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Feb 9, 2016 - redesign, this microbead based assay was incorporated into two new prototypes for point-of-care testing: a well test and a syringe test...
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Development of Aptamer-Based Point-of-Care Diagnostic Devices for Malaria Using Three-Dimensional Printing Rapid Prototyping Roderick M. Dirkzwager,† Shaolin Liang,† and Julian A. Tanner* School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong S. A. R. China S Supporting Information *

ABSTRACT: We present the adaption of an aptamer-tethered enzyme capture (APTEC) assay into point-of-care device prototypes with potential for malaria diagnosis. The assay functions by capturing the malaria biomarker Plasmodium falciparum lactate dehydrogenase (Pf LDH) from samples and using its intrinsic enzymatic activity to generate a visualizable blue color in response to Plasmodium positive samples. Using three-dimensional (3D) printing rapid prototyping, a paperbased syringe test and magnetic bead-based well test were developed. Both were found to successfully detect recombinant Pf LDH at ng mL−1 concentrations using low sample volumes (20 μL) and could function using purified or spiked whole blood samples with facile sample preparation. The syringe test was found to be more analytically sensitive but required more additional preparation steps, while the well test required fewer steps and hence may be better suited for future clinical testing. Additionally, the development reagents required for the color response were fully stabilized through desiccation with sugar stabilization agents and could withstand temperatures as high as 90 °C. This study demonstrates how a previously reported biochemical assay can be adapted into workable point-of-care device prototypes by using 3D printing rapid prototyping. This novel technology, intended for rapid diagnostic tests (RDTs) for malaria, is distinct and carries many potential advantages relative to established lateral flow immunochromatographic approaches. KEYWORDS: aptamer, malaria, biosensor, point-of-care, 3D printing

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160 million in 2013.1 However, since such technologies rely on antibodies as biorecognition agents, they can be prone to thermal degradation. Despite the push for cool chain distribution and storage of malaria RDTs, studies have shown that RDT supply chain conditions remain a significant challenge with temperatures regularly exceeding 30 °C (the typical recommended maximum storage temperature) and humidity levels often exceeding 94%.5,6 Commercially available point-of-care diagnostics typically rely on lateral flow assay formats. However, new emerging technologies such as microfluidic-based diagnostics (mChip) and paper-based microfluidics (μPADs) have demonstrated potential as alternative low tech platforms for point-of-care diagnosis.7,8 Moreover, recent combinations of 3D printing approaches with fluidic technologies are beginning to influence point-of-care device development.9,10 Also known as additive manufacturing, 3D printing is a process of joining polymers layerwise to form a solid object from computer generated designs. 3D printing is being increasingly adopted in the fields of engineering, manufacturing and design.

he assimilation of three-dimensional (3D) printing technology into diagnostic development gives the overall design process greater manufacturing and prototyping power. Such approaches may prove vital to developing world medicine where there is still a great need for innovative and cost-effective diagnostic tests for diseases such as malaria. 3.3 billion people are at risk of contracting malaria and an estimated 584 000 people die annually due to the disease despite effective treatments being available.1 Diagnosis remains a challenging obstacle in the ongoing fight against malaria with only 64% of the estimated 207 million annual cases receiving a confirmatory diagnostic test before treatment.2 The standard method for malaria diagnosis uses light microscopy which is able to sensitively quantify and differentiate between species of malaria infection. However, microscopy methods are often unfeasible in the field where facilities, supplies, and expertise can be limited.3 Rapid diagnostic tests (RDTs) in the form of lateral flow immunochromatographic assays have greatly helped many of the issues incurred by classical microscopy-based diagnosis since they are portable, simple to interpret, and require limited expertise, making them ideal for field use. RDTs are critical in combating the overprescription of antimalarials and antimalarial resistance emergence. One study found RDT use reduced overprescription levels by 73%.4 As a result, the total number of RDTs purchased has risen from 200 000 in 2005 to more than © XXXX American Chemical Society

Received: October 23, 2015 Accepted: February 9, 2016

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Figure 1. A 96-well plate based assay for malaria diagnosis (APTEC) was adapted into a microbead based assay for enhanced functionality. The assay functions by using aptamer functionalized microbeads to capture Pf LDH from patient blood samples. After washing, a development reagent is added which, using the enzymatic activity of Pf LDH, produces a blue response for malaria positive samples. Using 3D printing to aid rapid prototyping and redesign, this microbead based assay was incorporated into two new prototypes for point-of-care testing: a well test and a syringe test.

for malaria diagnosis into point-of-care device prototypes by using 3D printing rapid prototyping. These prototypes were designed to be suitable for future clinical testing and address many of the issues incurred by current commercially available antibody-based RDT technologies.

