A Microflow Cytometry-Based Agglutination Immunoassay For

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Article A Microﬂow Cytometry-Based Agglutination Immunoassay for Point-of-Care Quantitative Detection of SARS-CoV-2 IgM and IgG

Jianxi Qu 1 , Mathieu Chenier 2, Yushan Zhang 1 and Chang-qing Xu 1,2,*

1 School of Biomedical Engineering, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4L8, Canada; [email protected] (J.Q.); [email protected] (Y.Z.) 2 Department of Engineering Physics, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4L8, Canada; [email protected] * Correspondence: [email protected]; Tel.: +1-905-525-9140

Abstract: A rapid, sensitive and simple microﬂow cytometry-based agglutination immunoassay (MCIA) was developed for point-of-care (POC) quantitative detection of SARS-CoV-2 IgM and IgG antibodies. The antibody concentration was determined by using the transit time of beads aggregates. A linear relationship was established between the average transit time and the concentration of SARS-CoV-2 IgM and IgG, respectively. The limit of detection (LOD) of SARS-CoV-2 IgM and IgG by the MCIA measurement are 0.06 mg/L and 0.10 mg/L, respectively. The 10 µL sample consumption, 30 min assay time and the compact setup make this technique suitable for POC quantitative detection of SARS-CoV-2 antibodies.

Keywords: SARS-CoV-2 IgM; SARS-CoV-2 IgG; microﬂow cytometry; agglutination immunoassay; point-of-care

Citation: Qu, J.; Chenier, M.; Zhang, Y.; Xu, C.-q. A Microflow Cytometry-Based Agglutination Immunoassay for Point-of-Care 1. Introduction Quantitative Detection of Despite the COVID-19 pandemic persisting for more than a year, there is still a need SARS-CoV-2 IgM and IgG. for a fast and sensitive antibody test to detect SARS-CoV-2 infections. Unlike diagnostic Micromachines 2021, 12, 433. tests, antibody or serological tests are not intended to detect infections at their onset. Rather, https://doi.org/10.3390/mi12040433 they detect if an individual has produced antibodies in response to a specific foreign body, in this case, the SARS-CoV-2 virus. Since seroconversion occurs some time after initial Academic Editor: Stéphane Colin infection, antibody tests are not intended to replace diagnostic testing methods, namely PCR tests [1]. Rather, antibody tests are intended to complement diagnostic testing and Received: 24 February 2021 identify individuals previously infected with SARS-CoV-2. Further, due to their ability Accepted: 12 April 2021 Published: 14 April 2021 to detect past infections, antibody tests can be deployed for epidemiological purposes to monitor the spread of COVID-19 [2]. POC antibody tests are serological tests that do not require the use of laboratory Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in technicians or facilities to provide results [3]. POC testing, unlike intensive laboratory published maps and institutional affil- methods, is timely and convenient. A fast and simple POC antibody test could allow for iations. mass screening, assist in contact tracing and help monitor the spread of COVID-19 [2,4]. Further, a POC antibody test has the advantage of being accessible, allowing it to be used at health care facilities, at home, or in the field without the equipment required by some laboratory techniques [3]. For a POC antibody test to be an effective alternative to current laboratory methods, it should be fast, quantitative, portable, inexpensive, easy to operate Copyright: © 2021 by the authors. and require small sample volumes while remaining sensitive and specific. Licensee MDPI, Basel, Switzerland. This article is an open access article Currently, enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoas- distributed under the terms and says (LFIAs) are two common methods used to detect SARS-CoV-2 antibodies. ELISAs conditions of the Creative Commons must be performed by skilled operators, have long incubation times and require laboratory Attribution (CC BY) license (https:// equipment to quantify results, making them impractical for POC environments [5]. LFIAs, creativecommons.org/licenses/by/ although suitable for POC environments, are not traditionally quantifiable and can provide 4.0/). ambiguous results [6]. Tests of both formats have reported wide ranges of sensitivity and

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specificity and have been evaluated under high risk of patient selection bias, making it unclear if they are suitable for mass testing [2,7–9]. Other testing formats have been proposed for the detection of SARS-CoV-2 antibodies including flow cytometry. Grzelak et al. developed a technique to detect anti-spike (S) protein antibodies by expressing the S protein on the surface of human embryonic kidney 293T cells. The cells were then incubated with human serum samples and fluorescent anti- IgG or anti-IgM before being evaluated by flow cytometry [8]. Horndler et al. developed a similar method by expressing the SARS-CoV-2 S protein on the human leukemic T cell line [10]. These methods may not be suitable for POC detection, however, as they require flow cytometry equipment and cell cultures that must remain stable to provide sensitive testing. Fluorescent microsphere immunoassays (FMIAs) have also been developed for the detection of SARS-CoV-2 antibodies, with one method being granted an emergency use authorization (EUA) by the Food and Drug Administration (FDA). Typically, in the FMIA methods, SARS-CoV-2 antigens are bound to microbeads. These complexes are then mixed with serum samples and fluorescent secondary antibodies to produce a quantifiable signal. Cameron et al. describe an FMIA for the detection of anti-N, S and receptor-binding domain (RBD) antibodies [11]. These formats are currently destined for laboratory use only as they require large flow cytometry equipment. In this work, a fast and sensitive MCIA suitable for the POC quantitative detection of SARS-CoV-2 IgM and IgG was proposed. Microbeads were conjugated to anti-IgM and anti-IgG antibodies and incubated with SARS-CoV-2 antibodies. SARS-CoV-2 S protein was then added to cause microbeads to agglutinate. Using microflow cytometry, transit time was measured and used to distinguish monomers from larger aggregates. The relationship between the average transit-time of the aggregates and certain concentrations of SARS-CoV- 2 IgM and IgG was studied. A linear correlation was found. This work also discussed the potential application of the MCIA for the POC detection and quantification of SARS-CoV-2 IgM and IgG.

