A Piezoelectric Immunosensor Using Hybrid Self-Assembled Monolayers for Detection of Schistosoma japonicum

BACKGROUND: The parasite Schistosoma japonicum causes schistosomiasis disease, which threatens human life and hampers economic and social development in some Asian countries. An important lesson learned from efforts to reduce the occurrence of schistosomiasis is that the diagnostic approach must be altered as further progress is made towards the control and ultimate elimination of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Using mixed self-assembled monolayer membrane (mixed SAM) technology, a mixture of mercaptopropionic acid (MPA) and mercaptoethanol (ME) was self-assembled on the surface of quartz crystals by gold-sulphur-bonds. Soluble egg antigens (SEA) of S. japonicum were then cross-linked to the quartz crystal using a special coupling agent. As compared with the traditional single self-assembled monolayer immobilization method, S. japonicum antigen (SjAg) immobilization using mixed self-assembled monolayers exhibits much greater immunoreactivity. Under optimal experimental conditions, the detection range is 1:1500 to 1:60 (infected rabbit serum dilution ratios). We measured several infected rabbit serum samples with varying S. japonicum antibody (SjAb) concentrations using both immunosensor and ELISA techniques and then produced a correlation analysis. The correlation coefficients reached 0.973. CONCLUSIONS/SIGNIFICANCE: We have developed a new, simple, sensitive, and reusable piezoelectric immunosensor that directly detects SjAb in the serum. This method may represent an alternative to the current diagnostic methods for S. japonicum infection in the clinical laboratory or for analysis outside the laboratory

Linear regression analysis of immunosensors and ELISA methods.

<p>The correlation coefficient is 0.973.</p

Effect of <i>Sj</i>Ag concentration on <i>Sj</i>Ag immobilization.

<p>The MPA and ME of volume ratios in mixed SAM are 3∶7. The <i>Sj</i>Ag dilution ratio is 1∶100. The best concentration of <i>Sj</i>Ag for immobilization is 0.2 mg ml⁻¹.</p

Calibration curves for the relationship between the frequency shift of immunoreaction and the dilution ratio of <i>Sj</i>Ab.

<p>The dynamic dilution range of <i>Sj</i>Ab is ∼1∶1500 to 1∶60 with the detection limit of ∼1∶1800 dilution, estimated according to the 3σ (standard deviation) rule. A sample with 1∶100 dilution of <i>Sj</i>Ab was determined repeatedly for five times. The average response frequency shift value was 340±19 Hz, and the relative standard deviation (RSD) among five runs was 9.1%.</p

Effect of ME concentration in mixed SAM on <i>Sj</i>Ag immobilization.

<p>The best dose of ME in the mixed membrane was determined to be 70%.</p

Typical real time frequency response characteristics of the immunosensor in PBS solution.

<p>(a) NRS as negative control with PEG; (b) <i>Sj</i>Ab without PEG (dilution ratio 1∶100); (c) <i>Sj</i>Ab with PEG (dilution ratio 1∶100). Using PEG can significantly enhance the sensor response signals. Immune response-mediated frequency shift values increased from 225 Hz (b) to 343 Hz (c). The corresponding time of immune response decreased from 1600 s (b) to 1200 s (c). Considering the analysis time and reproducibility, the time for both analysis and detection of the sensors in this experiment was 1200 s.</p