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

Primers and synthetic DNA

Starting from the primers used in our previous work for developing the qPCR¹³, the primers in this work (Table 1), were produced by IDT (Leuven, Belgium), delivered in IDTE buffer (pH = 7) and were used without further processing, while all the dilutions were done using distilled water. The primers 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 RPA liquid basic kit and HybriDetect – Universal Lateral Flow Assay Kit strips purchased from TwistDx Limited (Maidenhead, UK). The synthetic DNA was purchased as double stranded DNA (geneBlock) in IDTE buffer (pH = 7).

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 TwistDx²³. 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 NTC and 0.5 µL of MgOAc (280 mM) were added to the tube lids. Once the tubes were closed, they were inverted 4 times and spun down for 5 s using a tabletop centrifuge at 6000 rotations per minute (rpm), i.e., at 2000 g. 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 min) 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, Supplementary Fig. 6) with two lines for colorimetric detection (i.e., test and control) were immersed into this solution, and the colorimetric results were visualized 10 min 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 Supplementary Fig. 6.

Images of the LFSs were captured using a fi-65F Fujitsu (Tokyo, Japan) flat desk scanner at 10 min 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 Eq. 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 (Eq. 1). The SNR was calculated according to Eq. 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 min, spun at 6000 rpm (2000 g) for 1 min 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 swiftly to ice for further manipulation. To avoid possible contamination sources in thermocycler and egg incubator testing, the equipment was washed with ethanol between tests, complemented with the use of sterilized material (i.e., gloves, PCR tubes, pipettes, pipette tips). 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 min 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^21,25. More specifically, the microfluidic network (Supplementary Fig. 4) was designed using Inkscape vectorial software (Version 1.3.2, Software Freedom Conservancy, USA) and fabricated using a manual layer-by-layer lamination method. Moreover, 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 3 M (Minnesota, USA). 125 μm thick PET was purchased from Pütz GmbH (Taunusstein, Germany), 1 mm thick PMMA layers from Pyrasied (Leeuwarden, The Netherlands), Versapor™ acrylic copolymer 3000 membrane for the liquid barriers from Cytiva (DC, USA), and Whatman Grade 3 paper from Sigma-Aldrich (Overijse, Belgium). These materials were cut using an 80-watt Speedy300 Trotec laser cutter (Trotec Lasers, Austria). Figure 4 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 (Fig. 4A).

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 Fig. 4A) contained the porous material (Whatman Grade 3), inserted into its chamber during assembly and secured with transfer tape. The bottom layer (V in Fig. 4A) 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 Fig. 4A) 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: HBVs and liquid barriers. HBVs were made in the sample processing unit top layer (II in Fig. 4A) following a mask-based strategy, based on a previous report²⁰. In short, both top and bottom or only bottom PET films were locally coated with 2 µL of hydrophobic solution Fluoropel 800 purchased at Cytonix (Maryland, USA) to create dc or scHBVs, respectively. After, the PET films were allowed to dry for 1 h at room temperature and masks were removed. The bottom layer of the sample processing unit (III in Fig. 4A) 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 Fig. 4B, C. Moreover, only the 3D mixing chambers span two layers (i.e., going through the first and second layers of the sample processing unit; Fig. 4A). 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 report²⁰. 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 described³⁰. The working liquid comprised a blue microfluidic dye obtained from Darwin Microfluidics (Paris, France) diluted in distilled water (1:20) mixed with 4% (w/v) PVA obtained from Sigma-Aldrich (Overijse, Belgium). 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 (Fig. 4A). 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 Video 2. 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 × 10^–5 ng/μL. For testing the SIMPLE-RPA chip, 2 µL of HINTW synthetic DNA sample in IDTE buffer or 2 µL of DI-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.

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• Source: https://www.nature.com/articles/s44328-024-00019-6