Key words

1 Introduction

Owing to their high sensitivity and specificity, nucleic acid amplification tests (NAATs) have become the preferred methods for many clinical diagnostics, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1, 2], human immunodeficiency virus (HIV) [3, 4], tuberculosis [5, 6], Chlamydia trachomatis and Neisseria gonorrhoeae infections [7, 8]. These tests involve a sample preparation step to extract and purify nucleic acids (DNA or RNA) from complex biological matrices (e.g., blood, urine, feces, nasopharyngeal/oropharyngeal swabs, and genital track swabs), prior to performing amplification and detection of the amplicons to obtain results. Although commercially available kits can be used, multiple pipetting, centrifugation, and washing steps are usually required [9]. This time-consuming, labor-intensive, and contamination-prone process prohibits the full potential use of NAATs for clinical diagnostics, especially in resource-constrained settings.

Utilizing microscale gates in combination with the surface tension properties of immiscible liquids (e.g., oil-aqueous), the immiscible filtration assisted by surface tension (IFAST) method offers an elegant way to perform sequential and multiple assays in one single device [10, 11]. Comprising a larger sample chamber (0.5–1 mL) and a series of smaller wash chambers (20–120 μL) interconnected via microscale trapezoidal gates (100–200 μm deep), fluidic barriers between immiscible liquids can be created, allowing side-by-side liquid compartmentation in credit-card size devices [10,11,12,13,14,15,16]. Filtration of analytes of interest from complex matrices within the sample chamber is achieved by the use of a permanent magnet to manipulate functionalized superparamagnetic particles across the immiscible barriers into a separate aqueous solution [12]. This rapid and robust microscale purification (< 1 min) eliminates extra essential steps for extraction of analytes from biological samples prior to detection [11].

IFAST has seen numerous applications in sample preparations for specific isolation of nucleic acids [10, 11, 13, 17,18,19], bacteria [14, 15], and rare cells [12] from various biological samples. To take advantage of the IFAST platform further, a detection chamber within the same device has been introduced after purification steps to allow for sample-in to answer-out analyses. Our group combined the specificity of the IFAST with the sensitive ATP assays for detections of Escherichia coli from wastewater [15], and Streptococcus agalactiae from urine samples [14], allowing rapid detection of bacteria within 20 min. This provides a promising platform for point-of-need pathogen analyses to replace the gold standard cell culture method which requires 24–48 h to obtain results. Additionally, all-in-one IFAST devices for extraction, amplification, and detection of nucleic acids were also reported, exploiting the commercially available isothermal amplification kits for nucleic acids by Shaw’s group for DNA identification of rare rhinoceros species [19], and by our group for SARS-CoV-2 RNA detection [11]. These robust platforms enable sample preparation and nucleic amplification tests to be integrated in one device, facilitating point-of-need/point-of-care testing in resource-limited settings.

Here, we discuss the application of the microfluidic IFAST platform as an example of an all-in-one device for RNA isolation, amplification, and detection for COVID-19 diagnostics (Fig. 1). However, the device is also largely applicable to other nucleic amplification assays for various pathogen analyses.

Fig. 1
3 schematic. 1. R N A capture. Oligimagnetic bead, SARS COV-2 R N A, and contaminants are visualized. 2. R N A isolation. Labels are aqueous phase, oil phase, and magnet. 3. R T LAMP detection. Negative and positive results are revealed.

Schematic demonstrating on-chip workflow for RNA isolation and detection utilizing immiscible liquids and functionalized magnetic particles, allowing sequential steps to take place in one device

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2 Materials

Prepare all reagents related to nucleic acid amplification using nuclease-free water, and wash buffer and reagents for gel electrophoresis with purifying deionized water with a sensitivity of 18 MΩ-cm at 25 °C. Prepare and store all reagents at room temperature unless otherwise stated. Reagents purchased should be stored following the assay instruction manuals. Waste disposal regulations should be diligently followed when disposing waste materials.

2.1 Biomolecules, RT-LAMP Reagents, Magnetic Particles, and Immiscible Liquids

  1. 1.

    Genomic RNAs: SARS-CoV-2 RNA (2019-nCoV/USA-WA1/2020, ATCC VR-1986D), (LGC standards, UK) (see Note 1).

  2. 2.

    RT-LAMP assay kit: WarmStart® DNA and RNA polymerase (MS1800S, NewEngland Biolabs) (see Note 2).

  3. 3.

