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.

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.

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.