SARS-CoV-2 detection via high-sensitivity RT-PCR in 15 minutes using microfluidic COP chips

SARS-CoV-2 detection via high-sensitivity RT-PCR in 15 minutes using microfluidic COP chips | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SARS-CoV-2 detection via high-sensitivity RT-PCR in 15 minutes using microfluidic COP chips Anna Lena Maisch, Sisi Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7453406/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Moving-Plug PCR System, developed by Fraunhofer IMM, is a portable device that dramatically accelerates PCR reaction speeds by eliminating the time-consuming heating and cooling cycles traditionally used in thermal cycling. Instead, the reaction sample is moved between preheated hot and cool zones. When combined with a SARS-CoV-2 PCR kit, we successfully conducted 40 PCR cycles and achieved quantitative detection of SARS-CoV-2 RNA in 15 minutes, which is more than 2x faster than using a thermocycler. This innovation demonstrates significant potential for rapid and sensitive diagnostic applications. The polymerase used in our SARS-CoV-2 PCR kit can transcribe RNA into cDNA during the first step of PCR. This omits the need for an additional reverse transcriptase step before PCR, therefore quantitative results could be achieved in 15 minutes. The use of polycarbonate as a chip material required the addition of betaine in the PCR to overcome its limitations, while cyclic olefin copolymer was evaluated as chip material without the addition of betaine. The sensitivity achieved using a cyclic olefin copolymer chip was equivalent to that obtained with a commercially PCR thermocycler. Notably, it was also possible to detect RNA effectively in heat-inactivated saliva samples, which further enhances its applicability in real-world settings. These findings can help overcome the disadvantages of traditional diagnostic methods, such as the long testing times of traditional PCR assays or the lack of sensitivity of Enzyme-linked Immunosorbent Assay. For future infectious disease outbreaks a fast Point-of-Care-PCR Technology with short result reporting times and adequate sensitivity is achieved. SARS-CoV-2 microfluidic device RT-PCR diagnosis COVID-19 lab-on-chip Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction As of 2023, the global landscape of COVID-19 has undergone significant changes since the initial outbreak in late 2019[ 1 ]. Widespread vaccination campaigns have been implemented worldwide, leading to a substantial decrease in severe cases and mortality rates. However, the virus continues to evolve, with new variants emerging that pose ongoing challenges to public health systems. Rapid and reliable diagnostic tools that can be used directly at the point of care are critical for the early detection of new infections and an effective response. Reverse transcription polymerase chain reaction (RT-PCR) is currently the gold standard for diagnosing viral diseases[ 2 – 5 ] due to its high sensitivity and specificity[ 6 – 8 ]. The COVID-19 pandemic has accelerated advancements in diagnostic technology, particularly in Point-of-Care Testing (POCT), which is crucial for controlling outbreaks and mitigating the impact on healthcare systems. During the pandemic, most SARS-CoV-2 tests based on polymerase chain reaction (PCR) methods were conducted in the centralized laboratories, leading to lengthy sample transportation, batch loading and result reporting times. POCT offers advantages such as ease of use, timely detection, and comparable accuracy and sensitivity, essential for daily epidemic control and early treatment[ 9 ]. While PCR tests are highly sensitive, the average 1–2 hours test duration, along with sampling and logistical challenges and complex sample preparation processes, has hindered their widespread adoption as point-of-care diagnostics. In contrast, lateral flow immunoassays (LFIA) were used for rapid mass testing, although they are generally less sensitive than PCR tests[ 10 – 12 ]. An ideal POCT solution would combine the speed of LFIA with the sensitivity of PCR for optimal epidemic control. Microfluidics-based PCR platforms has led to the development of miniaturized devices capable of nucleic acid amplification with reduced sample volumes and faster processing times[ 13 ]. Advances in microfluidics-based POCT technology have shown potential for diagnosing infectious diseases, including COVID-19. These devices offer rapid detection using low-cost and high-throughput systems, particularly beneficial in developing countries and low-resource settings[ 14 , 15 ]. The performance of these diagnostic tests continues to be optimized to prepare for future infectious disease outbreaks[ 16 , 17 ]. In current research focused on enhancing PCR technology, various innovative approaches have emerged. For instance, some systems utilize integrated heating mechanisms and rotating components to achieve rapid detection of multiple viruses simultaneously, significantly reducing analysis time[ 18 ]. Others utilize thermoplastic materials with infrared-mediated thermocycling to accelerate nucleic acid amplification, although yields may be lower than with traditional methods [ 19 ]. Furthermore, infrared light-emitting diodes activate heat-generating nanoparticles for isothermal PCR, and microfluidic platforms miniaturize PCR processes[ 20 , 21 ]. This enables rapid, cost-effective diagnostics with improved heat transfer and increased throughput, allowing multiple reactions to be executed simultaneously. Fraunhofer IMM has developed the Moving-Plug PCR System to address the need for rapid and sensitive diagnostic tools. This ultrafast microfluidic module uses oscillatory PCR to complete 30 cycles in six minutes[ 22 ], without requiring time-consuming up-and-down thermal cycling. Instead, the reaction sample is repeatedly moved forward and backward between the preheated hot and cool temperature zones. The settings of the MoPPS, such as the number of pump cycles and the temperatures of the heating zones, can be flexibly adjusted to accommodate the specific requirements of the respective PCR kits used. It is a highly compact and portable device, comparable in size to a syringe pump commonly used in laboratories. The disposable chip is featuring a simple design and can be fabricated from various thermoplastic materials. Polycarbonate (PC) has been widely used due to its favorable properties, including strength, toughness, and optical transparency. The material's excellent moldability make it a prevalent choice various engineering applications[ 23 ]. Cyclic Olefin Polymer (COP), as an alternative chip material, has also been developed specifically to meet high-performance requirements, including a high glass transition temperature, good mechanical properties, high transparency, and excellent film-forming abilities[ 24 ]. It provides strong mechanical performance, and excellent chemical resistance[ 25 ]. For RT- PCR a SARS-Cov-2 PCR kit from a third party is used. The polymerase used in this this kit combines wild-type Taq DNA polymerase with artificially induced reverse transcriptase activity[ 26 ]. This combination removes the requirement for a separate initial reverse transcriptase step, thereby simplifying the entire process by allowing the use of unprocessed patient samples. Consequently, the process time is significantly reduced, and handling procedures are made more efficient. This work aimed to establish a rapid and highly sensitive PCR system by validating and optimizing the compatibility of MoPPS with a SARS-CoV-2 PCR kit. This enables faster viral detection than conventional thermocyclers. The initial experiments on PC chips involved using RNA samples and focused on optimizing PCR duration. The use of COP as an alternative chip material to PC was also evaluated using purified RNA, followed by testing with human saliva samples to assess its practical applicability. Through these efforts, the aim of this research is to develop a robust, rapid and highly sensitive PCR system for reliable diagnostics in significantly reduced timeframes, thereby enhancing the accessibility and speed of point-of-care testing (POCT) compared to traditional methods. 2 Material and methods 2.1 DNA amplification procedure on the Bio-Rad PCR System The reactions were performed using the Volcano3G RT-PCR Probe Master Mix and the SARS-CoV2 Singleplex N1-Assay (FAM-Probe) (myPOLS Biotec). As a template, in the beginning 5 µL RNA (5x10 7 copies/ µL) for amplification were used. For testing against the MOPPS the RNA amount was reduced to 1 µl RNA sample. Each 30 µL RT-PCR reaction mix consisted of 15 µL of 2x Volcano3G RT-PCR Probe Master Mix, 3 µL SARS-CoV2 Singleplex N1-Assay, 5/1 µL of RNA and 7/11 µL PCR grade water according to the manufacturer's instructions. qPCR reactions were carried out in a Bio-Rad CFX96 Real-Time PCR Detection System with the following cycling parameters: reverse transcription step at 70°C for 15 min or initial denaturation at 95°C for 120 s, followed by 55 cycles of 5 s of denaturation at 95°C, 5s of annealing at 57°C and 30 s of elongation at 71°C. The used parameters were further shortened to the following: initial denaturation at 95°C for 120 s, followed by 55 cycles of 5 s of denaturation at 95°C and 30s of annealing + elongation at 65°C. The measured fluorescence dye was FAM. The fluorescence at 520 nm was monitored for real-time data collections during elongation. Thermal cycling, fluorescent data collection and data analysis were carried out using the Bio-Rad CFX Maestro 2.3 (Bio-Rad). The System automatically calculates at which PCR cycle the fluorescence exceeded the threshold. This was defined as quantification cycle (Ct). 2.2 Setup of the Moving-plug PCR System and manufacture of the microfluidic chips The Moving-plug PCR System was developed with three heating modules, which are located under the three zones of the PCR chip. Two heating modules ensure temperature control of the reaction mixture during amplification, while the third prevents condensation of liquid in the gas reservoir (dead end). The polymer chip was precisely positioned on the heaters with the help of magnets in a black sealing cap. Integrated light barriers facilitate the correct positioning of the stopper in the channel. This allowed the reaction mixture to be oscillated reproducibly at high speed between the two distinct temperature zones. The detection of the liquid by light barriers provided feedback to the customized syringe pump module (more information: [ 22 ]). The PCR microchip was manufactured from polycarbonate by injection moulding (microfluidic ChipShop, Jena, Germany) to ensure reproducibility. The design was developed by Brunklaus et al. The COP chips were produced on a milling machine (Charlyrobot S.A.S., Cruseilles, France). Firstly, the chips were cleaned by CO 2 blasting to remove the residues from the milling process. To remove the biological debris, the chips were placed in isopropanol and sonicated for 5 minutes. Next, the channels of the chip were sealed with a thin COP film (188 µm) by solvent bonding. The COP film was covered with the bonding solvent, which consist of one part cyclohexane mixed with one-part decalin. By spin coating the bonding solvent will be spread evenly on the COP film. Next the foil was pressed with 3.5 tons for 3 min on the COP chip. After evaporation of the solvents, the chip can be used. The chip was connected to a syringe pump via a welded-on Luer lock adapter. 