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Id Subject Object Predicate Lexical cue
T1 0-69 Sentence denotes Disposable DNA Amplification Chips with Integrated Low-Cost Heaters †
T2 71-79 Sentence denotes Abstract
T3 80-298 Sentence denotes Fast point-of-use detection of, for example, early-stage zoonoses, e.g., Q-fever, bovine tuberculosis, or the Covid-19 coronavirus, is beneficial for both humans and animal husbandry as it can save lives and livestock.
T4 299-373 Sentence denotes The latter prevents farmers from going bankrupt after a zoonoses outbreak.
T5 374-528 Sentence denotes This paper describes the development of a fabrication process and the proof-of-principle of a disposable DNA amplification chip with an integrated heater.
T6 529-789 Sentence denotes Based on the analysis of the milling process, metal adhesion studies, and COMSOL MultiPhysics heat transfer simulations, the first batch of chips has been fabricated and successful multiple displacement amplification reactions are performed inside these chips.
T7 790-890 Sentence denotes This research is the first step towards the development of an early-stage zoonoses detection device.
T8 891-979 Sentence denotes Tests with real zoonoses and DNA specific amplification reactions still need to be done.
T9 981-983 Sentence denotes 1.
T10 984-996 Sentence denotes Introduction
T11 997-1057 Sentence denotes Diseases were and can still be a major problem in the world.
T12 1058-1093 Sentence denotes Examples are outbreaks of zoonoses.
T13 1094-1189 Sentence denotes One very recent example is the Covid-19 coronavirus outbreak in the People’s Republic of China.
T14 1190-1253 Sentence denotes Zoonoses are also a widespread problem in animal husbandry [1].
T15 1254-1380 Sentence denotes This group encompasses diseases which can be transferred between animals (usually vertebrates) and between animals and humans.
T16 1381-1482 Sentence denotes They are transmitted through zoonotic agents (e.g., bacteria, viruses, fungi, and parasites) [2,3,4].
T17 1483-1746 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 1747-1818 Sentence denotes These diseases are of potential risk for humans and livestock of farms.
T19 1819-1938 Sentence denotes Poon et al. show that early-stage detection of coronaviruses positively influence the survival chances of patients [6].
T20 1939-2084 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 2085-2239 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 2240-2373 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 2374-2543 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 2544-2612 Sentence denotes A lab-on-a-chip platform can be used for this early-stage detection.
T25 2613-2801 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 2802-3085 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 3086-3447 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 3449-3453 Sentence denotes 1.1.
T29 3454-3470 Sentence denotes State-of-the-Art
T30 3471-3550 Sentence denotes In the past, several chip-based DNA and RNA amplification devices are reported.
T31 3551-3658 Sentence denotes It goes beyond the scope of this paper to discuss the state-of-the-art of DNA amplification chips in-depth.
T32 3659-3747 Sentence denotes There are several good review papers written on this topic [12,13,14,15,16,17,18,19,20].
T33 3748-3820 Sentence denotes Readers are referred to these for a comprehensive overview of the field.
T34 3821-4011 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 4012-4054 Sentence denotes These points will be discussed separately.
T36 4055-4121 Sentence denotes With respect to heat supply, different methods have been employed.
T37 4122-4234 Sentence denotes Almassian et al. give a comprehensive overview of different possible heating methods in their review paper [12].
T38 4235-4384 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 4385-4494 Sentence denotes Examples of these rather difficult methods are using heating via induction, infrared, or microwave radiation.
T40 4495-4622 Sentence denotes Others are not useful due ot their challenging temperature control, like with heating up the system using exothermic reactions.
T41 4623-4706 Sentence denotes Within the field of DNA amplification, different mechanisms of amplification exist.
T42 4707-4792 Sentence denotes Some are based on thermo cycling processes, e.g., PCR, whereas others are isothermal.
T43 4793-4880 Sentence denotes The use of an isothermal amplification technique puts less requirements on the heaters.
T44 4881-5074 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 5075-5224 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 5225-5357 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 5358-5482 Sentence denotes Furthermore, it eliminates the use of a continuous flow approach in systems with low thermal conductivities, e.g., polymers.
T48 5483-5557 Sentence denotes Therefore, it is easier to implement within lab-on-a-chip devices [17,21].
T49 5558-5715 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 5716-5777 Sentence denotes However, these heating systems are bulky and power-consuming.
T51 5778-5865 Sentence denotes Therefore, they are not useful for portable equipment or operation at remote locations.
T52 5866-5931 Sentence denotes Miniaturizing heaters lowers the bulkiness and power consumption.
T53 5932-6120 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 6121-6300 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 6301-6424 Sentence denotes The geometry of such a heater contributes significantly to the uniformity of the heat distribution within the chip [26,37].
T56 6425-6551 Sentence denotes One method to accurately control the temperature is the use of a proportional-integral-derivative (PID) controlled thermostat.
T57 6552-6684 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 6685-6794 Sentence denotes There are various materials that can be used to fabricate lab-on-a-chip devices for DNA or RNA amplification.
T59 6795-6930 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 6931-7010 Sentence denotes The major property playing a role here is the biocompatibility of the material.
T61 7011-7099 Sentence denotes The surface of the microfluidic structure should not inhibit the amplification reaction.
T62 7100-7253 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 7254-7384 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 7385-7502 Sentence denotes However, this is a fabrication technology used in academia and is not suitable for upscaling to mass production [40].
T65 7503-7606 Sentence denotes Fabrication methods suitable for mass production are thermoforming/embossing or injection molding [41].
T66 7607-7826 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 7827-8007 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 8008-8081 Sentence denotes Another benefit of COC is the possibility to shape it using micromilling.
T69 8082-8195 Sentence denotes This technique is a rapid prototyping technology and therefore very useful within proof-of-concept projects [41].
T70 8196-8284 Sentence denotes Integrating resistive metal tracks onto a COC substrate have also been done in the past.
T71 8285-8504 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 8505-8587 Sentence denotes Other papers describe the direct deposition of metal onto the COC surface [28,48].
T73 8588-8687 Sentence denotes Chung et al. specifically, fabricated an amplification chip in COC with integrated Au heaters [28].
T74 8688-8818 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 8819-8922 Sentence denotes This COC could not withstand the required heater temperatures to have enough heat flux into the system.
