Field of the invention.

The invention relates to methods and devices for the detection of nucleic acid molecules, particularly those intended for the detection of nucleic acids of disease-causing microorganisms, more particularly those that are portable and allow the efficient and reliable detection of small amounts of said molecules, which allows them to be versatile and to be used in any installation. The present invention is specifically related to the efficient and reliable identification of low amounts of the mRNA of the SARS-CoV-2 virus in samples from suspected patients, by using the methodology and the device described for the present invention.

Background of the invention.

Reverse Transcription quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) remains the gold standard for detecting the virus SARS-CoV-2 [1 ,2] and other infectious diseases [3], During the COVID-19 pandemic, early diagnosis (achieved mainly by RT-Qpcr) demonstrated to be crucial for proper disease and infection management [4], In highly industrialized countries where testing is not restricted to central laboratories (e.g., Germany), the number of cases and deaths has been remarkably reduced due to early detection strategies [5,6], In contrast, in developing countries, samples are transported to a handful of centralized laboratories for PCR diagnosis. For example, at the beginning of the COVID-19 pandemic, Nepal [7] and the Republic of Zimbabwe [8] had only a single testing facility for the whole country. The lack of laboratories reduces the number of tests, thus causing delays in identifying infected people, providing them with medical treatments, as well as to and isolate them at an appropriate time, therefore leading to inadequate control of the virus propagation [9], COVID-19 has exhibited marked global health inequalities and evidenced vulnerabilities in the supply chain of diagnostic devices [10,11], Since major lockdowns were imposed to prevent the spread of the disease, health supply chains have been disrupted, thus hampering shipping and procurement of diagnostic tests and instruments, leading to their scarcity [10], For qPCR, large-scale testing represents a global challenge because instruments are expensive, require frequent maintenance, and technical support is not readily available in every city [12], Therefore, rapidly deploying cost-effective and scalable technologies could be a practical solution to carry out a large number of diagnostic tests necessary for proper disease management, especially in countries with under-resourced healthcare systems [8,13],

Frugal innovations in healthcare aim to make medical technologies accessible in low- or middle- income countries or situations with constrained resources, such as rural areas. Frugal instruments and devices are characterized by low cost and reduced complexity, compared to their commercial counterparts, trying to maintain optimal test functionality and quality, in addition to offering sustainability and scalability [14], Progress in frugal innovations has been boosted by the proliferation and low cost of digital fabrication tools, such as 3D printing, micromilling, and laser cutting. These tools have been used to manufacture point-of-care (POC) instruments for nucleic acid amplification (including PCR)[15-19] or produce microfluidic molds or devices from different materials (glass [20] and plastics [21 ,22] included). Digital manufacturing technologies enable distributed manufacturing, where goods are produced locally, attuned to the requirements of the local market [23], This characteristic allows them to bypass the vulnerabilities in the supply chains of medical devices [24], which has never been more evident than in the current pandemic [25], Altogether, the relative accessibility and low cost of digital manufacturing tools (empowered by the maker movement [24]) enable health-care democratization.

Attempts at producing frugal instruments for qPCR with integrated fluorescence detection have been made. However, some instruments lack simplicity in design and construction because they integrate expensive optical components such as lasers, photodiodes, photomultipliers, lenses, and precision machined parts [26-28] or complicated electronic control boards [17,26-29], Although significant progress has been made in developing low-cost and simple fluorescence detection systems, for example, using LEDs and smartphone cameras equipped with optical filters [15,16,18,19,30-32], most of these platforms do not possess the thermal cycling capabilities required for PCR. Indeed, integrated thermal cyclers with fluorescence detection, still use complicated optical setups or require expensive semiconductor techniques to fabricate the PCR chambers [17,26], These features complicate their manufacturing, replication, and adoption, preventing their widespread implementation in resource- constrained settings. According with the mentioned above, the portable devices for the PCR analysis and further detection of amplified nucleic acids of interest, could be useful for the SARS-CoV-2 detection.

Among the detection methods developed for the same purpose described in the art, the following can be mentioned: Bravo-Gonzalez and col. [47] describe the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction. Such simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 pL of reaction mix. Gouilh and col. [48] describe a rapid and all-in-one SARS-CoV-2 RT-LAMP based molecular detection system, including RNA, for point-of-care or massive testing of nasopharyngeal swabs. The point-of- care format uses LoopX, a small portative device ensuring optimal LAMP reaction and automated reading with 95.2% and 95.5% sensitivity and specificity resp.

