An electrochemical aptasensor for Δ9-tetrahydrocannabinol detection in saliva on a microfluidic platform.

We present a novel "on-off", cost-effective, rapid electrochemical aptasensor combined with a microfluidics cartridge system for the detection of Δ9-THC (Δ9-tetrahydrocannabinol) in human saliva via differential pulse voltammetry. The assay relied on the competitive binding between the Δ9-THC and a soluble redox indicator methylene blue, using an aptamer selected via FRELEX. We found that the aptasensor can detected 1 nM of Δ9-THC in PBS in a three-electrode cell system, while the sensitivity and both the dissociation constant (Kd) and association constant (Kb) were dependent on the aptamer density. The aptamer also showed great affinity towards Δ9-THC when tested against cannabinol and cannabidiol. The same limit of detection of 1 nM in PBS was achieved in small volume samples (∼60 µL) using the aptamer-modified gold screen-printed electrodes combined with the microfluidic cartridge setup, however, the presence of 10% raw human saliva had a negative effect which manifested in a 10-fold increase in the LOD due to interfering elements. Filtering the saliva, improved the tested volume to 50% and the LOD to 5 nM of Δ9-THC which is lower than the concentrations associated with impairment (6.5-32 nM). The aptasensor showed a good storage capability up to 3 days, however, the reusability significantly dropped from 10 cycles (freshly prepared) to 5 cycles. The results clearly demonstrate the feasibility of the aptasensor platform with the microfluidics chamber towards a point-of-care testing application for the detection of Δ9-THC in saliva.

Electrochemical nutrient removal from natural wastewater sources and its impact on water quality.

In this study, a suite of natural wastewater sources is tested to understand the effects of wastewater composition and source on electrochemically driven nitrogen and phosphorus nutrient removal. Kinetics, electrode behavior, and removal efficiency were evaluated during electrochemical precipitation, whereby a sacrificial magnesium (Mg) anode was used to drive precipitation of ammonium and phosphate. The electrochemical reactor demonstrated fast kinetics in the natural wastewater matrices, removing up to 54% of the phosphate present in natural wastewater within 1 min, with an energy input of only 0.04 kWh.m-3. After 1 min, phosphate removal followed a zero-order rate law in the 1 min - 30 min range. The zero-order rate constant (k) appears to depend upon differences in wastewater composition, where a faster rate constant is associated with higher Cl- and NH4+ concentrations, lower Ca2+ concentrations, and higher organic carbon content. The sacrificial Mg anode showed the lowest corrosion resistance in the natural industrial wastewater source, with an increased corrosion rate (vcorr) of 15.8 mm.y-1 compared to 1.9-3.5 mm.y-1 in municipal wastewater sources, while the Tafel slopes (ß) showed a direct correlation with the natural wastewater composition and origin. An overall improvement of water quality was observed where important water quality parameters such as total organic carbon (TOC), total suspended solids (TSS), and turbidity showed a significant decrease. An economic analysis revealed costs based upon experimental Mg consumption are estimated to range from 0.19 $.m-3 to 0.30 $.m-3, but costs based upon theoretical Mg consumption range from 0.09 $.m-3 to 0.18 $.m-3. Overall, this study highlights that water chemistry parameters control nutrient recovery, while electrochemical treatment does not directly produce potable water, and that economic analysis should be based upon experimentally-determined Mg consumption data. Synopsis Statement: Magnesium-driven electrochemical precipitation of natural wastewater sources enables fast kinetics for phosphate removal at low energy input.

Electroless Production of Fertilizer (Struvite) and Hydrogen from Synthetic Agricultural Wastewaters.

