Split G-Quadruplexes Enhance Nanopore Signals for Simultaneous Identification of Multiple Nucleic Acids.

Assembly of DNA structures based on hybridization like split G-quadruplex (GQ) have great potential for the base-pair specific identification of nucleic acid targets. Herein, we combine multiple split G-quadruplex (GQ) assemblies on designed DNA nanostructures (carrier) with a solid-state nanopore sensing platform. The split GQ probes recognize various nucleic acid sequences in a parallel assay that is based on glass nanopore analysis of molecular structures. Specifically, we split a GQ into two asymmetric parts extended with sequences complementary to the target. The longer G-segment is in solution, and the shorter one is on a DNA carrier. If the target is present, the two separate GQ parts will be brought together to facilitate the split GQ formation and enhance the nanopore signal. We demonstrated detection of multiple target sequences from different viruses with low crosstalk. Given the programmability of this DNA based nanopore sensing platform, it is promising in biosensing.

Multiplex Sensing of Complex Mixtures by Machine Vision Analysis of TLC-SERS Images.

Thin layer chromatography in tandem with surface-enhanced Raman scattering (TLC-SERS) has demonstrated tremendous potentials as a new analytical chemistry tool to detect a wide range of substances from real-world samples. However, it still faces significant challenges of multiplex sensing from complex mixtures due to the imperfect separation by TLC and the resulting interference of SERS detection. In this article, we propose a multiplex sensing method of complex mixtures by machine vision analysis of the scanning image of the TLC-SERS results. Briefly, various pure substances in solution and the complex mixture solution are separated by TLC followed by one-dimensional SERS scanning of the entire TLC plate, which generates TLC-SERS images of all target substances along the chromatography path. After that, a machine vision method is employed to extract the template images from the TLC-SERS images of pure substance solutions. Finally, we apply a feature point matching strategy based on the Winner-take-all principle, which matches the template image of each pure substance with the mixture image to confirm the existence and derive the position of each target substance in the TLC plate, respectively. Our experimental results based on the mixture solution of five different substances show that the proposed machine vision analysis is highly selective, sensitive and does not require artificial analysis of the SERS spectra. Therefore, we envision that the proposed machine vision analysis of the TLC-SERS imaging is an objective, accurate, and efficient method for multiplex sensing of trace level of target substances from complex mixtures.

Recent advances in receptor-based optical biosensors for the detection of multiplex biomarkers.

Multiplex biosensors are highly sought-after tools in disease diagnosis. This technique involves the simultaneous sensing of multiple biomarkers, whose levels and ratios can provide a more comprehensive assessment of disease conditions compared to single biomarker detection. In most diseases like cancer due to its complexity, several biomarkers are involved in their occurrence. On the other hand, a single biomarker may be implicated in various diseases. Multiplex sensing employs various techniques, such as optical, electrochemical, and electrochemiluminescence methods. This comprehensive review focuses on optical multiplex sensing techniques, including surface plasmon resonance, localized surface plasmon resonance, fluorescence resonance energy transfer, chemiluminescence, surface-enhanced Raman spectroscopy, and photonic crystal sensors. The review delves into their mechanisms, materials utilized, and strategies for biomarker detection.

Multiplexed smFRET Nucleic Acid Sensing Using DNA Nanotweezers.

The multiplexed detection of disease biomarkers is part of an ongoing effort toward improving the quality of diagnostic testing, reducing the cost of analysis, and accelerating the treatment processes. Although significant efforts have been made to develop more sensitive and rapid multiplexed screening methods, such as microarrays and electrochemical sensors, their limitations include their intricate sensing designs and semi-quantitative detection capabilities. Alternatively, fluorescence resonance energy transfer (FRET)-based single-molecule counting offers great potential for both the sensitive and quantitative detection of various biomarkers. However, current FRET-based multiplexed sensing typically requires the use of multiple excitation sources and/or FRET pairs, which complicates labeling schemes and the post-analysis of data. We present a nanotweezer (NT)-based sensing strategy that employs a single FRET pair and is capable of detecting multiple targets. Using DNA mimics of miRNA biomarkers specific to triple-negative breast cancer (TNBC), we demonstrated that the developed sensors are sensitive down to the low picomolar range (≤10 pM) and can discriminate between targets with a single-base mismatch. These simple hybridization-based sensors hold great promise for the sensitive detection of a wider spectrum of nucleic acid biomarkers.

Smart Multiplex Point-of-Care Platform for Simultaneous Drug Monitoring.

Recently, illicit drug use has become more widespread and is linked to problems with crime and public health. These drugs disrupt consciousness, affecting perceptions and feelings. Combining stimulants and depressants to suppress the effect of drugs has become the most common reason for drug overdose deaths. On-site platforms for illicit-drug detection have gained an important role in dealing, without any excess equipment, long process, and training, with drug abuse and drug trafficking. Consequently, the development of rapid, sensitive, noninvasive, and reliable multiplex drug-detecting platforms has become a major necessity. In this study, a multiplex laser-scribed graphene (LSG) sensing platform with one counter, one reference, and three working electrodes was developed for rapid and sensitive electrochemical detection of amphetamine (AMP), cocaine (COC), and benzodiazepine (BZD) simultaneously in saliva samples. The multidetection sensing system was combined with a custom-made potentiostat to achieve a complete point-of-care (POC) platform. Smartphone integration was achieved by a customized application to operate, display, and send data. To the best of our knowledge, this is the first multiplex LSG-based electrochemical platform designed for illicit-drug detection with a custom-made potentiostat device to build a complete POC platform. Each working electrode was optimized with standard solutions of AMP, COC, and BZD in the concentration range of 1.0 pg/mL-500 ng/mL. The detection limit of each illicit drug was calculated as 4.3 ng/mL for AMP, 9.7 ng/mL for BZD, and 9.0 ng/mL for COC. Healthy and MET (methamphetamine) patient saliva samples were used for the clinical study. The multiplex LSG sensor was able to detect target analytes in real saliva samples successfully. This multiplex detection device serves the role of a practical and affordable alternative to conventional drug-detection methods by combining multiple drug detections in one portable platform.

