Method for Lysis and Paper-based Elution-free DNA Extraction with Colorimetric Isothermal Amplification

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Doeven, Dan Yuan, Rosanne M. Guijt This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3875247/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Nucleic acid amplification testing has great potential for point-of-need diagnostic testing with high detection sensitivity and specificity. Current sample preparation is limited by a tedious workflow requiring multiple steps, reagents and instrumentation, hampering nucleic acid testing at point of need. In this study, we present then use of mixed cellulose ester (MCE) paper for DNA binding by ionic interaction and fluid transport by wicking. The poly(ethylene) glycol-based (PEG) reagent simultaneously provides the alkalinity effect for alkaline lysis and crowding effects for ionic DNA binding of the DNA under high salt conditions. Using a narrow strip of paper, the freed DNA concentrates at the paper tip, while the wicking removes the sample matrix when briefly washing using 40% isopropanol, a 15 in process that is followed by on-paper amplification after a drying step. Colourimetric loop-mediated isothermal amplification enabled the detection of 10 2 CFU/mL of Escherichia coli ( E. coli ) from culture media and the detection of E. coli in milk < 10 3 CFU/mL (10 CFU) after incubation at 68°C for 60 min, demonstrating applicability of the method to complex biological samples. Biological sciences/Biological techniques/Analytical biochemistry Biological sciences/Biological techniques/Lab on a chip Biological sciences/Biological techniques/Sensors and probes Physical sciences/Chemistry/Analytical chemistry/Bioanalytical chemistry Physical sciences/Chemistry/Analytical chemistry/Lab on a chip Physical sciences/Chemistry/Analytical chemistry/Microfluidics Physical sciences/Chemistry/Analytical chemistry/Sensors Paper-based DNA extraction PASAP Nucleic acid amplification test (NAAT) Elution-free sample preparation colourimetric loop-mediated isothermal amplification (cLAMP) E. coli detection Milk Point-of-need PONT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nucleic acid amplification tests (NAATs) have gained popularity due to their high sensitivity and specificity for identifying target species in samples of interest. Research has focused on integrating NAAT into portable devices to meet the increasing demand for point-of-need testing (PONT). In parallel, paper analytical devices (PADs) have been demonstrated to provide an affordable substrate for chemical and biological assays due to accessible manufacture, low cost, and ease of disposal [ 2 – 5 ] . PADs can be as simple as the popular lateral flow-based immunoassays (LFA) that patients can self-administer for diagnostic tests, such as for pregnancy and coronavirus (COVID-19). In addition to clinical settings, PONT is also important in warranting food safety [ 6 ] , with recent progress in LFA and PAD-based technologies applied to chemical and biological food safety reviewed elsewhere [ 7 , 8 ] . The challenge in developing ' sample-to-answer' PADs for NAATs is in sample preparation [ 9 ] and to provide the more complex techniques compared to the immunoassay typically used in LFA tests, as NAAT typically relies on amplification before detection. In the traditional, laboratory-based workflow, sample preparation for NAAT comprises two main steps: lysis and solid-phase extraction (binding, washing, and elution). In the simplest form, PADs have been used for the detection of amplification products generated off-PAD, eliminating the need for complex instrumentation, such as fluorescence detection. More recently, more advanced PADs have use the paper as carrier for dried reagents that – following rehydration – can be used for isothermal amplification by loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) on the paper membrane [ 4 , 10 , 11 ] . Inspired by the traditional art of origami, folded PADs were introduced to bring complementary functionalised areas in close proximity to facilitate a sequence of sample preparation steps on a compact device [ 11 – 17 ] . Various papers have been utilised for PADs, including glass fibre, polyethersulfone (PES) filters, and Flinders Technology Associates (FTA) cards. In recently reported advances, a 3D-printed rotational device was developed to minimise manual handling by accommodating an assay using a glass fibre pad for NA capture and RT-LAMP using freeze-dried reagents [ 18 ] . A multi-layer, multi-material PAD where the NAs were captured on a PES filter, followed by LAMP on a 3 mm X 3 mm glass fibre pad after reconstitution dried reagents, including hydroxynaphthol blue for colourimetric detection [ 19 ] . In other work, FTA cards were placed in a polymer fluidic cartridge, allowing for the transfer of NAs on the paper disk to a vial for amplification to detect SARS-Cov-2-virus and Helicobacter pylori down to 4 X 10 2 copies/mL by fluorescence detection [ 3 ] . In PADs, however, lysis is often conducted off-device, and a lengthy drying step may be required before eluting the captured DNA. Additionally, complex PADs that embody multiple foldings and materials to accommodate the processing steps, impose a need for highly developed skills during manufacture and operation and associated increased cost and risk of contamination with manual handling during manufacture and use. Commercially available mixed cellulose ester (MCE) membrane is a paper consisting of about 70% cellulose nitrate and 30% cellulose acetate and is negatively charged at neutral pH. The paper is employed, for example to filter lactic acid bacteria to detect beer spoilage using Barocylcer™ treatment [ 20 ] or to capture environmental DNA from water samples where MCE outperforms PES in capturing and preservation capacity outperforming [ 21 ] , probably due to the ability of MCE paper to resist DNA digestion by DNase I and proteinase K [ 22 ] . Here, we propose to use of the DNA capturing capacity of MCE paper as stationary phase during sample preparation. We recently reported Abridged Solid-Phase Extraction with Alkaline Poly(ethylene) Glycol Lysis (ASAP), introducing a single reagent that combines cell lysis and NA immobilisation at the anionic surface of paramagnetic particles before on-bead amplification [ 23 ] . The method used the concentration-dependent alkalinity of poly(ethylene) glycol (PEG) to provide the alkalinity required for alkaline lysis and solubilisation of the NAs as well as its rapid decrease in pH upon dilution to bring the sample solution pH into the realms of the buffer capacity of amplification buffers [ 24 ] . Simultaneously, the molecular crowding effect of PEG facilitated DNA precipitation onto the anionic support under high salt conditions. However, the paramagnetic beads used in ASAP led to some experimental challenges, as bead recovery was compromised by the relatively high viscosity of the PEG - also limiting the sample volume - and the binding capacity limited by the bead mass tolerated during amplification. Here, the method is advanced by substituting the beads with paper for paper-based ASAP (PASAP), as schematically illustrated in Fig. 1 . The reagent was re-optimised to bind the DNA to a mixed MCE paper under high salt conditions and - after a washing step – directly deposited in a vial for colourimetric LAMP (cLAMP) in the presence of the paper disk. The PASAP method was applied to do DNA sample preparation for the detection of Escherichia coli (E. coli) in milk, a complex biological matrix comprising organic and inorganic small molecules, proteins, and lipids aggregated in a colloidal suspension [ 25 ] . The optimised method is conducted as a four-step assay: 1) wicking of the lysate into the paper, 2) a 10-sec wash using 40% isopropanol (IPA), 3) 30-sec air-drying, and 4) cLAMP following the release of the tip of the paper into the amplification vial by snapping the brittle paper using a cap of the tube. Detection of the target DNA was achieved through the colour change from pink to yellow due to decreased pH as H + ions are generated during amplification. The PASAP method resolves major challenges in sample preparation for NAAT by reducing the number of reagents and processing steps limiting and does not rely on external equipment other than a pipette (and a heater for amplification). After 15 min, a PCR tube containing the paper is ready to be loaded into a suitable heating system. The method produces minimal plastic waste (two PCR tubes and two pipette tips) and only relies on a pipette, eliminating the need for a magnet and associated handling challenges in comparison with the ASAP method. Isothermal amplification can be done in a thermocycler, or using a battery-powered heater. The PASAP method allowed for the detection of E. coli in milk samples with a limit of detection (LOD) of 10 3 colony forming unit (CFU)/mL (40% success rate at 10 2 CFU/mL), which is below the maximum allowable plate count of 10 4 -10 5 CFU/mL for raw and pasteurised milk intended for further processing [ 26 ] . Combined with a battery-powered heater, the approach has potential for rapid and affordable NA-PONT for applications including food safety. Methods Materials Absolute ethanol, isopropanol (IPA), potassium hydroxide, and sodium chloride were obtained from Chem Supply Australia (Gillman, Australia). Luria-Bertani Broth (LB), agar, and PEG 8000 were purchased from PhytoTechnology Laboratories (Lenexam USA). Ethylenediaminetetraacetic acid (EDTA) solution (03690), nuclease-free water (W4502), phosphate-buffered saline (PBS), Tween 20 (P7949), 2-dodecanol (D221503), and 1-decanol (239763) were obtained from Sigma-Aldrich® (Macquarie Park, Australia). Phenol red (32661) was obtained from Fluka (ChemSupply Australkia, Gilwell, Australia).) A hydrophilic MCE membrane filter with an 8 µm pore size (SCWP04700) was purchased from MF-Millipore™ Membrane Filter (Millipore, North Ryde, Australia) and cut into 45 mm long strips. Mili-Q water was obtained from a Cascada™ Lab water purification system (Pall Australia, MelbourneMelbourne) and autoclaved using Pratika S20 from Siltex (Bentley East, Australia) before its use. Luna® Universal qPCR Master mix (M3003) and WarmStart® Colorimetric LAMP 2X Mater Mix (DNA & RNA) (M1800) were purchased from New England Biolabs (Nottinghill, Australia). Molecular grade agarose (BIO-41025) was obtained from Bioline (Eveleigh, Australia). Purified E. coli genomic DNA (gDNA) was obtained using an ISOLATE II Genomic DNA extraction kit (Bioline, Eveleigh, Australia) following the manufacturer's instructions, and the eluate was stored in a 1.5-mL DNA LoBind® Tube (Eppendorf, part # 0030108051, Macquarie Park, Australia) at -20°C until further use. PASAP The 2x PASAP solution was prepared by mixing 0.1753 g of NaCl, 0.3 g of 50% (v/v) PEG8000, 2 µL of 100% Tween 20, 1 µL of 0.5 M EDTA, 100 µL of 20 mM phenol red, and water up to 1 mLTo perform PASAP, 49 µL of the PASAP stock solution, 1 µL of 700 mM KOH, and 50 µL sample were briefly mixed in a 1.5-mL centrifuge tube. A 20-µL drop of this lysate was pipetted on a petri dish surface and absorbed by a MCE paper strip (4 x 45 mm), a process that takes about 10 min, after which the tip of the paper was placed in a 20 µL drop of 40% isopropanol (IPA) for 10-sec for washing. To prevent residual IPA change the colour of the phenol red, the paper was air-dried for ~ 30 sec at