Phage coated magnetoelastic micro-biosensors for real-time detection of Bacillus anthracis spores

Sensors and Actuators B 137 (2009) 501–506

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Phage coated magnetoelastic micro-biosensors for real-time detection of Bacillus anthracis spores夽 Wen Shen a,∗ , Ramji S. Lakshmanan a , Leslie C. Mathison a , Valery A. Petrenko b , Bryan A. Chin a a b

Materials Research & Education Center, Auburn University, 275 Wilmore Labs, Auburn, AL 36849, USA Department of Pathobiology, Auburn University, Auburn, AL 36849, USA

a r t i c l e

i n f o

Article history: Received 14 August 2008 Received in revised form 25 December 2008 Accepted 13 January 2009 Available online 30 January 2009 Keywords: Magnetoelastic Biosensor Phage Bacillus anthracis Flowing test

a b s t r a c t A micro-scale, freestanding, magnetoelastic biosensor coated with phage has been developed for the real-time in vitro detection of Bacillus anthracis spores. The sensor exhibits a characteristic resonance frequency upon the application of an alternating external magnetic field. It has a high sensitivity to the change in mass when spores are attached. The frequency versus mass sensitivity increases significantly with a decrease in sensor length. Spore detection is realized by measuring the resonance frequency change due to the change in mass as spores are captured onto the sensor surface. B. anthracis spore suspensions in a range of concentration levels (5 × 101 to 5 × 108 spores/ml) was tested using a 1000 ␮m × 200 ␮m × 15 ␮m sensor in a flowing fluid at a flow rate of 40 ␮l/min. The binding kinetics was analyzed based on the attachment rate. The specificity of the sensor to B. anthracis spores was examined compared with other Bacillus species. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Bacillus anthracis spores have become well-known biological warfare agents because of their ability to cause mortality in humans and their long life under unfavorable environments [1]. To guard against this threat, it is necessary to have a method of detection that is very rapid, very sensitive, and small enough that it can be taken to the site of possible contamination and give results without requiring extensive training of operating personnel. The conventional methods of detection use antibodies and peptides as bio-molecular recognition elements. These methods tend to be very expensive, take a long time to obtain results, are not very sensitive or may not be selective [2–4]. An alternate and newer method has been developed at Auburn University for detecting the B. anthracis spores [5–7]. This new method uses phages (clone JRB7 from a phage library developed at Auburn University in Alabama) that are very specific for binding with B. anthracis spores. Compared with antibodies, these phages serve as excellent binding agents due to their low cost, simple operation, and high specificity to the target spore or bacterium type while rejecting other strains of anthracis or non-target bacterium. By using a phage developed to specifically

夽 Paper presented at the International Meeting of Chemical Sensors 2008 (IMCS12), July 13–16, 2008, Columbus, OH, USA. ∗ Corresponding author. E-mail address: [email protected] (W. Shen). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.01.027

capture B. anthracis spores, detection can be done in minutes using machines that can be transported to the site of interest [8,9]. Very often, bacteria and spores exist in liquids. To reliably detect low concentrations, it is necessary to bring the bacteria to the sensor. The method proposed here uses a flowing liquid to achieve a more rapid and reliable response than could be achieved by using sensors in static liquid. Previous research using static liquid was used to verify the change in frequency versus mass of very small sensors using phage [9]. Unlike traditional acoustic wave devices [10] that require complex wiring for power and measurement of the transducer, the sensor proposed here is wireless, free standing, robust in both air and liquid and has a rapid response which allows real-time detection of bacteria or spores. Since the new sensors are free-standing and have no wires or mechanical connections, they are easily adapted for use in flowing liquids without needing complex circuits or mechanical structures. 2. Principle of operation The operating principle of this magnetoelastic sensor is based on the phenomenon of magnetoelasticity. Simply stated, upon the application of a magnetic field, the material undergoes a corresponding shape change due to its internal magnetic moments, i.e., it gets longer, wider, thicker based on the magnetic direction [11,12]. Briefly, the platform of this sensor is a magnetoelastic material which has a characteristic resonance frequency that is a function

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Fig. 1. Schematic of the detection system.

of its material properties [13], shape, physical dimensions [14] and mass of the material [15]. If an alternating magnetic field is applied with a frequency that matches this characteristic resonance frequency, a maximum shape change occurs. This shape change can be sensed by an external frequency monitoring device as explained later in this paper. When temperature and humidity are constant, a small uniform mass loading on the sensor surface will cause a shift of the resonance frequency by an amount given by [15] f = −

f m 2 M

(1)

where f, f, M and m represent initial resonance frequency of the sensor, the shift in the resonance frequency, initial mass and change in mass of the sensor, respectively. If the increase in mass (m) is caused by a target biological analyte attaching to the sensor surface, the resonance frequency will decrease by a corresponding amount (f). Previous research tests in our lab have been done to verify the above equation. The results of these tests are given in the results and discussion.

