Search

Loop-mediated isothermal amplification (LAMP) is an outstanding method for molecular diagnostics, as the rapid, specific, and sensitive amplification of target genes is possible. However, it is necessary to measure fluorescence in the quantitative analysis of LAMP products, so a sophisticated optical setup is required. This study tried to develop a novel sensing method that can quantify target analytes with simple equipment, such as nonspectroscopic white light and a CMOS camera. To achieve this, a retroreflective Janus particle (RJP) as a probe and specially designed loop primers, fluorescein isothiocyanate (FITC)- and biotin-modified loop primers, were introduced into the LAMP system. By performing LAMP in the presence of designed primers, double-stranded amplicons possessing FITC and biotin labels at each end are generated in proportion to the quantity of the target pathogen. Using the anti-FITC antibody-modified sensing surface and streptavidin-conjugated RJP probes, the amplicons can be captured in sandwich-configuration and detected under nonspectroscopic conditions composed of white light and a camera. To confirm the feasibility of the sensing system, the

The detection of foodborne pathogenic microorganisms is an essential issue in molecular diagnostics. Fluorescence-based assays have been widely utilized in molecular diagnostics because of their ability to detect and measure low analyte concentrations. However, conventional fluorescence-based assays require sophisticated optics systems, such as a specific light source and light filter. To overcome these limitations, we developed an optical sensing system using a retroreflective Janus microparticle (RJP) as a signaling probe. Compared to fluorescent dyes, RJPs have the advantage of not requiring complicated optic systems because they can be observed using visible light without a filter. To confirm that RJPs can be used as a probe for molecular diagnostics, Salmonella was detected using a biotinylated stem-loop DNA probe to capture the target gene DNA and a streptavidin-conjugated RJP (SA-RJP) as the detection molecule. When the target gene DNA was present at the sensing surface where the stem-loop DNA probe was immobilized, the biotinylated stem-loop DNA probe was stretched, exposing biotin, which can react with SA-RJP. Since the amount of exposed biotin increased according to the concentration of the applied target gene DNA, the number of observed RJPs on the sensing surface increased with the concentration of the target gene DNA. Consequently, the concentration of Salmonella could be quantitated by counting the number of observed RJPs. Using this system, Salmonella at concentrations ranging from 0 to 100 nM could be analyzed, with high sensitivity and selectivity, with a limit of detection of 2.48 pM. Electronic supplementary material The online version of this article (10.1186/s40580-019-0186-1) contains supplementary material, which is available to authorized users.

Optical biochemical assays that utilize traditional optical signaling labels, such as fluorophores and fluorescent nanoparticles, have been extensively applied in the development of optical biosensors. However, traditional optical-label-based analytical approaches require expensive and sophisticated optical instruments; thus, the application of traditional optical-label-based biochemical assays to optical biosensors in point-of-care testing (POCT) concepts that require cost-effectiveness and user-friendliness remains challenging. Retroreflection-based optical biosensing technology that utilizes micro-sized retroreflectors as an optical signaling label is being studied as a promising technological alternative to overcome the drawbacks of conventional optical-label-based biosensors. Retroreflection is an optical phenomenon whereby light rays strike a specific surface, a retroreflector, and are redirected to the light source along the inverse direction of the incident light. Biosensors that involve the retroreflection principle and retroreflector-type optical label offer distinctive advantages, such as the cost-effective simplification of optical instrument configuration, highly flexible applicability to various biochemical assays, and high analytical capability; therefore, their further applications toward the biosensing platform for POCT is highly promising. This review introduces the fundamentals of retroreflection and summarizes recent research achievements of retroreflection-based optical biosensor development from the perspective of how retroreflectors can be coupled and utilized with the optical biosensing principle as optical signal labels. The expected future applications of retroreflection-based optical biosensor technology is also discussed.

This study aims to suggest a simple migratory cell monitoring method in the Transwell system by utilizing retroreflective Janus microparticles (RJPs) as an optical probe. The RJP could be internalized on cells without compromising the cell viability and can be registered as bright spots within the cell body by inducing retroreflection from nonspectroscopic light sources. Conventional optical probes (e.g., fluorophores, chromogens, and nanoparticles) have been extensively studied and applied across diverse platforms (e.g., Boyden chamber, wound closing, and microfluidic chips) for understanding in vitro kinetic cell behavior. However, the complexities of running such platforms and setting up analytical instruments are limiting. In this regard, we aimed to demonstrate a modified Transwell migration assay by introducing the retroreflection principle to the cell quantification procedures that ensure a simplified optical setup, assure easy signal acquisition, and are compatible with conventional platforms. To demonstrate retroreflection as a signaling principle, a half-metal-coated silica particle that can induce interior retroreflection was synthesized. Because the RJPs can concentrate incident light and reflect it back to the light source, retroreflection was distinctively recognizable and enabled sensitive visualization. To verify the applicability of the developed migration assay, cell quantification during the incremental progress of macrophage migration, and cell quantification under gradients of chemoattractant monocyte protein-1, was accomplished by obtaining phagocytosed RJP-mediated retroreflection signals. Considering that conventional assays are designed as endpoint measurements, we anticipate the proposed retroreflection-based cell quantification technique to be a promising solution, bypassing current limitations.

