Small-Size Polymerase Chain Reaction Device with Improved Heat

Article pubs.acs.org/IECR

Cite This: Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Small-Size Polymerase Chain Reaction Device with Improved Heat Transfer and Combined Feedforward/Feedback Control Strategy Michele Gregorini, Gediminas Mikutis, Robert N. Grass, and Wendelin J. Stark* Institute for Chemical and Bioengineering, ETH Zurich, Vladimir-Prelog-Weg 1, 8093 Zürich, Switzerland

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S Supporting Information *

ABSTRACT: We report improvements on the heat transfer and on the control strategy of a thermal cycler implemented on a novel device for performing a fast polymerase chain reaction (PCR). The reduction of the thermal mass of the sample holder and the direct contact with the sample allow for a significant reduction of the transition times, while the hybrid feedforward/feedback controller can rely on inexpensive components (actuators, sensors, processor) and still achieve minimal over/undershooting and good temperature stability. The design of the device has been improved by performing transient heat conduction analysis on the highly heatconductive sample holder and on the solid metal body of the device which rapidly dissipates the excess heat produced during the thermal cycling. In the current setup, the sample holder hosts nine 1 μL samples covered with mineral oil, which can be simultaneously read in real time by the detection module designed in-house and installed on the device. An increase in speed of the PCR amplification was achieved with a reduction of the transition time of 63.8% when compared against a commercial real-time PCR machine. Our work shows that a complete, stand-alone, and ready-to-use quantitative PCR instrument can be fabricated from inexpensive and easily available components and it can achieve fast thermal cycling thanks to a hybrid control strategy.

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INTRODUCTION The polymerase chain reaction (PCR) enables the exponential amplification of a specific sequence of deoxyribonucleic acid (DNA) in a sample. Since its invention,1 the PCR has become a laboratory tool used in a variety of fields, such as molecular biology (e.g., for DNA sequencing and DNA cloning), forensic analysis (e.g., for identifying genetic fingerprint and for parental testing), and medical diagnostics (e.g., for the formulation of individualized cancer therapy treatments). In order for PCR to occur, the sample’s temperature has to be repeatedly changed between different set points, namely the primer annealing temperature (typically around 56 °C), the polymerase-driven sequence extension temperature (72 °C), and the denaturation temperature (95 °C). Theoretically the amount of DNA doubles at every cycle; therefore, after 30−40 cycles more than a billion copies of DNA can be generated even starting from a single molecule. In standard (or conventional) PCR, the outcome of the amplification can be confirmed with several techniques, such as agarose gel electrophoresis or capillary electrophoresis, which provide end-point measures of the reaction’s product.2 Realtime analysis of the reaction’s progress is also possible by monitoring the signal emitted by fluorogenic compounds previously added in the PCR reactants (such as Taqman probe or SYBR green),3 which allows for a direct quantification of the number of DNA copies in the sample. This method of performing PCR is called “real-time quantitative PCR” (or © XXXX American Chemical Society

qPCR), and it requires the presence of both the thermal cycling and the quantification function in the same instrument. Since the invention of qPCR, a variety of thermal cycler and dedicated tools has appeared on the market.4 Commercial qPCR systems, such as those compared in Figure 1, are produced by major biotechnology product development companies (e.g., Roche, Thermo Fisher Scientific, Bio-Rad) and are typically designed for high-throughput applications. Therefore, dimensions and weight are of little importance while the price target is generally medium-high. Due to the high cost of commercial devices, in recent years several simplified machines have been progressively introduced in the market with a lower target price. Some examples of this trend are the OpenPCR project (sold at a price lower than 500 USD5) and the miniPCR device (price lower than 800 USD6), both providing only the thermal cycling function. Additional photodiode-based detection is offered only in more recent and more expensive devices, such as Open qPCR5 and Biomeme’s two37 (both sold at a price of 4000 USD or more). While reducing size and cost, this new generation of PCR devices in most cases relies on traditional PCR sample holders, namely plastic (polypropylene) tubes which are then placed in Received: March 4, 2019 Revised: May 8, 2019 Accepted: May 11, 2019

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DOI: 10.1021/acs.iecr.9b01209 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

Figure 1. Examples of qPCR machines developed in the past two decades and overview of some key characteristics. Early models were large and had a significant power consumption, while in recent years machines have become more affordable but still with a large footprint. PyPCR is smaller than commercial qPCR machines; it has higher heating and cooling rates and a lower total power consumption.

