Digital assay for rapid electronic quantification of clinical pathogens using DNA nanoballs

Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, β-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).


SARS-CoV-2 clinical samples
We obtained anonymized or pseudo anonymized surplus aliquots from 30 SARS-CoV-2 positive and 10 negative nasopharyngeal samples that had previously been clinically diagnosed for COVID-19 by RT-PCR in early February 2022 by demand of the Public Health Agency of Sweden.Specimens, originating from central Sweden, were collected in a fixed volume of 1 mL physiological saline (0.9% NaCl) and inactivated by heat (70°C for 50 min) upon arrival to the laboratory, and subsequently subjected to extraction-free SARS-CoV-2 RT-PCR.Samples were stored at 4 °C prior to use.The SARS-CoV-2 RT-PCR assay was an improved multiplex version of the extraction-free protocol developed by Smyrlaki et. al. (8) with increased sample input and reaction volume and increased sensitivity (29).For each reaction, 24 uL RT-PCR master mix was prepared, containing 7.5 μL TaqPath 1-Step RT-qPCR Master Mix, CG (Thermo Fisher, containing ROX as passive reference), 0.9 μL 10% Tween20 (Sigma), N1 primer-probe mix (forward: GACCCCAAAATCAGCGAAAT, reverse: TCTGGTTACTGCCAGTTGAATCTG, probe: FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1, Integrated DNA Technologies), S primer-probe mix (forward: ATATTCTAAGCACACGCCTATTATAG, reverse: CTACCAATGGTTCTAAAGCCGAA, probe: Cy5-GAGCCAGAAGATCTCCCTCAGGGT-BXQ2, Merck), RNaseP primer-probe mix (forward: AGATTTGGACCTGCGAGCG, reverse: GAGCGGCTGTCTCCACAAGT, probe:HEX-TTCTGACCTGAAGGCTCTGCGCG-BHQ1, Merck; detecting the Omicron BA.1 sub lineage), and nuclease free water up to 24 uL.Primer/Probe concentrations in the final reactions were 246/62 nM (N1), 491/125 nM (S), and 122/37 nM (RNaseP).For RT-PCR testing, 6 μL heat-inactivated nasopharyngeal swab sample (in 0.9% NaCl) was added to optical 96-well PCR plates (EnduraPlate, Applied Biosystems) containing 24 μL master mix.RT-PCR was performed on QuantStudio real-time PCR machines (Applied Biosystems) using the QuantStudio Design &amp; Analysis Software v1.5.2 and temperature cycles: 25 °C for 2 min, 50 °C for 10 min, 95 °C for 2 min, and 40 cycles of 95 °C for 3 s and 56 °C for 30 s. RT-PCR CT values are available in Table SYZ.Informed consent for the use of anonymized/ pseudo anonymized surplus aliquots obtained in routine clinical diagnostics was not obtained and not required, which is in accordance with the study permit obtained by the Swedish Ethical Review Authority (Dnr 2020-01945 and 2022-01139-02, Etikprövningsmyndigheten). All SARS-CoV-2 positive samples (n=30) used in our study had their viral genome sequenced using the Illumina COVIDSeq Test kit (Illumina) (See Table S3.for variant classifications,and Lentini et. al. (29) for details regarding sequencing and data analysis).

Fluorescent Microscopy imaging
To verify the production of DNA Nanoballs from our modified RT-LAMP reaction we subsequently imaged the products on a Nikon ECLIPSE Ti inverted research microscope using Plan Apo I 100x oil Ph3 DM (1.45 NA).1-2ul per sample was pipetted onto a glass slide, allowed to air dry and a cover slip applied.5-6-FAM (Fluorescein) fluorescence based images were captured using excitation and emission filters for GFP.Additionally, 1ul of the 1uM Dynabeads™ MyOne™ Streptavidin T1 (Thermo Fisher Scientific, Waltham, MA, USA) was imaged as a size reference.

Microfluidic chip
The microfluidic chip is made of PDMS on a glass surface with integrated gold electrodes.The first step for the formation of the microfluidic chip is patterning and fabricating the electrodes on the glass wafer.Electrodes are fabricated on glass using standard photolithography on a 3″ fused silica wafer.The process consists of photo-patterning resist on the fused silica wafer, electron beam metal evaporation, and liftoff processing.The process of photo-patterning includes wafer cleaning, spin coating the photoresist, soft bake of the resist, ultraviolet light exposure through a chromium mask printed on a 4″ × 4″ glass plate, resist development, and hard bake of the resist.Following the photo patterning process, a 100-nm-gold layer is deposited on the substrate using electron beam evaporation.A 10-nm layer of chromium is used to enhance the adhesion of gold to the glass wafer; otherwise, the gold film gets peeled off easily.We chose gold as the electrode due to its resistance to corrosion and its inert nature.The width of the electrodes was 20 μm and spacing between the two electrodes was 20 μm.
We fabricated the microfluidic channel itself in PDMS (Poly-dimethylsiloxane) by using soft lithography.A layer of SU-8 was patterned onto a 3″ Silicon wafer that acts as a master mold.The SU-8 photo-patterning process involves standard cleaning, spin coating, soft baking, exposure, development, and hard baking.After the master mold was fabricated, PDMS (10:1 prepolymer/curing agent) was poured onto the master mold and baked at 80°C over 2 h for curing.The PDMS channel was then peeled off from the mold.A 5-mm hole and a 3-mm hole were then punched to form the inlet and outlet, respectively.The PDMS substrate was then aligned and bonded to the electrode chip after both substrates have undergone oxygen plasma treatment.The bonded chip was then baked at 70 °C for 40 min to form the irreversible bond.Our microfluidic channel had a width of 20 μm and height of 15 μm.

Optimization of Electrical parameters of the Impedance Spectroscope
Multiple configurations of the electrical parameters of the impedance spectroscope were tested for optimizing the detection of the DNA nanoballs.We performed experiments by passing Dynabeads™ MyOne™ in 1 x Phosphate Buffered Saline solution (PBS) for 10 minutes and then recording the response.The data was analyzed for baseline voltage, the signal-to-noise ratio, the number of beads detected, and the peak amplitude.All the measurements were done in the Faraday cage to reduce noise and interference from external sources.The electrical parameters were changed in a progressive fashion.We first increased the excitation voltage.The increase in excitation voltage results in a better signal.However, the electrodes break down at very high voltage and therefore we progressively increased the voltage until it reached the maximum allowable limit.We also changed the transimpedance gain, the bandwidth, and the excitation frequency to find the optimal electrical parameters for the detection of 1 µm particles in the microfluidic chip.Although we optimized the parameters for the detection of 1 µm beads, experiments showed that there was a similar improvement for detection of the DNA nanoballs.

Fig. S3 :
Fig. S3: Simplified outline of full experimental protocol.A 60min RT-LAMP reaction using target specific LAMP primers and compaction oligos produces DNA Nanoballs which are subsequently detected.

Table S2 . Various configurations for electrical parameter optimizations.
Configuration 6 yielded the best results.Results are shown in Figure S1.