Hybrid Probe Research Articles

Hybrid multi-modal NDE sensing system for in-motion detection and localization of rolling contact fatigue damage in rails

This article presents a multi-modal hybrid-probe approach to nondestructive inspection of RCF cracks and damage in rails. A combination of electromagnetic (EM) and ultrasonic testing (UT) techniques are presented, which allows for complementary physics to be utilized to enhance detection and characterization of surface and sub-surface cracks in a non-contact manner at high speeds. A novel integrated design which combines the motion-induced eddy current (MIEC) effect and ultrasonic Rayleigh surface waves generated and detected using electromagnetic acoustic transducer (EMAT) is presented. The hybrid probe was tested at low speeds to demonstrate an increased damage localization capability. This was carried out using a data registration and fusion approach between the sensing modalities. Finally, the capability of MIEC effect at high-speeds is demonstrated. The results show that the hybrid probe has a high potential for in-motion, high-speed damage detection and characterization in the future.

Reductive carbon dots for reduction, ratiometric fluorescence determination, and intracellular imaging of Au3+

Many studies show that ortho-phenylenediamine (OPD) produces an oxidized fluorescent product when exposed to an oxidizing agent that enables the direct or indirect fluorescence detection of a range chemical and biochemical analytes. However, there is no report on this unique optical behavior for other two isomers of phenylenediamine. This study demonstrates that a simple hydrothermal treatment of para-phenylenediamine (PPD) in the presence of sulfuric acid results in the formation of fluorescent N, S-doped carbon dots (CDs) with triple functionalities including the reduction of Au3+ into gold nanoparticles (AuNPs), the stabilization of the produced AuNPs, and the determination of Au3+ concentration through an intrinsic ratiometric fluorescence signal. In the presence of Au3+, the blue emission of CDs at 437 nm quenched, and a green emission at 540 nm emerged. The linear concentration range for the determination of Au3+ was 20 nM–16 µM with a detection limit of 16 nM. Additionally, the dual emissive CDs-AuNPs hybrid probe showed potential for the indirect fluorescence ratiometric determination of cysteine and sulfide ions. The linear concentration range for cysteine and sulfide ions were 0.25–8 μM and 0.1–6 μΜ, with detection limits of 0.095 μM and 0.041 μM, respectively. Accordingly, CDs were applied to detect Au3+ and S2− in real water samples. Moreover, the synthesized CDs showed no cytotoxicity for HeLa cells up to 300 µg mL−1, as determined by the MTT assay. Therefore, their potential for intracellular imaging of Au3+ in living cells was also investigated.

A novel PEC-EC dual-mode biosensing platform for dual target detection of miRNA-133a and cTnI

MicroRNA-133a (miRNA-133a) and cardiac troponin I (cTnI) are different-type crucial biomarkers of acute myocardial infarction (AMI), whose levels are great significance for AMI diagnosis and treatment. Herein, a novel photoelectrochemical-electrochemical (PEC-EC) dual-mode biosensing platform for dual-target assays of miRNA-133a and cTnI was developed. In which, a PEC-EC dual-mode sensing platform for miRNA-133a was constructed based on the changes of the photocurrent inhibition effect and the electrochemical signal of Fc on the Fc-hairpin DNA probe (Fc-HP)/ZnCdS-quantum dots (QDs)/ITO electrode. Furthermore, under magnetic separation and the specific interaction between cTnI and its aptamer, the N-doped porous carbon-ZnO polyhedra (NPC-ZnO)-hemin-capture DNA probe hybrid (NH-CP) was obtained and introduced to the Fc-HP/ZnCdS-QDs/ITO electrode via hybridization between NH-CP and Fc-HP. The hemin molecules encapsulated in NH-CP could effectively induce the photocurrent-polarity-switching of the Fc-HP/ZnCdS-QDs/ITO electrode and generate a new electrochemical signal originating from hemin. Thus, cTnI was assayed sensitively and selectively by the PEC-EC dual-mode biosensing platform. Here, Fc and hemin not only serve as the electrochemical indicators, but also respectively inhibit the photocurrent and switch the photocurrent polarity of ZnCdS-QDs. Furthermore, the proposed biosensing platform could be easily expanded to the detection of other multiplex-type biomarkers via the change of the sequences of the related DNA probes, implying its significant potential in clinical diagnosis and biological analysis.

