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PMC:7143804 / 981-48862 JSONTXT 13 Projects

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Id Subject Object Predicate Lexical cue
T9 0-2 Sentence denotes 1.
T10 3-15 Sentence denotes Introduction
T11 16-76 Sentence denotes Diseases were and can still be a major problem in the world.
T12 77-112 Sentence denotes Examples are outbreaks of zoonoses.
T13 113-208 Sentence denotes One very recent example is the Covid-19 coronavirus outbreak in the People’s Republic of China.
T14 209-272 Sentence denotes Zoonoses are also a widespread problem in animal husbandry [1].
T15 273-399 Sentence denotes This group encompasses diseases which can be transferred between animals (usually vertebrates) and between animals and humans.
T16 400-501 Sentence denotes They are transmitted through zoonotic agents (e.g., bacteria, viruses, fungi, and parasites) [2,3,4].
T17 502-765 Sentence denotes 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].
T18 766-837 Sentence denotes These diseases are of potential risk for humans and livestock of farms.
T19 838-957 Sentence denotes Poon et al. show that early-stage detection of coronaviruses positively influence the survival chances of patients [6].
T20 958-1103 Sentence denotes 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].
T21 1104-1258 Sentence denotes 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.
T22 1259-1392 Sentence denotes Therefore, early-stage detection of this group of diseases, and other diseases as well, is often the key to save lives and livestock.
T23 1393-1562 Sentence denotes 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.
T24 1563-1631 Sentence denotes A lab-on-a-chip platform can be used for this early-stage detection.
T25 1632-1820 Sentence denotes In 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.
T26 1821-2104 Sentence denotes 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.
T27 2105-2466 Sentence denotes 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.
T28 2468-2472 Sentence denotes 1.1.
T29 2473-2489 Sentence denotes State-of-the-Art
T30 2490-2569 Sentence denotes In the past, several chip-based DNA and RNA amplification devices are reported.
T31 2570-2677 Sentence denotes It goes beyond the scope of this paper to discuss the state-of-the-art of DNA amplification chips in-depth.
T32 2678-2766 Sentence denotes There are several good review papers written on this topic [12,13,14,15,16,17,18,19,20].
T33 2767-2839 Sentence denotes Readers are referred to these for a comprehensive overview of the field.
T34 2840-3030 Sentence denotes 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.
T35 3031-3073 Sentence denotes These points will be discussed separately.
T36 3074-3140 Sentence denotes With respect to heat supply, different methods have been employed.
T37 3141-3253 Sentence denotes Almassian et al. give a comprehensive overview of different possible heating methods in their review paper [12].
T38 3254-3403 Sentence denotes 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.
T39 3404-3513 Sentence denotes Examples of these rather difficult methods are using heating via induction, infrared, or microwave radiation.
T40 3514-3641 Sentence denotes Others are not useful due ot their challenging temperature control, like with heating up the system using exothermic reactions.
T41 3642-3725 Sentence denotes Within the field of DNA amplification, different mechanisms of amplification exist.
T42 3726-3811 Sentence denotes Some are based on thermo cycling processes, e.g., PCR, whereas others are isothermal.
T43 3812-3899 Sentence denotes The use of an isothermal amplification technique puts less requirements on the heaters.
T44 3900-4093 Sentence denotes 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.
T45 4094-4243 Sentence denotes The switching between these temperature steps does not have to be as fast as with thermal cycling steps in, for example, PCR amplification reactions.
T46 4244-4376 Sentence denotes The use of less temperature variations makes it easier to maintain the set temperature as there is less heating an cooling involved.
T47 4377-4501 Sentence denotes Furthermore, it eliminates the use of a continuous flow approach in systems with low thermal conductivities, e.g., polymers.
T48 4502-4576 Sentence denotes Therefore, it is easier to implement within lab-on-a-chip devices [17,21].
T49 4577-4734 Sentence denotes Isothermal DNA amplification reactions can already be performed by putting the chip on a commercially available hotplate [22,23] or Peltier elements [24,25].
T50 4735-4796 Sentence denotes However, these heating systems are bulky and power-consuming.
T51 4797-4884 Sentence denotes Therefore, they are not useful for portable equipment or operation at remote locations.
T52 4885-4950 Sentence denotes Miniaturizing heaters lowers the bulkiness and power consumption.
T53 4951-5139 Sentence denotes 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].
T54 5140-5319 Sentence denotes 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].
T55 5320-5443 Sentence denotes The geometry of such a heater contributes significantly to the uniformity of the heat distribution within the chip [26,37].
T56 5444-5570 Sentence denotes One method to accurately control the temperature is the use of a proportional-integral-derivative (PID) controlled thermostat.
T57 5571-5703 Sentence denotes These PID controllers are coupled to the electrical heaters and use a thermocouple as feedback-loop to the controller [22,23,24,25].
T58 5704-5813 Sentence denotes There are various materials that can be used to fabricate lab-on-a-chip devices for DNA or RNA amplification.
T59 5814-5949 Sentence denotes In the past 15 years, more than ten polymers, ceramic materials, and metals have successfully been used to fabricate such devices [15].
T60 5950-6029 Sentence denotes The major property playing a role here is the biocompatibility of the material.
T61 6030-6118 Sentence denotes The surface of the microfluidic structure should not inhibit the amplification reaction.
T62 6119-6272 Sentence denotes 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].
T63 6273-6403 Sentence denotes One often used material is polydimethylsiloxane (PDMS) [22,23,31,32,34,35,38], which can be processed using soft lithography [39].
T64 6404-6521 Sentence denotes However, this is a fabrication technology used in academia and is not suitable for upscaling to mass production [40].
T65 6522-6625 Sentence denotes Fabrication methods suitable for mass production are thermoforming/embossing or injection molding [41].
T66 6626-6845 Sentence denotes 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].
T67 6846-7026 Sentence denotes 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].
T68 7027-7100 Sentence denotes Another benefit of COC is the possibility to shape it using micromilling.
T69 7101-7214 Sentence denotes This technique is a rapid prototyping technology and therefore very useful within proof-of-concept projects [41].
T70 7215-7303 Sentence denotes Integrating resistive metal tracks onto a COC substrate have also been done in the past.
T71 7304-7523 Sentence denotes 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].
T72 7524-7606 Sentence denotes Other papers describe the direct deposition of metal onto the COC surface [28,48].
T73 7607-7706 Sentence denotes Chung et al. specifically, fabricated an amplification chip in COC with integrated Au heaters [28].
