Ru-Qin Yu

A New Fluorescence Immunosensing Method Based on Aptamer-plasmid Complex Amplification

Abstract A novel simple, sensitive fluorescence immunosensing method based on aptamer-plasmid complex amplification was developed. This method used the specific recognition between antibody and antigen, as well as aptamer-plasmid complex, and the intercalation of fluorescence dye SYBR Green I in the groove of duplex plasmid DNA in detection of platelet-derived growth factor-BB (PDGF-BB). The immunoassay was performed in the microtiter wells in which rabbit anti-PDGF-BB antibody was immobilized. The PDGF-BB analyte was captured by the primary antibody and then sandwiched by the aptamer-plasmid DNA complex. The introduction of SYBR Green I fluorescence dye allowed for the detection of the sandwiched complex of antibody/antigen/aptamer-plasmid. The optimized salt concentration and ratio of aptamer to PUC19 and SYBR Green I concentration were also analyzed. A linear relationship for the detection of PDGF-BB was at the range from 0.2 to 200 ng ml −1 with a detection limit of 0.1 ng ml −1 .

Highly Sensitive Fluorescent Aptasensor for Thrombin Detection Based on Competition Triggered Rolling Circle Amplification

Abstract Based on the competition reaction of target protein, aptamer probe, padlock probe and the complementary sequence, a highly sensitive fluorescent aptasensor was developed in this study in combination with rolling circle amplification. In the absence of target protein, the ligation-rolling circle amplification reaction was repressed because the complementary sequence hybridized with aptamer probe to form double-stranded duplex. On the contrary, in the presence of target protein, the target molecules bond specifically with its aptamer probe, inducing the displacement of the complementary sequence and hybridization with padlock probe. The padlock probe was circularized with the assistance of Escherichia coli DNA ligase, and the rolling circle amplification process could be accomplished by Phi29 DNA polymerase. The amplification product contained thousands of repeated sequences which could hybridize with the loop of molecular beacons (the detection probes), resulting in a significant fluorescence signal. The effects of length of complementary DNA (CDNA) sequence and concentration of padlock probe were investigated. Under the optimized experimental conditions, the model target protein of thrombin was highly sensitively detected by the proposed aptasensing system in a linear range of 0.067–32.4 nM with a detection limit of 0.03 nM (approximately 90 amol target molecules). Moreover, the present sensing method was universal for other target analysis by skillful design of the sequence of aptamer probe and related oligonucleotides.

Aptamer-based Liquid Crystal Biosensor for Detection of Platelet-derived Growth Factor BB

Abstract A novel liquid crystal (LC) biosensor was developed for the detection of platelet-derived growth factor BB (PDGF-BB) based on the orientation changes of liquid crystals. One glass slide of the LC cell was first modified with the APTES/DMOAP ((3-aminopropyl)trimethoxysilane/N,N-dimethyl-N-octadecyl(3-aminopropyl) trimethoxysilyl chloride) self-assembled monolayer (SAM) to trigger the homeotropic alignment of LC molecules and thereby produced a black background optical image under the crossed polarized light, and then the specific aptamer of PDGF-BB was immobilized on SAM through glutaraldehyde crosslinking to construct a LC sensing substrate. In the presence of PDGF-BB, the stable triple helix structure of PDGF-BB aptamer was formed by the specific binding event and thus led to the target PDGF-BB was captured to the sensing substrate, making a visible optical change which could be observed under the crossed polarized light via the huge steric effect of the PDGF-BB on disrupting the LC alignment. However, in the absence of PDGF-BB, the low packing density of the PDGF-BB aptamer had little effect on disrupting the ordered alignment to the LC and caused the black background optical image which could also be observed under the crossed polarized light. An obvious optical change was observed when the concentration of target PDGF-BB was 5 nM. The proposed LC biosensing method permits the label-free detection of PDGF-BB with good selectivity and high sensitivity, making them sufficiently simple and particularly useful for low-cost screening bioassay performed away from central laboratories.

