This article describes the development of biosensors with chemically-amplified responses. In general, chemical amplification involves a reaction sequence for a substance to generate a relatively large amount of product. Thus a trace concentration of analyte can be caused to yield orders of magnitude higher product concentrations which may be more easily measured than the analyte itself. For biosensor systems to detect biochemical reactions on the transducer surfaces, chemical amplification procedures suitable for concentrating the reaction product on the transducer/test solution interface should be utilized to enhance the sensor response effectively. The amplification procedures, such as enzymatic cycling at the enzyme-modified electrode and the preconcentration of the biochemical reaction product on the electrode surface, are particularly useful for realizing highly-sensitive biosensors. The combination of such amplification techniques with immunoassay protocols has provided simple measuring systems for trace amounts of peptide hormones, such as A- and B-type natriuretic peptides and insulin. These simple and highly-sensitive immunoassay systems are suitable for the purpose of the point-of-care testing.
A novel electrochemical biosensor for hydrogen peroxide with high electron transfer efficiency was developed using a nanocomplex composed of horseradish peroxidase and Au-nanoparticle. The nanocomplex was prepared through adsorption of horseradish peroxidase on an Au-nanoparticle. The nanocomplexes were easily coagulated and immobilized on an ITO electrode as a monolayer film by simply casting the nanocomplex dispersion and drying below 10°C. The electrode worked as a hydrogen peroxide biosensor featuring high electron transfer efficiency without any soluble mediator at a mild electrode potential of +0.15 V vs. Ag/AgCl.
We developed a cell-based assay device for the detection of endotoxin, the potentially toxic compound that induces septic shock. Genetically-engineered cells that secrete alkaline phosphatase (SEAP) on exposure to endotoxin were cultured in an electrochemical cell device in medium containing p-aminophenyl phosphate and various concentrations of endotoxin. After 24 hr incubation, p-aminophenol (pAP), generated by SEAP-catalyzed hydrolysis, was detected by amperometry at +0.35 V. The amperometric response increased with the concentration of endotoxin in the range of 0.01–1 ng/ml.
Electrical stimulation has various effects on cellular functions. We previously reported a combined effect of electrical stimulation and cisplatin treatment on HeLa cell death. The present study investigates the signaling pathway behind this phenomenon. Decreased viability of HeLa cells after electrical stimulation plus cisplatin treatment was suppressed by addition of the caspase inhibitor zVAD-FMK. This showed that HeLa cell death induced by combining electrical stimulation and cisplatin treatment was caused by increased caspase activity. Moreover, mitochondrial cytochrome c release and c-Jun N-terminal kinases (JNK) activities were enhanced by this combined treatment. These results indicated that combined treatment-induced apoptosis was caused by increasing caspase activity via the JNK signal pathway.
An electrode was coated by double layer of electrodeposited polypyrroles (PPy). The inner thicker PPy was doped with p-toluene sulfonate to ensure larger electrical capacity that is required for less-invasive stimulation of neural cells. The outer thinner PPy was doped with polyacrylate to present carboxyl group on the surface. Nerve Growth Factor was immobilized by utilizing the carboxyl groups, and successfully induced the differentiation of neuronal PC12 cells and their neurite growth. The present layered structure is one of effective approach to modify the high capacity PPy-based stimulation electrode with biomolecules for controlling cellular functions.
For detection of endocrine disrupting chemicals (EDCs), a novel biosensor based on the degradation of cell membrane model by EDCs is proposed. As a cell membrane model, liposome cluster was prepared from cationic liposome and membrane protein bacteriorhodopsin. We fabricated a biosensor to detect EDCs by immobilization of the cluster on quartz crystal microbalance (QCM) through ionic alkyl thiol. Nonylphenol was examined as representative EDCs and the sensor detected nonylphenol at the range of 1.0 ppb-1.0 ppm.
Alternating electrical potentials consisting rectangular pulse waves of 0∼+1.0 V (vs. Ag/AgCl/Sat’d KCl) at 100 Hz were loaded onto MAGI/CCR5 (HeLa-CD4-LTR-β-gal) cells cultured on an ITO electrode. The ratio of cell membrane damage due to electrical potential loading increased with the expansion time of the potential loading, with a positive potential of more than +0.85 V. Furthermore, the induction of cell damage was very sensitive to the temperature of the potential loading. The membrane damage induced by the potential loading was irreversible.
