Lab-on-a-Chip Electrochemical Biosensors for Foodborne Pathogen Detection: Comparison
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Please note this is a comparison between Version 2 by Jason Zhu and Version 1 by Or Zolti.

Foodborne pathogens are an important diagnostic target for the food, beverage, and health care industries due to their prevalence and the adverse effects they can cause to public health, food safety, and the economy. The standards that determine whether a given type of food is fit for consumption are set by governments and must be taken into account when designing a new diagnostic tool such as a biosensor platform. In order to meet these stringent detection limits, cost, and reliability standards, recent research has been focused on developing lab-on-a-chip-based approaches for detection devices that use microfluidic channels and platforms. The microfluidics-based devices are designed, developed, and used in different ways to achieve the established common standards for food pathogen testing that enable high throughput, rapid detection, low sample volume, and minimal pretreatment procedures. Combining microfluidic approaches with electrochemical biosensing could offer affordable, portable, and easy to use devices for food pathogen diagnostics.

  • foodborne pathogens
  • food safety
  • biosensors
  • electrochemical sensors
  • standards

1. Foodborne Pathogen Statistics and Standards

The standard that governs the standard for the allowed concentration of foodborne pathogens is set by governmental organizations. The standard of whether or not a product is fit for human consumption is set for each specific type of food products separately such as dairy, shellfish, ready to eat foods, etc. While some countries like Great Britain set quantitative limits that correspond to consuming population susceptibility and the infective dose, other countries like the United States of America have mostly set the standard on any detectable trace of these pathogens in the products tested [35,36,37,38,39][1][2][3][4][5]. These standards are affected by the minimum infective dose (MID) of each foodborne pathogen, and describe the detectable amount in a specific food or drink. Both the standards, MID, products that contain the pathogen. Not all pathogens appear in both standards, which is due to its prevalence in that country. These standards will be mostly used during testing prior to the product reaching the consumer or during specific steps in the production and distribution chain [35,36,37,38,39,40][1][2][3][4][5][6]. It is important to also analyze the effect of the most common pathogens on the infected individuals. The biggest issue with such analysis is generated from the nature of the illness, which in most cases will be very mild and will not be diagnosed.

2. Microfluidic Channel Material Choice

The characteristics of a microfluidic channel are derived from the material used to fabricate them. The channel’s biocompatibility, reusability, fabrication simplicity, and cost are among the first characteristics that come to mind when considering a microfluidic channel for electrochemical biosensor use. In addition to the qualities that are important, their ability to be used in the field, the use of low sample volume, and compatibility to be used with multiple different pathogens (multiplexability) will make a huge impact when they are considered as a viable solution for detection in industrial settings [51,52,53,54][7][8][9][10]. Focus on microfluidics for foodborne pathogen electrochemical detection since 2017 has shown that the main materials used are glass [55][11], polydimethylsiloxane (PDMS) [16,17,19[12][13][14][15][16],20,56], thermosets [18][17], paper [57][18], and thread-based [58][19]. In addition, there are also other materials that should be considered as microfluidic channel fabrication materials, although not as common such as polymethylmethacrylate (PMMA), cycloolefin polymer (COP), cyclic olefin copolymer (COC), and silicon.

2.1. Glass

The word glass is used to describe various materials among them: borosilicate [51[7][20][21],59,60], Pyrex [61][22], soda lime [62][23], quartz [63][24], and others [64,65][25][26]. Glass microfluidic channels are usually fabricated by using photolithography to print complex patterns on them and etch specific areas to form channels of specific height and width. Due to its amorphic structure, glass etching usually results in a round cross-sectional profile, which can help create a more homogenous flow pattern but forms a challenge when a high aspect-ratio is required [51,59,61][7][20][22]. Other fabrication methods include micromachining, where material is removed from the substrate and bonding or adhering it to another substrate [60][21] or laser patterning, where a beam of high-energy laser is used to pattern the channel [62,63][23][24]. These fabrication methods are highly accurate, where their resolution is determined by the etching chemicals or the resolution of the lithography and laser machines. The machinery usually comes with a very high-price tag and the use of harsh chemicals increases the complexity of the fabrication process. Glass is also thermoconductive, which means that the temperature of the work environment will be limited to a range that will not affect either the sample or the sensor. Another limitation comes from the hardness of glass and its brittle nature, which makes it harder to use in field conditions and makes the addition of valves or bonding very challenging. Although glass is very biocompatible, it is not permeable to gases, which limits the time a live pathogenic sample can survive in it. Additionally, glass has some very attractive qualities. Its resistance to most organic solvents significantly improves its ability to be washed and reused for multiple experiments, and it is very compatible with metal deposition, high surface stability, and as a substrate, it is commonly found, which reduces the overall cost of the devices. Another quality of glass is its electro isolation property, which allows for the incorporation of electrophoresis within it [51,52,53,54,55,66][7][8][9][10][11][27].