High fidelity detection of miRNAs from complex physiological samples through electrochemical nanosensors empowered by proximity catalysis and magnetic separation

Electrochemical detection of miRNA biomarkers in complex physiological samples holds great promise for accurate evaluation of tumor burden in the perioperative period, yet limited by reproducibility and bias issues. Here, nanosensors installed with hybrid probes that responsively release catalytic DNAzymes (G-quadruplexes/hemin) were developed to solve the fidelity challenge in an immobilization-free detection. miRNA targets triggered toehold-mediated strand displacement reactions on the sensor surface and resulted in amplified shedding of DNAzymes. Subsequently, the interference background was removed by Fe3O4 core-facilitated magnetic separation. Binding aptamers of the electrochemical reporter (dopamine) were tethered closely to the catalytic units for boosting H2O2-mediated oxidation through proximity catalysis. The one-to-many conversion by dual amplification from biological-chemical catalysis facilitated sufficient homogeneous sensing signals on electrodes. Thereby, the nanosensor exhibited a low detection limit (2.08 fM), and high reproducibility (relative standard deviation of 1.99%). Most importantly, smaller variations (RSD of 0.51–1.04%) of quantified miRNAs were observed for detection from cell lysates, multiplexed detection from unprocessed serum, and successful discrimination of small upregulations in lysates of tumor tissue samples. The nanosensor showed superior diagnostic performance with an area under curve (AUC) of 0.97 and 94% accuracy in classifying breast cancer patients and healthy donors. These findings demonstrated the synergy of signal amplification and interference removal in achieving high-fidelity miRNA detection for practical clinical applications.

Hybrid Small-Molecule/Protein Fluorescent Probes.

Hybrid small-molecule/protein fluorescent probes are powerful tools for visualizing protein localization and function in living cells. These hybrid probes are constructed by diverse site-specific chemical protein labeling approaches through chemical reactions to exogenous peptide/small protein tags, enzymatic post-translational modifications, bioorthogonal reactions for genetically incorporated unnatural amino acids, and ligand-directed chemical reactions. The hybrid small-molecule/protein fluorescent probes are employed for imaging protein trafficking, conformational changes, and bioanalytes surrounding proteins. In addition, fluorescent hybrid probes facilitate visualization of protein dynamics at the single-molecule level and the defined structure with super-resolution imaging. In this review, we discuss development and the bioimaging applications of fluorescent probes based on small-molecule/protein hybrids.

Sensitively detection of ascorbic acid in orange samples with ionic liquid functionalized carbon dots and lanthanide complexes

In the food industry, ascorbic acid (AA) is widely used as an antioxidant. However, the concentration of AA is influenced by various factors, including temperature, time, and pH value during food preparation and storage, leading to its instability in content. This makes the accurate detection of AA content particularly important. Precise determination of AA content in food and pharmaceuticals is crucial for ensuring the adequate intake of essential vitamins and maintaining good health. Herein, we have developed a successful preparation method of an imidazole-based ionic liquid functionalized CDs hybrids APM-CDs. A novel photo functional hybrid probe Eu3+@APM-CDs was obtained via an ion-exchange reaction between APM-CDs and lanthanide complex Eu(DPA)3. The obtained hybrid probe was extensively characterized in detail. What's more, the results show that the fluorescent intensity of Eu3+@APM-CDs has a good linear response to AA concentrations in the range of 25–500 μM (R2 = 0.99), with a detection limit of 80 nM. The fluorescence probe demonstrates good selectivity and sensitivity toward AA and has the potential for significant application in AA detection by simplifying procedures and reducing interference from impurities.

A photoelectrochemical biosensor based on self-calibration platform of carbon-rich plasmonic probe with near-infrared driving signal amplification

Exploring the photochemical (PEC) method induced by low-energy light source makes great significance to achieve high stability and accurate analysis. A sensing platform driven by near-infrared (NIR) light was designed by making the biochemically encoded carbon rich plasmonic hybrid (CPH) probe, the peptide@C–Mo2C. The inherent plasmonic effect of C–Mo2C CPH can directly absorb NIR light, thus starting effective electronic-hole pairs separation. Moreover, the photothermal effect of C–Mo2C CPH also promoted the reaction yield of photothermal catalyst reaction on sensing interface to assist the PEC signal amplification. In the presence of target trypsin, it cleaves the peptides, resulting in the release of peptide@C–Mo2C probe from interface, which leads to a relative decrease in PEC signal. More importantly, a self-calibration system consisting of two independent PEC test channels attempted to eliminate the influence of background signal and baseline drift. The test channel was used to specify the recognition target, while the blank channel was used as a reference. Therefore, the signal difference between two channels was recorded, so as to obtain results with less error and higher stability. In this NIR driven PEC sensor, the carbon rich probe with direct and efficient NIR light conversion promoted the sensitivity and a self-calibration system guaranteed the stability which provided innovative thoughts for developing ingenious PEC sensor.