2. Materials and Methods Figure1 shows the microfluidic platform used to measure SARS-CoV-2 antibodies. Light emitted from a 532 nm green laser was coupled into the input waveguide of a microflow cytometry device and then focused into the center of a microfluidic channel through an embedded lens system. The microfluidic chip was made by the photolithography. It consists of four layers: Pyrex glass layer, SU-8 photoresist layer, polydimethylsiloxane (PDMS) layer and a glass layer. The bottom Pyrex glass layer was used as the substrate. The SU-8 layer, with a thickness of 50 µm, was patterned with microfluidic channels by photolithography. PDMS was used to seal the SU-8 layer to form microfluidic channels. The top glass layer was used to fix the metal pins which are used as the inlets and outlets of the chip. The cross-sectional area of the microchannel is 50 µm × 100 µm (height × width). The dimensions of the chip are approximately 3 cm × 2 cm × 0.5 cm (length × width × height). Assay mixtures from the immune reaction were introduced into the microfluidic channel by an external syringe pump and hydrodynamically focused into a narrow stream by two sheath flows. The aggregates passing through the interrogation region (the intersection of the laser beam and the microchannel) interacted with the coupled laser beam, resulting in scattered light. The scattered light was collected by an objective lens which was mounted perpendicular to both the light beam and the microchannels, then deflected by a mirror. The deflected light passed through a band pass filter and then was amplified by a photo- multiplier tube (PMT). A current-to-voltage amplifier was used to convert current signals from the PMT into amplified voltage signals. The amplified voltage signals were digitized by a data acquisition board (DAQ) and then processed by a custom LabVIEW program with a personal computer. The details of the microflow cytometry device were presented in a previous publication [12]. It is worth noting that, since both the microchannel and optical lens are integrated in the microflow cytometry device, the microfluidic platform shown in Figure1 can be easily packaged into a small box by employing an advanced, Micromachines 2021, 12, x FOR PEER REVIEW 3 of 9

a custom LabVIEW program with a personal computer. The details of the microflow cytometry device were presented in a previous publication [12]. It is worth noting that, since Micromachines 2021, 12, 433 3 of 9 both the microchannel and optical lens are integrated in the microflow cytometry device, the microfluidic platform shown in Figure 1 can be easily packaged into a small box by employingoff-the-shelf an micro-PMT.advanced, off-the-shelf micro-PMT.

