This article describes recent progress of cell-based electrochemical biosensors, especially focusing on the incorporation of micro-fabrication technology and gene-modified engineering. Modern electroanalytical techniques offer extremely low detection limits that are achievable using small sample volumes. Bio-MEMS (bio-micro-electromechanical system) technology allows on-line monitoring of cellular functions in which various analytical operations with microfluidics. Reporter gene systems are frequently used in gene-expression studies and applied to environmental monitoring and food safety analyses. These research trends have clearly demonstrated that electrochemical whole-cell devices will have strong impacts on modern biotechnology and become an indispensable tool to ensure the safety of our society.
We demonstrated an ion-sensitive field-effect transistor (IS-FET) based biosensor to directly monitor the hybridization of deoxyribonucleic acid (DNA) using peptide nucleic acid (PNA) as a probe. A single strand DNA or PNA was immobilized as probes on the IS-FET. To detect the hybridization of DNA, we carried out measurements of IS-FET’s drain current hybridized by complemental target DNA. Then, the difference voltage was evaluated to be about only 10 mV by using the probe DNA, furthermore monitored as shifts about 170 mV by attempting the probe PNA. The changes in threshold voltage observed for neutral PNA-immobilized IS-FETs were more than 17 times greater than those for negatively charged DNA-immobilized IS-FETs. This approach demonstrates that the PNA-modified IS-FET-based biosensor works more effectively as a signal transducer of genetic information.
A smart carbon dioxide gas sensor was developed by the combination of Al3+ ion conductor [(Al0.2Zr0.8)20/19Nb(PO4)3] and O2− ion conductor [(8 mol%)Y2O3–ZrO2, YSZ] with the refractory Li2CO3-doped La2O2SO4 solid solution as the gas detecting auxiliary electrode. Here, the two layered solid electrolyte pellet was preliminarily fabricated by using the Al3+ ion and the O2− ion conductor powders and then the pellet was pressed and finally sintered. The sensor response was very quick within several seconds, with showing a long term stability over 100 days. The size of the present sensor tip is designed as small as 2 mm×3 mm×1 mm.
The non-labeled and direct immunoassay of cytokine on a thin polyion complex film of poly-L-lysine and poly-styrenesulfonate was studied using a surface plasmon resonance sensor. The suppression of non-specific protein adsorption was investigated by changing the blend ratio of poly-L-lysine and poly-styrenesulfonate. With our film, the protein adsorption fell to less than 10% of that of an unmodified surface when the poly-L-lysine to poly-styrenesulfonate ratio was 4:6. We also measured the kinetic rates between tumor necrosis factor-α and anti-tumor necrosis factor-α antibodies, which were immobilized on the film. The binding constant was calculated to be 1.5×108 (M−1), which satisfies the binding constant level for a monoclonal antibody modified on a commercially available surface.
Multilayer thin films were prepared on the surface of a gold (Au) electrode using sodium carboxymethylcellulose (CMC) to elucidate the suitability of the polysaccharide-based thin film as a scaffold for constructing chemical sensors. A quartz crystal microbalance study revealed that an alternate deposition of CMC and poly(allylamine hydrochloride) (PAH) gives an exponentially growing multilayer films on the surface of a quartz resonator. The PAH-CMC film-coated Au electrodes exhibited a redox response to Fe(CN)63−/4− ions in solution, depending on the number of layers in the film. The PAH-CMC films were permeable to Fe(CN)63−/4− ions irrespective of the thickness and the sign of electric charges on the outermost surface of the film, suggesting a loosely-packed porous nature of the film. It was found that the film contained electrostatic binding sites to which Fe(CN)63−/4− ions can be strongly immobilized.
Metal electrodes for chemical sensors have been fabricated on an overhead-transparency film using the Line Patterning Method. This method comprises i) laser-printing of toner with negative pattern of the electrodes, ii) vapor-deposition of metal layers, and iii) removal of the toner and the upper metal layers. The resolution of the pattern was ca. 0.1–0.2 mm. We found that multilayered electrodes such as Au/Cr/film, Pt/Au/Cr/film, and Ag/Pt/Au/Cr/film satisfied conductivity, adhesiveness, and flexibility. Furthermore, a glucose sensor using the film electrode was fabricated.
Micropatterns of protein molecules were fabricated by a method based on anodic oxidation of Si surface by atomic force microscope (AFM). In this method, an arbitrary pattern of oxide is drawn on the Si surface by applying a voltage between the conducting AFM probe and the Si substrate. The oxide pattern is then used as a template for immobilization of protein molecules. The obtained patterns were investigated with AFM and scanning electron microscope (SEM).
