Pumpless three-dimensional photo paper-based microfluidic analytical device for automatic detection of thioredoxin-1 using enzyme-linked immunosorbent assay.

Microfluidic-based biosensors have been developed for their precise automatic reaction control. However, these biosensors require external devices that are difficult to transport and use. To overcome this disadvantage, our group made an easy-to-use, cheap, and light pumpless three-dimensional photo paper-based microfluidic analytical device (3D-µPAD; weight: 1.5 g). Unlike conventional paper-based microfluidic analytical devices, the 3D-µPAD can be used to control fluid flow in a 3D manner, thus allowing sophisticated multi-step reaction control. This device can control fluid flow speed and direction accurately using only the capillary-driven flow without an external device like a pump. The flow speed is controlled by the width of the microfluidic channel and its surface property. In addition, fluid speed control and 3D-bridge structure enable the control of fluid flow direction. Using these methods, multi-step enzyme-linked immunosorbent assay (ELISA) can be done automatically in sequence by injecting solutions (sample, washing, and enzyme's substrate) at the same time in the 3D-µPAD. All the steps can be performed in 14 min, and data can be analyzed immediately. To test this device, thioredoxin-1 (Trx-1), a biomarker of breast cancer, is used as the target. In the 3D-µPAD, it can detect 0-200 ng/mL of Trx-1, and the prepared 3D-µPAD Trx-1 sensor displays excellent selectivity. Moreover, by analyzing the concentration of Trx-1 in real patients and healthy individuals' blood serum samples using the 3D-µPAD, and comparing results to ELISA, it can be confirmed that the 3D-µPAD is a good tool for cancer diagnosis.

Protein Nanoparticle Fabrication for Optimized Reticuloendothelial System Evasion and Tumor Accumulation.

Nanoparticles (NPs) of protein-based materials have become one of the most promising candidates for drug carriers in drug-delivery systems because of their in vivo nontoxicity, biodegradability, compatibility with hydrophilic drugs, and adaptability to the human body. Many studies have investigated the fabrication of protein NPs from human serum albumin (HSA) as a new drug carrier. It is important for these NPs to remain in the blood until they reach their therapeutic target to achieve the desired effect; the quicker the clearance of drugs from the body, the shorter is the residence time of drugs in the body, which eventually reduces drug efficacy. Macrophage uptake is a major mechanism for clearance of NPs from the body, so, reducing the degree of macrophage uptake is a major challenge in drug-delivery systems. Original studies of HSA NP uptake by macrophages showed that denatured HSA and HSA NPs synthesized with 80% (v/v) ethanol showed a high degree of macrophage uptake. We found that HSA NPs synthesized with lower ethanol content at pH 7 showed lower macrophage uptake in in vitro macrophage cellular uptake experiments. The effects of the preparation parameters of ethanol concentration, pH, and glutaraldehyde on the macrophage uptake of NPs were thoroughly studied. This newly developed protein NP with lower macrophage uptake has potential application as a drug carrier for many delivery systems.

Cationic Surfactant-Induced Formation of Uniform Gold Nanoparticle Clusters with High Efficiency of Photothermal Conversion under Near-Infrared Irradiation.

A novel and simple method for the fabrication of gold nanoparticle (AuNP) clusters was introduced for use as an efficient near-infrared (NIR) photothermal agent. Cationic surfactants were employed to assemble AuNPs into clusters, during which polyvinylpyrrolidone (PVP) was used to stabilize the AuNP clusters. Through this manner, AuNP clusters with a uniform shape and a narrow size distribution (55.4 ± 5.0 nm by electron microscope) were successfully obtained. A mechanism for the formation of AuNP clusters was studied and proposed. Electrostatic interactions between AuNPs and cationic surfactants, hydrophobic interactions between hydrocarbon chains of cationic surfactants, and repulsive steric interactions of PVP were found to play an important role with regard to the formation mechanism. Photothermal effect in the NIR range of the AuNP clusters was demonstrated; results presented a highly efficient photothermal conversion (with a maximum η of 65%) of the AuNP clusters. The clusters could be easily coated by a silica layer, enabling their biocompatibility and colloidal stability in physiological fluids. The easy-to-fabricate AuNP clusters showed high potential of use as an NIR photothermal agent for cancer therapy.