In traditional colorimetric lateral flow immunoassay (LFI) using gold nanoparticles (AuNPs) as a probe, additional optical transducers are required to quantify the signal intensity of the test line because it presents as a single red-colored line. In order to eliminate external equipment, the LFI signal should be quantifiable by the naked eye without the involvement of optical instruments. Given this objective, the single line test zone of conventional LFI was converted to several spots that formed herringbone patterns. When the sandwich immunoassay was performed on a newly developed semi-quantitative (SQ)-LFI system using AuNPs as an optical probe, the spots were colorized and the number of colored spots increased proportionally with the analyte concentration. By counting the number of colored spots, the analyte concentration can be easily estimated with the naked eye. To demonstrate the applicability of the SQ-LFI system in practical immunoanalysis, microalbumin, which is a diagnostic marker for renal failure, was analyzed using microalbumin-spiked artificial urine samples. Using the SQ-LFI system, the calibration results for artificial urine-based microalbumin were studied, ranging from 0 to 500 &mu, g/mL, covering the required clinical detection range, and the limit of detection (LOD) value was calculated to be 15.5 &mu, g/mL. Thus, the SQ-LFI system provides an avenue for the realization of an efficient quantification diagnostic device in resource-limited conditions.

Herein, we report a novel lateral flow immunoassay (LFIA) system for detecting cardiac troponin I (cTnI) in serum using the time-resolved fluorescence resonance energy transfer (TR-FRET) technique and the fusion 5 membrane. The fusion 5 membrane is used as a strip for LFIA, and it is constructed without additional matrices (such as a sample or conjugation pad). Although this strategy for constructing the LFIA strip is quite simple and cost-effective, LFIA is still not suitable for the analysis of biomarkers that require high sensitivity, such as cTnI. Therefore, the highly sensitive TR-FRET technique is integrated with a fusion 5 membrane-based LFIA strip. To accomplish this, a microparticle covered with europium chelate-contained silica nanoparticles is synthesized as a raspberry-type particle and used as a fluorescence donor. A gold nanorod (GNR) is used as a fluorescence acceptor particle. In the TR-FRET-based LFIA system, the competitive immunoassay should be performed to satisfy the condition required for the FRET phenomenon to occur. Therefore, the fluorescence signal is proportional to the cTnI concentration, ensuring a quantitative analysis of cTnI can be accomplished by measuring the fluorescence signal between the raspberry-type europium particles and GNR. Using the developed TR-FRET-based LFIA system, sensitive detection of cTnI is successfully achieved with a limit of detection of 97 pg/mL in human serum. Moreover, because the result can be obtained using one matrix (the fusion 5 membrane), the developed LFIA system can be employed in cTnI diagnosis with a simple manufacturing process.

We have recently witnessed substantial advances in the microfluidic lab chip technology and its development toward stand-alone diagnostic devices. These efforts have been translated into commercialization-ready prototypes by incorporating smartphones and wearable devices as signal transducing and data processing equipment. Such integrated systems provide numerous advantages, including low-cost manufacturing, facile fabrication, and intuitive data processing. All the biosensing procedures, including signal generation, transducing, data processing, and displaying results, can be efficiently operated in the stand-alone device. Recent accomplishments in these efforts are reviewed and summarized.

Herein, we introduce a smartphone-integrated immunosensor based on non-spectroscopic optical detection. Sedimentation of the retroreflector and gentle inversion of the microfluidic chip was chosen as biosensing principles to ensure minimal human involvement. To realize this, wash-free immunosensing was implemented on a polymeric microfluidic chip device fabricated for light signal penetration in retroreflection signal acquisition. Applying a transparent chip and passive modulation of retroreflectors enabled the minimization of human error during sensing. In addition, a retroreflection-detectable optical gadget was constructed for integration with the commercial smartphone. The gadget had an optical chamber that induced retroreflection by integration with a smartphone. When the micro-sized reflector, named the retroreflective Janus microparticle, reacted on the sensing surface, the incident light was retroreflected towards the image sensor and quantified by a smartphone-installed Android application package. The developed application package features include time-lapse image capture performed by manipulating LED flash and camera modules, and quantification of retroreflected signal counts by image processing of time-lapse images. With this platform, the user could independently commence optical signal processing without a complicated optical setup and running software on a PC, and sensitive and reproducible immunosensing results could be obtained. The applicability test for creatine kinase-myocardial band detection from the buffer to serum was conducted and presented a calibration curve of 0–1000 ng/mL within 1 h. With the developed system, we believe that the applicability of the platform in bioanalytical detection can be expanded.

Loop-mediated isothermal amplification (LAMP) is a powerful gene amplification method, which has many advantages, including high specificity, sensitivity, and simple operation. However, quantitative analysis of the amplified target gene with the LAMP assay is very difficult. To overcome this limitation, we developed a novel biosensing platform for molecular diagnosis by integrating the LAMP method and retroreflective Janus particle (RJP) together. The final amplified products of the LAMP assay are dumbbell-shaped DNA structures, containing a single-stranded loop with two different sequences. Therefore, the concentration of the amplified products can be measured in a manner similar to the sandwich-type immunoassay. To carry out the sandwich-type molecular diagnostics using the LAMP product, two DNA probes, with complementary sequences to the loop-regions, were prepared and immobilized on both the sensing surface and the surface of the RJPs. When the amplified LAMP product was applied to the sensing surface, the surface-immobilized DNA probe hybridized to the loop-region of the LAMP product to form a double-stranded structure. When the DNA probe-conjugated RJPs were injected, the RJPs bound to the unreacted loop-region of the LAMP product. The number of RJPs bound to the loop-region of the LAMP product was proportional to the concentration of the amplified LAMP product, indicating that the concentration of the target gene can be quantitatively analyzed by counting the number of observed RJPs. Using the developed system, a highly sensitive and selective quantification of Salmonella was successfully performed with a detection limit of 102 CFU.

