Ultrafast detection of infectious bacteria using optoelectronic nose based on metallic nanoparticles

Highlights

• A rapid colorimetric assay was developed for detection of bacteria volatiles.

• 10 infectious bacteria strains can be detected in 10 min and discriminated in 50 min.

• Sensitive determination with the detection limit of 10² CFU/mL was obtained.

• Urinary tract infections can be detected without needing to urine culture.

Abstract

Developing an accurate and sensitive method for detection of waterborne pathogens and urinary tract infection are important environmental and clinical challenges. We report a colorimetric assay based on arrays of nanoparticles for identification of 10 infectious bacteria strains belonging to both gram positive and gram-negative species. The detection system works based on the interaction of the volatile metabolites emitted from bacteria with the arrays of metallic nanoparticles deposited on paper substrate. The color changes, monitored by a flatbed scanner, are analyzed by statistical pattern recognition methods. A unique colorimetric pattern is achieved for each bacteria strain. We could detect bacteria at very low concentrations (around 100 bacteria/mL) in less than 10 min, which is the fastest method reported ever. The sensor can be used successfully for detection of bacteria in drinking water as well as human urine (containing 177 healthy and 123 patient samples) in less than 50 min, which is dramatically faster than urine culture. The provided detection system opens a new way to develop simple, small, sensitive and rapid devices for detecting pathogens in various clinical samples.

Introduction

Existence of different types of bacteria in water and food are major reasons for endangering the lives of millions of people. Many infectious diseases like typhoid fever, cholera and diarrhea caused by waterborne pathogens is responsible for more than 2 million deaths annually [1]. Using sensitive and cost effective diagnostic tests for monitoring pathogen can help to prevent and control the related diseases and also contribute to improving public safety and health [2]. The conventional methods such as culturing [3], enzyme-linked immunosorbent assay (ELISA) [4] and polymerase chain reaction (PCR) [5] as well as some instrumental methods including chemiluminescence [6], chromatography [7] and mass spectroscopy [8] are used for bacterial detection.

Gas chromatography-mass spectrometry (GC–MS) is a well-known approach for screening volatile bacterial metabolites [9]. The volatile compounds represent a unique chemical signature for each species or even each strain and can be used to diagnose many acute infectious diseases from analysis of breath, blood, urine and sweat samples [10]. Bacterial VOCs can be classified in six categories including carboxylic acids, alcohols, aldehydes, esters, hydrocarbons and organic sulphur derivatives [11]. Many strains of bacteria can produce the same VOCs but with different concentrations. On the other hand, some VOCs can exist in the specific bacteria metabolism pathways. For example, the concentration of ethanol in gram positive bacteria is more than gram negative bacteria which is inverse for acetaldehyde and formaldehyde. Propanol, pentanol isomers, acetic acid, and dimethyl disulfide were shown only in the E. Coli VOCs profile while butyl butyrate, 3-Methyl butyrate and 2-Methyl butyrate were indicated in chemical composition of staphylococcus strains [11]. Therefore, the ability of GC–MS to find the difference in the concentration of VOCs or difference in the chemical composition of bacteria strains can help to discriminate and identification of different bacteria in environmental and biological samples.

The mentioned methods have many benefits such as detecting the trace amount of bacteria or consuming the volume of analyte as lower as nanoliter, but they are very costly, needing tedious sample preparation, complex instrumentation and trained personnel.

Developing electronic nose can help diminish the above limitations and provide user-friendly method by analyzing the odorous headspace of various materials such as food [12,13], explosive [14] and toxic materials [15]. These devices, which are fabricated by conductive polymers, metal oxides or chemical dyes, can also be used to identify the volatile metabolites of bacteria [[16], [17], [18], [19], [20], [21]] and fungi [[22], [23], [24], [25], [26]]. Based on colorimetric mode, Carey et al. identified 10 strains of bacteria classified in the Enterococcus faecalis and Staphylococcus aureus by using a colorimetric sensor array (CSA) including 36 chemoresposive dyes within 10 h [18]. In the other study, Lonsdale et al. developed a CSA based on 80 chemoresponsive dyes for discrimination of pathogenic bacteria which is classified in Yersinia pestis and Bacillus anthracis. The analysis were done in the range of time between 6−30 h based on type of strains [27]. Also, Lim et al. reported a CSA consisting of 73 chemoresponsive dyes for identifying 18 species of bacteria within 3 h of growth detection and with 91.9 % accuracy [28]. As can be seen, the sensitivity and response time of these E-nose has not been resolved because of weak interaction between analyte and sensor and the minimum time for detection of bacteria reported by these methods was 3 h [28].

Colorimetric detection based on nanoparticles has gained more attraction in the recent years due to many advantages such as unique physicochemical properties as well as high sensitivity and good stability [29]. Aggregation or changing in morphological surface of nanoparticles leads to change in their optical properties which are recorded as response of the chemical sensor [29]. Nanoparticles stabilized by different capping agent such as polymer and enzyme were applied to detect bacteria in solution [30]. These methods tried to monitor merely the presence of one or two strain of bacteria with high detection limit after preparing the used samples while the detection with electronic nose does not require any preparation.

Very recently, we introduced a new generation of the paper-based optoelectronic noses, which use arrays of silver and gold nanoparticles as sensing elements. Our nano-optoelectronic noses (NOENs) could detect and determine trace amounts of volatile compounds (VOCs) as low as 10 ppb [31]. In addition to this promising sensitivity, they exhibited differential selectivity to VOCs, resulted from different physicochemical properties of the used capping agents, such that 45 VOCs were accurately discriminated.

In this study, the paper-based NOENs were optimized to detect and discriminate different strains of bacteria based on their volatile metabolites. The studied bacteria are the main environmental, waterborne and foodborne pathogens that responsible for wide range of infectious diseases such as lung, kidney, intestine, stomach, bladder and the other part of urinary system. They also cause pneumonia and sepsis in children and old people [32]. We aim to investigate the ability of the proposed sensor for detecting the volatile metabolite emitted from bacteria by observing the changes in the color of sensing element after interaction with VOCs, and to evaluate sensitivity of the developed sensor for direct detection of small colonies of bacteria human urine without need to culture.