A Novel Microfluidic Chip for Fast, Sensitive Quantification of Plasma Extracellular Vesicles as Biomarkers in Patients With Osteosarcoma

Plasma circulating extracellular vesicle (EV) has emerged as a promising biomarker for diagnosis and prognosis of various epithelial tumors. However, fast and efficient capture of EVs with microfluidic chip in sarcoma remains to be established. Herein, we reported a ZnO-nanorods integrated (ZNI) microfluidic chip, where EV capture antibody was uniformly grafted to the surface of the ZnO-nanorods of the chip to enhance the plasma turbulence formation and the capture efficiency at the micro-scale. Based on osteosarcoma (OS) cell line, we demonstrated that a combination of CD81 and CD63 antibody on ZNI chip yielded the greatest amount of total EVs, with an extra sensitive limit of detection (LOD) of ~104 particles mL-1. Furthermore, the addition of fluorescent labeling of Vimentin (VIM), a previously reported sarcoma cell surface biomarker, could enabled the dual visualization of total plasma EVs and VIM-positive EVs from OS patients’ plasma. Based on our ZNI chip, we found that the amount of plasma total EVs was significantly different between OS and healthy donors (1562 a.u. versus 639 a.u., p< 0.05), but not between metastatic and nonmetastatic OS (p> 0.05). Interestingly, patients with metastatic disease had a significantly greater amount of VIM-positive EVs (1411 a.u. versus 231 a.u.., p< 0.05) and increased VIM-positive/total EVs ratio (0.943 versus 0.211, p< 0.05) in comparison with the nonmetastatic counterpart. Therefore, our ZNI microfluidic chip has great potential for the fast quantification of plasma EVs, and the microfluidic-based quantification of total and VIM-positive EVs might serve as a promising biomarker for the diagnosis and surveillance in OS patients.

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Characteristics and Clinical Application of Extracellular Vesicle-Derived DNA

Cancers ◽

10.3390/cancers13153827 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3827

Author(s):

Jae Young Hur ◽

Kye Young Lee

Keyword(s):

Therapeutic Potential ◽

Extracellular Vesicle ◽

Clinical Settings ◽

Biomarker Detection ◽

Diagnosis And Prognosis ◽

Double Stranded Dna ◽

Fundamental Research ◽

The Stability ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Extracellular vesicles (EVs) carry RNA, proteins, lipids, and diverse biomolecules for intercellular communication. Recent studies have reported that EVs contain double-stranded DNA (dsDNA) and oncogenic mutant DNA. The advantage of EV-derived DNA (EV DNA) over cell-free DNA (cfDNA) is the stability achieved through the encapsulation in the lipid bilayer of EVs, which protects EV DNA from degradation by external factors. The existence of DNA and its stability make EVs a useful source of biomarkers. However, fundamental research on EV DNA remains limited, and many aspects of EV DNA are poorly understood. This review examines the known characteristics of EV DNA, biogenesis of DNA-containing EVs, methylation, and next-generation sequencing (NGS) analysis using EV DNA for biomarker detection. On the basis of this knowledge, this review explores how EV DNA can be incorporated into diagnosis and prognosis in clinical settings, as well as gene transfer of EV DNA and its therapeutic potential.

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Fabrication of an Electrochemical Sensor for Glucose Detection using ZnO Nanorods

MRS Advances ◽

10.1557/adv.2016.149 ◽

2016 ◽

Vol 1 (13) ◽

pp. 861-867 ◽

Cited By ~ 2

Author(s):

Sanghamitra Mandal ◽

Mohammed Marie ◽

Omar Manasreh

Keyword(s):

