High-sensitivity Detection System Research Articles

Dual-recognition colorimetric platform based on porous Au@Pt nanozymes for highly sensitive washing-free detection of Staphylococcus aureus.

Adual-recognition strategy is reportedto construct a one-step washing and highly efficient signal-transduction tag system for high-sensitivity colorimetric detection of Staphylococcus aureus (S. aureus). The porous (gold core)@(platinum shell) nanozymes (Au@PtNEs) as the signal labels show highly efficient peroxidase mimetic activity and arerobust. For the sake of simplicity the detection involved the use of a vancomycin-immobilized magnetic bead (MB) and aptamer-functionalized Au@PtNEs for dual-recognition detection in the presence of S. aureus.In addition, we designed a magnetic plate to fit the 96-well microplate to ensure consistent magnetic properties of each well, which can quickly remove unreacted Au@PtNEs and sample matrix while avoiding tedious washing steps. Subsequently, Au@PtNEs catalyze hydrogen peroxide (H2O2) to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) generating a color signal. Finally, the developed Au@PtNEs-based dual-recognition washing-free colorimetric assay displayed a response in the range of S. aureus of 5 × 101-5 × 105 CFU/mL, and the detection limit was 40 CFU/mL within 1.5 h. In addition, S. aureus-fortified samples were analyzed to further evaluate the performance of the proposed method, which yielded average recoveries ranging from 93.66 to 112.44% and coefficients of variation (CVs) within the range 2.72-9.01%. These results furnish a novel horizon for the exploitation of a different mode of recognition and inexpensive enzyme-free assay platforms as an alternative to traditional enzyme-based immunoassays for the detection of other Gram-positive pathogenic bacteria.

High sensitivity and ultra-low concentration range photoacoustic spectroscopy based on trapezoid compound ellipsoid resonant photoacoustic cell and partial least square

A high sensitivity and ultra-low concentration range photoacoustic spectroscopy (PAS) gas detection system, which was based on a novel trapezoid compound ellipsoid resonant photoacoustic cell (TCER-PAC) and partial least square (PLS), was proposed to detect acetylene (C2H2) gas. In the concentration range of 0.5 ppm ∼ 10.0 ppm, the limit of detection (LOD) values of TCER-PAC-based PAS system without data processing was 66.4 ppb, which was lower than that of the traditional trapezoid compound cylindrical resonant photoacoustic cell (TCCR-PAC). The experimental results indicated that the TCER-PAC had higher sensitivity than of TCCR-PAC. Within the concentration range of 12.5 ppb ∼ 125.0 ppb, the LOD and limit of quantification (LOQ) of TCER-PAC-based PAS system combined with PLS regression algorithm were 1.1 ppb and 3.7 ppb, respectively. The results showed that higher detection sensitivity and lower LOD were obtained by PAS system with TCER-PAC and PLS than that of TCCR-PAC-based PAS system.

Ag+-stabilized DNA triplex coupled with catalytic hairpin assembly and CRISPR/Cas12a amplifications for sensitive metallothionein assay

Metallothionein (MT) is a protein biomarker secreted by liver in response to the treatment for heavy metal toxicity and oncological diseases. On the basis of a new Ag+-stabilized DNA triplex probe (Ag+-SDTP), we establish a fluorescent biosensing system for high sensitivity detection of MT by combining catalytic hairpin assembly (CHA) and the CRISPR/Cas12a signal enhancements. The MT analyte complexes with Ag+ in Ag+-SDTP to disrupt the triplex structure and to release the ssDNA strands, which trigger subsequent CHA formation of many protospacer adjacent motif (PAM)-containing dsDNAs from two hairpins. Cas12a/crRNA further recognizes these PAM sequences to activate its trans-catalytic activity to cyclically cleave the fluorescently quenched ssDNA reporters to recovery drastically amplified fluorescence for detecting MT down to 0.34 nM within the dynamic range of 1∼800 nM. Moreover, the sensing method is able to selectively discriminate MT from other non-specific molecules and can realize low level detection of MT in diluted human serums, manifesting its potentiality for monitoring the disease-specific MT biomarker at trace levels.