The potential of 3D printing for producing complex 3D designs using a one step process has also been recognized by biomedical scientists for rapid prototyping. 3D printed biochips with applications in bacteria detection and virus analysis have been successfully developed.11−13 One key advantage of these biochips is their easy adaptation to other applications with only small modifications.14 Therefore, the integration of 3D printing technology into lab-based medical device development enables researchers to design multiple prototypes and cycle through the “design−test−redesign” process within shorter time scales. This allows for the development of medical devices tailored for each individual application.15 Aptamers are single-stranded oligonucleotide-based biorecognition agents which bind to their targets through specific 3D conformations that are typically developed through an in vitro technique called Systematic Evolution of Ligands by Exponential Evolution (SELEX).16 For biosensing applications, aptamers can offer advantages when compared to their monoclonal antibody counterparts in terms of thermostability, reproducibility and cost.17,18 Previously, we reported the crystal structure of an aptamer sequence (2008s) which tightly and specifically binds to the malaria biomarker Plasmodium falciparum LDH (Pf LDH).19 A subsequent colorimetric aptamer-tethered enzyme capture (APTEC) assay in a 96well format was also developed and found to work successfully on patient samples with the same sensitivity (80%) as a commercially available antibody-based RDT.20 The test requires no additional enzymes or antibodies in its function, making it suitable for point-of-care implementation if adapted to a suitable format. Therefore, this study sought to develop point-of-care malaria diagnostic tests which use the APTEC sensing system by using 3D printing technology as a platform for rapid prototyping (Figure 1). Two prototypes were developed and fully assessed. In this way, the study aimed to adapt an aptamer-based assay

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EXPERIMENTAL SECTION

Chemicals and Materials. 2008s-biotin DNA aptamer (5′BiotinCTG GGC GGT AGA ACC ATA GTG ACC CAG CCG TCT AC3′) was purchased from Integrated DNA Technology Inc. (Singapore). Streptavidin coated microbeads (10 μm diameter) were purchased from Spherotech. Magnetic Dynabeads MyOne Streptavidin T1 were purchased from Invitrogen. Bovine serum albumin was purchased from Affymetrix. 3-Acetylpyridine adenine dinucleotide was purchased from Apollo Scientific (U.K).. Pullulan from Aureobasidium pullulans was purchased from Polysciences. PlasCLEAR photopolymer was purchased from Asiga. All other chemicals were purchased from Sigma-Aldrich. Rat blood was kindly provided by the Department of Pharmacology at The University of Hong Kong. Recombinant Pf LDH was expressed in E. coli BL21 and purified by HisTrap chromatography (GE Healthcare) as reported previously.19 Device prototypes were created using a stereolithography-type 3D printer (Freeform Pico, Asiga). Designs were produced as .stl files using Inventor 3D CAD software and printed with PlasCLEAR photopolymer. Development reagent was freshly prepared prior to use. For a 6 mL solution, 80 μL of 50 mg mL−1 APAD solution, 300 μL of 10 mg mL−1 NTB solution (for ×5 final NTB concentration), and 12.5 μL of 5 mg mL−1 MePMS solution were added to 6 mL of L-lactate buffer (0.2 M sodium L-lactate, 100 mM Tris HCl, 0.2% Triton X-100, pH 9.1) and kept shielded from light. Syringe Test Protocol. Streptavidin coated microbeads (10 μm diameter, 2.5 mg) were incubated in 1 mL of 2.5 μM 2008s-biotin in PBS for 2 h at room temperature. Beads were spun down at 5000 rpm for 5 min and then washed with 3 × 2 mL PBST (0.1% Tween-20). MB-2008s were blocked with 5% BSA in PBS overnight at 4 °C and then finally resuspended in 500 μL of PBST and stored at 4 °C. 3MM chromatography paper (Whatman) was blocked in 5% BSA for 3 h at 37 °C and dried. Paper test circles 6 mm in diameter were cut using a hole punch and inserted into the syringe device casing. For B

