Development of zinc oxide-based sub-micro pillar arrays for on-site capture and DNA detection of foodborne pathogen

Abstract

Prevention and early detection of bacterial infection caused by foodborne pathogens are the most important task to human society. Although currently available diagnostic technologies have been developed and designed for detection of specific pathogens, suitable capturing tools for the pathogens are rarely studied. In this paper, a new methodology is developed and proposed to realize effective capturing through touchable flexible zinc oxide-based sub-micro pillar arrays through genetic analysis. Zinc oxide coated pillar arrays have a high surface area, flexible, and adheres strongly to bacteria. Therefore, it contributes to enhance the bacterial capturability. An in-depth analysis on the sub-sequential capturing process at the bacterial cell-pillar interface is presented. By carefully observing the structural changes and performing numerical analysis under different reaction times, the results are presented. The resulting zinc oxide coated pillar arrays exhibited comprehensive capturability. These pillars were able to detect pathogenic bacteria due to a combination of complex structures, depletion force, and high surface electrostatics. The developed sub-micro pillars successfully captured and detected infectious foodborne bacteria of Escherichia coli in the range of 10⁶–10¹ CFU/mL.

Introduction

Effective capture and detection of infectious pathogenic bacteria is great of importance in the field of diagnostics, therapeutics, and healthcare [1], [2], [3], [4], [5]. In particular, the rapidly growing numbers of foodborne illnesses around the world, such as food poisoning, has lead researchers to focus on the prevention of pre-and post-contamination from potential foodborne bacteria [4], [6], [7], [8]. Bacterial foodborne pathogens commonly exist on the surface of kitchenware, silverware, as well as raw food materials. This is due to the continuous exposure to inadequate hygienic facilities and inadequate sanitation practices. Even though progresses have been made in the detection and analysis of pathogenic bacteria using both immunoassays and molecular diagnostics, effective capturing tools are rarely developed and the capturing mechanisms are not understood. Therefore, to manage food safety, on-site, cost-effective, sensitive, and reliable capturing tools via touching are highly demanded.

Up-to-date, antibody-based immunoassay with zero- and one-dimensional (0D and 1D) organic and/or inorganic nanomaterials that includes carbon dots [9], [10], silica [11], [12], magnetic particles [12], [13], and nanowires [14] have been widely adopted to bind with pathogenic bacteria. However, non-specific binding issues, low adhesiveness, low stability of antibodies, and complex steps to fabricate nanomaterials with target specificity hinder application in real life [15], [16]. A promising approach is to utilize interaction between topological structures of nanomaterials and the surface of bacteria [5], [17], [18], [19], [20]. As an example, Linklater et al. enhanced the interaction between vertically-aligned carbon nanotubes and bacterial cells to realize bactericidal properties [19]. In addition, Liu et al. proposed to upgrade the performance of the bacterial capturability in fluid bloodstreams by controlling mechanical properties and nano-topological interactivity of inorganic nanowires [17]. Liu et al. showed that polycrystalline nanowires with numerous grain boundaries exhibit excellent mechanical bending properties when compared to single crystals that resulted in higher bacterial capturability. Likewise, various types of inorganic nanostructures have been researched and among them, zinc oxide (ZnO)-based nanomaterials have received a lot of attention due to its potential of interactivity with various kinds of biomolecules, including protein, deoxyribonucleic acid (DNA), mammalian cells, and bacteria [21], [22], [23], [24]. Furthermore, ZnO-based nanomaterials have exhibited good biocompatibility, non-toxicity, and significant antibacterial activity [25], [26], [27], [28]. Although the ZnO proves its capability, the low stiffness, brittleness, and strong agglomeration of ZnO-based nanoparticles and wires are still major hurdle to be used as bacterial capturing tool [29]. More importantly, the nanotopological interactivity between the surface of ZnO-based materials and pathogenic bacteria is rarely investigated which could be a key to enhance its capturability.

To overcome the challenges mentioned in the paragraph above and to adopt the mechanical and chemical characteristics of the avorementioned inorganic materials, recent advances in nanotechnology and nanofabrication techniques with polymers have allowed the fabrication of vertical nanopillar arrays [30], [31], [32]. These approaches support mechanical stability and flexibility that can be served as a unique platform for adherent mammalian cells, microorganisms, and biomolecules through nano-topological interactions [33], [34]. Inspiring from previous findings, herein, we report the novel method to capture and detect pathogenic bacteria using ZnO with a wrinkled surface and introduce positive surface charge and topological effects leading to interaction of negatively charged bacteria. The sub-micro pillar arrays (PAs) are fabricated by photo/soft-lithography process to have a 700 nm diameter and it is gold sputtered by the sonochemical method to ensure even growth. During this process the time-dependent interactivity between pathogenic bacteria and ZnO-coated PAs (Z-PAs) are investigated to determine the nano-topological interactions. Finally, it is confirmed that Z-PAs has the capability to capture and detect bacteria by genetic analysis with the assistance of polymerase chain reaction (PCR) amplification.