Response Time ◽

Glass Substrate ◽

Glucose Sensor ◽

Limit Of Detection ◽

Sol Gel ◽

Zno Nanorods ◽

Phosphate Buffer Solution ◽

Buffer Solution ◽

Coated Glass Substrate ◽

Coated Glass

ABSTRACTAn electrochemical glucose sensor based on zinc oxide (ZnO) nanorods is fabricated, characterized and tested. The ZnO nanorods are synthesized on indium titanium oxide (ITO) coated glass substrate, using the hydrothermal sol-gel technique. The working principle of the sensor under investigation is based on the electrochemical reaction taking place between cathode and anode, in the presence of an electrolyte. A platinum plate, used as the cathode and Nafion/Glucose Oxidase/ZnO nanorods/ITO-coated glass substrate used as anode, is immersed in pH 7.0 phosphate buffer solution electrolyte to test for the presence of glucose. Several amperometric tests are performed on the fabricated sensor to determine the response time, sensitivity and limit of detection of the sensor. A fast response time less than 3 s with a high sensitivity of 1.151 mA cm-2mM-1 and low limit of detection of 0.089 mM is reported. The glucose sensor is characterized using the cyclic voltammetry method in the range from -0.8 – 0.8 V with a voltage scan rate of 100 mV/s.

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Protein biomarkers in breast cancer-derived extracellular vesicles for use in liquid biopsies

AJP Cell Physiology ◽

10.1152/ajpcell.00048.2021 ◽

2021 ◽

Author(s):

Dan Li ◽

Wenjia Lai ◽

Di Fan ◽

Qiaojun Fang

Keyword(s):

Breast Cancer ◽

Early Diagnosis ◽

Extracellular Vesicles ◽

Liquid Biopsy ◽

Breast Cancer Diagnosis ◽

Extracellular Vesicle ◽

Detection Methods ◽

Protein Markers ◽

Liquid Biopsies ◽

Diagnosis And Prognosis

Breast cancer is the most common malignant disease in women worldwide. Early diagnosis and treatment can greatly improve the management of breast cancer. Liquid biopsies are becoming convenient detection methods for diagnosing and monitoring breast cancer due to their non-invasiveness and ability to provide real-time feedback. A range of liquid biopsy markers, including circulating tumor proteins, circulating tumor cells, and circulating tumor nucleic acids, have been implemented for breast cancer diagnosis and prognosis, with each having its own advantages and limitations. Circulating extracellular vesicles are messengers of intercellular communication that are packed with information from mother cells and are found in a wide variety of bodily fluids; thus, they are emerging as ideal candidates for liquid biopsy biomarkers. In this review, we summarize extracellular vesicle protein markers that can be potentially used for the early diagnosis and prognosis of breast cancer or determining its specific subtypes.

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Enzyme Method-Based Microfluidic Chip for the Rapid Detection of Copper Ions

Micromachines ◽

10.3390/mi12111380 ◽

2021 ◽

Vol 12 (11) ◽

pp. 1380

Author(s):

Binfeng Yin ◽

Xinhua Wan ◽

Changcheng Qian ◽

A. S. M. Muhtasim Fuad Sohan ◽

Teng Zhou ◽

...

Keyword(s):

Metal Ions ◽

Microfluidic Chip ◽

Limit Of Detection ◽

Copper Ions ◽

Practical Applications ◽

Enzyme Method ◽

In Series ◽

High Concentrations ◽

Biochemical Detection ◽

Seawater Samples

Metal ions in high concentrations can pollute the marine environment. Human activities and industrial pollution are the causes of Cu2+ contamination. Here, we report our discovery of an enzyme method-based microfluidic that can be used to rapidly detect Cu2+ in seawater. In this method, Cu2+ is reduced to Cu+ to inhibit horseradish peroxidase (HRP) activity, which then results in the color distortion of the reaction solution. The chip provides both naked eye and spectrophotometer modalities. Cu2+ concentrations have an ideal linear relationship, with absorbance values ranging from 3.91 nM to 256 μM. The proposed enzyme method-based microfluidic chip detects Cu2+ with a limit of detection (LOD) of 0.87 nM. Other common metal ions do not affect the operation of the chip. The successful detection of Cu2+ was achieved using three real seawater samples, verifying the ability of the chip in practical applications. Furthermore, the chip realizes the functions of two AND gates in series and has potential practical implementations in biochemical detection and biological computing.

