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PMC:7143804 / 27292-43081 JSONTXT 10 Projects

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Id Subject Object Predicate Lexical cue
T207 0-2 Sentence denotes 3.
T208 3-25 Sentence denotes Results and Discussion
T209 27-31 Sentence denotes 3.1.
T210 32-70 Sentence denotes COMSOL MultiPhysics Simulation Results
T211 71-168 Sentence denotes In Figure 5, the results of heat transfer simulations of two different heater spacings are shown.
T212 169-293 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 294-421 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 422-608 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 609-786 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 787-978 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 979-1185 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 1186-1363 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 1364-1478 Sentence denotes This is evident as smaller heater spacings will result in a better coverage of the heated area by heater material.
T220 1479-1633 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 1634-1787 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 1788-1951 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 1952-2153 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 2154-2286 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 2287-2426 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 2427-2546 Sentence denotes It is known that a meandering heater structure does not give the optimal temperature distribution over the device [26].
T227 2547-2762 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 2763-2898 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 2899-2949 Sentence denotes This prevents curvature due to intrinsic stresses.
T230 2950-3045 Sentence denotes See Figure 7 for the outlines of both shadow masks, together with the resulting pattern on COC.
T231 3046-3144 Sentence denotes The use of two shadow masks will give a metal track in which small parts has the double thickness.
T232 3145-3181 Sentence denotes Here, the temperature will be lower.
T233 3182-3307 Sentence denotes The system is designed such that these thicker parts are outside the reaction chamber and temperature control chamber region.
T234 3309-3313 Sentence denotes 3.2.
T235 3314-3325 Sentence denotes Fabrication
T236 3327-3359 Sentence denotes Milling and Optical Transparency
T237 3360-3454 Sentence denotes The milling increased the surface roughness of the COC plates also increases the surface area.
T238 3455-3594 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 3595-3933 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 3934-3968 Sentence denotes The results are shown in Figure 8.
T241 3969-4028 Sentence denotes The surface roughness of pristine COC had a RRMS of 3.5 nm.
T242 4029-4101 Sentence denotes This increased two orders of magnitude after milling (RRMS of 310.1 nm).
T243 4102-4301 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 4302-4513 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 4514-4663 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 4664-4777 Sentence denotes As the grade number increase, the ratio changes towards more norbornene monomers and less linear ethene monomers.
T247 4778-4965 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 4966-5047 Sentence denotes Lowering the surface roughness also increased the optical transmittance fivefold.
T249 5048-5148 Sentence denotes Transmittance measurements in the visible range are done using a Woollam M-2000UI ellipsometer (J.A.
T250 5149-5180 Sentence denotes Woollam Co., Lincoln, NE, USA).
T251 5181-5231 Sentence denotes The results can be seen in Figure 9 and Figure 10.
T252 5232-5471 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 5472-5712 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 5714-5718 Sentence denotes 3.3.
T255 5719-5733 Sentence denotes Metal Adhesion
T256 5734-5849 Sentence denotes To get reliable heaters, four possible options are investigated for their adhesion properties to the COC substrate.
T257 5850-6036 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 6037-6295 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 6296-6348 Sentence denotes See Table 4 for the results of the Scotch tape test.
T260 6349-6664 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 6665-6831 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 6832-7012 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 7013-7082 Sentence denotes The bulk TCR values are 0.0034 K−1 and 0.0037 K−1 for Au and Pt [67].
T264 7083-7204 Sentence denotes The TCR characterizations of the metal strips on a COC substrate did not yield trustworthy TCR values at the first cycle.
T265 7205-7370 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 7371-7444 Sentence denotes After this first cycle, the values more or less show the linear behavior.
T267 7445-7570 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 7571-7706 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 7707-7824 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 7825-7922 Sentence denotes COC of the grade TOPAS 6017 has a coefficient of linear thermal expansion of 6.0 × 10−5 K−1 [42].
T271 7923-8017 Sentence denotes This mismatch in coefficients of linear thermal expansion can give strain in the metal layers.
T272 8018-8175 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 8176-8245 Sentence denotes Another effect influencing the TCR value of the metal layer is aging.
