An updated review on the development of a nanomaterial-based field-effect transistor-type biosensors to detect exosomes for cancer diagnosis.

Cancer, a life-threatening genetic disease caused by abnormalities in normal cell growth regulatory functions, poses a significant challenge that current medical technologies cannot fully overcome. The current desired breakthrough is to diagnose cancer as early as possible and increase survival rates through treatments tailored to the prognosis and appropriate follow-up. From a perspective that reflects this contemporary paradigm of cancer diagnostics, exosomes are emerging as promising biomarkers. Exosomes, serving as mobile biological information repositories of cancer cells, have been known to create a microtumor environment in surrounding cells, and significant insight into the clinical significance of cancer diagnosis targeting them has been reported. Therefore, there are growing interests in constructing a system that enables continuous screening with a focus on patient-friendly and flexible diagnosis, aiming to improve cancer screening rates through exosome detection. This review focuses on a proposed exosome-embedded biological information-detecting platform employing a field-effect transistor (FET)-based biosensor that leverages portability, cost-effectiveness, and rapidity to minimize the stages of sacrifice attributable to cancer. The FET-applied biosensing technique, stemming from variations in an electric field, is considered an early detection system, offering high sensitivity and a prompt response frequency for the qualitative and quantitative analysis of biomolecules. Hence, an in-depth discussion was conducted on the understanding of various exosome-based cancer biomarkers and the clinical significance of recent studies on FET-based biosensors applying them.

Advances in nanobiosensors during the COVID-19 pandemic and future perspectives for the post-COVID era.

The unprecedented threat of the highly contagious virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes exponentially increased infections of coronavirus disease 2019 (COVID-19), highlights the weak spots of the current diagnostic toolbox. In the midst of catastrophe, nanobiosensors offer a new opportunity as an alternative tool to fill a gap among molecular tests, rapid antigen tests, and serological tests. Nanobiosensors surpass the potential of antigen tests because of their enhanced sensitivity, thus enabling us to see antigens as stable and easy-to-access targets. During the first three years of the COVID-19 pandemic, a substantial number of studies have reported nanobiosensors for the detection of SARS-CoV-2 antigens. The number of articles on nanobiosensors and SARS-CoV-2 exceeds the amount of nanobiosensor research on detecting previous infectious diseases, from influenza to SARS-CoV and MERS-CoV. This unprecedented publishing pace also implies the significance of SARS-CoV-2 and the present pandemic. In this review, 158 studies reporting nanobiosensors for detecting SARS-CoV-2 antigens are collected to discuss the current challenges of nanobiosensors using the criteria of point-of-care (POC) diagnostics along with COVID-specific issues. These advances and lessons during the pandemic pave the way for preparing for the post-COVID era and potential upcoming infectious diseases.

Hydrogel-based technologies in liquid biopsy for the detection of circulating clinical markers: challenges and prospects.

Liquid biopsy, which promises noninvasive detection of tumor-derived material, has recently been highlighted because of its potential to lead us to an era of precision medicine. However, its development has encountered challenges owing to the extremely low frequency and low purity of circulating tumor markers, such as circulating tumor cells (CTCs), circulating exosomes, and circulating tumor nucleic acids (ctNAs). Much effort has been made to overcome this limitation over the last decade, and an increasing number of studies have shown interest in the special characteristics of hydrogels. This hydrophilic and biocompatible polymeric network, which absorbs a large amount of water, can aid in the isolation, protection, and analysis of these low-abundance and short-lived circulating biomarkers. The role of hydrogels in liquid biopsy is extensive and ranges from enrichment to encapsulation. This review provides an overview of hydrogel-based technologies to pave the way in liquid biopsy.

Fast-response electrochemical biosensor based on a truncated aptamer and MXene heterolayer for West Nile virus detection in human serum.

West Nile virus (WNV) is a mosquito-borne flavivirus that can cause West Nile fever, meningitis, encephalitis, and polio. Early detection of WNV is important to prevent infection spread on the field. To commercialize the electrochemical biosensor for WNV, rapid target detection with the cheap manufacture cost is essential. Here, we developed a fast-response electrochemical biosensor consisting of a truncated WNV aptamer/MXene (Ti3C2Tx) bilayer on round-type micro gap. To reduce the target binding time, the application of the alternating current electrothermal flow (ACEF) technology reduced the target detection time to within 10 min, providing a rapid biosensor platform. The MXene nanosheet improved electrochemical signal amplification, and the aptamer produced through systematic evolution of ligands by exponential enrichment process eliminated unnecessary base sequences via truncation and lowered the manufacturing cost. Under optimized conditions, the WNV limit of detection (LOD) and selectivity were measured using electrochemical measurement methods, including cyclic voltammetry and square wave voltammetry. The LOD was 2.57 pM for WNV diluted in deionized water and 1.06 pM for WNV diluted in 10% human serum. The fabricated electrochemical biosensor has high selectivity and allows rapid detection, suggesting the possibility of future application in the diagnosis of flaviviridae virus.

