biosensors Article Magnetic Bead Handling Using a Paper-Based Device for Quantitative Point-of-Care Testing Kevin Arias-Alpízar 1,2 , Ana Sánchez-Cano 1,2 , Judit Prat-Trunas 1 , Elena Sulleiro 2,3,4 , Pau Bosch-Nicolau 3,4 , Fernando Salvador 3,4 , Inés Oliveira 3,4 , Israel Molina 3,4 , Adrián Sánchez-Montalvá 2,3,4 and Eva Baldrich 1,4, * 1 Diagnostic Nanotools Group, Vall d’Hebron Institut de Recerca (VHIR), 08035 Barcelona, Spain 2 Universitat Autònoma de Barcelona (UAB), 08193 Barcelona, Spain 3 International Health Unit Vall d’Hebron-Drassanes, Vall d’Hebron Hospital Universitari, PROSICS Barcelona, 08035 Barcelona, Spain 4 Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, 28028 Madrid, Spain * Correspondence: eva.baldrich@vhir.org Abstract: Microfluidic paper-based analytical devices (µPADs) have been extensively proposed as ideal tools for point-of-care (POC) testing with minimal user training and technical require- ments. However, most µPADs use dried bioreagents, which complicate production, reduce device reproducibility and stability, and require transport and storage under temperature and humidity- controlled conditions. In this work, we propose a µPAD produced using an affordable craft-cutter and stored at room temperature, which is used to partially automate a single-step colorimetric magneto- immunoassay. As a proof-of-concept, the µPAD has been applied to the quantitative detection of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), a biomarker of malaria infection. In this system, detection is based on a single-step magneto-immunoassay that consists of a single 5-min incubation of the lysed blood sample with immuno-modified magnetic beads (MB), detection anti- Citation: Arias-Alpízar, K.; body, and an enzymatic signal amplifier (Poly-HRP). This mixture is then transferred to a single-piece Sánchez-Cano, A.; Prat-Trunas, J.; paper device where, after on-chip MB magnetic concentration and washing, signal generation is Sulleiro, E.; Bosch-Nicolau, P.; achieved by adding a chromogenic enzyme substrate. The colorimetric readout is achieved by the Salvador, F.; Oliveira, I.; Molina, I.; naked eye or using a smartphone camera and free software for image analysis. This µPAD afforded Sánchez-Montalvá, A.; Baldrich, E. quantitative Pf-LDH detection in <15 min, with a detection limit of 6.25 ng mL−1 when the result Magnetic Bead Handling Using a was interpreted by the naked eye and 1.4 ng mL−1 when analysed using the smartphone imaging Paper-Based Device for Quantitative system. Moreover, the study of a battery of clinical samples revealed concentrations of Pf-LDH that Point-of-Care Testing. Biosensors 2022, correlated with those provided by the reference ELISA and with better sensitivity than a commercial 12, 680. https://doi.org/10.3390/ bios12090680 rapid diagnostic test (RDT). These results demonstrate that magneto-immunoassays can be partly automated by employing a µPAD, achieving a level of handling that approaches the requirements of Received: 26 July 2022 POC testing. Accepted: 20 August 2022 Published: 25 August 2022 Keywords: immuno-modified magnetic beads; paper-based diagnostic device; low-cost assay automation; Publisher’s Note: MDPI stays neutral smartphone colorimetric detection; point-of-care testing; malaria quantitative diagnosis with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Paper and paper-like materials are extensively employed to produce analytical tools [1,2]. Copyright: © 2022 by the authors. Compared to other alternatives, such as glass-, silicon- or polymer-based chips, µPADs are Licensee MDPI, Basel, Switzerland. inexpensive, relatively easy to fabricate, and convenient to dispose of. The porous membrane This article is an open access article provides filtration and pre-treatment of complex samples, large surface areas for reagent in- distributed under the terms and corporation, and passive solution pumping without external power sources or equipment. conditions of the Creative Commons When coupled to the visual interpretation of a colorimetric readout, µPADs approach ideal Attribution (CC BY) license (https:// POC diagnostic devices, matching the ASSURED criteria established by the World Health creativecommons.org/licenses/by/ Organization (WHO): affordable, sensitive, specific, user-friendly, rapid and robust, equipment 4.0/). free, and deliverable to end-users [3]. Biosensors 2022, 12, 680. https://doi.org/10.3390/bios12090680 https://www.mdpi.com/journal/biosensors Biosensors 2022, 12, 680 2 of 13 The most widely used µPADs are lateral-flow assays (LFAs), which consist of 4 types of overlapping membranes (sample, conjugate, test and absorbent pads, respectively), two of them containing dried reagents [4]. To carry a test, the user dispenses the sample (and sometimes running buffer), which wicks along the device to interact with a labelled detection antibody (d-Ab) and the immobilized capture antibody (c-Ab), forming coloured bands that are interpreted visually or using hand-held readers [5]. More sophisticated µPADs incorporate hydrophobic barriers to define hydrophilic channels and chambers (such as by paper patterning, multi-component stacking, or folding into three-dimensional origami chips) [1,2]. When reagent-modified, µPADs display limited stability and should be distributed and stored under controlled temperature and humidity [2]. In addition, reagent-modified µPADs can be used only to detect the analyte they were produced for. Malaria is a disease caused by Plasmodium parasites that are transmitted to humans by the bite of infected female Anopheles mosquitoes [6]. Despite being curable, malaria took the life of 627,000 people and caused 241 million cases in 2020 [7]. Of the human-infecting Plasmodium species, P. falciparum is the most frequent and deadly, accounting for 94% of the cases and deaths reported worldwide. There is a general agreement that factors such as increasing parasite drug resistance [8], migration processes [9], climate change effects [10], growth of the population at risk, and the COVID-19 pandemic [8] combine to jeopardise the advances made over the last decades to control the disease in the endemic countries, increasing the risk of malaria reintroduction in areas from which it had been eliminated. Currently, the “gold standard” for the diagnosis of malaria is microscopy [11,12]. Although a well-trained microscopist can detect down to 50–100 parasites per µL of blood (parasite µL−1 ) under typical field conditions [13], microscopy relies on expertise and sub- jective result interpretation, requires >1 h per sample, and