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Laser Ablation - From Fundamentals to Applications || Matrix-Assisted Pulsed Laser Evaporation of Organic Thin Films: Applications in Biology and Chemical Sensors
摘要： Polymer and biomolecule processing for medical and electronics applications, i.e. the fabrication of sensors and biosensors, microarrays, or lab on chip devices is a cornerstone field which shows great promise. Laser based thin film deposition techniques such as pulsed laser deposition or matrix-assisted pulsed laser evaporation (MAPLE) are competing with conventional methods for integrating new materials with tailored properties for novel technological developments. Successful polymer and protein thin film deposition requires several key elements for depositing viable and functional thin films, i.e. the characteristics of the laser depositing system, the choice of targets and receiver substrates, etc. This chapter reviews the following topics: brief presentation of the MAPLE process including several examples of polymer materials deposited by MAPLE, thus illustrating the potential of the technique as a gentle laser-assisted deposition method. In particular, the “synthesis” of new materials, their analysis and correlation of the bulk and interface properties to its bio-environment shall be discussed as a method to tackle some bioengineering issues. We will also focus on recent breakthroughs of the MAPLE technique for the fabrication of functional devices, i.e. sensor devices based either on chemoresponsive polymers or on proteins.
关键词： saw,maple,hydroxyapatite nanoparticles,odorant-binding proteins,PEG-PCL-me,polyethylenimine,polyepichlorohydrin,ha,polyethylene glycol-co-polycaprolactone methyl ether,polyisobutylene,lactoferrin
更新于2025-09-19 17:13:59
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Femtomolar sensing of Alzheimer's tau proteins by water oxidation-coupled photoelectrochemical platform
摘要： Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder. A key pathogenic event of AD is the formation of intracellular neurofibrillary tangles that are mainly composed of tau proteins. Here, we report on ultrasensitive detection of total tau (t-tau) proteins using an artificial electron donor-free, BiVO4-based photoelectrochemical (PEC) analysis. The platform was constructed by incorporating molybdenum (Mo) dopant and iron oxyhydroxide (FeOOH) ad-layer into the BiVO4 photoelectrode and employing a signal amplifier formed by horseradish peroxidase (HRP)-triggered oxidation of 3,3′-diaminobenzidine (DAB). Despite the absence of additional electron suppliers, the FeOOH/Mo:BiVO4 conjugated with the Tau5 antibody produced strong current signals at 0 V (vs. Ag/AgCl, 3M NaCl) under the illumination of a white light-emitting diode. The Mo extrinsic dopants increased the charge carrier density of BiVO4-Tau5 by 1.57 times, and the FeOOH co-catalyst promoted the interfacial water oxidation reaction of Mo:BiVO4-Tau5 by suppressing charge recombination. The introduction of HRP-labeled Tau46 capture antibodies to the FeOOH/Mo:BiVO4-Tau5 platform produced insoluble precipitation on the transducer by accelerating the oxidation of DAB, which amplified the photocurrent signal of FeOOH/Mo:BiVO4-Tau5 by 2.07-fold. Consequently, the water oxidation-coupled, FeOOH/Mo:BiVO4-based PEC sensing platform accurately and selectively recognized t-tau proteins down to femtomolar concentrations; the limit of detection and limit of quantification were determined to be 1.59 fM and 4.11 fM, respectively.
关键词： Alzheimer’s disease,water oxidation,Femtomolar sensitivity,tau proteins,BiVO4
更新于2025-09-19 17:13:59
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Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy
摘要： Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump–probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.
关键词： time-resolved crystallography,fluorescent proteins,transient absorption spectroscopy,photoswitching,rsEGFP2
更新于2025-09-19 17:13:59
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In Situ Investigation on the Protein Corona Formation of Quantum Dots by Using Fluorescence Resonance Energy Transfer
摘要： A fundamental understanding of nanoparticle–protein corona and its interactions with biological systems is essential for future application of engineered nanomaterials. In this work, fluorescence resonance energy transfer (FRET) is employed for studying the protein adsorption behavior of nanoparticles. The adsorption of human serum albumin (HSA) onto the surface of InP@ZnS quantum dots (QDs) with different chirality (d- and l-penicillamine) shows strong discernible differences in the binding behaviors including affinity and adsorption orientation that are obtained upon quantitative analysis of FRET data. Circular dichroism spectroscopy further confirms the differences in the conformational changes of HSA upon interaction with d- and l-chiral QD surfaces. Consequently, the formed protein corona on chiral surfaces may affect their following biological interactions, such as possible protein exchange with serum proteins plasma as well as cellular interactions. These results vividly illustrate the potential of the FRET method as a simple yet versatile platform for quantitatively investigating biological interactions of nanoparticles.
