EC Detection Research Articles

Re-assessing the diagnostic value of the enhancing capsule in hepatocellular carcinoma imaging.

The enhancing capsule (EC) in hepatocellular carcinoma (HCC) diagnosis has received varying degrees of recognition across major guidelines. This study aimed to assess the diagnostic utility of EC in HCC detection. We retrospectively analyzed patients who underwent pre-surgical computed tomography (CT) and hepatobiliary agent-enhanced magnetic resonance imaging (HBA-MRI) between January 2016 and December 2019. A single hepatic tumor was confirmed based on the pathology of each patient. Three radiologists independently reviewed the images according to the Liver Imaging Reporting and Data System (LI-RADS) v2018 criteria and reached a consensus. Interobserver agreement for EC before reaching a consensus was quantified using Fleiss κ statistics. The impact of EC on the LI-RADS classification was assessed by comparing the positive predictive values for HCC detection in the presence and absence of EC. In total, 237 patients (median age, 60 years; 184 men) with 237 observations were included. The interobserver agreement for EC detection was notably low for CT (κ=0.169) and HBA-MRI (κ=0.138). The presence of EC did not significantly alter the positive predictive value for HCC detection in LI-RADS category 5 observations on CT (94.1% [80/85] vs. 94.6% [88/93], P=0.886) or HBAMRI (95.7% [88/92] vs. 90.6% [77/85], P=0.178). The diagnostic value of EC in HCC diagnosis remains questionable, given its poor interobserver agreement and negligible impact on positive predictive values for HCC detection. This study challenges the emphasis on EC in certain diagnostic guidelines and suggests the need to re-evaluate its role in HCC imaging.

P10.18.B JUGLONE AS A NOVEL EPIGENETIC DRUG FOR GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most common and aggressive primary brain tumor and one of the human malignancies with the highest mortality. Standard approaches for GBM, including, gross total resection, radiotherapy, and chemotherapy, cannot destroy all the cancer cells and, despite advances in its treatment, the prognosis for GBM remains poor. The, until now, the most successful chemotherapy with temozolomide (TMZ) for brain gliomas is not fully effective, and therefore new therapeutic strategies for GBM are needed. We found that juglone (J), which exhibits cytotoxic, anti-proliferative, and anti-invasive effects on various cells could be a promising agent for GBM therapy. MATERIAL AND METHODS We analyzed the effects of juglone alone and in combination with temozolomide on glioblastoma cell lines. In addition to the analysis of cell viability and cell cycle, we looked at the epigenetic effects of these compounds on cancer cells. We estimated the total DNA methylation in T98G, U138, U118, and HaCaT lines after treatment with different J and J/TMZ concentrations and incubation time using TLC separation of radioactively labeled nucleotides. The combination treatment with J and TMZ was evaluated with total DNA methylation analysis and 8-oxo-dG (oxidative stress marker) estimation using EC detection. RESULTS We noticed concentration and time-dependent changes in total DNA methylation. The highest increase was observed for the T98G cell line at the incubation time of 24hrs. Normal cell line (HaCaT) did not present significant changes. TMZ alone induced a lowering of DNA methylation, but juglone diminished that effect. J induces minute oxidative stress in low concentrations, but is quite high and rapid at concentrations over 50uM, concomitantly with none/slight decrease in DNA methylation. However, the combination of J/TMZ in increasing concentrations diminished the oxidative stress effect. CONCLUSION Our results strongly suggest that a combination of juglone and temozolomide can be applied for better GBM treatment. At certain incubation times and concentrations, juglone increases DNA methylation, which is a positive therapeutic effect in oncogenesis. Small doses of J keep a low level of 8-oxo-dG with an increase in DNA methylation. The combination of J/TMZ in high concentrations of both drugs decreases the oxidative stress effect of chemotherapy. The study was supported by OPUS 19 (2020/37/B/NZ5/03249) grant from the National Science Center, Poland.

GaEsSeer: Early detection of gastric and esophageal cancer by integrating methylation and fragmentomics signatures in cfDNA.