“PyPCR”, has been designed following some of the basic principles of chemical product design12 (such as understanding the needs of potential users, elaborating several design concepts, and selecting easily available components and established manufacturing techniques), and it is capable of thermal cycling and analyzing nine ∼1 μL samples. Real-time quantification is performed through a CCD sensor and it is rapid and simultaneous for all samples; therefore, it does not interfere with the thermal cycling speed and it provides a measure taken at the same time point across all samples. As opposed to other novel PCR systems developed in recent years,13−15 PyPCR is a fully enclosed and ready-to-operate instrument with features (such as easiness-to-use, simplicity of the sample preparation, compact size, and robust design) that go beyond the bare execution of the analysis, and it meets most of the requirements for point-of-care tests in resource-limited settings.16 Some of the key elements in the development of PyPCR were the availability of low-cost components, in particular electronics and easily producible three-dimensional (3D) printed parts, and the high computational power of microcomputers (such as Raspberry Pi or equivalent boards) which are available at low cost. The performance was evaluated by amplifying a synthetic DNA and comparing the results against amplification of the same reactants in a commercial Roche LightCycler 96 machine.

a thermal diffusive block that is in contact with the heating element. Even though these PCR tubes are widely used in the scientific community, they present intrinsic limitations to both the speed8 and the number of reactions that can be performed simultaneously in a small-size device. While the upper limit in the number of parallel reactions restricts only the throughput of the machine but has no impact on the reaction’s efficiency, the transition time between the PCR target temperatures should be minimized in order to avoid side reactions that could happen at intermediate temperatures. The overall time required by the qPCR analysis depends on the time required by the (bio)chemical reactions to occur and the time required to vary the temperature of the sample (i.e., heating and cooling rates), which in turn is related to the power provided by the thermal cycler as well as the physics of the sample holder combined with the thermal diffusive block. Traditional PCR tubes and thermal diffusive blocks present a significant thermal inertia and, therefore, limit the heating/ cooling rates of PCR devices to typical values of around 2−4 °C/s (Figure 1). The need for rapid thermal cycling has been addressed several times in recent years, and a variety of solutions were investigated, in particular by designing sample holders made of glass,9 silicon,10 and other polymers.11 Some of the drawbacks of these designs, however, lie in the high processing costs (such as for silicon) or in the low thermal conductivity (for glass and polymers), which hinders a rapid thermal response. In our work, we have performed transient heat conduction analysis to investigate the thermal inertia in a conventional PCR thermal cycler and we have designed a thin aluminum sample holder driven by a small-size device that can achieve a great reduction of the PCR transition times. The system is built with inexpensive and easily available components, and it is driven by a hybrid controller with a mixed feedforward/ feedback strategy that can deal with fast transition times as well as with relatively slow temperature readings from the sensors and a low control loop frequency. The device, named

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DESIGN PROCESS AND SYSTEM SETUP Improved Heat Transfer. With the aim of achieving low thermal inertia and simple manufacturing processes, we have selected a low-profile, flat sample holder (therefore called “thin sample holder”) made of aluminum (40 × 40 × 0.5 mm), where each reaction chamber is obtained by milling a well with a diameter of 2 mm and a depth of 0.35 mm. Each well can accommodate approximately 1 μL of reaction mix, and several B


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Figure 2. Comparison of the simulated transient thermal heat transfer in a conventional 0.2 mL polypropylene qPCR tube containing 13 μL of water (top) and our aluminum sample holder containing 1 μL of water covered with 0.2 mm of mineral oil (bottom). The sequence of images indicates the expected temperature distribution after applying 95 °C to the bottom surface of the sample holders (initially at 72 °C) after 0.1, 1, 10, and 15 s. In our aluminum sample holder, the sample approaches the target temperature after 1 s, whereas in a conventional qPCR tube this takes more than 15 s. Dimensions of the models in millimeters.