Atrazine is a widely used herbicide. The electrochemical sensing of atrazine was performed by the detection of its reductive current. In order to make the sensing system smaller and simpler, a gold electrode chip (detection area: 1.8×0.6 mm) was prepared and modified with an atrazine imprinted polymer layer, as the recognition element for atrazine. An atrazine sensing chip modified with molecularly imprinted polymer (MIP) layer was fabricated by spin-coating in this study. Using the atrazine-sensing chip, a cathodic current of atrazine was observed and the reductive current depended on the concentration of atrazine below 20 µM. The detection limit of the sensor was 50 nM (11 ppb). The atrazine-sensing chip was more selective to atrazine than other herbicides such as simazine, ametryn and MCC.
We report on an amperometric biosensor that is based on a nanocomposite of carbon nanotubes (CNT), a nano-thin plasma-polymerized film (PPF), and glucose dehydrogenase (GDH). A mixture of the GDH and a CNT film is sandwiched with 6-nm-thick acetonitrile PPFs. Under PPF layer was deposited onto a sputtered gold electrode. To facilitate the electrochemical communication between the CNT layer and GDH, CNT was treated with nitrogen plasma. The resulting device showed that the large oxidizing current response due to enzymatic reaction was observed at +0.4 V (vs. Ag/AgCl) polarized potential. This demonstrates that the CNT play a role as a catalysis for NADH oxidation caused by enzymatic reaction and as a electron transfer conductor. The optimized glucose biosensor showed sensitivity of 3.3 µA mM−1 cm−2 (correlation coefficient of 0.983, linear response range of 4.9–19 mM) and response of 7 s (reach 95% of maximum response).
Direct electron transfer-type bioelectrocatalytic oxidation of d-gluconate was observed with d-gluconate 2-dehydrogenase (GADH, EC 188.8.131.52, from Gluconobacter frateurii) at bare and thiols-modified gold electrodes. Thiols used have different charges and various lengths of the alkyl chains. The catalytic current at every electrode arose at −0.05 V, which would correspond to the formal potential of the heme c site in GADH. The GADH-loading examined through quartz crystal microbalance measurements was practically independent of the surface properties. However, the current-potential curves were strongly affected by the electrode surface properties, and were interpreted in views of kinetics of enzymatic reaction and electrode reaction, and states of GADH on the electrode surface. The interfacial electron transfer rate constants of GADH depended on the alkane-chain lengths.
In order to detect lactate, high sensitivity lactate sensor was completed by attaching lactate oxidase (LOD) to the hydrogen peroxide sensor. The hydrogen peroxide sensor was prepared as follows; poly(4-styrenesulfonate), peroxidase (POD), ferrocene and poly-l-lysine solutions were dropped on a glassy carbon electrode. After drying this electrode, LOD and glutaraldehyde solutions were dropped on the membrane to immobilize the enzyme. The electrode was immersed into a buffer solution (pH 6.5), and a potential of −0.2 V vs. Ag/AgCl was applied to detect the reduction current. Immediately after the addition of l-lactate (10 µM) to the test solution, the reductive current increased and reached a plateau within 20 s. The lower detection limit was 1 µM (S/N=3), and the ratio of the response for the interferent to that for l-lactate on the electrode was 0.18.
We describe a method to create cell-adhesive regions and to position adherent cells on the newly created regions in sequence within a microfluidic channel. One of the microelectrodes fabricated at the channel wall was used for locally electrogenerating hypobromous acid that renders the opposite face of the channel protein-adsorptive. After the fibronectin was immobilized on the treated region, an ac voltage (1 MHz, 20 Vpp) was applied to the microelectrodes array in the presence of suspended HeLa cells. Since a repulsive force of negative dielectrophoresis (DEP) directs the cells toward the weakest region of the nonuniform electric field, the cells were positioned on the fibronectin-patterned region to allow the cell adhesion, even in the presence of fluid flow (<0.1 µL min−1). By repeating the above process, two types of cells could be patterned in the microchannel.
The nano/micro-structured materials and solid substrates are novel and exciting field of research for biotechnology applications. Especially, the porous silicon (PSi) has many attractive characteristics and is expected to several applications and devices. In this study, influences of PSi with different surface structures for cell adhesion characteristics were investigated. For investigation of cell adhesion characteristics, different surface structures of PSi substrates were fabricated. Using these PSi substrates, evaluation of the cell adhesion characteristics were carried out. In addition, in this study, Hela and COS-7 cell strains were used for evaluation of the cell adhesive characteristics.