3. Detection system design and experimental methods 3.1. Sensor platform A sensor consists of the basic platform plus the bio-recognition element material used to make it sensitive for a specific bacteria or spore. The magnetoelastic material used for making the sensor platform was Metglas® 2826 MB alloy from Honeywell Inc. This alloy has a saturation magnetostriction of 12 ppm [16]. The basic sensor platform consists of a magnetoelastic device with a thin layer of gold on the surface. The sensors used in this study are free-standing rectangular pieces of 1000 ␮m × 200 ␮m × 15 ␮m in dimensions. The gold layer (100 nm thick) was used to protect the magnetoelastic material from corrosion and at the same time to provide a bioactive surface to which the biological probe may be easily attached. The detailed design and construction of the magnetoelastic sensor platform was published by Lakshmanan et al. [17] and Guntupalli et al. [18].

3.2. Target pathogen and bio-recognition probe selection After the platform is completed, it is coated with a biorecognition element. The bio-recognition element used on the magnetoelastic platform was filamentous phage, clone JRB7, derived from a landscape f8/8 phage library developed by the Department of Pathobiology at Auburn University [5,19]. This phage was designed to be a specific probe for B. anthracis spores while ignoring other spores. The JRB7 phage was immobilized onto the gold surface of the magnetoelastic sensor platform by immersing the gold-coated sensor platform particles in a JRB7 phage suspension of 1012 vir/ml for 1 h. The sensors were then washed with distilled water multiple times and dried in air. Once the phage is immobilized to the sensor surface it serves as the bio-recognition probe for B. anthracis spores. 3.3. Signal processing unit and fluid unit design 3.3.1. Detection system The basic structure of the detection system for our magnetoelastic sensors is presented in Fig. 1. The detection system consists of two main parts; the signal processing section and the fluid flow section. The signal processing section is used for measuring resonance frequency of the sensors. Coils are used for applying a magnetic field to the sensor and for receiving signals from the sensor. By measuring the signal amplitude at each applied frequency, maximum amplitude is measured at the resonance frequency. In our case we monitor the difference between the applied signal and the sensed signal to give a negative going peak. 3.3.2. Fluid unit The fluid flow section consisted of small reservoir to hold the test solution and another to hold the discard solution. Between the reservoirs is a glass tube containing the sensor and a peristaltic pump used to provide a controlled flow rate. Around the glass tube containing the sensor is a coil set used for applying the magnetic field and sensing the frequency. The reservoirs, pump and sensor tube were connected with flexible tubes to complete the path from start to finish. The sensing coil was connected to an analyzer to sense the resonance frequency change of the sensor as spores accumulated on its surface, adding mass to the sensor. The analyzer also provided a swept frequency signal to the excitation coil to provide

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the time-varying magnetic field necessary to excite the sensor and cause oscillation at its resonance frequency. 3.4. Real-time detection and dose response of B. anthracis spores in fluid Another condition needed by sensors is the ability to rapidly detect the presence of the target analyte, in this case, B. anthracis. Our system uses an analyzer system that continuously sweeps from a low to high frequency that includes the resonance frequency of the magnetoelastic sensor. Each sweep completed in seconds, much faster than the accumulation of mass as spores bind to the phage coated sensor surface. The flowing system exposes the sensor to the test solution continuously so that the spores are continuously brought to the sensor, providing a system that gives results within minutes or a few tens of minutes. The flow rate is adjusted to provide continuous exposure to the test solution. The sensor was initially exposed to distilled water, flowing at 40 ␮l/min, for at least 10 min before the introduction of a 108 spores/ml suspension of B. anthracis Sterne strain spores. To determine the effect in resonance frequency change caused by different concentration levels of B. anthracis suspension exposure to the sensor, a 1000 ␮m × 200 ␮m × 15 ␮m sized sensor was separately exposed to increasing concentrations of B. anthracis spores (5 × 101 to 5 × 108 spores/ml) suspensions in water. The flow rate was 40 ␮l/min and, for each concentration, a 0.8 ml suspension was used for detection. 3.5. Specificity tests of the sensors In order to verify the effectiveness of the bio-probe, it was necessary to verify the ability of the sensor to detect B. anthracis but reject similar strains of Bacillus [19]. To do this, we obtained a set of similar spores from the Department of Pathobiology at Auburn University. The spores of the Sterne strain of B. anthracis, B. megaterium, B. paratyphosus, B. cereus, B. subtilis and B. licheniformis were tested. Concentrated spore suspensions were stored in sterile distilled water at about 5 ◦ C and diluted in distilled water as needed. Suspensions of the different strains were tested under nearly identical conditions and the analyzer results were compared with the actual spore count using scanning electron microscopy (SEM) photographs of the sensor surface.