cannot detect low parasitemias (so-called submicroscopic malaria). In centralised laboratories, molecular techniques, such as the polymerase chain reaction (PCR), are gradually being incorporated, achieving excep- tional LODs (around 1 parasite µL−1 ), often in fully automated and species-specific assay formats. However, PCR demands long analysis time, experienced personnel, sophisticated infrastructure and high operational costs. Finally, RDTs have been positioned as fast, inex- pensive, and easy-to-use tools for malaria POC testing in low/middle-resource settings (such as the countries where malaria is more prevalent) or as the first test in high-resource non-endemic settings before performing confirmatory analysis [14–16]. Most RDTs are LFA-based devices that detect Plasmodium antigens in a drop of blood, in <30 min, at a cost of 1–5 € [17]. Nonetheless, RDTs display limitations as well, including variable performance depending on the storage and environmental conditions, subjective result interpretation, qualitative (yes/no) response, insufficient sensitivity, and limited detection of “non-falciparum” species [18,19]. Attempts have been made to produce upgraded µPADs. For instance, Pilon dos Santos et al. produced a µPAD to detect P. falciparum histidine-rich protein 2 (Pf-HRP2) [20]. The paper chip, cut using a CO2 laser plotter, displayed 3 discrete areas separated by narrow microfluidic channels. The first served for the addition of sample and peroxidase-labelled d-Ab; the second displayed immobilised c-Ab; and the third granted solution pumping and waste storage. The system displayed an LOD of 4.5–5.0 ng mL−1 of Pf-HRP2 in saline solution (equivalent to 59–65 parasites µL−1 ) but was not tested in clinical samples. Since Pf-HRP2 is only present in P. falciparum and deletions in the pfhrp2/3 genes may pre- vent detection by HRP2-based RDTs, alternative Pan Plasmodium biomarkers have been targeted [21–24]. A multiplexed µPADs was produced by Deraney and co-workers by stacking 8 layers of wax-patterned paper, two of them modified with gold-labelled d-Ab and immobilised c-Ab [25]. The device detected Pf-HRP2 and Pan Plasmodium lactate dehydrogenase (pLDH) in about 20 min, with LODs of 20.3 ng mL−1 and 80.2 ng mL−1 , respectively, and achieved detection in spiked lysed blood. Singh et al. employed aptamer- coated MB to trap pLDH and P. falciparum glutamate dehydrogenase (Pf-GDH), which were then incubated in a chromogenic enzyme substrate solution [26]. The supernatant was next transferred to a piece of chemically modified chromatographic paper for colour inter- Biosensors 2022, 12, 680 3 of 13 pretation by the naked eye (qualitative) or using image analysis (quantitative), achieving LODs down to 68–69 pM in spiked serum in about 95 min (equivalent to 2.3–2.4 ng mL−1 ). The electrochemical µPAD reported by Ruiz-Vega and co-workers included a sample filtration unit, a double-sided screen-printed paper electrode and absorbent pads in a low-cost magnetic holder [27]. The system facilitated the partial automation of a single-step magneto-immunoassay and detected Pf-LDH in lysed whole blood. Pf-LDH quantita- tion was afforded down to 2.47 ng mL−1 in about 20 min, identifying patients presenting malaria parasitemias > 0.3%. Here, we develop a µPAD for magneto-immunoassay POC operation. As a proof-of- concept, we report the fast and quantitative detection of Pf-LDH based on a single-step magneto-immunoassay, a generic single-piece microfluidic paper device and smartphone- based colorimetry. The system affords quantitative Pf-LDH detection in lysed whole blood samples in <20 min, with an LOD of 1.4 ng mL−1 and minor user intervention. However, the storage-stable paper device could be employed to detect other analytes by just adjusting the magneto-immunoassay reagents. 2. Material and Methods 2.1. Reagents and Biocomponents Recombinant Pf-LDH was provided by CTK Biotech (San Diego, CA, USA). Mono- clonal capture and detection antibodies to pLDH (C01834M and C01835M, c-MAb and d-MAb, respectively) were from Meridian Bioscience (Memphis, TE, USA). The latter were modified with biotin to produce biotinylated d-MAb (bd-MAb; Supporting Information). Carboxylic acid MB of 1 µm diameter (Dynabeads MyOne, Invitrogen Ref. 65011 and SpeedBead Magnetic Carboxylate, GEHealthcare Ref. 45152105050250), Streptavidin Poly- HRP (Ref. 21140), sulphuric acid 1 M and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were from Thermo Fisher Scientific (Barcelona, Spain). Bovine serum albumin (BSA), Triton X-100, Tween 20, 3,30 ,5,50 -Tetramethylbenzidine Liquid Substrate System (TMB; Ref. T0440), and 2-(N-morpholino)ethanesulfonic acid hydrate (MES) were from Sigma-Aldrich (Madrid, Spain). Phosphate-buffered saline tablets (PBS; Barcelona, Spain) produced KH2 PO4 250 mM sodium phosphate, NaCl 150 mM, and KCl 2.7 mM, pH 7.4. Reagent Diluent (10 × RD, Ref. DY995; equivalent to 10 × PBS, 10% BSA) was from R&D (Abingdon, UK). The membranes used for device production were Standard 17 and CF5 (Refs. 17114594 and 29008181; Cytiva Europe, Freiburg, Germany). For washing, PBS was supplemented with 0.05% of Tween 20 (PBS-T). Unless otherwise stated, blocking and incubation steps were carried out with PBS supplemented with BSA 1% and 0.05% of Tween 20 (PBST-BSA). 2.2. Device Fabrication The system for partial magneto-immunoassay operation and detection automation included two components, a disposable µPAD and a reusable magnetic holder, which were assembled as previously described in [28] (Figure 1a). Briefly, the main body of the µPAD was a single-piece device, which was designed using Silhouette Studio version 4.4.476 and cut on Standard 17 using an inexpensive craft plotter, Silhouette Cameo 3 (Silhouette America, Lehi, UT, USA; Figure 1b,c). It contained three distinguishable sections (Figure 1b). The first one was a tear-shaped washing pad (23 mm × 13.5 mm) for the addition of sample and assay reagents. Accordingly, this section was designed to display high-volume load capacity and efficient solution flow towards the second section. This was an MB concentration zone 5 mm in diameter, which was placed onto a magnet when the paper device was positioned in the magnetic holder. Apart from MB retention, this section also provided a lecture zone for the colorimetric readout. Finally, the third section was a circular end. This was allocated below an absorbent pad (26 × 16 mm), which was produced using a guillotine and CF5. This pad functioned as a flow-driving absorbent pump and a waste storage unit. MB concentration zone 5 mm in diameter, which