key: cord-0916773-vhmsx8mb
authors: Rodriguez-Mateos, Pablo; Ngamsom, Bongkot; Walter, Cheryl; Dyer, Charlotte E.; Gitaka, Jesse; Iles, Alexander; Pamme, Nicole
title: A lab-on-a-chip platform for integrated extraction and detection of SARS-CoV-2 RNA in resource-limited settings
date: 2021-06-14
journal: Anal Chim Acta
DOI: 10.1016/j.aca.2021.338758
sha: 9ee791fd8a8d6f96cad2a6584105b188337c888d
doc_id: 916773
cord_uid: vhmsx8mb

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the unprecedented global pandemic of coronavirus disease-2019 (COVID-19). Efforts are needed to develop rapid and accurate diagnostic tools for extensive testing allowing for effective containment of the infection, via timely identification and isolation of SARS-CoV-2 carriers. Current gold standard nucleic acid tests require many separate steps that need trained personnel to operate specialist instrumentation in laboratory environments, hampering turnaround time and test accessibility, especially in low-resource settings. We devised an integrated on-chip platform coupling RNA extraction based on immiscible filtration assisted by surface tension (IFAST), with RNA amplification and detection via colorimetric reverse-transcription loop mediated isothermal amplification (RT-LAMP), using two sets of primers targeting open reading frame1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2. Results were identified visually, with a colour change from pink to yellow indicating positive amplification, and further confirmed by DNA gel electrophoresis. The specificity of the assay was tested against HCoV-OC43 and H1N1 RNAs. The assay based on use of gene N primers was 100% specific to SARS-CoV-2 with no cross-reactivity to HCoV-OC43 nor H1N1. Proof-of-concept studies on water and artificial sputum containing genomic SARS-CoV-2 RNA showed our IFAST RT-LAMP device to be capable of extracting and detecting 470 SARS-CoV-2 copies mL(-1) within 1 h (from sample-in to answer-out). IFAST RT-LAMP is a simple-to-use, integrated, rapid and accurate COVID-19 diagnostic platform, which could provide an attractive means for extensive screening of SARS-CoV-2 infections at point-of-care, especially in resource-constrained settings.

An outbreak of Coronavirus Disease 2019 (COVID-19) caused by a novel human betacoronavirus SARS-CoV-2 was reported in Wuhan, Hubei province of China, in late December 2019 [1, 2] . Due to the rate of transmission and possible fatal progression, the World Health Organisation (WHO) declared a global health emergency on 31 January 2020 and a pandemic situation on 11 March 2020 [3] . The clinical spectrum of COVID-19 ranges from asymptomatic infection to acute respiratory distress syndrome with multisystem failures [4, 5] . The epidemiological update as of 4 February 2021 reported over 100 million cumulative cases and 2.2 million deaths globally since the start of the pandemic [6] . The increasing gravity of the situation has been attributed to the highly contagious nature of the disease from both asymptomatic and pre-symptomatic cases [7, 8] , in combination with the lack of effective point-of-care testing for rapid and accurate identification of SARS-CoV-2 carriers [9] .

Among rapid diagnostic tests developed for point-of-care and community purposes are the antigen lateral flow tests targeting the nucleocapsid protein of SARS-CoV-2. Despite showing great promise for ease-of-use and ≤ 30 min turnaround time, the low sensitivity (10 6 copies mL -1 ) is a major disadvantage of this approach [10] . Current gold standard COVID-19 tests still rely on nucleic acid amplification tests measuring viral nucleic acids based on quantitative reverse transcription polymerase chain reaction (RT-qPCR). Samples from the upper respiratory tract (nasopharyngeal swab, nasal aspirate, or pharyngeal swab) or lower respiratory tract (sputum, tracheal aspirate) are taken from suspected cases for RNA J o u r n a l P r e -p r o o f extraction followed by reverse transcription and cDNA amplification of a genomic specific region [4, 5] . The analytical limits of detection of RT-qPCR are usually around 10 3 viral RNA copies mL -1 with sample-to-result times of 24-48 h [11] . Several SARS-CoV-2 RT-PCR detection kits have been developed by different companies and institutions [9] .

