Punnee Asawatreratanakul

Label-free capacitive immunosensor for microcystin-LR using self-assembled thiourea monolayer incorporated with Ag nanoparticles on gold electrode.

A label-free immunosensor based on a modified gold electrode incorporated with silver (Ag) nanoparticles (NPs) to enhance the capacitive response to microcystin-LR (MCLR) has been developed. Anti-microcystin-LR (anti-MCLR) was immobilized on silver nanoparticles bound to a self-assembled thiourea monolayer. Interaction of anti-MCLR and MCLR were directly detected by capacitance measurement. Under optimum conditions, MCLR could be determined with a detection limit of 7.0 pgl(-1) and linearity between 10 pgl(-1) and 1 microgl(-1). The immobilized anti-MCLR on self-assembled thiourea monolayer incorporated with silver nanoparticles was stable and good reproducibility of the signal could be obtained up to 43 times with an R.S.D. of 2.1%. Comparing to the modified electrode without silver nanoparticles it gave 1.7-fold higher sensitivity and lower limit of detection. The developed immunosensor was applied to analyze MCLR in water samples and the results were in good agreement with those obtained by high-performance liquid chromatography (HPLC) (P < 0.05).

Comparison of surface plasmon resonance and capacitive immunosensors for cancer antigen 125 detection in human serum samples.

This paper presents a comparison between surface plasmon resonance (SPR) and capacitive immunosensors for a flow injection label-free detection of cancer antigen 125 (CA 125) in human serum. Anti-CA 125 was immobilized on gold surface through a self-assembled monolayer. Parameters affecting the responses of each system were optimized. Under optimal conditions, SPR provided a detection limit of 0.1 U ml(-1) while 0.05 U ml(-1) was obtained for the capacitive system. Linearity for SPR was between 0.1 and 40 U ml(-1) and 0.05-40 U ml(-1) for capacitive system. These immunosensors were applied to analyze CA 125 concentrations in human serum samples and compared with conventional enzyme linked fluorescent assay (ELFA). Both systems showed good agreement with ELFA (P<0.05). Moreover, these immunosensors were very stable and provided good reproducible responses after regeneration, up to 32 times for SPR and 48 times for capacitive system with relative standard deviation lower than 4%. The SPR immunosensor provided advantages in term of fast response and real-time monitoring while capacitive immunosensor offered a sensitive and cost-effective method for CA 125 detection.

Capacitive biosensor for quantification of trace amounts of DNA

A flow injection capacitive biosensor system to detect trace amounts DNA has been developed based on the affinity binding between immobilized histone and DNA. Histones from calf thymus and shrimp were immobilized on gold electrodes covered with self-assembled monolayer (SAM) of thioctic acid. Each of these histones was used to detect DNA from calf thymus, shrimp and Escherichia coli. The studies indicated that histones can bind better with DNA from the same source and give higher sensitivity than the binding with DNA from different sources. Under optimum conditions, both histones from calf thymus and shrimp provided the same lower detection limit of 10(-5) ng l(-1) for DNA from different sources, i.e., calf thymus, shrimp and E. coli. The standard curve for the affinity reaction between calf thymus histone and DNA shows sigmoidal behavior and two linear ranges, 10(-5) to 10(-2) ng l(-1) and 10(-1) to 10(2) ng l(-1), could be obtained. The immobilized histones were stable and after regeneration good reproducibility of the signal could be obtained up to 43 times with a %R.S.D. of 3.1. When applied to analyze residual DNA in crude protein extracted from white shrimp recoveries were obtained between 80% and 116%.

Affinity sensor using 3-aminophenylboronic acid for bacteria detection

Boronic acid that can reversibly bind to diols was used to detect bacteria through its affinity binding reaction with diol-groups on bacterial cell walls. 3-aminophenylboronic acid (3-APBA) was immobilized on a gold electrode via a self-assembled monolayer. The change in capacitance of the sensing surface caused by the binding between 3-APBA and bacteria in a flow system was detected by a potentiostatic step method. Under optimal conditions the linear range of 1.5×10(2)-1.5×10(6) CFU ml(-1) and the detection limit of 1.0×10(2) CFU ml(-1) was obtained. The sensing surface can be regenerated and reused up to 58 times. The method was used for the analysis of bacteria in several types of water, i.e., bottled, well, tap, reservoir and wastewater. Compared with the standard plate count method, the results were within one standard deviation of each other. The proposed method can save both time and cost of analysis. The electrode modified with 3-APBA would also be applicable to the detection of other cis-diol-containing analytes. The concept could be extended to other chemoselective ligands, offering less expensive and more robust affinity sensors for a wide range of compounds.