4069 Background: Esophageal and gastric cancer (EC and GC) are two common cancer types that severely impact patients’ health. The 5-year survival rate for EC and GC is as low as 19% and 31%, respectively. However, early detection will significantly increase the survival rate: stage-1 EC has a 5-year survival rate of 51%, while for stage-1 GC it’s 69%. Invasive screening methods, such as endoscopy and biopsy, caused low compliance. Computational tomography and carcinoembryonic antigen were limited by low sensitivity. To address this problem, we developed GaEsSeer, a non-invasive targeted-sequencing-based assay that utilizes multiple methylation and fragmentomics features of cell-free DNA (cfDNA) to accurately detect EC and GC signals in blood. Methods: cfDNA was tested using the GaEsSeer panel, which was developed using in-house genome-wide sequencing data on EC and GC samples, and public datasets from databases and literature. Methylation features, which was quantified as methylation haplotypes or methylation encoding score, and fragmentomics features including copy number and end motif ratio were taken for modeling. Separate sub-models were trained utilizing each type of feature, which were eventually combined via logistic regression to establish the final predicting model. Results: A total of 1770 participants were recruited from multiple centers. This included 787 healthy individuals, 448 cancers (209 EC, 239 GC; stage I:156, -II:120, -III:78, and -IV:58), 174 benign esophageal diseases, and 361 benign gastric diseases. For cancer detection, the methylation-only model had an AUC of 0.909 and 0.897 in training (618 total) and test sets (617 total), respectively; while the AUC of the fragmentomics-based model was 0.885 and 0.911, respectively. The combinatorial model further improved performances, which achieves an AUC of 0.940 and 0.931 in the training and test cohorts, respectively. While the specificity remained at 96.7%, GaEsSeer detected 81.1% EC and 70.3% GC cases in the test cohort. It had a sensitivity of 74.2% and 48.9% for stage-I EC and GC, respectively. GaEsSeer also has high specificities of 87.9% and 89.8% for benign esophageal and gastric diseases, respectively. Additionally, the performance of GaEsSeer was compared with known serum cancer markers such as CEA, CA19-9, and CA72-4; and the results show that it had significantly higher sensitivity than any of these serum markers (54.8% vs 6.4% when against CEA; 53.5% vs 7.1% when against CA19-9; 50% and 16.7% when against CA72-4). Conclusions: In this pilot study, we developed the blood-based GaEsSeer assay and a model for EC and GC detection with high accuracy by stacking multiple methylation- and fragmentomics-based submodules together. Further optimization and validation of GaEsSeer using larger prospective cohorts are needed to validate its potentials for clinical application.

Sulfur Quantum Dot-Based Electrochemiluminescence Resonance Energy Transfer System for a Dual-Mode Ultrasensitive Detection of MC-LR Using Ag+ as a Signal Amplification Switch.

Improving the sensitivity and accuracy of bioimmunoassays has been the focus of research into the development of electrochemiluminescence (ECL) sensing platforms, as this is a critical factor in their application to practical analysis. In this work, an electrochemiluminescence-electrochemistry (ECL-EC) dual-mode biosensing platform based on an "off-on-super on" signals pattern strategy was developed for the ultrasensitive detection of Microcystin-LR (MC-LR). In this system, sulfur quantum dots (SQDs) are a novel class of ECL cathode emitter with almost no potentially toxic effects. The sensing substrate is made from rGO/Ti3C2Tx composites, whose huge specific surface area greatly reduces the possibility of aggregation-caused quenching of SQDs. The ECL detection system was constructed based on the ECL-resonance energy transfer (ERET) strategy, where methylene blue (MB) with an ECL receptor function was bound to the aptamer of MC-LR by electrostatic adsorption and the center actual distance between the donor and the acceptor was calculated to be 3.84 nm, which was verified to be in accordance with the ERET theory. Meanwhile, the introduction of Ag+ as an ECL signal-amplifying molecule greatly improved the sensitivity of sensing analysis. Based on the specific binding of MC-LR to the aptamer, the concentration of MC-LR was found to have a positive correlation with the ECL signal. Also, EC detection was realized with the benefit of the excellent electrochemical properties of MB. The dual-mode biosensor greatly improves the confidence of the detection, examination areas of 0.001-100 pg/mL with MC-LR for ECL and EC were obtained, and the detection limits are 0.17 and 0.24 pg/mL, respectively.