    LAMP primers (Integrated DNA Technology) (see Note 2).

  4. 4.

    Superparamagnetic particles: Dynabeads Oligo dT(25) (Invitrogen, UK) (see Note 3).

  5. 5.

    Wash buffer: Phosphate buffer saline solution containing 0.005% Tween20 (PBST). Dissolve a PBS tablet in 200 mL water. Add 2.5%w/v Tween 20 to afford a final concentration of 0.005%w/v. Store at 4 °C (see Note 4).

  6. 6.

    Mineral oil (see Note 5).

2.2 Microfluidic Chip Design and Fabrication

  1. 1.

    In a suitable design software (e.g., AutoCAD, SolidWorks), reproduce the chip design shown in Fig. 2a. The chip design features a large sample chamber 1 (26 mm wide, 26 mm long); wash chambers 2, 4, 6 (3 mm wide, 3 mm long); wash chamber 3 (3 mm wide, 14 mm long); wash chambers 5, 7, 8 (3 mm wide, 8.5 mm long); and a detection chamber 9 (3 mm wide, 3 mm long). All chambers were interconnected via gates (3 mm to 0.5 mm wide, 3 mm long).

  2. 2.

    Fabricate the chip design by CNC machine milling (Daltron M7, Milton Keynes) using a polymethyl methacrylate (PMMA) sheet (Kingston Plastics), to a depth of 3.8 mm for all chambers, and 0.2 mm for the gates (Fig. 2b) (see Notes 6 and 7).

Fig. 2
A. An illustration of a 3-D model of the microfluidic device. B. A photograph of the Polymethyl methacrylate. D. Photograph of 2 sample tubes and 2 strings. D. A photograph of a hotplate required for on-chip assays.

(a) Design of the microfluidic device, featuring a large sample chamber (1); wash chambers (2–8), and a detection chamber (9), interconnected via gates. (b) Photograph of the device fabricated in PMMA. (c) All elements required for on-chip assays: 1 = micropipette tips, 2 = 0.005% Tween 20, 3 = mineral oil, 4 = oligo d(T) magnetic beads, 5 = RT-LAMP substrate, 6 = micropipettes, 7 = PMMA device, 8 = magnet assembly, 9 = adhesive tape. (d) A hotplate set at 65 °C was used as a heating source for on-chip assay. (Reprinted from Rodriguez-Mateos et al. [11], with permission from Elsevier)

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2.3 On-Chip Assay Setup

Figures 2c, d illustrate the elements required for on-chip assays.

  1. 1.

    Adhesive film for device sealing: Adhesive PCR plate seals (Thermofisher Scientific).

  2. 2.

    Neodemium-iron-boron (NdFeB) magnet (Magnet Sales) (see Note 8).

  3. 3.

    Guanidine hydrochloride (GuHCl) solution (Sigma-Aldrich): Dissolve GuHCl in PBST to afford a final concentration of 5 M. Keep at room temperature (see Note 9).

  4. 4.

    Inverted microscope (e.g., Nikon Eclipse Ti) with a suitable camera (e.g., Nikon DS-Fi3) and image acquisition software (e.g., NIS-Elements Freeware (F)) (see Note 10, Fig. 3).

  5. 5.

    RNase surface decontamination solution: RNase Away Surface Decontaminant (Thermo Scientific) (see Note 11).

  6. 6.

    Blu-Tack (Bostik).

  7. 7.

    Heating source, e.g., hot plate (Stuart) or incubator.

Fig. 3
Left. An illustration of a device used for amplification. The labels are aqueous, oil, and R T lamp substrate. Middle and Right. 2 micrographs. Left. Oil, interface, and oligo d t beads are marked. Right. R T-Lamp mix is marked.

Microscope images of interfaces between mineral oil and RT-LAMP reagents at 65 °C at 0- and 50-min incubation time. Scale bar = 1 mm. (Reprinted from Rodriguez-Mateos et al. [11], with permission from Elsevier)

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2.4 Agarose Gel Electrophoresis

  1. 1.

    Tris Acetate-EDTA (TAE) buffer: Dilute 20 mL of 50× TAE buffer stock (Sigma-Aldrich) into 980 mL water. Store at room temperature.

  2. 2.

    Agarose gel: add agarose (Melford) into 1× TAE buffer to afford a final concentration of 1%w/v.

  3. 3.

    DNA gel stain: Add appropriate volume of 10,000× SYBR-safe DNA gel stain (Invitrogen) into melted agarose prior to casting.