Figure 1. A diagram of the microfluidic platform for SARS-CoV-2 antibody detection. Figure 1. A diagram of the microfluidic platform for SARS-CoV-2 antibody detection. Figure2 presents the immune reaction steps of the MCIA. The protocols for the detectionFigure of2 presents SARS-CoV-2 the immune IgM and reaction IgG are st theeps same, of the except MCIA. for The the protocols secondary for antibodythe de- tectionused. of The SARS-CoV-2 following IgM protocols and IgG will are describe the same, the except SARS-CoV-2 for the secondary IgM measurement antibody used. and is Thea model following to illustrate protocols the will immunoassay. describe the SARS-CoV-2 The first step IgM is measurement secondary antibody and is a coupling. model toGoat illustrate anti-human the immunoassay. IgM secondary Theantibodies first step is (31136, secondary Invitrogen, antibody Thermo coupling. Fisher Goat Scientific, anti- humanRockford, IgM MD,secondary USA) were antibodies coupled (31136, to Dynabeads Invitrogen, M-270 Thermo Epoxy Fisher (14301, Scientific, Invitrogen, Rockford, Thermo MD,Fisher USA) Scientific, were coupled Carlsbad, to CA,Dynabeads USA) magnetic M-270 Epoxy beads (14301, (MBs) according Invitrogen, to theThermo user manual.Fisher Scientific,The lyophilized Carlsbad, powder CA, USA) of MBs magnetic has a concentrationbeads (MBs) according of 6.7 × 10to 7thebeads/mg. user manual. The The MBs lyophilizedneed to be powder washed of before MBs thehas antibody a concentration coupling of procedure.6.7 × 107 beads/mg. A total of The 5 mg MBs of need lyophilized to be washedbeads werebefore weighed the antibody out andcoupling resuspended procedure. in A 1 mLtotal of of 0.15 mg M of sodium lyophilized phosphate beads were buffer weighed(PB) (P5244, out and Millipore resuspended Sigma, Oakville,in 1 mL ON,of 0.1 Canada). M sodium The phosphate MBs were incubatedbuffer (PB) for (P5244, 10 min Milliporewith tilting Sigma, and Oakville, rotation andON, thenCanada). applied The toMBs a magnet. were incubated The supernatant for 10 min was with removed tilting andby rotation a pipette. and The then MBs applied were suspendedto a magnet. in The 1 mL supernatant of PB again was and removed vortexed by for a 30pipette. s. The TheMBs MBs were were ready suspended to couple in 1 to mL secondary of PB again antibodies and vortexed after removingfor 30 s. The the MBs supernatant. were ready A tototal couple of 5to mg secondary of MBs were antibodies used to after couple removi approximatelyng the supernatant. 100 µg of A secondary total of 5 mg antibody of MBs as wererecommended used to couple by the approximately user manual. 100 The µg concentration of secondary ofantibody goat anti-human as recommended IgM secondary by the userantibodies manual. was The2.32 concentration mg/mL. Therefore, of goat anti-human the volume ofIgM secondary secondary antibody antibodies needed was for 2.32 the mg/mL.coupling Therefore, procedure the was volume approximately of secondary 43 µ antibodyL. The washed needed MBs for werethe coupling suspended procedure in 43 µ L wasof PBapproximately with mixing. 43 Then, µL. The 43 µ washedL of goat MBs anti-human were suspended IgM secondary in 43 µLantibody of PB with was mixing. added Then,into 43 the µL solution of goat withanti-human thoroughly IgM secondar mixing.y An antibody equal volume, was added 43 µintoL of the 1 Msolution ammonium with thoroughlysulfate buffer mixing. was An added equal into volume, the solution 43 µL of with 1 M mixingammonium and thensulfate the buffer MB solution was added was ◦ intoincubated the solution at 37 withC for mixing 24 h. After and thethen incubation, the MB solution the MBs was were incubated separated at from 37 °C the for solution 24 h. Afterusing the the incubation, magnet separation the MBs procedure.were separated Then, from the MBsthe solution were washed using withthe magnet 1 mL phosphate separa- tionbuffered procedure. saline Then, (PBS, the pH MBs = 7.4) were four washed times. with The MBs1 mL were, phosphate finally, buffered suspended saline in 330(PBS,µ L 9 pHof = PBS 7.4) with four atimes. concentration The MBs ofwere, 1 × 10finally,beads/mL. suspended The in secondary 330 µL ofantibody PBS with coated a concen- MBs ◦ trationwere storedof 1 × 10 at94 beads/mL. to 8 C with The 0.02% secondary (w/v) sodiumantibody azide coated (08591, MBs Sigma-Aldrich,were stored at 4 Millipore to 8 °C withSigma, 0.02% Oakville, (w/v) sodium ON, USA) azide to provide(08591, Sigma-Aldrich, a long shelf-life Millipore for the MCIA Sigma, antibody Oakville, test. ON, The USA)recommended to provide ratioa long between shelf-life the for secondary the MCIA antibodies antibody andtest. the The MBs recommended from the user ratio manual be- tweenwasused the secondary in this study antibodies and the and antibody the MBs coupling from the efficiency user manual has been was verified used in bythis coupling study andMBs the to antibody anti-human coupling IgM secondary efficiency antibody-HRP has been verified instead by of coupling the goat anti-humanMBs to anti-human IgM. This IgMprovided secondary evidence antibody-HRP that the secondary instead of antibodies the goat wereanti-human successfully IgM. capturedThis provided by the evi- MBs denceusing that the the recommended secondary antibodies ratio. For were the antibody successfully test, captured the concentration by the MBs of using the secondary the rec- × 6 ommendedantibody conjugated ratio. For the MBs antibody was optimized test, the concentration and should be of higher the secondary than 5 antibody10 beads/mL. con- The storage conjugated MBs were washed with PBS containing 0.1% bovine serum albumin (BSA) (15561020, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) once and then diluted by PBS to the desired concentration. A total of 10 µL of diluted conjugated MBs µ µ were resuspended in a 600 L centrifuge vial. 10 L of SARS-CoV-2 RBD IgM humanized coronavirus monoclonal antibodies (MBS355900, MyBioSource, Inc., San Diego, CA, USA) Micromachines 2021, 12, x FOR PEER REVIEW 4 of 9

