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Highly Sensitive and Selective Aptamer-based Fluorescence Detection of a Malarial Biomarker using Single-Layer MoS2 Nanosheets * Kenry, Alisha Geldert, Xiao Zhang, Hua Zhang, and Chwee Teck Lim ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00449 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 13, 2016

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Highly Sensitive and Selective Aptamer-based Fluorescence Detection of a Malarial Biomarker using Single-Layer MoS2 Nanosheets Kenry1,2,3,#, Alisha Geldert3,#, Xiao Zhang5, Hua Zhang5,*, Chwee Teck Lim1,2,3,4,* 1

NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456

2

Centre for Advanced 2D Materials and Graphene Research Centre, National University of Singapore, Singapore 117543

3

Department of Biomedical Engineering, National University of Singapore, Singapore 117576 4

Mechanobiology Institute, National University of Singapore, Singapore 117411

5

Center for Programmable Materials, School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798 #

These authors contributed equally to this work. *Correspondence Chwee Teck Lim ([email protected]) Department of Biomedical Engineering National University of Singapore Singapore 117575, Singapore Hua Zhang ([email protected]) Center for Programmable Materials School of Materials Science and Engineering Nanyang Technological University Singapore 639798, Singapore

KEYWORDS: 2D nanomaterials; Transition metal dichalcogenides; MoS2; Aptamers; Malarial biomarker; Plasmodium lactate dehydrogenase (pLDH).

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Abstract We demonstrate a highly selective and sensitive aptamer-based “capture-release” fluorescence detection of a malarial biomarker, i.e., Plasmodium lactose dehydrogenase (pLDH) protein, using single-layer two-dimensional MoS2 nanosheets. The detection of the target pLDH protein utilizing the aptamer-nanosheet sensing platform is rapid and can be easily completed within 10 min. In addition, the developed biomolecular sensor is capable of detecting the target malarial biomarker in homogeneous protein solutions as well as in heterogeneous mixtures of proteins. We anticipate that the ultrathin MoS2 nanosheet-based biosensor will facilitate the further development of biomolecular nanosensors for the detection of a wide range of biomarkers for malaria and other diseases. Malaria, an infectious disease caused by the protozoan Plasmodium parasite, remains a prominent global health concern. In 2015, malaria posed a risk to over three billion people, causing approximately 214 million new infections and 438,000 deaths 1. Early malaria diagnosis has been recognized as an essential element for managing this infectious disease effectively to reduce its associated morbidity and mortality 2. In fact, prompt and accurate diagnosis of malaria is crucial not only to provide treatment to those who need it, but also to distinguish this infectious disease from other tropical diseases in order to prevent overprescription of antimalarial drugs which may lead to drug resistance 3. However in 2014, diagnostic tests were only performed on 78% of suspected malaria cases presented in public health facilities 1; let alone those cases which were not within reach of a healthcare provider. In addition to the difficult access to malaria diagnosis, the current suboptimal state of the diagnostic tools further compounds the problems. Unfortunately, early malaria diagnosis remains challenging because of the difficulty in distinguishing the scarcely available malaria parasites in blood at the early stage of infection. As a result, there is an urgent need to develop simple, fast, and cost-effective malaria diagnostic strategies with high sensitivity and specificity. The standard method of malaria diagnosis relies on Giemsa-staining and optical microscopy to identify parasite-infected red blood cells

4,5

. However, the lack of proper equipment and/or

skilled technicians limits the application of microscopy-based diagnosis in low-resource settings. Microscopy-based diagnosis requires elaborate sample preparation and time-consuming examination. Moreover, the poor sensitivity of this method at low parasitemia levels increases the false negative risk while artifacts, such as bacteria, fungi, and general debris, can be indistinguishable from parasites and cause false positive diagnosis. To overcome the limitations 2


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of the microscopy assay, rapid diagnostic tests (RDTs) which offer a simple and fast diagnostic readout have been developed in recent years and are becoming an increasingly common alternative method of malaria diagnosis