  4. 4.

    DNA loading dye (DNA Gel Loading Dye, Thermofisher Scientific).

  5. 5.

    DNA ladder (GeneRuler DNA Ladder Mix, Thermofisher Scientific).

  6. 6.

    DNA electrophoresis setup (Mini-Sub Cell GT Horizontal Electrophoresis System and PowerPac Basic Power Supply, Bio-Rad).

  7. 7.

    Molecular imager (Chemidoc XRS+, BioRAD).

  8. 8.

    Cooking microwave.

3 Methods

It is recommended that the preparation of microfluidic chip (see Subheading 3.1), magnetic beads (see Subheading 3.2), and RT-LAMP reagents (see Subheading 3.3) is performed in a pre-amplification area, separated from the amplification and post-amplification area (see Note 12).

3.1 Preparation of Microfluidic Chip

  1. 1.

    Wipe the chip with RNase decontamination solution and rinse with nuclease-free water. Leave the device to dry (ca. 5 min at room temperature) prior to use.

  2. 2.

    Cut the adhesive film to the same size of the chip perimeter. Gently press the bottom side of chip (microscale gates can be seen from this side) onto the adhesive film.

  3. 3.

    Prepare another piece of adhesive film (ca. 28 mm wide × 28 mm long) with pre-cut hole (ca. 3 mm in diameter) in the middle to cover the sample chamber.

3.2 Preparation of Magnetic Beads

  1. 1.

    Resuspend the magnetic particles in the stock bottle either by shaking or using a vortex mixer.

  2. 2.

    Pipette required volume (20 μL per assay, See Note 13) from bead stock into a 1.5 mL Eppendorf tube.

  3. 3.

    Attach a permanent magnet to the side of the Eppendorf tube using Blu-Tack. Allow 2 min for the magnetic particles to be pulled to the side of the tube.

  4. 4.

    While the magnet is still in place, remove supernatant using a pipette.

  5. 5.

    Add 1 mL PBST to the beads. Agitate/Vortex for 5 s.

  6. 6.

    Attach a NdFeB magnet to the side of the Eppendorf tube using Blu-Tack.

  7. 7.

    While the magnet is still in place, remove supernatant using a pipette.

  8. 8.

    Suspend the washed beads in PBST to the volume originally taken out from the stock bottle.

3.3 Preparation of RT-LAMP Reagents

  1. 1.

    Reconstitute the primers in nuclease-free water according to the instruction manual (see Note 2).

  2. 2.

    Follow the instruction manual to prepare RT-LAMP master mix; make up total volume of 20(x + 1) μL, where x = number of assays to be performed, and one assay requires 20 μL reagent. The master mix contains 16 mM forward inner primer (FIP) and backward inner primer (BIP), 2 mM forward outer primer (F3) and backward outer primer (B3), 4 mM forward loop primer (LF) and backward loop primer (LB), 1× LAMP reaction mix and nuclease-free water.

3.4 On-Chip RNA Capture, Purification, Concentration, Amplification, and Detection (RT-LAMP)

  1. 1.

    Add 2 μL mineral oil to chamber 2.

  2. 2.

    Add 974 μL of 5 M GuHCl in PBST into the sample chamber followed by 25 μL washed Oligo (dT) magnetic beads and gently agitate to disperse magnetic beads inside the liquid.

  3. 3.

    Add 1 μL of RNA into the mix to afford the desired concentration. Always perform on-chip control experiments where RNA is replaced by nuclease-free water.

  4. 4.

    Place a small piece of adhesive film with a small pre-cut hole above the sample chamber to prevent spillage. Agitate the device for 10 min (see Note 14).

  5. 5.

    Alternately fill the wash chambers (2–8) with immiscible liquids: chambers 2, 4, 6 = mineral oil (20 μL) mineral oil; chamber 8 = mineral oil (50 μL); chamber 3 = PBST (100 μL); chambers 5, 7 = PBST (50 μL). Fill the detection chamber (9) with 20 μL of RT-LAMP reagent (see Note 15).

  6. 6.

    Use a permanent magnet (see Note 8) to gather RNA-extracted magnetic beads in the sample chamber toward the gate to chamber 2.

  7. 7.

    Use the permanent magnet to move the beads from the sample chamber (1), through wash chambers (2–8), into chamber 9 (see Notes 16–18).

  8. 8.

