Detection of dengue NS1 antigen using long-range surface plasmon waveguides
Introduction
Dengue is a tropical mosquito borne disease affecting over half of the world population (Beatty et al., 2010) with about 390 million cases annually (Bhatt et al., 2013). The diagnosis of dengue can be difficult because its symptoms are nonspecific and current laboratory techniques are expensive, time consuming, and require highly skilled lab personnel. Current laboratory dengue diagnosis techniques include virus isolation, detection of virus components (RNA or antigen), and detection of dengue-specific antibodies (IgM or IgG) (Peeling et al., 2010, Vorndam et al., 1997, World Health Organization, 2009). Over the past years, much research has been done on a dengue diagnostic tool through the detection of nucleic acid (Baeumner et al., 2002, Zaytseva et al., 2005, Zhang et al., 2006), antigen (Camara et al., 2013, Linares et al., 2013, Silva et al., 2014b, Tai et al., 2005) or antibodies (Kumbhat et al., 2010, Lee et al., 2009, Wong et al., 2014a). However, none of the studies are able to fulfill the requirement of an “ideal” dengue diagnostic test which should be sensitive regardless of the stage of infection (Peeling et al., 2010). We previously argued that not all patients seek medical attention during early onset of symptoms and therefore presented a dengue biosensor which is able to detect dengue-specific antibodies in blood plasma (Wong et al., 2014a). Recent studies suggest that the combined detection of dengue non-structural 1 (NS1) antigen and dengue-specific antibodies improves the diagnostic sensitivity (Blacksell et al., 2011, Fry et al., 2011).
Non-structural 1 (NS1) protein, which is approximately 45 kDa in molecular weight (Allonso et al., 2011, Zhao et al., 1987), is secreted from dengue virus infected cells. Dengue NS1 antigen is an important diagnostic biomarker found circulating in patient blood samples up to 9 days after the onset of symptoms (Alcon et al., 2002). Furthermore, dengue serotypes can be identified by using serotype-specific anti-NS1 monoclonal antibody (Ding et al., 2011). Much of the research on biosensors has been done on the detection of purified dengue NS1 antigen in buffer (Camara et al., 2013, Figueiredo et al., 2015, Hu et al., 2013, Mishra et al., 2014, Silva et al., 2014a, Singh, 2012, Su et al., 2003, Tai et al., 2005) or on spiked NS1 antigen in serum samples (Cecchetto et al., 2015, Dias et al., 2013, Silva et al., 2014b, Yen et al., 2015). The detection of naturally occurring dengue NS1 antigen in serum was reported using electrochemical (Cavalcanti et al., 2012, Parkash et al., 2014), fluorescence (Linares et al., 2013) and quartz crystal microbalance (Wu et al., 2005) biosensors. In this paper we present the detection of purified dengue NS1 antigen in buffer, and naturally-occurring dengue NS1 antigen in patient blood plasma, using straight long-range surface plasmon polariton (LRSPP) waveguides. Five clinical plasma samples were tested for dengue NS1 antigen. We also compare the performance of three commercially-available anti-NS1 monoclonal antibodies to detect dengue NS1 antigen. This is the first biosensor to demonstrate the detection of naturally occurring dengue NS1 antigen in blood plasma. The detection of dengue NS1 in blood plasma is more challenging than the detection of dengue-specific antibodies (Wong et al., 2014a) because NS1 has a smaller molecular weight and occurs in lower concentration in blood (0.01–2 μg/ml (Alcon et al., 2002)).
LRSPPs are transverse magnetic polarized optical surface waves propagating along a thin metal slab or stripe bounded by dielectrics of similar refractive index (Berini, 2009). The excitation of LRSPPs can be easily achieved by an optical fiber butt-coupled to the metal waveguide. The ease of LRSPP excitation enables compact and miniaturized biosensors. LRSPPs have reduced confinement and lower modal sensitivity than single-interface SPPs but its greater propagation length provides better overall sensitivity (Berini, 2008). For biosensing applications, low-index claddings are used to match the refractive index of biologically compatible sensing fluids (~1.32) which then maintains the optical symmetry of the mode. Fluoropolymers such as Cytop (Asahi) and Teflon (Dupont) are the most common materials used as low index claddings (Joo et al., 2010, Slavík and Homola, 2007, Wark et al., 2005). The sensor used throughout this paper consists of a straight gold (Au) stripe embedded in Cytop claddings with an etched microfluidic channel for sensing. The sensitivity of the straight waveguide as a biosensor was discussed previously (Wong et al., 2015a).
Section snippets
Chemicals and reagents
16-Mercaptohexadecanioc acid (16-MHA), phosphate buffered saline (PBS) 0.01 M, pH 7.4, N-Hydroxysuccinimide sodium salt (NHS), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), human IgG kappa antibody, sodium dodecyl sulfate (SDS), 2-Isopropanol semiconductor grade (IPA), acetone HPLC grade ≥99.9%, heptane and glycerol (electrophoresis grade) were obtained from Sigma-Aldrich. Distilled water was deionized using Millipore filtering membranes (Millipore, Milli-Q water system at
Detection of dengue NS1 antigen using monoclonal antibody (MAb) in cell culture supernatant
The measurements obtained for the detection of NS1 antigen in clean fluid (Fig. 2a) and in patient blood plasma (Fig. 2c) using MAb in cell culture supernatant are summarized in Fig. 4. The anti-NS1 MAb was diluted 1:10 in PBS/Gly buffer. Fig. 4a shows the normalized power during the injection of antibodies (human IgG kappa antibody (control) or anti-NS1 MAb (positive)) over a clean NS1 functionalized surface. The measured power (in μW) was normalized to the baseline signal before the injection
Discussion
The results obtained using our biosensor were analyzed using a similar figure of merit as the one used in conventional dengue NS1 capture antigen enzyme-linked immunosorbent assay (ELISA) so that their performance can be compared. A test sample is considered positive if the time-averaged surface mass density is greater than twice the mean value of the negative samples. In other words, we define a positive-to-negative ratio as (Wong et al., 2014a):where 〈ΓPOS〉 is the
Conclusion
The detection of dengue NS1 antigen has been demonstrated using a compact, cost-effective, label-free and real-time LRSPP based biosensor. The complex composition of anti-NS1 MAb in cell culture supernatant causes nonspecific binding which resulted in one false negative measurement. Using purified anti-NS1 MAb for recognition overcomes the problem by showing almost no response for negative samples and distinguishable results for all of our positive samples. Although the positive-to-negative
Acknowledgment
The authors gratefully acknowledge Oleksiy Krupin, Anthony Olivieri, and Babu Ramanathan for assistance in carrying out the experiments. This work is supported by the Ministry of Education, Malaysia, under High Impact Research Grant (UM.C/625/1/HIR/MOHE/H-20001-00-E000053 and UM.0000005/HIR.C1) and by the Natural Sciences and Engineering Research Council (NSERC) of Canada.
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