Suspension Array Research Articles

Microsphere-based suspension array for simultaneous recognition and quantification of multiple cancer-associated miRNA via DNAzyme-Mediated signal amplification

Herein, a novel suspension array of polystyrene (PS) beads for simultaneous recognition and quantification of multiple cancer-associated microRNAs (miRNAs) using flow cytometry has been reported. The suspension array contained three moieties, streptavidin-modified PS beads, biotin-labeled substrate strands (17S) of the 17–8 DNAzyme and two split DNAzyme parts (PA, PB). 17S was labeled with 6-carboxyfluorescein (FAM) and Dabcyl on both sides of the ribonucleic acid. Once the target miRNAs appear, they can bind with the corresponding PA and PB to form an active secondary structure of DNAzyme. The active DNAzyme can cleave 17S and remove Dabcyl from the bead’s surface, thus recovering the FAM’s fluorescence intensity. Furthermore, the released target miRNA can autonomously move to the neighboring inactive DNAzyme for further cleavage, thus amplifying the fluorescence signal. Therefore, the target miRNAs can be quantified by reading the fluorescence intensity output from flow cytometry. The PS beads-based suspension array for the target miRNA in buffer shows good selectivity and high sensitivity. Via binding with a different pair of PA and PB, this suspension sensor array has successfully typed and quantified cancer-associated miRNAs of miR-21, miR-155, miR-335, and miR-122 in buffer and serum conditions.

Bead-based multiplex detection of dengue biomarkers in a portable imaging device.

Dengue is one of the most rapidly spreading mosquito-borne viral diseases in the world. Differential diagnosis is a crucial step for the management of the disease and its epidemiology. Point-of-care testing of blood-borne dengue biomarkers provides an advantageous approach in many health care settings, and the ability to follow more than one biomarker at once could significantly improve the management of the disease. Bead-based multiplex technologies (suspension array) can measure multiple biomarker targets simultaneously by using recognition molecules immobilized on microsphere beads. The overarching objective of our work is to develop a portable detection device for the simultaneous measurement of multiple biomarkers important in dengue diagnosis, monitoring and treatment. Here, we present a bead-based assay for the detection of one of the four serotypes of dengue virus non-structural protein (DENV-NS1) as well as its cognate human IgG. In this system, the fluorescent microspheres containing the classification fluorophore and detection fluorophore are imaged through a microfluidic chip using an infinity-corrected microscope system. Calibration curves were plotted for median fluorescence intensity against known concentrations of DENV-NS1 protein and anti-NS1 human IgG. The limit of quantitation was 7.8 ng/mL and 15.6 ng/mL, respectively. The results of this study demonstrate the feasibility of the multiplex detection of dengue biomarkers and present its analytical performance parameters. The proposed imaging device holds potential for point-of-care testing of biomarkers on a highly portable system, and it may facilitate the diagnosis and prevention of dengue as well as other infectious diseases.

The effect of apremilast therapy on skin cytokine levels in patients with psoriasis

Objective — Assessment of phosphodiesterase-4 inhibitor (apremilast) therapy’s influence on skin cytokine levels in patients with moderate-to-severe and severe psoriasis. Material and Methods — An open, uncontrolled study was conducted. 16 patients with plaque psoriasis (13 men, 3 women; mean ± standard deviation (SD) age 35.1±9.7 years, range 21-60) were enrolled. The mean Psoriasis Area and Severity Index (PASI) was 20.7±8.93 (range 10-47). All patients were prescribed apremilast 30 milligrams (mg) per os (PO) Bis In Die (BID). The efficacy of therapy was evaluated by PASI at 14 and 26 weeks of therapy. Lesional skin samples were collected at baseline and weeks 14 and 26. Levels of interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL -33, interferon (INF)-γ, Soluble CD40-ligand (sCD40L), tumor necrosis factor (TNF)-α were measured by microsphere-based suspension array technology (Luminex® xMAP™ system). Results — Levels of cytokines (except IL-4 and IL-33) in lesional skin samples were found to have decreased at week 14 compared with those at baseline. Similar decreases were seen for IL-23, IL-25, IL-31, sCD40L at week 26. In contrast, the levels of other cytokines increased again at week 26, in comparison with baseline. Levels of IL-4 and IL-33 rose throughout the follow-up period. Cytokine levels in lesional skin samples were compared with those of healthy controls both at baseline and during therapy. Conclusion — The results of our study show that administering apremilast therapy to patients with psoriasis can bring the levels of cytokines involved in the IL-23/IL-17 axis in the lesional skin to the level of cytokine in non-lesional skin and to the levels in the skin of healthy individuals.