Gonzalez-Gonzalez and col. [49] describe the use of the miniPCR, a compact and portable PCR device in combination with a common well-plate reader as a diagnostic system for detecting genetic material of the SARS-CoV-2 virus; such device is useful for amplify viral DNA sequences of three different regions that encode for the N protein; prior to amplification, samples were combined with a DNA intercalating reagent (i.e., EvaGreen Dye). Sample fluorescence after amplification was then read using a com. 96-well plate reader. This straightforward method allows the detection and amplification of SARS-CoV-2 nucleic acids in the range of ~625 to 2x105 DNA copies.

By other hand, microfluidic devices for PCR offer several advantages over conventional PCR tubes, such as small size, fast thermal cycling, reduced sample and reagent volumes, low cost, integration with sample preparation, among others [33], However, for several years a significant limitation of these devices has been the readily formation of air bubbles due to: incorrect pipetting, evaporation [34], or uneven surface wetting [35], which reduce PCR efficiency and throughput. Several strategies have recently been developed to mitigate or eliminate the appearance of bubbles in microfluidics [34-37], making their operation more reliable.

Therefore, it is important to have better devices for the detection of molecules of interest in order to solve the problems described above.

Brief description of the figures.

Figure 1. Engineering of the Hybrid Opto-Thermocycler (HybOT cycler) of the invention. Shows (a) a schematic of the essential components of the thermocycler (IR, infrared; Em, emission, Ex, excitation). Heat and light are generated from the bottom and travel upwards, (b) Cross-section view of the parts in the assembled instrument. Notice the components composing the lid. (1) Light sensors.

(2) Em filter. (3) Microfluidic device. (4) PCR chamber. (5) Ex filter. (6) High power LED. (7) Thermo- Electric Cooler. (8) IR sensor. (9) Heatsink. (10) Aluminum piece, (c) Photograph of the instrument. Inset shows the lid closed, (d) Block diagram of the components inside the instrument. Arrows show the communication direction between the sensors, LEDs, actuators, electronics, and the microcontroller.

Figure 2. Tablet interface. Shows the interface developed in App inventor for the control of the HybOT cycler of the invention. The display shows fluorescence intensity and temperature plots in real time.

Figure 3. Photograph of the components inside the HybOT Cycler of the invention. Shows the vacuum pump can be seen at the bottom, (a) H-Bridge. (b) Vacuum pump, (c) TEC Fans, (d) TEC Heatsink, (e) LED Driver, (f) Arduino Nano, (g) Bluetooth module, (h) DC-DC Booster.

Figure 4. Design and assembly of the main parts that make the HybOT Cycler of the invention.

Shows the (a) exploded view of the sensors lid and (b) the exploded view of the thermal and excitation module.

Figure 5. Optothermal characteristics of the HybOT cycler of the invention. Shows (a) a top-view photograph of the heating plate without (left) and with the device (right). The bottom schematic shows the location of temperature sensors. (3) Microfluidic device. (4) Reaction chamber. (5) Ex filter which acts as heating plate. (8) IR sensor, (b) Average temperature of the heating plate from 30 to 95°C. Inset shows a thermal picture of the heating plate with the dotted area highlighting the area analyzed, (c) The temperature profile of a single cycle was measured on the plate (IR) and in the microfluidic chambers (TC). The programmed temperature (PT) is shown in gray color, (d) Temperature profile of the heating plate under different external temperatures, (e) Correlation of fluorescence signals between the HybOT cycler and an inverted fluorescence microscope (Zeiss), (f) Fluorescence readings of the light sensors for three concentrations of fluorescein during 40 cycles.

Figure 6. Shows the variations of the light sensor over a PCR assay without active cooling. Figure 7. Bubble-free microfluidic device of the invention. Shows (a) a design of the microfluidic device of the present invention (3), the PCR chambers, and the degassing channels are denoted by yellow and blue colors, respectively, (b) Photograph of the device (3); scale bar: 3 mm. (8) IR sensor cavity. (4) Reaction chambers, (c) Step-by-step fabrication of the device. Cross-sectional view of the dotted line in panel a. (d) Dot graph shows the state of the chambers (empty, with bubbles, or filled) after degassing them for a range of times and pressures, (e) Representative photographs of the microfluidic device for each state.