T74 7707-7837 Sentence denotes However, their system required heating from both sides as the used grade of COC has a glass transition temperature (Tg) of 130 °C.
T75 7838-7941 Sentence denotes This COC could not withstand the required heater temperatures to have enough heat flux into the system.
T76 7942-8076 Sentence denotes 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.
T77 8077-8181 Sentence denotes With their double-sided heating they ensured that the reaction mixture had the desired PCR temperatures.
T78 8182-8336 Sentence denotes However, double-sided heating doubles the amount of metal required, increases the amount of fabrication steps, and therefore increases the price per chip.
T79 8338-8342 Sentence denotes 1.2.
T80 8343-8361 Sentence denotes The Presented Work
T81 8362-8715 Sentence denotes The 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.
T82 8716-8903 Sentence denotes 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].
T83 8904-8979 Sentence denotes The first step towards such a device is the development of the chip itself.
T84 8980-9079 Sentence denotes This paper focuses on the choice of substrate material, metal deposition method, and type of metal.
T85 9080-9237 Sentence denotes Although, it is mentioned above that PCR and HDA are sequence specific, the reaction chosen is the isothermal multiple displacement amplification (MDA) [50].
T86 9238-9516 Sentence denotes 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.
T87 9517-9656 Sentence denotes The use of an isothermal amplification technique also simplifies the final device and lowers its footprint, as there are no pumps required.
T88 9657-9812 Sentence denotes 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.
T89 9813-9939 Sentence denotes However, suggestions and comments on the integration of low-cost detection methods, which are ASSURED, are given in Section 5.
T90 9941-9945 Sentence denotes 1.3.
T91 9946-9981 Sentence denotes Multiple Displacement Amplification
T92 9982-10135 Sentence denotes The proof-of-principle amplification of choice is a MDA reaction, which is a non-specific isothermal method of amplification performed around 30 °C [50].
T93 10136-10226 Sentence denotes MDA is a method of whole genome amplification (WGA), as it amplifies all present DNA [52].
T94 10227-10297 Sentence denotes It is commonly used when the initial amount of DNA sample is very low.
T95 10298-10433 Sentence denotes After the WGA is performed, a sequence specific amplification can be done since the quality the amplified DNA by MDA is very high [53].
T96 10434-10570 Sentence denotes The amplification reaction is illustrated below in Figure 1 (the contour of the amplified sequence is highlighted in black for clarity).
T97 10571-10787 Sentence denotes 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.
T98 10788-10857 Sentence denotes The hexamers anneal themself to aleatory parts of the ssDNA sequence.
T99 10858-10926 Sentence denotes These hexamers work as initiation sites for the ϕ29 DNA polymerases.
T100 10927-11041 Sentence denotes After denaturation at 95 °C, the mixture is cooled down to ice temperature and the rest of the reagents are added.
T101 11042-11242 Sentence denotes 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.
T102 11243-11380 Sentence denotes Once this happens the polymerase will lift up that hexamer and starts to separate the amplified sequence formed from that annealing site.
T103 11381-11478 Sentence denotes As the polymerase displaces the formed strand ahead of it, it continues to complete the sequence.
T104 11479-11698 Sentence denotes 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.
T105 11699-11785 Sentence denotes Finally, the inactivation of the polymerase is done by heating up the system to 65 °C.
T106 11786-12030 Sentence denotes Even 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.
T107 12031-12111 Sentence denotes After the amplification reaction, the polymerase has to be inactivated at 65 °C.
T108 12112-12255 Sentence denotes However, this does not require fast temperature changes, as would be the case with, for example, the temperature cycling in PCR amplifications.
T109 12256-12472 Sentence denotes 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 devices.
T110 12474-12476 Sentence denotes 2.
T111 12477-12499 Sentence denotes Design and Fabrication
T112 12501-12505 Sentence denotes 2.1.
T113 12506-12535 Sentence denotes Microfluidic Structure Design
T114 12536-12648 Sentence denotes The microfluidic structure consists of two chambers, i.e., a reaction chamber and a temperature monitor chamber.
T115 12649-12700 Sentence denotes In Figure 2, a close-up of the final chip is shown.
T116 12701-12789 Sentence denotes For clarity reasons, the two microfluidic structures are colored with food coloring dye.
T117 12790-13238 Sentence denotes The reaction chamber is based on the work of Bruijns et al. [36] and its dimensions are chosen in such way that the internal volume of the reaction chamber is the same as the reaction volume of the used Illustra GenomiPhi V2 DNA amplification kit (GE Healthcare Life Sciences, Eindhoven, The Netherlands) together with the EvaGreen dye solution (Biotium, Fremont, CA, USA), while maintaining an as low as possible surface-area-to-volume ratio [44].
T118 13239-13584 Sentence denotes Using SolidWorks 2018 computer-aided design (CAD) software (Dassault Systemes, Vélizy-Villacoublay, France), the 3D image of the chip is drawn and with the use of the AutoDesk HSMWorks computer-aided manufacturing (CAM) plug-in (Autodesk Inc., San Rafael, CA, USA), this image is transferred into a computer numerical control (CNC) milling code.
T119 13585-13673 Sentence denotes The total chip size is 3 cm by 3 cm and contains an inlet and outlet of 1.5 mm diameter.
T120 13674-13771 Sentence denotes The inlet and outlet are of such size that the reaction chamber can be filled using pipette tips.
T121 13772-13864 Sentence denotes In between the inlet and outlet, a rectangular reaction chamber of 10 mm by 3 mm is located.
T122 13865-13966 Sentence denotes Two trapezoid structures are placed in the tapered channels between the inlet/outlet and the chamber.
T123 13967-14011 Sentence denotes The function of these trapezoids is twofold:
T124 14012-14172 Sentence denotes First, they minimize the dead volume between the inlet/outlet and the reaction chamber, locating as much as possible of the reaction mixture inside the chamber.
T125 14173-14226 Sentence denotes Second, they provide support for the chamber closure.
T126 14227-14384 Sentence denotes A stadium-shaped channel of 1.5 mm wide and 1.0 mm deep is located next to the reaction chamber, in such way that this channel is also covered by the heater.
T127 14385-14436 Sentence denotes This channel serves as temperature monitor chamber.
T128 14437-14540 Sentence denotes A thermocouple is inserted in this channel for real-time monitoring of the temperature inside the chip.
T129 14541-14635 Sentence denotes This way, a more accurate temperature of the reaction mixture inside the chip can be obtained.
T130 14636-14706 Sentence denotes Via a feedback loop, the input potential can be changed when required.