T76 8923-9057 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 9058-9162 Sentence denotes With their double-sided heating they ensured that the reaction mixture had the desired PCR temperatures.
T78 9163-9317 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 9319-9323 Sentence denotes 1.2.
T80 9324-9342 Sentence denotes The Presented Work
T81 9343-9696 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 9697-9884 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 9885-9960 Sentence denotes The first step towards such a device is the development of the chip itself.
T84 9961-10060 Sentence denotes This paper focuses on the choice of substrate material, metal deposition method, and type of metal.
T85 10061-10218 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 10219-10497 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 10498-10637 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 10638-10793 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 10794-10920 Sentence denotes However, suggestions and comments on the integration of low-cost detection methods, which are ASSURED, are given in Section 5.
T90 10922-10926 Sentence denotes 1.3.
T91 10927-10962 Sentence denotes Multiple Displacement Amplification
T92 10963-11116 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 11117-11207 Sentence denotes MDA is a method of whole genome amplification (WGA), as it amplifies all present DNA [52].
T94 11208-11278 Sentence denotes It is commonly used when the initial amount of DNA sample is very low.
T95 11279-11414 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 11415-11551 Sentence denotes The amplification reaction is illustrated below in Figure 1 (the contour of the amplified sequence is highlighted in black for clarity).
T97 11552-11768 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 11769-11838 Sentence denotes The hexamers anneal themself to aleatory parts of the ssDNA sequence.
T99 11839-11907 Sentence denotes These hexamers work as initiation sites for the ϕ29 DNA polymerases.
T100 11908-12022 Sentence denotes After denaturation at 95 °C, the mixture is cooled down to ice temperature and the rest of the reagents are added.
T101 12023-12223 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 12224-12361 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 12362-12459 Sentence denotes As the polymerase displaces the formed strand ahead of it, it continues to complete the sequence.
T104 12460-12679 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 12680-12766 Sentence denotes Finally, the inactivation of the polymerase is done by heating up the system to 65 °C.
T106 12767-13011 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 13012-13092 Sentence denotes After the amplification reaction, the polymerase has to be inactivated at 65 °C.
T108 13093-13236 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 13237-13453 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 13455-13457 Sentence denotes 2.
T111 13458-13480 Sentence denotes Design and Fabrication
T112 13482-13486 Sentence denotes 2.1.
T113 13487-13516 Sentence denotes Microfluidic Structure Design
T114 13517-13629 Sentence denotes The microfluidic structure consists of two chambers, i.e., a reaction chamber and a temperature monitor chamber.
T115 13630-13681 Sentence denotes In Figure 2, a close-up of the final chip is shown.
T116 13682-13770 Sentence denotes For clarity reasons, the two microfluidic structures are colored with food coloring dye.
T117 13771-14219 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 14220-14565 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 14566-14654 Sentence denotes The total chip size is 3 cm by 3 cm and contains an inlet and outlet of 1.5 mm diameter.
T120 14655-14752 Sentence denotes The inlet and outlet are of such size that the reaction chamber can be filled using pipette tips.
T121 14753-14845 Sentence denotes In between the inlet and outlet, a rectangular reaction chamber of 10 mm by 3 mm is located.
T122 14846-14947 Sentence denotes Two trapezoid structures are placed in the tapered channels between the inlet/outlet and the chamber.
T123 14948-14992 Sentence denotes The function of these trapezoids is twofold:
T124 14993-15153 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 15154-15207 Sentence denotes Second, they provide support for the chamber closure.
T126 15208-15365 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 15366-15417 Sentence denotes This channel serves as temperature monitor chamber.
T128 15418-15521 Sentence denotes A thermocouple is inserted in this channel for real-time monitoring of the temperature inside the chip.
T129 15522-15616 Sentence denotes This way, a more accurate temperature of the reaction mixture inside the chip can be obtained.
T130 15617-15687 Sentence denotes Via a feedback loop, the input potential can be changed when required.
T131 15688-15777 Sentence denotes In Figure 3, the SolidWorks design of the chamber-based chip with both chambers is shown.
T132 15778-15854 Sentence denotes In Figure A1, in Appendix A, the technical drawing of the chip can be found.
T133 15856-15860 Sentence denotes 2.2.
T134 15861-15894 Sentence denotes Resistive Heater Structure Design
T135 15895-16068 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 16069-16172 Sentence denotes A meandering heater design is chosen, as this minimizes the input power required to heat up the heater.
T137 16173-16526 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 16527-16725 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 16726-16960 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 16961-17038 Sentence denotes However, most amplification methods require a DNA denaturation step at 95 °C.
T141 17039-17728 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 17729-17770 Sentence denotes Values for κi can be found in Appendix B.
T143 17771-17851 Sentence denotes From Equation (3), the product Rth×A can be defined as the sum of 1/h and li/κi.
T144 17852-18074 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 18075-18267 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 18268-18528 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 18529-18642 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 18643-18763 Sentence denotes The only side note here is that at higher temperatures, the temperature gradient through the system also becomes larger.
T149 18764-18841 Sentence denotes This can be eliminated by using double-sided heating, like Chung et al. [28].
T150 18842-19092 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 19093-19253 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 19254-19338 Sentence denotes The meandering heater are assumed to be lines at the bottom side of the layer stack.
T153 19339-19477 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 19478-19546 Sentence denotes The heater temperature is set at a constant temperature of 303.15 K.
T155 19547-19669 Sentence denotes This makes the heater material independent and the model purely focused on the heat transfer inside the COC–H2O–COC stack.
T156 19670-19724 Sentence denotes All used values and equations are given in Appendix B.
T157 19725-19848 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 19849-20013 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 20014-20197 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 20198-20393 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 20394-20513 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 20514-20619 Sentence denotes The total amount of large and smaller interconnecting rectangles for all 4 cases is estimated in Table 3.
T163 20620-21014 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 21015-21304 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 21305-21622 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 21623-21676 Sentence denotes The subscript i denotes the material, being Au or Pt.
T167 21677-21828 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 21829-21955 Sentence denotes Therefore, any possible combination of heater width and heater spacing will give a resistive that can withstand its operation.
T169 21957-21961 Sentence denotes 2.3.
T170 21962-21973 Sentence denotes Fabrication
T171 21974-22146 Sentence denotes COC [42] is chosen as polymeric substrate because of its biocompatibility, optical transparency, physical resistance, chemical resistance, electrical insulation, and price.