Figure 8. Shows the acrylic mold for 25 PCR microfluidic devices.

Figure 9. Shows that an initial concentration of 10 ⁷ copies of the gene RNase P gene was amplified in the four chambers of the device at the same time. Resulting curves are shown. Four negative controls were also amplified (grey colors).

Figure 10. Amplification curves for the RNase P gene. Shows (a) the conventional thermocycler and (b) the hybrid thermocycler. The inset shows CT values for both thermocyclers (conventional in red and Hybrid in blue). Figure 11. Detection of SARS-CoV-2 N1 gene. Shows the representative qPCR amplification curves for different dilutions of cDNA of the N1 gene obtained with (a) a benchtop thermocycler (StepOne) and (b) the HybOT Cycler, (c) Standard curves for threshold cycle values (CT) as a function of cDNA concentration. Error bars show the standard deviation (n=3). (d) Analysis of PCR efficiency of both thermocyclers at various normalized thresholds to aid in defining proper thresholding. Figure 12. Detection of SARS-CoV-2 from clinical samples. Shows the (a) Workflow for detecting SARS-CoV-2 in 20 positive samples (+) and 10 negative controls (-). Samples are collected from a nasopharyngeal swab, prepared for RT-qPCR, then divided and assessed in both thermo cyclers, (b) Violin plots show the CT values for 20 COVID-19 positive samples (P) and ten negative controls (N) obtained with both thermocyclers, (c) Receiver operating characteristic (ROC) curves for HybOT (blue) and StepOne (red). AUCs: areas under the curve.

Figure 13. Shows the representative photographs of the microfluidic devices for three different degassing conditions (top to bottom). The assay conditions are shown to the right of each row. Left and right photographs show the beginning of the assay and the end of the initial denaturation stage, respectively. The red arrows point to the formation of bubbles in the chambers.

Detailed description of the invention.

Although RT-qPCR is the gold standard for detecting the virus SARS-CoV-2 and other pathogens, the COVID-19 pandemic has highlighted the scarcity of instruments and reagents for massive PCR testing. At least for under-resourced countries, it has become critical to deploy instruments that can be rapidly constructed and satisfy this demand. Here we describe a portable thermocycler for RT- qPCR (dubbed HybOT Cycler) that integrates thermal control, illumination, and fluorescence detection into a highly integrated hybrid module, simplifying its assembly. The HybOT Cycler of the present invention is wirelessly controlled from an application installed in a tablet. We characterized its thermal performance and fluorescence sensitivity and found it to behave similarly to benchtop thermocyclers. We developed a bubble-free microfluidic device that can be easily replicated from an acrylic mold to run the PCR assays. Degassing the PCR chambers for 3 min at 350 KPa with a portable vacuum pump prevented any bubbles during the assay. Both the instrument and the microfluidic mold are fabricated with digital fabrication tools and off-the-shelf electronics. Concentrations as low as 10 ³ copies/pL of the SARS-CoV-2 N1 gene were detected. We then used our platform to screen individuals infected with the virus, reaching a sensitivity of 95% and specificity of 100%. Our portable platform can assist in diagnosing of SARS-CoV-2 and other pathogens, and its simple configuration and assembly can lead to quick adoption by labs around the world.

Thus, we developed a portable instrument for RT-qPCR that integrates a thermocycler and a fluorescent detection system into a compact hybrid module. The instrument of the present invention runs bubble-free microfluidic devices to perform PCR assays. Both the instrument and the microfluidic device mold are made with digital fabrication tools. To demonstrate the robustness of our platform, we analyzed samples of patients infected with SARS-CoV-2 and compared the performance with a commercial thermocycler, achieving 95% sensitivity and 100% specificity. Design of the Hybrid Opto-Thermo cycler (HybOT Cycler) of the invention. Our portable qPCR instrument seamlessly integrates temperature control and illumination into a single hybrid module that simultaneously transfers heat and shines light to microfluidic chambers (Figure 1a,b). Each microchamber (4) is sandwiched between a light source (6) and a detector (1), thus: (i) simplifying the design of the Opto-mechatronic system, (ii) facilitating the alignment of all the components, and (iii) eliminating the need for external optical parts, such as lenses, optical fibers or pinholes. The hybrid module consists of an aluminum plate housing (10) four high-power LEDs (6) covered by an excitation glass filter (5) (483 nm). This machined aluminum plate (10) sits on top of a Thermoelectric Cooler (TEC) (7) that heats or cools the aluminum plate (10). The excitation filter (5) serves two purposes: it conducts heat generated by TEC (7) to the microfluidic chip (3) and filters the light from the LEDs.

