Specificity of polyclonal antibody-based magnetostrictive biosensors

Magnetoelastic (ME) biosensors were developed by immobilizing polyclonal antibody using Langmuir-Blodgett (LB) technique onto a magnetostrictive platform, which can offer wireless and remote detecting, a unique advantage over conventional sensor platforms. Due to the magnetoelastic nature of the amorphous magnetostrictive alloy, the sensor exhibits a physical resonance when it undergoes a time-varying magnetic field, a shift in resonance frequency occurs when its mass changes due to bacteria binding, and the bound bacterial cells were visibly confirmed by scanning electron microscopy (SEM) micrographs, then the density of bacteria bound on the biosensor was calculated. The specificity to detect different kinds of bacteria, including Salmonella species and nonSalmonella species was studied. The results show that the prepared polyclonal antibodybased biosensor shows excellent specificity to detect bacteria of Salmonella species, and especially prefers to bind Salmonella typhimurium, almost zero affinity for nonSalmonella species of Listeria monocytogenes.


INTRODUCTION
Magnetostrictive materials are made of amorphous ferromagnetic alloys composed of Iron, Nickel, Molybdenum and Boron. Due to the magnetoelastic nature of the amorphous alloy, the sensor exhibits a physical resonance when exposed to a longitudinal time-varying magnetic field and a DC biasing field. By employing a suitable bioprobe for detecting pathogens, a biosensor can be developed. This characteristic allows the biosensors to be monitored remotely without the use of direct physical connections, such as wires, enabling detection of environmental parameters from within sealed, opaque containers [1][2][3] . Antibodies are immune system-related proteins called immunoglobulins, which can selectively bind target antigen due to their special structure and different amino acid sequence [2][3][4] ; hence, it was selected as the bioprobe in this research.
As well known it is necessary for biosensors to have good specificity in practical applications. Therefore, an essential test for the characterization of any biosensor is to investigate the specificity of the bioprobe (the bio-molecular recognition element). Hence it is of important research value to investigate the specificity of the developed biosensors.
In this work, Polyclonal Antibody immobilized magnetostrictive biosensors with the size of 5mm x 1mm were prepared and the specificity of the magnetostrictive sensors for detecting different kinds of bacteria, including 8 kinds of Salmonella species and 1 kind of non-Salmonella species was studied.

Materials
The magnetostrictive material used in this work was purchased commercially from Honeywell International Corporation (2826 MB); its composition is Fe 40 Ni 38 Mo 4 B 18 with a thickness of about 30µm. The flat sensor samples were diced from this magnetostrictive material after dry polishing. Polyclonal Antibody used in this work was purchased from AbCam Company. All kinds of bacterial cells were prepared in the department of Nutrition and Food Science at Auburn University.

Sensor platform
The biosensor is comprised of two main parts: the first part is magnetostrictive substrate used as transducer; the second part is Polyclonal Antibody layer used as the bioprobe for binding bacterial cells, which was immobilized onto the sensor surface using the Langmuir-Blodgett (LB) method. The biosensor exhibits a physical resonance when it undergoes a time-varying magnetic field, thus emitting magnetic flux, this can be monitored remotely without the use of direct physical connections, due to the magnetoelastic nature of the amorphous magnetostrictive alloy [5] . Schematic illustration of the wireless nature of the magnetostrictive biosensors was shown in Figure 1.

Figure 1 : Schematic illustration of the wireless nature of the magnetostrictive biosensors and principle for bacteria detection Experimental
After biosensors were prepared, they were immersed in 1 ml solution of different kinds of bacterial cultures with a concentration of 1×10 9 cfu/mL for 30 minutes for binding bacterial cells. The resonance frequency of the sensors was measured using a HP network analyzer 8751A with S-parameter test set at 87511A before and after bacterial cells binding. Then the sensors were removed and exposed to Osmium tetra Oxide (OsO 4 ) vapor for one hour to fix the bacterial cell's wall. Finally, SEM images were taken using JEOL 7000F, operating at 5kV, which was used for investigating the physical distribution and density of the bacterial cells attached to the sensor surface.

Scanning electron microscopy (SEM)
Polyclonal antibody immobilized sensors with the size of 5mm x 1mm were exposed to the following eight kinds of   Figure 3 shows that the polyclonal antibody preferred to bind Salmonella typhimurium, though existed some affinity to other Salmonella species, but almost zero affinity was seen with Listeria monocytogenes. Thus, the conclusion could be drawn that the polyclonal antibody exhibited excellent specificity to Salmonella species bacteria.

CONCLUSIONS
Polyclonal antibody immobilized magnetostrictive biosensors were prepared with a unique advantage over conventionally used platforms, which needs no physical contact in order to obtain a response. The specificity to detect different kinds of bacteria, including Salmonella species and non-Salmonella species was studied. It was found that polyclonal antibody showed excellent specificity to detect bacteria of Salmonella species, and especially prefered to bind Salmonella typhimurium, almost zero affinity for non-Salmonella species of Listeria monocytogenes.