Ultrasensitive Simultaneous Detection of Multiple Rare Modified Nucleosides as Promising Biomarkers in Low-Put Breast Cancer DNA Samples for Clinical Multi-Dimensional Diagnosis

Early cancer diagnosis is essential for successful treatment and prognosis, and modified nucleosides have attracted widespread attention as a promising group of cancer biomarkers. However, analyzing these modified nucleosides with an extremely low abundance is a great challenge, especially analyzing multiple modified nucleosides with a different abundance simultaneously. In this work, an ultrasensitive quantification method based on chemical labeling, coupled with LC-MS/MS analysis, was established for the simultaneous quantification of 5hmdC, 5fdC, 5hmdU and 5fdU. Additionally, the contents of 5mdC and canonical nucleosides could be obtained at the same time. Upon derivatization, the detection sensitivities of 5hmdC, 5fdC, 5hmdU and 5fdU were dramatically enhanced by several hundred times. The established method was further applied to the simultaneous detection of nine nucleosides with different abundances in about 2 μg genomic DNA of breast tissues from 20 breast cancer patients. The DNA consumption was less than other overall reported quantification methods, thereby providing an opportunity to monitor rare, modified nucleosides in precious samples and biology processes that could not be investigated before. The contents of 5hmdC, 5hmdU and 5fdU in tumor tissues and normal tissues adjacent to the tumor were significantly changed, indicating that these three modified nucleosides may play certain roles in the formation and development of tumors and be potential cancer biomarkers. While the detection rates of 5hmdC, 5hmdU and 5fdU alone as a biomarker for breast cancer samples were 95%, 75% and 85%, respectively, by detecting these three cancer biomarkers simultaneously, two of the three were 100% consistent with the overall trend. Therefore, simultaneous detection of multiple cancer biomarkers in clinical samples greatly improved the accuracy of cancer diagnosis, indicating that our method has great application potential in clinical multidimensional diagnosis.     

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

Serum Proteomics in Biomedical Research: A Systematic Review

Proteins that are important indicators of physiological or pathological states may contribute to the early diagnosis of disease, which may provide a basis for identifying the underlying mechanism of disease development. Serum, contains an abundance of proteins, offers an easy and inexpensive approach for disease detection and possesses a high potential to revolutionize the diagnostics. These differentially expressed proteins in serum have become an important role to monitoring the state for disease. Availability of emerging proteomic techniques gives optimism that serum can eventually be placed as a biomedium for clinical diagnostics. Advancements have benefited biomarker research to the point where serum is now recognized as an excellent diagnostic medium for the detection of disease. Comprehensive proteome of human serum fluid with high accuracy and availability has the potential to open new doors for disease biomarker discovery and for disease diagnostics, providing insights useful for future study. Thus, this review presents an overview of the value of serum as a credible diagnostic tool, and we aim to summarize the proteomic technologies currently used for global analysis of serum proteins and to elaborate on the application of serum proteomics to the discovery of disease biomarkers, and discuss some of the critical challenges and perspectives for this emerging field.     

Methods of screening,monitoring and management of cardiac toxicity induced by chemotherapeutics

Cardiac toxicity is one of the most common side effects of anticancer drugs. Cardiac toxicity results dysfunction of heart including hypotension, heart failure, and even cause death in extreme cases. The potential risk of cardiotoxicity is a huge concern in chemotherapeutics mediated cancer treatment. The individual with any pre-existing cardiac issues are excluded from clinical trials due to the potential risk of cardiotoxicity. Because of the potential cardiotoxicity, there is an emerging need...     

Tellurium-Modified Nucleosides,Nucleotides, and Nucleic Acids with Potential Applications

Tellurium was successfully incorporated into proteins and applied to protein structure determination through X-ray crystallography. However, studies on tellurium modification of DNA and RNA are limited. This review highlights the recent development of Te-modified nucleosides, nucleotides, and nucleic acids, and summarizes the main synthetic approaches for the preparation of 5-PhTe, 2′-MeTe, and 2′-PhTe modifications. Those modifications are compatible with solid-phase synthesis and stable during Te-oligonucleotide purification. Moreover, the ideal electronic and atomic properties of tellurium for generating clear isomorphous signals give Te-modified DNA and RNA great potential applications in 3D crystal structure determination through X-ray diffraction. STM study also shows that Te-modified DNA has strong topographic and current peaks, which immediately suggests potential applications in nucleic acid direct imaging, nanomaterials, molecular electronics, and diagnostics. Theoretical studies indicate the potential application of Te-modified nucleosides in cancer therapy.     

Advent of the cancer methylome

DNA hypermethylation of CpG islands plays an important role in gene regulation during cancer development. Many techniques have been developed to detect global DNA methylation in cancer cells compared to normal tissues. This knowledge helps us to better understand cancer progression and also aids in the development of new biomarker for early cancer detection. New prognostic tools for monitoring drug efficacy during cancer treatment can also be developed. In this review, we will examine the different techniques that have been used to study DNA methylation, as well as the emerging high resolution, high throughput techniques for identification of methylated regions to defining cancer related genes in the cancer methylome.     

