Extended-Gate Field-Effect Transistor Consisted of a CD9 Aptamer and MXene for Exosome Detection in Human Serum.

Cancer progresses silently to the terminal stage of the impossible operable condition. There are many limitations in the treatment options of cancer, but diagnosis in an early stage can improve survival rates and low recurrence. Exosomes are the biomolecules released from cancer cells and are promising candidates for clinical diagnosis. Among them, the cluster of differentiation 9 (CD9) protein is an important exosomal biomarker that can be used for exosome determination. Therefore, here, a CD9 aptamer was first synthesized and applied to an extended-gate field-effect transistor (EGFET)-type biosensor containing a disposable sensing membrane to suggest the possibility of detecting exosomes in a clinical environment. Systematically evaluating ligands using the exponential enrichment (SELEX) technique was performed to select nucleic acid sequences that can specifically target the CD9 protein. Exosomes were detected according to the electrical signal changes on a membrane, which is an extended gate using an Au microelectrode. The fabricated biosensor showed a limit of detection (LOD) of 10.64 pM for CD9 proteins, and the detection range was determined from 10 pM to 1 µM in the buffer. In the case of the clinical test, the LOD and detection ranges of exosomes in human serum samples were 6.41 × 102 exosomes/mL and 1 × 103 to 1 × 107 exosomes/mL, respectively, showing highly reliable results with low error rates. These findings suggest that the proposed aptasensor can be a powerful tool for a simple and early diagnosis of exosomes.

Axion Dark Matter Search around 4.55 µeV with Dine-Fischler-Srednicki-Zhitnitskii Sensitivity.

We report an axion dark matter search at Dine-Fischler-Srednicki-Zhitnitskii sensitivity with the CAPP-12TB haloscope, assuming axions contribute 100% of the local dark matter density. The search excluded the axion-photon coupling g_{aγγ} down to about 6.2×10^{-16} GeV^{-1} over the axion mass range between 4.51 and 4.59 µeV at a 90% confidence level. The achieved experimental sensitivity can also exclude Kim-Shifman-Vainshtein-Zakharov axion dark matter that makes up just 13% of the local dark matter density. The CAPP-12TB haloscope will continue the search over a wide range of axion masses.

Introduction of Nanomaterials to Biosensors for Exosome Detection: Case Study for Cancer Analysis.

Exosomes have been gaining attention for early cancer diagnosis owing to their biological functions in cells. Several studies have reported the relevance of exosomes in various diseases, including pancreatic cancer, retroperitoneal fibrosis, obesity, neurodegenerative diseases, and atherosclerosis. Particularly, exosomes are regarded as biomarkers for cancer diagnosis and can be detected in biofluids, such as saliva, urine, peritoneal fluid, and blood. Thus, exosomes are advantageous for cancer liquid biopsies as they overcome the current limitations of cancer tissue biopsies. Several studies have reported methods for exosome isolation, and analysis for cancer diagnosis. However, further clinical trials are still required to determine accurate exosome concentration quantification methods. Recently, various biosensors have been developed to detect exosomal biomarkers, including tumor-derived exosomes, nucleic acids, and proteins. Among these, the exact quantification of tumor-derived exosomes is a serious obstacle to the clinical use of liquid biopsies. Precise detection of exosome concentration is difficult because it requires clinical sample pretreatment. To solve this problem, the use of the nanobiohybrid material-based biosensor provides improved sensitivity and selectivity. The present review will discuss recent progress in exosome biosensors consisting of nanomaterials and biomaterial hybrids for electrochemical, electrical, and optical-based biosensors.

Rapid electrochemical dual-target biosensor composed of an Aptamer/MXene hybrid on Au microgap electrodes for cytokines detection.

Rapid detection methods for cytokine storm markers, such as tumor necrosis factor α (TNF-α) and interferon gamma (IFN-γ), are required. Herein, we describe the fabrication of a rapid electrochemical dual-target biosensor composed of aptamer/MXene (Ti3C2) nanosheet on an Au microgap electrode. Alternating current electrothermal flow (ACEF) significantly reduced the detection time (<10 min) to achieve the rapid biosensor construction. Additionally, MXene nanosheet was synthesized to improve the detection sensitivity. A dual-type Au microgap electrode was designed to measure TNF-α and IFN-γ levels using a single biosensor. Moreover, it performs 12 measurements using a small sample volume. To reduce detection time with stable aptamer-target complex formation, various ACEF conditions were evaluated and optimized to 10 min. Using the optimal conditions, the limit of detection (LOD) and selectivity were determined by electrochemical impedance spectroscopy (EIS). A linear region was observed in the concentration range of 1 pg/mL to 10 ng/mL of TNF-α and IFN-γ. The LOD of TNF-α and IFN-γ were 0.15 pg/mL and 0.12 pg/mL within 10 min, respectively. Furthermore, the proposed biosensor detected TNF-α and IFN-γ diluted in 10% human serum in the concentration range of 1 pg/mL to 10 ng/mL with LODs of 0.25 pg/mL and 0.26 pg/mL, respectively.

