An addressable microelectrode array for electrochemical detection

Column and row electrodes on two different glass substrates were orthogonally arranged in order to assemble an addressable microelectrode device for the purpose of comprehensive electrochemical detection. Amperometric signal at the individual crossing point of the column and row electrodes was detected separately on the basis of redox cycling of localized electroactive species occurring between the electrodes. The addressable microelectrode device was simple and could be easily assembled; however, it comprised as many as 10 x 10 addressable detection points on a single chip. The basic electrochemical performance of the device was investigated by using the ferricyanide/ferrocyanide redox couple. Electrochemical responses at 100 individual points could be collected within 22 s. The present device was successfully used for imaging the spots of alkaline phosphatase on the array substrate. The results indicate that the device can be applied to comprehensive and high-throughput detection and imaging of biochemical species.     

Label-free electrochemical immunoassay for the detection of human chorionic gonadotropin hormone

Here we report on a new and rapid immunoassay for the label-free voltammetric detection of human chorionic gonadotropin hormone (hCG) in urine. Monitoring the changes in the current signals of antibodies (Abs) before and after the binding of the antigen (Ag) provides the basis for an immunoassay that is simple, rapid, and cost-effective. Since hCG is found at highly elevated levels in pregnant female urine with the range of 30,000-200,000 mIU/mL (approximately 30-200 nM) by 8-10 weeks into pregnancy, its label-free electrochemical detection was achieved by using our method. The coverage of the electrode surface with the Ab and the incubation time with the target Ag were optimized for the detection of hCG. The limit of detection of our method was calculated to be 15 pM (n = 3, approximately 15 mIU/mL) in synthetic hCG samples and 20 pM (n = 3, approximately 20 mIU/mL) in human urine. The electrochemical results for the detection of hCG in the urine samples were in agreement with the results obtained using a reference system, enzyme-linked immunosorbent assay. Further research about the intrinsic electroactivity of Abs and their target molecules would surely provide new and sensitive screening assays, as well as extensive data regarding their interaction mechanisms.     

Simple and highly enantioselective electrochemical aptamer-based binding assay for trace detection of chiral compounds

A new electrochemical methodology is reported for monitoring in homogeneous solution the enantiospecific binding of a small chiral analyte to an aptamer. The principle relies on the difference of diffusion rates between the targeted molecule and the aptamer/target complex, and thus on the ability to more easily electrochemically detect the former over the latter in a homogeneous solution. This electrochemical detection strategy is significant because, in contrast to the common laborious and time-consuming heterogeneous binding approaches, it is based on a simple and fast homogeneous binding assay which does not call for an aptamer conformational change upon ligand binding. The methodology is here exemplified with the specific chiral recognition of trace amounts of l- or d-tyrosinamide by a 49-mer d- or l-deoxyribooligonucleotide receptor. Detection as low as 0.1% of the minor enantiomer in a nonracemic mixture can be achieved in a very short analysis time (<1 min). The assay finally combines numerous attractive features including simplicity, rapidity, low cost, flexibility, low volume samples (few microliters), and homogeneous format.     

Label-free detection of telomerase activity using guanine electrochemical oxidation signal

Telomerase is an important biomarker for cancer cells and its activation in 85% of all cancer types confers a clinical diagnostic value. A label-free electrochemical assay based on guanine oxidation signal to measure telomerase activity is described. This developed technology combined with a disposable sensor, carbon graphite electrode (CGE), and differential pulse voltammetry (DPV) was performed by using PCR amplicons with/without telomeric repeats as the guanine oxidation signal observed at +1.0 V measured after the immobilization of PCR products. Guanine oxidation signal was chosen as a measure of telomerase activity because a substantial increase in the number of guanines was introduced by the action of telomerase which adds hexameric repeats (TTAGGG)n that contain 50% guanine. The developed assay was shown to specifically measure telomerase activity from cell extracts, and elongation rates increased linearly in a concentration dependent manner. Telomerase activity could be detected in cell extracts containing as low as 100 ng/microL of protein. All of the electrochemical measurements were also confirmed with the conventional TRAP-silver staining assay. Rapidity, simplicity, and the label-free nature of the developed assay make it suitable for practical use in quantitative determination of telomerase activity from clinical samples for diagnosis of cancer.     