was placed onto a magnet when the paper device was positioned in the magnetic holder. Apart from MB retention, this section also provided a lecture zone for the colorimetric readout. Finally, the third section was a circular end. This was allocated below an absorbent pad (26 × 16 mm), which was pro- Biosensors 2022, 12, 680 4 of 13 duced using a guillotine and CF5. This pad functioned as a flow-driving absorbent pump and a waste storage unit. Figure 1. (a) Schematic representation of the system developed, which resembled that described in Figure 1. (a) Schematic representation of the system developed, which resembled that described [28] but displayed different geometric features. (b) Dimensions of the single-piece paper device. (c) in [28] but displayed different geometric features. (b) Dimensions of the single-piece paper device. Picture of the reusable magnetic holder (left) and the disposable paper device produced with Stand- (c) ardPicture of the reusable 17 membrane (right). magnetic holder (left) and the disposable paper device produced with Standard 17 membrane (right). On the On the other other hand, hand,thethemagnetic magneticholder holderwas wasa areusable reusabledevice device made made of of a first layer a first of layer ethylene-vinyl acetate (purchased from a local retailer). It displayed of ethylene-vinyl acetate (purchased from a local retailer). It displayed a pit that was a pit that was made with awith made biopsy punchpunch a biopsy and accommodated and accommodated a neodymium magnet a neodymium (1 mm(1 magnet thick, mm 5thick, mm diam- 5 mm eter). Two layers diameter). of acetate, Two layers carvedcarved of acetate, with the Silhouette, with were secured the Silhouette, on top with were secured double- on top with sided adhesive. double-sided The bottom adhesive. one wasone The bottom plain wastoplain keeptothe magnet keep in placeinand the magnet placeprevent direct and prevent contactcontact direct with the withMBs.the The MBs.other displayed The other a cavity displayed to accommodate a cavity to accommodate the paper device the paper and device guarantee the correct alignment with the magnet. Once placed and guarantee the correct alignment with the magnet. Once placed in the holder, the in the holder, the paper device fit in this cavity and sat directly directly onto onto thethe first first acetate. acetate. Paper devices were blocked by immersion in BSA 5%, Tween Tween 0.5%0.5% for 15 min at room temperature, and were then washed twice with PBS-T for 3 min each. Finally, Finally, the devices were rinsed were rinsedwith withwater, water,dried driedforfor 3030minmin at 37.5 ◦ C and at 37.5 °C and stored stored in a in a ziplock ziplock pouch pouch until until used. used. 2.3. Smartphone Imaging System 2.3. Smartphone Imaging System imaging system was designed to take photographs of the A smartphone-integrated µPAD Acolorimetric readout (Figure smartphone-integrated S1 insystem imaging Supplementary was designedMaterial). This to take included a home- photographs of the made dark box produced using a cardboard box (28 × 16 × 10 μPAD colorimetric readout (Figure S1 in Supplementary Material). This included a home- cm), a dimmable strip of white 6000K LED lights with a power of 1200 lm for lighting the made dark box produced using a cardboard box (28 × 16 × 10 cm), a dimmable strip of chamber, and a smartphone placed on the LED white 6000K lid, which lightshadwitha hole for theofphone a power 1200 camera. For image lm for lighting theacquisition, chamber, and paper a devices were removed from the magnetic holders and were smartphone placed on the lid, which had a hole for the phone camera. For image acquisi-placed in the centre of the dark box. The tion, paper box was devices were closed, removed the lights from the were turned on, magnetic and images holders and were were obtained placed in theusing cen- the smartphone camera. When processing the images with ImageJ tre of the dark box. The box was closed, the lights were turned on, and images were ob- software, the signal at the detection tained using thepad was calculated smartphone camera. forWhen each processing paper device theby subtracting images the background with ImageJ software, registered for the whole device, converting the image into an the signal at the detection pad was calculated for each paper device by subtracting 8-bit format and setting the the colour threshold background to 235for registered to the obtain wholethedevice, mean grey value. the converting Thus, the colorimetric image signal was into an 8-bit format and taken settingusing the whole the colour sensortoarea. threshold 235 to obtain the mean grey value. Thus, the colorimetric signal 2.4. was taken Pf-LDH using the whole sensor area. Magneto-Immunodetection Unless Magneto-Immunodetection 2.4. Pf-LDH otherwise stated, MB modified with c-MAb (c-MAb-MB; Supporting infor- mation) were incubated in 1.5 mL Eppendorf tubes at 24 ◦ C, protected from light in a Unless otherwise thermoshaker (Thermalstated, ShakeMB lite,modified with c-MAbLeuven, VWR International, (c-MAb-MB; Supporting Belgium), infor- and washed mation) were incubated in 1.5 mL Eppendorf tubes at 24 °C, protected using a magnetic separator (BILATEST, Merck Life Science, Madrid, Spain). from light in a thermoshaker (Thermal Shake lite, VWR International, Leuven, Belgium), and washed us- ing a magnetic 2.4.1. separator (BILATEST, Magneto-Immunoassay in TubesMerck Life Approach) (Classical Science, Madrid, Spain). The magneto-immunoassay originated from a previous development (Figure S2) [27,28]. Briefly, c-MAb-MB were washed two times with PBS and resuspended in PBST-BSA to a final concentration of 5 mg mL−1. Pf-LDH was then agitated at 1500 rpm for 5 min with 4 µL of c- MAb-MB in PBST-BSA supplemented with bd-MAb (75 ng mL−1) and Poly-HRP (50 ng mL−1) in a final volume of 110 µL. For spectrophotometric assay detection, the c-MAb-MB/Pf-LDH/bd- MAb/Poly-HRP complexes were washed twice with 150 µL of PBST, resuspended in 100 µL of Biosensors 2022, 12, 680 5 of 13 TMB and stirred for 20 min at 1500 rpm. After this, MBs were concentrated, the supernatant was transferred to 96-well plates, and 50 µL of 1 M sulphuric acid was added to stop the reaction. Finally, absorbance was measured at 450 nm using a Sunrise plate reader (Tecan Group, Männedorf, Switzerland). 