Research

15 pages, 4298 KiB

Open AccessArticle

3D Printed Modular Immunofiltration Columns for Frequency Mixing-Based Multiplex Magnetic Immunodetection

by Stefan Achtsnicht, Julia Tödter, Julia Niehues, Matthias Telöken, Andreas Offenhäusser, Hans-Joachim Krause and Florian Schröper

Sensors 2019, 19(1), 148; https://0-doi-org.brum.beds.ac.uk/10.3390/s19010148 - 03 Jan 2019

Cited by 10 | Viewed by 4778

Abstract

For performing point-of-care molecular diagnostics, magnetic immunoassays constitute a promising alternative to established enzyme-linked immunosorbent assays (ELISA) because they are fast, robust and sensitive. Simultaneous detection of multiple biomolecular targets from one body fluid sample is desired. The aim of this work is [...] Read more.

For performing point-of-care molecular diagnostics, magnetic immunoassays constitute a promising alternative to established enzyme-linked immunosorbent assays (ELISA) because they are fast, robust and sensitive. Simultaneous detection of multiple biomolecular targets from one body fluid sample is desired. The aim of this work is to show that multiplex magnetic immunodetection based on magnetic frequency mixing by means of modular immunofiltration columns prepared for different targets is feasible. By calculations of the magnetic response signal, the required spacing between the modules was determined. Immunofiltration columns were manufactured by 3D printing and antibody immobilization was performed in a batch approach. It was shown experimentally that two different target molecules in a sample solution could be individually detected in a single assaying step with magnetic measurements of the corresponding immobilization filters. The arrangement order of the filters and of a negative control did not influence the results. Thus, a simple and reliable approach to multi-target magnetic immunodetection was demonstrated. Full article

(This article belongs to the Special Issue Immunosensors - 2018 Trends and Perspective)

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10 pages, 1839 KiB

Open AccessArticle

Nanobody-Alkaline Phosphatase Fusion Protein-Based Enzyme-Linked Immunosorbent Assay for One-Step Detection of Ochratoxin A in Rice

by Zhichang Sun, Xuerou Wang, Qi Chen, Yonghuan Yun, Zongwen Tang and Xing Liu

Sensors 2018, 18(11), 4044; https://0-doi-org.brum.beds.ac.uk/10.3390/s18114044 - 20 Nov 2018

Cited by 17 | Viewed by 4202

Abstract

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. [...] Read more.

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL⁻¹ and a limit of detection of 0.059 ng mL⁻¹, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice. Full article

(This article belongs to the Special Issue Immunosensors - 2018 Trends and Perspective)

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12 pages, 1965 KiB

Open AccessArticle

A Regenerative Immunoaffinity Layer Based on the Outer Membrane of Z-Domains Autodisplaying E. coli for Immunoassays and Immunosensors

by Daseul Jeon, Jae-Chul Pyun, Joachim Jose and Min Park

Sensors 2018, 18(11), 4030; https://0-doi-org.brum.beds.ac.uk/10.3390/s18114030 - 19 Nov 2018

Cited by 5 | Viewed by 2669

Abstract

Through orientation control of antibodies, Z-domains autodisplaying Escherichia coli outer cell membrane (OM) may be utilized to improve the sensitivity and limit of detection (LOD) of immunoassays and immunosensors. A regenerative immunoaffinity layer based on Z-domains autodisplaying E. coli OM was developed for [...] Read more.

Through orientation control of antibodies, Z-domains autodisplaying Escherichia coli outer cell membrane (OM) may be utilized to improve the sensitivity and limit of detection (LOD) of immunoassays and immunosensors. A regenerative immunoaffinity layer based on Z-domains autodisplaying E. coli OM was developed for the surface plasmon resonance (SPR) biosensor. Regeneration conditions for the Z-domains autodisplaying E. coli OM-based immunoassays and immunosensors were optimized by varying pH and detergent concentration. An E. coli cell-based HRP immunoassay was tested and validated in three sequential regenerative immunoassays under optimal conditions. The OM of Z-domains autodisplaying E. coli was isolated and coated on the two-dimensional substrate (microplate). The OM-based HRP immunoassay was tested and validated in four regenerative immunoassays. This regenerative OM layer was applied to the SPR biosensor. Z-domains autodisplaying OM layered onto the gold surface of SPR biosensors was developed, and the OM-based regenerative immunoaffinity layer with orientation control was tested using CRP analyte. The SPR biosensor regenerative immunoaffinity layer demonstrated that CRP biosensing was repeated for five regeneration cycles with less than 2% signal difference. Therefore, the newly developed regenerative immunoaffinity layer with antibody orientation control may improve biosensing sensitivity and reduce the cost of medical diagnosis. Full article

