Multiplexed, Patterned-Paper Immunoassay for Detection of Malaria

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A multiplexed, patterned-paper immunoassay for detection of malaria and dengue fever Rachel N. Deraney, Charles R. Mace, Jason P. Rolland, and Jeremy E. Schonhorn Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00854 • Publication Date (Web): 17 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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Analytical Chemistry

A multiplexed, patterned-paper immunoassay for detection of malaria and dengue fever Rachel N. Deraney,1 Charles R. Mace,2,* Jason P. Rolland,1 and Jeremy E. Schonhorn1

1

Diagnostics For All, 840 Memorial Drive, Cambridge, MA 02139 United States, 2Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, MA 02155 United States

*corresponding author: [email protected]

1 ACS Paragon Plus Environment


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Abstract Multiplex assays detect the presence of more than one analyte in a sample. For diagnostic applications, multiplexed tests save healthcare providers time and resources by performing many assays in parallel, minimizing the amount of sample needed, and improving the quality of information acquired regarding the health status of a patient. These advantages are of particular importance for those diseases that present with general, overlapping symptoms, which makes presumptive treatments inaccurate and may put the patient at risk. For example, malaria and dengue fever are febrile illnesses transmitted through mosquito bites, and these common features make it difficult to obtain an accurate diagnosis by symptoms alone. In this manuscript, we describe the development of a multiplexed, patterned paper immunoassay for the detection of biomarkers of malaria and dengue fever: malaria HRP2, malaria pLDH, and dengue NS1 type 2. In areas co-endemic for malaria and dengue fever, this assay could be used as a rapid, point-ofcare diagnostic to determine the cause of a fever of unknown origin. The reagents required for each paper-based immunoassay are separated spatially within a three-dimensional device architecture, which allows the experimental conditions to be adjusted independently for each assay. We demonstrate the analytical performances of paper-based assays for each biomarker and we show that there is no significant difference in performance between the multiplexed immunoassay and those immunoassays performed in singleplex. Additionally, we spiked individual analytes into lysed human blood to demonstrate specificity in a clinically relevant sample matrix. Our results suggest multiplex paper-based devices can be an essential component of diagnostic assays used at the point-of-care.


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Introduction Vector-borne diseases, or diseases where a pathogen is transmitted to a human through an intermediate host (e.g., mosquito, tick or flea), account for 17% of the estimated burden of infectious disease.1 Malaria, a disease caused by parasites of the Plasmodium species, is the most deadly vector-borne disease1 and was responsible for an estimated 207 million infections and 627,000 deaths in 2012 alone.2 Dengue fever, caused by the dengue virus, is the world's fastest growing vector-borne disease, with a 30-fold increase in incidence rates in the past 50 years.1 In order to reduce the burden of these diseases, accurate diagnoses are required to determine the course of treatment. Unfortunately, in resource-limited settings, diagnoses are often based on physiological symptoms alone (i.e., a presumptive diagnosis) due to the lack of affordable and reliable analytical assays.3 This approach fails to accurately diagnose diseases that present common symptoms. For example, the symptoms of malaria and dengue fever—fever, headache, and chills—are non-specific.1 These diseases are co-endemic in American and Asian intertropical regions4 and, more recently, Western Africa,5 which further complicates the use of only presumptive means to diagnose the cause of the symptoms. Furthermore, the treatments for these diseases are unique. The therapeutic options recommended by the WHO to treat infections by malaria are specific for the species of Plasmodium parasite: Uncomplicated P. falciparum infections are treated with artemisinine-based combination therapies. Infections by P. vivax, P. ovale, and P. malariae are treated with chloroquine, unless the diagnosis is made in Papua New Guinea or Indonesia where chloroquine-resistant parasites are found.2,6 In stark contrast, the current recommended treatment for non-severe dengue fever is bed rest and liquid consumption.7 An accurate test that is able to distinguish between each disease would allow for the selection of