2.4.2. Magneto-Immunoassay Using the µPAD (POC Approach) For on-chip MB washing and colorimetric assay detection, after the 5-min immuno- capture performed in tubes (as indicated in Section 2.4.1), the mixture of c-MAb-MB/Pf- LDH/bd-MAb/Poly-HRP was directly placed in the µPAD washing pad. Four consecutive washes were next carried out by adding 100 µL of PBST each time on the washing pad. Then, 50 µL of TMB was added to the detection pad, where the magnetic complexes had been retained by a magnet, and the device was incubated for 5 min without agitation at room temperature before image capture. 2.4.3. Pf-LDH Detection in Whole Blood Clinical Samples Malaria patients and healthy individuals were recruited during the period of 2018–2020 at Vall d’Hebron University Hospital. Peripheral blood samples were obtained in heparin collection tubes before the administration of any antimalarial drugs. Malaria acute infec- tions were confirmed by microscopy and/or PCR. The study was approved by the Ethics Committee of Vall d’Hebron University Hospital (PR(AG)30/2018), and the corresponding informed consent was obtained from all patients. For detection of Pf-LDH using the µPAD, samples were diluted 1:1 with lysis buffer (50 mM KH2 PO4 , 300 mM NaCl, 0.25 M imidazole, 1% Triton X-100) and were incubated for 5 min at room temperature. Samples were diluted 1:10–1:100 with PBST-BSA and were processed as described in Section 2.4.2. All samples were analysed 2 times independently and were assayed in parallel by commercial RDT, microscopy and ELISA [29]. 2.5. Data Analysis Paper devices were used just once (i.e., each device for a single Pf-LDH concentration). Except for the detection of clinical samples (n = 2), graphs show the average of the colori- metric signal readout registered for no less than three independent replicates. In the same way, error bars correspond to the standard deviation (SD) of those replicates. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated as the average of the signals registered for the blanks (i.e., experiments carried with all the reagents in the absence of Pf-LDH) plus 3 times and 10 times their SD, respectively. The sensitivity was calculated from the slope of the linear assay range. The variability was analysed in terms of coefficient of variation (% CV = (SD/mean) × 100). Signal-to-noise ratio (S/N) stands for the signal generated by each Pf-LDH concentration divided by the average signal registered for the blanks. 3. Results and Discussion The starting point for this work was a single-step magneto-immunoassay developed previously for Pf-LDH detection [27], with some improvement (Figures 2a and S2) [28]. This consisted of a single 5-min incubation of the sample (lysed whole blood) with a cocktail of three reagents in an Eppendorf tube: cAb-MBs, which allowed fast Pf-LDH capture and concentration from whole blood; bd-MAb to grant binding specificity in a sandwich assay format; and Poly-HRP, an enzymatic signal amplifier formed by streptavidin and hundreds of HRP molecules that provided large signals. In the classical approach, this incubation was followed by two consecutive washing steps with PBS-T. Each washing step entailed placing the tube for 2 min in a magnetic rack for MB concentration, removing the supernatant without disturbing the MB pellet, and resuspending the MBs in the appropriate solution using a pipette. After the last wash, MBs were resuspended in TMB substrate solution, the Poly-HRP enzymatic reaction was allowed to proceed for 20 min, the tube was placed once more in the magnet for 2 min, and the supernatant was transferred to a 96-well plate hundreds of HRP molecules that provided large signals. In the classical approach, this incubation was followed by two consecutive washing steps with PBS-T. Each washing step entailed placing the tube for 2 min in a magnetic rack for MB concentration, removing the supernatant without disturbing the MB pellet, and resuspending the MBs in the ap- Biosensors 2022, 12, 680 propriate solution using a pipette. After the last wash, MBs were resuspended in6 TMB of 13 substrate solution, the Poly-HRP enzymatic reaction was allowed to proceed for 20 min, the tube was placed once more in the magnet for 2 min, and the supernatant was trans- ferred to a 96-well to quantitate colourplate to quantitate intensity. colour Although intensity. fast and Although efficient fast and in the hands efficient of an expert,inthis the hands of an expert, this procedure required user training and was incompatible procedure required user training and was incompatible with POC testing carried out by with POC testing carried untrained out by untrained personnel. personnel. Figure Figure2.2.Single-step Single-stepmagneto-immunoassay magneto-immunoassay carried out manually carried out manually as as reported reportedin in[28] [28](a) (a)and andusing using the µ PAD developed here, which differed in size and shape from that in our previous work to better the µPAD developed here, which differed in size and shape from that in our previous work to better fit the requirements of colorimetric detection (b). (c) Scheme of TMB redox reaction (here, Poly-HRP fit the requirements of colorimetric detection (b). (c) Scheme of TMB redox reaction (here, Poly-HRP catalyses TMB oxidation coupled to H2O2 reduction for colour generation), and examples of nega- catalyses TMB oxidation coupled to H2 O2 reduction for colour generation), and examples of negative tive and positive µ PAD readouts. and positive µPAD readouts. The Theobjective objectiveofofthis thiswork workwaswas toto demonstrate demonstrate that thisthis that type of assay type cancan of assay be partially be par- automated using ausing tially automated simple and inexpensive a simple disposable and inexpensive µ PADµPAD disposable (Figure 2b). Here, (Figure the color- 2b). Here, the imetric readout colorimetric obtained readout has been obtained interpreted has been alternatively interpreted alternativelyby semi-quantitative by semi-quantitativevisual vi- inspection or using sual inspection a smartphone or using camera a smartphone and ImageJ camera free software, and ImageJ whichwhich free software, provided quan- provided titative results. quantitative Furthermore, results. this strategy Furthermore, entails this strategy the production entails the production of aof universal paper a universal de- paper vice, which device, could which be stored could be storedandand employed employed to detect alternatively to detect alternativelydifferent magneto-im- different magneto- immunoassays. munoassays. The The nextnext sections sections summarize summarize the optimization the optimization of µ of the the PADµPAD geometric geometric fea- features, tures, magneto-immunoassay magneto-immunoassay handling handling usingthe using theµµPAD, PAD, and and technology technology validation validationwith with clinicalsamples. clinical samples. 