2.2. Silicon

Silicon microfluidic channel fabrication is very similar to glass. Si microfluidic channels are fabricated by means of micromachining and photolithography and wet etch similarly to the fabrication of glass microfluidics. Another technique that is mostly used in Si is the buried channel technique, where a deep vertical trench is etched into the Si by deep reactive ion etching (DRIE), followed by isotropic etching of the bottom [67][28]. Another advantage of Si lies in its ability to fabricate thin membranes that can be used to form integrated micropumps [68][29] and microvalves [69][30] in the channel. Silicon and glass have very similar characteristics, but one major difference is due to the crystalline structure of silicon, which causes a rectangular cross-sectional shape, while glass has a round one. Another difference between the two is the fact that Si is opaque and will not let light pass through it [51][7].

2.3. PDMS

PDMS is the most popular material used for microfluidic fabrication, in general, and as a microfluidic channel for foodborne pathogen microfluidic electrochemical detection specifically [16,17,19,20,56][12][13][14][15][16]. PDMS microfluidic channels are fabricated using a mold, also known as soft lithography, along with low temperature curing, which makes it very repeatable and highly cost efficient. The advantages of PDMS includes the ease of bonding with other PDMS components to form complexed multi-level channels, and hard substrates such as glass or Si to provide mechanical stability. It is very compatible with a high concentration of valves, it is biocompatible, possesses very low toxicity, has a high permeability to gases, which allows a long biostability time of living pathogens in the channel, and supports a very low resolution and any cross-sectional profile, which depends on the mold used. While these advantages make it a very popular material to be used, it also possesses some significant disadvantages. PDMS is hydrophobic and tends to adsorb or absorb small hydrophobic molecules into its walls and cause swelling. It is very sensitive to most organic solvents, which restricts it to aqueous samples only and reduces its reusability. The rigid surface of PDMS can cause pathogens to be trapped in its surface, and its high permeability to gases can change the concentration of a sample due to water evaporation through its walls [51,52,53,54,66][7][8][9][10][27].

2.4. PMMA

PMMA is a transparent and rigid thermoplastic polymer, which makes it ideal for sustainable applications. It also possesses a glass-like quality with its clarity, UV resistance, low-toxicity, and transparency, with half the density and an order of magnitude better impact resistance. PMMA is chemical resistant and is not affected by aqueous solutions, detergents, inorganic acids, alkalis, and aliphatic hydrocarbons. Among its disadvantages are low impact resistance with respect to other polymers, very limited heat resistance, sensitive to some organic solvents, poor wearing resistance, and tends to crack under medium to high load [70,71][31][32]. One of the common fabrication methods is hot embossing, where a piece of PMMA is placed on a Si or metal negative master pattern. The system is then heated under continuous pressure [72][33]. Another technique is room-temperature imprinting, where the PMMA is placed on a silicon template and then pressed together under high pressure [73][34]. As with most polymers, PMMA microfluidic channels can also be made with injection molding, where PMMA pallets are melted and injected on a master template under high pressure and then cooled down to room temperature [74][35]. Finally, as in glass and silicon, PMMA can be molded into microfluidic channels by laser ablation or wet etching [75][36].

2.5. COP/COC

COP and COC are promising materials for microfluidic channels due to their chemical resistance for polar solvents, low water absorption, transparency, ease of fabrication, and bio-inertness [76,77][37][38]. COP and COC fabrication methods are laser ablation, injection molding, hot embossing, and nanoimprint lithography. They are also thermoplastic polymers like PMMA and possess a very high electric insulating capability. Their major disadvantage is their low chemical resistance to organic solvents [78,79][39][40].

2.6. Thermosets

Thermosets such as VisiJet® M2R-CL [18][17] are very limited in their microfluidic applications, mostly due to their high cost and high stiffness. Their advantages lie with their high chemical and thermal stability, which improves their field compatibility and reusability. Furthermore, their compatibility with 3D printing allows them to be shaped into highly complexed channels, but reduces their ability to work with a low sample volume. Thermosets can also support very high aspect ratio due to their high strength [51,52][7][8].