jugated MBs was optimized and should be higher than 5 × 106 beads/mL. The storage conjugated MBs were washed with PBS containing 0.1% bovine serum albumin (BSA) (15561020, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) once and then diluted by PBS to the desired concentration. A total of 10 µL of diluted conjugated MBs were resuspended in a 600 µL centrifuge vial. 10 µL of SARS-CoV-2 RBD IgM humanized coronavirus monoclonal antibodies (MBS355900, MyBioSource, Inc., San Diego, CA, USA) Micromachines 2021, 12, 433 were added into the vial and mixed with a pipette tip for 30 s. After a4 25 of 9 min incubation, during which SARS-CoV-2 IgM antibodies were captured by the IgM secondary antibodies, the conjugated MBs were sonicated for 10 s and then 10 µL of SARS-CoV-2 spike S1- were added into the vial and mixed with a pipette tip for 30 s. After a 25 min incubation, duringHis recombinant which SARS-CoV-2 protein IgM (40591-V08B1, antibodies were captured Sino Biological by the IgM Inc., secondary Beijing, antibodies, China) with a concen- thetration conjugated of 25 MBsmg/L were was sonicated added into for 10 the s and vial then and 10 mixedµL of SARS-CoV-2 for 30 s. The spike aggregates S1-His were formed recombinantin this incubation protein (40591-V08B1, procedure. Sino After Biological a 3 min Inc., incubation, Beijing, China) the with assay a concentration mixtures were introduced ofinto 25 mg/L the microflow was added intocytometer the vial by and the mixed external for 30 s.syringe The aggregates pump. wereThe flow formed rate in for sample and this incubation procedure. After a 3 min incubation, the assay mixtures were introduced intosheath the microflow flows were cytometer set as by 200 the µL/h external and syringe 800 µL/h, pump. respectively. The flow rate forA total sample of and 2 min of data were sheathcollected flows and were analyzed set as 200 byµL/h the and LabView 800 µL/h, progra respectively.m. For Athe total SARS-CoV-2 of 2 min of data IgG test, goat anti- wererabbit collected IgG (H and + analyzed L) antibodies by the LabView (31210, program. Invitrog Foren, the Thermo SARS-CoV-2 Fisher IgG Scientific, test, goat Carlsbad, CA, anti-rabbitUSA) were IgG coupled (H + L) antibodies to the MBs (31210, instead. Invitrogen, SARS-C ThermooV-2 Fisher spike Scientific, S1 antibodies Carlsbad, (40150-R007, Sino CA, USA) were coupled to the MBs instead. SARS-CoV-2 spike S1 antibodies (40150-R007, Biological, Inc., Beijing, China) were tested by the MCIA. It is worth mentioning that the Sino Biological, Inc., Beijing, China) were tested by the MCIA. It is worth mentioning thatdescribed the described immune immune reaction reaction step step of of the the MCIA MCIA cancan be be easily easily automated, automated, which which is is important importantfor its POC for its applications. POC applications.

FigureFigure 2. Schematic2. Schematic illustration illustration of the MCIA of the for MCIA the quantitative for the quantitative detection of SARS-CoV-2 detection IgM/IgG. of SARS-CoV-2 3.IgM/IgG. Results and Discussion The MCIA is derived from the particle counting immunoassay with the principle that agglutination3. Results causedand Discussion by an immune reaction decreases the number of free particles in assay mixturesThe [13 MCIA]. Microflow is derived cytometry from is the a competitive particle counting tool for particle immunoassay counting due with to its the principle that compacted size, low reagent consumption and relatively high sensitivity [14]. Generally, agglutination caused by an immune reaction decreases the number of free particles in as- light intensity of the side scattered light from microflow cytometry is analyzed. Large sizessay ofmixtures beads or aggregates[13]. Microflow tend to result cytometry in higher is scattered a competitive light intensities tool for and particle thus can counting due to beits used compacted to discriminate size, differentlow reagent sizes of consumption beads. However, and the relatively relatively high high deviations sensitivity [14]. Gener- ofally, scattered light lightintensity intensity of the affect side the scattered detection sensitivitylight from [15 microflow]. Additionally, cytometry using light is analyzed. Large intensitiessizes of beads to discriminate or aggregates between thetend sizes to ofresult single in beads higher and aggregatesscattered islight unfeasible. intensities In and thus can this proposed method, transit time was used to discriminate between the different sizes of beadsbe used and aggregates.to discriminate The time different it took for sizes beads of to beads. pass through However, the interrogation the relatively region high deviations wasof definedscattered as the light transit intensity time—the affect interval the between detection the intercepts sensitivity of the [15]. signal Additionally, pulse and using light theintensities threshold. to The discriminate threshold was between the standard the deviation sizes of ofsingle the background beads and noise, aggregates which is unfeasible. wasIn this used proposed to differentiate method, signals transit from noise.time was Details used regarding to discriminate how to use between the average the different sizes transit time to analyze data can be found from our previous publication [16]. of beadsFigure3 and shows aggregates. the side scattered The time light signalsit took of fo a 0.6r beads mg/L SARS-CoV-2to pass through IgM sample the interrogation re- recordedgion was by thedefined custom as program. the transit The xtime—the-axis is the timeinterval in milliseconds between andthe theinterceptsy-axis of the signal representspulse and the the amplitude threshold. of the signal.The threshold Red solid lines was are the waveforms standard of the deviation signals. Peaks of the background innoise, Figure which3a were was produced used when to differentiate aggregates of signal MBs passeds from through noise. the Details interrogation regarding how to use region.the average Figure3 btransit shows time the definition to analyze of the data transit can time. be found The signal from was our distinguished previous publication [16]. from the noise by the threshold, which is the green dashed line. The threshold value was the standardFigure deviation 3 shows of the backgroundside scattered noise. light signals of a 0.6 mg/L SARS-CoV-2 IgM sample recordedCompared by withthe monomers,custom program. aggregates The have x-axis longer is transit the time times. in Figure milliseconds4a shows and the y-axis therepresents transit time the distribution amplitude obtained of the signal. from a negativeRed solid control lines in are which waveforms PBS was usedof the signals. Peaks asin the Figure sample 3a and were Figure produced4b shows awhen sample aggregates containing 1of mg/L MBs SARS-CoV-2 passed through IgM. The the interrogation x-axis in Figure4 is the transit time and the y-axis in Figure4 is the number of monomers region. Figure 3b shows the definition of the transit time. The signal was distinguished

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from the noise by the threshold, which is the green dashed line. The threshold value was the standard deviation of the background noise.