Significantly, although both the optical and electronic components are subjected to high temperatures (~100°C), they do not exceed their maximum operating parameters. A separate 3D-printed lid houses the fluorescence detection system and consists of four digital light detectors (1) and a small fan (9). These detectors are mounted on a PCB that rests on an emission glass filter (2) (535 nm). Below this filter is an infrared (IR) sensor (8), located at the center of the light detectors, that measures the temperature of the heating plate (5) (/.e., the excitation filter). When closed, the lid pushes over the microfluidic device (3), securing it in place and blocking outside light into the system, Figure 1c. In summary, light travels from the LEDs (6), passes through the optical filter (5), the microfluidic chambers (4), the emission filter (2), in that order, and reaches the light sensors (1). Notably, in one of their embodiments, the instrument of the invention is assembled from standard optical and electronic components easily accessible in the market as can be seen in the table S1 (Table 1).

For the purposes of the invention, all the mechanical parts and the casing are 3D-printed, enabling the construction of our instrument even in resource-limited settings. An Android application downloaded into a tablet or smartphone communicates with our instrument via Bluetooth, relaying temperature cycle parameters and retrieving fluorescence data. The application features a graphical user interface to display the fluorescence values of each chamber, the temperature of the device, and fields to enter the PCR cycle parameters (Figure 2). The total weight of the instrument is 1 Kg. The system’s operation starts by turning a single power switch and placing a bubble-free microfluidic device loaded with the PCR solution.

Table 1. List of electronic and optical components used to assemble the HybOT cycler of the invention. Bold letters indicate the most expensive components: the optical filters.

Component Model Manufacturer

Fan THA0412AD Delta electronics

Power supply ALM150PS12 XP Power

Peltier module +Heatsink Assembly 1335 Adafruit Infrared Thermometers MLX90615 Melexis

Microcontroller Nano Arduino

Driver H-Bridge PWM For Arduino BTS7960B Infineon

Bluetooth module HC-06 Wavesen

Vacuum Pump SC3710PM SKOOCOM

DC-DC Voltage Boost Step Up 20w XL6019 Xlsemi

Fluorescence Emission filter 867-028 Edmund Optics

Fluorescence Excitation filter 867-031 Edmund Optics

Blue excitation LED LXML-PX02-A900 Lumileds

Light sensor VELM6030 Vishay

LED driver COM-13705 SparkFun

The instrument of the invention contains a microcontroller (Arduino) that runs a digital proportional- integral-derivative (PID) temperature controller to regulate the TEC actuation (Figure 1d). It also synchronizes the activation of the power LEDs and the readings of the light sensors. A miniature vacuum pump, placed inside the instrument, is connected to the microfluidic device to apply negative pressure during the PCR assay (Figure 3). A key advantage of the device of our invention is that it can be disassembled and reassembled relatively quickly, and thus maintenance would be straightforward. Furthermore, we would like to note that the main component of HybOT, which performs the management of temperature and fluorescence detection, consists of only 15 easy-to-assemble parts; an exploded view of the assembly of the 15 principal components of HybOT was included in figure 4.

Optothermal performance. We studied the thermal characteristics of the instrument of the invention by measuring the temperature of the heating plate and the microfluidic chambers (Figure 5a). Using an infrared camera, we found the temperature of the heating plate to be uniform (standard deviation of ±0.68°C)(Figure 5b). Next, we compared this temperature to that inside the microfluidic chambers using a thermocouple. As shown in Figure 5c, at steady state, the temperature difference between the plate and the PCR chambers is of -0.48°C and 1.29°C for the 95°C and 65°C temperature setpoints, respectively. The difference in temperature between all the chambers was ±0.8°C. Overall, 5 this characterization demonstrates a good temperature uniformity of the heating plate and in the microchambers.