Synthesis of Isolated DNA Aptamer and Its Application of AC-Electrothermal Flow-Based Rapid Biosensor for the Detection of Dengue Virus in a Spiked Sample.

Dengue fever is an infectious disease caused by the dengue virus (DENV) and is transmitted by mosquitoes in tropical and subtropical regions. The early detection method at a low cost is essential. To address this, we synthesized the isolated DENV aptamer for fabricating a rapid electrochemical biosensor on a Au interdigitated microgap electrode (AuIMGE). The DENV aptamers were generated using the SELEX (systemic evolution of ligands by exponential enrichment) method for binding to DENV surface envelope proteins. To reduce the manufacturing cost, unnecessary nucleotide sequences were excluded from the isolation process of the DENV aptamer. To reduce the detection time, the alternating current electrothermal flow (ACEF) technique was applied to the fabricated biosensor, which can shorten the detection time to 10 min. The performance of the biosensor was evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). In the diluted DENV protein solution, the linear range of the concentrations was from 1 pM to 1 µM and the LOD was 76.7 fM. Moreover, the proposed biosensor detected DENV in a diluted spiked sample at a linear range of 10-6 to 106 TCID50/mL, while the detection performance was proven with an LOD of 1.74 × 10-7 TCID50/mL along with high selectivity.

Fabrication of graphene oxide-based pretreatment filter and Electrochemical-CRISPR biosensor for the field-ready cyanobacteria monitoring system.

Microcystis aeruginosa (M. aeruginosa) cause the eutrophication of lakes and rivers. To effectively control the overgrowth of M. aeruginosa, a suitable measurement method should be required in the aquatic fields. To address this, we developed a field-ready cyanobacterial pretreatment device and an electrochemical clustered regularly interspaced short palindromic repeats (EC-CRISPR) biosensor. The cyanobacterial pretreatment device consists of a syringe, glass bead, and graphene oxide (GO) bead. Then, the M. aeruginosa dissolved in the freshwater sample was added to fabricated filter. After filtration, the purified gene was loaded onto a CRISPR-based electrochemical biosensor chip to detect M. aeruginosa gene fragments. The biosensor was composed of CRISPR/Cpf1 protein conjugated with MXene on an Au microgap electrode (AuMGE) integrated into a printed circuit board (PCB). This AuMGE/PCB system maximizes the signal-to-noise ratio, which controls the working and counter electrode areas requiring only 3 µL samples to obtain high reliability. Using the extracted M. aeruginosa gene with a pre-treatment filter, the CRISPR biosensor showed a limit of detection of 0.089 pg/µl in fresh water. Moreover, selectivity test and matrix condition test carried out using the EC-CRISPR biosensor. These handheld pre-treatment kit and biosensors can enable field-ready detection of CyanoHABs.

Electrochemical Detection of Different Foodborne Bacteria for Point-of-Care Applications.

Bacterial infections resulting from foodborne pathogenic bacteria cause millions of infections that greatly threaten human health and are one of the leading causes of mortality around the world. To counter this, the early, rapid, and accurate detection of bacterial infections is very important to address serious health issue concerns. We, therefore, present an electrochemical biosensor based on aptamers that selectively bind with the DNA of specific bacteria for the accurate and rapid detection of various foodborne bacteria for the selective determination of bacterial infection types. Different aptamers were synthesized and immobilized on Au electrodes for selective bindings of different types of bacterial DNA (Escherichia coli, Salmonella enterica, and Staphylococcus aureus) for the accurate detection and quantification of bacterial concentrations from 101 to 107 CFU/mL without using any labeling methods. Under optimized conditions, the sensor showed a good response to the various concentrations of bacteria, and a robust calibration curve was obtained. The sensor could detect the bacterial concentration at meager quantities and possessed an LOD of 4.2 × 101, 6.1 × 101, and 4.4 × 101 CFU/mL for S. Typhimurium, E. Coli, and S. aureus, respectively, with a linear range from 100 to 104 CFU/mL for the total bacteria probe and 100 to 103 CFU/mL for individual probes, respectively. The proposed biosensor is simple and rapid and has shown a good response to bacterial DNA detections and thus can be applied in clinical applications and food safety monitoring.

