamer-based biosensors for cancer diagnosis and their future perspectives in cancer diagnostics.

Cellulase-Linked Immunomagnetic Microbial Assay on Electrodes: Specific and Sensitive Detection of a Single Bacterial Cell.

Pathogen-associated infections represent one of the major threats to human health and require reliable methods for immediate and robust identification of pathogenic microorganisms. Here, an inexpensive cellulase-linked immunomagnetic methodology was developed for the specific and ultrasensitive analysis of bacteria at their single-cell levels within a 3 h procedure. Detection of a model bacterium, Escherichia coli, was performed in a sandwich reaction with E. coli-specific either aptamer or antibody (Ab)-modified magnetic beads (MBs) and Ab/aptamer reporter molecules linked to cellulase. The cellulase-labeled immuno-aptamer sandwich applied onto nitrocellulose-film-modified electrodes digested the film and changed its electrical conductivity. Electrode's chronocoulometric responses at 0.3 V, in the absence of any redox indicators, allowed a single E. coli cell detection and from 1 to 4 × 104 CFU mL-1 E. coli quantification. No interference/cross-reactivity from Salmonella enteritidis, Enterobacter agglomerans, Pseudomonas putida, Staphylococcus aureus, and Bacillus subtilis was observed when the assay was performed on Ab-modified MBs, and E. coli could be quantified in tap water and milk. This electrochemically label-free methodology is sufficiently fast, highly specific, and sensitive to be used in direct in-field applications. The assay can be adapted for specific detection of other bacterial strains of either the same or different species and offers new analytical tools for fast, specific, and reliable analysis of bacteria in the clinic, food, and environment.

Electrochemical Immuno- and Aptamer-Based Assays for Bacteria: Pros and Cons over Traditional Detection Schemes.

Microbiological safety of the human environment and health needs advanced monitoring tools both for the specific detection of bacteria in complex biological matrices, often in the presence of excessive amounts of other bacterial species, and for bacteria quantification at a single cell level. Here, we discuss the existing electrochemical approaches for bacterial analysis that are based on the biospecific recognition of whole bacterial cells. Perspectives of such assays applications as emergency-use biosensors for quick analysis of trace levels of bacteria by minimally trained personnel are argued.

Directional Preference of DNA-Mediated Electron Transfer in Gold-Tethered DNA Duplexes: Is DNA a Molecular Rectifier?

Electrical properties of self-assembling DNA nanostructures underlie the paradigm of nanoscale bioelectronics, and as such require clear understanding. DNA-mediated electron transfer (ET) from a gold electrode to DNA-bound Methylene Blue (MB) shows directional preference, and it is sequence-specific. During the electrocatalytic reduction of [Fe(CN)6 ]3- catalyzed by DNA-bound MB, the ET rate constant for DNA-mediated reduction of MB reaches (1.32±0.2)103 and (7.09±0.4)103  s-1 for (dGdC)20 and (dAdT)25 duplexes. The backward oxidation process is less efficient, making the DNA duplex a molecular rectifier. Lower rates of ET via (dGdC)20 agree well with its disturbed π-stacked sub-molecular structure. Such direction- and sequence-specific ET may be implicated in DNA oxidative damage and repair, and be relevant to other polarized surfaces, such as cell membranes and biomolecular interfaces.

Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via dA10 Tags.

Electrical properties of DNA critically depend on the way DNA molecules are integrated within the electronics, particularly on DNA-electrode immobilization strategies. Here, we show that the rate of electron transport in DNA duplexes spacer-free tethered to gold via the adenosine terminal region (a dA10 tag) is enhanced compared to the hitherto reported DNA-metal electrode tethering chemistries. The rate of DNA-mediated electron transfer (ET) between the electrode and methylene blue intercalated into the dA10-tagged DNA duplex approached 361 s-1 at a ca. half-monolayer DNA surface coverage ΓDNA (with a linear regression limit of 670 s-1 at ΓDNA → 0), being 2.7-fold enhanced compared to phosphorothioated dA5* tethering (6-fold for the C6-alkanethiol linker representing an additional ET barrier). X-ray photoelectron spectroscopy evidenced dA10 binding to the Au surface via the purine N, whereas dA5* predominantly coordinated to the surface via sulfur atoms of phosphothioates. The latter apparently induces the DNA strand twist in the point of surface attachment affecting the local DNA conformation and, as a result, decreasing the ET rates through the duplex. Thus, a spacer-free DNA coupling to electrodes via dA10 tags thus allows a perspective design of DNA electronic circuits and sensors with advanced electronic properties and no implication from more expensive, synthetic linkers.

