Microfluidic Chips for Detection of Microorganisms

Author: Burcu Yaldız, University of Ege Department of Bioengineering

   A Microorganism is a general name of a large living community. Bacteria, fungi, protozoa and many other species are accepted as microorganisms, which have critical roles in the ecosystem. However, pathogenic microorganisms harm both living beings and the economy.  Pathogens can spread from one host to another by air, body fluid, food, water, and other transport ways. Therefore, it is highly crucial to detect and identify pathogens quickly and accurately. Till today, various immunological and molecular diagnostic methods have been developed to detect them [1].

   Although these techniques are used in different fields of study, most are expensive and time-consuming. Some also have low sensitivity and specificity which decreases accuracy at mixed samples. When its advantages are considered, chip systems have become promising platforms for detection of microorganisms toward recent years.

Why Chip Systems?

   As mentioned in the above section, some of the current techniques used to detect microorganisms have some disadvantages. For example, by using a mass spectrometry method, only purified bacteria samples present at very high concentrations can be identified [1]. Another example is the semi-quantitative plate culture method, which is accepted as the gold standard for bacterial detection. Although it is highly preferred in the detection and identification of pathogenic microorganisms from clinical samples, this method is very time- consuming. In generally, culturing takes 3-4 day, particularly for pathogens that are difficult to culture.  Moreover, it cannot successfully differentiate bacteria at strain and species level [1].

  In addition, PCR is one of the most powerful techniques, it also has disadvantages. In the PCR method, thermocouples are needed to ensure fast heating/cooling temperature cycles [2]. This requirement increases the cost of instrumentation. By qPCR method, additionally, live/dead cells cannot be discriminated. Lab-on-a-Chip technology has the potential to overcome these kinds of problems. A microfluidic chip is a system allowing microliters and smaller volumes of fluids to be controlled and moved in micro-scale channels. The advantages of chip technology make working with these systems quite attractive to detect pathogen microorganisms at the clinical or industrial level or at the field of application far away from central laboratories. Studies performed on these platforms are carried out in high yield with the use of a small sample. This feature makes the chip system more flexible and cheaper than other techniques. Due to the easy standardization, automation, and control processes; the reproducibility of the work is high. Additionally, the integration of platforms enables chip systems to be combined with other techniques. In this way, miniature and portable devices can be obtained. Therefore, microorganism detection can be performed in situ and simultaneously. This property is quite substantial for pathogens that cause epidemics and deaths. To date, it has been observed that the performance of the detection methods used with the chip systems is increasing. 

Combining the microchannels with other sensors

   Until now, various techniques such as optical and electrochemical detectors, mass spectrometry and nuclear magnetic resonance have been combined with lab-on-a-chip devices [3]. Optical and electrochemical detectors are more widely chosen to be used with chip systems because of their selectivity and sensitivity such as chemiluminescence, fluorescence, dynamic light scattering, lens-free holograms etc. In studies using the CL method, chip platforms with an inactivated enzyme, antibody or nucleic acid were used to capture pathogenic microorganisms [4]. PCR method is also performed on chips to detect and identify microorganisms. Studies were carried out by carefully controlling the reaction conditions. Using PCR and RT-PCR techniques, the analysis of protected DNA or RNA sequences is widely used to detect pathogens [1]. There are specific temperatures in which the denaturation, binding and elongation steps occur in the PCR process. Consequently, the most crucial parameter in this process is the temperature and primer selection for target nucleic acid sequence.  The essential primers, buffers, enzymes, and other PCR reagents are introduced into the microchannel to carry out the detection reaction. This low volume system is theoretically more sensitive than immunoassay tests. It is also more portable than bench-top PCR devices with suitable equipment. Using PCR chips, detection was successfully performed in a clinical sample with a low concentration of influenza A [5]. In the MALDI-TOF MS method, biomolecules and large organic molecules are ionized. And then protein profiles are removed through electrical or magnetic fields. Graphical images of the profile spectra are compared with the reference organism in the database of the system. According to their harmony, microorganisms are identified at the basis of genus and species. It has been observed that combining chip systems with mass spectrometry improved the performance of MS-based approaches [6]. In a study, a chip has been developed for the detection of bacteria that exist with a big amount in the air [7]. Vibrio parahaemolyticus, Listeria monocytogenes, and Escherichia coli were identified from the samples collected in the chip using liquid chromatography and mass spectrometry [7].

