Electrochemical detection of glycan and protein epitopes of glycoproteins in serum

Aberrant protein glycosylation is associated with a range of pathological conditions including cancer and possesses diagnostic importance. Translation of glycoprotein biomarkers will be facilitated by the development of a rapid and sensitive analytical platform that simultaneously interrogates both the glycan and protein epitopes of glycoproteins in body fluids such as serum or saliva. To this end, we developed an electrochemical biosensor based on the immobilization of a lectin on the gold electrode surface to recognize/capture a target glycan epitope conjugated to glycoproteins, followed by detection of the protein epitope using a target protein-specific antibody. Electrochemical signals are generated by label-free voltammetric or impedimetric interrogation of a ferro/ferricyanide redox couple (e.g. [Fe(CN)(6)](3-/4-)) on the sensing surface, where the change in voltammetric current or interfacial electron transfer resistance was measured. The detection system was demonstrated using the model glycoprotein chicken ovalbumin with Sambucus nigra agglutinin type I (SNA lectin), and exhibits femtomolar sensitivity in the background of diluted human serum. The results obtained in this proof-of-concept study demonstrate the possibility of using electrochemical detection for developing cheap point-of-care diagnostics with high specificity and sensitivity for blood glycoprotein biomarkers

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes

Simple, sensitive and inexpensive regional DNA methylation detection methodologies are imperative for routine patient diagnostics. Herein, we describe eMethylsorb, an electrochemical assay for quantitative detection of regional DNA methylation on a single-use and cost-effective screen-printed gold electrode (SPE-Au) platform. The eMethylsorb approach is based on the inherent differential adsorption affinity of DNA bases to gold (i.e. adenine > cytosine ≥ guanine > thymine). Through bisulfite modification and asymmetric PCR of DNA, methylated and unmethylated DNA in the sample becomes guanine-enriched and adenine-enriched respectively. Under optimized conditions, adenine-enriched unmethylated DNA (higher affinity to gold) adsorbs more onto the SPE-Au surface than methylated DNA. Higher DNA adsorption causes stronger coulombic repulsion and hinders reduction of ferricyanide [Fe(CN)]ions on the SPE-Au surface to give a lower electrochemical response. Hence, the response level is directly proportional to the methylation level in the sample. The applicability of this methodology was tested by detecting the regional methylation status in a cluster of eight CpG sites within the engrailed (EN1) gene promoter of the MCF7 breast cancer cell line. A 10% methylation level sensitivity with good reproducibility (RSD = 5.8%, n = 3) was achieved rapidly in 10 min. Furthermore, eMethylsorb also has advantages over current methylation assays such as being inexpensive, rapid and does not require any electrode surface modification. We thus believe that the eMethylsorb assay could potentially be a rapid and accurate diagnostic assay for point-of-care DNA methylation analysis

Ionic liquids at electrified interfaces

Until recently, “room-temperature” (<100–150 °C) liquid-state electrochemistry was mostly electrochemistry of diluted electrolytes(1)–(4) where dissolved salt ions were surrounded by a considerable amount of solvent molecules. Highly concentrated liquid electrolytes were mostly considered in the narrow (albeit important) niche of high-temperature electrochemistry of molten inorganic salts(5-9) and in the even narrower niche of “first-generation” room temperature ionic liquids, RTILs (such as chloro-aluminates and alkylammonium nitrates).(10-14) The situation has changed dramatically in the 2000s after the discovery of new moisture- and temperature-stable RTILs.(15, 16) These days, the “later generation” RTILs attracted wide attention within the electrochemical community.(17-31) Indeed, RTILs, as a class of compounds, possess a unique combination of properties (high charge density, electrochemical stability, low/negligible volatility, tunable polarity, etc.) that make them very attractive substances from fundamental and application points of view.(32-38) Most importantly, they can mix with each other in “cocktails” of one’s choice to acquire the desired properties (e.g., wider temperature range of the liquid phase(39, 40)) and can serve as almost “universal” solvents.(37, 41, 42) It is worth noting here one of the advantages of RTILs as compared to their high-temperature molten salt (HTMS)(43) “sister-systems”.(44) In RTILs the dissolved molecules are not imbedded in a harsh high temperature environment which could be destructive for many classes of fragile (organic) molecules

Development of Extraction and Analytical Methods of Nitrite Ion from Food Samples: Microchip Electrophoresis with a Modified Electrode

Two simple and fast methods for the extraction of the nitrite ion (NO2 -) from food samples have been developed. The methods were characterized by UV-visible spectroscopic and electrochemical measurements, and their performance for NO2 - extraction was compared with a standard method. The extraction methods yielded relative recoveries between 100 and 120% with good reproducibility of 3.9% (RSD, n = 4) in UV-visible experiments. Microchip electrophoresis with electrochemical detection (MCE-ED) coupled with a copper (3-mercaptopropyl)trimethoxysilane [Cu(II)-MPS] complex- modified carbon paste electrode (CPE) has been employed to detect NO2 - in extracted samples. The Cu(II)-MPS complex was synthesized and characterized by voltammetry, XPS, and FT-IR analyses. Experimental parameters affecting the separation and detection performances of the MCE-ED method were assessed and optimized. The potential for the electrocatalytic reduction of NO2 - for MCE-ED was found to be -190 mV (vs Ag/AgCl). When extracted food samples were analyzed by the MCE-ED method, a reproducible response for the NO2 - reduction (RSD of 4.3%) at the modified-CPE reflected the negligible electrode fouling. A wide dynamic range of 1.0-160 ppm was observed for analyzing standard NO2 - with a sensitivity of 0.05106 ( 0.00141, and the detection limit, based on S/N = 3, was found to be 0.35 ( 0.05 ppm. No apparent interference from NO3 -, other inorganic ions, and biological compounds was observed under the optimal experimental conditions. A standard addition method for real samples showed wide concentration ranges of 1.10-155 and 1.2-150 ppm for analyzing NO2 - in ham and sausage samples, respectively