Micromachines 2021, 12, 433 (a) (b) 5 of 9 Figure 3. (a) One second of raw data showing the side scattered light signal obtained from a 0.6 mg/L SARS-CoV-2 IgM sample.and aggregates (b) A withdetailed different view transit of a side times scattered counted by light the signal microﬂow showing cytometer. the Each solid square represents the number of single MBs or aggregates. The decrease in the definition of the transit time.proportion of monomers and the increase of aggregates is obvious in terms of the transit time. The distribution of the transit time for the negative control in Figure4a is tapered Compared with andmonomers, symmetrical aggregates because most have of the longer beads in thetransit assay times. mixtures Figure were monomers. 4a shows The the transit time distributiondistribution obtained of the transit from time a negative for the 1 mg/L control SARS-CoV-2 in which IgM inPBS Figure was4b hasused a peak as Micromachines 2021, 12, x FOR PEER REVIEW that is short and wide. Compared with the distribution of the transit time of the negative5 of 9 the sample and Figurecontrol, 4b shows an obvious a sample peak shoulder containing was presented 1 mg/L inSARS-CoV-2 the 1 mg/L SARS-CoV-2 IgM. The IgM x-axis group, in Figure 4 is the transitwhich time was and attributed the y to-axis increased in Figure aggregate 4 is formation the number caused byof themonomers immune reaction. and aggregates with differentOther transit detected samplestimes coun containingted by different the microflow concentrations cytometer. of SARS-CoV-2 Each IgM andsolid IgG from the noise by the alsothreshold, showed similar which distributions is the green of the dashed transit time line. as shown The threshold in Figure4b. Byvalue calculating was square represents the thenumber average of transit single time, MBs a relationship or aggregates. can be established The decrease between in the the average proportion transit time theof monomers standard deviation and theand increase of the the concentration background of aggregates of SARS-CoV-2 noise. is obvious antibodies. in terms of the transit time. The distribution of the transit time for the negative control in Figure 4a is tapered and symmetrical because most of the beads in the assay mixtures were monomers. The distribution of the transit time for the 1 mg/L SARS-CoV-2 IgM in Figure 4b has a peak that is short and wide. Compared with the distribution of the transit time of the negative control, an obvious peak shoulder was presented in the 1 mg/L SARS-CoV-2 IgM group, which was attributed to increased aggregate formation caused by the immune reaction. Other detected samples containing different concentrations of SARS-CoV-2 IgM and IgG also

showed similar distributions(a) of the transit time as shown in Figure(b )4b. By calculating the average transit time, a relationship can be established between the average transit time Figure 3. (a) One second of raw data showing the side scattered light signal obtained from a 0.6 mg/L SARS-CoV-2 IgM Figureand the 3. concentration(a) One second of rawSARS-CoV-2 data showing antibodies. the side scattered light signal obtained from a 0.6 mg/Lsample. SARS-CoV-2 (b) A detailed IgM view sample. of a side scattered (b) A detailed light signal view showing of thea side deﬁnition scattered of the transitlight time.signal showing the definition of the transit time.

Compared with monomers, aggregates have longer transit times. Figure 4a shows the transit time distribution obtained from a negative control in which PBS was used as the sample and Figure 4b shows a sample containing 1 mg/L SARS-CoV-2 IgM. The x-axis in Figure 4 is the transit time and the y-axis in Figure 4 is the number of monomers and aggregates with different transit times counted by the microflow cytometer. Each solid square represents the number of single MBs or aggregates. The decrease in the proportion of monomers and the increase of aggregates is obvious in terms of the transit time. The distribution of the transit time for the negative control in Figure 4a is tapered and symmetrical because most of the beads in the assay mixtures were monomers. The distribution of the transit time for the 1 mg/L SARS-CoV-2 IgM in Figure 4b has a peak that is short (a) (b) and wide. Compared with the distribution of the transit time of the negative control, an obviousFigureFigure 4. 4.TransitpeakTransit shoulder time time distribution distribution was presented achieved achieved from in from the detection the1 mg/L detection of SARS-CoV-2 (a) negativeof (a) negative sample IgM (PBS) sample group, and (PBS) (bwhich) 1 mg/Land was SARS-CoV-2 IgM. attributed(b) 1 mg/L SARS-CoV-2to increased IgM. aggregate formation caused by the immune reaction. Other detected samples containingFigure different5 shows theconcen measuredtrations correlation of SARS-CoV-2 between the average IgM transit and timeIgG and also the showedFigure similar 5 shows distributions theSARS-CoV-2 measured of IgMthe concentrationcorrelationtransit time with between aslinear shown ﬁttings, the in averageFigure determined 4b. transit by By the calculating MCIA. time Theandx-axis the is SARS-CoV-2 IgM concentrationthe concentration with of SARS-CoV-2linear fittings, IgM and determined the y-axis is the by average the MCIA. transit time. The The x-axis stored average transit time, agoat relationship anti-human IgM can secondary be established antibody coatedbetween MBs werethe average used to detect transit SARS-CoV-2 time andis the the concentration concentration ofIgM of SARS-CoV-2 SARS-CoV-2 on different days. IgM antibodies. Different and colouredthe y-axis symbols, is the including average black transit squares, time. red triangles The stored goat anti-humanand IgM blue circles,secondary are used antibody in Figure5 coatedto represent MBs testing were results used obtained to detect from SARS- days 1, 8 CoV-2 IgM on differentand days. 15, respectively. Different Each coloured symbol and symbols, error bar areincluding the mean black value of squares, three detections red triangles and blue circles, are used in Figure 5 to represent testing results obtained from