T172 22147-22298 Sentence denotes This copolymer consists of two monomers, an apolar bridged cyclic hydrocarbon (norbornene) monomer and a linear, lesser apolar, linear ethene monomers.
T173 22299-22494 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 22495-22597 Sentence denotes This grade is chosen because of its high norbornene content, giving it a relatively high Tg of 170 °C.
T175 22598-22770 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 22771-22940 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 22941-23029 Sentence denotes Milling is a very fast prototyping technique and chosen because of its flexibility [41].
T178 23030-23120 Sentence denotes The milling creates a surface roughness, which increases the surface-area-to-volume ratio.
T179 23121-23263 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 23264-23415 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 23416-23500 Sentence denotes Therefore, a chemical post-treatment with cyclohexane vapor is done (see Figure 4c).
T182 23501-23731 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 23732-23830 Sentence denotes CNC milling and subsequent cyclohexane vapor post-treatment are less suitable for mass production.
T184 23831-23895 Sentence denotes However, COC has the possibility of being injection molded [42].
T185 23896-23943 Sentence denotes The used substrates are made using this method.
T186 23944-24184 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 24185-24309 Sentence denotes Guckenberger et al. mention production costs of $ 47 per simple microfluidic device when only 50 pieces are fabricated [41].
T188 24310-24387 Sentence denotes This price is expected to drop drastically when large numbers are fabricated.
T189 24388-24547 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 24548-24679 Sentence denotes Mo has a smaller coefficient of thermal expansion than stainless steel, and therefore gives less deformation during the deposition.
T191 24680-24782 Sentence denotes Metals of interest are Au or Pt, which are commonly used metals to function as resistive heaters [54].
T192 24783-25110 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 25111-25249 Sentence denotes Both deposition methods are capable of large-scale production, which will lower the production costs in the large-volume production stage.
T194 25250-25694 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 25696-25700 Sentence denotes 2.4.
T196 25701-25710 Sentence denotes Operation
T197 25711-25863 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 25864-25942 Sentence denotes Each cleaning step was done 3 times and the chips are blow dried using N2 gas.
T199 25943-26166 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 26167-26416 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 26417-26598 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 26599-26773 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 26774-26870 Sentence denotes The thermocouple is read out with a Tenma 72-7715 Thermometer (Premier Farnell Ltd., Leeds, UK).
T204 26871-26974 Sentence denotes The source and the read-out of the thermocouple are operated using a custom-programmed LabVIEW program.
T205 26975-27123 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 27124-27290 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 27292-27294 Sentence denotes 3.
T208 27295-27317 Sentence denotes Results and Discussion
T209 27319-27323 Sentence denotes 3.1.
T210 27324-27362 Sentence denotes COMSOL MultiPhysics Simulation Results
T211 27363-27460 Sentence denotes In Figure 5, the results of heat transfer simulations of two different heater spacings are shown.
T212 27461-27585 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 27586-27713 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 27714-27900 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 27901-28078 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 28079-28270 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 28271-28477 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 28478-28655 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 28656-28770 Sentence denotes This is evident as smaller heater spacings will result in a better coverage of the heated area by heater material.
T220 28771-28925 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 28926-29079 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 29080-29243 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 29244-29445 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 29446-29578 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 29579-29718 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 29719-29838 Sentence denotes It is known that a meandering heater structure does not give the optimal temperature distribution over the device [26].
T227 29839-30054 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 30055-30190 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 30191-30241 Sentence denotes This prevents curvature due to intrinsic stresses.
T230 30242-30337 Sentence denotes See Figure 7 for the outlines of both shadow masks, together with the resulting pattern on COC.
T231 30338-30436 Sentence denotes The use of two shadow masks will give a metal track in which small parts has the double thickness.
T232 30437-30473 Sentence denotes Here, the temperature will be lower.
T233 30474-30599 Sentence denotes The system is designed such that these thicker parts are outside the reaction chamber and temperature control chamber region.
T234 30601-30605 Sentence denotes 3.2.
T235 30606-30617 Sentence denotes Fabrication
T236 30619-30651 Sentence denotes Milling and Optical Transparency
T237 30652-30746 Sentence denotes The milling increased the surface roughness of the COC plates also increases the surface area.
T238 30747-30886 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 30887-31225 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 31226-31260 Sentence denotes The results are shown in Figure 8.
T241 31261-31320 Sentence denotes The surface roughness of pristine COC had a RRMS of 3.5 nm.
T242 31321-31393 Sentence denotes This increased two orders of magnitude after milling (RRMS of 310.1 nm).
T243 31394-31593 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 31594-31805 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 31806-31955 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 31956-32069 Sentence denotes As the grade number increase, the ratio changes towards more norbornene monomers and less linear ethene monomers.
T247 32070-32257 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 32258-32339 Sentence denotes Lowering the surface roughness also increased the optical transmittance fivefold.
T249 32340-32440 Sentence denotes Transmittance measurements in the visible range are done using a Woollam M-2000UI ellipsometer (J.A.
T250 32441-32472 Sentence denotes Woollam Co., Lincoln, NE, USA).
T251 32473-32523 Sentence denotes The results can be seen in Figure 9 and Figure 10.
T252 32524-32763 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 32764-33004 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 33006-33010 Sentence denotes 3.3.
T255 33011-33025 Sentence denotes Metal Adhesion
T256 33026-33141 Sentence denotes To get reliable heaters, four possible options are investigated for their adhesion properties to the COC substrate.
T257 33142-33328 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 33329-33587 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 33588-33640 Sentence denotes See Table 4 for the results of the Scotch tape test.
T260 33641-33956 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 33957-34123 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 34124-34304 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 34305-34374 Sentence denotes The bulk TCR values are 0.0034 K−1 and 0.0037 K−1 for Au and Pt [67].
T264 34375-34496 Sentence denotes The TCR characterizations of the metal strips on a COC substrate did not yield trustworthy TCR values at the first cycle.
T265 34497-34662 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 34663-34736 Sentence denotes After this first cycle, the values more or less show the linear behavior.
T267 34737-34862 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 34863-34998 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 34999-35116 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 35117-35214 Sentence denotes COC of the grade TOPAS 6017 has a coefficient of linear thermal expansion of 6.0 × 10−5 K−1 [42].
