Retroreflection-based sandwich type affinity sensing of isothermal gene amplification products for foodborne pathogen detection.

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 invA gene of Salmonella was selected as the target. It was possible to quantitatively analyze the Salmonella concentration from 0 to 106 colony-forming units, sufficiently covering the required detection range. In addition, quantitative analyses of pathogens in contaminated food sources, including milk and chicken meat, were successfully conducted with a limit of detection of 10 CFU.

Rapid Dot-Blot Immunoassay for Detecting Multiple Salmonella enterica Serotypes.

Salmonella, a major contributor to foodborne infections, typically causes self-limiting gastroenteritis. However, it is frequently invasive and disseminates across the intestinal epithelium, leading to deadly bacteremia. Although the genus is subdivided into >2,600 serotypes based on their antigenic determinants, only few serotypes are responsible for most human infections. In this study, a rapid dot-blot immunoassay was developed to diagnose multiple Salmonella enterica serotypes with high incidence rates in humans. The feasibility of 10 commercial antibodies (four polyclonal and six monoclonal antibodies) was tested using the 18 serotypes associated with 67.5% Salmonella infection cases in the United States of America (U.S.A) in 2016. Ab 3 (polyclonal; eight of 18 serotypes), Ab 8 (monoclonal; 13 of 18 serotypes), and Ab 9 (monoclonal; 10 of 18 serotypes) antibodies exhibited high detection rates in western blotting and combinations of two antibodies (Ab 3+8, Ab 3+9, and Ab 8+9) were applied to dot-blot assays. The combination of Ab 3+8 identified 15 of the tested 18 serotypes in 3 h, i.e., S. Enteritidis, S. Typhimurium, S. Javiana, S. I 4,[5],12:i:-, S. Infantis, S. Montevideo, S. Braenderup, S. Thompson, S. Saintpaul, S. Heidelberg, S. Oranienburg, S. Bareilly, S. Berta, S. Agona, and S. Anatum, which were responsible for 53.7% Salmonella infections in the U.S. in 2016. This cost-effective and rapid method can be utilized as an on-site colorimetric method for Salmonella detection.

Amplifying endogenous stem cell migration for in situ bone tissue formation: Substance P analog and BMP mimetic peptide-loaded click-crosslinked hyaluronic acid hydrogel.

Endogenous stem cell-driven in situ bone tissue formation has recently garnered increasing attention. Therefore, our study sought to refine methods to enhance the migration and subsequent osteogenic differentiation of these cells. Our innovative approach involves using an injectable hydrogel that combines click cross-linking sites and a BMP-2 mimetic peptide (BP) with hyaluronic acid (HA). This injectable formulation, hereinafter referred to as SPa + Cx-HA-BP, incorporates a substance P analog peptide (SPa) with Cx-HA-BP, proving versatile for in vitro and in vivo applications without cytotoxicity. The controlled release of SPa creates a gradient that guides endogenous stem cells towards the Cx-HA scaffold from specific tissue niches. Both Cx-HA and SPa+Cx-HA induced minimal changes in the expression of genes associated with osteogenic differentiation. In contrast, these genes were robustly induced by both SPa + Cx-HA+BP and SPa + Cx-HA-BP, in which BP was respectively integrated via physical and chemical methods. Remarkably, chemically incorporating BP (Cx-HA-BP) resulted in 4-9 times higher osteogenic gene expression than physically mixed BP in Cx-HA+BP. This study validates the role of SPa role in guiding endogenous stem cells toward the hydrogel and underscores the substantial impact of sustained BP presence within the hydrogel. Collectively, our findings offer valuable insights for the development of innovative strategies to promote endogenous stem cell-based tissue regeneration. The developed hydrogel effectively guides stem cells from their natural locations and facilitates sustained osteogenic differentiation, thus holding great promise for applications in regenerative medicine.

Coupling hCG-based protease sensors with a commercial pregnancy test strip for simple analyses of protease activities.