T274 8246-8367 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 8368-8436 Sentence denotes This could be due to adsorbed materials present on the surface [64].
T276 8437-8553 Sentence denotes However, in this device, the TCR is not of importance as the metal structure will not be used as temperature sensor.
T277 8554-8648 Sentence denotes Real-time temperature sensing is done using a thermocouple in the temperature monitor chamber.
T278 8649-8789 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 8790-8914 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 8915-9017 Sentence denotes The TCR can become more important when other (higher) temperatures are required for the amplification.
T281 9018-9168 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 9169-9300 Sentence denotes Sputtering is an industrial-scale technique that is already being used in, for example, the car mirror and headlight industry [75].
T283 9302-9306 Sentence denotes 3.4.
T284 9307-9323 Sentence denotes Chip Functioning
T285 9324-9464 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 9465-9627 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 9628-9834 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 9835-9935 Sentence denotes These measurements also gives a better insight of the heat distribution inside the reaction chamber.
T289 9936-10000 Sentence denotes The images are processed using the FLIR postprocessing freeware.
T290 10001-10057 Sentence denotes Results of these measurements are shown in Figure 11a,b.
T291 10058-10124 Sentence denotes The results are in good agreement with the estimations in Table 1.
T292 10125-10507 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 10508-10631 Sentence denotes The reliability of the heater is tested by inserting the thermocouple into the temperature control chamber (see Figure 3a).
T294 10632-10744 Sentence denotes A constant input potential of 4 V is applied using the Keithley source and the temperature is measured for 25 h.
T295 10745-10896 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 10897-10933 Sentence denotes The results are shown in Figure 11c.
T297 10934-11195 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 11197-11201 Sentence denotes 3.5.
T299 11202-11219 Sentence denotes DNA Amplification
T300 11220-11408 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 11409-11558 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 11559-11705 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 11706-12081 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 12082-12197 Sentence denotes This makes the functioning of the integrated resistive heater less critical than the stability shown in Figure 11c.
T305 12198-12400 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 12401-12515 Sentence denotes As heat supply the water bath of an IKA Rotary Evaporator RV 8V (IKA-Werke, Staufen im Breisgau, Germany) is used.
T307 12516-12619 Sentence denotes This water bath is according to its specification stable within a range of the set temperature ±0.1 °C.
T308 12620-12744 Sentence denotes The chip and an Eppendorf tube are loaded with the reaction mixture containing the DNA sample and the EvaGreen dye solution.
T309 12745-12878 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 12879-12969 Sentence denotes The inlet and outlet of the chip are sealed with the Microseal “B” PCR plate sealing foil.
T311 12970-13106 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 13107-13241 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 13242-13304 Sentence denotes The complete mixtures are according to Table A3 in Appendix D.
T314 13305-13478 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 13479-13589 Sentence denotes The MDA is also performed inside the chip, but with the integrated Au resistive heater serving as heat source.
T316 13590-13634 Sentence denotes The set up shown schematically in Figure 12.
T317 13635-13701 Sentence denotes The same procedure is followed as with the water bath heated test.
T318 13702-13748 Sentence denotes Denaturation is done in a separate water bath.
T319 13749-13972 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 13973-14043 Sentence denotes In Figure 14 the logged temperature during the amplification is shown.
T321 14044-14234 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 14235-14352 Sentence denotes Fluorescence measurements are done in a Horiba Scientific FluoroMax+ spectrofluorometer to verify each amplification.
T323 14353-14528 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 14529-14689 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 14690-14771 Sentence denotes No Enzyme was added to this mixture, therefore no amplification could take place.
T326 14772-14843 Sentence denotes See Figure 15 for the results obtained in the Eppendorf tube and chips.
T327 14844-14938 Sentence denotes Figure A4 in Appendix E shows the background signal which is subtracted from all measurements.
T328 14939-15214 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 15215-15284 Sentence denotes There is an order of magnitude difference in the fluorescence signal.
T330 15285-15377 Sentence denotes However, the fluorescence intensity cannot be used as a value to quantify the amount of DNA.
T331 15378-15518 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 15519-15661 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 15662-15789 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.