Capillary and Electrodynamic Forces-Driven Separation Detection of Metal Ions Using a Disposable Microfluidic Sensor with a Composite Electrode.

A disposable microfluidic channel sensor printed on a plastic platform was developed to analyze heavy metal ions (HMIs) as a model target species. Precise separation and detection of multiple targets were established by symmetrically applying a small AC potential on the carbon channel walls to induce an electrodynamic force. The separation device was constructed by covering it with a plastic lid to achieve capillary action in the channel. The sample flow rate was regulated by the hydrophilicity of the lid plastic and electrodynamic convection by the AC field, which was characterized by the contact angle measurement and the additional electrodynamic force. The flow variables and their relevance to the capillary phenomena were demonstrated, and the analytical parameters were optimized. The working electrode was modified with poly(diamino terthiophene) anchored with nanosized graphene oxide (pDATT/GO) to enhance the detection performance. The experimental variables for separating and detecting the target species were optimized according to the AC frequency and amplitude, sample flow rate, electrolytes, pH, temperature, and applied potential for detection. The linear dynamic ranges were between 0.1 and 200.0 ppb, with detection limits of 0.04 ± 0.023, 0.29 ± 0.05, 0.07 ± 0.011, and 0.14 ± 0.06 ppb for Cu2+ Cd2+, Hg2+, and Pb2+, respectively. Finally, the reliability of the proposed method was evaluated through analysis of HMIs in real water samples. The results were matched to those obtained through parallel analysis using ICP-MS at a 95% confidence level.

Conducting polymer composite-based biosensing materials for the diagnosis of lung cancer: A review.

The development of a simple and fast cancer detection method is crucial since early diagnosis is a key factor in increasing survival rates for lung cancer patients. Among several diagnosis methods, the electrochemical sensor is the most promising one due to its outstanding performance, portability, real-time analysis, robustness, amenability, and cost-effectiveness. Conducting polymer (CP) composites have been frequently used to fabricate a robust sensor device, owing to their excellent physical and electrochemical properties as well as biocompatibility with nontoxic effects on the biological system. This review brings up a brief overview of the importance of electrochemical biosensors for the early detection of lung cancer, with a detailed discussion on the design and development of CP composite materials for biosensor applications. The review covers the electrochemical sensing of numerous lung cancer markers employing composite electrodes based on the conducting polyterthiophene, poly(3,4-ethylenedioxythiophene), polyaniline, polypyrrole, molecularly imprinted polymers, and others. In addition, a hybrid of the electrochemical biosensors and other techniques was highlighted. The outlook was also briefly discussed for the development of CP composite-based electrochemical biosensors for POC diagnostic devices.

Exosomal microRNAs array sensor with a bioconjugate composed of p53 protein and hydrazine for the specific lung cancer detection.

For the early diagnosis of lung cancer, a novel strategy to detect microRNAs encapsulated in exosomes with immunomagnetic isolation was demonstrated for the selective extraction of exo-miRNAs from patient serum. Here, miRNA was captured from lysed exosomes in specially designed capture probe modified magnetic beads, followed by T4 DNA polymerase-mediated in situ formation of chimeric 5'-miRNA-DNA-3' (Target). The poly-(2,2':5',2''-terthiophene-3'-(p-benzoic acid)) (pTBA)-modified electrode harbors Probe-1 DNA that hybridizes to the 5' end of the chimera, followed by hybridization of Probe-2 DNA to the 3' end of the chimera, resulting in the formation of a 20-nucleotide-long dsDNA consensus sequence for p53 protein binding. A bioconjugate composed of p53 and hydrazine assembled on AuNPs (p53-AuNPs-Hyd) recruits the p53 protein to recognize a specific sequence, forming the final sensor probe (pTBA-Probe-1:Target/Probe-2:bioconjugate), where hydrazine functions as an electrocatalyst to generate amperometric signal from the reduction of H2O2. This sensor has double specificity via selective capture of the target in Probe-1 and p53 recognition, which shows excellent analytical performance, revealing a dynamic range between 100 aM and 10 pM with a detection limit of 92 (±0.1) aM. For practical applications, we prepared a multiplexed array sensor to simultaneously detect four exo-miRNAs (miRNA-21, miRNA-155, miRNA-205, and miRNA-let-7b) up to femtomolar levels from 1.0 mL to 125 µL of cell culture (A549, MCF-7 and BEAS-2B) media and lung cancer patient serum samples, respectively.

