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.

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.

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.

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.

Simple and portable on-site system for nucleic acid-based detection of Clostridium difficile in stool samples using two columns containing microbeads and loop-mediated isothermal amplification.

It is challenging to employ nucleic acid-based diagnostics for the in situ detection of Clostridium difficile from complex fecal samples because essential sample preparation and amplification procedures require various experimental resources. In this study, a simple and effective on-site nucleic acid-based detection system was used to detect C. difficile in stool samples. Two columns containing different microbeads, namely, glass and functionalized graphene oxide-coated microbeads, were designed to remove relatively large impurities by filtration and concentrate bacteria, including C. difficile, from stool samples by adsorption. The bacterial nucleic acids were effectively extracted using a small bead beater. The effectiveness of enzyme inhibitors remaining in the sample was efficiently reduced by the direct buffer developed in this study. This sample preparation kit consisting of two simple columns showed better performance in real-time polymerase chain reaction (PCR) and equivalent performance in loop-mediated isothermal amplification (LAMP) than other sample preparation kits, despite 90% simplification of the process. The amplification-ready samples were introduced into two microtubes containing LAMP pre-mixtures (one each for E. coli as an external positive control and C. difficile) by a simple sample loader, which was operated using a syringe. LAMP, which indicates amplification based on color change, was performed at 65 °C in a small water bath. The limit of detection (L.O.D) and analytical sensitivity/specificity of our simple and effective kit were compared with those of a commercial kit. C. difficile in stool samples could be detected within 1 h with 103 cfu/10 mg using LAMP combined simple on-site detection kit.

Circulating Tumor Marker Isolation with the Chemically Stable and Instantly Degradable (CSID) Hydrogel ImmunoSpheres.

Here, we present chemically stable and instantly degradable (CSID) hydrogel immunospheres for the isolation of circulating tumor cells (CTCs) and circulating tumor exosomes (CTXs). The CSID hydrogels, which are prepared by the hybridization of alginate and poly(vinyl alcohol), show an equilibrium swelling ratio (ESR) of at pH 7, with a highly stable pH-responsive property. The present hybrid hydrogel is not easily disassociated in the biological buffers, thus being suitable for use in "liquid biopsy", requiring a multistep, long-term incubation process with biological samples. Also, it is gradually degraded by the action of chelating agents; effortless retrieval of the circulating markers has been achieved. Then, we modified the CSID hydrogel spheres with the anti-EpCAM antibody ("C-CSID ImmunoSpheres") and the anti-CD63 antibody ("E-CSID ImmunoSpheres") to isolate two promising circulating markers in liquid biopsy: CTCs and CTXs. The immunospheres' capabilities for marker isolation and retrieval were confirmed by a fluorescence image, where the spheres successfully isolate and effortlessly retrieve the target circulating markers. Lastly, we applied the CSID hydrogel immunospheres to five blood samples from colorectal cancer patients and retrieved average 10.8 ± 5.9 CTCs/mL and average 96.5 × 106 CTXs/mL. The present CSID hydrogel immunospheres represent a simple, versatile, and time-efficient assay platform for liquid biopsy in the practical setting, enabling us to gain a better understanding of disease-related circulating markers.

Integrated microsystems for the in situ genetic detection of dengue virus in whole blood using direct sample preparation and isothermal amplification.

Owing to the frequent outbreak of dengue fever worldwide, a highly sensitive but in situ simple process diagnostic device is required to detect the dengue virus. However, the current immune affinity-based methods have sensitivity issues and nucleic acid-based diagnostic devices have not been suitable for field diagnosis due to the complexity in sample preparation. Here, a simple and fast nucleic acid-based diagnostic tool to directly detect dengue viruses in whole blood is demonstrated using a microbead-assisted direct sample preparation buffer (MB-buffer) and isothermal amplification (loop-mediated isothermal amplification, LAMP). To maximize the performance of the sample preparation process in the microfluidic chip platform, the chemical composition of the sample preparation buffer is simplified and combined with physical tools (heating and bead beating). The entire serial processes consisted of only (1) sample (whole blood) loading, (2) stirring for 90 s, (3) heating at 70 °C for 10 min, and (4) LAMP amplification in the simply designed microfluidic chip cartridge. A single syringe was utilized for sample loading and microfluidic solution transfer. Consequently, dengue viruses were qualitatively detected and discriminated with high sensitivity (LOD: 102 PFU per 200 µL of whole blood) in less than 1 hour without the use of any sophisticated system.

Robust Bioengineered Apoferritin Nanoprobes for Ultrasensitive Detection of Infectious Pancreatic Necrosis Virus.