Nanoparticle-Based Chimeric Antigen Receptor Therapy for Cancer Immunotherapy.

Adoptive cell therapy with chimeric antigen receptor (CAR)-engineered T cells (CAR-Ts) has emerged as an innovative immunotherapy for hematological cancer treatment. However, the limited effect on solid tumors, complex processes, and excessive manufacturing costs remain as limitations of CAR-T therapy. Nanotechnology provides an alternative to the conventional CAR-T therapy. Owing to their unique physicochemical properties, nanoparticles can not only serve as a delivery platform for drugs but also target specific cells. Nanoparticle-based CAR therapy can be applied not only to T cells but also to CAR-natural killer and CAR-macrophage, compensating for some of their limitations. This review focuses on the introduction of nanoparticle-based advanced CAR immune cell therapy and future perspectives on immune cell reprogramming.

Rapid electrochemical biosensor composed of DNA probe/iridium nanoparticle bilayer for Aphanizomenon flos-aquae detection in fresh water.

Toxic cyanobacteria pose a serious threat to aquatic ecosystems and require adequate detection and control systems. Aphanizomenon flos-aquae is a harmful cyanobacterium that produces the toxicant saxitoxin. Therefore, it is necessary to detect the presence of A. flos-aquae in lakes and rivers. We proposed a rapid electrochemical biosensor composed of DNA primer/iridium nanoparticles (IrNP) bilyer for the detection of A. flos-aquae in freshwater. The extracted A. flos-aquae gene (rbcL-rbcX) is used as a target, and it was fixed to the electrode using a 5'-thiolated DNA primer (capture probe). Then, Avidin@IrNPs complex for amplification of electrical signals was bound to the target through a 3'-biotinylated DNA primer (detection probe). To rapidly detect the target, an alternating current electrothermal flow technique was introduced in the detection step, which could reduce the detection time to within 20 min. To confirm the biosensor fabrication, atomic force microscopy was used to investigate the surface morphology. To evaluate the biosensor performance, cyclic voltammetry and electrochemical impedance spectroscopy were used. The target gene was detected at a concentration of 9.99 pg/mL in tap water, and the detection range was 0.1 ng/mL to 103 ng/mL with high selectivity. Based on the combined system, we employed A. flos-aquae in tap water. This rapid cyanobacteria detection system is a powerful tool for CyanoHABs in the field.

RGO-PANI composite Au microelectrodes for sensitive ECIS analysis of human gastric (MKN-1) cancer cells.

Microelectrode-based cell chip studies for cellular responses often require improved adhesion and growth conditions for efficient cellular diagnosis and high throughput screening in drug discovery. Cell-chip studies are often performed on gold electrodes due to their biocompatibility, and stability, but the electrode-electrolyte interfacial capacitance is the main drawback to the overall sensitivity of the detection system. Thus, here, we developed reduced graphene oxide-polyaniline-modified gold microelectrodes for real-time impedance-based monitoring of human gastric adenocarcinoma cancer (MKN-1) cells. The impedance characterization on modified electrodes showed 28-fold enhanced conductivity than the bare electrodes, and the spectra were modeled with proper equivalent circuits to extrapolate the values of circuit elements. The impedance of both time-and frequency-dependent measurements of cell-covered modified electrodes with equivalent model circuits was analyzed to achieve cellular behavior, such as adhesion, spreading, proliferation, and influence of anti-cancer agents. The normalized impedance at 41.5 kHz (|Z|norm 41 kHz) was selected to monitor the cell growth analysis, which was found linear with the proliferation of adherent cells along with the influence of the anticancer drug agent on the MKN-1 cells. The synergistic effects and biocompatible nature of PANI-RGO modifications improved the overall sensitivity for the cell-growth studies of MKN-1 cells.

Image-guided in situ cancer vaccination with combination of multi-functional nano-adjuvant and an irreversible electroporation technique.