2.3 DNA amplification procedure on the Moving-plug PCR System For on-chip amplification, the same reagents as for the tests with Bio-Rad PCR System were used. For initial tests DNA (20.000 copies/ µL) was used instead of RNA. First the 2-step PCR protocol was validated with PC chips on the MoPPS (Table I). The addition of betaine at a final concentration of 1 M was essential for this validation. Subsequently, the PCR protocol was progressively shortened, and RNA at a concentration of 1 × 10 6 copies/µL was utilized instead of DNA. The flow velocity within the microfluidic channel for fluid plug movement was chosen between 15–40 ml/min, respectively, as indicated for the individual experiments. The different PCR protocols and flow rates are listed in Table II. The fastest protocol was also tested with heat inactivated saliva. As template human saliva was heated for 10 minutes at 95°C and spiked with 1 µl RNA (concentration 1 × 10 6 copies/µL). For the tests with COP chips, the same reagents (excluding betaine) and the same PCR protocol (Table II, d) as for the tests with PC chips were used. This time, 1x 10 6 copies/ µL of Sars-CoV-2 RNA was used. Four runs on the MoPPS were tested against four runs on the Bio-Rad PCR System. For the tests involving real saliva samples, saliva was taken from the experimenter and heated for 10 minutes at 95°C. This will be used as the sample matrix for further experiments. For the experiments with spiked saliva, 1 µL of RNA with a concentration of 1 × 10⁶ copies/µL was used. The experiments to test the negative control were conducted using H₂O as the sample matrix. For all tests the temperatures inside the liquid were adjusted to approx. 95°C in the denaturation region and 66°C in the annealing and elongation region. The evaluation of the PCR data was conducted using a self-designed Python script, which analyses the fluorescence signal of each cycle. 3 Results 3.1 Optimization of the RT-PCR protocol The RT-PCR protocol for the MoPPS was adapted to minimize the PCR run time using the SARSCoV-2 myPOLS kit, which eliminates the need for an additional reverse transcription (RT) step. The polymerase in this kit performs RNA to cDNA transcription during denaturation. For confirmation, two standard protocols were tested on the Bio-Rad PCR System: one with the extra RT step and one without (Table I, protocol 1 and 2). Each protocol was tested three times using 1x10 7 copies of SARS-CoV-2 RNA. The results confirmed that the extra RT step could be omitted, yielding average Ct-values of 15.60 ± 0.23 with the RT step and 15.81 ± 0.07 without it (Fig. 1 ). Furthermore, the PCR protocol was adapted to a 2-step protocol, wherein annealing and elongation are combined, to further decrease the runtime. The protocol utilized was provided by myPOLS (Table I, protocol 3) which was subsequently validated on the Bio-Rad PCR System, resulting in an average Ct value of 15.27 (Fig. 1 ). 3.2 Shorten the PCR reaction time to 15 minutes The experiments conducted on the MoPPS aim to assess the possibility of shortening the PCR runtime to be faster than the conventional PCR runtime. The PCR protocol was successfully modified to a 2-step protocol, wherein annealing and elongation are combined. This protocol was initially validated in preliminary experiments on the Bio-Rad PCR System (Fig. 2 ). Following the incorporation of betaine as a PCR additive, the 2-step protocol (Table II, a) was effectively implemented on the MoPPS using PC chips. Additional testing was carried out to achieve a PCR runtime that is faster than that typically observed in laboratory settings. Various parameters were adjusted to optimize the PCR runtime: time of denaturation, time of combined annealing and elongation (AE), and the flow rate (FR). The FR is defined by the pumping velocity between the two individual heating zones. A higher flow rate results in a faster temperature change of the reagents in the chip. The initial denaturation time remained unchanged. Four different PCR protocols were evaluated and compared on the MoPPS (Table II). The PCR runtime decreased progressively with each protocol. The protocol, using 1 sec for denaturation and 5 sec for AE along with an increased FR, was the most efficient, reducing the runtime to 15 minutes. Following the initial successful PCR, the AE time was further reduced, and PCRs were conducted using RNA. The AE step was successfully shortened to 15 seconds (Table II, protocol b), and the FR was increased to 20 ml/min under this protocol (Table II, protocol c). This setup allowed for 40 PCR cycles to be completed in approximately 25 minutes. To further reduce the duration, the denaturation step was shortened to 1 second, and the AE step to 5 seconds, while also increasing the FR (Table II, protocol d). Each protocol indicates a positive PCR signal. Simultaneously, the total time decreased from 38 to 15 minutes, making it 2.5 times faster than the initial used PCR protocol, and the time for a positive signal decreased from 27 to 10 minutes. The fastest 15-minute PCR protocol was evaluated three times on both the MoPPS and the Bio-Rad PCR System (Fig. 2 , a-d). The MoPPS demonstrated consistent amplification curves (Fig. 2 , a). However, the amplification curves obtained from the MoPPS exhibited a significant rightward shift to higher Ct values compared to those from the Bio-Rad PCR System, indicating reduced sensitivity or efficiency in amplification (Fig. 2 , b). Specifically, the average Ct value on the MoPPS was 31 ± 1, while it was 21.23 ± 0.68 on the Bio-Rad PCR System, indicating a loss of sensitivity of approximately 10 Ct values. Direct comparisons are challenging due to the different Ct value thresholds of the individual devices. This variation can lead to discrepancies in the observed Ct values between the two systems. The use of the MoPPS significantly reduced the PCR duration time, achieving a runtime of 15:04 ± 0.03 minutes compared to 35:50 minutes on the Bio-Rad PCR System (Fig. 2 , c-d). This highlights the MoPPS's capability to detect SARS-CoV-2 RNA at a speed that is 2.4 times faster than that of the conventional thermocycler. 3.3 Evaluation of using COP as chip material The use of PC as a chip material has several drawbacks, including the need to add betaine for RT-PCR. Alternatively, COP serves as a beneficial chip material that can be used to mill chips in-house at our institute. The chips are milled, cleaned, and solvent bonded. Comparative testing on the MoPPS displayed that PC exhibits high intrinsic autofluorescence (Fig. 3 , a), while COP showed only low intrinsic autofluorescence (Fig. 3 , b). A significant advantage of using COP as a chip material is the elimination of additives in PCR, as tests demonstrated that RT-PCR can be performed effectively without them, using the same PCR protocol applied to PC chips (Table II, protocol d). Four runs were conducted on the MoPPS using COP chips. The average Ct value obtained was 21.19 ± 0.73. For comparison, the same protocol run on the Bio-Rad PCR System yielded an average Ct value of 19.94 ± 0.29 (Fig. 4 , a). COP as a chip material exhibited higher sensitivity on the MoPPS compared to PC. The difference in Ct values between the Bio-Rad PCR System and the MoPPS was approximately 1 Ct with COP chips (21.19 on the MoPPS compared to 19.94 on the Bio-Rad PCR System) and approximately 10 Ct with PC chips (31.00 on the MoPPS compared to 21.23 on the Bio-Rad PCR System, Fig. 4 , b). 3.4 Evaluation of heat-inactivated Saliva as a PCR template To evaluate the effectiveness of this method using patient samples, human saliva was used as the sample material. After validating the fastest PCR protocol (see Table II, protocol d), the tests were extended to include human saliva. First, the saliva was tested to establish its baseline characteristics. For this, the saliva was heat-inactivated and used as sample in the PCR. The saliva came from an experimenter who had previously had Covid-19. This is evident from the received Ct values. A high Ct value was obtained on both systems: 39.84 ± 2.01 on the MoPPS and 36.38 ± 1.13 on the reference system (Fig. 5 ). After testing the background, saliva RNA was added to the saliva to imitate a real patient sample. The heat-inactivated saliva was spiked with RNA and processed using the 15-minute rapid protocol (Table II, protocol d). Successful RNA detection in the saliva sample was achieved, resulting in an average Ct value of 24.41 ± 1.37 using the MoPPS system and an average Ct value of 25.09 ± 0.10 using the Bio-Rad PCR system (Fig. 6 ). 