Metabolite Profiling to Evaluate Metabolic Changes in Genetically Modified Protopanaxadiol-Enriched Rice.

Event DS rice producing protopanaxadiol (PPD) has been previously developed by inserting Panax ginseng dammarenediol-II synthase gene (PgDDS) and PPD synthase gene (CYP716A47). We performed a gas chromatography-mass spectrometry (GC-MS)-based metabolomics of the DS rice to identify metabolic alterations as the effects of genetic engineering by measuring the contents of 65 metabolites in seeds and 63 metabolites in leaves. Multivariate analysis and one-way analysis of variance between DS and non-genetically modified (GM) rice showed that DS rice accumulated fewer tocotrienols, tocopherols, and phytosterols than non-GM rice. These results may be due to competition for the same precursors because PPDs in DS rice are synthesized from the same precursors as those of phytosterols. In addition, multivariate analysis of metabolic data from rice leaves revealed that composition differed by growth stage rather than genetic modifications. Our results demonstrate the potential of metabolomics for identifying metabolic alterations in response to genetic modifications.

A highly sensitive and versatile transcription immunoassay using a DNA-encoding tandem repetitive light-up aptamer.

Highly sensitive and accurate measurements of protein biomarkers are crucial for early diagnosis and disease monitoring. Here we report a versatile detection platform for sensitive detection of a protein biomarker using a tandem repeat Spinach aptamer DNA-based transcription immunoassay, which is a immunoassay combined with transcription-assisted Spinach RNA aptamer generation. We designed a DNA template encoding spa tandem repetitive Spinach sequence for enhanced generation of an RNA aptamer. The tandem repeated Spinach DNA template is consist of multiple monomeric units which is composed of T7 promoter, Spinach-2 and terminator. After in vitro transcription, the fluorescence signal from the 16R (nR, n = number of repeats) DNA template was enhanced up to ~ 15-fold compared to a single form (1R) DNA template. Using tandem repeat DNA, the proposed transcription immunoassay showed a limit of detection (LOD) of 37 aM, which is 103-fold lower than that of the conventional enzyme-linked immunosorbent assay (ELISA). The results demonstrate substantial promise for the ultrasensitive detection of various biological analytes using simple ELISA techniques. The high sensitivity and reliability of the proposed transcription immunoassay offer great promise for clinical assays.

Reusable surface plasmon resonance biosensor chip for the detection of H1N1 influenza virus.

We developed a reusable magnetic surface plasmon resonance (SPR) sensor chip for detecting various target molecules repeatedly in a conventional SPR system. Here, ferromagnetic patterns on a SPR sensor chip were utilized to trap a layer of magnetic particles, and they were utilized as a solid substrate for SPR sensing in a conventional SPR system. After a sensing experiment, the used magnetic particles were removed by external magnetic fields, and a new layer of magnetic particles was immobilized to the SPR sensor chip for additional sensing measurements. Since magnetic particles were trapped on the ferromagnetic patterns, we could use our reusable SPR chip for SPR sensing measurements in a traditional SPR system without any applied magnetic fields. Significantly, ferromagnetic patterns on the sensor chip surface deflected the strong external fields, so that the large aggregation of magnetic particles on the sensor surface was reduced. We demonstrated using a single reusable SPR sensor chip to measure the nucleoprotein (NP) of H1N1 influenza virus solution ranging repeatedly for more than 7 times without significant signal degradation. Also, different target molecules could be repeatedly measured in a single SPR chip. Since our reusable SPR sensor chip can be repeatedly used in a conventional SPR system without any chemical processes for refreshment, the cost for SPR sensing should be significantly reduced. In this case, our reusable SPR sensor chip can be a major breakthrough and can be used for versatile practical applications of SPR sensors.

Transcription immunoassay: light-up RNA aptamer-based immunoassay using in vitro transcription.

Here, we present an ultra-enhanced immunoassay for sensitive and reliable biomarker detection using layer-by-layer assembly and transcription-assisted light-up aptamer generation to induce signal amplification. This dendrimer structure-based transcription immunoassay is ∼1500 times more sensitive than commercial fluorescence ELISA, achieving a detection limit of 108 aM.

A novel and highly specific phage endolysin cell wall binding domain for detection of Bacillus cereus.

Rapid, specific and sensitive detection of pathogenic bacteria is crucial for public health and safety. Bacillus cereus is harmful as it causes foodborne illness and a number of systemic and local infections. We report a novel phage endolysin cell wall-binding domain (CBD) for B. cereus and the development of a highly specific and sensitive surface plasmon resonance (SPR)-based B. cereus detection method using the CBD. The newly discovered CBD from endolysin of PBC1, a B. cereus-specific bacteriophage, provides high specificity and binding capacity to B. cereus. By using the CBD-modified SPR chips, B. cereus can be detected at the range of 10(5)-10(8) CFU/ml. More importantly, the detection limit can be improved to 10(2) CFU/ml by using a subtractive inhibition assay based on the pre-incubation of B. cereus and CBDs, removal of CBD-bound B. cereus, and SPR detection of the unbound CBDs. The present study suggests that the small and genetically engineered CBDs can be promising biological probes for B. cereus. We anticipate that the CBD-based SPR-sensing methods will be useful for the sensitive, selective, and rapid detection of B. cereus.