Development and application of a real-time capacitive sensor

A real-time capacitive sensor based on a potentiostatic step method was developed. It can display in real-time the evoked current waveform, capacitance and the electrical resistance of elements serially connected to the insulation layer on the electrode as a function of time as well as the ohmic resistance of the insulation layer. These features enable the user to observe the association and dissociation of the affinity binding pairs and to evaluate the insulating property of the electrode surface during measurement. The system allows the setting of potential pulse height, pulse interval, gain, filter, and sampling frequency, enabling the system to be more flexible. The performance of the system was firstly evaluated with equivalent circuits. Under suitable parameter settings it provided good accuracy of both the capacitance and resistance. Using the affinity binding pair of human serum albumin (HSA) and anti human serum albumin (anti-HSA) the measured capacitance change was used for the direct detection of HSA. The developed system provided the same sensitivity as the commercially available potentiostat (P>0.05). The proposed system was then applied to analyse HSA in real urine samples and the results agreed well with the immunoturbidimetric assay (P>0.05). The proposed system can be applied for capacitance measurement to directly detect other target analytes using different affinity binding pairs. Other applications such as kinetics analysis of the interaction between affinity bindings, thickness analysis, and the study of the insulation property of the modified layer are also promising.

Detection of staphylococcal enterotoxin A (SEA) at picogram level by a capacitive immunosensor

This work presents the use of a flow injection capacitive immunosensor to detect staphylococcal enterotoxin A (SEA). The study was based on the direct detection of a capacitance change due to the binding between SEA and anti-SEA immobilized on a gold electrode. The optimal regeneration solution, flow rate, sample volume and buffer conditions were studied. Under the optimum conditions, this label-free biosensor provided linearity between 1 × 10−12 g L−1 and 1 × 10−8 g L−1 of SEA and the limit of detection was 1 × 10−12 g L−1 which was much lower than the infectious dose (0.5 × 10−6 – 1 × 10−6 g L−1). Using the regeneration solution of, 15.0 mM glycine-HCl pH 2.20, to break the binding between SEA and the immobilized anti-SEA enabled the electrode to be reused up to 39 times. This technique was applied to analyze SEA in liquid and solid food samples. Any matrix effect can be eliminated by simple dilution. SEA contamination was found in three samples, iced tea with milk (28 ± 1 ng L−1), orange juice (113 ± 6 ng L−1) and fried chicken (1.1 ± 0.2 ng g−1); however, the concentrations were much lower than the infectious dose. The proposed method would be useful for rapid screening of SEA in various matrices.

Real-time label-free affinity biosensors for enumeration of total bacteria based on immobilized concanavalin A

This work presents the results of the use of flow injection surface plasmon resonance and impedimetric affinity biosensors for detecting and enumerating total bacteria based on the binding between E. coli and Con A, immobilized on a modified gold electrode. The single analysis time for both techniques was less than 20 min. Dissociation between the immobilized Con A and the E. coli using 200 mM of glucose in HCl at pH of 2.00 enabling the sensor to be reused for between 29–35 times. Impedimetric detection provided a much lower limit of detection (12 CFU mL−1) than the surface plasmon resonance method (6.1 × 107 CFU mL−1). Using the impedimetric system, real sample analysis was performed and the results were compared to the plate count agar method. Cell concentrations obtained by the biosensor were only slightly different from the result obtained from the plate count agar. The proposed system offers a rapid and useful tool for screening detection and enumeration of total bacteria.

Capacitive antibacterial susceptibility screening test with a simple renewable sensing surface

A simple renewable surface for a rapid antibacterial susceptibility test has been demonstrated. The 3-aminophenylboronic acid (3-APBA) modified electrode bind with cis-diol groups on the cell wall of both gram positive and gram negative bacteria. The detection of antibacterial susceptibility response by a capacitive system can be done within a short time, 2.5h for the whole process, with good repeatability of the electrodes preparation. An acid solution, could break the bonding between 3-APBA and the bacteria, which were then easily removed by the fluid flow, renewing the sensing surface for the next test. This modified electrode can be reused up to 35 times. This sensor is useful for testing the susceptibility of bacteria to antibacterial agents that affect their cell wall. Results from the capacitive sensor corresponded well with the antimicrobial information in the literature and to the morphology of the treated bacteria revealed by scanning electron microscopy. Antimicrobial susceptibility to natural products could also be easily tested.