Blocking Buffer Research Articles

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.

LSPR based on-chip detection of dengue NS1 antigen in whole blood.

The development of a biosensor for rapid and quantitative detection of the dengue virus continues to remain a challenge. We report a lab-on-chip device that combines membrane-based blood plasma separation and a localized surface plasmon resonance (LSPR) based biosensor for on-chip detection of dengue NS1 antigen from a few drops of blood. The LSPR effect is realized by irradiating UV-NIR light having a spectral peak at 655 nm onto nanostructures fabricated via thermal annealing of a thin metal film. We study the effect of the resulting metal nanostructures on the LSPR performance in terms of sensitivity and limit of detection, by annealing silver films at temperatures ranging from 100 to 500 °C. The effect of annealing temperature on the nanostructure size and uniformity and the resulting optical characteristics are investigated. Further, the binding between non-targeted blood plasma proteins and NS1-antibody-functionalized nanostructures on the LSPR performance is studied by considering different blocking mechanisms. Using a nanostructure annealed at 200 °C and 2X-phosphate buffer saline with 0.05% Tween-20 as the blocking buffer, from 10 μL of whole blood, the device can detect NS1 antigen at a concentration as low as 0.047 μg mL−1 within 30 min. Finally, we demonstrate the detection of NS1 in the blood samples of dengue-infected patients and validate our results with those obtained from the gold-standard ELISA test.

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate.

BackgroundDue to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus.MethodsHere, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated.FindingsWe report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.InterpretationThe results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2.FundingThis work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.

Efficient fully blade-coated perovskite solar cells in air with nanometer-thick bathocuproine buffer layer

Fully printed perovskite solar cells (PSCs) were fabricated in air with all constituent layers, except for electrodes, deposited by the blade coating technique. The PSCs incorporated, for the first time, a nanometer-thick printed bathocuproine (BCP) hole blocking buffer using blade coating and deposited at relative humidity up to 50%. The PSCs with a p-i-n structure (glass/indium tin oxide (ITO)/poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS)/CH3NH3PbI3/[6,6]-phenyl-C61-butyric acid methyl ester (PCBM)/BCP/Ag) delivered a maximum power conversion efficiency (PCE) of 14.9% on an active area of 0.5 cm2 when measured under standard test conditions. The PSCs with a blade coated BCP delivered performance of 10% and 63% higher (in relative terms) than those incorporating a spin coated BCP or without any BCP film, respectively. The atomic force microscopy (AFM) showed that blade coated films were more homogeneous and acted also as a surface planarizer leading to a reduction of roughness which improved BCP/Ag interface lowering charge recombination. The demonstration of 15% efficient devices with all constituent layers, including nanometer-thick BCP (∼ 10 nm), deposited by blade coating in air, demonstrates a route for industrialization of this technology.

Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25mg would be tested, starting with 10mg in cohort 1. No significant difference between Mirococept at 10mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80mg (equivalent to 120mg in human kidney) that provides a basis for further clinical development.

Development and optimization of thermal contrast amplification lateral flow immunoassays for ultrasensitive HIV p24 protein detection

Detection of human immunodeficiency virus (HIV) p24 protein at a single pg/ml concentration in point-of-care (POC) settings is important because it can facilitate acute HIV infection diagnosis with a detection sensitivity approaching that of laboratory-based assays. However, the limit of detection (LOD) of lateral flow immunoassays (LFAs), the most prominent POC diagnostic platform, falls short of that of laboratory protein detection methods such as enzyme-linked immunosorbent assay (ELISA). Here, we report the development and optimization of a thermal contrast amplification (TCA) LFA that will allow ultrasensitive detection of 8 pg/ml p24 protein spiked into human serum at POC, approaching the LOD of a laboratory test. To achieve this aim, we pursued several innovations as follows: (a) defining a new quantitative figure of merit for LFA design based on the specific to nonspecific binding ratio (BR); (b) using different sizes and shapes of gold nanoparticles (GNPs) in the systematic optimization of TCA LFA designs; and (c) exploring new laser wavelengths and power regimes for TCA LFA designs. First, we optimized the blocking buffer for the membrane and running buffer by quantitatively measuring the BR using a TCA reader. The TCA reader interprets the thermal signal (i.e., temperature) of GNPs within the membrane when irradiated by a laser at the plasmon resonance wavelength of the particle. This process results in higher detection and quantitation of GNPs than in traditional visual detection (i.e., color intensity). Further, we investigated the effect of laser power (30, 100, 200 mW), GNP size and shape (30 and 100 nm gold spheres, 150 nm gold-silica shells), and laser wavelength (532, 800 nm). Applying these innovations to a new TCA LFA design, we demonstrated that 100 nm spheres with a 100 mW 532 nm laser provided the best performance (i.e., LOD = 8 pg/ml). This LOD is significantly better than that of the current colorimetric LFA and is in the range of the laboratory-based p24 ELISA. In summary, this TCA LFA for p24 protein shows promise for detecting acute HIV infection in POC settings.

Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis.

Human leishmaniasis which is considered a neglected tropical parasitic disease presents in three main clinical forms (i.e., cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL)) that are mainly determined by its causative species. Leishmania donovani, the most virulent and visceralizing parasite, is increasingly reported to cause CL in many countries in the world. Although CL is generally not considered to evoke a humoral immune response except for a nonrobust and a variable response in minority of cases, VL is associated with a clear strong humoral response. However, humoral response in L. donovani-induced CL has not been well evaluated before. A suitable serology-based assay is an essential primary step in such a study. An indirect enzyme-linked immunosorbent assay (ELISA) based on Leishmania promastigote crude antigen (Ag) was designed and optimized in order to utilize in further serological studies on this new clinical entity. Optimization included quantification of crude Ag, checkerboard titration method for determination of optimal concentrations for coating Ag, human sera and secondary antibody (Ab) with suitable coating buffer, blocking buffer, and incubating temperatures. The selected coating buffer was 0.02 M phosphate buffer, pH 6.8, and the blocking buffer was 2% fetal bovine serum with 0.01 M phosphate-buffered saline. At least 1 μg of crude Ag was required for coating the ELISA plate, while 1 : 1000 serum was used as primary Ab. The optimized concentration of secondary Ab was 1 : 64000 which might be altered according to manufacturer recommendations. The assay specificity was pre-evaluated using sera (n = 20 from each category) from confirmed CL patients and controls (other skin diseases which mimic CL, other systemic diseases that mimic VL, nonendemic healthy controls, and endemic healthy controls). This procedure described an optimization procedure of an ELISA technique for detection of anti-Leishmania antibodies in patients with L. donovani caused CL.

Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P=.009), decreased expression of injury biomarker NGAL (P=.012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P=.019, medulla P=.001), downregulation of interleukin (IL)-1β (P=.050), and upregulation of IL-10 (P<.047) and Indolamine-2, 3-dioxygenase (P=.050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P<.001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

Development and evaluation of an indirect ELISA based on recombinant structural protein VP2 to detect antibodies against duck hepatitis A virus

An indirect enzyme-linked immunosorbent assay (I-ELISA) based on the VP2 protein of duck hepatitis A virus type 3 (DHAV-3) was established in this study. The optimal dilutions of antigen, serum and goat anti-duck IgG conjugate were 1:1600 (2.23 μg/mL), 1:160 and 1:2000, respectively. The optimal blocking buffer was 1% skim milk. The cut-off value for the method was 0.25, and the analytical sensitivity of the method was 1:5120. The results of specific evaluation showed that except for DHAV-1, DHAV-3 antisera did not cross-react with any other common duck-sensitive pathogens, indicating that this method can be used to detect DHAV-3 and DHAV-1 antibodies. The coefficients of variation (CVs) were lower than 10 %. The coincidence rate between the VP2-DHAV-3-ELISA and the neutralization test was 93.3 %. In summary, the I-ELISA method based on VP2 protein has high sensitivity, specificity, and coincidence rate compared with the neutralization test and has advantages in serum monitoring. The I-ELISA method based on VP2 protein provides a simple and rapid method for the detection of anti-DHAV antibodies and the epidemiological monitoring of DHAV.

Dilution of digestive fish proteases with protein-free blocking reagents prevents loss of catalytic activity during microquantification

The activity of proteases may be lost during microquantification because of their nonspecific binding to tubes and pipette tips. In this study, the effects of blocking reagents (blockers) in dilutions of digestive proteases of yellowtail, Seriola quinqueradiata, were compared. EzBlock Chemi (EBC; protein free), Western BLoT Blocking Buffer (WBB; protein free), casein-based blocker, and bovine serum albumin were evaluated by the measurement of trypsin, chymotrypsin, and aminopeptidase activities using fluorogenic substrates. Protease activities were linear in extracts of pyloric caeca at dilution rates of up to at least 1/6400 with the four blockers. However, the activities of pancreatic enzymes differed, and the highest levels were observed with EBC and WBB. Extracts diluted with EBC and WBB were incubated for up to 24 h at 25 °C. Pancreatic enzyme activities increased with time in extracts diluted with EBC, but for those diluted with WBB there was no clear trend. The activities of chymotrypsin and aminopeptidase in whole-body extracts of individual yellowtail larvae extracted with EBC were significantly higher than in those extracted with 150 mM NaCl, suggesting that EBC inhibits the loss of enzyme activity that may otherwise occur during extraction. Thus, EBC is considered to be the most effective diluent for the microquantification of proteases amongst the four blockers tested.