An electrochemically oxidized carbon fiber paper (CFP) electrode was fabricated and the direct electrochemical oxidation of NADH was examined for a biofuel cell application. The Raman spectra of the oxidized CFP electrode indicated that edge plane sites were generated by electrochemical activation. On a cyclic voltammogram of the electrode at pH 7.0, an oxidation-reduction peak with a redox potential of about 0 mV (vs. Ag/AgCl) appeared. The electrocatalytic oxidation of NADH with an anodic peak at low potential (+50 mV) occurred and the current density reached as large as a few mA/cm2. The quinoidal moieties which are generated at the edge plane site act as an electron transfer mediator. With NAD+-dependent alcohol dehydrogenase (ADH), the ADH catalyzed bioanode could be constructed by entrapment with poly(diallyldimethylammonium chloride) on the oxidized CFP electrode.
An electrochemical system equipped with dual electrodes was developed for the simultaneous monitoring of motility and phototaxis of immobilized single flagellate alga, Volvox carteri. The flagellate alga was immobilized on a polyion complex membrane. Photoinduced behavior of flagellate alga was recorded as changes in the redox currents for a coexisting redox marker ([Fe(CN)6]3−). From the amperometric measurements, the immobilized alga possessed its flagellar activity on the polyion complex membrane. In addition, it was found that changes of algal flagellar movement induced by phototaxis gave rise to different current changes at each electrode.
Polyamide microcapsule containing glucose oxidase (GOD) and tetrathiafulvalene (TTF) was prepared for the application to a molecular recognition element of screen-printed enzyme electrode. The microcapsules were entrapped in a polyion complex film or a carbon ink containing TTF, and then immobilized directly on the screenprinted carbon electrodes. The enzyme activity of the microcapsule was evaluated by the cyclic voltammetry in a phosphate buffer solution containing glucose. The enzyme electrodes were active to oxidize the dissolved glucose which is catalyzed by the entrapped GOD. In addition, the electroactive TTF molecules were proved to penetrate the polyamide membrane and to function as a mediator.
This work describes a disposable DNA-arrayed thin film transistor (TFT) photosensor integrating a miniaturized reaction unit and a photoelectronic effect-based internal detection sensor. Single stranded DNA was chemically conjugated and arrayed on the TFT photosensor, which was fabricated by the semiconductor integrated circuit (IC) technology, for the detection of specific target DNA by DNA-DNA hybridization. The photosensor surface can be modified by various silane coupling reagents for the optimization of oligonucleotide immobilization conditions and single nucleotide polymorphism (SNP) detection was successfully employed using the TFT photosensor. A microfluidic channels were constructed on the TFT photosensor to improve the hybridization efficiency. The developed TFT photosensor will further promote the development of disposable photodetecting devices applicable in genetic diagnosis.
We synthesized an α-helix forming polypeptide (CK30) and a random-coiled peptide (CdlK30) in order to clear the effect of secondary structure of the modifier on the electrochemical reaction in solution. By changing the surface structure of the CK30 from random-coil to α-helix, the electrochemical reaction of Fe(CN)64− was suppressed. In contrast, on the CdlK30 modified electrode, the oxidation and reduction peak currents of the cyclic voltammogram increased.
An immunoliposome encapsulating a Ru complex with two aminobutyl moieties was prepared to rapidly detect bovine serum albumin (BSA) protein. The detection was carried out within 80 min by electrochemiluminescence (ECL) from the Ru complex adsorbed on Au electrodes after immunoreaction. The adsorption of the complex is a key-step of this procedure in order to increase its sensitivity. Under optimal conditions, the present method can detect BSA in the concentration range from about 1×10−9 g/mL to 1×10−6 g/mL, with almost the same sensitivity as that obtained by conventional ELISA.
Actions of capsaicin glucosides transformed by using the cultured cells of Phytolacca americana on intestinal transport in rats were investigated. Transient increase in the transmural short-circuit current (Isc) related with intestinal transport were observed when capsaicin glucosides (50 µM) were applied to the mucosal side at jejunum and ileum, but capsaicin induced no Isc changes at jejunum and Isc decrease at ileum. Compared with actions of capsaicin, different action manners and lesser Isc changes by capsaicin glucosides against intestinal transport suggest the possibility of the use of capsaicin glucosides as food ingredients.
