Abstract
A DNA micro-optode for dengue virus detection was developed based on the sandwich hybridization strategy of DNAs on succinimide-functionalized poly(n-butyl acrylate) (poly(nBA-NAS)) microspheres. Gold nanoparticles (AuNPs) with an average diameter of ~20 nm were synthesized using a centrifugation-based method and adsorbed on the submicrometer-sized polyelectrolyte-coated poly(styrene-co-acrylic acid) (PSA) latex particles via an electrostatic method. The AuNP–latex spheres were attached to the thiolated reporter probe (rDNA) by Au–thiol binding to functionalize as an optical gold–latex–rDNA label. The one-step sandwich hybridization recognition involved a pair of a DNA probe, i.e., capture probe (pDNA), and AuNP–PSA reporter label that flanked the target DNA (complementary DNA (cDNA)). The concentration of dengue virus cDNA was optically transduced by immobilized AuNP–PSA–rDNA conjugates as the DNA micro-optode exhibited a violet hue upon the DNA sandwich hybridization reaction, which could be monitored by a fiber-optic reflectance spectrophotometer at 637 nm. The optical genosensor showed a linear reflectance response over a wide cDNA concentration range from 1.0 × 10−21 M to 1.0 × 10−12 M cDNA (R2 = 0.9807) with a limit of detection (LOD) of 1 × 10−29 M. The DNA biosensor was reusable for three consecutive applications after regeneration with mild sodium hydroxide. The sandwich-type optical biosensor was well validated with a molecular reverse transcription polymerase chain reaction (RT-PCR) technique for screening of dengue virus in clinical samples, e.g., serum, urine, and saliva from dengue virus-infected patients under informed consent.
Highlights
Dengue virus is regarded globally as the most important arboviral disease
Polyacrylate microspheres modified with NAS succinimide ester groups were chosen as a substrate carrier as they allowed strong covalent binding with aminated pDNA via a succinimide–amine coupling reaction
poly(styrene-co-acrylic acid) (PSA) microspheres, on the other hand, given their far smaller size, i.e., about 14 to 18 orders of magnitude compared to acrylic microspheres, provided a large specific surface area for the high loading capacity of the AuNP optical amplify reporter label through electrostatic interaction in order to generate the substantial optical amplification effect of the reflectance response upon sandwich DNA detection of targeted DNA sequences
Summary
Dengue virus is regarded globally as the most important arboviral disease. Dengue virus reproduces in temperate climate zones, e.g., in tropic and sub-tropic regions. Traditional laboratory techniques are laborious and time-consuming, especially for the cultivation of dengue viruses, and the confirmation of dengue infection in human normally takes several days up to two weeks, whereas the sample collection can only be done after five days of illness onset They are not able to provide real-time results. Due to the advent of technology, immunoglobulin G (IgG)-based ELISA assays and many rapid diagnostic test kits were developed, which are used for large-scale routine testing. They show flavivirus cross-reactivity, while they are less sensitive and not type-specific [3]. As such, miniaturized, portable and user-friendly detection kits are highly required for high-sensitivity and high-specificity diagnosis of populations sustaining mosquito-borne viral disease
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