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ACS Sensors the APTEC test, 20 μL of MB-2008s solution was added to each paper test zone and dried using syringe force. A volume of 20 μL of sample was applied to the paper test zone and incubated for 5 min. The paper test zone was then washed with 3 mL of PBST using a syringe attachment and then dried using syringe force. Then 5 μL of development reagent was added to the paper test zone and incubated for 45 min. Images were taken using a camera phone. Results could be qualitatively assessed by eye, or the image quantitatively analyzed. Relative intensity (AU) was calculated using ImageJ software and defined as I/I0 where I0 represents the intensity of blank paper. For whole blood lysis, Pf LDH spiked blood samples were diluted 1:1 in PBS (0.5% Triton X-100) lysis buffer and underwent sample preparation prior to testing. Using the spin down method, 40 μL of lysed blood sample was centrifuged at 5000 rpm for 5 s and 20 μL supernatant removed and tested using the syringe test protocol. Using the syringe filter method, 40 μL of lysed blood sample was added to a cotton wool packed syringe filter device and the sample expelled onto the paper test zone using syringe force. The protocol proceeded as before but only the wash step used 3 mL of PBST with 1% H2O2 and then 1 mL of PBST prior to color development. Well Test Protocol. The amount of 0.5 mg of streptavidin coated magnetic beads was incubated with 200 μL of 2.5 μM biotinylated 2008s in PBS for 1 h and then washed with 3 × 200 μL of PBST by magnetic separation before resuspension in 50 μL of PBS and storage at 4 °C. A volume of 1 μL of magnetic MB-2008s solution was deposited in W1 of the well test. Then 40 μL of sample (either 5% BSA PBS or 1:1 spiked whole blood to PBS 0.5% TX-100) was added and incubated for 5 min. Using the magnetic stick, beads were transferred from W1 and washed in wells W3−W5 consecutively (containing 300 μL PBST). Beads were deposited in W6 containing 50 μL of development reagent by removing the magnet stick from the sheath and incubated for 45 min. Solutions were removed, and absorbance at A570 nm recorded for quantitative analysis or could be qualitatively assessed by eye. Long-Term Stability Experiment. A volume of 5 μL of APAD (6.6 mg mL−1, 6% pullulan), NTB (0.4 mg mL−1, 6% pullulan), and MePMS (8 μg mL−1) was deposited as separate droplets onto the bottom of a microtiter well and then dried for 3 h using a silica bead chamber. Plates were stored at room temperature for 2 months and wells assayed each week. Wells were reconstituted with 50 μL of Llactate buffer and mixed thoroughly. Then 10 μL of PBS with or without 1 μg mL−1 Pf LDH was added and incubated for 45 min. The reaction was stopped with 50 μL of 5% acetic acid and absorbance recorded at 570 nm.

Figure 2. Steps taken during 3D rapid prototyping of (A) the syringe test and (B) well test to produce workable malaria diagnostic prototypes. (A) Redesign of the adaptor/membrane/filter cassette was required to ensure a tight seal during syringe operation. (B) An initial slide design for the well test was replaced by a sheathed magnet stick for improved washing strength.