Photolithographic Fabrication of Upconversion Barcodes for Multiplexed Molecular Detection

AbstractThe demands of clinical diagnostics and genetic analysis for high‐performance multiplex detection systems have driven substantial advancement from planar microarrays to suspension arrays for accurate encoding, inventorying, and detecting extensive databases of biomolecules. Despite the multitude of related works and the commercial availability of nanostructured arrays for biodetection, it remains challenging to develop biodetection modules of massive coding permutation with high production rates and fast reaction kinetics . Herein, the concurrent improvement in both production and coding capacities without compromising detection kinetics is reported using lithography‐based upconversion barcodes as the basis for bioassay detection modules. This method exploits microstructural forms of dense graphical encoding, with barcode production up to 109 per run at extensive color permutations. The upconversion barcoding technique requires a million times lower analyte consumption compared to currently available technologies. Moreover, the use of upconversion barcodes is demonstrated in a DNA hybridization test with high target specificity, low detection limit, and ease of flexible surface bioconjugation, enabling potential development of biomarkers in the analysis of massive proteomics, genomics, and metabolomics data.

Loss of Osteopontin Expression Reduces HSV-1-Induced Corneal Opacity.

PurposeCorneal opacity and neovascularization (NV) are often described as outcomes of severe herpes simplex virus type 1 (HSV-1) infection. The current study investigated the role of colony-stimulating factor 1 receptor (CSF1R)+ cells and soluble factors in the progression of HSV-1-induced corneal NV and opacity.MethodsMaFIA mice were infected with 500 plaque-forming units of HSV-1 in the cornea following scarification. From day 10 to day 13 post-infection (pi), mice were treated with 40 µg/day of AP20187 (macrophage ablation) or vehicle intraperitoneally. For osteopontin (OPN) neutralization experiments, C57BL/6 mice were infected as above and treated with 2 µg of goat anti-mouse OPN or isotypic control IgG subconjunctivally every 2 days from day 4 to day 12 pi. Mice were euthanized on day 14 pi, and tissue was processed for immunohistochemistry to quantify NV and opacity by confocal microscopy and absorbance or detection of pro- and anti-angiogenic and inflammatory factors and cells by suspension array analysis and flow cytometry, respectively.ResultsIn the absence of CSF1R+ cells, HSV-1-induced blood and lymphatic vessel growth was muted. These results correlated with a loss in fibroblast growth factor type 2 (FGF-2) and an increase in OPN expression in the infected cornea. However, a reduction in OPN expression in mice did not alter corneal NV but significantly reduced opacity.ConclusionsOur data suggest that CSF1R+ cell depletion results in a significant reduction in HSV-1-induced corneal NV that correlates with the loss of FGF-2 expression. A reduction in OPN expression was aligned with a significant drop in opacity associated with reduced corneal collagen disruption.

Microorganism@UiO-66-NH2 Composites for the Detection of Multiple Colorectal Cancer-Related microRNAs with Flow Cytometry.

High-throughput analyses of multitarget markers can facilitate rapid and accurate clinical diagnosis. Suspension array assays, a flow cytometry-based analysis technology, are among some of the most promising multicomponent analysis methods for clinical diagnostics and research purposes. These assays are appropriate for examining low-volume, complex samples having trace amounts of analytes due to superior elimination of background. Physical shape is an important and promising code system, which uses a set of visually distinct patterns to identify different assay particles. Here, we presented a morphology recognizable suspension arrays based on the microorganisms with different morphologies. In this study, UiO-66-NH2 (UiO stands for University of Oslo) metal-organic frameworks (MOFs), was wrapped on the microorganism surface to form an innovative class of microorganism@UiO-66-NH2 composites for suspension array assays. The use of microorganisms endowed composites barcoding ability with their different morphology and size. Meanwhile, the UiO-66-NH2 provided a stable rigid shell, large specific surface area, and metal(IV) ions with multiple binding sites, which could simplify the protein immobilization procedure and enhance detection sensitivity. With this method, simultaneous detection of three colorectal cancer-related microRNA (miRNA), including miRNA-21, miRNA-17, and miRNA-182, could be easily achieved with femtomolar sensitivity by using a commercial flow cytometer. The synergy between microorganisms and MOFs make the composites a prospective barcoding candidate with excellent characteristics for multicomponent analysis, offering great potential for the development of high throughput and accurate diagnostics.