thermal diffusivity of aluminum (9.7 × 10−5 m2/s), t is the time point, and x is the spatial coordinate. Assuming an initial temperature of 72 °C (e.g., during the extension step), once the lower surface is brought to 95 °C (temperature of the denaturation step) it would take less than 0.1 s for the aluminum sample holder to reach a temperature higher than 92 °C and less than 0.5 s to reach a temperature higher than 94 °C. The direct contact between the sample and the aluminum plate, moreover, eliminates any thermal contact resistance typical of most commercial PCR devices where the sample is located in a polymeric container (such as tradition polypropylene PCR tubes), which is then placed in contact with a thermal diffuser block that is coupled to the heating and cooling element. The behavior of the thin sample holder was compared against a traditional PCR sample holder in a simulation conducted by means of COMSOL Multiphysics Modeling Software, and the results indicate the clear advantages of the low-profile design (Figure 2). Another benefit of the thin sample holder lies in the high specular reflectivity of the wells milled in the aluminum plate,19 which facilitates the detection of the fluorescent signal (as discussed in under Real-Time Detection). Other techniques to fabricate wells in the aluminum plate, such as laser engraving, are currently being investigated in order to increase the number of reaction chambers and eventually perform digital PCR with the same device. The temperature of the sample holder, and thus of the sample contained in the wells, is controlled by the thermoelectric element (TEC, also called Peltier cell) which is

reaction chambers can be placed next to each other to form an array of wells. The limit to the number of wells is determined by the dimensions of the chambers, the spacing between them, and the field of view of the detection system. A thin silicone gasket with an internal diameter of 30 mm, a thickness of 2 mm, and a height of 2 mm is placed in the center of the sample holder in order to create an additional chamber on top of the reaction wells where mineral oil can be added to prevent evaporation of water from the sample.17 Compared to conventional qPCR sample holders, the thin aluminum plate used in PyPCR exhibits higher heating and cooling rates (due to its geometry and its thermal characteristics) and faster overall PCR time, thanks to a significant reduction in sample volumes (which in the case of 96-well plates is typically above 10 μL). In order to understand the technical advantage of our design, we have analyzed the thermal behavior of the thin sample holder by means of a one-dimensional transient conduction heat transfer model under the assumption of a semi-infinite solid with constant surface temperature. Losses on the other surfaces of the sample holder are expected to be considerably small due to its geometry, and therefore they are neglected. The analytical solution is given by the following equation:18 i x yz T (x , t ) − Ts zz = erfjjjj z Ti − Ts k 2 αt {

where Ts is the temperature of the bottom surface (constant), Ti is the initial temperature of the sample holder, α is the C