room temperature. The tip of the paper was laid flat on a PCR tube and snapped off by closing the lid, dropping the ~ 4 x 3 mm section in 20-µL LAMP reaction solution. The brittle MCE paper easily snaps, making the pressure applied by the lid of a PCR tube sufficient to cut off the tip. Finally, cLAMP is conducted at 68°C for 60 min without removal of the paper. During amplification, the generated protons change alkaline pH to acidic, causing the colour of phenol red to change from pink (pH > 8.2) to yellow (pH < 6.8) [3]. Real-time PCR (qPCR) analysis Amplification and real-time fluorescence readings were conducted on the CFX Connect Real-Time PCR System (Bio-Rad). The qPCR reactions included 10 µL of Luna® Universal qPCR Master mix, 0.25 µL of 10 µM each forward and revere primer, 5 µL of DNA template, and 4.5 µL of nuclease-free water, resulting in a 20 µL final reaction volume. Forward and reverse primers previously designed to target the Escherichia phage Lambda gene in E. coli strain BL21 were used [ 23 ] . Thermal cycling conditions were 1 min at 95°C for initial denaturation, followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec, with a melt-curve analysis from 65°C to 95°C at 0.5°C intervals. The Cq values obtained from qPCR amplification canbe used to calculate the DNA concentration using a previously constructed standard curve [ 23 ] . However, as most amplifiactions were conducted under attenuating conditions, all qPCR data is reported as number of cycles (Cq). cLAMP reactions All LAMP primers used in this study were taken based on an earlier literature report [ 27 ] and purchased from Integrated DNA Technologies (IDT). Before the LAMP reaction, a 10X primer mix containing 1.6 µM of each forward inner primer (FIP) and backward inner primer (FIP), 0.2 µM of each forward outer primer (F3) and backward outer primer (B3), and 0.4 µM of each forward loop primer (LF) and backward loop primer (BF) was prepared. The cLAMP reaction contained 10 µL of WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA, 2 µL of 10X primer mix, and 2 µL of nuclease-free water, making the final volume of 20 µL per reaction. Once the DNADNA-carrying MCE paper was immersed in the LAMP mixture, the PCR tubes were incubated at 68°C for 60 min in Eppendorf mastercycler X50s. The potential for in-field use was confirmed using a 12 V heater consisting of an Arduino Nano microcontroller (Seeed Studio), running a PID algorithm which controlled a 12V 2A cartridge heater via switching a transistor. Temperature feedback was provided by a DS18B20 digital temperature probe. The temperature probe and heater cartridge were mounted in drilled holes within an aluminium block also containing recesses for 8 PCR tubes. Using a laptop, an interface designed in MegunoLink was used to maintain the temperature at 68°C, start and stop the heating process, and view the temperature of the block over time graphically. The heater system was powered by a 12V lab power supply, or a portable power bank with a USB-C power-delivery (Comsol 25600 mAh 100 W power bank, Officeworks, Australia). Arduino and interface code are available on request. As the potable system lacked a cooled lid, a drop of silicone oil was added on top of the LAMP mixture to prevent evaporation. The colour change of the solution from pink to yellow was used as indication of amplification, and amplification, and the presence of amplicons was confirmed by gel electrophoresis on a 3% (w/v) agarose gel. E. coli detection from milk samples A colony of E. coli BL21 was cultured in 10 mL of sterile LB broth placed on an orbital shaker (Model: TU-400 Thermoline Scientific Equipment Pty Ltd, Wetherill Park, Australia) overnight at 37°C and 150 rpm. Subsequently, 100 µL of the liquid culture was transferred to fresh LB, incubated at 37°C and 220 rpm until the OD 600 (optical density at 600 nm) reached 0.5 using a GENESYS™ 30 Visible Spectrophotometer (Thermo Fisher Scientific, Scoresby, Australia). Next, 1 mL of the cell suspension was dispensed into a sterilised 1.5 mL microcentrifuge tube (NEST®, cat. No. 615001, Adelab Scientific, Thebarton, Australia)) and centrifuged for 2 min at 8,000 rpm using a PicoTM 21 centrifuge (Thermo Fisher Scientific, Scoresby, Australia). The resulting cell pellet underwent three washes with a sterile PBS solution. Finally, the washed cells were resuspended in 1 mL of PBS and promptly stored at -20°C for future use. Skim long-life milk (Coles Heat Treated Australian Milk, Coles Group limited, Hawthorn East, Australia, containing 120 mg calcium, 40 mg sodium, 4.8 g carbohydrate, 3.3 g protein, and less than 1 g of saturated fat) was purchased from a local supermarket. To perform E. coli detection in milk, 1 mL of the cultured E. coli was centrifuged to obtain a cell pellet and resuspended in 1 mL of 10-fold diluted skim milk. This spiked milk was then serially diluted with 10-fold diluted milk with the final concentrations ranging from 10 6 to 10 1 CFU/mL and used for PASAP. Statistical and Image analysis The data analysis for qPCR was conducted using Bio-Rad CFX Manager 3.1 software, with Cq determination in single threshold mode. Statistical analysis and graphing were executed using OriginPro 2022b Learning Edition. Each data point represents a minimum sample size of 3 (n = 3), and significance differences were assessed through the post-hoc method using Two-way ANOVA. To analyse photographs following cLAMP, raw images were subjected to RGB splitting, and the green and blue channels' grey values were measured using ImageJ following a published study [ 1 ] . Then, the mean intensity values of blue were subtracted from green, and the difference was plotted. Results and Discussion On-paper amplification The compatibility of commonly used amplification approaches - PCR and LAMP - with MCE paper was investigated, using MCE paper because of its mild negative charge, DNA binding capacity and quick drying owing to the thin film (130 µm). In our earlier work, the viscosity of the ASAP solution provided a challenge in magnetically recovering the beads during DNA extraction [28] . With the ambition of using the wicking by MCE paper for fluid transport, the wicking time of the viscous solution was examined with MCE paper with 1.2, 5.0, and 8.0 µm pore size, with the results presented in Figure S 1 . As expected for the limited height to be wicked [29] , smaller pores correlated with higher resistance and hence lower flow rates, with the 1.2 µm MCE paper showing the slowest speed, while the rates for 5.0 and 8.0 µm were comparable. Thus, 8 µm MCE paper was selected for faster processing. The compatibility of commonly used amplification approaches, PCR and LAMP, with MCE paper was investigated. A piece of 8.0 µm pore size MCE paper (3 x 4 mm) was introduced into PCR vials containing target DNA, primers, and an amplification mix, aiming for fluorescence detection of the amplification product using SYBR green as an intercalating dye. No fluorescence could be detected during qPCR and qLAMP (Figure S2 and Figure S3). However, amplicons were detected by gel electrophoresis following LAMP but not PCR, indicating the paper inhibited both amplification and detection during qPCR but only interfered with the fluorescence detection in qLAMP. Combined with an expected loss in sensitivity at higher salt levels because of an increase in dissociation constant (K d ) of SYBR green for DNA [1, 2], fluorescence detection was deemed unsuitable for the targeted extraction following PASAP. PASAP Colourimetric cLAMP has become a popular choice for in-field testing for non-quantitative detection. For example, NEB's cLAMP test contains phenol red, a pH indicator that confirms the generation of H + during amplification, and this approach was adopted here. In addition, the advantages of isothermal amplification include elimination of thermocycling and better salt tolerance of Bst 2.0 polymerase in LAMP amplification for strand displacement activity has better salt tolerance than Taq polymerase used in PCR (complete (full inhibition at 100 mM vs 40 mM NaCl)) [30] , making LAMP a better choice than qPCR for detection following PASAP. ASAP method combines lysis and extraction with direct, on-bead DNA amplification. The approach combines the reversible binding of NAs in the presence of poly(ethylene) glycol (PEG) for solid phase extraction onto magnetic beads, an approach conceptually similar to solid phase reversible immobilisation (SPRI) [23] , with DNA binding facilitated by the crowding effects of PEG under high salt conditions. Here, the aim is to use mixed cellulose ester paper to act as a stationary phase and fluid handling by wicking. To confirm binding of the DNA to the MCE paper, a strip of MCE paper was used to wick 20 µL of a sample containing 10 µL purified gDNA and 10 µL 15 % PEG8000 and 3.5 mM KOH in the presence and absence of 1 M NaCl. After wicking the 20 µL sample following the insertion of the short edge into the droplet, the strip was washed with 80 % ethanol before being cut into about 3 mm x 4 mm long at the top, middle, and bottom sections of the paper. Each of these was placed in 20 µL of water to elute the gDNA, after which 5 µL of the eluate was used for qPCR. When no salt was present, the larger gDNA concentrations found in the eluates from the MID and TOP sections indicating that the DNA was carried up the MCE paper by the flow caused by the wicking of the aqueous solution by the hydrophilic porous material ( Figure 2 A ) . As there was no strong DNA binding, the gDNA concentration increased with the distance wicked up the strip, as weakly bound DNA was transported upwards with the wicking of more liquid. As expected, in the presence of 1 M NaCl, the DNA was retained in the bottom section of the MCE paper. The high salt concentration facilitates electrostatic bonding between the slightly anionic surface [31,32] and partially neutralises the negative charge of the DNA in the presence of PEG8000, inducing DNA precipitation. The concentration of the gDNA in the tip of the paper strip in the presence of 1 M NaCl demonstrates the potential of using MCE paper for DNA extraction under ASAP conditions. Encouraged by these results, the effect of NaCl concentration was investigated using a reagent combining 15 % PEG8000 and 3.5 mM KOH with increasing NaCl concentrations (0, 0.5, 1.0, 1.5, and 2.0 M) to understand the correlation of the binding capability of paper with NaCl concentration. The gDNA in the eluate was analysed by qPCR. Under low NaCl concentrations (0 – 1 M) without washing, a decrease in Cq value with increasing salt concentration demonstrated the efficacy of DNA binding with increasing salt concentration ( Figure 2 B ). The Cq value, however, increased to 25.75 ± 1.21 at 1.5 M, and amplification was not detected at 2.0 M as a result of inhibition of amplification; NaCl is known to have an inhibitory effect on Taq polymerase preventing amplification when [Na + ] ≥ 40 mM [30] . The efficacy of the washing step was investigated using 70 % ethanol, commonly used in SPRI and DNA extraction protocols. Washing was performed by dipping the bottom section of the paper in a ~20 µL droplet of 70 % ethanol for 30 sec, followed by air-drying of the strip before eluting the DNA in 20 µL Milli-Q water. Disappointingly, the wash significantly increased Cq values of the eluates about 27.28, 25.68, and 26.00 for 0, 0.5, and 1.0 M NaCl, respectively, indicating considerable DNA loss during the