Poster W8 - Evaluation of the Ability to Use Shear Horizontal Surface Acoustic Wave Biosensors to Perform Sterility Testing of Nutrient Media

Introduction: This article describes a rapid method to evaluate media sterility using a shear-horizontal surface acoustic wave (SH-SAW) biosensor. Commercial SH-SAW biosensors were used to monitor the time-dependent phase responses to the solid-liquid interface. Phase changes resulted from a reduction in the acoustic wave velocity induced by the binding of bacterial cells to a monolayer of the 3-Mercaptopropanyl-N-hydroxysuccinimide ester layer. Increases in the phase shift equal to or less than 5.0 degrees, the average baseline signal measured for the 1x phosphate buffered solution used for suspending the cells, indicates no bacterial cells or a positive sterility test. Likewise, sterility tests that indicate a phase shift more significant than 5.0 degrees indicate a failed test. Our devices were applied to both sterility testing and to monitor Escherichia coli (E. coli) cell growth. Filtered 1x PBS was used as the negative control, and an undiluted 24-hour culture of E. coli was a positive control. The sensitivity for our 250 MHz quartz SH-SAW sensors was calculated based on the response from the E. coli solution over the linear dynamic range.

Materials and

Methods: Escherichia coli (E. coli) was purchased from Fisher Scientific; 3,3'-Dithiobis (succinimidyl) Propionate (DSP) was purchased from Fisher Scientific, 10x Phosphate buffered saline (PBS) was purchased from Fisher Scientific; Ultrapure water from Fisher Scientific, Saint Louis, MO; Tryptone; Yeast extracts; Sodium chloride; 200-proof ethanol; SYTO 59 red fluorescent dye (5 uM in DMSO) was purchased from Life Technologies; Taiwan, and electronics from TST Biomedical Electronics; Gold-coated SH-SAW sensors from the TST Biomedical, Tris (2-carboxyethyl)phosphine (TCEP) purchased from Fisher Scientific; 1x PBS solution; LB Broth; Zeiss Axioskop 2 Plus, which is equipped with a Teledyne Lumenera INFINITY5-5 microscopy camera.

Results, Conclusions, and Discussions: This report presented data demonstrating the successful analysis of E. coli solutions on a DSP-coated SH-SAW that can analyze as little as a 2x10^4 CFU per ml E. coli solution. While our study only considered 2x10^4 – 2x10^9 CFU ml-1, with the linear dynamic range only being in the 2x10^4 – 2x10^7 CFU per ml range, lower cell concentrations may be possible by optimizing the concentrations of the DSP cross-linker coating along with the incubation times of the cells on the biosensor surface. However, the practical limit of the signal cannot go below the baseline signal with 1x PBS, which gives an average phase shift of 5.0 degrees. Since the lowest signal measured was 13.1 degrees, we did not go below the practical limit since 13.1 degrees – 4.8 degrees = 8.3 degrees. We further postulate that the range of phase shifts between 8.3 degrees – 5.01 degrees = 3.29 degrees may be sufficiently large that we could realize one order of magnitude reduction in the bacterial concentration from 2x10^4 down to 2x10^3 CFU per ml. However, it is also possible that below 2x10^4, the SH-SAW may just be too insensitive to measure any bacterial cells, although this seems unlikely since it can measure the phase shift of 1x PBS with no cells. The net result is that we would be able to measure about 1,000 cells on the biosensor since the sensor only uses a 5ul sample. However, we developed a method called the E-F system, that allows us to analyze 50ul of solution while only using 5ul of solution for the analysis. We observed that an increase in the phase shift greater than 5.0 degrees would constitute a failed sterility test. We also demonstrated the sensor’s ability to monitor E. coli concentrations over a wide range from 2x10^4 – 2x10^9 per CFU per ml. The response from the biosensor takes the shape of a saturation curve with a linear dynamic range between 2x10^4 - 2x10^7 CFU per ml.

Acknowledgements (Optional): This work was funded by the United States National Science Foundation (NSF), grant number 1817282, and the United States Department of Energy (DOE), grant number 620035.