Infectious pancreatic necrosis virus (IPNV) has been identified as a viral pathogen for many fish diseases that have become a huge hurdle for the growing fishing industry. Thus, in this work, we report a label-free impedance biosensor to quantify IPNV in real fish samples at point-of-care (POC) level. High specificity IPNV sensor with a detection limit of 2.69 TCID50/mL was achieved by conjugating IPNV antibodies to portable Au disk electrode chips using human heavy chain apoferritin (H-AFN) nanoprobes as a binding agent. H-AFN probes were bioengineered through PCR by incorporating pET-28b(+) resulting in 24 subunits of 6 × his-tag and protein-G units on its outer surface to increase the sensitivity of the IPNV detection. The biosensor surface modifications were characterized by differential pulse voltammetry (DPV) and EIS methods for each modification step. The proposed nanoprobe based sensor showed three-fold enhancement in charge transfer resistance toward IPNV detection in comparison with the traditional linker approach when measured in a group of similar virus molecules. The portable sensor exhibited a linear range of 100-10000 TCID50/mL and sensitivity of 5.40 × 10-4 TCID50/mL in real-fish samples. The performance of the proposed IPNV sensor was fully validated using an enzyme-linked immunosorbent assay (ELISA) technique with a sensitivity of 3.02 × 10-4 TCID50/mL. Results from H-AFN nanoprobe based IPNV sensor indicated high selectivity, sensitivity, and stability could be a promising platform for the detection of similar fish viruses and other biological molecules of interest.

Semi-automatic instrumentation for nucleic acid extraction and purification to quantify pathogens on surfaces.

Public lavatories may cause the spread of infectious pathogens because they are enclosed spaces that both healthy people and patients can use. Thus, surface analysis for microbial contamination in public lavatories is of great importance because it is considered as an indicator of hygiene control. Herein, we developed polymerase chain reaction (PCR)-compatible surface sample preparation tools to increase the detection sensitivity and reproducibility within a short time using a semi-automatic detection system. The bacteria and viruses on different surfaces were collected using half A4-sized wipes. The wipes were treated through four different processes in a cartridge: (1) the pathogens were transferred from the wipes to the aqua phase using simple gentle vortexing; (2) the bacteria and viruses were concentrated by adsorption on the graphene surface; (3) the pathogens on the graphene layer were perfectly lysed using bead-beating tools and (4) the released DNA/RNA was collected in a microtube. The prepared nucleic acid sample was amplified using PCR or loop-mediated isothermal amplification (LAMP). At least one order of magnitude higher sensitivity was achieved using the wipe collecting method compared to that achieved using the normal swab method. This was confirmed using a semi-automatic cartridge for the wipe sampling in a lavatory hygiene test.

Quantitative evaluation of software packages for single-molecule localization microscopy.

The quality of super-resolution images obtained by single-molecule localization microscopy (SMLM) depends largely on the software used to detect and accurately localize point sources. In this work, we focus on the computational aspects of super-resolution microscopy and present a comprehensive evaluation of localization software packages. Our philosophy is to evaluate each package as a whole, thus maintaining the integrity of the software. We prepared synthetic data that represent three-dimensional structures modeled after biological components, taking excitation parameters, noise sources, point-spread functions and pixelation into account. We then asked developers to run their software on our data; most responded favorably, allowing us to present a broad picture of the methods available. We evaluated their results using quantitative and user-interpretable criteria: detection rate, accuracy, quality of image reconstruction, resolution, software usability and computational resources. These metrics reflect the various tradeoffs of SMLM software packages and help users to choose the software that fits their needs.

Short-Length DNA Adsorption on Graphene Oxide-Coated Microbeads for DNA Target Separation from Clinical Samples.

Nucleic acid preparation (concentration and purification of various nucleic acid targets) from biological samples is essential for personalized and precision medicine. The adsorption of short-length DNA on graphene oxide (GO) layers was investigated and compared with that on silica surfaces. GO was efficiently coated on glass beads to be used more easily and spatially. Surface of the GO bead was confirmed by field-emission scanning electron microscopy. GO-coated beads were packed and the adsorption conditions of short-length DNA were optimized under various pH and flow rate conditions. The amount of adsorbed DNA was confirmed by real-time polymerase chain reaction and visualized using fluorescence microscopy.

Elimination of Humic Acid from Aqueous Sample Using Zinc Oxide/Graphene Oxide-Coated Microbeads.