An influx of calcium ions into a living cell occurs when the cell is deformed mechanically by compressing, indenting or stretching. The calcium ions are taken up through mechanosensitive channels sensing deforming signals. The calcium ion is a key trigger for the reformation of cytoskeletal structure. Indentation of a capillary to a cell during microinjection causes influx of calcium ions, as does a typical AFM tip. A nanoneedle, which is a sharpened needle shaped AFM tip of 200 nm in diameter does not cause any influx of calcium ions when it is inserted into a cell. The mechanical stress from the nanoneedle insertion is negligible and its invasiveness is low.
We have previously reported the development of an artificial enzyme catalysing hydrolytic oxidation reaction of fructosyl-valine (Fru-val). The enzyme was prepared by molecularly imprinting a catalytic polymer composed of vinyl imidazole to form a molecular imprinting catalyst (MIC). In this study, we have attempted to improve both the selectivity and sensitivity of the MIC-based Fru-val sensor by altering the operational conditions. Assuming that the hydrophobic interaction between the valine residue of Fru-val and the cross-linker in the polymer increases with increasing ionic strength of the buffer solution, and that the hydrophilic interaction between N-epsilon-substituted fructosyl-lysine (Fru-ε-lys) and the polymer decreases, we have used a higher concentration of the buffer solution for sensor operation. The sensitivity towards Fru-val increased, whereas the sensitivity towards Fru-ε-lys decreased when a 100 mM potassium phosphate buffer was used for sensor operation instead of a 10 mM buffer. The selectivity of the MIC towards Fru-val against Fru-ε-lys increased drastically from 1.9 to 5.7. In addition, the sensor was able to measure a glycated peptide, fructosyl valyl histidyl, which is a characteristic of the N-terminal structure of glycated hemoglobin.
Pentacyanoferrate-polymer (PVI[FeIII(CN)5]) was synthesized from potassium hexacyanoferrate (II) and a backbone polymer, poly(1-vinylimidazole) (PVI). Bilirubin oxidase (BOD) from Myrothecium verrucaria and PVI[Fe(CN)5] were cross-linked with poly(ethylene glycol) diglycidyl ether. BOD-PVI[Fe(CN)5]-modified glassy carbon electrode showed a sigmoidal wave of a catalytic four-electron reduction of dioxygen (O2) with a half-wave potential of 0.2 V vs. Ag|AgCl|KCl(sat.) in rotating disk voltammetry. The current density as high as 15 mA cm−2 was achieved by immobilization of BOD-PVI[Fe(CN)5] on carbon paper electrodes at 4000 rpm under O2-saturated conditions.
The ion transport from one aqueous (W1) to another (W2) across a bilayer lipid membrane (BLM) in the presence of a hydrophobic ion, dipicrylaminate (DPA−), was investigated by cyclic voltammetry when the hydrophilic salt such as LiCl, NaCl, KCl or CsCl was used as a supporting electrolyte. Voltammograms for the ion transport at a lower scan rate than 10 mV s−1 were in steady state and showed sigmoid curves. The magnitude of the ion transport current density at a given potential was proportional to the hydrophobicity of the counter cation. The result supports our proposed mechanism that the hydrophobic ion serves as a carrier compound in the BLM for the ion transfer of the hydrophilic counter ion in the presence of the membrane potential gradient.
Histamine dehydrogenase (HmDH) from Nocardioides simplex was used in constructing mediated bioelectrochemical systems for histamine oxidation using Os complexes as mediators. Amperometric histamine sensor was prepared by immobilizing a mixture of HmDH and a mediator, poly(1-vinylimidazole) complexed with [Os(2,2′-dipyridylamine)2Cl], with a crosslinker, poly(ethylene glycol) diglycidyl ether on a glassy carbon electrode. The electrode responded linearly to histamine over the range from 2 to 30 µmol dm−3, became free from the substrate inhibition observed in solution, and was almost insensitive to ascorbate at pH 7.0. On the other hand, the charge in coulometry using carbon felt electrodes was proportional to the histamine amount over the range from 0.1 to 5 nmol and the slope indicates a 4-electron oxidation (2-electron in the enzyme reaction and 2-electron in follow-up electrochemical reaction).
To elucidate the mechanism of the electrochemical killing, the global gene expression of synchronously cultured Saccharomyces cerevisiae exposed to electrochemical stimulus was analyzed using DNA microarray. By applying a potential of 1.0 V vs. saturated calomel electrode (SCE), oxidative stress related genes were expressed after only 5 minutes. The gene for cta1 exhibited the highest expression level, while ROS gradually accumulated inside the cells. Genes for signal transduction which may be related to cellular apoptosis were also seen expressed.