4–6

. For instance, RDTs were used in 71% of diagnoses

of suspected malaria cases in Africa in 2014 1. While RDTs have improved access to malaria diagnosis, they can also suffer from subpar performance, especially in tropical conditions. Most of the current RDTs use monoclonal antibodies to bind and detect malarial biomarkers in blood. Nevertheless, these antibodies are typically sensitive to heat and humidity 7. To minimize the risk of antibody degradation, manufacturers of malaria RDTs generally recommend storage at 430 ⁰C. However, in practice, RDTs are often exposed to greater than 30 ⁰C and over 70% humidity 4. Consequently, the stability, accuracy, and clinical utility of these antibody-based malaria RDTs may be compromised, especially in the tropical conditions found in many malariaendemic regions. As an alternative to antibodies, nucleic acid aptamers have been progressively identified as potential molecular recognition elements for the construction of biosensors capable of detecting various biomolecular targets. Aptamers are short single-stranded oligonucleotides artificially generated to have high affinity and specific binding to target biomolecules 8. In comparison to antibodies, aptamers possess several intrinsic advantages, such as facile chemical synthesis and modification, robust long-term storage and thermal stability, and high target affinity and selectivity. These properties are favorable and crucial for effective clinical sensing and diagnostic applications

9

, particularly in tropical settings where malaria is prevalent and

antibody-based tests may be unstable. As such, the past decade has seen the rapid advancement of a large number of aptamer-based sensors, such as electrochemical and optical aptasensors 10–12. One simple and promising optical aptasensing technique utilizes fluorescence resonance energy transfer (FRET) which relies on the fluorescence quenching of an aptamer upon its adsorption to a quenching material and the subsequent fluorescence recovery when the aptamer releases from the material to bind to a target. The selection of a proper quenching material, therefore, is critical to this technique and several nanomaterials offer promising fluorescence quenching and biosensing capabilities. Over the last few years, two-dimensional (2D) atomically thin nanomaterials, notably, graphene and its related derivatives, have garnered tremendous interest due to their attractive physicochemical properties and potential utility, particularly in biosensing

13,14

. Graphene oxide

(GO), the oxygenated derivative of graphene with numerous bioapplications 3


15–19

, has been

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regarded as a promising component for the assembly of biosensors. In fact, GO-based biosensors have been actively demonstrated for the sensitive and specific fluorescence detection of different biomolecules, including proteins, nucleic acids, and small molecules, because of the intrinsic capability of GO in inducing long-range energy transfer and fluorescence quenching

20–22

. More recently, another class of layered 2D nanomaterials

analogous to graphene, i.e., transition metal dichalcogenide (TMD) nanosheets, such as ultrathin MoS2, WS2, TaS2, TiS2 nanosheets, have also generated significant attention because of their 2D geometrical structure, large specific surface area, unprecedented physicochemical properties, ease of large scale production and dispersion in aqueous solutions, and widespread potential bioapplications

23–25

. Interestingly, several studies have reported the high affinities of

these layered TMD nanosheets for various biomolecules and their superior fluorescence quenching capabilities 26,27 which may be manipulated for the development of rapid biomolecular sensing platforms with high sensitivity and selectivity. In fact, TMD nanosheet aptasensors have been found to have similar or lower limits of detection as compared to GO nanosheet aptasensors for the detection of both single-stranded DNA

27,28

and protein targets, such as

thrombin 29 and prostate specific antigen 30. Motivated by the strong affinity for nucleic acids and the robust fluorescence quenching capability of 2D TMD nanomaterials, we present a novel FRET-based aptasensing platform using single-layer MoS2 nanosheets for the detection of Plasmodium lactate dehydrogenase (pLDH) protein. In general, pLDH is a highly expressed malarial biomarker. It serves as a common target for malaria detection because it is produced by all Plasmodium species and pLDH concentrations have been found to correlate with parasitaemia levels

7,31

. The aptamer-

MoS2 nanosheet-assisted “capture-release” fluorescence detection of the target protein is rapid, sensitive, and highly specific. We anticipate that this study will further facilitate the application of nanomaterial and aptamer-based techniques to develop robust and stable biomolecular sensors for the detection of a variety of disease biomarkers.