Fabrication of MERS-nanovesicle biosensor composed of multi-functional DNA aptamer/graphene-MoS2 nanocomposite based on electrochemical and surface-enhanced Raman spectroscopy.

Middle East respiratory syndrome coronavirus (MERS-CoV) is one of the most harmful viruses for humans in nowadays. To prevent the spread of MERS-CoV, a valid detection method is highly needed. For the first time, a MERS-nanovesicle (NV) biosensor composed of multi-functional DNA aptamer and graphene oxide encapsulated molybdenum disulfide (GO-MoS2) hybrid nanocomposite was fabricated based on electrochemical (EC) and surface-enhanced Raman spectroscopy (SERS) techniques. The MERS-NV aptamer was designed for specifically binding to the spike protein on MERS-NVs and it is prepared using the systematic evolution of ligands by exponential enrichment (SELEX) technique. For constructing a multi-functional MERS aptamer (MF-aptamer), the prepared aptamer was connected to the DNA 3-way junction (3WJ) structure. DNA 3WJ has the three arms that can connect the three individual functional groups including MERS aptamer (bioprobe), methylene blue (signal reporter) and thiol group (linker) Then, GO-MoS2 hybrid nanocomposite was prepared for the substrate of EC/SERS-based MERS-NV biosensor construction. Then, the assembled multifunctional (MF) DNA aptamer was immobilized on GO-MoS2. The proposed biosensor can detect MERS-NVs not only in a phosphate-buffered saline (PBS) solution (SERS LOD: 0.176 pg/ml, EIS LOD: 0.405 pg/ml) but also in diluted 10% saliva (SERS LOD: 0.525 pg/ml, EIS LOD: 0.645 pg/ml).

Fabrication of a surface-enhanced Raman spectroscopy-based analytical method consisting of multifunctional DNA three-way junction-conjugated porous gold nanoparticles and Au-Te nanoworm for C-reactive protein detection.

C-Reactive protein (CRP) is a biomarker of inflammatory responses and an index for assessing the risk of cardiovascular disease and estimating prognosis. In this study, we constructed a surface-enhanced Raman spectroscopy (SERS) biosensor composed of a multifunctional DNA three-way junction (DNA 3WJ), porous gold nanoplates (pAuNPs), and an Au-Te nanoworm structure for detection of CRP. The pAuNP and Au-Te nanostructures were synthesized by galvanic replacement reactions, and the morphology was confirmed by transmission electron microscopy, scanning electron microscopy, and dynamic light scattering (DLS). To generate the SERS signal, the Au-Te nanostructure was immobilized on an indium-tin oxide substrate, and the thiol-modified CRP aptamer was then self-assembled onto the modified substrate for CRP recognition. To amplify the SERS signal and identify the Raman tag, the multifunctional DNA 3WJ was conjugated with the pAuNPs, and each fragment of 3WJ was functionalized to biotin (pAuNP conjugation), methylene blue (Raman reporter), and CRP aptamer (target binding). The results were confirmed by gel electrophoresis. For conjugation between pAuNPs and DNA 3WJ, avidin was encapsulated in pAuNPs, and the conjugation structure was confirmed by DLS. The fabricated SERS biosensor showed detection limits of 2.23 pM in phosphate-buffered saline and 3.11 pM in diluted human serum. Overall, the proposed biosensor may have potential applications as a SERS biosensor platform.

Recent Advances in Aptasensor for Cytokine Detection: A Review.

Cytokines are proteins secreted by immune cells. They promote cell signal transduction and are involved in cell replication, death, and recovery. Cytokines are immune modulators, but their excessive secretion causes uncontrolled inflammation that attacks normal cells. Considering the properties of cytokines, monitoring the secretion of cytokines in vivo is of great value for medical and biological research. In this review, we offer a report on recent studies for cytokine detection, especially studies on aptasensors using aptamers. Aptamers are single strand nucleic acids that form a stable three-dimensional structure and have been receiving attention due to various characteristics such as simple production methods, low molecular weight, and ease of modification while performing a physiological role similar to antibodies.

