Novel Stable DNA Nanoscale Material and Its Application on Specific Enrichment of DNA.

DNA nanostructures are a new type of technology for constructing nanomaterials that has been developed in recent years. By relying on the complementary pairing of DNA molecules to form a double-stranded property, DNA molecules can construct a variety of nanoscale structures of 2D and 3D shapes. However, most of the previously reported DNA nanostructures rely solely on hydrogen bonds to maintain structural stability, resulting in DNA structures that can be maintained only at low temperature and in the presence of Mg2+, which greatly limits the application of DNA nanostructures. This study designed a DNA nanonetwork structure (nanonet) and changed its topological structure to DNA nanomesh by using DNA topoisomerase to make it thermally stable, while escaping the dependence on Mg2+, and the stability of the structure can be maintained in a nonsolution state. Moreover, the nanomesh also has a large amount of ssDNA (about 50%), providing active sites capable of exerting biological functions. Using the above characteristics, we prepared the nanomesh into a device capable of adsorbing specific DNA molecules, and used the device to enrich DNA. We also tried to mount antibodies using DNA probes. Preliminary results show that the DNA nanomesh also has the ability to enrich specific proteins.

A sensitive sandwich-type immunosensor for the detection of MCP-1 based on a rGO-TEPA-Thi-Au nanocomposite and novel RuPdPt trimetallic nanoalloy particles.

A novel electrochemical immunosensor was proposed for the detection of monocyte chemoattractant protein-1 (MCP-1), a biomarker of cardiovascular disease. Due to thionine (Thi) possessing electroactive redox properties, a one-step approach was utilized to synthesize a reduced graphene oxide-tetraethylene-thionine-Au (rGO-TEPA-Thi-Au) nanocomposite at room temperature using the synergistic effect of Thi and rGO-TEPA towards HAuCl4. We obtained the excellent matrix material, which immobilized more primary antibody MCP-1-Ab1 on rGO-TEPA on a modified glassy carbon electrode (GCE). To further enhance the sensitivity of the sensor, a novel signal generation and amplification strategy was developed for detection. RuPdPt trimetallic nanoalloy particles (RuPdPt TNPs), a novel nanomaterial, were synthesized by a one-pot method, displayed a uniform morphology as well as good electrochemical activity and bound with the secondary antibodies against MCP-1 via the Pt-NH2 bond. Most importantly, RuPdPt TNPs have a significant ability to catalyze H2O2 to produce an electron. The electrochemical signal was highly amplified because the electrochemical signal was primarily derived from the synergistic catalysis of H2O2 by RuPdPt TNPs and recorded by chronoamperometry. Under the optimal conditions, this newly designed biosensor exhibited sensitive detection of MCP-1 in the range from 20 fg mL-1 to 1000 pg mL-1, with a detection limit of 8.9 fg mL-1 (based on a S/N = 3). Additionally, the designed immunosensor showed acceptable selectivity, reproducibility and stability. This immunosensor is a promising strategy for analyzing clinical serum samples in the future.

Efficient preparation and site-directed immobilization of VHH antibodies by genetic fusion of poly(methylmethacrylate)-binding peptide (PMMA-Tag).

A PMMA-binding peptide (PMMA-tag) was genetically fused with the C-terminal region of an anti-human chorionic gonadotropin (hCG) single-domain antibody (VHH). It was over-expressed in an insoluble fraction of E. coli cells, and recovered in the presence of 8 M urea via one-step IMAC purification. Monomeric and denatured PMMA-tag-fused VHH (VHH-PM) was successfully prepared via the reduction and oxidation of VHH-PM at a concentration less than 1 mg/mL in the presence of 8 M of urea. Furthermore, the VHH-PM was refolded with a recovery of more than 95% by dialysis against 50 mM TAPS at pH 8.5, because the genetic fusion of PMMA-tag resulted in a decrease in the apparent isoelectric point (pI) of the fusion protein, and its solubility at weak alkaline pH was considerably increased. The antigen-binding activities of VHH-PM in the adsorptive state were 10-fold higher than that of VHH without a PMMA-tag. The density of VHH-PM on a PMMA plate was twice that of VHH, indicating that the site-directed attachment of a PMMA-tag resulted in positive effects to the adsorption amount as well as to the orientation of VHH-PM in its adsorptive state. The preparation and immobilization methods for VHH-PM against hCG developed in the present study were further applied to VHH-PMs against four different antigens, and consequently, those antigens with the concentrations lower than 1 ng/mL were detected by the sandwich ELISA. Thus, the VHH-PMs developed in the present study are useful for preparation of high-performance and economical immunosorbent for detection of biomarkers.