3.5 Analysis of a Negative Control To ensure that the results were not influenced by unwanted factors or contaminants, a negative control was tested, which is crucial for validating the reliability of the assay. For this purpose, water was used as sample instead of RNA. Tests were conducted on both the MoPPS and the Reference System. Of the three runs for each system, one resulted in a positive outcome. Figure 7 a shows the average of three runs on the MoPPS, a fitted curve, and three runs on the Bio-Rad PCR System. To calculate the average Ct value, negative results were assigned a value of 41. High Ct values were obtained for both systems: 40.19 ± 1.41 for the MoPPS system and 38.69 ± 4.00 for the reference system (Fig. 7 , b). Table 1 Tested PCR protocols on the Bio-Rad PCR System Tested protocols RT Initial denaturation Denaturation Annealing Elongation Cycle number 1: 3- step PCR with RT 15 min, 70°C - 5 sec, 95°C 5 sec, 57 °C 30 sec, 71 °C 55 (later reduced to 45) 2: 3-step PCR without RT - 120 sec, 95°C 5 sec, 95°C 10 sec, 57 °C 60 sec, 71°C 55 (later reduced to 45) 3: 2-step PCR - 120 sec, 95°C 5 sec, 95°C 30 sec, 65°C 55 (later reduced to 45) Table 2 Tested PCR protocols on the MoPPS Tested protocols Sample material Initial denaturation Denaturation Annealing + Elongation Flow Rate Total PCR time (40 cycles) a DNA 120 sec, 95°C 5 sec, 95°C 30 sec, 65°C 15 ml/min 38:00 min b RNA 120 sec, 95°C 5 sec, 95°C 15 sec, 66°C 15 ml/min 29:00 min c RNA 120 sec, 95°C 5 sec, 95°C 15 sec, 66°C 20 ml/min 27:00 min d RNA 120 sec, 95°C 1 sec, 95°C 5 sec, 66°C 30 ml/min and 40 ml/min 15:00 min 4 Discussion The COVID-19 pandemic has underscored the need for innovative viral detection methods, especially in low-resource settings. POCT offers rapid results for effective disease management, but traditional methods often require significant time and resources. Advancements like RT-PCR and isothermal amplification show promise, though rapid antigen tests often sacrifice sensitivity. The ideal POCT system would combine the speed of antigen tests with the sensitivity of nucleic acid amplification. The development of lab-on-a-chip solutions, such as the MoPPS, represents a notable development in viral diagnostics. The MoPPS has shown the capability to complete 30 PCR cycles in just 6 minutes[ 22 ], facilitating quantitative rapid PCR. Preliminary tests with the SARS-CoV-2 detection kit revealed that the PCR runtime can be further reduced by eliminating the reverse transcription step and using a two-step PCR protocol. The polymerase utilized in this kit is a combination of a thermostable wild-type Taq DNA polymerase with an artificially induced reverse transcriptase activity[ 26 ]. Following the development of this innovative polymerase[ 27 , 28 ], it was compared to conventional RT-PCR methods [ 26 ], yielding promising results that warrant further investigation for viral detection applications[ 29 , 30 ]. This polymerase significantly simplifies the reagent process, allowing for the use of unprocessed patient samples. Its efficiency not only reduces processing time but also streamlines handling procedures, thereby minimizing the risk of cross-contamination. Consequently, this polymerase presents itself as a viable alternative in the field of viral diagnostics. Polymerase-integrated microfluidics can achieve ultra-fast PCR, making them ideal for rapid diagnostic applications. PC is a common thermoplastic used for PCR applications due to its favorable properties. However, the use of PC as a material for microfluidic chips presents several drawbacks. Notably, there is a requirement for betaine as a PCR additive, and PC has relatively high intrinsic autofluorescence (Fig. 3 ). The performance of the PCR with PC as chip material can be influenced by various factors, including its permeability to oxygen and moisture[ 31 ], which can degrade sensitive PCR reagents and affect reaction stability. Additionally, PC's sensitivity to notching can compromise its mechanical properties [ 23 ], which can be problematic under the thermal cycling conditions of PCR. Another concern is the adsorption of reagents onto the PC chip surface. Numerous studies have documented the application of surface treatments or coatings to PC substrates prior to their utilization in PCR[ 32 – 38 ]. For instance, UV treatment has been employed to enhance wettability and improve fluidic transport[ 32 , 33 ]. In the absence of such treatments, reagent adsorption can occur[ 34 ]. Coating the microfluidic channels is a common strategy to prevent nonspecific adsorption of PCR reagents to the channel walls[ 35 ]. Zhang et al. demonstrated that Taq polymerase can adsorb to the surface, emphasizing the need for surface modifications[ 36 ]. To prevent this, channels are often treated with BSA, glycerol, or other reagents[ 35 – 38 ]. The use of PCR additives, such as betaine, BSA or PEG, is often used across various PCR applications[ 39 , 40 ]. The necessity of coating the microfluidic channels aligns with the findings of this study, indicating that an additive to coat the channel walls is essential for achieving positive PCR results when using PC as chip material. Despite these challenges, the MoPPS was able to run 40 PCR cycles and deliver quantitative SARS-CoV-2 results in 15 minutes (Fig. 2 ), which is faster than a commercial thermocycler. The comparison of Ct values reveals that those obtained with PC are significantly higher than those from the commercial thermocycler. However, direct comparison is hindered by the differing threshold calculations used in the two devices. To address the limitations associated with PC, comparisons were made with COP as an alternative chip material. COP has high optical clarity, chemical resistance, and biocompatibility[ 25 ]. The high optical clarity is demonstrated in the lower intrinsic autofluorescence, which is advantageous for PCR detection. Tests with RNA on COP chips revealed that no addition of betaine was needed, and the resulting Ct values closely matched those from commercially thermocyclers (Fig. 4 ). The improvement is likely due to the low levels of extractable substances in COP, which reduces the risks of contaminations and reagent adsorption[ 41 ]. Several studies have indicated that COP surfaces exhibit minimal adsorption of reagents or moisture[ 25 , 41 – 44 ], preserving the integrity of PCR reagents[ 41 , 44 ]. Additionally, COP does not degrade or compromise assays due to structural weaknesses or chemical contamination[ 25 , 44 ]. In addition to evaluating COP, the study examined the effectiveness of using heat-inactivated human saliva as a sample material. Baseline saliva tests indicated a significant RNA load due to a prior SARS-CoV-2 infection (Fig. 5 ). Following the spiking of inactivated saliva with RNA, the achieved Ct values were comparable to those obtained from thermocyclers (Fig. 6 ). This demonstrates that RNA in saliva can be detected with similar sensitivity. However, it appears that saliva may have an inhibitory effect, as the Ct values obtained with spiked saliva samples (24.41 ± 1.37 using the MoPPS and 25.09 ± 0.10 using the Bio-Rad PCR System) were higher than those obtained with pure RNA (19.94 ± 0.29 using the Bio-Rad PCR System and 21.19 ± 0.73 using the MoPPS). This suggests that an additional purification step could effectively mitigate this inhibitory effect and improve the sensitivity of the assay. Furthermore, evaluating negative controls was crucial in validating the reliability of the assay (Fig. 7 ). Using water as sample for negative control tests on both systems demonstrated a low likelihood of contamination, indicating the robustness of the assay. The clear differentiation between negative control results and those from spiked saliva samples further emphasizes the reliability of the PCR system in detecting true positives. Various lab-on-a-chip devices exist for PCR applications. For example, Chen et al. developed the SWM-02 device, which processes six samples but requires a PCR runtime of 40 minutes[ 45 ] —considerably longer than that of the MoPPS. Kwon et al . tested the GENECHECKER™ Ultra-Fast PCR System, achieving influenza results in 23 minutes[ 46 ]. Most commercial products, such as those from Abbott, BioFire, and Cepheid, integrate nucleic acid extraction with RT-PCR in a single automated cassette but rarely achieve results in 15 minutes, with most COVID-19 tests taking 30 to 120 minutes[ 47 – 49 ]. The MoPPS can be developed into a fully autonomous by incorporating an RNA extraction step while maintaining detection times under 30 minutes. Its cost-effectiveness and reduced infrastructure requirements make it suitable for rapid diagnosis in developing countries, enhancing accessibility during public health emergencies. While the MoPPS has demonstrated significant advancements in PCR diagnostics, challenges with PC materials persist. The evaluation and transition to COP represents a crucial improvement in sensitivity and performance. Further enhancements, particularly in automated sample preparation, could elevate the MoPPS's usability, ensuring that rapid and accurate testing remains at the forefront of public health responses. The combination of speed, sensitivity, and cost-effectiveness could position the MoPPS to serve as a strong tool in modern diagnostics. 5 Conclusion In summary, the integration of microfluidic PCR technology into point-of-care diagnostics offers substantial advantages for detecting infectious diseases, including COVID-19. Our miniaturized, portable, and ultra-fast PCR device demonstrates the ability to complete 40 PCR cycles and deliver quantitative SARS-CoV-2 results in 15 minutes. Remarkably, this rapid PCR protocol is effective in detecting both purified RNA and RNA that has been added to saliva samples. This highlights its practical applicability in real-world scenarios. The evaluation and transition to COP as chip material significantly enhances sensitivity, aligning the device's performance with that of commercially available PCR thermocyclers and ensuring accurate and reliable diagnostics. Looking forward, the potential for incorporating freeze-dried PCR reagents directly within the chip presents promising opportunities for rapid and convenient application at the point of care. This advancement would further enhance the device's practicality for widespread use in various settings. Additionally, expanding detection capabilities to include a broader range of viruses and bacteria could enable comprehensive pathogen surveillance. Integrating this innovative PCR technology into existing healthcare systems has the potential to greatly improve pandemic management, providing rapid and reliable diagnostics that can effectively curb the spread of infectious diseases. As we continue to refine and enhance this technology, its role in public health could become increasingly helpful. Declarations Competing Interests The authors declare they have no financial or competing interests. Ethical Approval Not applicable. Funding Declaration This study was funded by the Fraunhofer Society (Grant No. 40–00440) Author Contribution A. was responsible for the conceptualization of the study, the development of the theoretical model, the execution of the experimental measurements, and the contribution to the data analysis. Additionally, A. authored the initial draft of the manuscript, and was responsible for revising and finalizing the manuscript.B. was responsible for revising the manuscript and supervising the project.All authors discussed the results and approved the final manuscript. Acknowledgement We acknowledge the InBaDtec project as a funding source provided by the Fraunhofer Society for supporting this research. We express our gratitude to Dr. Tobias Gerling for his support during the preparation of this manuscript. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. 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J, Höth J, Jung M, Latta D, Strobach X, Winkler C, Ritzi-Lehnert M and Drese K S 2012 Electrophoresis 33 3222–8, doi: 10.1002/elps.201200259 Kausar A 2018 Journal of Plastic Film & Sheeting 34 60–97, doi: 10.1177/8756087917691088 Kohara T 1996 Macromolecular Symposia 101 571–9, doi: 10.1002/masy.19961010163 Niles W D and Coassin P J 2008 Assay and drug development technologies 6 577–90, doi: 10.1089/adt.2008.134 Chovancova P, Merk V, Marx A, Leist M and Kranaster R 2017 Biology methods & protocols 2 bpx008, doi: 10.1093/biomethods/bpx008 Sauter K B M and Marx A 2006 Angewandte Chemie (International ed. in English) 45 7633–5, doi: 10.1002/anie.200602772 Blatter N, Bergen K, Nolte O, Welte W, Diederichs K, Mayer J, Wieland M and Marx A 2013 Angewandte Chemie (International ed. in English) 52 11935–9, doi: 10.1002/anie.201306655 Kuiper J W P, Baade T, Kremer M, Kranaster R, Irmisch L, Schuchmann M, Zander J, Marx A and Hauck C R 2020 PloS one 15 e0241740, doi: 10.1371/journal.pone.0241740 Zander J, Scholtes S, Ottinger M, Kremer M, Kharazi A, Stadler V, Bickmann J, Zeleny C, Kuiper J W P and Hauck C R 2021 Microbiology spectrum 9 e0036121, doi: 10.1128/spectrum.00361-21 Schaepkens M, Kim T W, Gün Erlat A, Yan M, Flanagan K W, Heller C M and McConnelee P A 2004 Journal of Vacuum Science & Technology A: Vacuum, Surfaces, and Films 22 1716–22, doi: 10.1116/1.1705646 Liu Y, Ganser D, Schneider A, Liu R, Grodzinski P and Kroutchinina N 2001 Analytical chemistry 73 4196–201, doi: 10.1021/ac010343v Li Y, Wang Z, Ou L M L and Yu H-Z 2007 Analytical chemistry 79 426–33, doi: 10.1021/ac061134j Bhurke A S, Askeland P A and Drzal L T 2007 The Journal of Adhesion 83 43–66, doi: 10.1080/00218460601102860 Ha M L and Lee N Y 2015 Food Control 57 238–45, doi: 10.1016/j.foodcont.2015.04.014 Zhang Y, Trinh K T L, Yoo I-S and Lee N Y 2014 Sensors and Actuators B: Chemical 202 1281–9, doi: 10.1016/j.snb.2014.06.078 Chen J, Wabuyele M, Chen H, Patterson D, Hupert M, Shadpour H, Nikitopoulos D and Soper S A 2005 Analytical chemistry 77 658–66, doi: 10.1021/ac048758e Ragsdale V, Li H, Sant H, Ameel T and Gale B K 2016 Biomedical microdevices 18 62, doi: 10.1007/s10544-016-0091-x Kim J, Byun D, Mauk M G and Bau H H 2009 Lab on a chip 9 606–12, doi: 10.1039/b807915c Yang J, Liu Y, Rauch C B, Stevens R L, Liu R H, Lenigk R and Grodzinski P 2002 Lab on a chip 2 179–87, doi: 10.1039/b208405h Waxman L, Erwin R L and Vilivalam V D 2017 BioTechniques 62 223–8, doi: 10.2144/000114546 Geissler M et al 2020 The Analyst 145 6831–45, doi: 10.1039/d0an01232g Nunes P S, Ohlsson P D, Ordeig O and Kutter J P 2010 Microfluid Nanofluid 9 145–61, doi: 10.1007/S10404-010-0605-4 Shin J Y, Park J Y, Liu C, He J and Kim S C 2005 Pure and Applied Chemistry 77 801–14, doi: 10.1351/PAC200577050801 Chen X, Liu Y, Zhan X, Gao Y, Sun Z, Wen W and Zheng W 2022 Bioengineering (Basel, Switzerland) 9, doi: 10.3390/bioengineering9100548 Kwon S-H, Lee S, Jang J, Seo Y and Lim H-Y 2018 Journal of medical virology 90 1019–26, doi: 10.1002/jmv.25046 Abbott ID NOW™ COVID-19: Interim Clinical Study Results from 1,003 People to Provide the Facts on Clinical Performance and to Support Public Health Cepheid Xpert xpress SARS-CoV-2 https://www.cepheid.com/de-CH/tests/respiratory/xpert-xpress-sars-cov-2.html Biofire COVID-19 test https://www.biofiredefense.com/covid-19test/ Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7453406","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513565421,"identity":"5e527f5d-df9b-4812-9362-39b25a43a456","order_by":0,"name":"Anna Lena Maisch","email":"","orcid":"","institution":"Fraunhofer Institute for Microengineering and Microsystems IMM","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"Lena","lastName":"Maisch","suffix":""},{"id":513565423,"identity":"4b3bc393-035e-45a0-a952-caa59368a99f","order_by":1,"name":"Sisi Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYDACZgaGAwwFEnJsIE4FiJBgYPgApBgb8GoxkDAGazkD0cI4A68WMDBgSGwgWot8O/tDoC0W6X1ipxMYDu6xS9wu3Xyw4eMeBtl+XOYf5jEAOSy3TTp3A8OBZ8mJO+ccS2yc8YzBeCYOawyYeRjgWpg/HGBO3HAjx/wxzwGGxA0HcDismf0BSEs6G9iWA/UgLYbNf4Ba9uPQwnCYAeywBKiWwxAtDCBb8PklwUDCEOSwAwcOHDfecCMtsbHngITxDFwO6z/++MOHijp5+dm5Gx8cOFAtu+FG8sGGHwdsZPtxeB8MEqA0srESeNSPglEwCkbBKCAEABnjZDCQ6ZqDAAAAAElFTkSuQmCC","orcid":"","institution":"Fraunhofer Institute for Microengineering and Microsystems IMM","correspondingAuthor":true,"prefix":"","firstName":"Sisi","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-08-25 11:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7453406/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7453406/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91336751,"identity":"6af99389-c827-4fa5-876a-5e5821f2cced","added_by":"auto","created_at":"2025-09-15 12:09:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":35390,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of a 3-step PCR protocol with and without extra reverse transcription step and a 2-step PCR protocol. Three different PCR protocols (Table I) were tested with 1x10\u003csup\u003e7\u003c/sup\u003e copies of SARS-CoV-2 RNA each. The graph displays the average values from three separate PCR wells. The average Ct values of the three different PCR protocols are close aligned. Each PCR was conducted independently, and therefore, the threshold for determining the Ct value was calculated separately for each PCR\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7453406/v1/44d91fc5fd0a45f9c02965e4.png"},{"id":91336752,"identity":"1068b576-4970-400e-a7b3-497a5ffdbfd5","added_by":"auto","created_at":"2025-09-15 12:09:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98495,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of a 15-minute PCR protocol on the MoPPS versus PCR on the Bio-Rad PCR System, a) amplification curves of the three distinct runs on the MoPPS, their average and the average of three runs on the Bio-Rad PCR System. The graph displays normalized RFU versus PCR cycle number, b) average Ct values of three runs on the MoPPS and Bio-Rad PCR System. The average Ct value on the MoPPS is 31 ± 1 while on the Bio-Rad PCR System it is 21.23 ± 0.68, c) exemplary amplification curves from a single run on the MoPPS and one run on the Bio-Rad PCR System, with normalized RFU plotted against time in minutes, d) total time required for 40 PCR cycles of the PCRs on the MoPPS (15:04 ± 0.03 min) compared to the Bio-Rad PCR System (35:50 min)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7453406/v1/20fb943317176d7eb6e3dd5d.png"},{"id":91336757,"identity":"2c779f05-0f19-44e8-9f5b-7a28dfe7cc35","added_by":"auto","created_at":"2025-09-15 12:09:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":335716,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of intrinsic fluorescence between COP and PC chips after 40 PCR cycles, a) shows the PC chip, while b) displays the COP chip. RT-PCR was performed on both chips, and fluorescence images were captured using the same exposure time\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7453406/v1/f99fa64cc79c4a8417eee12f.png"},{"id":91336754,"identity":"148fd220-7a20-40de-8e9a-d51454cf0be7","added_by":"auto","created_at":"2025-09-15 12:09:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34932,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of COP as chip material, a) average Ct values from four runs on the MoPPS with COP chips and the Bio-Rad PCR System. The average Ct value on the MoPPS is 21.19 ± 0.73, while on the Bio-Rad PCR System it is 19.94 ± 0.29, b) ∆Ct value of different chip materials. The average Ct value of the runs on the MoPPS was compared to the average Ct value of runs on the Bio-Rad PCR System\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7453406/v1/e7ea82070a4c4882047ff56d.png"},{"id":91336758,"identity":"aa265404-32e8-40c5-89b0-dfe8d7b7c95b","added_by":"auto","created_at":"2025-09-15 12:09:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":13286,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of heat-inactivated saliva as a sample material. Human saliva was heat-inactivated for 10 minutes at 95 °C and tested using the fast protocol. The average Ct value was found to be 39.84 ± 2.01 on the MoPPS and 36.38 ± 1.13 on the Bio-Rad PCR System\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7453406/v1/c987f71d21ca23ebeb3b7e96.png"},{"id":91336753,"identity":"416952af-c290-4d74-94b2-0aa438f6551d","added_by":"auto","created_at":"2025-09-15 12:09:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10746,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of heat-inactivated saliva spiked with RNA as sample material. Human saliva was heat-inactivated for 10 minutes at 95 °C, spiked with RNA, and tested using the fast protocol. The average Ct value was found to be 24.41 ± 1.37 using the MoPPS and 25.09 ± 0.10 using the Bio-Rad PCR System\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7453406/v1/a2e22688842b5a8598317f13.png"},{"id":91338008,"identity":"b98c0845-008a-45b8-b097-4a82e69c0ec0","added_by":"auto","created_at":"2025-09-15 12:25:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":43375,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of a Negative control, a) Amplification curves of the average of three runs on the MoPPS, a fitted curve, and the average of three runs on the Bio-Rad PCR System. The graph displays normalized relative fluorescence units (RFU) versus PCR cycle number. b) Average Ct values of three runs on the MoPPS and Bio-Rad PCR System. The average Ct value was 40.19 ± 1.41 on the MoPPS and 38.69 ± 4.00 on the Bio-Rad PCR System\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7453406/v1/c92eb604e60108d1bf4991f5.png"},{"id":92356456,"identity":"2efdff0d-90c5-43c6-85db-54847836c293","added_by":"auto","created_at":"2025-09-28 14:31:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1444191,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7453406/v1/95a328ad-425c-4985-9800-a8c61f1a04ad.