key: cord-0784184-crifn8ze
authors: Chen, Wei; Cai, Bo; Geng, Zhi; Chen, Fenghua; Wang, Zheng; Wang, Lin; Chen, Xiaoyuan
title: Reducing false negatives in COVID-19 testing by using microneedle-based oropharyngeal swabs
date: 2020-10-05
journal: Matter
DOI: 10.1016/j.matt.2020.09.021
sha: e347fa533cca9c7299f9434720a0354f856ff2fb
doc_id: 784184
cord_uid: crifn8ze

Coronavirus disease 2019 (COVID-19) has become a severe threat to human health worldwide. Early etiological diagnosis plays a critical role in controlling COVID-19 pandemic. However, etiological diagnosis has been largely compromised by high "false negative" rates of viral nucleic acid testing, resulting from limited sampling efficiency using conventional oropharyngeal swabs. Herein, we engineer regular swabs by using a microneedle (MN) patch to significantly improve the quality and quantity of virus collection. The combination of MNs with different crosslinking levels endows the patches with dual capability of mucus penetration and virus extraction. Moreover, the antibody (Ab) against viral spike protein was integrated into the patch, conferring MNs with an active virus capture potential. By taking advantage of the biological and engineered species, it is believed the designed MN/Ab swabs could serve as a promising tool to improve current sampling efficiency with less "false negatives", contributing to the containment of COVID-19 pandemic.

An outbreak of coronavirus disease 2019 , a severe respiratory illness, has 2 become one of the most-concerning threats to human health globally, 1 impacting 215 3 countries (including territory or area), causing over 30 million infected cases and more than 4 950,000 deaths by September 19, 2020. 2 Severe acute respiratory syndrome coronavirus 2 5 (SARS-CoV-2) has been identified as an etiological cause of the disease. 3,4 To effectively 6 contain this pandemic, early detection of SARS-CoV-2 is critical as it would facilitate timely 7 diagnosis and treatment, and implementation of public health measures. 5 Among the current 8 diagnostic approaches, viral tests and antibody detection are two main methods that have 9 been broadly applied. 6 As serological tests (blood antibody detection) mainly reflect the 10 infection at the mid-and late-stage of the illness, 7 viral nucleic acid 11 polymerase-chain-reaction (PCR) testing on specimens obtained through oropharyngeal (OP) 12 swab sampling, is a gold standard and widely used method for early diagnosis, due to its 13 practical convenience, simple equipment requirement, low cost, and negligible influence on 14 sample integrity. [8] [9] [10] However, early studies demonstrated that such tests using OP samples for 15 detecting COVID-19 might produce up to 30% "false negatives". [11] [12] [13] [14] This is thought to be 16 largely ascribed to ineffective sample collection when using regular swabs, as they could only 17 provide limited physical adsorption from and nearly no infiltration into mucosal tissues, with 18 which they have weak interactions, or show poor recovery for low-volume samples with low 19 transfer efficiency. 12, 13, 15 Moreover, regular swabs could only collect superficial tissues from 20 pharyngeal mucosa surface, which are readily contaminated or diluted by foods and 21 J o u r n a l P r e -p r o o f virus recruitment. [29] [30] [31] After optimization for base fabrication, a soft flexible patch was 1 attached to an OP swab using biomedical device adhesives, thus conferring the regular swab 2 with the abilities of mucus penetration and active virus extraction (Figure 1 ). Thanks to 3 chemically engineered MN swabs, the diagnostics of COVID-19 has been remarkably 4 improved with decreased "false negatives", bringing great hopes in controlling COVID-19 pa 5 6 RESULTS AND DISCUSSION 7