Electrochemical and Photoelectrochemical Detection of Hydrogen Peroxide Using Cu2O/Cu Nanowires Decorated with TiO2-x Deriving from MXenes.

H2O2 is a major transmitter of redox signals in electrochemical processes, whose detection is relevant for various industries. Herein, we developed a new fabrication method for a Cu2O/Cu nanowire-based nonenzymatic H2O2 electrochemical sensor that was decorated with irregular TiO2-x nanoparticles deriving form Ti3C2 MXene. The TiO2-x/Cu2O/Cu-NW electrodes possess excellent selectivity, stability, and reproducibility for H2O2 detection in both EC and PEC operational modes. In the EC detection of H2O2, the TiO2-x/Cu2O/Cu-NW electrode shows a linear relationship in the range from 10 μM to 42.19 mM and a low detection limit of 0.79 μM (S/N = 3), which has a similar sensitivity but a much broader linear range compared with the commercial H2O2 analyzer (0-5.88 mM, Q45H/84, US-QContums). It also shows excellent recovery in detecting H2O2 in the real orange juice and milk samples with the recovery ranging from 96.9 to 105%, indicating the potential for practical applications. In the PEC detection of H2O2, the TiO2-x/Cu2O/Cu-NW electrode shows a lower detection limit of 59 nM (S/N = 3), which is 13 times more sensitive than the EC electrode. The enhanced PEC performance can be attributed to the formation of p-n heterojunction between TiO2-x and Cu2O, which improves light utilization and inhibits the recombination of photo-induced electrons and holes. This work illuminates the extraordinary potential of MXene-derived TiO2 in electrochemical and photoelectrochemical applications.

Dual catalytic hairpin assembly and enzyme cascade catalysis amplification based sensitive dual-mode biosensor with significantly enhanced opposite signal readout

Herein, a sensitive photoelectrochemical-electrochemical (PEC-EC) dual-mode biosensor with opposite readout signals was constructed for sensitive and accurate detection of miRNA-21, which based on the dual catalytic hairpin assembly (dual CHA) and enzyme cascade catalysis amplification. Firstly, dual CHA could transform a small amount of target into substantial output DNA for achieving efficient conversion between target and output signal. Moreover, the multifunctional carbon nitride material (NaKCN-MB), synthetized through g-C3N4 co-doping by K+, Na+ ions and compounding with organic dye MB, could be not only as the ternary self-enhanced photoactive material, but also as glucose oxidase (GOx)-mimicking enzyme to generate in situ H2O2 as an intermediate that subsequently decomposed by hemin polymer for acquiring a direct electron transfer-based enzyme cascade electrocatalytic system. The proposed approach accomplished the simultaneous cascade amplification in PEC and EC dual-mode detection system with opposite readout signals, which effectively resolved the low sensitivity and accuracy in traditional biosensors. As a result, the proposed dual-mode detection approach exhibited a wide detection range of 0.1 fM to 1 nM for PEC detection and 1 fM to 1 nM for EC detection, which provided an efficient and universal biosensing avenue, possessing great meaning in biological analysis and disease diagnosis.

Detecting endometrial cancer

Key content Endometrial cancer (EC) is the most common gynaecological cancer in the UK. Ninety percent of women with EC present with postmenopausal bleeding (PMB), but less than 10% of women with PMB have a sinister underlying cause. National Institute for Health and Care Excellence guidance advises that symptomatic postmenopausal women undergo urgent investigation; however, guidance is unclear for premenopausal women. Current investigations for PMB, including transvaginal ultrasound scan, endometrial biopsy and/or outpatient hysteroscopy, have advantages and disadvantages. Novel detection tools are in development, which combine minimally invasive sampling with genomic, proteomic and single cell technologies. Learning objectives To understand who is at risk of EC and who should be referred for urgent investigations. To understand the evidence underpinning the current diagnostic pathway for EC. To highlight unique and promising perspectives for EC detection and their potential to transform clinical care. Ethical issues Current diagnostics for EC are invasive and often painful. There is an urgent need for high‐quality randomised controlled trials to inform effective pain relief options. Premenopausal women with suspected EC do not fit criteria for urgent investigations. How can we identify those at highest risk to ensure they are fast‐tracked appropriately? Novel diagnostic tools hold promise, but they must be robustly validated before being introduced into clinical practice.