T131 14707-14796 Sentence denotes In Figure 3, the SolidWorks design of the chamber-based chip with both chambers is shown.
T132 14797-14873 Sentence denotes In Figure A1, in Appendix A, the technical drawing of the chip can be found.
T133 14875-14879 Sentence denotes 2.2.
T134 14880-14913 Sentence denotes Resistive Heater Structure Design
T135 14914-15087 Sentence denotes A resistive heater structure will be placed at the bottom side of the chip using shadow masks and a metal deposition method capable of being used for large-scale production.
T136 15088-15191 Sentence denotes A meandering heater design is chosen, as this minimizes the input power required to heat up the heater.
T137 15192-15545 Sentence denotes This is evident from Equation (1), which is the relation between Joule’s law, Ohm’s law, and Pouillet’s law. (1) P=Across−section∗V2ρres,i∗lheater Here, P is the input power, Across−section is the cross-sectional area of the resistor, V is the input potential, ρres,i is the resistivity of resistor material i, and lheater is the length of the resistor.
T138 15546-15744 Sentence denotes This makes a meandering structure, or any other narrow line structure, a quite often used pattern for heaters or electrodes within micro-electromechanical structures and microfluidics [26,29,54,55].
T139 15745-15979 Sentence denotes MDA is being done at temperatures of around 30 °C [50], which is lower than, for example, temperatures required for HDA (64 °C) [9] or LAMP (65 °C) [11] and the required PCR temperatures of Chung et al. (95 °C, 54 °C, and 72 °C) [28].
T140 15980-16057 Sentence denotes However, most amplification methods require a DNA denaturation step at 95 °C.
T141 16058-16747 Sentence denotes Equation (2) is used to make an estimation of the required heating powers for a COC–H2O–COC stack (in the real device, the upper plate is an adhesive PCR foil, but the thermal properties of this foil are unknown). (2) P=ΔTRth=ΔT∗Aheated∗κCOClCOC,1+κH2OlH2O+κCOClCOC,2+h Here, Rth is defined as the sum of all thermal resistances in series: (3) Rth=1h×Aheated+∑isubstancesliκi×Aheated Here, P is the required power, ΔT is the temperature difference, Rth is the thermal resistance, Aheated is the heated area, h is the convective heat transfer coefficient (being 10 W m−2 K−1 for convection to air [56]), κi is the thermal conductivity of substance i, and li is the thickness of substance i.
T142 16748-16789 Sentence denotes Values for κi can be found in Appendix B.
T143 16790-16870 Sentence denotes From Equation (3), the product Rth×A can be defined as the sum of 1/h and li/κi.
T144 16871-17093 Sentence denotes Based on this summation, one can conclude that the convective heat transfer to the air is the most present heat transfer mechanism within the system (begin almost a factor 100 higher than the heat lost in the COC and H2O).
T145 17094-17286 Sentence denotes This is also evident from solving Equation (2) for every individual temperature differences within the system and also including convective heat transfer directly from the heater into the air.
T146 17287-17547 Sentence denotes If a heated area of 7.7 mm by 10.1 mm is assumed, which covers both the reaction chamber and the temperature monitor chamber, and a system consisting of 1 mm COC–0.5 mm H2O–0.1 mm COC is assumed, than the heater temperatures and powers in Table 1 are required.
T147 17548-17661 Sentence denotes These are all in the workable range when a COC of a proper grade is chosen (e.g., TOPAS 6017 has a Tg of 170 °C).
T148 17662-17782 Sentence denotes The only side note here is that at higher temperatures, the temperature gradient through the system also becomes larger.
T149 17783-17860 Sentence denotes This can be eliminated by using double-sided heating, like Chung et al. [28].
T150 17861-18111 Sentence denotes To determine the optimal heater width and heater spacing in the heated area, a parametric study using COMSOL Multiphysics 5.3a finite element method simulations with the Heat Transfer in Solids (ht) package is done (COMSOL Inc., Burlington, MA, USA).
T151 18112-18272 Sentence denotes The model is designed such that it consists of two parallel rectangles of COC (in the real device, the upper plate is an adhesive PCR foil) with H2O in between.
T152 18273-18357 Sentence denotes The meandering heater are assumed to be lines at the bottom side of the layer stack.
T153 18358-18496 Sentence denotes This reduces the required complexity of the mesh tremendously, as the heater in the real device will be approximately 100 nm in thickness.
T154 18497-18565 Sentence denotes The heater temperature is set at a constant temperature of 303.15 K.
T155 18566-18688 Sentence denotes This makes the heater material independent and the model purely focused on the heat transfer inside the COC–H2O–COC stack.
T156 18689-18743 Sentence denotes All used values and equations are given in Appendix B.
T157 18744-18867 Sentence denotes The layer stack is meshed with an extremely fine mapped mesh consisting of 280.650 elements with average quality of 0.9966.
T158 18868-19032 Sentence denotes A parametric sweep from 0.3 mm to 2.0 mm, in steps of 0.1 mm, is done for both the heater width (wheater) and the heater spacing (sheater), giving 324 combinations.
T159 19033-19216 Sentence denotes The simulations are solved by using the fully coupled, direct Pardiso solver on a custom-build and 40% CPU overclocked simulation computer, containing the equipment listed in Table 2.
T160 19217-19412 Sentence denotes To validate whether the metal tracks can withstand the required current, a quick analysis is done for the four extreme cases (i.e., wheater of 0.3 mm and 2.0 mm and sheater of 0.3 mm and 2.0 mm).
T161 19413-19532 Sentence denotes In the same heated area of 7.7 mm by 10.1 mm a 100 nm (theater) thick heater track consisting of rectangles is assumed.
T162 19533-19638 Sentence denotes The total amount of large and smaller interconnecting rectangles for all 4 cases is estimated in Table 3.
T163 19639-20033 Sentence denotes Based on the polynomial approximation equations for the resistivity of Au and Pt (ρres,i, where i is either Au or Pt) which COMSOL MultiPhysics 5.3a uses (Equations (4) and (5)) and Equation (6) an estimation is made for the required input currents and the created current densities (defined as Ii/Across−section, in A m−2) when the heater is operated at 129.4 mW to get a temperature of 95 °C.