For the purposes of the invention, we implemented and tuned a Proportional Integrative Derivative (PID) controller to precisely regulate the temperature in each step of the reaction. To achieve faster down ramps, we kept the TEC at 60°C using an on-off controller and a heat sink. Our PID controllerD achieved a heating rate of 4.11 °C/s and a cooling rate of 2.8°C/s (Figure 5c). Commercially available thermal cyclers report rates for the heating block from 3.8 to 6.4°C/s and 3.9 to 5.9°C/s for the up and down ramps [39], respectively. However, the sample temperature rates are typically lower, ranging from 3.4 to 4.6°C/s for the up ramp and from 2.8 to 4.3°C/s for the down ramp [39], similar to those achieved by the HybOT Cycler of the invention. 5 The temperature profiles do not present a noticeably overshooting (reaching a maximum temperature deviation of 0.48°C at the 95°C setpoint) with no undershooting observed. These results suggest we achieved a critically damped PID controller, exhibiting an optimal compromise between response time and temperature accuracy. However, an overdamped response when reaching 60°C effectively reduced the step length and could lead to poor performance due to incomplete elongation (Figure0 5c). To compensate for this reduction, we increased the step length by 5 s to guarantee that the reaction chambers stayed at 60°C for a sufficient time. Additionally, under different external temperatures: 5, 15, 27, and 40°C, the HybOT Cycler of the invention reached the same temperature profiles (Figure 5d). In summary, our thermal control system provides a similar performance to its benchtop counterparts. 5 Next, to characterize the fluorescence detection system, we compared the fluorescence signals from solutions of fluorescein (0 to 1 pM) measured with our instrument and a high-end inverted fluorescence microscope. As shown in Figure 5e, there is a strong correlation between both systems (R=0.98) demonstrating that our instrument performs similarly to a fluorescence microscope despite not having any optical components other than the light detector. We realized that overheating the light sensors0 led to inaccurate readouts (Figure 6) but adding a heatsink and a fan above them solved this issue.

To verify their proper performance, we simulated a complete PCR assay (40 cycles) for three different concentrations of fluorescein (0, 0.3, and 0.6 pM). As shown in Figure 5f, the fluorescence signals remain practically unchanged during the assay. These results indicate that changes in sensor readout will be due to PCR amplification and not as a result of temperature-induced noise.

Bubble-free microfluidic device. The appearance of air bubbles during a PCR assay in a microfluidic device can reduce its efficiency [40], modify the final injected volume and alter reagent concentrations [41], Also, bubble expansion can expel the reagents away from the chambers or lead to a thermal gradient within them [35], Finally, bubbles can interfere with the optical readout due to light reflection0 or refraction [42], Several strategies have been proposed to mitigate the formation of bubbles but are either too impractical [34,43,44] or not efficient [35],

To achieve bubble-free PCR assays, we engineered the device of the present invention that consisted of microfluidic chambers surrounded by a vacuum channel (Figure 7a, b). Our device contains four reaction chambers with a diameter of 3 mm and a height of 1 mm for a total volume of 7 pL (Figure5 7a). Each chamber has an inlet and an outlet and is surrounded by a horseshoe vacuum channel (1 mm x 1 mm) that degasses the PCR chambers during the thermal cycles preventing bubble formation. The chamber’s vacuum channels are joined to a shared inlet connected to a vacuum pump located inside the instrument (Figure 7b).