Here we are reporting a new and rapid immunoassay for the food allergen using disposable screen printed carbon electrode (SPCE) in connection with the differential pulse voltammetry (DPV). Changes in the current signals due to antibody-antigen interaction provide the basis for an immunoassay on the SPCE surface which is simple, rapid, and cost effective. Casein, which is one of the most potent allergens in milk, was utilized as the model target in our experiment. After the immobilization of the anti-casein antibody on the pre-treated SPCE surface, sample solution contained casein was applied on the electrode surface and measured with DPV. Peak current due to casein was comparable while with bovine serum albumin (BSA) in solution. Our protocol is readily transferable for application to other immunological tests of food testing, because of this system is derived from utilizing the intrinsic electrochemical activity of antibody and antigen.
We report on an electrochemical nicotin adenine dinucleotide (NADH) sensor that is based on carbon nanotubes (CNT) and plasma-polymerized film (PPF). The configuration of sensing electrode was CNTs sandwiched between two 6 nm thick PPFs made from acetonitrile on sputtered gold thin film. We optimized the CNT concentration for casting formation onto the under PPF layer. The sensor showed high sensitivity (a sensitivity of 240 µA mM−1 cm−2, a detection limit of 3.9 µM at S/N=3, +0.4 V vs. Ag/AgCl), wide dynamic range (a linear response range of 0.009–2.3 mM, a correlation coefficient of 0.993), and rapid response (<7 s in reaching 95% of maximum response). This high performance is attributed that CNTs offer excellent electrocatalytic activity and enhance electron transfer, therefore, PPF and/or plasma process are the electrochemistry-friendly platform for CNT applications.
The application of voltage has been used to control the movement of charged molecule such as DNA, some proteins and phospholipid in recent year. In this study, we first applied voltage to amyloidogenic protein, α-synuclein, which is related to Parkinson’s disease. To offer the new approach using electric field and new insight into aggregation and fibrillation mechanism of α-synuclein, we tried to control the aggregation of α-synuclein, which has a negative charge, by applying voltage to it. The aggregation of α-synuclein without conformational change occurred rapidly when a voltage of 1 V was applied. The protein did not form amyloid-like fibrils, but it did form small aggregates. These results demonstrate that this technique might be useful not only to efficiently control aggregation of α-syn but also to understand the mechanism of aggregation and fibrillation of α-syn.
The nanocomposite enzymatic cathode and anode with carbon nanotube were fabricated and optimized for the application to biofuel cells. The nanocomposite electrodes consisted of polyion complex matrix where enzyme, mediator, and carbon nanotube as an electron transport enhancer were immobilized on the glassy carbon electrode. For the fabrication of anode and cathode, glucose oxidase and tetrathiafulvalene and bilirubin oxidase and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium, respectively, were immobilized together in the polymer matrix. Electrochemical observation confirmed the bioelectrocatalytic ability of glucose oxidation and oxygen reduction at the anode and cathode, respectively, due to the enzymatic activity. The biological-fuel cell fabricated by combining the nanocomposite enzyme electrodes demonstrated open circuit voltage of 0.65 V, and its maximum power was 150 µW cm−2.
Spatial distribution of intracellular Ca2+ concentration was measured with a spectro-imaging system composed of an image slicer (10×10 channels), a grism, and a high sensitive CCD camera. The Ca2+ concentration of each single protoplast was different from others both at a steady state (10–100 nmol dm−3) and after electric stimulation. Therefore traceable observation of each single-cell was essential, which contrasted conventional methods dealing with an average of many cells. When a pulsing electric stimulation was applied to single-cells of rice protoplast, the transient variation of intracellular Ca2+ concentration was observed and chitinase gene expression followed (16 out of 36 cells, 44.4%). Its significance level was estimated as 90% by χ2-test.
A mediated bioelectrocatalytic electrode for acetate detection was constructed using Escherichia coli (K-12) as a catalyst embedded in a carbon felt anode. E. coli cells were pre-cultured with acetate as a sole carbon source. Acetate is oxidized into CO2 through the metabolic pathway in the E. coli cells and the electron is transferred from the reducing equivalents in the cell to the anode by [Fe(CN)6]3−/4− as an electron transfer mediator. The linear range was from 0.05 to 0.5 mM in amperometric detection under anaerobic conditions. Aerobic detection was also feasible, although the sensitivity depended on the dissolved O2 concentration. The electrode was almost insensitive to most of other organic acids, alcohols and saccharides except glucose, mannose and lactate.