Detection of Bacillus anthracis spores and a model protein using PEMC sensors in a flow cell at 1 mL/min

doi:10.1016/j.bios.2005.12.002

Biosensors and Bioelectronics

Volume 22, Issue 1, 15 July 2006, Pages 78-85

https://doi.org/10.1016/j.bios.2005.12.002 Get rights and content

Abstract

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors of 4 mm² sensing area were immobilized with antibody specific to Bacillus anthracis (anti-BA) spores or bovine serum albumin (anti-BSA). Detection of pathogen (Bacillus anthracis (BA) at 300 spores/mL) and BSA (1 mg/mL) were investigated under both stagnant and flow conditions. Two flow cell designs were evaluated by characterizing flow-induced resonant frequency shifts. One of the flow cells labeled SFC-2 (hold-up volume of 0.3 mL), showed small fluctuations (±20 Hz) around a common resonant frequency response of 217 Hz in the flow rate range of 1–17 mL/min. The total resonant frequency change obtained for the binding of 300 spores/mL in 1 h was 90 ± 5 Hz (n = 2), and 162 ± 10 Hz (n = 2) under stagnant and flow conditions, respectively. Binding of antibodies, anti-BA and anti-BSA, were more rapid under flow than under stagnant conditions. The sensor was repeatedly exposed to BSA with an intermediate release step. The first and second responses to BSA were nearly identical. The total resonant frequency response to BSA was 388 ± 10 (n = 2) Hz under flow conditions. Kinetic analysis is carried out to quantify the effect of flow rate on antibody immobilization and the two types of detection experiments.

Introduction

Recently, we reported that detection sensitivity of piezoelectric-excited millimeter-sized cantilever (PEMC) sensors for pathogens and proteins is on the order 50 pg/Hz (Campbell and Mutharasan, 2005a, Campbell and Mutharasan, 2005c, Campbell and Mutharasan, 2006). We also showed that Escherichia coli O157:H7 can be detected using antibody immobilized PEMC sensors at as low a concentration as 700 cells/mL in batch with 1 mL samples. More recently, we showed that spores of Bacillus anthracis can be detected at 300 spores/mL in a batch cell using PEMC sensors (Campbell and Mutharasan, 2006). While batch measurement is effective when sample volume is small, it is not effective for applications where sample volume is large and the pathogen count is very low. An example of this application is the case of drinking water and source water analysis for pathogen content. The tolerable level for Cryptosporidium and Giardia is 0.2/L of drinking water (Szewzyk et al., 2000). Due to the size of the sensor (a few mm²), dipping it into a large volume of liquid is not an effective means of contacting the target analyte with the sensor surface, particularly for particulate antigen such as the spores. Hence, contacting in a flow cell configuration is a natural next step of development. Thus, investigating the effects of flow rate on sensor performance, and the design configurations of flow cell for PEMC sensor that yields good sensor performance are worthwhile goals from a practical perspective. In this paper, we address these two issues.

Sensors that rely on mechanical resonance such as quartz crystal microbalance (QCM) (Kim and Park, 2003), and microcantilevers (Pei et al., 2004) have been used in the flow cell configuration. Flow rates in these resonators and in plasma resonance (SPR) (Kawazumi et al., 2005, Yu and Lai, 2005) sensors use flow rates in the order of μL/min. Microcantilevers operated by Pei et al. (2004) used a fairly low flow rate of 33 μL/min. In general performance of both QCM and microcantilevers deteriorate at high flow rates such as 100 μL/min (Sota et al., 2002). Alternate flow cell design was proposed by Sota et al. (2002) that enabled a 27 MHz QCM to operate satisfactorily at 100 μL/min. In this investigation, we show that the PEMC sensor performance does not deteriorate at flow rates as high as 17 mL/min, which is approximately 200-fold higher than any of the cantilevers sensors.

Section snippets

Cantilever physics

The resonant frequency of a rectangular PEMC sensor in liquid exhibits high mass change sensitivity of 50 pg/Hz and is described in detail in our previous publications (Campbell and Mutharasan, 2005a, Campbell and Mutharasan, 2005b). Briefly, the resonant frequency of a cantilever is given by: ${f^{'}}_{n f} = k_{n} \sqrt{\frac{K}{{M^{'}}_{e} + Δ m}}$ where k_n are the eigen values (Elmer and Dreier, 1997, Naik et al., 2003). The parameter K is the effective spring constant and depends on the thickness, density, and modulus of the cantilever

Chemicals

All chemicals were purchased from Sigma–Aldrich (Allentown, PA). Deionized water used was from a Milli-Q plus ultra-pure water system (18.2 MΩ cm).

Flow cell fabrication

Several sensor flow cells were designed, fabricated, and tested. In this paper, we report on two cells, labeled SFC-1 and SFC-2, which showed good response at high flow rates. The flow cells were constructed of Plexiglas^® and dimensional details are given in Fig. 1. The central contacting chamber is cylindrical in shape of 7.0 mm diameter. Once PEMC

Modeling fluid flow

The flow patterns through the sensor flow cell were investigated by modeling the flow using finite element modeling platform, FEMLAB^®. The two-dimensional Navier Stokes equation (ρ = 1000 kg/m³ and μ = 0.001 Pa s) was solved in conjunction with continuity equation for various inlet flow rates (1–17 mL/min). The sensor flow cells used in the model were identical in shape and dimension to the actual ones used in the experiments within ±0.05 mm.

Resonance characterization of PEMC sensors

Typical resonance spectra, a plot of phase angle versus excitation frequency, in air of PEMC sensor may be found in our earlier publication (Campbell and Mutharasan, 2005a). For the PEMC sensor used in this study the fundamental and second mode resonant frequencies were at 23.05 ± 0.01 and 91.02 ± 0.01 kHz in air, respectively. In this study, the fundamental mode was used for detection because the peak was very stable and remained sharp under various flow rates. Each experiment was repeated at least

Conclusion

In this paper, we have shown that a flow cell system comprised of a PEMC sensor functionalized with the appropriate antibody can be used to detect low concentrations of proteins and pathogens in real time. The most significant result is that sensor response is not only more rapid for spore detection, but also for protein binding. Furthermore, the total sensor response was significantly higher (∼100%) for both B. anthracis spores and protein (BSA) under 1 mL/min flow conditions compared to

Acknowledgements

The authors gratefully acknowledge Dan Luu for the development of data acquisition programs and the sensor flow cell fabrication. The work was partially supported by the Environmental Protection Agency Grant R8296041, the National Institutes of Health Grant 5R01EB000720, and the United States Department of Transportation (Grant PA-26-0017-00 Federal Transit Administration) in the interest of information exchange. The United States Government assumes no liability for the contents or use thereof.

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Detection of Bacillus anthracis spores and a model protein using PEMC sensors in a flow cell at 1 mL/min

Abstract

Introduction

Section snippets

Cantilever physics

Chemicals

Flow cell fabrication

Modeling fluid flow

Resonance characterization of PEMC sensors

Conclusion

Acknowledgements

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