Highly sensitive and stable fluorescent aptasensor based on an exonuclease III-assisted amplification strategy for ATP detection

Fluctuations in intracellular adenosine triphosphate (ATP) concentration are closely associated with some cancer diseases. Thus, it is a worthwhile undertaking to predict sickness by monitoring changes in ATP levels. However, the detection limits of current fluorescent aptamer sensors for ATP detection are in the range of nmol L−1 to μmol L−1. It has become crucial to employ amplification strategies to increase the sensitivity of fluorescent aptamer sensors. In the current paper, a duplex hybrid aptamer probe was developed based on exonuclease III (Exo III)-catalyzed target recycling amplification for ATP detection. The target ATP forced the duplex probe configuration to change into a molecular beacon that can be hydrolyzed with Exo III to achieve the target ATP cycling to amplify the fluorescence signal. Significantly, many researchers ignore that FAM is a pH-sensitive fluorophore, leading to the fluorescence instability of FAM-modified probes in different pH buffers. The negatively charged ions on the surface of AuNPs were replaced by new ligands bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP) to improve the drawback of FAM instability in alkaline solutions in this work. The aptamer probe was designed to eliminate the interference of other similar small molecules, showing specific selectivity and providing ultra-sensitive detection of ATP with detection limits (3σ) as low as 3.35 nM. Such detection limit exhibited about 4-500-fold better than that of the other amplification strategies for ATP detection. Thus, a relatively general high sensitivity detection system can be established according to the wide target adaptability of aptamers, which can form specific binding with different types of targets.

Optical design and stray RF investigation of ITER EC H&CD equatorial launcher using mockup

The aim of this study is to assess power transmission characteristics of ITER EC H&CD equatorial launcher (EL) which will deliver 20 MW of millimeter wave power to the ITER tokamak. A precise optical mockup of the launcher is fabricated in order to experimentally verify the characteristics of 170 GHz beam transmission. To observe the intensity distribution of output beams, a mobile high-sensitivity power detection system is used for millimeter wave monitoring, which is in good agreement with quasi-optical calculations. Internal stray RF generation presents an additional concern regarding heat load inside the launcher especially in case of deteriorated input mode purity. Therefore, firstly, the stray RF power is quantified by observation after the beam duct (i.e., launcher exit) of the mockup. The results show that a significant portion of stray RF (>20%) is emitted and does not contribute to the heat load. For another approach to estimate the heat load, geometric optics is performed with various degrees of scattering. This approach indicates that nearly half of the stray RF power is emitted from the launcher exit. Thus, the internal heat load due to stray RF is calculated 44 kW/m2, which is an important result in relaxing the design conditions of the EL.

Kinetics of Enzymatic Reactions at the Solid/Liquid Interface in Nanofluidic Channels.

Nanostructures can realize highly efficient reactions due to their structural advantages. However, the mechanism of accelerating enzyme reactions in a nanospace is still unknown from a kinetic perspective because it is difficult to control a well-defined nanospace, enzyme density, and reaction time. Here, we investigated kinetic parameters of an immobilized enzyme in micro- and nanochannels using nanofabrication, partial enzyme patterning, fluidic control, and a high sensitivity detection system. Devices with channel depths of 300 nm, 4.4 μm, and 13.6 μm were fabricated. Kinetic parameters were determined by the Michaelis-Menten model. Compared to the bulk reaction, all kcats for immobilized enzyme reactors were decreased, although the kcats were approximately the same for the immobilized enzyme reactors of different depths. An ultrafast enzyme reaction could overcome the drawback due to immobilization by an increase of the apparent [E]0 due to the decreased channel depth.

Yoctonewton force detection based on optically levitated oscillator

Optically levitated oscillators in high vacuum have excellent environmental isolation and low mass compared with conventional solid-state sensors, which makes them suitable for ultrasensitive force detection. The force resolution usually scales with the measurement bandwidth, which represents the ultimate detection capability of the system under ideal conditions if sufficient time is provided for measurement. However, considering the stability of a real system, a method based on the Allan variance is more reliable to evaluate the actual force detection performance. In this study, a levitated optomechanical system with a force detection sensitivity of 6.33 ± 1.62 zN/Hz1/2 was demonstrated. And for the first time, the Allan variance was introduced to evaluate the system stability due to the force sensitivity fluctuations. The force detection resolution of 166.40 ± 55.48 yN was reached at the optimal measurement time of 2751 s. The system demonstrated in this work has the best force detection performance in both sensitivity and resolution that have been reported so far for optically levitated particles. The reported high-sensitivity force detection system is an excellent candidate for the exploration of new physics such as fifth force searching, high-frequency gravitational waves detection, dark matter research and so on.