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the correct treatment option, enable faster patient recovery, ensure the proper distribution of limited medications and limit the spread of drug-resistant pathogens. Immunochromatographic lateral flow tests are used commonly in resource-limited settings because of their portability, low cost, and easy-to-interpret, visual read-out.8,9,10 Lateral flow tests for malaria and dengue fever are commercially available,11,12 but a multiplexed test that can distinguish between the two febrile illnesses does not exist. In fact, there are relatively few commercial immunochromatographic assays that test for multiple markers simultaneously from a single, small volume sample,13,14 and some may require the use of an electronic reader to assist with the interpretation of the results of an assay.15,16 Ideally, multiple assays could be performed in parallel using a single device. Although increases in sample and wash volumes from singleplex assays are required, multiplex assays eliminate the compromises (e.g., pH and conjugate load) made when detecting multiple assays in serial using a common fluidic pathway. Three-dimensional, paper-based microfluidic devices—combining the advantages of lateral flow with vertical flow of fluids between multiple layers—have the potential to enable these parallel, multiplexed assays.17,18 We developed a three-dimensional microfluidic device prepared from patterned paper (Figure 1) that is capable of performing multiplexed assays. We demonstrate the use of this device by performing three immunochromatographic assays simultaneously: malaria histidine-rich protein 2 (HRP2),19 malaria Plasmodium lactate dehydrogenase (pLDH)20 and dengue non-structural protein 1 (NS1) type 2.21 The analytical performance of a multiplexed assay for each of these three markers is comparable to those assays conducted individually (i.e., in singleplex). In addition, the multiplexed assay can detect each marker specifically when using lysed whole blood as a sample matrix. These results suggest that multiplexed assays performed on pattered paper can be a tool to differentiate between the two


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most common vector borne diseases without sacrificing the simplicity or convenience of current point-of-care tests.

Experimental Design Paper-based device architecture We have previously reported the development of a three-dimensional patterned paper device for use in singleplex immunoassays.22 For the experiments described in this manuscript, we modified the design of our earlier device architecture to include a layer for sample distribution that is not required for a singleplex immunoassay but is necessary for multiplexed assays (Figure S1). Using this approach, we could directly compare the performances of singleand multiplexed immunoassays because they shared a common microfluidic network. The multiplex devices discussed here can detect three different biomarkers for malaria and dengue fever; however, the flexibility of the paper-based microfluidic platform enables designs to be altered easily to include any desired number of detection zones.23 Additional details related to the design and fabrication of singleplex and multiplexed devices can be found in the Supporting Information. We considered a number of channel geometries for the development of the multiplex platform, which included branched, split, and parallel tracks. Our initial design criteria for these configurations included preservation of symmetry and control of the overall footprint of the device. Since capillary action (i.e., wicking) transports fluids in paper-based microfluidic devices, it is advantageous for the multiple fluidic networks patterned within the device to have equal pathlengths. This approach assures: (i) a uniform distribution of sample volume between channels, (ii) consistent rehydration of stored reagents and mixing with an applied sample, and



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(iii) equal timing for all assays. These characteristics influence the reproducibility and performance of paper-based immunoassays, and, critically, ensure that the performance of an assay is not related to its spatial position within a multiplexed device. Ultimately, for the multiplexed immunoassays described in this manuscript, we chose a device design where assays are conducted in parallel channels that are fed by a large, common sample application zone (Figure 1). Future efforts will be dedicated to investigating and comparing the performance of alternative channel geometries for multiplexed immunoassays. Selection of sample matrices All three antigens—HRP2, pLDH, and NS1—are present in the plasma of infected patients, but hemolysate (i.e., whole blood with completely lysed cells) is needed to improve the sensitivity of an assay by releasing the intracellular malaria antigens found in parasitized erythrocytes,24,25 which ensures a more accurate diagnosis. All preliminary experiments with singleplex and multiplex paper-based immunoassays used purified antigens in buffer as the sample (Supporting Information). Once the device design and experimental conditions were finalized, we transitioned to antigens spiked in hemolysate to model the realistic and complex sample matrix required for analysis at the point-of-care.

Results and Discussion Comparison of singleplex and multiplex immunoassays We began by systematically varying the concentrations of both the capture and conjugate antibodies to determine treatment conditions for best performance of each immunoassay in the singleplex platform (Supporting Information). Once these conditions were determined, we tested the limit of blank (LOB) for our assays. The LOB sample panels comprised 11 concentrations of