Analytical performances of aptamer-based sensing for thrombin detection

Aptamer-based assays represent a modern and attractive approach in bioanalytical chemistry. The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to critically evaluate all the parameters that can influence the sensor performances by using the thrombin aptamer immobilized onto piezoelectric quartz crystals. The optimization of the immobilization and the binding protocol was of paramount importance, and improvements in analytical performances could be obtained by optimizing simple steps in immobilization and assay conditions. Moreover, the work demonstrated the possibility of using aptamer-based sensors in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.     

Label-free electrochemical detection of protein based on a ferrocene-bearing cationic polythiophene and aptamer

Two label-free electrochemical methods for the detection of human alpha-thrombin using a water-soluble, ferrocene-functionalized polythiophene transducer and a single-stranded oligonucleotide aptamer probe are described. The first approach is a direct method in which the recorded current decreases upon addition of the targeted protein. The second one requires more steps and the additional utilization of PNA probes and nuclease enzyme. This indirect method leads to an increase of the electrical signal as a function of the concentration of human alpha-thrombin with a detection limit of 75 fmol.     

A microfluidic paper-based electrochemical biosensor array for multiplexed detection of metabolic biomarkers

Abstract

Paper-based microfluidic devices have emerged as simple yet powerful platforms for performing low-cost analytical tests. This paper reports a microfluidic paper-based electrochemical biosensor array for multiplexed detection of physiologically relevant metabolic biomarkers. Different from existing paper-based electrochemical devices, our device includes an array of eight electrochemical sensors and utilizes a handheld custom-made electrochemical reader (potentiostat) for signal readout. The biosensor array can detect several analytes in a sample solution and produce multiple measurements for each analyte from a single run. Using the device, we demonstrate simultaneous detection of glucose, lactate and uric acid in urine, with analytical performance comparable to that of the existing commercial and paper-based platforms. The paper-based biosensor array and its electrochemical reader will enable the acquisition of high-density, statistically meaningful diagnostic information at the point of care in a rapid and cost-efficient way.     

Ultrasensitive aptamer-based multiplexed electrochemical detection by coupling distinguishable signal tags with catalytic recycling of DNase I

This work reports an aptamer-based, disposable, and multiplexed sensing platform for simultaneous electrochemical determination of small molecules, employing adenosine triphosphate (ATP) and cocaine as the model target analytes. The multiplexed sensing strategy is based on target-induced release of distinguishable redox tag-conjugated aptamers from a magnetic graphene platform. The electronic signal of the aptasensors could be further amplified by coupling DNase I with catalytic recycling of self-produced reactants. The assay was based on the change in the current at the various peak potentials in the presence of the corresponding signal tags. Experimental results revealed that the multiplexed electrochemical aptasensor enabled the simultaneous monitoring of ATP and cocaine in a single run with wide working ranges and low detection limits (LODs: 0.1 pM for ATP and 1.5 pM for cocaine). This concept offers promise for rapid, simple, and cost-effective analysis of biological samples.     