Catalytic SrMoO4 nanoparticles and conducting polymer composite sensor for monitoring of K+-induced dopamine release from neuronal cells.

Octahedral SrMoO4 nanoparticles (NPs) with a high degree of crystallinity and controlled size (250-350 nm) were synthesized for the first time by employing a facile hydrothermal method. The prepared NPs were composited with a carboxyl group bearing conducting polymer (2,2:5,2-terthiophene-3-(p-benzoic acid, TBA)) to attain a stable sensor probe (pTBA/SrMoO4) which was analyzed using various surface analysis methods. The catalytic performance of the composite electrode was explored as an oxidation catalyst for biological molecules through anchoring on the conducting polymer layer, which functioned as a matrix to enhance the stability and selectivity of the sensor probe. The pTBA/SrMoO4 coated on glassy carbon displayed excellent electrocatalytic performance for the oxidation of some biologically important molecules, including dopamine (DA) in neuronal cells. The sensor immobilized with the catalyst showed an excellent response for DA with a dynamic range between 0.2 and 500 µM and a detection limit of 5 nM. The proposed sensor demonstrates the detection of trace DA released from PC12 cells under K+ stimulation, followed by inhibition of the release of exogenic DA by a Ca2+ channel blocker (nifedipine). The developed method provides an interesting way to monitor the effect of extracellular substances on living cells and the drug potency test.

Disposable amperometric immunosensor with a dual monomers-based bioconjugate for granzyme B detection in blood and cancer progress monitoring of patients.

A disposable amperometric biosensor with a dual monomers-based bioconjugate was developed for granzyme B (GzmB) detection and for monitoring of the cancer progression of patients before and after immunotherapy. The biosensor was fabricated by immobilizing a GzmB monoclonal antibody (Ab1) on a poly3'-(2-aminopyrimidyl)-2,2':5',2''-terthiophene/gold nanoparticle (pPATT/AuNP) layer. The bioconjugate nanoparticles were synthesized through self-assembly of a monomer mixture of 2,2:5,2-terthiophene-3-(p-benzoic acid) (TBA) and PATT onto AuNPs, followed by chemical binding of brilliant cresyl blue (BCB) on TBA and GzmB polyclonal antibody (Ab2) on the PATT layer. Each sensing layer was investigated by surface analysis and electrochemical experiments. The sensor performance was assessed with selectivity, stability, reproducibility, detection limit, and real sample analysis. Under the optimized conditions, the dynamic range of GzmB was in two slopes from 3.0 to 50.0 pg/ml and from 50.0 to 1000.0 pg/ml with a detection limit of 1.75 ± 0.14 pg/ml (RSD ≤5.2%). GzmB monitoring was performed for the patient's serum samples, where a low level of GzmB was observed for lung cancer patients before immunotherapy (10.51 ± 0.99 pg/ml, RSD ≤6.2%), and the level was increased after therapy (17.19 ± 2.22 pg/ml, RSD ≤2.6%). Moreover, a significantly higher level was present in healthy persons (34.40 ± 3.92 pg/ml, RSD ≤1.4%). The cancer progression of patients before and after therapy was evaluated by monitoring GzmB levels in human serum using the proposed sensor.

Hydrogen Evolution and Oxygen Reduction Reactions in Acidic Media Catalyzed by Pd4 S Decorated N/S Doped Carbon Derived from Pd Coordination Polymer.