(a) (b) Figure 4. Transit time distribution achieved from the detection of (a) negative sample (PBS) and (b) 1 mg/L SARS-CoV-2 IgM.

Figure 5 shows the measured correlation between the average transit time and the SARS-CoV-2 IgM concentration with linear fittings, determined by the MCIA. The x-axis is the concentration of SARS-CoV-2 IgM and the y-axis is the average transit time. The stored goat anti-human IgM secondary antibody coated MBs were used to detect SARS- CoV-2 IgM on different days. Different coloured symbols, including black squares, red triangles and blue circles, are used in Figure 5 to represent testing results obtained from

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Micromachines 2021, 12, 433 6 of 9 days 1, 8 and 15, respectively. Each symbol and error bar are the mean value of three detections from duplicated samples and the standard deviation of the three detections, from duplicated samples and the standard deviation of the three detections, respectively. respectively. As shown in Figure 5, the average transit time has a strong linear relationship As shown in Figure5, the average transit time has a strong linear relationship with the with the concentration of SARS-CoV-2 IgM when the test is performed on different days, concentration of SARS-CoV-2 IgM when the test is performed on different days, although although the slope of the linear curve decreases with the increase of anti-IgM conjugated the slope of the linear curve decreases with the increase of anti-IgM conjugated MBs MBs storage time. Therefore, the performance of the conjugated MBs decreased with the storage time. Therefore, the performance of the conjugated MBs decreased with the elapse elapse of conjugated MB storage time. For days 1, 8 and 15, the R2 was 0.9984, 0.9982 and of conjugated MB storage time. For days 1, 8 and 15, the R2 was 0.9984, 0.9982 and 0.9941, 0.9941, respectively. The LOD of SARS-CoV-2 IgM for the MCIA was determined as 3.3 respectively. The LOD of SARS-CoV-2 IgM for the MCIA was determined as 3.3 times the times the residual standard deviation of the regression line divided by the slope. Day 1 residual standard deviation of the regression line divided by the slope. Day 1 data were data were used to calculate the LOD, which was determined to be 0.06 mg/L. Iyer et al. used to calculate the LOD, which was determined to be 0.06 mg/L. Iyer et al. reported reported a LOD of 0.28 mg/L for anti-RBD SARS-CoV-2 IgM for their in-house ELISA a LOD of 0.28 mg/L for anti-RBD SARS-CoV-2 IgM for their in-house ELISA method, whichmethod, is comparablewhich is comparable to the LOD to the achieved LOD achieved by the MCIA by the [17 MCIA]. Additionally, [17]. Additionally, the average the transitaverage time transit deviated time fromdeviated the linearfrom the relationship linear relationship as the concentration as the concentration of SARS-CoV-2 of SARS- IgM surpassedCoV-2 IgM 1.0 surpassed mg/L due 1.0 tomg/L the due prozone to the effect prozone [18]. effect Therefore, [18]. Therefore, it is necessary it is necessary to keep the to concentrationkeep the concentration of SARS-CoV-2 of SARS-CoV-2 IgM below IgM 1.0 mg/Lbelow to1.0 avoid mg/L false to avoid positive false results. positive This results. may beThis done may by be diluting done by biological diluting biological samples. samples.

FigureFigure 5. TheThe measured measured relationship relationship between between the the aver averageage transit transit time time and and the theconcentration concentration of of SARS-CoV-2SARS-CoV-2 IgM detected at different different days days of of secondary secondary antibody antibody conjugated conjugated MB MB storage. storage. Each Each symbolsymbol isis thethe meanmean valuevalue of three detections from duplicated samples. Erro Errorr bars are are the standard deviation of the three detections. deviation of the three detections.