Label-free detection of bacteria by electrochemical impedance spectroscopy: comparison to surface plasmon resonance

The low but known risk of bacterial contamination has emerged as the greatest residual threat of transfusion-transmitted diseases. Label-free detection of a bacterial model, Escherichia coli, is performed using nonfaradic electrochemical impedance spectroscopy (EIS). Biotinylated polyclonal anti-E. coli is linked to a mixed self-assembled monolayer (SAM) on a gold electrode through a strong biotin-neutravidin interaction. The binding of one antibody molecule for 3.6 neutravidin molecules is determined using the surface plasmon resonance (SPR). The detection limit of E. coli found by SPR is 10(7) cfu/mL. After modeling the impedance Nyquist plot of E. coli/anti-E. coli/mixed SAM/gold electrode for increasing concentrations of E. coli (whole bacteria or lysed bacteria), the main parameter that is modified is the polarization resistance RP. A sigmoid variation of RP is observed when the log concentration of bacteria (whole or lysed) increases. A concentration of 10 cfu/mL whole bacteria is detected by EIS measurements while 103 cfu/mL is detected for lysed E. coli.     

Label-free electrochemical monitoring of DNA ligase activity

This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase.     

Electrochemical aptamer-based sandwich assays for the detection of explosives

Electrochemical impedance spectroscopy (EIS) is used to detect 2,4,6-trinitrotoluene (TNT) in a novel sandwiched structure which relies on the specific interactions between (i) primary amine with TNT and (ii) TNT and anti-TNT aptamer. With pure targets, the assay has a sensitivity of 10(-14) M, a dynamic range of 10(-14)-10(-3) M, and employs a small sample volume (25 μL). The method's sensitivity is comparable to state of the art optical methods with the added advantages of electrochemical detection, which can be easily miniaturized and implemented into a hand-held device.     

Label-free detection of aflatoxin M1 with electrochemical Fe3O4/polyaniline-based aptasensor

The selective detection of ultratrace amounts of aflatoxin M1 (AFM1) is extremely important for food safety since it is the most toxic mycotoxin class that is allowed to be present on cow milk with strictly low regulatory levels. In this work, Fe₃O₄ incorporated polyaniline (Fe₃O₄/PANi) film has been polymerized on interdigitated electrode (IDE) as sensitive film for AFM1 electrochemical biosensor. The immobilized aptamers as an affinity capture reagent and magnetic nanoparticles for signal amplification element have been employed in the sensing platform. Label-free and direct detection of the aptamer-AFM1 on Fe₃O₄/PANi interface were performed via electrochemical signal change, acquired by cyclic and square wave voltammetries. With a simplified strategy, this electrochemical aptasensor shows a good sensitivity to AFM1 in the range of 6–60 ng·L^? 1, with the detection limit of 1.98 ng·L^? 1. The results open up the path for designing cost effective aptasensors for other biomedical applications.     

Label-free electrochemical hybridization genosensor for the detection of hepatitis B virus genotype on the development of Lamivudine resistance

The resistance analysis related to the hepatitis B virus (HBV) genotyping and treatment procured key information for the study of infected patients. The aim of this study was to develop a novel assay for the voltammetric detection of DNA sequences related to the HBV genotype on the development of lamuvidine resistance by monitoring the oxidation signal of guanine. This new technique not only provides a rapid, cost-effective, simple analysis but also gives information concerning both genotyping and lamivudine resistance. Synthetic single-stranded oligonucleotides ("probe") including YMDD (HBV wild type) YVDD, or YIDD (mutations in the YMDD) variants have been immobilized onto pencil graphite electrodes with the adsorption at a controlled potential. The probes were hybridized with different concentrations of their complementary ("target") sequences such as synthetic complementary sequences, clonned PCR products, or real PCR samples. The formed synthetic hybrids on the electrode surface were evaluated by a differential pulse voltammetry technique using a label-free detection method. The oxidation signal of guanine was observed as a result of the specific hybridization between the probes and their synthetic targets and specific PCR products. The response of the hybridization of the probes with their single-base mismatch oligonucleotides at PGE was also detected. Control experiments using the noncomplementary oligonucleotides were performed to determine whether the DNA genosensor responds selectively. Numerous factors, affecting the probe immobilization, target hybridization, and nonspecific binding events, were optimized to maximize the sensitivity and reduce the assay time. Under the optimum conditions, 457 fmol/mL was found as the detection limit for target DNA. With the help of the appearance of the guanine signal, the new protocol is based on the electrochemical detection of HBV genotype for the development of lamuvidine resistance for the first time. Features of this protocol are discussed and optimized.     