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analyte that were prepared by serial dilution spanning the range from 4096 ng/mL to 4 ng/mL in sample buffer (Figure S2). Each dilution was tested twice in quadruplicate along with a companion buffer blank (i.e., a negative). Therefore, eight replicate measurements were made for each concentration of analyte. Negative samples were assessed using 22 replicate measurements. We fit the resulting data to logistic curves and determined the LOB for each singleplex assay: 20.3 ng/mL (malaria HRP2), 80.2 ng/mL (malaria pLDH), and 79.4 ng/mL (dengue NS1). After completion of singleplex experiments for each antigen, we performed identical experiments using the multiplex paper-based device architecture. We performed these assays using five serial dilutions of antigen from 31.25 to 2000 ng/mL, which were each assessed using eight replicates. We tested samples of buffer only (i.e., a negative sample) using a total of 16 replicates (Figure S3). An additional sample at a concentration of 7.8 ng/mL was evaluated for the malaria HRP2 assay in an attempt to observe a low positive result. For these multiplexed experiments, we introduced samples containing only a single antigen (i.e., only HRP2 spiked into buffer), which allowed us to compare the performance of the fully integrated device against those of the singleplex immunoassays and determine if any cross-reactivity between reagents or other sample interferences existed in this format. The LOB for single antigens detected in a multiplexed format were: 46.1 ng/mL (malaria HRP2), 100.7 ng/mL (malaria pLDH), and 57.9 ng/mL (dengue NS1). We thus observed that singleplex and multiplex immunoassays have comparable sensor response profiles (Figure 2) and LOB, which indicate that test accuracy was not compromised with the inclusion of additional diagnostic markers or a radical change to the architecture of the paper-based microfluidic device. In addition to determining LOB, the analysis of results from multiplexed immunoassays provides critical information regarding the specificities of the assays and overall performance of the device (Figure S4). We observed that



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non-specific signals (i.e., a false positive) for omitted antigens only appear at very high concentrations of antigen (2000 ng/mL). Limit of blank analyses The clinically relevant range of parasitemia in cases of malaria is expected to be substantial. While many patients begin to appear symptomatic (i.e., febrile) at parasite densities of several hundred to several thousand/µL,26,27 hyperparasitemia in severe cases of nonfalciparum malaria can reach an overall burden of 2% (i.e., 100000 parasites/µL) and up to 20% or higher in severe falciparum malaria.28 Using the technical results presented in a WHO report on malaria diagnoses, we were able to convert the antigen concentration from our LOB studies to an estimated number of parasites detectable by our devices. For a sample obtained from a patient that is infected with P. falciparum at a total of 200 parasites/µL, the mean antigen concentration for both HRP2 and pLDH was reported to be 15.3 ng/mL.29 At an identical parasitic burden in malaria instead caused by P. vivax, the concentration of HRP2 is not expected to be significantly different (17.4 ng/mL).29 We can thus estimate the limits of detection for our multiplexed immunoassay to be 600 parasites/µL using HRP2 and 1300 parasites/µL using pLDH. The standard clinical method for diagnosis of malaria and quantification of parasitemia is the blood smear.30 The limit of detection for this approach is approximately 10–50 parasites/µL when using thick blood smears, but is considerably higher when using thin blood smears.31 Immunochromatographic lateral flow tests have the potential to improve the availability of healthcare options at the point-of-care. For example, the BinaxNOW malaria test can be used for a differential diagnosis of infections by Plasmodium species.32 Here, two assays are performed within a shared fluidic pathway and are useful for the detection of moderate parasitic burden in both falciparum-malaria (1000–1500 parasites/µL) and non-falciparum malaria (5000–5500


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parasites/µL for P. vivax), but the well-documented concerns over a lack of reliable analytical performance of current immunochromatographic devices make them unlikely candidates to replace blood smears.26,33 The performance of our paper-based multiplexed immunoassay—with LODs between those of blood smears and immunochromatographic tests—suggests it may find use for determination of febrile illness at parasitic burdens that are consistent with the onset of symptoms. Future efforts must be directed at improving the sensitivity and specificity of the multiplexed immunoassay in order to compete with standard blood smears and address the desirable diagnosis of high parasitemias (> 25000 parasites/µL). Previous studies have determined the median level of circulating NS1 in serum for patients infected with dengue virus (major serotype DENV-1) is 126 ng/mL,34 but may be as low as 4 ng/mL.35 Our experimental result of 57.9 ng/mL for NS1 falls within this clinically relevant range of concentrations and could thus be used for the effective determination of infection by the dengue virus. Differentiation in relevant sample matrices In addition to characterizing the function of the multiplexed immunoassay using samples of antigens in aqueous buffer, we wanted to demonstrate its performance using a clinically relevant sample matrix—lysed human blood. The procedure used to perform a multiplexed immunoassay is shown in Figure S5. For these experiments, we assessed all possible combinations of the three antigens: all positive, all negative, and combinations of pLDH-, HRP2-, and NS1-positive (Figure 3). Human blood was first lysed in a lysis buffer (Supporting Information) and then spiked with 500 ng/mL of a desired antigen or combination of antigens, which corresponds to approximately 6500 parasites/µL when considering samples that are positive for malaria. Although an additional step is required to mix the blood sample with the