T164 20034-20323 Sentence denotes These estimations are also given in Table 3. (4) ρres,Au(T)=−2.210068×10−9+9.057611×10−11∗Tfor60≤T<400−4.632985×10−14∗T2+6.950205×−17∗T3 (5) ρres,Pt(T)=−1.927892×10−8+5.233699×10−10∗Tfor160≤T<600−4.107885×10−13∗T2+6.694129×−16∗T3−4.447775×10−19∗T4 (6) Ii=PRi=P∗Across−sectionρres,i∗lheater
T165 20324-20641 Sentence denotes In which ρres,i is the resistivity, T is the temperature, Ii is the current going through the resistor, Pi is the input power, R is the resistance of the resistor, lheater is the length of the resistor, and Across−section is the cross-sectional area of the resistor defined as width times thickness (wheater×theater).
T166 20642-20695 Sentence denotes The subscript i denotes the material, being Au or Pt.
T167 20696-20847 Sentence denotes All these current densities are below the critical current densities for Au and Pt, which are around 1010 A m−2 [57] and 1011 A m−2 [58], respectively.
T168 20848-20974 Sentence denotes Therefore, any possible combination of heater width and heater spacing will give a resistive that can withstand its operation.
T169 20976-20980 Sentence denotes 2.3.
T170 20981-20992 Sentence denotes Fabrication
T171 20993-21165 Sentence denotes COC [42] is chosen as polymeric substrate because of its biocompatibility, optical transparency, physical resistance, chemical resistance, electrical insulation, and price.
T172 21166-21317 Sentence denotes This copolymer consists of two monomers, an apolar bridged cyclic hydrocarbon (norbornene) monomer and a linear, lesser apolar, linear ethene monomers.
T173 21318-21513 Sentence denotes Injection molded COC plates (10 cm by 10 cm and 1.5 mm thickness) of the grade TOPAS 6017 (see Figure 4a) are obtained via Kunststoff-Zentrum Leipzig (Kunststoff-Zentrum gGmbH, Leipzig, Germany).
T174 21514-21616 Sentence denotes This grade is chosen because of its high norbornene content, giving it a relatively high Tg of 170 °C.
T175 21617-21789 Sentence denotes This minimizes the chance of melting during the milling process and decreases the chance of heater failure due to a deforming substrate during operation of the heater [28].
T176 21790-21959 Sentence denotes The microfluidic structure explained in Section 2.1 is CNC-milled using a Mikron WF 21C milling machine (Mikron SA Agno, Agno, Switzerland), as can be seen in Figure 4b.
T177 21960-22048 Sentence denotes Milling is a very fast prototyping technique and chosen because of its flexibility [41].
T178 22049-22139 Sentence denotes The milling creates a surface roughness, which increases the surface-area-to-volume ratio.
T179 22140-22282 Sentence denotes This roughness increases the chance of inhibition during the amplification due to the interaction of the used chemicals with the surface [44].
T180 22283-22434 Sentence denotes It also causes a considerable loss of optical transparency, which could obstruct the potential use of in situ fluorescence detection in future devices.
T181 22435-22519 Sentence denotes Therefore, a chemical post-treatment with cyclohexane vapor is done (see Figure 4c).
T182 22520-22750 Sentence denotes Such treatment dissolves a thin outer layer of the COC substrate and causes reflowing of the surface roughness due to the surface tension of the material, restoring the optical transparency and reducing the surface roughness [59].
T183 22751-22849 Sentence denotes CNC milling and subsequent cyclohexane vapor post-treatment are less suitable for mass production.
T184 22850-22914 Sentence denotes However, COC has the possibility of being injection molded [42].
T185 22915-22962 Sentence denotes The used substrates are made using this method.
T186 22963-23203 Sentence denotes This is a large-scale production method and could lower the costs of the eventual product and it eliminates the cyclohexane vapor post-treatment, as injection-molded chips would have the same optical transparency as the pristine substrates.
T187 23204-23328 Sentence denotes Guckenberger et al. mention production costs of $ 47 per simple microfluidic device when only 50 pieces are fabricated [41].
T188 23329-23406 Sentence denotes This price is expected to drop drastically when large numbers are fabricated.
T189 23407-23566 Sentence denotes A metal is deposited on the backside of the substrate using two laser-cut metal (Mo) shadow masks to outline the shape of the resistive heater (see Figure 4d).
T190 23567-23698 Sentence denotes Mo has a smaller coefficient of thermal expansion than stainless steel, and therefore gives less deformation during the deposition.
T191 23699-23801 Sentence denotes Metals of interest are Au or Pt, which are commonly used metals to function as resistive heaters [54].
T192 23802-24129 Sentence denotes The deposition methods studied are DC magnetron sputtering using a custom-build machine (Techno Centrum voor Onderwijs en Onderzoek, University of Twente, Enschede, The Netherlands) and e-beam physical vapor deposition (evaporation) using a Balzers BAK 600 CE (Oerlikon Balzers limited, Balzers, Principality of Liechtenstein).
T193 24130-24268 Sentence denotes Both deposition methods are capable of large-scale production, which will lower the production costs in the large-volume production stage.
T194 24269-24713 Sentence denotes The metal and deposition method will be chosen based on the metal adhesion performances on the COC substrate, which is studied using the Scotch tape test [60,61], and the resistance versus temperature behavior in the range 20 °C to 100 °C, which is measured in a Heraeus T5025 oven (Heraeus Holding GmbH, Hanau, Germany), customized with electrical readout and connected to a custom-build National Instruments LabVIEW program (Austin, TX, USA).
T195 24715-24719 Sentence denotes 2.4.
T196 24720-24729 Sentence denotes Operation
T197 24730-24882 Sentence denotes The chambers with the resistive heater on the backside, are intensively cleaned by rinsing with acetone, MilliQ DI water, ethanol, and isopropanol [45].
T198 24883-24961 Sentence denotes Each cleaning step was done 3 times and the chips are blow dried using N2 gas.
T199 24962-25185 Sentence denotes After drying, the chambers are closed using Microseal “B” PCR plate sealing foil from Bio-Rad (Bio-Rad Inc., Hercules, CA, USA), which is cut in the proper size and manually attached on top of the substrate (see Figure 4e).
T200 25186-25435 Sentence denotes The DNA, reactants and buffer solutions from the Illustra GenomiPhi V2 DNA amplification kit and an EvaGreen fluorescence dye are pipetted inside the chip using the inlet aperture, after which the inlet and outlet are closed using the same PCR foil.
T201 25436-25617 Sentence denotes An input potential is applied on the resistive heater using a Keithley 2602 SYSTEM SourceMeter (Cleveland, OH, USA) until they acquire the desired temperature for the amplification.
T202 25618-25792 Sentence denotes The temperature is real-time monitored by inserting a 162 series RS Technics thermocouple K (RS Components B.V., Haarlem, The Netherlands) in the temperature monitor chamber.