Label-free protein recognition two-dimensional array using nanomechanical sensors

We demonstrate two-dimensional multiplexed real-time, label-free antibody-antigen binding assays by optically detecting nanoscale motions of two-dimensional arrays of microcantilever beams. Prostate specific antigen (PSA) was assayed using antibodies covalently bound to one surface of the cantilevers by two different surface chemistries, while the nonreaction surfaces were passivated by poly(ethylene glycol)-silane. PSA as low as 1 ng/mL was detected while 2 mg/microl of bovine serum albumin induced only negligible deflection on the cantilevers.     

Label-free and substrate-free potentiometric aptasensing using polycation-sensitive membrane electrodes

A potentiometric label-free and substrate-free (LFSF) aptasensing strategy which eliminates the labeling, separation, and immobilization steps is described in this paper. An aptamer binds specifically to a target molecule via reaction incubation, which could induce a change in the aptamer conformation from a random coil-like configuration to a rigid folded structure. Such a target binding-induced aptamer conformational change effectively prevents the aptamer from electrostatically interacting with the protamine binding domain. This could either shift the response curve for the potentiometric titration of the aptamer with protamine as monitored by a conventional polycation-sensitive membrane electrode or change the current-dependent potential detected by a protamine-conditioned polycation-sensitive electrode with the pulsed current-driven ion fluxes of protamine across the polymeric membrane. Using adenosine triphosphate (ATP) as a model analyte, the proposed concept offers potentiometric detection of ATP down to the submicromolar concentration range and has been applied to the determination of ATP in HeLa cells. In contrast to the current LFSF aptasensors based on optical detection, the proposed strategy allows the LFSF biosensing of aptamer/target binding events in a homogeneous solution via electrochemical transduction. It is anticipated that the proposed strategy will lay a foundation for development of potentiometric sensors for LFSF aptasensing of a variety of analytes where target binding-induced conformational changes such as the formation of folded structures and the opening of DNA hairpin loops are involved.     

Metallophthalocyanine-nanofibre-based electrodes for electrochemical sensing of biomolecules

Metal phthalocyanines, possessing rich redox chemistry due to the presence of the central metal cation and pyrrolic nitrogen atoms of the macrocycle, are explored as electrochemical sensors. Nickel phthalocyanine nanofibres (NiPc NF) prepared by a simple chemical route are coated on a pencil graphite rod and the electrocatalytic performance of NiPc NF electrode is investigated for quantitative detection of ascorbic acid (AA) in 0.2 M phosphate buffer solution. The performance of NiPc NFs is shown to be superior to that of commercial NiPc and is attributed to the high electrochemically active surface area available for fibres. The electrode exhibits linearity for the detection over a wide concentration range of AA from \(5.5\,\upmu \hbox {M}\) to 5.2 mM. The detection limit for AA sensing with NiPc-NF-modified electrode is \(1.5\,\upmu \hbox {M}\). The higher performance of NiPc fibres due to its nanostructure morphology may be utilized for the quantitative detection of other biomolecules.     

Microfluidic electrochemical array for detection of reactive metabolites formed by cytochrome P450 enzymes

A novel, simple, rapid microfluidic array using bioelectronically driven cytochrome P450 enzyme catalysis for reactive metabolite screening is reported for the first time. The device incorporates an eight-electrode screen-printed carbon array coated with thin films of DNA, [Ru(bpy)(2)(PVP)(10)](ClO(4)) {RuPVP}, and rat liver microsomes (RLM) as enzyme sources. Catalysis features electron donation to cyt P450 reductase in the RLMs and subsequent cyt P450 reduction while flowing an oxygenated substrate solution past sensor electrodes. Metabolites react with DNA in the film if they are able, and damaged DNA is detected by catalytic square wave voltammetry (SWV) utilizing the RuPVP polymer. The microfluidic device was tested for a set of common pollutants known to form DNA-reactive metabolites. Logarithmic turnover rates based on SWV responses gave excellent correlation with the rodent liver TD(50) toxicity metric, supporting the utility of the device for toxicity screening. The microfluidic array gave much better S/N and reproducibility than single-electrode sensors based on similar principles.     