Results and discussion The single-layer 2D TMD MoS2 nanosheets were first synthesized using the electrochemical lithium-intercalation method as reported previously

26,32

. They were then morphologically

characterized using atomic force microscope (AFM), transmission electron microscope (TEM), selected area electron diffraction (SAED), and high-resolution TEM (HRTEM) (Fig. 1). We observed that the thickness of the as-prepared ultrathin nanosheets was about 0.8 nm (Fig. 1a 4


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and Inset of Fig. 1a), demonstrating their single-layer thickness, while the size of a typical MoS2 nanosheet was approximately hundreds of nm (Fig. 1b). We further observed that the nanosheets possessed a hexagonal lattice structure with a lattice spacing of 0.27 nm, corresponding to (100) plane of MoS2, from the obtained SAED pattern (Fig. 1c) and HRTEM image (Fig. 1d). In fact, the single-crystalline nature of MoS2 nanosheets could be additionally noted from their continuous lattice fringes (Fig. 1d).

Figure 1 | Morphology of single-layer MoS2 nanosheets. (a) AFM height image of MoS2 nanosheets with (Inset) their corresponding sectional profile (i.e., S1 and S2 in Fig. 1a), showing their height of approximately 0.8 nm. (b) TEM image and (c) the corresponding SAED pattern of a typical MoS2 nanosheet. (d) HRTEM image of a typical MoS2 nanosheet revealing its single-crystalline nature from the continuous lattice fringes.

The proposed aptamer-based “capture-release” biomolecular sensing strategy using the singlelayer MoS2 nanosheets is depicted in Fig. 2. In brief, the fluorescently-labeled aptamer (Fig. 2a) is initially incubated with the MoS2 nanosheets. Upon the spontaneous adsorption of the 5


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aptamer to the nanosheet, the fluorescence of the aptamer will be quenched (Fig. 2b). Nevertheless, upon the introduction of the target pLDH protein to the system, the aptamer will be released from the nanosheets and bind to the target due to the high affinity between the aptamer and target protein (Fig. 2c). Consequently, the quenched fluorescence of the aptamer will be restored, indicating the selective sensing of the target biomarker. This “capture-release” sensing strategy is simple and enables rapid sensing as it eliminates the probe-sample preincubation step.

Figure 2 | Schematic illustration of the aptamer-based “capture-release” sensing assay for the fluorescence detection of a malarial biomarker, i.e., Plasmodium lactate dehydrogenase (pLDH), using single-layer MoS2 nanosheets. (a) Pure pLDH aptamer solution with its original fluorescence. (b) Quenching of the aptamer fluorescence by MoS2 nanosheets. (c) Recovery of the aptamer fluorescence in the presence of the target pLDH protein. The pLDH aptamer will change its conformation upon binding to the target pLDH protein, inducing release of aptamer from the nanosheet surface and subsequently, recovery of the aptamer fluorescence. All representative fluorescence intensity profiles possess the same fluorescence intensity (a.u.) and wavelength (nm) scales.

Here, the malarial biomarker, i.e., pLDH protein, was selected as the target biomolecule as it is highly expressed in both sexual and asexual parasitic stages 6. In fact, the pLDH levels correspond directly to the presence of the metabolically active Plasmodium parasites

31

. As

such, pLDH is highly preferred as one of the target biomarkers in the development of biosensor for the early and accurate diagnosis of malaria. We then used a single-stranded aptamer probe labeled with the fluorescent dye, fluorescein (FAM), with a sequence of 6


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5'-GTTCGATTGGATTGTGCCGGAAGTGCTGGCTCGAAC-FAM-3' which was found to have a high affinity to the pLDH protein, to target this malarial biomarker 33.