Fig. 2. Calibration of resonance frequency as a function of sensor length (with a length to width ratio of 5).

first order resonance frequency (“resonance frequency”, for short) is 1 f = 2L

df dm

4.1.1. Resonance characteristics of the sensors The sensors used in this work have a length to width ratio of 5 and only the first order of the longitudinal vibration was of interest. It has been demonstrated [14] that under the above conditions, the sensor undergoes a plane-stress or biaxial state, and the observed

(3)

Substituting Eqs. (1) and (2), the mass sensitivity is Sm

1 =− 2L2 Wt



E (1 − )

(4)

where L, W and t are the length, width and thickness of the sensor, respectively. Since all the sensors have a length to width ratio of 5, the sensitivity can be simplified to Sm = −

4.1. Theories

(2)

4.1.2. Mass sensitivity Mass sensitivity (Sm ) is essential to determine how well our sensor responds to a small mass change. It is defined as the change in frequency caused by each unit of mass change, i.e. Sm =

4. Results and discussion

E (1 − )

where L, E,  and  are the length, Young’s modulus, Poisson’s ratio  and density of the material of the sensor, respectively, and E/(1 − ) is defined as the acoustic wave propagation speed a . Since all the tests were conducted at room temperature and one atmosphere, E was considered constant. Therefore, a is also a constant (4498.4 m/s for the sensors with a length to width ratio of 5), and the resonance frequency of the as-fabricated sensor is proportional to the reciprocal of the length of the sensor. The first order resonance frequency of different length magnetoelastic sensors ranging from 500 ␮m to 5 mm is shown in Fig. 2. The length to width ratio was kept as 5 and the thicknesses of the sensors were kept as 15 ␮m. Ten sensors were measured for each size selected. The theoretical prediction was calculated from Eq. (2). The experimental values were very close to the theoretical prediction.

3.6. Microscopic analysis of sensors SEM was used to confirm the phage-spore binding at the sensor surface. Before exposing the spores to the SEM, a preparation process is necessary to protect the spores from cellular structure damage. This process is called “fixation” and is accomplished by exposing the previous assayed biosensors to osmium tetroxide (OsO4 ) vapor for 30 min. The “fixed” sensors were then mounted onto aluminum stages and then sputter coated with gold (30 nm thick) at 0.8 mbar Argon atmosphere. A JEOL-7000F SEM, operating at 15 keV, was used to examine the sensor surface.



5 2L3 t



E (1 − )

(5)

Since L, E,  and  are all material constants, the mass sensitivity depends only on the length and thickness of the sensor. For determining the mass sensitivity experimentally, a magnetoelastic ribbon of two different thicknesses (15 ␮m and 30 ␮m) was cut into rectangular pieces of various sizes, identified individually and their resonance frequencies measured. Afterwards, the particles were coated with a 3 ␮m thickness of gold and the mass change was calculated from the volume of the sputtered gold. Then

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Fig. 3. Plot of mass sensitivity for various lengths and thickness of the sensor platform.

each sensor was tested for its resonance frequency after the gold coat. Fig. 3 shows the results of an early test to verify that the magnetostrictive sensors follow the mass sensitivity equations presented in Eqs. (1) and (5). The 30 ␮m devices were measured in a first test and the 15 ␮m devices were measured at a later date as we learned to work with very small sensors. The 30 ␮m devices were considered as a group and individual devices were not tagged. As a result of the variations in dicing and in gold coating and measuring, we noted that there were variations in the measured mass sensitivity, but the overall test results followed the trend curve reasonably well. Later, as we learned to make smaller sensors, and began the polishing process to convert from 30 to 15 ␮m, we devised a specific test to verify that smaller sensors followed the trend curve. The 15 ␮m devices were not considered as a group. They were very carefully selected and tagged in preparation for this specific test. Those with slight variations in size were discarded. Also each device was numbered so that their initial and final weights were pertinent to that specific device only. As the chart shows, the data for the thinner devices follows the theoretical curve more closely than the larger devices. From the curves, we note that the small-sized magnetoelastic sensors have a very large frequency change for a small mass change. Theoretically, by detecting this change in frequency, it is possible to detect a single spore or bacterium.