T271 35215-35309 Sentence denotes This mismatch in coefficients of linear thermal expansion can give strain in the metal layers.
T272 35310-35467 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 35468-35537 Sentence denotes Another effect influencing the TCR value of the metal layer is aging.
T274 35538-35659 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 35660-35728 Sentence denotes This could be due to adsorbed materials present on the surface [64].
T276 35729-35845 Sentence denotes However, in this device, the TCR is not of importance as the metal structure will not be used as temperature sensor.
T277 35846-35940 Sentence denotes Real-time temperature sensing is done using a thermocouple in the temperature monitor chamber.
T278 35941-36081 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 36082-36206 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 36207-36309 Sentence denotes The TCR can become more important when other (higher) temperatures are required for the amplification.
T281 36310-36460 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 36461-36592 Sentence denotes Sputtering is an industrial-scale technique that is already being used in, for example, the car mirror and headlight industry [75].
T283 36594-36598 Sentence denotes 3.4.
T284 36599-36615 Sentence denotes Chip Functioning
T285 36616-36756 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 36757-36919 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 36920-37126 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 37127-37227 Sentence denotes These measurements also gives a better insight of the heat distribution inside the reaction chamber.
T289 37228-37292 Sentence denotes The images are processed using the FLIR postprocessing freeware.
T290 37293-37349 Sentence denotes Results of these measurements are shown in Figure 11a,b.
T291 37350-37416 Sentence denotes The results are in good agreement with the estimations in Table 1.
T292 37417-37799 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 37800-37923 Sentence denotes The reliability of the heater is tested by inserting the thermocouple into the temperature control chamber (see Figure 3a).
T294 37924-38036 Sentence denotes A constant input potential of 4 V is applied using the Keithley source and the temperature is measured for 25 h.
T295 38037-38188 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 38189-38225 Sentence denotes The results are shown in Figure 11c.
T297 38226-38487 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 38489-38493 Sentence denotes 3.5.
T299 38494-38511 Sentence denotes DNA Amplification
T300 38512-38700 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 38701-38850 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 38851-38997 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 38998-39373 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 39374-39489 Sentence denotes This makes the functioning of the integrated resistive heater less critical than the stability shown in Figure 11c.
T305 39490-39692 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 39693-39807 Sentence denotes As heat supply the water bath of an IKA Rotary Evaporator RV 8V (IKA-Werke, Staufen im Breisgau, Germany) is used.
T307 39808-39911 Sentence denotes This water bath is according to its specification stable within a range of the set temperature ±0.1 °C.
T308 39912-40036 Sentence denotes The chip and an Eppendorf tube are loaded with the reaction mixture containing the DNA sample and the EvaGreen dye solution.
T309 40037-40170 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 40171-40261 Sentence denotes The inlet and outlet of the chip are sealed with the Microseal “B” PCR plate sealing foil.
T311 40262-40398 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 40399-40533 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 40534-40596 Sentence denotes The complete mixtures are according to Table A3 in Appendix D.
T314 40597-40770 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 40771-40881 Sentence denotes The MDA is also performed inside the chip, but with the integrated Au resistive heater serving as heat source.
T316 40882-40926 Sentence denotes The set up shown schematically in Figure 12.
T317 40927-40993 Sentence denotes The same procedure is followed as with the water bath heated test.
T318 40994-41040 Sentence denotes Denaturation is done in a separate water bath.
T319 41041-41264 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 41265-41335 Sentence denotes In Figure 14 the logged temperature during the amplification is shown.
T321 41336-41526 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 41527-41644 Sentence denotes Fluorescence measurements are done in a Horiba Scientific FluoroMax+ spectrofluorometer to verify each amplification.
T323 41645-41820 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 41821-41981 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 41982-42063 Sentence denotes No Enzyme was added to this mixture, therefore no amplification could take place.
T326 42064-42135 Sentence denotes See Figure 15 for the results obtained in the Eppendorf tube and chips.
T327 42136-42230 Sentence denotes Figure A4 in Appendix E shows the background signal which is subtracted from all measurements.
T328 42231-42506 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 42507-42576 Sentence denotes There is an order of magnitude difference in the fluorescence signal.
T330 42577-42669 Sentence denotes However, the fluorescence intensity cannot be used as a value to quantify the amount of DNA.
T331 42670-42810 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 42811-42953 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 42954-43081 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 43083-43085 Sentence denotes 4.
T335 43086-43097 Sentence denotes Conclusions
T336 43098-43321 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 43322-43460 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 43461-43548 Sentence denotes As proof-of-principle the MDA reaction and ex-situ fluorescence measurements were used.
T339 43549-43650 Sentence denotes With the proposed fabrication process, low-cost and biocompatible chips (Figure 12b) were fabricated.
T340 43651-43905 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 43906-44018 Sentence denotes The main cause of this period of lowered temperature was due to the fact that the measurement was run overnight.
T342 44019-44144 Sentence denotes With the proof-of-principle device, successful DNA amplifications using MDA inside a disposable polymeric chip were achieved.
T343 44145-44233 Sentence denotes The heat for the reaction was applied using the integrated low-cost Au-resistive heater.
T344 44234-44403 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 44404-44492 Sentence denotes A distinct peak is visible in the reaction mixtures which is absent in the NTC mixtures.
T346 44493-44597 Sentence denotes The operating temperature for MDA reactions is around 30 °C, which is comparable with a nice summer day.
T347 44598-44710 Sentence denotes Using amplification reactions which such low reaction temperatures could encounter problems at warmer locations.
T348 44711-44813 Sentence denotes However, as MDA is not sequence specific, this reaction will not be integrated in the final protocols.
T349 44814-44947 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 44948-45074 Sentence denotes Sequence specific amplifications, e.g., HDA and LAMP, are performed at higher temperatures, as will be discussed in Section 5.
T351 45075-45135 Sentence denotes This makes the system less sensitive to the hot summer days.
T352 45136-45287 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 45288-45343 Sentence denotes However, the first steps are made to an ASSURED device.
T354 45344-45440 Sentence denotes Future steps which will make the device fully ASSURED are given in the next section (Section 5).
T355 45442-45444 Sentence denotes 5.
