Multiplexed Opto-Microfluidic Biosensing: Advanced Platform for Prostate Cancer Detection.

Cancer stands as a prominent global cause of mortality, necessitating early detection to augment survival rates and alleviate economic burdens on healthcare systems. In particular, prostate cancer (PCa), impacting 1.41 million men globally in 2020, accentuates the demand for sensitive and cost-effective detection methods beyond traditional prostate-specific antigen (PSA) testing. While clinical techniques exhibit limitations, biosensors emerge as compact, user-friendly alternatives to traditional laboratory approaches. However, existing biosensors predominantly concentrate on PSA detection, prompting the necessity for advancing toward multiplex sensing platforms. This study introduces a compact opto-microfluidic sensor featuring a substrate of gold nanospikes, fabricated via electrodeposition, for enhanced sensitivity. Embedded within a microfluidic chip, this nanomaterial enables the precise and concurrent measurement of PSA, alongside two complementary PCa biomarkers, matrix metalloproteinase-2 (MMP-2) and anti-α-methylacyl-CoA racemase (anti-AMACR) in diluted human plasma, offering a comprehensive approach to PSA analysis. Taking advantage of the localized surface plasmon resonance principle, this biosensor offers robustness and sensitivity in real sample analysis without the need for labeling agents. With the limit of detection at 0.22, 0.37, and 0.18 ng/mL for PSA, MMP-2, and anti-AMACR, respectively, this biosensing platform holds promise for point-of-care analysis, underscoring its potential impact on medical diagnostics.

EGR1 Regulates SHANK3 Transcription at Different Stages of Brain Development.

The expression levels of SHANK3 are associated with autism spectrum disorder (ASD). The dynamic changes in SHANK3 expression during different stages of brain development may impact the progression of ASD. However, no studies or detailed analyses exploring the upstream mechanisms that regulate SHANK3 expression have been reported. In this study, we employed immunofluorescence to examine the expression of SHANK3 in brain organoids at various stages. Our results revealed elevated levels of SHANK3 expression in brain-like organoids at Day 60. Additionally, we utilized bioinformatics software to predict and analyze the SHANK3 gene's transcription start site. Through the dual luciferase reporter gene technique, we identified core transcription elements within the SHANK3 promoter. Site-directed mutations were used to identify specific transcription sites of SHANK3. To determine the physical binding of potential transcription factors to the SHANK3 promoter, we employed electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Our findings demonstrated that the transcription factor EGR1 regulates SHANK3 expression by binding to the transcription site of the SHANK3 promoter. Although this study did not investigate the pathological phenotypes of human brain organoids or animal model brains with EGR1 deficiency, which could potentially substantiate the findings observed for SHANK3 mutants, our findings provide valuable insights into the relationship between the transcription factor, EGR1, and SHANK3. This study contributes to the molecular understanding of ASD and offers potential foundations for precise targeted therapy.

Preferred penetration of active nano-rods into narrow channels and their clustering.

In a channel connected to a reservoir, passive particles prefer staying in the reservoir than the channel due to the entropic effect, as the size of the particles is comparable to that of the channel. Self-propelled rods can exhibit out-of-equilibrium phenomena, and their partition behavior may differ from that of passive rods due to their persistent swimming ability. In this work, the distribution of active nano-rods between the nanoscale channel and reservoir is explored using dissipative particle dynamics. The ratio of the nano-rod concentration in the slit to that in the reservoir, defined as the partition ratio Ψ, is a function of active force, channel width, and rod length. Although passive nano-rods prefer staying in bulk (Ψ < 1), active rods can overcome the entropic barrier and show favorable partition toward narrow channels (Ψ > 1). As the slit width decreases to about the rod's width, active rods entering the slit behave like a quasi-two-dimensional system dynamically. At sufficiently high concentrations and Peclet numbers, nano-rods tend to align and move together in the same direction for a certain time. The distribution (PM) of the cluster size (M) follows a power law, PM ∝ M-2, for small clusters.

Detection of antibodies against SARS-CoV-2 spike protein by gold nanospikes in an opto-microfluidic chip.