Comparison of serum human epididymis protein 4 and CA125 on endometrial cancer detection: A meta-analysis

BackgroundEndometrial cancer (EC) is common type of gynecologic malignancy affecting a large number of females around the world. While most early stage cases are well managed with a relatively benign prognosis, the late stage cases have poor survival. Among the many biomarkers identified, serum human epididymis protein 4 (HE4) and CA125 are most promising surrogates for EC detection. MethodsWe performed a meta-analysis to estimate the diagnostic accuracy of HE4 and CA125 and compared their performance. A literature research was performed in Medline, Cochrane Literature Library and CNKI. After filtering, twelve studies evaluating the diagnostic value of serum HE4, alone or in comparison with CA125, were included. The total sample size was 1106 patients and 1480 controls. Pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR) were calculated and summary receiver operating characteristic (SROC) curves were plotted to assess the diagnostic accuracy. ResultsThe pooled estimates for HE4 were sensitivity: 0.71 (95%CI 0.56–0.82), specificity: 0.87 (95%CI 0.80–0.92), and area under ROC curve: 0.88 (0.85–0.91), compared to 0.35 (95% CI 0.25–0.46), 0.83 (95% CI 0.71–0.91), and 0.58 (95% CI 0.54–0.63), respectively, of CA125. Subgroup analysis demonstrated a better performance of HE4 in Caucasian population, compared to Chinese population. ConclusionThis analysis suggested that when stage and histological type are not specifically considered, serum HE4 is generally a better tool than CA125 in EC diagnosis by its significantly higher sensitivity than CA125.

P 146 - The effect of anti-aging agents in skin oxidative stress induced by UV Radiation in vivo

UV radiation is related to oxidative stress and skin aging. UV induced reactive oxygen species lead to progressive degradation of dermal collagen and elastin. Four of the most known anti-aging agents were examined in the photoaged skin of hairless SKH-hr2 mice. 50 mice were irradiated with increasing UVR doses (0.5–2 MED) 3 times per week for 4 weeks. After the end of the irradiations, 5 formulations containing retinoic acid 0.05%, lactic acid 10%, salicylic acid 3%, epidermal growth factor 3.3% and the vehicle were applied on the back of the mice for 3 weeks. Hydration, transepidermal water loss (TEWL), elasticity and skin thickness, were frequently measured. After the end of applications, oxidative stress was evaluated in skin biopsies and strippings of stratum corneum using fluorescence spectroscopy, as well endogenous antioxidants using HPLC–EC detection. Epidermal thickening was evident in all mice after UV exposure. After treatment with lactic acid the thickness decreased significantly. Highest TEWL was observed in the retinoic acid group. The skin treated with salicylic acid demonstrated the lowest levels of ascorbic and uric acid. In strippings, lactic acid icreased the levels of antioxidants. Highest elasticity was measured in the retinoic acid group. In conclusion, retinoic and lactic acids seem to be the most effective treatments for the photodamaged skin.