关键词： serum proteins,quantum dots,chirality,nanoparticle–protein interactions,protein corona,fluorescence resonance energy transfer
更新于2025-09-19 17:13:59
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White-emitting protein-metal nanocluster phosphors for highly performing bio-hybrid light-emitting diodes
摘要： This work presents a simple in-situ synthesis and stabilization of fluorescent gold nanoclusters (AuNCs) with different sizes using engineered protein scaffolds in water. The protein-AuNC hybrids show a dual emission (450 and 700 nm) with a record photoluminescence quantum yield of 20%. These features impelled us to apply them to bio-hybrid light-emitting diodes (Bio-HLEDs) as color down-converting filters or bio-phosphors. Efficient white emission (x/y CIE color coordinates of 0.31/0.29) and stabilities of >800 h were achieved. This represents a two orders of magnitude enhancement compared to the prior art. Besides the outstanding performance, the protein scaffold also infers a unique anisotropic emission character that is considered as a proof-of-concept of high interest for single-point lighting and display.
关键词： bio-phosphors,protein design,Light-emitting diodes,repeat proteins,bio-hybrid materials,metal nanocluster
更新于2025-09-19 17:13:59
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Synthesis and Characterization of Thiolate-Protected Gold Nanoparticles of Controlled Diameter
摘要： Gold nanoparticles (AuNPs) have been the focus of many studies owing to their unique optical and electronic properties and versatile applications. However, synthesis of stable and homogeneous AuNPs with a particular choice of size is still a challenge. In this study we describe a direct synthesis approach to produce stable and monodisperse water-soluble AuNPs with a tightly controlled diameter in the 1.7?2.4 nm range. We controlled the size by changing only the sodium hydroxide (NaOH) concentration in the synthesis. Gel electrophoresis, transmission electron microscopy (TEM), and solution X-ray scattering showed that the AuNPs had narrow size-distributions. We further showed that AuNPs of the di?erent sizes were clearly distinguishable in TEM micrographs, paving the way to dual-target labeling. The reactivity of the AuNPs toward DNA and proteins was also demonstrated. We utilized this reactivity to label tail-anchored proteins embedded in the membrane of the anticancer nanodrug Doxil as a means to target it to speci?c cell types. The gold-labeling enabled the precise localization of the tail-anchored proteins in cryo-TEM images of the therapeutic liposomes.
关键词： thiolate-protected,bioconjugation,cryo-TEM,tail-anchored proteins,synthesis,size control,Gold nanoparticles,Doxil
更新于2025-09-16 10:30:52
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Multiplexed surface plasmon imaging of serum biomolecules: Fe3O4@Au Core/shell nanoparticles with plasmonic simulation insights
摘要： Nano-biosensors that are not only sensitive and selective, but also enable multiplex detection of ultra-low levels of both large and small biomolecules in clinical sample matrices are essential for in vitro diagnostics. We present herein a multiplex surface plasmon microarray design that employs citrate-stabilized Fe3O4@Au core/shell nanoparticles (NPs) as the plasmon signal ampli?cation label for combined detection of serum proteins and nucleotide markers. The multiplex sensing is demonstrated using two interleukins (IL-6 and IL-8) and two microRNAs (miRNA-21 and miRNA-155) in 10% serum, which is clinically relevant than simple bu?er solution based biosensors. We observed that the surface plasmon signal change for larger proteins even at higher concentrations was less than the relatively smaller miRNA molecules. We draw two conclusions from this result: (i) the number of selectively bound analytes onto the sensor (i.e., antigen for an antibody or miRNA for a capture nucleotide) in?uences the signal change, and (ii) the extent of interaction of the detection probe carrying core/shell NP labels with the sensor surface plasmons in?uences the amount of signal change. Results indicate that both factors, (i) and (ii), are greater for small oligonucleotide hybridization assembly than a large sandwich protein immunoassembly. The core/shell NPs o?ered several fold enhanced sensitivity and wider dynamic range of detection over assays without using them. With recently growing attention on in vitro diagnostics for painless/minimally-invasive detection of diseases and abnormalities, ?ndings presented herein are important for designing novel multiplex biosensors for real sample analysis in complex matrices.
