PMC:7143804 / 981-13453 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7143804","sourcedb":"PMC","sourceid":"7143804","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7143804","text":"1. Introduction\nDiseases were and can still be a major problem in the world. Examples are outbreaks of zoonoses. One very recent example is the Covid-19 coronavirus outbreak in the People’s Republic of China. Zoonoses are also a widespread problem in animal husbandry [1]. This group encompasses diseases which can be transferred between animals (usually vertebrates) and between animals and humans. They are transmitted through zoonotic agents (e.g., bacteria, viruses, fungi, and parasites) [2,3,4]. Examples of bacterial zoonoses are the infections caused by Coxiella burnetii (Q-fever), Mycobacterium bovis (bovine tuberculosis), and by species of the Salmonella (Salmonellosis), Campylobacter (Campylobacteriosis), and Escherichia (Escherichiasis) genus [2,5]. These diseases are of potential risk for humans and livestock of farms. Poon et al. show that early-stage detection of coronaviruses positively influence the survival chances of patients [6]. An outbreak among the livestock of a farm is often disastrous to the owner of that farm, and for people living in the proximity of that farm [7]. Often, more animals of the livestock are infected and the whole livestock is exterminated out of precaution, which could lead to bankruptcy of the farmer. Therefore, early-stage detection of this group of diseases, and other diseases as well, is often the key to save lives and livestock. As these diseases are also encountered at remote locations and in developing countries, it is desired that such detection equipment is portable and as cheap as possible. A lab-on-a-chip platform can be used for this early-stage detection.\nIn the early stage of diseases, the agent, and therefore its genetic material, is only present in low concentrations within the infected human or animal, making detection rather difficult. One way to overcome this low concentration is to amplify the genetic material of the agent, i.e., deoxyribonucleic acid (DNA) in case of bacteria and DNA or ribonucleic acid (RNA) in case of viruses, until a certain threshold is reached and detection of the disease is made possible. When this amplification reaction is specific to certain DNA or RNA sequences, for example, by using polymerase chain reaction (PCR) [6,8], helicase-dependent amplification (HDA) [9,10], or loop-mediated isothermal amplification (LAMP) [11], and when a fluorescent DNA or RNA binding dye is used, a simple yes-or-no answer for a specific disease can be obtained.\n\n1.1. State-of-the-Art\nIn the past, several chip-based DNA and RNA amplification devices are reported. It goes beyond the scope of this paper to discuss the state-of-the-art of DNA amplification chips in-depth. There are several good review papers written on this topic [12,13,14,15,16,17,18,19,20]. Readers are referred to these for a comprehensive overview of the field. In this paper, the state-of-the-art is divided into several discussion points, i.e., the heating method, the temperature control method, and the substrate material and fabrication technique. These points will be discussed separately.\nWith respect to heat supply, different methods have been employed. Almassian et al. give a comprehensive overview of different possible heating methods in their review paper [12]. Not all of the mentioned methods are easy to implement in low-cost and portable lab-on-a-chip devices due to their bulkiness or implementation costs. Examples of these rather difficult methods are using heating via induction, infrared, or microwave radiation. Others are not useful due ot their challenging temperature control, like with heating up the system using exothermic reactions. Within the field of DNA amplification, different mechanisms of amplification exist. Some are based on thermo cycling processes, e.g., PCR, whereas others are isothermal. The use of an isothermal amplification technique puts less requirements on the heaters. Isothermal processes are either truly isothermal or consisting of three different temperatures, as they have a thermal denaturation step before and a termination step after the elongation step. The switching between these temperature steps does not have to be as fast as with thermal cycling steps in, for example, PCR amplification reactions. The use of less temperature variations makes it easier to maintain the set temperature as there is less heating an cooling involved. Furthermore, it eliminates the use of a continuous flow approach in systems with low thermal conductivities, e.g., polymers. Therefore, it is easier to implement within lab-on-a-chip devices [17,21]. Isothermal DNA amplification reactions can already be performed by putting the chip on a commercially available hotplate [22,23] or Peltier elements [24,25]. However, these heating systems are bulky and power-consuming. Therefore, they are not useful for portable equipment or operation at remote locations. Miniaturizing heaters lowers the bulkiness and power consumption. Miniaturized heaters can be integrated as integrated resistive heaters, e.g., as deposited thin-film metal [26,27,28,29] or as laminated Cu foil [30], or as micro-Peltier elements [31,32]. These miniaturized heaters can be implemented directly onto the microfluidic chip [28] or on a different substrate and leter incorporated onto the microfluidic chip [33,34,35,36]. The geometry of such a heater contributes significantly to the uniformity of the heat distribution within the chip [26,37]. One method to accurately control the temperature is the use of a proportional-integral-derivative (PID) controlled thermostat. These PID controllers are coupled to the electrical heaters and use a thermocouple as feedback-loop to the controller [22,23,24,25].