Cancer immunotherapy is a next-generation treatment strategy; however, its side effects limit its clinical translation. Here, a novel combination of a multi-functional nano-adjuvant (M-NA) prepared with an iron oxide/gold core and a cationic polymer shell via multilayer synthesis with CpG oligodeoxynucleotide (CpG-ODN) electrostatically complexed on its surface, and irreversible electroporation (IRE) technique was developed for effective image-guided in situ cancer vaccination. The M-NA can be retained long-term in the dense tumoral extracellular matrix after intratumoral injection and internalized by antigen-presenting cells (APCs). The IRE can induce immunogenic cell death. Indeed, in a mouse tumor model, the M-NA showed longer tumor retention time than free CpG-ODN. Compared with other treatments, the combined treatment significantly inhibited tumor growth with 100% survival rate for ∼60 days. The therapy induced the activation of cytotoxic lymphocytes and the maturation of APCs in vivo. This treatment could be effective in image-guided local cancer immunotherapy.

Selection of DNA aptamer and its application as an electrical biosensor for Zika virus detection in human serum.

Zika virus is a highly infectious virus that is part of the flavivirus group. Precise diagnosis of the Zika virus is significant issue for controlling a global pandemic after the COVID-19 era. For the first time, we describe a zika virus aptamer-based electrical biosensor for detecting Zika virus in human serum. The electrical biosensor composed of a Zika virus aptamer/MXene nanoparticle heterolayer on Au micro-gap electrode (AuMGE)/print circuit board (PCB) system. The Zika virus aptamer was designed to bind the envelope protein of the Zika virus by systematic evolution of ligands by exponential enrichment (SELEX) technique. The binding affinity of the aptamer was determined by fluorescence. For improving the sensor signal sensitivity, Ti3C2Tx MXene was introduced to surface of Au micro-gap electrode (AuMGE). The immobilization process was confirmed by atomic force microscopy (AFM). The prepared aptamer/MXene immobilized on AuMGE can detect the Zika virus through capacitance change according to the target concentration. The capacitance signal from the biosensor increased linearly according to increment of envelope proteins in the human serum. The limit of detection was determined to 38.14 pM, and target proteins could be detected from 100 pM to 10 µM. Thus, the developed electrical aptabiosensor can be a useful tool for Zika virus detection.

Electrochemical biosensor with aptamer/porous platinum nanoparticle on round-type micro-gap electrode for saxitoxin detection in fresh water.

Cyanotoxins are toxins produced by cyanobacteria; they negatively impact water resources used by humans and disrupt ecosystems worldwide. Among cyanotoxins, saxitoxin (STX) is a small molecule that causes paralysis in humans and contamination in freshwater resources. To monitor low concentration of STX levels, a sensitive and high fidelity detection system is required. In this study, a round-type micro-gap electrode (RMGE) was fabricated that provides the high signal fidelity for STX detection in real freshwater sample. The RMGE has the 15 pairs of identical electrode wire length between gap that gives the high signal fidelity. In addition, the sensitivity for STX detection was improved by introducing the porous platinum nanoparticle (pPtNP) that enahced the electrochemical sensitivity and the STX aptamer was used as the bioprobe. An electrochemical measurement method (square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS)) was introduced to construct STX biosensor. To evaluate the biosensor performance, the limit of detection (LOD) and selectivity test were performed on real freshwater samples. The biosensor demonstrated high selectivity even in freshwater samples over a wide linear concentration range of 10 pg/mL to 1 µg/mL and a detection limit of 4.669 pg/mL. These results suggest that the designed biosensor shows a wide range of possibilities for the detection of toxicants in freshwater that provide the new direction to the biosensor electrode design.

Differences in the gut microbiome composition of Korean children and adult samples based on different DNA isolation kits.

Recent studies have revealed that the composition of human gut microbiota varies according to region, race, age, diet, living environment, and sampling and DNA extraction method. The purpose of this study was to broaden our understanding of the intestinal microbial composition of Koreans by conducting a 16S rRNA amplicon sequencing on 78 Korean samples composed of adults, children, normal and obese groups. We compared the microbiome composition and diversity of these groups at different levels including the phylum and genus level using two different stool DNA extraction kits of QIAamp® PowerFecal® DNA Kit (Qiagen, Hilden, Germany) and CT Max Fecal DNA Kit (Ct bio, Korea). We found that Ct bio (Ct) kit recovered higher DNA yields and OTUs than QIAamp® PowerFecal® DNA Kit (Qia). The Ct kit, which adopted more rigorous bead beating method, detected the most Gram-positive (G+) bacteria, Firmicutes, at the Phylum level, whereas the Qia kit, which used a less rigorous cell lysis method, found the most Gram-negative (G-) bacteria, Bacteroidetes. The Firmicutes-to-Bacteroidetes (F/B) ratio showed no significant difference between the obese and the normal groups of same kit; however, they were significantly different with two different kits. There was a difference in the intestinal flora between healthy Korean adults and children. The taxa that differed significantly between the adults and children were Bacteroides, Bifidobacterium, Prevotella, and Subdoligranulum. There was no significant difference in the intestinal flora between the normal weight group and the obese group in adults and children, respectively. This is probably because the difference in body mass index (BMI) between the sample groups collected in this study is statistically significant, but it is not large enough to show a clear difference in the flora. Therefore, these results should be interpreted with caution while considering the BMI values and Korean obesity criterion together.