However, such assays are mostly limited to highly specialized laboratories with trained personnel [4, 12] . As opposed to conventional PCR assays, isothermal nucleic acid amplification tests utilise a single temperature and involve no expensive instrumentation, requirement for calibrated internal controls nor trained personnel for operation and result interpretation. Specifically, loop-mediated isothermal amplification (LAMP) relies on autocycling strand displacement DNA synthesis using four to six primers, resulting in exponential amplification [13] . Colorimetric RT-LAMP based on pH change can offer rapid and sensitive detection of viral RNAs and requires only a heat source [14, 15] . RT-LAMP has been applied for detection of many viral infectious diseases [15] . Several groups have explored RT-LAMP for SARS-CoV-2 RNA detection, showing comparable results to the gold standard RT-PCR with lowest sensitivity being ca. 20 -200 RNA copies per reaction, while achieving faster turnaround times (30 -40 min) [16] [17] [18] [19] [20] [21] [22] . Despite the successful detection of SARS-CoV-2 RNA from crude cell lysates reported by Zhang et al. [16] , the majority of the reported RT-LAMP assays required RNA extraction and purification steps to be performed on clinical samples prior to amplification using laboratory based techniques and procedures. A lower sensitivity of the assays was also reported when clinical samples were directly subjected to RT-LAMP without an RNA isolation step [23] . In addition, investigations on direct RT-LAMP of respiratory samples without RNA extraction using the Variplex™ system [24] demonstrated high false negative rates as well as failure to reliably detect SARS-CoV-2 [25] .

A lab-on-a-chip process for a combined workflow of nucleic acid extraction and purification, namely 'IFAST', utilises 'pinned' aqueous/organic liquid interfaces in microchannels to streamline the extraction mechanism, replacing all washing steps with a single traverse of an immiscible fluid barrier [26] . By attaching magnetically-responsive particles to a target cell/ molecule via immunocapture, the target bound magnetic particles can be selectively transported across the immiscible barrier and into a separate solution.

Contaminants are prevented from inadvertently crossing the immiscible phase by the high interfacial energy associated with the immiscible phase/aqueous phase boundaries [27] .

IFAST has been successfully employed for preconcentration and purification of DNAs [26, 28, 29] and RNAs [29, 30] from various samples. The platform is simple and requires no additional laboratory infrastructure, and yet maintains comparable or better purity, yield and J o u r n a l P r e -p r o o f scalability to existing methods [26] . Previously, our group combined the versatility of the IFAST device with sensitive molecular detection via adenosine triphosphate (ATP) bioluminescence assay, and devised on-chip IFAST/ATP platforms for rapid detections of Escherichia coli O157:H7 from wastewater samples [31] , and Group B Streptococcus from urine samples [32] for point-of-need testing in resource-limited settings. Very recently, Wimbles et al. reported the use of IFAST RT-LAMP platform for on-site extraction of DNA from animal dung, enabling identification of Ceratotherium simum, a near threatened species [33] .

In this communication, we present for the first time, a lab-on-a-chip device based on Oligo (dT)-coated magnetic beads, SYBR Safe and nuclease-free water were supplied by Thermofisher Scientific, UK. Mineral oil and guanidine hydrochloride (GuHCl) were purchased from Sigma-Aldrich.

This work utilised polymethyl methacrylate (PMMA) chips fabricated via CNC machine milling (Datron M7, Milton Keynes, UK), rather than the polydimethylsiloxane (PDMS) chips employed previously for IFAST/ATP platforms [31, 32] . The chip features a large sample chamber 1 (26 mm wide, 26 mm long); wash chambers 2, 4, 6 (3 mm wide × 3 mm long); wash chamber 3 (3 mm wide× 14 mm long); wash chambers 5, 7, 8 (3 mm wide × 8.5 mm long); and a detection chamber 9 (3 mm wide × 3 mm long). All chambers used had a J o u r n a l P r e -p r o o f 5 depth of 3.8 mm, and were interconnected via gates (3 mm to 0.5 mm wide, 3 mm long, 0.2 mm deep) as shown in Fig. 2 . To reduce carry-over and provide more effective washing, the previous chip designs were modified to include more wash chambers (Section B, Supplementary Data). To prevent contamination with RNases, devices were sprayed with RNase decontamination solution (ThermoFisher Scientific), followed by rinsing with nuclease-free water (ThermoFisher Scientific) and were left to dry prior to use. The bottom of the chip was sealed with PCR adhesive film (ThermoFisher Scientific).