Relationship between retinal blood flow and cytokines in central retinal vein occlusion

BackgroundWe evaluated the relationship between retinal blood flow and aqueous humor levels of cytokines/growth factors in patients with central retinal vein occlusion (CRVO).MethodsIn an observational study, 64 eyes of 64 CRVO patients were examined before anti-vascular endothelial growth factor (VEGF) therapy. Blood flow was assessed in large vessels around and at the optic disk by determining the mean blur rate using laser speckle flowgraphy. Aqueous humor samples were obtained from the patients during anti-VEGF therapy and levels of the following molecules were measured by the suspension array method: soluble VEGF receptor (sVEGFR)-1, sVEGFR-2, VEGF, plancental growth factor (PlGF), platelet-derived growth factor (PDGF)-AA, soluble intercellular adhesion molecule (sICAM)-1, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6, IL-8, IL-12(p70), and IL-13.ResultsThe mean blur rate of the affected eye was significantly lower than that of the unaffected eye. The mean blur rate showed a significant negative correlation with the log-transformed aqueous humor levels of PlGF, sICAM-1, and IL-8, but not VEGF.ConclusionsThese findings suggest that retinal blood flow velocity might be more strongly correlated with inflammatory factors than VEGF in patients with nonischemic CRVO and macular edema.

Antibody responses to the RTS,S/AS01E vaccine and Plasmodium falciparum antigens after a booster dose within the phase 3 trial in Mozambique

The RTS,S/AS01E vaccine has shown consistent but partial vaccine efficacy in a pediatric phase 3 clinical trial using a 3-dose immunization schedule. A fourth-dose 18 months after the primary vaccination was shown to restore the waning efficacy. However, only total IgG against the immunodominant malaria vaccine epitope has been analyzed following the booster. To better characterize the magnitude, nature, and longevity of the immune response to the booster, we measured levels of total IgM, IgG, and IgG1-4 subclasses against three constructs of the circumsporozoite protein (CSP) and the hepatitis B surface antigen (HBsAg, also present in RTS,S) by quantitative suspension array technology in 50 subjects in the phase 3 trial in Manhiça, Mozambique. To explore the impact of vaccination on naturally acquired immune responses, we measured antibodies to P. falciparum antigens not included in RTS,S. We found increased IgG, IgG1, IgG3 and IgG4, but not IgG2 nor IgM, levels against vaccine antigens 1 month after the fourth dose. Overall, antibody responses to the booster dose were lower than the initial peak response to primary immunization and children had higher IgG and IgG1 levels than infants. Higher anti-Rh5 IgG and IgG1-4 levels were detected after the booster dose, suggesting that RTS,S partial protection could increase some blood stage antibody responses. Our work shows that the response to the RTS,S/AS01E booster dose is different from the primary vaccine immune response and highlights the dynamic changes in subclass antibody patterns upon the vaccine booster and with acquisition of adaptive immunity to malaria.