FigureFigure6 6 shows shows the the measured measured correlation correlation between between the the average average transit transit time time and and the the concentrationconcentration ofof SARS-CoV-2 SARS-CoV-2 IgG, IgG, determined determined by by the the MCIA. MCIA. The Thex-axis x-axis is the is the concentration concentra- oftion SARS-CoV-2 of SARS-CoV-2 IgGantibody IgG antibody and the andy -axisthe y is-axis the is average the average transit transit time. Goattime. anti-rabbitGoat anti- IgGrabbit (H IgG + L) (H secondary + L) secondary antibody antibody coated coated MBs MBs with with different different storage storage times times were were used used for thefor the detection detection of SARS-CoV-2 of SARS-CoV-2 IgG. IgG. Different Differen colouredt coloured symbols symbols including including black black squares, squares, red trianglesred triangles and and blue blue circles, circles, are used are used in Figure in Figure6 to represent 6 to represent testing testing results results obtained obtained from daysfrom 1,days 8 and 1, 15,8 and respectively. 15, respectively. Each symbol Each sy andmbol error and bar error represent bar represent the mean the value mean of value three detectionsof three detections from duplicated from duplicated samples samples and the and standard the standard deviation deviation of the three of the detections, three de- respectively.tections, respectively. Similar to Similar the results to the for results SARS-CoV-2 for SARS-CoV-2 IgM shown IgM in shown Figure in5, theFigure average 5, the transitaverage time transit has time a strong has lineara strong relationship linear relationship with the with concentration the concentration of SARS-CoV-2 of SARS-CoV- IgG on different2 IgG on daysdifferent of storage, days of although storage, the although slope of the the slope linear of curve the linear decreases curve with decreases the increase with ofthe conjugated increase of MBs conjugated storage time.MBs storage The performance time. The ofperformance the conjugated of the MBs conjugated decayed withMBs thede- elapsecayed with of time, the butelapse the of R2 time,for each but curvethe R2 wasfor each consistent; curve was for daysconsistent; 1, 8 and for 15, days the 1, R 28was and 0.9981,15, the R 0.99632 was 0.9981, and 0.9987, 0.9963 respectively. and 0.9987, Therespectively. LOD of SARS-CoV-2The LOD of SARS-CoV-2 IgG for the MCIA IgG for was the determinedMCIA was determined as 3.3 times theas 3.3 residual times standard the residual deviation standard of the deviation regression of linethe regression divided by line the slope.divided Day by 1the data slope. were Day used 1 todata calculate were used the LOD,to calculate which wasthe LOD, determined which towas be determined 0.10 mg/L. Iyer et al. reported a LOD of 0.04 mg/L for anti-RBD SARS-CoV-2 IgG for their in-house

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to be 0.10 mg/L. Iyer et al. reported a LOD of 0.04 mg/L for anti-RBD SARS-CoV-2 IgG for ELISAtheir in-house method, ELISA which method, is comparable which is to comparab the LOD achievedle to the LOD by the achieved MCIA by [17 the]. In MCIA addition, [17]. theIn addition, average transit the average time deviated transit time from deviated the linear from relationship the linear as relationship the concentration as the of concen- SARS- CoV-2tration IgG of SARS-CoV-2 exceeded 1mg/L IgG exceeded due to the 1 mg/L prozone due effect to the [ 18prozone]. Therefore, effect it[18]. is necessaryTherefore, to it keepis necessary the concentration to keep the of concentration SARS-CoV-2 IgGof SARS-CoV-2 below 1.0 mg/L IgG forbelow the MCIA.1.0 mg/L Again, for the this MCIA. may beAgain, accomplished this may be by accomplished dilution. by dilution.

FigureFigure 6.6. TheThe measured measured relationship relationship between between the the aver averageage transit transit time time and and the the concentration concentration of of SARS-CoV-2SARS-CoV-2 IgGIgG detecteddetected at different days days of of se secondarycondary antibody antibody conjugat conjugateded MB MB storage. storage. Each Each symbolsymbol isis thethe meanmean valuevalue ofof threethree detectionsdetections fromfrom duplicatedduplicated samples.samples. ErrorError bars are the standard deviation of the three detections. deviation of the three detections.

AccordingAccording to to the the results results presented presented in in Figures Figure5 sand 5 and6, the 6, slopesthe slopes of the of linear the linear curves curves for thefor SARS-CoV-2the SARS-CoV-2 IgM IgM measurements measurements were were greater greater than than those those for for SARS-CoV-2 SARS-CoV-2 IgG. IgG. This This is likelyis likely due due to theto the pentameric pentameric structure structure of IgM, of IgM, which which increases increases the probability the probability of aggregates of aggre- togates form. to form. ToTo testtest thethe performanceperformance of of the the MCIAMCIA forfor itsits practicalpractical application,application, a a certaincertain numbernumber ofof SARS-CoV-2SARS-CoV-2 antibodiesantibodies diluteddiluted byby humanhuman serumserum (H4522,(H4522, HumanHuman serum,serum, Sigma-Aldrich,Sigma-Aldrich, Oakville,Oakville, ON,ON, Canada)Canada) werewere spikedspiked intointo thethe samplessamples andand thenthen testedtested byby thethe MCIA.MCIA. TheThe recoveryrecovery indexindex was was calculated calculated by by the the following following equation, equation, where whereC spikedCspiked sample sample is thethe concen-concen- trationtration ofof thethe sample after after the the addition addition of of a aconcentrated concentrated standard, standard, which which was was diluted diluted by bythe the human human serum. serum. CunspikedCunspiked sample sampleis theis concentration the concentration of the of sample the sample prepared prepared with withPBS. PBS.Cadded Cisadded the isconcentration the concentration of the of added the added standard standard in the in whole the whole volume. volume.

