Optical DNA Biosensor Based on Square-Planar Ethyl Piperidine Substituted Nickel(II) Salphen Complex for Dengue Virus Detection

Abstract

A sensitive and selective optical DNA biosensor was developed for dengue virus detection based on novel square-planar piperidine side chain-functionalized N,N'-bis-4-(hydroxysalicylidene)-phenylenediamine-nickel(II), which was able to intercalate via nucleobase stacking within DNA and be functionalized as an optical DNA hybridization marker. 3-Aminopropyltriethoxysilane (APTS)-modified porous silica nanospheres (PSiNs), was synthesized with a facile mini-emulsion method to act as a high capacity DNA carrier matrix. The Schiff base salphen complexes-labelled probe to target nucleic acid on the PSiNs renders a colour change of the DNA biosensor to a yellow background colour, which could be quantified via a reflectance transduction method. The reflectometric DNA biosensor demonstrated a wide linear response range to target DNA over the concentration range of 1.0 × 10^-16-1.0 × 10^-10 M (R² = 0.9879) with an ultralow limit of detection (LOD) at 0.2 aM. The optical DNA biosensor response was stable and maintainable at 92.8% of its initial response for up to seven days of storage duration with a response time of 90 min. The reflectance DNA biosensor obtained promising recovery values of close to 100% for the detection of spiked synthetic dengue virus serotypes 2 (DENV-2) DNA concentration in non-invasive human samples, indicating the high accuracy of the proposed DNA analytical method for early diagnosis of all potential infectious diseases or pathological genotypes.

Keywords: dengue virus detection; nickel(II) salphen complex; optical DNA biosensor; porous silica nanospheres; reflectance measurement; synthetic DNA binder.

Figure 1

The schematic diagram representing the development of reflectance dengue virus DNA biosensor based on the use of optical Schiff base metal salphen complex synthetic DNA label.

Figure 2

The chemical reactions involved in the synthesis of ethyl piperidine substituted nickel(II) salphen complex: (a) Chemical reaction between 1,2-diamino-benzene and 2,4-dihydroxybenzaldehyde to form the salphen ligand followed by reaction with Ni(OAc)₂•4H₂O to form the nickel(II) salphen complex; (b) The nickel(II) salphen complex further reacted with 1-(2-chloroethyl) piperidine hydrochloride in the presence of potassium carbonate to obtained the desired piperidine side chain functionalized nickel(II) salphen complex.

Figure 3

FESEM micrograph of the as-synthesized APTS-functionalized silica particles captured by JEOL FESEM at 15 kV acceleration voltage and 30 kX magnification.

Figure 4

Dengue virus DNA biosensor reflectance spectra before (a) and after (b) hybridization with 1 × 10⁻⁷ µM target containing 1 mM ethyl piperidine substituted nickel(II) salphen complex at pH 7.0.

Figure 5

(a) Effect of PSiNs loading on the reflectometric response of the dengue virus DNA biosensor, (b) the reflectance response curve of the DNA biosensor with immobilized DNA probe between 0.5 µM and 4.0 µM towards the detection of 1 × 10⁻⁷ µM target DNA and (c) the effect of ethyl piperidine substituted nickel(II) salphen complex loading on the DNA hybridization response using 2.5 µM immobilized DNA probe and 1 mM metal complex binder.

Figure 5

(a) Effect of PSiNs loading on the reflectometric response of the dengue virus DNA biosensor, (b) the reflectance response curve of the DNA biosensor with immobilized DNA probe between 0.5 µM and 4.0 µM towards the detection of 1 × 10⁻⁷ µM target DNA and (c) the effect of ethyl piperidine substituted nickel(II) salphen complex loading on the DNA hybridization response using 2.5 µM immobilized DNA probe and 1 mM metal complex binder.

Figure 6

(a) Effect of Na-phosphate buffer capacity on the DNA hybridization response of the reflectance DNA biosensor at pH 7.0 using 1 × 10⁻⁷ µM cDNA and 2 mM metal complex marker and (b) pH profile of the optical DNA biosensor from pH 5.0–pH 8.0 using 0.07 M Na-phosphate buffer in the determination of 1 × 10⁻⁷ µM cDNA containing 2 mM synthetic DNA label.

Figure 7

(a) DNA hybridization duration study carried with 0.07 M Na-phosphate buffer (pH 7.0) containing 1 × 10⁻⁷ µM cDNA and 2 mM synthetic DNA binder, (b) long-term stability profile of the optical dengue virus DNA biosensor in the quantification of 1 × 10⁻⁷ µM cDNA containing 2 mM metal complex at neutral pH, (c) dynamic linear response range of the optical dengue virus DNA biosensor (inset shows the corresponding reflectance spectra generated by the DNA biosensor in the cDNA concentration range of 1 × 10⁻¹⁰–1 × 10⁻² µM and 2 mM ethyl piperidine substituted nickel(II) salphen complex DNA hybridization marker) and (d) the selectivity of the DNA biosensor towards target DNA, ncDNA and mismatch DNA (mmDNA) at 1 × 10⁻⁸ µM at pH 7.0.