To fabricate microneedle patches, a well-designed master mold was printed following the 8 standard protocol (Figure 2A and 2B). A poly(dimethylsiloxane) (PDMS) mold (negative) 9 was constructed from the master mold ( Figure 2C ) by mixing two-part resin systems 10 containing vinyl groups and hydrosiloxane groups. To capture the SARS-CoV-2, the antibody 11 targeting the spike protein ( Figure 2D and Figure S1 ) on the virus surface was selected as the 12 source for virus acquirement. 33 After being loaded into the PDMS mold, alginate polymer 13 was cast to form the MNs via distinct crosslinking processes ( Figure 2E) . The high-level 14 crosslinking (hard) was expected to provide extensive tissue penetration capability, while low 15 crosslinking-level (soft) was to offer potent virus extraction and ease of release. Fluorescein 16 isothiocyanate (FITC) and rhodamine dyes were respectively used to label the microneedle 17 patches with different crosslinking levels ( Figure S2 ). These patches were combined by 18 biomedical device adhesives ( Figure 2F ). Scanning electron microscopy (SEM) images 19 exhibited a recognizable distinction between the high and low cross-linked MNs ( Figure 2G ), 20 consistent with the observation from fluorescence microscopy ( Figure 2H ). The obvious 21 boundary between the MNs could be readily identified by fluorescence distribution (Figure  1 2I), suggesting the successful fabrication of the dually functionalized MN patches. The 2 combination of MNs with diverse strengths could complement each other's deficiencies by 3 preventing low virus migration (high cross-linked MNs) and poor penetration (low 4 cross-linked MNs), providing a synergetic and interdependent profile. With this smart design, 5 a dually functionalized MN patch was invented, which would hold great promise to improve 6 the SARS-CoV-2 collection. 7 8 After MN formation, the assembly of the patches onto the regular swab was optimized 9 by adjusting the base intensity. 1% alginate cross-linked by 50 mM CaCl 2 was applied to 10 construct the base, which would provide sufficient strength and toughness to allow flexible 11 bending in a controllable fashion ( Figure 3A -C). This bendable property is indispensable for 12 the MN patch to fit swab heads as brittle or hard materials would hinder shape alteration, as 13 well as the subsequent attachment. Notably, the transparent patch with a thin base (1 mm) 14 could be effortlessly curled by hand and perfectly wrapped on the head of a regular swab 15 ( Figure 3D -F). When using the swab, a rotated scratch was suggested owing to the interlaced 16 distribution of dually functionalized MNs on the patch. In such a way, the high cross-linked 17 ones would penetrate the mucus introducing numerous micro-channels (slightly bent, Figure  18 3G), while low cross-linked ones would facilitate virus infiltration and release in virus 19 preservation solution (VPS) by controllable dissolution ( Figure 3H ). The strength difference 20 ( Figure 3I ) between the patches and controlled dissolving behaviors ( Figure S3 ) of the 21 patches verified the observations, further suggesting a tunable and precisely manageable 1 sampling process. 2 3 To evaluate the sampling efficiency of engineered MN swabs, FITC-labeled streptavidin 4 was introduced in the mouth of rats to act as a drug model for the in vivo test ( Figure 4A ). 5

After biotin was loaded in the MNs, dually functionalized MN (high and low cross-linked) 6 patches were combined and attached to regular swabs. Similar to the treatment of patients, 7 the swabs were inserted into the oral cavity of rats and rotated to collect samples for seconds 8 ( Figure 4B ), then transferred to VPS, followed by fluorescence measurement. Apparently, the 9 MN/biotin swab turned green, indicating a successful capture of FITC-streptavidin ( Figure  10 4C). Importantly, compared with empty MN and regular swabs, a marked increase in FITC 11 fluorescence intensity was observed after MN/biotin swab treatment ( Figure 4D ), revealing a 12 synergistic effect of MN insertion and antigen-antibody interactions in sampling. After MN 13 treatment, micro-scaled channels were generated as evidenced by tissue staining ( Figure 4E ), 14

suggesting an in-depth tissue sampling (regular swab could not penetrate tissues.). It should 15 be emphasized that MN treatment is minimally invasive while the micro-channels induced by 16 sampling effect ( Figure 4G ). This was mainly because within this range, the advantages of 1 tissue insertion and active binding could be both maximized, synergizing sampling efficiency. 2 Similar to momentary sampling using regular swabs, this process with engineered MN swabs 3 was also swift (less than 10 seconds, Figure 4H ), thus retaining convenience while 4 guaranteeing effectiveness. In such a way, it is believed that this well-designed MN swab 5 might rapidly substitute regular swabs given its superior sampling efficiency and negligible 6 side effects. 7 8 Facing the challenge of high "false negative" rates during COVID-19 detection, we 9

applied the microneedle-based oropharyngeal swabs on a COVID-19 rat model to evaluate 10 their sampling efficiency. Briefly, a COVID-19 pseudovirus developed by the YEASON 11 Company was introduced in the oral cavity of the rat model, followed by sampling using 12 different swabs ( Figure 5A ). After treatment in VPS, the samples were tested within 24 h by 13 RT-PCR ( Figure 5B ). Three typical targets for COVID-19 testing, nucleocaspid (N), envelope 14 (E), and RNA-dependent RNA polymerase (ORF1ab/RdRp) genes, were analyzed by using 15 real-time RT-PCR assays. As shown in Figure 5C , regular swab sampling needed a higher Ct 16 value (> 30) to gain an obvious signal for the detection, indicating a low virus payload. 17