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Industrial & Engineering Chemistry Research coupled to the bottom surface of the sample holder. Among other alternatives, a thermoelectric element was selected thanks to its robustness, the absence of moving parts, and the ability to provide both active heating and active cooling. The main drawbacks of Peltier cells, such as the low efficiency and the need to rapidly dissipate the waste heat generated by the module, are overcome by the low-profile design of the sample holder and its small thermal inertia, which in turns reduces the power requirements from the Peltier module. Thanks to its limited thickness, the aluminum plate has a very low thermal time constant and therefore it almost instantaneously reaches the temperature of the TEC element. For this reason, the heating and cooling rates of the sample holder can be approximated with those of the Peltier element and exceed 13 °C/s (in both heating and cooling), and therefore PyPCR heats and cools considerably faster than other commercial qPCR machines. Power modulation is provided to the TEC by varying the duty cycle of power pulses, according to the pulsewidth-modulation principle (or PWM). As previously reported,20 the frequency of the pulses does not substantially affect the ac resistance of the TEC module; therefore, the value can be set within a large interval. The temperature of the upper surface of the Peltier element is measured with a thin (0.2 mm) negative temperature coefficient thermistor (NTC) glued in a thin slot created on the upper surface of the module. Control Strategy. The control system is implemented in an off-the-shelf microcomputer (Raspberry Pi, model 3B), and the values of the temperature sensors are read with a standard analog-to-digital converter (ADC) with 16-bit precision (module ADS1115). As previously reported,8 the Peltier element can be used to provide both active heating and cooling of the sample holder, simply by switching the voltage across the two faces. This can be easily achieved by means of a relay-based H-bridge (as shown in Figure 3). The control system of our thermal cycler takes advantage of this functionality and switches the polarity of the power applied to the TEC in order to provide a fast transition in the cooling step of the PCR cycle (from denaturation to annealing). Very high heating (>16 °C/s) and cooling rates (>13 °C/s) can be achieved by providing the maximum rated current to the Peltier device (as shown in Figure 4). As opposed to what was disclosed in previous designs,21 our thermal cycler assembly consists only of inexpensive, commercially available components and it requires no movable parts, as the heat produced by the TEC is passively dissipated by the device’s metal body. In order to achieve quick transitions between the desired temperatures, as well as to minimize overshooting or undershooting when the temperature approaches the desired value, we have developed a hybrid control strategy that includes a feedforward component (coupled to a bang−bang control action) and a conventional proportional−integral (PI) controller, with an approach similar to what was previously reported by Qiu et al.22 During the initial part of the temperature transient, full power is provided to the Peltier element for a predetermined amount of time (bang−bang action). Immediately after, the power is completely switched off for a fraction of a second in order for the TEC to rapidly settle (this step is defined as “backlash transient”). Afterward, the temperature is adjusted (if necessary) and kept constant by means of a PI controller. A representation of the control strategy and the effect on the temperature transition is given in Figure 4. The controller therefore consists of a feedforward part (the timing of the full-power and backlash transients) and

Figure 3. PyPCR schematics. The TEC element is connected to a modified H-bridge, realized with mechanical relays, which acts as a polarity reverse switch. The modulation of the power is achieved by means of a N-channel MOSFET, controlled by the Raspberry Pi (control lines in orange). The two main temperature sensors (TEC and device’s case) are read by means of an ADS1115 module (analogto-digital converter), that communicates to Raspberry Pi by means of the Inter-Integrated Circuit (I2C) protocol. Blue LEDs, controlled by means of a second N-channel MOSFET, are used in combination with a blue emission filter to excite the sample. The detection is performed by taking a picture with the Raspberry camera module combined with a 6 mm optical lens and an amber emission filter.

a feedback loop (the traditional PI controller). In order to account for changes in the device’s body temperature, which affect the time response of the TEC element, the parameters of the feedforward controller are progressively adjusted by analyzing the behavior of the system during each cycle and therefore temperature overshoots and undershoots are minimized. This modified controller can overcome the limitations of a conventional PI controller introduced by the nature of the TEC device, namely the fact that the polarity of the voltage applied to the element cannot be switched too frequently and therefore the regulation value is limited in the closed interval [0, 100%]. Moreover, due to the timing required to read the temperature sensors (around 100 ms), the low loop frequency of the PI controller would not be suitable to achieve fast temperature transients without considerable output oscillations. The lower side of the thermoelectric element is placed directly in contact with the body of our device, which consists entirely of aluminum; thanks to its high heat conduction, the metal body rapidly dissipates the heat produced (Figure 5c) and this prevents the temperature of the lower surface of the TEC element from increasing considerably, which in turn would reduce the speed of the thermal transients. Since part of the waste heat is absorbed by the device’s body, the temperature of the case is expected to vary during the thermal cycling process. This, in turn, has an effect on the behavior of the TEC element, and therefore an additional NTC sensor in installed on the metal case of the device (in proximity of the lower surface of the Peltier element) to record the behavior of the system and to establish a correlation between the case’s temperature and the performance of the TEC element (Figure 4). These data, D


