Development of a microfluidic biosensor for detection of environmental mycobacteria

Abstract

In this paper, we describe the development of a microfluidic culture-based biosensor for detecting mycobacteria in environmental samples. The biochips rely upon the unique paraffinophilic (i.e., wax-loving) nature of mycobacteria to rapidly and selectively differentiate them from non-target microorganisms. A new method of depositing and patterning paraffin was developed to fabricate the prototype biosensors using Opticlear™ as solvent to generate liquid paraffin and pattering with positive photoresist lift-off. The prototype biochips were experimentally tested to demonstrate the concept of rapid and selective detection of mycobacteria using pure cultures and epifluorescence microscopy to visualize microorganisms on the surface. Our successful demonstration of the culture-based biochip technology presents an alternative approach for developing new technology to track microorganisms in complex environmental samples. A microfluidic chip to selectively culture mycolic acid containing actinomycetes from environmental samples will improve screening for mycobacteria to protect public health as well as screening for mycobacteria to reduce the costs associated with treating nocardiafoam at sewage treatment plants.

Introduction

For environmental engineers, microorganisms are important for a number of reasons. First, pathogenic microorganisms represent a threat to public health and must be eliminated from potable water; second, biocatalytic microorganisms are used to treat toxic pollutants (i.e., bioremediation) and thus should be encouraged to thrive; and third, nuisance microorganisms upset the reliable performance of bioprocesses. For environmental engineers, mycobacteria represent all three types. For instance, Gordonia (formerly Nocardia) amarae have been shown to be a causative agent for the formation of filamentous nuisance biological foam (e.g., nocardiafoam) on the surfaces of aeration basins, secondary clarifiers, and anaerobic digesters in municipal sewage treatment around the world [1], [2], [3]. The problem of nocardiafoam has been studied for more than 30 years, and the estimated total accumulative cost of incidences of nocardiafoam disrupting the acceptable performance of sewage treatment plants is of the order of 1 billion dollars [4].

Mycobacteria are slim, rod-shaped microorganisms that are 1–10 μm long [5] with a High Guanine plus Cytosine content of DNA (High G + C Gram positive bacteria). The mycobacteria include six distinct genera: Corynebacterium, Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella [6]. Perhaps the most notable of these microorganisms are the pathogens Mycobacterium tuberculosis (i.e., the etiological agent of the disease “TB”) and Mycobacterium leprae (i.e., the etiological agent of the disease “leprosy”). Although TB was nearly eradicated during the 20th century, the occurrence of illness is rising, with more than 2 million annual deaths attributed to TB especially in less developed countries—the largest number of deaths attributed to a single cellular microorganism. This resurgence of TB is significantly more dangerous because the wide-spread use of antibiotics over the past 50 years has lead to the emergence of Mycobacterium demonstrating multiple-antibiotic resistance [7]. Furthermore, with the outbreak of human immunovirus (HIV)-induced acquired immunodeficiency syndrome (AIDS) in the industrialized world beginning in the 1980s, the infection of immunocompromised individuals with opportunistic pathogens belonging to the Mycobacterium avium complex (MAC) has risen dramatically [8], [9]. Although a historically well-known opportunistic pathogen, MACs generally do not infect individuals with healthy immune systems. Instead, individuals suffering from HIV infection, patients on a regimen of immuno-suppressant medication, as well as children and the elderly are the most commonly infected individuals. Because MAC infection primarily occurs due to oral ingestion, with inhalation as a secondary route, and because MACs are ubiquitous in nature the United States Environmental Protect Agency (US EPA) listed MACs as one often microbiological agents of interest on the Drinking Water Contaminant Candidate List (CCL) originally released in 1998 [10], [11] and updated in 2005 [12].

Due to their preeminent role in a variety of important environmental systems, robust, rapid, and easy-to-use technology to monitor mycobacteria is needed. The traditional methods to identify and monitor mycobacteria in environmental samples include direct observation using microscopy, often coupled with staining procedures or cultivation on semi-selective media [13], [14], [15]. Although these traditional methods have been used successfully to track mycobacteria in a variety of environmental samples, they suffer a number of limitations. For example, direct observation using microscopy requires significant training to recognize staining properties and the morphology of individual populations of microorganisms. Furthermore, it is accepted that the staining properties and morphology of microbial populations can change dramatically depending upon the environmental conditions experienced by the microorganisms. Thus, the robust nature of direct observation using microscopy can be questioned. Traditional cultivation of mycobacteria can require 3–5 weeks for the growth of colonies on agar surfaces. Obviously, such a turnaround time for an assay is extensive and cannot be classified as “rapid” by practical standards.

Alternatively, significant efforts have been invested in the development of molecular biology-based assays to identify and enumerate mycobacteria in environmental samples, including the detection of mycobacteria with antibodies [16], [17], [18], fluorescence in situ hybridization (FISH) targeting 16S ribosomal ribonucleic acid (16S rRNA) [16], [19], [20], [21], [22], and fatty acid methyl ester (FAME) analysis [23]. These methods show promise for identifying mycobacteria because they are sensitive, specific, and rapid. However, molecular biology-based techniques often lack the ability to distinguish between living and dead organisms, do not generate cultures that may be assayed subsequently for additional phenotypic traits (e.g., including antibiotic sensitivity), and are yet to be integrated into a user-friendly format. These drawbacks have caused cultivation to remain the primary means of screening for mycobacteria in clinical and environmental samples.

To improve culture-based techniques for monitoring specific microbial populations in environmental samples, we have developed a novel approach using microfabrication to create devices to rapidly screen for mycobacteria. Rather than using molecular biology-based assays to identify mycobacteria, we have miniaturized the culture-based assay to rapidly identify individual microbial populations adhered to a semi-selective surface material inside a microfluidic microchannel. In this paper we present the development of such a microfluidic biosensor and the application of this approach to rapid detection of mycobacteria in samples removed from a municipal activated sludge sewage treatment system.