A new alcohol oxidase (AOX) enzyme-based formaldehyde biosensor based on acrylic microspheres has been developed. Hydrophobic poly(n-butyl acrylate-N-acryloxy-succinimide) [poly(nBA-NAS)] microspheres, an enzyme immobilization matrix, was synthesized using photopolymerization in an emulsion form. AOX-poly(nBA-NAS) microspheres were deposited on a pH transducer made from a layer of photocured and self-plasticized polyacrylate membrane with an entrapped pH ionophore coated on a Ag/AgCl screen printed electrode (SPE). Oxidation of formaldehyde by the immobilized AOX resulted in the production of protons, which can be determined via the pH transducer. Effects of buffer concentrations, pH and different amount of immobilization matrix towards the biosensor's analytical performance were investigated. The formaldehyde biosensor exhibited a dynamic linear response range to formaldehyde from 0.3-316.2 mM and a sensitivity of 59.41 ± 0.66 mV/decade (R(2) = 0.9776, n = 3). The lower detection limit of the biosensor was 0.3 mM, while reproducibility and repeatability were 3.16% RSD (relative standard deviation) and 1.11% RSD, respectively (n = 3). The use of acrylic microspheres in the potentiometric formaldehyde biosensor improved the biosensor's performance in terms of response time, linear response range and long term stability when compared with thick film immobilization methods.
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 (R(2) = 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 μM. 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.
We report the potential of carbon nanodots (CNDs) as a molecular scaffold for enhancing the antimicrobial activities of small dendritic poly(amidoamines) (PAMAM). Carbon nanodots prepared from sago starch are readily functionalized with PAMAM by using N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Electron microscopy images of these polyaminated CNDs show that they are approximately 30-60nm in diameter. Infrared and fluorescence spectroscopy analyses of the water-soluble material established the presence of the polyamidoaminated moiety and the intrinsic fluorescence of the nanodots. The polyaminated nanodots (CND-PAM1 and CND-PAM2) exhibit in vitro antimicrobial properties, not only to non-multidrug resistant bacteria but also to the corresponding Gram-negative multidrug bacteria. Their minimum inhibitory concentration (MIC) ranges from 8 to 64μg/mL, which is much lower than that of PAMAM G1 or the non-active PAMAM G0 and CNDs. Additionally, they show synergistic effect in combination with tetracycline or colistin. These preliminary results imply that CNDs can serve as a promising scaffold for facilitating the rational design of antimicrobial materials for combating the ever-increasing threat of antibiotic resistance. Moreover, their fluorescence could be pertinent to unraveling their mode of action for imaging or diagnostic applications.
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.
Cell-internalizing peptides (CIPs) can be used to mediate specific delivery of nanoparticles across cellular membrane. The objective of this study was to develop a display technique using hepatitis B virus (HBV) capsid-binding peptide as a "nanoglue" to present CIPs on HBV nanoparticles for cell-targeting delivery. A CIP was selected from a phage display library and cross-linked specifically at the tips of the spikes of the HBV capsid nanoparticle via the "nanoglue" by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Fluorescent oligonucleotides packaged in the nanoparticles and the fluorescein molecules conjugated on the nanoparticles were delivered to cells by using this display technique. This study demonstrated a proof of principle for cell-targeting delivery via "nanoglue" bioconjugation.
One way sunlight causes skin cancer is by suppressing anti-tumor immunity. A major mechanism involves altering mast cell migration via the C-X-C motif chemokine receptor 4-C-X-C motif chemokine ligand 12 (CXCR4-CXCL12) chemokine pathway. We have discovered that pharmacologically blocking this pathway with the CXCR4 antagonist AMD3100 prevents both UV radiation-induced immune suppression and skin cancer. The majority of control mice receiving UV-only developed histopathologically confirmed squamous cell carcinomas. In contrast, skin tumor incidence and burden was significantly lower in AMD3100-treated mice. Perhaps most striking was that AMD3100 completely prevented the outgrowth of latent tumors that occurred once UV irradiation ceased. AMD3100 protection from UV immunosuppression and skin cancer was associated with reduced mast cell infiltration into the skin, draining lymph nodes, and the tumor itself. Thus a major target of CXCR4 antagonism was the mast cell. Our results indicate that interfering with UV-induced CXCL12 by antagonizing CXCR4 significantly inhibits skin tumor development by blocking UV-induced effects on mast cells. Hence, the CXCR4-CXCL12 chemokine pathway is a novel therapeutic target in the prevention of UV-induced skin cancer.