Elsevier

Biosensors and Bioelectronics

Volume 26, Issue 4, 15 December 2010, Pages 1612-1617
Biosensors and Bioelectronics

Surface functionalization of electrospun nanofibers for detecting E. coli O157:H7 and BVDV cells in a direct-charge transfer biosensor

https://doi.org/10.1016/j.bios.2010.08.028Get rights and content

Abstract

Electrospinning is a versatile and cost effective method to fabricate biocompatible nanofibrous materials. The novel nanostructure significantly increases the surface area and mass transfer rate, which improves the biochemical binding effect and sensor signal to noise ratio. This paper presents the electrospinning method of nitrocellulose nanofibrous membrane and its antibody functionalization for application of bacterial and viral pathogen detection. The capillary action of the nanofibrous membrane is further enhanced using oxygen plasma treatment. An electrospun biosensor is designed based on capillary separation and conductometric immunoassay. The silver electrode is fabricated using spray deposition method which is non-invasive for the electrospun nanofibers. The surface functionalization and sensor assembly process retain the unique fiber morphology. The antibody attachment and pathogen binding effect is verified using the confocal laser scanning microscope (CLSM) and scanning electronic microscope (SEM). The electrospun biosensor exhibits linear response to both microbial samples, Escherichia coli O157:H7 and bovine viral diarrhea virus (BVDV) sample. The detection time of the biosensor is 8 min, and the detection limit is 61 CFU/mL and 103 CCID/mL for bacterial and viral samples, respectively. With the advantage of efficient antibody functionalization, excellent capillary capability, and relatively low cost, the electrospinning process and surface functionalization method can be implemented to produce nanofibrous capture membrane for different immuno-detection applications.

Introduction

Nano-structured materials, such as nanowires and nanofibers, which have unique biochemical properties, are promising candidates to fabricate biosensors with high sensitivity, rapid response and low cost. Biocompatible materials, such as nitrocellulose, polyvinylidene fluoride, and polyethersulfone, are known for their excellent binding ability to separate and capture biomolecules and pathogenic cells from biological samples. Based on these unique biochemical properties, low cost and high sensitivity biosensors have been developed based on immunochromatography and immunoassay for detecting infectious disease organisms (Lin et al., 2008), analyzing drugs of abuse (Zhang et al., 2006), veterinary testing (Beck et al., 2000), agricultural applications, environmental testing, and product quality evaluation (Wong and Tse, 2008). In recent years, many technological advances have been directed towards the development of nano-structured biosensors to obtain enhanced surface-area-to-volume ratios and reduced cross-sections, offering more effective bio-target immobilization capabilities (Basu et al., 2004, Mehdinia et al., 2009). Most development efforts are focused on conventional planar structured mesoporous layers.

New type of sensor material with one-dimensional (1D) structure, such as carbon nanotubes and metal oxide nanowires, has been introduced which provide unparalleled fast mass transfer capability of analyte molecules (Claussen et al., 2009). The 1D nanostructure has exceptional advantages in surface to volume ratio which determines the binding effect and sensor response. However, sensors based on single nanowire suffer from inherent statistical variation which induces sensing property deviation and excessive noise levels. Instead of random approach, sensor structure with multiple nanowire networks has the advantages of high reproducibility, low noise, and high reliability.

Electrospinning is a versatile and cost effective method to produce nanofibrous membranes with uniform fiber diameters in nanoscale (Wang et al., 2002). The process is based on the principle of electrostatic to draw ultrafine solid threads from material solutions, which does not require coagulation chemistry or high temperatures. This non-invasive method is particularly suited to fabricate nanofibers using biological materials. The surface area is significantly increased by the novel nanostructure compared to conventional planar materials, which enhance binding effect and reaction rate (Huang et al., 2003). Moreover, the electrospinning process provides many attractive advantages to develop high performance nano-structured materials for biomedical and biosensor applications, including inherent stability, compatibility with micro-fabrication process, high production yield and relatively low cost (Kim et al., 2006). The electrospun nanofiber with biomimetic structures has been developed in different morphologies for tissue engineering (Li et al., 2006), drug delivery (Zeng et al., 2003), and artificial organ implant applications (Venugopal et al., 2008). The excellent binding capability improved the performance in molecular absorption (Nama et al., 2009) and cell adhesion (Hou et al., 2009). In recent years, more research efforts have been directed towards biosensor applications, such as biomolecular detection and enzyme functionalization (Wang et al., 2009, Ren et al., 2006, Sawicka et al., 2005). However, its application in whole cell detection of microbial or viral pathogens has not been reported in the literature.

This paper presents electrospinning method to produce nitrocellulose nanofibrous membrane, and its optimization and functionalization with biological treatment for biosensor applications. A biosensor based on the electrospun capture membrane is designed by integrating magnetic separation, capillary immunoassay, and direct-charge electrical measurement for rapid and quantitative detection of bacterial and viral pathogens. In this experimental study, the process condition and parameters of electrospinning and biological functionalization were optimized to improve capillary action and biomolecular capture capability to achieve enhanced sensor response and sensitivity. The electrospun nanofibrous membrane was synthesized from nitrocellulose polymer solution with ultrafine fiber diameter around 150 nm. And surface functionalization method was developed and optimized according to the specific pathogen target. The high surface area of the material allowed more biological events to occur and facilitated assay kinetics. The electrospun biosensor was tested using both bacterial and viral samples to verify the binding and separation performance of the bio-modified nanofibrous membrane.

Section snippets

Electrospun material synthesis

The electrospun nanofibrous membrane was fabricated using the Nanofiber Electrospinning Unit (NEU, Kato Tech Co., Japan), which is shown in Fig. 1A. The electrospinning process uses the principles of electrostatic generated by high voltage power source between the needle spinneret and collector to produce its fine polymer jet from polymer or composite material liquid (Fig. 1B).

The electrospun material was fabricated using nitrocellulose polymer due to its excellent biocompatibility and

Results and discussion

The effect of surface functionalization method was verified using a confocal laser scanning microscopy (CLSM). Fluorescein isothiocyanate (FITC) conjugated antibody was functionalized on the electrospun nanofibers using a bio-modification process described above. After several wash steps to remove unbound antibodies, the CLSM was used to observe the antibody attachment by measuring the laser excited fluorescein emission at 530 nm (Fig. 4A). The untreated nanofiber was also analyzed to compensate

Conclusion

An electrospun biosensor based on immunochromatography, nanoparticle based magnetic separation, and immunoassay was designed and optimized for rapid and sensitive detection of microbial and viral pathogens. Due to the unique nanostructure and biocompatibility of electrospun nitrocellulose membranes, the biosensor had linear detection response for E. coli O157:H7 and BVDV virus samples. In an 8 min detection process, sensitivity of the portable and low cost biosensor was 61 CFU/mL and 103 CCID/mL

Acknowledgments

This project is funded by the Michigan Economic Development Corporation through the Michigan 21st Century Jobs Fund under contract number 06-1-P1-0262. The authors wish to thank Dr. Daniel Grooms for providing the BVDV samples and BVDV antibodies.

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