Modulation of Apoptosis by the Dithiolethione ACDT as a Neuroprotective Mechanism Against Manganese‐Induced Toxicity

IntroductionManganese (Mn) is an essential trace metal involved in metabolic processes in both animals and plants. Overexposure to this metal can cause oxidative stress leading to a neurological disorder known as manganism. Excessive accumulation of Mn is believed to cause apoptosis in cells. Apoptosis is a tightly regulated process in which cell death is induced by the activation of specific signaling pathways through numerous regulatory proteins such as Bax and Bcl‐2. Dithiolethiones are a class of sulfur‐containing heterocyclic molecules. Among them, ACDT (5‐amino‐3‐thioxo‐3H‐(1,2) dithiole‐4‐carboxylic acid ethyl ester) has been shown to induce cellular production of the natural antioxidant glutathione. It has been proven that ACDT provides neuroprotection against Mn toxicity by activating the Nrf2‐Keap1 pathway. In this study, we hypothesized that ACDT also causes upregulation of anti‐apoptotic protein Bcl‐2 and downregulation of pro‐apoptotic protein Bax as an additional mechanism of neuroprotection.MethodsSH‐SY5Y cells were treated with ACDT at concentrations of 50, 75 and 100 μM. After 24 hours, the cells were either treated with MnCl2 (300 μM) or media for an additional 24 hours. Eight experimental groups included ‐ Control, MnCl2 (300 μM) alone, ACDT (50, 75, and 100 μM) alone and MnCl2 + ACDT (50, 75, and 100 μM) in combination.The BCA assay was conducted for the quantification of total protein in each sample. A Western blot analysis was performed to detect and measure the levels of Bcl‐2 and Bax proteins. Denatured samples were loaded onto polyacrylamide gels. Subsequently, gel electrophoresis was performed, and separated proteins were transferred onto a nitrocellulose membrane. The membrane was blocked with blocking buffer and then treated sequentially with primary and secondary antibodies, followed by a chemiluminescent substrate. The protein bands on the membranes were then detected using a C‐Digit scanner, and quantified using the ImageStudio Lite® software. Beta actin served as a loading control.ResultsA significant decrease in Bax protein levels (4‐fold) was observed in groups that were pretreated with ACDT (75 μM and 100 μM) and then exposed to Mn. A significant decrease in Bax was also observed in the ACDT (50 μM) + Mn group when compared to ACDT (50 μM) alone. No significant changes were observed in Bcl‐2 levels for any treatment group. However, a trend towards a dose‐dependent decrease in Bcl‐2 levels was observed in cells treated with the ACDT (50, 75 and 100 μM) and MnCl2 in combination.ConclusionsACDT was found to cause a downregulation of the pro‐apoptotic protein Bax, which likely contributed to its neuroprotective effects. As no changes in levels of the Bcl‐2 protein were observed, the data obtained only partially met our hypothesis. Additional research is currently underway to conclusively prove the role of apoptosis in ACDT’s neuroprotective pathway.Support or Funding InformationMCPHS University School of Pharmacy‐ SURF Program

Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

BackgroundHaemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat.ResultsPurified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21–103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 μg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%.ConclusionThese results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

Whole-mount Immunohistochemistry of Adult Zebrafish Retina for Advanced Imaging.

Immunohistochemistry is a widely used technique to examine the expression and subcellular localization of proteins. This technique relies on the specificity of antibodies and requires adequate penetration of antibodies into tissues. The latter is especially challenging for thick specimens, such as embryos and other whole-mount preparations. Here we describe an improved method of immunohistochemistry for retinal whole-mount preparations. We report that a cocktail of three reagents, Triton X-100, Tween-20, and DMSO, in blocking and antibody dilution buffers strongly enhances immunolabeling in whole-mount retinas from adult zebrafish. In addition, we establish that in whole retinal tissues, a classic epitope retrieval method, based on citrate buffer, is effective for immunolabeling membrane-associated proteins. Overall, this simple modification allows precise and reproducible immunolabeling of proteins in retinal whole-mounts.