Multiplex detection platform for tumor markers and glucose in serum based on a microfluidic microparticle array

We present a multiplex detection platform based on a microfluidic microparticle array to detect proteins and glucose in serum simultaneously. Multiplex detection of proteins and glucose was performed using biofunctionalized microparticles arrayed on gel-based microstructures integrated in microfluidics. The microparticles immobilized on these microstructures showed high stability under microfluidic flow conditions. With arrays of antibody-coated microbeads, microfluidic quantitative immunoassays for two protein tumor markers, human chorionic gonadotropin (hCG) and prostate specific antigen (PSA) were performed in serum samples with detection limits bellow the cut-off values for cancer diagnosis. Parallel to the immunoassays, quantitative enzymatic assays for glucose in the physiological concentration range were performed. Multiplex detection was achieved by using a spatially encoded microarray. By patterning antibody-coated microbeads and enzyme-containing microparticles on a novel mixed structure array, we successfully demonstrated simultaneous immunoassays (binding based assay) for proteins and an enzymatic assay (reaction kinetic based assay) for glucose. Our microparticle arrays could be potentially used for the detection of multiple categories of biomolecules (proteins, small metabolites and DNA) for clinical diagnostics and other biological applications.     

Thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection

Detection of point mutations in driver genes is of great significance for the early diagnosis, treatment, and prognostic evaluation of cancer. However, current detection methods do not offer versatility, specificity,and rapid performance simultaneously. Thus, multiple mutation detection processes are necessary, which results in long processing times and high costs. In this study, we developed a thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detec...     

Precise nanomedicine for intelligent therapy of cancer

Precise nanomedicine has been extensively explored for efficient cancer imaging and targeted cancer therapy, as evidenced by a few breakthroughs in their preclinical and clinical explorations. Here, we demonstrate the recent advances of intelligent cancer nanomedicine, and discuss the comprehensive understanding of their structure-function relationship for smart and efficient cancer nanomedicine including various imaging and therapeutic applications, as well as nanotoxicity. In particular, a few emerging strategies that have advanced cancer nanomedicine are also highlighted as the emerging focus such as tumor imprisonment, supramolecular chemotherapy, and DNA nanorobot. The challenge and outlook of some scientific and engineering issues are also discussed in future development. We wish to highlight these new progress of precise nanomedicine with the ultimate goal to inspire more successful explorations of intelligent nanoparticles for future clinical translations.     

Applications of capillary electrophoresis on microchip

CE on microchip is an emerging separation technique that has attracted wide attention and gained considerable popularity. Because of miniaturization of the separation format, CE on chip typically offers shorter analysis time and lower reagent consumption with potential development of portable analytical instrumentation. This review with 143 references is focused on proteins and peptides analysis, DNA separation including fragment sizing, genotyping, mutation detection and sequencing, and also the analysis of low-molecular-weight compounds, namely explosive residues and warfare agents, pharmaceuticals and drugs of abuse, and various small molecules in body fluids.     

Electrophoresis mobility shift assay and biosensor used in studying the interaction between bleomycin A5 and DNA

DNA is the potential target of numerous drugs utilized widely in clinical cancer therapy. Here we employed bleomycin A5, with its deactivated form as contrast, to investigate the interaction between small pharmaceutical and DNA. Electrophoresis mobility shift assay (EMSA) which is a common technique used in studying specific interactions between DNA and proteins is applied in visualizing the binding of bleomycin A5 with DNA intuitively. More accurate association equilibrium constant for native and deactivated bleomycin A5 to DNA achieved on biosensor IAsys AUTO⁺ is 1.25 × 10⁴ and 1.3 × 10³ M⁻¹, respectively. With combination of EMSA and biosensor, a qualitative and quantitative method is described, which can be extended to studying the binding of most small molecules with targeting DNA and serves as a powerful tool in designing and screening for novel drugs.     

Analyte‐Triggered DNA‐Probe Release from a Triplex Molecular Beacon for Nanopore Sensing

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex‐forming molecular beacon and a stem‐forming DNA component that is modified with a host–guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α‐hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point‐of‐care diagnostics with a portable nanopore kit in the future.     

High Throughput Isolation and Data Independent Acquisition Mass Spectrometry (DIA-MS) of Urinary Extracellular Vesicles to Improve Prostate Cancer Diagnosis