Rapid Determination of Bitertanol Content in Banana Using Three-dimensional Fluorescence Coupled with Second-order Calibration

Abstract A novel approach was developed for the direct quantitative analysis of bitertanol in banana by the incorporation of three-dimensional fluorescence with second-order calibration methodologies based on alternating trilinear decomposition (ATLD), alternating fitting residue (AFR), and self-weighted alternating trilinear decomposition (SWATLD) algorithms, respectively. The average recoveries of bitertanol in banana extract by using ATLD, AFR and SWATLD with an estimated component number of two were 98.2% ± 2.6%, 97.6% ± 2.1%, and 91.5% ± 3.5%, respectively. Moreover, the accuracy of these three algorithms was also evaluated through elliptical joint confidence region tests (EJCR) as well as figures of merit, such as sensitivity (SEN), selectivity (SEL), and limit of detection (LOD). The results demonstrate that these algorithms have been successfully applied to determine the content of bitertanol in banana extract.

Simultaneous Determination of Warfarin and Aspirin Contents in Biological Fluids Using Excitation-Emission Matrix Fluorescence Coupled with a Second-order Calibration Method

In the present work, a practical method that combines excitation-emission matrix fluorescence with a second-order calibration method based on an alternating trilinear decomposition (ATLD) algorithm was developed in order to simultaneously and directly determine the contents of warfarin (WAR) and aspirin (ASA) in human plasma and urine samples, even in the presence of unknown interferences. With the pre-estimated component number of 4, the obtained average spiked recoveries were 105.4 ± 7.8 and 104.2 ± 8.3% for WAR, 96.5 ± 2.8 and 91.2 ± 2.3% for ASA in human plasma and urine samples, respectively. Furthermore, the figures of merit were calculated and also inter- and intra-day experiments were performed that proved the proposed method is of great significance to the monitoring of clinical administration and also being a simple sample pretreatment at low-cost.

Application of Inorganic Layered Materials in Electrochemical Sensors

Abstract Inorganic layered materials with low dimensional opened structure and good biological compatibility and stability are considered as one of the most promising materials for immobilizing biomolecules, which can not only adjust the spacing of layers according to guest molecule size to prevent leakage of guest molecule, but also reduce the effects of surrounding elements on immobilized molecules significantly. Moreover, they can promote electron transfer between the enzyme and the electrode. Therefore, the inorganic layered materials have great potential application in electrochemical sensors. This paper reviews the ways of immobilizing guest molecules on inorganic layered materials and the applications and development prospects of the different types of inorganic layered materials including cationic, anionic and nonionic inorganic layered materials in electrochemical sensors.

Influence of pH Value and Anion on Surface-Enhanced Raman Scattering of 2,6-Pyridinedicarboxylic Acid on Gold Nanoparticle Surface

Abstract Surface-enhanced Raman scattering (SERS) was used for the detection of 2,6-pyridinedicarboxylic acid (DPA), a biomarker for bacterial spores. By immobilizing gold nanoparticles of diameter 60 nm on a polished Au electrode using PVP as an adhesive layer, the steady and highly sensitive SERS substrates were fabricated. By using strong acid and alkali to adjust the pH value of DPA molecules, the molecular adsorption configuration of DPA via the SERS characteristics under various pH values were studied and the changes of SERS intensity were compared when HCl, H 2 SO 4 and HNO 3 were separately added to the DPA solution to get a pH value of 1.3. The changes of the adsorption form were also analyzed when NaCl, MgSO 4 and NaNO 3 were separately added to the DPA solution. The results showed that the anion of acids could play a role as a bridge when DPA molecules were protonated by the addition of acids, which could greatly enhance the SERS signal; however, when the pH value is greater than the second dissociation constant of DPA, because of steric effect and the disappearance of the bridge, the SERS signal decreases gradually. Compared with NO 3 − and SO 4 2- , Cl − has a much stronger binding force with protonated N on DPA; consequently, the addition of HCl can get the maximum SERS signal enhancement.