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"SARS-CoV-2 detection via high-sensitivity RT-PCR in 15 minutes using microfluidic COP chips","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAs of 2023, the global landscape of COVID-19 has undergone significant changes since the initial outbreak in late 2019[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Widespread vaccination campaigns have been implemented worldwide, leading to a substantial decrease in severe cases and mortality rates. However, the virus continues to evolve, with new variants emerging that pose ongoing challenges to public health systems. Rapid and reliable diagnostic tools that can be used directly at the point of care are critical for the early detection of new infections and an effective response. Reverse transcription polymerase chain reaction (RT-PCR) is currently the gold standard for diagnosing viral diseases[\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] due to its high sensitivity and specificity[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe COVID-19 pandemic has accelerated advancements in diagnostic technology, particularly in Point-of-Care Testing (POCT), which is crucial for controlling outbreaks and mitigating the impact on healthcare systems. During the pandemic, most SARS-CoV-2 tests based on polymerase chain reaction (PCR) methods were conducted in the centralized laboratories, leading to lengthy sample transportation, batch loading and result reporting times. POCT offers advantages such as ease of use, timely detection, and comparable accuracy and sensitivity, essential for daily epidemic control and early treatment[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. While PCR tests are highly sensitive, the average 1\u0026ndash;2 hours test duration, along with sampling and logistical challenges and complex sample preparation processes, has hindered their widespread adoption as point-of-care diagnostics. In contrast, lateral flow immunoassays (LFIA) were used for rapid mass testing, although they are generally less sensitive than PCR tests[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. An ideal POCT solution would combine the speed of LFIA with the sensitivity of PCR for optimal epidemic control.\u003c/p\u003e\u003cp\u003eMicrofluidics-based PCR platforms has led to the development of miniaturized devices capable of nucleic acid amplification with reduced sample volumes and faster processing times[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Advances in microfluidics-based POCT technology have shown potential for diagnosing infectious diseases, including COVID-19. These devices offer rapid detection using low-cost and high-throughput systems, particularly beneficial in developing countries and low-resource settings[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The performance of these diagnostic tests continues to be optimized to prepare for future infectious disease outbreaks[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn current research focused on enhancing PCR technology, various innovative approaches have emerged. For instance, some systems utilize integrated heating mechanisms and rotating components to achieve rapid detection of multiple viruses simultaneously, significantly reducing analysis time[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Others utilize thermoplastic materials with infrared-mediated thermocycling to accelerate nucleic acid amplification, although yields may be lower than with traditional methods [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, infrared light-emitting diodes activate heat-generating nanoparticles for isothermal PCR, and microfluidic platforms miniaturize PCR processes[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This enables rapid, cost-effective diagnostics with improved heat transfer and increased throughput, allowing multiple reactions to be executed simultaneously.\u003c/p\u003e\u003cp\u003eFraunhofer IMM has developed the Moving-Plug PCR System to address the need for rapid and sensitive diagnostic tools. This ultrafast microfluidic module uses oscillatory PCR to complete 30 cycles in six minutes[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], without requiring time-consuming up-and-down thermal cycling. Instead, the reaction sample is repeatedly moved forward and backward between the preheated hot and cool temperature zones. The settings of the MoPPS, such as the number of pump cycles and the temperatures of the heating zones, can be flexibly adjusted to accommodate the specific requirements of the respective PCR kits used. It is a highly compact and portable device, comparable in size to a syringe pump commonly used in laboratories.\u003c/p\u003e\u003cp\u003eThe disposable chip is featuring a simple design and can be fabricated from various thermoplastic materials. Polycarbonate (PC) has been widely used due to its favorable properties, including strength, toughness, and optical transparency. The material's excellent moldability make it a prevalent choice various engineering applications[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cyclic Olefin Polymer (COP), as an alternative chip material, has also been developed specifically to meet high-performance requirements, including a high glass transition temperature, good mechanical properties, high transparency, and excellent film-forming abilities[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It provides strong mechanical performance, and excellent chemical resistance[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor RT- PCR a SARS-Cov-2 PCR kit from a third party is used. The polymerase used in this this kit combines wild-type Taq DNA polymerase with artificially induced reverse transcriptase activity[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This combination removes the requirement for a separate initial reverse transcriptase step, thereby simplifying the entire process by allowing the use of unprocessed patient samples. Consequently, the process time is significantly reduced, and handling procedures are made more efficient.\u003c/p\u003e\u003cp\u003eThis work aimed to establish a rapid and highly sensitive PCR system by validating and optimizing the compatibility of MoPPS with a SARS-CoV-2 PCR kit. This enables faster viral detection than conventional thermocyclers. The initial experiments on PC chips involved using RNA samples and focused on optimizing PCR duration. The use of COP as an alternative chip material to PC was also evaluated using purified RNA, followed by testing with human saliva samples to assess its practical applicability. Through these efforts, the aim of this research is to develop a robust, rapid and highly sensitive PCR system for reliable diagnostics in significantly reduced timeframes, thereby enhancing the accessibility and speed of point-of-care testing (POCT) compared to traditional methods.\u003c/p\u003e"},{"header":"2 Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 DNA amplification procedure on the Bio-Rad PCR System\u003c/h2\u003e\u003cp\u003eThe reactions were performed using the Volcano3G RT-PCR Probe Master Mix and the SARS-CoV2 Singleplex N1-Assay (FAM-Probe) (myPOLS Biotec). As a template, in the beginning 5 \u0026micro;L RNA (5x10\u003csup\u003e7\u003c/sup\u003e copies/ \u0026micro;L) for amplification were used. For testing against the MOPPS the RNA amount was reduced to 1 \u0026micro;l RNA sample. Each 30 \u0026micro;L RT-PCR reaction mix consisted of 15 \u0026micro;L of 2x Volcano3G RT-PCR Probe Master Mix, 3 \u0026micro;L SARS-CoV2 Singleplex N1-Assay, 5/1 \u0026micro;L of RNA and 7/11 \u0026micro;L PCR grade water according to the manufacturer's instructions. qPCR reactions were carried out in a Bio-Rad CFX96 Real-Time PCR Detection System with the following cycling parameters: reverse transcription step at 70\u0026deg;C for 15 min or initial denaturation at 95\u0026deg;C for 120 s, followed by 55 cycles of 5 s of denaturation at 95\u0026deg;C, 5s of annealing at 57\u0026deg;C and 30 s of elongation at 71\u0026deg;C. The used parameters were further shortened to the following: initial denaturation at 95\u0026deg;C for 120 s, followed by 55 cycles of 5 s of denaturation at 95\u0026deg;C and 30s of annealing\u0026thinsp;+\u0026thinsp;elongation at 65\u0026deg;C. The measured fluorescence dye was FAM. The fluorescence at 520 nm was monitored for real-time data collections during elongation. Thermal cycling, fluorescent data collection and data analysis were carried out using the Bio-Rad CFX Maestro 2.3 (Bio-Rad). The System automatically calculates at which PCR cycle the fluorescence exceeded the threshold. This was defined as quantification cycle (Ct).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Setup of the Moving-plug PCR System and manufacture of the microfluidic chips\u003c/h2\u003e\u003cp\u003eThe Moving-plug PCR System was developed with three heating modules, which are located under the three zones of the PCR chip. Two heating modules ensure temperature control of the reaction mixture during amplification, while the third prevents condensation of liquid in the gas reservoir (dead end). The polymer chip was precisely positioned on the heaters with the help of magnets in a black sealing cap. Integrated light barriers facilitate the correct positioning of the stopper in the channel. This allowed the reaction mixture to be oscillated reproducibly at high speed between the two distinct temperature zones. The detection of the liquid by light barriers provided feedback to the customized syringe pump module (more information: [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]). The PCR microchip was manufactured from polycarbonate by injection moulding (microfluidic ChipShop, Jena, Germany) to ensure reproducibility. The design was developed by Brunklaus et al. The COP chips were produced on a milling machine (Charlyrobot S.A.S., Cruseilles, France). Firstly, the chips were cleaned by CO\u003csub\u003e2\u003c/sub\u003e blasting to remove the residues from the milling process. To remove the biological debris, the chips were placed in isopropanol and sonicated for 5 minutes. Next, the channels of the chip were sealed with a thin COP film (188 \u0026micro;m) by solvent bonding. The COP film was covered with the bonding solvent, which consist of one part cyclohexane mixed with one-part decalin. By spin coating the bonding solvent will be spread evenly on the COP film. Next the foil was pressed with 3.5 tons for 3 min on the COP chip. After evaporation of the solvents, the chip can be used. The chip was connected to a syringe pump via a welded-on Luer lock adapter.