wash. In contrast, improved Cq values of 24.49 ± 0.62 and 24.23 ± 003 were obtained after the wash for 1.5 and 2.0 M NaCl, correspondingly showing that the inhibitory effect of high NaCl can be alleviated by washing provided sufficient NaCl was present to ensure strong binding of the DNA to the paper. The lowest Cq values were obtained using 1.0 M NaCl without washing. However, it is unlikely that the washing step can be eliminated when targeting complex biological samples like milk. As no improvement in sensitivity was found using 2 M NaCl compared with 1.5 M NaCl for binding (Cq values 24.23 ± 0.03 vs 24.49 ± 0.62), 1.5 M NaCl was selected for further method optimisation to minimise salt-induced inhibition. The increase in Cq value in comparison with input DNA (dashed line) suggests there is room for improvement, increasing binding efficiency or decreasing DNA loss and/or attenuation of amplification. Optimisation of the wash The results presented above suggest that the eluting strength of 70 % ethanol may be too high for it to be a suitable washing reagent. Washing reagents during DNA extraction on anionic stationary phases has included water-miscible solvents like isopropanol to remove the salt. Non-polar solvents were used in the two-phase wash to limit the carryover of aqueous inhibitors but were typically added between the wash step and the final elution [27] . Four washing liquids were examined, 70 % ethanol, 70 % IPA and two long-chain alcohols (2-dodecanol and 1-decanol) as used in two phase wash [27] . In line with the experiments discussed above, 20 µL of a solution containing 10 mL each of the gDNA standard and PSAP reagent was wicked up a 45 mm long MCE strip, after which the bottom 3-4 mm was immersed in a 20 µL-drop of the washing liquid for 30 sec. The MCE paper was air-dried before elution into 20 µL water. The number of qPCR cycles determined the efficacy in reducing loss and/or attenuation. As shown in Figure 3 , DNA was detected following a wash with 70 % IPA, with Cq values of 25.92 ± 0.27 in the eluate. No washing increased the Cq value (27.49 ± 033), indicating an inhibition equivalent to a 3.3-fold DNA loss. Slightly delayed Cq values of 26.93 ± 0.33 using 70 % ethanol and 27.12 ± 0.30 using 100 % 2-dodecanol were found, whereas for 100 % 1-decanol the Cq values were significantly increased to 28.37 ± 0.74, a higher value than no wash . The poor washing efficiency of both hydrophobic solvents may the due to the decreased efficacy of the salt removal owing to the poor solubility of NaCl in the washing solvent is vital at the high NaCl concentration used for binding. The solubility of DNA in IPA is lower than in 70 % ethanol [33] , preventing the elution of immobilised DNA while still providing good solubility for NaCl to be an effective washing agent. To optimise the capacity of the washing liquid to remove salt without eluting the DNA, the IPA content was optimised (Figure S4), indicating that 40 % IPA was most effective in mitigating attenuation with minimal processing time (10 sec). For longer washes (60 sec), decreasing Cq values suggest the washing efficacy of IPA>40% increased; however, with the objective of fast sample preparation, 40 % IPA was selected. Lysis and Inhibitory Effects of Tween 20 in PASAP During ASAP, Tween 20 was required to prevent magnetic particles from aggregation but may also have contributed to the lytic activity. As the proposed PASAP method replaces the beads with the MCE paper, aggregation of particles is no longer a risk. Hence, the necessity of Tween 20 was investigated. Using E. coli as a biological sample, 20 µL of 10 6 CFU/mL bacteria was resuspended in the PASAP solutions with or without 0.2 % Tween 20, wicked by the MCE paper and washed with 40 % IPA for 10 sec or directly eluted in water to further determination of the selected washing steps for E. coli samples. As shown in Figure 4 A , the inclusion of Tween 20 led to a statistically significant improvement in DNA concentration in the eluate, increasing from 7.6 × 10 3 to 61 × 10 3 copies/µL (nearly 10 times) after washing. Previous studies also reported that Tween 20 is non-inhibitory in PCR reactions and can enhance the amplification efficiency under a range of concentrations (4 – 10 %) by reducing the inhibitory effect and stimulating Taq DNA polymerase activity [34-36] . It is, therefore, anticipated that the inhibition observed without washing in the presence of Tween 20 is the result of the salt, not the Tween 20. The duration of cell lysis was optimised by incubating a mixture of PASAP and cultured E. coli (10 4 CFU) at room temperature for different durations (0 to 15 min) before the DNA binding step using the MCE membrane. The measured DNA concentrations, as determined by qPCR, following elution are presented in Figure 4 B . Overall, a trend of decreasing DNA concentration in the eluate was obtained with an increasing incubation period, with recovery drastically worsened when the E. coli mixture was incubated for 15 min. The denaturation of DNA can explain this by extended gDNA exposure time to the highly alkaline conditions (> pH 12) [37] . The extended lysis time could have damaged the DNA decreasing the amplification efficiency. Considering the 10 min wicking time and the similar DNA concentrations for 0- and 5- min incubation, it was concluded there was no need to for sample incubation with the PASAP reagent before starting the wicking, allowing combining lysis and binding steps into a single step. Effect of size of MCE paper on direct on-paper cLAMP Having demonstrated LAMP amplification in presence of 3 x 4 mm MCE paper by gel electrophoresis, it was confirmed the addition of phenol red allowed for cLAMP. The PASAP method was then used to bind 100 pg/µL DNA to the MCE paper under ASAP conditions, followed by wash with 70% ethanol, or no wash. The size of MCE paper that can be used for direct on-paper cLAMP is a compromise between binding capacity and attenuation of amplification. To optimise the paper size, MCE paper was cut into squares of 3 x 3, 4 x 4, 5 x 5, and 6 x 6 mm . The squares were exposed to 20 µL ASAP lysis/binding reagent and with/without a wash with 70% ethanol for 30 sec introduced to 20 µL LAMP reaction mix containing the gDNA. After heating to 68 °C for 50 min, amplification was visualised by the colour change and confirmed by gel electrophoresis, as shown in Figure 5 . A colour change from pink to yellow/orange was observed for all paper sizes and gel electrophoresis confirmed that the colour change of LAMP reaction was indeed the result of amplification ( Figure 5 ). However, from the intensity of gel bands, it is apparent that the quantity of amplified DNA decreased gradually when increase the size of the MCE paper. This agrees with an earlier report using 2-mm glass fibre disc placed in 50 µL cLAMP mix for the detection of down to 10 3 copies /mL of rotavirus A within 30 min, but larger discs would inhibit amplification [38] . Here, cutting the brittle MCE paper with scissors limited the size to strips that were 3-4 mm wide. Recognising this is a crude and manual approach, scissors were selected after we found a laser cutter burned the edges, impeding wicking efficiency and a paper guillotine fragmented the membrane in irregular sizes. The importance of the optimised washing for cLAMP was confirmed conducing experiments with 100 pg/µL gDNA with and without the 10 s wash in 40 % IPA, and resulting colour changes and gels are shown in Error! Reference source not found. , showing significant attenuation without washing as a result of inhibition. Please note that while a positive was detected after 30 min for a high concentration gDNA sample, decreasing the amplification time would lead to a decrease in sensitivity. The benign PAPAS reagent and limited supplies (a pipette with 2 pipette tips, 2 test tubes and the MCE strip) make the PASAP method attractive for in-field testing, particularly in low resource settings. With all amplifications listed above conducted in a PCR machine, compatibility with in0field use was confirmed using a purpose-designed heater, accommodating the PCR vials in recesses CNC-milled in an aluminium block (Figure S9A, B). The temperature was controlled using a laptop using the feedback from a temperature sensor. Evaporation of the amplification mixture when heated in the aluminium block prevented amplification, probably unlike in PCR machines the block did not have a heated lid, leading to condensation. A drop (~ 20 mL) of silicone oil provided an effective inert barrier to prevent evaporation of the amplification solution. Preliminary testing of the 12 V heater yielded following PASAP of a gDNA standard yielded comparable results to those obtained using the PCR instrument, showing a colour change from yellow to pink following 60 min at 68°C for 100 pg/µL gDNA (Figure S9C). No differences were found between the heater powered using a power supply or battery pack, conforming suitability for in-field use. Assay sensitivity and E. coli detection in milk With the PASAP/cLAMP method established, E. coli was used as biological target to understand applicability, and milk was selected as sample matrix. Recently, multienzyme isothermal rapid amplification was used for the detection of Streptococcus in milk following its extraction on filter paper following lysis [39] . Here, first cultured bacteria were serially diluted in PBS from 10 4 to 10 1 CFU/mL, and 50 µL of the diluted samples were used for the PASAP method following the optimised workflow with negative control (fresh PBS without E. coli ). The PASAP method combined with cLAMP successfully allowed for the detection of E. coli from PBS solutions with a LOD at 10 2 CFU/mL, or 1 CFU present in the 10 µL sample, with the presence of amplified DNA for all positive colourimetric results confirmed by gel electrophoresis ( Figure 6 A ). Semi-quantitative data were obtained by image analysis [1] , ( Figure 6 B ) showing the calculated colour intensity of LAMP solution for a range of E. coli concentrations . We previously reported that 15 CFU/50 µL could be detected using a magnetic bead kit, and 150 CFU/50 µL using a spin column [28] , making 1 CFU/10 µL slightly between than but inferior to the 0.15 CFU detected in a 50 µL using the ASAP method. When using milk as the sample, the yellow hue indicative of a positive test shifted slightly towards orange, but a change from the pink in the absence of DNA could still observed at 10 2 CFU/mL; an evident change was only found at 10 3 CFU/mL ( Figure 6 C ). While detection through colour change and gel bands at 10 2 CFU/mL was demonstrated twice in two of five replicates, the high failure rate means using the PASAP method E. Coli can be detected in milk at 10 3 CFU/mL ( Figure 6 D ). This decreased sensitivity compared with other work [14,39] should be contrasted by a significant reduction in reagents, consumables, waste and manual handling. The sensitivity of PASAP/cLAMP may be improved by increasing the sample volume processed, and future research will focus on the introduction of a wicking pad to increase the sample volume, as currently the sample volume is limited by low wicking rate of the PASAP solution in the MCE paper. The presented PASAP method provides a fast, low-cost and easy sample preparation method compatible with on-paper cLAMP that can be applied to complex biological samples including for the detection of bacteria in milk. The simple workflow is