The ongoing global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to active research in its associated diagnostics and medical treatments. While quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most reliable method to detect viral genes of SARS-CoV-2, serological tests for specific antiviral antibodies are also important as they identify false negative qRT-PCR responses, track how effectively the patient's immune system is fighting the infection, and are potentially helpful for plasma transfusion therapies. In this work, based on the principle of localized surface plasmon resonance (LSPR), we develop an opto-microfluidic sensing platform with gold nanospikes, fabricated by electrodeposition, to detect the presence and amount of antibodies specific to the SARS-CoV-2 spike protein in 1µL of human plasma diluted in 1mL of buffer solution, within ∼30min. The target antibody concentration can be correlated with the LSPR wavelength peak shift of gold nanospikes caused by the local refractive index change due to the antigen-antibody binding. This label-free microfluidic platform achieves a limit of detection of ∼0.08ng/mL (∼0.5pM), falling under the clinical relevant concentration range. We demonstrate that our opto-microfluidic platform offers a promising point-of-care testing tool to complement standard serological assays and make SARS-CoV-2 quantitative diagnostics easier, cheaper, and faster.

Size-dependence and interfacial segregation in nanofilms and nanodroplets of homologous polymer blends.

The size-dependent behavior of nanofilms and nanodroplets of homologous polymer blends was explored by many-body dissipative particle dynamics. Although a homologous blend can be regarded as a completely miscible and athermal system, enrichment of the surface in short polymers always takes place. First, liquid-gas and solid-liquid interfacial tensions of polymer melts were acquired. It is found that they increase and approach asymptotes with increasing chain lengths. The molecular weight dependence can be depicted using two semi-empirical expressions. Second, the variation of surface tension and surface excess of polymer blend nanofilms with the thickness was observed. Surface tension of the blend is observed to increase but the extent of surface segregation decreases upon increasing the film thickness. Finally, the wetting phenomenon of nanodroplets of homologous blends was examined. The contact angle is found to increase as the droplet size is reduced. Our simulation results indicate that the size-dependence of nanofilms and nanodroplets is closely related to surface segregation in homologous blends.

Size-dependent behavior and failure of young's equation for wetting of two-component nanodroplets.

HYPOTHESIS: For macroscopic systems, the interfacial properties are size-independent and Young's equation is generally valid for smooth substrates. For nanoscale systems, however, size-dependence and failure of Young's equation may emerge. EXPERIMENTS: The wetting behavior of a nanodroplet containing two miscible liquids on a smooth substrate is explored by many-body dissipative particle dynamics simulations. The size-dependent surface tension of nanofilms is investigated as well. FINDINGS: It is found that Young's equation is valid for nanodroplets of pure fluids but fails for two-component nanodroplets. The actual contact angle is always larger than the Young's contact angle, and their difference is getting smaller as the composition approaches pure fluids or the compatibility of the mixture is increased. The failure of Young's equation is closely associated with the size-dependent behavior in two-component nanodroplets and nanofilms. As the nanodroplet size is increased, the actual contact angle is found to decline but approaches a constant expected in macroscopic systems. Similarly, as the nanofilm thickness is increased, surface tension decreases and reaches its macroscopic value. The change of surface tension is attributed to the size-dependent surface composition, which is responsible for the failure of Young's equation.

Pressure-gated capillary nanovalves based on liquid nanofilms.

HYPOTHESIS: Nanoscale valving is essential to expand the potentiality of the nanodevices. However, it is difficult to fabricate valves with movable control elements in nanoscale systems and thus it is desirable to design a nanovalve which can manipulate opening and blocking a gate without moving parts. EXPERIMENTS: A pressure-gated capillary valve which contains a nanoscale liquid layer sandwiched between two plates with two aligned orifices was designed and the proof-of-concept demonstration was achieved by Many-body Dissipative Particle Dynamics. FINDINGS: The concave or convex meniscus appears naturally within the orifice of the capillary valve and can be controlled by the pressure difference between liquid and gas phases based on Young-Laplace equation and the edge effect. The closed state can be transformed into the open state (hole) when the two concave menisci are allowed to touch each other. The on/off switch is reversible by Laplace pressure manipulation and the passing and blocking of the fluid particles through the valve is verified.

Favorable partition of nanoswimmers toward a confined slit.

The partition of nanoswimmers between a narrow channel and a reservoir is explored by dissipative particle dynamics. In contrast to passive colloids, nanoswimmers prefer to stay in the slit rather than in the reservoir for sufficiently large active force (F_{a}) or run time (τ). The partition ratio (φ) increases with F_{a} and τ. Interestingly, as the slit height decreases, φ grows accordingly until the confinement effect dominates. Three types of the concentration profile in the slit are identified as a consequence of the competition between surface accumulation and entropic barrier.

Real-time monitoring of DNA immobilization and detection of DNA polymerase activity by a microfluidic nanoplasmonic platform.