A specific, ultra-sensitive and rapid naked-eye on-site recognition of CN– and Cu2+ ions in tap water using an isonicotinohydrazide-salisaldehyde hybrid probe

Schiff base 1, namely (E)-N'-(2-hydroxy-3-methoxybenzylidene)isonicotinohydrazide, exhibits amide-iminol tautomerism in its molecular structure and functions as a colorimetric sensor for detecting cyanide (CN–) and copper ions (Cu2+) in ethanol/H2O (1:1). Compound 1 can distinguish CN– and Cu2+ ions from various competing ions based on unique color and spectral alterations. Spectral analyses reveal the prevalence of the iminol-form of 1 over the amide-form, enabling the selective detection of CN– and Cu2+ ions. When CN– is present, deprotonation occurs in both the phenolic and iminolic groups, leading to increased conjugation along molecule 1, as confirmed by 1H NMR spectroscopy. On the other hand, Cu2+ sensing involves coordination with 1 through a tridentate moiety, specifically interacting with the azomethine nitrogen atom as well as the deprotonated phenolic and iminolic groups. This mechanism allows for superior detection of Cu2+ compared to CN–. Key features of this sensor include its selectivity, sensitivity (with detection limits of 4.05 nM for CN– and 1.7 nM for Cu2+), a rapid response (less than 5 s), and the capability for on-site monitoring of CN– and Cu2+ ions in tap water using either solution or solid kits. Additionally, DFT calculations have validated the influence of Cu2+ and CN– on the system's electronic structure, as demonstrated by the results of MEP maps and changes in gap energy values.

Protein-encapsulated long-wavelength fluorescent probe hybrid for imaging lipid droplets in living cells and mice with non-alcoholic fatty liver

Non-alcoholic fatty liver disease (NAFLD) is prevalent worldwide as a chronic liver disease that not only gives rise to hepatic complications, but leads to other chronic diseases such as type 2 diabetes and atherosclerosis. The aberrant accumulation of lipid droplets (LDs) in hepatocytes is a prominent signature of NAFLD. However, conventional techniques lack the capability to effectively monitor the dynamic changes in LD levels during NAFLD with living organisms. Hence, it is imperative to develop LD-specific long-wavelength fluorescent probes with high imaging contrast for the in-situ diagnosis of NAFLD. In this study, we synthesized a new LD-selective long-wavelength fluorescent probe, denoted as LD-1, based on the twisted intramolecular charge transfer (TICT) mechanism. The probe exhibits a large Stokes shift and intensive fluorescence emission in nonpolar and viscous solutions. By self-assembling LD-1 with bovine serum albumin (BSA), a biocompatible, long-wavelength fluorescent probe hybrid, LD-1@BSA, was formed, allowing for LDs to be selectively imaged in hepatocytes. Moreover, LD-1@BSA successfully discriminates NAFLD cells before and after drug treatment, and achieves non-invasive and real-time monitoring of LD accumulation in a mouse model of NAFLD.

Rational design of NT-PSMA heterobivalent probes for prostate cancer theranostics.