which could attach to a luer lock syringe attachment (adapted from open source 3D printing designs) to allow for buffer to be pushed through the paper. The casing was designed to hold a 6 mm diameter paper test zone so that they could be cut using a hole punch to simplify assembly. This early spin column design incurred problems in maintaining a tight seal around the paper test zone. This was solved in the second generation prototype which enclosed the paper test zone with a tight seal (Figure 2A). This allowed for stringent paper washing without any leakage. A magnetic bead-based well test was chosen for the second design. This more closely mimicked the original 96-well plate assay system but used aptamer-functionalized magnetic beads instead. Using beads in this way is preferable to the 96-well plate format since a high surface area for antigen binding can be used and combined with dynamic mixing despite using small sample volumes. Beads could be moved by a magnet between wells which perform different functions: W1, incubation well; W2−5, wash wells; W6−development well (Figure 2B). Beads could be released into the development well and agitated for optimal color production by removing the magnet. Initial prototypes used a slide mechanism as a method to transfer the magnetic beads through lateral motion (Figure 2B). However, this design suffered from occasional instability in slide movement and did not allow for stringent washing to be implemented. Instead, a second generation design used a magnet stick design which allowed the beads to be manually moved between wells. By stirring the stick in the wash wells, high stringency washing was possible. The second generation design was also minimized to optimize portability. Grooves were also added to the development well (W6) so that reagents could be dried to the well bottom and reconstituted before use. Device Performance Using Spiked Serum. The two test designs were initially tested using spiked serum samples which mimic the protein density of whole blood and confirm selectivity with respect to Pf LDH sensing. Bovine serum

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RESULTS AND DISCUSSION Device Design and Fabrication. During the steps of the APTEC assay, aptamers first capture Pf LDH from samples, then all other biomolecules are washed away, and then finally development reagent is added and a blue color response is produced using Pf LDH activity coupled to the reduction of a tetrazolium dye (Figure 1). The original assay used a microtiter plate format.20 During device design, it was therefore important to ensure each step could be carried out effectively within the new format. First, a paper-based sensor was developed. Paper has some key advantages for use in low cost diagnostics, such as its price, homogeneity, disposability, and white color which is suited for colorimetric test interpretation. Paper was adapted into a syringe test design so that large volumes of wash buffer could be applied to the paper test zone without having to rely on the limited wash ability of wicking action. Paper test zones were functionalized with aptamers by embedding aptamer decorated microbeads (10 μm diameter) into the cellulose matrix as a one-step paper biofunctionalization method.21 The first generation design was based on a spin column (Figure 2a) C

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otherwise qualitative color response.23 The response showed a large dynamic range (4 orders of magnitude) and was capable of detecting Pf LDH concentrations as low as 5 ng mL−1 by visual analysis (Figure S2). This is encouraging sensitivity since the 96-well plate APTEC assay had a limit of detection (LOD) of 4.9 ng mL−1 for recombinant Pf LDH (where LOD was defined as 3σ/m of the response curve) and was found to work on patient samples with similar sensitivity to a commercial antibody-based RDT.20 Previous reports have also found typical Plasmodium LDH blood levels for infected patients to be around 3900 ng mL−1.24 This indicates that, despite adaption to a paper-based format and with lower sample volume, APTEC sensitivity was not reduced. Since all reagents can be applied by syringe, the syringe test also shows potential to be adapted to a fully closed test system for future clinical application. For the well test, similarly encouraging results were observed with a similar response for Pf LDH being observed as was seen for the syringe test (Figure 3B). Unlike the syringe test, blocking steps were not necessary since magnetic beads could be moved between wash buffers easily and washed stringently. Sample concentrations of 50 ng mL−1 Pf LDH could be distinguished by eye (Figure S2). The decrease in sensitivity between the syringe test and well test was attributed to the signal being more diffuse in solution in the well test (50 μL of development reagent), whereas the syringe test response was concentrated in a small test area (5 μL). Additionally, the well test may not be as easily adapted to a closed system when compared to the syringe test. Higher concentrations gave intense color responses which saturated at around 500 ng mL−1. For very high Pf LDH concentrations, dye began to precipitate out of solution after 45 min on account of the poor solubility of the dye product. Water-soluble tetrazolium salts could be incorporated in future to solve such issues.25 Device Performance Using Spiked Whole Blood. Biosensing within whole blood can often be challenging on account of its molecular complexity, viscosity and intense red color (from hemoglobin). Since malaria is diagnosed molecularly using lysed whole blood, it was therefore critical to confirm the two test devices could analyze Pf LDH spiked lysed whole blood samples with a similar efficacy as the platebased APTEC assay.20 The Triton X-100 lysis buffer used in the plate-based assay was also used here since it is suitable for incorporation into point-of-care devices. For the syringe test, lysed whole blood caused initial problems with large amounts of red particulates becoming stuck in the matrix of the paper even after stringent washing. To solve this, two blood sample preparation techniques were developed. The first method used a syringe filter (created using 3D printing) which incorporated a cotton wool filter into a luer lock attachment (Figure 4A). The second removed large particulate by brief centrifugation (5000 rpm, 5 s) prior to application onto the paper test zone. Centrifugation is a simple approach to blood separation and has been previously incorporated in automatic microfluidic devices for field use.26 As an additional method to remove background red color from the paper test zones, H2O2 was incorporated into the wash buffer to bleach the paper without affecting APTEC functionality (Figure S3). Previous reports have used H2O2 in blood sample preparation prior to RDT analysis in order to prevent the visual hindrance often caused by hemoglobin.27 Paper test zones were compared using all these methods (Figure 4B). Without any sample preparation steps, significant amounts of red particulate is observed even with H2O2 washing.