To begin with, the fluorescence quenching capability of the MoS2 nanosheets was evaluated (Fig. 3). The pLDH aptamer was prepared at 25 nM while the MoS2 nanosheets were prepared at different concentrations ranging from 0 to 12 µg/mL. Next, these MoS2 nanosheets were mixed with the aptamer followed by an excitation at 495 nm, the maximal absorption wavelength of the FAM dye conjugated to the aptamer. The fluorescence emission with a peak at 527 nm was then recorded from 525 to 650 nm. Here, we observed that the aptamer exhibited a strong fluorescence emission and it was robustly quenched upon the addition of MoS2 nanosheets (Fig. 3a and Fig. 3b). In fact, the quenching of the aptamer fluorescence was highly dependent on the concentration of MoS2 nanosheets. Interestingly, close to 90% of the aptamer fluorescence was quenched even at a low MoS2 concentration of 8 µg/mL. Beyond this concentration, complete fluorescence quenching was noted (Fig. 3a). As such, the 8 µg/mL of MoS2 nanosheets was recognized as the optimal nanosheet concentration for the fluorescence quenching of 25 nM aptamer and this aptamer:nanosheet concentration ratio was used in subsequent experiments. Further to this, the fluorescence quenching capability of MoS2 nanosheets over a period of time was evaluated (Fig. 3c and Fig. 3d). We noted that upon the aptamer-MoS2

nanosheet interactions, the aptamer fluorescence was quenched almost

instantaneously. This suggests the strong molecular interactions between the aptamer and MoS2 nanosheets as well as the robust fluorescence quenching capability of the nanosheets. Next, the recovery of the aptamer fluorescence upon the introduction of the target pLDH protein was examined (Fig. 4). The fluorescence recovery was observed in the presence of pLDH protein with various concentrations spanning from 0 to 500 nM (Fig. 4a and Fig. 4b). Interestingly, even at low nanomolar concentrations of pLDH protein, it was evident that the fluorescence recovery induced by pLDH protein was concentration-dependent. Specifically, we noted a linear relationship in the low pLDH concentration range of between 0 and 62.5 nM (Fig. 4c). Based on these data, the limit of detection of the aptamer MoS2 nanosheet-based biomolecular sensor was computed to be approximately 550 pM. Impressively, this sensitivity has far exceeded that required for clinical applications as the mean pLDH level of malariainfected patients is typically found to be on the order of hundreds of nM 34. We further evaluated 7


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the kinetics of the fluorescence recovery in the presence of the pLDH protein over a period of 25 min (Fig. 4d). The fluorescence intensity in the presence of the target pLDH protein was noted to increase consistently with time while that of the nanosheet-aptamer sample in the absence of the target protein decreased steadily over the same period of observation. The sharpest respective increase and decrease, however, occurred within the first 5-10 min of incubation. The single-time-point fluorescence measurements were therefore performed after 10 min of incubation to ensure that the difference between the quenched and recovered samples was sufficiently large and had generally stabilized. Thus, MoS2 nanosheet-based detection of the malarial biomarker pLDH protein is rapid and can be easily accomplished within 10 min in most cases.

Figure 3 | Concentration-dependent quenching of the aptamer fluorescence by the single-layer MoS2 nanosheets. (a, b) Quenching of the aptamer fluorescence by MoS2 nanosheets with different concentrations (i.e., 0, 2, 4, 6, 8, 10, and 12 µg/mL), as shown by: (a) full spectra over wavelengths from 525 to 650 nm, and (b) peak emission at 527 nm. Fluorescence of the aptamer decreases when mixed with increasing concentrations of MoS2 nanosheets, indicating the quenching of aptamer fluorescence as

8


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a function of the concentration of MoS2 nanosheets. (c) Quenching of the aptamer fluorescence by MoS2 nanosheets with different concentrations (i.e., 0, 2, 4, 6, 8, 10, and 12 µg/mL) recorded over a period of 25 min. All fluorescence emissions were observed to stabilize rapidly. (d) Efficiency of the quenching of aptamer fluorescence by MoS2 nanosheets with different concentrations (i.e., 2, 4, 6, 8, 10, and 12 µg/mL). The proportion of aptamer fluorescence that was quenched by MoS2 nanosheets is concentration-dependent but rapidly approaches peak quenching in all cases. All fluorescence measurements were taken with an excitation wavelength of 495 nm and an emission wavelength of 527 for peak emission readings, or an emission wavelength range of 525 to 650 nm for full spectra.