T356 45445-45452 Sentence denotes Outlook
T357 45453-45709 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 45710-45844 Sentence denotes HDA utilizes DNA helicase (an enzyme also used in vitro during DNA replication) to separate the dsDNA instead of thermal denaturation.
T359 45845-46083 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 46084-46253 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 46254-46333 Sentence denotes LAMP is more similar to MDA in the way it also uses heat to denature the dsDNA.
T362 46334-46518 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 46519-46728 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 46729-46847 Sentence denotes Despite not being a truly isothermal technique, LAMP offers the possibility to use turbidity as detection method [76].
T365 46848-47007 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 47008-47074 Sentence denotes Different amplification techniques require different temperatures.
T367 47075-47199 Sentence denotes Based on Table 1 one can conclude that a higher temperature would also give a larger temperature gradient within the system.
T368 47200-47331 Sentence denotes This can be disadvantageous for amplification reactions, as optimal denaturation temperatures are in the range 92 °C to 94 °C [77].
T369 47332-47447 Sentence denotes The denaturation in this research was done in a separate water bath, so this temperature gradient was circumvented.
T370 47448-47656 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 47657-47754 Sentence denotes This can be done by using different heater geometries [26,37] or using double-sided heating [28].
T372 47755-47842 Sentence denotes The third step that has to be optimized in the sample collection and work-up procedure.
T373 47843-48024 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 48025-48092 Sentence denotes Such crude samples contain full cells, with the DNA present within.
T375 48093-48179 Sentence denotes There are different approaches to perform cell lysis in order to extract the DNA [78].
T376 48180-48314 Sentence denotes Various components of bodily fluids, and reagents and products of the lysis are well-known to inhibit the amplification reaction [79].
T377 48315-48386 Sentence denotes However, MDA [80] and HDA [9] could also be performed on crude samples.
T378 48387-48520 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 48521-48761 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 48762-48862 Sentence denotes The lamp and photodiode could both be used for fluorescence measurements and turbidity measurements.
T381 48864-48879 Sentence denotes Acknowledgments
T382 48880-49005 Sentence denotes The authors would like to thank Brigitte Bruijns from Micronit Microtechnologies for her help during the brainstorm sessions.
T383 49006-49128 Sentence denotes Jörg Strack from TOPAS Advanced Polymers and Thomas Wagenknecht from KUZ Leipzig are thanked for their information on COC.
T384 49129-49549 Sentence denotes Pieter Post, Rob Dierink, and Sip Jan Boorsma of TCO (Technical Center for Education and Research of the University of Twente) are thanked for their work in the milling and laser cutting processes, Christian Bruinink for his assistance in the transparency measurements, Daniel Monteiro Cunha, M.Sc. for his assistance in the AFM measurements, and Nikki Stroot for her assistance with the initial amplification reactions.
T385 49550-49570 Sentence denotes Sample Availability:
T386 49571-49637 Sentence denotes Samples of the amplification chips are available from the authors.
T387 49639-49659 Sentence denotes Author Contributions
T388 49660-50098 Sentence denotes Conceived and designed the experiments: H.-W.V., F.A.M., and R.S.; developed the measurement setup and software, and provided technical help: R.S.; developed the methodology: H.-W.V. and F.A.M.; performed the experiments: F.A.M.; visualized the results: H.-W.V. and F.A.M.; wrote the first draft of the paper: H.-W.V.; reviewed and edited the first draft of the paper: H.-W.V., F.A.M., R.S., R.W. and J.L.; supervised the project: H.-W.V.
T389 50099-50175 Sentence denotes All authors have read and agreed to the published version of the manuscript.
T390 50177-50184 Sentence denotes Funding
T391 50185-50234 Sentence denotes This work is financed with institutional funding.
T392 50236-50257 Sentence denotes Conflicts of Interest
T393 50258-50302 Sentence denotes The authors declare no conflict of interest.
T394 50303-50316 Sentence denotes Abbreviations
T395 50317-50401 Sentence denotes The following abbreviations are used in this manuscript: AFM atomic force microscopy
T396 50402-50409 Sentence denotes Au gold
T397 50410-50435 Sentence denotes CAD computer-aided design
T398 50436-50468 Sentence denotes CAM computer-aided manufacturing
T399 50469-50499 Sentence denotes CNC computer numerical control
T400 50500-50527 Sentence denotes COC cyclic olefin copolymer
T401 50528-50545 Sentence denotes DC direct current
T402 50546-50559 Sentence denotes DI de-ionized
T403 50560-50585 Sentence denotes DNA deoxyribonucleic acid
T404 50586-50611 Sentence denotes dsDNA double stranded DNA
T405 50612-50621 Sentence denotes H2O water
T406 50622-50658 Sentence denotes HDA helicase-dependent amplification
T407 50659-50702 Sentence denotes LAMP loop-mediated isothermal amplification
T408 50703-50742 Sentence denotes MDA multiple displacement amplification
T409 50743-50756 Sentence denotes Mo molybdenum
T410 50757-50768 Sentence denotes N2 nitrogen
T411 50769-50793 Sentence denotes NTC non template control
T412 50794-50823 Sentence denotes PCR polymerase chain reaction
T413 50824-50849 Sentence denotes PDMS polydimethylsiloxane
T414 50850-50886 Sentence denotes PID proportional-integral-derivative
T415 50887-50898 Sentence denotes Pt platinum
T416 50899-50928 Sentence denotes PVD physical vapor deposition
T417 50929-50949 Sentence denotes RNA ribonucleic acid
T418 50950-50982 Sentence denotes SEM scanning electron microscopy
T419 50983-51008 Sentence denotes ssDNA single stranded DNA
T420 51009-51065 Sentence denotes SDI Sexually Transmitted Diseases Diagnostics Initiative
T421 51066-51107 Sentence denotes TCR temperature coefficient of resistance
T422 51108-51139 Sentence denotes Tg glass transition temperature
T423 51140-51170 Sentence denotes WGA whole genome amplification
T424 51171-51200 Sentence denotes WHO World Health Organization
T425 51202-51213 Sentence denotes Appendix A.
T426 51214-51236 Sentence denotes Technical Drawing Chip
T427 51237-51296 Sentence denotes Figure A1 Technical drawing of the DNA amplification chip.
T428 51297-51322 Sentence denotes All dimensions are in mm.
T429 51323-51359 Sentence denotes The total chip size is 3 mm by 3 mm.
