Expression of stress-induced phosphoprotein1 (STIP1) is associated with tumor progression and poor prognosis in epithelial ovarian cancer.

Stress-induced phosphoprotein1 (STIP1) is a candidate biomarker in epithelial ovarian cancer (EOC). In this study, we investigated in detail the expression of STIP1, as well as its functions, in EOC. STIP1 expression was assessed by immunohistochemistry (IHC) and the results were compared with clinicopathologic factors, including survival data. The effects of STIP1 gene silencing via small interfering RNA (siRNA) were examined in EOC cells and a xenograft model. The expression of STIP1 protein in EOC was significantly higher than in the other study groups (P < 0.001), and this increase of expression was significantly associated with tumor stage (P = 0.005), tumor grade (P = 0.029), and lymph node metastasis (P = 0.020). In multivariate analysis, overall survival in EOC was significantly shorter in cases with high STIP1 expression (HR = 2.78 [1.01-7.63], P = 0.047). STIP1 silencing in EOC cells resulted in inhibition of cell proliferation and invasion. In addition, in vivo experiments using STIP1 siRNA clearly showed a strong inhibition of tumor growth and a modulation of expression of prosurvival and apoptotic genes, further suggesting that STIP1 silencing can prevent cell proliferation and invasion. In conclusion, increased STIP1 expression is associated with poor survival outcome in EOC, and STIP1 may represent a useful therapeutic target in EOC patients.

Development of 1,1'-oxalyldiimidazole chemiluminescent biosensor using the combination of graphene oxide and hairpin aptamer and its application.

Highly sensitive biosensor with 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) detection was developed to rapidly quantify Vibrio (V) parahaemolyticus without time-consuming procedures such as multiple long-incubations and washings. When V. parahaemolyticus in Tris-HCl (pH 7) and hairpin DNA aptamer conjugated with TEX615 in DNA free deionized water were consecutively added in PBS buffer (pH 7.4) containing graphene oxides (GOs), V. parahaemolyticus and GOs bind competitively to hairpin DNA aptamer conjugated with TEX615 during 10 min of incubation at room temperature. Brightness of light immediately emitted with the addition of ODI-CL reagents (e.g., ODI, H2O2) after the incubation was dependent on the concentration of V. parahaemolyticus in a sample. The dynamic range of linear calibration curve for the quantification of V. parahaemolyticus in a sample was from 4375 to 70,000 cells/ml. The limit of detection (LOD = background + 3 × standard deviation, 2230 cells/ml) of the biosensor operated with good accuracy, precision, and recovery was lower than those of conventional assay methods such as time-consuming and expensive enzyme-linked immunosorbent assays.

Rapid and simple G-quadruplex DNA aptasensor with guanine chemiluminescence detection.

Cost-effective and sensitive aptasensor with guanine chemiluminescence detection capable of simply quantifying thrombin in human serum was developed using thrombin aptamer (TBA), one of the G-quadruplex DNA aptamers, without expensive nanoparticles and complicated procedures. Guanines of G-quadruplex TBA-conjugated carboxyfluorescein (6-FAM) bound with thrombin do not react with 3,4,5-trimethoxylphenylglyoxal (TMPG) in the presence of tetra-n-propylammonium hydroxide (TPA), whereas guanines of free TBA- and TBA-conjugated 6-FAM immobilized on the surface of graphene oxide rapidly react with TMPG to emit light. Thus, guanine chemiluminescence in 5% human serum with thrombin was lower than that without thrombin when TBA-conjugated 6-FAM was added in two samples and incubated for 20 min. In other words, the brightness of guanine chemiluminescence was quenched due to the formation of G-quadruplex TBA-conjugated 6-FAM bound with thrombin in a sample. High-energy intermediate, capable of emitting dim light by itself, formed from the reaction between guanines of TBA and TMPG in the presence of TPA, transfers energy to 6-FAM to emit bright light based on the principle of chemiluminescence energy transfer (CRET). G-quadruplex TBA aptasensor devised using the rapid interaction between TBA-conjugated 6-FAM and thrombin quantified trace levels of thrombin without complicated procedures. The limit of detection (LOD = background + 3 × standard deviation) of G-quadruplex TBA aptasensor with good linear calibration curve, accuracy, precision, and recovery was as low as 12.3 nM in 5% human serum. Using the technology reported in this research, we expect that various types of G-quadruplex DNA aptasensors capable of specifically sensing a target molecule such as ATP, HIV, ochratoxin, potassium ions, and thrombin can be developed.