Herein, we report an optical sensing platform for mercury ions (Hg2+) in water based on the integration of Hg2+-mediated thymine-thymine (T-T) stabilization, a biotinylated stem-loop DNA probe, and a streptavidin-modified retroreflective Janus particle (SA-RJP). Two oligonucleotide probes, including a stem-loop DNA probe and an assistant DNA probe, were utilized. In the absence of Hg2+, the assistant DNA probe does not hybridize with the stem-loop probe due to their T-T mismatch, so the surface-immobilized stem-loop DNA probe remains a closed hairpin structure. In the presence of Hg2+, the DNA forms a double-stranded structure with the loop region via Hg2+-mediated T-T stabilization. This DNA hybridization induces stretching of the stem-loop DNA probe, exposing biotin. To translate these Hg2+-mediated structural changes in DNA probe into measurable signal, SA-RJP, an optical signaling label, is applied to recognize the exposed biotin. The number of biospecifically bound SA-RJPs is proportional to the concentration of Hg2+, so that the concentration of Hg2+ can be quantitatively analyzed by counting the number of RJPs. Using the system, a highly selective and sensitive measurement of Hg2+ was accomplished with a limit of detection of 0.027 nM. Considering the simplified optical instrumentation required for retroreflection-based RJP counting, RJP-assisted Hg2+ measurement can be accomplished in a much easier and inexpensive manner. Moreover, the detection of Hg2+ in real drinking water samples including tap and commercial bottled water was successfully carried out.

According to recent increases in public healthcare costs associated with diabetes mellitus, the development of new glycemic monitoring techniques based on the biosensing of glycated hemoglobin A1c (HbA 1c ), a promising long-term glycemic biomarker, has become a major challenge. In the development of HbA 1c biosensors for point-of-care applications, the selection of an effective biorecognition layer that provides a high reaction yield and specificity toward HbA 1c is regarded as the most significant issue. To address this, we developed a novel HbA 1c biosensing interfacial material by the integration of boronate hydrogel with glass fiber membrane. In the present study, a new boronate-functionalized hydrogel was designed and spatio-selectively photopolymerized on a hydrophilic glass fiber membrane by using N-hydroxyethyl acrylamide, 3-(acrylamido)phenylboronic acid, and bis( N , N ′-methylene-bis-acrylamide). Using this approach, the boronic acid group, which specifically recognizes the cis-diol residue of glucose on the HbA 1c molecule, can be three-dimensionally coated on the surface of the glass fiber network with a high density. Because this network structure of boronate hydrogel-grafted fibers enables capillary-driven fluid control, facile HbA 1c biosensing in a lateral flow assay concept could be accomplished. On the proposed HbA 1c biosensing interface, various concentrations of HbA 1c (5–15%) in blood-originated samples were sensitively measured by a colorimetric assay using horseradish peroxidase, a glycoenzyme can generate chromogenic signal after the competitive binding against HbA 1c to the boronic acid residues. Based on the demonstrated advantages of boronate hydrogel-modified membrane including high analytical performance, easy operation, and cost-effectiveness, we expect that the proposed biorecognition interfacial material can be applied not only to point-of-care HbA 1c biosensors, but also to the quantitative analysis of other glycoprotein biomarkers.

We present a hand-held optical biosensing system utilizing a smartphone-embedded illumination sensor that is integrated with immunoblotting assay method. The smartphone-embedded illumination sensor is regarded as an alternative optical receiver that can replaces the conventional optical analysis apparatus because the illumination sensor can respond to the ambient light in a wide range of wavelengths, including visible and infrared. To demonstrate the biosensing applicability of our system employing the enzyme-mediated immunoblotting and accompanying light interference, various types of ambient light conditions including outdoor sunlight and indoor fluorescent were tested. For the immunoblotting assay, the biosensing channel generating insoluble precipitates as an end product of the enzymatic reaction is fabricated and mounted on the illumination sensor of the smartphone. The intensity of penetrating light arrives on the illumination sensor is inversely proportional to the amount of precipitates produced in the channel, and these changes are immediately analyzed and quantified via smartphone software. In this study, urinary C-terminal telopeptide fragment of type II collagen (uCTX-II), a biomarker of osteoarthritis diagnosis, was tested as a model analyte. The developed smartphone-based sensing system efficiently measured uCTX-II in the 0–5 ng/mL concentration range with a high sensitivity and accuracy under various light conditions. These assay results show that the illumination sensor-based optical biosensor is suitable for point-of-care testing (POCT).

Here, we proposed a retroreflective optical immunoassay platform by introducing the intrinsic sedimentation characteristics of a micro-retroreflector, namely retroreflective Janus particles (RJPs), wherein the sediment-based passive movement of RJPs minimised the random errors due to human involvement and resulted in a simple procedure that does not require the washing step, to follow the concept of point-of-care testing. The transparent sensing interface and the sedimentation property of RJPs were combined to develop a practical retroreflective immunoassay platform. For the sensing surface, transparent silanized poly(methyl methacrylate) was applied to the inverted focusing method. In the retroreflection phenomenon, as the incident light returns to its source by the retroreflector, efficient design of the retroreflective optical path between the light source and retroreflector can be crucial in signal registration. While preparing the RJP-bound transparent substrate on the microfluidic channel, the signal could be achieved more efficiently by directly focusing on the sensing interface, and not via the fluidic channels. To integrate this to build an immunoassay protocol, the sedimentation property of RJPs was employed for microfluidic chip inversion-based particle movement control, which was utilised for both luring and separating RJPs on the sensing surface, resulting in a wash-free immunoassay without any human involvement. To ensure accurate analysis, a time-lapse imaging-based image processing was conducted to eliminate the non-specific signals. To validate the applicability of the proposed immunoassay platform, quantification of acute cardiac infarction marker creatine kinase-MB was performed.