% Recovery index = Cspiked sample − Cunspiked sample× 100% % Recovery index = × 100% Cadded For the detection of SARS-CoV-2 IgM, 16 µL samples prepared by PBS were split into two Forequals. the One detection was added of SARS-CoV-2 with 2 µL IgM,of deionized 16 µL samples water and prepared the other by was PBS added were splitwith into2 µL two of the equals. concentrated One was standard added with of SARS-CoV-2 2 µL of deionized IgM. Both water samples and the were other measured was added by withthe MCIA.2 µL of The the concentrated concentrated standard ofwas SARS-CoV-2 diluted by the IgM. human Both samples serum sample were measured and Cadded bywas the calculated MCIA. The to concentratedbe 0.4 mg/L. C standardunspiked sample was was diluted diluted by by the PBS human to a concentration serum sample of and 0.4 Cmg/L.added was In this calculated case, C tospiked be sample 0.4 should mg/L. Cbeunspiked 0.8 mg/L, sample wasprovided diluted that by the PBS recovery to a concentration index was of100%. 0.4 mg/L.After measurement In this case, ofCspiked the samples, sample should the co bencentrations 0.8 mg/L, of provided the samples that were the recovery obtained indexfrom the was calibration 100%. After curve measurement of the SARS-CoV-2 of the samples, IgM day the 1 concentrationsmeasurement (Y of = the 24.964 samples × X + were105.73), obtained which fromwas shown the calibration in Figure curve5. The ofexperiments the SARS-CoV-2 were repeated IgM day once 1 measurement and the aver- (Yages = 24.964 were used× X +to 105.73), calculatewhich the recovery was shown index, in Figure which5 .was The determined experiments to werebe 96.85%. repeated The oncerecovery and theindex averages of SARS-CoV-2 were used IgG to calculate was calc theulated recovery in a index,similar which manner was to determined SARS-CoV-2 to beIgM. 96.85%. The calibration The recovery curve index of SARS-CoV-2 of SARS-CoV-2 IgG IgG day was 1 detection calculated in Figure in a similar 6 was manner Y = 13.442 to SARS-CoV-2 IgM. The calibration curve of SARS-CoV-2 IgG day 1 detection in Figure6 was

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Y = 13.442 × X + 107.99. The calculated recovery index for SARS-CoV-2 IgG was 98.98%. From a practical point of view, both recovery indexes were acceptable. The proposed MCIA can be used for the quantitative detection of SARS-CoV-2 antibodies and other kinds of antibodies as well. Compared with other POC antibody tests such as LFIAs, the MCIA can provide quantitative results instead of qualitative ones. The quantitative measurement of antibodies provides more information regarding the degree of the immune response, which may be helpful for monitoring the progression of COVID-19. The assay time of the MCIA is about half an hour, which meets the requirement of a POC test. The 10 µL sample volume consumption of the MCIA is comparable to most POC devices. Although the cost per analysis of the MCIA is dependent on the reagents needed, the MCIA is based on the microflow cytometry format, so the inherent advantages of microflow cytometry, such as cost-efficiency, also exist for the MCIA. The linear range of antibody concentrations detected by the MCIA is restricted by the prozone effect, but dilution can be performed when the concentration of the sample exceeds this range. Furthermore, it is important to note that there is about a 75% similarity between the genomes of SARS-CoV and SARS-CoV-2 [19]. The MCIA for SARS-CoV-2 detection did have cross-reactivity with IgG from SARS-CoV based on the results from our experiments.

4. Conclusions A MCIA has been developed for the POC quantitative detection of SARS-CoV-2 IgM and IgG antibodies. A linear relationship has been established between the average transit time and the concentration of SARS-CoV-2 antibodies up to 1.0 mg/L. 0.06 mg/L and 0.10 mg/L are the LODs for SARS-CoV-2 IgM and IgG, respectively. These values are comparable to those reported for in-house ELISAs, indicating that the device could ade- quately perform if tested against serum samples. The proposed protocol has demonstrated to be rapid, simple and sensitive for the POC quantitative measurement of SARS-CoV-2 antibodies. The 30 min assay time, 10 µL sample consumption, relatively simple proce- dures and compact core devices make the MCIA a promising tool for the POC detection of SARS-CoV-2 IgM and IgG. Furthermore, the developed protocol is readily adaptable to be applied for the detection of other antibodies. However, there are still some limitations of this study. The detection ranges of SARS-CoV-2 IgM and IgG were not large enough for the method to be conceivably performed without dilution of serum samples. The manual steps for the immune reaction need to be integrated into the MCIA to improve the detection efﬁciency. Further investigation of this study should focus on reducing the mentioned limitations and testing the sensitivity and speciﬁcity of the MCIA performance using serum specimens from individuals who have been infected with SARS-CoV-2.

Author Contributions: Conceptualization, C.-q.X. and J.Q.; methodology, C.-q.X., J.Q. and M.C.; validation, J.Q.; resources, C.-q.X. and Y.Z.; writing—original draft preparation, J.Q. and M.C.; writing—review and editing, J.Q., M.C. and C.-q.X.; supervision, C.-q.X.; project administration, C.-q.X.; funding acquisition, C.-q.X. and J.Q. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data available on request due to restrictions of privacy. Acknowledgments: The authors would like to thank Forsee Instruments Ltd. and the MITACS VIP grant for the support of the project. Conﬂicts of Interest: The authors declare no conﬂict of interest. Micromachines 2021, 12, 433 9 of 9

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