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Detection of Plasmodium lactate dehydrogenase (pLDH) antigen in buffer using aptamer-modified magnetic microparticles for capture, oligonucleotide-modified quantum dots for detection, and oligonucleotide-modified gold nanoparticles for signal amplification Chloe Kim, and Peter C Searson Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00328 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017

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Bioconjugate Chemistry

Detection of Plasmodium lactate dehydrogenase (pLDH) antigen in buffer using aptamermodified magnetic microparticles for capture, oligonucleotide-modified quantum dots for detection, and oligonucleotide-modified gold nanoparticles for signal amplification.

Chloe Kim1,2 and Peter C. Searson1,2* 1

Department of Materials Science and Engineering, Johns Hopkins University, 3400 North

Charles Street, Baltimore, MD 21218 2

Institute for Nanobiotechnology Johns Hopkins University, 3400 North Charles Street,

Baltimore, MD 21218

Abstract To overcome the limitations associated with antibody-based sensors, we describe a proof-ofconcept of an aptamer-based sandwich assay for detection of lactate dehydrogenase, an antigen associated with malaria. We show a detection limit of Plasmodium falciparum lactate dehydrogenase and Plasmodium vivax lactate dehydrogenase of 0.5 fmole in buffer, comparable to an antibody-based assay, using a magnetic particle - aptamer construct for capture, and a quantum dot - aptamer construct for detection. We then demonstrate a detection limit of 10 amole (50-fold amplification) using oligonucleotide-functionalized gold nanoparticles to allow conjugation of multiple quantum dots for each target antigen.

Keywords: lactate dehydrogenase, malaria, aptamer, oligonucleotide, nanoparticle, quantum dots, signal amplification, sandwich assay

*[email protected]

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Malaria is an infection of red blood cells caused by parasites that puts nearly half of the world’s population at risk.1, 2 Malaria occurs in over 100 countries but is mainly confined to poorer, tropical areas with limited resources.1, 2 Although the rapid diagnostic test (RDT) is the state-ofthe-art method for rapid diagnosis of malaria in low-resource settings, there remain issues with cost, sensitivity, and stability in tropical climates that are associated with the use of antibodies for recognition of the parasite antigen.3-6 Recent studies show that commercial RDTs are not reliable for measurement of low-density parasites (< 200 parasites µL-1) and low antigen concentrations (< 1 ng mL-1), which account for many false-negative results.5, 7-9 Molecular analysis tools, such as PCR, are more sensitive and reliable, since they do not involve the use of antibodies, but they are complex, expensive, labor intensive, and cannot be implemented in lowresource settings.3 Therefore, there is a need for a sensitive, accurate, and more affordable diagnostic test for the developing world.

Here we address two issues associated with assays for malaria: the need for more stable alternatives to antibodies for detection, and the need for detection of low antigen concentrations (< 1 ng mL-1). We have developed a nanoparticle-based assay for detection of malaria antigens using aptamer - modified magnetic microparticles (MMPs) for capture and oligonucleotide modified quantum dots (QD) for detection (Fig. 1A). To increase the sensitivity, we have developed a strategy for signal amplification using oligonucleotide - modified gold nanoparticles (AuNPs) to increase the number of QDs for each target antigen (Fig. 1A). Magnetic particles functionalized with targeting moieties enable efficient collection and separation of target molecules in a simple and rapid process without any filtration or centrifugation. Quantum dots enable quantitative optical detection with higher photostability than fluorescence or colorimetric detection. In this study, we use an aptamer as a capture and detection moiety as an alternative to antibodies, and oligonucleotide hybridization for QD conjugation.

Aptamers are single-stranded oligonucleotide sequences that can bind with high specificity to various molecular targets with high affinity (KD = 1 - 100 nM).10-12 A unique aptamer is generally selected from a library of oligonucleotides over multiple rounds of in vitro selection, and a number of aptamer sequences for detection of malaria antigens have been reported in the literature.13-16 Aptamers are attractive alternatives to antibodies in diagnostic tests because they


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are thermally stable, cheaper to synthesize, and can be tailored for specificity and sensitivity.17-19 In addition, aptamers can be easily modified with additional oligonucleotides to give additional functionality, for example, to ensure that the binding site can be isolated from the conjugation site (Fig. 1D). In contrast, bioconjugation of antibodies is often achieved at surface residues (e.g. at amines on lysine residues) that can interfere with the binding site.