Two Ways to Achieve the Same Goal-Two Validated Quantitative NMR Strategies for a Low-Abundance Natural Product in Standardized Extracts: The Case of Hepatodamianol in Turnera diffusa.

The quantification of low-abundance secondary metabolites in plant extracts is an analytical problem that can be addressed by different analytical platforms, the most common being those based on chromatographic methods coupled to a high-sensitivity detection system. However, in recent years nuclear magnetic resonance (NMR) has become an analytical tool of primary choice for this type of problem because of its reliability, inherent simplicity in sample preparation, reduced analysis time, and low solvent consumption. The versatility of strategies based on quantitative NMR (qNMR), such as internal and external standards and electronic references, among others, and the need to develop validated analytical methods make it essential to compare procedures that must rigorously satisfy the analytical well-established acceptance criteria for method validation. In this work, two qNMR methods were developed for the quantification of hepatodamianol, a bioactive component of T. diffusa. The first method was based on a conventional external standard calibration, and the second one was based on the pulse length-based concentration determination (PULCON) method using the ERETIC2 module as a quantitation tool available in TopSpin software. The results show that both procedures allow the content of the analyte of interest in a complex matrix to be determined in a satisfactory way, under strict analytical criteria. In addition, ERETIC2 offers additional advantages such as a reduction in experimental time, reagent consumption, and waste generated.

A sliver deposition signal-enhanced optical biomolecular detection device based on reduced graphene oxide

The development of high-sensitive biomolecular detection system is of great significance for diseases early diagnosis. The novel optical sensor based on the polarization-sensitive absorption of graphene has a great potential in biological detection. However, the detection sensitivity of the device can hardly meet the needs of clinical analysis currently. This study applies sliver deposition signal amplification to the optical biomolecular detection device based on reduced graphene oxide for the sensitive immunoassay. In redox cycling enzymatic silver deposition reaction, the more alkaline phosphatase label bound on chip surface will cause a faster silver deposition rate. The specific antibody detection confirms that the sliver deposition can enhance the detection signal significantly. In cardiac biomarker Creatine Kinase-MB measurement, the minimum detection concentration is 0.1 ng/mL. To be more important, within the range from detection limit to 10 ng/mL, the signal intensity is highly correlated with target protein concentration, so the biomolecular detection device can meet clinical assay requirements. The signal-enhanced optical biomolecular detection device based on reduced graphene oxide shows excellent sensitivity and selectivity, and provides a new strategy for biomolecules detection, which can be applied in diseases accurate prediction and diagnosis.

High sensitivity and fast detection system for sensing of explosives and hazardous materials

We demonstrate a high sensitivity and fast detection system for sensing of explosives and hazardous materials based on the external cavity quantum cascade laser (ECQCL). In this system, one galvanometer is utilized as the tuning component of the ECQCL to get high wavelength scanning speed. Meanwhile, the driving module of the galvanometer and the analog to digital conversion (ADC) module for data acquisition are synchronized, which brings in great significance for fast high sensitivity detection. For one thing, this synchronization exactly defines the data of each scanning period during data acquirement and processing, which enables our system to avoid data dislocation and cumulative error. For another, enough averaging times are allowed to obtain high sensitivity while detection speed is high enough for practical application. To verify the practical application effect, this system was utilized to detect trace amount of adsorbed trinitrotoluene (TNT) on the People's Republic of China Resident Identity (ID) Card. Although the wavelength tuning rate was as high as 17400 cm−1/s over the range of 1280–1454 cm−1(∼6.88–7.81 µm) with scanning frequency of 100 Hz, a TNT detection sensitivity of 1 μg/cm2 was achieved with detection speed of one result per second through multiple averaging technique.