2.7. Paper

The fabrication of paper-based microfluidics is conducted by creating hydrophobic barriers on selected areas on the paper substrate to force the sample to flow in a specific path. Paper-based microfluidics are very cost effective. Paper-based microfluidics are also very simple to make, have high porosity and physical absorption, which makes them more compatible with the field. They are easily sterilized and modified, which assists in letting only specific biocomponents through, are biocompatible, and do not require a pump or any other supporting equipment. Alternatively, paper-based systems have very low mechanical properties, and in a more complex design, the sample flow might experience some challenges [54,80][10][41].

2.8. Thread Based

Thread-based materials for microfluidics have some great advantages with respect to paper-based ones. Thread-based microfluidics are very cost efficient since they do not require a clean room, complex fabrication methods, or expensive machinery. They are therefore also very simple to fabricate, while their hydrophilic and capillary nature make pumps and hydrophobic barriers redundant. Most threads used for bio detection are very biocompatible, easily modified with different biorecognition elements, and can be easily shaped to almost any planar or 3D structure. Their low weight and handling simplicity make them relatively suitable for field work. Additionally, threads cannot be reused, and while they do have high strength compared to paper, overall, they are very sensitive to mechanical strain. Thread-based microfluidics are still only available in laboratory settings and have not yet been used for any commercial applications [58,81,82][19][42][43].

3. Lab-on-a-Chip Electrochemical Biosensors

The combination of microfluidics and electrochemical detection on one LOC platform offers the benefits of both worlds such as the small fluid volume and quick processing of microfluidics, along with the sensitivity and specificity of electrochemical biosensors. Electrochemical biosensors can be easily implemented into a microfluidic chip by utilizing modern integrated circuit fabrication techniques [55][11], microfluidic paper-based technology [57][18], and 3D printing techniques [18][17]. LOC portability and field compatibility is an important factor when assessing a device. In addition to the material, the microfluidic channel is made from, auxiliary equipment such as pumps, readers or a potentiostat, and biorecognition element stability. In most cases, the required auxiliary devices are larger than the LOC device and take up significant space [106][44], and therefore limits the application for on-field use. The miniaturization of pumps [68,69,107,108][29][30][45][46] and the use of microcapillaries [109,110][47][48] have enabled their integration into the device. In some instances, hand held potentiostats [111,112][49][50] integrated with a smart phone reader have been shown to provide better field compatibility [107,109][45][47], therefore significantly reducing the need for auxiliary equipment. The stability of the biomolecules of the biosensors presents the biggest challenge for widely used LOC biosensors in the field. Most natural biorecognition elements such as peptides, antibodies, bacteriophage, etc. are very sensitive to environmental conditions. To overcome their stability, molecularly imprinted polymers (MIPs) have been rising as an alternative. MIPs are artificially prepared materials the show advantages with respect to natural biorecognition elements such as reversable adsorption/release of the target pathogen. MIPs can also be imprinted with different nanomaterials to improve their magnetic, optic, or electric characteristics [113,114,115][51][52][53]. LOC offers not only the combination of microfluidics and detection, but also the automation of the whole process. For example, a device using microfluidics to bring the sample to the electrochemical biosensor that utilizes amperometric tests to detect Escherichia coli O157:H7 by using horse radish labeled antibody as the biorecognition element and forms an immobilized Ab/bacteria/anti-E. coli antibody structure following exposure to the bacteria [124][54]. Another interesting approach is the use of paper science to fabricate LOC devices. Paper offers an easier way to transport the fluid and allows for complex 2D structures to be utilized with the use of a printer and specialized ink. They are very portable and can not only deliver the sample to the sensing area, but can also merge multiple reagents, split samples, and delay the delivery by creating hydrophilic or hydrophobic areas on the paper [104,125][55][56]. This technology was shown to create a disposable impedimetric biosensor with immobilized antibodies as its biorecognition element. The paper itself acted as the microfluidic channel and carried the sample to the desired location [57][18]. On the other hand, paper-based LOC platforms tend to lack in sensitivity and their reproducibility is also a big issue that has yet to be solved [126][57]. LOC platforms can also utilize auxiliary forces such as dielectrophoresis [17,55][11][13] or magnetic field [16][12] to manipulate, focus, and concentrate the sample. They can do so by adding focused electrodes to form the electric field for dielectrophoresis, while an electric coil or a strong magnet can form the magnetic field. Applying such forces have been reported to enhance signal response by up to 18-fold when compared to the reaction without them [55][11]. Although these methods offer significant advantages and their fabrication is relatively established and common, they also add significant cost and energy requirements to the device.

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