3.1.Production 3.1. Productionofofthe the µ µPAD PAD A µPAD was designed to carry out MB washing on-chip and under flow conditions. A µ PAD was designed to carry out MB washing on-chip and under flow conditions. This device displayed three sections: a washing pad for the serial addition of the mixture This device displayed three sections: a washing pad for the serial addition of the mixture of sample and reagents (c-MAb-MB, poly-HRP and bd-MAb) and the washing buffer; a pad for MB magnetic concentration and colour readout; and the final end with a staked absorbent pad (Figure 1). For its utilization, the µPAD was placed onto a reusable magnetic holder, which consisted of a piece of ethylene-vinyl acetate with an embedded magnet and a plastic cover that facilitated the alignment of the paper device. The µPAD initially displayed a rectangular washing pad with smooth edges in order to facilitate solution flow and limit non-specific biocomponent retention. Figure 3a,b show 4 devices with washing pads of increasing area after adding the MBs in 100 µL of a absorbent pad (Figure 1). For its utilization, the µ PAD was placed onto a reusable mag- netic holder, which consisted of a piece of ethylene-vinyl acetate with an embedded mag- net and a plastic cover that facilitated the alignment of the paper device. The µ PAD initially displayed a rectangular washing pad with smooth edges in order Biosensors 2022, 12, 680 to facilitate solution flow and limit non-specific biocomponent retention. Figure 3a,b show 7 of 13 4 devices with washing pads of increasing area after adding the MBs in 100 µ L of a stained solution, equivalent to the volume of sample and reagents used in the magneto-immuno- stained assay. Thesolution, equivalent two smallest to the washing volume pads of (A1–A2) tested sample and hadreagents used inabsorption a theoretical the magneto- capac- ityimmunoassay. of 30 and 60 µThe twowere L and smallest ablewashing to absorbpads tested 100 (A1–A2) µ L of hadonly solution a theoretical absorption if these were pipetted capacity very slowly,of which 30 andwas60 µL notand were able practical. Ontothe absorb other100 µL of hand, solution faster only ifproduced pipetting these weresolu- pipetted very slowly, which was not practical. On the other hand, tion overflow, with the corresponding loss of MBs. The absorption pad in thefaster pipetting produced A3 device solution overflow, with the corresponding loss of MBs. The absorption pad in the A3 device absorbed this volume of solution in about 15 s, soaking the absorption and detection pads absorbed this volume of solution in about 15 s, soaking the absorption and detection pads completely. In contrast, the largest absorption pad (A4), which had a theoretical adsorp- completely. In contrast, the largest absorption pad (A4), which had a theoretical adsorption tion capacity capacity of 125 of 125 µ L, was µL, was not completely not completely wetthis wet with with this volume volume of solution, of solution, which in- which increased creased the amount of washing buffer needed the amount of washing buffer needed later on. later on. Figure Figure 3. 3.Production Production of themicrofluidic of the microfluidic paper paper device. device. (a) Design (a) Design of devices of paper paper devices with pads with washing washing pads of increasing of increasing areasareas (A1–A4) (A1–A4) and (b)and (b)absorption their their absorption capacity capacity after after adding MBsadding in 100MBs µL ofina 100 µ L of a stained stained solution. (c) Device A3, in which the 100 µL of MB stained solution has been washed away after fouraway solution. (c) Device A3, in which the 100 µ L of MB stained solution has been washed after four consecutive consecutive additionsadditions of washing of buffer washing buffer of, from leftof,tofrom right,left 50, to right, 100, 50, 100, and 200 and while µL each, 200 µthe L each, while MBsthe areMBs are magnetically retained retained magnetically in the in the detection detection zone (Figure S3).zone (Figure (d,e) S3). (d,e) Tear-shaped andTear-shaped drop-shaped and drop-shaped devices before devices before (d) and after (d) andstained (e) the after (e) the stained solution has been solution washed has been away (4washed washes, away 100 µL(4 washes, each). 100(f)µ Background L each). (f) noise Background obtained noise for theobtained for the magneto-immunoassay, magneto-immunoassay, carried carried in 100 µL of lysed wholein 100 bloodμL of lysed whole blood (diluted 1:100) after washing 4 times (100 μL each) and adding (diluted 1:100) after washing 4 times (100 µL each) and adding TMB substrate solution. Pictures of TMB substrate solution. drop- andPictures of drop- tear-shaped and tear-shaped devices devices are not to scale. are not to scale. In In A3,A3,3–4 3–4serial serial additions additions of of100 100µL ofof µL washing washingbuffer werewere buffer enough to wash enough to the wash100the µL 100 of stained solution and concentrate the MB in the retention zone (Figures 3c and S3). Lower µ L of stained solution and concentrate the MB in the retention zone (Figures 3c and S3). washing volumes required more additions to accomplish washing and recover the MBs Lower washing volumes required more additions to accomplish washing and recover the in the detection area, and larger volumes extended the washing steps unnecessarily. In MBsall in the detection cases, area,zones the peripheral and larger of the volumes extended washing pad the washing were washed steps unnecessarily. less efficiently than the In centre all cases, the peripheral zones of the washing pad were washed (Figure S3). This was concerning because the assay had to be carried less efficiently than the out in lysed centre whole (Figure blood,S3). and This was concerning any unwashed because cell debris the assay and reagents hadincrease could to be carried out in lysed the background whole blood, and any unwashed cell debris and reagents could increase noise. Accordingly, the washing pad was re-designed to be either drop- or tear-shaped the background 2 with noise. Accordingly, (Figure 3d,e). These thetwo washing pad was new designs hadre-designed washing padtoareas be either drop- of around 250orcmtear-shaped elongated (Figure 3d,e).shapes Theseand twovery newsmooth designsedges to minimise had washing padMB dispersion. areas of around These 250 geometries cm2 with elon- granted fast adsorption of the 100 µL of stained solution gated shapes and very smooth edges to minimise MB dispersion. These(Figure 3d), improved thegeometries flow of the MBs towards the magnet, and were washed efficiently (Figure 3e). Finally, the best-performing designs (A1–A3, drop-shaped and tear-shaped) were used to determine MB on-chip washing efficiency. For this, lysed whole blood (Pf-LDH negative) was diluted at 1:100 in PBST-BSA and was incubated with c-MAb-MBs, bd-MAb and Poly-HRP for 5 min, and the mixture was transferred to the device washing pad. Four washes were carried out (100 µL each), the adsorption pads were removed, and TMB was Biosensors 2022, 12, 680 8 of 13 added to the detection area (Figure 3f). Blue colour, attributed to poor washing, formed mainly in the vicinity of the lecture zone, with a significant decrease in colour intensity and dispersion as the washing pad area increased. The lowest background noise was observed for the drop and tear-shaped designs. This indicated that these washing pads provided the best MB washing and removal of leftover components. 