Abstract:Encoded hydrogel microparticles synthesized via flow lithography have drawn attention for multiplex biomarker detection due to their high multiplex capability and solution-like hybridization kinetics. However, the current methods for preparing particles cannot achieve a flexible, rapid probe-set modification, which is necessary for the production of various combinations of target panels in clinical diagnosis. In order to accomplish the unmet needs, streptavidin was incorporated into the encoded hydrogel microp… Show more
“…The detailed calculation procedures and related datas were presented in the supporting information and Figure S2. These unreacted acrylate groups are highly reactive with various functionalized molecules through chemical reactions, including Michael addition reactions 10,16,24 . In particular, considering that various cell‐adhesion promoters (e.g., PLL, fibronectin, collagen, and dopamine) contain amine groups in their molecular structures, the aza‐Michael addition of cell‐adhesion promoter molecules to microparticles can be directly achieved without requiring the preconjugation of linker molecules 2 …”
Section: Resultsmentioning
confidence: 99%
“…Considering that there are highly abundant amine groups in the PLL structures, we induced the aza‐Michael addition reaction between electrophilic α,β‐unsaturated double bonds in the particles and nucleophilic nitrogen donor groups in the PLL (Figure 1B). 24,31 Among diverse acrylate‐based conjugation chemistries, the aza‐Michael addition reaction has distinct advantages, including commercial availability, no requirement for catalyst, and low productivity of reactive by‐products during polymerization 32 . To confirm the conjugation of the PLL to particles by a proposed manner, we used PLL labeled with fluorescein isothiocyanate (PLL‐FITC) to qualitatively observe and quantitatively analyze the fluorescent images and signals of attached PLL in the particles.…”
Section: Resultsmentioning
confidence: 99%
“…To confirm the conjugation of the PLL to particles by a proposed manner, we used PLL labeled with fluorescein isothiocyanate (PLL‐FITC) to qualitatively observe and quantitatively analyze the fluorescent images and signals of attached PLL in the particles. We selected a fluorescent labeling method for the cell‐adhesion promoter since it has powerful advantages for molecular conjugation analysis in hydrogel microparticles, such as the visualization of distributed molecules and the facile quantification of the loaded amount of the molecules by fluorescent intensity 14,21,22,24 …”
Section: Resultsmentioning
confidence: 99%
“…Accordingly, we characterized the amount of conjugated PLL‐FITC in unit particles by fluorescent signals based on the pH of the incubation buffer and incubation time during the aza‐Michael reaction process. First, we optimized the pH of the incubation solution considering that pH is one of the critical factors for efficient aza‐Michael addition reactions as it affects the nucleophilicity of electron donors 24 . We screened optimal pH conditions from 7 to 10 using the fluorescent signals of the particles conjugated with the PLL‐FITC for 4 h (Figure 2A).…”
Section: Resultsmentioning
confidence: 99%
“…To avoid using additional linker molecules and complicated preconjugation steps, the direct conjugation of cell‐adhesion promoter molecules containing amine groups can be an alternative by inducing the aza‐Michael reaction between unreacted acrylate groups in the particles and the nucleophilic amine groups in the cell‐adhesion promoters. Recently, there has been high interest in utilizing unreacted acrylate groups in PEG crosslinkers during polymerization for a high‐performance and simple bio‐conjugation process, in which the remnant acrylate groups enable Michael addition reactions with amine and thiol groups in various biomolecules 10,16,24,25 . It has already been demonstrated that the unreacted acrylate group‐based functionalization methods have powerful advantages, such as the high conjugation capacity, 25 simplicity of process because of the unnecessity of linker molecules, 26 and prevention of the aggregation of biomolecules inside the gel structures 16 .…”
Polyethylene glycol (PEG) hydrogel microparticles generated via stop‐flow lithography can be utilized for efficient microparticle‐based cell culture processes because of their high biocompatibility, the molecular diffusion capability in the gel structure, and the tunability of their shape and size. However, the typical functionalization process of PEG microparticles with cell‐adhesion promoters has inevitable limitations, requiring additional linker molecules and the preconjugation of linkers to cell‐adhesion promoters and microparticles. In this study, a simple and direct cell‐adhesion promoter functionalization process of the PEG microparticles is presented by use of aza‐Michael reaction between remnant unreacted acrylate groups in particles and amine groups in cell‐adhesion promoters. On the basis of proposed process, particles are directly conjugated with poly‐l‐lysine (PLL), a typical cell‐adhesion promoter that can electrostatically interact with cellular membranes, in a controllable manner. We demonstrate enhanced cell‐adhesion capabilities of the particles along with the increased amount of conjugated PLL in the particles. Furthermore, to validate extended applicability, the particles are directly conjugated with Gly‐Arg‐Gly‐Asp‐Ser (GRGDS) peptides, in which RGD sequence is involved in the cell‐adhesion behavior of extracellular matrix proteins, including fibronectin. The introduced GRGDS peptides increase the cell‐adhesion capacity of the microparticles binding to integrin proteins in cellular membranes.