T430 51361-51372 Sentence denotes Appendix B.
T431 51373-51431 Sentence denotes Equations and Values Used in the COMSOL Multiphysics Study
T432 51433-51446 Sentence denotes Appendix B.1.
T433 51447-51452 Sentence denotes Water
T434 51453-51713 Sentence denotes For H2O, the build-in temperature-dependent equations for the density (ρH2O, see equations in Equation (A1)), heat capacity at constant pressure (CP,H2O, see equations in Equation (A2)), and thermal conductivity (κH2O, see equations in Equation (A3)) are used.
T435 51714-51918 Sentence denotes The ratio (γH2O) of the specific heats at constant pressure (CP,H2O) and constant volume (CV,H2O) is calculated manually according to the table from the Engineering Toolbox website [81] and Equation (A4).
T436 51919-52293 Sentence denotes The values for γH2O are listed in Table A1. (A1) ρH2O(T)=972.7584+0.2084T−4×10−4T2for273≤T<283345.28+5.749816T−0.0157244T2+1.264375×10−5T3for283≤T<373 (A2) CP,H2O(T)=12010.1471−80.4072879T+0.309866854T2for273.15≤T<553.75−5.38186884×10−4T3+3.62536437×10−7T4 (A3) κH2O(T)=−0.869083936+0.00894880345T−1.58366345×10−5T2for273.15≤T<553.75+7.97543259×10−9T3 (A4) γH2O=CP,H2OCV,H2O
T437 52294-52341 Sentence denotes Table A1 The ratio of specific heats of water.
T438 52342-52359 Sentence denotes T γH2O T γH2O
T439 52360-52378 Sentence denotes [K] [-] [K] [-]
T440 52379-52419 Sentence denotes 273.16 1.000592782 393.15 1.157465496
T441 52420-52460 Sentence denotes 283.15 1.001073729 413.15 1.199809492
T442 52461-52501 Sentence denotes 293.15 1.006591292 433.15 1.246234334
T443 52502-52542 Sentence denotes 298.15 1.010560913 453.15 1.297534537
T444 52543-52583 Sentence denotes 303.15 1.015203400 473.15 1.355013713
T445 52584-52624 Sentence denotes 313.15 1.025996023 493.15 1.420794975
T446 52625-52665 Sentence denotes 323.15 1.038520763 513.15 1.498241758
T447 52666-52706 Sentence denotes 333.15 1.052405261 533.15 1.592792563
T448 52707-52747 Sentence denotes 343.15 1.067512483 553.15 1.714447794
T449 52748-52788 Sentence denotes 353.15 1.083658241 573.15 1.883524402
T450 52789-52829 Sentence denotes 363.15 1.100748613 593.15 2.148448797
T451 52830-52870 Sentence denotes 373.15 1.118756966 613.15 2.666580033
T452 52871-52911 Sentence denotes 383.15 1.137648990 633.15 4.550527720
T453 52913-52926 Sentence denotes Appendix B.2.
T454 52927-52950 Sentence denotes Cyclic Olefin Copolymer
T455 52951-53265 Sentence denotes Values for the density (ρCOC = 1020 kg m−3), specific heat (cCOC, see Table A2), and thermal conductivity (κCOC,23°C = 0.17 W m−1 K−1 and κCOC,320°C = 0.24 W m−1 K−1, linear fit in between these point) of TOPAS 6017 COC are obtained via TOPAS Advanced Polymers (TOPAS Advanced Polymers, Farmington Hills, MI, USA).
T456 53266-53462 Sentence denotes The heat capacity at constant pressure (CP,COC) is calculated assuming a homogeneous body of mass m, via Equation (A5), where COMSOL interpolated linearly in between the points. (A5) CP,COC=cCOC×m
T457 53463-53509 Sentence denotes Table A2 The specific heat of TOPAS 6017 COC.
T458 53510-53523 Sentence denotes T c T c
T459 53524-53560 Sentence denotes [°C] [W m−1 K−1] [°C] [W m−1 K−1]
T460 53561-53580 Sentence denotes 30 1333 180 2298
T461 53581-53600 Sentence denotes 70 1538 210 2412
T462 53601-53621 Sentence denotes 110 1754 250 2539
T463 53622-53642 Sentence denotes 150 1968 290 2645
T464 53643-53800 Sentence denotes 160 2047 330 2758 Convective heat loss to the air is also taken into account with Equation (A6), which is often used in simulations [56]. (A6) h=10Wm−2K−1
T465 53802-53813 Sentence denotes Appendix C.
T466 53814-53830 Sentence denotes TCR Measurements
T467 53831-53996 Sentence denotes The two subsections below show two effects on the TCR measurements, i.e., the effect of thermal annealing during the first temperature cycle and the effect of aging.
T468 53998-54011 Sentence denotes Appendix C.1.
T469 54012-54029 Sentence denotes Thermal Annealing
T470 54030-54118 Sentence denotes The graph below is an example of the measured thermal annealing of a metal layer on COC.
T471 54119-54216 Sentence denotes Here, only the graph for evaporated Au is shown, but all except evaporated Pt show this behavior.
T472 54217-54272 Sentence denotes Evaporated Pt had some contaminants in the metal track.
T473 54273-54321 Sentence denotes This is most probably caused by the shadow mask.
T474 54322-54384 Sentence denotes Figure A2 Thermal annealing of an evaporated 100 nm Au layer.
T475 54386-54399 Sentence denotes Appendix C.2.
T476 54400-54405 Sentence denotes Aging
T477 54406-54514 Sentence denotes The layer analyzed in Figure A2 is stored for two weeks in ambient conditions and the TCR is analyzed again.
T478 54515-54605 Sentence denotes From the graph it is visible that the aging has some effect on the TCR of the metal layer.
T479 54606-54690 Sentence denotes Figure A3 The effect of two weeks aging in ambient conditions on a 100 nm Au layer.
T480 54691-54793 Sentence denotes The TCR of the as deposited layer is 0.00161 K−1 and the TCR of the two week old layer is 0.00224 K−1.
T481 54795-54806 Sentence denotes Appendix D.
T482 54807-54827 Sentence denotes MDA Reaction Mixture
T483 54828-54905 Sentence denotes The reaction mixture for the on-chip MDA reaction consisted of the following.
