Fully automated sample-to-result SIMPLE-RPA microfluidic chip: towards in ovo sexing application

Fully automated sample-to-result SIMPLE-RPA microfluidic chip: towards in ovo sexing application | 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 Fully automated sample-to-result SIMPLE-RPA microfluidic chip: towards in ovo sexing application Simão Monteiro Belo dos Santos, Celine Wegsteen, Dries Vloemans, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5123850/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 Several European countries have implemented new legislations to eliminate the killing of day-old male chicks, pushing the laying hen industry to find animal-friendly solutions. Although embryo sexing during incubation ( in ovo sexing) is highly promising, no current solution meets the industry requirements of handling all egg colors with >98 % sexing accuracy, low cost and minimal embryo disturbance, while allowing sexing before day 13 of incubation and processing > 20 000 eggs/hour. Recombinase polymerase amplification (RPA) presents a promising alternative to PCR as a sensitive isothermal technique to be integrated into a microfluidic platform. In this work we developed a fully autonomous microfluidic cartridge (SIMPLE-RPA chip) for chick female-specific synthetic HINTW gene detection in 30 minutes at 37.7 °C inside an egg incubator. We first optimized off-chip RPA, allowing for highly sensitive DNA detection (1.6 x 10 -5 ng/µL), matching expected concentrations in allantoic fluid samples. Then, the SIMPLE-RPA chip was developed to accommodate and automate the RPA bioassay on-chip, requiring only sample introduction and finger press activation to reduce user errors and contamination risks. Importantly, the SIMPLE-RPA chip maintained the LOD achieved off-chip. The presented SIMPLE-RPA chip is inexpensive, has a small footprint, is compatible with upscaling, and can be easily transferred to other point-of-care applications. Agricultural Engineering Food Science & Technology Biotechnology and Bioengineering SIMPLE RPA in ovo sexing isothermal DNA amplification microfluidic platform lab-on-chip Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The killing of day-old male chicks is a widespread practice in the laying hen poultry industry. Approximately 372 million male chicks in the EU are culled upon hatching, as they neither lay eggs nor yield quality meat 1 . Several governments have banned male chick culling ( e.g. , Germany, France, and Italy) 2 , 3 , endorsing increasing consumer awareness of animal welfare issues and preventing the disadvantages for the industry ( i.e. , investing in a byproduct and image degradation) 4 . Among several solutions offered to date for solving this problem 1 , 5 , sex detection before hatching ( i.e. , in ovo sexing) and subsequent male embryo disposal is the preferred one by both the consumers and industry. With in ovo sexing, the incubation is stopped prior to the onset of pain perception (day 13 of incubation), while the industry avoids investing in males ( i.e. , male eggs can be removed from the incubator and do not require to be grown) 6 , 7 . However, in ovo sexing techniques must meet several requirements for industrialization, including: 1) compatibility with all colors of eggs, 2) high throughput (> 20 000 eggs/hour, complying with the market's high product demand), 3) high accuracy (> 98%), 4) high sensitivity to be applied early in incubation, imperatively before day 13 of incubation, 5) maintaining a high hatchability rate and 6) being low cost (around 2–3 €/day-old-chick) 5 . Both researchers and industry have made significant efforts to develop in ovo sexing methods that can meet these imposed requirements 5 . Current techniques include optical and non-optical methods 5 . Optical techniques employ visible near-infrared spectroscopy to distinguish the embryo's feather colors, however, one day after the embryo’s pain perception onset (day 13 of incubation) 8 . Another available optical technique is magnetic resonance imaging to detect the embryo's gonads on day 12 of incubation 9 , with low accuracy ( 4 €/day-old-chick). Non-optical methods currently include extracting allantoic fluid (AF) from the egg at day 9 of incubation to detect (1) a female-specific hormone (estrone sulfate) with an enzyme-linked immunosorbent assay (ELISA) that offers 98% accuracy but has a high cost (> 4–5 €/day-old-chick) 10 , or (2) a male-specific metabolite 11 with mass spectrometry that has only 95% accuracy 12 . Recently, DNA analysis proved to be the most accurate method, as shown in our recent report using quantitative PCR (qPCR) to target the HINTW female-specific gene 13 . Following this approach, it is possible to detect the embryos’ sex with 100% accuracy from days 6 to 9 of incubation using a small amount of AF (< 5 µL), while having minimal impact on embryo development (seen only when sampling AF on day 6 of incubation) 13 . However, despite this exceptional accuracy, qPCR is a lengthy (90 minutes) and expensive technique (> 3 €/test) that involves temperature cycling, making it unsuitable for non-laboratory settings like an incubator, hence introducing interruption in the eggs’ incubation. In this context, isothermal DNA amplification techniques, such as recombinase polymerase amplification (RPA), can offer advantages for developing a desired test 14 . Compared to other isothermal methods such as loop-mediated isothermal amplification (LAMP) or rolling circle amplification (RCA), RPA requires only two primers, and does not require an initial denaturation temperature. Meanwhile, it can be performed at low incubation temperatures (37–42°C) and is rapid (5–20 minutes) 14 . For instance, previous work showed that RPA can amplify Mycobacterium tuberculosis in urine samples at 37°C, close to the temperature of the egg incubation (37.7°C), offering high sensitivity (1 copy per sample) in less than 20 minutes 15 . Moreover, RPA can be combined with colorimetric detection methods like commercial lateral flow strips (LFSs) - which are not mainstream with PCR – thus avoiding the need for additional devices to carry out fluorescence read-out and instead generating results in an easy, practical, and rapid manner 16 . Because of all these advantages, RPA has been incorporated into hand-held microfluidic platforms aiming to deliver almost “one-pot” reactions. In Liu et al. 2021, a poly(methyl methacrylate) (PMMA) device containing an LFS was built to detect SARS-CoV-2 RNA sequences with a limit of detection (LOD) of 1 copy per µL. Other researchers developed a paper origami device to detect Salmonella enterica in food samples 17 in which the RPA amplification was performed in stages controlled by the user’s consecutive folding, resulting in an LOD of 6, 9 and 58 CFU/mL for lettuce, chicken breast and milk, respectively 17 . Despite the proven potential to perform on-chip RPA with high sensitivity, the existing devices require user intervention ( e.g. , inserting the correct amount of sample in the inlet, manipulating or folding the device), which makes these systems user-dependent and more prone to errors. Examples of sample-in-result-out types of microfluidic platforms that require less user manipulation have been reported, including centrifugal microfluidics 18 or pump-driven devices 19 that allow for complex liquid manipulations ( e.g. , sample mixing, RNA or DNA extraction). However, they rely on external devices (centrifuge or pumps), making them unsuitable for the point-of-care (POC). Hence, laying hen farms are still looking for new technologies that can provide fast, highly accurate but low-cost in ovo sexing tests with minimal to no disturbance of egg incubation. In this work, we present a microfluidic-based tool, named SIMPLE-RPA chip, to perform fully autonomous RPA for detecting the synthetic female-specific HINTW gene within an incubator at 37.7°C. To achieve this, we first establish an off-chip RPA bioassay for detecting synthetic DNA. Furthermore, we develop a SIMPLE-RPA chip, starting from our in-house developed SIMPLE (self-powered imbibing microfluidic pump by liquid encapsulation) technology. The SIMPLE is an obvious choice for the in ovo sexing application because it is a self-powered, cost-effective (< 1€/chip ) microfluidic technology that can perform complex liquid manipulations autonomously upon a single finger press activation step, while remaining small in size 20 – 22 (which is crucial for the limited space in egg incubators). Finally, we implement for the first time an RPA bioassay on the SIMPLE-RPA chip with an integrated LFS for qualitative colorimetric identification of the synthetic female-specific HINTW gene on the spot without the need for external read-out devices. Due to its low cost, complete automation and user-friendliness, the presented SIMPLE-RPA chip shows great prospect for final integration with AF sampling directly from eggs in the incubator and consequently in ovo sexing on genomic DNA, as well as for applications in other fields, such as medicine, veterinary sciences and agriculture. Materials and Methods Materials All sequences of primers and synthetic DNA used in this work (Table 1) were produced by IDT (Leuven, Belgium), delivered in IDTE buffer (pH = 7) and were used without further processing. All the dilutions were done using distilled water. RPA liquid basic kit and HybriDetect – Universal Lateral Flow Assay Kit strips were purchased from TwistDx Limited (Maidenhead, UK). PMMA with 1 mm thickness was purchased from Pyrasied (Leeuwarden, The Netherlands). Polyethylene terephthalate (PET) films of 125 μm thickness were bought at Pütz GmbH (Taunusstein, Germany). Double-sided pressure sensitive adhesive (PSA) tape (200 MP 7956MP) and transfer tape (467MP) with thicknesses of 153 and 50 μm, respectively, were acquired from 3M (Minnesota, USA). Microfluidic dyes were obtained from Darwin Microfluidics (Paris, France). Hydrophobic solution Fluoropel 800 was purchased at Cytonix (Maryland, USA). Whatman quantitative filter paper of grade 3 and Mowiol 8-88 (polyvinylalcohol; PVA) were obtained from Sigma-Aldrich (Overijse, Belgium). Versapor™ acrylic copolymer 3000 membrane was purchased from Cytiva (DC, USA). Table 1 – Primers and synthetic DNA sequences used in this work. HINTW synthetic DNA was used as double-stranded DNA (dsDNA) Primers and synthetic DNA Starting from the primers used in our previous work for developing the qPCR 13 , the primers in this work were modified to meet the RPA primer design requirements (≈ 30 base pairs, TwistDx), albeit maintaining the same target gene. Primers were selected using the NCBI BLAST engine to detect HINTW (GenBank accession number: NC_052571), a specific gene in the W chromosome of laying hen chickens ( Gallus Gallus ). IDT Oligoanalyzer 3.1 (IDT) was further used to verify the primers' annealing temperature, stability, and self-complementary. The forward primer was labeled at the 5' end with biotin, while the reverse primer was labeled with fluorescein (FAM) at the 5' end for the detection with the HybriDetect LFSs. The synthetic DNA was purchased as a dsDNA (geneBlock) in IDTE buffer (pH = 7). The sequences used are presented in Table 1. Off-chip optimization of the RPA bioassay The RPA bioassay was first optimized off-chip by testing different primer concentrations and amplification times (see further for more details). The RPA bioassay was performed based on the manual instructions provided by TwistDx 23 . TwistAmp TM liquid master mix was prepared by mixing 25 μL of 2x Reaction buffer, 2.25 μL dNTPs (40 mM), 5 μL 10x Basic E-mix, 2.4 μL of forward and reverse primer in different concentrations (0.5, 2.5, 5 and 10 μM), 7 μL of deionized (DI)-water and 2.5 μL of 20x Core Reaction Mix. This tube was briefly centrifuged and vortexed to achieve complete mixing of the reagents before dividing the solution into different PCR tubes with 9 μL of the prepared master mix. Before closing the tubes, 1 μL of the sample (0.01 ng/µL synthetic DNA) or DI water for non-template control (NTC) and 0.5 µL of magnesium acetate (MgOAc; 280 mM) were added to the tube lids. Once the tubes were closed, they were inverted 4 times and spun down for 5 seconds using a tabletop centrifuge. The tubes were then incubated in a Biometra TProfessional basic thermocycler (Analytik Jena GmbH, Jena, Germany) for a specific time (5, 10, 15 or 20 minutes) at 37.7 °C (mimicking egg incubation temperature). After the incubation time, the thermocycler temperature was decreased to 4 °C to stop the reaction. Next, the RPA amplification product from the PCR tubes (10 μL) was mixed with 40 μL of LFS buffer in an Eppendorf tube. The LFSs (HybriDetect – Universal Lateral Flow Assay Kit, Figure S1 from Supplementary Material) with two lines for colorimetric detection ( i.e., test and control) were immersed into this solution, and the colorimetric results were visualized 10 minutes later. The test line included a biotin-specific antibody, whereas the control line comprised antibodies specific to the reporter probe ( i.e., against FAM-specific antibodies immobilized on gold nanoparticles (AuNPs)). The RPA amplified product was drawn over the LFS, where reporter probes were lyophilized in the sample pad, thus binding the FAM label. The working method of the used LFS is further explained in Figure S1 from Supplementary Material. Images of the LFSs were captured using a fi-65F Fujitsu (Tokyo, Japan) flat desk scanner at 10 minutes after running the test. Band intensities were evaluated using ImageJ software, where the color image was transformed into a black-and-white 8-bit channel image. A rectangular area was drawn around the test line, and the average histogram intensity ( AvgTestHist in Equation 1) was calculated and normalized ( NormInt ) by subtracting it from 255 (the maximum intensity value for a pixel in an 8-bit image) and then dividing by 255 (Equation 1). The signal-to-noise ratio (SNR) was calculated according to Equation 2 using the mean intensity for each condition, where NormInt Noise and NormInt Sample are the average test line intensity value of the NTC and positive tests, respectively. Starting from the same post-amplified RPA samples evaluated with the LFSs, we simultaneously performed electrophoresis analysis with an Agilent 4150 TapeStation system (Agilent Technologies, Inc, USA). All the post-amplified RPA samples handling was performed in ice to avoid the reaction to continue. 