Nanostructure Modified Microelectrode for Electrochemical Detection of Dopamine with Ascorbic Acid and Uric Acid.

Dopamine (DA) is one kind of neurotransmitter in central nervous system which is indicator of neural disease. For this reason, determination of DA concentration in central nervous system is very important for early diagnosis of neural disease. In this study, we designed micro electrode array and fabricated by MEMS technology. Furthermore, we fabricated 3-D conducting nanostructure on electrode surface for enhanced sensitivity and selectivity due to increased surface area. Compared with macro and normal micro electrode, the 3-D nanostructure modified micro electrode shows better electrical performance. These surface modified pin type electrode was applied to detect low concentration of DA and successfully detect various concentration of DA from 100 µM to 1 µM with linear relationship in the presence of ascorbic acid and uric acid. From these results, our newly designed electrode shows possibility to be applied as brain biosensor for neural disease diagnosis such as Parkinson's diseases.

Highly Sensitive Electrical Detection of HIV-1 Virus Based on Scanning Tunneling Microscopy.

A highly sensitive immunosensor based on scanning tunneling microscopy (STM) was developed for the first time to detect living material such as HIV-1 virus by gold (Au) nanoparticle and fragmented antibody complex. Fragmented antibodies were pre-immobilized on the Au surface, then HIV-1 virus like particles (HIV-1 VLPs) and Au-nanoparticle and fragmented antibody complexes were applied to develop sandwich assay. The developed surface morphology and the current profile of fabricated immunosensing element were characterized by Raman spectroscopy and investigated with STM. The power spectrum derived from the current profile was found to be related with concentrations of HIV-1 VLPs. Using the electrical detection method based on current mapping profile of STM, living material such as virus, HIV-1 VLPs, was able to be detected successfully. The proposed technique can be a promising method to construct the highly sensitive and efficient sensor for detecting viruses and other living materials.

Development of a HIV-1 Virus Detection System Based on Nanotechnology.

Development of a sensitive and selective detection system for pathogenic viral agents is essential for medical healthcare from diagnostics to therapeutics. However, conventional detection systems are time consuming, resource-intensive and tedious to perform. Hence, the demand for sensitive and selective detection system for virus are highly increasing. To attain this aim, different aspects and techniques have been applied to develop virus sensor with improved sensitivity and selectivity. Here, among those aspects and techniques, this article reviews HIV virus particle detection systems incorporated with nanotechnology to enhance the sensitivity. This review mainly focused on four different detection system including vertically configured electrical detection based on scanning tunneling microscopy (STM), electrochemical detection based on direct electron transfer in virus, optical detection system based on localized surface plasmon resonance (LSPR) and surface enhanced Raman spectroscopy (SERS) using plasmonic nanoparticle.

Localized surface plasmon resonance-based label-free biosensor for highly sensitive detection of dopamine.

Localized surface plasmon resonance (LSPR) is the phenomenon that is observed on specific metal nanoparticles (NPs) like Au, Ag which can be used for sensitive detection for many kinds of biomaterials. Dopamine (DA) is a typical neurotransmitter considered as indicator of some neural diseases. Due to its small size, it is very difficult to detect DA at low concentrations directly and sensitively with conventional sensing techniques. In this research, we propose a DA detection sensor based on LSPR phenomenon. Electrochemical deposition technique was used to make LSPR substrates, where Au NPs were electrochemically deposited on ITO glasses and these substrates showed optical characteristic of LSPR phenomenon. Different concentrations of DA solution were deposited on antibody immobilized LSPR substrates. With additions of increasing concentrations of DA, LSPR peak intensity was increased linearly. These results could be applied to many fields of clinical trials for diseases caused by small molecules.

Enzyme-free glucose sensor based on Au nanobouquet fabricated indium tin oxide electrode.

In this study, we demonstrated a simple, rapid and inexpensive fabrication method to develop a novel gold nanobouquet structure fabricated indium tin oxide (GNB/ITO) electrode based on electrochemical deposition of gold ions onto ITO substrate. The morphology of the fabricated electrode surface was characterized by scanning electron microscopy (SEM) to confirm the GNB formation. Enzyme-free detection of glucose using a GNB/ITO electrode was described with high sensitivity and selectivity based on cyclic voltammetry assay. The results demonstrate a linear relation within wide concentration range (500 nM to 10 mM) of glucose, with a correlation coefficient of 0.988. The interference effect of uric acid was effectively avoided for the detection of glucose (1 µM to 10 mM). Moreover, the developed sensor was applied to determine the concentration of glucose in the presence of human serum to indicate the ability of GNB/ITO electrodes in real samples. Hence, newly developed GNB/ITO electrode has potential application in enzyme-free glucose sensor with highly sensitivity and selectivity.