A Paper-Based Reagent Storage Approach for Point-of-Care Electrochemical Sensing

In this work, we report on a paper-based reagent storage approach for electrochemical sensing applications. We use a paper pad to store dry reagents. Once re-hydrated, the pad provides proper aqueous environment for electrochemical measurement. This approach offers several advantages including low cost, no need for on-site sample preparation, ease of fabrication, operation, storage, and disposal. We expect this approach to be highly beneficial in point-of-care electrochemical sensing applications. We fabricated the three-electrode electrochemical sensor on a flexible polyethylene terephthalate (PET) film by evaporation of metal (Au or Cu) followed by standard photolithography and wet etching. Our electrochemical sensor consists of two shorted interdigitated array (IDA) working electrode (WE) with finger width/spacing of 10µm/5µm, an auxiliary electrode (AE) and a reference electrode (RE) (Fig. 1a). We arranged the electrode layout into USB style for convenient connection with a potentiostat. We fabricated paper-based reagent-storage pads by shaping Whatman No.1 filter paper with a paper puncher or a pair of scissors. We designed paper pads with a size of 12mm×10mm to cover the entire area of the three electrodes (Fig. 1b). We first demonstrate cyclic voltammetry by rehydrating a paper pad containing dry ferricyanide on gold IDA sensor. A 10mM ferricyanide solution was first dried on a paper pad. After contacting with the sensor surface, 50µL DI water was dispensed on the pad followed by cyclic voltammetry (CV) scanning from -0.5V to 0.3V with a scan rate of 100mV/s. As shown in Fig. 2a, the CV spectra exhibites two redox peaks as expected, while the CV spectra using a control paper pad without ferricyanide is flat. This indicates that the dry-form of ferricyanide in the paper pad can be dissolved into aqueous solution during re-hydration process supporting electrochemical reaction on the sensor surface. The paper-based dry-reagent platform can be used in anodic stripping voltammetry (ASV) for detection of lead (Pd) [1]. Reagents containing pH 5.5 acetate buffer with 50, 100 and 200ppb Pd were dispensed and dried on three different paper pads. After aligning a paper pad with a Cu-based sensor [2], 50µL liquid sample was introduced on the paper pad followed by ASV scanning. The experimental parameters were selected after optimization: -0.8 V and 300 s for preconcentration; 50 ms period, 50 mV amplitude, and 8 mV increment for square wave. As shown in Fig. 2b, the peak amplitude of ASV spectra increases with the concentration of Pd within the paper pad. This approach can provide supporting electrolyte for electrochemical impedimetric sensing. We first dried 10mM ferricyanide on a paper pad. After aligning the paper pad with a gold IDA sensor and rehydrating with PBS, electrochemical impedance scanning was conducted from 0.1Hz to 1MHz under a DC bias of -0.15V with an AC bias of 10mV. The Nyquist plot (Fig. 2c) appears as a semicircle indicating the background charge transfer resistance through bare Au electrode surface to be ~4000ohm. This result suggests the paper pad with dry ferricyanide can provide Fe2+/Fe3+to the sensor surface for electrochemical impedance spectroscopy without disrupting the measurement. In conclusion, we developed a paper-based platform capable of storing dry reagents for electrochemical sensing applications. Paper pads containing dry reagents can be easily re-hydrated to provide adequate aqueous environment for different types of electrochemical sensing techniques. We envision this platform can be valuable for low-cost, point-of-care electrochemical sensing applications. Reference [1] W. Kang, X. Pei, A. Bange, E. Haynes, W. R. Heineman, and I. Papautsky, Anal. Chem. 2014, in press. [2] X. Pei, W. Kang, W. Yue, A. Bange, W. R. Heineman, and I. Papautsky, Anal. Chem. 2014, 86, 4893-4900. Acknowledgements This work was supported in part by funds provided by the NIEHS award R01ES022933. Figure captions: Fig. 1 (a) Photography of IDA sensor fabricated on a flexible PET film. (b) Aligning a paper pad containing dry reagent on sensor surface. (c) Conventional way of dispensing samples for EC detection. (d) Operation of dry-reagent pad including (1) dispensing reagent on a paper pad, (2) drying reagent, (3) Aligning reagent pad with sensor, and (4) rehydrate dry pad with aqueous sample for EC measurements. Fig. 2 (a) Paper pad platform for CV scanning. (b) Paper pad platform for detection of Pd using ASV. (c) Paper pad platform for EIS measurement. Figure 1

Electrochemistry of nonconjugated proteins and glycoproteins. Toward sensors for biomedicine and glycomics.

In this review, we wish to show that in the recent years a significant progress was done in the EC analysis of practically all proteins, based on electroactivity of amino acid (aa) residues in proteins. Also electrochemistry of polysaccharides, oligosaccharides and glycoproteins greatly advanced in creating important steps for its larger application in the glycoprotein research. In recent decades, a great effort was devoted to the discovery and application of biomarkers for analysis of different diseases, including cancer. In the following paragraphs, special attention will be paid (i) to intrinsic electroactivity of peptides and proteins, including the sensitivity to changes in protein 3D structures, as well as to recent advances in EC investigations of DNA-protein interactions, (ii) to intrinsic electroactivity of glycans and polysaccharides, advances in EC detection of lectin-glycoprotein interactions and to introduction of electroactive labels to polysaccharides and glycans and finally (iii) to EC detection of protein biomarkers, based predominantly on application of antibodies in immunoassays, nucleic acid and peptide aptamers for construction of aptasensors, and lectin biosensors for detection of glycoprotein biomarkers.