Targeting the prostate-specific membrane antigen (PSMA) with radiopharmaceuticals for imaging and/or therapy has demonstrated significant advancement in the management of prostate cancer patients. However, PSMA targeting remains unsuccessful in prostate cancers with low expression of PSMA, which account for 15% of cases. The neurotensin receptor-1 (NTS1) has been highlighted as a suitable oncotarget for imaging and therapy of PSMA-negative prostate cancer lesions. Therefore, heterobivalent probes targeting both PSMA and NTS1 could improve the prostate cancer management. Herein, we report the development of a branched hybrid probe (JMV 7489) designed to target PSMA and/or NTS1 bearing relevant pharmacophores and DOTA as the chelating agent. The new ligand was synthesized with a hybrid approach, which includes both syntheses in batch and in the solid phase. Saturation binding experiments were next performed on HT-29 and PC3-PIP cells to derive K d and B max values. On the PC3-PIP cells, [68Ga]Ga-JMV 7489 displayed good affinity towards PSMA (K d = 53 ± 17 nM; B max = 1393 ± 29 fmol/106 cells) in the same range as the corresponding reference monomer. A lower affinity value towards NTS1 was depicted (K d = 157 ± 71 nM; B max = 241 ± 42 fmol/106 cells on PC3-PIP cells; K d = 246 ± 1 nM; B max = 151 ± 44 fmol/106 cells on HT-29 cells) and, surprisingly, it was also the case for the corresponding monomer [68Ga]Ga-JMV 7089. These results indicate that the DOTA macrocycle and the linker are critical elements to design heterobivalent probes targeting PSMA and NTS1 with high affinity towards NTS1.

Translocator Protein 18 kDa (TSPO): A Promising Molecular Target for Image-Guided Surgery of Solid Cancers.

The translocator protein 18-kDa (TSPO) is a mitochondrial membrane protein that is previously identified as the peripheral benzodiazepine receptor (PBR). Furthermore, it plays a significant role in a diverse range of biochemical processes, including steroidogenesis, mitochondrial cholesterol transport, cell survival and death, cell proliferation, and carcinogenesis. Several investigations also reported its roles in various types of cancers, including colorectal, brain, breast, prostate, and lung cancers, as well as melanoma. According to a previous study, the expression of TSPO was upregulated in cancer cells, which corresponds to an aggressive phenotype and/or poor prognosis. Consequently, the potential for crafting diagnostic and prognostic tools with a focus on TSPO holds great potential. In this context, several radioligands designed to target this protein have been identified, and some of the candidates have advanced to clinical trials. In recent years, the use of hybrid probes with radioactive and fluorescence molecules for image-guided surgery has exhibited promising results in animal and human studies. This indicates that the approach can serve as a valuable surgical navigator during cancer surgery. The current hybrid probes are built from various molecular platforms, including small molecules, nanoparticles, and antibodies. Although several TSPO-targeted imaging probes have been developed, their development for image-guided surgery of cancers is still limited. Therefore, this review aims to highlight recent findings on the involvement of TSPO in carcinogenesis, as well as provide a new perspective on the potential application of TSPO-targeted hybrid probes for image-guided surgery.

New hybrid radio-fluorescent probes [131I]-BPF-01 and [131I]-BPF-02 for visualisation of cancer cells: Synthesis and preliminary in vitro and ex vivo evaluations

We synthesised and biologically evaluated two new hybrid probes [131I]BPF-01 and [131I]BPF-02 which were built from three structural entities: benzothiazole-phenyl, fluorescein isothiocyanate (FITC), and iodine-131. These probes were designed for potential applications in assisting surgical procedures of solid cancers. The cytotoxicity study demonstrated that fluorescent probes BPF-01 (31.23 μg/mL) and BPF-02 (250 μg/mL) were relatively not toxic to normal immortalized human keratinocytes (HaCaT) cells, as indicated by the percentage of cell survival above 50 %. Furthermore, both probes displayed low to moderate anticancer activity against the breast cancer cells (MDA-MB-231) and prostate cancer cells (LNCaP and DU-145). The probe BPF-01 apparently showed an accumulation in the tumour tissues, as suggested by ex vivo fluorescence examinations. In addition, the cellular uptake study suggests that hybrid probe [131I]-BPF-01 was potentially accumulated in the MCF-7 cell line with the highest uptake of 16.11 ± 1.52 % after 2 h of incubation, approximately 50-fold higher than the accumulation of iodine-131 (control). The magnetic bead assay suggests that [131I]-BPF-02 and [131I]-BPF-02 showed a promising capability to interact with translocator protein 18 kDa (TSPO). Moreover, the computational data showed that the binding scores for ligands 7–8, BPF-01 and BPF-02, and [131I]-BPF-01 and [131I]-BPF-02 in the TSPO were considerably high. Accordingly, fluorescent probes BPF-01 and BPF-02, and hybrid probes [131I]BPF-01 and [131I]BPF-02 can be further developed for targeting cancer cells during intraoperative tumour surgery.