Development of an indirect ELISA for detecting humoral immunodominant proteins of Mycoplasma hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera

BackgroundMycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of porcine enzootic pneumonia, which has been associated with economic losses due to reduced daily weight gain and feed efficiency. Although it has a small genome and no more than 1000 genes, M. hyopneumoniae can be cultured in cell free media. However, some proteins were not expressed or were only expressed in negligible amounts under culture conditions. Nevertheless, some of these proteins can be expressed at a high level and induce a strong and rapid immune response after M. hyopneumoniae infection. The unexpressed or less expressed proteins may play critical roles in pathogenesis and/or immune response. In order to find the differentially expressed proteins of M. hyopneumoniae between culture condition and infected animals, we established an indirect ELISA for the detection of humoral immunodominant proteins which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera by using Mhp366 protein which did not react with sera from bacterin-immunized pigs, but revealed a strong immunoreaction with porcine convalescent sera.ResultsThe checkerboard titration method was done by using porcine convalescent sera as positive sera and inactivated bacterin-induced hyperimmune sera as negative sera. The bacterial lysates of fusion proteins and free GST protein without dilution were the optimal coating antigens. The optimal blocking buffer was PBS with 10% FBS and 2.5% skimmed milk. In the checkboard ELISAs, when the sera were diluted at 1:500 and the HRP-labeled rabbit anti-pig IgG were diluted at 1:20000, most positive result was obtained for the assay.ConclusionsThis established indirect ELISA can be used as a tool for the detection of humoral immunodominant proteins of M. hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

Analysis of Glycosphingolipid Glycans by Lectin Microarrays.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates of cell membranes. Identification of unknown GSL-glycan structures is still a major challenge. To address this challenge, we developed a novel strategy for analysis of GSL-glycans from cultured cells based on a lectin microarray that can directly detect and reveal glycopatterns of GSL extracts without the need for glycan release. There were six steps to perform the analysis of GSL-glycans: (i) extraction of GSLs from cell pellets, (ii) quantification of GSL-glycans using orcinol-sulfuric acid reaction, (iii) preparation of lyso-GSLs by using sphingolipid ceramide N-deacylase, (iv) fluorescence labeling of lyso-GSLs, (v) detection by a lectin microarray, (vi) data acquisition and analysis. Simultaneously, a supplementary verification analysis for GSL-glycans was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Optimized experimental conditions, which consisted of the blocking buffer, incubation buffer, and appropriate GSL concentration, were investigated by analyzing the glycopatterns of a standard ganglioside (GM1a) via lectin microarray. The analysis of GSL-glycans from human hepatocarcinoma cell lines (MHCC97L, MHCC97H, and HCCLM3) showed that there were 27 lectins (e.g., WFA, MAL-II, and LTL) to give significantly different signals compared with a normal human liver cell line (HL-7702), indicating up- and/or down-regulations of corresponding glycopatterns such as α1-2 fucosylation and α2-3 sialylation, and changes of certain glycostructures such as Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glc:Cer and GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glc:Cer. The lectin microarray analysis of lyso-GSLs labeled by fluorescence has proven to be credible, which can provide the glycopatterns and detailed linkage information on GSL-glycans.

Development and evaluation of an indirect ELISA based on recombinant nonstructural protein 3A to detect antibodies to duck hepatitis A virus type 1

To develop an indirect enzyme-linked immunosorbent assay(I-ELISA) method based on 3A protein of duck hepatitis A virus type 1(DHAV-1) for detection of DHAV-1 antibody, the recombinant protein 3A of DHAV-1 was expressed in E.coli and detected by Western blotting with DHAV-1 infected duck serum. A 3A-ELISA method using the expressed 3A protein as coating antigen for the detection of antibodies to DHAV-1 was developed. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:200(6.185 μg/ml), 1:20 and 1:2000, respectively. The optimal blocking buffer was 5% BSA. The cutoff value was determined to be 0.274, and the analytical sensitivity was 1:1280. There was no cross reaction between DHAV-1 infected duck serum and other common pathogenic duck serum, indicating that I-ELISA could be used to detect DHAV-1 infected duck serum. The coefficients of variation(CVs) were lower than 10%. The concordance between the I-ELISA based on the 3A subunit of DHAV-1 and that based on the whole DHAV-1 particle was 92.7%. Taken together, the 3A-ELISA method is a highly sensitive and specific test that could be used for screening for DHAV-1 infection and monitoring DHAV-1 antibody.