T203 25793-25889 Sentence denotes The thermocouple is read out with a Tenma 72-7715 Thermometer (Premier Farnell Ltd., Leeds, UK).
T204 25890-25993 Sentence denotes The source and the read-out of the thermocouple are operated using a custom-programmed LabVIEW program.
T205 25994-26142 Sentence denotes The initial potential is based on the heater characterization measurements, but will be adjusted according to the feedback-loop of the thermocouple.
T206 26143-26309 Sentence denotes Detection of the amplification is done ex-situ by using quartz cuvets and an Horiba Scientific FluoroMax+ spectrofluorometer (Horiba Scientific, Piscataway, NJ, USA).
T207 26311-26313 Sentence denotes 3.
T208 26314-26336 Sentence denotes Results and Discussion
T209 26338-26342 Sentence denotes 3.1.
T210 26343-26381 Sentence denotes COMSOL MultiPhysics Simulation Results
T211 26382-26479 Sentence denotes In Figure 5, the results of heat transfer simulations of two different heater spacings are shown.
T212 26480-26604 Sentence denotes The heater width for both geometries is 0.3 mm, while the heater spacing in Figure 5b,c are 0.3 mm and 2.0 mm, respectively.
T213 26605-26732 Sentence denotes In Figure 6a–d, tables with the results of the full parametric sweep for different heater widths and heater spacings are shown.
T214 26733-26919 Sentence denotes Figure 6a shows the temperature deviation between the highest and lowest temperature at the top of the chamber, i.e., the second H2O and COC interface (ΔTtopofchamber=Ttop,max−Ttop,min).
T215 26920-27097 Sentence denotes Figure 6b shows the deviation between the highest and lowest temperature inside the chamber, i.e., between the two COC and H2O interfaces (ΔTacrosschamber=Tbottom,max−Ttop,min).
T216 27098-27289 Sentence denotes Figure 6c shows the temperature deviation between the highest and lowest temperature at the bottom of the chamber, i.e., the first COC and H2O interface (ΔTbottomofchamber=Tbot,max−Tbot,min).
T217 27290-27496 Sentence denotes Figure 6d shows the deviation between the set heater temperature of 30 °C and the lowest temperature at the top of the chamber, i.e., the second H2O and COC interface (ΔTdeviationfromsetT=Theater−Ttop,min).
T218 27497-27674 Sentence denotes As can be seen from the results in Figure 6, a combination of small heater widths and heater spacings will result in smaller temperature differences inside the reaction mixture.
T219 27675-27789 Sentence denotes This is evident as smaller heater spacings will result in a better coverage of the heated area by heater material.
T220 27790-27944 Sentence denotes The smaller heater widths will result in a smaller heater cross-sectional area, and thus can be operated at lower powers, as is evident from Equation (1).
T221 27945-28098 Sentence denotes Resulting in the fact that a densely packed meander structure with small heater widths and small heater spacings can dissipate more heat into the system.
T222 28099-28262 Sentence denotes Based on these results and its simplicity, a meandering heater pattern of a heater with a width of 0.3 mm and a spacing of 0.3 mm in between the lines is designed.
T223 28263-28464 Sentence denotes A side note on the chosen heater width and heater spacing is that according to the simulations, the temperature differences within the chamber are less than ±0.3 °C for the most unfavorable dimensions.
T224 28465-28597 Sentence denotes This temperature difference is still well-accepted in the temperature window in which the MDA reaction takes place (25 °C to 35 °C).
T225 28598-28737 Sentence denotes However, as pointed out, a smaller cross-sectional area will result in a lower power consumption and therefore these dimensions are chosen.
T226 28738-28857 Sentence denotes It is known that a meandering heater structure does not give the optimal temperature distribution over the device [26].
T227 28858-29073 Sentence denotes Therefore, the heater lines are longer than the width of the reaction chamber, and thus also covering the bulk material outside the chamber in order to improve the temperature uniformity inside the reaction mixture.
T228 29074-29209 Sentence denotes The heater pattern is divided over two shadow masks to minimize the length of the narrow Mo tracks in between the meandering structure.
T229 29210-29260 Sentence denotes This prevents curvature due to intrinsic stresses.
T230 29261-29356 Sentence denotes See Figure 7 for the outlines of both shadow masks, together with the resulting pattern on COC.
T231 29357-29455 Sentence denotes The use of two shadow masks will give a metal track in which small parts has the double thickness.
T232 29456-29492 Sentence denotes Here, the temperature will be lower.
T233 29493-29618 Sentence denotes The system is designed such that these thicker parts are outside the reaction chamber and temperature control chamber region.
T234 29620-29624 Sentence denotes 3.2.
T235 29625-29636 Sentence denotes Fabrication
T236 29638-29670 Sentence denotes Milling and Optical Transparency
T237 29671-29765 Sentence denotes The milling increased the surface roughness of the COC plates also increases the surface area.
T238 29766-29905 Sentence denotes Inhibition of the amplification can be caused by large surface areas as the used chemicals have more surface to have interaction with [44].
T239 29906-30244 Sentence denotes The created surface roughness is visualized using a FEI Sirion high resolution scanning electron microscope (HR-SEM) (FEI Company, Hillsboro, OR, USA) and measured using a Bruker Icon Dimension AFM in tapping mode with Bruker Tespa-V2 cantilevers (Bruker Nano Surfaces, Santa Barbara, CA, USA) and Gwyddion 2.52 open source freeware [62].
T240 30245-30279 Sentence denotes The results are shown in Figure 8.
T241 30280-30339 Sentence denotes The surface roughness of pristine COC had a RRMS of 3.5 nm.
T242 30340-30412 Sentence denotes This increased two orders of magnitude after milling (RRMS of 310.1 nm).
T243 30413-30612 Sentence denotes With the reported surface treatment [44,59] we were capable of decreasing the surface roughness to a value even lower than that of pristine COC and the lowest reported in literature (RRMS of 0.9 nm).
T244 30613-30824 Sentence denotes For this grade of COC (TOPAS 6017) it worked the best to do four short exposures of 5 s, with N2 blow drying after each exposure, instead of one longer exposure, as is more common in other grades of COC [44,59].
T245 30825-30974 Sentence denotes The difference in duration for the cyclohexane vapor post-treatment can be explained by the different ratios of the copolymers present in each grade.
T246 30975-31088 Sentence denotes As the grade number increase, the ratio changes towards more norbornene monomers and less linear ethene monomers.
T247 31089-31276 Sentence denotes The norbornene is more apolar due to the bridged cyclic hydrocarbon present in its molecular structure and therefore, will dissolve faster in non-polar solvents, like cyclohexane (vapor).