The Detection of Anthrax Biomarker DPA by Ratiometric Fluorescence Probe of Carbon Quantum Dots and Europium Hybrid Material Based on Poly(ionic)- Liquid.

Bacillus anthracis has gained international attention as a deadly bacterium and a potentially deadly biological warfare agent. Dipicolinic acid (DPA) is the main component of the protective layer of anthracis spores, and is also an anthrax biomarker. Therefore, it is of great significance to explore an efficient and sensitive DPA detection method. Herein, a novel ratio hybrid probe (CQDs-PIL-Eu3+) was prepared by a simple one-step hydrothermal method using carbon quantum dots (CQDs) as an internal reference fluorescence and a covalent bond between CQDs and Eu3+ by using a polyionic liquid (PIL) as a bridge molecule. The ratiometric fluorescence probe was found to have the characteristics of sensitive fluorescence visual sensing in detecting DPA. The structure and the sensing properties of CQDs-PIL-Eu3+ were investigated in detail. In particular, the fluorescence intensity ratio of Eu3+ to CQDs (I616/I440) was linear with the concentration of DPA in the range of 0-50 μM, so the detection limit of the probe was as low as 32 nm, which was far lower than the DPA dose released by the number of anthrax spores in human body (60 μM) and, thus, can achieve sensitive detection. Therefore, the ratiometric fluorescence probe in this work has the characteristics of strong anti-interference, visual sensing, and high sensitivity, which provides a very promising scheme for the realization of anthrax biomarker DPA detection.

Identification of Autoantibodies to a Hybrid Insulin Peptide in Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that attacks the insulin-producing b cells of the pancreatic islets. Autoantibodies to b cell proteins typically appear in the circulation years before disease onset, and serve as the most accurate biomarkers of T1D risk. Our laboratory has recently discovered novel b cell proteins comprising hybrid proinsulin:islet amyloid polypeptide peptides (IAPP). T cells from a diabetic mouse model and T1D patients are activated by these hybrid peptides. In this study, we asked whether these hybrid molecules could serve as antigens for autoantibodies in T1D and prediabetic patients. We analyzed sera from T1D patients, prediabetics and healthy age-matched donors. Using a highly sensitive electrochemiluminescence assay, sera were screened for binding to recombinant proinsulin:IAPP probes or truncated derivatives. Our results show that sera from T1D patients contain antibodies that bind larger hybrid proinsulin:IAPP probes, but not proinsulin or insulin, at significantly increased frequencies compared to normal donors. Examination of sera from prediabetic patients confirms titers of antibodies to these hybrid probes in more than 80% of individuals, often before seroconversion. These results suggest that hybrid insulin peptides are common autoantigens in T1D and prediabetic patients, and that antibodies to these peptides may serve as valuable early biomarkers of the disease.

State of Aging Classification of Modified-HP Steel Tubes by Eddy Current Test Applying Machine Learning

In the petrochemical industry, steam reforming furnaces play a crucial role in large-scale hydrogen production. These furnaces are equipped with centrifugal cast HP-modified austenitic stainless-steel tubes, which are exposed to temperatures ranging from 600 to 1000ºC for extended periods. As a result, the wall temperature profile of these tubes exhibits a vertical gradient of distinct aging states, labelled as state I to VI in the literature, each characterized by specific microstructures. Given that the reformer tubes are expensive components of the furnace assembly, it is imperative to monitor their service life. Non-destructive testing is a vital tool for evaluating the structural integrity of industrial components. Hence, the objective of this study is to establish a real-time classification system for determining the aging states of HP-modified stainless-steel tubes using a non-destructive magnetic system and the machine learning technique Support Vector Machine (SVM). Two tubes, each measuring 12.6 meters in length, were removed from the same steam reforming furnace after 160,000 hours of service. The inspection was conducted using an eddy current hybrid probe adapted to an inspection vehicle, allowing for real-time data acquisition. The results demonstrated that the developed classification system was capable of accurately identifying the different aging states present along the studied tubes.