LC-MS/MS Analysis of Cerebrospinal Fluid Metabolites in the Pterin Biosynthetic Pathway.

The analysis of (6R)-5,6,7,8-tetrahydrobiopterin (BH4) and neopterin in cerebrospinal fluid (CSF) is often used to identify defects in the pterin biosynthetic pathway affecting monoamine metabolism that can lead to pediatric neurotransmitter diseases. Low levels of BH4 and neopterin alone may not be sufficient to determine the defect, and further testing is often required. We have developed a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of BH4, 7,8-dihydrobiopterin (BH2), neopterin, and sepiapterin in CSF, which provides a more comprehensive evaluation of the pterin pathway. The method utilizes labeled stable isotopes as internal standards and allows for a fast 10-minute analysis by LC/MS/MS over a linear working range of 3 to 200nmol/L. Total analytical imprecision is less than 14.4% for all pterin metabolites. Accuracy for BH4 and neopterin was determined by comparing data obtained by an alternative method using HPLC with EC and fluorescence detection. Excellent correlation was demonstrated for BH4 (r = 0.9646, 1/slope = 0.9397; n = 28; concentration range 3 to 63nmol/L) and neopterin (r = 0.9919, 1/slope = 0.9539; n = 13; concentration range 5 to 240nmol/L). CSF specimens from patients diagnosed with inborn errors of sepiapterin reductase (SR), 6-pyruvoyl-tetrahydropterin synthase (PTPS), dihydropteridine reductase (DHPR), and guanosine triphosphate cyclohydrolase (GTPCH) have been analyzed, and distinct pterin metabolite patterns were consistent with the initial diagnosis. This method differentiates patients with DHPR and SR deficiency from other pterin defects (GTPCH and PTPS) and will be useful for the diagnosis of specific defects in the pterin biosynthetic pathway.

Potential Diagnostic Value of Serum p53 Antibody for Detecting Esophageal Cancer: A Meta-Analysis

BackgroundMutant p53 protein overexpression has been reported to induce serum antibodies against p53. Various studies assessing the diagnostic value of serum p53 antibody in patients with esophageal cancer remain controversial. This study aims to comprehensively and quantitatively summarize the potential diagnostic value of serum p53 antibody in esophageal cancer.MethodsWe systematically searched PubMed and Embase until 31st May 2012, without language restriction. Studies were assessed for quality using QUADAS (quality assessment of studies of diagnostic accuracy). Positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were pooled separately and compared with overall accuracy measures diagnostic odds ratio (DOR) and symmetric summary receiver operating characteristic (sROC). The PLR and NLR and their 95% confidence interval (CI) were calculated using a fixed effects model according to the Mantel-Haensed method and random effects model based on the work of Der Simonian and laird, respectively.ResultsFifteen studies (cases = 1079, controls = 2260) met the inclusion criteria for the meta-analysis. Approximately 53.33% (8/15) of the included studies were of high quality (QUADAS score≥8), which were retrospective case-control studies. The summary estimates for quantitative analysis of serum p53 antibody in the diagnosis of esophageal cancer were PLR 6.95 (95% CI: 4.77–9.51), NLR 0.75 (95%CI: 0.72–0.78) and DOR 9.65 (95%CI: 7.04–13.22). However, we found significant heterogeneity between NLRs.ConclusionsThe current evidence suggests serum p53 antibody has a potential diagnostic value for esophageal cancer. However, its discrimination power is not perfect because of low sensitivity.ImpactThese results suggest that s-p53-antibody may be useful for monitoring residual tumor cells and for aiding in the selection of candidates for less invasive treatment procedures because of the high specificity of s-p53-antibody. Further studies may need to identify patterns of multiple biomarkers to further increase the power of EC detection.