Proteomic profiling of extracellular vesicles (EVs) represents a promising approach for early detection and therapeutic monitoring of diseases such as cancer. The focus of this study was to apply robust EV isolation and subsequent data-independent acquisition mass spectrometry (DIA-MS) for urinary EV proteomics of prostate cancer and prostate inflammation patients. Urinary EVs were isolated by functionalized magnetic beads through chemical affinity on an automatic station, and EV proteins were analyzed by integrating three library-base analyses (Direct-DIA, GPF-DIA, and Fractionated DDA-base DIA) to improve the coverage and quantitation. We assessed the levels of urinary EV-associated proteins based on 40 samples consisting of 20 cases and 20 controls, where 18 EV proteins were identified to be differentiated in prostate cancer outcome, of which three (i.e., SERPINA3, LRG1, and SCGB3A1) were shown to be consistently upregulated. We also observed 6 out of the 18 (33%) EV proteins that had been developed as drug targets, while some of them showed protein-protein interactions. Moreover, the potential mechanistic pathways of 18 significantly different EV proteins were enriched in metabolic, immune, and inflammatory activities. These results showed consistency in an independent cohort with 20 participants. Using a random forest algorithm for classification assessment, including the identified EV proteins, we found that SERPINA3, LRG1, or SCGB3A1 add predictable value in addition to age, prostate size, body mass index (BMI), and prostate-specific antigen (PSA). In summary, the current study demonstrates a translational workflow to identify EV proteins as molecular markers to improve the clinical diagnosis of prostate cancer.     

DNA conformational switches as sensitive electronic sensors of analytes

The electrical conductivity of DNA is dependent on its conformational state. We demonstrate here that such a dependence may be harnessed for the electronic sensing of external analytes, for instance, adenosine. Such a DNA sensor incorporates an analyte "receptor", whose altered conformation in the presence of bound analyte switches the conformation, and hence, the conductive path between two DNA double-helical stems. Two distinct designs for such sensors are described here, that permit significant electrical conduction through a "detector" double-helical stem only in the presence of the bound analyte. In the first design, current flows through the analyte receptor itself, whereas in the second, current flows in a path adjacent to the receptor. The former design may be especially suitable for certain categories of analytes, including heterocycle-containing compounds such as adenosine, whereas the latter design should be generally applicable to the detection of any molecular analyte, large or small. Since analyte detection in these DNA sensors is electronic, the potential exists for their application in rapid and automated chip-based detection of small molecules as well as of proteins and other macromolecules.     

Multiplexed detection of protein cancer markers on Au/Ag-barcoded nanorods using fluorescent-conjugated polymers

Integration of fluorescent-conjugated polymers as detection moiety with metallic striped nanorods for multiplexed detection of clinically important cancer marker proteins in an immunoassay format was demonstrated in this report. Specifically, cationic conjugated polymers were introduced to protein complexes through electrostatic binding to negatively charged double-stranded DNA, which was tagged on detection antibodies prior to antigen recognition. The intense fluorescence emission of conjugated polymers resulted in highly sensitive detection of cancer marker proteins wherein an undiluted bovine serum sample as low as ∼25 target molecules captured on each particle was detectable. Meanwhile, the use of polymer molecules as the detection probe did not obscure the optical pattern of underlying nanorods, i.e., the encoding capability of barcoded nanorods was preserved, which allowed simultaneous detection of three cancer marker proteins with good specificity.     

Assembling DNA through Affinity Binding to Achieve Ultrasensitive Protein Detection

Recent advances in DNA assembly and affinity binding have enabled exciting developments of nanosensors and ultrasensitive assays for specific proteins. 1 – 6 These sensors and assays share three main attractive features: 1 , 4 , 7 1) the detection of proteins can be accomplished by the detection of amplifiable DNA, thereby dramatically enhancing the sensitivity; 2) assembly of DNA is triggered by affinity binding of two or more probes to a single target molecule, thereby resulting in increased specificity; and 3) the assay is conducted in solution with no need for separation, thus making the assay attractive for potential point‐of‐care applications. We illustrate here the principle of assembling DNA through affinity binding, and we highlight novel applications to the detection of proteins.     

Advances in single-particle detection for DNA sensing

Rapid, accurate and sensitive detection of particular DNA sequence is critical in fundamental biomedical research and clinical diagnostics. However, conventional approaches for DNA assay often suffer from cumbersome procedures, long analysis time and insufficient sensitivity. Recently, single-particle detection technology has emerged as a powerful tool in the biosensing area due to its significant advantages of ultrahigh sensitivity, low sample-consumption and rapid analysis time. Especially, the introduction of novel nanomaterials has greatly promoted the development of single-particle detection and its applications for DNA sensing. In this review, we summarize the recent advance in single-particle detection strategies for DNA sensing, and focus mainly on metallic nanoparticle-and semiconductor quantum dot-based single-particle detection. We highlight the emerging trends in this field as well.     

The synthesis and bio-applications of magnetic and fluorescent bifunctional composite nanoparticles

Magnetic-fluorescent composite nanoparticles as a new kind of nanoparticle have attracted much attention in recent years. The composite nanoparticles combine the fluorescent properties, magnetic properties and the physical properties of nano-size, so they can offer a range of potential applications, such as bioseparation and bio-imaging, tumor cell localization, and even cancer treatment. This Minireview will introduce the main synthesis strategies for the fabrication of magnetic-fluorescent composite nanoparticles, the current and potential bio-application of magnetic-fluorescent nanocomposites, including protein and DNA separation and detection, bio-imaging and sorting in vitro and in vivo, drug delivery and the cancer treatment.     

Emerging optofluidic technologies for point-of-care genetic analysis systems: a review

This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for “sample-to-answer” systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA–DNA interactions in the context of point-of-care analysis systems.