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Wave-shaped microfluidic chip assisted point-of-care testing for accurate and rapid diagnosis of infections

10.21203/rs.3.rs-829274/v1 ◽

2021 ◽

Author(s):

Binfeng Yin ◽

Xinhua Wan ◽

Mingzhu Yang ◽

Changcheng Qian ◽

A S M Muhtasim Fuad Sohan

Keyword(s):

Microfluidic Chip ◽

Limit Of Detection ◽

Point Of Care ◽

Simultaneous Detection ◽

Accurate Diagnosis ◽

Point Of Care Testing ◽

Promising Alternative ◽

Reactive Protein ◽

Linear Relationships ◽

Multiple Biomarkers

Abstract Background: Simultaneous and timely detection of C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) provides effective information for the accurate diagnosis of infections. Early diagnosis and classification of infections increase the cure rate while decreasing complications, which is significant for severe infections, especially for war surgery. However, traditional methods rely on laborious operations and bulky devices. On the other hand, point-of-care (POC) methods suffer from limited robustness and accuracy. Therefore, it is of urgent demand to develop POC devices for rapid and accurate diagnosis of infections to fulfill on-site militarized requirements.Methods: We developed a wave-shaped microfluidic chip (WMC) assisted multiplexed detection platform (WMC-MDP). WMC-MDP reduces detection time and improves repeatability through premixing of the samples and reaction of the reagents. We further combined the detection platform with the streptavidin-biotin (SA-B) amplified system to enhance the sensitivity while using chemiluminescence (CL) intensity as signal readout. We realized simultaneous detection of CRP, PCT, and IL-6 on the detection platform and evaluated the sensitivity, linear range, selectivity, and repeatability. Finally, we finished detecting 15 samples from volunteers and compared the results with commercial ELISA kits.Results: Detection of CRP, PCT, and IL-6 exhibited good linear relationships between CL intensities and concentrations in the range of 1.25-40 μg/mL, 0.4-12.8 ng/mL, and 50-1600 pg/mL. The limit of detection (LOD) of CRP, PCT, and IL-6 were 0.54 μg/mL, 0.11 ng/mL, and 16.25 pg/mL, respectively. WMC-MDP is capable of good adequate selectivity and repeatability. The whole detection procedure takes only 22 minutes that meets the requirements of a POC device. Results of 15 samples from volunteers were consistent with the results detected by commercial ELISA kits.Conclusion: WMC-MDP allows simultaneous, rapid, and sensitive detection of CRP, PCT, and IL-6 with satisfactory selectivity and repeatability, requiring minimal manipulation. However, WMC-MDP takes advantage of being a microfluidic device showing the coefficients of variation less than 10% enabling WMC-MDP to be a type of POCT. Therefore, WMC-MDP provides a promising alternative to point-of-care testing (POCT) of multiple biomarkers. We believe the practical application of WMC-MDP in militarized fields will revolutionize infection diagnosis for soldiers.

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Methodological Approaches to Study Extracellular Vesicle miRNAs in Epstein–Barr Virus-Associated Cancers

International Journal of Molecular Sciences ◽

10.3390/ijms19092810 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2810 ◽

Cited By ~ 3

Author(s):

Li Sun ◽

David Meckes

Keyword(s):

Epstein Barr Virus ◽

Biomarker Discovery ◽

Immune Escape ◽

Human Cancer ◽

Biological Fluids ◽

Extracellular Vesicle ◽

Cellular Mirnas ◽

Barr Virus ◽

Diagnosis And Prognosis ◽

Epstein Barr

Epstein Barr-virus (EBV) was the first virus identified to be associated with human cancer in 1964 and is found ubiquitously throughout the world’s population. It is now established that EBV contributes to the development and progression of multiple human cancers of both lymphoid and epithelial cell origins. EBV encoded miRNAs play an important role in tumor proliferation, angiogenesis, immune escape, tissue invasion, and metastasis. Recently, EBV miRNAs have been found to be released from infected cancer cells in extracellular vesicles (EVs) and regulate gene expression in neighboring uninfected cells present in the tumor microenvironment and possibly at distal sites. As EVs are abundant in many biological fluids, the viral and cellular miRNAs present within EBV-modified EVs may serve as noninvasion markers for cancer diagnosis and prognosis. In this review, we discuss recent advances in EV isolation and miRNA detection, and provide a complete workflow for EV purification from plasma and deep-sequencing for biomarker discovery.