Ammonia gas detection based on polyaniline nanofibers coated on interdigitated array electrodes

Ammonia gas sensors were fabricated from polyaniline nanofibers, which were synthesized by a simple dilute polymerization method without external template. The films of polyaniline nanofibers were deposited on interdigitated array electrodes by drop casting method. The ammonia gas sensing mechanism arises from the deprotonation process of acid doped polyaniline. The sensors exhibited the sensitivity of 1.06 and response time of 10 s for 50 ppm of ammonia gas. Such high sensitivity and fast response are attributed to the large surface-to-volume ratio and interconnected network structures. These results suggest that polyaniline nanofibers are promising materials for ammonia gas sensors with high performance.     

Label-free detection of antibody-antigen interactions on (R)-lipo-diaza-18-crown-6 self-assembled monolayer modified gold electrodes

Novel (R)-diaza-18-crown-6 has been prepared by a simple two-step synthetic method and characterized for its ability to form a uniform self-assembled monolayer (SAM) on gold as well as to immobilize proteins using atomic force microscopy, quartz crystal microbalance, and electrochemical impedance spectroscopy (EIS) experiments. The (R)-lipo-diaza-18-crown-6 was shown to form a well-defined SAM on gold, which subsequently captures the antibody (Ab) molecules that in turn capture the antigen (Ag) molecules. The Ab molecules studied include antibody C-reactive protein (Ab-CRP) and antibody ferritin (Ab-ferritin) along with their Ag's, i.e., CRP and ferritin. Quantitative detection of the Ab-Ag interactions was accomplished by EIS experiments with a Fe(CN)6(3-/4-) redox probe present. The ratios of the charge-transfer resistances for the redox probe on the SAM-antibody-covered electrode to those with the antigen molecules attached show an excellent linearity for log[Ag] with lower detection limits than those of other SAMs for the electrochemical sensing of proteins.     

Label-free impedimetric detection of glycan-lectin interactions

A compact biosensor for a label-free, rapid (<80 s) detection of glycan-lectin interactions using ac impedance measurements was developed for the first time. A galactose-binding peanut agglutinin (PNA) and sialic acid-binding Sambucus nigra agglutinin (SNA) were covalently surface-immobilized on the layered Cu/Ni/Au printed circuit board (PCB) electrodes. Samples of artificial and natural glycoconjugates consisting of (1) gold glyconanoparticles encapsulated with approximately 90-100 copies of TF-antigen disaccharide Galalpha1-3GalNAc (TF-AuNP), (2) asialofetuin (ASF) containing both LacNAc (Galbeta1-4GlcNAc) and TF-antigen, and (3) fetuin (FET), the sialylated glycoform of ASF. The samples were run separately on PNA- and SNA-immobilized PCB electrodes. Our results indicate that TF-AuNP could be rapidly and reliably detected up to 1 pg/mL (13 fM) concentration on PNA electrode but, as expected, yielded no response on the SNA electrode. ASF and FET glycocoproteins were unambiguously detectable up to 10 pg/mL (150 fM) on PNA and SNA electrodes, respectively. Moreover, the technique allowed us to observe glyco-microheterogeneity of FET as well as establish the presence of two isoforms of SNA lectin, SNA-I and SNA-II, in one of the vendor's formulations. Further elaboration of the described technology into novel electrochemically driven lectin arrays may find applications in diagnosis of cancer and other diseases with multiple glycobiomarkers or as a rapid low-cost bioanalytical tool for glycoproteome analyses.