Instead of using photolithography to fabricate master molds, we made an acrylic mold with a milling5 machine using a single drill bit (Figure 8). (Entry-level milling machines are readily available in most countries at an affordable price [21]). 25 PDMS replicas can be made from this mold at once. The replicas are then bonded to a coverslip that fits into the heating plate (Figure 7c). Our PDMS/glass device possesses better thermal conductivity (coverslip 1.4 and PDMS 0.27 Wm ^-1 K' ¹ ) than polypropylene (0.22 Wnr ¹ K’ ¹ ), the material of conventional PCR tubes. In addition to a high5 surface/volume ratio (3.33 in our case) due to our chambers geometry, this property results in an improvement of heat transfer, an essential factor in thermocycling [45], Before each assay, the inlets and outlets are sealed with a plastic foil to prevent evaporation, step (iv). The device and the plastic foil contain a cavity in its center to measure the coverslip with the IR sensor. During PCR, the critical stage for the formation of bubbles is the initial denaturation step [46], This step is carried out at 90-95°C for up to 10 min [35], depending on the type of DNA polymerase used [46], Thus, we methodically studied the effect of applying different degassing times and pressure conditions on the vacuum channel. We observed three different behaviors during the denaturation step (Figure 7d,e). For 950 mBar, at least one chamber had all the solution expelled to the inlets/outlets, but it also occurred when no degassing was applied at 450 and 650 mBar. When the pressure was decreased to 650 mBar and 450 mBar for at least 3 min, air bubbles developed in at least one chamber and also occurred at 350 mBar when the chambers were not degassed. However, all the chambers remained bubble-free at 250 mBar and 350 mBar when sustaining this pressure for at least 3 min. We used a portable and low-cost vacuum pressure pump (350 mBar) for successive experiments and set the degassing time for 3 min. In combination with the microfluidic device, this simple setup, allowed us to carry out experiments without bubbles that could interfere with the optical sensor measurement. PCR assays. First, to demonstrate that the instrument of the present invention provided consistent results across the device, PCR was performed in all the chambers with the same initial cDNA concentration. We found the amplification curves to be identical, demonstrating the reliability of our device (Figure 9). Next, to assess the sensitivity of our system, we performed qPCR on cDNA dilutions of the human RNase P gene (Figure 10) and the SARS-CoV-2 N1 gene from 10 ⁷ to 10 ³ copies per pL (Figure 11a,b). Similar amplification curves were observed for the RNase P gene without significant differences in threshold cycle values (Cy)(Figure 10b). For the N1 gene, we observed that the amplification curves for the three highest concentrations reached a plateau in the fluorescence signal before those in the StepOne (Figure 11 b). This plateau can be attributed to the unspecific absorption of the enzymes and primers to the PDMS surface. We also noted higher variability in the amplification curves when performing inter-assay experiments than the StepOne (Figure 11c).

Nevertheless, the CT values are similar in both systems. Overall, we demonstrated that the HybOT Cycler of the present invention could amplify samples with as few copies as 10 ³ /pL together with the microfluidic device.

Finally, we evaluated the utility of our HybOT Cycler of the invention in the detection of the SARS- CoV-2 virus. We analyzed samples from 20 individuals previously diagnosed with the virus and ten negative controls. As shown in Figure 12b, we detected 19 of 20 samples with the virus, while all the controls were identified correctly; this translates into sensitivity and specificity of 95% and 100%, respectively. This experiment demonstrates the ability of the HybOT cycler of the invention to detect the presence of viral RNA from human samples. Thanks to the characteristics of the device of the present invention and the advantages it represents compared to the state of the art, in one of its modalities any nucleic acid sample in which it is intended to detect the presence of some nucleic acid of interest can be used and that can be carried out in any facility, given the portability potential of the present invention, such as nucleic acids from human, animal, and plant samples, from microorganisms such as, for example, fungi, bacteria and viruses to name a few, including any sample that contains nucleic acids and that are intended to be detected by means of the method and device of the present invention, either in specialized facilities for its detection or in some facility that meets the minimum conditions for its correct operation, such as hospitals, health centers, animal care centers, veterinarians, facilities for clinical diagnosis or in suitable rural facilities. The following examples are included below solely as a way to illustrate the invention, without implying limitations on its scope.