suitable for automation and combined with a battery-powered heater has potential to be integrated into hand-held-devices for PONT in food safety and diagnostics. Conclusions A new method for on-paper DNA extraction and cLAMP with minimal use of reagents, consumables and equipment is presented. The aqueous alkaline PEG-based reagent lyses the cell providing the high pH for alkaline lysis, enhancing the lytic activity with the surfactant Tween20. Owing to crowding conditions in presence of PEG and NaCl the DNA was immobilised at the tip of a strip of mixed cellulose ester paper used for wicking the lysate. Following a 10 sec wash conducted by dipping the strip in 40% isopropanol, the tip of the dried paper strip is snapped off into a vial for colorimetric LAMP. The proposed workflow takes 15-min and uses wicking for liquid handling. The instrumentation used was minimal (a pipette for depositing 20 µL drops of sample and washing reagent) and produces minimal waste (2 vials, 2 pipette tips and a 3 x 45 mm membrane). Supporting the isothermal amplification with a battery-powered 12 V heater (60 min at 68 °C), the method is compatible with in-field use. Recognising the quantitative limitations of cLAMP, the method allowed for the detection of E. Coli down to 10 CFU/mL in buffer, increasing to 10 3 CFU/mL in 1:10 diluted milk though occasionally a colour change was detected at 10 2 CFU/mL Future work will focus enhancing the sensitivity by processing larger sample volumes without compromising processing time, for example, connecting the paper with an absorbent pad. Declarations Acknowledgement We acknowledge Mr Richard Alexander for the fabrication of the heater. SML acknowledges the receipt of a scholarship from Deakin University's Faculty of Science, Engineering, and Built Environment and the graphical illustration from JML. DY acknowledges Alfred Deakin Postdoctoral Fellowship from Deakin University. Author Contributions SML performed all experiments, wrote manuscript, and analysed the data. DY, ED and RMG contributed through support in the experimental design, data analysis and interpretation and revised manuscript.{Varona, 2019 #273} Competing Interest Statement The authors have no competing financial and/or non-financial interests to declare in relation to the work described. Data availability Statement The data generated and/or analysed during the current study not presented in this paper are available from the corresponding author on reasonable request. 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Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 23, 4742–4743, doi: 10.1093/nar/23.22.4742 (1995). Green, M. R. & Sambrook, J. Precipitation of DNA with isopropanol. Cold Spring Harbor Protocols 2017, pdb.prot093385, doi: 10.1101/pdb.prot093385 (2017). Goldenberger, D., Perschil, I., Ritzler, M. & Altwegg, M. A simple" universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. Genome Res. 4, 368–370 (1995). Cunnington, J. H., Takamatsu, S., Lawrie, A. C. & Pascoe, I. G. Molecular identification of anamorphic powdery mildews (Erysiphales). Australasian Plant Pathology 32, 421–428, doi: 10.1071/AP03045 (2003). Coutlée, F. & Voyer, H. Effect of nonionic detergents on amplification of human papillomavirus DNA with consensus primers MY09 and MY11. J. Clin. Microbiol. 36, 1164, doi: 10.1128/jcm.36.4.1164-1164.1998 (1998). Ehrlich, P. & Doty, P. The alkaline denaturation of deoxyribose nucleic acid. J. Am. Chem. Soc. 80, 4251–4255 (1958). Ye, X. et al. Equipment-free nucleic acid extraction and amplification on a simple paper disc for point-of-care diagnosis of rotavirus A. Anal. Chim. Acta 1018, 78–85, doi: https://doi.org/10.1016/j.aca.2018.02.068 (2018). Zhu, L. et al. Integrating filter paper extraction, isothermal amplification, and lateral flow dipstick methods to detect Streptococcus agalactiae in milk within 15 min. Frontiers in Veterinary Science 10, doi: 10.3389/fvets.2023.1100246 (2023). Additional Declarations No competing interests reported. Supplementary Files MethodforLysisandPaperSciRepSI.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Feb, 2024 Reviews received at journal 21 Feb, 2024 Reviewers agreed at journal 14 Feb, 2024 Reviewers agreed at journal 12 Feb, 2024 Reviewers invited by journal 12 Feb, 2024 Editor assigned by journal 09 Feb, 2024 Editor invited by journal 09 Feb, 2024 Submission checks completed at journal 09 Feb, 2024 First submitted to journal 18 Jan, 2024 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. We do this by developing innovative software and high quality services for the global research community. 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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-3875247","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":272140847,"identity":"429d2e6d-0107-4350-a8b1-318267f3b406","order_by":0,"name":"Soo Min Lee","email":"","orcid":"","institution":"Deakin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Soo","middleName":"Min","lastName":"Lee","suffix":""},{"id":272140848,"identity":"303f823f-c430-461a-b82e-4b1eeacb01bb","order_by":1,"name":"Egan H. Doeven","email":"","orcid":"","institution":"Deakin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Egan","middleName":"H.","lastName":"Doeven","suffix":""},{"id":272140849,"identity":"00cbae8b-29f2-4547-ab80-f2f41dc086ba","order_by":2,"name":"Dan Yuan","email":"","orcid":"","institution":"The University of Queensland","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Yuan","suffix":""},{"id":272140850,"identity":"90641990-64e2-49be-b15d-ee7d96e622ed","order_by":3,"name":"Rosanne M. Guijt","email":"data:image/png;base64,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","orcid":"","institution":"Deakin University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Rosanne","middleName":"M.","lastName":"Guijt","suffix":""}],"badges":[],"createdAt":"2024-01-18 08:44:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3875247/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3875247/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50997295,"identity":"618c90c2-340d-4ac9-a682-777364fccee9","added_by":"auto","created_at":"2024-02-12 12:28:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61593,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the PASAP method for simultaneous cell lysis and DNA binding on paper. The sample and the PASAP solution are mixed in a 1:1 ratio, and 20-µL is absorbed by the MCE paper, wicking it from the short end. Under crowding conditions, the DNA binds to the bottom section o the paper. After 10 min the 20 mL is absorbed and the paper tip is washed with 40 % IPA for 10 sec and allowed to air-drie for 30 sec. The tip of the paper is snapped off using a cap of a PCR tube and used for direct on-paper cLAMP for 60 min. The amplification results are visualised by a colour change of the LAMP solution as a result of the drop in pH during amplification (pink=negative, yellow=positive).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3875247/v1/7e85d0e9f11f92ebf31ccea6.png"},{"id":50997296,"identity":"8e418cf9-b68a-4cea-b934-69c67eebe5d6","added_by":"auto","created_at":"2024-02-12 12:28:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45095,"visible":true,"origin":"","legend":"\u003cp\u003eExtraction of gDNA onto MCE paper. \u003cstrong\u003eA)\u003c/strong\u003e qPCR cycle number for gDNA in the eluate for the TOP (red), MID (green) and BTM (blue) sections of a 4 mm wide, 45 mm long strip of MCE paper. Data reported in absence of NaCl (0 M) and in presence of 1 M NaCl. BTM refers to the bottom 15 mm, MD to the middle section of 15 mm and TOP to the remaining 15 mm of the strip. \u003cstrong\u003eB)\u003c/strong\u003e qPCR cycle number for gDNA in the eluate from the BTM for NaCl concentration increasing from 0 to 2 M in the PASAP solution. Eluates were analysed with (red circle) and without (blue triangle) a washing step with 70 % ethanol. Black dashed line shows the Cq value for the purified gDNA input.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3875247/v1/02ce25ee513b74d5f52a435e.png"},{"id":50997297,"identity":"c320367b-730b-4ae8-a823-c6ba3caef1fd","added_by":"auto","created_at":"2024-02-12 12:28:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31746,"visible":true,"origin":"","legend":"\u003cp\u003eOptimisation of the washing solution. Number of qPCR cycles of the PASAP method including no washing (red), washing with 70% ethanol (green), 70% IPA (blue), 100 % 2-dodecanol (purple), and 100 % 1-decanol (yellow\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3875247/v1/881521a18630527331e1ae50.png"},{"id":50997298,"identity":"9dd5cbdb-3e11-41b1-859e-d1aa0adc9b75","added_by":"auto","created_at":"2024-02-12 12:28:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24445,"visible":true,"origin":"","legend":"\u003cp\u003eOptimisation of lysis conditions. \u003cstrong\u003eA)\u003c/strong\u003e Cycle number following lysis with or without Tween 20 for E. coli. The inhibitory effect was compared without (red) and with washing step. \u003cstrong\u003eB)\u003c/strong\u003e Cycle number for the eluate obtained for different lysis time (0 - 15 min).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3875247/v1/d83f3cc7e7457691824cfdfa.png"},{"id":50997693,"identity":"9abf4899-c912-45b3-b1ce-c26e45a696d4","added_by":"auto","created_at":"2024-02-12 12:36:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":80899,"visible":true,"origin":"","legend":"\u003cp\u003ecLAMP in presence of MCE paper. Squares with Increasing size (3 x 3 mm, 4 x 4 mm, 5 x 5 mm, and 6 x 6 mm) were introduced into cLAMP of gDNA. Colorimetric change and cropped gel images for each paper size are compared with amplification without paper (Ref Amplicon) and the negative control (NTC). Image of the full gel is provided in the Supplementary Information.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3875247/v1/ecf0acce40bd9467cd389c44.png"},{"id":50997300,"identity":"c678958f-2c6b-4eb5-88c2-272e1231127b","added_by":"auto","created_at":"2024-02-12 12:28:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":104140,"visible":true,"origin":"","legend":"\u003cp\u003eAssay sensitivity for the detection of E. coli (n=3). \u003cstrong\u003eA)\u003c/strong\u003e Colorimetric change and cropped gel images for E. coli detection in PBS and \u003cstrong\u003eB)\u003c/strong\u003e Intensity plot for E. coli detection in PBS. \u003cstrong\u003eC) \u003c/strong\u003eColorimetric change and cropped gel images E. coli detection in skim milk and \u003cstrong\u003eD)\u003c/strong\u003e Intensity plot for E. coli in milk. The intensity measurement was inspired by earlier study \u003csup\u003e[1]\u003c/sup\u003e. The white dashed line in B and D indicates the visible colour changes of the reaction tubes by naked eye. Full gel images are supplied in Supplementary Information.