Sandwich-Type DNA Micro-Optode Based on Gold–Latex Spheres Label for Reflectance Dengue Virus Detection

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.

Biosensing Technology for Dengue Virus Detection

“…检测登革热病毒的荧光生物传感器, 在各种光学生 物传感器中最受欢迎, 因为这种传感方法具有高准确性 和敏感性 [65] . Oliveira 等 [87] 将荧光信号分子和寡核苷酸 组合构建了一种适体/核酸酶传感技术, 当捕获探针和 靶核酸特异性结合时, 可在 20 min 后释放出荧光信号, 通过核酸酶切割, 可实现信号放大.…”

A Review on the Development of Gold and Silver Nanoparticles-Based Biosensor as a Detection Strategy of Emerging and Pathogenic RNA Virus

“…In biochemical sensors, an additional layer is added between the transducer and the recognition elements to reduce non-specific interaction, which is based on the white-light interference of visible radiation reflected on both interfaces of a layer [ 103 ]. A study by Jeningsih et al [ 104 ] has demonstrated a sandwich hybridization strategy of DNAs using a serotype-specific and ultrasensitive microspheres-based DNA opto-sensor to detect dengue virus (DENV). The succinimide-functionalized poly(n-butyl acrylate) (poly(nBA-NAS) microspheres were employed as the DNA hybridization platform and the gold-latex (AuNPs-PSA) sphere was employed as an optical amplification label.…”

“…Based on the table, all the biosensors demonstrated high sensitivity with a low LOD in the range of fM to nM. The lowest LOD can be observed by the reflectance-based biosensor, which showed the LOD value of 1 × 10 −29 M via a sandwich recognition procedure [ 104 ]. However, the biosensor was susceptible to nucleic acid degradation, resulting in unexpected errors in actual nucleic acid detection.…”

“… Schematic diagram showing the one-step sandwich hybridization recognition procedure, which involves a DNA capture probe (pDNA) immobilized on the acrylic microspheres, and target (cDNA) and reporter (rDNA) probes labeled with gold nanoparticle–poly(styrene- co -acrylic acid) latex (AuNP–PSA) spheres. Reproduced from Jeningsih et al [ 104 ]. …”

“…In biochemical sensors, an additional layer is added between the ducer and the recognition elements to reduce non-specific interaction, which is ba the white-light interference of visible radiation reflected on both interfaces of [103]. A study by Jeningsih et al [104] has demonstrated a sandwich hybrid The fabrication of the paper-based colorimetric DNA biosensor was assembled using the principles of origami, which consisted of two layers. A wax printing technique was used to create PADs, and the Whatman Grade 1 filter paper was used to print the wax design.…”