Supramolecular Recognition-Mediated Layer-by-Layer Self-Assembled Gold Nanoparticles for Customized Sensitivity in Paper-Based Strip Nanobiosensors. Huang Xiaolin,Zhou Yaofeng,Ding Lu,Yu Guocan,Leng Yuankui,Lai Weihua,Xiong Yonghua,Chen Xiaoyuan Small (Weinheim an der Bergstrasse, Germany) Herein, a smart supramolecular self-assembly-mediated signal amplification strategy is developed on a paper-based nanobiosensor to achieve the sensitive and customized detection of biomarkers. The host-guest recognition between β-cyclodextrin-coated gold nanoparticles (AuNPs) and 1-adamantane acetic acid or tetrakis(4-carboxyphenyl)porphyrin is designed and applied to the layer-by-layer self-assembly of AuNPs at the test area of the strip. Thus, the amplified platform exhibits a high sensitivity with a detection limit at subattogram levels (approximately dozens of molecules per strip) and a wide dynamic range of concentration over seven orders of magnitude. The applicability and universality of this sensitive platform are demonstrated in clinically significant ranges to measure carcinoembryonic antigen and HIV-1 capsid p24 antigen in spiked serum and clinical samples. The customized biomarker detection ability for the on-demand needs of clinicians is further verified through cycle incubation-mediated controllable self-assembly. Collectively, the supramolecular self-assembly amplification method is suitable as a universal point-of-care diagnostic tool and can be readily adapted as a platform technology for the sensitive assay of many different target analytes. 10.1002/smll.201903861
    Colorimetric Paper-Based Immunosensor for Simultaneous Determination of Fetuin B and Clusterin toward Early Alzheimer's Diagnosis. Brazaca Laís C,Moreto José R,Martín Aída,Tehrani Farshad,Wang Joseph,Zucolotto Valtencir ACS nano Alzheimer's disease is a devastating condition characterized by a progressive and slow brain decay in elders. Here, we developed a paper-based lateral flow immunoassay for simultaneous and fast determination of Alzheimer's blood biomarkers, fetuin B and clusterin. Selective antibodies to targeted biomarkers were immobilized on gold nanoparticles (AuNPs) and deposited on paper pads. After adding the sample on the paper-based device, the biofluid laterally flows toward the selective antibody, permitting AuNP-Ab accumulation on the test zone, which causes a color change from white to pink. Image analysis was performed using a customized algorithm for the automatic recognition of the area of analysis and color clustering. Colorimetric detection was compared to electrochemical methods for the precise quantification of biomarkers. The best performance was found for the color parameter "*". Good linearity ( = 0.988 and 0.998) and reproducibility (%RSD = 2.79% and 1.82%, = 3) were demonstrated for the quantification of fetuin B and clusterin, respectively. Furthermore, the specificity of the immunosensor was tested on mixtures of proteins, showing negligible cross-reactivity and good performance in complex environments. We believe that our biosensor has a potential for early-stage diagnosis of Alzheimer's disease and toward a better understanding of Alzheimer's developing mechanisms. 10.1021/acsnano.9b06571
    Wash- and Amplification-Free Digital Immunoassay Based on Single-Particle Motion Analysis. Akama Kenji,Iwanaga Niina,Yamawaki Koya,Okuda Masaki,Jain Krupali,Ueno Hiroshi,Soga Naoki,Minagawa Yoshihiro,Noji Hiroyuki ACS nano Digital enzyme-linked immunosorbent assay (ELISA) is a powerful analytical method for highly sensitive protein biomarker detection. The current protocol of digital ELISA requires multiple washing steps and signal amplification using an enzyme, which could be the potential drawback in diagnosis. In this study, we propose a digital immunoassay method, which we call "Digital HoNon-ELISA" (digital homogeneous non-enzymatic immunosorbent assay) for highly sensitive detection without washing and signal amplification. Target antigen molecules react with antibody-coated magnetic nanoparticles, which are then magnetically pulled into femtoliter-sized reactors. The antigens on the particles are captured by antibodies anchored on the bottom surface of the reactor molecular tethers. Magnetic force enhances the efficiency of particle