(This article belongs to the Special Issue Immunosensors - 2018 Trends and Perspective)

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16 pages, 3490 KiB

Open AccessArticle

How to Improve Sensitivity of Sandwich Lateral Flow Immunoassay for Corpuscular Antigens on the Example of Potato Virus Y?

by Shyatesa C. Razo, Vasily G. Panferov, Irina V. Safenkova, Yuri A. Varitsev, Anatoly V. Zherdev, Elena N. Pakina and Boris B. Dzantiev

Sensors 2018, 18(11), 3975; https://0-doi-org.brum.beds.ac.uk/10.3390/s18113975 - 15 Nov 2018

Cited by 23 | Viewed by 5421

Abstract

A simple approach was proposed to decrease the detection limit of sandwich lateral flow immunoassay (LFIA) by changing the conditions for binding between a polyvalent antigen and a conjugate of gold nanoparticles (GNPs) with antibodies. In this study, the potato virus Y (PVY) [...] Read more.

A simple approach was proposed to decrease the detection limit of sandwich lateral flow immunoassay (LFIA) by changing the conditions for binding between a polyvalent antigen and a conjugate of gold nanoparticles (GNPs) with antibodies. In this study, the potato virus Y (PVY) was used as the polyvalent antigen, which affects economically important plants in the Solanaceae family. The obtained polyclonal antibodies that are specific to PVY were characterized using a sandwich enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). For LFIA, the antibodies were conjugated with GNPs with a diameter of 17.4 ± 1.0 nm. We conducted LFIAs using GNP conjugates in a dried state on the test strip and after pre-incubation with a sample. Pre-incubating the GNP conjugates and sample for 30 s was found to decrease the detection limit by 60-fold from 330 ng∙mL⁻¹ to 5.4 ng∙mL⁻¹ in comparison with conventional LFIA. The developed method was successfully tested for its ability to detect PVY in infected and uninfected potato leaves. The quantitative results of the proposed LFIA with pre-incubation were confirmed by ELISA, and resulted in a correlation coefficient of 0.891. The proposed approach is rapid, simple, and preserves the main advantages of LFIA as a non-laboratory diagnostic method. Full article

(This article belongs to the Special Issue Immunosensors - 2018 Trends and Perspective)

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15 pages, 2267 KiB

Open AccessArticle

Significance Testing and Multivariate Analysis of Datasets from Surface Plasmon Resonance and Surface Acoustic Wave Biosensors: Prediction and Assay Validation for Surface Binding of Large Analytes

by Mihaela Puiu, Lucian-Gabriel Zamfir, Valentin Buiculescu, Angela Baracu, Cristina Mitrea and Camelia Bala

Sensors 2018, 18(10), 3541; https://0-doi-org.brum.beds.ac.uk/10.3390/s18103541 - 19 Oct 2018

Cited by 6 | Viewed by 3171

Abstract

In this study, we performed uni- and multivariate data analysis on the extended binding curves of several affinity pairs: immobilized acetylcholinesterase (AChE)/bioconjugates of aflatoxin B₁(AFB₁) and immobilized anti-AFB₁ monoclonal antibody/AFB₁-protein carriers. The binding curves were recorded [...] Read more.