    Add 10 μL mineral oil onto RT-LAMP reagent in chamber 9. Place the chip device on ice and transfer the chip to the post-amplification area (see Note 19).

  9. 9.

    Heat the device at 65 °C to initiate RT-LAMP reaction for 30–45 min using a preheated hotplate or incubator (see Note 20).

  10. 10.

    Remove the device from heating and allow to cool for result interpretation (see Note 21).

  11. 11.

    Observe color in the detection chamber: pink = negative amplification; yellow = positive amplification (Fig. 4a). The color of the RT-LAMP reagent in the control chip should remain pink.

  12. 12.

    Carefully pipette the amplification product from the RT-LAMP chamber (9) into a PCR tube, and keep on ice until ready for agarose gel electrophoresis.

Fig. 4
A. A photograph reveals the color in the detection chamber. The colors indicate the negative and positive amplification. B. A molecular image presents the bright paired patterns, marked positive and negative.

(a) Negative (−) and positive (+) results from on-chip assays. (b) Confirmation of on-chip results by gel electrophoresis. (Reprinted from Rodriguez-Mateos et al. [11], with permission from Elsevier)

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3.5 Agarose Gel Electrophoresis for Verification of On-Chip Detection Results

  1. 1.

    Add agarose to TAE buffer to afford a concentration of 1%w/v.

  2. 2.

    Dissolve agarose using a microwave (see Note 22).

  3. 3.

    Prepare the gel box (base plate + surrounding rubber molds) and comb.

  4. 4.

    Wait for the melted agarose to cool to ca. 60–65 °C, and add SYBR Safe to the required volume.

  5. 5.

    Pour agarose solution containing SYBR Safe into the gel box, use small pipette tips to remove bubbles, and leave the liquid to set at room temperature. During this time, prepare samples to be analyzed, by adding appropriate volume of DNA loading dye into each sample and keep on ice.

  6. 6.

    Once the gel is set, carefully remove the surrounding rubber molds to reveal the set gel on the base plate. Keep the comb inside the gel.

  7. 7.

    Carefully place the base plate carrying the gel into the gel tank pre-filled with appropriate volume of 1×TAE buffer. Gently remove the comb to reveal the wells into which amplification products will be loaded.

  8. 8.

    Use small pipette tips to load wells with DNA ladders (5 μL) and samples to be analyzed (20 μL per well).

  9. 9.

    Reattach the lid of the gel tank, and connect the power supply to the terminals on the lid. Set the voltage to 80 V and perform electrophoresis for 45 min.

  10. 10.

    Once finished, turn off the power supply. Remove the gel from the tank and wipe off excess liquid.

  11. 11.

    Image the gel (Fig. 4b) using a molecular imager.

4 Notes

  1. 1.

    Upon receipt, aliquot genomic RNAs to a volume required for each use and store at −80 °C. Avoid freeze-thaw cycles that can degrade RNA [20].

  2. 2.

    Follow instructions from manufacturers. Primer mix can be prepared in a large volume and aliquoted to a volume required for each use, and stored at −20 °C.

  3. 3.

    This work used Oligo dT(25) magnetic beads which selectively captured polyadenylated RNA species. However, they are fairly costly and require 2–8 °C storage. These magnetic beads can be replaced by the more economical silica paramagnetic particles. In this case, the selectivity of the on-chip assay will solely rely on the RT-LAMP primer specificity in the subsequent step.

  4. 4.

    Minute quantities of surfactant can be added to aqueous solutions to prevent sticking of magnetic beads to adhesive film used for sealing the chip, provided that the immiscible interfaces are not compromised. In our work, Tween 20 was added (final concentration of 0.005%w/v) in the sample chamber as well as wash buffer. Other surfactants, e.g., TritonX-100, can also be employed [21]; however, immiscible barriers between liquids should be checked under a microscope.

  5. 5.

    Mineral oil was employed as the immiscible phase. However, sunflower or olive oils among others can also be used, provided that they do not interfere with the downstream RT-LAMP assay (chamber 9). The quick way to check is to perform a tube-based assay overlaid with the oil of interest. These types of oils should provide cheaper and more accessible options for resource-constrained settings.

  6. 6.

    Polymer microfluidic devices can be fabricated via techniques such as hot embossing or injection molding to reduce the cost for mass production [22].

  7. 7.

    Polydimethylsiloxane devices with the same design were tested prior to PMMA devices. However, positive amplification to detect color change was longer than 60 min. PMMA devices allowed similar amplification to tube-based assays (30–40 min depending on initial RNA concentrations). Other thermoplastic materials such as polycarbonate can also be used.