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 DNA amplification procedure on the Moving-plug PCR System\u003c/h2\u003e\u003cp\u003eFor on-chip amplification, the same reagents as for the tests with Bio-Rad PCR System were used. For initial tests DNA (20.000 copies/ \u0026micro;L) was used instead of RNA. First the 2-step PCR protocol was validated with PC chips on the MoPPS (Table I). The addition of betaine at a final concentration of 1 M was essential for this validation. Subsequently, the PCR protocol was progressively shortened, and RNA at a concentration of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e copies/\u0026micro;L was utilized instead of DNA. The flow velocity within the microfluidic channel for fluid plug movement was chosen between 15\u0026ndash;40 ml/min, respectively, as indicated for the individual experiments. The different PCR protocols and flow rates are listed in Table II. The fastest protocol was also tested with heat inactivated saliva. As template human saliva was heated for 10 minutes at 95\u0026deg;C and spiked with 1 \u0026micro;l RNA (concentration 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e copies/\u0026micro;L).\u003c/p\u003e\u003cp\u003eFor the tests with COP chips, the same reagents (excluding betaine) and the same PCR protocol (Table II, d) as for the tests with PC chips were used. This time, 1x 10\u003csup\u003e6\u003c/sup\u003e copies/ \u0026micro;L of Sars-CoV-2 RNA was used. Four runs on the MoPPS were tested against four runs on the Bio-Rad PCR System. For the tests involving real saliva samples, saliva was taken from the experimenter and heated for 10 minutes at 95\u0026deg;C. This will be used as the sample matrix for further experiments. For the experiments with spiked saliva, 1 \u0026micro;L of RNA with a concentration of 1 \u0026times; 10⁶ copies/\u0026micro;L was used. The experiments to test the negative control were conducted using H₂O as the sample matrix. For all tests the temperatures inside the liquid were adjusted to approx. 95\u0026deg;C in the denaturation region and 66\u0026deg;C in the annealing and elongation region. The evaluation of the PCR data was conducted using a self-designed Python script, which analyses the fluorescence signal of each cycle.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Optimization of the RT-PCR protocol\u003c/h2\u003e\u003cp\u003eThe RT-PCR protocol for the MoPPS was adapted to minimize the PCR run time using the SARSCoV-2 myPOLS kit, which eliminates the need for an additional reverse transcription (RT) step. The polymerase in this kit performs RNA to cDNA transcription during denaturation.\u003c/p\u003e\u003cp\u003eFor confirmation, two standard protocols were tested on the Bio-Rad PCR System: one with the extra RT step and one without (Table I, protocol 1 and 2). Each protocol was tested three times using 1x10\u003csup\u003e7\u003c/sup\u003e copies of SARS-CoV-2 RNA. The results confirmed that the extra RT step could be omitted, yielding average Ct-values of 15.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 with the RT step and 15.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 without it (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, the PCR protocol was adapted to a 2-step protocol, wherein annealing and elongation are combined, to further decrease the runtime. The protocol utilized was provided by myPOLS (Table I, protocol 3) which was subsequently validated on the Bio-Rad PCR System, resulting in an average Ct value of 15.27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Shorten the PCR reaction time to 15 minutes\u003c/h2\u003e\u003cp\u003eThe experiments conducted on the MoPPS aim to assess the possibility of shortening the PCR runtime to be faster than the conventional PCR runtime. The PCR protocol was successfully modified to a 2-step protocol, wherein annealing and elongation are combined. This protocol was initially validated in preliminary experiments on the Bio-Rad PCR System (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Following the incorporation of betaine as a PCR additive, the 2-step protocol (Table II, a) was effectively implemented on the MoPPS using PC chips. Additional testing was carried out to achieve a PCR runtime that is faster than that typically observed in laboratory settings.\u003c/p\u003e\u003cp\u003eVarious parameters were adjusted to optimize the PCR runtime: time of denaturation, time of combined annealing and elongation (AE), and the flow rate (FR). The FR is defined by the pumping velocity between the two individual heating zones. A higher flow rate results in a faster temperature change of the reagents in the chip. The initial denaturation time remained unchanged. Four different PCR protocols were evaluated and compared on the MoPPS (Table II). The PCR runtime decreased progressively with each protocol. The protocol, using 1 sec for denaturation and 5 sec for AE along with an increased FR, was the most efficient, reducing the runtime to 15 minutes.\u003c/p\u003e\u003cp\u003eFollowing the initial successful PCR, the AE time was further reduced, and PCRs were conducted using RNA. The AE step was successfully shortened to 15 seconds (Table II, protocol b), and the FR was increased to 20 ml/min under this protocol (Table II, protocol c). This setup allowed for 40 PCR cycles to be completed in approximately 25 minutes. To further reduce the duration, the denaturation step was shortened to 1 second, and the AE step to 5 seconds, while also increasing the FR (Table II, protocol d). Each protocol indicates a positive PCR signal. Simultaneously, the total time decreased from 38 to 15 minutes, making it 2.5 times faster than the initial used PCR protocol, and the time for a positive signal decreased from 27 to 10 minutes.\u003c/p\u003e\u003cp\u003eThe fastest 15-minute PCR protocol was evaluated three times on both the MoPPS and the Bio-Rad PCR System (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, a-d). The MoPPS demonstrated consistent amplification curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, a). However, the amplification curves obtained from the MoPPS exhibited a significant rightward shift to higher Ct values compared to those from the Bio-Rad PCR System, indicating reduced sensitivity or efficiency in amplification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, b). Specifically, the average Ct value on the MoPPS was 31\u0026thinsp;\u0026plusmn;\u0026thinsp;1, while it was 21.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 on the Bio-Rad PCR System, indicating a loss of sensitivity of approximately 10 Ct values. Direct comparisons are challenging due to the different Ct value thresholds of the individual devices. This variation can lead to discrepancies in the observed Ct values between the two systems.\u003c/p\u003e\u003cp\u003eThe use of the MoPPS significantly reduced the PCR duration time, achieving a runtime of 15:04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 minutes compared to 35:50 minutes on the Bio-Rad PCR System (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, c-d). This highlights the MoPPS's capability to detect SARS-CoV-2 RNA at a speed that is 2.4 times faster than that of the conventional thermocycler.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Evaluation of using COP as chip material\u003c/h2\u003e\u003cp\u003eThe use of PC as a chip material has several drawbacks, including the need to add betaine for RT-PCR. Alternatively, COP serves as a beneficial chip material that can be used to mill chips in-house at our institute. The chips are milled, cleaned, and solvent bonded. Comparative testing on the MoPPS displayed that PC exhibits high intrinsic autofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, a), while COP showed only low intrinsic autofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA significant advantage of using COP as a chip material is the elimination of additives in PCR, as tests demonstrated that RT-PCR can be performed effectively without them, using the same PCR protocol applied to PC chips (Table II, protocol d).\u003c/p\u003e\u003cp\u003eFour runs were conducted on the MoPPS using COP chips. The average Ct value obtained was 21.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73. For comparison, the same protocol run on the Bio-Rad PCR System yielded an average Ct value of 19.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, a).\u003c/p\u003e\u003cp\u003eCOP as a chip material exhibited higher sensitivity on the MoPPS compared to PC. The difference in Ct values between the Bio-Rad PCR System and the MoPPS was approximately 1 Ct with COP chips (21.19 on the MoPPS compared to 19.94 on the Bio-Rad PCR System) and approximately 10 Ct with PC chips (31.00 on the MoPPS compared to 21.23 on the Bio-Rad PCR System, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Evaluation of heat-inactivated Saliva as a PCR template\u003c/h2\u003e\u003cp\u003eTo evaluate the effectiveness of this method using patient samples, human saliva was used as the sample material. After validating the fastest PCR protocol (see Table II, protocol d), the tests were extended to include human saliva. First, the saliva was tested to establish its baseline characteristics. For this, the saliva was heat-inactivated and used as sample in the PCR. The saliva came from an experimenter who had previously had Covid-19. This is evident from the received Ct values. A high Ct value was obtained on both systems: 39.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01 on the MoPPS and 36.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13 on the reference system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter testing the background, saliva RNA was added to the saliva to imitate a real patient sample. The heat-inactivated saliva was spiked with RNA and processed using the 15-minute rapid protocol (Table II, protocol d). Successful RNA detection in the saliva sample was achieved, resulting in an average Ct value of 24.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37 using the MoPPS system and an average Ct value of 25.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 using the Bio-Rad PCR system (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Analysis of a Negative Control\u003c/h2\u003e\u003cp\u003eTo ensure that the results were not influenced by unwanted factors or contaminants, a negative control was tested, which is crucial for validating the reliability of the assay. For this purpose, water was used as sample instead of RNA. Tests were conducted on both the MoPPS and the Reference System. Of the three runs for each system, one resulted in a positive outcome. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea shows the average of three runs on the MoPPS, a fitted curve, and three runs on the Bio-Rad PCR System. To calculate the average Ct value, negative results were assigned a value of 41. High Ct values were obtained for both systems: 40.