1 µL of post-amplified RPA sample or 1 µL of ladder were mixed in PCR tubes with 10 µL of sample buffer. The PCR tubes were vortexed for 1 minute, spun at 2000 rpm for 1 minute and placed in the TapeStation 4200 device. Data analysis was conducted using the TapeStation 4200 Analysis Software. Determining the sensitivity of the off-chip RPA bioassay Once primer concentration and incubation time of the RPA were optimized, HINTW synthetic DNA samples were diluted in 1:5 series, resulting in concentrations from 0.05 and 1.6 × 10 ‑5 ng/μL. The RPA on the dilution series was tested with two different heat sources: 1) the thermocycler like in the previous section and 2) the Rcom Max 50 DO incubator (Autolex Co., South Korea) used for the eggs’ incubation, which provided information about the amplification efficiency in the commercial egg incubator. After incubation at 37.7 °C the reaction was stopped by decreasing the thermocycler temperature to 4 °C whereas the tubes from the egg incubator were transferred to ice for further manipulation. The amplification product (10 μL) was mixed with 40 μL of LFS buffer in an Eppendorf tube. Subsequently, the LFSs were immersed into the tubes, and the colorimetric results were visualized 10 minutes later. Band intensity analysis was performed as described above. Fabrication of the SIMPLE-RPA chip The SIMPLE-RPA chip was fabricated using our previously established low-cost and rapid prototyping method 21,24 . More specifically, the microfluidic network (Figure S2 from Supplementary Material) was designed using Inkscape vectorial software (Version 1.3.2, Software Freedom Conservancy, USA) and fabricated using a manual layer-by-layer lamination method. PSA, transfer tape, PET, PMMA layers, liquid barriers and Whatman Grade 3 paper were cut using an 80-watt Speedy300 Trotec laser cutter (Trotec Lasers, Austria). Figure 1 shows different layers (A) together with SIMPLE-RPA chip workflow (B and C). The SIMPLE-RPA chip was divided into three vertically stacked units: the pumping unit, the sample processing unit, and the detection unit, which were linked with air connection holes and liquid connection holes, shown with the dashed and full arrows, respectively (Figure 1A). The pumping unit comprised 2 layers, each made of 2 stacked PSA layers sandwiched between 2 PET layers. The top layer of this unit (IV in Figure 1A) contained the porous material (Whatman Grade 3), inserted into its chamber during assembly and secured with transfer tape. The bottom layer (V in Figure 1A) comprised the microfluidic channels containing the working liquid, its prefilling hole and the activation site. The sample processing unit comprised 2 layers of single PSA sandwiched between 2 PET layers. The top layer (II in Figure 1A) comprised the microfluidic channels for sample processing, RPA and LFS reagents prefilling holes, liquid connection to the detection unit and air connection to the pumping unit. Two types of hydrophobic valves were created: hydrophobic burst valves (HBVs) and liquid barriers. HBVs were made in the sample processing unit top layer (II in Figure 1A) following a mask-based strategy, based on a previous report 20 . In short, both top and bottom or only bottom PET films were locally coated with 2 µL of Fluoropel 800 to create double-coated (dc) or single-coated (sc) HBVs, respectively. After, the PET films were allowed to dry for 1 hour at room temperature and masks were removed. The bottom layer of the sample processing unit (III in Figure 1A) holds the hydrophobic porous Versapor membranes ( i.e. , liquid membranes), allowing air passage but preventing liquid flow due to their high burst-pressure properties (> 0.7 bar) and working as switches for the liquid manipulations, as explained in detail in Figure 1B and C. Moreover, only the 3D mixing chambers span two layers ( i.e. , going through the first and second layers of the sample processing unit; Figure 1A). In these structures, the inlet and outlet of the chambers were located in different layers, i.e. , inlet from the top layer and outlet from the bottom layer or vice versa. More information on these structures can be found in a previous report 20 . The sample mixed with the RPA reagents proceeds through the mixing chambers and a series of meandering channels allowing for incubation and amplification of amplicons in the sample. The detection unit was designed to hold the LFS ( i.e. , a HybriDetect - Universal Lateral Flow Assay) by sandwiching a PSA layer between a 1 mm PMMA and PET layer. The PMMA layer contained a connection hole to the sample processing unit, while the pumping unit was connected through the LFS space. After the assembly of the microfluidic chip, 0.5 µL of MgOAc (280 mM) was loaded into the first mixing chamber and sealed with PSA. Next, the working liquid and reagents were prefilled through their respective prefilling holes, bringing the working liquid close to, but not in contact with, the porous material, as previously described 25 . The working liquid comprised a blue microfluidic dye diluted in distilled water (1:20) mixed with 4 % (w/v) PVA. The chips were used immediately after prefilling. When not prefilled, they were stored overnight at 4 °C in a closed bag with desiccant. On-chip RPA integration for synthetic HINTW DNA detection The SIMPLE-RPA chip performance was assessed using the off-chip optimized RPA conditions. The RPA master mix was prepared as explained in the section on off-chip optimization of the RPA bioassay, and 9 µL was subsequently introduced into the SIMPLE-RPA chip through its prefilling hole (Figure 1A). MgOAc (0.5 µL; 280 mM) was pipetted into the first 3D chamber and LFS buffer (40 µL) was loaded into the chip. All the reagents were inserted into the microfluidic channels using a micropipette. All the prefilling holes were sealed using a PSA membrane. A video showing the prefilling of all the liquids is shown in Supplementary material S7. The Rcom Max 50 DO incubator (Autolex Co) was set at 37.7 °C and 55 % humidity for incubation. The SIMPLE-RPA chip was placed inside the incubator and allowed to reach the set temperature. The synthetic HINTW DNA samples were prepared with a 1:5 dilution series between 0.05 and 1.6 x 10 -5 ng/μL. For testing the SIMPLE-RPA chip, 2 µL of HINTW synthetic DNA sample in IDTE buffer or 2 µL of water for NTC was added to the inlet with a micropipette and the SIMPLE-RPA chip was activated with a single finger press. The testing was done in triplicate. The autonomous SIMPLE-RPA chip operation happened inside the incubator without any disturbance or user intervention followed by its removal from the incubator only when all amplified RPA product with LFS buffer were absorbed by the LFS. The LFS was scanned using the fi-65F Fujitsu flat desk scanner and its band intensities were analyzed as previously described. Data analysis GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses and data visualization. One-way ANOVA (α = 0.05) was performed, followed by Tukey's multiple comparison tests. Results and Discussion Off-chip optimization of the RPA bioassay The RPA bioassay was first optimized off-chip using 4 distinct primer concentrations (0.5, 2.5, 5 and 10 µM), while keeping incubation time at 20 minutes (according to the manufacturer’s protocols 23 ). The selection of primer concentrations was based on a previous publication by Lobato et al. 2018 with suggested primer concentrations between 0.8 and 5 μM 14 . The primer sequences for this work were taken from our recent paper 13 , where they have been validated for high sensitivity and specificity towards HINTW synthetic DNA and genomic DNA (gDNA) sampled from AF at days 6 to 9 of incubation. Using the most efficient primer concentration from this first optimization step, we also tested 4 different incubation times (5, 10, 15, and 20 minutes), to evaluate when the amplification plateau was reached and thus to avoid incubating the samples for too long, risking primer dimer formation and non-specific amplification (as advised by the manufacturer’s manual 26 ). The incubation temperature of 37.7 °C was selected to match the incubation temperature of an egg incubator. Synthetic DNA in IDTE buffer, matching the targeted sequence from the HINTW gene, was used at 0.01 ng/μL, which corresponded to the lowest detected concentration achieved with qPCR detection of gDNA extracted from AF in our previous work 13 . NTC were added for each variation of primer concentration or incubation time, whereas all conditions were tested in triplicates. The results were visualized with LFSs (Figures 2 and 3) and electrophoresis using a TapeStation device (Figure S3 from Supplementary Material). Figure 2A shows examples of the obtained LFSs for different tested primer concentrations and Figure 2B shows the normalized intensity of the respective LFS test bands, analyzed using ImageJ. From all the tested concentrations, 0.5 μM was the only one that did not show a statistically significant difference compared to its NTC, which was also confirmed visually by the absence of a test line. Although the other three tested primer concentrations all gave statistically significant differences compared to the NTC (Tukey’s test for multiple comparisons, α = 0.05), the best SNR was obtained when using 2.5 µM (0.92), leading to the selection of this concentration as the most suitable to continue with. It is important to note that the visible NTC test lines when using 10 and 5 µM of primers (Figure 2A) possibly resulted from primer dimers and non-specific amplification. Starting from a selected primer concentration of 2.5 μM, we then tested different incubation times, with examples of the obtained LFSs in Figure 3A and the normalized intensity of the respective LFS test bands, analyzed using ImageJ in Figure 3B. For all tested incubation times, the NTCs showed minimal to no amplification. When compared to the positive samples, statistically significant differences were observed for all incubation times, except for 5 minutes (Figure 3B). Although the remaining incubation times (10, 15 and 20 minutes) did not reveal significant statistical differences among themselves, they revealed slightly different SNR values. Therefore, we selected 15 minutes as a condition with the best SNR of 0.98 for the remaining benchtop and on-chip experiments, since it enables a rapid bioassay, without hindering the sensitivity. The results presented in Figures 2 and 3 are further confirmed with obtained electrophoresis results in Figure S3 from Supplementary Material. Determining the sensitivity of the off-chip RPA bioassay The optimized RPA bioassay for HINTW synthetic DNA detection (with 2.5 µM of forward and reverse primers and 15 minutes of incubation) was subsequently tested with a serial dilution of the synthetic DNA in buffer (1:5 dilution ratio between 0.05 and 1.6 x 10 ‑5 ng/µL) and incubation at 37.7 °C using a PCR thermocycler (Figure 4A) or an egg incubator (Figure 4B) as the heat source (Figure S4 from Supplementary Material shows examples of the LFSs obtained from this experiment). For both heating conditions, amplification was achieved for all concentrations of the synthetic DNA between 0.05 and 4 x 10 ‑4 ng/μL with no statistical differences (Figure 4A and B; Tukey’s test for multiple comparisons, α = 0.05). Furthermore, 8 x 10 -5 (Figure 4A) and 1.6 x 10 -5 (Figure 4B) ng/μL were the two lowest concentrations that resulted in a significantly higher intensity compared to the NTC signal. Although we cannot explain the differences seen in the LOD when using the PCR thermocycler or the egg incubator, the fact that we obtained a lower LOD in the egg incubator is extremely promising due to the intended final application of the SIMPLE-RPA chip. Moreover, our previous work demonstrated that the acquired LOD of 1.6 x 10 -5 ng/μL is sufficient for detecting the HINTW gene in