DNA polymerase catalyzes the replication of DNA, one of the key steps in cell division. The control and understanding of this reaction owns great potential for the fundamental study of DNA-enzyme interactions. In this context, we developed a label-free microfluidic biosensor platform based on the principle of localized surface plasmon resonance (LSPR) to detect the DNA-polymerase reaction in real-time. Our microfluidic LSPR chip integrates a polydimethylsiloxane (PDMS) channel bonded with a nanoplasmonic substrate, which consists of densely packed mushroom-like nanostructures with silicon dioxide stems (~40 nm) and gold caps (~22 nm), with an average spacing of 19 nm. The LSPR chip was functionalized with single-stranded DNA (ssDNA) template (T30), spaced with hexanedithiol (HDT) in a molar ratio of 1:1. The DNA primer (P8) was then attached to T30, and the second strand was subsequently elongated by DNA polymerase assembling nucleotides from the surrounding fluid. All reaction steps were detected in-situ inside the microfluidic LSPR chip, at room temperature, in real-time, and label-free. In addition, the sensor response was successfully correlated with the amount of DNA and HDT molecules immobilized on the LSPR sensor surface. Our platform represents a benchmark in developing microfluidic LSPR chips for DNA-enzyme interactions, further driving innovations in biosensing technologies.

Strong competition between adsorption and aggregation of surfactant in nanoscale systems.

HYPOTHESIS: The size-dependent behavior including surface tension, surface density (Γ), and critical micelle concentration (CMC) of a nanoscale liquid film containing surfactant has not been investigated until now. EXPERIMENTS: Strong competition between surface adsorption and bulk aggregation of surfactant in nanoscale systems was explored by Many-body Dissipative Particle Dynamics simulations. FINDINGS: In nanoscale systems, as the surfactant concentration increases, Γ continues rising even after CMC is exceeded. The saturation level of Γ is achieved only when the surfactant bulk concentration is over ten times of CMC. Moreover, both surface micelles formed by adsorbed surfactant and the sublayer below the adsorbed layer are clearly identified. The former can reduce the contacts of adsorbed surfactant with water, while the latter has the surfactant concentration significantly higher than that in bulk. The strong coupling between adsorption and micellization is attributed to large surface-to-volume ratio compared to macroscopic systems, and can be simply realized by the fact that the ratio of the numbers of surfactant distributed in bulk (nbulk) and at interface (nads) is always less than unity (nbulk/nads < 1) in nanoscale systems.

Penetration dynamics through nanometer-scale hydrophilic capillaries: Beyond Washburn's equation and extended menisci.

HYPOTHESIS: Recently, the naoncapillary devices with the channel width about 2-3 water molecules have been fabricated. Water transport through these nanoslits showed unexpectedly fast flow, revealing the failure of Washburn's equation. EXPERIMENTS: Liquid penetration into a nanocapillary made of two parallel walls is explored by many-body dissipative particle dynamics. Both partial wetting and total wetting walls are considered and the no-slip boundary condition is satisfied. FINDINGS: The wicking velocity generally obeys Washburn's equation, but the dynamic contact angle (CA) has to be employed. The dynamic CA (θD) relies on the penetration rate and is always larger than the equilibrium CA. The breakdown of Washburn's equation occurs under two conditions, (i) the channel width close to molecular size and (ii) the positive spreading coefficient is large enough. Both cases come about when the wicking velocity in a nanoslit exceeds the maximum value corresponding to cos(θD) = 1. The failure of Washburn's equation is attributed to the invalidity of Young-Laplace equation associated with undefined meniscus. The extended meniscus will be developed as a wall of the nanoslit continues to extend outside the exit mouth. The shapes of extended menisci are discussed for both partial wetting and total wetting surfaces.

Nanoplasmonics for Real-Time and Label-Free Monitoring of Microbial Biofilm Formation.

Microbial biofilms possess intrinsic resistance against conventional antibiotics and cleaning procedures; thus, a better understanding of their complex biological structures is crucial in both medical and industrial applications. Existing laboratory methodologies have focused on macroscopic and mostly indirect characterization of mechanical and microbiological properties of biofilms adhered on a given substrate. However, the kinetics underlying the biofilm formation is not well understood, while such information is critical to understanding how drugs and chemicals influence the biofilm formation. Herein, we report the use of localized surface plasmon resonance (LSPR) for real-time, label-free monitoring of E. coli biofilm assembly on a nanoplasmonic substrate consisting of gold mushroom-like structures. Our LSPR sensor is able to capture the signatures of biofilm formation in real-time by measuring the wavelength shift in the LSPR resonance peak with high temporal resolution. We employ this sensor feature to elucidate how biofilm formation is affected by different drugs, including conventional antibiotics (kanamycin and ampicillin) as well as rifapentine, a molecule preventing cell adhesion yet barely affecting bacterial viability and vitality. Due to its flexibility and simplicity, our LSPR based platform can be used on a wide variety of clinically relevant bacteria, thus representing a valuable tool in biofilm characterization and drug screening.