Interestingly, a MN swab (without antibody) slightly enhanced the sampling by reducing the 18 Ct value, ascribing to the MN penetration to mucus tissue for more virus collection. Notably, 19

after antibody loading, MN/Ab swab exhibited a superior sampling potential by showing Ct 20 value lower than 30 ( Figure S5 ). In our test, the Ct ≤ 30 was evaluated to be more 21 significant compared to the value that was larger than 30, which was suggested based on the 1 protocol of the detection kit. Besides, in order to verify the clinical potential of the MN-based 2 swabs, a laboratory-confirmed COVID-19 patient with typical CT imaging characteristics 3 participated in the test ( Figure 5D and Figure S6 ). After swab sampling in patients or 4 volunteers (COVID-19 negative) by well-trained nurses ( Figure 5E ), degree of pain, itching, 5 nausea, saliva, cough, sputum and dyspnea were evaluated by using a questionnaire, which 6 was completed immediately after the tests. Four levels were provided (score 4, very serious; 7 score 3, serious; score 2, mild; score 1, very mild) and the data from each patient/volunteer 8 were presented. Generally, the regular swab did not induce intense discomfort and only one 9 volunteer feel severe nausea ( Figure 5F ). Importantly, with or without antibody loading, no 10 patient claimed severe discomfort to MN-based swabs, which is comparable to the regular 11 swab, suggesting minor or negligible adverse effects ( Figure 5F ). During the test on the 12 positive patient, the best candidate (the ratio of highly cross-linked MN was 0.5 and antibody 13 amount is 25 µg/patch) was applied to compare with the clinical used ones. As expectation, 14 significantly reduced cycle thresholds (Ct value) was found for all three genes after the 15 MN/Ab swab treatment compared to regular swab on the same patient ( Figure 5G and Figure  16 S7), indicating a higher amount of virus nucleic acid on the engineered swab. To normalize 17 the results, the Ct values of target genes were divided by that of the reference (added after 18 sampling), and MN/Ab swab treatment still presented remarkably lower Ct values relative to 19 regular ones ( Figure 5H ). It should be emphasized that this enhancement is essential for the 20 COVID-19 test, as the Ct values from regular swabs were close to the detection limit of the 21 J o u r n a l P r e -p r o o f method, which could easily mislead doctors. Evidently, the compelling improvement for the 1 detection allowed the collection of more viruses and widen the distance between Ct values of 2 positive patients and those of negative patients. In this regard, the MN/Ab swab could serve 3 as a useful tool for COVID-19 sampling compared to currently used ones. In negative 4 patients, the MN/Ab swabs would not induce undesired "false positives" as regular swabs did 5 ( Figure S8 ), suggesting the feasible and reliable detection profiles. was diluted in PBS with the concentration of 125 µg/ml. 2% FITC was added as the indicator. 20

After weighing the molds, 200 µl solution was added to the mold, followed by 10 min 21 vacuum (2.5 kPa) and 10 min centrifuge (2000 rpm). It should be mentioned that all the 1 solution could be added to the PDMS mold; thus each patch contained around 25 µg antibody. 2 Then 2 ml 2% alginate solution was introduced to the PDMS molds, which were placed in the 3 oven overnight (37 °C). After cooling to room temperature, the patches were carefully peeled 4 off from the PDMS molds by using a tweezer. All the patches were stored at 4 °C until use. 5 6

The fabricated patches were treated by different concentrated CaCl 2 solution to induce 8 distinct cross-linked levels. Briefly, for high cross-linked patches, the samples were 9 immersed in 1 M CaCl 2 for three minutes, followed by wiping with a tissue paper. Meanwhile, 10 low cross-linked samples were generated by using 20 mM CaCl 2 instead, and the treatment 11 time was controlled to be 5 seconds. High cross-linked samples were labeled by rhodamine 12 while low cross-linked ones were indicated by FITC. 13 14

The patches with different cross-linked levels were combined by using biomedical device 16 adhesives (Loctite 4061, Henkel, Germany). Then the patches were curved with the help of 17 rod-like fixture, followed by the assembly to the head of regular swab (Munkfoam, China) 18 with the help of biomedical device adhesives. All the samples were stored at 4 °C until use. 19 20

The mechanical strength of the MN arrays with high or low cross-linked levels was tested by 1 using an MTS 30 G tensile testing machine. The MNs were pressed against a stainless-steel 2 fixture. The initial gauge was set as 2 mm between the MN tips and the stainless-steel fixture, 3 with 10.00 N as the load cell capacity. The speed of the top stainless-steel plate movement 4 toward the MN arrays was set as 0.1 mm s −1 . The failure force of MNs was recorded as the 5 needle began to buckle. Hospital. After sampling in COVID-19 patients/volunteers, regular and MN/Ab swabs (the 19 ratio of highly cross-linked of MN was 0.5) were stored in VPS. A questionnaire was 20 completed by each patient/volunteer (one positive patient, one negative patient (recovery 21 from positive to negative) and seven volunteers participated the study), which included their 1 feelings about pain, itching, nausea, saliva, cough, sputum and dyspnea. A quantitative 2 analysis was conducted based on questionnaire ((score 4, very serious; score 3, serious; score 3 2, mild; score 1, very mild)). The clinical samples were analyzed by using the method 4 mentioned above. 5 6

All experiments were repeated at least three times and the data were presented as mean ± SD. 8 SPSS statistical software (SPSS 16.0) was applied for statistical analyses. Any significant 9 differences among mean values were evaluated by the two-tailed Student's t-test or ANOVA 10 (*P < 0.05,**P < 0.01, ***P < 0.001). 

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