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Figure 4. Temperature profile and controller value (“Duty cycle”) recorded by the device during a single PCR cycle (a). Rapid change in temperature without over/undershooting or oscillations, achieved by means of a bang−bang control action: duty cycle initially set to ±100% (“fullpower” step), then completely cut off (“backlash”) for a predetermined amount of time, and finally computed with a standard PI algorithm. The durations of the full-power and the backlash transients are determined by interpolating within values previously recorded in similar operating conditions and stored in “learning tables”, as shown on the right (b).

saved in arrays called “learning tables”, are used to initialize the thermal cycler when starting a PCR and they could also potentially be used to run the device without any temperature sensor on the Peltier element. In order to provide fast temperature transients, the chosen TEC device is operated at relatively high power (150 W peak). Nevertheless, since it is used at maximum power only for a limited amount of time, the average energy consumption of the thermal cycler is modest (theoretically below 1500 mAh per qPCR run, with an operating voltage of 15 V), and therefore the machine has the potential to be battery operated (more than to two full runs on an average laptop battery). For this reason, a portable version of the device is currently being developed. Real-Time Detection. The detection of the fluorescent signal generated by the sample is performed by exciting the dye (SYBR Green) with light at a central wavelength of 465 nm and observing the reemission from the dye at wavelengths above 500 nm. In previous studies,17,23 a combination of LEDs, filters, and photodiodes has been used to perform quantification during qPCR amplification. These setups, however, generally lacked the ability of simultaneously detecting several samples, unless a multitude of units is operated in parallel. We have designed a simple and inexpensive detection system where the excitation energy is provided by commercial off-theshelf blue LEDs (Figure 6). The signal generated by the sample is measured by means of an inexpensive CCD sensor (Raspberry Pi Camera Module V2) coupled with a standard off-the-shelf M12 6 mm optical lens. Additional excitation and emission filters (Clear Cast Acrylic Sheet, fluorescent blue and fluorescent amber, McMaster-Carr) help to increase the signalto-noise ratio and better quantify the signal emitted by the sample. The main advantages of such a system is the low cost of the components (in total below 40 USD), the total absence of moving parts, and the high number of wells that can be simultaneously detected, which in principle is limited only by the sensitivity of the CCD sensor. The excitation of the sample and the detection of the signal reemitted are facilitated by the

reflective properties of the aluminum sample holder. As shown in Figure 6b, the excitation rays (in blue) travel initially through the sample and excite the fluorescent dye in the liquid volume. Rays that are not absorbed by the dye reach the bottom of the sample holder and are reflected back into the sample, thus increasing the overall probability to generate a signal from the dye. Moreover, assuming an isotropic light generation in the dye, the walls of the reaction chambers reflect part of the energy emitted by the dye toward the CCD, thus increasing the amount of energy per well that reaches the sensor. These characteristics of the sample holder and of the detection system allow for simpler and more accurate detection of the signal emitted by the sample.

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EXPERIMENTAL SECTION The real-time PCR protocol that was chosen to validate the behavior of our machine consists of a hot start at 95 °C followed by 40 cycles of annealing (56 °C), extension (72 °C), and denaturation (95 °C). The synthetic DNA used in the PCR protocol consisted of the following 148 base pairs: Sequence (5′−3′): ACA CGA CGC TCT TCC GAT CTG ACT CTC ATC TAC TAG ATA GAT CTC CAC CTC GCA GTC TCG TCT TCA ACG GTG CTC ACG CGA TAT AGT TAG CTC GCG ACT ACC ATA GCG CTA CAT AGA AGT CAG CAA GAG ATC GGA AGA GCA CAC GTC T Sequence (5′−3′): AGA CGT GTG CTC TTC CGA TCT CTT GCT GAC TTC TAT GTA GCG CTA TGG TAG TCG CGA GCT AAC TAT ATC GCG TGA GCA CCG TTG AAG ACG AGA CTG CGA GGT GGA GAT CTA TCT AGT AGA TGA GAG TCA GAT CGG AAG AGC GTC GTG T The forward primer was AGA CGT GTG CTC TTC CGA TC, and the reverse primer was ACA CGA CGC TCT TCC GAT CT. The reaction was carried out with KAPA SYBR FAST qPCR Master Mix (2X), which contains KAPA SYBR FAST DNA polymerase, reaction buffer, dNTPs, SYBR Green I dye, and MgCl2 at a final concentration of 2.5 mM. In E