albumin (BSA, 5%) in PBS buffer was chosen for this purpose. Adapted from the 96-well plate protocol, sample incubation time was reduced from 1 h to 5 min to ensure the total test time of both tests was under 1 h. This incubation time was found to be sufficient for signal production. Several development reagent optimizations were undertaken. To enhance the color intensity at higher Pf LDH concentrations, the response limiting reagent nitrotetrazolium blue chloride (NTB) was increased to ×5 concentration from the original development reagent recipe (Figure S1, Supporting Information). Additionally, the cofactor NAD+ was substituted for 3-acetylpyridine adenine dinucleotide (APAD) which has been found to react specifically with Plasmodium LDH isoforms.22 Since background human LDH has the potential to cause false positive results using NAD+, use of APAD added specificity to the assay. For the syringe test, paper test circles (Whatman 3MM) were loaded with 2008s functionalized microbeads. Preblocking steps limited nonspecific adsorption of proteins to the cellulose matrix. The color responses for various concentrations of Pf LDH (in 5% BSA) were quantified from images taken using a mobile phone (Figure 3A). Telemedicine techniques show promise for enabling quantitative off-site analysis from an

Figure 3. Response of the APTEC point-of-care device prototypes using various concentrations of Pf LDH in serum samples (5% BSA). (A) The syringe test has a paper test zone which turns blue when positive. Response was quantified using image analysis of camera phone images. (B) The well test has a test well which turns blue when positive. Response was quantified using absorbance (A570 nm). D

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quickly expire when dried together (Figure S4).30 The last stabilization strategy used sugar stabilization agents to further stabilize APAD and NTB. Pullulan was chosen as a stabilization agent since it has been shown to be effective at protecting labile biomolecules from oxidation and thermal degradation.31 APAD, NTB, and MePMS were dried with or without pullulan and incubated for 16 h at 90 °C (Figure S5). APAD and NTB lost their activity (when assayed using Pf LDH) but maintained their activity when dried with pullulan. MePMS remained stable even without pullulan. These stabilization conditions were combined in a long-term stability experiment for 2 months at room temperature when APAD + pullulan, NTB + pullulan, and MePMS were dried separately in a microtiter plate well (Figure 5). No decrease in

Figure 4. Lysed whole blood sample preparation steps. (A) Design of the 3D printed syringe filter. (B) Syringe test zones after lysed whole blood samples are washed with/without H2O2 using different preparation methods. (C) APTEC syringe test response using Pf LDH spiked lysed whole blood with sample preparation. (D) APTEC well test response using Pf LDH spiked lysed whole blood and no sample preparation.