encapsulation in the reactors. Subsequent physical compartmentalization of the particles enhances the binding efficiency of antigen-carrying particles to the antibodies. The tethered particles show characteristic Brownian motion within a limited space by the molecular tethering, which is distinct from free diffusion or nonspecific binding of antigen-free particles. The number of tethered particles directly correlates with the concentration of the target antigen. Digital HoNon-ELISA was used with a prostate-specific antigen to achieve a detection of 0.093 pg/mL, which is over 9.0-fold the sensitivity of commercialized highly sensitive ELISA (0.84 pg/mL) and comparable to digital ELISA (0.055 pg/mL). This digital immunoassay strategy has sensitivity similar to digital ELISA with simplicity similar to homogeneous assay. Such similarity allows for potential application in rapid and simple digital diagnostic tests without the need for washing and enzymatic amplification. 10.1021/acsnano.9b05917
    The Role of Nanoparticle Design in Determining Analytical Performance of Lateral Flow Immunoassays. Zhan Li,Guo Shuang-Zhuang,Song Fayi,Gong Yan,Xu Feng,Boulware David R,McAlpine Michael C,Chan Warren C W,Bischof John C Nano letters Rapid, simple, and cost-effective diagnostics are needed to improve healthcare at the point of care (POC). However, the most widely used POC diagnostic, the lateral flow immunoassay (LFA), is ∼1000-times less sensitive and has a smaller analytical range than laboratory tests, requiring a confirmatory test to establish truly negative results. Here, a rational and systematic strategy is used to design the LFA contrast label (i.e., gold nanoparticles) to improve the analytical sensitivity, analytical detection range, and antigen quantification of LFAs. Specifically, we discovered that the size (30, 60, or 100 nm) of the gold nanoparticles is a main contributor to the LFA analytical performance through both the degree of receptor interaction and the ultimate visual or thermal contrast signals. Using the optimal LFA design, we demonstrated the ability to improve the analytical sensitivity by 256-fold and expand the analytical detection range from 3 log to 6 log for diagnosing patients with inflammatory conditions by measuring C-reactive protein. This work demonstrates that, with appropriate design of the contrast label, a simple and commonly used diagnostic technology can compete with more expensive state-of-the-art laboratory tests. 10.1021/acs.nanolett.7b02302
    An ultra-sensitive immunoassay detects and quantifies soluble Aβ oligomers in human plasma. Liu Lei,Kwak Hyunchang,Lawton Trebor L,Jin Shan-Xue,Meunier Angela L,Dang Yifan,Ostaszewski Beth,Pietras Alison C,Stern Andrew M,Selkoe Dennis J Alzheimer's & dementia : the journal of the Alzheimer's Association INTRODUCTION:Evidence strongly suggests that soluble oligomers of amyloid beta protein (oAβ) help initiate the pathogenic cascade of Alzheimer's disease (AD). To date, there have been no validated assays specific for detecting and quantifying oAβ in human blood. METHODS:We developed an ultrasensitive oAβ immunoassay using a novel capture antibody (71A1) with N-terminal antibody 3D6 for detection that specifically quantifies soluble oAβ in the human brain, cerebrospinal fluid (CSF), and plasma. RESULTS:Two new antibodies (71A1; 1G5) are oAβ-selective, label Aβ plaques in non-fixed AD brain sections, and potently neutralize the synaptotoxicity of AD brain-derived oAβ. The 71A1/3D6 assay showed excellent dilution linearity in CSF and plasma without matrix effects, good spike recovery, and specific immunodepletion. DISCUSSION:We have created a sensitive, high throughput, and inexpensive method to quantify synaptotoxic oAβ in human plasma for analyzing large cohorts of aged and AD subjects to assess the dynamics of this key pathogenic species and response to therapy. 10.1002/alz.12457