Homogeneous electron-transfer reaction between electrochemically generated ferrocenium ions and amine-containing compounds

The homogeneous catalytic oxidation of propylamine (PrA), diethylamine (DEA), pyrrolidine (Pyr), and triethylamine (TEA) has been investigated for the first time in the presence of electrochemically generated ferrocenium ions as the catalyst. Mechanistic details for this electrocatalytic process have been investigated by cyclic voltammetry and other electrochemical techniques. A one-electron oxidative process was observed for all amines. Deviation from this mechanism was only observed during controlled-potential bulk electrolysis of ferrocene in the presence of high concentrations of propylamine, where problems with electrode fouling and catalyst deactivation processes were encountered. The catalytic efficiency and the catalytic oxidation rate constants were estimated and found to follow the order TEA > Pyr > DEA > PrA. Interestingly, the catalytic reaction was not observed when ferrocene was replaced by decamethylferrocene. This observation was analyzed in terms of thermodynamic and structural effects, and a hypothesis is presented. The homogeneous catalytic oxidation reported opens a new avenue to achieve simple, low-cost, and efficient amine oxidation, which is potentially useful in several areas of chemistry

Large amplitude fourier transformed AC voltammetric investigation of the active state electrochemistry of a copper/aqueous base interface and implications for electrocatalysis

The higher harmonic components available from large-amplitude Fourier-transformed alternating current (FT-ac) voltammetry enable the surface active state of a copper electrode in basic media to be probed in much more detail than possible with previously used dc methods. In particular, the absence of capacitance background current allows low-level Faradaic current contributions of fast electron-transfer processes to be detected; these are usually completely undetectable under conditions of dc cyclic voltammetry. Under high harmonic FT-ac voltammetric conditions, copper electrodes exhibit well-defined and reversible premonolayer oxidation responses at potentials within the double layer region in basic 1.0 M NaOH media. This process is attributed to oxidation of copper adatoms (Cu*) of low bulk metal lattice coordination numbers to surface-bonded, reactive hydrated oxide species

Highly selective and sensitive DNA assay based on electrocatalytic oxidation of ferrocene bearing zinc(II)-cyclen complexes with diethylamine

A highly selective and sensitive electrochemical biosensor has been developed that detects DNA hybridization by employing the electrocatalytic activity of ferrocene (Fc) bearing cyclen complexes (cyclen = 1,4,7,10-tetraazacyclododecane, Fc[Zn(cyclen)H2O]2(ClO4)4 (R1), Fc(cyclen)2 (R2), Fc[Zn(cyclen)H2O](ClO4)2 (R3), and Fc(cyclen) (R4)). A sandwich-type approach, which involves hybridization of a target probe hybridized with the preimmobilized thiolated capture probe attached to a gold electrode, is employed to fabricate a DNA duplex layer. Electrochemical signals are generated by voltammetric interrogation of a Fc bearing Zn&minus;cyclen complexes that selectively and quantitatively binds to the duplex layers through strong chelation between the cyclen complexes and particular nucleobases within the DNA sequence. Chelate formation between R1 or R3 and thymine bases leads to the perturbation of base-pair (A&minus;T) stacking in the duplex structure, which greatly diminishes the yield of DNA-mediated charge transport and displays a marked selectivity to the presence of the target DNA sequence. Coupling the redox chemistry of the surface-bound Fc bearing Zn&minus;cyclen complex and dimethylamine provides an electrocatalytic pathway that increases sensitivity of the assay and allows the 100 fM target DNA sequence to be detected. Excellent selectivity against even single-base sequence mismatches is achieved, and the DNA sensor is stable and reusable.<br /

Amplification-free detection of gene fusions in prostate cancer urinary samples using mRNA-gold affinity interactions

A crucial issue in present-day prostate cancer (PCa) detection is the lack of specific biomarkers for accurately distinguishing between benign and malignant cancer forms. This is causing a high degree of overdiagnosis and overtreatment of otherwise clinically insignificant cases. As around half of all malignant PCa cases display a detectable gene fusion mutation between the TMPRSS2 promoter sequence and the ERG coding sequence (TMPRSS2:ERG) in urine, noninvasive screening of TMPRSS2:ERG mRNA in patient urine samples could improve the specificity of current PCa diagnosis. However, current gene fusion detection methodologies are largely dependent on RNA enzymatic amplification, which requires extensive sample manipulation, costly labels for detection, and is prone to bias/artifacts. Herein we introduce the first successful amplification-free electrochemical assay for direct detection of TMPRSS2:ERG mRNA in PCa urinary samples by selectively isolating and adsorbing TMPRSS2:ERG mRNA onto bare gold electrodes without requiring any surface modification. We demonstrated excellent limit-of-detection (10 cells) and specificity using PCa cell line models, and showcased clinical utility by accurately detecting TMPRSS2:ERG in a collection of 17 urinary samples obtained from PCa patients. Furthermore, these results were validated with the current gold standard reverse transcription (RT)-PCR approach with 100% concordance

Macrocyclic Zn complexes for DNA detection

Researchers from Monash University have developed an electrocatalytic method based on a charge transport through DNA films, which allows detection of complementary over non-complementary and mismatched DNA sequences in fully hybridized duplexes