Crustal Failure as a Tool to Probe Hybrid Stars

It is currently unknown if neutron stars (NSs) are composed of nucleons only or are hybrid stars, i.e., in addition to nucleonic crusts and outer cores, they also possess quark cores. Quantum chromodynamics allows for such a possibility, but accurate calculations relevant for compact stars are still elusive. Here we investigate some crust-breaking aspects of hybrid stars. We show that the crust-breaking frequency and maximum fiducial ellipticity are sensitive to the quark–hadron density jump and equation of state stiffness. Remarkably, the crust-breaking frequency related to static tides scales linearly with the mass of the star (for a given companion mass), and its slope encompasses information about the microphysics of the star. However, for precise crust-breaking frequency predictions, relativistic corrections to Kepler’s third law and the Newtonian tidal field should not be ignored. When a liquid quark core touches an elastic hadronic phase (the result of a significant energy density jump), the maximum ellipticity can increase by around an order of magnitude when compared to a liquid quark core touching a liquid hadronic phase. That is relevant because it would increase the odds of detecting continuous gravitational waves from NSs. Our order-of-magnitude analysis also suggests that a given upper limit to the ellipticity (crust-breaking frequency) could have representatives in stars with either small or intermediate (large) energy density jumps. Therefore, when upper limits to the ellipticity for isolated stars are better constrained or electromagnetic radiation (e.g., gamma-ray precursors) is detected along with gravitational waves in inspiraling binary systems, they may help constrain some aspects of phase transitions in NSs.

Ultra-sensitive and specific detection of pathogenic nucleic acids using composite-excited hyperfine plasma spectroscopy combs sensitized by Au nanoarrays functionalized with 2D Ta2C-MXene

Accurate and rapid screening techniques on a population scale are crucial for preventing and managing epidemics like COVID-19. The standard gold test for nucleic acids in pathogenic infections is primarily the reverse transcription polymerase chain reaction (RT-PCR). However, this method is not suitable for widespread screening due to its reliance on large-scale equipment and time-consuming extraction and amplification processes. Here, we developed a collaborative system that combines high-load hybridization probes targeting N and OFR1a with Au NPs@Ta2C-M modified gold-coated tilted fiber Bragg grating (TFBG) sensors to enable direct nucleic acid detection. Multiple activation sites of SARS-CoV-2 were saturable modified on the surface of a homogeneous arrayed AuNPs@Ta2C-M/Au structure based on a segmental modification approach. The combination of hybrid probe synergy and composite polarisation response in the excitation structure results in highly specific hybridization analysis and excellent signal transduction of trace target sequences. The system demonstrates excellent trace specificity, with a limit of detection of 0.2 pg/mL, and achieves a rapid response time of 1.5 min for clinical samples without amplification. The results showed high agreement with the RT-PCR test (Kappa index = 1). And the gradient-based detection of 10-in-1 mixed samples exhibits high-intensity interference immunity and excellent trace identification. Therefore, the proposed synergistic detection platform has a good tendency to curb the global spread of epidemics such as COVID-19.

Ultra-sensitive specific detection of nucleic acids in pathogenic infections by Ta2C-MXene sensitization-based ultrafine plasmon spectroscopy combs

Accurate and rapid population-scale screening techniques based on SARS-CoV-2 RNA are essential in preventing and controlling the COVID-19 epidemic. However, the sensitivity and specificity of the assay signal are challenged by the problems of target dilution and sample contamination inherent in high-volume pooled testing. Here, we reported a collaborative system of high-loaded hybrid probes targeting N and OFR1a coupling with the novel Ta2C-M/Au/TFBG biosensor, providing high-intensity vector signals for detecting SARS-CoV-2. The method relies on a segmental modification approach to saturable modify multiple activation sites of SARS-CoV-2 on the high-performance Ta2C-M surface. The coupling of multi-site synergy with composite excited TFBG results in excellent signal transduction, detection limits (0.2 pg/mL), and hybridization efficiency. Without relying on amplification, the collaborative system achieved specific differentiation of 30 clinical samples in an average diagnostic time of 1.8 min. In addition, for the first time, a kinetic determination of dilution mixed samples was achieved and showed a high-intensity carrier signal and fantastic stability. Therefore, it can be used as a collaborative, integrated tool to play a massive role in the screening, prevention, and control of COVID-19 and other epidemics.