real extracted gDNA 13 Development of the SIMPLE-RPA chip To implement the RPA bioassay for the first time onto the SIMPLE microfluidic chip, we designed a completely novel SIMPLE-RPA chip as shown in Figure 1, in which the different liquid operations are demonstrated using colored liquids (depicted in Figure 5 with supporting Video Supplementary Material in Section S8). To tackle the needs of the laying hen poultry industry, the chip was designed with a (1) reduced size ( i.e., 13x7 cm) and footprint complying with the limited space inside the egg incubators, (2) fully autonomous sample-to-result operation initiated with a single finger press (Figure 5-1), and hence a possibility for direct implementation within the egg incubator with low chick development disturbances, (3) low sample volume requirements (2 µL), being compatible with a limited volume of the AF sample available at the early stages of chick development, (4) low-cost materials (< 1 €/chip) compatible with scalable production methods ( i.e. , roll-to-roll manufacturing), to meet the high-volume testing requirements in this industrial sector, and (5) colorimetric read-out that facilitates sex detection and sorting, enabling high-throughput in ovo sexing. The cartridge is activated by a finger press, hereby, bringing the working liquid in contact with the porous filter, leading to its absorption through capillary action. Once the SIMPLE-RPA chip is activated, 1 µL of the sample is first accurately metered (Figure 5-2), which precludes errors from non-specialized users when introducing the sample. In particular, the coordinated burst action of sc and dcHBVs (purple and red squares in Figure 1, respectively) is used to first meter an exact sample volume in the sample channel, after which the sample excess is discarded to the waste channel. Once the sample excess reaches the liquid barrier at the end of the waste channel (V1 in Figure 5-2), the precisely metered sample volume first blocks the liquid barrier adjacent to that channel (V2) and bursts through de dcHBV (Figure 5-3), merging with the prefilled RPA reagents. The merged liquids proceed towards the first 3D mixing chamber (for more information on the measuring and merging systems, we refer to our previous work 20 ). Next, the reagents and sample go through a series of 3D chambers (Figures 5-4 and 5-5), allowing for efficient mixing due to the expansion effect 27 . In the first 3D chamber, the 9 µL of RPA reagents and 1 µL of sample are merged with the 0.5 µL MgOAc (280 mM), thereby initiating the RPA reaction. Adding the MgOAc only after the sample is introduced allows for accurately controlling the start of the RPA reaction, avoiding false positives or unwanted amplification. Moreover, MgOAc integration in the first 3D mixing chamber of the chip enables simple storage and addition, contrary to what was observed in RPA implementation in other platforms 17 , where this step required extra handling, thereby increasing the complexity of their devices. Furthermore, incubation and mixing happen simultaneously, which has been proven to increase the sensitivity in previous reports 14,28 . The four 3D chambers and the presented length of meandering channels, used for the incubation of the mixture, were adequate to reach 15 minutes of incubation with the SIMPLE-RPA chip and to have a fully mixed solution with a homogenous color at the fourth 3D mixing chamber, as shown in Figure 5-6. Next, we optimized the time the liquids reside in the channels to match the necessary 15 minutes of incubation. To achieve this, one option was to increase the number of 3D mixing chambers or length of the meandering channels, which, however, increased the amount of sample loss, retained by the hydrophilic PET surface (data not shown), thus hindering the RPA reaction. Hence, to avoid altering the already optimized chip design, we introduced here a completely novel way of controlling the on-chip incubation time when using the SIMPLE microfluidic chip. By increasing the working liquid’s viscosity with the addition of PVA, flow rates could be altered, which was very practical since the SIMPLE-RPA chip design could be kept constant throughout the experiments. The Lucas-Washburn equation 29 shows the inverse proportionality between the wetted distance of a porous media over time and the liquid viscosity (Supplementary Material Section S9). After testing increasing concentrations of PVA in the working liquid (Figure S6 from Supplementary Material shows different timings considering different amounts of PVA), we observed that 4 % (w/v) of PVA enabled an incubation time of approximately 15 minutes to match the requirements of the optimized RPA (the obtained timing can be seen in Video S8 from Supplementary Material). Following the incubation in the 3D mixing chambers and meandering channels, the amplified DNA reached and stopped at a scHBV (which prevented the loaded LFS buffer from flowing in the microfluidic channels during prefilling). At the same time, the amplified DNA blocked the liquid barrier adjacent to the channel (Figure 1B and Figure 5-7). Once the last liquid barrier was blocked, the amplified DNA bridged the scHBV to merge with the 40 µL of LFS buffer (Figure 5‑7), after which it was drawn over the dummy LFS inserted within the SIMPLE-RPA chip (Figure 5-8). Once the LFS buffer mixed with the RPA product was absorbed into the LFS sample pad, the SIMPLE pump did not exert any more pulling force on the liquid (as airflow can pass by above the liquid). The presented SIMPLE-RPA chip allows for fully autonomous on-chip implementation of the RPA assay without user dependency, which drastically reduces the risk of user errors, in less than 30 minutes. To perform all the steps in a reproducible manner, all the microfluidic structures had to be properly sealed, which was one of the biggest challenges. Moreover, because of the manual fabrication, slight differences ( e.g. , differences in metered samples) were sometimes observed, which, however, did not drastically influence the results ( e.g. , bioassay time or intensity of the bands) and can be further prevented by automating the chip fabrication process. Importantly, the chips provide a decreased risk of contamination from the environment and cross-contamination since the closed system reduces the chance of aerosol formation. Finally, even though the combination between the liquid barriers and dc/scHBVs allowed for complex liquid manipulations, introducing further complexity (such as other microfluidic components or multiplexing) within the platform would allow for an even greater degree of autonomy, thus bringing huge advantages to the field of autonomous POC devices 30 . On-chip RPA integration for synthetic HINTW DNA detection To test the SIMPLE-RPA chip performance, the previously optimized off-chip bioassay was implemented on the chip. The RPA master mix was prepared off-chip, from which 9 µL was loaded into each chip (matching the off-chip reaction volume), while 40 µL of the LFS buffer was directly inserted in the first 3D mixing chamber from the detection unit and MgOAc (0.5 µL, 280 mM) was inserted in the first 3D mixing chamber from the sample processing unit (Figure 1). Once the reagents were loaded and the SIMPLE-RPA chip was properly sealed, the same serial dilution (as off-chip) of the HINTW synthetic DNA target in IDTE buffer was tested with the on-chip RPA in triplicate. NTC was also included by replacing the HINTW synthetic DNA with 2 µL DI water. Because our data showed that using an egg incubator as a heating source resulted in equal if not better results compared to the PCR thermocycler (Figure 4), the chips were left inside the egg incubator until the formation of the control line and the absence of liquid in the chip. Band readout was done by the naked eye and subsequently, the chips were removed from the incubator to complement visual read-out with the tabletop scanner for intensity of test band quantification (Figure 6), as described earlier. Figure 6A shows images of the SIMPLE-RPA chips after the finished bioassay, revealing a relevant test band for concentrations ranging from 0.05 to 8 x 10 -5 ng/µL with no statistically significant differences among each other (Figure 6B; Tukey’s test for multiple comparisons, α = 0.05). However, a concentration of 1.6 x 10 -5 ng/µL presented a dimmer band similar to the NTC, further supported by the lack of statistically significant difference between them, shown in Figure 6B. Hence, 8 x 10 -5 ng/µL was considered the LOD of the SIMPLE-RPA chip, which was the same for the off-chip RPA when using PCR thermocycler (Figure 4A) but was slightly lower when using the egg incubator (Figure 4B) as a heating source. In this context, it is relevant to note that PET transparency allows for non-obstructed visualization of the band, which is easily identifiable by a human user as well as by a camera. This was the first time a complete SIMPLE‑RPA chip was presented with a sample-in-result-out workflow, which was achieved by using HINTW synthetic DNA in IDTE buffer. By further optimizing the RPA bioassay for detection of HINTW gene directly in the AF sample, this technology can be used for male embryo detection in laying hen hatcheries . As it was proven in our recent work 13 , HINTW in the gDNA extracted from AF of embryos incubated between days 6 and 9 allowed distinguishing the sexes with 100 % accuracy. Conclusion Currently, in ovo sexing is the preferred solution from consumers and industry to solve male chick culling. However, for in ovo sexing technologies to be applied in the industry, strict requirements must be followed (such as compatibility with all colors of eggs, high throughput, high accuracy, high sensitivity, maintaining a high hatchability rate and low cost 5 ), which are currently unmet. To meet these requirements, we developed a microfluidic chip integrated with an RPA bioassay, namely the SIMPLE-RPA chip that offers great prospect to be used for in ovo sexing. This innovative chip autonomously amplifies the female-specific HINTW synthetic DNA within the egg incubator, possibly avoiding to impact the embryos’ development and hatchability due to a reduced incubation disturbance. The RPA bioassay was first optimized off-chip regarding primer concentration and incubation time, achieving a high sensitivity that matched the necessary LOD for detecting HINTW in real samples. The off-chip optimized bioassay was subsequently integrated with the SIMPLE chip without an effect on the LOD, achieving the result in only 30 minutes, while using only the heating from the egg incubators to run RPA at 37.7°C. Because of its autonomous operation, almost no handling from the user is required, thus decreasing contamination risk. Importantly, to control the RPA incubation time of the SIMPLE-RPA chip, the working liquid viscosity properties were manipulated by adding PVA, which proved to be a practical and robust approach in this context. The presented SIMPLE-RPA chip has a small footprint, is built with low-cost materials, and can be produced in large numbers in an automated fashion. Moreover, the choice of RPA as an amplification method offers high sensitivity and specificity for target detection, while also allowing for an integrated colorimetric read-out with an LFS, thus enabling easy results interpretation by the naked eye, which is more practical than fluorescence-based alternatives. RPA also provides the possibility of lyophilization 17 for improved shelf life. As such, this kind of technology can be easily integrated into the hatcheries, as seen in Respeggt, GmbH 31 or In Ovo, B.V. 12 , where automated lines for sampling and testing AF samples were implemented. However, for a fully integrated solution, further advancements are expected, such as sample extraction and processing, as well as touchless activation outside the incubators. In addition, important improvements are expected in providing LFSs and RPA assay components by commercial suppliers at more competitive prices or through in-house production. Although the presented SIMPLE-RPA chip has been developed for in ovo sexing application, by simply changing the primers in the RPA reaction, other sequences or pathogens can be easily detected, such as sexually transmitted ( e.g. , HIV or HPV), respiratory diseases ( e.g. , SARS-Cov 2 or Influenza ), or even food pathogens ( e.g. , E. Coli or S. Enterica ). Moreover, this platform shows high potential to be applied in the various POC settings by untrained users with distinct heat sources ranging from egg incubators, like in this work, to even hot plates or heating pads. Declarations Acknowledgements This work was funded by the Flemish environment department, the Research Foundation – Flanders [1S54823N and S003923N], Flanders Innovation & Entrepreneurship – VLAIO (HBC.2022.0710) and the European Union (FORTIFIEDx and DECIPHER). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the granting authority European Union's Horizon Europe research and innovation programme. Neither the European Union nor the granting authority can be held responsible for them. The FORTIFIEDx and DECIPHER projects ha received funding under the Horizon Europe research and innovation programme (grant agreement No 101092049 and 101137242, respectively). Author Contributions S. M. B. D. S. – Conceptualization, investigation, methodology, funding acquisition, formal analysis, writing original draft; C. W. – Investigation, methodology, reviewing and editing; M. C. – Conceptualization, funding acquisition, reviewing and editing; B. D. K. - Conceptualization, funding acquisition, reviewing and editing; D. V. – Conceptualization, reviewing and editing; D. S. – Conceptualization, funding acquisition, reviewing and editing; J. L. – Conceptualization, funding acquisition, reviewing and editing. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at References Bruijnis, M. R. N., Blok, V., Stassen, E. N. & Gremmen, H. G. J. Moral “Lock-In” in Responsible Innovation: The Ethical and Social Aspects of Killing Day-Old Chicks and Its Alternatives. J. Agric. Environ. Ethics 28 , 939–960 (2015). Deutscher Bundestag. Entwurf eines gesetzes zur änderung des tierschutzgesetzes – verbot des kükentötens. https://www.bmel.de/SharedDocs/Downloads/DE/Glaeserne-Gesetze/Kabinettfassung/aenderung-tierschutzgesetz-kueken.html (2021). République Française. Journal officiel - lois et décerets. https://www.legifrance.gouv.fr/jorf/jo/2022/02/06/0031 (2022). Reithmayer, C., Mußhoff, O. & Danne, M. Alternatives to culling male chicks – the consumer perspective. Br. Food J. 122 , 753–765 (2020). Corion, M. et al. 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How to prevent chick culling in the poultry industry ? Discovery of a new biomarker for in ovo gender screening. BioRxiv 31 , 1–17 (2023). In Ovo. In Ovo. https://inovo.nl/ (2023). Monteiro Belo Santos, S., Corion, M., De Ketelaere, B., Lammertyn, J. & Spasic, D. Allantoic Fluid-Based qPCR for Early Onset In Ovo Sexing. J. Agric. Food Chem. (2023) doi:10.1021/acs.jafc.3c09418. Lobato, I. M. & O’Sullivan, C. K. Recombinase polymerase amplification: Basics, applications and recent advances. Trends Anal. Chem. 19–35 (2018). Law, I. L. G. et al. Automated real-time detection of drug-resistant Mycobacterium tuberculosis on a lab-on-a-disc by Recombinase Polymerase Amplification. Anal. Biochem. 544 , 98–107 (2018). Liu, D. et al. A microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay for rapid COVID-19 detection. Lab Chip 21 , 2019–2026 (2021). Chen, Y., Hu, Y. & Lu, X. Polyethersulfone-Based Microfluidic Device Integrated with DNA Extraction on Paper and Recombinase Polymerase Amplification for the Detection of Salmonella enterica. ACS Sensors 8 , 2331–2339 (2023). Choi, G. et al. A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria. Lab Chip 16 , 2309–2316 (2016). Nguyen, V. D., Nguyen, H. Q., Bui, H. K., Kang, Y. J. & Seo, T. S. A smartphone-controllable molecular diagnostic platform for SARS-CoV-2 detection by reverse-transcription recombinase polymerase amplification. Sensors Actuators B Chem. 398 , 134728 (2024). Vloemans, D. et al. Precise sample metering method by coordinated burst action of hydrophobic burst valves applied to dried blood spot collection. Lab Chip 21 , 4445–4454 (2021). Dal Dosso, F., Bondarenko, Y., Kokalj, T. & Lammertyn, J. SIMPLE analytical model for smart microfluidic chip design. 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W. The dynamics of capillary flow. Phys. Rev. 18 , 206–209 (1921). Wilkinson, A. F., Barra, M. J., Novak, E. N., Bond, M. & Richards-Kortum, R. Point-of-care isothermal nucleic acid amplification tests: progress and bottlenecks for extraction-free sample collection and preparation. Expert Rev. Mol. Diagn. 1–16 (2024) doi:10.1080/14737159.2024.2375233. Respeggt. Respeggt group, GmbH. https://www.respeggt.com/ (2024). Additional Declarations The authors declare no competing interests. Supplementary Files SantosFullyautomatedsampletoresultSIMPLERPAmicrofluidicchiptowardsinovosexingapplicatioSM.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-5123850","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":357141021,"identity":"517dddff-3a40-43fd-b260-f0c4d77e5d7d","order_by":0,"name":"Simão Monteiro Belo dos Santos","email":"","orcid":"https://orcid.org/0000-0002-9505-3943","institution":"KU Leuven, Department of Biosystems – Biosensors group, Willem de Croylaan 42, 3001 Leuven, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Simão","middleName":"Monteiro Belo dos","lastName":"Santos","suffix":""},{"id":357141022,"identity":"7ce97a87-9ae6-46c6-9205-209f300f39b0","order_by":1,"name":"Celine Wegsteen","email":"","orcid":"https://orcid.org/0009-0001-1423-564X","institution":"KU Leuven, Department of Biosystems – Biosensors group, Willem de Croylaan 42, 3001 Leuven, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Celine","middleName":"","lastName":"Wegsteen","suffix":""},{"id":357141023,"identity":"5cd390b5-7a03-4800-93e2-b1fc54ba2265","order_by":2,"name":"Dries Vloemans","email":"","orcid":"https://orcid.org/0000-0003-2885-5249","institution":"KU Leuven, Department of Biosystems – Biosensors group, Willem de Croylaan 42, 3001 Leuven, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Dries","middleName":"","lastName":"Vloemans","suffix":""},{"id":357141024,"identity":"a7753796-cf1e-4c18-8c09-c6fbd3def4db","order_by":3,"name":"Matthias Corion","email":"","orcid":"https://orcid.org/0000-0002-9984-7660","institution":"KU Leuven, Department of Biosystems – A2H Nutrition \u0026 Animal EcoSystems (NAMES) Lab, Kasteelpark Arenberg 30, 3001 Leuven, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"","lastName":"Corion","suffix":""},{"id":357141025,"identity":"292311f4-d818-4273-8261-1096b40993f5","order_by":4,"name":"Bart De Ketelaere","email":"","orcid":"https://orcid.org/0000-0002-5140-1643","institution":"KU Leuven, Department of Biosystems – MeBioS Biostatistics group, Kasteelpark Arenberg 30, 3001 Leuven, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Bart","middleName":"","lastName":"De Ketelaere","suffix":""},{"id":357141026,"identity":"f8a4af9e-5e8b-48d6-866d-c2476e7c0f62","order_by":5,"name":"Dragana Spasic","email":"","orcid":"https://orcid.org/0000-0001-9882-5091","institution":"KU Leuven, Department of Biosystems – Biosensors group, Willem de Croylaan 42, 3001 Leuven, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Dragana","middleName":"","lastName":"Spasic","suffix":""},{"id":357141027,"identity":"21d1ab3a-8b74-4f01-a975-f9acb3a10184","order_by":6,"name":"Jeroen Lammertyn","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8143-6794","institution":"KU Leuven, Department of Biosystems – Biosensors group, Willem de Croylaan 42, 3001 Leuven, Belgium","correspondingAuthor":true,"prefix":"","firstName":"Jeroen","middleName":"","lastName":"Lammertyn","suffix":""}],"badges":[],"createdAt":"2024-09-20 13:04:26","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5123850/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5123850/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65160753,"identity":"cf0da9e8-c633-49aa-8d31-dacd1bf904e4","added_by":"auto","created_at":"2024-09-24 09:07:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":407743,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic drawing of the SIMPLE-RPA chip and its layers and functions. \u003cstrong\u003eA)\u003c/strong\u003e The platform is built using layer-by-layer stacking. Distinct units were built to enable the whole bioassay, from sample metering to results' visualizations. The detection unit (I) mixes the LFS buffer and the amplified DNA target and draws it over the LFS. The sample processing unit (II and III) contains all the microfluidic structures, channels and sc and dcHBVs (purple and red, respectively) for the various liquid manipulations (metering 1 µL of sample, merging and mixing the sample with 9 µL of RPA master mix, mixing the liquids in 3D mixing chambers and performing incubation for DNA amplification). Under the microfluidic channel structure is a layer comprising the liquid barriers (III), allowing for complex liquid manipulations. The pumping unit comprised the porous material (IV) and the working liquid (V, blue color), which is used to create a negative pressure due to capillary wicking (after finger press activation). This drives the liquids inside the connected microfluidic channels. Dashed arrows represent the direction of the airflow. Normal arrows represent the direction of the fluid flow. \u003cstrong\u003eB)\u003c/strong\u003e and \u003cstrong\u003eC)\u003c/strong\u003e The liquid barriers are used as switches: (1) the user inserts the sample in the inlet, whereas the RPA and LFS reagents are already prefilled in their intended positions. The system is in the initial state and the pump produces the same pressure in the three channels: W (waste), M (sample and RPA reagents mix) and S (sample). Once the platform is activated with a finger press, the (2) sample is pulled towards the RPA reagents and is stopped between a scHBV (purple) and a dcHBV (red). The metered sample blocks the liquid barrier S. (3) 1 µL is measured and the remaining goes over the scHBV to the waste channel. After blocking a second liquid barrier W, the measured sample is (4) mixed with the RPA reagents inside the 4 3D mixing chamber. Following the incubation, the RPA product merges with the LFS buffer after blocking the last liquid barrier M, and (5) flows towards the LFS. LFS – Lateral flow strip; RPA – Recombinase polymerase amplification; HBV – Hydrophobic burst valve; sc – single coated; dc – double coated.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5123850/v1/161180372af6c96145146cf9.png"},{"id":65160430,"identity":"d9e00e56-513f-466a-bd0b-f55eacd370d3","added_by":"auto","created_at":"2024-09-24 08:59:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":461850,"visible":true,"origin":"","legend":"\u003cp\u003ePrimer concentration optimization, with four distinct primer concentrations: 10, 5, 2.5 and 0.5 μM, with 20 minutes incubation time and 0.01 ng/µL concentration of synthetic DNA from the targeted \u003cem\u003eHINTW\u003c/em\u003e gene. A) Example of the obtained LFSs; primer concentrations of 10, 5, 2.5 and 0.5 µM, and their respective NTC controls. B) Signal intensities were analyzed with ImageJ for all the different conditions and their respective NTCs, normalized between 0 and 1 and their calculated SNR, following the method explained in the Materials and methods, considering the 8-color bit image pixel maximum intensity of 255. Error bars represent one standard deviation (n ≥ 3). Statistical differences of the means analysed with one-way ANOVA are given by the different letters, followed by Tukey's multiple comparisons test (α = 0.05). NTC – Non-template control.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5123850/v1/799194f62d311e3b502cb113.png"},{"id":65160424,"identity":"c0beeba5-1823-4d81-920f-5cbb9df49e7a","added_by":"auto","created_at":"2024-09-24 08:59:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":374839,"visible":true,"origin":"","legend":"\u003cp\u003eIncubation time optimization, with four distinct times: 20, 15, 10 and 5 minutes, 2.5 µM primer concentration and 0.01 ng/µL concentration of synthetic DNA from the targeted \u003cem\u003eHINTW\u003c/em\u003e gene. A) Example of the obtained LFSs; incubation times of 20, 15, 10 and 5 minutes and their respective NTC controls. B) Signal intensities were analyzed with ImageJ for all the different conditions and their respective NTCs following the method explained in the Materials and methods, normalized between 0 and 1 and their SNR calculated, considering the 8-color bit image pixel maximum intensity of 255. Error bars represent one standard deviation (n ≥ 3). Statistical differences of the means analysed with one-way ANOVA are given by the different letters, followed by Tukey's multiple comparisons test \u003cstrong\u003e(\u003c/strong\u003eα = 0.05). NTC – Non-template control.