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3875247/v1/69963bacc83b9e7159fac2e7.png"},{"id":50998421,"identity":"36aad2c9-3e6f-4b05-a718-3fccc871dc87","added_by":"auto","created_at":"2024-02-12 12:44:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":703127,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3875247/v1/cc36a418-2df6-44b9-b1ad-90adb3f96df7.pdf"},{"id":50997301,"identity":"80e0d70d-198f-4810-b0ab-fcd33d6de756","added_by":"auto","created_at":"2024-02-12 12:28:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3663034,"visible":true,"origin":"","legend":"","description":"","filename":"MethodforLysisandPaperSciRepSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-3875247/v1/1d7e13bda3a3cb27160bbcb4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Method for Lysis and Paper-based Elution-free DNA Extraction with Colorimetric Isothermal Amplification","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNucleic acid amplification tests (NAATs) have gained popularity due to their high sensitivity and specificity for identifying target species in samples of interest. Research has focused on integrating NAAT into portable devices to meet the increasing demand for point-of-need testing (PONT). In parallel, paper analytical devices (PADs) have been demonstrated to provide an affordable substrate for chemical and biological assays due to accessible manufacture, low cost, and ease of disposal \u003csup\u003e[\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. PADs can be as simple as the popular lateral flow-based immunoassays (LFA) that patients can self-administer for diagnostic tests, such as for pregnancy and coronavirus (COVID-19). In addition to clinical settings, PONT is also important in warranting food safety \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, with recent progress in LFA and PAD-based technologies applied to chemical and biological food safety reviewed elsewhere \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The challenge in developing '\u003cem\u003esample-to-answer'\u003c/em\u003e PADs for NAATs is in sample preparation \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e and to provide the more complex techniques compared to the immunoassay typically used in LFA tests, as NAAT typically relies on amplification before detection. In the traditional, laboratory-based workflow, sample preparation for NAAT comprises two main steps: lysis and solid-phase extraction (binding, washing, and elution).\u003c/p\u003e \u003cp\u003eIn the simplest form, PADs have been used for the detection of amplification products generated off-PAD, eliminating the need for complex instrumentation, such as fluorescence detection. More recently, more advanced PADs have use the paper as carrier for dried reagents that \u0026ndash; following rehydration \u0026ndash; can be used for isothermal amplification by loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) on the paper membrane \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Inspired by the traditional art of origami, folded PADs were introduced to bring complementary functionalised areas in close proximity to facilitate a sequence of sample preparation steps on a compact device \u003csup\u003e[\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Various papers have been utilised for PADs, including glass fibre, polyethersulfone (PES) filters, and Flinders Technology Associates (FTA) cards. In recently reported advances, a 3D-printed rotational device was developed to minimise manual handling by accommodating an assay using a glass fibre pad for NA capture and RT-LAMP using freeze-dried reagents \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. A multi-layer, multi-material PAD where the NAs were captured on a PES filter, followed by LAMP on a 3 mm X 3 mm glass fibre pad after reconstitution dried reagents, including hydroxynaphthol blue for colourimetric detection \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In other work, FTA cards were placed in a polymer fluidic cartridge, allowing for the transfer of NAs on the paper disk to a vial for amplification to detect SARS-Cov-2-virus and \u003cem\u003eHelicobacter pylori\u003c/em\u003e down to 4 X 10\u003csup\u003e2\u003c/sup\u003e copies/mL by fluorescence detection \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn PADs, however, lysis is often conducted off-device, and a lengthy drying step may be required before eluting the captured DNA. Additionally, complex PADs that embody multiple foldings and materials to accommodate the processing steps, impose a need for highly developed skills during manufacture and operation and associated increased cost and risk of contamination with manual handling during manufacture and use. Commercially available mixed cellulose ester (MCE) membrane is a paper consisting of about 70% cellulose nitrate and 30% cellulose acetate and is negatively charged at neutral pH. The paper is employed, for example to filter lactic acid bacteria to detect beer spoilage using Barocylcer\u0026trade; treatment \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e or to capture environmental DNA from water samples where MCE outperforms PES in capturing and preservation capacity outperforming\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, probably due to the ability of MCE paper to resist DNA digestion by DNase I and proteinase K \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we propose to use of the DNA capturing capacity of MCE paper as stationary phase during sample preparation. We recently reported Abridged Solid-Phase Extraction with Alkaline Poly(ethylene) Glycol Lysis (ASAP), introducing a single reagent that combines cell lysis and NA immobilisation at the anionic surface of paramagnetic particles before on-bead amplification \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The method used the concentration-dependent alkalinity of poly(ethylene) glycol (PEG) to provide the alkalinity required for alkaline lysis and solubilisation of the NAs as well as its rapid decrease in pH upon dilution to bring the sample solution pH into the realms of the buffer capacity of amplification buffers \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Simultaneously, the molecular crowding effect of PEG facilitated DNA precipitation onto the anionic support under high salt conditions. However, the paramagnetic beads used in ASAP led to some experimental challenges, as bead recovery was compromised by the relatively high viscosity of the PEG - also limiting the sample volume - and the binding capacity limited by the bead mass tolerated during amplification. Here, the method is advanced by substituting the beads with paper for paper-based ASAP (PASAP), as schematically illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The reagent was re-optimised to bind the DNA to a mixed MCE paper under high salt conditions and - after a washing step \u0026ndash; directly deposited in a vial for colourimetric LAMP (cLAMP) in the presence of the paper disk. The PASAP method was applied to do DNA sample preparation for the detection of \u003cem\u003eEscherichia coli (E. coli)\u003c/em\u003e in milk, a complex biological matrix comprising organic and inorganic small molecules, proteins, and lipids aggregated in a colloidal suspension \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The optimised method is conducted as a four-step assay: 1) wicking of the lysate into the paper, 2) a 10-sec wash using 40% isopropanol (IPA), 3) 30-sec air-drying, and 4) cLAMP following the release of the tip of the paper into the amplification vial by snapping the brittle paper using a cap of the tube. Detection of the target DNA was achieved through the colour change from pink to yellow due to decreased pH as H\u003csup\u003e+\u003c/sup\u003e ions are generated during amplification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PASAP method resolves major challenges in sample preparation for NAAT by reducing the number of reagents and processing steps limiting and does not rely on external equipment other than a pipette (and a heater for amplification). After 15 min, a PCR tube containing the paper is ready to be loaded into a suitable heating system. The method produces minimal plastic waste (two PCR tubes and two pipette tips) and only relies on a pipette, eliminating the need for a magnet and associated handling challenges in comparison with the ASAP method. Isothermal amplification can be done in a thermocycler, or using a battery-powered heater. The PASAP method allowed for the detection of \u003cem\u003eE. coli\u003c/em\u003e in milk samples with a limit of detection (LOD) of 10\u003csup\u003e3\u003c/sup\u003e colony forming unit (CFU)/mL (40% success rate at 10\u003csup\u003e2\u003c/sup\u003e CFU/mL), which is below the maximum allowable plate count of 10\u003csup\u003e4\u003c/sup\u003e-10\u003csup\u003e5\u003c/sup\u003e CFU/mL for raw and pasteurised milk intended for further processing \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Combined with a battery-powered heater, the approach has potential for rapid and affordable NA-PONT for applications including food safety.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eAbsolute ethanol, isopropanol (IPA), potassium hydroxide, and sodium chloride were obtained from Chem Supply Australia (Gillman, Australia). Luria-Bertani Broth (LB), agar, and PEG 8000 were purchased from PhytoTechnology Laboratories (Lenexam USA). Ethylenediaminetetraacetic acid (EDTA) solution (03690), nuclease-free water (W4502), phosphate-buffered saline (PBS), Tween 20 (P7949), 2-dodecanol (D221503), and 1-decanol (239763) were obtained from Sigma-Aldrich\u0026reg; (Macquarie Park, Australia). Phenol red (32661) was obtained from Fluka (ChemSupply Australkia, Gilwell, Australia).) A hydrophilic MCE membrane filter with an 8 \u0026micro;m pore size (SCWP04700) was purchased from MF-Millipore\u0026trade; Membrane Filter (Millipore, North Ryde, Australia) and cut into 45 mm long strips. Mili-Q water was obtained from a Cascada\u0026trade; Lab water purification system (Pall Australia, MelbourneMelbourne) and autoclaved using Pratika S20 from Siltex (Bentley East, Australia) before its use. Luna\u0026reg; Universal qPCR Master mix (M3003) and WarmStart\u0026reg; Colorimetric LAMP 2X Mater Mix (DNA \u0026amp; RNA) (M1800) were purchased from New England Biolabs (Nottinghill, Australia). Molecular grade agarose (BIO-41025) was obtained from Bioline (Eveleigh, Australia).\u003c/p\u003e \u003cp\u003ePurified \u003cem\u003eE. coli\u003c/em\u003e genomic DNA (gDNA) was obtained using an ISOLATE II Genomic DNA extraction kit (Bioline, Eveleigh, Australia) following the manufacturer's instructions, and the eluate was stored in a 1.5-mL DNA LoBind\u0026reg; Tube (Eppendorf, part # 0030108051, Macquarie Park, Australia) at -20\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePASAP\u003c/h2\u003e \u003cp\u003eThe 2x PASAP solution was prepared by mixing 0.1753 g of NaCl, 0.3 g of 50% (v/v) PEG8000, 2 \u0026micro;L of 100% Tween 20, 1 \u0026micro;L of 0.5 M EDTA, 100 \u0026micro;L of 20 mM phenol red, and water up to 1 mLTo perform PASAP, 49 \u0026micro;L of the PASAP stock solution, 1 \u0026micro;L of 700 mM KOH, and 50 \u0026micro;L sample were briefly mixed in a 1.5-mL centrifuge tube. A 20-\u0026micro;L drop of this lysate was pipetted on a petri dish surface and absorbed by a MCE paper strip (4 x 45 mm), a process that takes about 10 min, after which the tip of the paper was placed in a 20 \u0026micro;L drop of 40% isopropanol (IPA) for 10-sec for washing. To prevent residual IPA change the colour of the phenol red, the paper was air-dried for ~\u0026thinsp;30 sec at room temperature. The tip of the paper was laid flat on a PCR tube and snapped off by closing the lid, dropping the ~\u0026thinsp;4 x 3 mm section in 20-\u0026micro;L LAMP reaction solution. The brittle MCE paper easily snaps, making the pressure applied by the lid of a PCR tube sufficient to cut off the tip. Finally, cLAMP is conducted at 68\u0026deg;C for 60 min without removal of the paper. During amplification, the generated protons change alkaline pH to acidic, causing the colour of phenol red to change from pink (pH\u0026thinsp;\u0026gt;\u0026thinsp;8.2) to yellow (pH\u0026thinsp;\u0026lt;\u0026thinsp;6.8) [3].