The drive toward sustainable phosphorus (P) recovery from agricultural and municipal wastewater streams has intensified. However, combining P recovery with energy conservation is perhaps one of the greatest challenges of this century. In this study, we report for the first time the simultaneous electroless production of struvite and dihydrogen from aqueous ammonium dihydrogen phosphate (NH4H2PO4) solutions in contact with either a pure magnesium (Mg) or a Mg alloy as the anode and 316 stainless steel (SS) as the cathode placed in a bench-scale electrochemical reactor. During the electroless process (i.e., in the absence of external electrical power), the open circuit potential (OCP), the formation of struvite on the anode, and the generation of dihydrogen at the cathode were monitored. We found that struvite is formed, and that struvite crystal structure/morphology and precipitate film thickness are affected by the concentration of the HnPO4n-3/NH4+ in solution and the composition of the anode. The pure Mg anode produced a porous 0.6-4.1 µm thick film, while the AZ31 Mg alloy produced a more compact 1.7-9.9 µm thick struvite film. Kinetic analyses revealed that Mg dissolution to Mg2+ followed mostly a zero-order kinetic rate law for both Mg anode materials, and the rate constants (k) depended upon the struvite layer morphology. Fourier-transform infrared spectrometry, X-ray diffraction, and scanning electron microscopy indicated that the synthesized struvite was of high quality. The dihydrogen and Mg2+ in solution were detected by a gas chromatography-thermal conductivity detector and ion chromatography, respectively. Furthermore, we fully demonstrate that the reactor was able to remove ∼73% of the HnPO4n-3 present in a natural poultry wastewater as mainly struvite. This study highlights the feasibility of simultaneously producing struvite and dihydrogen from wastewater effluents with no energy input in a green and sustainable approach.

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.

Sequence-Specific Electron Transfer Mediated by DNA Duplexes Attached to Gold through the Alkanethiol Linker.

Ability of the DNA double helix to transport electrons is its critical feature, underlying a number of important biological and biotechnological processes. Here, we show that electron transfer (ET) from the gold electrode to the DNA-bound methylene blue (MB) mediated by the DNA base-pair π-stack is less efficient in (dGdC)-rich duplexes compared to pure (dAdT) DNA. The ET rate constant ks extrapolated to the DNA surface coverage ΓDNA → 0 is 121 ± 8 s-1 for (dAdT)25, being almost twofold higher than 67 ± 3 s-1 shown for (dGdC)20, consistent with the electric-field-disturbed submolecular structure of the (dGdC)20 duplex earlier shown at electrified interfaces. DNA-mediated ET occurs both to MB intercalated and thus perfectly π-stacked into the (dGdC)20 duplex and to MB solely groove-bound to (dAdT)25. For both (dGdC)20 and (dAdT)25, ET is less efficient than ET in DNA duplexes of a mixed dA, dT, dG, dC composition. The results suggest new interpretations of the biological ET processes that may occur in dsDNA of different compositions at polarized interfaces.

Picomolar sensitive and SNP-selective "Off-On" hairpin genosensor based on structure-tunable redox indicator signals.

A robust and sensitive electrochemical assay for chrononocoulometric detection of nucleic acids at a single nucleotide polymorphism (SNP) level has been developed. The assay exploits hybridization-induced conformational switching of gold-tethered TP53-specific 33-mer and truncated 20-mer hairpin DNA probes and methylene blue (MB) as an intercalating redox indicator. We show that by fine tuning of MB-DNA intercations the enhanced binding of MB to hybrids formed with a cancer-biomarker sequence can be achieved, and that results in robust "off-on" sensing of hybridization, while the stem-loop probe design allows minimized, independent of the DNA length background signals. Both DNA probes were sensitive to the presence of SNP in the targeted DNA sequence already at 10 pM. DNA levels, and the robust "off-on" discrimination of 10 pM perfectly-matched DNA from 50 nM SNP-containing DNA was achieved by time-adjusted chronocoulometry. This label-free hairpin DNA strategy allows systematic design of DNA assays for fast, robust and inexpensive genetic analysis in excessive mixtures of structurally-related DNA sequences and was used for specific analysis of prostate-cancer-realted cellular microRNA in total RNA samples isolated from LNCaP and BPH1 cells.

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.

Submolecular Structure and Orientation of Oligonucleotide Duplexes Tethered to Gold Electrodes Probed by Infrared Reflection Absorption Spectroscopy: Effect of the Electrode Potentials.