Natural organic matter (NOM) is known to cause major problems with drinking water quality management, such as sedimentation of disinfectants during the purification process, microbial growth of water pipes, and corrosion of pipes. For efficient and continuous removal of NOM from drinking water, a packed bed-type platform containing microbeads based on nanostructured zinc oxide (ZnO) was developed. ZnO was synthesized on graphene oxide (GO)-coated microbeads by optimizing the ZnO concentration and reaction time. The morphology of the synthesized ZnO-coated microbeads was confirmed by scanning electron microscopy, and an adsorption test was conducted using a cationic dye. The ZnO/GO microbeads were packed in a microtube. A humic acid contaminated aqueous solution was allowed to flow through the microbeads, and its removal rate was measured by UV-vis spectroscopy. This study confirmed that the purification platform containing ZnO removed more than 90% of humic acid of about 1,000 ppm.

Fabrication of Electrochemical-Based Bioelectronic Device and Biosensor Composed of Biomaterial-Nanomaterial Hybrid.

The field of bioelectronics has paved the way for the development of biochips, biomedical devices, biosensors and biocomputation devices. Various biosensors and biomedical devices have been developed to commercialize laboratory products and transform them into industry products in the clinical, pharmaceutical, environmental fields. Recently, the electrochemical bioelectronic devices that mimicked the functionality of living organisms in nature were applied to the use of bioelectronics device and biosensors. In particular, the electrochemical-based bioelectronic devices and biosensors composed of biomolecule-nanoparticle hybrids have been proposed to generate new functionality as alternatives to silicon-based electronic computation devices, such as information storage, process, computations and detection. In this chapter, we described the recent progress of bioelectronic devices and biosensors based on biomaterial-nanomaterial hybrid.

Label-Free Impedance Sensing of Aflatoxin B1 with Polyaniline Nanofibers/Au Nanoparticle Electrode Array.

Aflatoxin B1 (AFB1) is produced by the Aspergillus flavus and Aspergillus parasiticus group of fungi which is most hepatotoxic and hepatocarcinogenic and occurs as a contaminant in a variety of foods. AFB1 is mutagenic, teratogenic, and causes immunosuppression in animals and is mostly found in peanuts, corn, and food grains. Therefore, novel methodologies of sensitive and expedient strategy are often required to detect mycotoxins at the lowest level. Herein, we report an electrochemical impedance sensor that selectively detects AFB1 at the lowest level by utilizing polyaniline nanofibers (PANI) coated with gold (Au) nanoparticles composite based indium tin oxide (ITO) disk electrodes. The Au-PANI nanocomposites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) spectroscopy, and electrochemical impedance spectroscopy (EIS). The composite electrode exhibited a 14-fold decrement in |Z|1Hz in comparison with the bare electrode. The Au-PANI acted as an effective sensing platform having high surface area, electrochemical conductivity, and biocompatibility which enabled greater loading deposits of capture antibodies. As a result, the presence of AFB1 was screened with high sensitivity and stability by monitoring the changes in impedance magnitude (|Z|) in the presence of a standard iron probe which was target specific and proportional to logarithmic AFB1 concentrations (CAFB1). The sensor exhibits a linear range 0.1 to 100 ng/mL with a detection limit (3) of 0.05 ng/mL and possesses good reproducibility and high selectivity against another fungal mycotoxin, Ochratoxin A (OTA). With regard to the practicability, the proposed sensor was successfully applied to spiked corn samples and proved excellent potential for AFB1 detection and development of point-of-care (POC) disease sensing applications.

Three-Dimensional Graphene-RGD Peptide Nanoisland Composites That Enhance the Osteogenesis of Human Adipose-Derived Mesenchymal Stem Cells.

Graphene derivatives have immense potential in stem cell research. Here, we report a three-dimensional graphene/arginine-glycine-aspartic acid (RGD) peptide nanoisland composite effective in guiding the osteogenesis of human adipose-derived mesenchymal stem cells (ADSCs). Amine-modified silica nanoparticles (SiNPs) were uniformly coated onto an indium tin oxide electrode (ITO), followed by graphene oxide (GO) encapsulation and electrochemical deposition of gold nanoparticles. A RGD-MAP-C peptide, with a triple-branched repeating RGD sequence and a terminal cysteine, was self-assembled onto the gold nanoparticles, generating the final three-dimensional graphene-RGD peptide nanoisland composite. We generated substrates with various gold nanoparticle-RGD peptide cluster densities, and found that the platform with the maximal number of clusters was most suitable for ADSC adhesion and spreading. Remarkably, the same platform was also highly efficient at guiding ADSC osteogenesis compared with other substrates, based on gene expression (alkaline phosphatase (ALP), runt-related transcription factor 2), enzyme activity (ALP), and calcium deposition. ADSCs induced to differentiate into osteoblasts showed higher calcium accumulations after 14-21 days than when grown on typical GO-SiNP complexes, suggesting that the platform can accelerate ADSC osteoblastic differentiation. The results demonstrate that a three-dimensional graphene-RGD peptide nanoisland composite can efficiently derive osteoblasts from mesenchymal stem cells.