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 for the MoPPS system and 38.69\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00 for the reference system (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTested PCR protocols on the Bio-Rad PCR System\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTested protocols\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInitial denaturation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDenaturation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAnnealing\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eElongation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCycle number\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1: 3- step PCR with RT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15 min, 70\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5 sec, 57\u0026nbsp;\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e30 sec, 71\u0026nbsp;\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e55 (later reduced to 45)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2: 3-step PCR without RT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10 sec, 57\u0026nbsp;\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e60 sec, 71\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e55 (later reduced to 45)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3: 2-step PCR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e30 sec, 65\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e55 (later reduced to 45)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTested PCR protocols on the MoPPS\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTested protocols\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSample material\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInitial denaturation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDenaturation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAnnealing\u0026thinsp;+\u0026thinsp;Elongation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFlow Rate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTotal PCR time (40 cycles)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30 sec, 65\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15 ml/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e38:00 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15 sec, 66\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15 ml/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e29:00 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15 sec, 66\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20 ml/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e27:00 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 sec, 95\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5 sec, 66\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e30 ml/min and 40 ml/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15:00 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe COVID-19 pandemic has underscored the need for innovative viral detection methods, especially in low-resource settings. POCT offers rapid results for effective disease management, but traditional methods often require significant time and resources.\u003c/p\u003e\u003cp\u003eAdvancements like RT-PCR and isothermal amplification show promise, though rapid antigen tests often sacrifice sensitivity. The ideal POCT system would combine the speed of antigen tests with the sensitivity of nucleic acid amplification.\u003c/p\u003e\u003cp\u003eThe development of lab-on-a-chip solutions, such as the MoPPS, represents a notable development in viral diagnostics. The MoPPS has shown the capability to complete 30 PCR cycles in just 6 minutes[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], facilitating quantitative rapid PCR. Preliminary tests with the SARS-CoV-2 detection kit revealed that the PCR runtime can be further reduced by eliminating the reverse transcription step and using a two-step PCR protocol. The polymerase utilized in this kit is a combination of a thermostable wild-type Taq DNA polymerase with an artificially induced reverse transcriptase activity[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Following the development of this innovative polymerase[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], it was compared to conventional RT-PCR methods [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], yielding promising results that warrant further investigation for viral detection applications[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This polymerase significantly simplifies the reagent process, allowing for the use of unprocessed patient samples. Its efficiency not only reduces processing time but also streamlines handling procedures, thereby minimizing the risk of cross-contamination. Consequently, this polymerase presents itself as a viable alternative in the field of viral diagnostics.\u003c/p\u003e\u003cp\u003ePolymerase-integrated microfluidics can achieve ultra-fast PCR, making them ideal for rapid diagnostic applications. PC is a common thermoplastic used for PCR applications due to its favorable properties. However, the use of PC as a material for microfluidic chips presents several drawbacks. Notably, there is a requirement for betaine as a PCR additive, and PC has relatively high intrinsic autofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The performance of the PCR with PC as chip material can be influenced by various factors, including its permeability to oxygen and moisture[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which can degrade sensitive PCR reagents and affect reaction stability. Additionally, PC's sensitivity to notching can compromise its mechanical properties [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], which can be problematic under the thermal cycling conditions of PCR.\u003c/p\u003e\u003cp\u003eAnother concern is the adsorption of reagents onto the PC chip surface. Numerous studies have documented the application of surface treatments or coatings to PC substrates prior to their utilization in PCR[\u003cspan additionalcitationids=\"CR33 CR34 CR35 CR36 CR37\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. For instance, UV treatment has been employed to enhance wettability and improve fluidic transport[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In the absence of such treatments, reagent adsorption can occur[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Coating the microfluidic channels is a common strategy to prevent nonspecific adsorption of PCR reagents to the channel walls[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Zhang \u003cem\u003eet al.\u003c/em\u003e demonstrated that Taq polymerase can adsorb to the surface, emphasizing the need for surface modifications[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To prevent this, channels are often treated with BSA, glycerol, or other reagents[\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe use of PCR additives, such as betaine, BSA or PEG, is often used across various PCR applications[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The necessity of coating the microfluidic channels aligns with the findings of this study, indicating that an additive to coat the channel walls is essential for achieving positive PCR results when using PC as chip material. Despite these challenges, the MoPPS was able to run 40 PCR cycles and deliver quantitative SARS-CoV-2 results in 15 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which is faster than a commercial thermocycler. The comparison of Ct values reveals that those obtained with PC are significantly higher than those from the commercial thermocycler. However, direct comparison is hindered by the differing threshold calculations used in the two devices.\u003c/p\u003e\u003cp\u003eTo address the limitations associated with PC, comparisons were made with COP as an alternative chip material. COP has high optical clarity, chemical resistance, and biocompatibility[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The high optical clarity is demonstrated in the lower intrinsic autofluorescence, which is advantageous for PCR detection. Tests with RNA on COP chips revealed that no addition of betaine was needed, and the resulting Ct values closely matched those from commercially thermocyclers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The improvement is likely due to the low levels of extractable substances in COP, which reduces the risks of contaminations and reagent adsorption[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Several studies have indicated that COP surfaces exhibit minimal adsorption of reagents or moisture[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], preserving the integrity of PCR reagents[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Additionally, COP does not degrade or compromise assays due to structural weaknesses or chemical contamination[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to evaluating COP, the study examined the effectiveness of using heat-inactivated human saliva as a sample material. Baseline saliva tests indicated a significant RNA load due to a prior SARS-CoV-2 infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Following the spiking of inactivated saliva with RNA, the achieved Ct values were comparable to those obtained from thermocyclers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This demonstrates that RNA in saliva can be detected with similar sensitivity. However, it appears that saliva may have an inhibitory effect, as the Ct values obtained with spiked saliva samples (24.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37 using the MoPPS and 25.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 using the Bio-Rad PCR System) were higher than those obtained with pure RNA (19.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 using the Bio-Rad PCR System and 21.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73 using the MoPPS). This suggests that an additional purification step could effectively mitigate this inhibitory effect and improve the sensitivity of the assay.\u003c/p\u003e\u003cp\u003eFurthermore, evaluating negative controls was crucial in validating the reliability of the assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Using water as sample for negative control tests on both systems demonstrated a low likelihood of contamination, indicating the robustness of the assay. The clear differentiation between negative control results and those from spiked saliva samples further emphasizes the reliability of the PCR system in detecting true positives.