Magnetic Particles Help to Detect Foodborne Pathogens

In March 2020, Professor Sung Yang from School of Mechanical Engineering, GIST, Republic of Korea and his colleagues published an article about magnetophoresis-based microfluidic device for sample concentration of foodborne pathogens on Food Control. Let’s see what they have studied.

The detection of foodborne pathogens is mainly with the help of medical, physical and chemical, biological, and other methods to comprehensively and deeply analyze the impact of microorganisms on human health. Scientific and effective foodborne pathogen test can timely grasp the situation of food contamination, determine whether food is safe or not, let people eat healthy and safe food, and avoid unnecessary economic losses and adverse social effects. Foodborne bacteria constitute a major public health problem in the world. CDC estimates that each year roughly 1 in 6 Americans (or 48 million people) gets sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases. At the same time, if there is no foodborne pathogen test, once there is a food safety accident, it is very difficult for medical staff to find out the cause timely and accurately, affecting the patient’s physical recovery. Accurate and complete foodborne pathogen test data can also provide an evaluation basis for hygiene.

Therefore, foodborne pathogen test is very important, and it must be carried out in strict accordance with relevant laws and regulations to minimize errors, ensure the accuracy and timeliness of test data, and really provide consumers with healthy and safe food. Finally, through the foodborne pathogen test, we can determine whether the whole food processing environment and process is up to the standard. According to the test results, we can also determine the degree of food contamination by bacteria in time, providing a basis for relevant departments to carry out health management work, and effectively preventing the occurrence of accidents such as food poisoning and infectious diseases. And it can also enable relevant departments to make targeted preventive measures and emergency plans in time.

The available methods for the detection of the foodborne pathogens:

• Culture-based method

The culture-based method is the oldest method for detecting microorganisms and even pathogens. This method gives definite results regarding the presence of specific pathogens. This method has a high success rate and is cost-effective. However, the biggest disadvantage of the culture-based method is slow growth. Due to the slow growth, it takes too much time to obtain the final result (often 18-24 hours), which may prove to be fatal.

• Immunoassays

Immunoassays were developed because they are easier to perform, gave faster results, and were cheaper. Therefore, an immunoassay is usually performed before a polymerase chain reaction (PCR)-based method. Enzyme-linked immunosorbent assay (ELISA) is one of the most widely used immunoassays so far. Antibody purity plays an important role in the success of immunoassays. In addition to purity, another factor that affects detection is the specificity of the antibody. Polyclonal antibodies are polyvalent (can react with multiple epitopes). This affects the assay, resulting in lower specificity and sensitivity. It must also be noted that this method may have false-positive results.

• PCR-based method

The main advantages of PCR are rapid and sensitive. It is faster than culture-based methods and immunoassays. It only takes 30 minutes to obtain the amplified product, and because multiple pairs of primers are used, the distinction between strains becomes much easier. With the lower detection limit of DNA amplicons as low as femtograms (10^-15 g), the detection limit of PCR can reach even lower. The PCR method can replace the tedious and time-consuming pathogen cultivation and identification procedures in food safety laboratories. But its difficulties include cell lysis and nucleic acid extraction, cross-contamination, and reaction failure due to the presence of inhibitory substances or competing DNA from non-target cells. This can lead to inconsistent results and reduce the appeal of PCR as a reliable method.

• Biosensors

The biosensor is the latest of all detection systems, some of which have better detection limits, greatly reducing and eliminating the disadvantages associated with PCR technology. A biosensor is a device used for pathogen detection. It usually includes three elements, namely, biological capture molecules (probes and antibodies), methods to convert capture molecule-target interactions into signals, and output data. Although the detection efficiency is higher, the results obtained using molecular biology methods may be affected by various food matrices.

• Other methods

DNA microarrays are currently receiving more and more attention due to their rapid, sensitive, specific, high-throughput analysis and other advantages, and have become a useful analysis tool. Other commonly used methods include ultrafiltration, immunomagnetic assays (IMS), immunochromatic assay (ICA), flow cytometry (FC), and lyophilization.

Methods such as cell culture, PCR, and immunoassay require skilled operators and expensive equipment. Therefore, to detect quickly, accurately, and simply, various biosensors have been developed. Most biosensors use a small amount of samples (a few tens of microliters) to detect bacteria. However, in Korea, the maximum acceptable limit for bacterial contaminants is 0 CFU/mL. Therefore, it is necessary to use at least a few milliliters of samples for bacterial testing to obtain statistically significant results. A small amount of low-concentration samples may cause false-negative results in bacterial tests because these samples may not contain bacteria. In this case, multiple tests must be performed to process a statistically significant volume of the sample (a few milliliters). In addition, the detection limit of most biosensors is very low, about 10¹-10² CFU/mL, which is still higher than the bacterial detection criterion for food samples. Therefore, it is necessary to concentrate food samples from a few milliliters to a few tens of microliters to obtain results that meet the criterion. At the same time, reducing the sample size can enhance statistical significance. Therefore, sample pretreatment (such as bacterial concentration) is essential for the rapid and accurate detection of foodborne bacteria.

In order to shorten the sample pretreatment time, the magnetophoresis-based bacteria concentration devices have been extensively studied. The target sample can be labeled with various biologically active groups combined with magnetic particles (MPS). MP-binding bacteria (MP/Bac) can be separated from the food matrix by an external magnetic field. In particular, the continuous sample pretreatment device based on microfluidic technology has been extensively studied in order to achieve rapid sample concentration. Professor Yang said, “The microfluidic platform presents multiple advantages such as small size, portability, low cost, and easy integration with various types of devices. However, most magnetophoresis-based concentration devices are fabricated as two-dimensional structures and have very short channel lengths. Therefore, the MPs cannot sufficiently move toward the channel wall near the magnet under high flow rate conditions, limiting the separation efficiency.” In addition, since the microfluidic channel and the magnet should be placed in a two-dimensional space, therefore, as the channel length increases, the device size and the distance between the channel and the magnet will also increase. Thus, the continuous bacterial concentration device based on the magnetophoresis method has low throughput.

Yang and his team developed a magnetophoresis-based microfluidic device to achieve rapid and continuous concentration of bacteria (Figure 1). The device is a three-dimensional structure composed of a commercial polyethylene tube and PDMS channel. By adjusting the length of the channel, the concentration of bacteria at a high flow rate is realized. For example, they conjugated Staphylococcus aureus with MPs (protein-A functionalized MPs, WHM-X032, CD Bioparticles, USA) before concentration, and found that the optimal channel length was 12 turns at a flow rate of 40 mL/h. Experiments with different channel lengths and flow rates with deionized water verified the calculated optimal channel length. When the flow rate was 40mL/h, the separation efficiency was greater than 96%, and the concentration factor was greater than 110 times. In addition, the performance of the device was evaluated using milk and homogenized cabbage samples. The separation efficiency and concentration factor obtained were greater than 92% and 117 times, respectively. However, due to the sudden expansion of the structure, the flow at the junction of the tube and the PDMS channel became unstable at high flow rates, so the performance of the proposed device was decreased. This problem can be overcome by using tubes of different diameters and designing the PDMS channel structure to reduce the associated vortex. In addition, the device can be easily tuned by adjusting the number of turns of the tube channel. Therefore, the device is expected to be used for bacterial concentration under high flow conditions in the fields of food, medicine, and industry.

Reference:
Jung, T., Jung, Y., Ahn, J., & Yang, S. (2020). Continuous, rapid concentration of foodborne bacteria (Staphylococcus aureus, Salmonella typhimurium, and Listeria monocytogenes) using magnetophoresis-based microfluidic device. Food Control, 107229.