Role of background observed in aptasensor with chemiluminescence detection.

One-step chemiluminescent aptasensor was developed using chemically initiated electron exchange luminescence (CIEEL) between high-energy intermediate formed from 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) reaction and G-quadruplex (ochratoxin A (OTA)-bound aptamer conjugated with TEX615) generated. The sensitivity of chemiluminescent aptasensor, optimized with various variables (e.g., property of microfibers fabricated with 3,4,9,10-perylenetetracarboxylic dimide, determination of fluorescent dye labeled with aptamer, physical properties of buffer solution), was dependent on the background (concentration of high-energy intermediate) generated in ODI-CL reaction. The limit of detection (LOD=background+3×standard deviation, 0.5 nM) of ODI-CL aptasensor with lower background was lower than that (3.7 nM) with 20 times higher background. Also, the ratio of signal to background (S/B) of ODI-CL aptasensor with low background was about 5-fold higher than that with high background. The sensitivities of ODI-CL aptasensors, with low as well as high background, capable of accurately and precisely quantifying OTA within 10 min, were better than those of fluorescent aptasensors and as good as those of highly sensitive but time-consuming competitive enzyme-linked immune-sorbent assays (ELISAs) using expensive antibody produced with the sacrifice of small animals.

Role of Triton X-100 in chemiluminescent enzyme immunoassays capable of diagnosing genetic disorders.

The use of Triton X surfactants in developing 1,1'-oxalylimidazole chemiluminescent enzyme immunoassays (ODI CEIs) with extended linear response range for the quantification of unconjugated estriol (uE3), alpha-fetoprotein (AFP), and human chorionic gonadotropin (hCG) is reported for the first time. The wider linear dynamic range in ODI CLEIA results from Triton X series (e.g., Triton X-100, -114, -405, -705) acting as an inhibitor in the interaction between Amplex Red (hydrophobic substrate) and horseradish peroxidase (hydrophilic enzyme) to produce resorufin (hydrophobic fluorescent dye). Triton X-100 acts as the appropriate inhibitor in ODI CLEIA. The maximum concentrations of AFP and hCG quantified with sandwich ODI CLEIA in the presence of Triton X-100 were 8 times higher than when analyzed with the same system in the absence of Triton X-100. In addition, the lowest concentration of uE3 determined using competitive ODI CLEIA in the presence of Triton X-100 was 20 times lower than that measured with competitive ODI CLEIA in the absence of Triton X-100. These results indicate that rapid quantification of AFP, uE3, and hCG using cost effective and highly sensitive ODI CLEIAs in the presence of Triton X-100 can be applied as an accurate, precise, and reproducible method to diagnose genetic disorders (e.g., trisomy 18 and trisomy 21) in fetuses.

Rapid monitoring of alkaline phosphatase in raw milk using chemiluminescence detection.

A simple biosensor with 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) detection capable of rapidly quantifying and screening alkaline phosphatase (ALP) in raw and pasteurized milk was developed as an indicator for confirming whether commercial milk is properly pasteurized. Fluorescein was formed when standards containing 1.0% milk with different activities of ALP and samples containing 1.0% raw milk were incubated with fluorescein diphosphate (FDP) for 15 min at room temperature. The relative CL intensity of fluorescein measured with the addition of 80 mM H2O2 and ODI formed from the reaction of 2.0 µM bis(2,4,6-trichlorophenyl) oxalate and 10.0 µM 4-methyl imidazole in ethyl acetate was proportional to the concentration of ALP in milk. The range (39∼2500 mU/L) of linear calibration curve (R2 = 0.998) for the quantification of ALP in milk using ODI-CL detection was wider than those using currently applied fluorescence and 1,2-dioxetane CL detections. Also, the limit of detection (3.7 mU/L) determined using the former detection, which has good precision, was lower than those reported using the latter detections. In conclusion, the cost-effective and highly sensitive biosensor with ODI-CL detection can be applied to monitor whether milk is pasteurized according to acceptable ALP activities threshold level (350 mU/L) for public safety newly adopted by US and EU and the internal investigation level (100 mU/L) proposed by EU.