“…The detailed calculation procedures and related datas were presented in the supporting information and Figure S2. These unreacted acrylate groups are highly reactive with various functionalized molecules through chemical reactions, including Michael addition reactions 10,16,24 . In particular, considering that various cell‐adhesion promoters (e.g., PLL, fibronectin, collagen, and dopamine) contain amine groups in their molecular structures, the aza‐Michael addition of cell‐adhesion promoter molecules to microparticles can be directly achieved without requiring the preconjugation of linker molecules 2 …”
Section: Resultsmentioning
confidence: 99%
“…Considering that there are highly abundant amine groups in the PLL structures, we induced the aza‐Michael addition reaction between electrophilic α,β‐unsaturated double bonds in the particles and nucleophilic nitrogen donor groups in the PLL (Figure 1B). 24,31 Among diverse acrylate‐based conjugation chemistries, the aza‐Michael addition reaction has distinct advantages, including commercial availability, no requirement for catalyst, and low productivity of reactive by‐products during polymerization 32 . To confirm the conjugation of the PLL to particles by a proposed manner, we used PLL labeled with fluorescein isothiocyanate (PLL‐FITC) to qualitatively observe and quantitatively analyze the fluorescent images and signals of attached PLL in the particles.…”
Section: Resultsmentioning
confidence: 99%
“…To confirm the conjugation of the PLL to particles by a proposed manner, we used PLL labeled with fluorescein isothiocyanate (PLL‐FITC) to qualitatively observe and quantitatively analyze the fluorescent images and signals of attached PLL in the particles. We selected a fluorescent labeling method for the cell‐adhesion promoter since it has powerful advantages for molecular conjugation analysis in hydrogel microparticles, such as the visualization of distributed molecules and the facile quantification of the loaded amount of the molecules by fluorescent intensity 14,21,22,24 …”
Section: Resultsmentioning
confidence: 99%
“…Accordingly, we characterized the amount of conjugated PLL‐FITC in unit particles by fluorescent signals based on the pH of the incubation buffer and incubation time during the aza‐Michael reaction process. First, we optimized the pH of the incubation solution considering that pH is one of the critical factors for efficient aza‐Michael addition reactions as it affects the nucleophilicity of electron donors 24 . We screened optimal pH conditions from 7 to 10 using the fluorescent signals of the particles conjugated with the PLL‐FITC for 4 h (Figure 2A).…”
Section: Resultsmentioning
confidence: 99%
“…To avoid using additional linker molecules and complicated preconjugation steps, the direct conjugation of cell‐adhesion promoter molecules containing amine groups can be an alternative by inducing the aza‐Michael reaction between unreacted acrylate groups in the particles and the nucleophilic amine groups in the cell‐adhesion promoters. Recently, there has been high interest in utilizing unreacted acrylate groups in PEG crosslinkers during polymerization for a high‐performance and simple bio‐conjugation process, in which the remnant acrylate groups enable Michael addition reactions with amine and thiol groups in various biomolecules 10,16,24,25 . It has already been demonstrated that the unreacted acrylate group‐based functionalization methods have powerful advantages, such as the high conjugation capacity, 25 simplicity of process because of the unnecessity of linker molecules, 26 and prevention of the aggregation of biomolecules inside the gel structures 16 .…”
Polyethylene glycol (PEG) hydrogel microparticles generated via stop‐flow lithography can be utilized for efficient microparticle‐based cell culture processes because of their high biocompatibility, the molecular diffusion capability in the gel structure, and the tunability of their shape and size. However, the typical functionalization process of PEG microparticles with cell‐adhesion promoters has inevitable limitations, requiring additional linker molecules and the preconjugation of linkers to cell‐adhesion promoters and microparticles. In this study, a simple and direct cell‐adhesion promoter functionalization process of the PEG microparticles is presented by use of aza‐Michael reaction between remnant unreacted acrylate groups in particles and amine groups in cell‐adhesion promoters. On the basis of proposed process, particles are directly conjugated with poly‐l‐lysine (PLL), a typical cell‐adhesion promoter that can electrostatically interact with cellular membranes, in a controllable manner. We demonstrate enhanced cell‐adhesion capabilities of the particles along with the increased amount of conjugated PLL in the particles. Furthermore, to validate extended applicability, the particles are directly conjugated with Gly‐Arg‐Gly‐Asp‐Ser (GRGDS) peptides, in which RGD sequence is involved in the cell‐adhesion behavior of extracellular matrix proteins, including fibronectin. The introduced GRGDS peptides increase the cell‐adhesion capacity of the microparticles binding to integrin proteins in cellular membranes.
Cytomegalovirus (CMV) is the most frequent cause of congenital infection worldwide; congenital CMV may lead to significant mortality, morbidity, or long-term sequelae, such as sensorineural hearing loss. The current study presents a newly designed surface plasmon resonance (SPR) biosensor for CMV-specific microRNAs that does not involve extra care for receptor immobilization or treatment to prevent fouling on bare gold surfaces. The modification-free approach, which utilizes a poly-adenine [poly(A)]−Au interaction, exhibited a high affinity that was comparable to that of the gold−sulfur (Au−S) interaction. In addition, magnetic nanoparticles (MNPs) were used to separate the analyte from complex sample matrixes that significantly reduced nonspecific adsorption. Moreover, the MNPs also played an important role in SPR signal amplification due to the binding-induced change in the refractive index. Our SPR biosensing platform was used successfully for the multidetection of the microRNAs, UL22A-5p, and UL112-3p, which were associated with CMV. Our SPR biosensor offered the detection limits of 108 fM and 24 fM for UL22A-5p and UL112-3p, respectively, with an R 2 of 0.9661 and 0.9985, respectively. The precision of this biosensor has an acceptable CV (coefficient of variation) value of <10%. In addition, our sensor is capable of discriminating between serum samples collected from healthy and CMV-infected newborns. Taken together, we believe that our newly developed SPR biosensing platform is a promising alternative for the diagnosis of CMV-specific microRNA in clinical settings, and its application for the detection of other miRNAs may be extended further.
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