The template-free synthesis and the characterization of an active electrocatalyst are performed for both the hydrogen evolution and oxygen reduction reactions in acidic media. In this work, the unique chelation mode of benzene-1,4-dithiocarboxamide (BDCA) is first used to synthesize a novel palladium-BDCA coordination polymer (PdBDCA) as a precursor of palladium sulfide nanoparticles-decorated nitrogen and sulfur doped carbon (Pd4 S-SNC). The newly synthesized PdBDCA and Pd4 S-SNC nanoparticles are characterized using chemical, electrochemical, and surface analysis methods. Notably, the nanoparticles obtained at 700 °C exhibit the remarkable catalytic property for the hydrogen evolution reaction in 0.5 m H2 SO4 , showing the overpotential of 32 mV (vs reversible hydrogen electrode (RHE)) and Tafel slope of 52 mV dec-1 , which are comparable to that of Pt/C. The catalyst also shows a high oxygen reduction activity, offering the half-wave and onset potentials of 0.92 and 0.77 V (vs RHE) in 0.5 m H2 SO4 , with improved methanol tolerance and long-term stability compared with Pt/C. The present study gives a way for the design of excellent electrocatalyst for the energy conversion devices in the corrosive acidic environment.

Nano-biosensor for the in vitro lactate detection using bi-functionalized conducting polymer/N, S-doped carbon; the effect of αCHC inhibitor on lactate level in cancer cell lines.

A robust amperometric sensor was developed for the lactate detection in the extracellular matrix of cancer cells. The sensor was fabricated by separately immobilizing nicotinamide adenine dinucleotide (NAD+) onto a carboxylic acid group and lactate dehydrogenase (LDH) onto an amine group of bi-functionalized conducting polymer (poly 3-(((2,2':5',2″-terthiophen)-3'-yl)-5-aminobenzoic acid (pTTABA)) composited with N, S-doped porous carbon. Morphological features of the composite layer and sensor performance were investigated using FE-SEM, XPS, and electrochemical methods. The experimental parameters were optimized to get the best results. The calibration plot showed a linear dynamic range between 0.5 µM and 4.0 mM with the detection limit of 112 ± 0.02 nM. The proposed sensor was applied to detect lactate in a non-cancerous (Vero) and two cancer (MCF-7 and HeLa) cell lines. Among these cell lines, MCF-7 was mostly affected by the administration of lactate transport inhibitor, α-cyano-4-hydroxycinnamate (αCHC), followed by HeLa and Vero, respectively. Furthermore, the effect of αCHC concentration and treatment time on the lactate level in the cell lines were demonstrated. Finally, cytotoxicity studies were also performed to evaluate the effect of αCHC on cell viability.

Electrodynamic Force Derived in-Channel Separation and Detection of Au Nanoparticles Using an Electrochemical AC Microfluidic Channel.

In this study, we have established the separation of Au nanoparticles (AuNPs) using a symmetrical AC electric field applied-electrochemical microfluidic device composed of carbon channel and detection electrodes. The lateral movement of AuNPs in the channel under the AC field was analyzed by simulation using the mathematically derived equations, which were formulated from Newtonian fluid mechanics. It shows that the nanoparticles are precisely separated according to their respective mass or size difference in a short time. The experimental parameters affecting the separation and detection of AuNPs were optimized in terms of applied frequency, amplitude, flow rate, buffer concentration, pH dependency, and temperature. The final separation was performed at 1.0 V amplitude with 8.0 MHz frequency at 0.4 µL/min flow rate for the separation, and the potential of 1.0 V was applied for the amperometric detection of AuNPs in a 0.1 M PBS. Before and after the separation, AuNPs (diameter range: 3-60 nm) were confirmed by UV-visible spectroscopy and transmission electron microscopy. In this case, the separation resolution was 3 nm with an enhanced separation efficiency of up to 597,503 plates/m for the AuNPs. In addition, the amperometric current response of the detection electrode under the AC field application was also enhanced by the sensitivity 5-fold compared with the absence of the AC field.

Separation detection of hemoglobin and glycated hemoglobin fractions in blood using the electrochemical microfluidic channel with a conductive polymer composite sensor.