Hydrodynamic interaction induced breakdown of the state properties of active fluids.

The mechanical pressure of active fluids in which swimmers are modeled by soft run-and-tumble spheres is investigated by dissipative particle dynamics simulations. The incremental pressure (Π) with respect to the system pressure with inactive swimmers comprises the direct contribution of the swimmers (π) and the indirect contribution of fluids associated with hydrodynamic interactions (HIs). The pressure can be determined from the bulk and confining wall and the former is always less than the latter. The π of dilute active dispersions is proportional to their active diffusivity while Π grows generally with propulsive force and run time. However, Π is always substantially less than π because of negative contributions to pressure by HIs. The wall pressure depends on the swimmer-wall interactions, verifying that pressure is not a state function for active spheres due to the HIs. Owing to the distinct flow patterns, Π varies with the swim-type (pusher and puller) subject to the same run-and-tumble parameters at high concentrations.

Diagnostic model generated by MRI-derived brain features in toddlers with autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder mainly showed atypical social interaction, communication, and restricted, repetitive patterns of behavior, interests and activities. Now clinic diagnosis of ASD is mostly based on psychological evaluation, clinical observation and medical history. All these behavioral indexes could not avoid defects such as subjectivity and reporter-dependency. Therefore researchers devoted themselves to seek relatively stable biomarkers of ASD as supplementary diagnostic evidence. The goal of present study is to generate relatively stable predictive model based on anatomical brain features by using machine learning technique. Forty-six ASD children and thirty-nine development delay children aged from 18 to 37 months were evolved in. As a result, the predictive model generated by regional average cortical thickness of regions with top 20 highest importance of random forest classifier showed best diagnostic performance. And random forest was proved to be the optimal approach for neuroimaging data mining in small size set and thickness-based classification outperformed volume-based classification and surface area-based classification in ASD. The brain regions selected by the models might attract attention and the idea of considering biomarkers as a supplementary evidence of ASD diagnosis worth exploring. Autism Res 2017, 0: 000-000. © 2016 International Society for Autism Research, Wiley Periodicals, Inc. Autism Res 2017, 10: 620-630. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

Two years changes in the development of caudate nucleus are involved in restricted repetitive behaviors in 2-5-year-old children with autism spectrum disorder.

Caudate nucleus volume is enlarged in autism spectrum disorder (ASD) and is associated with restricted and repetitive behaviors (RRBs). However, the trajectory of caudate nucleus volume in RRBs of young children remains unclear. Caudate nucleus volume was measured in 36 children with ASD and 18 matched 2-3-year-old subjects with developmentally delayed (DD) at baseline (Time 1) and at 2-year follow-up (Time 2). The differential growth rate in caudate nucleus volume was calculated. Further, the relationships between the development of caudate nucleus volume and RRBs were analyzed. Our results showed that caudate nucleus volume was significantly larger in the ASD group at both time points and the magnitude of enlargement was greater at Time 2. The rate of caudate nucleus growth during this 2-year interval was faster in children with ASD than DD. Right caudate nucleus volume growth was negatively correlated with RRBs. Findings from this study suggest developmental abnormalities of caudate nucleus volume in ASD. Longitudinal MRI studies are needed to explore the correlation between atypical growth patterns of caudate nucleus and phenotype of RRBs.

Generation of the first autosomal dominant osteopetrosis type II (ADO2) disease models.

Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl(-)/1H(+) antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene.

The involvement of MCT-1 oncoprotein in inducing mitotic catastrophe and nuclear abnormalities.

Centrosome amplification and chromosome abnormality are frequently identified in neoplasia and tumorigenesis. However, the mechanisms underlying these defects remain unclear. We here identify that MCT-1 is a centrosomal oncoprotein involved in mitosis. Knockdown of MCT-1 protein results in intercellular bridging, chromosome mis-congregation, cytokinesis delay, and mitotic death. Introduction of MCT-1 oncogene into the p53 deficient cells (MCT-1-p53), the mitotic checkpoint kinases and proteins are deregulated synergistically. These biochemical alterations are accompanied with increased frequencies of cytokinesis failure, multi-nucleation, and centrosome amplification in subsequent cell cycle. As a result, the incidences of polyploidy and aneuploidy are progressively induced by prolonged cell cultivation or further promoted by sustained spindle damage on MCT-1-p53 background. These data show that the oncoprotein perturbs centrosome structure and mitotic progression, which provide the molecular aspect of chromsomal abnormality in vitro and the information for understanding the stepwise progression of tumors under oncogenic stress.