Proteases play an essential role in many cellular processes, and consequently, abnormalities in their activities are related to various diseases. Methods have been developed to measure the activity of these enzymes, but most involve sophisticated instruments or complicated procedures, which hampers the development of a point-of-care test (POCT). Here, we propose a strategy for developing simple and sensitive methods to analyze protease activity using commercial pregnancy test strips that detect human chorionic gonadotropin (hCG). hCG was engineered to have site-specific conjugated biotin and a peptide sequence, which can be cleaved by a target protease, between hCG and biotin. hCG protein was immobilized on streptavidin-coated beads, resulting in a protease sensor. The hCG-immobilized beads were too large to flow through the membrane of the hCG test strip and yielded only one band in the control line. When the peptide linker was hydrolyzed by the target protease, hCG was released from the beads, and the signal appeared in both the control and test lines. Three protease sensors for matrix metalloproteinase-2, caspase-3, and thrombin were constructed by replacing the protease-cleavable peptide linker. The combination of the protease sensors and a commercial pregnancy strip enabled the specific detection of each protease in the picomolar range, with a 30-min incubation of the hCG-immobilized beads and samples. The modular design of the protease sensor and simple assay procedure will facilitate the development of POCTs for various protease disease markers.

A novel and cost-effective method for high-throughput 3D culturing and rhythmic assessment of hiPSC-derived cardiomyocytes using retroreflective Janus microparticles.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) gain attention as a potent cell source in regenerative medicine and drug discovery. With the necessity of the demands for experimental models to create a more physiologically relevant model of the heart in vitro we herein investigate a 3D culturing platform and a method for assessing rhythm in hiPSC-CMs. METHODS: The 3D cell culture PAMCELL™ plate is designed to enable cells to attach exclusively to adhesive patterned areas. These cell adhesive zones, named as micro-patterned pads, feature micron silica beads that are surface-modified with the well-known arginyl-glycyl-aspartic acid (RGD) peptide. RGD binding to the surface of hiPSC-CMs facilitates cell-cell attachment and the formation of uniform-size spheroids, which is controlled by the diameter of the micro-patterned pads. The assessment and evaluation of 3D hiPSC-CMs beating pattern are carried out using reflective properties of retroreflective Janus micro-particle (RJP). These RJPs are modified with an antibody targeting the gap junction protein found on the surface of hiPSC-CM spheroids. The signal assessment system comprises a camera attached to an optical microscope and a white light source. RESULTS: The 3D PAMCELL™ R100 culture plate efficiently generate approximately 350 uniform-sized hiPSC-CM spheroids in each well of a 96-well plate and supported a 20-day culture. Analysis of genes and protein expression levels reveal that iPSC-CM spheroids grown on PAMCELL™ R100 retain cardiac stem cell characteristics and functions, outperforming traditional 2D culture platform. Additionally, the RJPs enable monitoring and evaluation of in vitro beating properties of cardiomyocytes without using complex monitoring setup. The system demonstrates its capability to identify alteration in the rhythmic activity of cardiac cells when exposed to ion channel blockers, nifedipine and E4031. CONCLUSIONS: The integration of the 3D culture method and RJPs in this study establishes a platform for evaluating the rhythmic properties of 3D hiPSC-CMs. This approach holds significant potential for identifying arrhythmias or other cardiac abnormalities, ultimately contributing to the development of more effective therapies for heart diseases.

Chip-based cartilage oligomeric matrix protein detection in serum and synovial fluid for osteoarthritis diagnosis.

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.

Retroreflection-based optical biosensing: From concept to applications.

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.

Wash-free operation of smartphone-integrated optical immunosensor using retroreflective microparticles.

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.

Multienzyme-modified biosensing surface for the electrochemical analysis of aspartate transaminase and alanine transaminase in human plasma.

We investigated the electrochemical detection of aspartate transaminase (AST) and alanine transaminase (ALT) by using a multienzyme-modified electrode surface. Determination of the activities of transaminases in human serum is clinically significant because their concentrations and ratios indicate the presence of hepatic diseases or myocardial dysfunction. For electrochemical detection of AST and ALT, enzymes that participate in the reaction mechanism of AST and ALT, such as pyruvate oxidase (POX) and oxaloacetate decarboxylase, were immobilized on an electrode surface by using an amine-reactive self-assembled monolayer and a homobifunctional cross-linker. In the presence of suitable substrates such as L-aspartate (L-alanine) and α-ketoglutarate, AST and ALT generate pyruvate as an enzymatic end product. To determine the activities of AST and ALT, electroanalyses of pyruvate were conducted using a POX and ferrocenemethanol electron shuttle. Anodically generated oxidative currents from multienzyme-mediated reactions were correlated to AST and ALT levels in human plasma. On the basis of the electrochemical analysis, we obtained calibration results for AST and ALT concentrations from 7.5 to 720 units/L in human plasma-based samples, covering the required clinical detection range.