Electrochemical Assay for a Total Cellulase Activity with Improved Sensitivity.

Electrochemical methods allow fast and inexpensive analysis of enzymatic activities. Here, we report a simple and yet efficient electrochemical assay for the total activity of cellulase, a hydrolytic enzyme widely used in food and textiles industries, and for production of bioethanol. The assay exploits the difference in electrochemical signals from a soluble redox indicator, ferricyanide, on nitrocellulose films treated by cellulases. Ferricyanide electrochemistry is totally inhibited on graphite electrodes modified with an insulating nitrocellulose film and is evoked after the cellulase treatment. Ferricyanide voltammetric responses correlate with the increased permeability of the films and electrochemically active surface area of electrodes becoming accessible to the ferricyanide reaction after nitrocellulose digestion by cellulase. Trichoderma and Aspergillus niger cellulases activities were determined in a 5 min assay with a sensitivity of 10-8 U per assay, being 103-104-fold more sensitive than the standard commercially available optical assays. That makes the developed electrochemical approach the most prospective cost-effective alternative both for research and automated industrial applications.

A DNA sequence obtained by replacement of the dopamine RNA aptamer bases is not an aptamer.

A unique specificity of the aptamer-ligand biorecognition and binding facilitates bioanalysis and biosensor development, contributing to discrimination of structurally related molecules, such as dopamine and other catecholamine neurotransmitters. The aptamer sequence capable of specific binding of dopamine is a 57 nucleotides long RNA sequence reported in 1997 (Biochemistry, 1997, 36, 9726). Later, it was suggested that the DNA homologue of the RNA aptamer retains the specificity of dopamine binding (Biochem. Biophys. Res. Commun., 2009, 388, 732). Here, we show that the DNA sequence obtained by the replacement of the RNA aptamer bases for their DNA analogues is not able of specific biorecognition of dopamine, in contrast to the original RNA aptamer sequence. This DNA sequence binds dopamine and structurally related catecholamine neurotransmitters non-specifically, as any DNA sequence, and, thus, is not an aptamer and cannot be used neither for in vivo nor in situ analysis of dopamine in the presence of structurally related neurotransmitters.

Electrochemical Label-Free Aptasensor for Specific Analysis of Dopamine in Serum in the Presence of Structurally Related Neurotransmitters.

Cellular and brain metabolism of dopamine can be correlated with a number of neurodegenerative disorders, and as such, in vivo analysis of dopamine in the presence of structurally related neurotransmitters (NT) represents a holy grail of neuroscience. Interference from those NTs generally does not allow selective electroanalysis of dopamine, which redox transformation overlaps with those of other catecholamines. In our previous work, we reported an electrochemical RNA-aptamer-based biosensor for specific analysis of dopamine (Analytical Chemistry, 2013; Vol. 85, p 121). However, the overall design of the biosensor restricted its stability and impeded its operation in serum. Here, we show that specific biorecognition and electroanalysis of dopamine in serum can be performed by the RNA aptamer tethered to cysteamine-modified gold electrodes via the alkanethiol linker. The stabilized dopamine aptasensor allowed continuous 20 h amperometric analysis of dopamine in 10% serum within the physiologically important 0.1-1 µM range and in the presence of catechol and such dopamine precursors and metabolites as norepinephrine and l-DOPA. In a flow-injection mode, the aptasensor response to dopamine was ∼1 s, the sensitivity of analysis, optimized by adjusting the aptamer surface coverage, was 67 ± 1 nA µM(-1) cm(-2), and the dopamine LOD was 62 nM. The proposed design of the aptasensor, exploiting both the aptamer alkanethiol tethering to the electrode and screening of the catecholamine-aptamer electrostatic interactions, allows direct monitoring of dopamine levels in biological fluids in the presence of competitive NT and thus may be further applicable in biomedical research.

Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes.

Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures.

Surface state of the dopamine RNA aptamer affects specific recognition and binding of dopamine by the aptamer-modified electrodes.

Specific monitoring of dopamine, in the presence of structurally related neurotransmitters, is critical for diagnosis, treatment and mechanistic understanding of a variety of human neuropathologies, but nevertheless the proper tools are scarce. Recently, an electrochemical aptasensor for specific analysis of dopamine, exploiting dopamine biorecognition by the RNA aptamer electrostatically adsorbed onto a cysteamine-modified electrode, has been reported (Analytical Chemistry 85 (2013) 121). However it was not clear which way dopamine biorecognition and binding by such aptamer layers proceed and if they can be improved. Here, we show that the aptamer surface state, in particular the aptamer surface density, in a bell-shaped manner affects the dopamine binding, being maximal for the 3.5 ± 0.3 pmol cm(-2) monolayer coverage of the aptamer molecules lying flat on the surface. Therewith, the aptamer affinity for dopamine increases one order of magnitude due to electrostatically regulated immobilization, with the aptamer-dopamine dissociation constant of 0.12 ± 0.01 µM versus 1.6 ± 0.17 µM shown in solution. Under optimal conditions, 0.1-2 µM dopamine was specifically and 85.4 nA µM(-1) cm(-2) sensitively detected, with no interference from structurally related catecholamines. The results allow improvement of the robustness of dopamine monitoring by aptamer-modified electrodes in biological systems, within the 0.01-1 µM dopamine fluctuation range.

Electroanalysis of pM-levels of urokinase plasminogen activator in serum by phosphorothioated RNA aptamer.

Protein biomarkers of cancer allow a dramatic improvement in cancer diagnostics as compared to the traditional histological characterisation of tumours by enabling a non-invasive analysis of cancer development and treatment. Here, an electrochemical label-free assay for urokinase plasminogen activator (uPA), a universal biomarker of several cancers, has been developed based on the recently selected uPA-specific fluorinated RNA aptamer, tethered to a gold electrode via a phosphorothioated dA tag, and soluble redox indicators. The binding properties of the uPA-aptamer couple and interference from the non-specific adsorption of bovine serum albumin (BSA) were modulated by the electrode surface charge. A nM uPA electroanalysis at positively charged surfaces, complicated by the competitive adsorption of BSA, was tuned to the pM uPA analysis at negative surface charges of the electrode, being improved in the presence of negatively charged BSA. The aptamer affinity for uPA displayed via the binding/dissociation constant relationship correspondingly increased, ca. three orders of magnitude, from 0.441 to 367. Under optimal conditions, the aptasensor allowed 10(-12)-10(-9) M uPA analysis, also in serum, being practically useful for clinical applications. The proposed strategy for optimization of the electrochemical protein sensing is of particular importance for the assessment and optimization of in vivo protein ligand binding by surface-tethered aptamers.

Gated electron transfer reactions of truncated hemoglobin from Bacillus subtilis differently orientated on SAM-modified electrodes.

