Elsevier

Biosensors and Bioelectronics

Volume 78, 15 April 2016, Pages 132-139
Biosensors and Bioelectronics

Detection of dengue NS1 antigen using long-range surface plasmon waveguides

https://doi.org/10.1016/j.bios.2015.11.030Get rights and content

Highlights

  • A compact, label-free and real-time long range surface plasmon based biosensor.

  • The detection of dengue NS1 antigen in buffer and clinical blood plasma was presented.

  • The purified anti-NS1 monoclonal antibodies overcome the nonspecific problem.

  • The largest signal-to-noise ratio is 356 and the best detection limit is 5.73 pg/mm2.

Abstract

The non-structural 1 (NS1) protein of the dengue virus circulates in infected patients’ blood samples and can be used for early diagnosis of dengue infection. In this paper, we present the detection of naturally-occurring dengue NS1 antigen in infected patient blood plasma using straight long-range surface plasmon waveguides. Three commercially-available anti-NS1 monoclonal antibodies were used for recognition and their performance was compared and discussed. A similar figure of merit to the one used in conventional dengue NS1 capture using an enzyme-linked immunosorbent assay (ELISA) was applied to our results. In general, the positive patient samples can be clearly differentiated from the negative ones and the results agree with those obtained using ELISA. The largest signal-to-noise ratio observed during the experiments was 356 and the best detection limit observed is estimated as 5.73 pg/mm2.

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):P/N=ΓPOSΓNEG_Avgwhere 〈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.

References (44)

  • T.-Z. Wu et al.

    Piezoelectric immunochip for the detection of dengue fever in viremia phase

    Biosens. Bioelectron.

    (2005)
  • Y. Zhang et al.

    Toward the detection of single virus particle in serum

    Anal. Biochem.

    (2006)
  • S. Alcon et al.

    Enzyme-linked immunosorbent assay specific to Dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections

    J. Clin. Microbiol.

    (2002)
  • A.J. Baeumner et al.

    Biosensor for dengue virus detection: sensitive, rapid, and serotype specific

    Anal. Chem.

    (2002)
  • M.E. Beatty et al.

    Best practices in dengue surveillance: a report from the Asia-Pacific and Americas Dengue Prevention Boards

    Plos Negl. Trop. Dis.

    (2010)
  • P. Berini

    Bulk and surface sensitivities of surface plasmon waveguides

    New J. Phys.

    (2008)
  • P. Berini

    Long-range surface plasmon polaritons

    Adv. Opt. Photonics

    (2009)
  • S. Bhatt et al.

    The global distribution and burden of dengue

    Nature

    (2013)
  • S.D. Blacksell et al.

    Evaluation of six commercial point-of-care tests for diagnosis of acute dengue infections: the need for combining NS1 antigen and IgM/IgG antibody detection to achieve acceptable levels of accuracy

    Clin. Vaccine Immunol.

    (2011)
  • A.R. Camara et al.

    Dengue immunoassay with an LSPR fiber optic sensor

    Opt. Express

    (2013)
  • X. Ding et al.

    Full serotype-and group-specific NS1 capture enzyme-linked immunosorbent assay for rapid differential diagnosis of dengue virus infection

    Clin. Vaccine Immunol.

    (2011)
  • A. Figueiredo et al.

    Electrical detection of dengue biomarker using egg yolk immunoglobulin as the biological recognition element

    Sci. Rep.

    (2015)
  • Cited by (55)

    • G-quadruplex microspheres-based optical RNA biosensor for arthropod-borne virus pathogen detection: A proof-of-concept with dengue serotype 2

      2022, International Journal of Biological Macromolecules
      Citation Excerpt :

      Among the wide variety of biosensors designed for the determination of DENV, optical biosensors have driven a lot of advantages and broadly investigated by the researchers [17–20]. Recent studies on the optical biosensors for DENV include the detection of DENV DNA [21], DENV E protein [22,23] and detection of DENV NS1 antigen [24,25]. It is noted that most of the developed biosensors' technologies offer early detection, sensitive and low-cost analysis for diagnosis of DENV infection in comparison with above-mentioned conventional detection methods.

    • Human virus detection with graphene-based materials

      2020, Biosensors and Bioelectronics
      Citation Excerpt :

      An SPR sensor can also incorporate graphene-based materials, such as GO, for the detection of various forms of nucleic acids. Such devices rely on monitoring the change in plasmonic signal upon an antibody–antigen affinity reaction in real-time (Omar et al., 2020; Wong et al., 2016). Mahdi et al. deposited a thin film on glass slides containing a layer of gold, a layer of a composite of CdS quantum dots with amino-functionalized GO, and a layer of IgM immobilized via EDC-NHS (Au/CdS–NH2GO/EDC-NHS/IgM).

    View all citing articles on Scopus
    View full text