T484 54906-54946 Sentence denotes Table A3 On-chip MDA reaction mixtures.
T485 54947-55019 Sentence denotes Sample Reaction Buffer Sample Buffer DNA MilliQ DI Water EG Enzyme
T486 55020-55054 Sentence denotes [μL] [μL] [μL] [μL] [μL] [μL]
T487 55055-55086 Sentence denotes Tube with DNA 9 9 1 - 4 1
T488 55087-55113 Sentence denotes Tube NTC 9 9 - 1 4 1
T489 55114-55145 Sentence denotes Chip with DNA 9 9 1 - 4 1
T490 55146-55172 Sentence denotes Chip NTC 9 9 - 1 4 1
T491 55174-55185 Sentence denotes Appendix E.
T492 55186-55229 Sentence denotes Background Signal Fluorescence Measurements
T493 55230-55367 Sentence denotes All fluorescence measurements in the Horiba Scientific FluoroMax+ spectrofluorometer are normalized by subtracting the background signal.
T494 55368-55433 Sentence denotes This background signal is measured using the mixture in Table A4.
T495 55434-55491 Sentence denotes Table A4 Mixture used for background signal measurement.
T496 55492-55564 Sentence denotes Sample Reaction Buffer Sample Buffer DNA MilliQ DI Water EG Enzyme
T497 55565-55599 Sentence denotes [μL] [μL] [μL] [μL] [μL] [μL]
T498 55600-55628 Sentence denotes Background 9 9 1 1 4 -
T499 55629-55689 Sentence denotes Figure A4 The background signal of the mixture in Table A4.
T500 55691-55746 Sentence denotes Figure 1 Schematic representation of the MDA reaction.
T501 55747-55785 Sentence denotes In panel (a), a dsDNA strand is shown.
T502 55786-55830 Sentence denotes In panel (b), denaturation happens at 95 °C.
T503 55831-55946 Sentence denotes In panel (c), the random hexamer-primers (purple) and ϕ29 DNA polymerase (blue arrow) bind to the initiation sites.
T504 55947-56063 Sentence denotes In panel (d), the amplification is performed by the polymerase, which binds complementary bases to the ssDNA strand.
T505 56064-56184 Sentence denotes In panel (e), the polymerase encounters another hexamer binded to an initiation site and starts lifting up this hexamer.
T506 56185-56316 Sentence denotes In panel (f), a hexamer binds to the displaced ssDNA strands and the polymerase starts the amplification from this initiation site.
T507 56317-56371 Sentence denotes For clarity, the amplified DNA is bordered with black.
T508 56372-56594 Sentence denotes Figure 2 Close-up of the chip showing the Au resistive heater and alignment crosses, the DNA amplification chamber filled with blue food coloring dye, and the temperature monitor chamber filled with red food coloring dye.
T509 56595-56750 Sentence denotes Figure 3 The SolidWorks design of the DNA amplification chip with reaction chamber and chamber for thermocouple-assisted real-time temperature monitoring.
T510 56751-56808 Sentence denotes This drawing is used to set up the code for the CNC mill.
T511 56809-56848 Sentence denotes Total size of the chip is 3 cm by 3 cm.
T512 56849-56999 Sentence denotes Panel (a) shows the top view, panel (b) shows the bird’s eye view, and panel (c) the close-up of the chambers with the in-chamber trapezoid structure.
T513 57000-57121 Sentence denotes Figure 4 Schematic top views (top row) and cross-sectional representations (bottom row) of the full fabrication process.
T514 57122-57181 Sentence denotes In panel (a) the pristine COC substrate is shown as step 0.
T515 57182-57300 Sentence denotes Then, in panel (b) the first step is shown, i.e., the milling of the reaction chamber and temperature monitor chamber.
T516 57301-57361 Sentence denotes In panel (c), the cyclohexane vapor post-treatment is shown.
T517 57362-57439 Sentence denotes Then, the substrate is flipped and metal is deposited as step 3 in panel (d).
T518 57440-57539 Sentence denotes The substrate is flipped back and the microfluidic structure is closed using PCR foil in panel (e).
T519 57540-57610 Sentence denotes The images are not on scale and out of proportion for clarity reasons.
T520 57611-57769 Sentence denotes Figure 5 COMSOL Multiphysics 5.3a finite element method heat transfer simulations of a 0.75 mm deep chamber with different heater widths and heater spacings.
T521 57770-57934 Sentence denotes Panel (a) shows the schematics with the materials indicated in the figure and the heaters exaggerated in red (they are 1 dimensional lines in the simulation model).
T522 57935-58010 Sentence denotes Panel (b) shows the case for 0.3 mm heater width and 0.3 mm heater spacing.
T523 58011-58086 Sentence denotes Panel (c) shows the case for 0.3 mm heater width and 2.0 mm heater spacing.
T524 58087-58162 Sentence denotes The scale bar in panel (d) is in °C and applies to both panels (b) and (c).
T525 58163-58221 Sentence denotes Figure 6 Three temperature differences within the system.
T526 58222-58491 Sentence denotes In panel (a) the ΔTtopofchamber, in panel (b) the ΔTacrosschamber, in panel (c) the ΔTbottomofchamber, and in panel (d) the ΔTdeviationfromsetT are shown for different heater widths (0.3 mm to 2.0 mm, in the columns) and heater spacings (0.3 mm to 2.0 mm, in the rows).
T527 58492-58672 Sentence denotes The differences are obtained using a parametric sweeps for both the heater width and heater spacing in the COMSOL Multiphysics 5.3a finite element method heat transfer simulations.
T528 58673-58754 Sentence denotes The cells in dark indicate the smallest ΔT and the cells in white the largest ΔT.
T529 58755-58851 Sentence denotes Figure 7 Panels (a) and (b) show the SolidWorks drawings of shadow masks 1 and 2, respectively.
T530 58852-58911 Sentence denotes Panel (c) shows the resulting metal track after deposition.
T531 58912-58975 Sentence denotes The crosses are used for alignment under an optical microscope.
T532 58976-59126 Sentence denotes Figure 8 AFM measurements (a–c) and SEM images (d–f) showing the effect of a cyclohexane vapor post-treatment on the surface roughness of milled COC.
T533 59127-59258 Sentence denotes Panel (a,d) shows the pristine COC surface. (b,e) Shows the milled surface. (c,f) Shows the cyclohexane vapor post-treated surface.