Microfabrication of SiN membrane nanosieve using anisotropic reactive ion etching (ARIE) with an Ar/CF4 gas flow.

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).

Amyloid formation and disaggregation of α-synuclein and its tandem repeat (α-TR).

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.

Smartphone-integrated urinary CTX-II immunosensor based on wavelength filtering from chromogenic reaction.

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.

Nonspectroscopic Migratory Cell Monitoring Method Using Retroreflective Janus Microparticles.

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.

Instrumentation-Free Semiquantitative Immunoanalysis Using a Specially Patterned Lateral Flow Assay Device.

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 µg/mL, covering the required clinical detection range, and the limit of detection (LOD) value was calculated to be 15.5 µg/mL. Thus, the SQ-LFI system provides an avenue for the realization of an efficient quantification diagnostic device in resource-limited conditions.

Time-resolved fluorescence resonance energy transfer-based lateral flow immunoassay using a raspberry-type europium particle and a single membrane for the detection of cardiac troponin I.

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.

A surface acoustic wave-based immunosensing device using a nanocrystalline ZnO film on Si.

The novelty of this study resides in the fabrication of a bio-sensing device, based on the surface acoustic wave (SAW) on a nanocrystalline ZnO film. The ZnO film was deposited using an rf magnetron sputtering at room temperature on silicon. The deposited films showed the c-axis-oriented crystallite with grain size of approximately 40 nm. The immunosensing device was fabricated using photolithographic protocols on the film. As a model biomolecular recognition and immunosensing, biospecific interaction between a 6-(2,4-dinitrophenyl)aminohexanoic acid (DNP) antigen and its antibody was employed, demonstrating the shifts of resonant frequencies on SAW immunosensing device. The device exhibited a linearity as a function of the antibody concentration in the range of 20-20,000 ng/ml.

A non-spectroscopic optical biosensor for the detection of pathogenic Salmonella Typhimurium based on a stem-loop DNA probe and retro-reflective signaling.

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.

Wash-free non-spectroscopic optical immunoassay by controlling retroreflective microparticle movement in a microfluidic chip.

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.

Fluorescence affinity sensing by using a self-contained fluid manoeuvring microfluidic chip.

An application of a novel polymer microfluidic chip for sample exchange via natural capillary forces for immuno-analysis is described. The microfluidic device was designed to achieve sample replacement by capillary force only, which would therefore be suitable for point-of-care-testing. Complete and automatic replacement of the sample in the reaction chamber with another one makes the chip able to mimic affinity chromatography and immunoassay processes. The microfluidic chip was made using polymer replication techniques, which were suitable for fast and cheap fabrication. Micrometre-sized polystyrene beads were used for the functionalization of biomolecules. Dinitrophenyl (DNP) and anti-DNP antibody coordination was employed on the chip for fluorescence analysis. DNP was immobilized on the polymer beads via a pre-adsorbed dendrimer layer and the beads were placed in the reaction chamber. Fluorescein tagged anti-DNP was successfully observed by a fluorescence microscope after the completion of the entire flow sequence. A calibration curve was registered based on the anti-DNP concentration. A multiplex sensing was accomplished by adding biotin/streptavidin coordination to the system. DNP and biotin conjugated beads were placed in the reaction chamber in an ordered fashion and biospecific bindings of anti-DNP antibody and streptavidin were observed at their expected sites. A ratiometric analysis was carried out with different concentration ratios of anti-DNP/streptavidin. The microfluidic chip described in this work could be applied to various biological and chemical analyses using integrated washing steps or fluid replacement steps with minimum sample handling.

Salmonella Typhimurium Sensing Strategy Based on the Loop-Mediated Isothermal Amplification Using Retroreflective Janus Particle as a Nonspectroscopic Signaling Probe.

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.