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Development of a Sensitive Bead-Based Assay for Enhanced Monoclonal Antibody Detection

MRS Proceedings ◽

10.1557/opl.2011.1002 ◽

2011 ◽

Vol 1346 ◽

Author(s):

Manuel E. Ruidíaz ◽

Natalie Mendez ◽

Ana B. Sanchez ◽

Bradley T. Messmer ◽

Andrew C. Kummel

Keyword(s):

Monoclonal Antibody ◽

Limit Of Detection ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

Fluorescent Labeling ◽

Antibody Detection ◽

Serum Samples ◽

Mimetic Peptide ◽

Mimetic Peptides ◽

Zero Response

ABSTRACTMonoclonal antibodies are increasingly used in the treatment of cancer due to their enhanced targeting and immune system stimulation properties. Dosage guidelines typically do not account for personal cancer load or metabolism, thereby possibly affecting treatment outcome or causing unwanted side effects. The requirement for an assay that can quickly and precisely measure the concentration of the monoclonal antibody in a serum sample of a patient during therapy is unmet. A bead-based assay with peptide antigen mimetics has been developed to rapidly determine the concentration of antibody drug present in serum specimens with high sensitivity. Alemtuzumab (anti-CD52) and rituximab (anti-CD20) antigen mimetic peptides, as discovered by phage display, were synthesized on 10 um TentaGel resin beads using conventional solid phase peptide synthesis techniques. The beads were modified to allow for multiplexing and microfluidic handling via fluorescent labeling and magnetic functionalization. The antigen-displaying fluoromagnetic particles were incubated with spiked serum samples which allowed free antibody to be captured. Primary antibody detection was performed on alemtuzumab while rituximab detection was used to compensate for non-specific serum binding to the beads. After washing, the beads were incubated with a fluorescently tagged secondary label for detection by flow cytometry. (Results) A fast, low cost, specific assay has been developed with several key techniques which allows detection at low concentration (0.1ug/ml) of spiked samples. Primary to achieving this detection limit was the implementation of a compensation scheme where two antigen mimetic peptides behave linearly (R2=0.996) which enables the calculation of the zero response of the antigen mimetic peptide of interest (alemtuzumab antigen mimetic) while measuring the zero response of the compensatory antigen mimetic peptide (rituximab antigen mimetic) during primary assay measurement. This reduces fluorescence response variation due to variations present due to sample preparation, storage and different patients because of the equivalent interactions these effects have on the compensatory beads. The developed assay is therefore robust against serum variation and enables a lower limit of detection.

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Pre-concentration of microRNAs by LNA-modified magnetic beads for enhancement of electrochemical detection

Scientific Reports ◽

10.1038/s41598-021-99145-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Serife Ustuner ◽

Mark A. Lindsay ◽

Pedro Estrela

Keyword(s):

Electrochemical Detection ◽

Peptide Nucleic Acid ◽

Magnetic Beads ◽

Electrochemical Impedance ◽

Limit Of Detection ◽

Cancer Disease ◽

Circulating Micrornas ◽

Diagnosis And Prognosis ◽

Early Cancer Diagnosis ◽

Microrna Sequence

AbstractMicroRNAs are extremely promising candidates for early cancer diagnosis and prognosis. The levels of circulating microRNAs provide valuable information about cancer disease at its early stages. However, the levels of microRNAs that need to be detected are extremely low and difficult to discriminate from a large pool of oligonucleotides. There is the need for accurate, rapid and sensitive detection methodologies for detection of microRNAs. We developed electrochemical impedance spectroscopy peptide nucleic acid (PNA)-based sensors that can detect miRNAs in diluted serum with a limit of detection of 0.38 fM. In order to further improve the accuracy and reliability of the sensors, we developed an assay using magnetic beads for simple and rapid fishing of target microRNAs from solution and its pre-concentration prior to electrochemical detection. Our methodology utilizes magnetic beads for the capture of the target microRNA from solution and brings the concentrated sample to the sensor surface. We modify the magnetic beads with locked nucleic acids (LNA), which have high affinity and specificity to their complementary microRNA sequence. The separated and concentrated microRNA is then detected using the PNA-based sensors. By exposing the sensing electrodes only to the captured microRNAs, interferences from other nucleotides or biomolecules from the sample are eliminated.