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Figure 5. (a) CAD design of the PCR device: aluminum body, sample holder sitting on top of the thermoelectric element (not visible), and movable lid containing the detection module. (b) Aluminum sample holder (40 × 40 × 0.5 mm) with nine cylindrical reaction chambers (diameter, 2 mm; depth, 0.35 mm) milled in a 3 × 3 array pattern and a silicone gasket (30 × 34 × 2 mm). (c) Simulation of the thermal heat dissipation through the body of the device (section view) initially at 20 °C, assuming that maximum power (150 W) is provided continuously to the Peltier device for 10 s. (d, e) Results and time constants (τ, defined as the time needed to complete more than 63.2% of the temperature transaction) of the transient heat transfer study on (d) a water sample (blue) contained in traditional 0.2 mL polypropylene (PP) sample holder (orange) placed in an aluminum heat diffuser (gray) and (e) on an aluminum sample holder (gray) containing 13 μL (light blue) and 1 μL (blue) of water sample.

addition, bovine serum albumin (BSA) was added to the mix to a final concentration of 6 g/L. Three different concentrations of DNA (1.25 ng/nL diluted 10−5, 10−6, and 10−7) were used in order to evaluate the performance of the PCR amplification. One additional sample without DNA was also prepared as a negative control. The sample (up to 1 μL for each reaction well) was applied by means of a conventional laboratory pipet directly in each well of the sample holder. Once all the samples had been disposed in the wells, a thin silicone gasket was placed on the upper surface of the sample holder and a thin layer of mineral oil (PanReac, Switzerland) was poured on top of all the reaction wells to prevent evaporation from the samples. No sample drag between wells was observed (as quantified by fluorescence) when loading the oil. The sample holder was placed in contact with the Peltier device, and it was held in place by the pressure applied on top of the silicone gasket by closing the lid of the device. This

pressure also improves the thermal contact with the TEC element. The fluorescence signal was measured in the last 2 s of each extension step by activating the LEDs and consequently the CCD sensor and taking a picture through the Raspberry Pi microcomputer. The signal value was then assigned by calculating the average of the “green” pixel values (of the RGB image) in each well. In order to account for possible inhomogeneity in the illumination of the sample holder, the values calculated during the first cycle of the PCR procedure were used as baselines (individually assigned for each well) in every subsequent image. The temperature of the Peltier element was continuously monitored at intervals shorter than 200 ms, and during the constant-temperature periods a temperature variation of less than ±0.5 °C could be obtained on the top surface. In order to evaluate the advantage of the choice of the material for our sample holder, further experiments were performed with sample holders made of PTFE (Teflon) and F


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Figure 6. (a) Schematic illustration of the detection system, composed of a CCD sensor (included in the Raspberry Pi Camera Module V2), a 6 mm lens, excitation sources (blue LEDs with a central wavelength of 465 nm), and excitation and emission filters. (b) Path of excitation and detection rays through the sample.

producing meaningful comparison between wells with different contents. Therefore, an initial calibration was performed with a negative cotnrol and a positive control (i.e., samples without and with DNA in a large amount in two different wells), as shown in Figure 7. The default (automatic) settings of the camera turned out to be insufficiently optimized to detect the difference between the two samples, and several adjustments of exposure time, ISO, and white balance coefficients (auto white gains) have been necessary in order to produce a clear distinction not only between the two wells with different samples, but also between wells and background. The PCR protocol was initially performed with the following timing: hot start for 120 s, annealing for 30 s, extension for 30 s, and denaturation for 15 s. The same protocol and the same reaction mix was also cycled in a commercial Roche LightCycler 96. A second run was performed with shorter timing in order to reduce the overall reaction time: hot start for 120 s, annealing for 30 s, extension for 8 s, and denaturation for 1 s. The PCR amplification curves obtained with our device are plotted against the curve obtained with the commercial PCR machine in Figure 8. As shown in the plots, the