The highly selective detection of dopamine (DA) against 3,4-dihydroxyphenylacetic acid (DOPAC), L-ascorbic acid (AA), and uric acid (UA) was studied using a sputter deposited indium tin oxide electrode. The sensitivity of the electrode surface was more than 100 times higher for DA than for DOPAC, AA and UA as revealed by voltammograms obtained in a phosphate buffer. The DA response is linear from 10−6 to 10−3 M in the presence of 0.1 mM AA, suggesting that high selectivity is achieved in the mixed solution.
α-Human atrial natriuretic peptide (ANP), a marker peptide hormone for heart diseases, was determined at femtomolar levels by an electrochemical enzyme immunoassay. Alkaline phosphatase (ALP) was used as a labeling enzyme. Changes in ALP activity were monitored using a lipid-modified glucose oxidase electrode based on bioelectrocatalytic redox recycling as follows. p-Aminophenol (PAP), the product of the ALP enzyme reaction, was electrochemically oxidized at the electrode to p-iminoquinone (PIQ), and then reduced back by glucose oxidase which was stably immobilized on the electrode surface. The consumption/regeneration cycle of p-aminophenol resulted in a great enhancement in the sensitivity of the enzymatic immunoassay.
A simple and highly sensitive surface-plasmon-resonance (SPR) immunoassay has been demonstrated for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT). The immunoreaction between a home-made polyclonal anti-2,4,6-trinitrophenyl-keyhole-limpet-hemocyanine antibody (P-TNPh-KLH Ab) with a physically immobilized TNPh-ovalbumin (TNPh-OVA) conjugate was utilized for TNT quantification based on the principle of indirect competitive immunoreaction. The performance of the P-TNPh-KLH Ab was compared with three commercially-available antibodies and was observed that the home-made antibody exhibited the highest sensitivity to TNT (1 ppt) among the other antibodies with a response time of 2 min.
A new type of sensor was fabricated by using a stabilized zirconia (YSZ) tube as well as a potassium nitrite (KNO2)-based auxiliary sensing-electrode (SE), and its sensing performances were examined for detecting low concentration of NO2 at 450°C. The difference in potential (emf) between the nitrite-based SE and the Pt/air reference electrode (RE) was measured as a sensor signal. The device was found to detect NO2 rather sensitively down to 20 ppb, although the response time needed to be improved. The NO2 sensitivity of the sensor was hardly varied even if the concentration of CO2 in the sample gas was changed from 400 ppm to 1000 ppm, and the sensitivity varied logarithmically with the NO2 concentration in the range of 20–400 ppb. A new technique proposed here for measuring sensor response could shorten the response time of the present sensor.
A surface of gold electrode was modified with a self-assembled monolayer in order to protect the oxidation of ascorbic acid, which exists in biological fluid. The electrode was prepared by the modification with aqueous solution of 5-carboxypentanethiol (5C), 7-carboxyheptanethiol (7C) and 10-carboxydecanethiol (10C). At pH 7.0, alkanethiol modified electrodes did not show any anodic response to ascorbic acid but anodic peak to dopamine and serotonin. Dopamine and serotonin were most selectively oxidized with the 7C-modified electrode prepared in 0.15 mg/ml 7C. The anodic current to dopamine was 500 times higher than that of ascorbic acid. The selective oxidation of dopamine and serotonin was performed by the ionization of alkanethiol.
An immunosensor for human C-reactive protein (CRP) detection was developed by combining a portable surface plasmon resonance (SPR) machine with an anti-human C-reactive protein monoclonal antibody (AbCRP)-immobilized gold surface of the microfluidic SPR sensor chip (MSSC). The gold surface of MSSC was chemically modified using self-assembly monolayer technique with 4,4′-dithiodibutyric acid. Subsequently, AbCRP was immobilized on the chemically-activated gold surface of the MSSC by chemical adsorption and CRP determinations were performed using immunoreaction. The detection limit for CRP concentration by this developed SPR method was 1.0 µg/ml.
We investigated the resonant property of a quartz crystal microbalance (QCM) for C-reactive protein (CRP) detection using immunoagglutination technique of anti-CRP antibody immobilized latex beads. Serum containing CRP was added to the immunized latex-bead suspension. The immunized latex beads were agglutinated and adsorbed onto the QCM electrode surface. Based on the adsorption of agglutinated latex beads onto the QCM, the shifts of increasing impedance and decreasing resonance frequency were proportional to the CRP concentration in the serum.