Development of a rapid capture-cum-detection method for Escherichia coli O157 from apple juice comprising nano-immunomagnetic separation in tandem with surface enhanced Raman scattering.

A combined capture and detection method comprising of nano-immunomagnetic separation (NIMS) and surface enhanced Raman spectroscopy (SERS) was developed to detect Escherichia coli O157 from liquid media including apple juice. The capture antibodies (cAbs) were immobilized on magnetite-gold (Fe3O4/Au) magnetic nanoparticles (MNPs) which were used for separation and concentration of the E. coli O157 cells from model liquid food matrix. The capture efficiency (CE) for E. coli O157 using MNP was found to be approximately 84-94%. No cross reactivity was observed with background non-target organisms. There was a significant difference in the mean CE of bacteria captured by MNP and commercially sourced immunomagnetic microbeads (p<0.05). For the detection of target pathogen, SERS labels were prepared by conjugating gold nanoparticles with Raman reporter molecules and the detector antibody (dAb). Au-Raman label-dAb was interacted with gold coated MNP-cAb-E. coli O157 complex. The ability of this immunoassay to detect E. coli O157 in apple juice was investigated. We have successfully applied the synthesized Fe3O4/Au nanoclusters to E. coli O157 detection in apple juice using the SERS method. The lowest detectable bacterial cell concentration in apple juice was 10(2)CFU/mL with a total analysis time of less than an hour. This method presents a convenient way of preconcentration, separation, and detection of low levels of target pathogen from liquid food matrix.

A carbon nanotube screen-printed electrode for label-free detection of the human cardiac troponin T.

Label-free immunosensor based on amine-functionalized carbon nanotubes screen-printed electrode is described for detection of the cardiac troponin T, an important marker of acute myocardial infarction. The disposable sensor was fabricated by tightly squeezing an adhesive carbon ink containing carbon nanotubes onto a polyethylene terephthalate substrate forming a thin film. The use of carbon nanotubes increased the reproducibility and stability of the sensor, and the amine groups permitted nonrandom immobilization of antibodies against cardiac troponin T. Amperometric responses were obtained by differential pulse voltammetry in presence of a ferrocyanide/ferricyanide redox probe after troponin T incubation. The calibration curve indicated a linear response of troponin T between 0.0025 ng mL(-1) and 0.5 ng mL(-1), with a good correlation coefficient (r=0.995; p<0.0001, n=7). The limit of detection (0.0035 ng mL(-1) cardiac troponin T) was lower than any previously described by immunosensors and was comparable with conventional analytical methods. The high reproducibility and clinical range obtained using this immunosensor support its utility as a potential tool for point-of-care acute myocardial infarction diagnostic testing.

Establishment of an immunoaffinity chromatography for simultaneously selective extraction of Sudan I, II, III and IV from food samples.

The establishment of an immunoaffinity chromatography (IAC) for simultaneously selective extraction of four illegal colorants Sudan dyes (Sudan I, II, II and IV) from food samples was described. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against Sudan I to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. It was observed that IAC column was able to separately capture Sudan I, II, III and IV with maximum capacity of 295, 156, 184 and 173ng, respectively. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the extraction recoveries of the IAC column for Sudan I-IV at two different spiked concentrations were within 95.3-106.9%. After 50 times repeated usage, 64% of the maximum capacity was still remained. Six food samples randomly collected from local supermarket without spiking Sudan dyes were extracted with IAC column and detected by high performance liquid chromatography (HPLC). It was found that there was no detectable Sudan II, III and IV in all six food samples, but Sudan I with the content of 2.7-134.5ngg(-1) was detected in three food samples. To further verify the extraction efficiency, other three negative samples were spiked with Sudan I-IV at the concentrations of 20ngg(-1) and 50ngg(-1), which were then extracted with IAC column. The extraction recoveries and relative standard deviation (RSD) were 68.6-96.0% and 4.8-15.2%, respectively, demonstrating the feasibility of the prepared IAC column for Sudan dyes extraction.