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Recent Advances and Applications of Multiplexed Analysis and Multiplexed Nanobiosensors

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Special Issue Editor

Special Issue Information

Dear Colleagues,

Multiplex analysis has emerged as front-runner technology for the early and reliable diagnosis of diseases through its ability to achieve rapid simultaneous detection of multiple biomarkers, proteins and other clinically significant analytes in a single sample. This approach also enables the collection of more data, a significant reduction in cost, a lower likelihood of errors and a more rapid sample throughput. Recent developments in multiplexing strategies have enabled the clinical diagnosis of some diseases; rapid and reliable simultaneous detection of multiple SARS-CoV-2 mutations; and selective and simultaneous tagging in a single assay of bacteria, cancer cells and individual molecules, such as proteins and DNA. Smartphone-based multiplexed sensors have also emerged as excellent contenders for point-of-care diagnostics.

This Special Issue aims to collect and highlight recent advances in multiplex analysis based on the use of biosensors, nanobiosensors, PCR assays, fluorometric immunoassay, serological assay, CRISPR/Cas multiplexed biosensing, lab-on-chip assay, microfluidic array, lateral flow detection, multiplexed abundance assay, electrochemiluminescent immunoassay, ELISA-based multiplexing, SERS-based multiplex analysis, multiplexed targeted mass spectrometry assay, multiplexed abundance assay and any other related multiplexing methods that have or can be used for the early and reliable diagnosis of diseases or other health conditions, including those utilizing nanomaterials to improve multiple analyte detection. We welcome reviews and research articles in any of these areas.

Prof. Dr. Samuel B. Adeloju
Guest Editor

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Keywords

multiplex PCR assay, including real-time assay
multiplex tandem PCR
fluorometric immunoassay
multiplex bead-based assay
electrochemiluminescent immunoassay
ELISA-based multiplex method
multiplexed serological assay
multiplexed targeted mass spectrometry assay
SERS-based multiplex biomolecular analysis
CRISPR/Cas multiplexed biosensing
multiplexed electrohydrodynamic biosensor
multiple synchronized biosensors
multiplexed abundance assay
lab-on-chip assay
lateral flow detection
microfluidic array
SERS tags
nanomaterial supported and/or enhanced multiplexing

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Published Papers (3 papers)

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Research

17 pages, 2133 KiB

Open AccessArticle

A Truncated Multi-Thiol Aptamer-Based SARS-CoV-2 Electrochemical Biosensor: Towards Variant-Specific Point-of-Care Detection with Optimized Fabrication

by Sergio Roberto Molina Ramirez, Nafiseh Samiseresht, Mateo Alejandro Martínez-Roque, Ferdinando Catania, Kevin Graef, Martin Rabe, Andreas Offenhäusser, Dirk Mayer and Gabriela Figueroa-Miranda

Biosensors 2025, 15(1), 24; https://doi.org/10.3390/bios15010024 - 6 Jan 2025

Cited by 1 | Viewed by 1283

Abstract

With the goal of fast and accurate diagnosis of infectious diseases, this study presents a novel electrochemical biosensor that employs a refined aptamer (C9t) for the detection of spike (S) protein SARS-CoV-2 variants in a flexible multielectrode aptasensor array with PoC capabilities. Two aptamer modifications were employed: removing the primer binding sites and including two dithiol phosphoramidite anchor molecules. Thus, reducing fabrication time from 24 to 3 h and increasing the stability and sparseness for multi-thiol aptasensors compared to a standard aptasensor using single thiols, without a reduction in aptamer density. The biosensor fabrication, optimization, and detection were verified in detail by electrochemistry, QCM-D, SPR, and XPS. The analyte–receptor binding was further confirmed spectroscopically at the level of individual molecules by AFM-IR. The aptasensor possesses a low limit of detection (8.0 fg/mL), the highest sensitivity reported for S protein (209.5 signal per concentration decade), and a wide dynamic detection range (8.0 fg/mL–38 ng/mL) in nasopharyngeal samples, covering the clinically relevant range. Furthermore, the C9t aptasensor showed high selectivity for SARS-CoV-2 S proteins over biomarkers for MERS-CoV, RSV, and Influenza. Even more, it showed a three times higher sensitivity for the Omicron in comparison to the Wuhan strain (wild type), alpha, and beta variants of the SARS-CoV-2 virus. Those results demonstrate the creation of an affordable and variant-selective refined C9t aptasensor that outperformed current rapid diagnosis tests. Full article