Tube-based RT-LAMP was firstly performed targeting ORF1a and N genes using two sets of primers as described by Zhang et al., (Table S1 , Supplementary Data [16] ). Ten-fold serial dilutions of genomic SARs-CoV-2 RNA (4. However, the reaction was cooled to room temperature to allow colour intensification prior to photographing. Tubes or IFAST devices were placed on an A4 white printing paper to provide a clear background. Images were captured using a mobile phone camera (SAMSUNG Galaxy A3) taken from above the tubes/IFAST devices, under normal laboratory lighting. For comparison, images of tube/IFAST device with negative control were taken in the same frame as the investigated samples. 

On-chip RNA extraction was similarly performed as described above, followed by the 

Utilising the commercially available colorimetric LAMP kit and two primer sets targeting ORF1a and N genes [16] , the effectiveness of tube-based RT-LAMP for SARS-CoV-2 RNA detection was firstly assessed on a series of ten-fold dilutions performed on the initial genomic RNA (4.7×10 3 copies µL -1 ). The assay was capable of amplifying ≥ 470 copies of genomic RNA after 30 min (Fig. 3) , a reduced sensitivity compared with 120 copies being reported for RNA fragments using the same primer sets reported by Zhang et al. [16] .

Being amongst the largest viral genomes, with 30 kb size [35] , a longer time is expected for primers and enzymes to find the target sequences within the genome compared with much shorter RNA fragments.

In order to check the feasibility of performing on-chip RT-LAMP as a consecutive step after RNA extraction, it is vital to verify that (i) on-chip amplification occurs similarly to the tube-based assay, and (ii) the magnetic beads utilised for RNA extraction do not interfere with amplification. Consequently, for on-chip RT-LAMP, oligo (dT)-functionalised magnetic beads were added to the sample chamber and directed through the immiscible phases to combine with the RT-LAMP reaction mix in the last chamber prior to heating. Successful onchip amplification was achieved with no interference from the magnetic beads.

The specificity of the RT-LAMP primers for SARS-CoV-2 RNA detection was tested against Betacoronavirus HCoV-OC43, a ubiquitous human coronavirus in the environment responsible for up to one third of common colds [36, 37] , and influenza A virus H1N1, which shares substantial similarities in viral shedding, transmission dynamics and clinical features of viral respiratory illnesses [38] . RT-LAMP assays conducted on genomic HCoV-OC43 and H1N1 using their respective primers showed positive amplifications (Fig. 4a) .

Although demonstrating a slightly faster amplification than Gene N primers, ORF1a primers showed cross-reactivity with both HCoV-OC43 and H1N1 RNAs (Fig. 4b) , and were excluded from further investigations. In contrast, only samples with SARS-CoV-2 RNA resulted in positive amplifications exploiting Gene N primers, while HCoV-OC43 and H1N1 remained negative (Fig. 4b, c) , demonstrating specific pairing of Gene N primers to SARS-CoV-2 RNA, but not to HCoV-OC43 or H1N1 RNAs. This indicates the possibility of simultaneously diagnosing infection(s) of COVID-19 (SARS-CoV-2), a common cold virus (HCoV-OC43), and influenza A virus (H1N1) by paralleling on-chip RT-LAMP at a single amplification temperature and time, using primer sets specific to target viral genomes.

The microscale IFAST was next explored as a platform for one-step isolation and purification of SARS-CoV-2 RNA. Typically, multi-step solid phase extraction (SPE) processes for RNA extraction (e.g., Qiagen TurboCapture, Invitrogen FastTrack MAG 96) are labour-intensive and require expensive automated systems to facilitate the extensive washing that must be performed on individual samples [26] . The IFAST approach simplifies and expedites the cumbersome RNA extraction process, and enables direct interfacing with the amplification process, reducing overall labour and time-consuming pre-amplification steps. Oligo (dT)-functionalised magnetic beads were employed for selective isolation of polyadenylated RNA species. This specific capture discriminates ribosomal RNA, DNA, proteins and small RNA molecules. Although RNA fragmentation may occur during extraction, primers targeting the N gene region near to the 3' poly-A tail were used to ensure that the captured zone could be amplified and detected. One further advantage of using oligo (dT) magnetic beads is that this approach can also provide an opportunity to include a positive swab control, such as detection of RNAse P mRNA [39] . This abundant mRNA is an excellent sample control that is currently a typical internal standard for RT-PCR diagnostics, but is not incorporated in point-of-care COVID-19 lateral flow testing devices [40, 41] .