PP (Table 1), with the same geometrical characteristics as our aluminum sample holder but with intrinsically different thermal time constants (hundreds of times higher than aluminum). Table 1. Results of the PCR Amplification with Sample Holders Made of Different Materials or without the Addition of BSA in the PCR Mix sample holder plain aluminum (without BSA addition in PCR mix) aluminum (BSA concentration, 6 g/L) Teflon (PTFE) polypropylene (PP)

thermal diffusivity [10−5 m2/s]

results

9.728

0/3

9.728 0.01229 0.09630

3/3 0/3 0/3

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RESULTS AND DISCUSSION Both PCR amplification and detection have been carried out directly on the microcomputer inside PyPCR. Some settings of the Raspberry Pi camera module were particularly important in

Figure 7. Qualitative representation of the effect of different camera parameters on the detection capabilities of PyPCR. In each image the same two wells are marked with red dashed lines. The first well (top left) is filled with PCR mix before amplification (therefore without DNA), and the second well (bottom right) is filled with the PCR mix after PCR amplification (therefore with DNA). Careful selection of the camera settings (such as exposure time, ISO, and auto white gains, AWB) is essential in order to clearly identify the well that contains amplified DNA. G


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Figure 8. PCR amplification results of two experiments. Three different samples at different concentrations were tested (1.25 ng/nL diluted 10−5, 10−6, and 10−7), together with a negative control (sample without addition of DNA). On the left, amplification with the standard PCR timing (hot start 120 s, annealing 30 s, extension 30 s, and denaturation 15 s) carried out in both the commercial device and our machine. On the right, results for amplification with a faster protocol are shown (hot start 120 s, annealing 30 s, extension 8 s, and denaturation 1 s).

Figure 9. (left) Results of PCR amplification using three different sample holders: aluminum (red), Teflon (blue), and polypropylene (green). Cycling timing: hot start 120 s, annealing 30 s, extension 8 s, and denaturation 1 s. (right) Temperature of the device (green) during a full 40-cycle PCR program compared against Roche LightCycler (blue, nominal temperature profile only). After an initial temperature increase during the hot start step, the temperature of PyPCR exhibits small cyclic fluctuations in conjunction with the temperature of the Peltier element (red).

amplifications of the three samples match the results obtained with the commercial device, and the negative controls show a delay in the amplification in both experiments performed (negative samples still amplify due to either nonspecific amplification or background contamination when working with high concentrations of DNA). The total time for amplification required in the first run was 65.2 min for our device and 82.7 min for the LightCycler; therefore, we improved the total cycling time by 21.2%. In the second run the improvement was even higher (33.3%, 39.1 min against 58.7 min in the LightCycler) due to the smaller impact of constant-temperature phases over the entire cycling time. Given the desired PCR protocol timing, the absolute minimum time for a fixed number of cycles is simply the sum of the time required for the hot start and the time required for the PCR steps multiplied by the number of cycles, assuming

instantaneous transients between the different PCR temperatures. In our case the absolute minimum time required by the PCR protocol of the second run would be 120 + 40(30 + 8 + 1) = 1680 s = 28 min

Our device is able to complete the amplification in 39.1 min, therefore with an overall “time efficiency” of 28.0 = 71.6% 39.1

The difference between the minimum absolute time and the effective time required by the machine is due to the time spent to change the temperature of the samples, which in this case is equal to 11.1 min, approximately 16.6 s per cycle. The LightCycler has a lower time efficiency (47.7%) and more than half of the total time of the analysis is spent to vary the H