Electron transfer (ET) reactions of truncated hemoglobin from Bacillus subtilis (trHb-Bs) are suggested to be implicated in biological redox signalling and actuating processes that may be used in artificial environment-sensing bioelectronic devices. Here, kinetics of ET in trHb-Bs covalently attached via its surface amino acid residues either to COOH- or NH2-terminated (CH2)2-16 alkanethiol SAM assembled on gold are shown to depend on the alkanethiol length and functionalization, not being limited by electron tunnelling through the SAMs but gated by ET preceding reactions due to conformational changes in the heme active site/at the interface. ET gating was sensitive to the properties of SAMs that trHb-Bs interacted with. The ET rate constant ks for a 1e(-)/H(+) reaction between the SAM-modified electrode and heme of trHb-Bs was 789 and 110 s(-1) after extrapolation to a zero length SAM, while the formal redox potential shifted 142 and 31 mV, for NH2- and COOH-terminated SAMs, respectively. Such domain-specific sensitivity and responsivity of redox reactions in trHb-Bs may be of immediate biological relevance and suggest the existence of bioelectronic regulative mechanisms of ET proceeding in vivo at the protein-protein charged interfaces that modulate the protein reactivity in biological redox signalling and actuating events.

Electrocatalysis of water oxidation by H2O-capped iridium-oxide nanoparticles electrodeposited on spectroscopic graphite.

Electrocatalysis of water oxidation by 1.54 nm IrOx nanoparticles (NPs) immobilized on spectroscopic graphite electrodes was demonstrated to proceed with a higher efficiency than on all other, hitherto reported, electrode supports. IrOx NPs were electrodeposited on the graphite surface, and their electrocatalytic activity for water oxidation was correlated with the surface concentrations of different redox states of IrOx as a function of the deposition time and potential. Under optimal conditions, the overpotential of the reaction was reduced to 0.21 V and the electrocatalytic current density was 43 mA cm(-2) at 1 V versus Ag/AgCl (3 M KCl) and pH 7. These results beneficially compete with previously reported electrocatalytic oxidations of water by IrOx NPs electrodeposited onto glassy carbon and indium tin oxide electrodes and provide the basis for the further development of efficient IrOx NP-based electrocatalysts immobilized on high-surface-area carbon electrode materials.

DNA-mediated electron transfer in DNA duplexes tethered to gold electrodes via phosphorothioated dA tags.

The efficiency of DNA-based bioelectronic devices strongly depends on the way DNA molecules are linked to the electronic component. Commonly, DNA is tethered to metal electrodes via an alkanethiol linker representing an additional barrier for electron transport. Here we demonstrate that the replacement of the alkanethiol linker for a phosphorothioated adenosine tag increases the rate of DNA-mediated electron transfer (ET) up to 259 s(-1), representing the highest hitherto reported rate of electrochemically-modulated ET, and improves the stability of DNA-electrode surface binding. Both results offer pronounced technological and scientific benefits for DNA-based electronics.

Electrochemical analysis of the fibrillation of Parkinson's disease α-synuclein.

Amyloid formation of proteins and peptides is an important biomedical and biotechnological problem, intensively studied and yet not fully understood. In this context, the development of fast and reliable methods for real-time monitoring of protein misfolding is of particular importance for unambiguous establishment of disease-, drug- and environmentally induced mechanisms of protein aggregation. Here we show that the extent of aggregation of α-synuclein (αSN), involved in Parkinson's disease and other neurodegenerative disorders, can be electrochemically monitored by oxidizing tyrosine (Tyr) residues surface-exposed in monomeric αSN and buried in fibrillated αSN adsorbed onto graphite electrodes. Adsorption of αSN, analyzed through the Tyr electrochemistry, followed the Langmuir adsorption isotherm. The degree of electrooxidation of Tyr in αSN decreased upon protein fibrillation and correlated with the extent of αSN aggregation determined by the spectroscopic analysis of the fibrillation process. Minor changes in the adsorption state of αSN were followed through the shift of the Tyr oxidation potential, consistent with the compact and less-compact/unfolded conformation of αSN. Our results allow reliable electroanalysis of the extent of αSN fibrillation in vitro and offer an efficient tool for future in vivo monitoring of the protein conformational state.

Electrocatalytic aptasensor for bacterial detection exploiting ferricyanide reduction by methylene blue on mixed PEG/aptamer monolayers.