In this study, we performed uni- and multivariate data analysis on the extended binding curves of several affinity pairs: immobilized acetylcholinesterase (AChE)/bioconjugates of aflatoxin B₁(AFB₁) and immobilized anti-AFB₁ monoclonal antibody/AFB₁-protein carriers. The binding curves were recorded on three mass sensitive cells operating in batch configurations: one commercial surface plasmon resonance (SPR) sensor and two custom-made Love wave surface-acoustic wave (LW-SAW) sensors. We obtained 3D plots depicting the time-evolution of the sensor response as a function of analyte concentration using real-time SPR binding sensograms. These “calibration” surfaces exploited the transient periods of the extended kinetic curves, prior to equilibrium, creating a “fingerprint” for each analyte, in considerably shortened time frames compared to the conventional 2D calibration plots. The custom-made SAW sensors operating in different experimental conditions allowed the detection of AFB₁-protein carrier in the nanomolar range. Subsequent statistical significance tests were performed on unpaired data sets to validate the custom-made LW-SAW sensors. Full article

(This article belongs to the Special Issue Immunosensors - 2018 Trends and Perspective)

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9 pages, 1315 KiB

Open AccessArticle

Blocking-Free ELISA Using a Gold Nanoparticle Layer Coated Commercial Microwell Plate

by Ruijia Huang, Ke Zhang, Guoshuai Zhu, Zhencheng Sun, Songliang He and Wenwen Chen

Sensors 2018, 18(10), 3537; https://0-doi-org.brum.beds.ac.uk/10.3390/s18103537 - 19 Oct 2018

Cited by 6 | Viewed by 4428

Abstract

Enzyme-linked immunosorbent assays (ELISA) show extensive application in immunoassays, to detect and monitor protein biomarkers in clinical diagnosis. Nevertheless, the time required and its multiple steps limit its application. We take advantage of a polyethyleneimine (PEI) gold nanoparticle (GNP) coated microwell plate to [...] Read more.

Enzyme-linked immunosorbent assays (ELISA) show extensive application in immunoassays, to detect and monitor protein biomarkers in clinical diagnosis. Nevertheless, the time required and its multiple steps limit its application. We take advantage of a polyethyleneimine (PEI) gold nanoparticle (GNP) coated microwell plate to perform blocking-free ELISA, in which no nonspecific protein adsorption appears on the GNP layer. If the PEI-GNP coated microwell plate and immobilization of captured antibodies on the plate are prepared in advance, such as using an ELISA kit, the whole ELISA process can be finished in less than 2 h. Meanwhile, we have ensured that the GNP layer can preserve the precision and good linearity of ELISA without causing negative effects on the plate. Full article

(This article belongs to the Special Issue Immunosensors - 2018 Trends and Perspective)

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12 pages, 1869 KiB

Open AccessArticle

Glyphosate Determination by Coupling an Immuno-Magnetic Assay with Electrochemical Sensors

by Francesca Bettazzi, Aline Romero Natale, Eduardo Torres and Ilaria Palchetti

Sensors 2018, 18(9), 2965; https://0-doi-org.brum.beds.ac.uk/10.3390/s18092965 - 06 Sep 2018

Cited by 44 | Viewed by 5791

Abstract

Glyphosate (N-(phosphonomethyl)glycine) is the most frequently used broad-spectrum herbicide worldwide. Its mechanism of action is based on the inhibition of an enzyme that is essential to plant growth. Its intensive use has caused global contamination to occur, which has not only [...] Read more.

Glyphosate (N-(phosphonomethyl)glycine) is the most frequently used broad-spectrum herbicide worldwide. Its mechanism of action is based on the inhibition of an enzyme that is essential to plant growth. Its intensive use has caused global contamination to occur, which has not only affected the ecosystems, but even food and other objects of common use. Thus, there is a pronounced need for developing analytical methods for glyphosate determination in different matrices. Here, an electrochemical competitive immunoassay, based on the use of antibody-modified magnetic particles, has been developed. Tetramethylbenzidine (TMB) has been used as an enzymatic substrate. The extent of the affinity reaction has been achieved by monitoring the current value, due to the reduction of the enzymatic product. A disposable screen-printed electrochemical cell has been used. The calibration curve has been recorded in the 0–10,000 ng/L concentration range, with a detection limit of 5 ng/L and quantification limit of 30 ng/L. The electrochemical immunoassay has also been applied to the analysis of spiked beer samples. Full article

(This article belongs to the Special Issue Immunosensors - 2018 Trends and Perspective)

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15 pages, 2458 KiB

Open AccessArticle

An Impedimetric Biosensor Based on Ionic Liquid-Modified Graphite Electrodes Developed for microRNA-34a Detection

by Ece Kesici, Ece Eksin and Arzum Erdem

Sensors 2018, 18(9), 2868; https://0-doi-org.brum.beds.ac.uk/10.3390/s18092868 - 31 Aug 2018

Cited by 17 | Viewed by 4023

Abstract

In the present work, an impedimetric nucleic acid biosensor has been designed for the purpose of detection of microRNA (miRNA). Ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL))-modified chemically activated pencil graphite electrodes (PGEs) were used for the sensitive and selective detection of miRNA-34a. After covalent [...] Read more.