P1709THE ROLE OF CXCL9 AND CXCL13 IN PREDICTION OF POSTTRANSPLANT INFECTION IN KIDNEY TRANSPLANT RECIPIENTS

Abstract Background and Aims Chemokines are deeply involved in the process of inflammatory and immune responses. Interferon-γ-inducible chemokines C-X-C motif chemokine 9 and 10 (CXCL9 and CXCL10) are significantly associated with Th1 cells and monocytes and rise rapidly during early episode of renal allograft rejection and various infectious diseases. CXCL13 is one of the most potent B cells and T follicular helper (Tfh) cells chemoattractants when acts through its cognate receptor CXCR5. Recent work of CXCL13 indicated a critical immune regulatory role in both multiple infectious diseases and kidney transplantation. Additionally, C-C motif chemokine 2 (CCL2) is shown to be is critical for chronic kidney diseases. The aim of this study was to identify the predictive role of serum CXCL9, CXCL10, CXCL13 or CCL2 on kidney posttransplant infection. Method 95 kidney transplant recipients (KTRs) were enrolled in this study. 31 recipients experienced at least once infection episodes within the first posttransplant 12 months and 64 KTRs did not experience any infection episode during the follow-up period. Serum CXCL9, CXCL10, CXCL13 and CCL2 at the time points of pre-transplantation and post-transplant 30 days (POD 30) were analyzed with Bio-Plex® suspension array system. Results It was found that serum level of POD 30 CXCL9 and POD 30 CXCL13 was associated with infection within one year after transplantation (P=0.021, P=0.002, respectively, shown in Figure 1). The serum level of CXCL9 and CXCL13 before surgery, the serum level of CCL2 and CXCL10 before and after surgery were not associated with infection within posttransplant one year (P>0.05, shown in Figure 1). The combination of POD 30 CXCL9 plus POD 30 CXCL13 provided the best results with AUC of 0.721 (95%CI, 0.591-0.852), sensitivity of 71.4% and specificity of 68.5% at the optimal cut-off value of 52.72 pg/ml (shown in Figure 2). Conclusion Chemokines CXCL9 and CXCL13 as important chemokines could be used to predict the occurrence of infection within posttransplant one year in kidney transplant recipients.

Anti-VEGF Therapy Reduces Inflammation in Diabetic Macular Edema

Purpose: Correlations among the aqueous flare value (an indicator of inflammation), several functional-morphologic parameters, and aqueous humor levels of multiple cytokines or inflammatory factors were investigated in patients with diabetic macular edema (DME) receiving intravitreal rani­bizumab injection (IRI). Methods: Aqueous humor levels of 12 cytokines, growth factors, or inflammatory factors were measured in 46 DME patients who received IRI. Vascular endothelial growth factor (VEGF), soluble VEGF receptor (sVEGFR), and the other cytokines/inflammatory factors were measured by the suspension array method. In addition, aqueous flare values were measured with a laser flare meter, and central macular thickness (CMT) was examined by optical coherence tomography. Results: At 1 month after IRI therapy, the aqueous flare value showed a significant decrease compared with before treatment (baseline). Significant correlations were noted between the aqueous flare value and the aqueous humor levels of 6 factors/cytokines, including sVEGFR-1, placental growth factor, monocyte chemoattractant protein 1, soluble intercellular adhesion molecule-1, interleukin (IL)-6, and interferon-inducible 10-kDa protein (IP-10). There was also a significant correlation between the change in aqueous flare value and improvement in CMT 1 month after IRI. Conclusions: These findings suggest that IRI reduces subclinical inflammation and that the aqueous flare value is influenced by inflammatory factors/cytokines. In addition, the change in the aqueous flare value may be an indicator of the response of CMT to IRI in patients with DME.

Prevalence and clinical impact of malaria infections detected with a highly sensitive HRP2 rapid diagnostic test in Beninese pregnant women

BackgroundWhile sub-microscopic malarial infections are frequent and potentially deleterious during pregnancy, routine molecular detection is still not feasible. This study aimed to assess the performance of a Histidine Rich Protein 2 (HRP2)-based ultrasensitive rapid diagnostic test (uRDT, Alere Malaria Ag Pf) for the detection of infections of low parasite density in pregnant women.MethodsThis was a retrospective study based on samples collected in Benin from 2014 to 2017. A total of 942 whole blood samples collected in 327 women in the 1st and 3rd trimesters and at delivery were tested by uRDT, conventional RDT (cRDT, SD BIOLINE Malaria Ag Pf), microscopy, quantitative polymerase chain-reaction (qPCR) and Luminex-based suspension array technology targeting P. falciparum HRP2. The performance of each RDT was evaluated using qPCR as reference standard. The association between infections detected by uRDT, but not by cRDT, with poor maternal and birth outcomes was assessed using multivariate regression models.ResultsThe overall positivity rate detected by cRDT, uRDT, and qPCR was 11.6% (109/942), 16.2% (153/942) and 18.3% (172/942), respectively. Out of 172 qPCR-positive samples, 68 were uRDT-negative. uRDT had a significantly better sensitivity (60.5% [52.7–67.8]) than cRDT (44.2% [36.6–51.9]) and a marginally decreased specificity (93.6% [91.7–95.3] versus 95.7% [94.0–97.0]). The gain in sensitivity was particularly high (33%) and statistically significant in the 1st trimester. Only 28 (41%) out of the 68 samples which were qPCR-positive, but uRDT-negative had detectable but very low levels of HRP2 (191 ng/mL). Infections that were detected by uRDT but not by cRDT were associated with a 3.4-times (95%CI 1.29–9.19) increased risk of anaemia during pregnancy.ConclusionsThis study demonstrates the higher performance of uRDT, as compared to cRDTs, to detect low parasite density P. falciparum infections during pregnancy, particularly in the 1st trimester. uRDT allowed the detection of infections associated with maternal anaemia.