Structural Characterization of Plasma Metabolites Detected via LC-Electrochemical Coulometric Array Using LC-UV Fractionation, MS, and NMR

Liquid chromatography (LC) separation combined with electrochemical coulometric array detection (EC) is a sensitive, reproducible, and robust technique that can detect hundreds of redox-active metabolites down to the level of femtograms on column, making it ideal for metabolomics profiling. EC detection cannot, however, structurally characterize unknown metabolites that comprise these profiles. Several aspects of LC-EC methods prevent a direct transfer to other structurally informative analytical methods, such as LC-MS and NMR. These include system limits of detection, buffer requirements, and detection mechanisms. To address these limitations, we developed a workflow based on the concentration of plasma, metabolite extraction, and offline LC-UV fractionation. Pooled human plasma was used to provide sufficient material necessary for multiple sample concentrations and platform analyses. Offline parallel LC-EC and LC-MS methods were established that correlated standard metabolites between the LC-EC profiling method and the mass spectrometer. Peak retention times (RT) from the LC-MS and LC-EC system were linearly related (r(2) = 0.99); thus, LC-MS RTs could be directly predicted from the LC-EC signals. Subsequent offline microcoil-NMR analysis of these collected fractions was used to confirm LC-MS characterizations by providing complementary, structural data. This work provides a validated workflow that is transferrable across multiple platforms and provides the unambiguous structural identifications necessary to move primary mathematically driven LC-EC biomarker discovery into biological and clinical utility.

Determination of isoxanthohumol, xanthohumol, bitter acids, and trans and cis-iso- alpha acids by HPLC with UV and EC detection

Determination of isoxanthohumol, xanthohumol, bitter acids, and trans and cis-iso- alpha acids by HPLC with UV and EC detection

Dual amplified, sensitive electrochemical detection of pathogenic sequences based on biobarcode labels and functional graphene modified electrode

A dual signal amplified strategy for sensitive EC detection of pathogenic DNA sequences based on poly[G]20-conjugated biobarcode nanoparticles and carboxyl functionalized graphene is described. The capture probes are covalently linked to the graphene modified electrode. The addition of the target sequences and the biobarcode amplification labels leads to the formation of sandwich structures on the electrode surface. The presence of Ru(bpy)32+ in the detection buffer catalyzes the electro-oxidation of the numerous guanine bases captured on the electrode surface and generates significantly amplified current response for quantitation. Moreover, graphene can offer excellent heterogeneous electron transfer during electro-catalytic oxidation of guanine bases. This dual signal amplification strategy thus results in the determination of pathogenic DNA sequences down to the low picomolar level (1pM). Our approach also exhibits excellent discrimination between the target and single-base mismatch sequences, which provides great potential for highly sensitive and selective detection of different nucleic acid sequences in general.

Determination of Co-Q10 in plasma samples by dual-electrode amperometric detection and liquid chromatography

Co-Q10 is a lipid-soluble benzoquinone that is an important factor in free radical scavenging, mitochondrial membrane stability and ATP synthesis. Dietary Co-Q10 is a powerful antioxidant that has been useful in lessening the damage associated with ischemia-reperfusion injuries and aiding in the recovery of myocardial function after myocardial infarction. However, the role of dietary Co-Q10 in oxidative damage and repair is not well understood. Previous LC-EC methods have used packed carbon bed electrodes with high overpotentials that were sufficient to oxidize and reduce several biological compounds, thereby decreasing the selectivity that can be achieved with EC detection. Thin-layer cell dual electrode detection enables monitoring of reduced and oxidized forms of Co-Q10 simultaneously and selectively. The oxidation (+0.45 V vs. Ag/AgCl) and reduction (-0.4 V vs. Ag/AgCl) electrode potentials were optimized to oxidize and reduce the electroactive quinone moiety. The reduced form of Co-Q10 was prepared from the commercially available oxidized form using a Jones reductor. Confirmation of its formation was determined using the current ratios of the peak and half wave potentials from previously generated hydrodynamic voltammograms, using the oxidized form with electrodes in a series configuration. This analytical system was successfully applied to determine basal concentrations of oxidized (510 nM) and reduced (500 nM) Co-Q10 in human plasma. Peak identity of oxidized and reduced Co-Q10 was confirmed by two orthogonal methods: by the current ratios at +0.45 V and +0.25 V and -0.4 V and -0.2 V (vs. Ag/AgCl) as well as by retention time. Detection limits were determined to be 5 nM, with a linear range of three orders of magnitude.