The WO3 thin film nanosensors have been fabricated by dropping and calcination of H2WO4 suspension onto Au nano-gap electrode (100 nm of gap-size and 10 µm in width), which was fabricated by means of MEMS (Micro Electro-Mechanical Systems) techniques, photolithography and FIB. The amount of WO3-electrode interface was controlled by changing amount of WO3 grains packed in the nano-gap electrode. The sensitivity to dilute NO2 was increased with increasing amount of interface, suggesting the importance of oxide-electrode interface in NO2 sensing. Further, the response behavior was affected by the amount of WO3 grains packed in nano-gap electrode.
A new solid-electrolyte ion sensor device using a Na+-ion conductor (Na5DySi4O12: NDSO) as an impedancemetric transducer and a perovskite-type oxide thin-film as a receptor has been developed. The AC impedance of the device with a LaCoO3 receptor was found to vary logarithmically with increasing K2HPO4 concentration between 1.0×10−5 and 1.0×10−2 M at 10 kHz. The 90% response time was ca. 2 min at room temperature. The sensor showed a little sensitivity to NO3− at higher concentration, while no response was observed to the examined anions of Cl− and ClO4−.
The methanol dissociative adsorption at a Pt-loading carbon (Pt/C) electrocatalyst for direct methanol fuel cell has been investigated by electrochemical measurements using a porous microelectrode (PME) and porous/disk-dual microelectrode (P/D-ME). The proton elimination reaction based on the methanol dissociative adsorption during the primary process of the methanol electrooxidation on a Pt/C surface, especially at Pt(100), has been observed by PME measurements. Furthermore, by using the P/D-ME, we have succeeded for the first time in the electrochemical detection of protons dissociated from methanol molecules into the bulk solution due to the above reaction.
A flow-channel with the sub-micrometer depth was fabricated aiming highly sensitive SPR sensing. The channel structure and anodic bonding conditions based on a BK7 glass substrate were optimized. The formation of the submicrometer-depth fluidic channel was confirmed by scanning electron microscopy. Water and electrolyte solutions penetrated the prepared flow-channel by capillary force. SPR curves obtained with the fabricated flow-channel to the concentration of an electrolyte solution, which indicated that the fabricated nanofluidic channel is applicable for SPR sensing.
The electrochemical reaction of glucose on a thin gold film electrode was investigated using the surface plasmon resonance (SPR) method. A cyclic voltammogram of glucose on a thin gold film electrode showed anodic current in both anodic and cathodic scans. The potential dependence of the refractive index change was simultaneously measured by SPR and showed that the change in the gold electrode surface status switched the catalytic reaction. In addition, a potential step experiment revealed that the SPR signal showed the underlying electrode status whereas the electrode current showed the overall catalytic oxidation of glucose.
Highly stable, supramolecular assembled layers with 1:1 composition of C60 and C70 fullerenes were found to form on nickel(II) octaethylporphyrin (NiOEP)-modified Au(111) surface, whereas the replacement reaction of the first adlayer of nickel(II) tetraphenyl porphyrin (NiTPP) occurred upon adsorption of fullerene molecules as the second layer. This result shows that supramolecular assemblies of C60 and C70 are strongly influenced by the underlying layer of porphyrin. It is demonstrated that the stability of the first adlayer is an important factor in the surface design of host-guest selectivity of fullerenes on electrode surfaces.
Novel glutamate biosensor was fabricated by coupling of a glutamate oxidase (GlOx) to a copper ion embedded polyion complex membrane composed of double-stranded DNA and polyallylamine (PAA), which prepared on a surface of the glassy carbon (GC) electrode. A hydrogen peroxide (H2O2) produced during the GlOx reaction was cathodically detected, based on an electrocatalytic activity of a DNA-Cu(II) complex for a reduction of H2O2. The cathodic current responses to L-glutamate obtained at −0.2 V (vs. Ag/AgCl) and at pH 6.0 linearly increased up to 100 µM with a detection limit of 1 µM (S/N=3). The sensitivity (slope of the linear region) was 0.80 nA/µM, and the response time was less than 20∼30 s. The sensor retained its response characteristics for at least five days by the storage in dry state at 4°C.
Response of SnO2-based sensors to CO under extreme conditions was investigated. It was found that Pd and V2O5-loaded SnO2 device (thick film), after a reduction treatment in CO flow, showed reverse response to dilute CO (increase in resistance) under particular operating conditions, i.e., lower temperature below 325°C, low humidity less than 100 Pa water vapor pressure. The reverse response tended to be more conspicuous as the CO concentration decreased and the coexistent O2 partial pressure increased. Similar reduction-induced reverse response to CO was also observed with Pd-loaded SnO2 device. XPS analysis suggested that the reverse response should be related with the oxidation state of SnO2.