\nThere are various materials that can be used to fabricate lab-on-a-chip devices for DNA or RNA amplification. In the past 15 years, more than ten polymers, ceramic materials, and metals have successfully been used to fabricate such devices [15]. The major property playing a role here is the biocompatibility of the material. The surface of the microfluidic structure should not inhibit the amplification reaction. This biocompatibility can be an intrinsic property of the material or the surface can be modified or coated to achieve this [12,13,14,15,16,17,18,19,20]. One often used material is polydimethylsiloxane (PDMS) [22,23,31,32,34,35,38], which can be processed using soft lithography [39]. However, this is a fabrication technology used in academia and is not suitable for upscaling to mass production [40]. Fabrication methods suitable for mass production are thermoforming/embossing or injection molding [41]. One of the materials which is biocompatible and suitable for both industrial scale fabrication technologies is cyclic olefin copolymer (COC) [42], which is one of the materials used in the past as well [28,36,43,44,45]. Guckenberger et al. estimates the costs of injection molding of only 50 simple microfluidic devices on $47, but this becomes cheaper when the mass production stage is reached [41]. Another benefit of COC is the possibility to shape it using micromilling. This technique is a rapid prototyping technology and therefore very useful within proof-of-concept projects [41].\nIntegrating resistive metal tracks onto a COC substrate have also been done in the past. Some papers describe the use of a surface modification step done before metal deposition in order to enhance adhesion between the COC and the metal layer, like a pretreatment with plasma [46] or an organic solvent [47]. Other papers describe the direct deposition of metal onto the COC surface [28,48]. Chung et al. specifically, fabricated an amplification chip in COC with integrated Au heaters [28]. However, their system required heating from both sides as the used grade of COC has a glass transition temperature (Tg) of 130 °C. This COC could not withstand the required heater temperatures to have enough heat flux into the system. They had to heat up the heater to temperatures above 130 °C, which caused cracking of the heater tracks due to deformation of the COC. With their double-sided heating they ensured that the reaction mixture had the desired PCR temperatures. However, double-sided heating doubles the amount of metal required, increases the amount of fabrication steps, and therefore increases the price per chip.\n\n1.2. The Presented Work\nThe work presented at the 4th Microfluidic Handling Systems conference and which is extended in this paper aims at the development of a disposable, polymer-based DNA amplification lab-on-chip system with integrated resistive heater based on the World Health Organization (WHO) Sexually Transmitted Diseases Diagnostics Initiative (SDI) ASSURED criteria. Devices which are ASSURED are (A) affordable, (S) sensitive, (S) specific, (U) user-friendly, (R) robust and rapid, (E) equipment-free, and (D) deliverable to those who need them [20,49]. The first step towards such a device is the development of the chip itself. This paper focuses on the choice of substrate material, metal deposition method, and type of metal. Although, it is mentioned above that PCR and HDA are sequence specific, the reaction chosen is the isothermal multiple displacement amplification (MDA) [50]. This reaction is more straightforward [51], as it amplifies any present DNA, and is therefore better suitable as a proof-of-principle amplification reaction to show the functioning of the integrated heater and the biocompatibility of the substrate after the fabrication process. The use of an isothermal amplification technique also simplifies the final device and lowers its footprint, as there are no pumps required. In this research, external analysis methods are used which do not contribute to the WHO-SDI ASSURED criteria due to their bulkiness, costs, and difficulty. However, suggestions and comments on the integration of low-cost detection methods, which are ASSURED, are given in Section 5.\n\n1.3. Multiple Displacement Amplification\nThe proof-of-principle amplification of choice is a MDA reaction, which is a non-specific isothermal method of amplification performed around 30 °C [50]. MDA is a method of whole genome amplification (WGA), as it amplifies all present DNA [52]. It is commonly used when the initial amount of DNA sample is very low. After the WGA is performed, a sequence specific amplification can be done since the quality the amplified DNA by MDA is very high [53]. The amplification reaction is illustrated below in Figure 1 (the contour of the amplified sequence is highlighted in black for clarity). Starting with a double stranded DNA (dsDNA) molecule, a denaturation step at 95 °C is required, giving the random hexamer-primers and the ϕ29 DNA polymerase access to the bases of single stranded DNA (ssDNA) strands. The hexamers anneal themself to aleatory parts of the ssDNA sequence. These hexamers work as initiation sites for the ϕ29 DNA polymerases. After denaturation at 95 °C, the mixture is cooled down to ice temperature and the rest of the reagents are added. The mixture is heated up to ~30 °C so the polymerase starts to complete the complementary ssDNA sequence, creating again a dsDNA strand, eventually it encounters a hexamer from another annealing site. Once this happens the polymerase will lift up that hexamer and starts to separate the amplified sequence formed from that annealing site. As the polymerase displaces the formed strand ahead of it, it continues to complete the sequence. The displaced strand becomes a new ssDNA strand and therefore, it gives new sites for more primers to attach and initiation sites for the polymerase, continuing the amplification, and thus creating a web of DNA strands. Finally, the inactivation of the polymerase is done by heating up the system to 65 °C.\nEven though MDA is considered an isothermal process, prior to the reaction and to the addition of most reactants, the dsDNA and a buffer are heated up to 95 °C to denature the dsDNA to ssDNA and to give hexamers the initial access to the ssDNA. After the amplification reaction, the polymerase has to be inactivated at 65 °C. However, this does not require fast temperature changes, as would be the case with, for example, the temperature cycling in PCR amplifications. This, together with the robustness of the amplification (it is a self-limiting reaction that amplifies all present DNA [50]) makes MDA perfectly suitable as proof-of-principle amplification reaction for such 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