The integration of smart IT devices and biochemical assays with optical biosensing technology facilitates the development of efficacious optical biosensors for many practical diagnostic fields, owing to their minimized use of high-technical electronic components and simple operation. Herein, we introduced a simple optical biosensing system based on the specific wavelength filtering principle and count-based analysis method. The developed system uses a smartphone with a paper-based signal guide and a biosensing channel. The paper-based signal guide was prepared by printing red patterns of various brightness on a black background. Given that a blue product is generated as a result of horseradish peroxidase (HRP)-based enzymatic reaction in the biosensing channel, the channel could be used as a blue filter that absorbs red light. When red light reflected from the red pattern is absorbed by the channel, the pattern appears black. As such, the color of the patterns is assimilated with the black background, so it seems to disappear. Consequently, the amount of blue product relative to the concentration of the target analyte can be measured by counting the number of observed patterns on the paper-based signal guide. In this study, the concentration of urinary C-telopeptide fragment of type II collagen (uCTX-II, 0–10 ng/mL) was measured using the developed system without complicated equipment. In addition, the quantitative analysis of uCTX-II in the real urine sample was successfully performed. Therefore, we expect that the developed optical transducing system could be practically used for point-of-care testing (POCT) diagnosis under resource-limited environmental conditions.

We developed a simple and low-cost cell culture monitoring system utilizing a paper-based analytical device (PAD) and a smartphone. The PAD simultaneously analyses glucose and lactate concentrations in the cell culture medium. Focusing on the fact that animal cells consume glucose and produce lactate under anaerobic conditions, oxidase- and horseradish peroxidase (HRP) enzyme-mediated colorimetric assays were integrated into the PAD. The PAD was designed to have three laminated layers. By using a double-sided adhesive tape as the middle layer and wax coating, a bifurcated fluidic channel was prepared to manipulate sample flow. At the inlet and the outlets of the channel, a sample drop zone and two detection zones for glucose and lactate, respectively, were positioned. When sample solution is loaded onto the drop zone, it flows to the detection zone through the hydrophilic fluidic channel via capillary force. Upon reaching the detection zone, the sample reacts with glucose and lactate oxidases (GOx and LOx) and HRP, immobilized on the detection zone along with colorless chromophores. By the Trinder’s reaction, the colorless chromophore is converted to a blue-colored product, generating concentration-dependent signal. With a gadget designed to aid the image acquisition, the PAD was positioned to the smartphone-embedded camera. Images of the detection zones were acquired using a mobile application and the color intensities were quantified as sensor signals. For the glucose assay using GOx/HRP format, we obtained the limit of detection (LOD ∼0.3 mM) and the limit of quantification (LOQ ∼0.9 mM) values in the dynamic detection range from 0.3 to 8.0 mM of glucose. For lactate assay using LOx/HRP, the LOD (0.02 mM) and the LOQ (0.06 mM) values were registered in the dynamic detection range from 0.02 to 0.50 mM of lactate. With the device, simultaneous analyses of glucose and lactate in cell culture media were conducted, exhibiting highly accurate and reproducible results. Based on the results, we propose that the optical sensing system developed is feasible for practical monitoring of animal cell culture.

Matrix metalloproteinase-2 (MMP-2) is closely related to the proliferation and invasion of various types of cancers. The protease is secreted by malignant tumor cells, thus allowing the enzyme to serve as a biomarker for cancer diagnosis. Methods have been developed to analyze MMP-2 activities; however, their applications to disease diagnosis have not been widely demonstrated yet because of the need for highend analytical equipment and labor-intensive processes. In this study, we developed an MMP-2 activity assay system by integrating an engineered autoinhibited β-lactamase which can be activated by MMP-2 in an optical sensing system consisting of reassembled common electronic components, such as a laser diode, a solar cell, and a multimeter. The autoinhibited β-lactamase was immobilized on a polymeric biosensing channel by a polydopamine coating and self-assembled monolayer methods. In the presence of MMP-2, the immobilized autoinhibited β-lactamase was converted to an active form that hydrolyzed the chromogenic cephalosporin CENTA, thereby changing the substrate color from pale yellow (λmax=340 nm) to highly discernible chrome yellow (λmax=405 nm). By reading the interfered laser-light intensity, we were able to analyze MMP-2 activities precisely both with the samples prepared in a buffer solution and also those in urine. These results suggested that the developed system can be used for the quantitative analysis of enzyme activity related to cancer diagnosis.

