Portable Paper‐Based Nucleic Acid Enrichment for Field Testing

Abstract Point‐of‐care testing (POCT) can be the method of choice for detecting infectious pathogens; these pathogens are responsible for not only infectious diseases such as COVID‐19, but also for certain types of cancers. For example, infections by human papillomavirus (HPV) or Helicobacter pylori (H. pylori) are the main cause of cervical and stomach cancers, respectively. COVID‐19 and many cancers are treatable with early diagnoses using POCT. A variety of nucleic acid testing have been developed for use in resource‐limited environments. However, questions like unintegrated nucleic acid extraction, open detection systems increase the risk of cross‐contamination, and dependence on expensive equipment and alternating current (AC) power supply, significantly limit the application of POCT, especially for on‐site testing. In this paper, a simple portable platform is reported capable of rapid sample‐to‐answer testing within 30 min based on recombinase polymerase amplification (RPA) at a lower temperature, to detect SARS‐CoV‐2 virus and H. pylori bacteria with a limit of detection as low as 4 × 102 copies mL−1. The platform used a battery‐powered portable reader for on‐chip one‐pot amplification and fluorescence detection, and can test for multiple (up to four) infectious pathogens simultaneously. This platform can provide an alternative method for fast and reliable on‐site diagnostic testing.


Operation of Our POCT Platform 35
More detailed operation processes are shown in the Figure S1. 36 Sample collection: The collected swab or saliva samples are added to lytic buffers for simple 37 lysis at room temperature ( Figure 1a). 38 Paper-based nucleic acid enrichment: About 100 μL of the lysed sample was absorbed with 39 an dropper and added to the sample loading area of the enrichment chip. Nucleic acids were 40 extracted to the binding disc (which was made of the GE Whatman FTA card) through lateral 41 flow. Then the binding disc was washed with anhydrous ethanol to quickly remove residual 42 water. Then 20 μL of anhydrous ethanol is added to the sample loading area, allowing the 43 binding disc to quickly dry in 2 minutes. If the target is RNA, 20 μL oligo(dT)20 (10 μM) is 44 added to the sampling site prior to this step to weaken the RNA capture of cellulose, facilitating 45 the RNA releasing from paper for next amplification. 46 Recombinase polymerase amplification (RPA) reaction: The dried binding disc extracted 47 nucleic acids was transferred to the amplification chamber of the amplification chip. Primers, 48 probes and other RPA reagents in the pre-installed tube were loaded onto the amplification 49 chamber for one-pot reaction. Pre-installed reagent tube with several amplication reagents 50 spacing by air gap. All solutions are separated by air gap and are easily pushed out sequentially 51 using a dropper. The chip was sealed with clay and incubated in the heating chanmber of the 52 portable reader at 40 °C for 20 minutes. Before and after the constant temperature incubation, 53 the steel beads were stirred twice with a magnet stick, which is moved several times from top 54 of the chip to bottom and then back, to mix the system. 55 Result readout: The chip was removed and placed in the detecting chamber and observed 56 under 488 nm blue light excitation. The fluorescence results could be directly visualized from 57 the portable reader and further analyzed by a smartphone application ( Figure S2