Self-assembly of an upconverting nanocomplex and its application to turn-on detection of metalloproteinase-9 in living cells.

Upcoversion nanoparticles are an emerging luminescent nanomaterial with excellent photophysical properties that have great benefits in biological sensing. In this study, a luminescent turn-on biosensor for cell-secreted protease activity assay is established based on resonance energy transfer in an upconversion nanoparticle-graphene oxide nano-assembly. The proposed biosensor consists of a blue-emitting upconversion nanoparticle covered with a quenching complex, comprising gelatin as the proteinase substrate and graphene oxide nanosheets as luminescence acceptors. After enzymatic digestion, the upconversion nanoparticles lose the gelatin cover due to the disassembly of the quenching complex, thus the upconverting luminescence in the blue region is restored (a turn-on response). The recovered upconverting luminescence is proportional to the protease concentration; the limit of detection was 12 ng ml(-1). Finally, the upconversion-graphene oxide nanocomplex was successfully applied in the detection of cell-secreted protease-metalloproteinase in MCF-7 cancer cells with high sensitivity and specificity.

Addressing of LnCaP Cell Using Magnetic Particles Assisted Impedimetric Microelectrode.

In this study, we provide a facile, effective technique for a simple isolation and enrichment of low metastatic prostate tumor cell LNCaP using biocompatible, magnetic particles asissted impedimetric sensing system. Hydrophobic cell membrane anchors (BAM) were generated onto magnetic particles which diameters vary from 50 nm to 5 µm and were used to capture LNCaP cells from the suspension. Finally, magnetic particle-LNCaP complex were addressed onto the surface of the interdigitated microelectrode (IDM). Cell viability was monitored by our laboratory developed-technique Electrical Cell Substrate Impedance Sensing (ECIS). The results reavealed that 50 nm-magnetic particles showed best performance in terms of cell separation and cell viability. This technique provides a simple and efficient method for the direct addressing of LNCaP cell on the surface and enhances better understanding of cell behavior for cancer management in the near future.

Fabrication of Magnetic Upconversion Nanohybrid for Luminescent Resonance Energy Transfer-Based Detection of Glutathione.

This study introduces the facile fabrication of a bimodal nanohybrid for the luminescent ON/OFF detection of glutathione. The proposed nanosensor consists of magnetic (Fe3O4) and upconversion nanoparticles (UCP) co-encapsulated in a silica matrix, and decorated with gold nanoparticle (AuNP) as a luminescent quencher. The detection mechanism is based on the Luminescent Resonance Energy Transfer (LRET) between the donor (UCP) and the acceptor (AuNP) with the help of a disulfide bond as a bridging element. In the presence of glutathione, the disulfide bridges between AuNPs and Fe3O4/UCP@SiO2 was cleaved and the amount of glutathione could be traced by the restored luminescence (ON state) of the nanohybrids after magnetic separation.

Fatigue Test of Cytochrome C Self-Assembled on a 11-MUA Layer Based on Electrochemical Analysis for Bioelectronic Device.

A cytochrome c/11-MUA heterolayer was fabricated to analyze its electrochemical characteristics in harsh conditions for a stable bioelectronic device. Since a cytochrome c/11-MUA heterolayer has been applied to construct the bioelectronics device such as non-volatile biomemory device, an understanding of electrochemical property of the heterolayer in harsh conditions such as variation of temperature and pH, and repetition of usage is necessary to manufacture a stable platform of bioelectronic device. Cytochrome c, a metalloprotein to have a heme group, was self-assembled on the Au surface via the chemical linker 11-mercaptoundecanoic acid (11-MUA). Immobilization of the heterolayer was confirmed by surface-enhanced Raman spectroscopy (SERS) and scanning tunneling microscopy (STM). The fatigue test was done by investigating the redox properties based on cyclic voltammetry (CV) of the heterolayer. The retention time test and pH dependence, thermal test of the fabricated heterolayer were conducted by CV, which showed that the fabricated film retained redox properties for more than 33 days, and from pH 5.0 to pH 9.0, from 15 °C to 55 °C. Taken together, our results show that a cytochrome c/11-MUA heterolayer is very stable, which could be used as a platform of bioelectronic device.