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5123850/v1/4b6480dd9704027b6e5b0d8d.png"},{"id":65160752,"identity":"ad49e7e2-ba66-4c88-afaa-3cfeb3d6e535","added_by":"auto","created_at":"2024-09-24 09:07:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101406,"visible":true,"origin":"","legend":"\u003cp\u003eRPA performed with a serial dilution of synthetic DNA target using the optimized bioassay conditions (primer concentration of 2.5 μM and an incubation time of 15 minutes) and A) the thermocycler or B) the egg incubator as the heating source for achieving 37.7 °C. Error bars represent one standard deviation (n ≥ 3). Comparison of the test line average values of synthetic DNA concentration for each distinct heating sources was performed with one-way ANOVA (α = \u0026nbsp;0.05) followed by Tukey's multiple comparisons test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5123850/v1/ec012f1a7088f3afaae8b776.png"},{"id":65160431,"identity":"6799dcc7-bf80-4d0c-a147-4938a9622b12","added_by":"auto","created_at":"2024-09-24 08:59:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1809798,"visible":true,"origin":"","legend":"\u003cp\u003eSteps performed by the SIMPLE-RPA chip. Before the start, RPA reagents (red liquid), LFS buffer (green liquid) and working liquid (blue liquid behind the SIMPLE pump) are loaded inside the chip, and the inlet holes are sealed with PSA. Then, \u003cstrong\u003e1)\u003c/strong\u003e 2 µL of the sample is added to the inlet hole, and the SIMPLE-RPA chip is activated with a finger press, pushing the working liquid towards the SIMPLE pump, creating a negative pressure inside the microfluidic channels, driving the liquids. Using the liquid barriers (LB) together with the scHBVs (purple) and dcHBVs (red), it is possible to \u003cstrong\u003e2)\u003c/strong\u003e measure 1 µL of the sample. Due to an increase of the pressure inside the microfluidic channels, the measured 1 µL sample is pulled over the dcHBV and \u003cstrong\u003e3)\u003c/strong\u003emerged with the 9 µL RPA reagents, \u003cstrong\u003e4)\u003c/strong\u003e and flowing inside the first 3D mixing chamber where the solution is combined with 0.5 µL MgOAc (RPA reaction initiator) pre-loaded in that chamber. \u003cstrong\u003e5) \u003c/strong\u003eThe three solutions are efficiently mixed over the four subsequent 3D mixing chambers. \u003cstrong\u003e6) \u003c/strong\u003eSimultaneously, the mixed RPA reagents, sample and MgOAc are incubated. After incubation of the mixture (approximately 15 minutes with 4% w/v PVA in the working liquid), the amplified material is \u003cstrong\u003e7)\u003c/strong\u003e merged with 40 µL LFS buffer, mixed and \u003cstrong\u003e8)\u003c/strong\u003edrawn over the LFS RPA – Recombinase polymerase amplification; MgOAc – Magnesium acetate; LFS – Lateral flow strip; HBV – Hydrophobic burst valve.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5123850/v1/30d15477ba5e2a4f0023abc4.png"},{"id":65160429,"identity":"68d12a03-c9f4-4e90-9d2b-a8538a512839","added_by":"auto","created_at":"2024-09-24 08:59:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":615060,"visible":true,"origin":"","legend":"\u003cp\u003eOn-chip testing of a serial dilution of synthetic DNA target using the optimized RPA bioassay. A) Real images of the SIMPLE-RPA chip after finishing the bioassay for different tested target concentrations, including the NTC. B) ImageJ mean histogram analysis of the test colorimetric band from the LFSs as depicted in A. Error bars represent one standard deviation (n ≥ 3). Statistical differences of the means with one-way ANOVA (α = 0.05) are given by the different letters.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5123850/v1/be34d77108883a6fa7d5df1a.png"},{"id":65162119,"identity":"a936091c-2093-4167-b2c6-bad7cb8a7265","added_by":"auto","created_at":"2024-09-24 09:15:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5823726,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5123850/v1/47d5a250-1c55-4ed9-9e1e-bee516afec1d.pdf"},{"id":65160426,"identity":"9dfc1bac-2450-4d92-ade3-ab4f64faa3c0","added_by":"auto","created_at":"2024-09-24 08:59:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1483324,"visible":true,"origin":"","legend":"","description":"","filename":"SantosFullyautomatedsampletoresultSIMPLERPAmicrofluidicchiptowardsinovosexingapplicatioSM.docx","url":"https://assets-eu.researchsquare.com/files/rs-5123850/v1/be54f69688a973fd4da52457.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eFully automated sample-to-result SIMPLE-RPA microfluidic chip: towards \u003cem\u003ein ovo\u003c/em\u003e sexing application\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe killing of day-old male chicks is a widespread practice in the laying hen poultry industry. Approximately 372\u0026nbsp;million male chicks in the EU are culled upon hatching, as they neither lay eggs nor yield quality meat\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Several governments have banned male chick culling (\u003cem\u003ee.g.\u003c/em\u003e, Germany, France, and Italy)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, endorsing increasing consumer awareness of animal welfare issues and preventing the disadvantages for the industry (\u003cem\u003ei.e.\u003c/em\u003e, investing in a byproduct and image degradation)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Among several solutions offered to date for solving this problem\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, sex detection before hatching (\u003cem\u003ei.e.\u003c/em\u003e, \u003cem\u003ein ovo\u003c/em\u003e sexing) and subsequent male embryo disposal is the preferred one by both the consumers and industry. With \u003cem\u003ein ovo\u003c/em\u003e sexing, the incubation is stopped prior to the onset of pain perception (day 13 of incubation), while the industry avoids investing in males (\u003cem\u003ei.e.\u003c/em\u003e, male eggs can be removed from the incubator and do not require to be grown)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, \u003cem\u003ein ovo\u003c/em\u003e sexing techniques must meet several requirements for industrialization, including: 1) compatibility with all colors of eggs, 2) high throughput (\u0026gt;\u0026thinsp;20 000 eggs/hour, complying with the market's high product demand), 3) high accuracy (\u0026gt;\u0026thinsp;98%), 4) high sensitivity to be applied early in incubation, imperatively before day 13 of incubation, 5) maintaining a high hatchability rate and 6) being low cost (around 2\u0026ndash;3 \u0026euro;/day-old-chick)\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBoth researchers and industry have made significant efforts to develop \u003cem\u003ein ovo\u003c/em\u003e sexing methods that can meet these imposed requirements\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Current techniques include optical and non-optical methods\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Optical techniques employ visible near-infrared spectroscopy to distinguish the embryo's feather colors, however, one day after the embryo\u0026rsquo;s pain perception onset (day 13 of incubation)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Another available optical technique is magnetic resonance imaging to detect the embryo's gonads on day 12 of incubation \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, with low accuracy (\u0026lt;\u0026thinsp;98%) and high cost (\u0026gt;\u0026thinsp;4 \u0026euro;/day-old-chick). Non-optical methods currently include extracting allantoic fluid (AF) from the egg at day 9 of incubation to detect (1) a female-specific hormone (estrone sulfate) with an enzyme-linked immunosorbent assay (ELISA) that offers 98% accuracy but has a high cost (\u0026gt;\u0026thinsp;4\u0026ndash;5 \u0026euro;/day-old-chick)\u003csup\u003e10\u003c/sup\u003e, or (2) a male-specific metabolite\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e with mass spectrometry that has only 95% accuracy\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, DNA analysis proved to be the most accurate method, as shown in our recent report using quantitative PCR (qPCR) to target the \u003cem\u003eHINTW\u003c/em\u003e female-specific gene\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Following this approach, it is possible to detect the embryos\u0026rsquo; sex with 100% accuracy from days 6 to 9 of incubation using a small amount of AF (\u0026lt;\u0026thinsp;5 \u0026micro;L), while having minimal impact on embryo development (seen only when sampling AF on day 6 of incubation)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, despite this exceptional accuracy, qPCR is a lengthy (90 minutes) and expensive technique (\u0026gt;\u0026thinsp;3 \u0026euro;/test) that involves temperature cycling, making it unsuitable for non-laboratory settings like an incubator, hence introducing interruption in the eggs\u0026rsquo; incubation. In this context, isothermal DNA amplification techniques, such as recombinase polymerase amplification (RPA), can offer advantages for developing a desired test\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Compared to other isothermal methods such as loop-mediated isothermal amplification (LAMP) or rolling circle amplification (RCA), RPA requires only two primers, and does not require an initial denaturation temperature. Meanwhile, it can be performed at low incubation temperatures (37\u0026ndash;42\u0026deg;C) and is rapid (5\u0026ndash;20 minutes)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. For instance, previous work showed that RPA can amplify \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e in urine samples at 37\u0026deg;C, close to the temperature of the egg incubation (37.7\u0026deg;C), offering high sensitivity (1 copy per sample) in less than 20 minutes\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Moreover, RPA can be combined with colorimetric detection methods like commercial lateral flow strips (LFSs) - which are not mainstream with PCR \u0026ndash; thus avoiding the need for additional devices to carry out fluorescence read-out and instead generating results in an easy, practical, and rapid manner\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBecause of all these advantages, RPA has been incorporated into hand-held microfluidic platforms aiming to deliver almost \u0026ldquo;one-pot\u0026rdquo; reactions. In Liu et al. 2021, a poly(methyl methacrylate) (PMMA) device containing an LFS was built to detect SARS-CoV-2 RNA sequences with a limit of detection (LOD) of 1 copy per \u0026micro;L. Other researchers developed a paper origami device to detect \u003cem\u003eSalmonella enterica\u003c/em\u003e in food samples\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e in which the RPA amplification was performed in stages controlled by the user\u0026rsquo;s consecutive folding, resulting in an LOD of 6, 9 and 58 CFU/mL for lettuce, chicken breast and milk, respectively\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Despite the proven potential to perform on-chip RPA with high sensitivity, the existing devices require user intervention (\u003cem\u003ee.g.\u003c/em\u003e, inserting the correct amount of sample in the inlet, manipulating or folding the device), which makes these systems user-dependent and more prone to errors. Examples of sample-in-result-out types of microfluidic platforms that require less user manipulation have been reported, including centrifugal microfluidics\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e or pump-driven devices\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e that allow for complex liquid manipulations (\u003cem\u003ee.g.\u003c/em\u003e, sample mixing, RNA or DNA extraction). However, they rely on external devices (centrifuge or pumps), making them unsuitable for the point-of-care (POC). Hence, laying hen farms are still looking for new technologies that can provide fast, highly accurate but low-cost \u003cem\u003ein ovo\u003c/em\u003e sexing tests with minimal to no disturbance of egg incubation.\u003c/p\u003e \u003cp\u003eIn this work, we present a microfluidic-based tool, named SIMPLE-RPA chip, to perform fully autonomous RPA for detecting the synthetic female-specific \u003cem\u003eHINTW\u003c/em\u003e gene within an incubator at 37.7\u0026deg;C. To achieve this, we first establish an off-chip RPA bioassay for detecting synthetic DNA. Furthermore, we develop a SIMPLE-RPA chip, starting from our in-house developed SIMPLE (self-powered imbibing microfluidic pump by liquid encapsulation) technology. The SIMPLE is an obvious choice for the \u003cem\u003ein ovo\u003c/em\u003e sexing application because it is a self-powered, cost-effective (\u0026lt;\u0026thinsp;1\u0026euro;/chip ) microfluidic technology that can perform complex liquid manipulations autonomously upon a single finger press activation step, while remaining small in size\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e (which is crucial for the limited space in egg incubators). Finally, we implement for the first time an RPA bioassay on the SIMPLE-RPA chip with an integrated LFS for qualitative colorimetric identification of the synthetic female-specific \u003cem\u003eHINTW\u003c/em\u003e gene on the spot without the need for external read-out devices. Due to its low cost, complete automation and user-friendliness, the presented SIMPLE-RPA chip shows great prospect for final integration with AF sampling directly from eggs in the incubator and consequently \u003cem\u003ein ovo\u003c/em\u003e sexing on genomic DNA, as well as for applications in other fields, such as medicine, veterinary sciences and agriculture.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cp\u003eAll sequences of primers and synthetic DNA used in this work (Table 1) were produced by IDT (Leuven, Belgium), delivered in IDTE buffer (pH = 7) and were used without further processing. All the dilutions were done using distilled water. RPA liquid basic kit and HybriDetect \u0026ndash; Universal Lateral Flow Assay Kit strips were purchased from TwistDx Limited (Maidenhead, UK). PMMA with 1 mm thickness was purchased from Pyrasied (Leeuwarden, The Netherlands). Polyethylene terephthalate (PET) films of 125 \u0026mu;m thickness were bought at P\u0026uuml;tz GmbH (Taunusstein, Germany). Double-sided pressure sensitive adhesive (PSA) tape (200 MP 7956MP) and transfer tape (467MP) with thicknesses of 153 and 50 \u0026mu;m, respectively, were acquired from 3M (Minnesota, USA). Microfluidic dyes were obtained from Darwin Microfluidics (Paris, France). Hydrophobic solution Fluoropel 800 was purchased at Cytonix (Maryland, USA). Whatman quantitative filter paper of grade 3 and Mowiol 8-88 (polyvinylalcohol; PVA) were obtained from Sigma-Aldrich (Overijse, Belgium). Versapor\u0026trade; acrylic copolymer 3000 membrane was purchased from Cytiva (DC, USA).