EVALUATION OF A MULTIPLEX ARBOVIRUS SEROLOGY ASSAY AS A TOOL FOR SEROSURVEILLANCE

Abstract Arthropod-borne viruses (Arboviruses) constitute a serious threat to global health by causing significant mortality and morbidity. Recent years have seen the emergence of arboviruses due to climatic changes, the adaptation of mosquitoes, and other arthropods to a newer geographic area, urbanization, and increased global travel and trade. Currently, most serosurveillance programs focus on single pathogen testing that may miss the prevalent pathogen or misdiagnose due to the presence of cross-reacting antibodies from closely related arboviruses. In the wake of the 2015–2016 Zika virus outbreaks, we developed a multiplex suspension array for the detection of IgG antibodies and avidity to multiple analytes of arboviruses on the Luminex platform to overcome these limitations. We used optically coded magnetic microspheres to immobilize antigens from 10 different arboviruses, namely, Zika (ZIKV), Dengue all four serotypes (DENV 1–4), West Nile (WNV), Japanese Encephalitis (JEV), Yellow Fever (YFV), Tick-Borne Encephalitis (TBEV), and Chikungunya (CHIKV. This 18-plex assay also includes five different internal controls that assure the assay and reagent performance. Initial characterization of the platform was performed using experimentally infected primate samples collected longitudinally before and after infection with ZIKV. In this paper, we describe the results of the evaluation of this multiplex platform using over 400 well-characterized healthy and diseased state human serum and plasma samples from various commercial and collaborating resources. We conclude that this assay provides a relevant tool for efficient monitoring of population based arboviral antibodies in endemic areas and also for detection of outbreaks.

Optimisation and standardisation of a multiplex immunoassay of diverse Plasmodium falciparum antigens to assess changes in malaria transmission using sero-epidemiology.

Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX © qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodiumfalciparumrecombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX © platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample dilution, and bead storage conditions. Quality control procedures and data normalisation methods were developed to address high-throughput assay processing. Antigen-specific limits of quantification (LOQs) were also estimated using both in-house and WHO reference serum as positive controls. Results: Antigen-specific bead coupling was optimised across five serum dilutions and two positive controls, resulting in concentrations operational within stable analytical ranges. Coupled beads were stable after storage at room temperature (22⁰C) for up to eight weeks. High sensitivity and specificity for distinguishing positive and negative controls at serum sample dilutions of 1:500 (AUC 0.94 95%CI 0.91-0.96) and 1:1000 (AUC 0.96 95%CI 0.94-0.98) were observed. LOQs were also successfully estimated for all analytes but varied by antigen and positive control. Conclusions: This study demonstrates that developing a standardised malaria-specific qSAT protocol for a diverse set of antigens is achievable, though further optimisations may be required. Quality control and data standardisation methods may also be useful for future analysis of large sero-epidemiological surveys.

Spectral-Domain Phase Microscopy for Thickness Encoded Suspension Array

A system containing spectral-domain phase microscopy and fluorescence microscopy has been developed to decode and detect thickness-encoded suspension arrays. The spectral-domain phase imaging path is used for decoding the thickness of the micro glass pieces while the fluorescence imaging path is used for quantifying the targeted analytes. The spectral-domain phase microscopy uses a spectral domain phase retrieval technique so that it has a high-dynamic thickness imaging range and submicron-scale thickness resolution, increasing the encoding efficiency and decoding precision. The thickness decoding capability of the system has been demonstrated by multiplexed immunoassay experiments. The quantitative analysis capability has been verified by concentration gradient experiments.