In the present work, an impedimetric nucleic acid biosensor has been designed for the purpose of detection of microRNA (miRNA). Ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL))-modified chemically activated pencil graphite electrodes (PGEs) were used for the sensitive and selective detection of miRNA-34a. After covalent activation of the PGE surface using covalent agents (CAs), the ionic liquid (IL) was immobilized onto the surface of the chemically activated PGE by passive adsorption. The electrochemical and microscopic characterization of the IL/CA/PGEs was performed by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and scanning electron microscopy (SEM). DNA probe concentration, miRNA target concentration, and also the hybridization time and wet adsorption time were optimized by using the EIS technique. Then, the hybridization occurred between specific DNA probes and miRNA-34a was immobilized onto the surface of the IL/CA/PGEs. The impedimetric detection of miRNA-DNA hybrid was performed by EIS. The detection limit (DL) was calculated in a linear concentration range of 2–10 µg/mL miRNA-34a target, and it was found to be 0.772 µg/mL (109 nM) in phosphate buffer solution (PBS) and 0.826 µg/mL (117 nM) in diluted fetal bovine serum (FBS). The selectivity of impedimetric biosensor for miRNA-34a was also tested against to other non-complementary miRNA sequences both in buffer media, or diluted FBS. Full article

(This article belongs to the Special Issue Immunosensors - 2018 Trends and Perspective)

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12 pages, 4180 KiB

Open AccessArticle

Electrochemical Detection of E. coli O157:H7 in Water after Electrocatalytic and Ultraviolet Treatments Using a Polyguanine-Labeled Secondary Bead Sensor

by Michael G. Beeman, Ugochukwu C. Nze, Himanshu J. Sant, Hammad Malik, Swomitra Mohanty, Bruce K Gale and Krista Carlson

Sensors 2018, 18(5), 1497; https://0-doi-org.brum.beds.ac.uk/10.3390/s18051497 - 10 May 2018

Cited by 13 | Viewed by 4796

Abstract

The availability of clean drinking water is a significant problem worldwide. Many technologies exist for purifying drinking water, however, many of these methods require chemicals or use simple methods, such as boiling and filtering, which may or may not be effective in removing [...] Read more.

The availability of clean drinking water is a significant problem worldwide. Many technologies exist for purifying drinking water, however, many of these methods require chemicals or use simple methods, such as boiling and filtering, which may or may not be effective in removing waterborne pathogens. Present methods for detecting pathogens in point-of-use (POU) sterilized water are typically time prohibitive or have limited ability differentiating between active and inactive cells. This work describes a rapid electrochemical sensor to differentially detect the presence of active Escherichia coli (E. coli) O157:H7 in samples that have been partially or completely sterilized using a new POU electrocatalytic water purification technology based on superradicals generated by defect laden titania (TiO₂) nanotubes. The sensor was also used to detect pathogens sterilized by UV-C radiation for a comparison of different modes of cell death. The sensor utilizes immunomagnetic bead separation to isolate active bacteria by forming a sandwich assay comprised of antibody functionalized secondary magnetic beads, E. coli O157:H7, and polyguanine (polyG) oligonucleotide functionalized secondary polystyrene beads as an electrochemical tag. The assay is formed by the attachment of antibodies to active receptors on the membrane of E. coli, allowing the sensor to differentially detect viable cells. Ultravioloet (UV)-C radiation and an electrocatalytic reactor (ER) with integrated defect-laden titania nanotubes were used to examine the sensors’ performance in detecting sterilized cells under different modes of cell death. Plate counts and flow cytometry were used to quantify disinfection efficacy and cell damage. It was found that the ER treatments shredded the bacteria into multiple fragments, while UV-C treatments inactivated the bacteria but left the cell membrane mostly intact. Full article

(This article belongs to the Special Issue Immunosensors - 2018 Trends and Perspective)

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