Abstract 1025: OvaPrint - a novel cfDNA methylation liquid biopsy for cancer risk assessment in high grade serous ovarian carcinoma

Abstract Background: High grade serous ovarian carcinoma (HGSOC) is a highly lethal gynecologic malignancy with a typical 5-year survival rate of 40% or less when diagnosed with late-stage disease. There is currently no single effective screening tool for high risk or community-based risk assessment for HGSOC and multivariate tests for preselected patients are suboptimal. In women with a known pelvic mass, surgery followed pathological assessment is currently the only definitive way to diagnose HGSOC. About 15% of women with a suspicious mass will have HGSOC and less than 15% will present with stage 1 cancer. A liquid biopsy assay based on cell-free (cf)DNA enables non-invasive dynamic assessment of disease status in patients with cancer where cfDNA methylation is particularly promising for detection of early stage HGSOC. The goal of the current study is to develop a cfDNA methylation liquid biopsy that can be used for the screening and risk assessment for HGSOC. Methods: Tissue and plasma samples were acquired under informed consent from multiple commercial and academic sources. Reduced representation bisulfite sequencing (RRBS) was performed on 33 HGSOC tissues as well as 8 normal ovary and 13 normal fallopian tube controls. Differentially methylated regions (DMRs) were called between HGSOC and normal controls using metilene and DSS. DMR calls with p-values < 0.05 and |αβ| > 0.45 (n = 1677 DMRs) were used to generate a targeted tissue-based methylation assay using the myBaits® hybrid probe capture platform (Daicel Arbor Biosciences). Next, targeted enrichment was performed on bisulfite converted plasma cfDNA on an independent set of 84 HGSOC samples (27 stage 1, 20 stage 2, 37 stage 3) and 100 samples from patients with benign ovarian masses. Mean beta value across DMRs were used to train a novel cfDNA methylation classifier model called OvaPrintTM to discriminate HGSOC from benign masses using a repeated 3-fold cross validation approach to obtain an unbiased estimate of the performance of the classifier. The average cross validated score for each sample was used to generate a receiver operator characteristic (ROC) curve. Results. RRBS analysis on HGSOC tissue and normal tissue controls demonstrated excellent separation of HGSOC from normal tissue samples using the selected DMRs. OvaPrintTM testing in an independent cohort of 184 plasma samples discriminated HGSOC from benign tumors with a high sensitivity and specificity (area under curve (AUC) = 0.931) using cfDNA methylation. Conclusions: OvaPrintTM is a novel cfDNA methylation liquid biopsy that can be used for effective cancer risk assessment for HGSOC in women presenting with pelvic masses. Citation Format: David N. Buckley, Gerald Gooden, Lee Shulman, Monica Newman, Mona Guo, Ben Yi Tew, Lynda Roman, Juan Pablo Lewinger, Bodour Salhia. OvaPrint - a novel cfDNA methylation liquid biopsy for cancer risk assessment in high grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1025.

A DNA/RNA hybrid fluorescent probe for high-throughput quantification of the activity of human apurinic/apyrimidinic endonuclease 1 in subcellular extracts

As a multifunctional DNA repair enzyme, human apurinic/apyrimidinic endonuclease 1 (APE1) plays a pivotal role in many cellular processes. In recent years, it has been recognized as a potential prognostic biomarker and a therapeutic target for many cancers and other diseases. In this work, we report a DNA/RNA hybrid-based fluorescent probe which allows rapid and high-throughput detection of APE1 in the nuclear, cytoplasmic, and mitochondrial extracts of different types of cultured cells without any purification steps. As a noncanonical substrate, the DNA/RNA hybrid probe possesses inherent specificity toward APE1 without the need of additional chemical modification. A substantial acceleration effect of the 3′-flanking mismatches enabled rapid digestion of the abasic sites in the DNA/RNA hybrid by APE1 at a comparable rate to those in natural double-stranded DNA. The hybrid probe showed adequately high sensitivity to the target enzyme (linear working range: 0.02–1.0 U/mL; detection limit: 0.01 U/mL) and superior resistance to interference from cellular proteins. The mechanism for the 3′-mismatch enhancement was also clarified via kinetic study and single-molecule fluorescence analysis. By using the probe, significantly varied subcellular distribution of APE1 in different cells were disclosed, showing the importance of quantitative measurement of APE1 activity in subcellular compartments for drug screening and evaluation of the treatment efficacy. The method was simple and robust, which offered an easily accessible molecular tool for high-throughput quantification of the subcellular APE1 activity. It may also facilitate the development of effective anticancer drugs.