\u003c/p\u003e\n\u003cp\u003eTable 1 \u0026ndash; Primers and synthetic DNA sequences used in this work. \u003cem\u003eHINTW\u003c/em\u003e synthetic DNA was used as double-stranded DNA (dsDNA)\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ch2\u003ePrimers and synthetic DNA\u003c/h2\u003e\n\u003cp\u003eStarting from the primers used in our previous work for developing the qPCR\u003csup\u003e13\u003c/sup\u003e, the primers in this work were modified to meet the RPA primer design requirements (\u0026asymp; 30 base pairs, TwistDx), albeit maintaining the same target gene. Primers were selected using the NCBI BLAST engine to detect \u003cem\u003eHINTW\u003c/em\u003e (GenBank accession number: NC_052571), a specific gene in the W chromosome of laying hen chickens (\u003cem\u003eGallus Gallus\u003c/em\u003e). IDT Oligoanalyzer 3.1 (IDT) was further used to verify the primers\u0026apos; annealing temperature, stability, and self-complementary. The forward primer was labeled at the 5\u0026apos; end with biotin, while the reverse primer was labeled with fluorescein (FAM) at the 5\u0026apos; end for the detection with the HybriDetect LFSs. The synthetic DNA was purchased as a dsDNA (geneBlock) in IDTE buffer (pH = 7). The sequences used are presented in Table 1.\u003c/p\u003e\n\u003ch2\u003eOff-chip optimization of the RPA bioassay\u003c/h2\u003e\n\u003cp\u003eThe RPA bioassay was first optimized off-chip by testing different primer concentrations and amplification times (see further for more details). The RPA bioassay was performed based on the manual instructions provided by TwistDx\u003csup\u003e23\u003c/sup\u003e. TwistAmp\u003csup\u003eTM\u003c/sup\u003e liquid master mix was prepared by mixing 25 \u0026mu;L of 2x Reaction buffer, 2.25 \u0026mu;L dNTPs (40 mM), 5 \u0026mu;L 10x Basic E-mix, 2.4 \u0026mu;L of forward and reverse primer in different concentrations (0.5, 2.5, 5 and 10 \u0026mu;M), 7 \u0026mu;L of deionized (DI)-water and 2.5 \u0026mu;L of 20x Core Reaction Mix. This tube was briefly centrifuged and vortexed to achieve complete mixing of the reagents before dividing the solution into different PCR tubes with 9 \u0026mu;L of the prepared master mix. Before closing the tubes, 1 \u0026mu;L of the sample (0.01 ng/\u0026micro;L synthetic DNA) or DI water for non-template control (NTC) and 0.5 \u0026micro;L of magnesium acetate (MgOAc; 280 mM) were added to the tube lids. Once the tubes were closed, they were inverted 4 times and spun down for 5 seconds using a tabletop centrifuge. The tubes were then incubated in a Biometra TProfessional basic thermocycler (Analytik Jena GmbH, Jena, Germany) for a specific time (5, 10, 15 or 20 minutes) at 37.7 \u0026deg;C (mimicking egg incubation temperature). After the incubation time, the thermocycler temperature was decreased to 4 \u0026deg;C to stop the reaction.\u003c/p\u003e\n\u003cp\u003eNext, the RPA amplification product from the PCR tubes (10 \u0026mu;L) was mixed with 40\u0026nbsp;\u0026mu;L of LFS buffer in an Eppendorf tube. The LFSs (HybriDetect \u0026ndash; Universal Lateral Flow Assay Kit, Figure S1 from Supplementary Material) with two lines for colorimetric detection (\u003cem\u003ei.e.,\u003c/em\u003e test and control) were immersed into this solution, and the colorimetric results were visualized 10 minutes later. The test line included a biotin-specific antibody, whereas the control line comprised antibodies specific to the reporter probe (\u003cem\u003ei.e.,\u0026nbsp;\u003c/em\u003eagainst FAM-specific antibodies immobilized on gold nanoparticles (AuNPs)). The RPA amplified product was drawn over the LFS, where reporter probes were lyophilized in the sample pad, thus binding the FAM label. The working method of the used LFS is further explained in Figure S1 from Supplementary Material. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImages of the LFSs were captured using a fi-65F Fujitsu (Tokyo, Japan) flat desk scanner at \u0026nbsp;10 minutes after running the test. Band intensities were evaluated using ImageJ software, where the color image was transformed into a black-and-white 8-bit channel image. A rectangular area was drawn around the test line, and the average histogram intensity (\u003cem\u003eAvgTestHist\u003c/em\u003e in Equation 1) was calculated and normalized (\u003cem\u003eNormInt\u003c/em\u003e) by subtracting it from 255 (the maximum intensity value for a pixel in an 8-bit image) and then dividing by 255 (Equation 1). The signal-to-noise ratio (SNR) was calculated according to Equation 2 using the mean intensity for each condition, where \u003cem\u003eNormInt\u003csub\u003eNoise\u003c/sub\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;NormInt\u003csub\u003eSample\u003c/sub\u003e\u0026nbsp;\u003c/em\u003eare the average test line intensity value of the NTC and positive tests, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eStarting from the same post-amplified RPA samples evaluated with the LFSs, we simultaneously performed electrophoresis analysis with an Agilent 4150 TapeStation system (Agilent Technologies, Inc, USA). All the post-amplified RPA samples handling was performed in ice to avoid the reaction to continue. 1 \u0026micro;L of post-amplified RPA sample or 1 \u0026micro;L of ladder were mixed in PCR tubes with 10 \u0026micro;L of sample buffer. The PCR tubes were vortexed for 1 minute, spun at 2000 rpm for 1 minute and placed in the TapeStation 4200 device. Data analysis was conducted using the TapeStation 4200 Analysis Software.\u003c/p\u003e\n\u003ch2\u003eDetermining the sensitivity of \u0026nbsp;the off-chip RPA bioassay\u003c/h2\u003e\n\u003cp\u003eOnce primer concentration and incubation time of the RPA were optimized, \u003cem\u003eHINTW\u003c/em\u003e synthetic DNA samples were diluted in 1:5 series, resulting in concentrations from 0.05 and 1.6 \u0026nbsp; \u0026times; 10\u003csup\u003e‑5\u003c/sup\u003e ng/\u0026mu;L. The RPA on the dilution series was tested with two different heat sources: 1) the thermocycler like in the previous section and 2) the Rcom Max 50 DO incubator (Autolex Co., South Korea) used for the eggs\u0026rsquo; incubation, which provided information about the amplification efficiency in the commercial egg incubator. After incubation at 37.7 \u0026deg;C the reaction was stopped by decreasing the thermocycler temperature to 4 \u0026deg;C whereas the tubes from the egg incubator were transferred to ice for further manipulation. The amplification product (10 \u0026mu;L) was mixed with 40 \u0026mu;L of LFS buffer in an Eppendorf tube. Subsequently, the LFSs were immersed into the tubes, and the colorimetric results were visualized 10 minutes later. Band intensity analysis was performed as described above.\u003c/p\u003e\n\u003ch2\u003eFabrication of the SIMPLE-RPA chip\u003c/h2\u003e\n\u003cp\u003eThe SIMPLE-RPA chip was fabricated using our previously established low-cost and rapid prototyping method\u003csup\u003e21,24\u003c/sup\u003e. More specifically, the microfluidic network (Figure S2 from Supplementary Material) was designed using Inkscape vectorial software (Version 1.3.2, Software Freedom Conservancy, USA) and fabricated using a manual layer-by-layer lamination method. PSA, transfer tape, PET, PMMA layers, liquid barriers and Whatman Grade 3 paper were cut using an 80-watt Speedy300 Trotec laser cutter (Trotec Lasers, Austria). Figure 1 shows different layers (A) together with SIMPLE-RPA chip workflow (B and C). The SIMPLE-RPA chip was divided into three vertically stacked units: the pumping unit, the sample processing unit, and the detection unit, which were linked with air connection holes and liquid connection holes, shown with the dashed and full arrows, respectively (Figure 1A).\u003c/p\u003e\n\u003cp\u003eThe pumping unit comprised 2 layers, each made of 2 stacked PSA layers sandwiched between 2 PET layers. The top layer of this unit (IV in Figure 1A) contained the porous material (Whatman Grade 3), inserted into its chamber during assembly and secured with transfer tape. The bottom layer (V in Figure 1A) comprised the microfluidic channels containing the working liquid, its prefilling hole and the activation site.\u003c/p\u003e\n\u003cp\u003eThe sample processing unit comprised 2 layers of single PSA sandwiched between 2 PET layers. The top layer (II in Figure 1A) comprised the microfluidic channels for sample processing, RPA and LFS reagents prefilling holes, liquid connection to the detection unit and air connection to the pumping unit. Two types of hydrophobic valves were created: hydrophobic burst valves (HBVs) and liquid barriers. HBVs were made in the sample processing unit top layer (II in Figure 1A) following a mask-based strategy, based on a previous report\u003csup\u003e20\u003c/sup\u003e. In short, both top and bottom or only bottom PET films were locally coated with 2\u0026nbsp;\u0026micro;L of Fluoropel 800 to create double-coated (dc) or single-coated (sc) HBVs, respectively. After, the PET films were allowed to dry for 1 hour at room temperature and masks were removed. The bottom layer of the sample processing unit (III in Figure 1A) holds the hydrophobic porous Versapor membranes (\u003cem\u003ei.e.\u003c/em\u003e, liquid membranes), allowing air passage but preventing liquid flow due to their high burst-pressure properties (\u0026gt; 0.7 bar) and working as switches for the liquid manipulations, as explained in detail in Figure 1B and C. Moreover, only the 3D mixing chambers span two layers (\u003cem\u003ei.e.\u003c/em\u003e, going through the first and second layers of the sample processing unit; Figure 1A). In these structures, the inlet and outlet of the chambers were located in different layers, \u003cem\u003ei.e.\u003c/em\u003e, inlet from the top layer and outlet from the bottom layer or vice versa. More information on these structures can be found in a previous report\u003csup\u003e20\u003c/sup\u003e. The sample mixed with the RPA reagents proceeds through the mixing chambers and a series of meandering channels allowing for incubation and amplification of amplicons in the sample.\u003c/p\u003e\n\u003cp\u003eThe detection unit was designed to hold the LFS (\u003cem\u003ei.e.\u003c/em\u003e, a HybriDetect - Universal Lateral Flow Assay) by sandwiching a PSA layer between a 1 mm PMMA and PET layer. The PMMA layer contained a connection hole to the sample processing unit, while the pumping unit was connected through the LFS space.\u003c/p\u003e\n\u003cp\u003eAfter the assembly of the microfluidic chip, 0.5 \u0026micro;L of MgOAc (280 mM) was loaded into the first mixing chamber and sealed with PSA. Next, the working liquid and reagents were prefilled through their respective prefilling holes, bringing the working liquid close to, but not in contact with, the porous material, as previously described\u003csup\u003e25\u003c/sup\u003e. The working liquid comprised a blue microfluidic dye diluted in distilled water (1:20) mixed with 4 % (w/v) PVA. The chips were used immediately after prefilling. When not prefilled, they were stored overnight at 4 \u0026deg;C in a closed bag with desiccant.\u003c/p\u003e\n\u003ch2\u003eOn-chip RPA integration for synthetic \u003cem\u003eHINTW\u003c/em\u003e DNA detection\u003c/h2\u003e\n\u003cp\u003eThe SIMPLE-RPA chip performance was assessed using the off-chip optimized RPA conditions. The RPA master mix was prepared as explained in the section on off-chip optimization of the RPA bioassay, and 9 \u0026micro;L was subsequently introduced into the SIMPLE-RPA chip through its prefilling hole (Figure 1A). MgOAc (0.5 \u0026micro;L; 280 mM) was pipetted into the first 3D chamber and LFS buffer (40 \u0026micro;L) was loaded into the chip. All the reagents were inserted into the microfluidic channels using a micropipette. All the prefilling holes were sealed using a PSA membrane. A video showing the prefilling of all the liquids is shown in Supplementary material S7. The Rcom Max 50 DO incubator (Autolex Co) was set at 37.7 \u0026deg;C and 55 % humidity for incubation. The SIMPLE-RPA chip was placed inside the incubator and allowed to reach the set temperature. The synthetic \u003cem\u003eHINTW\u003c/em\u003e DNA samples were prepared with a 1:5 dilution series between 0.05 and 1.6 x 10\u003csup\u003e-5\u003c/sup\u003e ng/\u0026mu;L. For testing the SIMPLE-RPA chip, 2 \u0026micro;L of \u003cem\u003eHINTW\u003c/em\u003e synthetic DNA sample in IDTE buffer or 2 \u0026micro;L of water for NTC was added to the inlet with a micropipette and the SIMPLE-RPA chip was activated with a single finger press. The testing was done in triplicate. The autonomous SIMPLE-RPA chip operation happened inside the incubator without any disturbance or user intervention followed by its removal from the incubator only when all amplified RPA product with LFS buffer were absorbed by the LFS. The LFS was scanned using the fi-65F Fujitsu flat desk scanner and its band intensities were analyzed as previously described.