We developed a cost-effective and convenient optical transducing platform for use in gold nanoparticle-based immunosensing applications. Two types of gold nanoparticles were prepared and used as optical signaling probes: spherical gold nanoparticles (GNPs) exhibiting maximal absorbance at 519 nm and gold nanorods (GNRs) exhibiting maximal absorbance at 620 nm. Common components used in electronics, including two types of laser diodes (λmax = 532- and 635-nm), solar cells, and a multimeter, were reassembled to construct the optical transducing platform. Under the condition in which the emission spectra of lasers and the absorption spectra of gold nanoparticles are matched, laser intensity is decreased. In the designed transducing system, these changes in light intensity are directly indicated as alterations in the DC voltage signal. Based on this principle, we demonstrated the immunosensing applicability of the optical transducing platform. As model immunoreaction pairs, we used anti-mouse IgG/mouse IgG and anti-human IgG/human IgG; superparamagnetic microparticles (MPs) were conjugated with capture antibodies (anti-mouse IgG and anti-human IgG) and the GNPs and GNRs were conjugated with the target mouse IgG and human IgG (model antigens). When the capture antibody-modified MPs and the optical probes mixed in the platform's reaction channel, the optical probes bound to the MPs through an affinity-based reaction. Under a magnetic field, the biospecifically conjugated MP/GNP and MP/GNR complexes could be localized in the reaction channel. Consequently, the amounts of the GNPs and GNRs remaining in the solution were inversely proportional to the rate of the MP/GNP and MP/GNR reactions, and could be registered as DC voltage signals. Based on these results, we expect the novel transducing system and immunosensing strategy presented herein to be applied in practical optical immunosensing performed using key disease markers and their antibodies.

Recently, much effort has been focused on developing three-dimensional, paper-based microfluidic analytical devices (3D-μPADs) targeting in vitro diagnostics. However, 3D-μPAD fabrication typically requires tedious assembly that hinders mass production. Here, we report on a fabrication method for 3D-μPADs made of plastics without the need for additional assembly. Both sides of the paper were printed via liquid resin photopolymerization using a digital light processing (DLP) printer. The sample reservoir and detection zones are located on the top of the 3D-μPADs, and three microchannels are located on the bottom. The detection limits for glucose, cholesterol, and triglyceride (TG) in phosphate-buffered saline (PBS) were 0.3 mM, 0.2 mM, and 0.3 mM, respectively. The detectable ranges of glucose, cholesterol, and TG in human serum were 5–11 mM, 2.6–6.7 mM, and 1–2.3 mM. These results suggest that our fabrication method is suitable to mass produce 3D-μPADs with relative ease using simple fabrication processes.

To overcome the time and space constraints in disease diagnosis via the biosensing approach, we developed a new signal-transducing strategy that can be applied to colorimetric optical biosensors. Our study is focused on implementation of a signal transduction technology that can directly translate the color intensity signals—that require complicated optical equipment for the analysis—into signals that can be easily counted with the naked eye. Based on the selective light absorption and wavelength-filtering principles, our new optical signaling transducer was built from a common computer monitor and a smartphone. In this signal transducer, the liquid crystal display (LCD) panel of the computer monitor served as a light source and a signal guide generator. In addition, the smartphone was used as an optical receiver and signal display. As a biorecognition layer, a transparent and soft material-based biosensing channel was employed generating blue output via a target-specific bienzymatic chromogenic reaction. Using graphics editor software, we displayed the optical signal guide patterns containing multiple polygons (a triangle, circle, pentagon, heptagon, and 3/4 circle, each associated with a specified color ratio) on the LCD monitor panel. During observation of signal guide patterns displayed on the LCD monitor panel using a smartphone camera via the target analyte-loaded biosensing channel as a color-filtering layer, the number of observed polygons changed according to the concentration of the target analyte via the spectral correlation between absorbance changes in a solution of the biosensing channel and color emission properties of each type of polygon. By simple counting of the changes in the number of polygons registered by the smartphone camera, we could efficiently measure the concentration of a target analyte in a sample without complicated and expensive optical instruments. In a demonstration test on glucose as a model analyte, we could easily measure the concentration of glucose in the range from 0 to 10 mM.

This study reports a new strategy for simultaneous detection of the C-telopeptide fragments of type II collagen (CTX-II) as a biomarker of osteoarthritis (OA) using a fluoro-microbeads guiding chip. As osteoarthritis progresses, the joint components including matrix and cartilage are degraded by proteases. The degraded products such as CTX-II are released into the serum and urine, and the CTX-II concentration in body fluids reflects OA progression. Because the CTX-II has heterogeneous epitope structure in serum (sCTX-II; homodimers) and urine (uCTX-II; monomers or variant monomers), a multiple-sensing device enabling both sandwich and competitive-type immunoassays is required. For multiple assessments of serum and urinary CTX-II, we designed a fluoro-microbeads guiding chip (FMGC) containing multiple sensing areas and connecting channels. Using the approach, the sandwich (sCTX-II) and competition (uCTX-II) assays could be simultaneously performed on a single chip. We designed a fluidic control device enabling selective control of the open–close function of FMGC channels. The immune-specific signal was quantitatively analyzed by counting the number of fluorescent microbeads from the registered images. The results from the developed FMGC assay showed high correlation with those obtained in ELISA. The completion time of the FMGC assay was 24-fold and 3.5-fold shorter than the ELISA for urinary and serum CTX-II. Taken together, it enabled the simultaneous detection of both sCTX-II and uCTX-II. This FMGC-based assay would be a promising tool for monitoring of osteoarthritis.