Fabrication of electrochemical biosensor composed of multi-functional DNA 4 way junction for TNF-α detection in human serum.

Tumor necrosis factor (TNF-α) is a representative cytokine family known to induce multiple signaling cascades leading to various cellular responses, such as cell death, survival, and differentiation. It has been reported that blocking the action of TNF-α in various diseases can improve disease prognosis. Therefore, it is important to monitor TNF-α in patient plasma and properly regulate its action. In this study, we report a label-free electrochemical biosensor consisting of a multifunctional DNA 4-way junction (MF-4WJ) for TNF-α detection in human serum. MF-4WJ does not require additional labeling and signal amplification processes. The electrochemical properties of functionalized MF-4WJ were evaluated by cyclic voltammetry (CV) in the presence of Ag+ intercalated between the mismatched sequences of MF-aptamers as redox-active species. Afterward, CV was carried out to evaluate the performance of the fabricated biosensor. The proposed label-free electrochemical biosensor was able to effectively detect TNF-α in a dynamic range of 0.15 pg/ml to 150 ng/ml. Limit of detection (LOD) was at 0.07 pg/ml in HEPES. Moreover, it was confirmed that even in 10% diluted human serum, TNF-α could be detected in an excellent dynamic range of 0.15 pg/ml to ∼ 15 ng/ml and LOD was at 0.14 pg/ml in 10% diluted human serum.

Fabrication of an Electrochemical Aptasensor Composed of Multifunctional DNA Three-Way Junction on Au Microgap Electrode for Interferon Gamma Detection in Human Serum.

Interferon gamma (IFN-γ) is an important cytokine with antiviral, antibacterial, and immunosuppressive properties. It has been used as a biomarker for the early detection of several diseases, including cancer, human immunodeficiency virus (HIV), tuberculosis, and paratuberculosis. In this study, we developed an electrochemical biosensor composed of multifunctional DNA 3WJ to detect IFN-γ level with high sensitivity. Each multifunctional triple-stranded aptamer (MF-3WJ) was designed to have an IFN-γ aptamer sequence, anchoring region (thiol group), and 4C-C (cytosine-cytosine) mismatch sequence (signal generation), which could introduce silver ions. To generate the electrochemical signal, four Ag+ ions were intercalated (3wj b-3wj c) in the 4C-C mismatch sequence. MF-3WJ was assembled through the annealing step, and the assembly of MF-3WJ was confirmed by 8% tris-boric-EDTA native polyacrylamide gel electrophoresis. The Au microgap electrode was manufactured to load sample volumes of 5 µL. The reliability of electrochemical biosensor measurement was established by enabling the measurement of seven samples from one Au microgap electrode. MF-3WJ was immobilized on the Au microgap electrode. Then, cyclic voltammetry and electrochemical impedance spectroscopy were performed to confirm the electrochemical properties of MF-3WJ. To test the electrochemical biosensor's ability to detect IFN-γ, the limit of detection (LOD) and selectivity tests were performed by square wave voltammetry. A linear region was observed in the concentration range of 1 pg/mL-10 ng/mL of IFN-γ. The LOD of the fabricated electrochemical biosensor was 0.67 pg/mL. In addition, for the clinical test, the LOD test was carried out for IFN-γ diluted in 10% human serum samples in the concentration range of 1 pg/mL-10 ng/mL, and the LOD was obtained at 0.42 pg/mL.

Recent Advances in CRP Biosensor Based on Electrical, Electrochemical and Optical Methods.

C-reactive protein (CRP) is an acute-phase reactive protein that appears in the bloodstream in response to inflammatory cytokines such as interleukin-6 produced by adipocytes and macrophages during the acute phase of the inflammatory/infectious process. CRP measurement is widely used as a representative acute and chronic inflammatory disease marker. With the development of diagnostic techniques measuring CRP more precisely than before, CRP is being used not only as a traditional biomarker but also as a biomarker for various diseases. The existing commercialized CRP assays are dominated by enzyme-linked immunosorbent assay (ELISA). ELISA has high selectivity and sensitivity, but its limitations include requiring complex analytic processes, long analysis times, and professional manpower. To overcome these problems, nanobiotechnology is able to provide alternative diagnostic tools. By introducing the nanobio hybrid material to the CRP biosensors, CRP can be measured more quickly and accurately, and highly sensitive biosensors can be used as portable devices. In this review, we discuss the recent advancements in electrochemical, electricity, and spectroscopy-based CRP biosensors composed of biomaterial and nanomaterial hybrids.

Fabrication of an electrochemical biosensor composed of multi-functional Ag ion intercalated DNA four-way junctions/rhodium nanoplate heterolayer on a micro-gap for C-reactive protein detection in human serum.