Figure 1. Particle-based aptasensor. (A) Aptamer-based sandwich assay for detection of malaria antigens. (B) Aptamer-functionalized magnetic microparticle (MMP). (C) Oligonucleotidefunctionalized gold nanoparticle. (D) Aptamer functionalized with hybrid oligonucleotide for conjugation. (E) Oligonucleotide-functionalized quantum dot. (F) The assay involves: (1) capture of the target antigen by the aptamer-functionalized MMPs, (2) binding of oligonucleotide-functionalized gold nanoparticles to the MMP-aptamer-antigen complex, and (3) binding of oligonucleotide-functionalized quantum dots to the remaining hybridization sites on the gold nanoparticles for readout.

In this study, we target the Plasmodium lactate dehydrogenase (pLDH) antigen, which is one of the most widely studied protein biomarkers for malaria infection.3, 20, 21 Since the LDHs of different malaria species exhibit 90% identity, pLDH has potential for detection of all types of malaria parasite.2, 4, 20 pLDH is a tetrameric protein composed of four subunits, which form two Q-axis dimers. From X-ray crystallography, an aptamer with high affinity (KD = 42 nM) and specificity to Plasmodium falciparum lactate dehydrogenase (PfLDH) was shown to bind to both



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dimers of pLDH, exhibiting 1:2 pLDH:aptamer molar stoichiometry.14, 22 These features enable the use of a single aptamer to replace an antibody pair in a sandwich assay (Fig. 1A).

In previous work, we have developed a magnetic bead-quantum dot (MB-QD) immunoassay for detection of target antigens.23 The assay had a lower limit of detection of approximately 1 pM of target antigen and a 3 order of magnitude detection range. This detection limit corresponds to about 0.1 to 1 fmole of quantum dots, depending on the emission peak, quantum yield, and resolution of detector. To increase the sensitivity of the assay, signal amplification is critical. Recently reported QD signal amplification methods involve coupling of QDs on to different particles or encapsulation of multiple QDs.24, 25 However, these methods require additional synthesis and purification steps, and accurate measurement of the concentration remains challenging since it is difficult to control the number of QDs per antigen.

Our approach for signal amplification involves loading multiple QDs onto a gold nanoparticle (AuNP) without chemical crosslinking. The AuNPs function as carriers of the detection aptamer and multiple QDs (Fig. 1A). For this purpose, a unique pair of complementary oligonucleotides was designed: a conjugation-oligonucleotide (cOligo) (Fig. 1C) and a hybridizationoligonucleotide (hOligo) (Fig. 1D). Here our goal is to demonstrate the feasibility of the technology, and hence all experiments are performed in buffer spiked with known concentrations of PfLDH or PvLDH.

This proof-of-concept study is organized into three parts: (1) confirmation of pLDH capture using a protein capture assay, (2) demonstration of QD-aptamer detection following MMPaptamer capture, and (3) demonstration of signal amplification using oligonucleotidefunctionalized gold nanoparticles. We show: (1) efficient antigen capture with aptamers conjugated to oligonucleotide-functionalized quantum dots, (2) a limit of detection of 0.5 fmole pLDH in a sandwich assay with QD-aptamer detection, and (3) a limit of detection of 10 amole pLDH (50-fold amplification) using gold nanoparticles to conjugate multiple QDs per antigen. The detection limit (10 amole in 100 µL) in buffer corresponds to a concentration of 1 pg mL-1. To confirm the functionality of the selected aptamer and the efficiency of oligonucleotide hybridization, we performed a capture assay using micro-well plates (Fig. 2). First, we


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immobilized different concentrations of Plasmodium falciparum lactate dehydrogenase (PfLDH) and Plasmodium vivax lactate dehydrogenase (PvLDH) on a 96-well plate. Then, lipid coated QDs 23, 26, 27 were conjugated to the thiolated cOligo using a maleimide group. An average of 9 (± 3.4) cOligos were loaded onto a single QD. To determine the optimum number of aptamers per QD, the oligo-modified aptamers were hybridized to the cOligo-QD construct at QD:aptamer molar ratios of 1:1, 1:3, 1:6 and 1:9 (Fig. 2A). Next, the QD-cOligo-aptamer construct was incubated in pLDH-immobilized wells for 1 hour at room temperature. After removing unbound QDs and washing the wells three times with buffer, the fluorescence was measured using a plate reader (Fig. 2B).

Figure 2. Protein capture assay. (A) Schematic illustration of a pLDH-coated plate and detection by the aptamer-cOligo-QD construct. QDs are functionalized with the conjugationoligonucleotides (cOligo) and hybridized with different molar ratios of oligonucleotide-modified aptamers for detection. (B) Fluorescence detection of PfLDH and PvLDH on a plate using 1:1, 1:3, 1:6 and 1:9 molar ratios of the QD:aptamer construct. N = 6. Bars represent mean ± SD. p < 0.01 ** (student’s t-test).



