Designing DNA probe from HPV 18 and 58 in the E6 region for sensing element in the development of genosensor-based gold nanoparticles.

The E6 region has higher protuberant probability annealing than consensus probe focusing on another region in the human papillomavirus (HPV) genome in terms of detection and screening method. Here, we designed the first multiple virus single-stranded deoxyribonucleic acid (ssDNA) for multiple detections in an early phase of screening for cervical cancer in the E6 region and became a fundamental evolution of detection electrochemical HPV biosensor. Gene profiling of the virus ssDNA sequences has been carried by high-end bioinformatics tools such as GenBank, Basic Local Alignment Searching Tools (BLAST), and Clustal OMEGA in a row. The output from bioinformatics tools resulted in 100% of similarities between our virus ssDNA probe and HPV complete genome in the databases. The cross-validation between HPV genome and our designed virus ssDNA provided high specificity and selectivity during screening methods compared with Pap smear. The DNA probe for HPV 18, 5' COOH-GAT CCA GAA GGT ACA GAC GGG GAG GGC ACG 3', while 5'COOH-GGG CGC TGT GCA GTG TGT TGG AGA CCC CGA3' as DNA probe for HPV 58 designed with 66.77% guanine (G) and cytosine (C) content for both. Our virus ssDNA probe for the HPV biosensor promises high sensitivity, specificity, selectivity, repeatability, low fluid consumption, and will be useful in mini-size diagnostic devices for cervical cancer detection.

Cardiac Biomarkers: Invasive to Non-invasive Assessments.

Cardiovascular disease (CVD) is a major threat to global health, estimated to be the cause 30 % (17.3 million in 2008) of deaths every year, and the number of deaths caused by CVD is expected to increase further, reaching 23.3 million by 2030. Hence, there is a growing demand for simpler sample extraction, rapid screening results, and intervention of the subsequent analysis in emergency units. In this paper, we reviewed CVD biomarkers in blood- and saliva-based specimens. The history of cardiac biomarkers indicates that in the beginning, cardiac troponin I (cTnI) was a widely accepted 'gold standard' marker due to its high specificity and selectivity. Considering the advantages of salivary-based cardiac biomarkers, we examined correlations between non-invasive (salivary) and invasive (blood) diagnoses, and it was found that C-reactive protein (CRP) provides a better correlation. Despite the low abundance of salivary CRP, several reports displayed the detection limit down to pg/ml using existing technologies. Thus, salivary CRP has the potential to be used for future forefront diagnostics for the early assessment of cardiac risks.

Biotechnological Aspects and Perspective of Microbial Keratinase Production.

Keratinases are proteolytic enzymes predominantly active when keratin substrates are available that attack disulfide bridges in the keratin to convert them from complex to simplified forms. Keratinases are essential in preparation of animal nutrients, protein supplements, leather manufacture, textile processing, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical and biomedical industries, and waste management. Accordingly, it is necessary to develop a method for continuous production of keratinase from reliable sources that can be easily managed. Microbial keratinase is less expensive than conventionally produced keratinase and can be obtained from fungi, bacteria, and actinomycetes. In this overview, the expansion of information about microbial keratinases and important considerations in keratinase production are discussed.

Platelet-derived growth factor oncoprotein detection using three-dimensional carbon microarrays.

The potential of aptamers as ligand binding molecule has opened new avenues in the development of biosensors for cancer oncoproteins. In this paper, a label-free detection strategy using signaling aptamer/protein binding complex for platelet-derived growth factor (PDGF-BB) oncoprotein detection is reported. The detection mechanism is based on the release of fluorophore (TOTO intercalating dye) from the target binding aptamer's stem structure when it captures PDGF. Amino-terminated three-dimensional carbon microarrays fabricated by pyrolyzing patterned photoresist were used as a detection platform. The sensor showed near linear relationship between the relative fluorescence difference and protein concentration even in the sub-nanomolar range with an excellent detection limit of 5pmol. This detection strategy is promising in a wide range of applications in the detection of cancer biomarkers and other proteins.

