Piezoelectric inkjet printing of a cross-hatch immunoassay on a disposable nylon membrane

The development of a cost-effective method for manufacturing immunoassays is a key step towards their commercial use. In this study, a piezoelectric inkjet printer and a nylon membrane were used to fabricate a disposable immunoassay. Using a piezoelectric inkjet printer, a cross-hatch pattern of goat anti-mouse antibody (GαM) and rabbit anti-horseradish peroxidase (RαHRP) antibody were deposited on the nylon membrane. These patterns were subsequently treated with a solution containing rabbit anti-goat antibody labeled with horseradish peroxidase (RαG-HRP). The effectiveness of the immobilization process was examined using tetramethylbenzidine (TMB), which oxidizes in the presence of HRP to form a visible precipitate. Optical evaluation of the TMB precipitate was used to assess the precision of the features in the inkjet-printed pattern as well as antibody functionality following inkjet printing. Uniform patterns that contained functional antibodies were fabricated using the piezoelectric inkjet printer. These results suggest that piezoelectric inkjet printing may be used to fabricate low-cost disposable immunoassays for biotechnology and healthcare applications.     

Piezoelectric crystal immunosensor for the detection of staphylococcal enterotoxin B

The work of fabricating a piezoelectric (PZ) immunosensor for the detection of staphylococcal enterotoxin B (SEB) is presented in this paper. Three different immobilization methods using anti-SEB antibody onto a gold electrode of the PZ have been conducted. The electrode coated with polyethyleneimine (PEI) has shown the best result. The fabricated PZ sensor can be used for SEB determination in the range of 2.5–60 μg/ml with a correlation coefficient of 0.997. Milk samples spiked with various concentrations of SEB gave an average of 111% recovery of the toxin. The SEB assay is specific. For example the presence of staphylococcal enterotoxin A (SEA) at 40 μg/ml gave 6.44% of the signal while staphylococcal enterotoxin D (SED) appeared to give no detectable signal. After regeneration with 1.2 M NaOH, the coated crystal could be reused three times with retention of 66% of the initial signal. The crystal has also been found to be stable for 3 days when stored at 4 °C in a dry atmosphere without appreciable loss of activity.     

Surface plasmon resonance immunosensor for detection of<Emphasis Type="Italic">Legionella pneumophila</Emphasis>

An immunosensor based on surface plasmon resonance (SPR) onto a protein G layer by self-assembly technique was developed for detection ofLegionella pneumophila. The protein G layer by self-assembly technique was fabricated on a gold (Au) surface by adsorbing the 11-mercaptoundecanoic acid (MUA) and an activation process for the chemical binding of the free amine (-NH₂) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of the protein G layer by self-assembly technique on the Au substrate and the binding of the antibody and antigen in series were confirmed by SPR spectroscopy. The surface topographies of the fabricated thin films on an Au substrate were also analyzed by using an atomic force microscope (AFM). Consequently, an immunosensor for the detection ofL. pneumophila using SPR was developed with a detection limit of up to 10² CFU per mL.     

Nano-scale probe fabrication using self-assembly technique and application to detection of<Emphasis Type="Italic">Escherichia coli</Emphasis> O 157∶H7

A self-assembled monolayer of protein G was fabricated to develop an immunosensor based on surface plasmon resonance (SPR), thereby improving the performance of the antibody-based biosensor through immobilizing the antibody molecules (IgG). As such, 11-mercaptoundecanoic acid (11-MUA) was adsorbed on a gold (Au) support, while the non-reactive hydrophilic surface was changed through substituting the carboxylic acid group (-COOH) in the 11-MUA molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrocholide (EDAC). The formation of the self-assembled protein G layer on the Au substrate and binding of the antibody and antigen were investigated using SPR spectroscopy, while the surface topographies of the fabricated thin films were analyzed using atomic force microscopy (AFM). A fabricated monoclonal antibody (Mab) layer was applied for detectingE. coli O157∶H7. As a result, a linear relationship was achieved between the pathogen concentration and the SPR angle shift, plus the detection limit was enhanced up to 10² CFU/mL.     

Study on the interaction between DNA and protein induced by anticancer drug carboplatin

The interaction of DNA and human serum albumin (HSA) in the presence of anticancer drug carboplatin was studied with piezoelectric quartz crystal impedance (PQCI) and electrochemistry techniques. In the PQCI analysis, the correlative parameters including the frequency (f0), the motional resistance (R(m)), and the static capacitance (C0) in the experiment were obtained and discussed in detail. Additionally, the kinetics parameters of the cross-linking process were investigated and a response kinetics model was deduced. The values of association rate constant k1, dissociation rate constant k(-1) and the reaction equilibrium constant K were estimated to be 1.895 mg/ml(-1) s(-1), 4.7 x 10(-5) s(-1) and 4.03 x 10(4) (mg/ml)-1, respectively. Furthermore, cyclic voltammetry (CV) and electrochemical AC impedance techniques were employed to testify the cross-linking process.     