The syringe filter improved results but required an additional H2O2 wash to fully remove the red color. The spin down method worked the best with minimal red color remaining either with or without the H2O2 wash buffer. It was therefore decided to use the spin down method combined with the H2O2 wash as the blood sample preparation step for the syringe test. Using this method, the subsequent response for whole blood spiked with or without Pf LDH (1 μg mL−1) (Figure 4C). Nonetheless, the syringe filter + H2O2 wash could still remain a viable option for sample preparation if no means for centrifugation is available. For the well test, no additional issues when using whole blood were encountered. Since the magnetic beads are removed from the blood sample and transferred between different buffer wells, high stringency washing could be achieved. After washes in W2−W5, no remaining red color was observed and so APTEC color development proceeded unimpeded (Figure 4D). Thanks to the use of magnetic beads, the APTEC wash step could be completed with more efficiency than the 96-well plate format (where biorecognition agents remain fixed in one well). In this way, the well test performed favorably when using whole blood samples and with respect to producing a qualitative point-of-care device, outperformed the syringe test. Reagent Stabilization. Using aptamers as biorecognition agents in RDTs confers advantages in terms of test thermostability compared to using antibodies.28 In this way, all components of the test system must be equally stable if this advantage is to be exploited. This therefore required the stabilization of APTEC development reagent components. First, the light and temperature sensitive electron transporter reagent phenazine ethosulfate (PES) was replaced with a stable alternative methoxy methylphenazinium methyl sulfate (MePMS).29 Second, reagents were desiccated separately for storage and reconstituted with L-lactate buffer prior to use. Spatial separation of APAD, NTB, and MePMS was found to be key to maintaining reagent integrity since the reagents

Figure 5. APTEC development reagent stability over 2 months when development reagent chemicals are dried separately in a microtiter well. Each week reagent wells were reconstituted with L-lactate buffer and incubated with or without Pf LDH.

response or false positive results were observed. This is a significant improvement on the initial solution phase stability experiments and a report by Kannan et al. which used paper sensors printed with pullulan stabilized LDH development reagents.20,32 Both these reports observed reagent expiration after only 1 week storage at room temperature. The results here are a strong indication that the point-of-care APTEC tests could survive for a long time even at elevated temperatures. These reagent stabilization steps were incorporated into device kits for the syringe test and well test which contain all the constituents required for use in the field (Figure S6). Cost Analysis. As a final investigation into the economic viability of using APTEC devices for point-of-care malaria diagnosis, the cost per APTEC test was calculated and compared to the average antibody-based RDT price found in malaria endemic countries in the private sector which has been found to vary greatly (Table 1).33 Even though this is a comparison between production cost and commercial price, APTEC technology shows strong potential to be less expensive than immunochromatographic approaches since the aptamers and chemicals per test only cost a few United States cents. The major contributor to APTEC cost (between 67% and 93%) was found to be for the streptavidin support for aptamer immobilization. This shows potential for cost to be decreased E

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Table 1. APTEC Cost Analysis

component aptamers + development reagent streptavidin-well/ MBs paper/plastics total

APTEC syringe test

APTEC well test

0.03

0.02

0.07

0.39

1.64

0.24

0.42

0.10 1.76

0.05 0.36

ASSOCIATED CONTENT

S Supporting Information *

price (USD)a APTEC 96-well assay

Article

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.5b00175. Development reagent concentration optimization; minimum observable response concentrations; effect of H2O2 on APTEC assay response; development reagent longterm stability; APAD, NTB, and MePMS thermotolerance; APTEC test kit contents (PDF)

antibody-based RDT

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1−1733

a

APTEC costs were calculated according to typical supplier pricings and RDT price estimated from average commercial prices.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +852 3917 9472. Author Contributions †

R.M.D. and S.L. contributed equally.

in future as alternative, more cost-effective DNA immobilization techniques could be employed such as rolling circle amplification (RCA) for paper immobilization.34,35 Nonetheless, since the underlying biorecognition agents and signal amplification technique are so cost-effective even at this research stage, APTEC malaria tests have the potential to be produced at low cost in the future.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding for this research was provided by the Hong Kong University Grants Council under General Research Fund Grant HKU778813M. We acknowledge the HKU emerging Strategic Research Theme in Integrative Biology.