As inflammation plays a role in the pathogenesis of acute coronary syndromes, C-reactive protein (CRP) can be used as a biomarker. To detect CRP precisely, the authors prepared a CRP electrochemical biosensor consisting of an eight Ag ion-intercalated multifunctional DNA four-way junction (MF-DNA-4WJ) and a porous rhodium nanoparticle (pRhNP) heterolayer on a micro-gap electrode. To increase conductivity, we used eight Ag+ ion-inserted DNA four-way junctions through a C-C mismatch. Each DNA 4WJ was designed to have the CRP aptamer sequence, an anchoring region (thiol group), and two of four C-C mismatch regions at the end of the fragments. After an annealing step, the MF-DNA-4WJ assembly configuration and selective binding of CRP were confirmed through native TBM-PAGE (Tris-borate-magnesium chloride-polyacrylamide gel electrophoresis). The Au micro-gap electrode was fabricated to load 5 µl of the sample, and this was performed during eight experiments on one chip to establish the accuracy of the data. Then, pRhNPs were immobilized on a Au micro-gap electrode using cysteamine. To confirm the electrochemical properties, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were conducted. The durability of pRhNPs was confirmed through CV. To test the sensing performance of the prepared CRP biosensor, the limit of detection (LOD) and selectivity tests were conducted using EIS. The results indicated that charge transfer resistance (Rct) can be used efficiently to probe these interactions within the variable CRP concentration range, from 1 pM to 100 nM (0.23 ng L-1-23 µg L-1). The LOD of this sensor was 0.349 pM (0.08 ng L-1) (at S/N = 3). As a result of diluting the CRP to the same concentration range in a 20% human serum sample, the LOD was 3.55 fM (0.814 pg L-1) (at S/N = 3).

Fabrication of Electrochemical Influenza Virus (H1N1) Biosensor Composed of Multifunctional DNA Four-Way Junction and Molybdenum Disulfide Hybrid Material.

The outbreak of the influenza virus (H1N1) has symptoms such as coughing, fever, and a sore throat, and due to its high contagious power, it is fatal to humans. To detect H1N1 precisely, the present study proposed an electrochemical biosensor composed of a multifunctional DNA four-way junction (4WJ) and carboxyl molybdenum disulfide (carboxyl-MoS2) hybrid material. The DNA 4WJ was constructed to have the hemagglutinin aptamer on the head group (recognition part); each of the two arms has four silver ions (signal amplification part), and the tail group has an amine group (anchor). This fabricated multifunctional DNA 4WJ can specifically and selectively bind to hemagglutinin. Moreover, the carboxyl-MoS2 provides an increase in the sensitivity of this biosensor. Carboxyl-MoS2 was immobilized using a linker on the electrode, followed by the immobilization of the multifunctional 4WJ on the electrode. The synthesis of carboxyl-MoS2 was confirmed by field emission scanning electron microscopy (FE-SEM), and the surface of the electrode was confirmed by atomic force microscopy. When H1N1 was placed in the immobilized electrode, the presence of H1N1 was confirmed by electrochemical analysis (cyclic voltammetry, electrochemical impedance spectroscopy). Through selectivity tests, it was also possible to determine whether this sensor responds specifically and selectively to H1N1. We confirmed that the biosensor showed a linear response to H1N1, and that H1N1 could be detected from 100 nM to 10 pM. Finally, clinical tests, in which hemagglutinin was diluted with human serum, showed a similar tendency to those diluted with water. This study showed that the multi-functional DNA 4WJ and carboxyl-MoS2 hybrid material can be applied to a electrochemical H1N1 biosensor.

Recent Development of Aptasensor for Influenza Virus Detection.

In nowadays, we have entered the new era of pandemics and the significance of virus detection deeply impacts human society. Viruses with genetic mutations are reported nearly every year, and people have prepared tools to detect the virus and vaccines to ensure proper treatments. Influenza virus (IV) is one of the most harmful viruses reporting various mutations, sub-types, and rapid infection speed for humans and animals including swine and poultry. Moreover, IV infection presents several harmful symptoms including cough, fever, diarrhea, chills, even causing death. To reduce the IV-induced harm, its proper and rapid detection is highly required. Conventional techniques were used against various IV sub-types including H1N1, H3N2, and H5N1. However, some of the techniques are time-consuming, expensive, or labor-intensive for detecting IV. Recently, the nucleic acid-based aptamer has gained attention as a novel bioprobe for constructing a biosensor. In this review, the authors discuss the recent progress in aptasensors for detecting IV in terms of an electrochemical and an optical biosensor.