T248 31277-31358 Sentence denotes Lowering the surface roughness also increased the optical transmittance fivefold.
T249 31359-31459 Sentence denotes Transmittance measurements in the visible range are done using a Woollam M-2000UI ellipsometer (J.A.
T250 31460-31491 Sentence denotes Woollam Co., Lincoln, NE, USA).
T251 31492-31542 Sentence denotes The results can be seen in Figure 9 and Figure 10.
T252 31543-31782 Sentence denotes Having a high optical transparency in the visible range can be desired when in situ fluorescence detection will be implemented (e.g., EvaGreen fluorescence dye has an excitation wavelength of 500 nm and emission wavelength of 525 nm [63]).
T253 31783-32023 Sentence denotes However, as in situ fluorescence detection is not used yet in this system and can also be done through the transparent PCR plate sealing foil, no further effort is put into optimizing this procedure to get even better optical transmittance.
T254 32025-32029 Sentence denotes 3.3.
T255 32030-32044 Sentence denotes Metal Adhesion
T256 32045-32160 Sentence denotes To get reliable heaters, four possible options are investigated for their adhesion properties to the COC substrate.
T257 32161-32347 Sentence denotes The adhesion of Au and Pt deposited by either evaporation or DC magnetron sputtering is investigated using the Scotch tape test [60,61] before and after temperature cycling up to 100 °C.
T258 32348-32606 Sentence denotes Test patterns consisting of rectangular metal strips of 2 mm by 14 mm are fabricated by depositing 100 nm of metal using a hand-made shadow mask made out of DuPont Kapton® HN polyimide film of 0.05 mm thickness (RS Components B.V., Haarlem, The Netherlands).
T259 32607-32659 Sentence denotes See Table 4 for the results of the Scotch tape test.
T260 32660-32975 Sentence denotes Normally, heating up a glass or Si substrate with thin metal strips while measuring the resistance (RT) in these metal strips at certain temperature intervals (T) yields directly a linear relation, which can be fitted with RT/R0=1+αT−T0 [64], in which α is the the temperature coefficient of resistance (TCR) value.
T261 32976-33142 Sentence denotes The thin-film TCR values have to be measured as they differ from the bulk TCR values due to its dependency on layer purity, grain size, and deposition method [65,66].
T262 33143-33323 Sentence denotes Belser and Hicklin also lists other attributes, such as surface roughness, porosity, and adsorbed materials present in or on the substrate which could influence the TCR value [64].
T263 33324-33393 Sentence denotes The bulk TCR values are 0.0034 K−1 and 0.0037 K−1 for Au and Pt [67].
T264 33394-33515 Sentence denotes The TCR characterizations of the metal strips on a COC substrate did not yield trustworthy TCR values at the first cycle.
T265 33516-33681 Sentence denotes The first temperature cycle can be seen as a kind of thermal annealing, and therefore gives an hysteresis in the graphs, as can be seen in Figure A2 in Appendix C.1.
T266 33682-33755 Sentence denotes After this first cycle, the values more or less show the linear behavior.
T267 33756-33881 Sentence denotes The resulting TCR of this linear part is in agreement with the TCR ranges of Belser and Hicklin [64] and is given in Table 4.
T268 33882-34017 Sentence denotes Belser and Hicklin used for their experiments substrates with coefficients of linear thermal expansion lower than 1.2 × 10−5 °C−1 [64].
T269 34018-34135 Sentence denotes The coefficient of linear thermal expansion for Au and Pt are 1.42 × 10−5 K−1 and 0.88 × 10−5 K−1, respectively [68].
T270 34136-34233 Sentence denotes COC of the grade TOPAS 6017 has a coefficient of linear thermal expansion of 6.0 × 10−5 K−1 [42].
T271 34234-34328 Sentence denotes This mismatch in coefficients of linear thermal expansion can give strain in the metal layers.
T272 34329-34486 Sentence denotes Both Au [69,70,71] and Pt [72,73,74] are used as strain-sensitive gauges, and thus are sensitive to strain-induced geometry changes due to thermal expansion.
T273 34487-34556 Sentence denotes Another effect influencing the TCR value of the metal layer is aging.
T274 34557-34678 Sentence denotes As can be seen in Figure A3 in Appendix C.2, the TCR value already changes after two weeks storing in ambient conditions.
T275 34679-34747 Sentence denotes This could be due to adsorbed materials present on the surface [64].
T276 34748-34864 Sentence denotes However, in this device, the TCR is not of importance as the metal structure will not be used as temperature sensor.
T277 34865-34959 Sentence denotes Real-time temperature sensing is done using a thermocouple in the temperature monitor chamber.
T278 34960-35100 Sentence denotes The resistance of the heater structure changes with temperature; thus, the dissipated power changes when a fixed voltage or current is used.
T279 35101-35225 Sentence denotes However, the results in Section 3.4 show a 25 h stability test with a constant input potential and only a ±1.5 °C deviation.
T280 35226-35328 Sentence denotes The TCR can become more important when other (higher) temperatures are required for the amplification.
T281 35329-35479 Sentence denotes Based on the results in Table 4, the choice of heater material and deposition method to be used in the actual device is Au deposited using sputtering.
T282 35480-35611 Sentence denotes Sputtering is an industrial-scale technique that is already being used in, for example, the car mirror and headlight industry [75].
T283 35613-35617 Sentence denotes 3.4.
T284 35618-35634 Sentence denotes Chip Functioning
T285 35635-35775 Sentence denotes Characterization of the actual heat distribution is done using a FLIR One Pro iOS thermal camera (FLIR Systems, Inc., Wilsonville, OR, USA).
T286 35776-35938 Sentence denotes Thermal images of the heat distribution are made at the side of the substrate without the resistor, whereas different input powers are used to heat up the heater.
T287 35939-36145 Sentence denotes Au reflects the infrared radiation of the environment directly, therefore an image with the resistor facing the camera would give a heat map of the surrounding and not of the real temperature of the heater.
T288 36146-36246 Sentence denotes These measurements also gives a better insight of the heat distribution inside the reaction chamber.
T289 36247-36311 Sentence denotes The images are processed using the FLIR postprocessing freeware.
T290 36312-36368 Sentence denotes Results of these measurements are shown in Figure 11a,b.
T291 36369-36435 Sentence denotes The results are in good agreement with the estimations in Table 1.