T534 59259-59301 Sentence denotes The scale bars in the SEM images are 2 μm.
T535 59302-59499 Sentence denotes Figure 9 The effect of the cyclohexane vapor post-treatment on the optical transparency of milled COC. (a) shows a milled surface and (b) the same surface, but post-treated with cyclohexane vapor.
T536 59500-59572 Sentence denotes The graph in Figure 10 shows the transmittance data of these substrates.
T537 59573-59763 Sentence denotes Figure 10 Graph of the transmittance data for a pristine substrate, and the substrates of Figure 9a,b showing the improvement in optical transparancy after cyclohexane vapor post-treatment.
T538 59764-59886 Sentence denotes The red line is for pristine COC, the green line for milled COC, and the blue line for cyclohexane vapor post-treated COC.
T539 59887-59968 Sentence denotes Measurements are done with a Woollam M-2000UI ellipsometer in transmittance mode.
T540 59969-60099 Sentence denotes Figure 11 Characterization of the 100 nm thick Au heater structure deposited by sputtering using the two sequential shadow masks.
T541 60100-60299 Sentence denotes Panel (a) depicts the thermal images on the other side of the 1.5 mm thick substrate, while operating the heater at different input powers. (b) The graph with the recorded heat profiles of panel (a).
T542 60300-60386 Sentence denotes Panel (c) shows the durability test in which a constant potential is applied for 25 h.
T543 60387-60712 Sentence denotes Figure 12 The set up for MDA reacions, (a) the schematics in which the voltage source is a Keithley SourceMeter, the thermocouple is connected to a Tenma thermometer. (b) The DNA amplification chip (both the amplification chamber and temperature control chamber are filled with food coloring dye for visualization purposes).
T544 60713-60830 Sentence denotes Figure 13 Fluorescence signal of MDA reactions performed at 25 °C and 30 °C, together with their (overlapping) NTCs.
T545 60831-60982 Sentence denotes Figure 14 Temperature profile recorded with the thermocouple inside the water-filled temperature control chamber during the on-chip MDA amplification.
T546 60983-61061 Sentence denotes Figure 15 Fluorescence signal of DNA-binded EvaGreen dye after MDA reactions.
T547 61062-61127 Sentence denotes The reactions are performed inside an Eppendorf tube and on-chip.
T548 61128-61274 Sentence denotes As heating, two different methods are used, namely a water bath (for both the Eppendorf tube and chip) and the on-chip resistive heater (3 chips).
T549 61275-61363 Sentence denotes The fluorescence intensity cannot be used as a value to quantify the amount of DNA [44].
T550 61364-61491 Sentence denotes The two blue lines with circular points are the MDA (continuous line) and NTC (dashed line) performed inside an Eppendorf tube.
T551 61492-61628 Sentence denotes The two orange lines with circular points are the MDA (continuous line) and NTC (dashed line) performed inside a water bath heated chip.
T552 61629-61809 Sentence denotes The three continuous orange lines with asterisks, squares, and diamonds represent the signals obtained after MDA reactions performed inside chips while using the integrated heater.
T553 61810-61934 Sentence denotes Table 1 Calculated required heater temperatures and powers when a desired temperature at the top of the chamber is assumed.
T554 61935-61987 Sentence denotes The calculations are based on Equations (2) and (3).
T555 61988-62043 Sentence denotes Desired Temperature Heater Temperature Required Power
T556 62044-62060 Sentence denotes [°C] [°C] [mW]
T557 62061-62075 Sentence denotes 30 31.7 17.3
T558 62076-62090 Sentence denotes 54 59.1 58.6
T559 62091-62105 Sentence denotes 65 71.8 77.7
T560 62106-62120 Sentence denotes 72 79.8 89.6
T561 62121-62137 Sentence denotes 95 106.3 129.4
T562 62138-62185 Sentence denotes Table 2 Specifications of simulation computer.
T563 62186-62205 Sentence denotes Component Hardware
T564 62206-62246 Sentence denotes Motherboard ASUS RoG Rampage VI Extreme
T565 62247-62300 Sentence denotes CPU Intel Core i9 7900X processor, 10 cores, 3.3 GHz
T566 62301-62346 Sentence denotes CPU cooler NZXT Kraken X62 AM4 water cooling
T567 62347-62402 Sentence denotes RAM G.Skill Trident Z RGB LED 4 × 16 GB DDR4, 3200 MHz
T568 62403-62431 Sentence denotes GPU Radeon Pro WX2100, 2 GB
T569 62432-62468 Sentence denotes Power supply Corsair HX1000i 1000 W
T570 62469-62542 Sentence denotes Table 3 Estimated current densities for the 4 extreme heater geometries.
T571 62543-62618 Sentence denotes sheater wheater lheater IAu IAu/Across−section IPt IPt/Across−section
T572 62619-62673 Sentence denotes [mm] [mm] [mm] [mA] [109 A m−2] [mA] [108 A m−2]
T573 62674-62709 Sentence denotes 0.3 0.3 134.9 32 1.06 14 4.73
T574 62710-62744 Sentence denotes 0.3 2.0 46.4 54 1.84 24 8.06
T575 62745-62780 Sentence denotes 2.0 0.3 30.9 172 8.60 77 3.83
T576 62781-62816 Sentence denotes 2.0 2.0 22.2 203 1.01 90 4.51
T577 62817-62904 Sentence denotes Table 4 Results of the Scotch tape metal adhesion tests before and after heat cycling.
T578 62905-63037 Sentence denotes Here, + means passing the tape test, − means failing the tape test, and +/− means that not all test structures failed the tape test.
T579 63038-63079 Sentence denotes Material Method Tape Test Initial TCR
T580 63080-63114 Sentence denotes Before After Resistance [Ω] K−1
T581 63115-63150 Sentence denotes Au Evaporation + − 5.0 0.00161
T582 63151-63185 Sentence denotes Au Sputtering + + 8.2 0.00192
T583 63186-63226 Sentence denotes Pt Evaporation + + 400 ∗ −0.00440 ∗
T584 63227-63263 Sentence denotes Pt Sputtering + +/− 6.8 0.00207
T585 63264-63354 Sentence denotes * These results are not reliable as the shadow mask gave contamination in the metal track.