Recent Advances in Biomolecule-Nanomaterial Heterolayer-Based Charge Storage Devices for Bioelectronic Applications.

With the acceleration of the Fourth Industrial Revolution, the development of information and communications technology requires innovative information storage devices and processing devices with low power and ultrahigh stability. Accordingly, bioelectronic devices have gained considerable attention as a promising alternative to silicon-based devices because of their various applications, including human-body-attached devices, biomaterial-based computation systems, and biomaterial-nanomaterial hybrid-based charge storage devices. Nanomaterial-based charge storage devices have witnessed considerable development owing to their similarity to conventional charge storage devices and their ease of applicability. The introduction of a biomaterial-to-nanomaterial-based system using a combination of biomolecules and nanostructures provides outstanding electrochemical, electrical, and optical properties that can be applied to the fabrication of charge storage devices. Here, we describe the recent advances in charge storage devices containing a biomolecule and nanoparticle heterolayer including (1) electrical resistive charge storage devices, (2) electrochemical biomemory devices, (3) field-effect transistors, and (4) biomemristors. Progress in biomolecule-nanomaterial heterolayer-based charge storage devices will lead to unprecedented opportunities for the integration of information and communications technology, biotechnology, and nanotechnology for the Fourth Industrial Revolution.

Fabrication of electrochemical biosensor composed of multi-functional DNA/rhodium nanoplate heterolayer for thyroxine detection in clinical sample.

Thyroxine (T4) contains four iodine atoms and is a major thyroid hormone synthesized in the thyroid gland. Abnormal levels of T4 in the body cause various endocrine diseases. The present study describes the fabrication of an electrochemical biosensor composed of a multi-functional DNA structure/rhodium nanoplates heterolayer for precise detection of T4 concentration. A DNA 3-way junction (3WJ) structure was designed as a multi-functional bioprobe to perform several functions (including target detection, electrochemical signal reporting, and immobilization) simultaneously. Binding between T4 and the T4 DNA aptamer was confirmed through enzyme-linked aptamer assays (ELAAs) and filtration experiments. The multi-functional DNA was immobilized on porous rhodium nanoplates (pRhNPs)-heterolayer modified Au micro-gap electrode. The pRhNPs provided an increment in the surface area and amplification of the electrochemical signal. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to detect T4. Under optimal conditions, the limit of detection of T4 was found to be 10.33 pM. Furthermore, up to 11.41 pM of T4 could be detected in clinical samples. This study demonstrates the possibility of label-free detection of the T4 with multi-functional DNA/pRhNPs heterolayer that can be applied to small molecule detection platform in the near future.

Fabrication of Troponin I Biosensor Composed of Multi-Functional DNA Structure/Au Nanocrystal Using Electrochemical and Localized Surface Plasmon Resonance Dual-Detection Method.

In the present study, we fabricated a dual-mode cardiac troponin I (cTnI) biosensor comprised of multi-functional DNA (MF-DNA) on Au nanocrystal (AuNC) using an electrochemical method (EC) and a localized surface plasmon resonance (LSPR) method. To construct a cTnI bioprobe, a DNA 3 way-junction (3WJ) was prepared to introduce multi-functionality. Each DNA 3WJ arm was modified to possess a recognition region (Troponin I detection aptamer), an EC-LSPR signal generation region (methylene blue: MB), and an anchoring region (Thiol group), respectively. After an annealing step, the multi-functional DNA 3WJ was assembled, and its configuration was confirmed by Native-TBM PAGE for subsequent use in biosensor construction. cTnI was also expressed and purified for use in biosensor experiments. To construct an EC-LSPR dual-mode biosensor, AuNCs were prepared on an indium-tin-oxide (ITO) substrate using an electrodeposition method. The prepared multi-functional (MF)-DNA was then immobilized onto AuNCs by covalent bonding. Field emission scanning electron microscope (FE-SEM) and atomic force microscopy (AFM) were used to analyze the surface morphology. LSPR and electrochemical impedance spectroscopy (EIS) experiments were performed to confirm the binding between the target and the bioprobe. The results indicated that cTnI could be effectively detected in the buffer solution and in diluted-human serum. Based on the results of these experiments, the loss on drying (LOD) was determined to be 1.0 pM in HEPES solution and 1.0 pM in 10% diluted human serum. Additionally, the selectivity assay was successfully tested using a number of different proteins. Taken together, the results of our study indicate that the proposed dual-mode biosensor is applicable for use in field-ready cTnI diagnosis systems for emergency situations.