The evolution of schematic representations of flow shop scheduling problems

The schematic representation of schedules has been a useful tool for researchers; in this paper, we review existing network-based schematics for flow shop models. We also propose new schematics for flow shops with blocking, synchronous transfers, no-wait processing, no-idle time and limited buffer space. Using schematics, we explain some of the past findings and present new results. We also explain flow shop anomalies/paradoxes. Finally, we propose schematics for four flow shop models using the concept of string analog.

Remote Estimation of Correlated Sources Under Energy Harvesting Constraints

Remote estimation with an energy harvesting sensor with a limited data and energy buffer is considered. The sensor node observes an unknown temporally correlated field and communicates its observations to a remote fusion center using the energy it harvested. The fusion center employs linear minimum mean-square error estimation to reconstruct the unknown field. We provide performance guarantees for the estimation error under a block transmission scheme, where at each transmission block, data and energy buffers are completely emptied. Our bounds provide insights into how statistical properties of the energy harvesting process and buffer sizes may affect the estimation error. In particular, these bounds suggest insensitivity of the performance to buffer sizes for signals with low degree of freedom and suggest performance improvements with increasing buffer sizes for signals with relatively higher degree of freedom. Depending only on the mean, variance, and finite support of the energy arrival process, these results provide insights for the energy and data buffer sizes for deployment in future energy harvesting wireless sensing systems.

Cu/Cu2O nanocomposite films as a p-type modified layer for efficient perovskite solar cells

Cu/Cu2O films grown by ion beam sputtering were used as p-type modified layers to improve the efficiency and stability of perovskite solar cells (PSCs) with an n-i-p heterojunction structure. The ratio of Cu to Cu2O in the films can be tuned by the oxygen flow ratio (O2/(O2 + Ar)) during the sputtering of copper. Auger electron spectroscopy was performed to determine the elemental composition and chemical state of Cu in the films. Ultraviolet photoelectron spectroscopy and photoluminescence spectroscopy revealed that the valence band maximum of the p-type Cu/Cu2O matches well with the perovskite. The Cu/Cu2O film not only acts as a p-type modified layer but also plays the role of an electron blocking buffer layer. By introducing the p-type Cu/Cu2O films between the low-mobility hole transport material, spiro-OMeTAD, and the Ag electrode in the PSCs, the device durability and power conversion efficiency (PCE) were effectively improved as compared to the reference devices without the Cu/Cu2O interlayer. The enhanced PCE is mainly attributed to the high hole mobility of the p-type Cu/Cu2O film. Additionally, the Cu/Cu2O film serves as a protective layer against the penetration of humidity and Ag into the perovskite active layer.

Development of a sensitive Luminex xMAP-based microsphere immunoassay for specific detection of Iris yellow spot virus

BackgroundIris yellow spot virus (IYSV) is an Orthotospovirus that infects most Allium species. Very few approaches for specific detection of IYSV from infected plants are available to date. We report the development of a high-sensitive Luminex xMAP-based microsphere immunoassay (MIA) for specific detection of IYSV.ResultsThe nucleocapsid (N) gene of IYSV was cloned and expressed in Escherichia coli to produce the His-tagged recombinant N protein. A panel of monoclonal antibodies (MAbs) against IYSV was generated by immunizing the mice with recombinant N protein. Five specific MAbs (16D9, 11C6, 7F4, 12C10, and 14H12) were identified and used for developing the Luminex xMAP-based MIA systems along with a polyclonal antibody against IYSV. Comparative analyses of their sensitivity and specificity in detecting IYSV from infected tobacco leaves identified 7F4 as the best-performed MAb in MIA. We then optimized the working conditions of Luminex xMAP-based MIA in specific detection of IYSV from infected tobacco leaves by using appropriate blocking buffer and proper concentration of biotin-labeled antibodies as well as the suitable ratio between the antibodies and the streptavidin R-phycoerythrin (SA-RPE). Under the optimized conditions the Luminex xMAP-based MIA was able to specifically detect IYSV with much higher sensitivity than conventional enzyme-linked immunosorbent assay (ELISA). Importantly, the Luminex xMAP-based MIA is time-saving and the whole procedure could be completed within 2.5 h.ConclusionsWe generated five specific MAbs against IYSV and developed the Luminex xMAP-based MIA method for specific detection of IYSV in plants. This assay provides a sensitive, high-specific, easy to perform and likely cost-effective approach for IYSV detection from infected plants, implicating potential broad usefulness of MIA in plant virus diagnosis.