The amount of covalently immobilized horseradish peroxidase (HRP), ΓHRP, and its electrocatalytic current, ired, are measured with changing the monolayer composition for two kinds of mixed self-assembled monolayers (SAMs) of COOH- and OH-terminated alkanethiols with different domain structures. In phase-separated SAMs of mercaptopropionic acid (MPA) and 11-mercaptoundecanol (MUOL), HRP is selectively immobilized on the MPA domains and, therefore, both ΓHRP and ired values increase in proportion to the surface mole fraction of MPA. In homogeneously mixed SAMs of 11-mercaptoundecanoic acid (MUA) and MUOL, HRP is homogeneously immobilized on the entire surface of the SAM, and ΓHRP and ired values reach constant values at the mole fraction of MUA, χMUA=0.4.
An approach to detect urinary proteins has been examined. An electrochemical surface plasmon resonance (SPR) sensor was used to nonspecifically detect proteins such as albumin by observing the direct protein adsorption onto the sensor surface. The new method enabled us to measure the concentration of proteins by just contacting the sample solution with the sensor surface. The adsorption of human serum albumin (HSA) seemed to be regulated by applying potential on the Au electrode that is the sensing element of the sensor. The concentration of the protein was obtained from the change in the SPR signal caused by the protein adsorption. Furthermore, the potential application facilitated detergent to remove the adsorbed protein, which enabled us to reuse the sensor.
Slab optical waveguide (SOWG) spectroscopy was carried out for an in situ observation of ferroin and ferriin formed at an electrode/solution interface during electrolysis on an indium-tin oxide (ITO) electrode. A new spectroelectrochemical cell for SOWG spectroscopy was developed to observe in situ absorption spectra of species in the vicinity of the ITO electrode surface. A cyclic voltammogram (CV) of 5 mmol·dm−3 ferroin in 1 mol·dm−3 potassium nitrate aqueous solution was obtained as the potential swept from +1.4 to +0.4 V and then from +0.4 to +1.4 V. Furthermore a cathodic and anodic peaks appeared at +0.82 and +0.92 V, respectively. The SOWG spectra were obtained every 0.05 mV during the sweep. The SOWG spectra at the potential of +0.4 and +1.4 V were identical to the UV-VIS spectra of ferroin and ferriin, respectively. The concentration profiles near the electrode during cyclic voltammetry of ferroin were discussed based on the absorbance at 510 and 650 nm on SOWG spectra. Furthermore, in situ real-time monitoring of absorption during potential step experiment of ferroin was made at various incident angles from 70 to 77° using SOWG spectroscopy.
A planar NO2 sensor was fabricated by using a yttria-stabilized zirconia (YSZ) plate and Cr2O3 sensing-electrode (SE). The NO2 sensing performances were evaluated at 700°C in the presence of 5 vol.% water vapor for the sensor attached with each of Cr2O3-SE having different thicknesses. It was found that the NO2 sensitivity of the sensor was highest when the thinnest Cr2O3-SE was used and decreased with increasing thickness of SE. A linear correlation between the sensitivity and the NO2 concentration was observed at 700°C in the range of 50–400 ppm for each sensor. Based on the results of the measurements for the NO2 conversion to NO on each of Cr2O3 layers having different thicknesses, it was confirmed that the catalytic activity to the gas-phase NO2 decomposition on the Cr2O3-SE matrix played an important role in deciding the NO2 sensitivity of the present sensor.
11-Ferrocenyl-1-undecanethiol (FcC11SH) self-assembled monolayer and poly(divinylferrocene) film (PDVF) on gold electrodes were used for the electrochemically-controllable catalysts for chemiluminescence of luminol. When the electrode potential became positive, ferrocene species of both monomolecular layer and polymer film on gold were oxidized to ferricinium cation species and then they catalyzed the chemiluminescence of luminol. In the case of FcC11SH monolayer, the number of surface attached ferrocene groups was two orders smaller than that in the case of the PDVF film estimated from the electrochemical measurements, although magnitudes of luminosities were similar. This result indicates that the FcC11SH monolayer modified gold electrode showed more efficient luminosity than that of polymer film modified gold electrode. Ferrocene groups on the FcC11SH/electrode surface can be re-oxidized to ferricinium cation species much easily. The catalytic reaction of chemiluminescence takes place only at the surface layer on the polymer film modified gold electrode.