Separation and detection of hemoglobin (Hb) and glycated hemoglobin fractions (HbA1c, HbAld1+2, HbAle, HbAld3a, HbAla+b, HbA2, and HbAld3b) was performed using an electrochemical AC field modulated separation channel (EMSC) coupled with a sensor probe. The sensor was fabricated based on immobilization of a redox mediator on the poly(2,2':5',5″-terthiophene-3'-p-benzoic acid, pTTBA) and N,S-doped porous carbon (NSPC) nanocomposite. The different types of catalytic redox mediators such as Nile Blue (NB), toluidine blue O (TBO), and Neutral Red (NR) were evaluated to achieve the efficient detection. Of these, the NB-based sensor showed the best analytical signal for Hb and HbA1c, thus it was characterized using various electrochemical and surface analysis methods. After that, the sensor was coupled with the EMSC to achieve the separation detection of the Hb family. The frequency and amplitude of the AC electrical field applied onto the EMSC walls were the main driving forces for the separation and sensitive detection of the analytes. Under optimized conditions, linear dynamic ranges for Hb and HbA1c among their fractions were obtained between 1.0 × 10-6 to 3.5 mM and 3.0 × 10-6 to 0.6 mM with the detection limit of 8.1 × 10-7 ± 3.0 × 10-8 and 9.2 × 10-7 ± 5 × 10-8 mM, respectively. Interference effects of other biomolecules were also investigated and the clinical applicability of the device was evaluated by the determination of total Hb and % HbA1c in real human blood samples.

Comparison of enzymatic and non-enzymatic glucose sensors based on hierarchical Au-Ni alloy with conductive polymer.

Enzymatic and non-enzymatic amperometric glucose sensors based on nanostructured Au-Ni alloy were prepared and compared in their performance. The hierarchically structured Au-Ni surface was merely used for the non-enzymatic glucose sensor, while glucose oxidase attached poly-3'(benzoic acid) -2,2':5',2'- terthiophene (pTBA) formed on the alloy surface was used as the enzymatic sensor. The fabricated sensor was characterized using surface analysis and electrochemical experiments. In case of the enzymatic sensor, the anodic current of H2O2 generated from the enzyme reaction was used as the analytical signal, while the direct oxidation of glucose was observed on a mere Au-Ni alloy electrode without enzyme immobilization, which shows an excellent catalytic oxidation of glucose even in physiological pH. The potential pulse pretreatment of the sensor surfaces improved the performance, which allowed both the sensors reproducible and reusable (enzymatic sensor: coefficient of variation = 1.82%, n = 5, non-enzymatic: coefficient of variation = 2.93%). The enzymatic biosensor reveals the advantages of increased sensitivity, selectivity, and stability, compared with the non-enzymatic sensor. The linear range of enzymatic sensor was attained from 1.0 µM to 30.0 mM with a detection limit of 0.29 µM. The reliabilities of the sensors were also demonstrated through the glucose analysis in human blood samples, and the result was compared with the commercially available glucometer.

Simultaneous detection of ATP metabolites in human plasma and urine based on palladium nanoparticle and poly(bromocresol green) composite sensor.

A sensitive voltammetric sensor based on palladium nanoparticles (PdNPs) and poly-bromocresol green (pBG) composite layer immobilized on amide functionalized single-walled carbon nanotubes (AmSWCNTs) modified pyrolytic graphite (PdNPs:pBG/AmSWCNTs/PG) has been prepared for the simultaneous determination of adenosine triphosphate (ATP) catabolites, inosine (INO), hypoxanthine (HX), xanthine (XT), and uric acid (UA). The modified PdNPs:pBG/AmSWCNTs/PG was characterized by electrochemical experiments and surface analysis, which exhibited exceptional electrocatalytic effects towards the oxidation of INO, HX, XT, and UA with a significant enhanced peak current and well resolved peaks separation for all the analytes. The linear calibration curves were obtained in the concentration range of 0.001-175 µM, 0.001-200 µM, 0.001-150 µM, and 0.001-200 µM and limits of detection were found as 0.95 nM, 1.04 nM, 1.07 nM, and 0.43 nM corresponding to INO, HX, XT, and UA, respectively. The common metabolites present in the biological fluids did not interfere in the determination. The applicability of the proposed sensor was successfully demonstrated by determining INO, HX, XT, and UA in the human plasma and urine and the obtained results were validated by using HPLC.

Immunosensors for detection of Annexin II and MUC5AC for early diagnosis of lung cancer.