(This article belongs to the Special Issue Recent Advances and Applications of Multiplexed Analysis and Multiplexed Nanobiosensors)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
QCM-D measurements show the change of frequency in blue, and dissipation energy in orange. The aptamer solution flowed into the chamber in a 30 min window and was then cleaned with a buffer for 10 min. This procedure was repeated three times. For the PEG solution, two of these procedures were conducted. No change after the first buffer cleaning was observed for either. B stands for Buffer. Full article ">Figure 2
SPR and AFM-IR investigations. (a) SPR measurements with concentration series of 55, 166, 500, 1500, and 3000 nM of the truncated C9t aptamer for binding affinity determination to wild-type (dash line) and Omicron (continuous line) S proteins. (b) AFM-IR topography (inset) and spectra of aptamer C9t/PEG layer and S protein. The inset shows the AFM height image with the S protein–C9t aptamer complexes (circled) resolved. Spectra recorded of the S protein–C9t complex (circled) exhibit a strong feature assigned to the amide I band, followed by a broad feature at lower wave numbers assigned to the amide II band of protein. The spectra are averaged from the four circled positions and four positions on the receptor layer. Full article ">Figure 3
Calibration curves. The calibration curves represent the response of the aptasensor in a concentration ranging from 1 fg/mL to 100 ng/mL of the target proteins: against (a) wild-type S protein of the SARS-CoV-2 virus in ferri/ferrocyanide solution and (b) wild-type S protein of the SARS-CoV-2 in a spiked negative control (non-infectious) nasal swab, (c) the S protein of the Omicron variant in ferri/ferrocyanide solution, and (d) the S protein of the Omicron variant in a spiked negative (non-infectious) control nasal swab. The overlay figure shows the Langmuir–Freundlich curve representation of the biosensor’s response without logarithmic axis modifications, whilst the inset shows the logarithmic representation of the obtained data, and the dashed line represents the LoD for the individual media–analyte combination. Hereby, all calibration curves were determined at 16 individual electrodes per chip, for three chips per condition. Full article ">Figure 4
Selectivity Tests for the Aptasensor. The aptasensor’s response to analog analytes (selectivity tests) was conducted at an analyte concentration of 10 ng/mL. The selectivity test was performed as the responses to (a) wild-type response for viral proteins of other respiratory viruses with similar symptoms, (b) different strains of the SARS-CoV-2 virus, and (c) between wild-type and the Omicron variant, in ferri/ferrocyanide solution (Buffer), as well as in spiked negative nasal swab samples (N-Swab). (d) Comparison of the detection response in buffer for Omicron S protein by the C9t aptasensor versus a previously reported malaria aptamer LDHp11 [<a href="#B54-biosensors-15-00024" class="html-bibr">54</a>]. Hereby, all selectivity tests were conducted at 16 individual electrodes per chip, for two chips per condition. Full article ">

Abstract

The drug detection technology plays a pivotal role in the domains of pharmaceutical regulation and law enforcement. In this study, we introduce a method that combines thermal desorption corona discharge ionization (TD-CDI) with mass spectrometry for efficient drug detection. The TD-CDI module, characterized by its compact and simple design, enables the separation of analytes within seconds and real-time presentation of one or two analyte peaks on the mass spectrum most of the time, which reduces matrix interference and improves detection performance. Through experimental investigation, we studied the characteristics of TD-CDI for analyte separation and detection, even with the same mass number, and optimized the TD-CDI approach. TD-CDI-MS was employed for the rapid detection of drugs in various traditional medicine, food products, and human samples. Additionally, by utilizing TD-CDI for segmented hair direct analysis, it becomes possible to trace the drug usage cycle of individuals. This underscores the feasibility of the proposed analytical method within the realm of drug detection. Full article

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