Facile and foldable point-of-care biochip for nucleic acid based-colorimetric detection of murine norovirus in fecal samples using G-quadruplex and graphene oxide coated microbeads.

Norovirus is one of the most common causes of gastroenteritis, a disease characterized by diarrhea, vomiting, and stomach pain. A rapid on-site identification of the virus from fecal samples of patients is a prerequisite for accurate medical management. Here, we demonstrate a rapid nucleic acid-based detection platform as an on-site biosensing tool that can concentrate viruses from fecal samples. Moreover, it can perform RNA extraction and identification, and signal amplification using G-quadruplex and hemin containing DNA probes (G-DNA probes) and graphene oxide (GO)-coated microbeads. Briefly, murine noroviruses are lysed without chemicals on the surface of the GO microbeads. Subsequently, the target RNA is hybridized with G-DNA probes, and the resultant RNA/G-DNA probe complex is separated from unbound G-DNA probes using GO beads and is mixed with the detection buffer (ABTS/H2O2). Presence of murine noroviruses causes a colorimetric change of the buffer from colorless to green. Thus, we integrated all processes required to detect murine noroviruses in stool samples in a simple foldable microfluidic chip. Moreover, it can detect 101 pfu of the virus in 30 min in a fecal sample.

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.

Rapid, multiplexed, and nucleic acid amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor.

Considering the worldwide health crisis associated with highly contagious severe respiratory disease of COVID-19 outbreak, the development of multiplexed, simple and rapid diagnostic platforms to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in high demand. Here, a nucleic acid amplification-free electrochemical biosensor based on four-way junction (4-WJ) hybridization is presented for the detection of SARS-CoV-2. To form a 4-WJ structure, a Universal DNA-Hairpin (UDH) probe is hybridized with two adaptor strands and a SARS-CoV-2 RNA target. One of the adaptor strands is functionalized with a redox mediator that can be detected using an electrochemical biosensor. The biosensor could simultaneously detect 5.0 and 6.8 ag/µL of S and Orf1ab genes, respectively, within 1 h. The biosensor was evaluated with 21 clinical samples (16 positive and 5 negative). The results revealed a satisfactory agreement with qRT-PCR. In conclusion, this biosensor has the potential to be used as an on-site, real-time diagnostic test for COVID-19.

Mismatch-introduced DNA probes constructed on the basis of thermodynamic analysis enable the discrimination of single nucleotide variants.

Genotyping of single nucleotide variants (SNVs) has enabled the assessment of disease-related risk factors and significantly improved the potency of diagnosis and prognosis. Meanwhile, genotyping of SNVs is challenging due to the high sequence similarity between wild-type (WT) and SNV. To increase the discrimination between WT and SNV, probes are modified with nucleic acid analogues such as locked nucleic acid (LNA), or deliberate mismatches are introduced to the probe sequence. However, nucleic acid analogues have limitation in high cost and complexity in their synthesis. And a generalized methodology has not been proposed for determining the position and type of deliberate mismatches at the designated experimental conditions to the best of our knowledge. Herein, we propose a reliable workflow for designing mismatch-introduced probes (MIPs) based on nucleic acid thermodynamic analysis and rejection sampling. The theoretical hybridization state of MIP was calculated using nucleic acid thermodynamics, and the detectability was estimated by rejection sampling that simulates the errors from experimental environments. We fabricated MIPs for SNVs in epidermal growth factor receptor, and experimentally demonstrated optimized detectability. The detectability increased up to 7.19-fold depending on the position and type of mismatch; moreover, the optimized MIP showed higher detectability than the LNA probe. This indicates that the workflow can be broadly applied to the optimization of probe sequence for the detection of various disease-related SNVs.