Pathogen-triggered infections are the most severe global threat to human health, and to provide their timely treatment and prevention, robust methods for rapid and reliable identification of pathogenic microorganisms are required. Here, we have developed a fast and inexpensive electrocatalytic aptamer assay enabling specific and ultrasensitive detection of E. coli. E. coli, a biomarker of environmental contamination and infections, was captured on the mixed aptamer/thiolated PEG self-assembled monolayers formed on electrochemically pre-treated gold screen-printed electrodes (SPE). Signals from aptamer - E. coli binding were amplified by electrocatalytic reduction of ferricyanide mediated by methylene blue (MB) adsorbed on bacterial and aptamer surfaces. PEG operated as an antifouling agent and inhibited direct (not MB-mediated) discharge of ferricyanide. The assay allowed from 10 to 1000 CFU mL-1E. coli detection in 30 min, with no interference from B. subtilis, in buffer and artificial urine samples. This electrocatalytic approach is fast, specific, sensitive, and can be used directly in in-field and point-of-care applications for analysis of bacteria in human environment.

Antibacterial Action of Zn2+ Ions Driven by the In Vivo Formed ZnO Nanoparticles.

Antibacterial formulations based on zinc oxide nanoparticles (ZnO NPs) are widely used for antibiotic replacement in veterinary medicine and animal nutrition. However, the undesired environmental impact of ZnO NPs triggers a search for alternative, environmentally safer solutions. Here, we show that Zn2+ in its ionic form is a more eco-friendly antibacterial, and its biocidal action rivals that of ZnO NPs (<100 nm size), with a minimal biocidal concentration being 41(82) µg mL-1 vs 5 µg mL-1 of ZnO NPs, as determined for 103(106) CFU mL-1 E. coli. We demonstrate that the biocidal activity of Zn2+ ions is primarily associated with their uptake by E. coli and spontaneous in vivo transformation into insoluble ZnO nanocomposites at an internal bacterial pH of 7.7. Formed in vivo nanocomposite then damages E. coli membrane and intracellular components from the inside, by forming insoluble biocomposites, whose formation can also trigger ZnO characteristic reactions damaging the cells (e.g., by generation of high-potential reactive oxygen species). Our study defines a special route in which Zn2+ metal ions induce the death of bacterial cells, which might be common to other metal ions capable of forming semiconductor oxides and insoluble hydroxides at a slightly alkaline intracellular pH of some bacteria.

RNA aptamer-based electrochemical biosensor for selective and label-free analysis of dopamine.

The inherent redox activity of dopamine enables its direct electrochemical in vivo analysis ( Venton , B. J.; Wightman, M. R. Anal. Chem. 2003, 75, 414A). However, dopamine analysis is complicated by the interference from other electrochemically active endogenous compounds present in the brain, including dopamine precursors and metabolites and other neurotransmitters (NT). Here we report an electrochemical RNA aptamer-based biosensor for analysis of dopamine in the presence of other NT. The biosensor exploits a specific binding of dopamine by the RNA aptamer, immobilized at a cysteamine-modified Au electrode, and further electrochemical oxidation of dopamine. Specific recognition of dopamine by the aptamer allowed a selective amperometric detection of dopamine within the physiologically relevant 100 nM to 5 µM range in the presence of competitive concentrations of catechol, epinephrine, norepinephrine, 3,4-dihydroxy-phenylalanine (L-DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), methyldopamine, and tyramine, which gave negligible signals under conditions of experiments (electroanalysis at 0.185 V vs Ag/AgCl). The interference from ascorbic and uric acids was eliminated by application of a Nafion-coated membrane. The aptasensor response time was <1 s, and the sensitivity of analysis was 62 nA µM(-1) cm(-2). The proposed design of the aptasensor, based on electrostatic interactions between the positively charged cysteamine-modified electrode and the negatively charged aptamer, may be used as a general strategy not to restrict the conformational freedom and binding properties of surface-bound aptamers and, thus, be applicable for the development of other aptasensors.