3.2. Magneto-Immunoassay Handling and Detection Using the µPAD The operation of the magneto-immunoassay using the µPAD is illustrated in Figure 2b. The assay started with a single 5-min incubation of the sample/Pf-LDH and reagents (c-MAb-MB, poly-HRP and bd-MAb), which was carried out in a tube. The mixture was then transferred to the washing pad of the paper device. Washing buffer was added, which pushed the sample and reagents across the retention zone, where the MBs (and thus cMAb-MB/Pf-LDH/bd-MAb/Poly-HRP complexes in positive samples) were trapped magnetically. This stage was crucial because the washing buffer had to eliminate unbound reagents and non-targeted blood components, directing them towards the absorption pad in order to minimize background noise. Finally, 50 µL of TMB enzyme substrate solution was added to the detection zone, and the device was incubated for 5 min in the dark for colour development. The formation of a blue product was indicative of the presence of Pf-LDH, and the colour intensity was proportional to the concentration of Pf-LDH. The optimization of magneto-immunoassay performance under flow conditions in- cluded the study of the amount and location of TMB addition (Figure S4) and the concen- tration and type of c-MAb-MBs (Figures S5 and S6). Dispensing the TMB in the centre of the detection zone directly onto the MBs pro- duced MB random rearrangement, which reduced signal generation and reproducibility (Figure S4a). The highest colour intensity and reproducibility were achieved if the substrate solution was dispensed upstream from the magnet location in the stem that separated the washing and detection areas. Colour intensity also increased with the amount of TMB dispensed (30, 50 and 100 µL), but both in the positive and the negative controls (back- ground noise; Figure S4b). Therefore, the best S/N was achieved using 50 µL of TMB (Figure S4). The amount of MBs used per sample was next decreased in an attempt to reduce the background noise, but the signals and S/N registered in the presence of Pf-LDH decreased as well, as did the assay sensitivity (slope) (Figure S5). A concentration of 20 µg of c-MAb-MB per sample was selected for subsequent experiments. Invitrogen Dynabeads were tentatively substituted by GE SeraMag MB, which, according to the provider, display higher magnetic content. Although these exhibited faster magnetic concentration, they also provided slightly higher background noise and lower signals for all the concentrations of Pf-LDH studied. Accordingly, work continued with Dynabeads, which displayed higher signals for the Pf-LDH positive controls, lower background noise, and higher S/N in the magneto-immunoassay (Figure S6). 3.3. Quantitative Detection of Pf-LDH Using a Smartphone Detection System Upon optimization, the paper-based magneto-immunoassay took less than 13 min, including single-step immunocapture, washing and detection. Colour intensity was propor- tional to Pf-LDH concentration between 3.13 ng mL−1 and 100 ng mL−1 , with an LOD of 6 ng mL−1 (Figures 4a and S7), demonstrating the feasibility of carrying a semi-quantitative naked eye evaluation. The following step was implementing objective data acquisition and analysis using a smartphone. Biosensors 2022, 12, x FOR PEER REVIEW 9 of 13 of 6 ng mL−1 (Figures 4a and S7), demonstrating the feasibility of carrying a semi-quanti- Biosensors 2022, 12, 680 9 of 13 tative naked eye evaluation. The following step was implementing objective data acquisi- tion and analysis using a smartphone. Figure Figure 4. 4. Signals Signals registered the paper-based registered in the paper-basedmagneto-immunoassay magneto-immunoassayfor fordifferent different concentrations concentrations of ofPf-LDH. Pf-LDH. (a)(a) Photographs Photographs of of the the µPADs µ PADsandandresults resultsofofsemi-quantitative semi-quantitativeinterpretation interpretation (Pf-LDH (Pf-LDH in inPBST-BSA). PBST-BSA). (b) (b) Signals obtained for Signals obtained for the themagneto-immunoassay magneto-immunoassaycarried carriedinintubes tubesusing using anan ELISA ELISA reader reader (classical approach)and (classical approach) andthethe µPADµ PAD using using a smartphone a smartphone and ImageJ and ImageJ (n = 4;S7). (n = 4; Figure Figure S7). (c) (c) Signals Signals registered registered in the in the µPAD forµPf-LDH PAD forspiked Pf-LDH spiked in or in PBST-BSA PBST-BSA or (1:10 lysed blood lysedandblood (1:10 1:100 and 1:100 in in PBST-BSA). PBST-BSA). Smartphone-based densitometry can be challenging for POC applications due to the difficulty of controlling the Smartphone-based positioningcan densitometry of the camera and for be challenging compensating for the due POC applications variable to the background difficulty noise. Accordingly, of controlling controlling the positioning a number of the cameraofand parameters, such asfor compensating distance and the variable angle between background the camera noise. and the Accordingly, paper device, controlling light intensity, a number and area of parameters, measured, such is key as distance and to producing reproducible images and data. Here, an inexpensive dark angle between the camera and the paper device, light intensity, and area measured, is key box was produced forproducing to image acquisition reproducibleusingimages a cardboard and data. (28 ×an boxHere, × 10 cm), modified 16 inexpensive dark box with wasaproduced strip of for image acquisition using a cardboard box (28 × 16 × 10 cm), modified with aAstrip LEDs, used for lighting the chamber and controlling light exposure (Figure S1). holeof (2 × 2 cm) in the lid