The successful use of IFAST was demonstrated for extraction and purification of genomic SARS-CoV-2 RNA from aqueous samples containing 470 copies mL -1 within 10 min, validated by positive amplification of bead-bound isolated RNA via off-chip RT-LAMP assays (Fig. 5) . In these experiments, the magnetically isolated RNAs would be between 470 and 47 copies, as suggested by Fig. 3 (the same reaction time amplified 470 copies, Gene N primers). The level of detected genomic RNA isolated on-chip was significantly lower than the reported median viral loads of 7.99×10⁴ copies mL -1 and 7.52×10⁴ copies mL -1 in throat swab and sputum samples, respectively [42] . This on-chip IFAST purification process uses only minute quantities of mineral oil that can effectively filter contaminants from clinical samples in a single step, thereby eliminating multiple washing or centrifugation normally needed for RNA purification. The positive amplifications of the magnetically-isolated RNA by off-chip RT-LAMP confirmed successful purification with no adverse effect on RNA integrity. The current protocol was performed manually, demonstrating its simplicity with no requirement for additional laboratory infrastructure. However, improved capacity can be achieved by automation [43] . This on-chip RNA extraction platform is not limited by the use of oligo (dT) magnetic beads, it can also be applied with other suitable surface chemistries for J o u r n a l P r e -p r o o f magnetic isolation, e.g., silica paramagnetic particles [30, 33, 44] , and can be further explored for RNA extraction from clinical samples.

Having shown the two on-chip processes separately, i.e. RNA extraction via IFAST and RT-LAMP, the combined workflow for on-chip extraction and on-chip RT-LAMP was next investigated with water samples containing genomic RNA (Fig. 6a) . The platform was capable of detecting 470 RNA copies from 1 mL sample in 40 min. The entire process took less than 1 h to complete (2 min sample loading, 10 min RNA extraction and 40 min amplification), with the negative/positive results being clearly distinguishable by the naked eye.

The performance of the platform was further tested with artificial sputum spiked with genomic RNAs due to clinical sample inaccessibility during the investigation. The device is ultimately aimed for point-of-care testing, with swab samples being directly loaded into the sample chamber to mix with lysis reagents, followed by RNA extraction, amplification and visual detection for negative/positive results. With this in mind, the RNA-spiked artificial sputum samples were diluted with GuHCl, a chaeotropic reagent commonly used for isolation of intact mRNA from cells [45] , as lysis buffer containing strong surfactant can destroy the immiscible interfaces of the IFAST device. In addition, GuHCl can act as Ribonuclease (RNase) inhibitor which helps to maintain RNA integrity, a common challenge in analysis of clinical samples. By using GuHCl, mRNAs could be isolated from potential viral capsids without any additional steps, extracted with oligo (dT) magnetic beads and amplified via RT-LAMP. The compatibility of the GuHCl with RNA extraction by oligo (dT) magnetic beads, as well as RT-LAMP, was shown by successful specific detection of SARS-CoV-2 from samples containing SARS-CoV-2, H1N1 and HCoV-OC43 RNAs (Fig. 6b) . This interpretation. Additionally, these platforms have low sensitivities (2×10 4 and 9×10 3 copies mL -1 , for Abbott and Luminex systems, respectively) compared to conventional RT-qPCR [46] . The herein proposed RNA-based platform is also much more sensitive than the rapid antigen-based lateral flow assays aiming for community and point-of-care testing, whose positive results normally require confirmation from nucleic acid amplification test(s) [9, 47] .

The cost of our device is currently ca. $10 (small scale device fabrication = $1.8, reagents = $8.3; the FDA EUA approved Abbott BinaxNOW™ COVID-19 Ag Card rapid test costs $5). This estimation excluded the cost of a block heater and NdFeB magnet assembly as they can be reused. This figure is anticipated to be substantially reduced by mass production, i.e., using injection moulding process to replace the CNC-machined fabrication.

J o u r n a l P r e -p r o o f

We have devised a low complexity, high sensitivity and specificity lab-on-a-chip platform based on IFAST RT-LAMP for SARS-CoV-2 RNA detection, integrating two consecutive steps of RNA extraction and amplification into a single device. The current setup allowed detection of as little as 470 copies of genomic RNA within 1 h. This platform has the potential to increase COVID-19 screening speed and expand testing capacity for disease surveillance as well as point-of-care testing in resource-limited settings, enabling timely isolation prior to unwitting viral transmission. 

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