Namsoo Kim

Thiolated Salmonella antibody immobilization onto the gold surface of piezoelectric quartz crystal

An improved antibody-coated sensor system based on quartz crystal microbalance analysis on Salmonella spp. was developed making use of thiolated antibody immobilization onto one gold electrode of the piezoelectric quartz crystal surface. The best results in sensitivity and stability were obtained with the thin layer of a thiol-cleavable, heterobifunctional cross-linker, sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP). The long bridge of this reagent could function as a spacer, facilitating antibody-Salmonella interaction on the gold electrode. After the addition of a S. typhimurium suspension into a reaction cell with 0.1 M sodium phosphate buffer, pH 7.2, the resonant frequency decreased conspicuously. The time required for maximum frequency shift was about 30-90 min. Sensor response was observed for the microbial suspensions ranging from 9.9 x 10(5) to 1.8 x 10(8) CFU/ml.

Operational characteristics of an antibody-immobilized QCM system detecting Salmonella spp.

A quartz crystal microbalance (QCM) system detecting Salmonella spp. was developed by an anti-Salmonella antibody immobilization onto one gold surface of a piezoelectric quartz crystal surface with sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP) thiolation. The optimum temperature and pH for the antibody-immobilized sensor were 35 degrees C and 7.2, respectively. The frequency shifts obtained were correlated with the Salmonella concentrations in the range 3.2 x 10(6)-4.8 x 10(8) CFU per ml. The system was quite specific to Salmonella spp. and applicable for repetitive use after a regeneration step employing 1.2 M NaOH. A model sample measurement was done for a market milk spiked with Salmonella typhimurium.

Development of a direct-binding chloramphenicol sensor based on thiol or sulfide mediated self-assembled antibody monolayers

A batch-type antibody-immobilized quartz crystal microbalance (QCM) system for detecting chloramphenicol (CAP) was developed. To bind an anti-CAP antibody onto the gold electrode surface of piezoelectric crystals, self-assembled monolayers (SAMs) of different thiols or sulfides were formed by a chemisorption procedure. Then, the anti-CAP antibody was covalently linked to the pre-formed monolayers by an activation procedure using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide. The antibody-immobilized QCM chip thus prepared was installed in a well holder and was measured for sensor response. Compared with the bare QCM chip and the QCM chip only coated with 3-mercaptopropionic acid (MPA), the antibody-immobilized sensor showed greatly enhanced frequency shifts by 10-50-fold after CAP injection. In this case, CAP detection which was indicated by steady-state resonant frequency shift was accomplished within 10 min. When CAP solution was injected into the reaction cell in 50mM concentration, the frequency shifts obtained were, respectively, 530 and 505 Hz in case of thiosalicylic acid and MPA immobilization. Repeated use of the sensor chips up to eight times was possible after 1 min regeneration with 0.1M NaOH. This system demonstrated a potential application of thiol or sulfide mediated SAMs as the pre-coatings of a real-time detection on CAP in solution.

Application of a flow-type antibody sensor to the detection of Escherichia coli in various foods

A flow-type biosensor system which uses a broad-spectrum anti-Escherichia coli antibody and quartz crystal microbalance as biological component and transducer was developed. Biosensor responses were initiated by injecting viable E. coli suspensions through a flow cell and the sensor system was optimized for response time according to flow rate and injection time, followed by the measurement of responses for various E. coli strains. As expected, the sensor system showed a characteristic broad binding feature against E. coli strains. A linear sensor response in double-logarithmic scale was observed for the microbial suspensions ranging from 1.7 x 10(5) to 8.7 x 10(7) CFU/ml. Sample measurements could be done within 20-30 min after Stomacher treatment followed by spiking or enrichment.

Urease-based biosensor for mercuric ions determination

Abstract Urease immobilized into Nafion film on the surface of an ion sensitive field effect transistor (ISFET) has been used for heavy metal salt measurement. Enzyme immobilization into a negatively charged polymer seems to cause an increase in the effect of heavy metal inhibition due to cation accumulation in the polymeric matrix. Fifty percent urease inactivation was observed with 0.2 μM Ag (I), 1.5 μM Hg (II) or 5 μM Cu (II). To make the urease-based biosensor sensitive only to mercuric ions, it is proposed to add small amounts (up to 100 μM) of NaI into the sample (to suppress sensitivity to Ag ions) and to rewash the sensor in 100 mM solution of EDTA for 5 min to remove Cu ions. Restoration of enzyme activity after Hg (II) inhibition can be obtained by sensor rewashing in a 300 mM solution of NaI for 5 min.