Application Areas

   Recently, chip-based microorganism detection systems have been used in food control, environmental monitoring, and various clinical applications. Food safety is a serious and global problem for human and animal health. Pathogens in foods cause various epidemic outbreak and deaths. HPLC, GC, ELISA, and PCR are the most preferred methods for detecting pathogens as the gold standard. Although these methods have high precision, most are time-consuming and expensive and also there is a need for skilled technicians. After samples are taken for control, they are sent to an analysis laboratory. Here, obtaining results vary from a few hours to a few days. Therefore, it is not possible to perform the controls on-site and on time. On the other hand, since lab-on-a-chip systems can be miniaturized and automated, they have a strong potential for detection. They are also fast and sensitive. With the designed chip systems, detection can be performed at farms, packaging and distribution location and operation facilities. Furthermore, it might be possible to detect pathogens by consumers using these chip systems. The use of chip systems is also highly promising for improved environmental monitoring of microorganisms. For example, bioaerosols in the air include microorganisms such as fungi, bacteria, and viruses. Exposure to these bioaerosols can cause immunological, infectious and toxic lung diseases. Therefore, detection and identification of microorganisms in environmental samples are required to take necessary precautions against epidemics. Recently, a new study was conducted using chip systems to capture and identify airborne pathogenic microorganisms [8]. In this study, the capture chip was combined with the detection chip. 100% capture efficiency was achieved in the capture chip in approximately 9 minutes. And then detection was performed based on immunofluorescence analysis [8]. In addition to food and environmental analyzes, there are also studies conducted with chip systems for clinical studies. In a study, a lab-on-a-chip platform was designed to detect pathogens in enteric and diarrheal diseases. These pathogens cause death in children under five years of age. The designed platform can make rapid and sensitive immunological tests by using direct excretions without any sample preparation. As a result of a study, simultaneous detection of four bacterial pathogens was performed with 6 μL of sample in only 20 minutes [9].

In Consequent

   Today, various microfluidic chips are designed to detect microorganisms such as bacteria and fungi. In recent years, different studies have been carried out for this purpose. Still, chip technology for the detection of microorganisms is a new and growing area. Due to its advantages chip systems appear to be very suitable platforms for detecting microorganisms. In particular, the presence of pathogenic microorganisms is becoming more dangerous as the world population increases day by day. Nowadays, technology is developing rapidly and in parallel, the time of data transfer is shortened by the internet of things. Therefore, the detection of microorganisms from foods to clinical samples in a short time provides a great advantage for employees, consumers, and patients. Although it is a growing area nowadays, it is possible to say that the use of chip systems in microorganism detection will be quite common.

References

[1] Zhang D., et.al. (2018), doi: 10.1021/acs.analchem.8b00399 
[2] Huang G., et.al. (2017), doi: 10.1038/s41598-017-06739-2
[3] Mairhofer J., et.al. (2008), 10.3390/s90604804
[4] Yakovleva J., et.al. (2002), doi: 10.1021/ac015645b
[5] Cao Q., et.al. (2013), doi: 10.3791/50325
[6] Feng X., et.al. (2015), doi: 10.1002/mas.21417
[7] Bian X., et.al. (2016), doi: 10.1021/acs.analchem.6b02708
[8] Jing W., et.al. (2013), doi: 10.1021/ac400590c
[9] Phaneuf C.R., et.al. (2016), 10.3390/bios6040049