Figure 4 | Concentration-dependent recovery of the aptamer fluorescence in the presence of the target pLDH biomarker and sensitivity of single-layer MoS2 nanosheet-based biomolecular sensor. (a, b) Recovery of the aptamer fluorescence upon the introduction of the target pLDH protein with different concentrations (i.e., 0, 3.9, 7.8, 15.6, 23.425, 31.25, 62.5, 125, 250, and 500 nM), as shown by: (a) full spectra recorded over wavelengths from 525 to 650 nm, and (b) peak emission at 527 nm. Upon the addition of increasing concentrations of pLDH protein to an aptamer-MoS2 solution, higher fluorescence is measured, indicating that the recovery of the aptamer fluorescence is a function of the concentrations of pLDH protein. (c) Profile showing the linear aptamer fluorescence recovery at low

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concentrations of pLDH protein (i.e., 0, 3.9, 7.8, 15.6, 23.425, 31.25, 62.5 nM). (d) Time-dependent recovery of the aptamer fluorescence in the presence of the 250 nM pLDH protein recorded over a period of 25 min. The fluorescence of the quenched sample decreased over time while the fluorescence of the sample with pLDH protein increased and stabilized over time. All fluorescence measurements were taken with an excitation wavelength of 495 nm and an emission wavelength of 527 for peak emission readings, or an emission wavelength range of 525 to 650 nm for full spectra.

In addition to systematically probing the fluorescence quenching capability and sensitivity of the single-layer MoS2 nanosheet-based biomolecular sensor, we examined its specificity against various proteins in homogeneous protein solutions as well as heterogeneous mixture of proteins (Fig. 5). First, the target biomarker, i.e., pLDH protein, and several non-specific blood plasma proteins, i.e., insulin, albumin, globulin, and fibrinogen, were prepared. Both pLDH and nonspecific proteins were tested at a concentration of 250 nM, yielding a target-to-non-specific protein concentration ratio of 1:1 (Fig. 5a). Here, it was clear that the pLDH protein-induced fluorescence recovery was significantly higher than those caused by the non-specific plasma proteins. In fact, these non-specific fluorescence recoveries were negligible and in the same intensity range as that in the absence of any proteins. Specifically, pLDH induced about 4.8 – 6.1 times higher fluorescence recovery than an equivalent concentration of albumin, globulin, or fibrinogen, over 12 times higher recovery than insulin, and over 38 times higher recovery than a sample without any protein. This strongly indicates the excellent specificity of the aptamer-MoS2 nanosheet-based biomolecular sensing platform. In addition to this, we prepared heterogeneous mixtures of the non-specific plasma proteins of albumin, globulin, and fibrinogen at 250 nM, both in the presence and absence of 250 nM target pLDH biomarker, in order to mimic the heterogeneous nature of clinical samples. This yielded a target-to-non-specific protein concentration ratio of 1:3. We then evaluated the selectivity of our MoS2 nanosheet-based sensor against the different samples (Fig. 5b). Intriguingly, we observed that the fluorescence recoveries recorded from the pure target pLDH protein and the heterogeneous mixtures of pLDH and plasma proteins were significantly higher than those of non-specific protein mixture or solution with no proteins. More clearly, pLDH in the protein mixture induced a 3.3 times higher fluorescence recovery as compared to the protein mixture itself. In addition, homogeneous pLDH protein and heterogeneous protein mixture comprising the target induced similar fluorescence recovery. This further highlights the exceptional selectivity of the single-layer MoS2 nanosheet-based “capture-release” sensor for the fluorescence detection of the malarial biomarker. 10


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Figure 5 | Selectivity of single-layer MoS2 nanosheet-based biomolecular sensor in homogeneous protein solutions and heterogeneous mixtures of proteins. Fluorescence intensities recorded in (a) homogeneous solutions of individual proteins at 250 nM and (b) heterogeneous solutions comprising nonspecific plasma proteins in the presence and absence of target pLDH protein. All proteins in the heterogeneous solution were also prepared at a final concentration of 250 nM. Excitation was fixed at 495 nm while peak emission was recorded at 527 nm.