facilitated the correct positioning of any smartphone LEDs, used for lighting the chamber and controlling light exposure (Figure S1). A hole (2 camera. Finally, pictures were processed using ImageJ, an open-source image analysis software that allowed × 2 cm) in the lid facilitated the correct positioning of any smartphone camera. Finally, subtracting the background noise before calculating colour intensity. pictures were processed using ImageJ, an open-source image analysis software that al- Image analysis of the results obtained in the µPAD for increasing concentrations of lowed subtracting the background noise before calculating colour intensity. Pf-LDH (in PBST-BSA; Figures 4a and S7) allowed producing a calibration plot (Figure 4b). Image analysis of the results obtained in the µ PAD for increasing concentrations of Compared to the assay in tubes, the smartphone-based µPAD produced higher background Pf-LDH (in PBST-BSA; noise, attributed FiguresMB to less efficient 4a and S7) allowed washing, producing which resulted a calibration in higher LOD (0.37 plot (Figure ng mL −1 4b). Compared to the assay in tubes, − 1 the smartphone-based µ PAD produced in the assay in tubes; 1.4 ng mL in the µPAD). Signal saturation was attained at a higher higher back- ground Pf-LDHnoise, attributed concentration (50toinstead less efficient of 25 ngMB mLwashing, −1 ), withwhich resulted variability in higher between LOD (0.37 independent ng mL −1 in the assay in tubes; 1.4 ng mL−1 in the µ PAD). Signal saturation was attained at replicates below 18% (compared to <10% in the classical approach). a higher Pf-LDH A similar concentration experiment (50 insteadon was performed of spiked 25 ng mL −1), with variability between inde- blood. For this, whole lysed blood pendent from healthyreplicates below 18% individuals was (compared to <10% diluted at 1:10 in thewith and 1:100 classical approach). PBST-BSA and spiked with 0–100Ang ·mL−1experiment similar was performed of Pf-LDH. Finally, on spiked it was analysed usingblood. For this, whole lysed the smartphone-based µPAD.blood As from shown healthy individuals in Figure S7, bloodwas wasdiluted washedataway1:10 efficiently, and 1:100 with and thePBST-BSA andnot devices did spiked with display reddish blood leftovers. Furthermore, the calibration plots obtained in PBST-BSA and spiked blood (diluted 1:10 and 1:100) displayed comparable trends, with a linear response 0–100 ng·mL−1 of Pf-LDH. Finally, it was analysed using the smartphone-based µ PAD. A shown in Figure S7, blood was washed away efficiently, and the devices did not display reddish blood leftovers. Furthermore, the calibration plots obtained in PBST-BSA and Biosensors 2022, 12, 680 spiked blood (diluted 1:10 and 1:100) displayed comparable trends, with a linear 10 of 13 respons between 3.15 ng mL and 25 ng mL of Pf-LDH concentration (R > 0.99) and signal satu −1 −1 2 ration around 50 ng mL−1 (Figure 4c). In addition, the LODs were, in all cases, below 2. ng mL−13.15 between of Pf-LDH. ng mL−1Considering and 25 ng mLthat−1 of1 parasite µ L−1 corresponds Pf-LDH concentration (R2 > to approximately 0.99) and signal 0.2 n mL −1 of Pf-LDH [29], the− LOD 1 was under 11 parasites µ L −1. This was equivalent to 11 saturation around 50 ng mL (Figure 4c). In addition, the LODs were, in all cases, below 2.2 ng mL−µ1 Lof−1Pf-LDH. parasites in prediluted samples Considering when that testing 1 parasite −1 corresponds µLblood at 1:10, which is below the thresh to approximately oldngvalue 0.2 − 1 mL ofof200 parasites Pf-LDH µ LLOD [29], the −1 recommended was under 11 by WHOµL parasites −1RDTs for [13].equivalent to . This was 110 parasites µL−1 in prediluted samples when testing blood at 1:10, which is below the threshold valueofof 3.4. Analysis 200 parasites Clinical SamplesµL−1 recommended by WHO for RDTs [13]. The µ PAD 3.4. Analysis wasSamples of Clinical finally employed to study a battery of whole blood samples (diluted 1:10The andµPAD 1:100), was9 from finallypatients employedinfected to studywith P. falciparum a battery and 8 samples of whole blood from individuals (diluted eithe 1:10 and 1:100), 9 from patients infected with P. falciparum and 8 from individuals Samples healthy or displaying infections unrelated to malaria (Figures 5a and S8). either wer studiedorindisplaying healthy parallel by standard infections microscopy unrelated and/or to malaria PCR5a (Figures atand VallS8). d’Hebron Samples Hospital were and were also studied analysed in parallel by ELISA by standard and one of microscopy the commercial and/or RDT accredited PCR at Vall d’Hebron Hospitalby andthe WHO were alsoS9, (Figure analysed Tablesby S1 ELISA and one of the commercial RDT accredited by the WHO and S2). (Figure S9, Tables S1 and S2). Figure5. 5. Figure Detection Detection of clinical of clinical wholesamples whole blood blood using samples using(a)the the µPAD. µ PAD. Images (a)colour of the Images of the colou readouts obtained in the µPAD for 9 blood samples obtained from patients infected with P. falciparum (lysed and P. falcipa readouts obtained in the µ PAD for 9 blood samples obtained from patients infected with rum (lysed diluted andPBST-BSA). 1:10 with diluted 1:10 (b)with PBST-BSA). Colorimetric readout(b)obtained Colorimetric readout in samples obtained negative (C1–8)inorsamples positive negativ (P1–9) for P. falciparum (the horizontal line in the graph represents the analytical LOD, calculated fromthe (C1-8) or positive (P1-9) for P. falciparum (the horizontal line in the graph represents theanalytica LOD, and signals calculated fromfor SD registered thethe signals and negative SD registered clinical forCorrelation samples). (c) the negative of theclinical samples). concentration (c) Correlatio of Pf-LDH of the concentration of Pf-LDH detected by the smartphone-based µ PAD and detected by the smartphone-based µPAD and the reference ELISA (samples diluted 1:10, except for sample the reference ELISA P7, which was diluted to 1:100 due to signal saturation in the 1:10 dilution). Biosensors 2022, 12, 680 11 of 13 All the negative control samples displayed colour readouts that were below the POC LOD (Figure 5b). In contrast, 7 of the 9 malaria samples were clearly positive, exhibiting colour readouts above the test LOD, while the remaining two, which corresponded to submicroscopic malarias (negative by microscopy and confirmed by PCR and ELISA), displayed faint positives or negatives depending on the replicate. In positive samples, colour intensity visual interpretation allowed semi-quantitative result determination, with result agreement