Highly sensitive localized surface plasmon resonance immunosensor for label-free detection of HIV-1.

A highly sensitive label-free immunosensor for the detection of HIV-1 is newly developed based on localized surface plasmon resonance (LSPR) method. Uniform nanopattern of circular Au-dots (10-20nm) was fabricated on indium tin oxide (ITO) coated glass substrate by simple electrochemical deposition method. The surface of Au nanopattern was modified with HIV-1 neutralizing gp120 monoclonal antibody fragments. The modified substrate was employed to measure various concentrations of HIV-1 particles quantitatively based on the shift of longitudinal wavelength in the UV-Vis spectrum which results from the changes of local refractive index induced by specific antigen-antibody recognition events. The detection limit of the HIV-1 particles was estimated to be 200fg/mL, which is 10 fold higher than that of previously reported virus detection method based on LSPR. Since fabricated LSPR immunosensor has high sensitivity and selectivity, it is a promising approach for biological/medical sample analysis and various kinds of virus detection. FROM THE CLINICAL EDITOR: A localized surface plasmon resonance-based virus detection method is shown to have an order of magnitude improvement in detectable concentration of HIV-1 particles. Similar approaches may be used for screening other viral particles as well.

Horseradish Peroxidase-Encapsulated Fluorescent Bio-Nanoparticle for Ultra-Sensitive and Easy Detection of Hydrogen Peroxide.

Hydrogen peroxide (H2O2) has been a fascinating target in various chemical, biological, clinical, and industrial fields. Several types of fluorescent protein-stabilized gold nanoclusters (protein-AuNCs) have been developed for sensitive and easy detection of H2O2. However, its low sensitivity makes is difficult to measure negligible concentrations of H2O2. Therefore, to overcome this limitation, we developed a horseradish peroxidase-encapsulated fluorescent bio-nanoparticle (HEFBNP), comprising bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) and horseradish peroxidase-stabilized gold nanoclusters (HRP-AuNCs). The fabricated HEFBNP can sensitively detect H2O2 owing to its two properties. The first is that HEFBNPs have a continuous two-step fluorescence quenching mechanism, which comes from the heterogenous fluorescence quenching mechanism of HRP-AuNCs and BSA-AuNCs. Second, the proximity of two protein-AuNCs in a single HEFBNP allows a reaction intermediate (•OH) to rapidly reach the adjacent protein-AuNCs. As a result, HEFBNP can improve the overall reaction event and decrease the loss of intermediate in the solution. Due to the continuous quenching mechanism and effective reaction event, a HEFBNP-based sensing system can measure very low concentrations of H2O2 up to 0.5 nM and show good selectivity. Furthermore, we design a glass-based microfluidic device to make it easier use HEFBNP, which allowed us to detect H2O2 with the naked eye. Overall, the proposed H2O2 sensing system is expected to be an easy and highly sensitive on-site detection tool in chemistry, biology, clinics, and industry fields.

Detection of dopamine in dopaminergic cell using nanoparticles-based barcode DNA analysis.

Nanotechnology-based bio-barcode-amplification analysis may be an innovative approach to dopamine detection. In this study, we evaluated the efficacy of this bio-barcode DNA method in detecting dopamine from dopaminergic cells. Herein, a combination DNA barcode and bead-based immunoassay for neurotransmitter detection with PCR-like sensitivity is described. This method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA, and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated in order to remove the conjugated barcode DNA. The DNA barcodes were then identified via PCR analysis. The dopamine concentration in dopaminergic cells can be readily and rapidly detected via the bio-barcode assay method. The bio-barcode assay method is, therefore, a rapid and high-throughput screening tool for the detection of neurotransmitters such as dopamine.

Cell chip-based monitoring of toxic effects of cosmetic compounds on skin fibroblast cells.