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The fluorescence intensity of the wells after washing shows that the QD-cOligo can efficiently load up to six oligo-functionalized aptamers and detect pLDH proteins immobilized on the plate. Saturation of the fluorescence intensity at a 1:6 QD:aptamer ratio indicates that there are free, unbound aptamers that compete for pLDH and block QD binding. This assay shows that the modified aptamer can efficiently hybridize to the conjugation site of the cOligo and preserve the functionality of the binding site to the pLDH.

Having demonstrated antigen capture in a half-sandwich assay using a protein-immobilized plate, we next demonstrated a sandwich assay. Magnetic microparticles (MMP) functionalized with aptamers (1:6 ratio) were first added to different concentrations of pLDH in buffer for 30 minutes for antigen capture. Next, aptamer-modified QDs were added to the solution for sandwich binding and detection (Fig. 3A). The MMP-QD assay was designed to detect 0.033 to 3 ng of PfLDH, corresponding to 0.51 to 46 fmole. After magnetic separation, washing, and removing unbound excess reagents, the QDs were eluted from the sandwich complex (MMPpLDH-QD) for fluorescence measurements (Fig. 3B). Details of the assay protocol are provided in the Supplemental Information.

The results show that the MMP-QD assay has a limit of detection of 0.51 fmole and an upper saturation limit of 15 fmole. The performance of the MMP-QD aptamer assay is comparable to that of our previously reported MBQD antibody sandwich assay 23 and implies that the aptamer can be used as an alternative to antibodies in a malaria sandwich assay.

Having successfully demonstrated detection using an MMP-QD sandwich assay, we then explored strategies for signal amplification to lower the detection limit. While many nanoparticles can be used for this purpose, we selected gold nanoparticles (AuNP) for amplification for the ease of conjugation with thiolated hybridization oligos (Fig 1). To incorporate AuNPs into the sandwich assay as a carrier of multiple QDs, the spatial relationship between the particles must to be taken into account. Based on the size of the respective particles, we calculated the surface densities to approximate the maximum loading of AuNPs on MMP and QDs on AuNP (Table 1). The diameters of AuNPs and QDs are obtained from dynamic light


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scattering (DLS) measurements after oligonucleotide functionalization, and the maximum capacity determined by assuming that the footprint is the projected area of the particle. Using this approach, we estimated that about 940 AuNPs can be loaded onto the MMP surface and 147 QDs on the AuNP surface.

Figure 3. MMP-QD aptamer sandwich assay. (A) Schematic illustration of the MMP-QD assay using aptamers for detection of PfLDH and PvLDH. (B) Fluorescence intensity versus pLDH concentration for spiked samples with 0.033 ng to 3 ng in 100 µL buffer (0.51 - 46 fmole) PfLDH and PvLDH. The fluorescence intensities are background subtracted.

MMP Diameter (nm)

AuNP

1000 2

Surface area (nm )

3.1 x10

70 ± 5.8 6

1.5 x10

2

Footprint (nm ) Max capacity (surface area / footprint)

QD

34,000 940 AuNPs per MMP

14 ± 3.7

4

110

150 QDs per AuNP

Table 1. Estimated surface capacity of magnetic microparticles (MMPs), gold nanoparticles (AuNPs), and quantum dots (QDs). To detect 109 pLDH proteins (approximately 110 pg or 1.7 fmole), we need a minimum of 109 AuNPs. Based on the calculated surface capacity, a minimum of 106 MMPs is required to



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accommodate 109 AuNPs. The AuNPs can load a maximum of 1.5 x 1011 QDs, providing constraints on the minimum amount of materials required for the particle-based assay. For our MMP-AuNP-QD assay (Fig. 4A), we used a 50-fold excess of the minimum amount of MMPs and a 20-fold excess of QDs. The AuNPs were prepared at a 20-fold molar excess to the highest target protein concentration.

Figure 4. MMP-AuNP-QD aptamer sandwich assay. (A) Schematic illustration of the MMPAuNP-QD assay using aptamers for capture and detection of pLDH. (B) Fluorescence intensity versus concentration for the MMP-AuNP-QD assay for 0.66 to 100 pg in 100 µL buffer (10 amole - 1.5 fmole) PfLDH. The fluorescence intensity is background subtracted. The arrow indicates a detection limit achieved by MMP-QD aptamer sandwich assay without AuNP amplification.