Effective surface functionalization of nanocrystalline diamond films by direct carboxylation for PDGF detection via aptasensor.

An aptasensor was designed on a nanocrystalline diamond (NCD) surface that combined with biological recognition elements, PDGF-binding aptamers, which inherently possess high affinity to PDGF-BB proteins. Functional components such as carboxylic acids (-COOH) and amines (-NH2) were directly introduced onto the NCD surface and used as probing units for immobilization of PDGF-binding aptamers. The surface coverage of different components on the NCD was analyzed by X-ray photoelectron spectroscopy (XPS) measurements, and the effects of various functionalizations on the NCD biosensor performance were investigated via fluorescence observations. The coverages of carboxyl and amine groups achieved were 12 and 23%, respectively, for the directly aminated and carboxylated NCD; however, the lower density of carboxyl groups on the functionalized surface did not deteriorate the performance of the COOH-NCD biosensor. Fluorescence investigations demonstrated comparable performance in sensitivity and selectivity for PDGF protein detection on COOH-NCD and NH2-NCD biosensors. Multiple regeneration tests clearly showed that the COOH-NCD biosensor as well as the NH2-NCD biosensor retained a high performance without exhibiting any noticeable degradation.

Highly sensitive detection of platelet-derived growth factor on a functionalized diamond surface using aptamer sandwich design.

Aptamer-based fluorescence detection of platelet-derived growth factor (PDGF) on a functionalized diamond surface was demonstrated. In this work, a sandwich design based on the ability of PDGF to bind with aptamers at its two available binding sites was employed. It was found that this sandwich design approach significantly increases the fluorescence signal intensity, and thereby a very low detection limit of 4 pM was achieved. The effect of the ionic strength of MgCl(2) buffer solution was also investigated, and the most favourable binding for PDGF-BB occurred at a Mg(2+) concentration of 5.5 mM. Since the aptamers bind to the target PDGF with high affinity, fluorescence detection exhibited high selectivity towards different biomolecules. The high reproducibility of detection was confirmed by performing three cycles of measurements over a period of three days.

Boron δ-doped (111) diamond solution gate field effect transistors.

A solution gate field effect transistor (SGFET) using an oxidised boron δ-doped channel on (111) diamond is presented for the first time. Employing an optimised plasma chemical vapour deposition (PECVD) recipe to deposit δ-layers, SGFETs show improved current-voltage (I-V) characteristics in comparison to previous similar devices fabricated on (100) and polycrystalline diamond, where the device is shown to operate in the enhancement mode of operation, achieving channel pinch-off and drain-source current saturation within the electrochemical window of diamond. A maximum gain and transconductance of 3 and 200µS/mm are extracted, showing comparable figures of merit to hydrogen-based SGFET. The oxidised device shows a site-binding model pH sensitivity of 36 mV/pH, displaying fast temporal responses. Considering the biocompatibility of diamond towards cells, the device's highly mutable transistor characteristics, pH sensitivity and stability against anodic oxidation common to hydrogen terminated diamond SGFET, oxidised boron δ-doped diamond SGFETs show promise for the recording of action potentials from electrogenic cells.

Aptamer-based biosensor for sensitive PDGF detection using diamond transistor.

The detection of platelet-derived growth factor (PDGF) via a solution-gate field-effect transistor (SGFET) has been demonstrated for the first time using aptamers immobilized on a diamond surface. Upon introduction of PDGF to the immobilized aptamer, a shift of 31.7 mV in the negative direction is observed at a source-drain current of -50 µA. A shift of 32.3 mV in the positive direction is detected after regeneration by SDS solution, indicating that the static measurement returns to its original value. These SGFETs operate stably within the large potential window of diamond (>3.0 V), and hence the surface channel does not need passivating with a thick insulating layer. Thereof, the immobilized aptamer channels have been exposed directly to the electrolyte solution without a gate insulator. Immobilization is achieved via aptamers covalently bonding to amine sites, thereby increasing the sensitivity of the biosensors. Diamond SGFETs have potential for the detection of PDGF and show durability against biological degradation after repeated usage and regeneration.