Point‐of‐Care Diagnostics: Multiplexed Luminescence Oxygen Channeling Immunoassay Based on Dual‐Functional Barcodes with a Host–Guest Structure: A Facile and Robust Suspension Array Platform (Small 17/2020)

In article number 1907521, Hong Xu, Hongchen Gu, and co-workers present a suspension array platform that combines barcode technology and a luminescent oxygen channeling immunoassay. A unique dual-functional barcode based on a host-guest structure is constructed as the microcarrier to facilitate multiplexed detection with simple “mix-and-measure” operability. This work provides an ideal platform for the development of point-of-care diagnostics.

Evidence of altered Th17 pathway signatures in the cerebrospinal fluid of patients with Guillain Barré Syndrome

Data indexing the contribution of various immuno-inflammatory components in the cerebrospinal fluid (CSF) towards the pathophysiology of Guillain Barré Syndrome (GBS) are limited. Th17 pathway plays crucial role in many immune mediated disorders of the nervous system. This study was aimed at exploring the role of Th17 pathway related cytokines in the CSF of patients with GBS. Levels of multiple key cytokines of Th17 pathway in CSF of patients with GBS (N = 37) and controls (N = 37) were examined in this prospective study using Bio-plex Pro Human Th17 cytokine assays in a Multiplex Suspension Array platform. The findings were correlated with clinical features and electrophysiological subtypes. Three key cytokines of Th17 pathway (IL-6, IL-17A and IL-22) were significantly elevated in CSF of patients with GBS as compared to controls. There was a positive correlation between the levels of IL-6 and IL-17A as well as between the levels of IL-17A and IL-22 in the CSF of patients with GBS. The CSF levels of IL-6 and IL-22 were negatively correlated with the duration of symptoms of GBS. None of the studied cytokines correlated with functional disability scores at admission to hospital or with the electrophysiological subtypes. Identification of Th17 pathway signatures in CSF sheds more insights into the pathogenic role of Th17 cells in GBS. These findings complement the contemporary knowledge and tender further support towards the involvement of Th17 pathway in GBS.

Multiplexed Luminescence Oxygen Channeling Immunoassay Based on Dual-Functional Barcodes with a Host-Guest Structure: A Facile and Robust Suspension Array Platform.

The development of a powerful immunoassay platform with capacities of both simplicity and high multiplexing is promising for disease diagnosis. To meet this urgent need, for the first time, a multiplexed luminescent oxygen channeling immunoassay (multi-LOCI) platform by implementation of LOCI with suspension array technology is reported. As the microcarrier of the platform, a unique dual-functional barcode with a host-guest structure composed of a quantum dot host bead (QDH) and LOCI acceptor beads (ABs) is designed, in which QDH provides function of high coding capacity while ABs facilitate the LOCI function. The analytes bridge QDH@ABs and LOCI donor beads (DBs) into a close proximity, forming a QDH@ABs-DBs "host-guest-satellite" superstructure that generates both barcode signal from QDH and LOCI signal induced by singlet oxygen channeling between ABs and DBs. Through imaging-based decoding, different barcodes are automatically distinguished and colocalized with LOCI signals. Importantly, the assay achieves simultaneous detection of multiple analytes within one reaction, simply by following a "mix-and-measure" protocol without the need for tedious washing steps. Furthermore, the multi-LOCI platform is validated for real sample measurements. With the advantages of robustness, simplicity, and high multiplexing, the platform holds great potential for the development of point-of-care diagnostics.

Hydrogel-based microbeads for Raman-encoded suspension array using the reversed-phase suspension polymerization method and ultraviolet light curing.