Amperometric immunosensors were developed to diagnose lung cancer through the detection of Annexin II and MUC5AC. To fabricate the sensor probe, a conducting polymer (poly-terthiophene carboxylic acid; poly-TTCA) was electropolymerized onto a gold nanoparticle/glassy carbon electrode (AuNP/GCE) and a dendrimer (Den) was covalently bonded to the poly-TTCA through amide bond formation, where AuNPs were doped onto the dendrimer. To obtain the final sensor probe, an antibody (anti-Annexin II) and hydrazine (Hyd), which is a catalyst for the reduction of H(2)O(2) generated by glucose oxidase (GOx), were covalently attached onto the Den/AuNP-modified surface. Each surface was then characterized by SEM, impedance spectroscopy and XPS. The final sensor probe was examined before and after interaction with Annexin II and MUC5AC using impedance-spectroscopic, quartz crystal microbalance and amperometric methods. The performance of the immunosensor for the Annexin II was evaluated for the apical surface fluid labeled with GOx by the standard addition method. In this case, the detection limit of the proposed method was 0.051 ng/mL (k=3, n=5). The Annexin II concentration in the secretions collected from squamous metaplastic cells was determined to be 280+/-8.0 pg/mL (n=5).

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 (NO(2)(-)) from food samples have been developed. The methods were characterized by UV-visible spectroscopic and electrochemical measurements, and their performance for NO(2)(-) 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 NO(2)(-) 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 NO(2)(-) 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 NO(2)(-) 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 NO(2)(-) 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 NO(3)(-), 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 NO(2)(-) in ham and sausage samples, respectively.

Water sensor for a nonaqueous solvent with poly(1,5-diaminonapthalene) nanofibers.

A water sensor for a nonaqueous solvent was fabricated using poly(1,5-diaminonapthalene (DAN) nanofibers, which were prepared through a catalytic chemical polymerization of the DAN monomer using Fe(III) salt as the catalyst. Poly(1,5-DAN) nanofibers were characterized by atomic force microscope (AFM), transmission electron microscope (TEM), scanning electron microscope (SEM), and UV-vis spectroscopy. The electrochemical properties of poly(1,5-DAN) nanofibers were investigated using cyclic voltammetry (CV). The electrochemical activity of poly(1,5-DAN) nanofibers was utilized for water sensing. The fabrication of water sensor was followed by placing one drop (about 2 microL) of 0.01% poly(1,5-DAN) nanofibers solution in the gap between two split gold electrodes (PBSA) and completely dried. The response of the water sensor in an acetonitrile solution was evaluated under optimized conditions. The linear dynamic range was from 0.05 to 20%, and the detection limit was determined to be 0.01%. The response of this sensor was shown to be comparable to that obtained with the Karl Fischer titration method.

Electrochemical Sensors Based on Organic Conjugated Polymers.

Organic conjugated polymers (conducting polymers) have emerged as potentialcandidates for electrochemical sensors. Due to their straightforward preparation methods,unique properties, and stability in air, conducting polymers have been applied to energystorage, electrochemical devices, memory devices, chemical sensors, and electrocatalysts.Conducting polymers are also known to be compatible with biological molecules in aneutral aqueous solution. Thus, these are extensively used in the fabrication of accurate,fast, and inexpensive devices, such as biosensors and chemical sensors in the medicaldiagnostic laboratories. Conducting polymer-based electrochemical sensors and biosensorsplay an important role in the improvement of public health and environment because rapiddetection, high sensitivity, small size, and specificity are achievable for environmentalmonitoring and clinical diagnostics. In this review, we summarized the recent advances inconducting polymer-based electrochemical sensors, which covers chemical sensors(potentiometric, voltammetric, amperometric) and biosensors (enzyme based biosensors,immunosensors, DNA sensors).

An amperometric bilirubin biosensor based on a conductive poly-terthiophene-Mn(II) complex.

An amperometric bilirubin biosensor was fabricated by complexing the Mn(II) ion with a conducting polymer and the final biosensor surface was coated with a thin polyethyleneimine (PEI) film containing an enzyme, ascorbate oxidase (AsOx). The complexation between poly-5,2'-5',2''-terthiophene-3-carboxylic acid (PolyTTCA) and Mn(II) through the formation of Mn-O bond was confirmed by XPS. The PolyTTCA-Mn(II) complex was also characterized using cyclic voltammetry. The PolyTTCA-Mn(II)/PEI-AsOx biosensor specifically detect bilirubin through the mediated electron transfer by the Mn(II) ion. To optimize the experimental condition, various experimental parameters such as pH, temperature, and applied potential were examined. A linear calibration plot for bilirubin was obtained between 0.1 microM and 50 microM with the detection limit of 40+/-3.8 nM. Interferences from other biological compounds, especially ascorbate and dopamine were efficiently minimized by coating the biosensor surface with PEI-AsOx. The bilirubin sensor exhibited good stability and fast response time (<5s). The applicability of this bilirubin sensor was tested in a human serum sample.