of 80–91% among untrained individuals (Figure S10). Furthermore, the concentration of Pf-LDH detected using the smartphone-based µPAD correlated with that obtained by ELISA (Figure 5c), providing quantitative results faster and with less handling. Lastly, the samples were analysed using a commercial RDT, which detected in par- allel Pf-HRP2 and pLDH. Although most commercial RDTs afford LODs in the range of 0.4–1.6 ng mL−1 when detecting Pf-HRP2, the few tests that detect pLDH display LODs spanning 10–1000 ng mL−1 [30]. This difference has also been noticed by several authors. For example, the multiplexed ELISA reported by Jang et al. exploited Quansys Q-Plex technology and exhibited LODs of 2.3, 47.8, and 75.1 pg mL−1 for HRP2, pLDH and P. vivax LDH (Pv-LDH), respectively [31]. The Luminex fluorescent assay developed by Martiáñez-Vendrell and co-workers detected HRP2, Pf-LDH and Pv-LDH with LODs of 6, 56 and 1093 pg mL−1 [32], respectively. Additionally, the multiplexed µPAD produced by Deraney and co-workers detected Pf-HRP2 and pLDH with LODs of 20.3 ng mL−1 and 80.2 ng mL−1 , respectively [25]. Here, of the 9 malaria samples tested with the RDT, only 2 provided faintly positive test lines for pLDH, while the HRP2 result was positive in 7 and negative (P5) or faintly positive (P1) in the other 2 (Figure S9). In summary, the µPAD developed here achieved remarkable results when tested against a commercial RDT, detecting malaria samples better than the RDT pLDH test and similarly to the Pf-HRP2 test. Furthermore, the device provided quantitative readouts that correlated with those obtained using standard methods (Table S1). Despite the small number of samples studied, these results show that the technology developed provides fast quantitative results, entailing little user intervention and a cost per test below 0.5 € (Table S3). 4. Conclusions A µPAD has been developed that provides the smooth operation of single-step magneto-immunoassays, including MB magnetic concentration, washing and incubation with a chromogenic enzyme substrate. As has been shown, visual inspection of the col- orimetric readout provides a semi-quantitative interpretation, while image capture and analysis using a smartphone camera and ImageJ deliver analyte quantification. As a proof-of-concept, the system has been employed to detect Pf-LDH, a biomarker of malaria infection. Malaria antibody-based RDTs display turnaround times of 15–30 min and prices in the range of (1–5 €), delivering qualitative results. The µPAD developed in this work afforded quantitative Pf-LDH detection in lysed blood diluted 1:10 in <20 min (including 5 min of sample lysis), with LODs of 6.25 ng·mL−1 and 1.4 ng·mL−1 when inter- preted by the naked eye and imaging analysis, respectively. Considering 1 parasite µL−1 equivalent to 0.06–0.2 ng·mL−1 , these LODs correspond to 7–23 and 31–104 parasites µL−1 , which is below the cut-off of 200 parasites µL−1 recommended by the WHO for RDTs. Furthermore, Pf-LDH quantitation provided by the µPAD correlated with that granted by the reference ELISA, but the µPAD was faster and easier to use. Using the paper device to automate a magneto-immunoassay, rather than incorporat- ing the biocomponents on-chip, converts the system into a versatile universal platform, easier to tune than classical RDTs by just changing the analyte in the single-step magneto- immunoassay step. With a low estimated production cost and result interpretation using a common smartphone, this µPAD is a versatile platform that could facilitate cost-effective POC testing in remote and low-resource settings. Biosensors 2022, 12, 680 12 of 13 Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/bios12090680/s1, Figure S1: Optimization home-made light controlled box; Figure S2: Comparison of the single-step magneto-immunoassay developed in this work versus the assay previously developed; Figure S3: Geometrical evolution, washing strategy and signal intensity in the negative controls; Figure S4: Optimization of dispensing position and amount of TMB used in the µPAD for magneto-immunoassay detection; Figure S5: Optimization of the concentration of MBs in the paper-based magneto-immunoassay; Figure S6: Comparative performance of MBs from Invitrogen and GE Healthcare; Figure S7: Detection of Pf-LDH in PBST- BSA and blood using the POC device; Figure S8: Study of clinical samples using the POC device; Figure S9: Study of clinical samples using a commercial RDT; Figure S10: External evaluation of semiquantitative naked eye colour scale; Table S1: Summary of the results obtained in the analysis of clinical samples from malaria patients; Table S2: Summary of the results obtained for the control blood samples; Table S3: Estimate of the production cost of the retail-purchased reagents of µPAD. Author Contributions: Conceptualization, K.A.-A. and E.B.; methodology and investigation, K.A.-A., A.S.-C. and J.P.-T.; validation, K.A.-A., P.B.-N., F.S. and I.O.; data curation, E.S. and A.S.-M.; writing— original draft preparation, K.A.-A.; writing—review and editing, A.S.-M. and E.B.; supervision, E.B.; funding acquisition, I.M. and E.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Fondo de Investigaciones Sanitarias of Instituto de Salud Car- los III (co-funded by the European Regional Development Fund; grants CPII18/00025, IFI18/00020, JR18/00022 and QUPID EuroNanoMed AC21_2/00021) and La Caixa Foundation (ID 100010434, fellowship LCF/BQ/DI18/11660061). Diagnostic Nanotools is a consolidated group supported by Secretaria d’Universitats i Recerca, Generalitat de Catalunya (2017 SGR 240). Institutional Review Board Statement: This study was conducted in accordance with the Decla- ration of Helsinki, and approved by the Ethics Committee of Vall d’Hebron University Hospital (PR(AG)30/2018). Informed Consent Statement: Informed consent was obtained from all subjects involved in this study. Acknowledgments: The authors thank the 483 volunteers who kindly answered the 3 questioners shown in Figure S10. Conflicts of Interest: The authors declare no conflict of interest. References 1. Wang, C.M.; Chen, C.Y.; Liao, W.S. Enclosed Paper-Based Analytical Devices: Concept, Variety, and Outlook. Anal. Chim. Acta 2021, 1144, 158–174. [CrossRef] [PubMed] 2. Nishat, S.; Jafry, A.T.; Martinez, A.W.; Awan, F.R. Paper-Based Microfluidics: Simplified Fabrication and Assay Methods. Sens. Actuators B Chem. 2021, 336, 129681. [CrossRef] 3. Heidt, B.; Siqueira, W.F.; Eersels, K.; Diliën, H.; Van Grinsven, B.; Fujiwara, R.T.; Cleij, T.J. 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