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lysis buffer, this allows for the controlled formulation and application of the sample to the paperbased device. Our test produces clean backgrounds for negative samples of hemolysate (Figure 3A). Additional samples that contained at least one antigen (i.e., positive for either one, two or three antigens) had equally as clean backgrounds for negative immunoassays while producing a strong positive colorimetric signal for positive immunoassays (Figure 3B–H). Assessing all possible outcomes for a specific sample of blood allows us to demonstrate the potential of the multiplexed immunoassay as a diagnostic tool for a fever of unknown origin. Further, these promising results indicate that the paper-based microfluidic device is compatible with hemolysate, which is the clinically relevant sample for these biomarkers.

Conclusions When comparing the singleplex test to the multiplex test, there was an improvement in LOB for the NS1 antigen (79.4 ng/mL vs. 57.9 ng/mL) and a slight increase in LOB for the HRP2 and pLDH antigens (20.3 ng/mL vs. 46.1 ng/mL and 80.2 ng/mL vs. 100.7 ng/mL, respectively). Additionally, we have demonstrated that the multiplexed device architecture— through the spatial separation of assay-specific reagents—introduces no cross-reactivity when assays are performed in buffer or hemolysate. These results validate a long-stated advantage of the paper-based platform: by spatially separating reagents within a device, assays performed in singleplex and multiplex are functionally interchangeable. There are additional, tangible benefits to performing assays in multiplex rather than through multiple, singleplex devices: (i) The configuration of assays performed within a multiplexed device can be altered radically by including or excluding individual tests depending on the desired application. This flexibility


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allows a single sample to be used to conduct multiple tests, as opposed to requiring a set of samples to perform several singleplex tests. Further, the resulting assay conditions do not need to be re-optimized because regents are stored in separate, parallel fluidic networks. (ii) Multiplexed assays can greatly assist with the management of testing campaigns at the point-of-care by minimizing the waste associated with performing many individual assays simultaneously and simplifying the workflow in a clinic where many patients may be tested for many diseases. Our multiplexed paper-based immunoassays show clean backgrounds when exposed to negative samples of hemolysate, which is important to minimize the potential for misinterpretation of results (i.e., false positives) that could result from either non-specific binding of assay reagents or the accumulation of colored blood components (e.g., hemoglobin or erythrocyte fragments). Currently, however, these assays require the lysis of whole blood in a separate, preparative step. In order to improve the efficacy of this diagnostic assay in point-ofcare applications, future versions of this multiplexed device will likely require the integration of lysis capabilities (e.g., by treating a layer of paper with a lytic agent). We have demonstrated proof-of-concept for a three dimensional paper based test that can detect antigens for malaria (HRP2 and pLDH) and dengue fever (NS1) sensitively as well as distinguish between them accurately. This multiplexed test has the potential to aid health workers in the accurate diagnosis and differentiation of malaria and dengue fever, two febrile diseases that are characterized with common symptoms, are geographically co-endemic, and require drastically different treatments.

Acknowledgements This work was supported by Defense Advanced Research Projects Agency (DARPA) Cooperative Agreement HR001-12-2-0010 and in part by Tufts University.



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Supporting Information Details of materials used, experimental methods, and results of preliminary assays; Figures S1– S5; Table S1.


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Figure 1. Schematic of three-dimensional, paper-based devices for multiplexed immunoassays. Hydrophobic, wax barriers (black) are used to define hydrophilic channels and zones within each layer. Double-sided adhesive films (omitted from schematic) are used to affix layers to each other. Labels describe the function of each layer in the multiplexed assay.



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Figure 2. Results for paper-based immunoassays for (A) HRP2, (B) pLDH, and (C) NS1. Assays for each antigen were performed using singleplex and multiplex device architectures. Data points for each concentration represent the average of eight replicates and responses were fit to a Hill binding curve. Error bars have been omitted to assist with the comparison of the results of each approach.

A

B

C


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Figure 3. Results of multiplexed, paper-based immunoassays performed using hemolysate. HRP2, pLDH, and NS1 antigens were spiked into samples of lysed human blood in all possible combinations at final concentrations of 500 ng/mL. Histograms represent the results of the mean of three replicates while error bars represent the standard error of the mean for each condition.



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Table of Contents Graphic


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Multiplexed, Patterned-Paper Immunoassay for Detection of Malaria

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