Unique electronic and ligand recognition properties of the DNA double helix provide basis for DNA applications in biomolecular electronic and biosensor devices. However, the relation between the structure of DNA at electrified interfaces and its electronic properties is still not well understood. Here, potential-driven changes in the submolecular structure of DNA double helices composed of either adenine-thymine (dAdT)25 or cytosine-guanine (dGdC)20 base pairs tethered to the gold electrodes are for the first time analyzed by in situ polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) performed under the electrochemical control. It is shown that the conformation of the DNA duplexes tethered to gold electrodes via the C6 alkanethiol linker strongly depends on the nucleic acid sequence composition. The tilt of purine and pyrimidine rings of the complementary base pairs (dAdT and dGdC) depends on the potential applied to the electrode. By contrast, neither the conformation nor orientation of the ionic in character phosphate-sugar backbone is affected by the electrode potentials. At potentials more positive than the potential of zero charge (pzc), a gradual tilting of the double helix is observed. In this tilted orientation, the planes of the complementary purine and pyrimidine rings lie ideally parallel to each other. These potentials do not affect the integral stability of the DNA double helix at the charged interface. At potentials more negative than the pzc, DNA helices adopt a vertical to the gold surface orientation. Tilt of the purine and pyrimidine rings depends on the composition of the double helix. In monolayers composed of (dAdT)25 molecules the rings of the complementary base pairs lie parallel to each other. By contrast, the tilt of purine and pyrimidine rings in (dGdC)20 helices depends on the potential applied to the electrode. Such potential-induced mobility of the complementary base pairs can destabilize the helix structure at a submolecular level. These pioneer results on the potential-driven changes in the submolecular structure of double stranded DNA adsorbed on conductive supports contribute to further understanding of the potential-driven sequence-specific electronic properties of surface-tethered oligonucleotides.

Chronopotentiometric sensing of specific interactions between lysozyme and the DNA aptamer.

Specific DNA-protein interactions are vital for cellular life maintenance processes, such as transcriptional regulation, chromosome maintenance, replication and DNA repair, and their monitoring gives valuable information on molecular-level organization of those processes. Here, we propose a new method of label-free electrochemical sensing of sequence specific binding between the lysozyme protein and a single stranded DNA aptamer specific for lysozyme (DNAapta) that exploits the constant current chronopotentiometric stripping (CPS) analysis at modified mercury electrodes. Specific lysozyme-DNAapta binding was distinguished from nonspecific lysozyme-DNA interactions at thioglycolic acid-modified mercury electrodes, but not at the dithiothreitol-modified or bare mercury electrodes. Stability of the surface-attached lysozyme-DNAapta layer depended on the stripping current (Istr) intensity, suggesting that the integrity of the layer critically depends on the time of its exposure to negative potentials. Stabilities of different lysozyme-DNA complexes at the negatively polarized electrode surface were tested, and it was shown that structural transitions of the specific lysozyme-DNAapta complexes occur in the Istr ranges different from those observed for assemblies of lysozyme with DNA sequences capable of only nonspecific lysozyme-DNA interactions. Thus, the CPS allows distinct discrimination between specific and non-specific protein-DNA binding and provides valuable information on stability of the nucleic acid-protein interactions at the polarized interfaces.

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.

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.

Determination of zinc in vegetal tissue microsamples by platinum-wire loop in flame atomization atomic absorption spectrometry.

The zinc content of 3 microL of vegetal samples (tree leaves, lichens and grape sap) atomized from a Pt-wire in the methane-air flame has been determined by atomic absorption spectrometry. The effect of gas flow rates and the atomization height in the flame on the absorption of zinc was evaluated at 213.9 nm. The best results were obtained at a height of 5 mm and gas flow rates of 200 L/h air and 26 L/h methane, respectively. The effect of Na, K, Ca, Mg, SO4(2-), and PO4(3-) on the absorption of zinc was studied too. The detection limit of 0.40+/-0.21 ng was obtained at a significance level of 0.05, using the two-step Neyman-Pearson criterion. The zinc content of the samples has been determined with continuous nebulization and by atomization from the Pt-wire, using both the standard calibration curve and the standard addition method. The results of the two procedures agree within the determination errors.