We report a new smartphone-based immunosensing system that integrates an immunoblotting assay and a built-in illumination sensor to assay an osteoarthritis marker. The simple optical biosensing system developed in this study effectively uses smartphone-embedded components such as a white light-emitting diode and an illumination sensor as the light source and optical receiver, respectively. In contrast to conventional optical sensors, which utilize a specific spectrum and focused wavelength, the illumination sensor sensitively responds to the variations in external light intensity over a wide range of wavelengths. This functionality of the illumination sensor in the smartphone was employed as a signal transducer in the optical system. The immunoblotting technique, which uniformly changes the intensity of light because of the precipitation reaction, was introduced into the developed optical system. The horseradish peroxidase-induced insoluble precipitate interferes with the penetration of incident light, thereby facilitating the variation of applied light intensity. Subsequently, the quantity of light passing through the biosensing channel was immediately analysed using a lux meter in the mobile application. Herein, the urinary C-terminal telopeptide fragment of type II collagen (uCTX-II) was selected as an osteoarthritis biomarker and analysed to demonstrate the feasibility of the developed illumination sensor. The results indicate an obvious change in the lux value in accordance with the uCTX-II concentration ranging from 0 to 10 ng mL−1. The results were highly reproducible and sensitive to the variations in the concentration of the analyte. This suggests the potential use of the developed illumination sensor as a promising tool for the quantitative diagnosis of target analytes and point-of-care testing.

Herein, we report an optical sensing platform for mercury ions (Hg

The nanoporous free-standing graphene membrane is of great interest in high performance separation technology. In particular, the separation of biological molecules with similar sizes is one of the key challenges in the purification of biomaterials. Here, we report a reliable, cost-effective, and facile method for the fabrication of a graphene-based nanosieve and its application in the separation of similar-size proteins. A suspended reduced graphene oxide (rGO) nanosieve with ultra-thin, large-area, well-ordered, and dense 15 nm-sized pores was fabricated using block copolymer (BCP) lithography. The fabricated 5 nm-ultrathin nanosieve with an area of 200 μm × 200 μm (an ultra-high aspect ratio of ∼40 000) endured pressure up to 1 atm, and effectively separated hemoglobin (Hb) from a mixture of hemoglobin and immunoglobulin G (IgG), the common proteins in human blood, in a highly selective and rapid manner. The use of the suspended rGO nanosieve is expected to provide a simple and manufacturable platform for practical biomolecule separation offering high selectivity and a large throughput.

In this study, we developed a novel time-resolved fluorescence (TRF)-based immunosensing platform that consisted with the physically stable europium chelate-encapsulated silica nanoparticle and the simplified TRF analyzer. To verify the applicability of developed TRF immunosensing platform toward practical immunoassay, the sandwich immunosensing of cardiac troponin I (cTnI) was accomplished using the synthesized europium-encapsulated silica nanoparticles as a signaling probe and homemade TRF analysis system as an optical transducer.

According to recent increases in public healthcare costs associated with diabetes mellitus, the development of new glycemic monitoring techniques based on the biosensing of glycated hemoglobin A1c (HbA

A simple paper-based optical biosensor for glucose monitoring was developed. As a glucose biosensing principle, a colorimetric glucose assay, using glucose oxidase (GOx) and horseradish peroxidase (HRP), was chosen. The enzymatic glucose assay was implanted on the analytical paper-based device, which is fabricated by the wax printing method. The fabricated device consists of two paper layers. The top layer has a sample loading zone and a detection zone, which are modified with enzymes and chromogens. The bottom layer contains a fluidic channel to convey the solution from the loading zone to the detection zone. Double-sided adhesive tape is used to attach these two layers. In this system, when a glucose solution is dropped onto the loading zone, the solution is transferred to the detection zone, which is modified with GOx, HRP, and chromogenic compounds through the connected fluidic channel. In the presence of GOx-generated H2O2, HRP converts chromogenic compounds into the final product exhibiting a blue color, inducing color change in the detection zone. To confirm the changes in signal intensity in the detection zone, the resulting image was registered by a digital camera from a smartphone. To minimize signal interference from external light, the experiment was performed in a specifically designed light-tight box, which was suited to the smartphone. By using the developed biosensing system, various concentrations of glucose samples (0–20 mM) and human serum (5–17 mM) were precisely analyzed within a few minutes. With the developed system, we could expand the applicability of a smartphone to bioanalytical health care.

Lysostaphin is a Staphylococcus aureusspecific bacteriolytic enzyme that recognizes and cleaves the pentaglycine linkage of peptidoglycan layers in the cell wall of S. aureus. Its target-specific lytic property to the staphylococcal cell wall has attracted interest for the application of lysostaphin as natural antibiotics against S. aureus. However, studies that consider its applicability in the detection of S. aureus have not been reported yet. Based on this, lysostaphin-mediated peptidoglycan fragmentation principles were implemented in the label-free immunoassay of S. aureus. In this study, peptidoglycan layers of S. aureus were fragmented by lysostaphin and the obtained fragments were quantitatively analyzed by electrochemical impedance spectroscopy (EIS). A linear relationship between the logarithmic value of the S. aureus concentration and the increase in the EIS signal was acquired in a range between 102 and 107 colony-forming units (CFU)/mL. Compared to the whole cell-based analysis, the developed lysostaphin-mediated assay for S. aureus showed a 90% increase in sensitivity because the fragments can interact with the immunoanalytical surface more efficiently than the whole cell. In contrast to previous EIS-based bacterial immunoassays that utilize complex electrodes and/or additional signaling probes such as labeled antibodies for signal amplification, we could detect signals with high sensitivity on conventional electrodes without any signaling probes. In addition, by using the developed method, S. aureus was selectively determined in bacterial samples that also contained 106 CFU/mL of Escherichia coli and Corynebacterium glutamicum.