Identification and quantification of lignans in wheat bran by gas chromatography-electron capture detection

Whole grain cereals are an important source of bioavailable lignans, the group of compounds with potential anti-cancerogenic, antioxidant, anti-proliferative, pro-apoptotic, and antiangiogenic properties. The aim of this work was to develop a sensitive method for determination of wheat bran lignans. The analysis of lignans secoisolariciresinol, hydroxymatairesinol, lariciresinol, matairesinol, pinoresinol, syringaresinol is based on derivatization with pentafluoropropionic anhydride (PFPA) and gas chromatography-electron capture detection (GC-ECD), using styrene glycol as internal standard. To our knowledge, this is the first time that EC detection has been used for lignan analysis. The results show that the technique is reproducible and sensitive enough for detecting lignans in wheat at parts-per-billion (ppb) levels, except for hydroxymatairesinol. The method developed showed good recovery (85–105%) and precision (4–20%) for five types of lignans and thus represents a simpler and more affordable alternative to state-of-the-art wheat lignan liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis.

The rapid simultaneous tricyclic antidepressant determination by plasma deproteinization and liquid chromatography

Background: Imipramine, desipramine, amitriptyline and nortriptyline are widely prescribed antidepressant drugs in Asia because of their low price and high efficacy. The concentrations of these drugs in plasma need to be monitored for achieving efficient therapy. A simple, rapid and practical analytical method is required. Objective: To develop a plasma deproteinization process for rapid determination of imipramine, desipramine, amitriptyline or nortriptyline in plasma using high-performance liquid chromatography (HPLC). Methods: A deproteinizing agent, with an acceptable accuracy and sensitivity, to precipitate plasma protein containing imipramine, desipramine, amitriptyline and nortriptyline was investigated along with the HPLC condition for resolving each of these compounds. The developed method was verified by performing a bio-analytical method validation, and by analyzing plasma samples from volunteers who received imipramine or amitriptyline. Results: Using clomipramine as an internal standard, the concentrations of imipramine, desipramine, amitriptyline and nortriptyline in plasma could easily be determined by precipitating plasma protein with acetonitrile and concentrated aqueous potassium phosphate solution prior to HPLC analysis. The mobile phase comprised of acetonitrile and 70 mM phosphate buffer (pH 6.1) (60/40 v/v). A separation was achieved on a C 18 column, and the effluent was measured by UV absorption at 251 nm or by EC detection at +1.0 V of glassy carbon against silver/ silver chloride reference electrode. The chromatographic separation was excellent, without interference from endogenous plasma constituents. All compounds were resolved in a run time of 12 minutes. The method was suitable for quantifying drug concentrations in the ranges of 40-900 ng/ml for UV detection or 4-900 ng/mL for EC detection. The relative standard intra-day and inter-day deviations for each compound in the plasma were less than 8%. The percentage of bias was within ±4% which confirmed the accuracy of the method. The method proved to be efficient and practical for determining the plasma concentrations of each compound, following the administration of imipramine or amitriptyline to volunteers. Conclusion: This developed method is very simple, rapid and inexpensive and is practical for routine therapeutic drug monitoring and forensic toxicological screening.

MICROFLUIDIC FLOW-THROUGH REACTOR WITH ELECTROCHEMICAL SENSOR ARRAY FOR REAL-TIME PCR

We developed an integrated microfluidic flow-through EC-PCR (EC-PCR) microdevice for the concurrent DNA amplification, PCR products EC detection and PCR products quantification instead of the current available fluorescence detection scheme. The microfluidic flow-through EC-PCR microdevice was fabricated with the state-of-the-art microfabrication technology, by bonding a bottom glass substrate having a microelectrode array to a top glass cover having the microchannels made of PDMS material. Both the amplification of the target DNA sequence and the subsequent EC detection of the PCR products were carried out concurrently on the integrated device by real-time monitoring. The underlying principle of the microfluidic flow-through EC-PCR method was based on the changes of current signal of methylene blue (MB), which worked as an electrochemically active species DNA intercalator in the PCR mixture, during the amplification process at the extension phase. The results shown in this work indicated that the nucleic acid analysis could be performed in a fast thermal cycling and true real-time quantitative electrochemical detection. The signal variation trends of the EC detection and the fluorescence detection were the same in our verification measurements for both methods, which suggested that the EC detection method was feasible for this application.