Example 1. Instrument. All the components resided in a 3D-printed housing (24 x 14 x 10 cm). The heating system consisted of a stack of a Thermo Electric Cooler (TEC1-12715, Wakefield-Vette) (7, Figure 1a), a heat sink (1335, Adafruit) (Figure 3d), and a tangential fan (THA0412AD, Delta electronics) (Figure 3c). The optical excitation configuration comprised an array of four high-power LEDs (LXML-PB02, Phillips Lumileds) (6, Figure 1a) with a peak intensity at 470-nm and a 483-nm optical filter (67-028, Edmund Optics). An LED driver (COM-13705, Sparkfun) (Figure 3e) controlled the LEDs and was powered by a DC-DC converter (XL6019, Unit Electronics) (Figure 3h). A 3D- printed head housed an infrared sensor (MLX90615, Melexis) (8, Figure 1b), an array of 4 digital photodetectors (VEML6030, Vishay) (1 , Figure 1b), and a 435-nm optical glass filter (67-031 , Edmund optics) (2, Figure 1 b). The 3D-printed parts included self-aligning structures that facilitated the assembly of all the pieces. A microcontroller (ABX00034, Arduino) (Figure 3f) incorporating a Bluetooth RF transceiver (HC-06, Wavesen) (Figure 3g) controlled the electronics and the sensors. A Graphical User Interface (GUI) was programmed (MIT App Inventor) to input the PCR parameters and display the results (Figure 2). The GUI was installed in a Tablet (Galaxy Tab A, Samsung). A 12- VDC 12.5-A power supply (ALM150PS12, XP Power) energized the whole system, while an H-bridge driver circuit (DC 43A Double BTS7960B, Diymore) (Figure 3a) drove the TEC. Finally, the microfluidic device was connected to a vacuum pump (MR370BPM, MIROU) (Figure 3b).

Example 2. Microfluidic device. An acrylic mold (1 ,3mm thickness, ME303018, GoodFellow) (Figure 8) was fabricated using a CNC milling machine (MDX-40A, Roland) with a 0.5 mm drill bit (1600.0197.118, Kyocera) at a one mm/s feed rate and 15000 rpm spindle speed. The mold was placed in a petri dish with 3 mL of chloroform for 5 min in order to eliminate any roughness on the structures caused by the milling process [38], Devices were fabricated by pouring polydimethylsiloxane (PDMS, Sylgard 184, Corning) on the mold at a 10:1 base to curing agent ratio (w/w) (Figure 7c). The devices were cured for 40 min at 80 °C in a convection oven. Replicas were cut out, peeled off, and the inlets and outlets were punched using a punching machine (Accu-Punch MP, Syneo) (Figure 7c). The PDMS chip and the coverslip (2850-18, Corning) were plasma-activated (Zepto, Diener) for 1 min and then sealed. A thermocouple (CHAL-003-BW, Omega, USA) and a thermocouple-to-digital converter (MAX6675, Maxim Integrated, USA) were used to monitor the temperature in the chambers (Figure 5a).

Example 3. PCR assay. 20 pL qPCR reactions were set as follows: 10 pL of master mix (EvaGreen Supermix ddPCR, QX200, 1864034, Bio-Rad) were added to 6 pL of molecular grade sterile distilled water (7732-18-5, Fisher BioReagents), 1 pL of Reverse primer (10 pM), 1 pL of Forward primer (10 pM) and 2 pL of target DNA. The sequences of primers were as follows: SARS-CoV2:

N1 F-primer: 5’-GAC-CCC-AAA-ATC-AGC-GAA-AT-3’; N1 R-primer: 5’-RTCT-GGT-TAC-TGC-CAG-TTG-AAT-CTG-3’;

Human RNase P:

F-primer: 5’-AGA-TTT-GGA-CCT-GCG-AGC-G-3’,

R-primer: 5’-GAG-CGG-CTG-TCT-CCA-CAA-GT-3’, and RNA sample. The PCR components were mixed and centrifuged for 10 s at 6000 rpm. 6.5 pL of the mixture was then pipetted into each of the reaction chambers of the microfluidic device. The inlet and outlet ports of the device were sealed with an optical adhesive film (4311971 , Applied Biosystems). A drop of mineral oil (M5904, Sigma) was placed between the device and the thermocycler plate. The thermal cycling program includes a degasification step for 5 min at 30°C, an initial denaturation step for 3 min at 95°C, and 40 amplification cycles (denaturation: 95°C, 30 s; annealing 60°C, 45 s; extension 72°C, 45 s).

Example 4. Degassing experiment. 100 pL of master mix (TaqMan, Universal PCR Master Mix, 4304437, Roche) and 2 pL of blue food dye (Food dye, McCormick, USA) were mixed with 100 pL of sterile Milli-Q water and centrifuged for 10 s at 6000 rpm. 7 pL of the mixture were injected into the four reaction chambers and sealed with optical adhesive film. Four different times (0, 3, 6, and 9 min) were tested at five pressure conditions (250, 350, 450, 650, and 950 mBar) using a vacuum transducer (901 P-11034, Micro Pirani-Piezo Loadlock Vacuum Transducer) and three different vacuum pumps (ROB-10398, Sparkfun; 400-3910 Barnant Company; and H004C-11 , Parker). The degassing conditions were performed at 30°C, followed by the initial denaturation step for 10 min at 95°C. Photographs were acquired throughout the experiment (Figure 13).