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR (qPCR) analysis\u003c/h2\u003e \u003cp\u003eAmplification and real-time fluorescence readings were conducted on the CFX Connect Real-Time PCR System (Bio-Rad). The qPCR reactions included 10 \u0026micro;L of Luna\u0026reg; Universal qPCR Master mix, 0.25 \u0026micro;L of 10 \u0026micro;M each forward and revere primer, 5 \u0026micro;L of DNA template, and 4.5 \u0026micro;L of nuclease-free water, resulting in a 20 \u0026micro;L final reaction volume. Forward and reverse primers previously designed to target the Escherichia phage Lambda gene in \u003cem\u003eE. coli\u003c/em\u003e strain BL21 were used \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Thermal cycling conditions were 1 min at 95\u0026deg;C for initial denaturation, followed by 40 cycles at 95\u0026deg;C for 15 sec and 60\u0026deg;C for 30 sec, with a melt-curve analysis from 65\u0026deg;C to 95\u0026deg;C at 0.5\u0026deg;C intervals.\u003c/p\u003e \u003cp\u003eThe Cq values obtained from qPCR amplification canbe used to calculate the DNA concentration using a previously constructed standard curve \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. However, as most amplifiactions were conducted under attenuating conditions, all qPCR data is reported as number of cycles (Cq).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ecLAMP reactions\u003c/h2\u003e \u003cp\u003eAll LAMP primers used in this study were taken based on an earlier literature report \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e and purchased from Integrated DNA Technologies (IDT). Before the LAMP reaction, a 10X primer mix containing 1.6 \u0026micro;M of each forward inner primer (FIP) and backward inner primer (FIP), 0.2 \u0026micro;M of each forward outer primer (F3) and backward outer primer (B3), and 0.4 \u0026micro;M of each forward loop primer (LF) and backward loop primer (BF) was prepared.\u003c/p\u003e \u003cp\u003eThe cLAMP reaction contained 10 \u0026micro;L of WarmStart\u0026reg; Colorimetric LAMP 2X Master Mix (DNA \u0026amp; RNA, 2 \u0026micro;L of 10X primer mix, and 2 \u0026micro;L of nuclease-free water, making the final volume of 20 \u0026micro;L per reaction. Once the DNADNA-carrying MCE paper was immersed in the LAMP mixture, the PCR tubes were incubated at 68\u0026deg;C for 60 min in Eppendorf mastercycler X50s.\u003c/p\u003e \u003cp\u003eThe potential for in-field use was confirmed using a 12 V heater consisting of an Arduino Nano microcontroller (Seeed Studio), running a PID algorithm which controlled a 12V 2A cartridge heater via switching a transistor. Temperature feedback was provided by a DS18B20 digital temperature probe. The temperature probe and heater cartridge were mounted in drilled holes within an aluminium block also containing recesses for 8 PCR tubes. Using a laptop, an interface designed in MegunoLink was used to maintain the temperature at 68\u0026deg;C, start and stop the heating process, and view the temperature of the block over time graphically. The heater system was powered by a 12V lab power supply, or a portable power bank with a USB-C power-delivery (Comsol 25600 mAh 100 W power bank, Officeworks, Australia). Arduino and interface code are available on request. As the potable system lacked a cooled lid, a drop of silicone oil was added on top of the LAMP mixture to prevent evaporation.\u003c/p\u003e \u003cp\u003eThe colour change of the solution from pink to yellow was used as indication of amplification, and amplification, and the presence of amplicons was confirmed by gel electrophoresis on a 3% (w/v) agarose gel.\u003c/p\u003e \u003cp\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003edetection from milk samples\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA colony of \u003cem\u003eE. coli\u003c/em\u003e BL21 was cultured in 10 mL of sterile LB broth placed on an orbital shaker (Model: TU-400 Thermoline Scientific Equipment Pty Ltd, Wetherill Park, Australia) overnight at 37\u0026deg;C and 150 rpm. Subsequently, 100 \u0026micro;L of the liquid culture was transferred to fresh LB, incubated at 37\u0026deg;C and 220 rpm until the OD\u003csub\u003e600\u003c/sub\u003e (optical density at 600 nm) reached 0.5 using a GENESYS\u0026trade; 30 Visible Spectrophotometer (Thermo Fisher Scientific, Scoresby, Australia). Next, 1 mL of the cell suspension was dispensed into a sterilised 1.5 mL microcentrifuge tube (NEST\u0026reg;, cat. No. 615001, Adelab Scientific, Thebarton, Australia)) and centrifuged for 2 min at 8,000 rpm using a PicoTM 21 centrifuge (Thermo Fisher Scientific, Scoresby, Australia). The resulting cell pellet underwent three washes with a sterile PBS solution. Finally, the washed cells were resuspended in 1 mL of PBS and promptly stored at -20\u0026deg;C for future use.\u003c/p\u003e \u003cp\u003eSkim long-life milk (Coles Heat Treated Australian Milk, Coles Group limited, Hawthorn East, Australia, containing 120 mg calcium, 40 mg sodium, 4.8 g carbohydrate, 3.3 g protein, and less than 1 g of saturated fat) was purchased from a local supermarket. To perform \u003cem\u003eE. coli\u003c/em\u003e detection in milk, 1 mL of the cultured \u003cem\u003eE. coli\u003c/em\u003e was centrifuged to obtain a cell pellet and resuspended in 1 mL of 10-fold diluted skim milk. This spiked milk was then serially diluted with 10-fold diluted milk with the final concentrations ranging from 10\u003csup\u003e6\u003c/sup\u003e to 10\u003csup\u003e1\u003c/sup\u003e CFU/mL and used for PASAP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical and Image analysis\u003c/h2\u003e \u003cp\u003eThe data analysis for qPCR was conducted using Bio-Rad CFX Manager 3.1 software, with Cq determination in single threshold mode. Statistical analysis and graphing were executed using OriginPro 2022b Learning Edition. Each data point represents a minimum sample size of 3 (n\u0026thinsp;=\u0026thinsp;3), and significance differences were assessed through the post-hoc method using Two-way ANOVA.\u003c/p\u003e \u003cp\u003eTo analyse photographs following cLAMP, raw images were subjected to RGB splitting, and the green and blue channels' grey values were measured using ImageJ following a published study \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Then, the mean intensity values of blue were subtracted from green, and the difference was plotted.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003ch2\u003eOn-paper amplification\u003c/h2\u003e\n\u003cp\u003eThe compatibility of commonly used amplification approaches - PCR and LAMP - with MCE paper was investigated, using MCE paper because of its mild negative charge, DNA binding capacity and quick drying owing to the thin film (130 \u0026micro;m). In our earlier work, the viscosity of the ASAP solution provided a challenge in magnetically recovering the beads during DNA extraction \u003csup\u003e[28]\u003c/sup\u003e. With the ambition of using the wicking by MCE paper for fluid transport, the wicking time of the viscous solution was examined with MCE paper with 1.2, 5.0, and 8.0 \u0026micro;m pore size, with the results presented in \u003cstrong\u003eFigure S 1\u003c/strong\u003e. As expected for the limited height to be wicked \u003csup\u003e[29]\u003c/sup\u003e, smaller pores correlated with higher resistance and hence lower flow rates, with the 1.2 \u0026micro;m MCE paper showing the slowest speed, while the rates for 5.0 and 8.0 \u0026micro;m were comparable. Thus, 8 \u0026micro;m MCE paper was selected for faster processing. \u003c/p\u003e\n\u003cp\u003eThe compatibility of commonly used amplification approaches, PCR and LAMP, with MCE paper was investigated. A piece of 8.0 \u0026micro;m pore size MCE paper (3 x 4 mm) was introduced into PCR vials containing target DNA, primers, and an amplification mix, aiming for fluorescence detection of the amplification product using SYBR green as an intercalating dye. No fluorescence could be detected during qPCR and qLAMP (Figure S2 and Figure S3). However, amplicons were detected by gel electrophoresis following LAMP but not PCR, indicating the paper inhibited both amplification and detection during qPCR but only interfered with the fluorescence detection in qLAMP. Combined with an expected loss in sensitivity at higher salt levels because of an increase in dissociation constant (K\u003csub\u003ed\u003c/sub\u003e) of SYBR green for DNA [1, 2], fluorescence detection was deemed unsuitable for the targeted extraction following PASAP. PASAP Colourimetric cLAMP has become a popular choice for in-field testing for non-quantitative detection. For example, NEB\u0026apos;s cLAMP test contains phenol red, a pH indicator that confirms the generation of H\u003csup\u003e+ \u003c/sup\u003eduring amplification, and this approach was adopted here. In addition, the advantages of isothermal amplification include elimination of thermocycling and better salt tolerance of \u003cem\u003eBst\u003c/em\u003e 2.0 polymerase in LAMP amplification for strand displacement activity has better salt tolerance than \u003cem\u003eTaq\u003c/em\u003e polymerase used in PCR (complete (full inhibition at 100 mM vs 40 mM NaCl)) \u003csup\u003e[30]\u003c/sup\u003e, making LAMP a better choice than qPCR for detection following PASAP. \u003c/p\u003e\n\u003cp\u003eASAP method combines lysis and extraction with direct, on-bead DNA amplification. The approach combines the reversible binding of NAs in the presence of poly(ethylene) glycol (PEG) for solid phase extraction onto magnetic beads, an approach conceptually similar to solid phase reversible immobilisation (SPRI) \u003csup\u003e[23]\u003c/sup\u003e, with DNA binding facilitated by the crowding effects of PEG under high salt conditions. Here, the aim is to use mixed cellulose ester paper to act as a stationary phase and fluid handling by wicking. \u003c/p\u003e\n\u003cp\u003eTo confirm binding of the DNA to the MCE paper, a strip of MCE paper was used to wick 20 \u0026micro;L of a sample containing 10 \u0026micro;L purified gDNA and 10 \u0026micro;L 15 % PEG8000 and 3.5 mM KOH in the presence and absence of 1 M NaCl. After wicking the 20 \u0026micro;L sample following the insertion of the short edge into the droplet, the strip was washed with 80 % ethanol before being cut into about 3 mm x 4 mm long at the top, middle, and bottom sections of the paper. Each of these was placed in 20 \u0026micro;L of water to elute the gDNA, after which 5 \u0026micro;L of the eluate was used for qPCR. \u003c/p\u003e\n\u003cp\u003eWhen no salt was present, the larger gDNA concentrations found in the eluates from the MID and TOP sections indicating that the DNA was carried up the MCE paper by the flow caused by the wicking of the aqueous solution by the hydrophilic porous material (\u003cstrong\u003eFigure 2\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e) . As there was no strong DNA binding, the gDNA concentration increased with the distance wicked up the strip, as weakly bound DNA was transported upwards with the wicking of more liquid. As expected, in the presence of 1 M NaCl, the DNA was retained in the bottom section of the MCE paper. The high salt concentration facilitates electrostatic