The biosensor based on the pyruvate oxidase modified conducting polymer for phosphate ions determinations.

An enzymatic biosensor was fabricated by the covalent immobilization of pyruvate oxidase (PyO) onto the nano-particle comprised poly-5,2':5',2''-terthiophene-3'-carboxylic acid, poly-TTCA (nano-CP) layers on a glassy carbon electrode (GCE) for the amperometric detection of the phosphate ions. The direct electron transfer reaction of the immobilized PyO onto the nano-CP layers was investigated and the electron transfer rate constant was determined to be 0.65 s(-1). The electrochemically prepared nano-CP lowered the oxidation potential (+0.40 V versus Ag/AgCl) of an enzymatically generated H(2)O(2) by PyO in a phosphate solution. Experimental parameters affecting the sensitivity of the biosensors, such as amounts of the cofactors, the pH, the applied potential, and the temperature were optimized. A linear response for the detection of the phosphate ion was observed between 1.0 microM and 100 microM and the detection limit was determined to be about 0.3 microM. The response time of the biosensors was about 6s. The biosensor showed good selectivity towards other interfering anions. The long-term storage stability of the phosphate biosensor was studied and the sensor was applied in a human serum sample for the phosphate ions detection.

Detection of polymerase chain reaction fragments using a conducting polymer-modified screen-printed electrode in a microfluidic device.

A simple and fast method for electrochemical detection of amplified fragments by PCR was successfully developed using CE in a microfluidic device with a modified screen-printed carbon electrode (SPCE). The surfaces of the SPCE were modified with poly-5,2'-5',2''-terthiophene-3'-carboxylic acid, which improves the analysis performance by lowering the detection potential, enhancing the S/N characteristics, and avoiding electrode poisoning. DNA fragments amplified by PCR were separated within 210 s in a 75.5 mm-long coated-separation channel at a separation field strength of -200 V/cm. To minimize the sample adsorption into the inner surface of the capillary wall, which disturbs the separation, a dynamically coated capillary with an acrylamide solution was used. Furthermore, the analysis procedure was simplified and rendered reproducible by using 0.50% w/v hydroxyethylcellulose as a separation matrix in a coated channel. The reproducibility of the analysis employing the coated channel yielded RSD of 4.3% for the peak areas and 1.4% for the migration times in eight repetitive measurements at a modified electrode, compared with 21.3 and 9.4% for a bare electrode. The sensitivity of the assay was 18.74 pAs/(pg/microL) with a detection limit of 584.31 +/- 1.3 fg/microL.

Detection of protein-DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA-protein interaction.

A simple, direct method for the detection of DNA-protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purbeta (mPurbeta), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurbeta and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurbeta were 25 and 3 microg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.

Development of an immunosensor for the detection of vitellogenin using impedance spectroscopy.

A simple and direct immunosensor for the determination of carp (Carassius auratus) vitellogenin (Vtg), a female-specific protein, has been proposed based on an antibody-captured conducting polymer-coated electrode. The monoclonal antibody specific to carp (C. auratus) Vtg was immobilized by covalent coupling to the carboxylic acid group on the polymer. The antibody immobilization and antibody-antigen interaction have been demonstrated by means of quartz crystal microbalance and impedance spectroscopic techniques. The impedance change occurred at the sensor surface due to the specific immuno-interaction was utilized to determine Vtg. The sensor showed high selectivity and sensitive response to Vtg in a buffered medium without redox probe. Vtg was determined in the linear range from 1.0 to 8.0 microg/l with the standard deviation of +/-0.13 (n =3) and the detection limit was determined to be 0.42 microg/l. This method was applied to the determination of Vtg in real male and female carp (C. auratus) serum samples.