The present study estimated the efficacy of electrochemical detection of imidazolidinyl urea-induced cell toxicity in skin human fibroblast cells (HFF cells). The gold nanopunct structures were fabricated through a nanoporous alumina mask, and the structural formations were confirmed via scanning electron microscopy. The HFF cells were allowed to attach to RGD (Arg-Gly-Asp) peptide nanopatterned surfaces, and electrochemical tools were applied to skin cells attached to the chip surface. The HFF cells evidenced inflammation responses to allergens such as imidazolidinyl urea. The cells were subsequently treated with different concentrations of imidazolidinyl urea for 24 h in culture, which induced a change in the cyclic voltammetry (CV) current peak. Treatment with imidazolidinyl urea induced a loss of cell viability and accelerated inflammation in a concentration-dependent manner. The expression level of inflammation-related proteins such as IL-1 beta were increased in imidazolidinyl urea-treated cells. The CV results demonstrated that imidazolidinyl urea significantly reduced the current peaks in a dose-dependent manner. The results showed that the current peak was reduced in accordance with the increases in imidazolidinyl urea-induced inflammation. In conclusion, the results of this study suggest that the electrochemical-based chip provides crucial information for improvements to a cell chip system for drug screening applications.

Detection of dopamine using a silicon nanowire patterned by nanoimprint lithography.

Dopamine is one of the most important catecholamine neurotransmitter in the nucleus accumbens of wide variety of animals, including humans. In this study, silicon nanowire FET device was fabricated by UV-assisted NIL method and dopamine was successfully measured by conductance versus time characteristics within 10 pM to 100 nM.

Electrochemical sensor to detect neurotransmitter using gold nano-island coated ITO electrode.

Parkinson disease is a chronic neurodegenerative disorder characterized by the loss of dopamine, which is a neurotransmitter in the substantia nigra. In this study, a simple, rapid and inexpensive method to fabricate gold nano-island film (GNIF) coated ITO electrode has been developed based on electrochemical deposition of Au onto ITO substrate. The nanostructured film surface was characterized by scanning electron microscopy (SEM). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to evaluate the electrochemical behavior of induvidul dopamine and uric acid solution were studied. Moreover, GNIF/ITO electrode was applied to detecte DA in the presence of Bovine Serum Albumin (50 microM) as an interference. These results demonstrate that, interfering component has no effect on the determination of DA at GNIF electrode, hence this GNIF electrode is suitable for the determination of DA with high sensitivity and selectivity. Then, GNIF coated ITO electrode was applied to monitor the electrochemical simultaneous detection of dopamine and uric acid mixtures based on CV and DPV with high sensitivity. GNIF-modified ITO electrode showed a linear range for the determination of dopamine concentration from 0.1 microM to 40 microM in the presence of 50 microM of uric acid. Based on these results, the proposed technique can be a promising method to construct a highly sensitive biosensor as well as highly efficient protein chip.

Fabrication of nanoconcave surface for cell immobilization in cell-based chip.

Nanostructured surface such as nanoconcave shape can be utilized as a bioplatform to immobilize cells. In this study, we present fabrication of Au-coated nanoconcave surface and some possibility of cell immobilization. Long-range ordered periodic patterns with concave shape were formed on aluminum substrate by electrochemical anodization process. The morphology and topography of nanoconcave surface was investigated by atomic force microscopy and scanning electron microscopy. The pore-pore distance and the pore depth of nanoconcave pattern were measured at 105 +/- 5 nm and 30 +/- 2 nm, respectively. After Au deposition, the pore depth within Au-coated concave surface was 15 +/- 2 nm. The topography of HeLa cells immobilized on the nanoconcave surface was observed by AFM combined with confocal microscopy. The result expected that the Au-coated nanoconcave surface may be used as new culture substrate for cells immobilization in cell-based chip.

Detection of beta-amyloid (1-42) on protein array based on electrical detection technique using scanning tunneling microscopy.

In this study an immuno-array for Abeta42 based on scanning tunneling microscopy (STM) was developed using conjugated gold nanoparticle (Au-NP) and antibody (Ab) complex. Fragmented monoclonal Ab against Abeta42 was allowed to immobilize on the Au-dot arrays followed by its target protein Abeta42 and Au NP and Ab complex. The surface structure of Au-NP and Ab complex on Au-dots was investigated with Atomic Force Microscopy and the current profile of fabricated immunosensing element was investigated with STM. The power spectrum derived from the current profile was found to be increasing with higher concentrations of Abeta42 having a detection limit of 100 fg/ml. The proposed technique can be a promising method to construct the highly sensitive and efficient protein chip of immunosensors arrays.