A fluorescence-based biosensor for measuring the quantity of C-telopeptide fragments of type II collagen (CTX-II) was developed as an osteoarthritis biomarker in urine. During osteoarthritis progression, joint components such as cartilage collagen are degraded by collagenase-protease and secreted into the serum as CTX-II. To detect CTX-II in urine (uCTX-II) having structural heterogeneity, conventional sandwich format assay does not provide sufficient accuracy. As an alternative, a competitive immunoassay was developed for the quantification of uCTX-II, which uses antibody-conjugated fluoro-microbeads to generate an optical signal. After preparing the biosensing surface, the uCTX-II sample and antibody-optical probe conjugates were reacted on the PEG4-EKGPDP exposed biosensing surface. The optical probes competed with the uCTX-II in the sample for binding to the immobilized PEG4-EKGPDP. This assay was able to detect uCTX-II concentrations between 200 ng/mmol (corrected value vs. creatinine) and 1,400 ng/mmol, encompassing the clinical detection ranges required for osteoarthritis diagnosis. The competitive immunoassay developed for uCTX-II detection was rapid and exhibited good correlation with conventional ELISA methods. This novel competition assay is a promising tool for the diagnosis and monitoring of osteoarthritis using urine samples.

Accurate detection of tumor-associated biomarkers remains a challenge in the diagnosis of cancer. To address the issue, we developed a sensitive biosensing system using lectins for breast cancer diagnosis based on the analysis of tumor-associated O-linked glycoprotein such as cancer antigen 15-3 (CA15-3). In cancerous cells, aberrant short-terminated glycan moieties such as TF-antigen and sialic acid are expressed on cell surfaces, becoming cancer antigens. Based on the difference between the glycan moieties in normal and cancer cells, CA15-3 was biospecifically detected using Sambucus nigra agglutinin (SNA) and peanut agglutinin (PNA) lectins. These lectins were conjugated with fluoro-microbeads and used as a detection molecule for an antibody–lectin sandwich assay. A fluoro-microbead guiding chip (FMGC) containing multiple sensing and fluidic channels was designed to measure CA15-3 proteins using the lectin-based assay. A capture antibody against CA15-3 that binds to the peptide backbone was immobilized on the gold-patterned sensing surface. On the modified FMGC, antibody–lectin sandwich assay for target CA15-3 was conducted. The developed PNA- and SNA-based assays, which exhibited a detection limit of 1.2 and 0.4 U/mL, worked well over the clinically important range from 1.25 to 25 U/mL CA15-3. The developed biosensor showed good sensitivity, fast response and reproducibility with a small volume of sample. This lectin-based sandwich assay provides a promising tool for the quantitative diagnosis of CA15-3 and glycated markers in clinical applications.

We developed retroreflective Janus microparticles (RJPs) as a novel optical immunosensing probe for use in a nonspectroscopic retroreflection-based immunoassay. By coating the metals on the hemispherical surface of silica particles, highly reflective RJPs were fabricated. On the basis of the retroreflection principle, the RJPs responded to polychromatic white light sources, in contrast to conventional optical probes, which require specific monochromatic light. The retroreflection signals from RJPs were distinctively recognized as shining dots, which can be intuitively counted using a digital camera setup. Using the developed retroreflective immunosensing system, cardiac troponin I, a specific biomarker of acute myocardial infarction, was detected with high sensitivity. On the basis of the demonstrated features of the retroreflective immunosensing platform, we expect that our approach may be applied for various point-of-care-testing applications.

This study reports a simple and quantitative analytical method for detecting cartilage oligomeric matrix protein (COMP) using a lateral-flow immunoassay (LFI) format. An immuno-chromatographic assay was developed using a gold nanoparticle (AuNP)-conjugated monoclonal antibody (Ab) probe for the quantitative detection of COMP in human synovial fluid (SF). The detection antibody was conjugated with the AuNP (∼30 nm) to enable detection, and the capture antibody was immobilized within a multi-spot pattern in the test zone of a membrane strip. COMP in the human SF binds to the AuNP-conjugated detection antibody, and the COMP-Ab complex then binds to the capture antibody. The immunoassay test can be analyzed by visualizing the test strip with a commercial scanner and an imaging software program. The color density of the multi-spot pattern at the test zone increased in proportion to the concentration of COMP. The developed immunoassay exhibited a dynamic detection range from 0.6 to 20 μg/mL of COMP. The detection limit of the proposed LFI system was approximately 0.2 μg/mL. The test results showed good correlation with those obtained using a conventional enzyme-linked immunosorbent assay (ELISA) to detect COMP. We expect that the proposed immunoassay method, based on the quantification of COMP, is suitable for osteoarthritis diagnosis using SF samples.

The novelty of this study resides in the fabrication of stoichiometric and stress-reduced Si3N4/SiO2/Si3N4 triplelayer membrane sieves. The membrane sieves were designed to be very flat and thin, mechanically stressreduced, and stable in their electrical and chemical properties. All insulating materials are deposited stoichiometrically by a low-pressure chemical vapor deposition system. The membranes with a thickness of 0.4 µm have pores with a diameter of about 1 µm. The device is fabricated on a 6” silicon wafer with the semiconductor processes. We utilized the membrane sieves for plasma separations from human whole blood. To enhance the separation ability of blood plasma, an agarose gel matrix was attached to the membrane sieves. We could separate about 1 µL of blood plasma from 5 µL of human whole blood. Our device can be used in the cell-based biosensors or analysis systems in analytical chemistry.