T292 36436-36818 Sentence denotes The slight deviation between the values can be explained by the fact that the heated area in the calculations had an assumed value, the thermal camera measurements used 1.5 mm thick COC substrates without a water-filled chamber, the actual resistors have small parts wich have a double thickness due to the two used shadow masks, and rounding of the values used in the calculations.
T293 36819-36942 Sentence denotes The reliability of the heater is tested by inserting the thermocouple into the temperature control chamber (see Figure 3a).
T294 36943-37055 Sentence denotes A constant input potential of 4 V is applied using the Keithley source and the temperature is measured for 25 h.
T295 37056-37207 Sentence denotes This exceeds the required operation time at least twelve-fold, meaning that it is a good indication for the reliability of the heater and thermocouple.
T296 37208-37244 Sentence denotes The results are shown in Figure 11c.
T297 37245-37506 Sentence denotes To perform on-chip amplifications, the resistive heater on the chip is connected to the Keithley source using crocodile connections and the thermocouple is inserted in the temperature control chamber and connected to a Tenma 72-7715 Thermometer (see Figure 12).
T298 37508-37512 Sentence denotes 3.5.
T299 37513-37530 Sentence denotes DNA Amplification
T300 37531-37719 Sentence denotes First, to determine the temperature window of operation, MDA reactions are performed at 25 °C and 30 °C using the Illustra GenomiPhi V2 DNA amplification kit and EvaGreen fluorescence dye.
T301 37720-37869 Sentence denotes From the literature, we know that this reaction does not work above 35 °C due to degradation of the protein activity in presence of a substrate [44].
T302 37870-38016 Sentence denotes In Figure 13, a graph of the fluorescence signal during MDA reactions at 25 °C and 30 °C, together with their non template control (NTC) is shown.
T303 38017-38392 Sentence denotes These reactions are carried out in a conventional Bio-Rad CFX96 Touch Real-Time PCR machine (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the results show that the chosen proof-of-principle DNA amplification reaction is temperature dependent to some extent, but that there is a wide range of temperatures at which the amplification can be performed, i.e., 25°C to 35°C.
T304 38393-38508 Sentence denotes This makes the functioning of the integrated resistive heater less critical than the stability shown in Figure 11c.
T305 38509-38711 Sentence denotes MDA reactions are also performed inside an Eppendorf tube (Eppendorf AG, Hamburg, Germany) and inside the chip, again using the Illustra GenomiPhi V2 DNA amplification kit and EvaGreen fluorescence dye.
T306 38712-38826 Sentence denotes As heat supply the water bath of an IKA Rotary Evaporator RV 8V (IKA-Werke, Staufen im Breisgau, Germany) is used.
T307 38827-38930 Sentence denotes This water bath is according to its specification stable within a range of the set temperature ±0.1 °C.
T308 38931-39055 Sentence denotes The chip and an Eppendorf tube are loaded with the reaction mixture containing the DNA sample and the EvaGreen dye solution.
T309 39056-39189 Sentence denotes Here, the Eppendorf tube is serving as a control to show that the fabrication steps of the chips are not inhibiting the MDA reaction.
T310 39190-39280 Sentence denotes The inlet and outlet of the chip are sealed with the Microseal “B” PCR plate sealing foil.
T311 39281-39417 Sentence denotes The closed chip and tube are heated up in a separate water bath to 95 °C and kept at that temperature for 3 min to denaturate the dsDNA.
T312 39418-39552 Sentence denotes Subsequently, the chip and tube are cooled down by placing it in an ice bath for 5 min after which the rest of the reagents are added.
T313 39553-39615 Sentence denotes The complete mixtures are according to Table A3 in Appendix D.
T314 39616-39789 Sentence denotes After closing the chip and tube again, they are placed in the water bath of the rotary evaporater and left there for 90 min, after which the reaction is terminated at 65 °C.
T315 39790-39900 Sentence denotes The MDA is also performed inside the chip, but with the integrated Au resistive heater serving as heat source.
T316 39901-39945 Sentence denotes The set up shown schematically in Figure 12.
T317 39946-40012 Sentence denotes The same procedure is followed as with the water bath heated test.
T318 40013-40059 Sentence denotes Denaturation is done in a separate water bath.
T319 40060-40283 Sentence denotes The heater is driven by an input potential of 3.2 V to get to a temperature of 30 °C and at the end of the reaction, the system is heated up to 65 °C by applying a potential of 9.2 V in order to terminate the amplification.
T320 40284-40354 Sentence denotes In Figure 14 the logged temperature during the amplification is shown.
T321 40355-40545 Sentence denotes After the amplifications, the reaction mixtures are pipetted out of the chips and tubes and into 1 mL quartz cuvettes containing 55 μL MilliQ DI water (Merck Millipore, Burlington, MA, USA).
T322 40546-40663 Sentence denotes Fluorescence measurements are done in a Horiba Scientific FluoroMax+ spectrofluorometer to verify each amplification.
T323 40664-40839 Sentence denotes The mixture is excitated at a wavelength of 500 nm and the emission spectrum is measured at wavelengths from 510 nm to 550 nm (bounded EvaGreen dye has a peak at 525 nm [63]).
T324 40840-41000 Sentence denotes The measured spectra are normalized by subtracting the background signal of a mixture containing only the reaction buffer, the sample buffer, EvaGreen, and DNA.
T325 41001-41082 Sentence denotes No Enzyme was added to this mixture, therefore no amplification could take place.
T326 41083-41154 Sentence denotes See Figure 15 for the results obtained in the Eppendorf tube and chips.
T327 41155-41249 Sentence denotes Figure A4 in Appendix E shows the background signal which is subtracted from all measurements.
T328 41250-41525 Sentence denotes As can be seen in Figure 15, the spectra of the amplification performed inside the chip, and by applying heat with the water bath as well as with the integrated Au-resistive heater, show the same trend as the amplification performed in the Eppendorf and heated by water bath.
T329 41526-41595 Sentence denotes There is an order of magnitude difference in the fluorescence signal.
T330 41596-41688 Sentence denotes However, the fluorescence intensity cannot be used as a value to quantify the amount of DNA.
T331 41689-41829 Sentence denotes EvaGreen is a bis-intercalating cyanine fluorescence dye consisting of two monomeric DNA-binding dyes which are linked by a flexible spacer.
T332 41830-41972 Sentence denotes These two DNA-binding dyes bind each in between two base pairs, which make them simple and fast, but also nonuniform and non-specific [44,63].
T333 41973-42100 Sentence denotes However, with this dye, a simple yes-or-no answer can be obtained if the amplification took place, as can be seen in Figure 15.
T334 42102-42104 Sentence denotes 4.