Multiplex Sensing Based on Plasmonic Optics of Noble Metallic Nanostructures.

Since the colorimetric method has the characteristics of being simple and low cost, the fluorescence spectrum has the characteristics of a strong signal, and Surface-enhanced Raman scattering (SERS) detection has the characteristics of high sensitivity and strong specificity, people usually use these three methods for detection, but the detection of a single sample takes more time. If multiple samples can be tested at the same time, the detection efficiency and sensitivity can be improved, and the selectivity and reliability will be greatly improved. Multiplex sensing also provides a new direction for researchers. To fully understand the research of multiplex sensing based on the plasmonic optics of noble metal nanostructures, this review summarizes all the results previously reported in this field. It also discusses the principles of various detection methods and the biochemical application of multiple detections and finally summarizes the challenges and prospects.

Validation of an in vivo electrochemical immunosensing platform for simultaneous detection of multiple cytokines in Parkinson's disease mice model.

Parkinson's Disease (PD) is a neurodegenerative chronic disorder which destroys brain tissue and result in impaired movement. Early diagnosis of PD before the appearance of clinical symptom is vital for effective treatment. High levels of proinflammatory cytokines found in PD patient's brains, as natural inflammation response product, are potential biomarkers for PD detection in the early stage. Herein, we developed an in vivo electrochemical immunosensing device based on glassy carbon rod to simultaneously detect three proinflammatory cytokines (IL-1ß, IL-6 and TNF-α). The levels of IL-1ß, IL-6 and TNF-α secreted by N2a cells significantly increased within 24 h after lipopolysaccharide (LPS) stimulation. Under in vivo conditions, the concentrations of IL-1ß, IL-6 and TNF-α in PD model group achieved by injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally, were significantly higher than those in the control mouse group. The concentrations of three cytokines in vivo/vitro detected by this immunosensing device was comparable to that obtained by ELISA. Furthermore, this deployable immunosensing device was proved to be highly sensitive with the limits of detection (LODs) of 5 pg mL-1 for each cytokine, specific and reliable, suggesting its potential to be a universal immunosensing platform for early identification and diagnosis of PD in vivo in the future.

Data on characterization of glass biochips and validation of the label-free biosensor for detection of autoantibodies in human serum.

The data represent in-depth characterization of a novel method for highly sensitive simultaneous measuring in human serum of both critical parameters of autoantibodies: concentration and native kinetics. The latter refers to autoantibody interaction with free, not immobilized, antigen. The method and related biosensors are based on the spectral-correlation and spectral-phase interferometry. The data cover: multi-factor optimization and quantitative characterization of the developed affordable single-used biochips, including X-ray photoelectron spectroscopy (XPS) control of chemical modifications of the surface during fabrication; antibody screening; optimization and verification of protocols for label-free biosensing in human serum; mathematical model for fitting experimental data and calculation of kinetic constants of interaction of autoantibodies with free antigen; comprehensive verification of the method specificity; correlation between the data obtained with the developed biosensor and with enzyme linked immunosorbent assay (ELISA); comparison of analytical characteristics of the developed biosensor with the most advanced label-based methods. The data importance is confirmed by a companion paper (DOI 10.1016/j.bios.2020.112187), which shows that the combination of mentioned autoantibody parameters is promising for more accurate criteria for early diagnostics and efficient therapy of autoimmune disorders. The obtained data can be used in development of a wide range of biosensors, both label-free and based on various labels.

Electrochromic Sensor for Multiplex Detection of Metabolites Enabled by Closed Bipolar Electrode Coupling.

Biosensors based on converting electrochemical signals into optical readouts are attractive candidates as low-cost, high-throughput sensor platforms. Here, we described a closed bipolar electrode (CBE)-based two-cell electrochromic device for sensing multiple metabolites, using the simultaneous detection of lactate, glucose, and uric acid as a model system. In the two-cell configuration, an analytical cell contains a redox mediator combined with a specific oxidase, e.g., lactate oxidase, glucose oxidase, or uricase, to form an electrochemical mediator-electrocatalyst pair that supports redox cycling. A closed bipolar electrode couples the electron transfer event in the analytical cell to an electrochromic reaction in a separate reporter cell, such that the magnitude of the color change is related to the concentration of metabolites in the analytical cell. To demonstrate multiplex operation, the CBE-based electrochromic detector is modified by integrating three sets of detection chemistries into a single device, in which simultaneous determination of glucose, lactate, and uric acid is demonstrated. Device sensitivity can be tuned by using reporter cells with different volumes. Furthermore, the analytical cell of this device can be fabricated as a disposable, paper-based carbon electrode without any pretreatment, demonstrating the potential to screen phenotypes that require multiple biomarkers in a point-of-care format.