A one-step synthesis using the reversed-phase suspension polymerization method and ultraviolet light curing is proposed for preparing the Raman-encoded suspension array (SA). The encoded microcarriers are prepared by doping the Raman reporter molecules into an aqueous phase, and then dispersing the aqueous phase in an oil phase and curing by ultraviolet light irradiation. The multiplexed biomolecule detection and various concentration experiments confirm the qualitative and quantitative analysis capabilities of the Raman-encoded SA with a limit of detection of 52.68pM. The narrow bandwidth of the Raman spectrum can achieve a large number of codes in the available spectral range and the independence between the encoding channel and the fluorescent label channel provides the encoding method with high accuracy. This preparation method is simple and easy to operate, low in cost, and high in efficiency. A large number of hydrogel-based encoding microbeads could be quickly obtained with good biocompatibility. Most importantly, concentrating plenty of Raman reporter molecules inside the microbeads increases the signal intensity and means the molecular assembly is not limited by the functional groups; thus, the types of materials available for Raman encoding method are expanded. Furthermore, the signal intensity-related encoding method is verified by doping different proportions of Raman reporter molecules with our proposed synthesis method, which further increases the detection throughput of Raman-encoded SA. Graphical Abstract.

Carbon nanotube-mediated antibody-free suspension array for determination of typical endocrine-disrupting chemicals.

Acarbon nanotube (CNT)-mediated antibody-free suspension array (CASA) by integration of functionalized CNTs and aptamer (Apt) into xMAP® technology for simultaneous determination of typical endocrine-disrupting chemicals(EDCs) was developed. The interaction between CNTs and Apt acts as an effective and straightforward signal recognition, transformation, and amplification strategy. The amino-functionalized CNTs are covalently modified on the carboxyl-functionalized magnetic bead (MB) and further physically bridging with biotinylated Apt. CNTs on the surface of MBs not only increase the amount of Apt for target binding andsignal amplification but also maintain the biological activity of Apt. After magnetic separation, the encoded MB address was distinguished and the concentration of the target in the liquid was negatively correlated with median fluorescence intensity. A series of environmental water samples were analysed by CASA, traditional immuno-SA, and competitive inhibition enzyme-linked immunosorbent assay for validation. The results obtained using CASA well matched for the multiplexed detection of various targets with dynamic concentrationrange from 6.40 × 10-5 to 4.00μgL-1 within 1h. The method also confirmed good selectively, accuracy, and consistency with high-performance liquid chromatography. Graphical abstractSchematic representation of amino-functionalized carbon nanotube (CNT)-bridged antibody-free suspension array for detecting of three typical endocrine-disrupting chemicals. The amino-functionalized CNTs are covalently modified and further physically interacted with biotinylated aptamer featuring in the recognition and binding with the target of interest.

A unique ex vivo tumor model: 3D cocultured system with cancer and stromal cells including blood microvessels.

211 Background: Importance of interaction between cancer and stromal cells has been widely recognized in tumor progression and tolerance against treatment. Although 2D culture and spheroid consisting only cancer cells still remains the preferred platform for most laboratory preclinical studies while these provide only limited information about tumor microenvironment. In order to mimic the patient tumor tissue, ex vivo model which recaptures the tumor microenvironment is required. Methods: Layered 3D stromal tissues comprising microvascular network were produced by culturing fibroblasts and endothelial cells coated with the extra-cellular matrix (ECM) and natural polysaccharide, namely collagen and heparin. The layered 3D stromal tissues and co-cultured tumor were morphologically evaluated by HE stain, immunohistochemistry and immunofluorescence (IF). Their gene expression and secretome profile were characterized by RNA-sequencing and bio-plex suspension array technologies. Furthermore, drug sensitivity assay were conducted using popular colorectal cancer cell lines, and patient-derived cell lines (PDCs) established in the laboratory of JFCR. Remaining cancer cells post drug treatment were quantified by IF and imaging analysis. Results: The 3D stromal tissues including CD31 positive luminal structure were multi-layered (approximately 20 layers), and the tendency that dense microvascular network was formed nearby cancer cells was observed. In comparison with 2D culture or 3D mono-cultured spheroid model, decreased drug sensitivities were represented in the layered 3D co-cultured model. Omics profiles difference among models suggest that our 3D model has some similarity to in vivo tumor. Conclusions: We developed the layered 3D stromal tissue culture system including blood micro-vessels. Drug sensitivity in the co-cultured tumors may reflect the response of cancer cells in in vivo. Our unique 3D ex vivo model appear to be a valuable tool for drug evaluation, and thus testing approved and/or developing compounds with patient-derived cells would enable better prediction their efficacy.