Fig. 4. Resonance frequency response of a magnetoelastic biosensor when exposed to B. anthracis spores suspension (5 × 108 spores/ml).

and spores were uniformly distributed on most of the sensor surface, while the density of the spores was found to be higher at the edges of the sensor than in the more central areas. This would result in a frequency shift different from the theoretical value from Eq. (1) where it was assumed that the loaded mass was uniformly distributed [15]. However, we have found that by using the actual spore count from the SEM picture there was a close correlation to the frequency change. For example, for the 1 mm sensor detection shown above, the attached spore number counted from SEM pictures is 1.80 × 104 bound spores. This number is very close to the spore number calculated from Eq. (1), which is 2.02 × 104 spores. 4.4. Kinetics of spore binding The rate of spore/bacterium attachment to the sensor was quantitatively determined by a first order kinetic model [20] for antibody and antigen reaction as −kC0 t = ln

 f − f  ∞ f∞

(6)

where f is the change in frequency, f∞ is the change in frequency when all the attachment sites are filled, k is the attachment rate constant, C0 is the initial spore/bacterial concentration and t is the time spent when the frequency change is f.

4.2. Detection results of B. anthracis spores The response of a 1000 ␮m × 200 ␮m × 15 ␮m sensor for detecting B. anthracis spores in flowing liquid is shown in Fig. 4. The resonance frequency decreased continuously until saturation occurred, within 55 min. A lag of approximately 2 min occurs between the introduction of the spore suspension into the fluid delivery system and when the spores reach the sensor location. The change in resonance frequency as a result of spore capture is 1.83 kHz, corresponding closely to 2.02 × 104 bound spores according to Eq. (1) where each spore is assumed to have a mass of 2 pg. The dose response curve is shown in Fig. 5. The detection limit for the sensor is about 103 spores/ml. A linear response was found between the concentrations of 5 × 103 and 5 × 107 spores/ml. 4.3. Microscopic analysis of sensors SEM pictures were taken to confirm the spore capture on the sensor surface. Fig. 6(a) is the SEM pictures of bio-activated sensor surface coated with JRB7 phage and Fig. 6(b) shows the spore bound sensor surface after detection. It was found that the phage

Fig. 5. Resonance frequency shift as a function of various concentrations of B. anthracis spore (5 × 101 to 5 × 108 spores/ml). The smooth lines are the sigmoid fit for the experimental data (R2 = 0.9886).

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Fig. 6. Typical SEM images of a phage immobilized magnetoelastic sensor surface (a) before B. anthracis suspensions (phage distribution on sensor surface); (b) after B. anthracis suspensions (5 × 108 spores/ml).

The attachment rate of the B. anthracis spores to the magnetoelastic sensors is very similar to the above case. But unlike the antibody–antigen reaction (each antibody site captures only one antigen), each spore can be bound with more than one phage. Theoretically, the upper boundary of the number of spore binding would occur when the (phage) binding sites distributed all over the sensor surface, that is, the entire sensor surface was covered with spores. However, the condition when the entire sensor surface is covered with spores does not exist in reality, because the phages are normally positively charged and form a certain distribution on the sensor surface [21,22]. The theoretical determination of the actual amount of binding sites on the sensor surface was extremely difficult. However, it was observed from each detection curve (e.g. Fig. 4) that the sensor reaches saturation after a certain period of time. It is believed that this saturation was reached when all the active binding sites were occupied by the spores. The f∞ should be the frequency change between the sensor’s resonance frequency before spore binding and after saturation. The attachment constant determined based on this f∞ is 3.47 × 10−10 min−1 (spores/ml)−1 as shown in Fig. 7. 4.5. Specificity Suspensions of the different strains were tested under nearly identical conditions and the analyzer results were compared with

Fig. 7. Kinetic analysis for the binding of Bacillus anthracis spores using a 1000 ␮m × 200 ␮m × 15 ␮m magnetoelastic sensor at a flow rate of 40 ␮l/min. The attachment rate constant was determined from the slop of the curve.