High-sensitivity acetylene detection system using ellipsoid multi-pass cell (EMPC) based on the opposite dual optical source

A multi-pass cell with ellipsoid structure used dual optical source of opposite side was proposed firstly in this paper. The EMPC was composed of double concave spherical mirrors and an ellipsoidal resonator. The radius of curvature of the two symmetric concave spherical mirrors was 200 mm and the length of the cavity was 120 mm. The size of the resonator of the photoacoustic (PA) cell in the experiment is Φ28mm×100mm and the size of the buffer cell at both ends of the PA cell is Φ70mm×10mm. The two laser beams were injected from the two sides of the concave spherical mirrors by the optical fiber. The absorption spectrum line with the acetylene wavelength of 1531.58 nm was selected. The simulation results show that the light beam can be reflected 37 times in EMPC and the optical path length can reach 8.64 m. In the experiment, the 2f signal peak value fitting curve obtained at different concentrations with good linearity. Allan deviation analysis of experimental results shows the detection limit of EMPC was 60 ppb when the average time was 2 s. When the average time was 2 s, the normalized noise equivalent absorption (NNEA) coefficient of the system was9.84×10-8cm-1·W·Hz-1/2. The whole system has high detection sensitivity and stability.

Cyan-emitting silicon quantum dots as a fluorescent probe directly used for highly sensitive and selective detection of chlorogenic acid

As an important bioactive component in plants, chlorogenic acid (CGA) has been widely studied for its potential role in human health. In this work, cyan fluorescent silicon quantum dots were successfully synthesized via a simple one-pot method for the rapid detection of CGA. The optimal excitation and emission wavelength of the obtained SiQDs was 350 nm and 470 nm, respectively. When the CGA was added, the maximum emission intensity of the SiQDs can be effectively quenched due to dynamic and static mixed quenching mechanisms. More significantly, there was a remarkable linear correlation between fluorescence quenching efficiency and a broad concentration of CGA solution range from 10 to 150 μmol/L with a limit of detection (LOD) of 0.43 μmol/L. Furthermore, the proposed SiQDs were successfully applied to analyze CGA in coffee beans and instant coffee after simple pretreatment with satisfactory results. Based on these, a high sensitivity and excellent selectivity fluorescent probe detection system was constructed, and it provides a valuable platform for the detection of CGA and has broad application prospects in the biological and pharmaceutical analysis field.

脉冲激光器高灵敏功率检测系统设计

脉冲激光器高灵敏功率检测系统设计

Scanning single-molecule counting system for Eprobe with highly simple and effective approach.

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

Paper-based chemiluminescence enzyme-linked immunosorbent assay enhanced by biotin-streptavidin system for high-sensitivity C-reactive protein detection

High-sensitivity C-reactive protein (hs-CRP) has been regarded as a risk predictor of cardiovascular disease (CVD). Despite there are many methods to detect hs-CRP, quantitative, rapid, convenient, multiplex and highly sensitive measurement of it is still a challenge for point-of-care applications. In this study, we developed a paper-based ELISA to detect hs-CRP and the sensitive chemiluminescence was applied as detection signal. In this developed assay method, CRP concentration and chemiluminescence intensity were linearly correlated (r = 0.999) with a limit of detection (LOD) as low as 0.49 ng mL−1, which was comparable to that of conventional ELISA and superior to most of the current reported POCT methods for detection of hs-CRP. The precision of the assay was confirmed for low coefficient of variations, less than 7% for intra-assay and less than 10% for inter-assay. In clinical sample analysis, the results of hs-CRP detected by this assay were in good accordance with which obtained by commercial high sensitivity ELISA kit for in vitro diagnosis (r = 0.975). This assay required only 4 μL of sample and could be finished in less than 30 min. It may therefore be employed as a rapid pre-screening tool to identify patients with elevated risk of CVD.