Optimization of Paper-based Sample Pretreatment 72
In view of the unstable effect of the paper-based nucleic acid enrichment method extract 73 RNA from low concentration samples (< 10 4 copies/mL) ( Figure S4a, b), we first considered 74 the purification of RNA inhibitors in the sample. We tried washing the binding disc with the 75 FTA purification reagent to reduce the influence of RNase and amplification inhibitors, but the 76 fluorescence signal intensity did not improve ( Figure S4c). Then we try to add an FTA Elute 77 card before the FTA card in the flow channel ( Figure S5). As the sample fluid flows through 78 the FTA Elute card, the repressor proteins are tightly bound, and the RNA is captured by the 79 FTA card downstream, to further purify RNA. However, the amplification signal intensity was 80 not improved either ( Figure S4d). 81 Cellulose can strongly capture the nucleic acids and retain them following purification steps 82 [1] .Typical RNA amplification requires an additional elution step to release RNA from the paper 83 into the amplification solution [2] . Considering the omitted elution step and the small amount of 84 target RNA in our procedure, RNA may be strongly captured inside the paper and difficult to 85 be released into the solution. Thus, it may be insufficient to trigger the next amplification. We 86 designed the passivation steps by BSA and oligo nucleic acids to pre-occupy the cellulose 87 and/or to weaken the RNA binding via competition, effectively facilitating the RNA releasing 88 from paper for the next amplification step. Through repeated experiments, we found that the 89 FTA card was blunted by pre-soaking with 1% BSA. After RNA extraction, the remaining 90 nucleic acid binding sites in FTA card were blocked by single stranded oligo(dT)20 (10 μM), 91 weaken the RNA capture of cellulose, facilitating the RNA releasing from paper for next 92 amplification. This treatment method could stably detect 10 4 copies/mL of RNA in samples 93 with low concentration (Figure 2b,c). And pre-passivation with higher concentration (10%) of 94 BSA did not further increase the amplification efficiency and BSA might precipitate when 95 contacting the lysis buffer ( Figure S4e). 96 After nucleic acid extraction, the binding disc must be dried for further amplification. It took 97 about 20 minutes for our binding discs to dry naturally at room temperature. Heating up the 98 binding discs by a handwarmer can shorten this time to 8 ~ 10 minutes. We also tried rinsing 99 the binding discs with anhydrous ethanol to quickly volatilize the moisture. The effects of 100 different drying methods were compared ( Figure S6c

Optimization of Recombinase Polymerase Amplification (RPA) Reaction 138
To optimize the RPA reaction, we introduced exo probe for real-time detection of 139 amplification by fluorescence. Probes intended for the use in PCR and other nucleic acid 140 amplification processes (e.g. Taqman®) will not work in RT-RPA exo reactions. Therefore we 141 designed and screened of a series of candidate primers and probes, probe concentrations. We 142 evaluated their ability to produce an effective amplification of nucleic acids samples with 30 143 minutes of incubation through fluorescence instrument ( Figure S8). After optimization of 144 parameters, we found that 120 nM of the probe worked well ( Figure S8a). The reverse 145 transcriptase activity is essential for RNA detection, especially at low RNA concentrations. So, 146 we screened of the reverse transcriptase and selected RevertAid for next test because of its good 147 performance ( Figure S8b). Furthermore, detection of RNA typically requires reverse 148 transcription and amplification that can be performed sequentially (two-step) or simultaneously 149 in a one-pot system (one-step). Despite that the two-step reaction was more sensitive than one-150 step reaction as revealed by our results, the one-step method, omitting the opening of the chip, 151 was easier to handle and time saving (Figure S9a, b). 152 In addition, human ribonucnase P gene (RNase P) was selected as an Internal control (IC) to 153 monitor the quality of samples collected and the effectiveness of sample pretreatment, namely 154 the accuracy of the experimental operation process, to avoid possible false negative results [3][4][5] . 155 Multiple pairs of primer pairs were designed for H. pylori, SARS-CoV-2 and RNase P 156 respectively, and the combination with high amplification efficiency was selected, as shown in 157 Figure S8c, d and Table S1.

Construction of the Portable Reader 176
The isothermal incubation unit of the portable reader was mainly two heating pads, controlled 177 by a temperature circuit, sitting at the bottom and on the top lid of the chamber. Eight magnets 178 on and around the lid are used to secure the heating pads to be fully in contact with the chip. A 179 thermal insulating pad surrounding the chamber is employed to maximally prevent the heat loss 180 from chip edges. The temperature probe was attached to the chamber bottom. With these 181 improvements ( Figure S10), our data suggested that the temperature of the heating chamber has 182 been steadily kept at 40 ± 1 ºC for RPA, even when the environmental temperature changed 183 from 4 to 37 ºC.
We further validated the temperature stability of our system. We conducted repeated test of 185