bonding between the slightly anionic surface \u003csup\u003e[31,32]\u003c/sup\u003e and partially neutralises the negative charge of the DNA in the presence of PEG8000, inducing DNA precipitation. The concentration of the gDNA in the tip of the paper strip in the presence of 1 M NaCl demonstrates the potential of using MCE paper for DNA extraction under ASAP conditions. \u003c/p\u003e\n\u003cp\u003eEncouraged by these results, the effect of NaCl concentration was investigated using a reagent combining 15 % PEG8000 and 3.5 mM KOH with increasing NaCl concentrations (0, 0.5, 1.0, 1.5, and 2.0 M) to understand the correlation of the binding capability of paper with NaCl concentration. The gDNA in the eluate was analysed by qPCR. Under low NaCl concentrations (0 \u0026ndash; 1 M) without washing, a decrease in Cq value with increasing salt concentration demonstrated the efficacy of DNA binding with increasing salt concentration (\u003cstrong\u003eFigure 2\u003c/strong\u003e\u003cstrong\u003eB\u003c/strong\u003e). The Cq value, however, increased to 25.75 \u0026plusmn; 1.21 at 1.5 M, and amplification was not detected at 2.0 M as a result of inhibition of amplification; NaCl is known to have an inhibitory effect on \u003cem\u003eTaq\u003c/em\u003e polymerase preventing amplification when [Na\u003csup\u003e+\u003c/sup\u003e ] \u0026ge; 40 mM \u003csup\u003e[30]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe efficacy of the washing step was investigated using 70 % ethanol, commonly used in SPRI and DNA extraction protocols. Washing was performed by dipping the bottom section of the paper in a ~20 \u0026micro;L droplet of 70 % ethanol for 30 sec, followed by air-drying of the strip before eluting the DNA in 20 \u0026micro;L Milli-Q water. Disappointingly, the wash significantly increased Cq values of the eluates about 27.28, 25.68, and 26.00 for 0, 0.5, and 1.0 M NaCl, respectively, indicating considerable DNA loss during the wash. In contrast, improved Cq values of 24.49 \u0026plusmn; 0.62 and 24.23 \u0026plusmn; 003 were obtained after the wash for 1.5 and 2.0 M NaCl, correspondingly showing that the inhibitory effect of high NaCl can be alleviated by washing provided sufficient NaCl was present to ensure strong binding of the DNA to the paper. \u003c/p\u003e\n\u003cp\u003eThe lowest Cq values were obtained using 1.0 M NaCl without washing. However, it is unlikely that the washing step can be eliminated when targeting complex biological samples like milk. As no improvement in sensitivity was found using 2 M NaCl compared with 1.5 M NaCl for binding (Cq values 24.23 \u0026plusmn; 0.03 vs 24.49 \u0026plusmn; 0.62), 1.5 M NaCl was selected for further method optimisation to minimise salt-induced inhibition. The increase in Cq value in comparison with input DNA (dashed line) suggests there is room for improvement, increasing binding efficiency or decreasing DNA loss and/or attenuation of amplification. \u003c/p\u003e\n\u003ch2\u003eOptimisation of the wash\u003c/h2\u003e\n\u003cp\u003eThe results presented above suggest that the eluting strength of 70 % ethanol may be too high for it to be a suitable washing reagent. Washing reagents during DNA extraction on anionic stationary phases has included water-miscible solvents like isopropanol to remove the salt. Non-polar solvents were used in the two-phase wash to limit the carryover of aqueous inhibitors but were typically added between the wash step and the final elution \u003csup\u003e[27]\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003eFour washing liquids were examined, 70 % ethanol, 70 % IPA and two long-chain alcohols (2-dodecanol and 1-decanol) as used in two phase wash \u003csup\u003e[27]\u003c/sup\u003e. In line with the experiments discussed above, 20 \u0026micro;L of a solution containing 10 mL each of the gDNA standard and PSAP reagent was wicked up a 45 mm long MCE strip, after which the bottom 3-4 mm was immersed in a 20 \u0026micro;L-drop of the washing liquid for 30 sec. The MCE paper was air-dried before elution into 20 \u0026micro;L water. The number of qPCR cycles determined the efficacy in reducing loss and/or attenuation. As shown in \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3\u003c/strong\u003e, DNA was detected following a wash with 70 % IPA, with Cq values of 25.92 \u0026plusmn; 0.27 in the eluate. No washing increased the Cq value (27.49 \u0026plusmn; 033), indicating an inhibition equivalent to a 3.3-fold DNA loss. Slightly delayed Cq values of 26.93 \u0026plusmn; 0.33 using 70 % ethanol and 27.12 \u0026plusmn; 0.30 using 100 % 2-dodecanol were found, whereas for 100 % 1-decanol the Cq values were significantly increased to 28.37 \u0026plusmn; 0.74, a higher value than \u003cem\u003eno wash\u003c/em\u003e. The poor washing efficiency of both hydrophobic solvents may the due to the decreased efficacy of the salt removal owing to the poor solubility of NaCl in the washing solvent is vital at the high NaCl concentration used for binding. \u003c/p\u003e\n\u003cp\u003eThe solubility of DNA in IPA is lower than in 70 % ethanol \u003csup\u003e[33]\u003c/sup\u003e, preventing the elution of immobilised DNA while still providing good solubility for NaCl to be an effective washing agent. To optimise the capacity of the washing liquid to remove salt without eluting the DNA, the IPA content was optimised (Figure S4), indicating that 40 % IPA was most effective in mitigating attenuation with minimal processing time (10 sec). For longer washes (60 sec), decreasing Cq values suggest the washing efficacy of IPA\u0026gt;40% increased; however, with the objective of fast sample preparation, 40 % IPA was selected. Lysis and Inhibitory Effects of Tween 20 in PASAP\u003c/p\u003e\n\u003cp\u003eDuring ASAP, Tween 20 was required to prevent magnetic particles from aggregation but may also have contributed to the lytic activity. As the proposed PASAP method replaces the beads with the MCE paper, aggregation of particles is no longer a risk. Hence, the necessity of Tween 20 was investigated. Using \u003cem\u003eE. coli\u003c/em\u003e as a biological sample, 20 \u0026micro;L of 10\u003csup\u003e6\u003c/sup\u003e CFU/mL bacteria was resuspended in the PASAP solutions with or without 0.2 % Tween 20, wicked by the MCE paper and washed with 40 % IPA for 10 sec or directly eluted in water to further determination of the selected washing steps for \u003cem\u003eE. coli\u003c/em\u003e samples. \u003c/p\u003e\n\u003cp\u003eAs shown in \u003cstrong\u003eFigure \u003cem\u003e4\u003c/em\u003e\u003c/strong\u003eA\u003cstrong\u003e,\u003c/strong\u003e the inclusion of Tween 20 led to a statistically significant improvement in DNA concentration in the eluate, increasing from 7.6 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e to 61 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e copies/\u0026micro;L (nearly 10 times) after washing. Previous studies also reported that Tween 20 is non-inhibitory in PCR reactions and can enhance the amplification efficiency under a range of concentrations (4 \u0026ndash; 10 %) by reducing the inhibitory effect and stimulating \u003cem\u003eTaq \u003c/em\u003eDNA polymerase activity \u003csup\u003e[34-36]\u003c/sup\u003e. It is, therefore, anticipated that the inhibition observed without washing in the presence of Tween 20 is the result of the salt, not the Tween 20. The duration of cell lysis was optimised by incubating a mixture of PASAP and cultured \u003cem\u003eE. coli \u003c/em\u003e(10\u003csup\u003e4\u003c/sup\u003e CFU) at room temperature for different durations (0 to 15 min) before the DNA binding step using the MCE membrane. The measured DNA concentrations, as determined by qPCR, following elution are presented in \u003cstrong\u003eFigure \u003cem\u003e4\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eB\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eOverall, a trend of decreasing DNA concentration in the eluate was obtained with an increasing incubation period, with recovery drastically worsened when the \u003cem\u003eE. coli\u003c/em\u003e mixture was incubated for 15 min. The denaturation of DNA can explain this by extended gDNA exposure time to the highly alkaline conditions (\u0026gt; pH 12) \u003csup\u003e[37]\u003c/sup\u003e. The extended lysis time could have damaged the DNA decreasing the amplification efficiency. Considering the 10 min wicking time and the similar DNA concentrations for 0- and 5- min incubation, it was concluded there was no need to for sample incubation with the PASAP reagent before starting the wicking, allowing combining lysis and binding steps into a single step.\u003c/p\u003e\n\u003ch2\u003eEffect of size of MCE paper on direct on-paper cLAMP\u003c/h2\u003e\n\u003cp\u003e\u003cbr\u003eHaving demonstrated LAMP amplification in presence of 3 x 4 mm MCE paper by gel electrophoresis, it was confirmed the addition of phenol red allowed for cLAMP. The PASAP method was then used to bind 100 pg/\u0026micro;L DNA to the MCE paper under ASAP conditions, followed by wash with 70% ethanol, or no wash. The size of MCE paper that can be used for direct on-paper cLAMP is a compromise between binding capacity and attenuation of amplification. To optimise the paper size, MCE paper was cut into squares of 3 x 3, 4 x 4, 5 x 5, and 6 x 6 mm . The squares were exposed to 20 \u0026micro;L ASAP lysis/binding reagent and with/without a wash with 70% ethanol for 30 sec introduced to 20 \u0026micro;L LAMP reaction mix containing the gDNA. After heating to 68 \u0026deg;C for 50 min, amplification was visualised by the colour change and confirmed by gel electrophoresis, as shown in \u003cstrong\u003eFigure \u003cem\u003e5\u003c/em\u003e\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003eA colour change from pink to yellow/orange was observed for all paper sizes and gel electrophoresis confirmed that the colour change of LAMP reaction was indeed the result of amplification (\u003cstrong\u003eFigure \u003cem\u003e5\u003c/em\u003e\u003c/strong\u003e). However, from the intensity of gel bands, it is apparent that the quantity of amplified DNA decreased gradually when increase the size of the MCE paper. This agrees with an earlier report using 2-mm glass fibre disc placed in 50 \u0026micro;L cLAMP mix for the detection of down to 10\u003csup\u003e3\u003c/sup\u003e copies /mL of rotavirus A within 30 min, but larger discs would inhibit amplification \u003csup\u003e[38]\u003c/sup\u003e. Here, cutting the brittle MCE paper with scissors limited the size to strips that were 3-4 mm wide. Recognising this is a crude and manual approach, scissors were selected after we found a laser cutter burned the edges, impeding wicking efficiency and a paper guillotine fragmented the membrane in irregular sizes. The importance of the optimised washing for cLAMP was confirmed conducing experiments with 100 pg/\u0026micro;L gDNA with and without the 10 s wash in 40 % IPA, and resulting colour changes and gels are shown in \u003cstrong\u003eError! Reference source not found.