Abstract 3331: Development of a sensitive method to detect aberrant DNA methylation in cfDNA for pancreas cancer diagnosis

Abstract Pancreatic cancer is a highly lethal malignancy despite recent clinical advances. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop noninvasive biomarkers for detection. Epigenetic alterations have been recognized as a common hallmark of many types of human cancers as well as pancreatic cancer. The cell-free DNA (cfDNA) in blood possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system may be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first we performed genome-wide DNA methylation analysis of 38 endoscopic ultrasound-guided fine-needle aspiration specimens of pancreatic cancer using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. Analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated in at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We next examined the DNA methylation status of these markers in the validation cohort (n=46) by bisulfite pyrosequencing or quantitative methylation-specific PCR and found that 42/46 (91.3%) cases were methylated in at least one marker. Finally, we analyzed methylation status in cfDNA from pancreatic cancer patients. We performed bisulfite sequencing using next-generation sequencer and detected methylation in only 5/13 (38.5%) samples in one of the markers. Since the amount of DNA from cancer tissue is very small, we need to develop more a sensitive technique to detect aberrant DNA methylation to diagnose cancer patients. Now we are developing a new sensitive method to detect DNA methylation in cfDNA. Citation Format: Keiko Shinjo, Keisuke Katsushima, Miho Suzuki, Genta Nagae, Hiroyuki Aburatani, Kenji Yamao, Yutaka Kondo. Development of a sensitive method to detect aberrant DNA methylation in cfDNA for pancreas cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3331.

Development of High-Sensitive Detection System for Redox Analytes Having a Standard Potential Higher Than O2 Evolution by Using Micropatterned Conductive Boron-Doped DLC Electrodes

1. Introduction Recently, cobalt, manganese and cerium are widely used in mass-produced products such as a lithium ion battery. It has been reported that the excessive consumption of these metal ions causes various diseases like neurotoxicity. In industrial wastewater discharge standards, Co2+, Mn2+ and Ce3+contents of wastewater has been regulated below several micro-molar. These redox species are generally analyzed using photochemical method. Some time, however, is required for analysis using the method and initial measures cannot be taken promptly when problems such as the content of metal ions exceeding the discharge standard occur. Therefore, electrochemical monitoring system which can shorten time of analysis should be developed. The standard potential of Co2+, Mn2+ and Ce3+ are higher than that of O2evolution potential. Redox reactions of these analytes cannot be observed at conventional metal electrodes. In this study, boron-doped diamond-like carbon (B-DLC) [1] with higher overpotential for O2evolution was selected as the electrode that redox reaction of the analytes can be observed. The detection limit of Ce3+ (model analyte) at a flat-disk electrode of B-DLC was 39 mM. Our research group tried to fabricate interdigitated microarray electrodes (IDA) because it shows one or two orders of magnitude higher sensitivity than that of the flat-disk electrode. IDA are composed of two sets of microband array neighbored (called generator and collector). The potential of one microband pairs (collector) is set at reduction potential of redox analyte, and that of the other is swept toward oxidation potential (dual mode). Redox cycling occurs between two sets of the microband pairs. The generator exhibits higher amplification of oxidation current by redox cycling compared to the current without applying the potential on the collector (single mode). We have reported that B-DLC-IDA with 2 mm/2 mm of bandwidth/gap exhibited ca.10 times amplification factor corresponding to the theoretical value [2]. The amplification factor of Ce3+, however, was half of the theoretical value. As a result, the detection limit was 8.8 mM. The reduction of Ce4+ is not promoted at the collector because the sample solution is a reductant. Moreover, Ce3+generated at the collector is hardly supplied to generator by diffusion, which causes inefficient redox cycling. When the collector is set at an oxidation potential, Ce3+ are constantly supplied to the collector by nonplanar diffusion. Ce4+generated at the collector is considered to be supplied to the generator at a higher diffusion rate. In this report, amplification factor of Ce3+was tried to be improved by changing the potential set at the collector. 2. Experimental B-DLC was synthesized on quartz substrates with RF plasma CVD method using a mixed solution of n-hexane and trimethyl borate as a source material. RF power was 175 W and the deposition time was 40 minutes. The alminium pattern of IDA was fabricated by the lift-off technique using photoresist and alminium deposition. Alminium on B-DLC-IDA was removed in a sodium hydroxide solution after O2plasma etching of B-DLC. The structure of B-DLC-IDA was designed to be 65 pairs of microbands with a length of 200 μm and bandwidth/gap of 2.0 mm. Single-dual mode measurements were carried out using the B-DLC-IDA in 1 mM Ce(NO3)3 + 0.1 M HNO3solution. 3. Results and discussion Figure1 shows the optical micrographs of the B-DLC-IDA. The bandwidths and gaps were observed to be 1.96 and 2.04 mm, respectively. The deviation between the actual gap and the designed value was less than 2%. Raman spectra of B-DLC were identical before and after the processing of IDA. It was confirmed that amorphous structure and composition ratio of sp2/sp2+sp3 (= 75%) in B-DLC did not change during IDA processing. The working potential range in 0.1 M sulfuric acid was constant after IDA processing. These results suggest that the redox reaction of Ce3+can be served at the B-DLC-IDA. In the dual mode of Ce3+, the potential of the collector was set at 1.37 V, and the potential of generator was swept from 0.8 to 1.7 V. Amplification factor closed to the theoretical value could be obtained. The detection limit of Ce3+was decreased from 8.8 to 4.4 mM by changing the applied potential of the collector from 1.25 to 1.37 V. 4. Summary Fabrication of the B-DLC-IDA and optimizing method of electrochemical measurement can establish highly sensitive electrochemical system that can analyze redox species with a standard potential higher than O2evolution. 5.