Conclusions We reported the novel application of single-layer MoS2 nanosheets in developing a “capturerelease” aptamer-based biomolecular sensor for the fluorescence detection of a highly expressed malarial biomarker pLDH protein. The developed aptamer-MoS2 nanosheet-based sensing platform was simple, fast, sensitive, and highly specific to the target malarial biomarker. On top of its excellent selectivity in differentiating the target biomolecule from individual random proteins, such as insulin, albumin, globulin, and fibrinogen, the MoS2 nanosheet-based biomolecular sensor was highly capable of distinguishing the target pLDH protein in a heterogeneous mixture of proteins. We believe that this simple and rapid aptamer-MoS2 nanosheet-based “capture-release” sensing platform will be beneficial for the sensitive, selective, and robust detection of a wide range of biological markers related to infectious and other severe diseases.

Methods Materials MoS2 nanosheets were prepared using the electrochemical lithium-intercalation method as reported previously

26,32

. They were deposited on freshly cleaved mica and morphologically 11


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characterized using tapping mode AFM. TEM, HRTEM. Fluorescently-labeled pLDH aptamer was synthesized by Integrated DNA Technologies (sequence: 5’- GTT CGA TTG GAT TGT GCC GGA AGT GCT GGC TCG AAC - FAM - 3’). Recombinant falciparum pLDH protein was obtained from Sino Biological; other nonspecific proteins were purchased from Sigma Aldrich. A pH 8 buffer with 20 mM Tris-HCl, 50 mM NaCl, 5 mM KCl, and 5 mM MgCl2 was used as an aptamer solvent. Nonspecific proteins were dissolved in PBS, while MoS2 and pLDH protein were dissolved in water. A Tecan Infinite 200 microplate reader was used for fluorescence measurements.

Fluorescence Quenching and Recovery Assays MoS2 quenching was assessed by measuring the fluorescence of 25 nM pLDH aptamer incubated with different concentrations of MoS2 (i.e., 2, 4, 6, 8, 10, and 12 µg/mL). All concentrations were reported as the final values in each 200 µL sample. After determining 8 µg/ml MoS2 to be the optimal quencher concentration, fluorescence recovery experiments were performed. 25 nM pLDH aptamer and 8 µg/mL MoS2 were mixed and incubated for 10 min before the addition of either pLDH solutions of differing concentrations (to assess sensitivity) or heterogeneous or non-specific protein solutions (to assess specificity). The fluorescence of the sample obtained 10 min after the addition of analyte was used to evaluate the recovery. Fluorescence was measured from the emission at 527 nm (for single readings) or 515-650 nm (for full spectra) upon an excitation at 495 nm. Fluorescence quenching efficiency was defined as ிబ ିி೜ ிబ

(1)

while fluorescence recovery was defined as ிିி೜ ிబ ିி೜

(2)

where F0 is the fluorescence of the pure aptamer solution, Fq is the fluorescence of the aptamerMoS2 solution, and F is the fluorescence of the solution upon the addition of the analyte. The limit of detection, i.e., 3σ/m, was calculated using the standard deviation of the MoS2-quenched aptamer fluorescence (σ) and the slope of the linear portion of the sensitivity plot (m). 12


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Acknowledgements This research was supported by the National Research Foundation, Prime Minister’s Office, Singapore under its medium-sized centre programme, Centre for Advanced 2D Materials and its Research

Centre

of

Excellence,

Mechanobiology

Institute,

as

well

as

the

MechanoBioEngineering Laboratory of the Department of Biomedical Engineering of the National University of Singapore. H.Z. thanks the financial support from MOE under AcRF Tier 2 (ARC 26/13, No. MOE2013-T2-1-034; ARC 19/15, No. MOE2014-T2-2-093; MOE2015-T2-2057) and AcRF Tier 1 (RG5/13), NTU under Start-Up Grant (M4081296.070.500000) and iFood Research Grant (M4081458.070.500000), and Singapore Millennium Foundation in Singapore. Kenry and A. Geldert would like to acknowledge the NUS Graduate School for Integrative Sciences and Engineering Scholarship and Whitaker International Program Fellowship, respectively.

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