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Reusable, Noninvasive, and Sensitive Fluorescence Enhanced ZnO-Nanorod-Based Microarrays for Quantitative Detection of AFP in Human Serum

BioMed Research International ◽

10.1155/2021/9916909 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Saima Rafique ◽

Farukh Kiyani ◽

Sumbal Jawaid ◽

Rubina Nasir ◽

Mahmoosh Ahmad ◽

...

Keyword(s):

Human Serum ◽

Dynamic Range ◽

Limit Of Detection ◽

Zno Nanorods ◽

Protein Microarrays ◽

Great Promise ◽

Quantitative Detection ◽

Zno Nanorod ◽

Serum Samples ◽

Protein Assay

The fabrication of sensitive protein microarrays such as PCR used in DNA microarray is challenging due to lack of signal amplification. The development of microarrays is utilized to improve the sensitivity and limitations of detection towards primal cancer detection. The sensitivity is enhanced by the use of ZnO-nanorods and is investigated as a substrate which enhance the florescent signal to diagnose the hepatocellular carcinoma (HCC) at early stages. The substrate for deposition of ZnO-nanorods is prepared by the conventional chemical bath deposition method. The resultant highly dense ZnO-nanorods enhance the fluorescent signal 7.2 times as compared to the substrate without ZnO-nanorods. The microarray showed sensitivity of 1504.7 ng ml-1 and limit of detection of 0.1 pg ml-1 in wide dynamic range of 0.05 pg-10 μg ml-1 for alpha fetoprotein (AFP) detection in 10% human serum. This immunoassay was successfully applied for human serum samples to detect tumor marker with good recoveries. The ZnO-nanorod substrate is a simple protein microarray which showed a great promise for developing a low-cost, sensitive, and high-throughput protein assay platform for several applications in both fundamental research and clinical diagnosis.

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Highly Sensitive Picric Acid Chemical Sensor Based on Samarium (Sm) Doped ZnO Nanorods

Journal of Nanoscience and Nanotechnology ◽

10.1166/jnn.2019.16105 ◽

2019 ◽

Vol 19 (6) ◽

pp. 3637-3642 ◽

Cited By ~ 3

Author(s):

Yas Al-Hadeethi ◽

Ahmad Umar ◽

Kulvinder Singh ◽

Ahmed A Ibrahim ◽

Saleh. H Al-Heniti ◽

...

Keyword(s):

Large Scale ◽

Chemical Sensor ◽

Limit Of Detection ◽

Picric Acid ◽

Zno Nanorods ◽

Hydrothermal Process ◽

High Sensitivity ◽

Highly Sensitive ◽

Doped Zno ◽

Facile Hydrothermal Process

Herein, we report the synthesis, characterization and picric acid chemical sensing application of samarium (Sm) doped ZnO nanorods. The Sm-doped ZnO nanorods were synthesized by facile hydrothermal process and characterized using various analytical methods which confirmed the large-scale synthesis and wurtzite hexagonal crystal structure for the synthesized nanorods. The doping of Sm ions in the lattices of the synthesized nanorods was evaluated by the energy dispersive X-ray spectroscopy (EDS). The synthesized Sm-doped ZnO nanorods were used as potential scaffold to fabricate high sensitive and reproducible picric acid chemical sensor based on I–V technique. The fabricated picric acid chemical sensor based on Sm-doped ZnO nanorods exhibited a high sensitivity of 213.9 mA mM−1 cm−2 with the limit of detection of ∼0.228 mM and correlation coefficient of R═0.9889. The obtained results revealed that the facile grown Sm-doped ZnO nanorods can efficiently be used to fabricate high sensitive and reproducible chemical sensors.

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