\u003c/p\u003e\n\u003ch2\u003eData analysis\u003c/h2\u003e\n\u003cp\u003eGraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses and data visualization. One-way ANOVA (\u0026alpha; = 0.05) was performed, followed by Tukey\u0026apos;s multiple comparison tests. \u0026nbsp;\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003ch2\u003eOff-chip optimization of the RPA bioassay\u003c/h2\u003e\n\u003cp\u003eThe RPA bioassay was first optimized off-chip using 4 distinct primer concentrations (0.5, 2.5, 5 and 10\u0026nbsp;\u0026micro;M), while keeping incubation time at 20 minutes (according to the manufacturer\u0026rsquo;s protocols\u003csup\u003e23\u003c/sup\u003e). The selection of primer concentrations was based on a previous publication by Lobato et al. 2018 with suggested primer concentrations between 0.8 and 5 \u0026mu;M\u003csup\u003e14\u003c/sup\u003e. The primer sequences for this work were taken from our recent paper\u003csup\u003e13\u003c/sup\u003e, where they have been validated for high sensitivity and specificity towards \u003cem\u003eHINTW\u003c/em\u003e synthetic DNA and genomic DNA (gDNA) sampled from AF at days 6 to 9 of incubation. Using the most efficient primer concentration from this first optimization step, we also tested 4 different incubation times (5, 10, 15, and 20 minutes), to evaluate when the amplification plateau was reached and thus to avoid incubating the samples for too long, risking primer dimer formation and non-specific amplification (as advised by the manufacturer\u0026rsquo;s manual\u003csup\u003e26\u003c/sup\u003e). The incubation temperature of 37.7 \u0026deg;C was selected to match the incubation temperature of an egg incubator. Synthetic DNA in IDTE buffer, matching the targeted sequence from the \u003cem\u003eHINTW\u003c/em\u003e gene, was used at 0.01 ng/\u0026mu;L, which corresponded to the lowest detected concentration achieved with qPCR detection of gDNA extracted from AF in our previous work\u003csup\u003e13\u003c/sup\u003e. NTC were added for each variation of primer concentration or incubation time, whereas all conditions were tested in triplicates. The results were visualized with LFSs (Figures 2 and 3) and electrophoresis using a TapeStation device (Figure S3 from Supplementary Material).\u003c/p\u003e\n\u003cp\u003eFigure 2A shows examples of the obtained LFSs for different tested primer concentrations and Figure 2B shows the normalized intensity of the respective LFS test bands, analyzed using ImageJ. From all the tested concentrations, 0.5 \u0026mu;M was the only one that did not show a statistically significant difference compared to its NTC, which was also confirmed visually by the absence of a test line. Although the other three tested primer concentrations all gave statistically significant differences compared to the NTC (Tukey\u0026rsquo;s test for multiple comparisons, \u0026alpha; = \u0026nbsp;0.05), the best SNR was obtained when using 2.5 \u0026micro;M (0.92), leading to the selection of this concentration as the most suitable to continue with. It is important to note that the visible NTC test lines when using 10 and 5 \u0026micro;M of primers (Figure 2A) possibly resulted from primer dimers and non-specific amplification.\u003c/p\u003e\n\u003cp\u003eStarting from a selected primer concentration of 2.5 \u0026mu;M, we then tested different incubation times, with examples of the obtained LFSs in Figure 3A and the normalized intensity of the respective LFS test bands, analyzed using ImageJ in Figure 3B. For all tested incubation times, the NTCs showed minimal to no amplification. When compared to the positive samples, statistically significant differences were observed for all incubation times, except for 5 minutes (Figure 3B). Although the remaining incubation times (10, 15 and 20 minutes) did not reveal significant statistical differences among themselves, they revealed slightly different SNR values. Therefore, we selected 15 minutes as a condition with the best SNR of 0.98 for the remaining benchtop and on-chip experiments, since it enables a rapid bioassay, without hindering the sensitivity. The results presented in Figures 2 and 3 are further confirmed with obtained electrophoresis results in Figure S3 from Supplementary Material.\u003c/p\u003e\n\u003ch2\u003eDetermining the sensitivity of the off-chip RPA bioassay\u003c/h2\u003e\n\u003cp\u003eThe optimized RPA bioassay for \u003cem\u003eHINTW\u003c/em\u003e synthetic DNA detection (with 2.5 \u0026micro;M of forward and reverse primers and 15 minutes of incubation) was subsequently tested with a serial dilution of the synthetic DNA in buffer (1:5 dilution ratio between 0.05 and 1.6 x 10\u003csup\u003e‑5\u003c/sup\u003e ng/\u0026micro;L) and incubation at 37.7 \u0026deg;C using a PCR thermocycler (Figure 4A) or an egg incubator (Figure 4B) as the heat source (Figure S4 from Supplementary Material shows examples of the LFSs obtained from this experiment). For both heating conditions, amplification was achieved for all concentrations of the synthetic DNA between 0.05 and 4 x 10\u003csup\u003e‑4\u003c/sup\u003e ng/\u0026mu;L with no statistical differences (Figure 4A and B; Tukey\u0026rsquo;s test for multiple comparisons, \u0026alpha; = 0.05). Furthermore, 8 x 10\u003csup\u003e-5\u003c/sup\u003e (Figure 4A) and 1.6 x 10\u003csup\u003e-5\u003c/sup\u003e (Figure 4B) ng/\u0026mu;L were the two lowest concentrations that resulted in a significantly higher intensity compared to the NTC signal. Although we cannot explain the differences seen in the LOD when using the PCR thermocycler or the egg incubator, the fact that we obtained a lower LOD in the egg incubator is extremely promising due to the intended final application of the SIMPLE-RPA chip. Moreover, our previous work demonstrated that the acquired LOD of 1.6 x 10\u003csup\u003e-5\u003c/sup\u003e ng/\u0026mu;L is sufficient for detecting the \u003cem\u003eHINTW\u003c/em\u003e gene in real extracted gDNA\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003eDevelopment of the SIMPLE-RPA chip\u003c/h2\u003e\n\u003cp\u003eTo implement the RPA bioassay for the first time onto the SIMPLE microfluidic chip, we designed a completely novel SIMPLE-RPA chip as shown in Figure 1, in which the different liquid operations are demonstrated using colored liquids (depicted in Figure 5 with supporting Video Supplementary Material in Section S8). To tackle the needs of the laying hen poultry industry, the chip was designed with a (1) reduced size (\u003cem\u003ei.e.,\u0026nbsp;\u003c/em\u003e13x7 cm) and footprint complying with the limited space inside the egg incubators, (2) fully autonomous sample-to-result operation initiated with a single finger press (Figure 5-1), and hence a possibility for direct implementation within the egg incubator with low chick development disturbances, (3) low sample volume requirements (2 \u0026micro;L), being compatible with a limited volume of the AF sample available at the early stages of chick development, (4) low-cost materials (\u0026lt; 1 \u0026euro;/chip) compatible with scalable production methods (\u003cem\u003ei.e.\u003c/em\u003e, roll-to-roll manufacturing), to meet the high-volume testing requirements in this industrial sector, and (5) colorimetric read-out that facilitates sex detection and sorting, enabling high-throughput \u003cem\u003ein ovo\u003c/em\u003e sexing. The cartridge is activated by a finger press, hereby, bringing the working liquid in contact with the porous filter, leading to its absorption through capillary action.\u003c/p\u003e\n\u003cp\u003eOnce the SIMPLE-RPA chip is activated, 1 \u0026micro;L of the sample is first accurately metered (Figure 5-2), which precludes errors from non-specialized users when introducing the sample. In particular, the coordinated burst action of sc and dcHBVs (purple and red squares in Figure 1, respectively) is used to first meter an exact sample volume in the sample channel, after which the sample excess is discarded to the waste channel. Once the sample excess reaches the liquid barrier at the end of the waste channel (V1 in Figure 5-2), the precisely metered sample volume first blocks the liquid barrier adjacent to that channel (V2) and bursts through de dcHBV (Figure 5-3), merging with the prefilled RPA reagents. The merged liquids proceed towards the first 3D mixing chamber (for more information on the measuring and merging systems, we refer to our previous work\u003csup\u003e20\u003c/sup\u003e). Next, the reagents and sample go through a series of 3D chambers (Figures 5-4 and 5-5), allowing for efficient mixing due to the expansion effect\u003csup\u003e27\u003c/sup\u003e. In the first 3D chamber, the 9 \u0026micro;L of RPA reagents and 1 \u0026micro;L of sample are merged with the 0.5 \u0026micro;L MgOAc (280 mM), thereby initiating the RPA reaction. Adding the MgOAc only after the sample is introduced allows for accurately controlling the start of the RPA reaction, avoiding false positives or unwanted amplification. Moreover, MgOAc integration in the first 3D mixing chamber of the chip enables simple storage and addition, contrary to what was observed in RPA implementation in other platforms\u003csup\u003e17\u003c/sup\u003e, where this step required extra handling, thereby increasing the complexity of their devices. Furthermore, incubation and mixing happen simultaneously, which has been proven to increase the sensitivity in previous reports\u003csup\u003e14,28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe four 3D chambers and the presented length of meandering channels, used for the incubation of the mixture, were adequate to reach 15 minutes of incubation with the SIMPLE-RPA chip and to have a fully mixed solution with a homogenous color at the fourth 3D mixing chamber, as shown in Figure 5-6. Next, we optimized the time the liquids reside in the channels to match the necessary 15 minutes of incubation. To achieve this, one option was to increase the number of 3D mixing chambers or length of the meandering channels, which, however, increased the amount of sample loss, retained by the hydrophilic PET surface (data not shown), thus hindering the RPA reaction. Hence, to avoid altering the already optimized chip design, we introduced here a completely novel way of controlling the on-chip incubation time when using the SIMPLE microfluidic chip. By increasing the working liquid\u0026rsquo;s viscosity with the addition of PVA, flow rates could be altered, which was very practical since the SIMPLE-RPA chip design could be kept constant throughout the experiments. The Lucas-Washburn equation\u003csup\u003e29\u003c/sup\u003e shows the inverse proportionality between the wetted distance of a porous media over time and the liquid viscosity (Supplementary Material Section S9). After testing increasing concentrations of PVA in the working liquid (Figure S6 from Supplementary Material shows different timings considering different amounts of PVA), we observed that 4 % (w/v) of PVA enabled an incubation time of approximately 15 minutes to match the requirements of the optimized RPA (the obtained timing can be seen in Video S8 from Supplementary Material).\u003c/p\u003e\n\u003cp\u003eFollowing the incubation in the 3D mixing chambers and meandering channels, the amplified DNA reached and stopped at a scHBV (which prevented the loaded LFS buffer from flowing in the microfluidic channels during prefilling). At the same time, the amplified DNA blocked the liquid barrier adjacent to the channel (Figure 1B and Figure 5-7). Once the last liquid barrier was blocked, the amplified DNA bridged the scHBV to merge with the 40 \u0026micro;L of LFS buffer (Figure 5‑7), after which it was drawn over the dummy LFS inserted within the SIMPLE-RPA chip \u0026nbsp;(Figure 5-8). Once the LFS buffer mixed with the RPA product was absorbed into the LFS sample pad, the SIMPLE pump did not exert any more pulling force on the liquid (as airflow can pass by above the liquid).\u003c/p\u003e\n\u003cp\u003eThe presented SIMPLE-RPA chip allows for fully autonomous on-chip implementation of the RPA assay without user dependency, which drastically \u0026nbsp;reduces the risk of user errors, in less than 30 minutes. To perform all the steps in a reproducible manner, all the microfluidic structures had to be properly sealed, which was one of the biggest challenges. Moreover, because of the manual fabrication, slight differences (\u003cem\u003ee.g.\u003c/em\u003e, differences in metered samples) were sometimes observed, which, however, did not drastically influence the results (\u003cem\u003ee.g.