Loss of p53 and MCT-1 overexpression synergistically promote chromosome instability and tumorigenicity.

MCT-1 oncoprotein accelerates p53 degradation by means of the ubiquitin-dependent proteolysis. Our present data show that induction of MCT-1 increases chromosomal translocations and deregulated G(2)-M checkpoint in response to chemotherapeutic genotoxin. Remarkably, increases in chromosome copy number, multinucleation, and cytokinesis failure are also promoted while MCT-1 is induced in p53-deficient cells. In such a circumstance, the Ras-mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-mitogen-activated protein kinase signaling activity and the expression of metastatic molecules are amplified. Given a p53-silencing background, MCT-1 malignantly transforms normal breast epithelial cells that are satisfactory for stimulating cell migration/adhesion and tumorigenesis. Detailed analyses of MCT-1 oncogenicity in H1299 p53-null lung cancer cells have shown that ectopically expressed MCT-1 advances xenograft tumorigenicity and angiogenesis, which cannot be completely suppressed by induction of p53. MCT-1 counteracts mutually with p53 at transcriptional levels. Clinical validations confirm that MCT-1 mRNA levels are differentially enriched in comparison between human lung cancer and nontumorigenic tissues. The levels of p53 mRNA are comparatively reduced in a subset of cancer specimens, which highly present MCT-1 mRNA. Our results indicate that synergistic promotions of chromosomal imbalances and oncogenic potency as a result of MCT-1 expression and p53 loss play important roles in tumor development.

CLCN7 polymorphisms and bone mineral density in healthy premenopausal white women and in white men.

INTRODUCTION: Mutations in the chloride channel 7 gene (CLCN7) cause osteopetrosis, and polymorphisms of CLCN7 in the non-disease allele are associated with penetrance of the autosomal dominant osteopetrosis (ADO) phenotype. Studies have also shown an association between CLCN7 polymorphisms and bone mineral density (BMD) in women. However, there is no study to date that has examined whether CLCN7 polymorphisms underlie normal variation of peak BMD in healthy premenopausal white women and in white men. METHODS: Six single nucleotide polymorphisms (SNPs) and one variable number tandem repeat (VNTR) polymorphism in the CLCN7 gene were genotyped. Association was tested between CLCN7 gene polymorphisms and both lumbar spine and femoral neck BMD. Healthy premenopausal white sisters (age 33.1+/-7.2, n=1692) and healthy white brothers (age 33.6+/-10.9, n=715) were studied. RESULTS: No significant association between CLCN7 gene polymorphisms and BMD at the lumbar spine or femoral neck was found in white women or white men. CONCLUSIONS: Genetic variation in the CLCN7 gene is not a major contributor to the variability in peak BMD at the femoral neck and lumber spine in healthy premenopausal white women and in white men.

Efficient and stable gene expression into human osteoclasts using an HIV-1-based lentiviral vector.

Since osteoclasts are terminally differentiated cells without proliferating activity, efficient and stable gene expression into these cells remains a difficulty. In the current study, we investigate gene transduction into human preosteoclasts by a replication defective lentivirus-based vector containing a modified HIV-1 genome. Human preosteoclasts (differentiating osteoclasts) were transduced with lentiviruses bearing an enhanced green fluorescent protein (EFGP) reporter gene. Transduction efficiencies were measured by flow cytometry for EGFP protein expression. Sorted human transduced preosteoclasts were replated and differentiated under human macrophage colony-stimulating factor and human receptor activator of NF-kappaB ligand. Mature osteoclasts were then analyzed by the cell viability assay, TRACP assay, and pit formation assay. Efficient gene transduction was obtained at multiplicity of infection of 10, and gene expression lasted for over 4 weeks using our protocol. Lentiviral transduction did not affect osteoclast survival, formation, or function. These results establish an efficient method for gene transduction into human preosteoclasts using a lentiviral vector. Importantly, these transduced preosteoclasts could differentiate into mature osteoclasts without a negative impact from the lentiviruses. This protocol provides a new tool for studies of osteoclast biology. Further work in this area may open new avenues for the study of osteoclast gene signaling and gene therapy of disorders of osteoclast function.