A novel liquid-phase piezoelectric immunosensor for detecting Schistosoma japonicum circulating antigen

Aims

A new liquid-phase piezoelectric immunosensor (LP-PEIS), which can detect Schistosoma japonicum (Sj) circulating antigens (SjCAg) quantificationally, was developed.

Methods

The IgG antibodies were purified from the sera of rabbits which had been infected or immunized by Sj and were immobilized on the surface of piezoelectric quartz crystal in LP-PEIS by staphylococcal protein A (SPA). It was used to detect SjCAg in sera of rabbits which had been infected by Sj in order to acquire some optimum conditions for detecting SjCAg. Finally, the LP-PEIS with optimum conditions was used to detect SjCAg in sera of patients who had been infected by Sj, and was compared with sandwich ELISA.

Results

A lot of optimum conditions of LP-PEIS for detecting SjCAg had been acquired. In the detection of patients' sera with acute Schistosomiasis, LP-PEIS has higher positive rate (100%) and lower false positive rate (3.0%) than sandwich ELISA (92.8%, 6.0%). However, there were no significant difference between LP-PEIS and sandwich ELISA.

Conclusions

LP-PEIS can quantificationally detect SjCAg in patients' sera as well as sandwich ELISA.     

Improvement of antibody immobilization using hyperbranched polymer and protein A

For the construction of a well-defined antibody surface, protein A was used as a binding material to immobilize antibodies onto gold-derivatized transducers. The traditional method tends to assemble protein A directly onto the gold-derivatized transducers. In this paper, we tried to indirectly bind protein A onto sensors through hyperbranched polymer (HBP) which was synthesized from p-phenylenediamine and trimesic acid. The three-dimensional structure of HBP and the characteristics including orientation control and biocompatibility of protein A led to highly efficient immunoreactions and enhanced detection system performance. With this strategy, cysteamine monolayer was first assembled onto Au electrodes associated with the piezoelectric quartz crystal; secondly, the cysteamine-modified gold electrode was further modified by the activated HBP; thirdly, protein A was immobilized onto the HBP film; and finally, antibodies were immobilized onto the surface of protein A film for detecting the corresponding antigen. The quartz crystal microbalance immunosensor thus fabricated was applied to detect hepatitis B surface antigen in solutions that ranged from 0.71 to 300 μg mL⁻¹. The detection limit was estimated to be 0.53 μg mL⁻¹. The immunosensor holds good selectivity, sensitivity, and repeatability.     

Detection of Bacillus thuringiensis Cry1Ab protein based on surface plasmon resonance immunosensor

Two novel surface plasmon resonance immunosensors were fabricated for detection of the Bacillus thuringiensis Cry1Ab protein and to demonstrate their performance in analyzing Cry1Ab protein in crop samples. Sensor 2 was modified by 1,6-hexanedithiol, Au/Ag alloy nanoparticles, 3-mercaptopropionic acid, and protein A (or not [sensor 1]), with Cry1Ab monoclonal antibody. As a result, both of the immunosensors exhibited satisfactory linear responses in the Cry1Ab protein concentration ranges of 10 to 500 ng ml⁻¹ and 8 to 1000 ng ml⁻¹, and the detection limits were 5.0 and 4.8 ng ml⁻¹, respectively. The immunosensors possessed good specificity and acceptable reproducibility. In addition, crop samples could be analyzed after a simple treatment. The transgenic crops could be easily identified from the conventional ones by the two immunosensors.     

Electrochemical immunosensor with aptamer-based enzymatic amplification

An electrochemical immunosensor is reported by using aptamer-based enzymatic amplification with immunoglobulin E (IgE) as the model analyte. In this method, the IgE antibody is covalently immobilized as the capture probe on the gold electrode via a self-assembled monolayer of cysteamine. After the target is captured, the biotinylated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinylated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry. The results obtained demonstrated that the electrochemical immunosensor possesses high specificity and a wide dynamic range with a low detection limit that possibly arises from the combination of the highly specific aptamer and the highly sensitive stripping determination of enzymatically deposited silver.     

Piezoelectric printing and probing of Lectin NanoProbeArrays for glycosylation analysis

Glycans have great potential as disease biomarkers and therapeutic targets. However, the major challenge for glycan biomarker identification from clinical samples is the low abundance of key glycosylated proteins. To demonstrate the potential for glycan analysis with nanoliter amounts of glycoprotein, we have developed a new technology (Lectin NanoProbeArray) based on piezoelectric liquid dispensing for non-contact printing and probing of a lectin array. Instead of flooding the glycoprotein probe on the lectin array surface, as in conventional microarray screening, a piezoelectric printer is used to dispense nanoliters of fluorescently labeled glycoprotein probe over the lectin spots on the array. As a proof-of-concept, the ability of Lectin NanoProbeArrays to precisely identify and reliably distinguish between the closely related glycoforms of fetuin is illustrated here. Sensitivity levels comparable to lectin arrays that use evanescent-field scanners was achieved along with several orders of magnitude reduction in the amount of probe required for glycosylation analysis.