\u003c/em\u003e, bioassay time or intensity of the bands) and can be further prevented by automating the chip fabrication process. Importantly, the chips provide a decreased risk of contamination from the environment and cross-contamination since the closed system reduces the chance of aerosol formation. Finally, even though the combination between the liquid barriers and dc/scHBVs allowed for complex liquid manipulations, introducing further complexity (such as other microfluidic components or multiplexing) within the platform would allow for an even greater degree of autonomy, thus bringing huge advantages to the field of autonomous POC devices\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eOn-chip RPA integration for synthetic \u003cem\u003eHINTW\u003c/em\u003e DNA detection\u003c/h2\u003e\n\u003cp\u003eTo test the SIMPLE-RPA chip performance, the previously optimized off-chip bioassay was implemented on the chip. The RPA master mix was prepared off-chip, from which 9 \u0026micro;L was loaded into each chip (matching the off-chip reaction volume), while 40 \u0026micro;L of the LFS buffer was directly inserted in the first 3D mixing chamber from the detection unit and MgOAc (0.5 \u0026micro;L, 280 mM) was inserted in the first 3D mixing chamber from the sample processing unit (Figure 1). Once the reagents were loaded and the SIMPLE-RPA chip was properly sealed, the same serial dilution (as off-chip) of the \u003cem\u003eHINTW\u003c/em\u003e synthetic DNA target in IDTE buffer was tested with the on-chip RPA in triplicate. NTC was also included by replacing the \u003cem\u003eHINTW\u003c/em\u003e synthetic DNA with 2 \u0026micro;L DI water. Because our data showed that using an egg incubator as a heating source resulted in equal if not better results compared to the PCR thermocycler (Figure 4), the chips were left inside the egg incubator until the formation of the control line and the absence of liquid in the chip. Band readout was done by the naked eye and subsequently, the chips were removed from the incubator to complement visual read-out with the tabletop scanner for intensity of test band quantification (Figure 6), as described earlier.\u003c/p\u003e\n\u003cp\u003eFigure 6A shows images of the SIMPLE-RPA chips after the finished bioassay, revealing a relevant test band for concentrations ranging from 0.05 to 8 x 10\u003csup\u003e-5\u0026nbsp;\u003c/sup\u003eng/\u0026micro;L with no statistically significant differences among each other (Figure 6B; Tukey\u0026rsquo;s test for multiple comparisons, \u0026alpha; = 0.05). However, a concentration of 1.6 x 10\u003csup\u003e-5\u003c/sup\u003e ng/\u0026micro;L presented a dimmer band similar to the NTC, further supported by the lack of statistically significant difference between them, shown in Figure 6B. Hence, 8 x 10\u003csup\u003e-5\u0026nbsp;\u003c/sup\u003eng/\u0026micro;L was considered the LOD of the SIMPLE-RPA chip, which was the same for the off-chip RPA when using PCR thermocycler (Figure 4A) but was slightly lower when using the egg incubator (Figure 4B) as a heating source. In this context, it is relevant to note that PET transparency allows for non-obstructed visualization of the band, which is easily identifiable by a human user as well as by a camera.\u003c/p\u003e\n\u003cp\u003eThis was the first time a complete SIMPLE‑RPA chip was presented with a sample-in-result-out workflow, which was achieved by using \u003cem\u003eHINTW\u003c/em\u003e synthetic DNA in IDTE buffer. By further optimizing the RPA bioassay for detection of \u003cem\u003eHINTW\u003c/em\u003e gene directly in the AF sample, this technology can be used for male embryo detection in laying hen hatcheries\u003cem\u003e.\u003c/em\u003e As it was proven in our recent work\u003csup\u003e13\u003c/sup\u003e, \u003cem\u003eHINTW\u003c/em\u003e in the gDNA extracted from AF of embryos incubated between days 6 and 9 allowed distinguishing the sexes with 100 % accuracy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCurrently, \u003cem\u003ein ovo\u003c/em\u003e sexing is the preferred solution from consumers and industry to solve male chick culling. However, for \u003cem\u003ein ovo\u003c/em\u003e sexing technologies to be applied in the industry, strict requirements must be followed (such as compatibility with all colors of eggs, high throughput, high accuracy, high sensitivity, maintaining a high hatchability rate and low cost\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e), which are currently unmet. To meet these requirements, we developed a microfluidic chip integrated with an RPA bioassay, namely the SIMPLE-RPA chip that offers great prospect to be used for \u003cem\u003ein ovo\u003c/em\u003e sexing. This innovative chip autonomously amplifies the female-specific \u003cem\u003eHINTW\u003c/em\u003e synthetic DNA within the egg incubator, possibly avoiding to impact the embryos\u0026rsquo; development and hatchability due to a reduced incubation disturbance. The RPA bioassay was first optimized off-chip regarding primer concentration and incubation time, achieving a high sensitivity that matched the necessary LOD for detecting \u003cem\u003eHINTW\u003c/em\u003e in real samples. The off-chip optimized bioassay was subsequently integrated with the SIMPLE chip without an effect on the LOD, achieving the result in only 30 minutes, while using only the heating from the egg incubators to run RPA at 37.7\u0026deg;C. Because of its autonomous operation, almost no handling from the user is required, thus decreasing contamination risk. Importantly, to control the RPA incubation time of the SIMPLE-RPA chip, the working liquid viscosity properties were manipulated by adding PVA, which proved to be a practical and robust approach in this context.\u003c/p\u003e \u003cp\u003eThe presented SIMPLE-RPA chip has a small footprint, is built with low-cost materials, and can be produced in large numbers in an automated fashion. Moreover, the choice of RPA as an amplification method offers high sensitivity and specificity for target detection, while also allowing for an integrated colorimetric read-out with an LFS, thus enabling easy results interpretation by the naked eye, which is more practical than fluorescence-based alternatives. RPA also provides the possibility of lyophilization\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e for improved shelf life. As such, this kind of technology can be easily integrated into the hatcheries, as seen in Respeggt, GmbH\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e or In Ovo, B.V.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, where automated lines for sampling and testing AF samples were implemented.\u003c/p\u003e \u003cp\u003eHowever, for a fully integrated solution, further advancements are expected, such as sample extraction and processing, as well as touchless activation outside the incubators. In addition, important improvements are expected in providing LFSs and RPA assay components by commercial suppliers at more competitive prices or through in-house production.\u003c/p\u003e \u003cp\u003eAlthough the presented SIMPLE-RPA chip has been developed for \u003cem\u003ein ovo\u003c/em\u003e sexing application, by simply changing the primers in the RPA reaction, other sequences or pathogens can be easily detected, such as sexually transmitted (\u003cem\u003ee.g.\u003c/em\u003e, HIV or HPV), respiratory diseases (\u003cem\u003ee.g.\u003c/em\u003e, SARS-Cov 2 or \u003cem\u003eInfluenza\u003c/em\u003e), or even food pathogens (\u003cem\u003ee.g.\u003c/em\u003e, \u003cem\u003eE. Coli\u003c/em\u003e or \u003cem\u003eS. Enterica\u003c/em\u003e). Moreover, this platform shows high potential to be applied in the various POC settings by untrained users with distinct heat sources ranging from egg incubators, like in this work, to even hot plates or heating pads.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Flemish environment department, the Research Foundation \u0026ndash; Flanders [1S54823N and S003923N], Flanders Innovation \u0026amp; Entrepreneurship \u0026ndash; VLAIO (HBC.2022.0710) and the European Union (FORTIFIEDx and DECIPHER). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the granting authority European Union\u0026apos;s Horizon Europe research and innovation programme. Neither the European Union nor the granting authority can be held responsible for them. The FORTIFIEDx and DECIPHER projects ha received funding under the Horizon Europe research and innovation programme (grant agreement No 101092049 and 101137242, respectively).\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eS. M. B. D. S. \u0026ndash; Conceptualization, investigation, methodology, funding acquisition, formal analysis, writing original draft; C. W. \u0026ndash; Investigation, methodology, reviewing and editing; M. C. \u0026ndash; Conceptualization, funding acquisition, reviewing and editing; B. D. K. - Conceptualization, funding acquisition, reviewing and editing; D. V. \u0026ndash; Conceptualization, reviewing and editing; D. S. \u0026ndash; Conceptualization, funding acquisition, reviewing and editing; J. L. \u0026ndash; Conceptualization, funding acquisition, reviewing and editing.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eAdditional information\u003c/p\u003e\n\u003cp\u003eSupplementary information\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBruijnis, M. R. N., Blok, V., Stassen, E. N. \u0026amp; Gremmen, H. G. J. Moral \u0026ldquo;Lock-In\u0026rdquo; in Responsible Innovation: The Ethical and Social Aspects of Killing Day-Old Chicks and Its Alternatives. \u003cem\u003eJ. Agric. Environ. Ethics\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 939\u0026ndash;960 (2015).\u003c/li\u003e\n \u003cli\u003eDeutscher Bundestag. 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Respeggt group, GmbH. https://www.respeggt.com/ (2024).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"03a69e54-d193-4308-9881-994818d5d28d","identifier":"10.13039/501100003130","name":"Fonds Wetenschappelijk Onderzoek","awardNumber":"1S54823N","order_by":0},{"identity":"91132b95-bb27-4522-bf1b-bb09c83d7594","identifier":"10.13039/501100003130","name":"Fonds Wetenschappelijk Onderzoek","awardNumber":"S003923N","order_by":1},{"identity":"c4e5054a-dcde-4920-aba5-16af7fbe1a85","identifier":"10.13039/100012331","name":"Agentschap Innoveren en Ondernemen","awardNumber":"HBC.2022.0710","order_by":2},{"identity":"0e05b935-aeb8-4316-b620-47bb476504c3","identifier":"10.13039/100010661","name":"Horizon 2020 Framework Programme","awardNumber":"101092049","order_by":3},{"identity":"79c29eed-1f46-4951-bb1d-1bde62586002","identifier":"10.13039/100010661","name":"Horizon 2020 Framework Programme","awardNumber":"101137242","order_by":4}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"KU Leuven","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":"SIMPLE, RPA, in ovo sexing, isothermal DNA amplification, microfluidic platform, lab-on-chip","lastPublishedDoi":"10.21203/rs.3.rs-5123850/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5123850/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeveral European countries have implemented new legislations to eliminate the killing of day-old male chicks, pushing the laying hen industry to find animal-friendly solutions. Although embryo sexing during incubation (\u003cem\u003ein ovo\u003c/em\u003e sexing) is highly promising, no current solution meets the industry requirements of handling all egg colors with \u0026gt;98\u0026nbsp;% sexing accuracy, low cost and minimal embryo disturbance, while allowing sexing before day 13 of incubation and processing \u0026gt; 20 000 eggs/hour. Recombinase polymerase amplification (RPA) presents a promising alternative to PCR as a sensitive isothermal technique to be integrated into a microfluidic platform. In this work we developed a fully autonomous microfluidic cartridge (SIMPLE-RPA chip) for chick female-specific synthetic \u003cem\u003eHINTW\u003c/em\u003e gene detection in 30 minutes at 37.7 °C inside an egg incubator. We first optimized off-chip RPA, allowing for highly sensitive DNA detection (1.6 x 10\u003csup\u003e-5\u003c/sup\u003e ng/µL), matching expected concentrations in allantoic fluid samples. Then, the SIMPLE-RPA chip was developed to accommodate and automate the RPA bioassay on-chip, requiring only sample introduction and finger press activation to reduce user errors and contamination risks. Importantly, the SIMPLE-RPA chip maintained the LOD achieved off-chip. The presented SIMPLE-RPA chip is inexpensive, has a small footprint, is compatible with upscaling, and can be easily transferred to other point-of-care applications.\u003c/p\u003e","manuscriptTitle":"Fully automated sample-to-result SIMPLE-RPA microfluidic chip: towards in ovo sexing application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-24 08:58:59","doi":"10.21203/rs.3.rs-5123850/v1","editorialEvents":[{"type":"communityComments","content":0}],"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":"ebbbf300-9fbc-4247-8eb5-f49ae306cab3","owner":[],"postedDate":"September 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":37993998,"name":"Agricultural Engineering"},{"id":37993999,"name":"Food Science \u0026 Technology"},{"id":37994000,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2024-09-24T08:58:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-24 08:58:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5123850","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5123850","identity":"rs-5123850","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}