    Comparison between electrochemical and photoelectrochemical detection of dopamine based on titania-ceria-graphene quantum dots nanocomposite. Ahmadi Nasrin,Bagherzadeh Mojtaba,Nemati Ali Biosensors & bioelectronics In this study, titania-ceria-graphene quantum dot (TC-GQD) nanocomposite was synthesized by hydrothermal method for the first time. The prepared nanomaterials were characterized by XRD, FTIR dynamic light scattering (DLS), FESEM, HRTEM, and EDX spectroscopy along with elemental mapping. The synergistic effect of the nanocomposite components was studied by diffuse reflectance spectroscopy (DRS) and electrical conductivity meter. The results showed that band gap of TC-GQD nanocomposite was shifted to visible lights relative to its components (1.3 eV), and electrical conductivity of the sample was significant increased to 89.5 μS cm. After chemical and physical characterization, prepared new nanocomposites were used to design a new electrochemical (EC) and photoelectrochemical (PEC) dopamine (DA) sensors. In both EC and PEC methods effecting experimental parameters were optimized. Due to the synergic effect of the nanocomposite components, an outstanding photocurrent response was observed for DA based on PEC sensor. A linear calibration curve with a lower detection limit of 22 nM DA, and sensitivity of 13.8 mA/mM, in a wider range of 0.3-750 μM DA, was obtained for TC-GQD/GCE electrode in PEC. While, the TC-GQD/GCE electrode detected DA in the range of 1-500 μM DA, with two linear calibration curve, detection limit of 0.22 μM DA, and sensitivity of 4.9 mA/mM, in the EC. Observed results from EC and PEC sensors are presented and compared. 10.1016/j.bios.2019.111977
    The sweet detection of rolling circle amplification: Glucose-based electrochemical genosensor for the detection of viral nucleic acid. Ciftci Sibel,Cánovas Rocío,Neumann Felix,Paulraj Thomas,Nilsson Mats,Crespo Gaston A,Madaboosi Narayanan Biosensors & bioelectronics Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125 μg mL of GOx with 5 mM glucose using phosphate buffer saline (PBS), monitored for 1 min were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings. 10.1016/j.bios.2019.112002
    Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum. Bosch Irene,de Puig Helena,Hiley Megan,Carré-Camps Marc,Perdomo-Celis Federico,Narváez Carlos F,Salgado Doris M,Senthoor Dewahar,O'Grady Madeline,Phillips Elizabeth,Durbin Ann,Fandos Diana,Miyazaki Hikaru,Yen Chun-Wan,Gélvez-Ramírez Margarita,Warke Rajas V,Ribeiro Lucas S,Teixeira Mauro M,Almeida Roque P,Muñóz-Medina José E,Ludert Juan E,Nogueira Mauricio L,Colombo Tatiana E,Terzian Ana C B,Bozza Patricia T,Calheiros Andrea S,Vieira Yasmine R,Barbosa-Lima Giselle,Vizzoni Alexandre,Cerbino-Neto José,Bozza Fernando A,Souza Thiago M L,Trugilho Monique R O,de Filippis Ana M B,de Sequeira Patricia C,Marques Ernesto T A,Magalhaes Tereza,Díaz Francisco J,Restrepo Berta N,Marín Katerine,Mattar Salim,Olson Daniel,Asturias Edwin J,Lucera Mark,Singla Mohit,Medigeshi Guruprasad R,de Bosch Norma,Tam Justina,Gómez-Márquez Jose,Clavet Charles,Villar Luis,Hamad-Schifferli Kimberly,Gehrke Lee Science translational medicine The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses. 10.1126/scitranslmed.aan1589
    Engineering Staphylococcal Protein A for high-throughput affinity purification of monoclonal antibodies. Amritkar Vinod,Adat Satish,Tejwani Vijay,Rathore Anurag,Bhambure Rahul Biotechnology advances Protein A chromatography is one of the most widely used purification steps in the manufacturing of the various classes of recombinant and non-recombinant antibodies. Due to the higher cost, lower binding capacity, and limited life cycle of Protein A ligand, this affinity-based purification step is often one of the most significant contributors to the cost of manufacturing of monoclonal antibody (mAb) products. In the last decade, there has been significant progress in improving the Protein A chromatography throughput by designing new engineered Staphylococcal Protein A (SPA) variants with higher dynamic binding capacity, considerable alkaline tolerance, and mild acidic elution pH. This review aims at summarizing the various protein engineering approaches used for improving the throughput of the Protein A-based affinity purification of various immunoglobulins. With biopharmaceutical producers operating under ever-increasing pressure towards reducing the cost of manufacturing, these advances in engineered protein A variants will help in processing larger cell culture volumes with high throughput and thereby significantly lower the cost of raw materials. 10.1016/j.biotechadv.2020.107632