A regenerable electrochemical DNA biosensor based on a new type of acrylic microspheres and gold nanoparticles (AuNPs) composite coated onto a screen printed electrode (SPE) has been successfully developed for specific determination of the 35 S promoter from cauliflower mosaic virus (CaMV 35S) gene in soybean. DNA probe was immobilised onto acrylic microspheres via covalent bonding. The presence of modified gene in soybean can be detected via hybridisation of CaMV 35S gene-modified DNA with immobilised DNA probe, which was monitored by differential pulse voltammetry of anthraquinone-2-sulfonic acid monohydrate sodium salt (AQMS) as redox indicator during hybridisation event. The peak current signal of AQMS was linearly related to the target CaMV 35S gene concentration over the range of 2×10−15 to 2×10−9M (R2=0.982) with a very low concentration detect limit (7.79×10−16M). The recovery test showed satisfactory results of 94.6±5.1–105.4±4.9% (n=5) when the biosensor was used for the determination of genetically modified (GM) DNA sequences extracted from GM soybean samples. The DNA biosensor showed good reproducibility (relative standard deviation (RSD) below 5.0%, n=5) and regenerability (RSD below 5.0%, n=7). The biosensor response was stable up to 45 days of storage period at 4°C. The main advantages of this biosensor design are very low detection limit and capability of reusing the biosensor for at least seven times after regeneration with mild sodium hydroxide

Molecularly imprinted polymers (MIPs) are generally a more stable material for sensing application. The high selectivity and sensitivity of MIPs for sensors can be achieved if the template molecule is imprinted in the polymer and this makes them an ideal alternative as a recognition element for sensors. A new electrochemical sensor based on molecularly imprinted polymeric microspheres (MIPs) and multi-walled carbon nanotube/gold nanoparticle (MIP–MWCNT–AuNP) modified carbon screen-printed electrodes (SPEs) for the rapid detection of 17β-estradiol (E) hormone in serum samples has been successfully developed. Hydrophobic MIPs were synthesized using photopolymerization in emulsion form. The multi-walled carbon nanotube grafted with gold nanoparticles was firstly deposited onto a carbon screen-printed electrode for the purpose of accelerating electron transfer to the surface of the electrode. The MIP microspheres specific to the 17β-estradiol hormone, prepared via a facile photopolymerization technique, were coated onto the MWCNT–AuNP modified SPE. The presence of 17β-estradiol in biological samples could be detected with the sensor via absorption of 17β-estradiol into the deposited MIPs and this was monitored by differential pulse voltammetry (DPV) at 0.6 V for the reduction of 17β-estradiol. Under optimal conditions, the sensor could detect the concentrations of 17β-estradiol from 1.0 × 10⁻¹⁵ to 1.0 × 10⁻⁶ M (R² = 0.9921), with a detection limit of 2.5 × 10⁻¹⁶ M. The sensor based on MIP microspheres and the MWCNT–AuNP modified electrode demonstrated a stability of 55 days with good reproducibility (RSD < 5%, n = 5) and regenerability (RSD < 4%, n = 5). Using this sensor, the gender of the arowana fish determined via the level of 17β-estradiol using fish serum samples demonstrated good agreement with a conventional test kit based on the immuno-assay method.

An electrochemical microbiosensor for DNA has been fabricated based on new acrylic microspheres modified with reactive N-acryloxysuccinimide (NAS) functional groups. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesized in an emulsion form with a simple one-step photopolymerization technique. Aminated DNA probe was attached to the succinimde functional group of the acrylic microspheres via covalent bonding. The hybridization of the immobilized DNA probe with the complementary DNA was studied by differential pulse voltametry using anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) as the electroactive hybridization label. The influences of many factors such as duration of DNA probe immobilization and hybridization, pH, type of ions, buffer concentrations, ionic strength, operational temperature and non-complementary DNA on the biosensor performance were evaluated. Under optimized conditions, the DNA microbiosensor demonstrated a linear response range to target DNA over a wide concentration range of 1.0 × 10⁻¹⁶ and 1.0 ´ 10⁻⁸ M with a lower limit of detection (LOD) of 9.46 ´ 10⁻¹⁷ M (R² = 0.97). This DNA microbiosensor showed good reproducibility with 2.84% RSD (relative standard deviation) (n = 3). Application of the NAS-modified acrylic microspheres in the construction of DNA microbiosensor had improved the overall analytical performance of the resultant DNA microbiosensor when compared with other reported DNA biosensors using other nano-materials for membranes and microspheres as DNA immobilization matrices.

New acrylic microspheres were synthesised by photopolymerisation where the succinimide functional group was incorporated during the microsphere preparation. An optical biosensor for urea based on reflectance transduction with a large linear response range to urea was successfully developed using this material. The biosensor utilized succinimide-modified acrylic microspheres immobilized with a Nile blue chromoionophore (ETH 5294) for optical detection and urease enzyme was immobilized on the surface of the microspheres via the succinimide groups. No leaching of the enzyme or chromoionophore was observed. Hydrolysis of the urea by urease changes the pH and leads to a color change of the immobilized chromoionophore. When the color change was monitored by reflectance spectrophotometry, the linear response range of the biosensor to urea was from 0.01 to 1,000 mM (R² = 0.97) with a limit of detection of 9.97 mM. The biosensor response showed good reproducibility (relative standard deviation = 1.43%, n = 5) with no interference by major cations such as Na⁺, K⁺, NH₄⁺ and Mg²⁺. The use of reflectance as a transduction method led to a large linear response range that is better than that of many urea biosensors based on other optical transduction methods.

Ammonia analysis based on complexing ammonia with Cu²+ ions by UV-Vis spectrophotometry. This research aims to obtain uptake at the maximum wavelength of the copper-ammonia complex, optimum conditions and validation of UV-Vis spectrophotometry. The reaction between Cu²⁺ ions which are blue with ammonia colorless occur in solution to form complex compounds [Cu(NH3)4]²+ dark blue, the maximum absorbance is obtained at a wavelength of 615 nm. Optimization of the ammonia complexing the Cu²+ ions provide optimum conditions a solution of Cu²+ concentrations of 0.01 M in ammonia concentration of 0.04 M, pH 7, and the optimum time to form a complex occurred in the 30th minute and complex compounds can be stable for 90 minutes (± 1 hours 30 minutes). The analytical method validation ammonia using Cu²⁺ ions provide linear regression equation y = 4,772 x + 0.333 with R² = 0.989 ammonia concentration range of 0.003 M to 0.08 M; LOD 0.01 M; LOQ 0.04 M; % RSD = 1.32%; and% recovery = 102.03%. The concentration of ammonia in the wastewater sample application obtained the ammonia concentration of 0.04 M. Based on the results of the validation, Ammonia solution with Cu²⁺ ion as a complex used for the determination of ammonia levels by UV-Vis spectrophotometry.

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.