\u003c/strong\u003e, showing significant attenuation without washing as a result of inhibition. Please note that while a positive was detected after 30 min for a high concentration gDNA sample, decreasing the amplification time would lead to a decrease in sensitivity. \u003c/p\u003e\n\u003cp\u003eThe benign PAPAS reagent and limited supplies (a pipette with 2 pipette tips, 2 test tubes and the MCE strip) make the PASAP method attractive for in-field testing, particularly in low resource settings. With all amplifications listed above conducted in a PCR machine, compatibility with in0field use was confirmed using a purpose-designed heater, accommodating the PCR vials in recesses CNC-milled in an aluminium block (Figure S9A, B). The temperature was controlled using a laptop using the feedback from a temperature sensor. Evaporation of the amplification mixture when heated in the aluminium block prevented amplification, probably unlike in PCR machines the block did not have a heated lid, leading to condensation. A drop (~ 20 mL) of silicone oil provided an effective inert barrier to prevent evaporation of the amplification solution. Preliminary testing of the 12 V heater yielded following PASAP of a gDNA standard yielded comparable results to those obtained using the PCR instrument, showing a colour change from yellow to pink following 60 min at 68\u0026deg;C for 100 pg/\u0026micro;L gDNA (Figure S9C). No differences were found between the heater powered using a power supply or battery pack, conforming suitability for in-field use. \u003c/p\u003e\n\u003ch2\u003eAssay sensitivity and E. coli detection in milk\u003c/h2\u003e\n\u003cp\u003eWith the PASAP/cLAMP method established, \u003cem\u003eE. coli\u003c/em\u003e was used as biological target to understand applicability, and milk was selected as sample matrix. Recently, multienzyme isothermal rapid amplification was used for the detection of Streptococcus in milk following its extraction on filter paper following lysis\u003csup\u003e[39]\u003c/sup\u003e. Here, first cultured bacteria were serially diluted in PBS from 10\u003csup\u003e4\u003c/sup\u003e to 10\u003csup\u003e1\u003c/sup\u003e\u003csub\u003e \u003c/sub\u003eCFU/mL, and 50 \u0026micro;L of the diluted samples were used for the PASAP method following the optimised workflow with negative control (fresh PBS without \u003cem\u003eE. coli\u003c/em\u003e). \u003c/p\u003e\n\u003cp\u003eThe PASAP method combined with cLAMP successfully allowed for the detection of \u003cem\u003eE. coli \u003c/em\u003efrom PBS solutions with a LOD at 10\u003csup\u003e2\u003c/sup\u003e CFU/mL, or 1 CFU present in the 10 \u0026micro;L sample, with the presence of amplified DNA for all positive colourimetric results confirmed by gel electrophoresis (\u003cstrong\u003eFigure 6\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e). Semi-quantitative data were obtained by image analysis\u003csup\u003e[1]\u003c/sup\u003e, (\u003cstrong\u003eFigure 6\u003c/strong\u003e\u003cstrong\u003eB\u003c/strong\u003e) showing the calculated colour intensity of LAMP solution for a range of \u003cem\u003eE. coli\u003c/em\u003e concentrations\u003cstrong\u003e. \u003c/strong\u003eWe previously reported that 15 CFU/50 \u0026micro;L could be detected using a magnetic bead kit, and 150 CFU/50 \u0026micro;L using a spin column \u003csup\u003e[28]\u003c/sup\u003e, making 1 CFU/10 \u0026micro;L slightly between than but inferior to the 0.15 CFU detected in a 50 \u0026micro;L using the ASAP method. \u003c/p\u003e\n\u003cp\u003eWhen using milk as the sample, the yellow hue indicative of a positive test shifted slightly towards orange, but a change from the pink in the absence of DNA could still observed at 10\u003csup\u003e2\u003c/sup\u003e CFU/mL; an evident change was only found at 10\u003csup\u003e3\u003c/sup\u003e CFU/mL (\u003cstrong\u003eFigure 6\u003c/strong\u003e\u003cstrong\u003eC\u003c/strong\u003e). While detection through colour change and gel bands at 10\u003csup\u003e2\u003c/sup\u003e CFU/mL was demonstrated twice in two of five replicates, the high failure rate means using the PASAP method E. \u003cem\u003eColi \u003c/em\u003ecan be detected in milk at 10\u003csup\u003e3\u003c/sup\u003e CFU/mL (\u003cstrong\u003eFigure 6\u003c/strong\u003e\u003cstrong\u003eD\u003c/strong\u003e). This decreased sensitivity compared with other work \u003csup\u003e[14,39]\u003c/sup\u003e should be contrasted by a significant reduction in reagents, consumables, waste and manual handling. The sensitivity of PASAP/cLAMP may be improved by increasing the sample volume processed, and future research will focus on the introduction of a wicking pad to increase the sample volume, as currently the sample volume is limited by low wicking rate of the PASAP solution in the MCE paper. \u003c/p\u003e\n\u003cp\u003eThe presented PASAP method provides a fast, low-cost and easy sample preparation method compatible with on-paper cLAMP that can be applied to complex biological samples including for the detection of bacteria in milk. The simple workflow is suitable for automation and combined with a battery-powered heater has potential to be integrated into hand-held-devices for PONT in food safety and diagnostics. \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA new method for on-paper DNA extraction and cLAMP with minimal use of reagents, consumables and equipment is presented. The aqueous alkaline PEG-based reagent lyses the cell providing the high pH for alkaline lysis, enhancing the lytic activity with the surfactant Tween20. Owing to crowding conditions in presence of PEG and NaCl the DNA was immobilised at the tip of a strip of mixed cellulose ester paper used for wicking the lysate. Following a 10 sec wash conducted by dipping the strip in 40% isopropanol, the tip of the dried paper strip is snapped off into a vial for colorimetric LAMP. The proposed workflow takes 15-min and uses wicking for liquid handling. The instrumentation used was minimal (a pipette for depositing 20 \u0026micro;L drops of sample and washing reagent) and produces minimal waste (2 vials, 2 pipette tips and a 3 x 45 mm membrane). Supporting the isothermal amplification with a battery-powered 12 V heater (60 min at 68 \u0026deg;C), the method is compatible with in-field use. Recognising the quantitative limitations of cLAMP, the method allowed for the detection of E. Coli down to 10 CFU/mL in buffer, increasing to 10\u003csup\u003e3\u003c/sup\u003e CFU/mL in 1:10 diluted milk though occasionally a colour change was detected at 10\u003csup\u003e2\u003c/sup\u003e CFU/mL Future work will focus enhancing the sensitivity by processing larger sample volumes without compromising processing time, for example, connecting the paper with an absorbent pad.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe acknowledge Mr Richard Alexander for the fabrication of the heater. SML acknowledges the receipt of a scholarship from Deakin University\u0026apos;s Faculty of Science, Engineering, and Built Environment and the graphical illustration from JML. DY acknowledges Alfred Deakin Postdoctoral Fellowship from Deakin University.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eSML performed all experiments, wrote manuscript, and analysed the data. \u0026nbsp;DY, ED and RMG contributed through support in the experimental design, data analysis and interpretation and revised manuscript.{Varona, 2019 #273}\u003c/p\u003e\n\u003ch2\u003eCompeting Interest Statement\u003c/h2\u003e\n\u003cp\u003eThe authors have no competing financial and/or non-financial interests to declare in relation to the work described.\u003c/p\u003e\n\u003ch2\u003eData availability Statement\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe data generated and/or analysed during the current study not presented in this paper are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMalpartida-Cardenas, K., Baum, J., Cunnington, A., Georgiou, P. \u0026amp; Rodriguez-Manzano, J. 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Frontiers in Veterinary Science 10, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fvets.2023.1100246\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2023.1100246\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Paper-based DNA extraction, PASAP, Nucleic acid amplification test (NAAT), Elution-free sample preparation, colourimetric loop-mediated isothermal amplification (cLAMP), E. coli detection, Milk, Point-of-need, PONT","lastPublishedDoi":"10.21203/rs.3.rs-3875247/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3875247/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNucleic acid amplification testing has great potential for point-of-need diagnostic testing with high detection sensitivity and specificity. Current sample preparation is limited by a tedious workflow requiring multiple steps, reagents and instrumentation, hampering nucleic acid testing at point of need. In this study, we present then use of mixed cellulose ester (MCE) paper for DNA binding by ionic interaction and fluid transport by wicking. The poly(ethylene) glycol-based (PEG) reagent simultaneously provides the alkalinity effect for alkaline lysis and crowding effects for ionic DNA binding of the DNA under high salt conditions. Using a narrow strip of paper, the freed DNA concentrates at the paper tip, while the wicking removes the sample matrix when briefly washing using 40% isopropanol, a 15 in process that is followed by on-paper amplification after a drying step. Colourimetric loop-mediated isothermal amplification enabled the detection of 10\u003csup\u003e2\u003c/sup\u003e CFU/mL of \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) from culture media and the detection of \u003cem\u003eE. coli\u003c/em\u003e in milk\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e CFU/mL (10 CFU) after incubation at 68\u0026deg;C for 60 min, demonstrating applicability of the method to complex biological samples.\u003c/p\u003e","manuscriptTitle":"Method for Lysis and Paper-based Elution-free DNA Extraction with Colorimetric Isothermal Amplification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-12 12:27:58","doi":"10.21203/rs.3.rs-3875247/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-23T15:17:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-21T20:07:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16c847a2-0455-4d2e-a99f-01725e0121df","date":"2024-02-14T16:20:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b5aa698c-d529-42ec-944c-a067d5c4220a","date":"2024-02-12T19:09:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-12T18:46:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-09T13:27:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-09T12:39:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-09T12:37:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-18T08:30:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"884fe729-974b-4e34-bc08-04af7a31ba18","owner":[],"postedDate":"February 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":28694217,"name":"Biological sciences/Biological techniques/Analytical biochemistry"},{"id":28694218,"name":"Biological sciences/Biological techniques/Lab on a chip"},{"id":28694219,"name":"Biological sciences/Biological techniques/Sensors and probes"},{"id":28694220,"name":"Physical sciences/Chemistry/Analytical chemistry/Bioanalytical chemistry"},{"id":28694221,"name":"Physical sciences/Chemistry/Analytical chemistry/Lab on a chip"},{"id":28694222,"name":"Physical sciences/Chemistry/Analytical chemistry/Microfluidics"},{"id":28694223,"name":"Physical sciences/Chemistry/Analytical chemistry/Sensors"}],"tags":[],"updatedAt":"2024-04-15T07:28:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-12 12:27:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3875247","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3875247","identity":"rs-3875247","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}