Table 1 Specificity of a 500 ␮m magnetoelastic sensor over a range of Bacillus species. Bacillus species

f (Hz)

Spore capture (vir)

B. anthracis B. megaterium B. paratyphosus B. cereus B. subtilis B. licheniformis

5250 54 57 132 37 89

6980 72 76 175 49 118

the actual spore count using SEM photographs of the sensor surface. Table 1 shows the results of this test. Note that the spore count of B. anthracis is many times greater than that of any of the other Bacillus strains, with the next nearest being only about 2.5% in comparison. 5. Conclusions This paper presented a magnetoelastic sensor coated with JRB7 phage for the real-time in vitro detection of B. anthracis spores. The first order resonance frequency of the sensor was characterized. It was demonstrated both experimentally and theoretically that the frequency versus mass sensitivity of the sensor is high for millimeter and micrometer sized sensors and that it increases significantly as the sensor length decreases. The very good frequency versus mass sensitivity allows the detection of a small mass change when the target pathogens are captured by the sensor. In further study, single spore detection may be realized by decreasing the size of the sensors. The free-standing and wireless nature of the sensor allows it to be easily used in flowing liquid without the demands of a complex circuit structure. The real-time detection of B. anthracis spore in a water suspension (×108 spores/ml) at a flow rate of 40 ␮l/min using a 1 mm sensor showed that the resonance frequency of the sensor decreased continuously until the saturation of the binding occurs. The saturation occurs within 1 h after the exposure to the 108 spores/ml suspension of B. anthracis spores. The total frequency change was around 1.83 kHz, corresponding to a theoretical bound spore number of 2.02 × 104 , which is very close to actual spore counting results (1.80 × 104 bound spores) using a series of SEM pictures. For the detection of B. anthracis spores in water suspensions using a 1 mm sensor, the detection limit was found to be around 103 spores/ml and a linear response was found between the concentrations of 5 × 103 and 5 × 107 spores/ml. The binding kinetics was analyzed using a first order kinetic model. The attachment constant of a 1 mm sensor at a flow rate of 40 ␮l/min is 3.47 × 10−10 min−1 (spores/ml)−1 . The sensor was shown to be

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Biographies Wen Shen received her bachelors degree (2005) in materials science and engineering from Shanghai Jiao Tong University in Shanghai, China. She is currently a doctoral student in the Materials Engineering Department at Auburn University where she is doing research in new methodologies for food-borne pathogens and ways to enhance reliability of detection using statistical methods. In addition to her research in materials engineering, she is also pursuing a master’s degree in math and statistics. Ramji S. Lakshmanan received a BTech in metallurgical engineering and materials science and a MS degree in ceramics and composites from the Indian Institute of Technology (IIT), Mumbai, India. He is currently a doctoral student in the Materials Engineering Department at Auburn University and is presently carrying out research in the field of development of novel methodologies for detection of food-borne pathogens. L.C. Mathison is currently a senior research engineer, electronics design engineer in the Materials Engineering Department at Auburn University. He received his degree in electrical engineering from Auburn University. His experience includes electronics design for aerospace, military, computers, consumer and industrial controls and the telephone and cable TV industries. His designs include electronic circuits used on outer space satellites, aircraft, computers, power supplies, battery support systems, gas chromatographs, and battery powered vehicles. He has four patents and received outstanding engineer award from an international company for the high-speed gas chromatograph electronics. He is currently involved in research to provide new detection methods for magnetostrictive sensors and is designing data acquisition systems for use with various sensors used in materials research. Dr. Valery A. Petrenko is a professor at Auburn University—received MS in chemistry (Moscow State University, Russia, 1972), PhD in chemistry (the Institute of Organic Chemistry, Moscow, Russia, 1976), DSc in chemistry (Moscow State University, Russia, 1988) and Honor rank of professor in molecular biology (1992). In 1977–1993 he worked as Junior and Senior Scientist, Laboratory Head, Scientific Director, Director of Institute and Professor in Scientific Association “Vector” (Novosibirsk, Russia). In 1993 he moved to USA and worked as visiting and research professor at University of Missouri-Columbia and Professor at Auburn University (since 2000). He is recipient of grants from ARO and NIH and the Pfizer Animal Health Award for Research Excellence (2006). He is author of 95 peer reviewed journal articles and 4 book chapters. His research interests include monitoring and detection of biological threats, diagnosis of infectious and cancer diseases and tumor targeting. Dr. Bryan A. Chin is the director of the Auburn University Detection and Food Safety Center (AUDFS) and the chairman of the Materials Research and Education Center where he is also a professor in materials engineering at Auburn University. He has received numerous national awards from the US government and industry and is one of 50 U.S. scientists inducted into the Russian Academy of Engineering Sciences as a foreign member. He is the author of over 200 journal articles, 150 scientific reports and chapters in various major reference books. His current research is focused on the development of biosensors for food safety.