Example 5. Fluorescence experiment. Solutions of Fluorescein (2321-07-5, Sigma-Aldrich) were prepared in PBS (14040-133, ThermoFisher) at 1 , 0.8, 0.6, 0.5, 0.4, 0.3, and 0.2 pM and injected into the microfluidic chambers. Fluorescence micrographs were obtained with an inverted microscope using a 10x objective (Axio Observer A1 , Carl Zeiss) with an exposure time of 100 ms.

Example 6. COVID-19 samples. Total viral RNA was extracted from nasopharyngeal swabs with the Biopure kit (Mexico City) from COVID-19 patients. We used the CDC protocol for RT-qPCR to confirm the presence of SARS-CoV-2. 20 pl RT-qPCR reactions were set as follows: 10 pL of master mix (KAPA SYBR FAST qPCR 2X, KK4650, KAPA Biosystems,) were added to 6.4 pL of molecular grade sterile distilled water (7732-18-5, Fisher BioReagents), 0.4 pL of RT Mix (50X KAPA RT Mix, KK4650, KAPA Biosystems), 0.4 pL of ROX (ROX, KK4650, KAPA Biosystems), 0.4 pL of Reverse primer (10 pM), 0.4 pL of Forward primer (10 pM) (Same sequences of primers that example 3) and 1 pL of target RNA. Samples were obtained from 20 individuals who tested positive for the virus and a control cohort consisting of 10 samples from healthy individuals. This study was reviewed and approved by The Committee on Bioethics for Research in Human Beings (COBISH) of Cinvestav.

COVID-19 has emphasized the worldwide scarcity of tests and instruments to detect the virus SARS- CoV-2, in part because of an unexpected high demand and also because some of them are •S manufactured in countries where governments banned companies from exporting to other nations until satisfying their internal demand. Under these circumstances, it is clear that we need to develop alternatives that can be (i) manufactured locally (ii) without much training, (iii) ideally from off-the-shelf parts, and (iv) using digital fabrication tools (that are now available worldwide). Our contribution in this direction was to develop a simple yet reliable instrument to perform RT-qPCR (the gold standard to0 detect SARS-COV-2). As we laid out in the present invention, our HybOT Cycler consists of a hybrid module that works simultaneously as a heating element and as an illumination source. This simple configuration also enables detecting fluorescence signals by regular light sensors, with no additional optics. We also developed a microfluidic device to carry out 4 PCR assays in parallel. The device is connected to a portable vacuum pump to eliminate any bubbles during PCR. Notably, the mold of this 5 microfluidic device can be manufactured with a milling machine and does not require access to a clean room facility. Finally, and more importantly, we correctly identified 19 out of 20 patients infected with the virus, while no false positives were identified.

The thermocycler of the present invention can be repurposed for other similar assays, such as isothermal amplification methods. The HybOT Cycler’s low weight (D 1 Kg) and portability open the0 door to myriad applications, from its use in remote locations, homes, and clinics, to field applications such as monitoring water pathogens. Its wireless operation, together with GPS localization, means that real-time reports can be communicated to a centralized site (e.g., government facility, data center). The information can be employed to enact measures or to build epidemiological models [29], Furthermore, remote monitoring and control could enable field deployment while minimizing the risk5 of exposure and play an important role in halting the spread of COVID-19.

The HybOT Cycler of the invention has future possibilities, for example the concentration of some reagents in the master mix (BSA, enzymes, nucleotides) could be optimized to obtain more reliable amplification curves and the dimensions of the chambers and their volume could be re-engineered to reduce the unspecific absorption of reagents. A shortcoming of our device could be the few assays0 that can run in parallel (4 reaction chambers), but we are working on a future version to increase the number of chambers to 16. Nevertheless, the cost of the HybOT components (□ US$700), a fraction of a standard available commercial thermocycler ($>US10,000), and the facility to build it, shows the high potential of the invention for its efficient portable use for detect nucleic acid molecules, for example from the SARS-CoV2 virus.

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