We have developed a method to detect cartilage oligomeric matrix protein (COMP) as a specific biomarker of osteoarthritis (OA). In pathological conditions of the cartilage, COMP is released first into the synovial fluid (SF) and from there into the blood. Thus, measurement of COMP in the blood and SF facilitates OA diagnosis. To determine COMP, we developed a fluoro-microbead guiding chip (FMGC)-based immunoassay. The FMGC has four immunoreactive regions, each with five patterns, to allow multiple assays. A COMP-specific capture antibody was immobilized to the FMGC surface to create a self-assembled interfacial layer. SF or serum samples from patients with OA possessing the target COMP were applied to the COMP-sensing monolayer. To generate binding signal, COMP detection antibody-conjugated fluoro-microbeads were applied and the numbers of fluoro-microbeads bound specifically were counted to determine COMP concentrations. This FMGC-based immunoassay clearly distinguished immunospecific from nonspecific binding by comparing optical signals from inside and outside of the patterns. The optical signals showed linear correlations with serum and SF COMP concentrations. Optical detection and quantification of COMP using fluorescence microscopy correlated well with results from commercial enzyme-linked immunosorbent assay (ELISA). This FMGC-based immunoassay offers a new approach for detecting a clinically relevant biomarker for OA in human blood and SF.

We have designed, fabricated, and characterized a low-stressed silicon nitride (SiN) membrane nanosieve (100 microm x 100 microm) using an anisotropic reactive ion etching (ARIE) combining with gas mixture, thus maintaining compatibility with the complementary metal-oxide semiconductor integrated circuit (CMOS IC) processes. The holes pattern of this nanosieve membrane was precisely controlled under 30 nm diameter by the electron beam writing. By employing mainly anisotropic reactive ion etching plus diffusion to the depth direction, the etch holes size was controlled to be the same with patterns on the e-beam resist (ER). This nanosieve membrane has proper mechanical strength withstanding up to one bar of transmembrane pressure. And it can endure harsh treatments such as high temperature up to 800 degrees C. In addition, it is inert to a number of strong chemicals including the piranha (H2SO4 + H2O2) solution, highly-concentrated potassium hydroxide (KOH), hydrogen fluoride (HF), hydrogen chloride (HCI), and nitric acid (HNO3).

We have developed a fluoro-microbead guiding chip (FMGC) to perform an optical immunoassay of cardiac troponin I (cTnI). The plasma marker protein cTnI is the currently preferred marker to use for a definitive diagnosis and prognosis of myocardial infarction. The FMGC has four immunoreaction regions on a silicon oxide substrate, with five gold patterns imprinted on each region for multiple simultaneous assays. The FMGC assay clearly distinguished immunospecific binding from nonspecific binding by comparing optical signals from inside and outside of the patterns. To detect cTnI, a sandwich immunoassay was performed using antibody-tagged fluoro-microbeads. The cTnI-specific capture antibody was conjugated to the FMGC surface by reaction with 3-3'-dithiobis-propionic acid N-hydroxysuccinimide ester to create a self-assembling antigen-sensing monolayer (DTSP SAM) on the chip. A sample containing cTnI was applied to the antigen-sensing monolayer and allowed to react. To generate a binding signal, a cTnI detection antibody-linked fluoro-microbead preparation was added. The cTnI concentration in a sample was determined by counting the number of biospecifically bound fluoro-microbeads on the corresponding five patterns on the FMGC. The optical signal showed a linear correlation with cTnI concentrations in plasma samples containing from 3.4 pM to 3.4 nM (0.1-100 ng/ml) cTnI. The sensitivity of cTnI detection could be increased by reducing the non-specific binding of the beads to the antigen-sensing surfaces of the chip. Optical detection and quantification of binding by fluorescence microscopy gave results that correlated well with results from a commercial ELISA for cTnI in human plasma. Based on these findings, we propose that the FMGC-based immunoassay system may be adapted to detect and quantify a variety of clinically important targets in human samples.

The aggregation of α-synuclein is clearly related to the pathogenesis of Parkinson's disease. Therefore, detailed understanding of the mechanism of fibril formation is highly valuable for the development of clinical treatment and also of the diagnostic tools. Here, we have investigated the interaction of α-synuclein with ionic liquids by using several biochemical techniques including Thioflavin T assays and transmission electron microscopy (TEM). Our data shows a rapid formation of α-synuclein amyloid fibrils was stimulated by 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [BIMbF(3)Im], and these fibrils could be disaggregated by polyphenols such as epigallocatechin gallate (EGCG) and baicalein. Furthermore, the effect of [BIMbF(3)Im] on the α-synuclein tandem repeat (α-TR) in the aggregation process was studied.

The method to detect the organophosphorus (OP) pesticide was developed using an acetylcholine esterase (AChE) and choline oxidase (ChOx) bienzyme system. AChE, which is inhibited by the OP pesticide, was enzymatically linked with ChOx in solution as well as on electrodes. By employing acetylcholine as the substrate for AChE, we supplied the enzyme product choline to ChOx in order to generate electrocatalyzed current. For electrochemical signaling with AChE/ChOx, ferrocene (Fc)-mediated bioelectrocatalysis through a flavin adenine dinucleotide (FAD) cofactor of ChOx was verified experimentally. An immobilization method for ChOx on a gold electrode was designed to generate amplified and controlled signal by using poly-L-lysine charge interaction and glutaraldehyde cross-linking. Using diazinon-oxon (DZN) as a detection target among the OP pesticides, we successfully conducted an electrochemical detection on the basis of AChE inhibition mediated by the ChOx bioelectrocatalysis.