Cyanide determination by an ISFET-based peroxidase biosensor

Horseradish peroxidase immobilized on the surface of an Ion Sensitive Field Effect Transistor (ISFET) can be used for the determination of cyanide ions in aquatic media. When the enzyme is immobilized in BSA gel, the resulting sensor can determine 10(-5)-10(-3) M cyanide. Enzyme immobilization into a positively charged polymer, poly(4-vinylpyridine-co-styrene) (PVPy), seems to cause an increase in cyanide inhibition effects because of anion accumulation in the polymeric matrix, and the resulting sensor can measure cyanide concentration in the range 10(-7)-10(-5) M. Fifty percent peroxidase inactivation was observed with 80 microM KCN in the case of BSA-entrapped enzyme, and with only 0.6 microM KCN when the enzyme was covered by PVPy film. Because of the reversible nature of peroxidase inhibition with cyanide ions, restoration of the enzyme activity after inhibition can be obtained by sensor rewashing in fresh buffer.

Determination of glucose, ascorbic and citric acids by two-ISFET multienzyme sensor

Two-ISFET biosensor, containing horseradish peroxidase immobilized into BSA gel as well as glucose oxidase and urease, co-immobilized under a polymeric film, can be used for determination of glucose, ascorbic and citric acids in fruit juices and beverages. Dynamic range of the probe for glucose is 1–10 mM; for ascorbic acid 0.25–2 mM; and for citric acid 5–100 mM. As the contents of the noted species in fruit beverages are relatively high, the present biosensor can be a promising detector for routine drink analysis.

Carp vitellogenin detection by an optical waveguide lightmode spectroscopy biosensor

A label-free carp vitellogenin sensor has a strong potential for on-site monitoring on the possible contamination of edible fish with endocrine disruptors as a sum parameter in an inland carp farm. In this study, we performed a sensitive detection for carp vitellogenin with a direct-binding optical waveguide lightmode spectroscopy-based immunosensor. Carp vitellogenin bound over the sensor surface quite specifically, judging from the sensor responses according to stepwise antibody immobilization. This was also supported by a negligible sensor response found at bovine serum albumin immobilization. When plotted in double-logarithmic scale for carp vitellogenin concentrations of 0.00675-67.5 nM, a linear relationship was found between analyte concentration and sensor response, together with the limit of detection of 0.00675 nM. The reusability of the immunosensor after the regeneration with 10mM HCl was reasonably good, as presumed from the coefficient of variability of 6.02% for nine repetitive measurements. The model sample prepared by spiking a purified carp vitellogenin into a 10-fold diluted vitellogenin-free carp serum in 9.45 nM showed the response ratio of 96.70% against 9.45 nM of the purified carp vitellogenin. When a female and male carp sera induced with 17beta-estradiol injection were analyzed, biomarker induction was even identifiable at 2000-fold serum dilution.

Sulfamethazine detection with direct-binding optical waveguide lightmode spectroscopy-based immunosensor

A direct-binding optical waveguide lightmode spectroscopy-based immunosensor detecting sulfamethazine (SMZ) was prepared, followed by the measurement of its specificity and sensitivity. System construction was undertaken with a peristaltic pump, an injector and the main unit comprising a sensor holder, two signal-harvesting photodiodes, a beam mirror, shutter and He-Ne laser source emitting a monochrome light (λ=632.8nm), plus a PC. Antibody immobilization was performed in situ by covalent binding of an anti-SMZ antibody over the surface of a glutaraldehyde-activated 3-aminopropyltriethoxysilane-treated sensor chip. The reaction buffer for the system was 4mM Tris-HCl (pH 7.2) that showed a medium surface coverage and stable baseline. Sensor response was quite specific to antibody-antigen complexation, as judged from no sensor response caused by bovine serum albumin immobilization. The sensor responses according to SMZ concentrations from 10(-8) to 10(-2)M increased linearly in a semi-logarithmic scale, with the limit of detection of 10(-8)M. The immunosensor was favorably reusable for SMZ screening.

EDTA Determination by Urease-Based Inhibition Biosensor

Urease immobilized into poly(vinylpyridine-co-styrene) film on the surface of ion sensitive field effect transistor (ISFET) has been used for determination of chelating agents (sequestrants). These species, particularly EDTA, restored catalytic activity of the enzyme if it was inhibited with cupric ions. Enzyme immobilization into a positively charged polymer seems to cause an increase of the EDTA restoration effect because of anion accumulation in the polymeric matrix. The resulting biosensor can measure EDTA concentration in the range of 5–500 ppm. The influence of other sequestrants (citrate and phosphate) were also examined.