\u003c/p\u003e\u003cp\u003eVarious lab-on-a-chip devices exist for PCR applications. For example, Chen \u003cem\u003eet al.\u003c/em\u003e developed the SWM-02 device, which processes six samples but requires a PCR runtime of 40 minutes[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] \u0026mdash;considerably longer than that of the MoPPS. Kwon \u003cem\u003eet al\u003c/em\u003e. tested the GENECHECKER\u0026trade; Ultra-Fast PCR System, achieving influenza results in 23 minutes[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Most commercial products, such as those from Abbott, BioFire, and Cepheid, integrate nucleic acid extraction with RT-PCR in a single automated cassette but rarely achieve results in 15 minutes, with most COVID-19 tests taking 30 to 120 minutes[\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe MoPPS can be developed into a fully autonomous by incorporating an RNA extraction step while maintaining detection times under 30 minutes. Its cost-effectiveness and reduced infrastructure requirements make it suitable for rapid diagnosis in developing countries, enhancing accessibility during public health emergencies.\u003c/p\u003e\u003cp\u003eWhile the MoPPS has demonstrated significant advancements in PCR diagnostics, challenges with PC materials persist. The evaluation and transition to COP represents a crucial improvement in sensitivity and performance. Further enhancements, particularly in automated sample preparation, could elevate the MoPPS's usability, ensuring that rapid and accurate testing remains at the forefront of public health responses. The combination of speed, sensitivity, and cost-effectiveness could position the MoPPS to serve as a strong tool in modern diagnostics.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn summary, the integration of microfluidic PCR technology into point-of-care diagnostics offers substantial advantages for detecting infectious diseases, including COVID-19. Our miniaturized, portable, and ultra-fast PCR device demonstrates the ability to complete 40 PCR cycles and deliver quantitative SARS-CoV-2 results in 15 minutes. Remarkably, this rapid PCR protocol is effective in detecting both purified RNA and RNA that has been added to saliva samples. This highlights its practical applicability in real-world scenarios. The evaluation and transition to COP as chip material significantly enhances sensitivity, aligning the device's performance with that of commercially available PCR thermocyclers and ensuring accurate and reliable diagnostics.\u003c/p\u003e\u003cp\u003eLooking forward, the potential for incorporating freeze-dried PCR reagents directly within the chip presents promising opportunities for rapid and convenient application at the point of care. This advancement would further enhance the device's practicality for widespread use in various settings. Additionally, expanding detection capabilities to include a broader range of viruses and bacteria could enable comprehensive pathogen surveillance.\u003c/p\u003e\u003cp\u003eIntegrating this innovative PCR technology into existing healthcare systems has the potential to greatly improve pandemic management, providing rapid and reliable diagnostics that can effectively curb the spread of infectious diseases. As we continue to refine and enhance this technology, its role in public health could become increasingly helpful.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare they have no financial or competing interests.\u003c/p\u003e\n\u003ch2\u003eEthical Approval\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eDeclaration This study was funded by the Fraunhofer Society (Grant No. 40\u0026ndash;00440)\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eA. was responsible for the conceptualization of the study, the development of the theoretical model, the execution of the experimental measurements, and the contribution to the data analysis. Additionally, A. authored the initial draft of the manuscript, and was responsible for revising and finalizing the manuscript.B. was responsible for revising the manuscript and supervising the project.All authors discussed the results and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe acknowledge the InBaDtec project as a funding source provided by the Fraunhofer Society for supporting this research. We express our gratitude to Dr. Tobias Gerling for his support during the preparation of this manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHuang X, Wei F, Hu L, Wen L and Chen K 2020 \u003cem\u003eArchives of Iranian medicine\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e 268\u0026ndash;71, doi: 10.34172/aim.2020.09\u003c/li\u003e\n\u003cli\u003eLan L, Xu D, Ye G, Xia C, Wang S, Li Y and Xu H 2020 \u003cem\u003eJAMA\u003c/em\u003e \u003cstrong\u003e323\u003c/strong\u003e 1502\u0026ndash;3, doi: 10.1001/jama.2020.2783\u003c/li\u003e\n\u003cli\u003evan Kasteren P B, van der Veer B, van den Brink S, Wijsman L, Jonge J de, van den Brandt A, Molenkamp R, Reusken C B E M and Meijer A 2020 \u003cem\u003eJournal of clinical virology : the official publication of the Pan American Society for Clinical Virology\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e 104412, doi: 10.1016/j.jcv.2020.104412\u003c/li\u003e\n\u003cli\u003eVogels C B F\u003cem\u003e et al\u003c/em\u003e 2020 \u003cem\u003eNature microbiology\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e 1299\u0026ndash;305, doi: 10.1038/s41564-020-0761-6\u003c/li\u003e\n\u003cli\u003eCorman V M\u003cem\u003e et al\u003c/em\u003e 2020 \u003cem\u003eEuro surveillance: bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin\u003c/em\u003e \u003cstrong\u003e25, doi: \u003c/strong\u003e10.2807/1560-7917.ES.2020.25.3.2000045\u003c/li\u003e\n\u003cli\u003eGupta N, Augustine S, Narayan T, O\u0026apos;Riordan A, Das A, Kumar D, Luong J H T and Malhotra B D 2021 \u003cem\u003eBiosensors\u003c/em\u003e \u003cstrong\u003e11, doi: \u003c/strong\u003e10.3390/bios11050141\u003c/li\u003e\n\u003cli\u003eMackay I M, Arden K E and Nitsche A 2002 \u003cem\u003eNucleic acids research\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e 1292\u0026ndash;305, doi: 10.1093/NAR/30.6.1292\u003c/li\u003e\n\u003cli\u003eFilchakova O, Dossym D, Ilyas A, Kuanysheva T, Abdizhamil A and Bukasov R 2022 \u003cem\u003eTalanta\u003c/em\u003e \u003cstrong\u003e244\u003c/strong\u003e 123409, doi: 10.1016/j.talanta.2022.123409\u003c/li\u003e\n\u003cli\u003eSong Q, Sun X, Dai Z, Gao Y, Gong X, Zhou B, Wu J and Wen W 2021 \u003cem\u003eLab on a chip\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e 1634\u0026ndash;60, doi: 10.1039/d0lc01156h\u003c/li\u003e\n\u003cli\u003eHsieh W-Y, Lin C-H, Lin T-C, Lin C-H, Chang H-F, Tsai C-H, Wu H-T and Lin C-S 2021 \u003cem\u003eDiagnostics (Basel, Switzerland)\u003c/em\u003e \u003cstrong\u003e11, doi: \u003c/strong\u003e10.3390/diagnostics11101760\u003c/li\u003e\n\u003cli\u003eLambert-Niclot S, Cuffel A, Le Pape S, Vauloup-Fellous C, Morand-Joubert L, Roque-Afonso A-M, Le Goff J and Delaugerre C 2020 \u003cem\u003eJournal of clinical microbiology\u003c/em\u003e \u003cstrong\u003e58, doi: \u003c/strong\u003e10.1128/JCM.00977-20\u003c/li\u003e\n\u003cli\u003eNicholson B D\u003cem\u003e et al\u003c/em\u003e 2023 \u003cem\u003ePloS one\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e e0288612, doi: 10.1371/journal.pone.0288612\u003c/li\u003e\n\u003cli\u003eLee D, Chen P-J and Lee G-B 2010 \u003cem\u003eBiosensors \u0026amp; 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COVID-19: Interim Clinical Study Results from 1,003 People to Provide the Facts on Clinical Performance and to Support Public Health\u003c/em\u003e \u003c/li\u003e\n\u003cli\u003eCepheid \u003cem\u003eXpert xpress SARS-CoV-2\u003c/em\u003e https://www.cepheid.com/de-CH/tests/respiratory/xpert-xpress-sars-cov-2.html\u003c/li\u003e\n\u003cli\u003eBiofire \u003cem\u003eCOVID-19 test\u003c/em\u003e https://www.biofiredefense.com/covid-19test/\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2, microfluidic device, RT-PCR, diagnosis, COVID-19, lab-on-chip","lastPublishedDoi":"10.21203/rs.3.rs-7453406/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7453406/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Moving-Plug PCR System, developed by Fraunhofer IMM, is a portable device that dramatically accelerates PCR reaction speeds by eliminating the time-consuming heating and cooling cycles traditionally used in thermal cycling. Instead, the reaction sample is moved between preheated hot and cool zones. When combined with a SARS-CoV-2 PCR kit, we successfully conducted 40 PCR cycles and achieved quantitative detection of SARS-CoV-2 RNA in 15 minutes, which is more than 2x faster than using a thermocycler. This innovation demonstrates significant potential for rapid and sensitive diagnostic applications. The polymerase used in our SARS-CoV-2 PCR kit can transcribe RNA into cDNA during the first step of PCR. This omits the need for an additional reverse transcriptase step before PCR, therefore quantitative results could be achieved in 15 minutes. The use of polycarbonate as a chip material required the addition of betaine in the PCR to overcome its limitations, while cyclic olefin copolymer was evaluated as chip material without the addition of betaine. The sensitivity achieved using a cyclic olefin copolymer chip was equivalent to that obtained with a commercially PCR thermocycler. Notably, it was also possible to detect RNA effectively in heat-inactivated saliva samples, which further enhances its applicability in real-world settings. These findings can help overcome the disadvantages of traditional diagnostic methods, such as the long testing times of traditional PCR assays or the lack of sensitivity of Enzyme-linked Immunosorbent Assay. For future infectious disease outbreaks a fast Point-of-Care-PCR Technology with short result reporting times and adequate sensitivity is achieved.\u003c/p\u003e","manuscriptTitle":"SARS-CoV-2 detection via high-sensitivity RT-PCR in 15 minutes using microfluidic COP chips","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 12:08:52","doi":"10.21203/rs.3.rs-7453406/v1","editorialEvents":[{"type":"communityComments","content":1}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c6882e3-5956-45e5-a287-6298adb9f6c5","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-28T14:23:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-15 12:08:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7453406","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7453406","identity":"rs-7453406","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}