关键词： serum proteins,wide dynamic range,serum miRNAs,Multiplex imaging,core/shell nanoparticles,FDTD simulation
更新于2025-09-16 10:30:52
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Estimation of Flexible and Rigid Residues of Disulfide-Bridged and Phosphorylated Proteins Using Matrix-Assisted Laser Desorption/Ionization in-Source Decay Mass Spectrometry
摘要： Flexible and rigid residues in disul?de-bridged and phosphorylated proteins have been estimated by using MALDI in-source decay mass spectrometry (ISD MS). The MALDI-ISD spectra of bovine α-lactoalbumin, β-lactoglobulin A, and β-casein predict that the backbone amide of Xxx-Asp/Asn/Cys/Ser/pSer and Gly-Xxx residues has higher hydrogen accessibility than other residues, while Xxx-Ile/Val residues have less accessibility. The higher hydrogen-accessible and lower accessible residues as measured by MALDI-ISD are consistent with the ?exible and rigid residues determined by X-ray, nuclear magnetic resonance, and ?uorescence decay methods. The disul?de bridges and phosphate groups do not prevent the estimation of ?exible or rigid residues, whereas some other disul?de bridges inhibit the identi?cation because of decreased sensitivity of ISD fragment ions. The estimation of ?exible and rigid residues by means of the matrix-hydrogen accessibility can be explained by exposure or lack thereof to the hydrogen-accessible sites of intact proteins. It is proposed that MALDI-ISD is a powerful tool for identifying ?exible and rigid residues of posttranslational modi?ed proteins without the conformation information of the protein data bank.
关键词： flexible and rigid residues,phosphorylated proteins,disul?de-bridged proteins,hydrogen accessibility,MALDI in-source decay mass spectrometry
更新于2025-09-12 10:27:22
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Unraveling the origin of chirality from plasmonic nanoparticle-protein complexes
摘要： Plasmon-coupled circular dichroism has emerged as a promising approach for ultrasensitive detection of biomolecular conformations through coupling between molecular chirality and surface plasmons. Chiral nanoparticle assemblies without chiral molecules present also have large optical activities. We apply single-particle circular differential scattering spectroscopy coupled with electron imaging and simulations to identify both structural chirality of plasmonic aggregates and plasmon-coupled circular dichroism induced by chiral proteins. We establish that both chiral aggregates and just a few proteins in interparticle gaps of achiral assemblies are responsible for the ensemble signal, but single nanoparticles do not contribute. We furthermore find that the protein plays two roles: It transfers chirality to both chiral and achiral plasmonic substrates, and it is also responsible for the chiral three-dimensional assembly of nanorods. Understanding these underlying factors paves the way toward sensing the chirality of single biomolecules.
关键词： chiral nanoparticle assemblies,single-particle circular differential scattering spectroscopy,plasmonic aggregates,biomolecular conformations,chiral proteins,Plasmon-coupled circular dichroism
更新于2025-09-12 10:27:22
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Dynamic Color‐Switching of Plasmonic Nanoparticle Films
摘要： Single‐housed stress elicits a range of social isolation‐related behavioral and neurobiological abnormalities. To investigate single housing‐induced behavioral changes and sex differences on stress outcomes, we examined single‐housed stress‐induced learning and memory impairment, depression‐like behaviors, neuroplasticity abnormalities and underlying mechanism. The results showed that male and female mice socially isolated for 8 weeks had significantly decreased memory acquisition, as demonstrated in the learning curve of the Morris water maze task. Memory consolidation and retrieval were also decreased in both the single‐housed male and female mice. These findings were corroborated further by the two classical animal models, Y‐maze and novel object recognition tests, as demonstrated by reduced spontaneous alternation and recognition index in both sexes of single‐housed mice. Subsequent studies suggested that single‐housed male mice exhibited increased immobility time in both the forced swim and tail suspension tests, while the female mice only exhibited increased immobility time in the tail suspension test. Moreover, single‐housed stress significantly decreased the apical and basal branch points, dendritic length, and spine density in the CA1 of hippocampal neurons in both male and female mice. These effects were consistent with decreased neuroplasticity and neuroprotective‐related molecules such as synaptophysin, PSD95, PKA, pCREB and BDNF expression. These findings suggest that loss of neuronal remodeling and neuroprotective mechanisms due to single housing are involved in behavioral changes in both male and female mice. The results provide further evidence that neuroplasticity‐related signaling plays a crucial role in isolation‐induced effects on neuropsychiatric behavioral deficits in both sexes.
关键词： neuroplasticity,synaptic plasticity‐associated proteins,depression,cognition,single housing stress
更新于2025-09-11 14:15:04