Development of High-Sensitive Detection System for Redox Analytes Having a Standard Potential Higher Than O2 Evolution by Using Micropatterned Conductive Boron-Doped DLC Electrodes

High sensitive electroanalytical system for redox analytes with standard potential higher than O2 evolution was developed by realizing interdigitated microarray electrodes based on boron-doped DLC (B-DLC-IDA). For Ce3+/4+ with the standard potential of 1.6 V, the amplification of response current derived from redox cycling was observed at B-DLC-IDA in the dual mode measurement. The amperometric current was amplified 180-fold by applying the potential of 1.7 V and 0.8 V to the collector and the generator electrodes in chronoamperometriy (CA) at the dual mode measurement. Theoretical detection limit (S/N = 3) toward Ce3+ was 0.13 μM. This detection limit was able to be improved to the value 2 orders of magnitude lower than that at B-DLC flat electrode by developing the electroanalytical system using B-DLC-IDA electrodes.

Abstract 4448: Development of DNA methylation biomarkers for pancreatic cancer diagnosis

Abstract Pancreatic cancer is highly lethal malignancy despite the recent clinical advancement. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop minimally invasive biomarkers for detection. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancer. The presence of cell-free DNA (cfDNA) in blood stream has been known and cfDNA possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system might be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens of pancreatic cancer with clinical stage III-IV using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database, TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. In addition, analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We validated these methylation markers in different FNA sample set (n = 19) by bisulfite pyrosequencing or quantitative methylation specific PCR and found that 18/19 (95%) cases were methylated in at least one marker (AUC = 0.95). Finally, we performed methylation analysis in cfDNA from pancreatic cancer patients. From our preliminary data, we could detect aberrant DNA methylation in cfDNA from pancreatic cancer patients. We identified potential DNA methylation biomarkers for the diagnosis of pancreatic cancers and these markers might be applicable for detecting cancer in cfDNA. Citation Format: Keiko Shinjo, Keisuke Katsushima, Genta Nagae, Hiroyuki Aburatani, Kenji Yamao, Yutaka Kondo. Development of DNA methylation biomarkers for pancreatic cancer diagnosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4448.

Abstract B14: Use of DNA methylation in circulating free DNA as a potent biomarker for pancreatic cancer detection

Abstract Pancreatic cancer is highly lethal malignancy despite the recent clinical advancement. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop novel biomarkers for early detection with blood tests. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancer. Studies have shown that epigenetic dysregulation is associated with multiple steps during carcinogenesis. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system may be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic system, first, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens of pancreatic cancer with clinical stage III-IV using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database, TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. In addition, analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We next examined the DNA methylation status in the markers in the validation cohort (n=19) by bisulfite pyrosequencing and quantitative methylation specific PCR and found that 18/19 (95%) cases were methylated in at least one marker (AUC=0.95). Currently, our preliminary data showed that more than 95% of cancer patients have aberrant DNA methylation in circulating free DNA (cfDNA). Our results indicate that detection of DNA methylation in the cfDNA might be a promising and potent tool for diagnosis of pancreatic cancers. Citation Format: Keiko Shinjo, Yutaka Kondo. Use of DNA methylation in circulating free DNA as a potent biomarker for pancreatic cancer detection. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B14.