  8. 8.

    We have found that a NdFeB magnet assembly worked well with our system. A bar magnet (20 mm × 10 mm × 5 mm) was used for gathering of beads in the sample chamber after incubation with RNA sample. A smaller disc magnet (4 mm diameter × 2 mm height) was used to manipulate the RNA-captured magnetic beads through the gates to the subsequent wash and RT-LAMP chambers. Other strong NdFeB magnets should also work.

  9. 9.

    The compatibility of GuHCl to on-chip assay [11] suggests the possible use for lysis of viral particles in real samples. While 5 M GuHCl (aq) was used in our investigation, the powder GuHCl can also be pre-stored inside the sample chamber [16].

  10. 10.

    The rapid and robust analyte isolation and purification utilizing this device relies on the filtration assisted by the surface tension properties of immiscible liquids. Therefore, it is crucial that the immiscible interfaces (oil aqueous) are kept intact to prevent carry-over of the solution from the previous chamber into the next chamber. When first setting up the device and assay, monitor the placement and stability of the immiscible interfaces while considering the different buffer solutions, temperatures, and incubation times to be employed. This can be observed under an inverted microscope connected to a CCD camera. Figure 3 illustrates the immiscible interfaces between oil and RT-LAMP reagents during 65 °C from start up to 50 min.

  11. 11.

    Clean work surfaces prior to conducting experiments to eliminate RNase and DNA from laboratory surfaces using an RNase/DNA decontamination product.

  12. 12.

    It is advisable to perform experiments in two separate rooms for pre- and post-amplification steps to prevent DNA amplicon aerosol contamination. Change gloves regularly and avoid using the same pipettes for pre- and post-amplification steps.

  13. 13.

    The quantity of magnetic beads used for RNA isolation is dictated by (i) the gate size, which allows traverse of magnetic beads (too many beads can cause blockage in the gates) and (ii) capture efficiency (too few beads might lead to low capture efficiency and difficulty in visualization). The chip design herein can take up to 25 μL oligo(dT) magnetic beads (Thermofisher Scientific). Optimization is recommended for other types of beads.

  14. 14.

    When performing on-chip magnetic capture in the sample chamber, a small piece of adhesive film can be placed on top of the chamber to prevent spillages either during manual or automated mixing. Automated mixing can be achieved in a rotator (Stuart, UK; set at 40 rpm at 10–20° angle). The piece of adhesive tape also helps to avoid evaporation and movement of liquids during amplification. A small hole in the adhesive tape center helps to release building up of pressure during heating.

  15. 15.

    Fill the aqueous wash chambers (3, 5, 7) with solution before their oil counterparts (chambers 2, 4, 6, 8) to prevent oil penetration into adjacent chamber. Care should be taken when filling the RT-LAMP reagent chamber (9) as this contains some surfactant, that may result in overspill into the adjacent oil chambers. This can cause lower color intensity, and thus compromised result interpretation.

  16. 16.

    The integration of an on-chip amplification step after RNA isolation and purification eliminates the pipetting of beads-bound RNA into a separate amplification tube, reducing analyte loss and maintaining analyte integrity.

  17. 17.

    While particle loss can be expected during magnetic manipulation across chambers, this can be largely reduced by: (i) adding appropriate surfactant concentration, (ii) using a smaller magnet for passing through the gates, and (iii) maintaining a slow but steady movement, allowing beads to catch up with the magnet and move together as a group.

  18. 18.

    In rare occasions, small bubbles can appear when loading the aqueous and oil phases in the device chambers. Gently press the adhesive tape from the bottom side around the gate to move the bubble away into the chamber to rise to the surface.

  19. 19.

    The RT-LAMP reagent overlaid with mineral oil (10 μL) in chamber 9 is to prevent evaporation as well as contamination from DNA aerosol after amplification.

  20. 20.

    The block heater/incubator used for heating the on-chip RT-LAMP assay should be placed in the post-amplification area, and set at required temperature (65 °C) prior to performing the assay.

  21. 21.

    Ensure that color readouts are taken at room temperature. RT-LAMP color develops more strongly at lower temperature. Intensification of color can be achieved by placing the device on ice.

  22. 22.

    Care should be taken when preparing agarose gel using a microwave heating. Always use a container with volume ca. two times larger than actual volume to prevent spillage whilst heating.