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Industrial & Engineering Chemistry Research temperature of the sample (58.1 − 28 = 30.1 min). In the experiments performed, therefore, PyPCR has shown a total reduction of transient time of 63.8% (11.1 min instead of 30.1 min). Improvements on the PCR mix are currently being investigated in order to reduce the minimum absolute time for amplification and therefore lead to shorter total PCR time. The experiment performed with the sample holder made of PTFE (hot start 120 s, annealing 30 s, extension 8 s, denaturation 1 s) showed high noise and no clear amplification (Figure 9, left). PTFE is an inert synthetic fluoropolymer, which has already been used in microfluidics PCR;24 therefore we believe that the sample does not reach the required temperatures for PCR amplification due to the different thermal properties of the sample holder which are unsuitable for the high speed of the thermal cycler. Similarly, a sample holder made of polypropylene (PP) was also tested, but it did not produce meaningful results. Moreover, it was confirmed that the addition of BSA in the PCR mix plays an important role when an aluminum sample holder is used,25−27 as no results could be achieved without increasing the concentration of this protein (Table 1). The strategy implemented to control the temperature of the sample holder is evaluated by analyzing the temperature profile recorded by the device (Figure 9, right). The transients are characterized by a steep profile that rapidly flattens before settling at the desired temperature, overshooting and undershooting are minimized, and fast transitions can be achieved. Even though the temperature of the case increases slowly over time, the PCR cycling proceeds at the same temperatures thanks to the action of the hybrid controller.

distinct PCR runs. The absence of moving parts further reduces the device’s complexity and makes it suitable also for environments where air movement should be minimized, such as biological laboratories. The device does not require additional instruments or specific training to be operated. Thanks to the lean design, it can be manufactured with inexpensive, off-the-shelf components at a low price. In comparison to other PCR machines available on the market (Figure 1), PyPCR is smaller and it achieves higher heating and cooling rates, with a lower power consumption. Further improvements of the instrument and of the sample holder are being investigated in order to perform also real-time digital PCR analysis with a portable version of PyPCR.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.9b01209. Additional figures and tables further describing the PCR device, cycling performance, and comparison against commercial devices (PDF) Simulation report for the results shown in Figure 2, bottom (PDF) Simulation report for the results shown in Figure 2, top (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or [email protected].

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ORCID

CONCLUSIONS We have designed a PCR device with improved heat transfer properties and a hybrid controller capable of conducting multiple simultaneous reactions in a metallic sample holder. The device is able to perform rapid thermal cycling, and it is equipped with a simple, inexpensive detection module for providing real-time quantification of the PCR amplification. The great availability of low-cost electronics and rapid prototyping technologies has been exploited to increase the speed of the design iterations and constantly improve the quality of our device. The PCR analysis is carried out on an innovative thin sample holder that consists of an aluminum plate where the reaction wells are manufactured by conventional methods (such as milling). Thanks to the small thermal mass of the sample holder and to the direct contact with the liquid sample, fast temperature transients can be achieved and the system can perform 40 PCR cycles in less than 40 min. A hybrid controller has been designed in order to accurately control the temperature transients and to reduce temperature oscillations. Real-time quantification of the reaction products is achieved by means of a compact detection system that comprises commercial LEDs, an excitation filter, a detection filter, and a low-cost, high-definition CCD sensor. We have demonstrated the performance of our device by amplifying three samples of synthetic DNA together with a negative control, and by comparing the results to those obtained with a commercial PCR device (Roche LightCycler 96). Our system was able to produce comparable amplification in a considerably shorter time. This real-time PCR device is compact, robust, and fully assembled, and it has been stress-tested with at least 300

Michele Gregorini: 0000-0002-1091-5939 Robert N. Grass: 0000-0001-6968-0823 Wendelin J. Stark: 0000-0002-8957-7687 Notes

The authors declare the following competing financial interest(s): Michele Gregorini, Robert N. Grass, and Wendelin Jan Stark declare financial interest in the form of a patent application.

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ACKNOWLEDGMENTS Financial support by the ETH Zurich is kindly acknowledged. REFERENCES

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Small-Size Polymerase Chain Reaction Device with Improved Heat

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