In the MMP-AuNP-QD assay (Fig. 1A), MMPs were functionalized with aptamers (Fig. 1B), AuNPs loaded with the cOligo (Fig. 1C), and QDs functionalized with the hOligo (Fig. 1E). The loading of oligonucleotides on the AuNPs was achieved by thiol-gold conjugation and saltinduced aging to maximize packing.28 An average of 600 oligonucleotides were loaded onto a


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50 nm – AuNP.28 Initially, the AuNP-cOligo was hybridized with oligo-aptamer at a 1:50 molar ratio, leaving approximately 550 sites available for QDs. The MMP-aptamer construct was first incubated with pLDH proteins for capture. After 30 minutes, the AuNP-cOligo-aptamer construct was added for sandwich binding. After magnetic separation and washing to remove unbound reagents, the MMP-pLDH-AuNP construct was incubated with QD-hOligo to bind to available cOligo sites on the AuNPs. The MMP-pLDH-AuNP-QD complex was then magnetically separated and washed three times. For fluorescence detection, QDs were eluted from the complex since scattering and autofluorescence of MMPs increase the background signal and decreases the sensitivity of the assay. In our previous work, we developed an effective elution method to enhance the detection resolution of the MMP-QD sandwich assay.23

The MMP-AuNP-QD aptamer sandwich assay has a response curve that allows detection from 10 amole to about 1000 amole for both PfLDH and PvLDH (Fig. 4B). Lower concentrations (< 0.66 pg) were tested but the signal-to-noise ratio was less than 2 and did not show significant signal over the control. The lowest concentration detected (0.66 pg) is well above the background control (no antigen), demonstrating a detection limit of 10 amole (10 fM). The upper limit of about 1000 amole (66 pg) shows that the assay has a 2 order of magnitude detection range with the design conditions implemented here. Compared to the MMP-QD aptamer sandwich assay (Fig. 3), the sensitivity is increased by 50-fold. The average fluorescence intensity measured for 100 amole of PfLDH is 290 RFU, which corresponds to 11 fmole of QDs. This implies that about 91 QDs are bound to a single AuNP. A fit to the fluorescence - pfLDH curves in the range from 10 - 150 amole corresponds to an average of 83 QDs per AuNP.

The fact that the measured amplification is within a factor of two (55%) of the calculated maximum confirms the high efficiency of the steps involved in the assay. Signal attenuation can be derived from many steps in the assay, including: non-uniform coverage of the conjugation oligos on the AuNPs, non-uniform coverage of the capture aptamer on the AuNPs, a hybridization efficiency less than one, and loss of QDs during isolation and elution. The amplification achieved using QDs (Table 1) provides a significant improvement in the sensitivity of the assay and overcomes the limitation of detector resolution.



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In summary, we have performed a proof-of-concept demonstration of an antibody-free sandwich assay for detection of both PfLDH and PvLDH using aptamers, and demonstrated a unique signal amplification technique through application of oligonucleotides for simple and specific conjugation. Incorporation of gold nanoparticles allows binding of multiple QDs and has improved the sensitivity of the sandwich assay by 50-fold and achieved the detection limit of 10 amole. The detection limit (0.66 pg or 10 amole in 100 µL buffer) corresponds to a concentration of around 1 pg mL-1.

While the results presented here highlight the potential of the aptamer-based sandwich assay in the laboratory, there are several important issues that need to be assessed for clinical use. First, validation experiments need to be performed with relevant samples, such as urine. Possible problems associated with patient samples, such as non-specific binding, can be reduced by separating the capture and detection steps: MMPs incubated with patient samples for capture can be magnetically isolated and re-suspended in buffer for QD detection. Further improvements in sensitivity can be achieved by re-suspending the MMPs in a smaller volume of buffer thereby concentrating the sample. For example, MMPs in a 1 mL urine sample can be isolated and resuspended in 100 µL of buffer, achieving a 10-fold concentration. Second, while the magnetic beads, gold nanoparticles, and QDs can be pre-prepared for use, their stability and shelf-life remain to be established. Third, in low resource settings a fluorimeter is an expensive method for optical detection, however, a smart phone could be used for this purpose.29 Finally, for use in the field a wider range of detection may be necessary. Here we designed a detection range of two orders of magnitude. The upper limit of detection can be increased by increasing the concentration of MMPs in the sample, although this may also impact the detection limit. An alternative is to design separate assays for detection over different concentration ranges.

Supporting Information. Assay protocol and optimization of the binding buffer.

Conflict of Interest The authors declare no competing financial interest.


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