T335 42105-42116 Sentence denotes Conclusions
T336 42117-42340 Sentence denotes The aim of this study was to fabricate biocompatible, low-cost, and disposable chips with integrated heater, which should be able to perform DNA amplification, and possible in situ fluorescence detection in the near future.
T337 42341-42479 Sentence denotes In this case there is no interest in quantification of the DNA, but only in amplification of DNA until the detection threshold is reached.
T338 42480-42567 Sentence denotes As proof-of-principle the MDA reaction and ex-situ fluorescence measurements were used.
T339 42568-42669 Sentence denotes With the proposed fabrication process, low-cost and biocompatible chips (Figure 12b) were fabricated.
T340 42670-42924 Sentence denotes The integrated resistive heaters on the chips were characterized and showed a temperature stability of ±2 °C over a time period of 25 h, which is at least twelve-fold longer than the required operating times for DNA amplification reactions [6,8,9,10,11].
T341 42925-43037 Sentence denotes The main cause of this period of lowered temperature was due to the fact that the measurement was run overnight.
T342 43038-43163 Sentence denotes With the proof-of-principle device, successful DNA amplifications using MDA inside a disposable polymeric chip were achieved.
T343 43164-43252 Sentence denotes The heat for the reaction was applied using the integrated low-cost Au-resistive heater.
T344 43253-43422 Sentence denotes The device was operated at a suitable temperature for MDA reactions and the amplified DNA was measured using EvaGreen fluorescence dye and an ex situ spectrofluorometer.
T345 43423-43511 Sentence denotes A distinct peak is visible in the reaction mixtures which is absent in the NTC mixtures.
T346 43512-43616 Sentence denotes The operating temperature for MDA reactions is around 30 °C, which is comparable with a nice summer day.
T347 43617-43729 Sentence denotes Using amplification reactions which such low reaction temperatures could encounter problems at warmer locations.
T348 43730-43832 Sentence denotes However, as MDA is not sequence specific, this reaction will not be integrated in the final protocols.
T349 43833-43966 Sentence denotes MDA was only used as proof-of-principle reaction to show the biocompatibility of the device and functioning of the integrated heater.
T350 43967-44093 Sentence denotes Sequence specific amplifications, e.g., HDA and LAMP, are performed at higher temperatures, as will be discussed in Section 5.
T351 44094-44154 Sentence denotes This makes the system less sensitive to the hot summer days.
T352 44155-44306 Sentence denotes The device in its current state is not fully conform the WHO-SDI ASSURED criteria [49] as it still relies on the use of (expensive) external equipment.
T353 44307-44362 Sentence denotes However, the first steps are made to an ASSURED device.
T354 44363-44459 Sentence denotes Future steps which will make the device fully ASSURED are given in the next section (Section 5).
T355 44461-44463 Sentence denotes 5.
T356 44464-44471 Sentence denotes Outlook
T357 44472-44728 Sentence denotes Future steps, which will result in a device for early-stage detection of, for example, zoonoses, include studies on the optimization of this device for sequence specific DNA amplifications (e.g., primer design and reaction optimization), i.e., HDA or LAMP.
T358 44729-44863 Sentence denotes HDA utilizes DNA helicase (an enzyme also used in vitro during DNA replication) to separate the dsDNA instead of thermal denaturation.
T359 44864-45102 Sentence denotes After separation, ssDNA binding proteins hybridize on the ssDNA strands for stabilization, ensuring that the next primer will have time to bind to the ssDNA stripe and a DNA polymerase will extend the primers with the complementary bases.
T360 45103-45272 Sentence denotes This method is a truly isothermal technique in which the separation of the dsDNA can be performed at the same temperature as the amplification reaction, i.e., 64 °C [9].
T361 45273-45352 Sentence denotes LAMP is more similar to MDA in the way it also uses heat to denature the dsDNA.
T362 45353-45537 Sentence denotes After denaturation, a set of four primers (six can be used as well to achieve better selectivity) and a DNA polymerase is used at isothermal conditions (65 °C) to amplify the DNA [11].
T363 45538-45747 Sentence denotes When used in combination with reverse transcriptase, LAMP becomes a RNA amplification method, which could be used for RNA-containing viruses [11], like virus-based zoonoses diseases as the corona viruses [22].
T364 45748-45866 Sentence denotes Despite not being a truly isothermal technique, LAMP offers the possibility to use turbidity as detection method [76].
T365 45867-46026 Sentence denotes Such a detection method would simplify the required equipment even further as a decrease in transmitted light through the chip can be used as detection method.
T366 46027-46093 Sentence denotes Different amplification techniques require different temperatures.
T367 46094-46218 Sentence denotes Based on Table 1 one can conclude that a higher temperature would also give a larger temperature gradient within the system.
T368 46219-46350 Sentence denotes This can be disadvantageous for amplification reactions, as optimal denaturation temperatures are in the range 92 °C to 94 °C [77].
T369 46351-46466 Sentence denotes The denaturation in this research was done in a separate water bath, so this temperature gradient was circumvented.
T370 46467-46675 Sentence denotes However, when on-chip denaturation and/or another amplification technique will be used, a second step will be the optimization of the heater in order to create better temperature uniformity within the system.
T371 46676-46773 Sentence denotes This can be done by using different heater geometries [26,37] or using double-sided heating [28].
T372 46774-46861 Sentence denotes The third step that has to be optimized in the sample collection and work-up procedure.
T373 46862-47043 Sentence denotes One has to think of what kind of samples to collect in order to have the biggest chance of having the agent of the disease present in that sample (i.e., blood, mucus, saliva, etc.).
T374 47044-47111 Sentence denotes Such crude samples contain full cells, with the DNA present within.
T375 47112-47198 Sentence denotes There are different approaches to perform cell lysis in order to extract the DNA [78].
T376 47199-47333 Sentence denotes Various components of bodily fluids, and reagents and products of the lysis are well-known to inhibit the amplification reaction [79].
T377 47334-47405 Sentence denotes However, MDA [80] and HDA [9] could also be performed on crude samples.
T378 47406-47539 Sentence denotes A fourth step in the near-future is the development of a first prototype with all hardware integrated in a single piece of equipment.
T379 47540-47780 Sentence denotes Such a device in its pure essence will consist of a battery to power the heater and detection, a chip holder to firmly keep the chip in its place, a thermocouple for real-time monitoring of the temperature, and a LED light and a photodiode.
T380 47781-47881 Sentence denotes The lamp and photodiode could both be used for fluorescence measurements and turbidity measurements.