Fabrication of silicon nanowire for detecting p-amyloid (1-42) by nanoimprint lithography.

Ultraviolet nanoimprint lithography (UV-NIL) is a high volume and cost-effective patterning technique with sub-10 nm resolution. It has great potential as a candidate for next generation lithography. Using UV-NIL, nanowire patterns were successfully fabricated on a four-inch silicon-on-insulator (SOI) wafer under moderate conditions. The fabricated nanowire patterns were characterized by FE-SEM. Its electrical properties were confirmed by semiconductor parameter analysis. Monoclonal antibodies against beta-amyloid (1-42) were immobilized on the silicon nanowire using a chemical linker. Using this fabricated silicon nanowire device, beta-amyloid (1-42) levels of 1 pM to 100 nM were successfully determined from conductance versus time characteristics. Consequently, the nanopatterned SOI nanowire device can be applied to bioplatforms for the detection of proteins.

Nano-sized graphene oxide coated nanopillars on microgroove polymer arrays that enhance skeletal muscle cell differentiation.

The degeneration or loss of skeletal muscles, which can be caused by traumatic injury or disease, impacts most aspects of human activity. Among various techniques reported to regenerate skeletal muscle tissue, controlling the external cellular environment has been proven effective in guiding muscle differentiation. In this study, we report a nano-sized graphene oxide (sGO)-modified nanopillars on microgroove hybrid polymer array (NMPA) that effectively controls skeletal muscle cell differentiation. sGO-coated NMPA (sG-NMPA) were first fabricated by sequential laser interference lithography and microcontact printing methods. To compensate for the low adhesion property of polydimethylsiloxane (PDMS) used in this study, graphene oxide (GO), a proven cytophilic nanomaterial, was further modified. Among various sizes of GO, sGO (< 10 nm) was found to be the most effective not only for coating the surface of the NM structure but also for enhancing the cell adhesion and spreading on the fabricated substrates. Remarkably, owing to the micro-sized line patterns that guide cellular morphology to an elongated shape and because of the presence of sGO-modified nanostructures, mouse myoblast cells (C2C12) were efficiently differentiated into skeletal muscle cells on the hybrid patterns, based on the myosin heavy chain expression levels. Therefore, the developed sGO coated polymeric hybrid pattern arrays can serve as a potential platform for rapid and highly efficient in vitro muscle cell generation.

Noble Metal Nanomaterial-Based Biosensors for Electrochemical and Optical Detection of Viruses Causing Respiratory Illnesses.

Noble metal nanomaterials, such as gold, silver, and platinum, have been studied extensively in broad scientific fields because of their unique properties, including superior conductivity, plasmonic property, and biocompatibility. Due to their unique properties, researchers have used them to fabricate biosensors. Recently, biosensors for detecting respiratory illness-inducing viruses have gained attention after the global outbreak of coronavirus disease (COVID-19). In this mini-review, we discuss noble metal nanomaterials and associated biosensors for detecting respiratory illness-causing viruses, including SARS-CoV-2, using electrochemical and optical detection techniques. this review will provide interdisciplinary knowledge about the application of noble metal nanomaterials to the biomedical field.

Engineering Pseudomonas putida KT2440 to convert 2,3-butanediol to mevalonate.

Biological production of 2,3-butanediol (2,3-BDO), a C4 platform chemical, has been studied recently, but the high cost of separation and purification before chemical conversion is substantial. To overcome this obstacle, we have conducted a study to convert 2,3-BDO to mevalonate, a terpenoid intermediate, using recombinant Pseudomonas putida and this biological process won't need the separation and purification process of 2,3-BDO. The production of mevalonate when 2,3-BDO was used as a substrate was 6.61 and 8.44 times higher than when glucose and glycerol were used as substrates under the same conditions, respectively. Lower aeration contributed to higher yields of mevalonate in otherwise identical conditions. The maximum mevalonate production on the shaking flask scale was about 2.21 g/L, in this study (product yield was 0.295, 27% of theoretical yield (1.10)). This study was the first successful attempt for mevalonate production by P. putida using 2,3-BDO as the sole carbon source and presented a new metabolic engineering tool and biological process for mevalonate synthesis.