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SUMMARY AND CONCLUSIONS In conclusion, point-of-care device prototypes were developed for malaria diagnosis using an aptamer-based colorimetric assay for Pf LDH detection (APTEC assay). The devices were fabricated and optimized using 3D printing which allowed for rapid prototyping during the process. A paper-based syringe test and a magnetic bead-based well test were developed. Both tests were found to function with a comparable sensitivity for recombinant Pf LDH as our previously reported 96-well plate APTEC assay but they use a smaller sample volume (20 μL) and do not require special equipment or expertise to operate. In this way, the study aims were fulfilled. The syringe test showed a large dynamic range and better analytical sensitivity; however, it required additional sample preparation steps prior to testing whole blood which added complication to the protocol. On the contrary, the well test had no issues with testing whole blood but showed a smaller dynamic range. Therefore, as an overall qualitative and simple to use point-of-care diagnostic test, the well test outperformed the syringe test at this early development stage, especially since dried reagents can be stored within the well and foil sealed (Figure S6). In contrast to previous reports of aptamer-based Pf LDH sensing, here we present simple colorimetric point-of-care devices which can be interpreted by visual analysis and do not require any further equipment.36−38 Cost analysis showed the price for the aptamers and chemicals required per test was a few United States cents and the overall test cost could be reduced further by using more cost-effective aptamer immobilization techniques. In addition, the development reagents for the APTEC assay were fully stabilized using desiccation and pullulan stabilization with no change in response after storage for 2 months at room temperature. Such strategies may prove useful in other tests which use tetrazolium dyes such as human LDH detection and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency diagnosis.32,39 Future work will seek to test these devices in the field and further confirm their usability, sensitivity, and long-term stability in a point-of-care setting.

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ABBREVIATIONS APAD, 3-acetylpyridine adenine dinucleotide; APTEC, aptamer-tethered enzyme capture; BSA, bovine serum albumin; G-6-PD, glucose-6-phosphate dehydrogenase; LOD, limit of detection; MePMS, methoxy methylphenazinium methyl sulfate; NAD+, nicotinamide adenine dinucleotide; NTB, nitrotetrazolium blue chloride; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PES, phenazine ethosulfate; Pf LDH, Plasmodium falciparum lactate dehydrogenase; RCA, rolling circle amplification; RDT, rapid diagnostic test; SELEX, systematic evolution of ligands by exponential evolution

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REFERENCES

(1) WHO World Malaria Report; World Health Organization: Geneva, Switzerland, 2014. (2) WHO Malaria Rapid Diagnostic Test Performance − Results of the WHO Product Testing of Malaria RDTs: Round 4; World Health Organization: Geneva, Switzerland, 2012. (3) Coleman, R. E.; Maneechai, N.; Rachaphaew, N.; Kumpitak, C.; Miller, R. S.; Soyseng, V.; Thimasarn, K.; Sattabongkot, J. Comparison of Field and Expert Laboratory Microscopy for Active Surveillance for Asymptomatic Plasmodium falciparum and Plasmodium vivax in Western Thailand. Am. J. Trop. Med. Hyg. 2002, 67 (2), 141−144. (4) Mbonye, A. K.; Magnussen, P.; Lal, S.; Hansen, K. S.; Cundill, B.; Chandler, C.; Clarke, S. E. A Cluster Randomised Trial Introducing Rapid Diagnostic Tests into Registered Drug Shops in Uganda: Impact on Appropriate Treatment of Malaria. PLoS One 2015, 10 (7), e0129545. (5) Albertini, A.; Lee, E.; Coulibaly, S. O.; Sleshi, M.; Faye, B.; Mationg, M. L.; Ouedraogo, K.; Tsadik, A.; Feleke, S. M.; Diallo, I.; Gaye, O.; Luchavez, J.; Bennett, J.; Bell, D. Malaria Rapid Diagnostic Test Transport and Storage Conditions in Burkina Faso, Senegal, Ethiopia and the Philippines. Malar. J. 2012, 11 (1), 406. (6) Jorgensen, P.; Chanthap, L.; Rebueno, A.; Tsuyuoka, R.; Bell, D. Malaria Rapid Diagnostic Tests in Tropical Climates: the Need for a Cool Chain. Am. J. Trop. Med. Hyg. 2006, 74 (5), 750−754. (7) Chin, C. D.; Cheung, Y. K.; Laksanasopin, T.; Modena, M. M.; Chin, S. Y.; Sridhara, A. A.; Steinmiller, D.; Linder, V.; Mushingantahe,

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DOI: 10.1021/acssensors.5b00175 ACS Sens. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acssensors.5b00175 ACS Sens. XXXX, XXX, XXX−XXX